大肠杆菌O157VT2 B亚基基因的克隆与表达

Cloning and expression of the VT2 B subunit gene of Escherchia coli O157

目的对大肠杆菌O157VT2毒素B亚基基因进行克隆、表达与鉴定,为VT2毒素的抗原、抗体大规模制备及疾病的预防、诊断和疫苗研究打下基础。方法设计寡核苷酸引物,利用PCR技术从出血性大肠杆菌O157的染色体基因组中扩增出VT2毒素B亚基基因,连接到表达载体pET-28a以构建重组质粒。将重组质粒转入表达宿主菌BL21感受态细胞,通过IPTG诱导进行表达,对表达蛋白进行SDSPAGE电泳分析和Western检测。结果用PCR方法扩增出了预期大小的VT2B亚基基因,克隆得到重组质粒pET28aVT2B,转化后宿主菌成功表达分子量为8kD的目的蛋白,用特异抗体经Western检测该条带为阳性反应。结论目的基因成功克隆到宿主菌内,目的蛋白得到高效表达,最佳诱导时间为4h,目的蛋白表达量可达总蛋白量45.72%。

In order to provide for the development of a detection method for entero-hemorrhagic E.coli (EHEC) O157 and preparation of the antigens and antibodies of the VT2B subunit on a large scale as well as for the preparation of vaccines, a pair of primers was designed based on the sequences of the VT2B subunit gene of E.coli O157, and the primers were ended with Nco I and Xho I restriction endonuclease sites at the 5' ends. By using the PCR technique, the VT2B subunit gene was amplified from genomic DNA of E.coli O157, and the PCR product was ligated to the pET28a vector. The recombinant plasmid pET28a-VT2B was then transferred into the host strain BL21 (DE3), and the transformed host strain was induced via IPTG.The expressed proteins were detected by SDS-PAGE and Western immunoblotting. It was found that the expected size of the VT2B gene was amplified by PCR, and the PCR product was cloned. A recombinant plasmid pET28a-VT2B was thus produced, and after transformation, the host strain of bacteria could express successfully the target protein with a molecular size of 8 kDa as demonstrated by SDS-PAGE and Western immunoblotting. The target protein was expressed at high level, accounting for 45.72% of total proteins of BL 21(DE3) after induction with IPTG for 4 hours.