布鲁氏菌16M BtpA和BtpB真核表达载体的构建及在293T细胞中的表达  

Construction of Brucella 16M BtpA and BtpB Eukaryotic Expression Vectors and Its Expression in 293T Cells

作　　者：乔连江 杨森 张萍 杨艳玲[1] QIAO Lian-jiang;YANG Sen;ZHANG Ping;YANG Yan-ling(Key Laboratory of Special Animal Epidemic Disease,Ministry of Agricultural,Institute of Special Economic Animal and Plant Sciences,Chinese Academy of Agricultural Sciences,Changchun,Jilin,130122,China)

机构地区：[1]中国农业科学院特产研究所农业部经济动物疫病重点实验室,吉林长春130122

出　　处：《动物医学进展》2021年第7期12-17,共6页Progress In Veterinary Medicine

基　　金：吉林省科技厅重点研发计划资助项目(2016YFD0500907);“十三五”国家重大科研专项(2016YFD0500900)。

摘　　要：为了构建布鲁氏菌16M BtpA和BtpB真核表达载体,便于深入研究其在布鲁氏菌逃逸宿主免疫机制中发挥的作用。根据GenBank公布的布鲁氏菌16M BtpA和BtpB序列设计了特异性引物,并提取布鲁氏菌16M总RNA,将其逆转录成cDNA为模板,进行了PCR扩增,获得BtpA和BtpB目的片段,连接至pMD18-T进行克隆纯化,再与pcDNA3.1载体进行连接,经PCR、酶切和测序分析等方法鉴定后,利用Lipofectamine TM 2000脂质体转染至293T细胞,Western blot检测BtpA和BtpB蛋白的表达。结果显示,PCR扩增出了873 bp的BtpA基因片段和924 bp的BtpB基因片段,依次连至克隆载体pMD18-T和真核表达载体pcDNA3.1,经限制酶Eco RⅠ和XbaⅠ酶切验证正确,测序分析显示与GenBank公布的序列信息完全一致;Western blot检测结果显示,pcDNA3.1-BtpA-HA和pcDNA3.1-BtpB-HA分别在32 ku和35 ku处出现了条带,与预期结果完全相符,说明能在293T细胞中表达。成功构建了pcDNA3.1-BtpA-HA和pcDNA3.1-BtpB-HA真核表达载体,并证实了其能在293T细胞中表达,为后续研究其作用与机制提供了材料。To construct Brucella abortus 16M BtpA and BtpB eukaryotic expression vectors to facilitate an in-depth study of their role in the mechanism of Brucella abortus escape from host immunity,in this experiment,specific primers were designed based on the sequence information of Brucella 16M BtpA and BtpB published by GenBank,and the total RNA of Brucella 16M was extracted,which was reversely transcribed into cDNA as a template,and PCR amplification was performed to obtain BtpA and BtpB.The target fragment was ligated into PMD18T for cloning and purification,and then ligated with pcDNA3.1 vector.After identification by PCR,enzyme digestion and sequencing analysis,Lipofectamine 2000 TM liposome was used to transfect into 293T cells,and BtpA and BtpA protein expression were detected by Western blot.The results showed that PCR amplified 873 bp BtpA gene fragment and 924 bp BtpB gene fragment,which were connected to the cloning vector PMD18T and eukaryotic expression vector pcDNA3.1 in sequence,verified by restriction enzyme Eco RⅠ and Xba Ⅰ digestion,sequencing analysis showed that the sequence information published by GenBank is completely consistent,indicating that pcDNA3.1-BtpA-HA and pcDNA3.1-BtpB-HA eukaryotic expression vectors were successfully constructed.Western blot detection results showed that pcDNA3.1-BtpA-HA and pcDNA3.1- BtpB-HA showed bands at 32ku and 35ku respectively,which is completely consistent with the expected results,indicating that it can be expressed in 293T cells.In this study,pcDNA3.1-BtpA-HA and pcDNA3.1-BtpB-HA eukaryotic expression vectors were successfully constructed,and it was confirmed that they can be expressed in 293T cells.The result provided materials for subsequent studies of their effects and mechanisms.

关 键 词：布鲁氏菌 BtpA和BtpB基因 真核表达 293T细胞 

分 类 号：S852.614[农业科学—基础兽医学]