期刊文献+

乙肝表面抗原突变体的表达及其初步应用 被引量:3

Expression and preliminary applications of hepatitis B surface antigen mutants
下载PDF
导出
摘要 目的:构建和表达乙肝表面抗原(HBsAg)突变体用于HBsAg抗原性的深入研究。方法:利用定点突变技术构建HBsAg突变体,然后转化毕赤酵母GS115,菌落PCR、高浓度Zeocin抗性筛选鉴定转化子,表达产物经SDS-PAGE分析和Western blot分析,利用AxSYM HBsAgV2(Abbott)酶免试剂盒检测重组表面抗原的活性。结果:通过序列分析确定突变体构建成功,SDS-PAGE显示突变体能在毕赤酵母中有效表达,在Western blot试验中HBsAg突变体被特异性多克隆抗体识别,相对分子质量(Mr)约为38000,AxSYM试剂盒检测结果表明HBsAg突变体具有一定生物活性。结论:利用毕赤酵母表达系统高效表达出具有一定免疫反应性的HBsAg突变体,对于目前市售试剂盒的质量控制和临床应用有较高的实用价值。 AIM: To clone and express HBsAg mutant in the Pichia pastoris. METHODS: The cloned wild type pGAP-S was used as the DNA template to generate mutant type pGAP-MS with a single or double nucleotide changes incorporated in complementary oligonucleotide primers. The product was linearized with BspH I and transformed into Pichia pastods strain GS115, and stable multicopy integrants were screened in medium containing different concentrations of Zeocin. RESULTS, The pGAP-MS expression vector was successfully constructed and stable numbers integrated strains with high copy number were obtained. The expression of HBsAg mutant protein was identified by SDS-PAGE and Westem blot with specific polyclonal antibody. The molecular weight of recombinant HBsAg mutant was 38 kDa. AxSYM HBsAg V2(Abbott)assays demonstrated all 10 HBsAg mutants were reactive. CONCLUSION: The recombinant HBsAg mutant with immunoreactivity was successfully expressed in Pichia pastoris, and it was of practical value on the quality control and clinical applications of commercial assays.
出处 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2007年第12期1136-1139,共4页 Chinese Journal of Cellular and Molecular Immunology
基金 上海市科学技术委员会科研计划项目(07DZ19502)
关键词 乙肝表面抗原突变体 毕赤酵母 免疫反应性 Hepatitis B surface antigen mutant Pichia pastoris immunoreactivity
  • 相关文献

参考文献10

  • 1Schochetman G, Kuhns MC. HIV variants and hepatitis B surface antigen mutants : diagnostic challenges for immunoassays [ J ]. J Med Virol, 2006, 78(Suppl 1): S3-6.
  • 2Mangold CM, Streeek RE. Mutational analysis of the eysteine residues in the hepatitis B virus small enelope protein[J]. J Virol, 1993, 67 (8) : 4588 -4597.
  • 3Minami M, Okanoue T, Nakajima E, et al. Significance of Pre-S region-defective hepatitis B virus that emerged during exacerbation for chronic type B hepatitis[J]. Hepatology, 1993, 17(4) : 558 -563.
  • 4Jolivet-Reynaud C, Lesenechal M, O'donnell B, et al. Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies [J]. J Med Virol, 2001,65(2): 241 -249.
  • 5Ni F, Fang D, Gan R, et al. A new immune escape mutant of hepatitis B virus with an Asp to Ala substitution in aa144 of the envelop major protein[J]. Res Virol, 1995, 146(6) : 397 -407.
  • 6Ana Vassileva, Dipti A, Sathyamangalam Swaminthan et al. Expression of hepatitis B surface antigen in the methylotrophic yeast pichia pastoris using the GAP promoter[ J]. J Biotechnol, 2001, 88 (1) : 21 - 35.
  • 7白玉,夏国良,郭瑜,丛郁,贾志远,郭敏卓,赵洪兰,詹美云.乙型肝炎表面抗原不同突变体对细胞免疫的影响[J].中华实验和临床病毒学杂志,2005,19(2):124-127. 被引量:1
  • 8Hou J, Wang Z, Cheng J, et al. Prevalence of naturally occuring surface gene variants of hepatitis B virus in nonimmunized surface antigennegative Chinese carriers [ J ]. Hepatology, 2001, 34 ( 5 ) : 1027 - 1034.
  • 9Cooreman MP, Van Roosmalen MH, Morsche Rte, et al. Characterization of the reactivity pattern of murine monoclonal antibodies against wild-type hepatitis B surface antigen to G145R and other naturally occurring "a" loop escape mutations[J]. Hepatology, 1999, 30(5) : 1287 - 1292.
  • 10Paul F. Detectiong hepatitis B surface antigen mutants [ J ]. Emerg Infect Dis, 2006, 12(2) : 198 -203.

二级参考文献8

  • 1任红,TimHarrison.重组HBsAg免疫逃逸突变体的表达及其抗原性分析[J].中华微生物学和免疫学杂志,1996,16(2):130-132. 被引量:7
  • 2Hildt E, Saher G, Bruss V, et al.The hepatitis B virus larger surface protein (LHBs) is a transcriptional activator.Virology, 1996, 225: 235-239.
  • 3Milich DR, Schodel F, Hughes JL, et al.The hepatitis B virus core and e antigens elicit different Th cell subsets :antigen structure can affect Th cell phenotype.J Virol, 1997,71:2192-2201.
  • 4Cacciarelli TV, Martinez OM, Gish RG, et al.Immuno- regulatory cytokines in chronic hepatitis C virus infection: pre- and posttreatment with interferon alfa.Hepatology, 1996, 24:6-9.
  • 5Modlin RL, Nutman TB.Type 2 cytokines and negtive immune regulation in human infections.Curr Opin Immunol, 1993,5:511-514.
  • 6Wang Y, Menne S, Jacob JR, et al.Role of type 1 versus type 2 immune responses in liver during the onset of chronic woodchuck hepatitis virus infection.Hepatology, 2003,37:771-780.
  • 7Dong Y, Xi H, Yu Y, et al.Effects of oxymatrine on the serum levels of T helper cell 1 and 2 cytokines and the expression of the S gene in hepatitis B virus S gene transgenic mice: a study on the anti-hepatitis B virus mechanism of oxymatrine.J Gastroenterol Hepatol, 2002,17:1299-1306.
  • 8白玉,夏国良,贾志远,丛郁,王晓黎,郭敏卓,詹美云.常见乙型肝炎病毒表面抗原突变体的抗原性分析[J].中华实验和临床病毒学杂志,2003,17(1):31-34. 被引量:16

同被引文献21

  • 1李金明.乙型肝炎病毒血清标志物测定及结果解释的若干问题[J].中华检验医学杂志,2006,29(5):385-389. 被引量:154
  • 2李金明.感染性疾病血清学检验中应重视对弱反应性标本的确认[J].中华检验医学杂志,2006,29(7):577-580. 被引量:126
  • 3Cooreman MP,Van roosmalen MH,et al. Characterization of the reactivity pattern of murine monoclonal antibodies against wildtype hepatitis B surface antigen to C-145R and other naturally occurring "a" loop escape mulalions[J]. Hepalology, 1999,30(5) 1287-1292.
  • 4Weinbaum CM,Williams I,Mast EE,et al.enters for Disease Control and Prevention.Recommendations for identification and public health management of persons with chronic hepatitis B virus infection[J].MMWR Recomm Rep,2008,57(RR-8):1-20.
  • 5Hou J,Liu Z,Gu F. Epidemiology and prevention of hepatitis B virus infection[J].International Journal of Medical Sciences,2005,(01):50-57.
  • 6Raimondo G,Allain JP,Brunetto MR. Statements from the Taormina expert meeting on occult hepatitis B virus infection[J].Journal of Hepatology,2008,(4):652-657.doi:10.1016/j.jhep.2008.07.014.
  • 7Schochetman G,Mary C. HIV variants and hepatitis b surface antigen mutants:diagnostic challenges for immunoassays[J].Journal of Medical Virology,2006,(Suppl 1):S3-S6.
  • 8Levrero M,Pollicino T,Petersen J. Control of cccDNA function in hepatitis B virus infection[J].Journal of Hepatology,2009.581-592.
  • 9Jolivet-Reynaud C,Lésenéchal M,O'Donnell B. Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies[J].Journal of Medical Virology,2001,(02):241-249.
  • 10Jinlin H,Zhanggui W,Jinjun C. Prevalence of naturally occurring surface gene variants of hepatitis B virus in nonimmunized surface antigen-negative Chinese carriers[J].Hepatology,2001,(05):1027-1032.

引证文献3

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部