金葡菌多重耐药基因norA的原核表达及其抗体制备

Prokaryotic expression of the multi-drug resistant norA gene of Staphylococcus aureus and its antibody preparation

按金葡菌多重耐药转运蛋白N orA的编码序列设计引物,以金葡菌基因组DNA为模板,扩增出基因norA中约1155bp的cDNA片段,将所得片段与pM D 18-T载体连接,转化到感受态大肠埃希菌JM 109中,成功地筛选到阳性克隆,其质粒测序结果与文献报道一致。从阳性克隆中提取质粒,经N d eⅠ和X hoⅠ酶切,回收1155bp的目的片段,定向克隆到pET-28a(+)表达载体中,转化至感受态大肠埃希菌DH 5α中,提取质粒,再次转化到BL 21(DE 3)中,成功筛选到阳性克隆。经IPTG诱导阳性菌,通过SDS-PAGE检测出norA部分基因的表达。利用得到的纯化蛋白免疫家兔,并通过间接EL ISA法检测抗体效价,证明得到相应抗体。

A construction of norA-pMD18-T was generated by inserting the 1155bp fragment cDNA obtained by PCR into pMD18-T vector and selecting the positive clones. It was showed that the cloned sequence coincided with the reported sequence in other document. The norA-pMD18-T and pET-28a （＋） were digested with same enzymes （NdeⅠ and Xho Ⅰ ）, and fragments were ligated with T4 DNA ligase, further, the plasmid norA-pET-28a （＋） was transformed to competent cell BL21 （DE3）. Positive bacteria were induced with IPTG. The expression production of norA gene was observed on SDS-PAGE. The purified protein was obtained and immuned to rabbits. The antibody blood serum was detected by ELISA. It was concluded that the Nora antibody was obtained.