猪源戊型肝炎病毒基因IV型ORF3编码蛋白的表达及鉴定

通过PCR从已构建的猪源戊型肝炎病毒全基因克隆扩增ORF3全基因，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-ORF3表达载体，转入E.coli BL21 (DE3)，IPTG诱导表达。Ni-NTA层析柱纯化表达蛋白，用SDS-PAGE、免疫印迹、ELISA等方法分析鉴定表达产物。结果成功扩增到345 bp的目的基因；构建了重组表达载体pET28a-ORF3；转化宿主菌E.coli BL21 (DE3)后表达产物的相对分子质量在6.50~16.5 kDa之间，与预期表达的目的蛋白相对分子质量相符；表达的目的蛋白能与阳性猪源和人源血清发生特异性反应,证实其具有较好的反应原性。

Prokaryotic Expression and Identification of Protein Encoded by Open Reading Frame 3 of Genotype IV Swine Hepatitis E Virus

The ORF3 gene from a full-length swine HEV genomic clone was amplified by polymerase chain reaction(PCR) and inserted into vector pMD18-T, then subcloned to prokaryotic expression vector pET28a(+). The recombinant plasmid pET28a-ORF3 was transformed to E.coli BL21 (DE3) for expression under induction of IPTG. The expressed product was identified by SDS-PAGE, Western blot, then purified by Ni-NTA affinity chromatography and used as an antigen for the determination of HEV antibody in swine and human serum by ELISA. It indicated that the target gene was amplified by PCR. The results of restriction and DNA sequencing confirmed that the prokaryotic expression vector pET28a-ORF3 was constructed correctly. The expressed product, with a relative molecular weight 6 500~16 500, was consistent with the designed. Western blot showed specific reaction of the expressed fusion protein with mouse anti-His Tag serum. ELISA indicated that the expressed fusion protein can reacted with the HEV positive swine and human serum. The expressed product showed good reactionogenicity, which laid a foundation of developing a swine HEV diagnostic reagent.