非洲猪瘟病毒VP73基因克隆及在大肠杆菌中的高效表达

Cloning and High Level Expression of VP73 Gene From African Swine Fever Virus in E.coli

参照Genebank中非洲猪瘟VP73基因序列,人工合成的VP73全基因克隆至pMD18-T克隆载体质粒中,采用PCR扩增得到1188bp的VP73基因;将VP73基因片段亚克隆插入pBAD/Thio表达载体,经测序鉴定,筛选获得VP73基因正向插入、有正确读码框的阳性克隆,成功构建了非洲猪瘟病毒VP73基因重组表达载体。经L-Arabinose诱导表达,可稳定、高效地表达VP73蛋白抗原。SDS-PAGE结果表明,以终浓度为0.002%的L-Arabinose进行诱导,4h后表达量最高,表达蛋白为融合蛋白,分子量约60kDa,表达产量约占菌体总蛋白的30%。Western blotting和ELISA检测表明,表达的融合蛋白能与非洲猪瘟阳性血清发生特异性反应,说明表达获得的产物为非洲猪瘟病毒VP73融合蛋白,且具有良好的反应原性,这为应用该表达蛋白抗原制备ASFV免疫血清学诊断试剂和疫苗研究奠定了基础。

According to the gene sequence of African swine fever virus （ASFV） VP73 in the gene bank , VP73 was synthesized and cloned into pMD18-T vector. The VP73 gene of African Swine Fever Virus （ASFV） was amplified from DNA by PCR, the product of which was a 1188 bp DNA segment. The purified VP73 gene was subcloned into pBAD/Thio TOPO vector. The recombinant plasmid was identified by PCR. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted VP73 gene. The recombinant protein will be purified by proBond TM protein. The results of SDS-PAGE revealed that the VP73 protein was expressed in pBAD/Thio TOPO vector in a high level . The optimal amount of the expressed fusion protein is 30% of total bacterial protein after being induced with L-arabinose at 0. 002% concentration for 4 hours. It had a molecular mass of approximately 60 kDa and immunologically reactive activity. The VP73 protein was expressed in pBAD/Thio TOPO vector in a high level . The recombinant protein purified by proBond TM protein was tested in an enzymelinked immunosorbent assay （ ELISA ）, and the result showed that the VP73 fusion protein had good reactinogenicity, which based the using of the recombinant protein to prepare ASFV serological diagnostic reagent and vaccines.