- SHI Yan-chunΔ,WENG Bin,ZHENG Yuan-qiang,et al ( ΔThe Key Laboratory of Department of Education of Inner Mongolia Autonomous Region for Molecular Biology, Inner Mongolia University,Huhhot 010059,China)
Objective To explore the influence of combined immunization with the synthetic oligonucleotide containing CpG motif(CpG ODN) and rHBsAg on the histological change and lymphocyte proliferation of murine spleen.Methods Twenty-five BALB/c mice were randomly divided into four test groups and one control group,five for each,and immunized twice with rHBsAg,HB vaccine,rHBsAg+CpG ODN,HB vaccine+CpG ODN and physiological saline respectively,at an interval of 2 weeks.Prepare the murine spleens into routine paraffin sections for observation of histological change by H-E staining.Determine the proliferation of lymphocytes by 3H-TdR incorporation method.Results Significant histological change was observed in the murine spleens in rHBsAg+CpG ODN group.Compared with those of mice in rHBsAg and HB vaccine groups,the lymphocyte proliferations of mice in rHBsAg+CpG ODN and HB vaccine+CpG ODN groups were significantly enhanced.However,compared with those of mice in control group,the lymphocyte proliferations of mice in all the four test groups were significantly enhanced.Conclusion CpG ODN caused the histological change of murine spleen,such as extension of germinal center,and enhanced the lymphocyte proliferation significantly.It indicated that CpG ODN might be a potential Th1 type adjuvant of HB vaccine.
2006 02 [Abstract][OnlineView][Download 284K] - JIA Jian-an△,ZHOU Bo,QIAN Bao-hua,et al ( △Department of Microbiology,Second Military Medical University,Shanghai 200433,China)
Objective To express HIV-1 HXB2 subtype in prokaryotic cells,then purify and identify the expressed product for the screening of HIV-1 Gag CAP2/NC protein phage displayed library with randomized P2/NC protease cleavage site.Methods The primers were designed according to the PR DNA sequence of HIV-1 HXB2 subtype,and the DNA sequence encoding HIV-1 PR,with E.coli-preferred codon,was synthesized by overlapping PCR,then inserted into pMD18-T vector by T/A cloning.After identification by sequencing,the amplified HIV-1 PR DNA was cloned into prokaryotic expression vector pQE30 and expressed in E.coli M15 under induction of IPTG.The expressed product was purified by Ni-NTA affinity column chromatography and identified by SDS-PAGE,then analyzed for immunoreactivity with HIV-positive serum.Results The sequence of amino acids encoded by the synthesized DNA was completely identical to the original amino acid sequence of HIV-1 HXB2 subtype.The HIV-1 PR,with a relative molecular weight of 14 000 ,was expressed in the constructed prokaryotic expression vector pQE30.The purified target protein reached a purity of 7.74 mg/ml .ELISA proved specific reaction of the expressed protein with HIV-positive serum.Conclusion The DNA sequence encoding HIV-1 PR,with E.coli-preferred codon,was successfully synthesized and cloned,and the HIV-1 PR with immunological character was expressed in E.coli and purified.
2006 02 [Abstract][OnlineView][Download 484K] - CHEN YueΔ,LI Jing-hua,HE Kan,et al ( ΔResearch Center of Vaccine,College of Life Science,Jilin University,Changchun 130012,China)
Objective To clone human stathmin gene and express in prokaryotic cells.Methods Stathmin gene was amplified from the cDNA library of Hela cells by PCR.After TA cloning and sequencing,the amplified gene was cloned into vector pGEX-4T-1 and transformed to competent E.coli BL21 for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results A 460 bp cDNA fragment was amplified by PCR,and its sequence was consistent with that of human stathmin(NM_203401) reported in GenBank.The expressed product was proved as GST fusion protein by SDS-PAGE and Western blot.Conclusion Human stathmin was expressed in prokaryotic cells.It laid a foundation of further study on the structure and function of stathmin.
2006 02 [Abstract][OnlineView][Download 230K] - YUE Li-qinΔ, SHEN Xu-zhuang, ZHOU Yu-sen, et al ( ΔLaboratory of Microiology & Immunology, Beijing Children′s Hospital, Beijing 100045, China)
Objective To express the functional domain of C5a peptidase of streptococcus group B and lay a foundation of further development of vaccine.Methods Amplify four gene fragments encoding the different functional domains of C5a peptidase of streptococcus group B from the genomic DNA of standard GBSⅡ strain by PCR using the designed primers and insert into T vector respectively. Digest the recombinant plasmid with restriction endonuclease and insert tne obtained target gene fragments into prokaryotic expression vector pET32 for fusion expression. The expressed products were determined for immunogenicities and purified by Ni 2+ -NTA column chromatography.Results Four recombinant plasmids for expression of C5a peptidase of streptococcus group B was successfully constructed. SDS-PAGE showed that the relative molecular weights of four kinds of expressed products were 80 000, 78 000, 70 000 and 36 000, and their probability based mowse scores of C5a peptidase were 82, 152, 113 and 79, respectively. Western blot showed the specific reactions of all the four kinds of expressed products with antibody against C5a peptidase. After purification, the expressed product reached a purity of 90%.Conclusion Four functional domains of C5a peptidase of streptococcus group B were successfully expressed. It laid a foundation of study on immune epitopes and virulence mechanism of C5a peptidase and development of streptococcus group B subunit vaccine.
2006 02 [Abstract][OnlineView][Download 591K] - WANG Li-chanΔ,ZHANG Shu-min,YU Mo-song,et al ( ΔNational Institute for The Control of Pharmaceutical and Biological Products,Beijing 100050,China)
Objective To construct the prokaryotic expression vector of staphylococcal enterotoxin A(SEA) gene and express the gene in E.coli.Methods Amplify SEA gene by PCR,ligate to cloning vector pGEM T-easy,then insert into expression vector pET-30a and transform to E.coli for expression.Results A gene fragment at a length of 770 bp was amplified,and its sequence was identical to that reported.The protein with a relative molecular weight of 31 000 was expressed and identified as SEA after purification.Conclusion SEA protein was successfully expressed.It laid a foundation of further study and application of superantigen SEA.
2006 02 [Abstract][OnlineView][Download 450K] - XIAO Jian,ZHU Hua-song,YIN Juan,et al (Wuhan Institute of Biological Products,Wuhan 430060,China)
Objective To truncate the trans-membrane proteins gp41 of HIV-1 and gp36 of HIV-2 and express in E.coli.Methods Truncate the genes encoding gp41 and gp36 by PCR,purify the PCR product and clone into vector pGEM-T.Digest the recombinant plasmid with BamHⅠ,EcoRⅠ and SalⅠ and insert the obtained target gene into expression vector pGEX-4T3.Transform the constructed recombinant plasmid to E.coli BL21 for expression under induction of IPTG.Identify the expressed product by SDS-PAGE and Western blot.Results Restriction map proved that the lengths of truncated gp41 and gp36 genes were identical to those expected.SDS-PAGE profile revealed a fusion expression band with a relative molecular weight of 66 000.Western blot showed specific reactions of expressed product with the corresponding antibodies.Conclusion The HIV-1 gp41 and HIV-2 gp36 genes were successfully truncated and expressed in E.coli.It laid a foundation of further application of trans-membrane protein.
2006 02 [Abstract][OnlineView][Download 523K] - DU Guo-li,SONG Chang-zheng,ZHANG Geng-lin,et al (Key Laboratory for Biotech-Drugs,Ministry of Health,Shandong Medical Biotechnology Center,Jinan 250062,China)
Objective To construct a transgenic Lycium barbarum L. expression system for HIV capsid protein(CA).Methods The gene encoding MA_4-CA fusion protein was amplified by PCR using plasmid pET-3a-MA_4-CA as a template,then cloned into vector pGEM-T and transformed to E.coli DH5α.The recombinant plasmid was digested with BglⅡ and BstEⅡ,and the obtained MA_4-CA gene was inserted into secretory expression vector pCAMBIA1305.2.The constructed plasmid pCAMBIA1305.2-MA_4-CA was transferred into Lycium barbarum L. by Agrobacterium tumefaciens-mediated transformation,and the transgenic plant was cultivated for budding and rooting.Results Budding and rooting of the transformants was successfully induced in the growth chamber.PCR amplification of DNA from transgenic plant confirmed the presence of the fusion gene under the control of promoter p35S and the ligation between the MA_4-CA fusion gene and the promoter.ELISA and Western blot proved the presence of CA in transformed leaf extracts.Conclusion The findings indicated that the constructed transgenic Lycium barbarum L. expression system can be used to produce immunogenic CA.
2006 02 [Abstract][OnlineView][Download 569K] - HUANG Gang, HE Feng-tian, LI Rong-fen, et al (Department of Biochemistry & Molecular Biology, Third Military Medical University, Chongqing 400038, China)
Objective To clone the cDNA encoding the mutant(sΔBAFF) of human B cell activating factor to the TNF family(BAFF), with the amino acid residues at sites 142-160 of extracellular domain(sBAFF142-160) deleted, then express the gene in prokaryotic cells and purify the expressed product.Methods The nucleic acid sequence encoding amino acids 142-160 of sBAFF was deleted by one-step reverse PCR using the constructed recombinant plasmid pUC19/sBAFF(containing the cDNA encoding sBAFF_ 134-285 ) as a template. The amplified cDNA was identified by sequencing and cloned into prokaryotic expression vector pQE-80L for expression under induction of IPTG. The expressed product was analyzed by SDS-PAGE and Western blot, then purified by Ni 2+ -NTA chromatography.Results A cDNA at length of 401 bp was amplified by one-step reverse PCR, and its sequence was consistent with that encoding human sΔBAFF amino acids reported in GeneBank. Human sΔBAFF with a relative molecular weight of 18 000 was highly expressed in a form of inclusion body in E. coli and reached a purity of 95.5% after purification by Ni 2+ -NTA chromatography.Conclusion The successful expression of human sΔBAFF laid a foundation of further study on its function.
2006 02 [Abstract][OnlineView][Download 394K] - XIAO Bin,YANG Jun,TIAN Wen-biao,et al (Department of Clinical Microbiology and Immunology,The Third Military Medical University,Chongqing 400038,China)
Objective To clone and express human interleukin-24 gene and purify the expressed product.Methods Isolate human peripheral blood mononuclear cells(PBMCs) and culture under stimulation of Con A.Extract the total RNA of the cultured PBMCs for amplification of the gene encoding the mature peptide of hIL-24 by RT-PCR.Directly clone the amplified gene into fusion expression vector pGEX-4T-1.Identify the recombinant plasmid pGEX-4T-1/hIL-24 by DNA sequencing,then transform to E.coli BL21(DE3) for expression under induction of IPTG.Purify the expressed product by anion exchange chromatography and identify by SDS-PAGE and Western blot.Results The sequence of the cloned hIL-24 gene was consistent with that reported in GenBank.The identification by sequencing showed that fusion expression vector pGEX-4T-1/hIL-24 was correctly constructed.The fusion protein,with a relative molecular weight of 44 000,was expressed in form of inclusion body and contained 30.63% of total somatic protein.The purity of the expressed protein after purification reached more than 85%.Western blot showed specific reaction of the expressed fusion protein with rabbit anti-human IL-24 polyclonal antiserum.Conclusion The fusion expression vector pGEX-4T-1/hIL-24 was successfully constructed,and hIL-24 was highly expressed in E.coli.The fusion protein with high purity was obtained.It laid a foundation of study on function and activity of hIL-24.
2006 02 [Abstract][OnlineView][Download 705K] - LIU Qi-wei,LI Qun-liang,CAI Hai-bo,et al (The State Key Laboratory of Bioreactor Engineering,East China University of Science and Technology,Shanghai 200237,China)
Objective To screen the differentially expressed genes of cord blood CD34~+ cells before and after culture in vitro.Methods Analyze the gene expressions of cord blood CD34~+ cells cultured in static and dynamic systems by DDRT-PCR,using the freshly isolated CD34+ cells as control.The differentially expressed genes were cloned,sequenced and analyzed for bioinformatics.The authenticity of differential expression of genes was verified by semi-quantitative RT-PCR.Results Five differentially expressed genes were screened by DDRT-PCR,of which 4 were genes with known function and one was gene with unknown function.Two of the five genes,RAN and DC24,were analyzed by semi-quantitative RT-PCR.The result showed that,compared with that of freshly isolated cells,the expression levels of RAN mRNA of CD34+ cells cultured in static and dynamic systems increased 1.521 and 3.978 folds,and those of DC24 mRNA increased 14.275 and 2.374 folds,respectively.Conclusion Some important genes related to the proliferation in vitro of CD34~+ cells were discovered.It provided a basis for the regulation of proliferation in vitro of CD34~+ cells at molecular biological level.
2006 02 [Abstract][OnlineView][Download 358K] - GAO Yu-long,GAO Hong-lei,DENG Xiao-yun,et al (Harbin Veterinary Research Institute,Chinese Academy of Agricultural Science,Harbin 150001,China)
Objective To express the VP2 protein of attenuated infectious bursal disease virus(IBDV) strain and study the antigenicity of expressed product.Methods Amplify VP2 gene by RT-PCR using the genomic RNA of attenuated IBDV strain Gt as a templet and insert into vector pUC18.Identify the recombinant plasmid by PCR,digestion with restriction endonuclease and sequencing,then subclone into prokaryotic expression vector pET30a.After identification by digestion with restriction endonuclease,PCR and sequencing,the positive recombinants were transformed to E.coli BL21(DE3) for expression under induction of IPTG.Identify the expressed product by SDS-PAGE and Western blot.Results The expressed product contained 15% of total somatic protein and reacted with IBDV-positive serum.However,it showed no reaction with negative control serum.Conclusion The VP2 protein of attenuated IBDV strain was successfully expressed in E.coli and showed good antigenicity.
2006 02 [Abstract][OnlineView][Download 267K] - LIU Yi,HAN Jin-xiang,SHAO Jin-hui,et al (Key Laboratory for Biotech-Drugs,Ministry of Health,Key Laboratory for Modern Medicine and Technology of Shandong Province,Shandong Medical Biotechnology Center,Jinan 250062,China)
Objective To construct the Pichia pastoris expression vector of the glycoprotein G(gG) ectodomain gene of herpes simplex virus type 1(HSV-1) stocker strain and analyze its sequence.Methods Amplify the ectodomain of HSV-1 gG gene by PCR and clone into vector pGEM-T,then transform to competent E.coli DH5α.Extract recombinant plasmid pGEM-T-gG1 by alkaline lysis and ligate to expression vector pPIC9K,then transform to E.coli DH5α.Screen recombinant plasmid pPIC9K-gG1,analyze its sequence and predict the physiochemical property,antigenicity and form of expressed product.Results Recombinant yeast expression vector pPIC9K-gG was constructed.Sequencing result proved that the cloned fragment was highly conserved ectodomain of HSV-1-gB gene.By the prediction using bio-software,the expressed product contain the six strong antigenic determinants of the whole ectodomain and existed in a form of soluble protein,and its relative molecular weight and isoelectric point were 15 870 and 4.72 respectively.Conclusion The Pichia pastoris expression vector of ectodomain of gG gene of HSV-1 stocker strain was successfully constructed.
2006 02 [Abstract][OnlineView][Download 525K] - ZENG Jin,WANG Yu-jiong,XU Chong-bo,et al (Key Laboratory of Biotech,Ningxia University,Yinchuan 750021,China)
Objective To construct the β_2-β_1 fusion gene of Clostridium perfrigens and express in E.coli.Methods Amplify β_2 toxin gene from plasmid CPB2-9 containing the gene by PCR and identify by digestion with NcoⅠ and BamHⅠ.Amplify β_1 toxin gene from plasmid pXETB_1 by PCR and identify by the same method.Ligate the two gene fragments and transform to E.coli BL21(DE3) for expression.Identify the expressed product by SDS-PAGE and Western blot.Results SDS-PAGE and Western blot showed that the β_2-β_1 fusion gene was successfully expressed in E.coli BL21(DE3),and the expressed product was recognized by the corresponding antibody.Conclusion The β_2-β_1 fusion gene of Clostridium perfrigens was successfully constructed and expressed in E.coli.
2006 02 [Abstract][OnlineView][Download 310K] - LIU Jian-juΔ,WU Ya-zhen,LI Hui-nan,et al ( ΔDepartment of Ophthalmology,Second Hospital of Jilin University,Changchun 130041,China)
Objective To construct eukaryotic expression vector pcDNA3.1(+)-Vasostatin and express Vasostatin in 293T cells.Methods Clone the Vasostatin gene fragment containing a signal peptide into eukaryotic expression vector pcDNA3.1(+). Identify the constructed recombinant plasmid pcDNA3.1(+)-Vasostatin by restriction analysis and sequencing,then transfect 293T cells in the mediation of liposome.Identify the expressed product by Western blot.Results A eukaryotic expression vector pcDNA3.1(+)-Vasostatin was successfully constructed.Western blot proved the expression of Vasostatin in the supernatant of lysate of 293T cells transfected with the recombinant plasmid.Conclusion The constructed recombinant plasmid pcDNA3.1(+)-Vasostatin could be used for the expression of Vasostatin in eukaryotic cells.
2006 02 [Abstract][OnlineView][Download 273K] - PENG Qi-shengΔ,ZHANG Guo-li,WU Guang-mou,et al ( ΔCollege of Veterinary,Jilin University,Changchun 130062,China)
Objective To analyze the mechanism of secretory expression of recombinant pseudomonas extoxin.Methods Transform the six constructed recombinant plasmids expressing pseukomonas extoxin,i.e.pET-20b-IL-10_ 23-57 -PE40,pET-20b-EGF-PE40,pET-20b-LHRH-PE35,pET-20b-EGF-PE38,pET-20b-MSH-PE40 and pET-20b-LHRH-PE40,into E.coli Rosetta(DE3) for expression under induction of IPTG.Identify the expressed product by SDS-PAGE and Western blot.Results Only LHRH-PE40 and LHRH-PE35 were expressed.Western blot showed that the expressed product of pET-20b-LHRH-PE35 was recognized by the specific antibody against LHRH-PE40.The analysis of the six kinds of recombinant pseudomonas extoxin by DNAStar and ANThewin software showed that not all the recombinant pseudomonas extoxin fused with signal peptide sequence could expressed in a form of seretory protein.The secretory expression of recombinant pseudomonas extoxin was determined by the property of leader signal.Conclusion The mechanism of secretory expression of recombinant pseudomonas extoxin was preliminarily studied.
2006 02 [Abstract][OnlineView][Download 509K] - XUE Li-juan,BU Li-sha,YANG Shao-juan,et al (Central Laboratory,Japan-China Union Hospital,Jilin University,Changchun 130033,China)
Objective To explore the influence of 5-Fu on the expression of gene related to the repairing of DNA damage during the apoptosis of human colon cancer HCT/8 cells.Methods Induce the apoptosis of HCT/8 cells with 5-Fu and observe the morphological change of the cells.Determine the apoptosis of HCT/8 cells by genome electrophoresis,acridine orange staining and flow cytometer,and analyze the expression of gene related to the repairing of DNA damage by gene chip technique.Results The number of apoptotic cells 48 h after induction with 5-Fu increased significantly as compared with that of control cells,and typical “bladder bands” were shown on genome electrophoretic profile.The analysis by gene chip technique showed that the expression of 3 of the 12 genes related to the repairing of DNA damage was up-regulated,and that of the rest 9 genes were down-regulated.Conclusion 5-Fu induced the changes of many genes related to the repairing of DNA damage of human colon cancer cells,which could not repair the DNA damage in time and caused the cell apoptosis finally.
2006 02 [Abstract][OnlineView][Download 204K] - LI Chun-huiΔ,DU Bao-dong,SUN Yang,et al ( ΔDepartment of Otorhinolaryngology,Head and Neck Surgery,The First Hospital of Jilin University,Changchun 130021,China)
Objective To explore the role of Survivin,an apoptosis inhibitor,in the development of inner ears of mice.Methods Observe the morphological change of inner ear of mice during development by HE staining,and determine the expressions of Survivin in 12-17-day-old mrine embryo and the inner ears of mice within 14 days after birth by immunohistochemical method.Results Survivin was widely expressed during the proliferation period of otic vesicle epidermis,and its expression level increased gradually during the differentiation and maturation periods of cochlea and vestibule.However,the range of expression of Survivin decreased during the three periods.Conclusion Survivin played an important role in the development of inner ears of mice,including the proliferation and differentiation of cells and the mold of organ.It laid a foundation of further study on the regeneration of hair cells and the prevention and treatment of deaf.
2006 02 [Abstract][OnlineView][Download 226K] - ZHANG Hong-yan,HE Ying-qin,LIU Su-wen,et al (Jilin Institute of Family Planning,Changchun 130041,China)
Objective To explore the molecular mechanism of induction of testicular cell apoptosis by nitrofurazolidone(NFZ) and provide theoretical basis for guiding the application of drugs for human and animals.Methods Observe the morphological change of testicular cells under induction of NFZ by immunohistochemical method,cell culture,separation of subcellular organelles,cell-free system and immuno-precipitation.Examine the DNA electrophoretic profile of testicular cells after induction with NFZ as well as the expressions of Bcl-2 and caspase-3,using cell-free system as control.Results The morphological change of testicular cells induced with NFZ showed the characteristic of apoptosis.Scalariform bands were shown on the DNA electrophoretic profile of the induced cells,which indicated the fragmentation of DNA.The expressions of Bcl-2 and caspase-3 met the regulation of apoptosis.Conclusion NFZ induced the apoptosis of testicular cells.
2006 02 [Abstract][OnlineView][Download 831K] - XIANG MeiΔ,XIANG Jun,MA Yan,et al ( ΔDepartment of Gynecology,The Second Hospital,Jilin University,Changchun 130041,China)
Objective To observe the binding capacity of LHRH-PE40 to cervical carcinoma tissues of various physiological types and provide theoretical basis for the clinical application of LHRH-PE40.Methods Determine the binding capacity of LHRH-PE40 to cervical carcinoma tissue by IFA.Results The binding capacities of LHRH-PE40 to cervical connective tissue and squamous epithelium were 0%,and that to cervical carcinoma tissue was 74%.Of weak IFA-positive and IFA-negative specimens,67% were high differential squamous cell carcinoma tissues,and 15% were low differential squamous cell carcinoma tissues(P<0.05).However,of moderate IFA-positive specimens,37% were high differential squamous cell carcinoma tissues,and 85% were moderate and low differential squamous cell carcinoma tissues(P<0.05).All the three strong IFA-positive specimens were high differential adenocarcinoma tissues.Conclusion LHRH-PE40 showed the lowest binding capacity to high differential squamous cell carcinoma tissue and the highest binding capacity to cervical carcinoma tissue.The binding capacity of LHRH-PE40 to moderate and low differential squamous cell carcinoma tissues was 86%.
2006 02 [Abstract][OnlineView][Download 237K] - JIN Li-jieΔ,ZHAO Xiao-lin,FANG Yan-qiu,et al ( ΔChangchun Institute of Biological Products,Changchun 130062,China)
Objective To evaluate the influence of Hepatitis B(HB)-BCG combined vaccine on the cellular immunity in mice.Methods Divide BALB/c mice into three test groups(HB-BCG combined vaccine,BCG and HB vaccine),one blank control group(unimmunized) and one parallel control group(protective agent).Take the spleens of mice for lymphocyte transformation test and the determination of cytokine and T lymphocyte surface marker 1 and 2 months after immunization separately.Results Both the lymphocyte transformation stimulating index and IL-2 content of mice in HB-BCG combined vaccine group were significantly higher than those of BCG and HB vaccine groups.However,the CD4+/CD8+ value of HB-BCG combined vaccine group was significantly higher than that of HB vaccine group,but showed no significant difference from that of BCG group.Conclusion HB-BCG combined vaccine induced cellular immunity effectively.
2006 02 [Abstract][OnlineView][Download 104K] - ZHANG Yi-zheΔ,JIANG Chun-lai,WANG Zhong-cheng,et al ( ΔVaccine Research Center of Jilin University,Changchun 130021,China)
Objective To prepare the stabilizer of freeze-dried recombinant MVA virus vector vaccine.Methods Add the stabilizers prepared with various formula into recombinant MVA virus vector vaccine,then lyophilize the vaccine under appropriate condition.Screen the optimal formula of stabilizer according to the appearance,virus titer and thermostability of the freeze-dried vaccine.Results The stabilizer prepared with trehalose,mannitol,dextran and inositol showed good protective effect on recombinant MVA virus vector vaccine.The decrease of virus titer after lyophilization was less than 0.12 LgPFU/ml,and those after storage at 37℃ for 1 and 3 weeks were about 0.35 LgPFU/ml and not more than 1 LgPFU/ml respectively.However,the virus titers of stabilizer-free liquid vaccine decreased by 1 LgPFU/ml and 3.5 LgPFU/ml under the same condition respectively.Conclusion The stabilizer consisting of trehalose,mannitol,dextran and inositol provided good protective effect for the freeze-dried recombinant MVA virus vector vaccine.
2006 02 [Abstract][OnlineView][Download 91K]
2006 02 [Abstract][OnlineView][Download 142K] - FAN Yu-hong,QI Feng-chun,HUI Qi,et al (Changchun Intitute of Biological Products,Changchun 130062,China)
Objective To study the antigenicity variation of influenza virus.Methods Perform cross hemagglutination inhibition test on the standard antigens of influenza virus strains A1,A3 and B epidemic since 2000 with the corresponding immunosera.Calculate the antigenic ratio using the obtained data and analyze the variation of antigenicity.Results The antigenic ratios of two A1 strains,four A3 strains and five B strains were 2.6,1.2-13.2 and 1.8-22.6 respectively.Conclusion Both A1 and A3 strains showed no significant variation in their antigenicties,however,the antigenicity of B strain showed significant variation.
2006 02 [Abstract][OnlineView][Download 90K] - YANG Wan-juanΔ,GAO Kai,RAO Chun-ming,et al ( ΔNational Institute for The Control of Pharmaceutical and Biological Products,Beijing 100050,China)
Objective To prepare the McAb against DNA and use for the development of a specific,sensitive and simple method for the determination of residual exogenous DNA.Methods DNA-bovine serum albumin(BSA) antigen was prepared by coupling calf thymus DNA fragment with cationized BSA by Mannish reaction and used for the immunization of BALB/c mice.The hybridoma cell strains stably secreting the McAb against DNA was obtained by the fusion of murine splenocytes with SP2/0 plasmacytoma cells,and the secreted McAb was purified,concentrated and characterized.Results Three hybridoma cell strains of subtypes IgM/κ,IgM/λ and IgM/κ respectively were obtained.The indirect ELISA titers of the three strains were 1∶4×103,1∶8×104 and 1∶1×103,and the affinity constants of them were 3.96×108,4.04×109 and 1.26×108 L/mol,respectively.All the three strains showed strong binding capacity to ctDNA,DH5αDNA,CS115 DNA and H.S.DNA but showed weak or no binding capacity to RNA,BSA and casein.The three strains recognized 3 different epitopes.The detection limit of DNA with the prepared McAb by sandwich ELISA was 4 ng/ml.Conclusion Three hybridoma cells secreting McAb against DNA was successfully established.It laid a foundation of development of immunological method for determination of residual exogenous DNA.
2006 02 [Abstract][OnlineView][Download 226K] - ZHANG Nan,WANG Zhi-you,ZHANG Ai-hua,et al (Wuhan Institute of Biological Products,Wuhan 430060,China)
Objective To establish the hybridoma cell strains stably secreting McAb against SARS-associated coronavirus(SARS-CoV).Methods Immunize BALB/c mice with inactivated SARS-CoV and establish six hybridoma cell strains stably secret McAbs against SARS-CoV.Perform overall control tests on the six strains.Results Double immunodiffusion test proved the McAbs secreted by strain 1C6 as IgG2a subclass,and those secreted by the rest five strains as IgG1 subclass.All the titers of McAbs secreted by the six strains in ascites reached more than 10 -6 .None of the six kinds of McAbs showed cross reaction with other respiroviruses.Western blot showed that all the six kinds of McAbs recognized the S protein with a relative molecular weight of 150 000 and its lytic fragment with a relative molecular weight of 120 000.ELISA additivity test proved that all the six kinds of McAbs recognized the corresponding antigen epitopes.The elution with thiocyanate showed that,in the order of relative affinity,the six kinds of McAbs were as follows:1A4,4F6,1C6,1B10,2H2 and 1F3.Both the neutralizing titers of McAbs 4F6 and 1F3 were 1∶10.The hybridoma cell strains grew well after continuous culture for more than 3 months or after being stored lyophilized for 6 months,and the titers of secreted McAbs were stable.Conclusion The McAbs against SARS-CoV was obtained.It laid a foundation of early diagnosis and further study of SARS.
2006 02 [Abstract][OnlineView][Download 243K] - ZHANG LingΔ,DONG Tao,SONG Li-bing ( ΔState Key Laboratory of Oncology in Southern China,Department of Experimental Research,Cancer Center,Sun Yat-sen University,Guangzhou 510060,China)
Objective To prepare the antibodies against three kinds of common bacteria causing sore throat,i.e.β-streptococcus hemolyticus,streptococcus hemolyticus group G and staphylococcus aureus,and anlayze their properties.Methods Prepare the three kinds of bacteria into soluble antigens and immunize to hens.Extract IgY antibodies from the yolk,analyze their property by immunoeletrophoresis,ELISA and SDS-PAGE and study their thermostability.Results The IgY against three kinds of bacteria were isolated from the yolk only 10 d after immunization.Sixty days after immunization,the IgY titer reached 1∶102 400 and showed no significant change 5-6 months after storage at room temperature.Conclusion The prepared IgY antibodies showed high titers,specificities and stabilities.
2006 02 [Abstract][OnlineView][Download 258K] - DAI Xu-fangΔ,LIAN Ji-qin,ZHANG Yu-jing,et al ( ΔSpecial Education College of Chongqing Normal University,Chongqing 400047,China)
Objective To optimize the condition for expression of human laminin alpha 4 LG3-4 module(hLNα4LG3-4) gene in E.coli.Methods Study the optimal condition for expression of recombinant E.coli strain hLNα4LG3-4/pET28a,such as temperature and time for induction and the concentration of IPTG as an inducer.The expressed product was purified by Ni-NTA affinity column chromatography and determined for its effect on the adhesion and proliferation of human lung cancer A549 cells by MTT method.Results After induction with 1 mmol/L IPTG at 20℃ for 6 h,hLNα4LG3-4 was highly expressed in a form of soluble protein;however,after induction with 2 mmol/L IPTG at 37℃ for 4 h,the protein was highly expressed in a form of inclusion body.The expressed product reached a purity of 96% after purification and enhanced the adhesion and proliferation of A549 cells significantly.Conclusion The condition for expression of hLNα4LG3-4 in E.coli was optimized.
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2006 02 [Abstract][OnlineView][Download 52K] - CHA LiΔ,GAO Jun,HOU Jian-ying,et al ( ΔLiaoning Chengda Biotechnology Co.Ltd,Shenyang 110026,China)
Objective To observe the safety and immune effect of adjuvant-free rabies vaccine.Methods Inoculate Pasteur PV2061 strain of rabies virus into Vero cells less than 143 passages and incubate in bioreactor by perfused culture.Harvest the virus bulk continuously,then concentrate,inactivate and purify to prepare rabies vaccine.Immunize 502 victims(group A) with the vaccine by post-exposure schedule,using 100 victims(group B) immunized with the imported vaccine as control.Observe the adverse reactions and immune effects of the two groups.Results No severe local or systemic reactions were observed in the two groups.On day 14 after the first dose,both the antibody positive conversion rates of groups A and B reached 100%,and the GMTs of antibody were 5.2 and 5.6 IU/ml respectively.However,on day 45,the GMTs of groups A and B increased to 9.5 and 9.8 IU/ml respectively.Conclusion Adjuvant-free rabies vaccine showed good safety and immunogenicity.
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