- HE Yong-lin, LI Na, YANG Chun, et al (Department of Microbiology and Immunology,Chongqing University of Medical Sciences,Chongqing 400016, China)
Objective To construct the recombinant plasmid for co-expression of human granulysin active peptide(Mr 9 000) and murine interleukin-12(mIL-12) and identify the expressed product.Methods Amplify the gene encoding human granulysin active peptide by PCR using the plasmid containing the cDNA sequence of granulysin as template and insert into eukaryotic expression vector pBudCE4.1.Identify the constructed recombinant plasmid pBudCE4.1-S9K by restriction analysis and DNA sequencing,to which mIL-12 gene was subcloned.Transfect RAW264.7 cells with the constructed co-expression vector pBudCE4.1-S9K/mIL-12 and identify the expressed granulysin active peptide by RT-PCR,immunocytochemical method and Dot-ELISA,and the expressed mIL-12 by ELISA.Results The results of restriction analysis,PCR and DNA sequencing proved that human granulysin active peptide gene was correctly inserted into plasmid pBudCE4.1.RT-PCR and immunocytochemical method showed transient expression of human granulysin active peptide in RAW264.7 cells.However,Dot-ELISA showed extracellular secretion of expressed human granulysin active peptide.ELISA proved expression of mIL-12 in culture supernatant of RAW264.7 cells.Conclusion The recombinant plasmid pBudCE4.1-S9K/mIL-12 was successfully constructed and expressed in vitro,which laid a foundation of gene therapy of tumors with granulysin and mIL-12.
2007 03 [Abstract][OnlineView][Download 468K] - ZHOU Wei-ying,WU Chao,SHI Yun,et al (Department of Clinical Microbiology and Clinical Immunology,College of Medical Laboratory,Third Military Medical University,Chongqing 400038,China)
Objective To construct and express the fusion protein epitopes of Helicobactor pylori(Hp) urease B subunit(UreB) concatemer and E.coli heat-labile toxin B subunit(LTB),and analyze the biological and immunological characteristics of expressed product.Methods Amplify the genes encoding UreB epitope polypeptide and LTB by PCR respectively and link by overlap extension PCR.After T-A cloning,the linked gene Uepi-LTB was cloned into prokaryotic expression vector pET-22b(+),and the constructed recombinant plasmid pET-22b(+)-Uepi-LTB was transformed to E.coli BL21(DE3) for expression under induction of IPTG.Identify the expressed product by Tricine-SDS-PAGE and Western blot,and purify by SP Sepharose XL and Butyl Sepharose FF chromatography.Results Two gene fragments at the lengths of 206 bp and 336 bp respectively were amplified by PCR.However,by overlap extension PCR,a fusion gene fragment at the length of 524 bp was amplified.Both the results of restriction analysis and DNA sequencing of prokaryotic expression vector pET-22b(+)-Uepi-LTB were consistent with those designed.The expressed product,with a relative molecular weight of about 20 000,existed in a form of inclusion body and contained about 25% of total somatic protein.After purification,the expressed protein reached a purity of 96%.Western blot showed specific reaction of expressed fusion protein with rabbit anti-LTB serum.Conclusion Fusion protein Uepi-LTB was successfully expressed in prokaryotic cells.The expressed product showed good immunogenicity,which laid a foundation of developing novel Hp vaccine.
2007 03 [Abstract][OnlineView][Download 523K] - LIU Kai,WANG Xing-long,REN Lin-zhu,et al (College of Animal and Veterinary Sciences,Jilin University,Changchun 130062,China)
Objective To clone and sequence the full-length cDNA of genomic segment B of infectious bursal disease virus(IBDV) vaccine strain B87 and lay a foundation of study on the function as well as antigen and virulence variations of genome of IBDV strain B87 at a molecular level.Methods Extract the genomic RNA of IBDV by protease K digestion and phenol/chloroform extraction,then purify the genomic dsRNA by LiCl fractional precipitation for amplification of full-length cDNA of genomic segment B of IBDV strain B87 by one-step RT-PCR.Insert the amplified cDNA into pGEM-T vector,then sequence and analyze the result by using DNAStar software.Results The cDNA of genomic segment B of IBDV,at a full length of 2 827 bp,was cloned,which showed homologies of 89.80% and 89.30% to high virulent reference strains UK661 and HK46,90.2% and 89.5% to virulent reference strains Harbin-1 and ZJ2000,97.8% to variant strain GLS,both 99.8% to avirulent reference strains CEF94 and Gt,and 100% to strain P2,respectively.Conclusion The analysis of deduced amino acid sequences of 9 IBDV strains indicated that the 10 characteristic amino acid sites of B segment might be related to virulence.
2007 03 [Abstract][OnlineView][Download 430K] - CUI Da-wei, WANG Wei, ZHOU Xiao-lin, et al (Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi University, Taiyuan 030006, China)
Objective To express human myoglobin and prepare its polyclonal antibody.Methods The gene sequence encoding human myoglobin(hMb) from the total RNA of human skeletal muscle by RT-PCR and clone into prokaryotic expression vector pRSETc for expression in E.coli BL21(DE3).Purify the expressed product by affinity chromatography and gel filtration chromatography.Immunize mice with the purified protein to prepare polyclonal antibody.Test the prepared antibody for specificity by Western blot.Results The sequence of amplified hMb gene was consistent with that reported in GenBank(NM203377).The gene was highly expressed in a soluble form in E.coli BL21(DE3),and the expressed product was electrophoretically pure and contained 12.8% of total somatic protein.Western blot proved the expressed product as hMb protein.A portion of 6.438 mg of target protein was purified from 1 L of fermentation liquid.The polyclonal antibody induced in mice,with an ELISA titer of 1∶12 800,showed good specificity as proved by Western blot.Conclusion Human myoglobin gene was successfully cloned and expressed in a soluble form in E.coli,and the polyclonal antibody against hMb was successfully prepared.
2007 03 [Abstract][OnlineView][Download 510K] - HUANG Shi-he,PENG Xiang-bing,ZHANG Ai-hua,et al (Wuhan Institute of Biological Products,Wuhan 430060,China)
Objective To construct a baculovirus expression vector for complete IgG antibody against HBsAg.Methods Amplify L and Fd(VH+CH1) genes from plasmid L3rd6 containing the gene encoding Fab fragment of antibody against HBsAg by PCR.Insert the amplified L gene into baculovirus expression vector pAC-k-Fc to construct transient expression vector pAC-k-L-Fc,in which Fd gene was then inserted.After identification by restriction analysis and DNA sequencing,the constructed recombinant plasmid pAC-HBs-Fc was transfected to sf9 cells,and the expressed product was identified by IFA.Results The length of amplified fragments were about 650 bp,which were identical to those expected.Both the lengths and DNA sequences of gene fragments extracted from recombinant plasmids pAC-k-L-Fc and pAC-HBs-Fc were also consistent with those expected.The sf9 cells transfected with pAC-HBs-Fc was positive in IFA,while those untransfected were negative.Conclusion The baculovirus expression vector pAC-HBs-Fc for complete IgG antibody against HBsAg was successfully constructed.
2007 03 [Abstract][OnlineView][Download 547K] - LI Dong,YANG Wei,YU Chun-lei,et al (School of Public Health,Jilin University,Changchun 130021,China)
Objective To explore the expression level of promiscuous gene and MHC-Ⅱ molecules in the thymus of mice with nonobese diabetic(NOD) and their roles in central immunotolerance.Methods Determine the blood sugar of NOD mice by GA-PAG kit,urinary sugar by test paper and IAA content by indirect ELISA.Observe the histological change of pancreatic island by light microscopy.Determine the function of thymic lymphocytes by MTT method and the secretions of IL-4 and IFN-γ in thymic lymphocytes by sandwich ELISA.Test for the expression of MHC-Ⅱ molecules by flow cytometry,and that of promiscuous gene by RT-PCR.Results The blood sugar level of NOD mice showed no significant difference from that of normal BALB/c mice,and all the determination results of their urinary surgar were negative.Compared with that of normal BALB/c mice,the autoantibody level of NOD mice showed no significant difference.However,the pancreatic islands of NOD mice decreased significantly in quantity,and were scattered in distribution.The number of pancreatic island cells also decreased significantly,accompanied with the infiltration of a large quantity of inflammatory cells.No significant difference was observed between the secretion level of IFN-γ in thymic lymphocytes of NOD and normal mice,while the secretion level of IL-4 of the former was significantly lower than that of the latter.The insulin level in thymus of NOD mice decreased significantly,while GAD67 and PLP levels showed no significant change.The expression level of MHC-Ⅱ molecules decreased significantly.Conclusion The defect in the expression of promiscuous gene and MHC-Ⅱ molecules in thymus of NOD mice at stage of prediabete indicated their important roles in central immunotolerance.
2007 03 [Abstract][OnlineView][Download 368K] - LI Zhen△,JIN Ning-yi,JIN Hong-tao,et al (△College of Animal Science and Veterinary,Jilin University,Changchun 130062,China)
Objective To design a novel multiple-epitope DNA vaccine against HIV and study its immunogenicity in mice.Methods The full-length of MEGN gene was synthesized according to the designed sequence by chemical method and used for the construction of recombinant plasmid pVAX1-MEGNp24.Transfect BHK-21 cells with the constructed recombinant plasmid and identify the expressed product by IFA.Immunize BALB/c mice with the recombinant plasmid and determine the serum antibody by ELISA,and the T lymphocyte subgroup by flow cytometry.Results Restriction analysis and DNA sequencing proved that recombinant plasmid pVAX1-MEGNp24 was successfully constructed.IFA showed that the constructed recombinant plasmid was expressed in BHK-21 cells.Both humoral and cellular immunities were induced in mice,as proved by ELISA and flow cytometry respectively.Conclusion Recombinant plasmid pVAX1-MEGNp24 as a multiple-epitope DNA vaccine against HIV was successfully constructed and showed good immunogenicity in mice.
2007 03 [Abstract][OnlineView][Download 241K] - BAI Mu-qun,ZHOU Xu,AN Jing,et al (Lanzhou Institute of Biological Products,Lanzhou 730046,China)
Objective To clone the open reading frame 2(ORF2) gene of transfusion transmitted virus(TTV) Lanzhou strain and construct a prokaryotic expression vector.Methods The ORF2 gene of TTV was amplified from the serum of a blood donor in Lanzhou region by nest PCR and inserted into vector pGEM-T then,after identification by DNA sequencing,subcloned to expression vector pET22b(+).The constructed recombinant plasmid ORF2-pET22 was transformed to E.coli BL21 for expression under induction of IPTG.The expressed product was identified by Western blot,then purified by affinity chromatography and used as an antigen for the determination of TTV-IgG antibody in human serum specimens by ELISA.Results The homology of ORF2 gene of TTV Lanzhou strain to that of TA287 strain isolated in Japan was 99.3%.The ORF2 fusion protein with 6 histidine labels was expressed.Western blot showed specific reaction of expressed product with TTV-PCR positive serum.The ELISA result using ORF2 protein as antigen showed good accuracy.Conclusion The ORF2 gene of TTV was successfully cloned and expressed in prokaryotic cells.
2007 03 [Abstract][OnlineView][Download 443K] - LI Jin-long, WANG Quan, SUO Jian (Department of General Surgery, First Hospital, Jilin University, Changchun 130021, China)
Objective To test the expression of melanoma antigen-4(MAGE-4) gene in human colonic adenocarcinoma tissue.Methods The expression of MAGE-4 gene in 44 human colonic adenocarcinoma tissue specimens and 15 normal colon tissue specimens were tested by RT-PCR.Results The positive rate of MAGE-4 was 38.64% in 44 human colonic adenocarcinoma tissue specimens.However,no expression of MAGE-4 gene was detected in 15 normal colon tissue specimens.The RT-PCR product of specimens positive for MAGE-4 was sequenced and proved as the full length of MAGE-4 gene.Conclusion Specific expression of MAGE-4 gene was proved in human colonic adenocarcinoma tissue,which indicated that MAGE-4 gene might be used for the development of colonic carcinoma vaccine.
2007 03 [Abstract][OnlineView][Download 150K] - ZHANG Xi-zhen, YU Xiang-hui, ZHAO Dong-hai, et al (Vaccine Research Center,College of Life Science,Jilin University, Changchun 130012, China)
Objective To screen the 293 cell line for stable and high expression of virus-like particles(VLPs) containing structural proteins gag,pol and env of HIV-1.Methods Co-transfect 293 cell line with eukaryotic expression vector D-GPEi for HIV-1 gag,pol and env genes and plasmid pcDNA3.1 carrying neomycin resistant gene,and screen the cell lines for stable expression with G418 resistant medium.Results The major structural proteins gag,pol and env of HIV-1 were expressed in 293 cells.The expression levels in screened cell lines of various passages showed no significant difference.Conclusion The 293 cell lines for stable expression of VLPs of HIV-1 were successfully screened,which laid a foundation of development of HIV vaccine.
2007 03 [Abstract][OnlineView][Download 291K] - MA Ming-xiao△, JIN Ning-yi, LIU Hui-juan, et al (△College of Animal Science and Veterinary Medicine, Jinlin University, Changchun 130062, China)
Objective To construct expression cassette OAAT for multiple-epitope antigen of foot-and-mouth disease virus(FMDV).Methods The cassette OAAT for multiple-epitope antigen of FMDV was constructed using the structural protein VP1 genes of FMDV serotypes A and O and 5 antigenic epitope genes of two genotypes of FMDV serotype Asial as major immunogen genes,and two Th2 epitope genes of non-structural protein 3ABC and one Th2 epitope gene of structural protein VP4 as accessory genes,then cloned downstream to eukaryotic expression vector pVAX1 PCMV.Transfect HeLa cells with the constructed trivalent FMDV DNA vaccine pVAX1-OAAT and identify the expressed product by Western blot and IFA.Results The modeling of homology by InsightⅡ software and analysis by DNAStar5.0 software proved that the design of constructed expression cassette OAAT was consistent with that exprected in theory.Western blot and IFA showed correct expression of target gene in Hela cells.Conclusion The expression cassette OAAT for multiple-epitope antigen of FMDV was successfully designed,and trivalent FMDV DNA vaccine pVAX1-OAAT was successfully constructed.
2007 03 [Abstract][OnlineView][Download 988K] - ZHAO Chun-yan△, LIAN Pei-wen, WEI Wei, et al (△Department of Clinical Biochemistry, Dalian Medical University, Dalian 116027, China)
Objective To observe the influence of formation of Rap1GAP dimer on the intracellular localization of Rap1GAP.Methods Induce the site-directed mutations of nucleotides encoding the amino acids at sites 90 and 99 of green fluorescent protein-labeled wild Rap1GAP to obtain mutants M90 and M99 with Rap1GAP dimer deleted respectively.HEK293 cells were transfected with wild Rap1GAP,mutant M90,mutant M99,wild Rap1GAP+Gα,mutant M90+Gα and M99+Gα respectively.The distributions of wild Rap1GAP and mutants were observed by fluorescent microscopy.Results When HEK293 cells were transfected with wild Rap1GAP,mutant M90 or mutant M99,both wild Rap1GAP and mutants were diffused in cytoplasm.However,when the cells were co-transfected with wild Rap1GAP+Gα,M90+Gα or M99+Gα,both wild Rap1GAP and mutants were mainly distributed on cell membrane.Conclusion The formation of Rap1GAP dimer showed no significant influence on the intracellular localization of Rap1GAP.
2007 03 [Abstract][OnlineView][Download 266K] - YAO Wei△, LIU Guang-yang, REN Ya-ping, et al (△Lanzhou Institute of Biological Products, Lanzhou 730046, China)
Objective To observe the humoral and cellular immune effects of recombinant HBcAg and HBsAg mixed antigen in mice.Methods Immunize BALB/c mice with the prepared adjuvant-free and aluminium hydroxide adjuvant-containing recombinant HBcAg and HBsAg mixed antigens respectively.Determine the serum antibody and cytokine levels of the mice by ELISA,and analyze the T cell subgroups by flow cytometry.Results The IFN-γ,CD3+ and CD4+ levels of mice immunized with mixed antigen were significantly higher than those with HBcAg or HBsAg only.The CD8+ level of mice immunized with aluminium hydroxide adjuvant-containing mixed antigen was significantly higher than that with physiological saline as control.The serum anti-HBs level of mice immunized with mixed antigen showed no significant difference from that with HBsAg at double dosage.No significant difference was observed between the immune effects of adjuvant-free and aluminium hydroxide adjuvant-containing antigens.Conclusion Recombinant HBcAg and HBsAg mixed antigen showed good humoral and cellular immune effects,which provided a basis for the development of therapeutic hepatitis B vaccine.
2007 03 [Abstract][OnlineView][Download 104K] - WANG Guo-fu, WU Li-xian,BAI Li, et al (Department of Microbiology and Immunology, Dali College, Dali 671000, China)
Objective To explore the immune response induced by oral administration of fusion protein HpaA-CtxB-VacA(HCTV) of Helicobacter pylori(Hp) in mice.Methods Immunize mice by oral route with the HCTV purified by Ni2+-NTA column chromatography,using HpaA and VacA as parallel controls and PBS as blank control.Determine the lymphocytes in gastric mucosa and Peyer patches(PP) as well as specific antibody-secreting cells(ASC) by ELISPOT,and the IgG and IgA in sera as well as sIgA in intestinal mucus and fecal by ELISA.Results The numbers of sIgA-ASC and IgG-ASC,especially that of sIgA-ASC,in mucosa and PP increased significantly.Meanwhile,the IgA and IgG levels in sera and the sIgA levels in fecal and intestinal mucus of mice immunized with HCTV were significantly higher than those with HpaA,VacA or PBS.Conclusion Purified fusion protein HCTV was successfully prepared.It induced mucosal immune response in mice effectively and might be a candidate of oral Hp vaccine.
2007 03 [Abstract][OnlineView][Download 163K] - WU Yong-lin△, ZHAO Yu, HUANG Yu-xian, et al (△Chengdu Institute of Biological Products, Chengdu 610023, China)
Objective To study the dynamic distribution of attenuated Japanese encephalitis virus(JEV) strain SA14-14-2 in mice.Methods Inject mice with JEV strain SA14-14-2 by intracerebral and subcutaneous routes respectively, using virulent JEV strain SA14 as control. Determine the JEV in various organs including brain, liver, spleen, lung, heart, muscle and kidney of mice by plaque formation test with BHK21 cells on days 1-7, 14, 21 and 28 respectively.Results After injection with SA14-14-2 strain by intracerebral route, JEV was found only in brains of mice on days 1-6, but not found in any other organs. However, after injection with strain SA14-14-2 by subcutaneous route, no JEV was detected in any organs. As a contrast, high JEV titers were detected in various organs of mice injected with SA14 strain, either by intracerebral or by subcutaneous route.Conclusion JEV SA14-14-2 strain could not be replicated in the tissues of mice after subcutaneous infection, which proved the safety of live attenuated JE vaccine.
2007 03 [Abstract][OnlineView][Download 74K] - XIE Zhong-ping, CHEN Han-bo,SHEN Dong, et al (Institute of Medical Biology,Chinese Academy of Medical Sciences,Peking Union Medical College,Kunming 650118, China)
Objective To analyze the pathological lesion of monkeys caused by neurovirulence test of oral poliovirus vaccine(OPV) and evaluate the two injection methods for the test.Methods Analyze the results of pathological examinations of monkeys used for neurovirulence tests of OPV during 1988-2004.Results The qualification rates of OPV in neurovirulence tests by intracerebral and intraspinal injections during the past 18 years were 99.38% and 98.14% respectively,which showed no significant difference.The 3 batches of vaccines unqualified in the test by intraspinal injection were also unqualified in that by intracerebral injection.By intraspinal injection,more than 90% of the scores for pathological lesion caused by OPV type Ⅲ(Zhong Ⅲ_2 strain) were significantly lower than those by SabinⅢ reference strain,which indicated that,compared with SabinⅢ reference strain,Zhong Ⅲ_2 strain was further attenuated.No significant difference was observed between the scores of neurovirulence tests on a single batch and on the pooling of several batches of OPV.By intracerebral injection,neither pathological lesion rate nor pathological lesion constitution caused by original vaccine showed significant difference from those by 1∶10 diluted vaccine.The pathological lesion rate caused by OPV prepared with Sabin Ⅰ strain(4.88%) was significantly higher than those with SabinⅡ(0.65%) and Zhong Ⅲ_2(0.67%) strains.However,the lesion rates caused by OPV prepared with SabinⅡ and Zhong Ⅲ_2 strains showed no significant difference.The lesion rate in retest(6%) was significantly higher than that in primary test(0.66%).Conclusion Both intracerebral and intraspinal injections were sensitive methods for neurovirulence test of OPV.The results of pathological examinations were accurate and reliable.
2007 03 [Abstract][OnlineView][Download 131K] - SONG Ai-jing,XU Si-hong,LI Xiu-hua,et al (National Institute for the Control of Pharmaceutical and Biological Products,Beijing 100050,China)
Objective To establish a national reference for evaluation of confirmation reagent of HIV antibody.MethodsCollect plasma sample from the patients or doubtful patients with HIV infection,as well as those from donors without HIV infection,in various regions for determination of HIV antibody,RNA and antibody band.Establish the national reference by screening the representative samples and calibrate with the HIV confirmation reagents from 4 manufacturers.Results The national reference consisting of 27 samples was established,of which 10 were confirmed as HIV antibody-positive,12 as negative and 5 as indefinite.After 12 calibrations,all the 10 HIV antibody-positive references were confirmed as positive,and 12 negative samples as negative.The 5 indefinite samples were confirmed as positive or indefinite,but not negative.Conclusion The established national reference might be used for the evaluation of quality of HIV antibody confirmation reagent.
2007 03 [Abstract][OnlineView][Download 131K] - WANG Ze-jun,HU Qiao-ling,XU Ge-lin (Wuhan Institute of Biological Products,Wuhan 430060,China)
Objective To explore the method for internal quality control(IQC) of diagnostic kit for rabies virus nucleoprotein in laboratory.Methods Test for rabies virus nucleoprotein antigen by double antibody sandwich ELISA,and plot IQC figures by Levey-Jennings method and Instant Method.Results All the test results on IQC figure by Levey-Jennings method was under control,however,one test result on the IQC figure by Instant Method was out of control.Conclusion The IQC shall be performed by double methods to overcome the shortage in test result by a single method.
2007 03 [Abstract][OnlineView][Download 98K] - DU Li-juan△,WANG Shi-guang,ZHANG Li,et al (△College of Environmental and Life Sciences,Dalian University of Technology,Dalian 116023,China)
Objective To compare the purification effects of influenza virus by two methods.Methods The bulk of influenza virus was concentrated by ultrafiltration,then purified by Sepharose 4FF gel chromatography and sucrose density gradient centrifugation respectively.Results The purity as well as removal rates of egg albumin and foreign protein of virus bulk purified by Sepharose 4FF gel chromatography were slightly higher than those by sucrose density gradient centrifugation.However,the recovery of virus of the latter was higher than that of the former.Conclusion Both Sepharose 4FF gel chromatography and sucrose density gradient centrifugation were suitable for the purification of influenza virus.
2007 03 [Abstract][OnlineView][Download 124K] - LI Jian-ping△,ZHOU Chang-jun,WANG Yu,et al (△Changchun Institute of Biological Products,Changchun 130062,China)
Objective To compare the culture effects of CHO-C28 cells by domestic and imported DMEM.Methods Culture the CHO-C28 cells of the same batch by domestic and imported DMEM respectively and collect the culture supernatant for determination of expressed HBsAg.The HBsAg in culture supernatant was purified by the same method,and their chromatographic and electrophoretic profiles were compared.Hepatitis B(HB) vaccine was prepared with the purified HBsAg and tested for abnormal toxicity and potency.Results The expression level of HBsAg in culture supernatant of domestic DMEM was slightly higher than that of imported DMEM.Both the chromatographic and electrophoretic profiles of HBsAg expressed in culture supernatants of domestic and imported DMEM were in agreement.Both the HB vaccines prepared by domestic and imported DMEM were qualified in abnormal toxicity test,and showed no significant difference in potency.Conclusion Domestic DMEM was safe and effective for cell culture and reached the level of imported DMEM,thus was suitable for large-scale production of HB vaccine.
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2007 03 [Abstract][OnlineView][Download 121K] 下载本期数据