猪链球菌2型mrp基因ORF1序列的克隆及原核表达

Cloning and prokaryotic expression of ORF1 sequence of mrp gene of Streptococcus suis type 2

根据猪链球菌2型溶菌酶释放蛋白基因(mrp)的序列,设计并合成了1对特异性引物,以青海株的基因组DNA为模板扩增了mrp基因ORF1序列。将PCR产物进行了T/A克隆,转化大肠杆菌,鉴定成功获得目的片段后,将其定向亚克隆到pET-28a(+)中,构建了原核表达质粒pET-28a-mrp,并将其转化至大肠杆菌BL21感受态细胞中,经1 mmol/L IPTG诱导和SDS-PAGE分析,出现了与预期目的蛋白一致的外源蛋白带(27.0 ku)。Western-blot分析表明,该融合蛋白具有MRP的抗原表位。研究结果为今后开展该病的免疫学研究奠定了一定基础。

The ORF1 sequence of gene mrp encoding muramidase-released protein （MRP） was amplified from genomic DNA of Streptococcus suis type 2 Qinghai strain by PCR with specific primers. The PCR product was cloned into pMD18-T vector and transformed into host strain Escherichia coli, and the plasmid of positive clone was extracted, digested with enzymes and purified. Then the fragment was sub-cloned into expression vector pET-28a（＋） and the prokaryotic expression vector pET-28a-mrp was constructed. The recombinant plasmid was transformed into E. coli BL21 competent cells and then induced by 1 mmol/L IPTG. SDS-PAGE analysis revealed that a band of approximately 27. 0 ku in molecular weight was expressed from the induced E. coli BL21 component cells. Western-blot analysis indicated that the protein possessed antigenic epitopes of MRP. The result provided foundation for immunological studies of MRP.