摘要
目的 构建微小脲原体UP3-00830基因原核表达载体,诱导表达蛋白并进行生物信息学分析。方法 通过PCR方法扩增UP3-00830基因。双酶切目的基因和载体质粒pGEX-6P-2,经T4连接酶连接,连接产物转化XL1-Blue感受态,经10 mmol/L IPTG诱导表达GST-UP3-00830融合蛋白,运用生物信息学软件预测UP3-00830基因编码的假设蛋白的功能。结果 成功构建了pGEX-6p-2-UP3-00830重组质粒,测序后与NCBI中录入的序列进行比对,同源性为99.85%。将重组质粒转染大肠埃希菌后经10 mmol/L IPTG诱导,表达相对分子质量为51×10^(3)的GST-UP3-00830融合蛋白,与预期相符。采用生物信息学软件分析UP3-00830基因编码的假设蛋白,其分子式为C_(1150)H_(1846)N_(302)O_(356)S_(4),理论相对分子质量为25.73×10^(3),理论等电点(pI)为5.19,氨基酸数目为218,氨基酸组成中的赖氨酸(Lys)所占比例最高。含有α-螺旋、延伸链、无规则卷曲和β-转角,无跨膜区域和信号肽区域,第178-216位为卷曲螺旋区域。高级结构分析显示,UP3-00830基因编码的假设蛋白与蛋白库中1zxj.1相似性最高,为31.05%;结构及功能预测显示该蛋白具有蛋白转运和蛋白折叠的功能,与其相邻的蛋白分别为UU067、fecE(UU069)、hmuU-1(UU070)和ABCsbp-4(UU071)。结论 成功构建UP3-00830基因原核表达载体,并诱导表为51×10^(3)的目的蛋白,该蛋白可能参与物质转运和信号转导。
Objective To construct the prokaryotic expression vector of Ureaplasma minutiae UP3-00830 gene, induce the expression of protein and conduct bioinformatics analysis. Methods The UP3-00830 gene was amplified by PCR. The target gene and vector plasmid pGEX-6P-2 were double-digested, ligated by T4 ligase, and the ligated product was transformed into XL1-Blue competent, induced to express GST-UP3-00830 fusion protein by 10mmol/L IPTG,the functions of hypothetical proteins encoded by the UP3-00830 gene were predicted using bioinformatics software. Results The pGEX-6p-2-UP3-00830 recombinant plasmid was successfully constructed, and compared with the sequence entered in NCBI after sequencing, the homology was 99.85%. After the recombinant plasmid was transfected into Escherichia coli and induced by 10mmol/L IPTG,the GST-UP3-00830 fusion protein with a relative molecular mass of 51×10^(3) was expressed, which was in line with expectations. Using bioinformatics software to analyze the hypothetical protein encoded by the UP3-00830 gene, its molecular formula is C_(1150)H_(1846)N_(302)O_(356)S_(4),the theoretical relative molecular mass is 25.73×10^(3),the theoretical isoelectric point(pI) is 5.19,the number of amino acids is 218,and lysine in the amino acid composition Acid(Lys) has the highest proportion. Contains α-helix, extended chain, random coil and β-turn, no transmembrane region and signal peptide region, the 178th-216th is the coiled-coil region. Advanced structural analysis shows that the hypothetical protein encoded by the UP3-00830 gene has the highest similarity with 1zxj.1 in the protein library, at 31.05%;structure and function predictions show that this protein has the functions of protein transport and protein folding, and its adjacent proteins are respectively for UU067,fecE(UU069),hmuU-1(UU070) and ABCsbp-4(UU071). Conclusion The prokaryotic expression vector of UP3-00830 gene was successfully constructed, and the target protein of 51×10^(3) was induced to express, which may be involved in material transport and signal transduction.
作者
白一童
贾泽玮
贾晓晖
李瑞平
贾天军
BAI Yi-tong;JIA Ze-wei;JIA Xiao-hui;LI Rui-ping;JIA Tian-jun(Institute of Pathogenic Biology and Immunology,Key Laboratory of Clinical Laboratory and Diagnostics,Hebei North University,Zhangjiakou,075000;Department of Clinical Laboratory,the Second Affiliated Hospital of Hebei North University)
出处
《中国病原生物学杂志》
CSCD
北大核心
2023年第2期162-167,共6页
Journal of Pathogen Biology
作者简介
白一童(1996-),女,河北保定人,硕士研究生,主要从事解脲脲原体相关研究。E-mail:772041530@qq.com;通讯作者:贾天军,E-mail:452871832@qq.com
