Secretory Expression of Mycoplasma hyopneu-moniae P97R1 Gene in Pichia pastoris and Primary Application of the Expression Product

作     者：刘茂军 祝永琴 冯志新 吴叙苏 邵国青 Maojun LIU;Yongqin ZHU;Zhixin FENG;Xusu WU;Guoqing SHAO

作者机构：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江苏南京210014 

出 版 物：《Agricultural Science & Technology》 (农业科学与技术（英文版）)

年 卷 期：2013年第14卷第5期

页      面：710-715页

学科分类：090601[农学-基础兽医学] 09[农学] 0906[农学-兽医学] 

基　　金：Supported by the National Natural Science Foundation of China(31100136) the Special Fund for Independent Innovation of Agricultural Science and Technology in Jiangsu Province[CX(12)5051] 

主　　题：Mycoplasma hyopneumoniae P97R1 Pichia pastoris Indirect ELISA method 

摘      要：[Objective] This study aimed to investigate the secretory expression of P97R1 gene of Mycoplasma hyopneumoniae（Mhp） in Pichia pastoris expression system and the primary application; of the expression product. [Method] A pair of specific primers was designed to conduct PCR according to the Mhp P97R1 gene sequence in Genbank, and the amplified P97R1 gene was cloned into the pPICZa-A yeast expression vector to construct the secretory recombinant expression vector pPICZa-A-P97R1. The plasmid pPICZa-A-p97R1 linearized by Sac I was transformed into P. pastoris GSl15 by electroporation. Positive transformant identified by PCR was incubated to express P97R1 protein after methanol induction. And the expression product was identified using SDS-PAGE and Western-blotting ***. [Result] P97R1 protein was successfully expressed in the P. pastoris system, with a secre- tory amount of 499μg/ml, and revealed good reactogenicity. Meanwhile, an indirect ELISA method was established with P97R1 protein after the optimization of each reaction factor, which showed good specificity and repeatability according to repeated tests. [Conclusion] This study provides bases for developing the ELISA Kit for anti- body detection and genetically engineered vaccine to Mhp.