2009年05期目次

  • Expression of Gene Encoding Alkaline Cellulose in Pichia pastoris and Fermentation of Recombinant Pichia pastoris

    TIAN Sheng-li, SHAO Rui, LIU Gang, et al(College of Life Science, Shenzhen University, Shenzhen Key Laboratory of Microbial Gene Engineering, Shenzhen 518060, Guangdong Province, China)

    Objective To clone the gene encoding alkaline cellulose, construct a yeast integrative expression plasmid and express in Pichia pastoris, and optimize the procedure for fermentation of recombinant P. pastoris. Methods The gene encoding alkaline cellulose was amplified from Bacillus sp. ATCC21833 by PCR and cloned into yeast integrative expression vector pGAPZαA. The constructed recombinant plasmid pGAPZαA-ATCC21833 was transformed to P. pastoris strain GS115. The medium for fermentation of recombinant P. pastoris was optimized by single factor test and orthogonal test. The high density fermentation of recombinant P. pastoris was performed in 20 L fermentor, and the effect of carbon source on batch fermentation was observed. The dry weights of P. pastoris and enzyme activity in fermentation liquid by 4 methods for fed-batch, i.e. continuous fed-batch at a constant speed, intermittent fed-batch at a constant speed, intermittent fed-batch at a degressive speed and fed-batch by maintaining a constant substrate concentration, were analyzed and compared. Results Both restriction analysis and DNA sequencing proved that recombinant plasmid pGAPZαA-ATCC21833 was constructed correctly. The gene sequence of recombinant plasmid pGAPZαA-ATCC21833 was consistent with that of alkaline cellulose gene of Bacillus sp. KSM-635. The optimal fermentation medium consisted of 6% glucose, 2% ammonium sulfate and 12 g / L postassium dihydrogen phosphate. The concentration of carbon source showed significant effect on the growth and enzyme production of recombinant P. pastoris. SDS-PAGE showed that the relative molecular mass of expressed product was about 103 000. The dry weight of P. pastoris and enzyme activity of fermentation liquid with fed-batch by maintaining a constant substrate concentration were 29. 8 g / L and 24 U / ml respectively, which were significantly higher than those by the other three methods for fed-batch. Conclusion The recombinant P. pastoris for expression of alkaline cellulose gene was successfully established, and the fed-batch by maintaining a constant substrate concentration was screened as optimal for fermentation.

    2009 05 v.22 [Abstract][OnlineView][Download 446K]

  • RI Silencing by Small Interfering RNA Promotes Metastasis and Invasion of Human Bladder Cancer Cells

    OUYANG Xi, CHEN Jun-xia, HE Xiao-yan, et al(Cell Biology and Genetics Division, Chongqing Medical University, Chongqing 400016, China)

    Objective To construct the siRNA expression vectors and investigate the effect of RI expression silencing caused by vector-mediated RNAi on the metastasis and invasion of human bladder cancer BIU-87 cells. Methods Two siRNA expression vectors specific to RI mRNA and one non-homologous negative control vector were designed and constructed, then identified by restriction analysis and DNA sequencing and transfected to BIU-87 cells. The efficiency of RNAi was determined by RT-PCR, Western blot and immunocytochemical method. The cells were observed for morphological change by light microscopy and HE staining, and determined for adhesion ability by adhesion test, for metastasis ability by wound healing assay, and for invasion ability by Transwell invasion assay. The microfilament structure of cytoskeleton was observed by laser scanning confocal microscopy. Results Both restriction analysis and sequencing proved that the siRNA expression vectors were constructed correctly. The expression level of RI protein in BIU-87 cells transfected with specific siRNA expression vectors decreased significantly, while that with negative control vector showed no significant change. The cells transfected with specific siRNA expression vectors overlapped severely, and their adhesion ability decreased significantly, while metastasis and invasion abilities increased significantly. Assembled cytoskeleton microfilament and several extended lamellipodia were observed in cells transfected with specific siRNA expression vectors. Conclusion The siRNA expression vectors specific to RI gene was successfully constructed, which increased the metastasis and invasion abilities of bladder cancer cells, indicating that RI might be used as an effective target for inhibiting the metastasis and invasion of tumors.

    2009 05 v.22 [Abstract][OnlineView][Download 515K]

  • Site-directed Mutagenesis of Human Endostatin Gene and Expression of Mutant in E. coli

    DU Cui-hong, CAO Min-jie, YOU Pin-sheng, et al (College of Biological Engineering, Jimei University, Xiamen 361021, Fujian Province, China)

    Objective To increase the soluble expression level of human endiostatin (hES)gene in E. coli by site-directed mutagenesis. Methods The mutation sites were designed according to the information on structure as well as relationship between structure and function of hES from internet sites. The rationality of the designed mutation sites was confirmed by predicting the three-dimensional structure of hES mutant in relevant internet sites of bioinformatics. The site-directed mutagenesis of hES was achieved by overlapping extension PCR, and the mutated and original hES genes were subcloned into vector pGEX-4T-3 respectively for fusion expression in E. coli . The soluble expression levels of target protein before and after mutation were compared. Results The prediction of three-dimensional structure of hES showed rational design of mutation sites. Sequencing result proved the site-directed mutation of hES gene. Of the expressed product of hES gene mutant, 40% existed in a soluble form. However, almost all the expressed product of original hES gene were in forms of inclusion bodies. Conclusion The site-directed mutagenesis of hES gene was successfully achieved, which increased the soluble expression level of target protein.

    2009 05 v.22 [Abstract][OnlineView][Download 304K]

  • Construction and Identification of Eukaryotic Expression Vectors for shRNA Targeting Human Makorin Ring Finger Protein 1 Gene

    LIU Tao, SHI Yan-yan, YUAN Cheng-fu, et a(lMolecular Medicine and Tumor Research Center, Chongqing Medical University, Chongqing 400016, China)

    Objective To construct eukaryotic expression vectors for shRNA targeting human Makorin ring finger protein 1 (MKRN1)gene and determine their effects on expression of the said gene in HEK293 cells. Methods The oligonucleotides of shRNA were designed and synthesized according to the sequence of human MKRN1 gene and directionally cloned into plasmid pGenesil-1 with kanamycin-resistance and enhanced green fluorescence protein (GFP)genes. The constructed recombinant plasmids were identified by restriction analysis and DNA sequencing, then transfected to HEK293 cells in mediation of liposome. The effects of recombinant plasmids on the transcription of MKRN1 mRNA and expression of MKRN1 protein were observed. Results Restriction analysis and DNA sequencing proved that three recombinant plasmids for expression of shRNA targeting human MKRN1 gene and one recombinant plasmid as negative control were constructed correctly. The inhibiting rates of three recombinant plasmids to transcription of MKRN1 mRNA were 52. 4%, 26. 2% and 42. 9%, and those to expression of MKRN1 protein were 69. 1%, 27. 9% and 50. 0%, respectively. Conclusion The eukaryotic expression vectors for shRNA targeting human MKRN1 gene were successfully constructed, which laid a foundation of study on function of MKRN1 and the relationship between MKRN1 and human telomerase reverse transcriptase.

    2009 05 v.22 [Abstract][OnlineView][Download 256K]

  • Prokaryotic Expression and Biological Activity of Poly-Arg Protein Transduction Domain-Apoptin Fusion Protein

    ZHAO Jian, XIAO Wei, FU Wen-bin, et al(State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China)

    Objective To express poly-Arg protein transduction domain(PTD)-apoptin fusion protein in prokaryotic cells and determine its biological activity. Methods Arg9-VP3 sequence was amplified by PCR and inserted into vector pET-43.1a, and the constructed recombinant plasmid pET43.1a-Arg9-VP3 was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was purified by Ni2+-NTA chromatography, lysed with enterokinase, concentrated by ultrafiltration and determined for biological activity. Results Both restriction analysis and sequencing proved that recombinant plasmid pET43.1a-Arg9-VP3 was constructed correctly. Recombinant fusion protein was expressed in a soluble form, reached a purity of more than 90% after purification and showed inhibitory effect on the proliferation of HeLa cells. Conclusion Poly-Arg PTD-apoptin fusion protein was successfully expressed in E. coli and purified , which induced the apoptosis of HeLa cells.

    2009 05 v.22 [Abstract][OnlineView][Download 201K]

  • Prokaryotic Expression and Purification of Microcystis viridis Lectin Protein against HIV

    LI Yu-qin, ZHANG Xue-wu(College of Light Industry and Food Sciences, South China University of Science and Technology, Guangzhou 510640, China)

    Objective To express Microcystis viridis lectin(MVL)protein against HIV in prokaryotic cells, purify the expressed product and lay a foundation of further study on its biological activity and application. Methods The open reading frame (ORF)sequence of MVL gene was amplified by PCR using the genomic DNA of Microcystis viridis NIES-103 as a template and cloned into vector pET-30b (+). The constructed recombinant plasmid was identified by PCR and sequencing, then transformed to competent E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was purified by Ni2+-NTA affinity chromatography then re-naturalized. Results The gene fragment at a full-length of 345 bp was inserted into recombinant plasmid pET30b-MVL, with an identical sequence to that of MVL gene reported in GenBank. The condition for induction of recombinant E. coli and expression of target protein was optimized as follows: the recombinant E. coli at an original concentration (A600)of about 0.5 was induced with 0. 5 mmol / L IPTG in LB medium at 37℃ for 4 h. The expressed product under the optimized condition, with a relative molecular mass of about 20 000, mainly existed in a soluble form and contained 47% of total somatic protein. The purified recombinant protein reached a purity of more than 95% and was identified as mannose-bound MVL by mass spectrography. Conclusion MVL protein against HIV was successfully expressed in prokaryotic cells and purified.

    2009 05 v.22 [Abstract][OnlineView][Download 282K]

  • Protective Effect of Sucralfate on Activity of IgY against Recombinant VacA of Helicobacter pylori

    GUO Li-yuan, YANG Zhi-bang, TIAN Yi-ling, et a(lDepartment of Pathobiology, Institute of Neuroscience, College of Preclinical Medicine, Chongqing University of Medical Sciences, Chongqing 400016, China)

    Objective To observe the protective effect of sucralfate on activity of IgY against recombinant vacuolating cytotoxin antigon (VacA)of Helicobacter pylori. Methods Recombinant VacA was expressed in a large quantity in recombinant E. coli DH5α-vacA-pQE30 under induction of IPTG, purified by Ni2+-NTA affinity chromatography and used for the immunization of Lohman hens, based on which VacA IgY was prepared and purified. The sucralfate and pepsin, at various concentrations, were added into IgY at various pH values; the IgY containing various concentrations of sucralfate were subjected to 7 cycles of freeze-thawing; the IgY containing 30% sucralfate was stored at room temperature for 1 d to 4 weeks; the relative activities(AT / AC)of IgY under the above-mentioned conditions were determined by ELISA. Results At pH 1. 5, 2. 0 and 3. 0, the relative activities of IgY containing 30% ～ 60% sucralfate were significantly higher than those containing no sucralfate. At pH 1. 5, the relative activity of IgY containing 60% sucralfate was 68. 7%. However, both the relative activities of IgY containing 50% sucralfate at pH 2. 0 and that containing 40% sucralfate at pH 3. 0 were nearly 100%. At pH 2. 0 and 3. 0 and a normal pepsin concentration (0. 02 mg / ml), all the relative activities of IgY containing more than 30% sucralfate were protected effectively. The sucralfate at concentrations of more than 30% also enhanced the ability of IgY against freeze-thawing. After storage at room temperature for 4 weeks, the relative activity of IgY containing 30% sucralfate was more than 80%. Conclusion The sucralfate at a concentration of more than 30% enhanced the resistance to low pH value and pepsin as well as ability against freeze-thawing of VacA IgY, thus was an ideal protective agent of IgY.

    2009 05 v.22 [Abstract][OnlineView][Download 237K]

  • Prokaryotic expression and Activity of E. coli Heat-Labile Enterotoxin Subunit B

    DU Lian-feng, SUN Wan-bang, SONG Ming-ying (Department of Immunology, Zhuhai Campus of Zunyi Medical College, Zhuhai 519041, Guangdong Province, China)

    Objective To clone E. coli heat-labile enterotoxin subunit B(ltB)gene, construct its prokaryotic expression vector and determine the activity of expressed product. Methods ltB gene was amplified from the genome of toxigenic E. coli strain 129 by PCR and cloned into vector pET-32a (+), and the constructed recombinant plasmid pET-32a (+)-ltB was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was purified by Ni2+-NTA chromatography and determined for binding activity to bovine GM1 by ELISA. Results PCR proved that recombinant plasmid pET-32a(+)-ltB was constructed correctly. The homology of nucleotide sequence of cloned ltB gene to that reported in GenBank was 99. 9%. After induction of recombinant E. coli for 4 h, the expression level of LTB protein reached a peak value of about 25% of total somatic protein. The purified LTB protein, with a purity of about 96%, showed specific binding activity to bovine GM1. Conclusion ltB gene was successfully cloned, and its prokaryotic expression vector was constructed. The expressed LTB protein showed a certain biological activity.

    2009 05 v.22 [Abstract][OnlineView][Download 171K]

  • Construction of Shuttle Plasmid for Co-expression of Ipr1 and GFP Genes in Human Lung Cancer Cells

    WANG Yu-wei, ZHU Dao-yin, ZHANG Li, et a(lDepartment of Immunology, School of Basic Medicine, Chongqing Medical University, Chongqing 400016, China)

    Objective To construct a shuttle plasmid for co-expression of intracellular pathogen resistance gene 1(Ipr1)and green fluorescent protein(GFP)gene in human lung cancer A549 cells. Methods Ipr1 and GFP genes were amplified from plasmids pEGFP-C1-Ipr1 and pEGFP-C1 by PCR respectively. The amplified Ipr1 and GFP genes as well as mycobacterium replicon OriM were cloned into eukaryotic co-expression vector pBudCE4.1, and the constructed shuttle plasmid pBud-GFP-OriM-Ipr1 was transfected to A549 cells in mediation of liposome. The expression of GFP was observed by fluorescent microscopy, and that of Ipr1 protein was determined by immunohistochemical assay. Results Both restriction analysis and sequencing proved that shuttle plasmid pBudGFP-OriM-Ipr1 was constructed correctly. Expressed GFP was observed by fluorescent microscopy in transfected A549 cells. Immunohistochemical assay proved that Ipr1 protein was expressed in transfected A549 cells and located in cell nucleus. Conclusion A shuttle plasmid for eukaryotic co-expression of Ipr1 and GFP genes was successfully constructed, which laid a foundation of further study on function of Ipr1 against tuberculosis.

    2009 05 v.22 [Abstract][OnlineView][Download 218K]

  • Stability of Three Hepatitis C Virus Markers

    LONG Run-xiang, LI Hua, CUI Ping-fang, et al (Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650118, China)

    Objective To observe the stability of three kinds of hepatitis C virus(HCV)markers. Methods The HCV-Ab, HCV-RNA and HCV-Ag in serum samples were determined by using HCV-Ab, RT-PCR and HCV-Ag kits respectively, based on which the stabilities of the 3 markers after storage at various temperatures for various times and after freeze-thawing were compared. Results Of the 2 036 samples, 85 were positive for HCV-Ab, 51 for HCV-Ag, and 12 for HCV-RNA. All the 85 HCV-Ab positive samples were still positive for HCV-Ab after storage at 4℃ for 14 d, at -20℃ for 3 years and after 5 cycles of freeze-thawing. However, only 33. 33% of 12 HCV-RNA positive samples were positive for HCV-RNA after storage at -20℃ for 3 years, and 83.33% after freeze-thawing. The HCV-Ag positive rate of 51 HCV-Ag positive samples decreased to 68. 63% after storage at -20℃ for 3 years, while showed no significant change after storage at 4℃ for 14 d or after freeze-thawing. Conclusion In the turn of stability, the three HCV markers were HCV-Ab, HCV-Ag and HCV-RNA.

    2009 05 v.22 [Abstract][OnlineView][Download 139K]

  • Prokaryotic Expression of Murine Apolipoprotein A-Ⅰ and Preparation of Its Antiserum

    JU Chuan-jing , SONG Cheng-wei, WAN Jia-yu, et al (The Forth Hospital of Jilin University, Changchun 130011, China)

    Objective To express murine apolipoprotein A-Ⅰ(ApoA-Ⅰ)in prokaryotic cells and prepare its antiserum. Methods ApoA-Ⅰgene was amplified from the liver tissue of mice by RT-PCR and subcloned into prokaryotic expression vector pET-28a(+). The constructed recombinant plasmid pET-28a-ApoA-Ⅰwas transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed fusion protein was purified by Ni2+-NTA chromatography and used for immunization of rabbits to prepare antiserum. Results The sequence of amplified ApoA-Ⅰ gene was completely identical to that reported in GenBank (NM_009692). Restriction analysis proved that recombinant plasmid pET-28a-ApoA-Ⅰwas constructed correctly. The expressed fusion protein, with a relative molecular mass of about 32 000, contained 17% of total somatic protein, reached a purity of 90. 7% after purification and showed specific reaction with anti-His·Tag McAb. The prepared antiserum, at a titer of 1 ∶ 105, recognized ApoA-Ⅰfusion protein specifically. Conclusion Murine ApoA-Ⅰwas highly expressed in E. coli, and its antiserum at a high titer was prepared.

    2009 05 v.22 [Abstract][OnlineView][Download 197K]

  • Synthesis and Cloning of Gene Sequence of Tetravalent Recombinant Group A Streptococcal Polypeptide Vaccine

    YU Gang, YANG Jing-sheng, QUAN Jia-wu(Wuhan Institute of Biological Products, Wuhan 430060, China)

    Objective To design a recombinant polypeptide vaccine specific to group A streptococcus serotypes M1, 3, 6 and 18, which may cause rheumatic fever, and synthesize and clone the gene sequence of the designed vaccine. Methods The serotypespecific amino acid sequences of M protein of group A streptococcus were obtained from the CDC emm typing center website, from which the fragments without homologies to human tissue proteins were screened by the methods reported in documents as well as bioinformatics method. A recombinant polypeptide vaccine was designed by linking the screened fragments and a common conservative sequence J14 which showed a certain immunogenicity but no cross reaction with human tissue protein in the order of M1-3-6-18J14, and analyzed for homology of amino acids to that of human tissue protein by BlastP, then analyzed for hydrophility, secondary structure and spatial structure and predicted for B cell epitopes. A group of oligonucleotides were designed by DNAWORK2. 0 software, based on which the oligonucleotide sequence of designed vaccine was synthesized by overlap PCR, in which the restriction sites of BamHⅠand Hind Ⅲ were introduced at 5′ and 3′terminus respectively. The synthetic sequence was digested with BamHⅠand Hind Ⅲ and cloned into cloning vector pUC18, and the constructed recombinant plasmid was identified by sequencing. Results The designed vaccine showed no homology to human tissue protein. However, the vaccine showed good hydropility, and its secondary and spatial structures were similar to those of natural M protein. B cell epitopes might exist in the fragments of each serotype. The DNA sequence of designed vaccine was successfully cloned, and a recombinant cloning vector carrying designed DNA sequence was obtained. Conclusion A tetravalent recombinant group A streptococcal polypeptide vaccine was successfully designed, which laid a foundation of study on expression and study on immunogenicity of the vaccine.

    2009 05 v.22 [Abstract][OnlineView][Download 273K]

  • Preparation of Bacteriophage-Displayed Diabete Type 1 Vaccine in Chitosan Microspheres by Spray Drying

    WANG Dao-jun, ZENG Li-wen, HU Ning-zhu, et al (Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650118, China)

    Objective To prepare bacteriophage-displayed diabete type 1 vaccine in chitosan microspheres and observe its thermal stability. Methods Bacteriophage-displayed diabete type 1 vaccine in chitosan microspheres was prepared by spray drying using a protective agent composed of water-soluble chitosan, trehalose and glycine, then observed for shape and size by scanning electron microscopy. The effects of inlet temperature and trehalose concentration on activity of the prepared microsphere vaccine were observed. The vaccine sample after spray drying was stored at 37℃ for 6 d and observed for thermal stability. Results The prepared vaccine was in shape of irregular spheres at a mean size of about 20 μm under scanning electron microscope. The titer of vaccine after spray drying was lower than that of bulk before spray drying, and increased with the increasing inlet temperature then decreased. However, the titer of vaccine sample after spray drying increased with the increasing trehalose concentration. Compared with the bulk before spray drying, the microsphere vaccine showed good thermal stability. Conclusion Bacteriophage-displayed diabete type 1 vaccine in chitosan microspheres was successfully prepared and showed good thermal stability.

    2009 05 v.22 [Abstract][OnlineView][Download 147K]

  • Immuneprotective Effect of Intranasal Immunization with Recombinant Human IFNγ and Soluble Tachyzoite antigen of Toxoplasma gondii in Mice

    MENG Xiao-li, GONG Hong-fei, YIN Guo-rong, et al(Institute of Medical Parasitology, Shanxi Medical University, Taiyuan 030001, China)

    Objective To observe the mucosal and systemic immune responses as well as anti-infectious immunity against Toxoplasma gondii induced by intranasal immunization with recombinant human IFNγ and soluble tachyzoite antigen (STAg)of Toxoplasma gondii in mice. Methods BALB / c mice were divided into STAg, STAg + IFNγ and control groups and immunized i.n. with 20 μg of STAg, 20 μg of STAg + 1 000 U IFNγ and 20 μl of PBS for 2 times at an interval of 14 d respectively. The mice were challenged intragastrically with tachyzoite of Toxoplasma gondii strain RH on day 10 after the 2nd immunization, 4 × 104 tachyzoites per mouse, and observed for survival, based on which the survival rate was calculated. All the mice were killed on day 43 after challenge and counted for tachyzoites in liver and brain tissues, and their intestinal intraepithelial lymphocytes(iIELs)and lymphocytes in Peyer patches(PP)and spleen were isolated and counted. The Toxoplasma gondii-specific sIgA level in feces and IgG level in sera were determined by ELISA. Results The intranasal immunization with IFNγ and STAg protected the mice against the challenge with tachyzoites of Toxoplasma gondii. The survival rate of mice in STAg + IFNγ group(93%)was significantly higher than those in STAg (60%)and control(47%)groups. The counts of iIELs(1. 81 × 105)as well as lymphocytes in PP(3. 21 × 107)and spleen(3. 01 × 107)of mice in STAg + IFNγ group were also significantly higher than those in STAg and control groups. The sIgA level (A492)in feces of mice in STAg + IFNγ group(0. 435)was significantly higher than that in control group(0. 047), while the IgG level(A492)in sera(0. 233)was significantly higher than those in STAg(0. 193)and control(0. 115)groups. Compared with those in control group, the numbers of tachyzoites in liver and brain tissues of mice in STAg + IFNγ group decreased by 80. 90% and 64. 50% respectively. Conclusion The immune effect of intranasal immunization with STAg + IFNγ was superior to that with STAg alone. IFNγ as an adjuvant enhanced both the mucosal and systemic immune response levels induced by STAg, increased the survival rate of mice after challenge with tachyzoites of Toxoplasma gondii and decreased the counts of tachyzoites in liver and brain tissues.

    2009 05 v.22 [Abstract][OnlineView][Download 189K]

  • Construction, Expression and Purification of Integrated Interferon Mutant

    ZHANG Jing, LIU Jin-yi, ZHOU Min-yi, et a(lInstitute of Traditional Chinese Drugs, Chengde Medical College, Chengde 067000, Hebei Province, China)

    Objective To construct, express and purify a highly effective integrated interferon mutant(IIFNm108)for site-directed modification of mPEG-MAL. Methods The mutant site was designed by homologous sequence alignment and spatial structure modeling combined with the characteristic of binding of IFNα to its receptor. IIFNm108 gene was amplified by overlap PCR and inserted into vector pET-23b, and the constructed recombinant plasmid pET-23b-IIFNm108 was transformed to E. coli BL21(DE3)for expression under induction of IPTG. The expressed product was de-naturalized, re-naturalized and purified by DEAE anion exchange and gel filtration chromatography, then subjected to overall control tests. Results Both restriction analysis and sequencing proved that recombinant plasmid pET-23b-IIFNm108 was constructed correctly. The expressed IIFNm108 with a relative molecular mass of 19 500 mainly existed in a form of inclusion body and contained more than 30% of total somatic protein. The purified IIFNm108 reached a purity of more than 95% and a specific activity of about(2. 08 ± 0. 17)× 108 IU / mg. The amino acid sequences at Nand C-terminus of expressed IIFNm108 were consistent with those in theory. SDS-PAGE showed successful crosslink of expressed IIFNm108 with mPEG-MAL. Conclusion Integrated interferon mutant (IIFNm108) was obtained, which was suitable for the site-directed modification of mPEGMAL.

    2009 05 v.22 [Abstract][OnlineView][Download 203K]

  • Establishment of National Ovalbumin Quantitation Reference for Quality Control of Influenza Vaccine

    CHEN Zhen, ZHANG Guo-qiang, GAO Qiang, et al (National Institute for the Control of Pharmaceutical and Biological Products, Beijing 100050, China)

    Objective To establish a national ovalbumin quantitation reference for quality control of influenza vaccine. Methods The ovalbumin samples of various grades manufactured by various manufacturers were quantitatively determined by imported kit, from which the sample with the highest consistence rate of determination result with that of standard in the kit were screened as the material for preparation of reference, and the protective agent of reference was verified. The stock solution of prepared reference was diluted serially and calibrated collaboratively by three laboratories to determine the stated ovalbumin content. Results The sample Sigma GⅡ was screened as the material for preparation of ovalbumin quantitation reference. The protective agent showed no effect on the determination result and increased the stability of reference. The stated contents of ovalbumin in reference samples at concentrations of 15, 10, 5 and 2. 5 ng / ml were(17. 96 ± 1. 00),(12. 25 ± 1. 51),(6. 79 ± 1. 14)and(3. 11 ± 0. 31)ng / ml respectively. Conclusion The national ovalbumin quantiation reference with accurate content as well as good reproducibility and linearity was established.

    2009 05 v.22 [Abstract][OnlineView][Download 129K]

  • Safety of Simultaneous Inoculation with Two Kinds of Vaccines Included in National Immunization Program

    PANG Hong, WU Mei-hua(Changning District Center for Disease Control and Prevention, Shanghai City, Shanghai 200051, China)

    Objective To analyze the incidence rate of adverse events following immunization(AEFI)and evaluate the safety of simultaneous inoculation with two kinds of vaccines included in National Immunization Program (NIP). Methods The data on subjects inoculated with the 3rd dose of hepatitis B vaccine (HepB), the 1st dose of group A meningococcus polysaccharide vaccine (MCV-A), the 1st dose of measles vaccine(MV)and the 1st dose of Japanese encephalitis vaccine(JEV)during March 2006 to December 2007 in 9 immunization clinics in Changning District, Shanghai City were collected, based on which the incidence rates of AEFI of vaccines inoculated alone and simultaneously were calculated. Results The incidence rates of the 3rd dose of HepB and the 1st dose of MCV-A inoculated alone were 5. 86‰ and 3. 74‰ respectively, while that of the two kinds of vaccine inoculated simultaneously was 1. 18‰. However, the incidence rates of the 1st dose of MV and the 1st dose of JEV inoculated alone were 3. 59‰ and 9. 24‰ respectively, while that of the two kinds of vaccine inoculated simultaneously was 1. 93‰. Significant differences were observed in the incidence rates of same vaccines manufactured by various manufacturers or by the same manufacturer in various packages. The incidence rate of AEFI in infants aged 6 ～ 12 months was relatively high. Conclusion The incidence rate of AEFI of two kinds of vaccines included in NIP inoculated simultaneously showed no significant increase as compared with those inoculated alone. Two kinds of vaccines included in NIP may be recommended to be inoculated simultaneously to decrease the doses.

    2009 05 v.22 [Abstract][OnlineView][Download 125K]

  • Analysis of Determination Result of Serological Marker of HBV in 280 Patients Positive for HBV-DNA

    LI Gui-ling, LI Wei, JIA Li, et al(Department of Laboratory Medicine, China-Japan Union Hospital of Jilin University, Changchun 130021, China)

    Objective To analyze the determination results of serological markers of HBV(HBVM)in patients positive for HBV-DNA and explore its clinical significance. Methods The subjects receiving physical examination in China-Japan Union Hospital of Jilin University were determined for HBV-DNA by fluorescent quantitative PCR and for HBVM by ELISA. Results A total of 13 serological marker modes were detected in 280 patients positive for HBV-DNA, of which HBsAg positive mode in 272 and HBsAg negative mode in 8 patients. Conclusion HBV-DNA may be detected in patients with various HBVM modes, thus is considered as a more direct, sensitive and specific index of HBV infection.

    2009 05 v.22 [Abstract][OnlineView][Download 121K]

  • Verification of Method for Microbial Limit Test on Raw and Subsidiary Materials of Biologics

    CHEN Bo, XU Cheng-lin, LIU Gui-fang, et a(lBeijing Tiantan Biological Products Co., Ltd, Beijing 100024, China)

    Objective To develop and verify a method for microbial limit test on raw and subsidiary materials of biologics. Methods The microbial limits of 6 kinds of raw and subsidiary materials of biologics, i.e. lactose, glucose, gel, starch, trypsin and purified water, were tested by Petri dish culture and membrane filtration methods separately, and the developed methods were verified by adding standard microorganisms with known counts into the test sample and determining their recovery rates. Results All the recovery rates of standard Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Candida albicans and Aspergillus niger in the test samples of 6 kinds of raw and subsidiary materials were more than 80%, which met the requirements (more than 70%)in Chinese Pharmacopoeia (Volume Ⅱ, 2005 edition). Conclusion The developed Petri dish culture method was suitable for microbial limit test on lactose, glucose, gel, starch and trypsin, and membrane filtration method for that on purified water.

    2009 05 v.22 [Abstract][OnlineView][Download 125K]

  • Preliminary Study on Condition for Lysis of Human Respiratory Syncytial Virus

    CHEN Si-jin, XIE Zhong-ping, LI Hua, et al(Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Kunming 650118, China)

    Objective To explore the condition for lysis of human respiratory syncytial virus(HRSV)and lay a foundation of preparing purified HRSV cleavage vaccine. Methods HSRV was lysed with various lytic agents at various concentrations and temperatures for various time, then identified by reduced electrophoresis and Western blot and observed by electron microscopy. Results After lysis with 1% Triton X-100 at 4℃ for 90 min, HRSV were nebular particles under electron microscope, and specific F protein band was proved by Western blot. Conclusion The condition for lysis of HRSV was preliminarily screened.

    2009 05 v.22 [Abstract][OnlineView][Download 188K]

  • Development and Clinical Application of Double Antibody Sandwich ELISA for WuTac Concentration in Serum

    ZHANG Nan, LI Qian, GUO Chang-fu, et al(Wuhan Institute of Biological Products, Wuhan 430060, China)

    Objective To develop a double antibody sandwich ELISA for WuTac concentration in serum and preliminarily apply in clinic. Methods The WuTac concentration in sera was determined quantitatively by double antibody sandwich ELISA, based on which the concentration of coating antibody and the dilution of enzyme-labeled antibody were optimized and a standard curve was plotted. The developed ELISA was verified and used for the determination of serum specimens from 36 healthy volunteers at 14 time points before and after injection with WuTac at various dosages(0. 05, 0. 1 and 0. 2 mg / kg). Results The optimal concentration of coating antibody was 0. 2 μg / ml, and the optimal dilution of enzyme-labeled antibody was 1 ∶ 15 000. The correlation coefficient (r value)of standard curve was not less than 0. 99. The linear determination range of developed double antibody sandwich ELISA was 3. 9 ～ 125 ng / ml. The variation coefficient(CV)of determination results of high concentration WuTac standard by the developed ELISA was less than 15%. The developed ELISA showed a accuracy of 99. 05% ± 5. 00%(92. 43% ～ 110. 02%)and good specificity. The determination results of clinical serum specimens proved that WuTac showed a characteristic of linear pharmacokinetics within a concentration range of 0. 05 ～ 0. 2 mg / kg. Conclusion A double antibody sandwich ELISA for WuTac concentration in serum was developed, with sensitivity, precision and accuracy meeting the requirements of pharmacokinetic study on biologics.

    2009 05 v.22 [Abstract][OnlineView][Download 167K]

  • Development of Protein Chip Technique for Detection of Abrin and Recin

    LU Hao, WANG Xing-long, LI Xiao-yan, et al (The Military Veterinary Institute, Military Academy of Medical Science, Changchun 130062, China)

    Objective To develop a protein chip technique for detection of abrin and recin. Methods The McAbs against abrin and recin were prepared and purified, based on which a protein chip for detection of antigens by competitive immunoassay was prepared and used for detection of mock samples of abrin and recin. Results No fluorescent signals were observed in corresponding regions of mock samples of abrin, recin and their mixture by using the prepared protein chip, indicating positive results consistent with those designed. Conclusion The protein chip technique for detection of abrin and recin was successfully developed, which was of a broad prospect in application.

    2009 05 v.22 [Abstract][OnlineView][Download 196K]

  • Development of Research on Novel Schistosomiasis japonicum Vaccine

    ZHAI Yu-jia, LIU Quan (The Military Veterinary Institute, Academy of Military Medical Sciences, Changchun 130062, China)

    Schistosomiasis japonica is one of the most serious parasitic disease that threaten the health of humans and livestock. The development of vaccine is an effective measure for the prevention and control of the infectious disease. It is necessary to develop novel Schistosomiasis japonicum vaccines since the application of traditional vaccines is restricted due to their high cost, low immunogenicity and low safety. With the rapid progresses in immunology and molecular biology, various novel Schistosomiasis japonicum vaccine have been developed in recent years. The development of research on recombinant subunit vaccine, synthetic peptide vaccine, DNA vaccine, recombinant live vector vaccine and anti-idiotype antibody vaccine as well as advantages, shortcomings and prospects of the vaccines are reviewed in this paper.

    2009 05 v.22 [Abstract][OnlineView][Download 137K]

  • Progress in Study on Virus-like Particles as Vaccine and Vector

    ZHAO Pu, ZHENG Yu-shu, LIU Xing-you(Department of Animal Science and Technology, Henan Institute of Science and Technology, Xinxiang 453003, Henan Province, China)

    Virus-like particles(VLPs)are highly organized viral capsid proteins that self-assemble from virus-derived structural antigens, which resemble the overall structure of virus particles and thus preserve the native antigenic conformation of the immunogenic proteins. VLPs as vaccine and vector have been paid considerable attention. The progress in study on VLPs as vaccine and vector is reviewed in this paper.

    2009 05 v.22 [Abstract][OnlineView][Download 116K]

  • Progress in Study on p63 Gene Mutation and Its Relationship to Developmentassociated Diseases

    YAN Jun-xi, PAN Hai-long, ZHANG Xue-me(iChengdu Institute of Biological Products, Chengdu 610023, China)

    As an important functional gene associated with tissue development as well as cell apoptosis and differentiation, p63 is a key regulator of ectodermal, orofacial and limb developments. The mutation of p63 gene may lead to at least five syndromes in humans. The progress in study on p63 gene mutation and its relationship to development-associated diseases is reviewed in this paper.

    2009 05 v.22 [Abstract][OnlineView][Download 141K]

  • Progress in Study on Regulation of Hemopoiesis and Differentiation of Yolk Sac

    WU Ying, LIU Nian, YAO Cheng(Department of Blood Tumor, The First Hospital of Jilin University, Changchun 130021, China)

    The yolk sac was considered the original hematogenic organ during the ontogenesis of mammals, and the yolk sac hemopoietic stem cel(lYS-HSC)was differentiating pluripotent and multipotent. The new experimental approach showed that the yolk sac was the true source of adult haematopoiesis. The multiplication and differentiation of YS-HSC was regulated by multitude cytokines and colonization factors in the embryonic hematopoietic microenvironment, while it was a complicated process involved in multiple gene regulation. The progresses in study on yolk sac hemopoiesis as well as the proliferation, differentiation and regulation of the YSHSC are reviewed in this paper.

    2009 05 v.22 [Abstract][OnlineView][Download 117K]
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