【摘要】 以猪水泡病病毒RNA为模板,应用反转录聚合酶链式反应(RT-PCR)技术,扩增了849bp的VP1基因,通过T-A克隆技术,将VP1基因片段克隆至pMD18-T克隆载体质粒中,构建SVDVVP1基因克隆重组质粒,进行核苷酸序列分析。然后亚克隆插入pBAD/ThioTOPO表达载体,经测序鉴定,筛选获得VP1基因正向插入、有正确读码框的阳性克隆,成功构建了猪水泡病病毒VP1基因重组表达载体。经L-Arabinose诱导表达,可稳定、高效地表达VP1蛋白抗原。SDS-PAGE结果表明,以终浓度为0.002%的L-阿拉伯醛糖进行诱导,5h后表达可达到高峰,表达蛋白为融合蛋白,质量约47.13kDa,表达产量约占菌体总蛋白的16%。Westernblotting检测表明,诱导的蛋白能与猪水泡病阳性血清发生特异性反应。融合蛋白中含有猪水泡病病毒VP1蛋白抗原,为应用该表达蛋白抗原制备SVD免疫血清学诊断试剂和新型疫苗构建奠定基础。更多还原

【Abstract】 The VP1 gene of Swine vesicular disease virus(SVDV)was amplified by reverse transcription-polymerase chain reaction(RT-PCR)yielding a product of 849bp cDNA fragment.Using T-A cloning technique,the PCR product was cloned into pMD18-T vector.The purified VP1 gene was subcloned into the pBAD/Thio TOPO vector and the plasmid was identified by PCR.It was sequenced to confirm the authenticity of the sequence and orientations.SDS-PAGE and Western blotting revealed that the VP1 protein was expressed in Escherichia coli LGM194 at a high level and the recombinant fusion protein contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag.The optimal amount of the expressed fusion protein was 16% of total bacterial protein after being induced with L-arabinose at 0.002%concentration for 5 hours.It had a molecular mass of approximately 47.13 kDa and was immunologically reactive.The recombinant protein will be characterized and tested in an enzyme-linked immunosorbent assay(ELISA)format for potential application in the serodiagnosis of swine vesicular disease.更多还原