期刊文献+

G145R突变后HBsAg“a”决定簇合成肽的免疫学特性分析 被引量:5

Immunogenicity of G145R mutant HBsAg "a" determinant with synthetic peptides
原文传递
导出
摘要 目的研究G145R突变后乙肝表面抗原(HBsAg)“a”决定簇合成肽的免疫学特性改变情况。方法首先合成2条短肽P1wt和P2145R,分别代表野毒株和G145R突变后HBsAg“a”决定簇合成肽。然后用β巯基乙醇(2ME)变性试验以及用乙肝疫苗标准品制备的小鼠多抗血清研究2条合成肽的空间构象以及抗原性异同。最后用等量合成肽免疫小鼠,用酶免疫法、竞争抑制试验和Westernblot试验等研究G145R突变后“a”决定簇合成肽的免疫原性改变。结果合成肽P1wt和P2145R用2ME温和变性后,PAGE结果表现为相对分子质量(Mr)为4×103左右的单一条带,而变性前2条合成肽均表现为Mr是从4×103到30×103的弥漫条带,主带位置在5×103和10×103,分别相当于二聚体和四聚体位置;用HBsAg的多克隆抗体(antiHBs)检测合成肽的抗原性,结果在固定抗原量的前提下,在抗体1∶32000稀释时仍可检测到P1wt的阳性结果,而当同一抗体稀释到1∶8000时,P2145R检测结果即为阴性。合成肽P2145R免疫小鼠产生的抗体与P1wt合成肽的反应滴度比与P2145R合成肽本身的反应低4~8倍。结论针对HBsAg“a”决定簇合成肽可自发形成一定的空间构象,G145R突变株的HBsAg“a”决定簇的抗原性和免疫原性与野毒株相比发生了明显改变,为正确评价G145R变异株的流行危害以及现行疫苗的保护效果提供了实验依据。 Objective To study the change of antigenicity and immunogenicity of G145R mutant HBsAg. Methods Two peptides were synthesized which were specific to HBsAg “a” determinant. Pl-wt represents wild type and P2-145R represents G145R mutant. The conformation structure was analyzed with 2-ME denature test. Then the antigenicity and immunogenicity of G145R mutant HBsAg were studied with ELISA, competitive inhibition test and Western blot. Results SDS-PAGE results revealed that in the absence of reducing agents, both peptides had a heterogeneous mixture of multiple forms with molecular masses ranging from 4kD to 30kD and the main bands were at 5kD and 10kD, whereas in denatured condition, it showed one band of 4kD. In a direct ELISA, peptide 1(P1-wt) showed a high degree of cross-reactivity with polyclonal anti-HBsAg antisera, the highest titer of antisera could reach as high as 1/32 000, whereas the antigenicity of peptide 2(P2-145R) was significantly reduced, that means when the liter of antisera was 1/8000, the result became negative. And 6 weeks after being immunized with these peptides, mice produced a high level of serum antibody against respective peptides such as P1-wt and P2-145R, but the anti-peptide antibodies bound to the corresponding native peptide with an apparent affinity higher than the others. Conclusion All of the cysteine sulfhydryl groups may spontaneously form disulfide bonds to yield a conformational, disulfide-dependent “a” determinant of HBsAg. And the antigenicity and immunogenicty changed significantly after amino acid mutate at amino acid 145. This result suggest that it is necessary to reevaluate the effect of present hepatitis B vaccine, set up G145R mutant HBV specific diagnostic methods and to develop new vaccines.
出处 《中华微生物学和免疫学杂志》 CAS CSCD 北大核心 2005年第8期642-647,共6页 Chinese Journal of Microbiology and Immunology
基金 国家"十五"科技攻关计划资助项目(编号:2001BA705B05)
关键词 “a”决定簇合成肽 乙肝表面抗原 G145R变异株 免疫学特性 HBSAG 合成肽 决定簇 突变株 特性分析 多克隆抗体 Synthetic peptides to “a” determinant HBsAg G145R mutant strain Antigenicity and immunogenicity
  • 引文网络
  • 相关文献

参考文献11

  • 1Carman WF, Zanetti AR, Karayiannis P, et al. Vaccine-induced escape mutant of hepatitis B virus. Lancet, 1990, 336: 325-329.
  • 2Ogata N, Zanetti AR, Yu M, et al. Infectivity and pathogenicity in chimpanzees of a surface gene mutant of hepatitis B virus that emerged in a vaccinated infant. J Infect Dis, 1997, 175: 511-523.
  • 3Hsu HY, Chang MH, Liaw SH, et al. Changes of hepatitis B surface antigen variants in carrier children before and after universal vaccination in Taiwan. Hepatology, 1999, 30: 1312-1317.
  • 4Cooreman MP, van Roosmalen MH, te Morsche R, 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. Hepatology, 1999, 30(5): 1287-1292.
  • 5钟熙,王佑春,RLing,倪健,梁争论,祁自柏,TJHarrison,李河民.用现行国产HBsAg EIA检测乙肝S基因突变体问题的研究[J].中华微生物学和免疫学杂志,1998,18(2):85-87. 被引量:27
  • 6Manivel V, Ramesh R, Panda SK, et al. A synthetic peptide spontaneously self-assembles to reconstruct a group-specific, conformational determinant of hepatitis B surface antigen. J Immunol, 1992, 148(12): 4006-4011.
  • 7Dreesman GR, Sanchez Y, Ionescu-Matiu I, et al. Antibody to hepatitis B surface antigen after a single inoculation of uncoupled synthetic HBsAg peptides. Nature, 1982, 195: 158-160.
  • 8Bhatnagar PK, Papas E, Blum HE, et al. Immune response to synthetic peptide analogues of hepatitis B surface antigen specific for the "a" determinant. Proc Natl Acad Sci USA, 1982, 79: 4400-4404.
  • 9Wu L, He JW, Yao X, et al. A novel hepatitis virus variants S129(Gln-Leu): lack of correlation between antigencity and immunogenicity. J Med Virol, 1999, 59: 424-430.
  • 10Shizuma T, Hasegawa K, Ishikawa K, et al. Molecular analysis of antigenicity and immunogenicity of a vaccine-induced escape mutant of hepatitis B virus. J Gastroenterol, 2003, 38(3): 244-253.

二级参考文献3

共引文献26

同被引文献41

引证文献5

二级引证文献5

;
使用帮助 返回顶部