猪肺炎支原体P216基因片段的表达及黏附活性研究

畜牧兽医学报 ›› 2012, Vol. 43 ›› Issue (8): 1324-1329.

猪肺炎支原体P216基因片段的表达及黏附活性研究

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*

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

收稿日期:2012-01-06 修回日期:1900-01-01 出版日期:2012-08-24 发布日期:2012-08-24
通讯作者: 邵国青

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

DU Haixia ^1,2，LIU Maojun ^1,2，FENG Zhixin¹，XIONG Qiyan¹， BAI Fangfang¹，WANG Haiyan ¹，SHAO Guoqing^1*

1. Key Laboratory of Veterinary Biological Engineering and Technology Ministry of Agriculture National Center for Engineering Research of Veterinary Bioproducts, Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2. College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China

Received:2012-01-06 Revised:1900-01-01 Online:2012-08-24 Published:2012-08-24
Contact: SHAO Guoqing

摘要/Abstract

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 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。

关键词: 猪肺炎支原体, P216基因片段, 黏附活性

Abstract: 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.

Key words: Mycoplasma hyopneumoniae, P216 gene fragment, adhesion

杜海霞;刘茂军;冯志新;熊祺琰;白方方;王海燕;邵国青. 猪肺炎支原体P216基因片段的表达及黏附活性研究[J]. 畜牧兽医学报, 2012, 43(8): 1324-1329.

DU Haixia;LIU Maojun;FENG Zhixin;XIONG Qiyan;BAI Fangfang;WANG Haiyan;SHAO Guoqing . Expression of P216 Gene Fragment from Mycoplasma hyopneumoniae and Research on Adhesion Activity[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2012, 43(8): 1324-1329.

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