布鲁氏菌L7/L12和GroES真核表达载体的构建及免疫效果分析  

The Construction and Immune Effect Analysis of The Eukaryotic Expression Vector of L7/L12 and GroES Brucella

作　　者：周诗淼 于秀剑 冀梦瑶 肖丽荣 张志强 史秋梅[1] 吴同垒[1] ZHOU Shimiao;YU Xiujian;JI Mengyao;XIAO Lirong;ZHANG Zhiqiang;SHI Qiumei;WU Tonglei(College of Animal Science and Technology, Hebei Normal University of Science &Technology,Key Laboratory of Preventive Veterinary Medicine in Hebei Province, Qinhuangdao Hebei,066600,China)

机构地区：[1]河北科技师范学院动物科技学院河北省预防兽医学重点试验室,河北秦皇岛066600

出　　处：《河北科技师范学院学报》2022年第1期32-37,共6页Journal of Hebei Normal University of Science & Technology

基　　金：国家自然基金项目(项目编号:31902310);秦皇岛市科学技术研究与发展计划项目(项目编号:201803B006);河北省重点研发计划项目(项目编号:19226628D,19226629D);河北省二期农业产业技术体系创新团队肉牛防控岗位项目(项目编号:HBCT2018130203-WSZ)。

摘　　要：利用Overlap PCR方法连接基因L7/L12和GroES,分别利用pET32a和pcDNA3.1构建原核和真核表达质粒,分别命名为pET32a-LG和pcDNA3.1-LG。原核表达并纯化的重组蛋白免疫小鼠,制备多克隆抗体,用于验证真核表达载体目的基因的表达;重组真核表达质粒pcDNA3.1-LG转染293T细胞,经间接免疫荧光和Western blot验证蛋白成功表达。真核质粒免疫小鼠后,利用ELISA方法检测血清抗体水平,淋巴细胞转化试验检测细胞免疫水平,结果显示,该重组质粒具有良好的免疫效果。The overlap PCR was used to connect L7/L12 and GroES,and prokaryotic and eukaryotic expression plasmids were constructed with pET32a and pcDNA3.1,respectively,which were named pET32a-LG and pcDNA3.1-LG.After prokaryotic expression,the purified recombinant protein was used to immunize mice,and polyclonal antibodies were prepared to verify the expression of the target gene of eukaryotic expression vector.The recombinant eukaryotic expression plasmid pcDNA3.1-LG was transfected into 293T cells,and the protein expression was verified by indirect immunofluorescence and Western blot.After immunizing mice with eukaryotic plasmid,the serum antibody level was detected by ELISA,and the cellular immunity level was detected by lymphocyte transformation test.The results showed that the recombinant plasmid had a good immune effect against Brucellosis.

关 键 词：布鲁氏菌 L7/L12基因 GroES基因 真核表达 

分 类 号：S852.61[农业科学—基础兽医学]