2013年02期目次

  • Construction and identificaiton of avian influenza H5N1 virus-like particles

    SHI Jin-rong,YANG Xiao-ming,DU Hui,QIU A-ming,WANG Ni-dan,HUANG Xiao-yuan Wuhan Institute of Biological Products Co.Ltd.,Wuhan 430060,Hubei Province,China

    Objective To construct a recombinant baculovirus containing hemagglutinin(HA),neuraminidase(NA) and matrix protein(M1) genes of avian influenza H5N1 virus NIBRG-14 strain,and investigate the biological characteristics of expressed product.Methods HA,NA and M1 gene fragments were amplified from NIBRG-14 strain by RT-PCR,and subcloned into baculovirus shuttle plasmid pFastBacDual simultaneously.The constructed recombinant baculovirus shuttle plasmid was transfected to Sf9 cells for packaging of recombinant baculovirus Bacmid-HA-NA-M1a.The expression of HA,NA and M1 proteins in Sf9 cells was determined by Western blot and hemagglutination test.The expressed recombinant protein was concentrated by ultracentrifugation and purified by sucrose density gradient centrifugation,then observed for morphology of virus-like particles(VLPs) by transmission electron microscopy and determined for HA content by single radiation immune diffusion(SRID) test.Results The recombinant baculovirus for co-expression of HA,NA and M1 protein was successfully constructed.The three kinds of proteins could be self-assembled into VLPs and secreted into supernatant,each with a hemagglutination titer of 1 ∶ 64.The VLPs assembled by expressed protein formed precipitation ring with standard antiserum against NIBRG-14.The HA contents of concentrated and purified recombinant protein were 36 and 27 μg / ml respectively.Typical VLPs of influenza virus,each at a diameter of about 100 nm,were observed under transmission electron microscope.Conclusion The constructed recombinant baculovirus may be used for expression of VLPs,which lays a foundation of preparation of novel avian influenza vaccine.

    2013 02 v.26 [Abstract][OnlineView][Download 380K]

  • Prokaryotic expression,purification and immunogenicity of truncated G protein of respiratory syncytial virus

    LI Guang,JING Shen-rong,TIAN Jun,ZENG Wei-kun,WEI Yun-lin College of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500, Yunnan Province,China

    Objective To construct a prokaryotic expression vector for truncated G protein of respiratory syncytial virus(RSV),and determine the immunogenicity of expressed and purified protein.Methods Overlapping PCR was used to substitute the CX3C motif nucleotides in G protein partly synthesized with one of CTL epitope nucleotides from M protein of RSV.Recombinant plasmids GCX3C / pET22b and GCTL / pET22b were constructed and transformed into E.coli BL21(DE3) for expression under induction of IPTG.The expressed product was identified by SDS-PAGE,and purified by nickel ion affinity chromatography.The purified protein was identified by SDS-PAGE and Western blot.Kunming mice were divided into three groups,and immunized with 100 μg GCX3C,100 μg GCTL and 100 μl PBS(negative control) respectively.Each mouse was injected i.m.for 3 times,at weeks 0,1 and 4 respectively.Venous blood samples were collected at weeks 1 ~ 5,once a week,and determined for IgG level and counted for eosinophils.RSV-specific CTL response was determined by in vitro lymphocyte killing test in mice.Results Restriction analysis and sequencing proved that the G gene in recombinant prokaryotic expression vector was successfully modified.Recombinant protein,with a relative molecular mass of about 14 000,contained about 40% of total somatic protein and mainly existed in soluble form.Purified recombinant protein reached a purity of more than 90%,and showed specific reaction with RSV antiserum.The specific IgG level in sera of mice increased with the increasing doses(P < 0.01).The GMTs of IgG of mice in GCX3C and GCTL groups were 1584.89 and 1995.26 respectively,while the eosinophil count of the former was significantly higher than that of the latter(P < 0.01).GCTL induced RSV-specific CTL response in mice.Conclusion GCX3C and GCTL proteins were successfully expressed in prokaryotic cells,both of which showed good immunogenicity.GCTL eliminated the increase of eosinophils caused by CX3C motif and enhanced the CTL response.Expected results was observed after modification of G gene.

    2013 02 v.26 [Abstract][OnlineView][Download 456K]

  • Expression of F1 fragment of human respiratory syncytial virus protein in E.coli and purification of expressed product

    PENG Zheng-hua,CAI Lu-kui,JI Qiu-yan,BI Yan-wei,LUO Na,XIAO Hong-jian, YAN Ling-mei,GAO Dan-dan,LI Zhi-hua Institute of Medical Biology,Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650118,Yunnan Province,China

    Objective To express the 137 ~ 523 aa of F1 protein of respiratory syncytial virus(RSV),in which major antigenic epitopes were centralized,in E.coli,purify the expressed product and determine its immunogenicity.Methods Human RSV F1 gene fragment was amplified from viral RNA by RT-PCR and inserted into vector pThioHisA.The constructed recombinant plasmid pThioHisA-RSV F1 was transformed to E.coli Top10 which was induced with IPTG.The expressed product was identified by SDS-PAGE,refolded by dilution and purified by ion exchange and affinity chromatography,then analyzed by SDS-PAGE and Western blot.ICR mice were divided into three groups,and immunized with 50 μg purified fusion protein RSV F1,50 μg RSV F1 + 5 μg nOMV adjuvant and 100 μl PBS(negative control) respectively,by subcutaneous injection in several sites and boosted at week 3.Venous blood samples were collected at week 4,from which sera were separated and determined for immunogenicity by indirect ELISA.Results Restriction enzymes analysis and DNA sequencing proved that recombinant plasmid pThioHisA-RSV F1 was constructed correctly.The expressed fusion protein,with a relative molecular mass of about 56 000,mainly existed in a form of inclusion body and contained about 36% of total somatic protein.The purified fusion protein reached a purity of 95% and showed specific reaction with polyclonal antibody against RSV F1.The serum specific IgG levels of mice immunized with RSV F1 were significantly higher than that in negative control group(P < 0.05).Conclusion RSV F1 was successfully expressed in E.coli,and the purified fusion protein showed good immunogenicity in mice,which laid a foundation of further study on RSV F protein subunit vaccine.

    2013 02 v.26 [Abstract][OnlineView][Download 440K]

  • Effect of dnaJ gene defect on natural immune response in mice infected with Streptococcus pneumoniae

    CUI Jin,DONG Jie,JIANG Hui,ZHOU Ai-e,DONG Shan-shan,ZHANG Xue-mei,YIN Yi-bing,WANG Hong Key Laboratory of Medical Diagnostics of Ministry of Education,Department of Laboratory Medicine,Chongqing Medical University,Chongqing 400016,China

    Objective To investigate the effect of dnaJ gene defect on natural immune response in mice infected with Streptococcus pneumonia(S.pn).Methods BALB / c mice were randomly divided into blank control,D39 infection and △dnaJ groups,and infected with 30 μl of sterile PBS,30 μl(2 × 107 cfu) of D39 strain and 30 μl(2 × 107 cfu) of △dnaJ strain(dnaJ gene defective strain) of S.pn by intranasal drip respectively to copy mouse model of S.pn infection.Four mice were killed 6,12,24,36 and 48 h after infection respectively,of which the lung tissues were collected and homogenized.The supernatants were collected and determined for the expression levels of TNF-α,IL-1β,IL-6 and IFNγ by ELISA.The inflammatory reactions in lung tissues of mice 12 h after infection were observed by HE staining.S.pn D39 and △dnaJ strains were infected to mouse macrophage RAW264.7 strain in vitro,each at a ratio of 100 ∶ 1.The culture supernatant of RAW264.7 strain was collected 1,2 and 3 h after infection and determined for expression levels of TNF-α and IL-6 by ELISA.The transcription levels of mRNAs of TLRs in mouse lung tissue 12 h after infection were determined by real-time quantitative PCR,while the phosphorylation level of p38MAPK by Western blot.Results As compared with those in D39 infection group,the lung inflammatory response of mice in △dnaJ infection group was down-regulated,the appearance of secretion peaks of TNF-α,IL-6 and IL-1β were delayed,and the peak values decreased,while the secretion levels of TNF-α and IL-6 3 h after infection with RAW264.7 cells decreased significantly(P < 0.01 or P < 0.05);meanwhile,the transcription levels of Tlr2 and Tlr13 mRNAs(P < 0.05) as well as the phosphorylation level of p38MAPK decreased significantly.Conclusion The dnaJ gene defect of S.pn down-regulated the inflammatory response,secretion of pro-inflammatory cytokines and activation of celluar signal molecules,and affected the expressions of Tlr2 and Tlr13 mRNAs in lungs of mice infected with S.pn,which laid a foundation of further study on the molecular mechanism of natural immune response to DnaJ protein of S.pn.

    2013 02 v.26 [Abstract][OnlineView][Download 664K]

  • Protection level of neutralizing antibody against enterovirus 71

    MAO Qun-ying,GAO Fan,HAO Chun-sheng,DONG Cheng-hong,GAO Qiang,WANG Chao, YAO Xin,WU Xing,SHAO Jie,ZHU Feng-cai,LIANG Zheng-lun,WANG Jun-zhi National Institutes for Food and Drug Control,Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products,Beijing 100050,China

    Objective To measure the neutralizing antibody titer against enterovirus 71(EV71) in suckling mouse model,healthy infants and the target population after immunization and provide a basic data for determination of protection level of EV71 vaccine.Methods The neutralizing antibodies in six serum samples against various EV71 strains were quantified accurately,and evaluated for protection level by challenge test in two suckling mouse models.The neutralizing antibodies in serum samples of healthy infants and the target population after immunization were quantified accurately by standardized neutralization test in vitro and the standard substances for quantification of EV71 neutralizing antibody.Results The protection levels of EV71 neutralizing antibodies in 6 serum samples were similar in challenge tests in two suckling mouse models,with ED50 values of 10 ~ 50 U and 6.1 ~ 54.2 U,respectively.The antibody positive rates of infants at ages of 7,12 and 24 months were 45.0%,48.8% and 56.8%,while the neutralizing antibody titers in antibody positive infants were 113.8,120.3 and 330.6 U / ml(with a GMT of 173.2 U / ml),respectively.However,the antibody positive rate and the neutralizing antibody titer were 86.7% and 114.3 U / ml respectively in the infant's mothers.After immunization with two doses of vaccine at high and moderate dosages,the neutralizing antibody positive rates in infants were 100%,while the neutralizing antibody titers were 356.0 and 1 136.2 U / ml,respectively,which were 2.1 ~ 6.6 times higher than those in infants infected naturally.Conclusion The protection levels of EV71 neutralizing antibody on suckling mouse model,infants and the target population after EV71 vaccination were quantified as the unit of U / ml by using standardized neutralization test in vitro and the standard substances for quantification of EV71 neutralizing antibody,which provided a basis for determination of protection level of neutralizing antibody in vaccinated population.

    2013 02 v.26 [Abstract][OnlineView][Download 427K]

  • Fusion expression and purification of fusion protein of S.agalactiae GapC and S.typhimurium FliC

    WANG He,LIANG Hong-ru,HU Xu,ZHAO Da,JIANG Dong-jun,YIN Hui,GAO Jia-bin, CHEN Wei-hong,CUI Yu-dong,ZHU Zhan-bo College of Animal Science and Veterinary Medicine,Heilongjiang Bayi Agricultural University, Daqing 163319,Heilongjiang Province,China

    Objective To construct a fusion gene gapC-fliC of S.typhimurium fliC gene and S.agalactiae gapC gene,and express in E.coli.Methods The fliC and gapC genes were amplified by PCR respectively using the genomic DNA of S.typhimurium and plasmid of gapC-pQE30 as template,based on which fusion gene fliC-gapC was amplified by overlap PCR using the linker of(Gly4Ser)2 coding sequence,and cloned into vector pQE30(+).The constructed recombinant plasmid fliC-gapC-pQE30 was transformed to E.coli XL1-Blue and induced with IPTG,and the expressed product was identified by SDS-PAGE,purified by nickel ion affinity chromatography,and analyzed for activity by Western blot.Results Restriction analysis and sequencing proved that recombinant plasmid fliC-gapC-pQE30 was constructed correctly.The expressed fusion protein,with a relative molecular mass of about 95 000,contained about 58% of total somatic protein,mainly existed in a form of inclusion body,and showed specific reactions with both mouse polyclonal sera against S.typhimurium and S.agalactiae.Conclusion Recombinant plasmid flic-gapC-pQE30 was successfully constructed,and fusion protein was expressed in E.coli XL1-Blue,which laid a foundation of animal immune protection test in further study.

    2013 02 v.26 [Abstract][OnlineView][Download 515K]

  • Expression and characterization of carboxypeptidase mature peptide gene from Aspergillus usamii

    MIN Rou,HUANG Fang,ZENG Yan,WU Min-chen,WU Jing,CHEN Wei School of Medicine and Pharmaceutics,Jiangnan University,Wuxi 214122,Jiangsu Province,China

    Objective To clone and express the carboxypeptidase mature peptide gene of Aspergillus usamii and analyze its enzymatic property.Methods Primers were designed based on the previously cloned carboxypeptidase sequence of Aspergillus usamii,with which carboxypeptidase mature peptide gene AucpA was cloned and inserted into vector pPIC9K.The constructed recombinant plasmid pPIC9K-AucpA was transformed to Pichia pastori GS115 by electroporation for expression under induction of methanol.The expressed protein was purified by Sephadex-50 chromatography,and determined for activity and its influencing factors.Results Restriction analysis and sequencing proved that recombinant plasmid pPIC9K-AucpA was constructed correctly.The expressed recombinant carboxypeptidase,with a relative molecular mass of 81 000,reached a specific activity of 57.15 nkat / mg at most,of which the optimal temperature and pH value were 45 ℃ and 3.5 respectively.Metal ions and EDTA showed no effect on the activity of recombinant carboxypeptidase.However,the inhibiting rate of PMSF to the activity was more than 50%.Conclusion The carboxypeptidase gene of Aspergillus usamii,with a high activity,was successfully expressed in P.pastoris,which laid a foundation of further study on its application.

    2013 02 v.26 [Abstract][OnlineView][Download 490K]

  • Construction and identification of eukaryotic expression vector for Flap endonuclease 1 gene

    PAN Wan-long,FANG Yan,XU Ge,SHAN Xue-feng,XU Lei,TAO Ying,HUANG Yuan,HU Jie-li Key Laboratory of Molecular Biology on Infection Diseases,Ministry of Education,Chongqing Medical University,Chongqing 400016,China

    Objective To construct and identify the eukaryotic expression vector for flap endonuclease 1(FEN1) gene.Methods Total RNA was extracted from L02 cells and reversely transcribed to cDNA,with which FEN1 gene was amplified by RT-PCR and cloned into the eukaryotic expression vector pcDNA3.1.The constructed recombinant plasmid was identified by restriction analysis and sequencing,then transfected to 293T cells.The expressed FEN1 was identified by Western blot.Results Restriction analysis and sequencing proved that FEN1 gene was successfully inserted into plasmid pcDNA3.1.The target protein band with a relative molecular mass of about 47 000 was observed on Western blot profile.As compared with those in 293T cells transfected with empty vector and the 293T cells untransfected,the expression level of FEN1 in 293T cells transfected with the constructed recombinant plasmid increased by about 3 folds.Conclusion The eukaryotic expression vector for FEN1 was successfully constructed,and FEN1 was over-expressed in 293T cells.

    2013 02 v.26 [Abstract][OnlineView][Download 412K]

  • Construction and identification of recombinant lentivirus for high expression of transcription factor ZNF191

    LIANG Yan-jie,LIU Dong-yang,LU Fan,LI Jian-zhong Graduate Management Battalion,The Second Military Medical University,Shanghai 200433,China

    Objective To construct a recombinant lentivirus expression vector for transcription factor ZNF191 and to evaluate its potential role in gene therapy of tumor.Methods ZNF191 cDNA was amplified by RT-PCR using the total RNA isolated from HEK293 cells as a template,and subcloned into plasmid pLVX-AcGFP-N1.The constructed recombinant lentivirus plasmid pLVX-ZNF191 was identified by restriction analysis and sequencing,then transfected to 293T cells in combination with helper plasmid for packaging.The expression of ZNF191 protein in 293T cells infected with the lentivirus was determined by Western blot.Results Recombinant plasmid pLVX-ZNF191 was constructed correctly as proved by restriction analysis and sequencing.Western blot showed that the expression level of ZNF191 protein in HEK293T cells infected with recombinant lentivirus increased significantly.Conclusion Recombinant lentivirus expression vector pLVX-ZNF191 was successfully constructed,which laid a foundation of further study on the biological function of ZNF191 and its potential role in gene therapy of tumors.

    2013 02 v.26 [Abstract][OnlineView][Download 402K]

  • Cloning and prokaryotic expression of vjbR gene of Brucella melitensis

    BU Zhao-yang,WANG Jing-long,LI Xiao-yan,WAN Li-na,LANG Xu-long,WAN Zhong-hai, MENG ke-yin,WANG Xing-long Institute of Military Veterinary,Academy of Military Medical Sciences,Changchun 130122,Jilin Province,China

    Objective To clone the vjbR gene of virulent Brucella melitensis 16M strain and express in prokaryotic cells.Methods The vjbR gene of 16M strain was amplified by PCR and subcloned into prokaryotic expression vector pET-28a.The constructed recombinant plasmid pET-28a-vjbR was transformed to competent E.coli BL21(DE3) and induced with IPTG.The expressed recombinant protein was purified by His Trap FF column chromatography and analyzed by Western blot.Results The length of amplified vjbR gene was 708 bp,which was consistent with that expected.Restriction analysis and sequencing proved that recombinant plasmid pET-28a-vjbR was construced correctly.The expression level of recombinant protein,with a relative molecular mass of about 29 000,reached the maximum of 6.84 mg / ml 4 h after induction.The purified recombinant protein reached a purity of 65.7% and was recognized by B.melitensis-positive sera.Conclusion The VJBR protein of B.melitensis was successfully expressed,which laid a foundation of study on function of the protein,preparation of diagnostic kit for B.melitensis infection and development of subunit vaccine.

    2013 02 v.26 [Abstract][OnlineView][Download 404K]

  • Kinetics of IgA-secreting cells and sIgA antibody in small intestine of mice induced with heat shock protein 70 of Toxoplasma gondii by various immunization routes

    YIN Li-tian,SHEN Jin-yan,MENG Xiao-li,WANG Hai-long,LIU Hong-li,YIN Guo-rong Department of Physiology,Key Laboratory of Cellular Physiology Co-constructed by Province and Ministry of Education,Shanxi Medical University,Taiyuan 030001,Shanxi Province,China

    Objective To investigate the kinetics of IgA antibody-secreting cells(IgA-ASCs) in small intestinal mucosa of and specific sIgA level in small intestine washes of mice induced with recombinant Toxoplasma gondii heat shock protein 70(rTgHSP70) by intranasal and subcutaneous immunizations,and analyze the correlation between IgA-ASCs and sIgA level as well as their roles in up-regulation of mucosal immune response in small intestine.Methods Ninety BALB / c mice were randomly divided into 3 groups.The mice in intranasal immunization group were immunized twice with 20 μg rTgHSP70 by intranasal drip at an interval of 2 weeks,and those in subcutaneous immunization group were injected s.c.with two times of 80 μg rTgHSP70 at an internal of 2 weeks,while those in control group were unimmunized.Five mice in each group were killed at weeks 1,2,3,4,5 and 6 after the last immunization respectively,of which the quantity of IgA-ASCs in mucosa of duodenum,jejunum and ileum were determined by immunohistochemical assay,and the sIgA level in intestinal washes by ELISA.Results The IgA-ASCs were found in lamina propria of small intestine mucosa.The quantity of IgA-ASCs in small intestine mucosa of mice immunized by intranasal drip was significantly higher than those in subcutaneous immunization and control groups(P < 0.001).The sIgA level in intestinal washes of mice increased 1 ~ 4 weeks and reached the maximum at week 4 after immunization by intranasal drip,which were significantly higher than those in subcutaneous immunization and control groups(P < 0.001).The sIgA level in intestinal washes of mice in intranasal and subcutaneous immunization groups were positively related to the quantity of IgA-ASCS in duodenum,jejunum and ileum mucosa lamina propria.Conclusion Immunization with rTgHSP70 by both intranasal and subcutaneous routes induced sustained high levels of IgA-ASCs in intestinal mucosal lamina propria and sIgA in intestinal washes.Positive relation was observed between the two levels.However,intranasal immunization approach is superior to subcutaneous injection in up-regulation of mucosal immunization in small intestine.

    2013 02 v.26 [Abstract][OnlineView][Download 497K]

  • Prokaryotic expression and purification of leucine aminopeptidase of Schistosoma japonicum

    ZHONG Zheng-rong,XU Yun-xia,GONG Ji-yong,TIAN Wan-ling,GUO Pu,LI Xiao-yue,LUO Qing-li,SHEN Ji-long Department of the Clinical Medical Laboratory,The First Affiliated Hospital of Bengbu Medical College, Bengbu 233004,Anhui Province,China

    Objective To express leucine aminopeptidase(LAP) of Schistosoma japonicum(Sj) in prokaryotic cells and purify the expressed product.Methods LAP gene was amplified from the adult worm of Sj by RT-PCR and inserted into prokaryotic expression vector pET-28a.The constructed recombinant plamid pET-28a / SjLAP was transformed to competent E.coli for expression under induction of IPTG.The expressed recombinant SjLAP(rSjLAP) was identified by SDS-PAGE and Western blot,then purified by His Binding Purification Kit,and analyzed for purity by SDS-PAGE,and for concentration by Bradford method.Results Compared with the SjLAP gene reported in GenBank,two base substitutions were observed in the cloned gene,resulting the substitutions of two corresponding amino acids.However,neither the substitutions was located in dominant domain.Restriction analysis proved the recombinant plasmid pET-28a / SjLAP was constructed correctly.The expression level of rSjLAP,with a relative molecular mass of about 45 000,reached the maximum 4 h after induction.The purified rSjLAP reached a high purity and a concentration of 5.0 mg / ml,and was recognized by monoclonal antibody against mouse His-tag as well as human and rabbit sera infected with Sj,indicating a high reactogenicity.Conclusion Recombinant SjLAP was successfully expressed in E.coli and reached a high purity after purification,which laid a foundation of diagnosis and prevention of schistosomiasis.

    2013 02 v.26 [Abstract][OnlineView][Download 486K]

  • Differentiation of rat marrow stroma cells into neuron-like cells induced by resveratrol in vitro

    HUANG Jia-gui,SHEN Chang-bo,LIU Shu,XU Lan,YANG Qin Department of Neurology,The First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China

    Objective To investigate the differentiation of rat bone marrow stromal cells(MSCs) into neuron-like cells induced by resveratrol in vitro.Methods MSCs were isolated from the whole bone marrow of rats by adherent culture in vitro.The MSCs of passage 3 were divided into resveratrol,control and normal groups.The MSCs were pre-induced with DMEM / F12 containing 10 μg / L bFGF for 24 h,and induced with DMEM / F12 containing 15 μmol / L resveratrol for 6 h,then added with maintain medium(DMEM / F12 containing 15 μmol / L resveratrol,10 μg / L bFGF and 2% B27) and further cultured until 72 h.The MSCs in control group were pre-induced by the same method,the induced with DMEM / F12 for 6 h,added with maintain medium(DMEM / F12 containing 10 μg / L bFGF and 2% B27) and further cultured until 72 h.However,the MSCs in normal group were untreated.The MSCs were observed for morphology under invert microscope 72 h after induction,in which the expression levels of nestin and NSE proteins and mRNAs were determined by indirect IFA,Western blot and RT-PCR before and 2,6,24 and 72 h after induction.Results The MSCs after induction with resveratrol were shrunk,on which neuron-like enations were observed.IFA showed that the MSCs after induction were positive for nestin and NSE,while those in control group were negative.The expression levels of nestin and NSE proteins and mRNAs in MSCs induced with resveratrol were significantly higher than those in control group.The expression levels of nestin protein and mRNA reached the peak values 2 h after induction(P < 0.01) then decreased gradually.However,the expression levels of NSE protein and mRNA increased gradually and reached the peak values 72 h after induction(P < 0.01).The expression levels in control group showed no significant change.Conclusion Resveratrol induced the differentiation of MSCs into neuron-like cells in vitro,which provided an experimental basis for application of resveratrol to stem cell transplantation.

    2013 02 v.26 [Abstract][OnlineView][Download 657K]

  • Construction of retroviral vector for over-expression of mouse Hes1 gene and its expression in mouse Lin- hematopoietic cells

    TIAN Chen,ZHENG Guo-guang,ZHANG Yi-zhuo,LI Qiao,YUAN Wei-ping,CHENG Tao Tianjin Key Laboratory for Prevention and Treatment of Tumors,Department of Haematology,Tumor Hospital Affiliated to Tianjin Medical University,Tianjin 300060,China

    Objective To construct a retroviral vector for over-expression of mouse Hes1 gene,and determine the infection efficiency through infection of relatively primitive mouse Lin-hematopoietic cells.Methods Hes1 domain was amplified from the bone marrow cells of B6 mice by RT-PCR and inserted into retroviral vector MSCV-ICN1-IRES-GFP.The constructed recombinant retroviral vector MSCV-Hes1-IRES-GFP was transfected to 293T cells.The transfection efficacy was determined by flow cytometry,using those of cells transfected with empty vector as control.for the expression level of Hes1 was determined by realtime PCR.Recombinant retroviral vector MSCV-Hes1-IRES-GFP and empty vector were mixed with Lipofectmaine 2000 respectively,and transfected to 293T cells for packaging of recombinant retrovirus.Mouse Lin-hematopoietic cells were stably infected with recombinant retrovirus,of which the transfection efficacy was determined by flow cytometry,using those infected with empty vector virus as control.The expression level of Hes1 in GFP+ cells was determined by realtime PCR.Results DNA sequencing confirmed that the recombinant retroviral vector was constructed correctly.The transfection efficacy of recombinant retrovirus in 293T cells on day 3 after tranfection was about 95%,while the expression level of Hes1 was about 5 times of that in control group.The infection efficacy of recombinant retrovirus in Lin-cells on days 4 and 5 after infection with virus supernatant was about 7%,while the expression level of Hes1 gene was 6 times of that in control group.Conclusion Recombinant retrovirus vector for over-expression of Hes1 gene was successfully constructed and expressed stably in mouse Lin-cells,which laid a foundation of study on effect of Hes1 on hemopoietic stem / progenitor cells of patients with leukemia.

    2013 02 v.26 [Abstract][OnlineView][Download 520K]

  • Bioinformatics of human interleukin-29

    ZHENG Hai-jun,ZHU Rong,GE Chun-lei,CAI Xian,XU Wang,CHEN Wei,LU Yuan, School of Biotechnology,Jiangnan University,Wuxi 214122,Jiangsu Province,China

    Objective To analyze the bioinformatics of human interleukin-29(hIL-29) and lay a foundation of directed mutation of hIL-29 so as to improve its bioactivity.Methods The cloned hIL-29 gene was analyzed by bioinformatics tools.The physicochemical characteristics,structures,functions,and the key amino acid sites of protein encoded by the gene were predicted.The three-dimensional structure of the protein was predicated and compared with the crystal structure of IFNλ3.The homologies of IL-29 genes of nine species were compared,based on which a phylogenetic tree was plotted.Results The protein encoded by hIL-29 gene consisted of 200 amino acids,with a signal peptide containing 19 amino acids at N-terminus.The relative molecular mass of mature peptide was 20 018,while the theoretical isoelectric point was 9.08.The hIL-29 protein contained an intracellular-extracellular transmembrane domain.The spatial conformation was composed with six α-helixes(A to F) and some random coils.The helixes A,B and F might be the activity sites in which hIL-29 was bound to its specific receptor and gave play to its biological effect.It was predicted that four key amino acid sites might exist in helixes A and F.The IL-29 from human origin were highly homologous to those form dog,cat,panda,horse,wild boar,chimpanzee and gibbon origins,which belonged to the same big branch on phylogenetic tree.However,the IL-29 gene from human,chimpanzee and gibbon origins belonged to the same small branch,of which the relationship was the closest.Conclusion The variation of amino acids at key sites might have significant effect on bioactivity of hIL-29,indicating that the sites might be used as the mutation sites for molecular modification of hIL-29.It laid a theoretical foundation of further study on directed mutation as well as relationship between structure and function of hIL-29.

    2013 02 v.26 [Abstract][OnlineView][Download 1058K]

  • Endothelial injury and OX40L expression of human umbilical vein endothelial cells stimulated by oxidized low-density lipoprotein

    DONG Qian,MA Kang-hua,WU Qiao Department of Cardiology,First Affiliated Hospital of Chongqing Medical University,Chongqing 400016,China

    Objective To observe the endothelial injury and the OX40L expression of human umbilical vein endothelial cells(HUVECs) stimulated by oxidiaed low-density lipoprotein(ox-LDL).Methods HUVECs were treated with ox-LDL at various concentrations,using those with the medium at an equal volume of ox-LDL as control.The effect of various concentrations of ox-LDL on proliferation of HUVECs was determined by CCK-8 method,while the cell cycle and apoptosis rate of HUVECs treated with 100 mg / L ox-LDL by flow cytometry,the expression levels of OX40L in HUVECs treated with ox-LDL at various concentrations(50,100 and 150 mg / L) by Western blot,and the location of OX40L in HUVECs treated with 100 mg / L ox-LDL by IFA and laser confocal microscopy.Results As compared with those in control group,the proliferative activity of HUVECs showed a significantly dose-dependent decrease(P < 0.01).The cell cycle of HUVECs treated with 100 mg / L ox-LDL was arrested at S stage,of which the apoptosis rate increased significantly as compared with that in control group(P < 0.05).The expression level of OX40L in HUVECs increased gradually with the increasing concentration of ox-LDL(P < 0.05).The OX40L in HUVECs treated with 100 mg / L ox-LDL showed a significantly increased fluorescence intensity(P < 0.01),which was located in cell membrane and cytoplasma.Conclusion The ox-LDL damaged HUEVCs significantly,while promoted the expression of OX40L and affected OX40 / OX40L inflammatory signaling pathway.

    2013 02 v.26 [Abstract][OnlineView][Download 549K]

  • Prediction of secondary structure and B-cell antigenic epitopes of Mycoplasma suis ORF2 protein

    SHAN Si,BA Cai-feng Center of Laboratory Animals,Liaoning Medical College,Jinzhou 121001,Liaoning Province,China

    Objective To predict the secondary structure and B-cell antigenic epitopes of Mycoplasma suis ORF2.Methods The nucleotide sequence of ORF2 of M.suis was translated to amino acid sequence by using the Editseq program in DNAstar software,and compared with that reported in GenBank,based on which the secondary structure,hydrophilic region,flexible region,antigenic index,surface probability as well as prominent antigenic epitopes of B cells were predicted by Protean module.Results The ORF2 protein showed regular secondary structure,abundant hydrophilic and flexible regions,as well as the regions with large surface probability and the high antigenic index.The potential prominent antigenic epitopes of B cells were located in 17 ~ 23,121 ~ 127,138 ~ 144 and 192 ~ 200 aa.Conclusion The secondary structure and prominent antigenic epitopes of B cells in ORF2 of M.suis was predicted,which provided a theoretical basis for design of M.suis epitope vaccine and development of serological diagnostic kit.

    2013 02 v.26 [Abstract][OnlineView][Download 659K]

  • Plasmid stability and viable count of recombinant Lactobacillus casei during storage period

    CAO Ning,HOU Xi-lin,TAN Jun,LIU Cai-xia,WU Long-yu,YU Li-yun College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163319, Heilongjiang Province,China

    Objective To observe the plasmid stability and viable count of recombinant Lactobacillus casei with enterotoxigenic E.coli(ETEC) gene during storage period.Methods Recombinant L.casei pLA-K88 / L.casei was inoculated to MRS medium then transferred to WPC medium,and the bacterial liquid was collected and stored at 4,15 and 37 ℃ respectively.The stabilities of recombinant plasmid after storage at 4 ℃ for 1,15,30,60,90 and 120 d were tested by colony counting on MRS(+) and MRS(-) plates and PCR.The immune activity of foreign protein was determined by Dot ELISA.The viable counts of recombinant L.casei 1,2,3,7,15,30,60,90,120,210 and 240 d after storage at 4,15 and 37 ℃ were determined by plate counting.Results The recombinant plasmid showed high stability within 120 d after storage at 4 ℃.No target gene deletion was observed,while the activity of foreign protein was stable.Lactoalbumin showed no significant effect on the stability of recombinant L.casei or the immunogenicity of foreign protein.The recombinant L.casei stored at 4 ℃ survived for the longest time,of which the viable count 240 d after storage was more than 9 × 106 cfu / ml.However,the storage periods at 15 and 37 ℃ were only 90 and 15 d respectively.Conclusion The plasmid in recombinant L.casei with ETEC gene showed high stability at 4 ℃,while the foreign gene was expressed stably.

    2013 02 v.26 [Abstract][OnlineView][Download 528K]

  • Procyanidin regulates lipid metabolism through gut microbiota

    FU Ying,MEI Song,LIU Dong-ying,WU Jiu-sheng Zhejiang Academy of Medical Sciences,Hangzhou 310013,Zhejiang Province,China

    Objective Objectives To investigate the possible mechanism of procyanidin(PC) in regulating lipid metabolism through gut microbiota.Methods Male adult SD rats and Meriones unguiculatus were fed with basal forage for one week,of which the serum samples were collected and determined for total cholesterol(TC) level.These animals were divided into six groups according to their TC levels and bodyweights.The animals in normal control group were fed with common forage,while those in the other five groups with high lipid forage.The animals in three test groups were treated with PC by oral route at low(25 mg / kg),moderate(100 mg / kg) and high(150 mg / kg) dosages respectively,while those in positive control group with Fenofibrate at a dosage of 80 mg / kg,once a day for 8(rats) and 2(M.unguiculatus) weeks.The rats were killed at end of 8 weeks,while M.unguiculatus at end of 2 weeks,of which serum samples were collected.However,the fecal samples of animals within 72 h before being killed were collected.The TC,triglyceride(TG),high density lipoproteinprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C) and total bile acid(TBA) levels in sera were determined by full-automatic biochemical analyzer,while the TBA level in feces by enzymatic cycling assay kit,and the lecithin cholesterol acyl transferase(LCAT) activity by two-step double antibody sandwich ELISA.The histopathology of killed animals was observed.The genomic DNAs were extracted from bacteria in small intestines and intestinum caecum of the animals,and analyzed for the changes of predominant bacteria of gut microbiota by PCR-denaturing gradient gel electrophoresis(DGGE).Results The TC and TG levels of animals in model control group were significantly higher than those in normal control group(P < 0.05),indicating successful establishment of animal model of hyperlipidemia.As compared with those in model control group,the serum TC,TG,LDL-C and TBA levels in three test groups decressed significantly,while LCAT activity and fecal TBA level increased significantly(each P < 0.05).The liver lesion,hepatic cell swelling and degeneration in three test groups significantly improved.DGGE and imaging showed that the diversity of predominant bacteria from gut microbiota in three test groups increased significantly,while decreased significantly with the increasing dosage of TC.The bacterial profile of M.unguiculatus treated with 25 mg / kg PC was similar to that in model control group.However,the diversity of gut microbiota decreased significantly,and the predominant bacteria showed an obvious tendency of restoration,in 100 and 150 mg / kg group.Conclusion The predominant bacterial profile of gut microbiota of experimental animal model showed an obviouse tendency of restoration after intervention by PC and dosages of 100 and 150 mg / kg,indicating the PC might regulate the lipid metabolism via gut microbiota pathway.

    2013 02 v.26 [Abstract][OnlineView][Download 605K]

  • Role of Hedgehog signaling pathway in angiopoiesis in vitro of human gastric cancer SGC-7901 cells

    KOU Yao,AN Chang-yong,CHEN Zhi-xiong,ZHANG Liu-ping,OUYANG Xiao-bo,WEI Zheng-qiang Department of Gastrointestinal Surgery,The First Affiliated Hospital of Chongqing Medical University, Chongqing 400016,China

    Objective To investigate the role of Hedgehog(Hh) signaling pathway in angiopoiesis of human gastric cancer SGC-7901 cells in vitro as well as the relevant mechanism.Methods SGC-7901 and Human umbilical vein endothelial cell(HUVEC) were cultured by routine method.SGC-7901 cells were divided into control group A and test groups B and C.The cells in group A were untreated,while those in groups B and C with cyclopamine at final concentrations of 5 and 10 μmol / L respectively,and further cultured for 48 h.The expressions of glioma-associated oncogene homolog(Gli1) and platelet-derived growth factor D(PDGF-D) in SGC-7901 cells of various groups were determined by IFA,while the ability of angiopoiesis before and after treatment by tube formation assay and vasculogenic mimicry(VM) formation assay.The expressions of Gli1 and PDGF-D at mRNA and protein levels were determined by RT-PCR and Western blot respectively,between which the relationship was analyzed.Results Gli1 and PDGF-D were expressed in SGC-7901 cells of various groups.As compared with those in control group,the abilities of angiopoiesis in vitro of SGC-7901 cells in test groups showed a dose-dependent decrease(P < 0.05),while the expressions of Gli1 and PDGF-D at mRNA and protein levels decreased significantly(P < 0.05).However,the expressions of Gli1 at both mRNA and protein levels were positively related to those of PDGF-D,with r values of 0.829 and 0.786 respectively(P < 0.05).Conclusion Hh signaling pathway may promote the angiopoiesis of tumor by regulating the expression of PDGF-D with Gli1.

    2013 02 v.26 [Abstract][OnlineView][Download 700K]

  • Relationship between therapeutic effect of glucocorticoids or combination of antibacterial drugs on sepsis in rats and serum level of quinolinic acid

    QIU Hong-mei,XIONG Ying,ZHOU Qi-xin Department of Pharmacology,Chongqing Key Laboratory of Biochemistry and Molecular Pharmacology, Chongqing Medical University,Chongqing 400016,China

    Objective To investigate the relationship between therapeutic effect of dexamethasone or combination of antibacterial drugs(amoxicillin,gentamicin and metronidazole) on sepsis in rats and the serum level of quinolinic acid(QUIN).Methods Rat model of sepsis was copied by cecum ligation and puncture.The model rats were treated by dexamethasone and combination of antibacterial drugs(amoxicillin + gentamicin + metronidazole) respectively,using those receiving sham operation as control.The deaths of rats were recorded 4,12,36,72 and 144 h after operation,based on which the survival rate was calculated.The total white blood cells(WBCs) and neutrophils were counted,and the serum QUIN level was determined by gas chromatography-mass spectrometry.Results Both the WBC count and percentage of neutrophils in model rats increased significantly 4 h and reached a peak value 12 h after operation when deaths appeared.However,the serum QUIN level increased significantly 12 h and reached a peak value 72 h after operation,while maintained at a high level afterwards.The combination of antibacterial drugs decreased the death rate,WBC count and percentage of neutrophils significantly,and inhibited the increase of serum QUIN level significantly.However,the death rate of rats treated with dexamethasone alone showed no significant decrease,while the WBC count and QUIN level decreased significantly,and the percentage of neutrophils increased.Conclusion Serum QUIN level was closely related to the inflammatory progress of sepsis,which might be used as an effective index for prediction of prognosis of the disease.

    2013 02 v.26 [Abstract][OnlineView][Download 559K]

  • Prokaryotic expression and biologic characteristics of recombinant human interferon α2a protein in intein-mediated system

    WAN Yi,ZHANG Kun,ZHANG Yue-juan,HE Li-qing,ZHAO Ning,ZI Jing Research Center of Molecular Biology,Shaanxi Microbiology Institute,Xi'an 710043,Shaanxi Province,China

    Objective To express recombinant human interferon α2a protein(rhIFNα2a) in CBD(chitin-binding-domain)-intein / pTWIN 1 expression system and determine its biological activity.Methods The coding sequence of rhIFN-α2a was amplified by PCR using plasmid pBV220-IFNα2a as a template and inserted into expression vector pTWIN1 fused in frame with an upstream Ssp DnaB intein gene.The constructed recombinant plasmid pTWIN1-rhIFNα2a was transformed into E.coli BL21(DE3),and protein expression was induced with IPTG.The expressed product was identified by SDS-PAGE and Western blot,and the biological activity of lysis supernatant was determined.Results Both restriction analysis and sequencing proved that the fusion gene was cloned into expression vector pTWIN1.The expressed rhIFNα2a,with a relative molecular mass of about 44 300,contained 30% of total somatic protein,and reached an expression level of 25% in lysis supernatant.The expressed product showed specific binding to mouse McAb against hIFN.The rhIFNα2a activity was 5.57 × 107 IU / ml in lysis supernatant,indicating significantly antiviral activity of the recombinant protein.Conclusion Soluble fusion protein of rhIFNα2a and Ssp DnaB,with biological activity,was successfully expressed in E.coli BL21(DE3),which provided a new idea for large-scale production of IFNα2a.

    2013 02 v.26 [Abstract][OnlineView][Download 633K]

  • Preparation and identification of polyclonal antibody against phosphoprotein of Borna disease virus

    MA Li-hua,HUANG Rong-zhong,ZHANG Liang,XU Xiao-yan,Zeng Li,LIU Zhao, DENG Jing,LI Wen-juan,XIE Peng Department of Neurology,The First Affiliated Hospital,Chongqing Medical University,Chongqing 400016,China

    Objective To prepare and identify the polyclonal antibody against recombinant phosphoprotein of Borna disease virus(BDV),and provide a basis for development of seroimmunological detection method for BDV.Methods New Zealand rabbits were immunized with the recombinant BDV p24 prepared previously by subcutaneous injection in several sites for 4 times,of which the sera were collected on day 7 after the last immunization,purified by affinity chromatography,determined for titer by ELISA,and identified for specificity by Western blot and IFA.Results The prepared polyclonal antibody against BDV p24 showed a purity of 98%,an ELISA titer of 1︰128 000,and specific reaction with BDV phosphoprotein expressed either in prokaryotic or in eukaryotic cells.Conclusion The polyclonal antibody against BDV p24,with high sensitivity and specificity,was successfully prepared,which laid a foundation of development of relevant diagnostic kits and study on pathogenic mechanism of BDV.

    2013 02 v.26 [Abstract][OnlineView][Download 466K]

  • Expressions of Wnt-signaling upstream and downstream factors in liver cancer tissue and their clinical significance

    HU Man,ZENG Wei-zheng,JIANG Ming-de,WU Xiao-ling Department of Gastroenterology,General Hospital of Chengdu Military Region of PLA, Chengdu 610000,Sichuan Province,China

    Objective To investigate the expressions of the mRNA and protein of Wif-1,C-myc and Cycling-D1 of Wnt signal pathway in normal liver,cirrhosis and hepatocellular carcinoma tissues as well as their relationship to clinical pathological parameters in hepatocellular carcinoma(HCC).Methods The expressions of Wif-1,C-myc and Cycling-D1 mRNAs and proteins in 100 samples of HCC,50 samples of liver cirrhosis and 50 samples of normal liver tissue were determined by immunohistochemical assay and in situ hybridization.In addition,the positive rates,sensitivity and specificity of Wif-1,C-myc and Cycling-D1 in HCC were determined.Results The expression rates of Wif-1 mRNA and protein in liver cirrhosis and HCC tissues were significantly lower,while those of C-myc and Cyclin-D1 mRNAs and proteins were significantly higher,than those in normal liver tissues(each P < 0.05).The expression rates of Wif-1,C-myc and Cycling-D1 mRNA and proteins were significantly related to HBV grade,TNM stage and β-catenin heterogeneous expression(P < 0.05).Positive correlations were observed between the mRNA and protein expressions of Wif-1,C-myc and Cycling-D1 in HHC tissue(rWif-1 = 0.892,rC-myc = 0.354,rCycling-D1 = 0.378).The sensitivities of Wif-1 mRNA and protein in HCC were 9.09% and 11.11%,while those of C-myc were 91.38% and 89.66%,those of Cyclin-D1 were 91.67% and 82.5%,respectively.However,the specificities of Wif-1 mRNA protein in HCC were 97.75% and 95.6%,while those of C-myc were 82.5% and 85.71%,and those of Cycling-D1 were 92.86% and 81.82%,respectively.Conclusion The invasion of HCC and heterogeneous expression of β-catenin were related to the down-regulation of Wif-1 expression and up-regulation of expressions of C-myc and Cycling-D1.

    2013 02 v.26 [Abstract][OnlineView][Download 751K]

  • Development of an indirect ELISA method for hepatitis B virus PreS2 antibody in mouse serum

    JIANG Li-ming,TAN Chang-yao,LI Jiong Research Lab of Biotechnology,Chengdu Institute of Biological Products Co.Ltd.,Chengdu 610023,Sichuan Province, China

    Objective To develop an indirect ELISA method for hepatitis B virus(HBV) PreS2 antibody in mouse serum.Methods Microtiter wells were coated with HBV PreS2 polypeptides(1-26),based on which the reaction condition of indirect ELISA was optimized by block titration.The developed method was verified for precision,sensitivity and specificity,and the results were compared with those by commercial kit.Results The optimal antigen concentration for coating was 2 μg / ml,and the optimal dilution of serum was 1︰10,while the optimal reaction temperature and time of serum were 37 ℃ and 30 min respectively.However,the optimal dilution reaction temperature and time of HRP-labeled goat anti-mouse IgG were 1︰10 000,37 ℃ and 30 min respectively.The optimal temperature and time for coloration of TMB substrate were 37 ℃ and 15 min respectively.The 2.1 folds of A450 value of negative control serum was served as cut-off value.The A450 value of negative control serum less than 0.05 was calculated as 0.05.The developed method showed high intra-and inter-precisions,of which the sensitivity and specificity were 100% and 97.3% respectively.The coincidence rate of detection results by the developed method and the commercial kit was 98.5%.Conclusion An indirect ELISA method suitable for detection of HBV PreS2 antibody in mouse serum was successfully developed.

    2013 02 v.26 [Abstract][OnlineView][Download 507K]

  • Production of enterovirus 71 in Vero cells grown on microcarriers in a bioreactor

    XIA De-zhen,YANG Zhi-jian,ZHOU-zheng,GAO-han,ZHANG Gao-xia Vaccine Research and Development Center,Shanghai Zerun Biotechnology Co.,Ltd.,Shanghai 201203,China

    Objective To develop a procedure for production of enterovirus 71(EV71) in Vero cells grown on microcarriers in a bioreactor.Methods Vero cells were grown on Cytodex-1 microcarriers in a 5 L NBS CelliGen 310 bioreactor filled with MEM containing 10% new calf serum.The effect of concentrations(3,10,15 and 20 g / L) of Cytodex-1 microcarriers on growth,metabolism and density of cells were investigated,while the proliferation profile of virus in bioreactor was compared with that in cell factory(CF).The harvested virus liquid was pooled into three fractions as supernatant,wash and elution,in which the antigen contents and virus titers(CCID50) were compared.Results The density of Vero cells reached 5.47 × 106 cells / ml 120 h after batch culture on 10 g / L micorocarriers.When the concentration of microcarriers was more than 15 g / L,perfusion culture mode was adopted because of rapid consumption of glucose.The harvest time of EV71 inoculated at a MOI of 0.2 in bioreactor was 48 h later than that in CF.However,the titer of EV71 in bioreactor reached 8.8 log10CCID50 / ml,which was 5 times higher than that in CF.Since the ion exchange adsorption between EV71 and microcarriers,the virus was harvested by combining the supernatant and the eluent fractions,in which the antigen content(12 61 U / ml) was about 3 times higher than that harvested from CF.Conclusion The procedure for production of EV71 in Vero cells grown on microcarriers in a bioreactor was successfully developed,which laid a foundation of further large-scale culture of EV71 and development of vaccine.

    2013 02 v.26 [Abstract][OnlineView][Download 631K]

  • Development and preliminary application of quadruple PCR for four pathogenic bacteria of porcine respiratory disease complex

    ZHAO Hong-wu,LI Yu,SUN Pei,LIU Wen,WEI Jian-zhong College of Animal Science and Technology,Anhui Agricultural University,Hefei 230036,Anhui Province,China

    Objective To develop a quadruple PCR method for four pathogenic bacteria,i.e.Mycoplasma hyopneumoniae(Mhp),Streptococcus suis(SS),Haemophilus parasuis(HPS) and Pasteurella multocida(Pm) of porcine respiratory disease complex(PRDC).Methods Specific primers were designed upon the P36 gene of Mhp reported in GenBank as well as the genes of SS,HPS and Pm reported in references,based on which a quadruple PCR method was developed through optimization of reaction system by orthogonal test on four factors(Taq,Mg2+,primers and dNTPs) at four levels,and verified for specificity,sensitivity and reproducibility.The developed method was used for determination of nasal swab and lung tissue samples of pigs with PRDC,and for routine isolation and identification of SS,HPS and Pm in lung tissue,using single PCR as control.Results The reaction system and condition of the quadruple PCR were optimized.Specific bands at lengths of 568,294,821 and 457 bp respectively were amplified by the developed quadruple PCR,which were consistent with those expected.The electrophoretic results of amplified products of 8 repeat PCR were in agreement,and the minimum detection limits of Mhp,SS,HPS and Pm were 560,260,280 and 200 pg respectively.It indicated high specificity,sensitivity and reproducibility of the developed method.The determination results of nasal swab and lung tissue samples of pigs with PRDC by the developed quadruple PCR showed no significant difference with those by single PCR(P > 0.05).However,the positive rate of HPS determined by both quadruple and single PCR were significantly higher than that by routine isolation and identification of bacteria(P < 0.05).Conclusion The developed quadruple PCR method may be directly used for identification of four pathogenic bacteria of PRDC,which provided an effective technique for epidemiological survey of PRDC.

    2013 02 v.26 [Abstract][OnlineView][Download 661K]

  • Development and preliminary application of one step RT-PCR assay for Newcastle disease virus

    LIANG Jin,CHENG Fu-liang,CHEN Tian-tian,SU Zhi-rui,FAN Qun-ping Shandong Boly-lely Bioengineering Cooperation,Tai'an 271000,Shandong Province,China

    Objective To develop a one-step RT-PCR assay for Newcastle disease virus(NDV).Methods A pair of specific primers was designed according to the sequences of F gene of NDV,based on which a one step RT-PCR assay was developed and verified for specificity and sensitivity.Twenty-three clinic suspected samples were detected by the developed RT-PCR method,of which the results were compared with those by hemagglutination(HA) and hemagglutination inhibition(HI) tests.Results By developed RT-PCR method,the detection result of NDV was positive,while those of infectious bursal disease virus(IBDV) and avian influenza virus(AIV) subtype H9 were negative.The minimum detection limit of the method was 4 pg NDV RNA.Of the 23 clinic suspected samples,11 were judged as positive by RT-PCR,and 13 by HA and HI tests,indicating a positive coincidence rate of 85%.Conclusion One-step RT-PCR assay for NDV was developed,which was specific,sensitive and time-saving,and might be used for early rapid diagnosis and epidemiological investigation of NDV at molecular level.

    2013 02 v.26 [Abstract][OnlineView][Download 521K]

  • Optimization of fermentation condition for production of γ-aminobutyric acid by Enterococcus faecalis HX-3-6

    QIAN Sen-he,HUANG Zu-yao,XUE Zheng-lian,ZHAO Shi-guang Institute of Biologic & Chemical Engineering of Anhui Polytechnic University,Microorganism Fermentation Engineering and Technology Research Center of Anhui Province,Wuhu 241000,Anhui Province,China

    Objective To optimize the fermentation condition for production of γ-aminobutyric acid(GABA) by Enterococcus faecalis HX-3-6.Methods The fermentation condition of E.faecalis HX-3-6 producing GABA was optimized by single factor test and orthogonal test,based on which the main influencing factors of medium for production of GABA were screened by Plackett-Burman(PB) method,and optimized by Box-Behnken design and response surface method.Three validation tests were performed by using the optimized formula.Results The optimal substrate concen-tration,pH value,time and temperature for fermentation were 30 g / L,5.5,72 h and 35 ℃ respectively.The main influencing factors of medium were MnSO4﹒H2O,sodium chloride,citric acid triamide and glucose,of which the optimal concentrations were 0.100 785,0.224 459,0.215 355,and 12.335 95 g / L respectively.The mean yield of GABA in three validation tests reached 16.027 g / L,which increased by 45.62% compared with that before optimization(11.006 g / L).Conclusion The yield of GABA increased significantly by under the optimized fermentation condition,which laid a foundation of industrial production of GABA.

    2013 02 v.26 [Abstract][OnlineView][Download 797K]

  • Development of a real-time fluorescent quantitative PCR assay for BF1 and BF2 genes in chickens

    JIN Yuan-chang,LI Yu-feng,YUAN Zhi-dong College of Life Science,Hunan University of Science and Technology,Xiangtan 411201,Hunan Province,China

    Objective To develop a real-time fluorescent quantitative PCR assay for of BF1 and BF2 genes of major histocompatibility complex(MHC) in chickens.Methods Total RNA was extracted from chicken peripheral blood leukocytes(PBLs) and used as a template for amplification of BF1,BF2 and GAPDH genes by RT-PCR using the designed primers.The amplified genes were cloned into vector pGM-T respectively,and the constructed standard recombinant plasmids were identified by PCR,digestion with EcoR I and sequencing.The reaction system and condition were optimized,based on which a standard curve was plotted.The developed real-time FQ-PCR method was verified for sensitivity,specificity and reproducibility.Results PCR,restriction analysis and sequencing proved that the standard recombinant plasmids were constructed correctly.The threshold cycle(Ct) of standard curve showed good linear relationship to the log of original copy numbers of plasmid DNAs of BF1,BF2 and GAPDH genes,with coefficients of correlation of 1.00,0.98 and 0.99,and amplification efficiencies of 0.8,1.1 and 1.1,respectively.The developed method showed a sensitivity of 101 copies / μl.Melting curve showed consistent amplification efficiencies of the genes,indicating high specificity of the method.The CV values of 3 test results of standard plasmid DNA were not more than 0.5.Conclusion A real-time fluorescent quantitative PCR assay for of BF1 and BF2 genes in chickens was successfully developed,which laid a foundation of further application of the method to determination of transcription levels of BF1 and BF2 genes.

    2013 02 v.26 [Abstract][OnlineView][Download 703K]

  • Comments and recommendations on comparability of vaccines related to manufacturing site changes: quality issues

    LI Min,GAO En-ming,YIN Hong-zhang,LUO Jian-hui Center for Drug Evaluation,State Food and Drug Administration,Beijing 100038

    Increase in product yield and complementation of the requirement of national regulatory agency are driving to site changes of vaccines.Comparability exercise is the bridge to a successful change,which would ascertain that introduction of the manufacturing site change did not alter the safety and efficacy profile of the product.This paper introduced the core element of the international guideline,and put forwards considerations on designing and fulfilling comparability study regards to vaccine quality assessment.

    2013 02 v.26 [Abstract][OnlineView][Download 464K]

  • Advances in research on PEGylation of interferon

    ZHENG Bo,WANG Jin-feng School of Pharmaceutical Science and Technology,Tianjin University,Tianjin 300072,China

    Interferon(IFN) contains a family of cytokines that display a wide range of antiviral,immunomodulatory,and antiproliferative activities,but unfortunately the short circulating half-life limits their clinical applications.PEGylation may improve the physicochemical and biological characteristics,and extend the plasma circulating half-life of IFN.Recent advances in PEGylation of IFNs were reviewed in this paper,while the development of PEGylation of IFNs α,β,and γ were particularly introduced.Finally,the future trends and prospects in PEGylation of IFNs were predicted.

    2013 02 v.26 [Abstract][OnlineView][Download 597K]
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