奶牛黏附分子MadCam-1基因的克隆及原核表达

原核表达MadCam-1基因、纯化MadCam-1黏附蛋白,并制备其多克隆抗体,为进一步功能研究奠定基础。根据MadCam-1的已知基因序列设计并合成1对引物,应用PCR技术扩增MadCam-1基因片段插入到原核表达载体 pGEX-4T-1中,构建重组表达质粒 pGEX-4T-1-MadCam-1,转化入E.coliBL21(DE3),经IPTG诱导,对其产物进行SDS-PAGE分析和Western blotting鉴定,以纯化后的融合蛋白为抗原,免疫家兔,获得抗血清。结果表明,重组表达质粒pGEX-4T-1-MadCam-1经PCR及双酶切鉴定证明构建正确,测序结果与GenBank中登录的基因序列同源性为100%。表达产物经Western blotting鉴定,结果证实成功表达了分子质量约为50 ku 的蛋白质。纯化的蛋白质免疫家兔后,经ELISA检测多克隆抗体效价为1∶32000。因此,成功获得了奶牛 MadCam-1多克隆抗体,为进一步研究奶牛黏附分子 MadCam-1的功能奠定了基础。

Cloning and Prokaryotic Expression of MadCam-1 Gene from the Dairy Cows

To construct expression vertor of MadCam-1 gene, to expression and purify recombination protein and to prepare polyclonal antibody, which have laid a foundation of studying its function. A pair of primers was designed according to the sequence of MadCam-1 from GenBank. The MadCam-1 gene was amplified by PCR. The expression vector pGEX-4T-1-MadCam-1 was reconstructed and transformed into E.coli BL21,then it was induced by IPTG.The expressed product was identified by SDS-PAGE and Western blotting. Rabbits were immunized with the purified MadCam-1 to generate antisera. The results showed that the homology of 100% of the cloned MadCam-1 gene to that reported in GenBank. The recombinant plasmid pGEX-4T-1-MadCam-1 was constructed successfully. The expression product was identified by Western blotting. The results showed that the molecular weight of recombinant protein pGEX-4T-1-MadCam-1 was 50 ku.Then rabbits were injected with purified protein to induce immunoreactions and the potency of the antibody was as high as 1∶32000. Therefore,the polyclonal antibody of MadCam-1 was induced.The results of this study lay a foundation for further study of the function of dairy cows MadCam-1.