猪唾液酸黏附素氨基端结构域基因的克隆表达及其多克隆抗体制备

Cloning and Expression of the Amino Structure Domain Gene of Sialoadhesin from Swine and Preparation of Polyclonal Antibody against it

本试验旨在克隆出猪唾液酸黏附素的近氨基端结构域基因,进而构建原核表达载体并诱导表达重组蛋白,以制备多克隆抗体。试验中应用RT-PCR技术从健康仔猪肺巨噬细胞中克隆出猪唾液酸黏附素的近氨基端结构域的cDNA序列,并将其亚克隆到原核表达载体pET-32a中,构建重组表达质粒pET-Sn150,成功表达了分子质量大小约为43.1ku的γ亚单位重组蛋白。将重组蛋白pET-Sn150免疫小鼠,制备获得鼠抗pET-Sn150重组蛋白多克隆抗体,将得到的重组蛋白多克隆抗体采用间接ELISA和Western blotting试验方法检测,ELISA测定多克隆抗体的血清效价为1∶12800;Western blotting试验结果证明,制备的鼠抗pET-Sn150多克隆抗体可以与重组蛋白进行特异性结合,从而证明重组蛋白具有较好的免疫原性。本试验克隆出猪唾液酸黏附素的近氨基端结构域基因并成功表达,为进一步研究其结构与功能奠定了基础。

The pig sialoadhesin near the amino structure domain cDNA sequence which was cloned from the 90-day age lung health piglets macrophages with RT-PCR was subcloned into a prokaryotic expression vector pET-32a,to construct a recombinant expression plasmid pET-Sn150 which was successfully used to express a gamma recombinant protein subunits whose molecular weight was about 43 ku.By using the recombinant protein pET-Sn150 to immune the mouse,the mice sialoadhesin resistance gamma-irradiation His recombinant protein polyclonal antibody which could be tested by Western blotting and indirect ELISA could be achieved.And the result of indirect ELISA showed that the titer of the polyclonal antibody was 1∶12800.The specific binding of the mice sialoadhesin resistance gamma-irradiation His recombinant protein polyclonal antibody with the restructuring γ and unit protein revealed by the result of Western blotting proved that the recombinant protein had good immunogenicity,and that layed the foundation for a further study of its construction and function as the pig sialoadhesin near the amino structure domain gene was successfully cloned and expressed.