猪肺炎支原体P216基因片段的表达及黏附活性研究  

Expression of P216 Gene Fragment from Mycoplasma hyopneumoniae and Research on Adhesion Activity

作　　者：杜海霞[1,2] 刘茂军[1,2] 冯志新[1] 熊祺琰[1] 白方方[1] 王海燕[1] 邵国青[1] 

机构地区：[1]江苏省农业科学院兽医研究所农业部兽用生物制品工程技术重点实验室国家兽用生物制品工程技术研究中心,南京210014 [2]南京农业大学动物医学院,南京210095

出　　处：《畜牧兽医学报》2012年第8期1324-1329,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：农业科技成果转化资金(2009GB2C100129);江苏省农业科技自主创新资金(cx(10)215)

摘　　要：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P216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l 636bp;SDS-PAGE检测重组蛋白相对分子质量为80.1ku;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。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 l 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 immunogenicity.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 research of the other adhesions from Mycoplasma hyopneumoniae.

关 键 词：猪肺炎支原体 P216基因片段 黏附活性 

分 类 号：S852.62[农业科学—基础兽医学]