新型戊型肝炎病毒ORF2部分基因的构建表达及表达产物的抗原性分析

目的对新型戊型肝炎病毒ORF2的3′端部分基因进行重组质粒构建及融合表达，并对表达蛋白进行抗原性分析。方法用PCR方法从含有HEVORF2基因的pGEM-T载体上扩增表达片段，双酶切后与原核表达载体pThioHisA构建重组质粒pThioHisA-T11。诱导表达后进行SDS-PAGE和免疫印迹分析。并用纯化的融合蛋白建立EIA检测方法，对40份抗-HEVIgG国家参比血清进行检测。结果含有pThioHisA-T11重组质粒的菌株表达相对分子质量为40×103的可溶性融合蛋白T11，免疫印迹证明融合蛋白T11能与抗HEVIgG发生特异反应。EIA检测结果显示，阳性参比血清符合率为80%(8/10)，阴性参比血清符合率为87%(26/30)，总符合率为85%。结论表达了新型HEVORF2羧基端278氨基酸并证明有抗原活性，为今后提高HEV检出率奠定了基础。

Construction and expression of partial ORF2 from a novel hepatitis E virus and antigenicity analysis of the expression products

Objective To construct a recombinant plasmid expressing partial ORF2 of a novel HEV genotype and to analyse the antigenicity of expression protein. Methods Using PCR, the expression fragment was amplified from pGEM T vector containing HEV ORF2. The PCR products and expression vector pThioHisA, after digestion by restriction enzymes, were constructed into one recombinant plasmid: pThioHisA T11. The host bacteria containing the recombinant plasmid was induced to express. The fusion protein was analyzed by SDS PAGE and Western bolt. EIA was established based on the purified fusion protein and forty national reference samples of anti HEV IgG were assayed. Results The recombinant plasmid, pThioHisA T11, expressed a new soluble fusion protein with molecular weight 40 000, which can specifically react with anti HEV IgG by Western blot. The EIA results showed that 80% (8/10) of the positive reference samples and 87% (26/30) of the negative reference samples were detected by T11 antigen, respectively. The total accordance rate was 85% (34/40). Conclusions Recombinant protein containing amino acid sequences from open reading frame 2 of a novel HEV strain classified as a new genotype were constructed as fusions with Thioredoxin and the antigenicity was demonstrated by both Western blot and EIA. This has laid a necessary foundation for future research on improving the detection rate of HEV.