2型猪链球菌酪氨酸激酶Cps2C基因过表达株的构建  

Construction of Tyrosine Kinase Cps2C Overexpression Strain in Streptococcus suis serotype 2

作　　者：王悄悄 郑峰 刘旭苗 林苗 倪华 王怡雯 曹祥荣[2] 汪春晖 潘秀珍 WANG Qiao-qiao;ZHENG Feng;LIU Xu-miao;LIN miao;Ni Hua;WANG Yi-wen;CAO Xiang-rong;WANG Chun-hui;PAN Xiu-zhen(Hua Dong Research Institute for Medicine and Biotechnics,Nanjing 210002,China;College of Life Sciences,Nanjing Normal University;Key Laboratory of Biological Resources and Ecology of Pamirs Plateau in Xinjiang Uygur Autonomous Region,College of Life and Geographic Sciences,Kashi University)

机构地区：[1]东部战区疾病预防控制中心,江苏南京210002 [2]南京师范大学生命科学学院 [3]新疆帕米尔高原生物资源与生态重点实验室,喀什大学生命与地理科学学院

出　　处：《中国病原生物学杂志》2022年第7期770-773,778,共5页Journal of Pathogen Biology

基　　金：国家自然科学基金项目(No.82072256,81571965);江苏省自然科学基金项目(No.BK20201129,BK20151091)。

摘　　要：目的构建2型猪链球菌酪氨酸激酶Cps2C基因过表达株,为cps2C基因功能研究提供实验材料。方法使用重叠延伸PCR技术将延伸因子TufA(又名EF-Tu,编码基因为SSU05_0530)的启动子(命名为P0530)与cps2C基因连接为P0530cps2C片段,将P0530cps2C酶切后连接至猪链球菌-大肠埃希菌穿梭质粒pSET2,构建过表达质粒pSET2::P0530cps2C。将pSET2::P0530cps2C电转化导入S.suis 2野生毒株05ZYH33感受态细胞,抗性及组合PCR筛选得到cps2C过表达株。qRT-PCR检测过表达株cps2C基因的转录水平。结果重叠延伸PCR成功将P0530启动子片段(300 bp)和cps2C基因片段(696 bp)拼接为P0530cps2C(996 bp),片段及载体经BamH I和EcoR I酶切后连接,成功构建出过表达质粒pSET2::P0530cps2C;将pSET2::P0530cps2C电转化导入S.suis 205ZYH33感受态细胞,抗性筛选及组合PCR检测结果显示cps2C过表达株构建成功。提取野生株05ZYH33和cps2C过表达株总RNA后进行qRT-PCR实验,结果显示过表达株cps2C基因的相对表达水平较05ZYH33株上调2.4倍。结论本研究选用S.suis 205ZYH33的延伸因子TufA强启动子P0530作为启动子,成功构建过表达质粒pSET2::P0530cps2C并导入S.suis 2感受态细胞,成功获得cps2C过表达株,为进一步研究酪氨酸激酶Cps2C功能提供实验材料。Objective To construct type 2 Streptococcus suis tyrosine kinase Cps2 C gene overexpression strain and provide experimental materials for the study of Cps2 C gene function.Methods The promoter of TufA(also known as EF-TU,encoded by SSU05_0530,named P0530)was connected with cps2 C gene as P0530 cps2 C fragment by overlapping stretch PCR.P0530 cps2 C was digested by enzyme and ligated to shuttle plasmid pSET2 to construct overexpression plasmid pSET2::P0530 cps2 C.PSET2::P0530 cps2 C was electrically transformed into S.suis 205 ZYH33 strain receptor cells,and cps2 C overexpressed strain was screened by resistance and combined PCR.The transcription level of cps2 C gene was detected by qRT-PCR.Results The p0530 promoter fragment(300 bp)and cps2 C gene fragment(696 bp)were successfully spliced into p0530 cps2 C(996 bp)by overlapping extended PCR.The fragment and vector were digested by BamH I and EcoR I and ligated to construct the overexpressed plasmid pSET2::p0530 cps2 C.The pSET2::P0530 cps2 C was electrically transformed into S.suis 205 ZYH33 cells,and the results of resistance screening and combined PCR showed that cps2 C overexpressed strain was successfully constructed.Total RNA was extracted from wild strain 05 ZYH33 and cps2 C overexpressed strain,respectively.qRT-PCR results showed that the relative expression level of cps2 C gene in overexpressed strain was 2.4 times higher than that of 05 ZYH33.Conclusion In this study,the TufA promoter P0530 of S.suis 205 ZYH33 was selected as the strong promoter,and the overexpression plasmid pSET2::P0530 cps2 C was successfully constructed and cps2 C overexpression strain was successfully obtained.It provides good material support for further study of cps2 C gene function.

关 键 词：2型猪链球菌 酪氨酸激酶Cps2C 过表达株 

分 类 号：R378.12[医药卫生—病原生物学]