微小脲原体UP3-00750基因编码蛋白的表达及生物信息学分析  

Expression and bioinformatics analysis of UP3-00750 gene encoding of Ureaplasma parvum

作　　者：刘燕[1] 贾泽玮 白一童 贾晓晖[1] 周艳[1] 李萍[1] LIU Yan;JIA Ze-wei;BAI Yi-tong;JIA Xiao-hui;ZHOU Yan;LIPing(Key Laboratory of Clinical Laboratory and Diagnostics,Hebei North University,Zhangjiakou,075000,Hebei,China;Department of Clinical Laboratory,the Second Affiliated Hospital of Hebei North University)

机构地区：[1]河北北方学院临床检验诊断学重点实验室,河北张家口075000 [2]河北北方学院附属第二医院检验科

出　　处：《中国病原生物学杂志》2022年第7期751-756,共6页Journal of Pathogen Biology

摘　　要：目的构建微小脲原体UP3-00750基因的原核表达载体,对其编码的蛋白进行表达并进行生物信息学分析。方法利用Primer Premier 5.0软件设计引物,利用PCR方法扩增UP3-00750基因,经限制性内切酶双酶切后与同酶消化的pGEX-6p2于16℃在T4连接酶作用下连接16 h,连接产物转入大肠埃希菌XL1-Blue中,IPTG诱导GST-UP3-00750融合蛋白的表达,并采用相关生物信息学软件对其进行分析。结果对构建的pGEX-6p2-UP3-00750重组质粒测序,所得序列与NCBI中序列比对,与U.parvum strain hebnu uu3序列的同源性为100%。诱导的融合蛋白经SDS-PAGE分析显示其分子质量与GST-UP3-00750融合蛋白的预期大小一致。生物信息学分析该假设蛋白由159个氨基酸组成,分子式为C_(86)0_(H1369)N_(223)O_(254)S_(2),理论等电点(pI)为6.43,不稳定指数为36.70,脂肪族指数为112.14;其二级结构中α-螺旋(Hh)占39.62%,β-折叠(Ee)占19.50%,无规则卷曲(Cc)占40.88%,无β-转角;属于无信号肽的跨膜蛋白;蛋白质互作提示该基因编码的蛋白与atpA-1、atpD-1、UU044、UU045、UU046、UU047、UU048、UU049、UU050、UU052相关联。结论成功构建了GST-UP3-00750原核表达载体,在大肠埃希菌中表达GST融合蛋白。该蛋白为无信号肽的跨膜蛋白,可能参与ATP合成代谢途径。Objective To construct a prokaryotic expression vector of the UP3-00750 gene in Ureaplasma parvum and to bioinformatically analyze the protein it encodes.Methods In accordance with the UP3-00750 gene sequence of Ureaplasma parvum published in NCBI,primers were designed using Primer Premier 5.0 software and the UP3-00750 gene(480 bp)was amplified using PCR.The PCR products were purified by gel recovery kit.The purified PCR products of UP3-00750 and prokaryotic expression vector pGEX-6 p2 were respectively digested by restriction endonucleases BamH I and Not I.The vector was ligated with T4 ligase at 16℃for 16 h,and the ligate products were transformed into Escherichia coli XL1-Blue via the heat shock method.The transformed competent bacteria were coated on LB solid plates containing ampicillin and cultured overnight at 37℃,and the recombinant plasmid was screened by colony PCR.After confirmed by sequencing,the positive colony was induced to produce fusion protein GST-UP3-00750 with IPTG.The protein was analyzed by bioinformatics software.Results The UP3-00750 gene was amplified by PCR,and the amplification fragment was 500 bp detected by gel electrophoresis.The recombinant plasmid pGEX-6 p2-UP3-00750 was identified by universal primers,and the product of amplification was 600 bp in length.After sequencing,the recombinant plasmid pGEX-6 p2-UP3-00750 was compared with NCBI sequence and its homology was 100%similar to the Ureaplasma parvum strain hebnu uu3.Expression of the recombinant plasmid transfected into E.coli was induced with IPTG for 3 h to express the fusion protein GST-UP3-00750.SDS-PAGE analysis showed that the molecular weight of the induced fusion protein was 44 ku.Bioinformatics analysis showed that the protein coded for by the UP3-00750 was composed of 159 amino acids,its molecular formula was C_(86)0_(H1369)N_(223)O_(254)S_(2),its relative molecular weight was 18.96 ku,its theoretical isoelectric point(PI)was 6.43,its instability index was 36.70,and its aliphatic amino acid index was 112.14.Its a

关 键 词：微小脲原体 UP3-00750 原核表达 生物信息学 

分 类 号：R37[医药卫生—病原生物学]