- MING Ping-gang,SUN Wen,XU Ge-lin,et al (Wuhan Institute of Biological Products,Wuhan 430060,China)
Objective To analyze the sequence of glycoprotein(GP) gene of three passages of rabies vaccine strain CTN-1-V in China and compare with that of street virus strain.Methods The GP cDNA fragments were amplified from the murine brain infected with CTN-1-V strain by RT-PCR then sequenced.Results The homologies of nucleic acid and amino acid sequences of three passages of CTN-1-V strain to those of street strain were 83.2%-96.8% and 90.0%-97.4% respectively.The nucleic acid sequences of three passages of CTN-1-V strains were basically identical.Conclusion The structure of GP gene of CTN-1-V strain was bascially stable during passage,and the homology of CTN-1-V to street strain was higher than those of aG and PV strains.
2006 03 [Abstract][OnlineView][Download 163K] - FU YongΔ,SU Xue-mei,LIU Yan-fang,et al ( ΔDepartment of Pathology,Urumqi General Hospital,Lanzhou Military Region,Urumqi 830000,China)
Objective To prepare a novel recombinant immunotoxin with specific killing activity to human hepatocarcinoma.Methods Recombinant immunotoxin hdsFv-hEDN against hepatocarcinoma was expressed by using E.coli expression system,and the expressed product was purified by Ni-NTA affinity chromatography and determined for specific killing activity.Results Recombinant immunotoxin hdsFv-hEDN against hepatocarcinoma was expressed in a form of soluble protein and purified effectively.ELISA proved the specific binding capacity of expressed product to the corresponding antigen.Cytotoxicity test showed the killing activity of expressed product to hepatocarcinoma cells but no damage to normal hepatic cells.Conclusion The recombinant immunotoxin hdsFv-hEDN with specific binding capacity and killing activity to hepatocarcinoma cells was successfully prepared.It laid a foundation of further application of immunotoxin hdsFv-hEDN.
2006 03 [Abstract][OnlineView][Download 142K] - ZHENG Yuan-qiangΔ,SHI Yan-chun,YANG Gui-zhen ( ΔThe Key Laboratory of Department of Education of Inner Mongolia Autonomous Region for Molecular Biology,Inner Mongolia Medical College,Huhhot 010059,China)
Objective To explore the influence of synthetic CpG ODN2006 on the production of specific antibody in mice immunized with hepatitis B(HB) vaccine.Methods Divide BALB/c mice into test and control groups and immunize twice with CpG ODN2006+HB vaccine and HB vaccine respectively.Separate murine sera for determination of anti-HBs IgG titers by ELISA.Results Compared with those in control group,the anti-HBs IgG titer of mice in test group was significantly high and persistent.Conclusion CpG ODN2006 enhanced the production of anti-HBs IgG in mice.
2006 03 [Abstract][OnlineView][Download 59K] - ZHENG Li-shu,LI Wu-ping,WANG Gang, et al (State Key Laboratory for Molecular Virology and Genetic Engineering,National Institute for Viral Disease Control and Prevention,China CDC,Beijing 100052,China)
Objective To clone the Listeriolysin O(LLO) gene of Listeria monocytogenes(Lm),construct a prokaryotic expression system of the gene and identify the expressed product for antigenicity and hemolytic activity.Methods Amplify hlyA gene from the total DNA of Lm by PCR and compare its sequence with those of hlyA genes of the other 9 Lm strains in GenBank.Construct a prokaryotic expression vector pET30a-hlyA of LLO by using plasmid pET30a.Transform the constructed recombinant plasmid to E.coli BL21(DE3) for expression under induction of IPTG.Purify the expressed product by Ni 2+ column chromatography and identify its antigenicity by Western blot.Determine the hemolytic activity of expressed product with human erythrocyte.Results At most three amino acid substitutions were found in the PEST-like structure of amplified hlyA gene as compared with the corresponding amino acid sequences of the other 9 Lm strains in GenBank.LLO fusion protein was highly expressed in E.coli.After purification,the expressed protein reached a high purity and showed good antigenicity.At pH 5.5,LLO showed the maximum hemolytic activity of 1.41×104 HU/mg.Conclusion The prokaryotic expression system of LLO was successfully constructed.The expressed LLO protein showed good antigenicity and hemolytic activity.
2006 03 [Abstract][OnlineView][Download 237K] - LIAN HaiΔ,JIN Ning-yi,LI Xiao,et al ( ΔCollege of Animal Science and Veterinary Medicine,Jilin University,Changchun 130062,China)
Objective To construct a eukaryotic recombinant plasmid for coexpression of apoptin and hIL-18 genes,determine its effect on human liver cancer BEL-7402 cells in vitro and provide a basis for exploring a new gene therapeutics for tumor.Methods Clone chicken anemia virus VP3 gene and human interleukin-18(hIL-18) gene into eukaryotic expression vector pVAX1 and transfect the recombinant plasmid pVVP3IL-18 to human liver cancer BEL-7402 cells in the mediation of liposome.Observe the morphological change,DNA breakage,DNA content at different stages of cell cycle and ultrastructure of the apoptotic cells by AO/EB staining,terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling(TUNEL) assay,flow cytometry and transmission electron microscopy respectively.Results Along with the increasing hours after transfection,the killing effect of expressed product of recombinant plasmid pVVP3IL-18 on BEL-7402 cells was enhanced gradually.The morphological apoptosis of cells was observed 48 h after transfection.The apoptotic rate reached(56.5±11.9)% and the majority of cells were TUNEL positive.Typical apoptotic sub-G1 peak was observed by flow cytometry.Typical ultrastructure change of apoptotic cells,such as pyknosis and chromatin condensation,were observed by transmission electron microscopy.Conclusion The combined application of apoptin and hIL-18 genes induced the apoptosis of human liver cancer BEL-7402 cells in vitro significantly.
2006 03 [Abstract][OnlineView][Download 357K] - ZHANG Zhi-yuΔ,JIN Ning-yi,LI Xiao,et al ( ΔThe First Hospital,Jilin University,Changchun 130021,China)
Objective To explore the induction of apoptosis of osteosarcoma S180 cells by recombinant fowl pox virus(vFV3) with Apoptin,hemagglutinin-neuraminidase(HN) and interleukin 18(IL-18) genes in vitro.Methods S180 cells were infected with vFV3,then determined for activity by MTT method and analyzed for cell cycle by flow cytometry(FCM).The transmembrane potential of chondriosome was determined by Rhodamine123 staining combined with FCM to explore the mechanism of inducing the apoptosis of S180 cells by vFV3.Results The killing rate of S180 cells by vFV3 was 46.8%.The infection with vFV3 decreased the transmembrane potential of chondriosome and induced the apoptosis of S180 cells.Conclusion vFV3 induced the apoptosis of S180 cells by chondriosome route.
2006 03 [Abstract][OnlineView][Download 97K] - YUE Li-qinΔ, SHEN Xu-zhuang, ZHANG Hua-jie, et al ( ΔLaboratory of Microiology & Immunology, Beijing Children′s Hospital, Beijing 100045, China)
Objective To express the gene encoding the surface immunogenic protein(SIP) of streptococcus group B and provide target protein for further immunological study.Methods Amplify SIP gene from the genomic DNA of standard GBS strain type Ⅱ by PCR and insert into pMD18-T vector by T/A cloning. Digest the recombinant plasmid with restriction endonuclease Bgl Ⅱ and Hind Ⅲ, and clone the obtained gene fragments into pET32a vector. Transform the constructed recombinant plasmid pET32a-SIP into BL21(DE3)/pET system for expression of Trix-SIP fusion protein. The expressed product was identified by SDS-PAGE and peptide mass fingerprinting(PMF). Results The homology of amplified SIP gene sequence to that of SIP of GBS type Ia reported in GenBank was 99%. SDS-PAGE showed a protein band with relative molecular weight of 66 000. The probability based mowse score of SIP was 74 by PMF analysis. Conclusion GBS SIP fusion protein was successful expressed. It laid a foundation of study on role of SIP in pathopoiesis of bacteria and development of relevant vaccine.
2006 03 [Abstract][OnlineView][Download 338K] - DING PengΔ,WANG Jia-ning,YANG Bo,et al ( ΔRenmin Hospital,Wuhan University,Wuhan 430060,China)
Objective To construct prokaryotic expression vector pET15b-PEP-1-SOD1 and express and purify PEP-1-SOD1 fusion protein.Methods The SOD1 cDNA was amplified by PCR using plasmid pBluscript Ⅱ SK-SOD1 as a template,identified by restriction analysis and used for construction of prokaryotic expression vectors pET15b-SOD1 and pET15b-PEP-1-SOD1.The constructed recombinant plasmids were identified by sequencing and transformed to E.coli BL21(DE3) for expression of SOD1 protein and PEP-1-SOD1 fusion protein.The expressed products were identified by SDS-PAGE and Western blot.Results SOD1 and PEP-1-SOD1 proteins,with relative molecular weights of 22 000 and 26 000 respectively,were expressed.The expressed product existed in a nature and soluble form and contained 30% of total somatic protein.Conclusion SOD1 protein and PEP-1-SOD1 fusion protein were successfully prepared.
2006 03 [Abstract][OnlineView][Download 409K] - LI Xiang-hongΔ,ZHAO Jian-zeng,CUI Yun-long,et al ( ΔBeijing Tianshikang Medicine and Technology Co.Ltd,Beijing 100037,China)
Objective To clone gassericin T gene,express in prokaryotic cells and identify the microbiostatic activity of expressed product.Methods Amplify the DNA fragments encoding the mature peptide of gassericin T from three L.gasseri strains isolated in Beijing by PCR.Analyze the homology of the amplified gene fragments V1-1/gatA and V44-1/gatA to the gassericin T gene of L.gasseri strain isolated in Japan.Insert the two amplified gene fragments into expression vector pGEX-6P-1 to construct recombinant plasmids pGEX-gatA/V1-1 and pGEX-gatA/V44-1 respectively.Transform the constructed recombinant plasmids to E.coli BL21(DE3) PlysS for expression under induction of IPTG.Results The homologies of V1-1/gatA and V44-1/gatA to the gassericin T gene of L.gasseri strain isolated in Japan were 98% and 99% respectively.The expressed product,existing in a form of inclusion body,contained about 29% of total somatic protein.Recombinant gassericin T showed significant inhibiting activity to the growth of Staphylococcus aureaus,Pseudomonas aeruginosa,Shigella dysenteriae and Tyuphoidal salmonellosis.Conclusion The obtained recombinant gassericin T might be a novel antibiotic.
2006 03 [Abstract][OnlineView][Download 377K] - ZHANG Jing-min△,JIN Ning-yi,LIAN Hai,et al ( △School of Pharmacy,Jilin University,Changchun 130021,China)
Objective To analyze the distribution of CD109 gene in normal and brain cancer tissues.Methods Analyze the expression of CD109 in normal human and murine tissues as well as brain cancer tissue by real-time quantitative PCR,using 18S rRNA as an internal standard.Results The expression level of mRNA of CD109 protein was the highest in testis tissue but was low in the other tissues.However,the expression was up-regulated in 9 of the 12 brain cancer specimens.Conclusion CD109 gene was rarely expressed in most normal tissues.It might be a potential molecular target for the treatment of malignant tumors.
2006 03 [Abstract][OnlineView][Download 272K] - WU Guang-mou,ZHANG Guo-li,YUE Yu-huan,et al (Institute of Military Veterinary,Academy of Military Medical Sciences,Changchun 130062,China)
Objective To study the LHRH-PE40 antibody production induced by continuously intravenous injection with LHRH-PE40 in rabbits and the influence of the antibody on cytotoxicity.Methods The test rabbits were divided into three groups and intravenously injected every other day with LHRH-PE40,Freund adjuvant plus LHRH-PE40(positive control) and human serum albumin(negative control) respectively.Determine the LHRH-PE40 antibody level in sera by ELISA,and the cytotoxicity of LHRH-PE40 to Hela cells by XTT method.Results The LHRH-PE40 antibody was detected 8 d and reached a peak value 24 d after injection.After incubation with serum,the IC 50 of LHRH-PE40 to Hela cells increased slightly and was 1-2 times higher than that of negative control.However,the IC 50of positive control was 14 times higher than that of negative control.Conclusion Continuously intravenous injection with LHRH-PE40 induced low antibody level which showed no significant influence on the IC 50 of LHRH-PE40 to Hela cells.It provided a basis for continuous administration of LHRH-PE40 in clinic.
2006 03 [Abstract][OnlineView][Download 93K] - SHAO Li-jun,LIU Zhao-hui,JIN Li-jie,et al (Changchun Institute of Biological Products,Changchun 130062,China)
Objective To express HBV PreS2-MBP fusion protein in E.coli then identify and purify the expressed product.Methods Clone the full length of HBV PreS2 gene into prokaryotic expression vector pMAL-P2x by recombinant DNA technique,then transform to E.coli for expression under induction of IPTG.Identify the expressed product by SDS-PAGE and Western blot then purify by anion-exchange and Amylose Resin affinity chromatography.Results Recombinant plasmid pMAL-P2x/S2 was successfully constructed.The MBP-labeled PreS2-MBP fusion protein with good antigenicity was expressed in a soluble form and successfully purified by Amylose Resion affinity chromatography.Conclusion HBV PreS2-MBP fusion protein was successfully expressed in pMAL-P2x system.
2006 03 [Abstract][OnlineView][Download 428K] - LIU Da-weiΔ,LIU Jing-ye,WANG Peng-fu,et al ( ΔVaccine Research Center,College of Life Science,Jilin University,Changchun 130012,China)
Objective To clone the gene encoding late protein L1 of human papillomavirus 16(HPV16) from pathological tissue of cervical cancer,construct an eukaryotic expression vector and lay a foundation of preparation of vaccine for prophylactic use.Methods Amplify HPV16 L1 gene from the pathological tissue of cervical cancer and insert into vector pMD18-T.Identify the recombinant plasmid by sequencing,then digest with restriction endonuclease and ligate to vector pVAX1.Transiently transfect Cos-7 cells with the constructed eukaryotic expression plasmid pVAX1-L1 and identify the expressed product by immunohistochemical technique.Immunize mice with the constructed recombinant plasmid pVAX1-L1 and determine the immunogenicity by lymphocyte transformation test,using plasmid pVAX1 as control.Results The amplified L1 gene sequence showed several variations as compared with that reported in GenBank,which caused the corresponding changes of amino acids.Brownish-yellow immune complex was detected in the transfected Cos-7 cells by immunohistochemical technique,which indicated the expression of L1 protein.The stimulating index of lymphocyte of mice immunized with plasmid pVAX1-L1 was significantly higher than that with plasmid pVAX1.Conclusion The HPV16 L1 gene sequence amplified from cervical cancer tissue showed a certain variation.However,the expressed L1 protein show biological activity.
2006 03 [Abstract][OnlineView][Download 183K] - ZHENG Meng-liΔ,LUO Cheng-hua,ZHANG Xiang-hua,et al ( ΔDepartment of Thoracic and Cardiac Surgery,309th Clinical Division of Chinese PLA General Hospital,Beijing 100091,China)
Objective To explore the organic microenvironmental effect of skeletal muscles on the proliferation of malignant cells and evaluate its significance in the rarity of metastases in skeletal muscles.Methods Prepare the primary culture of new born Wistar rat skeletal muscle cells and collect the culture supernatant as murine skeletal muscle conditioned medium(MMCM).Analyze the suppressive effect in vitro of MMCM on the proliferation of malignant tumor cells including murine myeloma cell strain SP2/0,nasopharyngeal carcinoma cell strain CNE,pulmonary cytomegalic carcinoma cell strain PLA-801C,human oesophageal squamous cell carcinoma strain Eca109 and cervical carcinoma cell strain Hela.Results MMCM showed significant suppressive effect on the proliferations of all the five tumor cell strains.However,it showed no significant effect on normal cells.Conclusion The tumor inhibitors derived from new born Wistar rat skeletal muscle cells selectively suppressed the proliferation of malignant tumors and provided a biological basis for the study on rare metastases in skeletal muscles.
2006 03 [Abstract][OnlineView][Download 97K] - CHUAI Xia, ZHANG Yong-hong, JIN Yu-huai, et al (Department of Pathogenic Biology, School of Basic Medical Sciences, Hebei Medical University, Shijiazhuang 050017,China)
Objective To construct an eukaryotic expression system and express recombinant murine IFN-γ(mIFN-γ) in cos-7 cells.Methods Amplify mIFN-γ cDNA from ConA-activated murine splenocytes by RT-PCR and clone into pGEM-T vector.Identify the recombinant plasmid by sequencing and directly ligate to eukaryotic expression vector pcDNA3,then transfect cos-7 cells in the mediation of DEAE-Dextran and determine the intracellular and extracellular expressions of mIFN-γ.Results The sequence of amplified mIFN-γ cDNA was identical to that reported in GenBank.The transcription and expression of mIFN-γ was confirmed in the cos-7 cells transfected with pcDNA3/mIFN-γ.Conclusion The eukaryotic expression system of mIFN-γ was successfully constructed.It laid a foundation of development of antivirus gene vaccine.
2006 03 [Abstract][OnlineView][Download 248K] - JIN Yu-huai,ZHAO Zhan-sheng,CONG Bin,et al (Department of Pathophysiology,Hebei Medical University,Shijiazhuang 050017,China)
Objective To explore the influence of cholecystokinin octapeptide(CCK-8) on the expression of TNF receptor(TNFR) mRNA in rat synovial cells(RSC-364).Methods Detect the expression of TNFR mRNA under induction of TNF-α in the RSC-364 cells containing different concentrations of CCK-8 by RT-PCR.Results The CCK-8 at concentrations of 10 -6 mol/L and 10 -7 mol/L showed dose- and time-dependent inhibitory effect on the expression of TNFR2 mRNA in RSC-364 cells under induction of TNF-α,which could be reversed by a CCK receptor antagonist proglumide.Conclusion CCK-8 inhibited the expression of TNFR2 mRNA in RSC-364 cells significantly.
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2006 03 [Abstract][OnlineView][Download 36K] - JIN Yuan-changΔ,LIU Li,SU Xiao-yan,et al ( ΔHunan University of Science and Technology,Xiangtan 411201, China)
Objective To synthesize the gene encoding human GnRH and its transporter(TRS).Methods Design a pair of nucleotide sequences according to the reported gene encoding human GnRH and its transporter and purify by PAGE with denaturing polyacrylamide gel containing 7 mol/L urea.Synthesize a 90 bp target sequence GnRH/TRS by filling-in of a short complementary sequence at 3′-terminus of this pair of nucleotide sequences.Subclone the synthetic sequence to pMD18-T vector to construct recombinant plasmid pYC1.Identify the positive clones by restriction analysis and sequencing.Results The synthetic GnRH/TRS sequence consisted 90 nucleotides and showed a homology of 100% to that designed.Conclusion Human GnRH/TRS gene was successfully synthesized.It laid a foundation of further study and application of GnRH/TRS.
2006 03 [Abstract][OnlineView][Download 127K] - JIANG Xiao-ling,ZHANG Hong-mei,XU Liu-mei,et al (Shenzhen East Lake Hospital,Shenzhen Institute of Hepatopathy,Shenzhen 518020,China)
Objective To construct bovine α-S1 casein gene regulating sequence guiding the specific expression of exogenous gene in animal mammary gland tissue.Methods Extract DNA from the liver tissue of newborn calf for fractional amplification of bovine α-S1 casein gene regulating sequence by PCR.Identify the amplified product by agarose gel electrophoresis and sequencing,then ligate the fragments into a intact one.Subclone the exogenous gene ALB into the regulating sequence for transfection of C127 cells.Identify the expressed product by immunohistochemical method.Results The bovine α-S1 casein gene regulating sequence,consisting of 1.8 kb upstream sequence,exon 1,intron 1,the exon 2 with translation initiation site,a part of intron 2,a large part of the last intron,the last exon(containing poly A signal) which could not be translated and the whole flanking region at a length of 5.8 kb,was constructed.The exogenous gene ALB was highly expressed in the regulating sequence.Conclusion The bovine α-S1 casein gene regulating sequence positively regulating the expression of exogenous gene in animal mammary gland bioreactor was successfully constructed.
2006 03 [Abstract][OnlineView][Download 387K] - XIN Jiu-qingΔ,GAO Yu-long,LI Yuan,et al ( ΔHarbin Veterinary Research Institute of CAAS,Harbin 150001,China)
Objective To construct the expression vector of gene at N-terminus of lipoprotein LppQ of Mycoplasma mycoide subsp.Mycoides SC(MmmSC),purify the expressed product and study its immunogenicity.Methods The gene fragment encoding the N-terminal domain of LppQ was amplified from MmmSC HVRI-X strain by PCR then mutated from a Mycoplasma specific TGA(Trp) codon into a universal TGG(Trp) codon by one-step overlap extension PCR.The mutated gene was inserted into expression vector pET32a then transformed to E.coli.The expressed product was purified and determined for immunogenicity.Results The expressed product contained 53.7% of total somatic protein and showed good immunogenicity as proved by Western blot and ELISA.Conclusion Recombinant LppQ protein was successfully expressed and might be used as an antigen for serological diagnosis.
2006 03 [Abstract][OnlineView][Download 325K] - LI Yue-xi,TAO Kai-hua,ZHANG Jin-hai,et al (East-China Institute of Medicine and Biotechniques,Nanjing 210002,China)
Objective To construct the recombinant plasmid and engineering bacteria for expression of antigenic epitope of Toxoplasma P30 protein and use the expressed product for detection of Toxoplasma specific antibody.Methods Design a pair of primers and amplify the gene fragment encoding antigenic epitope of Toxoplasma P30 protein from the genomic DNA of Toxoplasma by PCR then insert into vector pET28a(+).Transform the contructed recombinant plasmid to E.coli BL21(DE3) for expression under induction of IPTG.Identify the expressed product by SDS-PAGE,purify by ion exchange chromatography and analyze for antigenicity by ELISA.Results The expressed product existed in a form of inclusion body and contained about 30% of total somatic protein.It showed good antigenicity and could be used for detection of Toxoplasma antibody.Conclusion The engineering bacteria for high expression of antigenic epitope of Toxoplasma P30 protein was successfully constructed.It laid a foundation of development of Toxoplasma antibody detection kit or Toxoplasma vaccine.
2006 03 [Abstract][OnlineView][Download 232K] - XIANG MeiΔ,XIANG Jun,CUI Man-hua,et al ( ΔSecond Hospital of Jilin University,Changchun 130041,China)
Objective To explore the binding capacity of LHRH-PE40 to the corresponding receptor on the surface of cancer cells.Methods Add LHRH and LHRH-PE40 into Hela cell culture,stain with crystal violet and determine the A value.Add LHRH and LHRH-PE40 onto the section of cervical carcinoma tissue,then add rabbit anti-PE40 serum and fluorescin isothiocyanate-labeled goat anti-rabbit IgG and observe the fluorescence intensity under fluorescent microscope.Results Along with the increasing ratio of molar number of LHRH to LHRH-PE40,the cytotoxicity was attenuated.When the ratio reached 500/1,the receptors on surface of cancer cells were saturated.When the ratio was 750/1,the A value of cell culture was close to that when the ratio was 500/1.The fluorescence intensity of cervical carcinoma tissue added with both LHRH and LHRH-PE40 was significantly lower than that added with LHRH-PE40 alone.Conclusion LHRH-PE40 showed binding capacity to LHRH receptor.
2006 03 [Abstract][OnlineView][Download 234K] - CHA LiΔ,GAO Jun,HOU Jian-ying,et al ( ΔLiaoning Chengda Biotechnology Co.Ltd,Shenyang 110026,China)
Objective To prepare rabies vaccine for human use in a large scale by cell culture in bioreactor.Methods Prepare rabies vaccine by perfused culture of Vero cells within 143 passages inoculated with PV2061 strain in bioreactor containing 25 g/L of microcarrier.The virus bulk was continuously harvested,then concentrated,inactivated and purified.Results The density of cultured cells reached 1.2×107~1.5×107 cells/ml.The virus bulk could be continuously harvested for 18~22 d after inoculation and reached a highest titer of 8.50 LogLD 50/ml and a mean titer of 7.2~7.6 LogLD 50/ml.After purification by column chromatography,the removal rate of foreign protein reached more than 99.95%.The total protein,DNA and GP contents of final bulk were not more than 80 μg,not more than 10 pg and 3.5~4.5 IU respectively,and its potency was not less than 4.5 IU/0.5 ml.Conclusion The cell culture in bioreactor could be used for the large-scale production of rabies vaccine for human use.
2006 03 [Abstract][OnlineView][Download 203K] - QI Feng-chunΔ,WANG Chun-yi,ZHANG Xue-mei,et al ( ΔCollege of Life Science,Jilin University,Changchun 130012,China)
Objective To explore the feasibility of culture of influenza virus type A1 in Vero and MDCK cells and optimize the culture condition.Methods Inoculate influenza virus type A1 into Vero and MDCK cells and culture in media containing fetal bovine serum(FBS) or trypsin at different concentrations.Harvest the virus liquid at different time for determination of HA titers.Results FBS inhibited the proliferation of influenza virus significantly.The addition of about 5 μg/ml of trypsin increased the virus yield.The optimal time for harvesting virus was 72-96 h after cultivation.The HA titer of influenza virus in MDCK cells was significantly higher than that in Vero cells.Conclusion Both Vero and MDCK cells were suitable for culture of influenza virus and development of influenza vaccine.
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2006 03 [Abstract][OnlineView][Download 58K] - DU Lin,TAN Xiao-mei,XUAN Jun-wen,et al (Lanzhou Institute of Biological Products,Lanzhou 730046,China)
Objective To analyze the biochemical and immunological characters of different fractions during the purification of Haemophilus influenzae type b(Hib) conjugate.Methods Purify Hib conjugate by Sepharose CL-4B column chromatography and collect five different fractions for the determination of biochemical and immunological characters.Results The ratios of polysaccharide to protein in fractions 1,2 and 3 were about 0.4,and the high molecular conjugate contents were more than 80%.HPLC proved that all the relative molecular weights of the three fractions were more than 1 400 000.Good immune responses were induced in the mice immunized with the three fractions.The fraction 4,with a high molecular conjugate content of about 70% and a relative molecular weight of about 670 000,also induced good immune response in mice.However,the fraction 5,with a high molecular conjugate content of 23.7% and a relative molecular weight of 50 000,was mainly polysaccharide and vector protein in free form and only induced low immune response in mice.Conclusion The fractions 1,2 and 3 were needed to be collected during purification of Hib conjugate.The fraction 4 was conjugate with a low relative molecular weight.However,the fraction 5 was mainly free polysaccharide and protein.
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2006 03 [Abstract][OnlineView][Download 51K] - CHEN Ke-jin,LIU Jian,YU Gang,et al (Wuhan Institute of Biological Products,Wuhan 430046,China)
Objective To study the influence of Kanserin,a preparation of group A hemoloytic streptococci,on the human hepatoma cells cultured in vitro and the enhancement of immunity of mice by combined vaccination with Kanserin and hepatitis B vaccine.Methods Add the Kanserin at various concentrations into the human hepatoma cells Hep3B and HepG22.2.2.15 cultured in vitro,observe the cell morphology and determine the cell activity by MTT method.Analyze the HBsAg and HBeAg contents in culture supernatant by EIA,the cell DNA fragments by Southern blot and the cell apoptosis by direct actinide orange staining.Inject i.p. NIH mice with Kanserin and hepatitis B vaccine and determine the anti-HBs antibody and transaminase levels in sera.Inject i.p. common mice with Kanserin and hepatitis B vaccine and observe the change of bodyweight before and after injection.Results Kanserin inhibited the secretion of HBsAg and HBeAg,caused the regular chromosome DNA fragmentation and leaded the cells to apoptosis.The combined vaccination with Kanserin and hepatitis B vaccine increased the anti-HBs level in murine sera.Neither combined vaccination with Kanserin and hepatitis B vaccine nor vaccination with hepatitis B alone caused adverse reactions.Conclusion Kanserin showed significant influence on human hepatoma cells cultured in vitro.The combined vaccination with Kanserin and hepatitis B vaccine enhanced the immunity of mice.
2006 03 [Abstract][OnlineView][Download 507K] - LI Ru-bing,FU Yong-hang,LI Ji-dong,et al (The 458th Hospital of PLA,Guangzhou 510600,China)
Objective To separate and purify the recombinant human soluble TRAIL(rhsTRAIL) expressed in Pichia pastoris and study its bioactivity.Methods Express rhsTRAIL in Pichia pastoris secretary expression system and purify by ammonium sulfate precipitation and column chromatography.Identify the purity and specificity of purified rhsTRAIL by SDS-PAGE,electrospray ionization-mass specectrometry(ESI-MS) and Western blot.Determine the bioactivity of rhsTRAIL in inducing the apoptosis of tumor cells by transmission electron microscopy,DNA ladder and MTT method.Results The expressed product with a relative molecular weight of 22 000 existed in a soluble form,reached a purity of 95% after purification and was identified as TRAIL protein by Western blot.Transmission electron microscopy,DNA ladder and MTT method showed bioactivity of rhsTRAIL in inducing the apoptosis of tumor cells.Conclusion The rhsTRAIL expressed in Pichia pastoris could be purified by column chromatography and showed bioactivity in inducing the apoptosis of tumor cells.
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