猪肺炎支原体P46基因的原核表达与间接ELISA方法的建立  

Expression of P46 Protein of Mycoplasma hyopneumoniae in Escherichia coli and Used for Indirect ELISA

作　　者：沈青春[1,2] 王芳[2] 韩明远[2] 覃青松[2] 范学政[2] 杨汉春[1] 宁宜宝[2] 

机构地区：[1]中国农业大学农业部人畜共患病重点开放实验室,北京100193 [2]中国兽医药品监察所,北京100081

出　　处：《畜牧兽医学报》2012年第3期431-437,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

摘　　要：猪肺炎支原体是猪喘气病的病原体,本研究选择猪肺炎支原体P46膜蛋白基因亲水区序列进行克隆,并将其内3个编码Trp的TGA突变成TGG,然后再克隆到pET28a(+)载体中,在大肠杆菌BL21(DE3)细胞内实现了高效表达,表达产物相对分子质量约为31ku,约占菌体总蛋白35%,表达形式为包涵体,通过Western blotting证明表达产物与猪肺炎支原体高免血清具有很好的反应原性和特异性。将大肠杆菌表达的猪肺炎支原体P46重组蛋白经过洗涤、过柱纯化后,作为间接ELISA包被抗原用于检测猪血清中猪肺炎支原体抗体,通过对各参数和试剂的优化建立了rP46-ELISA方法,获得了较好的效果,通过与现有ELISA检测方法的比较,结果表明二者间具有较高的符合率。Mycoplasrna hyopneumoniae is the agent of Enzootic pneumonia of swine. Part of the hydrophilic region of the surface protein P46 of M. hyopneumoniae was cloned, and three TGA coding Trp in the ORF of P46 gene were point mutated to TGG. The modified DNA fragement was coloned to vector pET28a（＋）, and high-efficiency expressed in E. coli BL21 （DE3）. SDS- PAGE showed that the target DNA was expressed in inclusion body, the molecular weight of the recombinant protein was 31 kDa, owed 35~//oo of the total proteins. The recombinant protein was proved to be well reacted with the high immune serum against M. hyopneumoniae from rabbit specially by western blotting. After wash and purification with Ni2＋ column, the recombinant protein was used as coating antigen of indirect ELISA for detection the antibody against M. hyo- pneumoniae in swine serum. The ELISA detection kid was developed by optimizing the relational parameters and reagents. The further compare detection test indicated the recombinant P46 pro- tein based ELISA was satisfactory and high complicated with the marketable products.

关 键 词：猪肺炎支原体 P46蛋白 原核表达 ELISA 

分 类 号：S852.62[农业科学—基础兽医学]