微小脲原体UP3-00235基因原核载体的构建表达和生物信息学分析  

Construction and expression of a prokaryotic vector of the Ureaplasma parvum UP3—00235 gene

作　　者：张荣荣 贾泽玮 李雪燕 王鑫 贾晓晖[1,2] 张雪飞 贾天军[1,2] ZHANG Rong-rong;JIA Ze-wei;LI Xue-yan;WANG Xin;JIA Xiao-hui;ZHANG Xue-fei;JIA Tian-jun(College of Medical Laboratory Science,Hebei North University,Hebei Zhangjiakou China 075000;Key Laboratory of Clinical Laboratory Diagnosis,Hebei North University)

机构地区：[1]河北北方学院医学检验学院,河北张家口O75OOO [2]河北北方学院临床检验诊断学重点实验室

出　　处：《中国病原生物学杂志》2020年第2期183-188,共6页Journal of Pathogen Biology

基　　金：省级大学生创新创业训练计划项目(No.2017023)。

摘　　要：目的构建微小脲原体UP3-00235基因的原核表达载体,并对其编码蛋白质进行生物信息分析。方法利用Primer Premier 5.0软件设计引物,利用PCR方法克隆UP3-00235基因(片段大小为483bp)。使用限制性核酸内切酶双酶切UP3-00235基因和pGEX-6p-2载体质粒,室温下用T4连接酶连接2.5h,连接后转入大肠埃希菌感受态中,IPTG诱导GST-UP3-00235蛋白的表达,并对其进行生物信息学分析。结果成功构建了pGEX-6p-2-UP3-00235重组质粒,测序后与NCBI中已录入的Ureaplasma parvum strain hebnu uu3序列进行比对,同源性100%。重组质粒转染大肠埃希菌后经IPTG诱导3h,表达出GST-UP3-00235融合蛋白,与预期大致相符。经生物信息学软件分析,UP3-00235基因编码蛋白质含有160个氨基酸,分子式为C823H1339N241O251S3,相对分子质量为18.73×103,等电点(PI)为9.43。二级结构中含有α-螺旋、β-折叠、无规则卷曲,分别占59.38%、17.50%和23.12%。无β-转角和跨膜区域,但含有信号肽区域。高级结构分析显示,UP3-00235基因编码蛋白分子与蛋白库中3r3t.1蛋白的B链相似性高,为36.56%;结构及功能预测显示该蛋白为UU-554,rpS6,与其相邻的蛋白分别为rpS18和rpL9。结论成功构建了GST-UP3-00235原核表达载体,并诱导表达45×103的目的蛋白,生物信息学预测该蛋白含有信号肽,属于核蛋白家族,为进一步研究微小脲原体UP3-00235基因的功能奠定了基础。Objective To construct a prokaryotic expression vector for the UP3-00235 gene in Ureaplasma parvum and to bioinformatically analyze the protein it encodes. Methods In accordance with the UP3-00235 gene sequence of U.parvumpublished in NCBI,specific primers were predicted using Primer Premier 5.0,and the UP3-00235 gene(483 bp)was cloned using PCR.The target gene and pGEX-6 p-2 vector were digested by the restriction endonucleases BamH I and Not I.The vector was ligated with T4 ligase at room temperature,and the recombinant plasmid was transformed into Escherichia coli XL1-Blue via the heat shock method.The transformed bacteria were inoculated on LB solid medium with ampicillin and cultured overnight,and the recombinant vector was identified with colony PCR.Protein expression was induced with IPTG.The protein was analyzed using bioinformatic software. Results The UP3-00235 gene was cloned with PCR,and the product of amplification was 510 bp in length.The recombinant plasmid pGEX-6 p-2-UP3-00235 was identified using universal primers,and the target fragment was 626 bp in length.After sequencing,the recombinant plasmid sequence of pGEX-6 p-2-UP3-00235 was 100%similar to the U.parvumstrain hebnu uu3 sequence registered in NCBI.Expression of the recombinant plasmid transfected into E.coli was induced with IPTG for 3 h to express the fusion protein GST-UP3-00235,which was roughly in line with expectations.Bioinformatic software indicated the following:the protein coded for by the UP3-00235 gene had 160 amino acids,its molecular formula was C823H1339N241O251S3,its relative molecular weight was 18.73×103,its aliphatic amino acid index was 82.94,its isoelectric point(pI)was 9.43,its molar extinction coefficient was 14,900,and its instability index was 36.67.Its total average hydrophilicity was-0.832,so the protein was predicted to be hydrophobic.The protein’s secondary structure consisted of α-helices(59.38%),β-folds(17.50%),and irregular curls(23.12%)but noβ-turns.The protein had no transmembrane regions but did con

关 键 词：微小脲原体 UP3-00235 原核表达 生物信息学分析 

分 类 号：R37[医药卫生—病原生物学]