G145R突变后HBsAg"a"决定簇合成肽的免疫学特性分析

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P1-wt和P2-145R,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(2-ME)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Western blot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P1-wt和P2-145R用2-ME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(anti-HBs)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∶32 000稀释时仍可检测到P1-wt的阳性结果,而当同一抗体稀释到1∶8000时,P2-145R检测结果即为阴性.合成肽P2-145R免疫小鼠产生的抗体与P1-wt合成肽的反应滴度比与P2-145R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.

Immunogenicity of G145R mutant HBsAg "a" determinant with synthetic peptides

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. P1 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 4 kD to 30 kD and the main bands were at 5 kD and 10 kD, whereas in denatured condition, it showed one band of 4 kD. 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 titer 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.