人乳头瘤病毒6型L1蛋白在原核表达体系中的表达

Expression of recombinant HPV6 L1 protein in prokaryotic expressing system

目的:比较人乳头瘤病毒6型(HPV6)L1序列的变异,并筛选有代表性的分离标本进行重组HPV6L1蛋白的表达和抗原性检测。方法:根据不同临床特征选择主要致病型HPV6型8个分离标本,扩增L1基因,构建重组测序质粒。分析L1区的氨基酸序列变异状况,选择L1序列变异较大的临床分离标本,通过对HPV6L1基因的起始和终止端进行基因定点改造,连于表达载体质粒pET32a,在原核表达体系表达重组HPV6L1蛋白,并进行L1蛋白的抗原性检测。结果:HPV6型8个分离标本中有6个分离标本L1区的蛋白质一级结构中分别有1～3个氨基酸发生变异,原核表达重组L1蛋白可与HPV6L1抗体发生特异反应。说明HPV6L1序列的变异并未影响L1蛋白的抗原性。结论:本临床分离标本可提供发展HPV6L1蛋白和病毒样颗粒(VLP)疫苗的候选目的基因,并可通过原核表达体系表达重组HPVL1蛋白进行L1蛋白的免疫学分析,以及L1蛋白疫苗的研究。

Objective:To investigate the intratype sequence variation of HPV6L1ORF,the expression of recombinant L1protein from typical HPV6isolate and the detection of their antigenicity.Methods:Eight isolates of HPV6with different clinical manifestation were chosen.L1genes were amplified and inserted into sequencing plasmid.Intratype amino acid se-quence variation was assessed.After site-directed mutagenesis of some rare codons near the L1start and final sequence,a typical variation of HPV6L1ORF was cloned into the plasmid pET32a.Results:HPV6L1capsid protein was expressed within prokaryotic system.The antigenicity of the recombinant L1protein was tested.Results revealed there was1to3amino acids changed in different L1sequences in six isolates respectively.Recombinant L1capsid protein was expressed and com-bined with HPV6L1antibody.Conclusions:L1intratype amino sequence variation didn't affect L1protein antigenicity.The isolate could be selected as a target gene for recombinant HPV6L1virus-like particles (VLP).Prokaryotic expression system could be used to express recombinant HPV6L1protein for immunogenicity analysis and vaccine development.