重组猪源戊型肝炎病毒衣壳蛋白的原核表达及鉴定

目的原核表达猪源戊型肝炎病毒衣壳蛋白,并对其反应原性进行分析。方法通过PCR从已构建的猪源戊型肝炎病毒全基因克隆扩增ORF2编码382~674位氨基酸序列的基因片段,将扩增产物插入到pMD18-T载体中,亚克隆至原核表达载体pET28a(+),构建pET28a-293表达载体,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经Ni-NTA层析柱纯化后,进行SDS-PAGE、Western blot及ELISA鉴定。结果成功扩增到904bp的目的基因。重组载体pET28a-293经双酶切鉴定及序列测定,证实构建正确。SDS-PAGE分析表明表达产物的相对分子质量与预期相符,表达量可占菌体总蛋白的66.15%。Western blot分析表明目的蛋白具有较好的反应原性。ELISA试验证实目的蛋白能与阳性猪源和标准HEV血清发生反应。结论已成功构建了猪源戊型肝炎病毒衣壳蛋白的pET28a-293原核表达载体,并得到了有效表达,为进一步建立猪源戊型肝炎的诊断方法奠定基础。

Prokaryotic Expression and Identification of Recombinant Swine Hepatitis E Virus Capsid Protein

Objective To express recombinant swine hepatitis E virus capsid protein in prokaryotic cells and analyze the reactogenicity of expressed product.Methods The ORF2 gene fragment encoding the amino acids 382～674 was amplified from the full-length of swine HEV genomic clone by PCR and inserted into vector pMD18-T,then subcloned to prokaryhotic expression vector pET28a(+).The constructed recombinant plasmid pET 28a-293 was transformed to E.coliBL21(DE3)for expression under induction of IPTG.The expressed product was purified by Ni-NTA chromatography and identified by SDS-PAGE,Western blot and ELISA.Results The target gene fragment at a length of 904 bp was amplified.Both restriction analysis and sequencing result proved that recombinant plasmid pET 28a-293 was constructed correctly.SDS-PAGE showed that the expressed product contained 66.15% of total somatic protein,and its relative molecular mass was consistent with that expected.Western blot showed good reactogenicity of expressed protein.ELISA showed specific reactions of expressed protein with both HEV positive swine serum and standard HEV positive serum.Conclusion The prokaryotic expression vector for swine hepatitis E vinus capsid protein was saccessfully constructed,and the target protein was expressed,which laid a foundation of further derelopment of diagnostic kit for swine hepatitis E.