伪结核棒状杆菌pld和cp40基因的克隆、序列分析及原核表达  

Cloning,Sequence Analysis and Prokaryotic Expressions of pld and cp40 Genes of Corynebacterium pseudotuberculosis

作　　者：尹峥 刘刚[1] 王理想 金剑 苏红 付明哲[1] 张琪[1] 许信刚[1] YIN Zheng;LIU Gang;WANG Li-xiang;JIN Jian;SU Hong;FU Ming-zhe;ZHANG Qi;XU Xin-gang(College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi,712100,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100

出　　处：《动物医学进展》2020年第10期25-29,共5页Progress In Veterinary Medicine

基　　金：2018年陕西省农业专项资金支持科技创新驱动项目(K3360218035);国家绒毛用羊产业技术体系项目(CARS-39-14);陕西省重点研发计划重点项目(2018ZDXM-NY-039)。

摘　　要：为了解伪结核棒状杆菌(Corynebacterium pseudotuberculosis,CP)陕西分离株的pld和cp40基因的遗传变异情况及制备检测抗原,利用GenBank收录CP的pld(CP001829.1)和cp40(KY041980.1)基因序列,设计合成特异性PCR引物,分别对pld和cp40基因进行扩增、序列分析和原核表达。结果表明,成功克隆了CP陕西分离株的pld和cp40全基因序列。核苷酸序列分析表明,陕西分离株pld基因与国内外已报道的伪结核棒状杆菌核苷酸同源性在98%~100%之间,cp40基因核苷酸同源性在91%~99%之间。pld和cp40基因重组原核表达菌株经IPTG诱导后可分别表达约为33 ku和41 ku的重组融合蛋白,重组蛋白均能与CP的阳性血清特异性结合。试验结果可为CP陕西分离株分子生物学特性研究提供资料,为进一步研制CP抗体ELISA检测试剂盒提供基础。In order to investigate the genetic variation of pld and cp40 genes of Corynebacterium pseudotuberculosis(CP)isolates from Shaanxi province,and to prepare and detecting antigens,the synthetic specific PCR primers were designed by using the pld(CP001829.1)and cp40(KY041980.1)gene sequences from GenBank,and the pld and cp40 genes were amplified,sequenced and prokaryotically expressed respectively.The results showed that the pld and cp40 whole-gene sequences of CP-Shaanxi isolate were successfully cloned,and the nucleotide sequence analysis indicated that the homology of the pld gene of Shaanxi isolate and the reported strains at home and abroad was between 98%and 100%,and that of the cp40 gene was between 91%and 99%.The recombinant prokaryotic expressions of pld and cp40 genes were induced by IPTG to express recombinant fusion proteins of about 33 ku and 41 ku,respectively.The recombinant proteins could specifically bind to the positive serum of CP.This test provided information for the study of molecular biological characteristics of CP Shaanxi isolate and further development of CP antibody ELISA detection kit.

关 键 词：伪结核棒状杆菌 PLD cp40 序列分析 原核表达 

分 类 号：S826.9[农业科学—畜牧学] S852.61[农业科学—畜牧兽医]