猪肺炎支原体P216基因片段的表达及黏附活性研究

摘要

This experiment was conducted to study the adhesion activity of P216 protein and establish the model of adhesion protein of Mycoplasma hyopneumoniae (Mhp). According to analysis, the fragment of P216 gene with hydrophilicity, antigenicity and good adhesion was chose. P216 gene fragment was amplified by PCR from Mhp NJ strain and inserted into expression vector pET-32a( + ) , and the recombinant plasmid pET-32a( + )/P216 was constructed. After IPTG induction, the immunological and adhesion activity of the recombined protein was detected by Western blot and indirect immunofluorescence assay. The results showed that, the PCR product of the target gene was 1 636 bp, the molecular weight of recombinant protein was 80. 1 kDa by SDS-PAGE, and Western blot results showed that recombinant protein had satisfactory immuno-genicity. Indirect immunofluorescence assay showed that the recombinant protein could produce occupied inhibition to the Mhp adhering with SJPL cells. These results indicated that the P216 protein had good adhesion activity, and could adhere SJPL cells. It provides new ideas for re-search of the other adhesions from Mycoplasma hyopneumoniae.%本研究旨在研究猪肺炎支原体P216蛋白的黏附活性并初步建立猪肺炎支原体蛋白黏附模型.根据软件分析和文献报道从P216全基因中选取亲水性、抗原性、黏附性较好的目的片段,利用PCR从Mhp NJ株扩增P 216基因片段,克隆到表达载体pET-32a(+)中,获得重组质粒pET-32a(+ )/P216,经IPTG诱导获得重组蛋白,通过Western blot和间接免疫荧光试验检测重组蛋白的免疫原性及黏附活性.结果表明,PCR扩增的目的基因大小为1 636 bp;SDS-PAGE检测重组蛋白相对分子质量为80.1 ku;Western blot检测表明重组蛋白能与Mhp阳性血清发生特异性反应；间接免疫荧光试验表明该蛋白对猪肺炎支原体黏附SJPL细胞产生占位抑制作用.结果提示,P216蛋白具有良好的黏附活性,能黏附SJPL细胞,从而为猪肺炎支原体其他黏附因子的研究奠定基础.