2019年01期目次

  • Construction and evaluation of recombinant adenovirus expressing glycoprotein E gene against varicella zoster virus

    CHEN Wei-wei;CUI Hai-yan;WEN Ye;PENG Shao-dan;ZHU Tao;College of Biotechnology,Tianjin University of Science and Technology;

    Objective To construct a recombinant adenovirus expressing glycoprotein E(gE)gene against varicella zoster virus(VZV)and evaluate its immunogenicity. Methods Recombinant adenovirus plasmid pAdEasy-gE carrying gE gene was constructed by using AdEasy system. The recombinant adenovirus rAd-gE packed in AD293 cells was identified by PCR and Western blot and purified by anion exchange and Capto Core 700 chromatography. NIH mice were injected i. m.with rAd-gE at dosages of 3. 3 × 10~6,1 × 10~7 and 3 × 10~7 ifu respectively,of which the serum antibody titer was determined. Results PCR and restriction analysis with PacⅠproved that pAdEasy-gE was constructed correctly. PCR and Western blot proved that rAd-gE was packaged successfully in AD293 cells. The recovery of rAd-gE purified by anion exchange and Capto Core 700 chromatography was 18. 2%. Humoral immune response was induced in mice by r AD-gE at dosages of 1 × 10~7 and 3 × 10~7 ifu. Conclusion The rAd-gE highly expressing gE gene was successfully constructed,which induced effective humoral immunity and laid a foundation of further development of recombinant adenovirus vaccine against VZV.

    2019 01 v.32 [Abstract][OnlineView][Download 436K]

  • Optimization of condition for culture of cold-adapted influenza H3N2 virus strain in Vero cells

    JI Yan-yan;FAN Jia-you;College of Pharmacy,Guizhou University;

    Objective To optimize the condition for culture of influenza H3N2 virus in Vero cells. Methods Influenza H3N2 virus was cultured in Vero cells,and the culture condition including MOI,adsorption time,sodium hydrogen carbonate concentration in maintenance medium,final concentration of TPCK-trypsin and CPE were optimized. Results The hemagglutination(HA)titer of influenza H3N2 virus reached the maximum when the MOI was 0. 05,the adsorption time was 1. 5 h,the 6. 6% sodium hydrogen carbonate concentration in maintenance medium was 2%,final concentration of TPCK-trypsin was 1 mg/L,and 40% ～ 50% CPE appeared. Conclusion The optimized condition was suitable for large-scale production of influenza vaccine.

    2019 01 v.32 [Abstract][OnlineView][Download 189K]

  • E. coli outer membrane vesicles as a novel exogenous plasmid delivery vector

    SHU Cong-yan;WANG Shi-jie;GAO Fu-lan;HUANG Wei-wei;MA Yan-bing;Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Diseases,Institute of Medical Biology, Chinese Academy of Medical Science and Peking Union Medical College;

    Objective To investigate the potential and significance of E. coli outer membrane vesicles(OMVs) as a novel delivery vector for exogenous plasmid. Methods OMVs were collected from the supernatant of E. coli DH5α strain by filtration, ultrafiltration and ultracentrifugation, then purified by density gradient centrifugation. The purified OMVs were co-incubated with phagocytic cells and non-phagocytic cells, from animal origin, in vitro to observe the uptake rate of OMVs in different cells. OMVs were transformed with the eukaryotic expression plasmid p IRES2-EGFP with green fluorescent protein by electroporation, and incubated with RRAW264. 7 cells to observe the entrance of OMVs carrying eukaryotic expression plasmids into phagocytic cells and expression of green fluorescent protein. Results OMVs were purified effectively by density gradient centrifugation. The efficiencies of purified OMVs in entering various mammalian cells were different, which was higher in entering phagocytic cells than in entering non-phagocytic cells. After electrotransformation, plasmid DNA was introduced into OMVs, and ingested and expressed with phagocytic cells. Conclusion The eukaryotic expression plasmid at a length of about 5 300 bp may be introduced to OMVs by electroporation and express in phagocytic cells, indicating the potential of OMVs as a novel delivery vector of plasmid DNA.

    2019 01 v.32 [Abstract][OnlineView][Download 444K]

  • Comparison of potential effects of natural cyclic dinucleotides as adjuvant of vaccines for human use

    WANG Zhi-biao;SHAN Pu;WEI Duo-qian;XU Jing;National Vaccine & Serum Institude,Beijing;

    Objective To compare the potential of several natural cyclic dinucleotides(CDN)as adjuvant of vaccines for human use. Methods THP-1 cells were co-incubated with various CDNs(c-di-GMP,c-di-AMP,3′3′cGAMP and 2′3′cGAMP) at an equal concentration in vitro. Meanwhile,c-di-GMP and 2′ 3′ cGAMP were mixed with liposome and coincubated with THP-1 cells. After incubation for 5 h,the cell pellets and supernatant were collected and determined for expression of IFNβ gene and protein by real-time quantitative polymerase chain reaction(q PCR)and ELISA respectively.The influence of liposome on stimulatory effect of various CDNs was evaluated. Results The CDNs enhanced the expressions of IFNβ gene and protein in THP-1 cells. In the turn of effect intensities,the CDNs were 2′3′cGAMP,3′3′cGAMP,c-di-AMP and c-di-GMP,with significant differences(P < 0. 01). Liposomes enhanced the effects of both noncanonical CDN and the canonical CDN on THP-1 cells significantly(P < 0. 01). Conclusion All the natural CDNs showed potential adjuvanticity to vaccines for human use. Specifically,2′ 3′ cGAMP was the optimal choice in terms of effect intensity,and liposome enhanced the effect. It laid a foundation of further study on the adjuvanticity of CDNs and their analogues.

    2019 01 v.32 [Abstract][OnlineView][Download 229K]

  • Prokaryotic expression,purification and immunogenicity of tree shrew stem cell factor

    LONG Qiong;HAO Jia-mao;XU Meng;BI Yan-wei;XIAO Hong-jian;LI Zhi-hua;YAN Ling-mei;DING Chen;LI Yu-zhong;YAO Yue-ting;CUN Wei;Institute of Medical Biology,Chinese Academy of Medical Sciences & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research and Development on Severe Infectious Disease;

    Objective To express the stem cell factor(SCF) of tree shrew in prokaryotic cells,purify the expressed product and determine its immunogenicity. Methods Primers were designed according to the predicted SCF gene sequence in GenBank,with which SCF gene was amplified and cloned into prokaryotic expression vector p ET-30a(+).The constructed recombinant plasmid pET-30a(+)-SCF was transformed to E. coli BL21(DE3) and induced with IPTG.The expressed fusion protein was purified by nickel ion column chromatography and immunized to rabbits,of which the immunogenicity was analyzed by Western blot and ELISA. Meanwhile,the distribution of SCF in various tissues of tree shrew was determined. Results Restriction analysis and sequencing proved that recombinant plasmid pET-30a(+)-SCF was constructed correctly. The expressed recombinant protein,with a relative molecular mass of about 34 500,contained about 35. 6% of total somatic protein,existed in a form of inclusion body,reached a purity of more than 90% after purification,and showed specific binding to anti-human SCF polyclonal antibody. The protein showed good immunogenicity,of which the antiserum titer was 1:256 000. SCF was detected in nose,trachea,lung,spleen,kidney and esophagus of tree shrew. Conclusion Recombinant SCF was highly expressed in E. coli,which showed good immunogenicity after purification and re-naturalization.

    2019 01 v.32 [Abstract][OnlineView][Download 326K]

  • Isolation,identification and phylogenetic analysis of a bovine parainfluenza virus type 3 strain

    CUI Yi-long;YANG Da-han;SHI Yun;YIN You-qin;XUE Jiang-dong;MA De-huin;College of Animal Science and Technology,Inner Mongolia University for the Nationalities;

    Objective To isolate,identify and perform phylogenetic analysis on a suspected bovine parainfluenza virus type 3(BPIV3)strain. Methods A suspected BPIV3 strain was isolated from the nose fluid of a cow with respiratory disease in a cattle farm in Inner Mongolia Mongolia Autonomous Region,China,propagated on MDBK cells,and identified by hemagglutination test and RT-PCR. The N gene of isolated BPIV3 strain was sequenced and analyzed,based on which a phylogenetic tree was constructed. Results The isolated BPIV3 strain was propagated on MDBK cells and caused specific CPE. The virulence of isolated strain of passage 6 was 10~(7. 1) TCID_(50)/100 μL,while the homology was 99. 9% to the N gene fragment of BPIV3 strain in GenBank,indicating the closest relation to the BPIV3 with sequence number of D84095. 1.Conclusion The strain isolated in this study was BPIV3 with high homology to a strain(D84095. 1)isolated in Japan.

    2019 01 v.32 [Abstract][OnlineView][Download 214K]

  • Expression of epitope chimaera of porcine circovirus type 2 ORF2 and porcine reproductive and respiratory syndrome virus ORF5 in Lactococcus lactis

    SHEN Ke-fei;YANG Liu;YANG Rui;ZHOU Xue;ZHANG Su-hui;FU Li-zhi;Chongqing Academy of Animal Sciences;

    Objective To express the epitope chimaera of porcine circovirus type 2(PCV2) ORF2 and porcine reproductive and respiratory syndrome virus(PRRSV)ORF5 in Lactococcus lactis. Methods Tandem antigenic epitope gene of PCV2 ORF2 and PRRSV ORF5 was designed and synthesized according to the codon preference of Lactococcus lactis,and inserted into vector pNZ8148. The constructed recombinant plasmid was transformed into L. lactis NZ9000 for expression under induction of nisin. The expressed product was purified by Ni-NTA affinity chromatography and analyzed by 12% SDS-PAGE. Results Restriction analysis showed that recombinant plasmid was constructed correctly. The chimaera,with a relative molecular mass of about 25 000,was mainly expressed in a soluble form,and reached a purity of more than 96% after purification,of which the concentration was about 1 mg/m L,and the yield was 60 mg/L bacterial culture. Conclusion The epitope chimaera of PCV2 ORF2 and PRRSV ORF5 was highly expressed in a soluble form in L. lactis.

    2019 01 v.32 [Abstract][OnlineView][Download 241K]

  • Action mechanism of LPS in proliferation of skin fibroblasts after inhibition of JNK

    WANG Yong-xiang;WANG Chun-yan;ZHAO Peng-wei;Department of Laboratory Medicine,The Affiliated Hospital of Inner Mongolia Medical University;

    Objective In this study,we studied the effect of Anisomycin and LPS on the growth of skin fibroblasts through MAPK pathway. Methods Skin fibroblasts were treated with 400 ng/mL Anisomycin and 20 ng/mL lipopolysaccharide(LPS),using those treated with LPS alone as control. The growth rates of fibroblasts 0,24,48 and 72 h after treatment were determined by MTT assay,while the phosphorylation of ERK,JNK and p38 0,20,40 and 60 min after treatment by Western blot. Results The growth rate of fibroblasts after treatment with Anisomycin and LPS for more than 24 h were significantly higher than that with LPS alone(P < 0. 05). The expressions of phosphorylated JNK,ERK and p38 showed no changes. However,the expression level of phosphorlyated JNK in fibroblasts 20 and 40 min after treatment with LPS alone decreased gradually(P < 0. 05). Conclusion The study provides an evidence that LPS inhibits the growth of skin fibroblasts mainly via JNK-dependent pathway,which lays a foundation of further study on role of LPS in pathogenic change of skin fibroblasts.

    2019 01 v.32 [Abstract][OnlineView][Download 161K]

  • Effect of sleep deprivation on expression levels of IL-6 and TNF-α in kidney of mice

    LIU Gang;HOU Liang-juan;Department of Infectious Disease,Chongqing Three Gorges Central Hospital;

    Objective To investigate the effect of sleep deprivation on expressions of IL-6 and TNF-α in kidney of mice.Methods Male C57 mice were randomly divided into four groups. The mice in three test groups were subjected to deprivation for 24,48 and 72 h respectively,while those in control group were untreated. The urea nitrogen(BUN),creatinine(CREA) and uric acid(URCA) levels in sera of mice were determined by automatic biochemical analyzer,while the activity of superoxide dismutase(SOD) in renal tissue by xanthine oxidase method,and the malondialdehyde(MDA) content by thiobarbituric acid method. The histological features of renal tissue was analyzed by H&E staining.The expression levels of IL-6 and TNF-α in renal tissue was determined by ELISA. Results Compared with those in control group,with the increasing time for SD,the BUN,CREA and URCA contents in sera of mice increased significantly 24 h(P < 0. 01)and continued to increase 48 and 72 h after SD(P < 0. 01),while the SOD activity in renal tissue decreased continuously(P < 0. 01),and the MDA content increased continuously(P < 0. 01). HE staining showed vacuolar degeneration of renal tubular epithelial cells,mild edema,glomerular and interstitial inflammatory cell infiltration and visible red blood cells,as well as renal capsule obliteration. However,the IL-6 and TNF-α contents increased continuously with the increasing time for SD(P < 0. 01). Conclusion SD induced the oxidative damage of renal tissues which elevated lipid peroxidation and impaired the structure and function of kidney. Massive release of inflammatory cytokines IL-6 and TNF-α activated the inflammatory responses which may further to contribute to kidney damage.

    2019 01 v.32 [Abstract][OnlineView][Download 300K]

  • High expression of Cap protein of porcine circovirus type 2 in recombinant baculovirus and immune property of expressed product

    ZHANG Yan-yan;ZHANG Jing-yuan;MIAO Fa-ming;CHEN Teng;YANG Jin-jin;RUAN Xiao-feng;ZHANG Shou-feng;HU Rong-liang;Jilin Agricultural University;

    Objective To highly express the capsid(Cap) protein of porcine circovirus type 2(PCV2) by baculovirus expression system and analyze the immune property of expressed product. Methods PCV2 Cap gene was cloned into transfer vector pFastBacⅠ. The constructed recombinant plasmid was transformed to competent E.coli DH10 Bac,and the obtained recombinant bacmid was transfected to Sf9 cells. The recombinant baculovirus was identified by SDS-PAGE and Western blot,and expressed in High Five(H5) cells. Kunming mice were randomly divided into four groups and immunized with PBS(control),wild baculovirus,recombinant baculovirus and commercid PCV2 vaccine respectively by intramuscular injection for 2 times at an interval of 2 weeks,100 μL for each. Serum samples were collected on days 21,28,35 and 42 after the first injection and determined for specific IgG level against recombinant protein by indirect ELISA. Results PCR analysis and sequencing showed that the recombinant transfer vector and recombinant bacmid were constructed correctly. Recombinant PCV2 Cap protein,with a relative molecular mass of about 28 000,showed specific binding to anti-His tag monoclonal antibody and anti-PCV2/Cap polyclonal antibody. The expression level of PCV2 Cap protein reached the maximum of about 150 μg/mL in H5 cells 72 h after infection with the recombinant baculovirus at MOIs of 5 and 1. The serum antibody levels of mice on days 21,28,35 and 42 after the first immunization with recombinant baculovirus were significantly higher than those with PBS and with wild baculovirus(P < 0. 001). However,serum antibody levels of mice on days 21,28 and 35 after the first immunization with recombinant baculovirus was significantly higher than those with commercial PCV2 vaccine(P < 0. 05),while showed no significant difference on day 42(P > 0. 05). Conclusion PCV2 Cap was highly expressed in baculovirus,which induced high antibody level in mice.It laid a foundation of preparation of PCV vaccine.

    2019 01 v.32 [Abstract][OnlineView][Download 369K]

  • Effect of EB virus on growth of intestinal epithelial cells via ERK1/2 pathway

    HU Xiang-zhen;ZHANG Yan-fei;Department of Gastroenterology,Affiliated Hospital of Inner Mongolia Medical University;

    Objective To analyze the mechanism of EB virus(EBV)in inhibiting the proliferation of intestinal epithelial cells and causing diarrhea. Methods Intestinal epithelial cells were inoculated with EBV for 0,12,24,36 and 48 h,and determined for proliferation by MTT assay. The expression of MAPK(phosphorylated ERK1/2,phosphorylated JNK and phosphorylated p38) pathway was determined by Western blot. Results The survival rates of intestinal epithelial cells 36 and 48 h were significantly lower than those 12 and 24 h after inoculation with EBV as well as that before inoculation(0 h,P < 0. 05). However,the expression levels of phosphorylated ERK1/2(p-ERK1/2) 36 and 48 h were significantly lower than those at the other three time points after inoculation(P < 0. 05). Conclusion EBV inhibited the proliferation of intestinal epithelial cells via ERK1/2 pathway. It laid a foundation of further study on the pathological mechanism of diarrhea caused by EBV.

    2019 01 v.32 [Abstract][OnlineView][Download 181K]

  • Regulatory effect of recombinant human interferon α1b lozenges on immune function of rats

    HE Li-si;JIANG Wei;WEI Kang;TIAN Jian-ming;WANG Yan;School of Applied Chemistry and Biology,Shenzhen Polytechnic;

    Objective To investigate the regulatory effect of recombinant human interferon α1b(rhIFNα1b)lozenges on immune function of rats. Methods Rats were divided into five groups by weight,14 for each. The rats in model,positive control and three test groups were treated with empty matrix powder,pidotimod and rIFNα1b at dosages of 1 000,10 000 and 50 000 IU/lozenge respectively,once a day for 3 weeks,and injected i.p. with cyclophosphamide at a dosage of 60 mg/kg on days 7 and 9,using ten normal rats as control. The rats were fasted 8 h and weighed 24 h after the last administration,of which the thymus and spleen coefficients were calculated,and the IL-2,IL-4,tumor necrosis factor-α(TNF-α) and IFNα levels in sera and spenocytes as well as IgG and IgA levels in sera and saliva were determined.Results As compared with those of normal control,the bodyweight and thymus coefficient(P < 0. 05),the IL-2,IL-4,TNF-α,IFNα,IgG and IgA levels in sera(each P < 0. 01),as well as IL-4,TNF-α and IFNα levels in spleen(P < 0. 01)and IgG and IgA levels in saliva(P < 0. 01)of rats in model group decreased significantly. However,as compared with those in model group,the bodyweight and thymus and spleen coefficients of rats treated with rhIFNα1b lozenges at various dosages showed no significant difference(P > 0. 05),while the IL-2,IL-4,TNF-α and IFNα levels in sera and spleen increased at different degrees,and the IgA levels in sera and saliva increased significantly(P < 0. 05). Conclusion The IFNα1b lozenges at moderate and high dosages improved the immune function of rats significantly.

    2019 01 v.32 [Abstract][OnlineView][Download 200K]

  • Evaluation of applicability of viral nucleic acid screening reagent for human plasma as a raw material

    SHI Yan-hong;DENG Kun;YANG Bang-ling;LI Qian;Wuhan Institute of Biological Products Co.,Ltd.;

    Objective To evaluate the applicability of viral nucleic acid screening reagent for human plasma as a raw material. Methods A total of 18 636 plasma samples qualified in ELISA were tested by mixed screening models of 6 samples and 96 samples in cobas s 201 system separately. The screened positive samples were subjected to COBAS AmpliPrep/COBAS TaqMan HBV/HCV/HIV-1(CAP/CTM) test to evaluate the applicability of the two screening models in practical test. Results Twelve positive samples were screened by mixed screening model of 6 samples,while no positive samples were screened by mixed screening model of 96 samples. HBV nucleic acid at low concentrations(less than 20 IU/mL)were detected in 6 of the 12 samples by CAP/CTM test,while no HIV and HCV nucleic acid positive samples were detected. Conclusion Viral nucleic acid positive samples may be effectively screened by mixed screening model of 96 samples,while no high concentration samples are missed. The undetected low concentration samples may be effectively inactivated by the further virus inactivation procedure,which shows no influence on the safety of blood products. The screening model shows high throughout,which is suitable for the routine nucleic acid screening of plasma as a raw material.

    2019 01 v.32 [Abstract][OnlineView][Download 124K]

  • Characteristics of adverse events following immunization of Haemophilus influenzae b-containing vaccines in Pudong New District,Shanghai during 2012～2016

    YANG Lai-bao;ZHOU Cui-ping;XIAO Shao-tan;DENG Peng-fei;CHEN Hong-ying;FEI Yi;Pudong New Area Center for Disease Control and Prevention,Shanghai;

    Objective To analyze the characteristics of adverse events following immunization(AEFI)reaction of Haemophilus influenzae b(Hib)containing vaccines. Methods The data on AEFI of Hib containing vaccines reported in Pudong New District,Shanghai were collected through China AEFI Information Management System during 2012 ～ 2016,while that on vaccination through the Shanghai Immunization Program Information System,and subjected to statistical analysis by descriptive epidemiological methods. Results A total of 1 766 cases of AEFI of Hib were reported during 2012 ～2016,indicating an incidence rate of 318. 78/100 000 doses,of which 1 735 cases were general reactions(98. 24%),indicating an incidence rate of 313. 36/100 000 doses,while 30 cases of abnormal reaction were reported,indicating an incidence rate of 5. 42/100 000 doses. One case of coincidence was observed,indicating an incidence rate of 0. 18/100 000 doses. The incidence rates of AEFI of Hib,epidemic encephalitis-HI,DTaP-Hib,DTaP-IPV-Hib were 160. 69/100 000 doses,203. 28/100 000 doses,358. 24/100 000 doses and 590. 59/100 000 doses respectively,which showed significant difference(P < 0. 001). A total of 473 223 doses of various vaccines were inoculated for primary immunization(three doses),of which the incidence rate of AEFI was 224. 84/100 000 doses. However,a total of 80 457 doses were inoculated for booster immunization,of which the incidence rate of AEFI was 871. 27/100 000 doses,indicating a significant difference with that of primary immunization(P < 0. 001). The incidence rates of AEFI of Hib,DTap-Hib and DTaP-IPV-Hib in booster immunization were significantly higher than those in primary immunization(each P < 0. 001). The incidence rates of AEFI of various vaccines in various injection sites showed significant difference(each P < 0. 001). The AEFI of Hib and epidemic encephalitis-HI mainly appeared in upper arm,with incidence rates of118. 71/100 000 doses and 203. 28/100 000 doses respectively. The AEFI of DTaP-IPV-Hib mainly appeared in thigh,with an incidence rate of 545. 75/100 000 doses,while that of DTaP-Hib mainly appeared in buttocks,with an incidence rate of 259. 41/100 000 doses. Most of the AEFI of various vaccines appeared 6 ～ 24 and 24 ～ 48 h after immunization,with an incidence rate of 255. 92/100 000 doses. The incidence rates of AEFI at various time intervals after immunization showed significant difference(each P < 0. 001). Conclusion The incidence of AEFI of Hib containing vaccines was low,indicating a high safety. It is recommended that the vaccines should be vaccinated in upper arm or thigh,and the monitoring and disposal of AEFI should be strengthened 1 ～ 2 d after vaccination.

    2019 01 v.32 [Abstract][OnlineView][Download 239K]

  • Change trend of epidemiological characteristics of rubella before and after implementation of Expanded Program on Immunization in Liaoning Province,China

    FANG Xing;CHEN Tao;WANG Yan;YAO Wen-qing;Liaoning Provincial Center for Disease Control and Prevention;

    Objective To analyze the change trend of epidemiological characteristics of rubella before and after implementation of the Expanded Program on Immunization(EPI)in Liaoning Province,China,and evaluate the effect of incorporation of rubella-containing vaccines(RCVs) into the EPI. Methods Descriptive epidemiology and analytical epidemiology were used to analyze and compare the epidemiological characteristics of rubella in Liaoning Province before(2005 ～ 2010)and after(2011 ～ 2016)the implementation of EPI with Excel and SPSS 23 software. Results The average annual incidence of rubella in Liaoning Province was 15. 24/100 000 before implementation of EPI,while decreased to 5. 88/100 000 after implementation(P < 0. 05). The proportion of cases appearing from April to June before and after implementation of EPI were 75. 29%(29 418/39 074)and 68. 39%(10 576/15 465)respectively,which showed significant difference(P < 0. 001),and the median age of onset were 18. 12 and 17. 83 years respectively.Conclusion After the implementation of EPI,the average annual incidence of rubella in Liaoning Province decreased significantly,but did not break the inherent regularity of rubella epidemic. The age of onset showed no significant backward shift,while the risk of congenital rubella syndrome(CRS) showed no increase,indicating the remarkable achievement of EPI.

    2019 01 v.32 [Abstract][OnlineView][Download 166K]

  • Optimization of medium for Mycoplasma pneumoniae by design of experiment

    LI Shuang-li;LI Ze-hua;ZHU Wen-yong;CHEN Feng;LI Wei-dong;LIAO Guo-yang;Institute of Medical Biology,Chinese Academy of Medical Science & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research & Development on Severe Infection Disease;

    Objective To optimize the medium for Mycoplasma pneumoniae(MP)by design of experiment(DOE)so as to increase the yield of MP. Methods Seven addition ingredients,i. e. extraction of brain and heart,lactoalbumin hydrolysate,peptone 3,arginine,calf thymus DNA,glucose and tryptone,were screened and optimized by PlackettBurman test,steepest ascent test and Box-Behnken design,using MP FH strain as test strain,PPLO as basic medium,and bacterial protein content as response value. The effect of culture of MP by using the modified medium was verified serving bacterial protein content and color change unit(CCU)as indicators. Results In the turn of effect on the growth of MP,the addition ingredients were glucose,tryptone,extraction of brain and heart,arginine,peptone 3,lactoalbumin hydrolysate and calf thymus DNA. Three additives,extract of brain and heart,glucose and peptone 3,were screened,of which the concentrations were 5. 68,1. 34 and 2. 64 g/L respectively. The bacterial protein content and CCU of MP cultured in the modified medium for 96 h reached the maximums of 851. 29 μg/mL and 10~9 respectively. Conclusion The medium for MP was optimized by DOE,while the significant factors influencing MP growth as well as their optimal concentrations were obtained,and the yield of MP increased.

    2019 01 v.32 [Abstract][OnlineView][Download 343K]

  • Preparation of antiserum against RhlR in Pseudomonas aeruginosa

    ZI Jing;WANG Jun;GAO Lei;ZHANG Kun;ZHANG Xu;WAN Yi;Research Center of Molecular Biology,Shaanxi Institute of Microbiology;

    Objective To express the Rhl R protein of Pseudomonas aeruginosa PAO1 in prokaryotic cells and specific rabbit antiserum against RhlR. Methods Recombinant plamids pGEX-4T-1-rhlR and pET-22 b-rhlR were constructed and transformed to competent E. coli BL21 and induced with IPTG. The expressed GST-RhlR and His-RhlR were purified by GST or nickelion affinity chromatography. Purified His-RhlR protein was immunized to New Zealand rabbits to prepare antiserum against RhlR,while purified GST-RhlR protein was determined for titer by ELISA. Results Restriction analysis and sequencing proved that recombinant plasmids pGEX-4T-1-rhlR and pET-22 b-rhlR were constructed correctly.The expressed GST-RhlR and His-RhlR,with relative molecular masses of 29 000 and 54 000 respectively,contained about 85% of total somatic proteins and reached purities of more than 90%. The prepared antiserum against RhlR reached a titer of 1 ∶ 80 000. Conclusion High titer antiserum against RhlR was obtained,which laid a foundation of study on the rhl regulator system of PA.

    2019 01 v.32 [Abstract][OnlineView][Download 233K]

  • Establishment of and application of standard system for detection of copy number of dengue virus type Ⅰ

    LIN Yao;HONG Shan;WEN Song-jiao;XI Jue-min;WANG Xiao-dan;CHEN Jun-ying;PAN Yue;YE Chao;GUAN Jiao-qiong;SUN Qiang-ming;Institute of Medical Biology,Chinese Academy of Medical Science & Peking Union Medical College,Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Disease;

    Objective To establish a standard system for detection of copy number of dengue virus-Ⅰ(DENV-Ⅰ).Methods Partial prM gene sequence of DENV-Ⅰwas cloned into vector pMD19T,and the constructed standard plasmid was detected for Ct value of copy number by real-time fluorescence quantitative PCR,based on which a standard curve of virus copy number against Ct was plotted,and the corresponding equation was obtained. Three serum samples of patients with DENV-Ⅰ infection in Xishuangbanna Region,Yunnan Province were detected by the established detection system using the standard plasmid. Results Restriction analysis and sequencing proved that the standard plasmid for detection of copy number of DENV-1 was constructed correctly. The equation of standard curve for real-time fluorescence quantitative PCR was as follows:y =-0. 300 5 x + 12. 133,R~2 = 0. 991 9. The viral copy numbers in three serum samples were 9 417 703. 12,21 949 591. 01 and 19 027 096. 91 copies/μL respectively. Conclusion Viral copy number standard of DENV-Ⅰ was established,which showed high accuracy and sensitivity and might be used for determination of DENV-Ⅰin serum samples.

    2019 01 v.32 [Abstract][OnlineView][Download 198K]

  • Construction of phage display technology for complete human single-chain antibody fragment library

    ZHANG Yu-chao;DING Long-fei;CHEN JIAN;ZHANG Xiao-yan;XU Jian-qing;School of Laboratory Medicine and Life Sciences,Wenzhou Medical University;

    Objective To construct the phage display technology for complete human single-chain fragment(scFv)library. Methods Peripheral blood samples were collected from 100 healthy elderly volunteers,5 mL for each,and pooled,from which peripheral blood mononuclear cells(PBMCs)were separated and subjected to RNA extraction followed by cDNA synthesis. The heavy chain variable region(V_H)and light chain variable region(V_L)were amplified by PCR using the obtained cDNA as a template and the special sequence of IgG antibody as primer,and used as templates to obtain scFv through overlapping PCR. The constructed scFv was digested with sfi Ⅰand inserted into pComb3 xss vector,based on which a primary antibody library was constructed by electrotransformation and co-cultured with auxiliary phage VCSM13 overnight. The supernatant was precipitated with PEG-sodium chloride, and then the bacteriophage was resuspended in sterile PBS to complete the construction of phage antibody library. Phage ELISA was used to characterize the specificity of monoclonal antibody against phage. Results The human scFv library with capacity of 2. 6 × 10~9 was constructed. Two suspected phage antibodies against HA antigen of H1N1 strain were screened from 100 randomly selected phage monoclonal clones. Conclusion The technology platform of phage antibody library is successfully constructed,which may be used for screening various functional antibodies.

    2019 01 v.32 [Abstract][OnlineView][Download 271K]

  • Determination of m-cresol content in recombinant human interferon α2b injection by high performance liquid chromatography

    SUN Rui-xin;WANG Jiang-lin;LIU Han;LIU Yun-nan;LIU Lin-lin;ZHANG Bo;ZHU Qiu-mei;LI Li;Changchun Institute of Biological Products Co.,Ltd.;

    Objective To develop and verify a high performance liquid chromatography(HPLC)method for determination of m-cresol content in recombinant human interferon alpha 2b injection. Methods Reverse phase(RP)-HPLC was performed on a ZORBAX 300 SB-C18 column(4. 6 mm × 250 mm 5-Micron),serving water-methanol(50 ∶ 50) as a mobile phase at a flow rate of 0. 5 mL/min and a column temperature of 45 ℃. The sample load was 20 μL while the detection wavelength was 270 nm. The method was verified for linearity,specificity,accuracy,precision quantitative limit and durability. Results The method showed good linearity at a cresol concentration of 1 000 ～ 0. 1 μg/mL,with a R~2 value of 0. 999 3. Neither medicinal excipients nor IFN showed interference to the determination of cresol content. The mean recovery rates of samples at high,moderate and low concentrations were 97. 10%,99. 00% and 103. 94% respectively,while the total mean recovery rate of 100. 01%. The RSD in reproducibility verification was 0. 105%,while that in intermediate precision verification was 0. 26%. The quantitative limit of the method was 0. 1 μg/mL. Under the same chromatographic condition,the mobile phase prepared using various apparatuses at various time showed no significant influence on determination result. However,numbers of theoretical plates used for chromatographic columns at various use degrees showed significant difference. Conclusion The develop HPLC method showed high specificity,precision and durability,which might be used for determination of cresol content in rh IFNα2b injection.

    2019 01 v.32 [Abstract][OnlineView][Download 278K]

  • Titers of rabbit antisera against enterovirus 71 prepared by two immunization routes

    HU Yun-guang;SHEN Dong;LI Ya-dong;YANG Xiao-lei;HONG Chao;FU Jie;WANG Xi;Institute of Medical Biology Chinese Academy of Medical Sciences;

    Objective To compare the titers of rabbit antisera against enterovirus 71(EV71) prepared by two immunization routes. Methods Two groups of rabbits,five for each,were immunized with inactivated EV71 vaccine by subcutaneous and intramuscular routes respectively,each at a dosage of 5 μg,on days 0,7,14 and 18. The antigen for the first dose was mixed well with an equal volume of complete Freund adjuvant,while those for the following doses with incomplete Freund adjuvant. Serum samples were collected before immunization and 7 d after each dose,and determined for neutralizing antibody titer. Results High antibody titers were induced by both subcutaneous and intramuscular routes.The antibody titers of more than 1 ∶ 10 000 were induced in seven rabbits,three in subcutaneous immunization group and four in intramuscular immunization group. The antibody titers induced by intramuscular route were higher than those by subcutaneous route,especially those on day 21 after the first dose(P < 0. 05). Conclusion The titer of rabbit antisera prepared by intramuscular route was higher than that by subcutaneous route, which provided a reference for the preparation of high titer rabbit antiserum against EV71.

    2019 01 v.32 [Abstract][OnlineView][Download 179K]

  • Isolation and identification of human placenta-derived hematopoietic stem cells and mesenchymal stem cells

    LI Xin;ZHAO Zhong-li;LUO Xiao-tong;LI Jia-ying;CAO Yang;YU Yong-sheng;Branch of Animal Husbandry,Jilin Academy of Agricultural Sciences;

    Objective To develop a method for isolation of human placenta-derived hematopoietic stem cells and mesenchymal stem cells,identify the isolated cells and analyze their components. Methods Ten placental tissues of healthy newborns were collected,from which placenta hematopoietic stem cells(hP-HSCs) were isolated by mechanical disruption combined with magnetic bead sorting,while placenta-derived mesenchymal stem cells(hP-MSCs)by placental chorionic membrane tissue block adherence method,observed for morphology,and identified by colony culture and flow cytometry. Results The total nucleated cell(TNC) number of hP-HSCs was(11. 82 ± 2. 46) × 10~8,while the TNC recovery was not less than 80%,the cell viability was(99. 7 ± 0. 3)%,the proportion of CD34~+CD45 dim cells was(8. 69 ± 0. 36)%,the total CD34~+ cell number was(108. 0 ± 6. 48)× 10~6,and the total number of colonies formed was(1. 88 ± 1. 07) × 10~6. However,the total number of frozen hP-MSCs was(40. 78 ± 9. 35) × 10~7,while the cell viability was(99. 0 ± 1. 5)%,and the proportions of CD34~+CD45~+,CD45~+CD105~+,CD14~+CD19~+ and CD90~+CD73~+ cells were(0. 1 ± 0. 1)%,(99. 6 ± 0. 2)%,(0. 1 ± 0. 1)% and(98. 9 ± 0. 2)%,respectively. The hP-MSCs showed potential in osteogenic and adipogenic differentiations. Conclusion The methods were isolation and culture of h P-hSCs and hP-MSCs were successfully developed,which laid a foundation of clinical application of placenta and provided a resource of cell seeds.

    2019 01 v.32 [Abstract][OnlineView][Download 401K]

  • Advance in study on T cell response induced by Staphylococcus aureus in various models

    ZENG Jiang-min;ZHAO Zhuo;Department of Microbiology and Biochemical Pharmacy,College of Pharmacy,The Third Military Medical University;

    Staphylococcus aureus is a major cause of hospital infection and community-acquired infection in the world.Specific antibody response against S. aureus is dominant in the previous studies. However,the latest studies show that the various subsets of T cells involve in the immune response caused by S. aureus. This paper reviews the advance in study on T cell response induced by S. aureus in various models.

    2019 01 v.32 [Abstract][OnlineView][Download 272K]

  • Separation,purification and clinical application of α2-macroglobulin

    HUANGFU Chao-ji;DANG Ming-an;ZHAO Xiong;ZHANG Jin-gang;Institute of Health Service and Transfusion Medicine,Academy of Military Medical Sciences;

    α2-Macroglobulin(α2-M)is a large molecular glycoprotein which mainly exists in human plasma. As a broad spectrum inhibitor of proteases,α2-M has the ability of removing both endogenous and exogenesis proteases. The original material for preparation of α2-M is mainly human plasma,while the laboratory preparation procedure mainly includes immuno-affinity chromatography,gel filtration and Cibacron blue agarose affinity chromatography. However,the pilot preparation procedure usually consists of crude separation with ammonium sulfate,rivanol or polyethylene glycol and further purification by zinc ion affinity chromatography or gel filtration. α2-M presents good curative effect on radiation induced ulcer of skin and mucosa,radiological pneumonia,radiological cystitis,keratitis and corneal ulcer. Meanwhile,α2-M may be used as a biomarker for judgment of stage of liver fibrosis and the severity of pancreatitis. This paper reviews the preparation procedure and clinical application of α2-M.

    2019 01 v.32 [Abstract][OnlineView][Download 161K]

  • Research on vaccine for nasopharyngeal carcinoma

    LI Hai-long;YANG Yu-fei;ZHANG Rui;Henan Grand Biologic Pharmaceutical INC;

    Nasopharyngeal carcinoma(NPC) is one of the most common malignant tumor in the south of China. Researches show that the tumor is statistically related to Epstein-Barr virus(EBV). The article introduces NPC,EBV as well as the current situation of diagnosis and treatment of NPC,while emphasis on the prospect of preventive and therapeutic vaccines.

    2019 01 v.32 [Abstract][OnlineView][Download 195K]

  • Impediments to development of Dengue fever vaccine

    WANG Wei-shan;SHEN Shuo;Changsha Reproductive Medical Hospital;

    Dengue fever(DF)is among the most prevalent and important arbovirus diseases of humans,which is caused by Dengue virus(DENV) infection and mainly transmitted by Aedes aegypti and Aedes albopictus. It is endemic in Southeast Asia,the Western Pacific,Central and South America,the Caribbean,and Africa. At present,about 3. 6 billion people live in dengue-endemic areas,and the virus causes approximately 400 million infectious and 100 million symptomatic cases annually. It is estimated that there are more than 2 million cases of severe dengue disease and more than 20 000 cases of deaths each year. Vaccination is an effective measure to prevent the disease. Because multiple difficulties have hindered the development of DENV vaccine,there is no ideal vaccine available at the moment. This article reviews the main impediments in the development of dengue virus vaccine.

    2019 01 v.32 [Abstract][OnlineView][Download 189K]

  • Progress in research on rabies vaccine for human use

    ZHOU Meng-chong;LI Chun-ming;LI Hai-long;YANG Yu-fei;Henan Grand Bio-Pharm.Co.,Ltd.;

    Rabies is a zoonosis and an acute infectious disease,which is caused by rabies virus and threats the health of humans. Currently,the vaccination of rabies vaccine is still the most effective measures to prevent rabies. This paper reviews the progress in research on the vaccine.

    2019 01 v.32 [Abstract][OnlineView][Download 213K]


@2012 Editorial Department of "Chinese Journal of Biologicals"