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

摘要

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.%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HEV ORF2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-HEV IgG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HEV IgG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HEV ORF2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。