类鼻疽伯克霍尔德菌groEL基因的克隆、原核表达及其蛋白的生物信息学分析

Cloning,Prokaryotic Expression and Protein Bioinformatics Analysis of groEL Gene of Burkholderia pseudomallei

试验旨在对类鼻疽伯克霍尔德菌groEL基因进行克隆与原核表达,并对其表达蛋白进行生物信息学分析。提取该菌基因组DNA作为模板,参考GenBank中类鼻疽伯克霍尔德菌groEL基因序列,设计1对引物。通过PCR扩增得到大小为1 641bp的groEL基因片段,将其连接至pMD19-T载体,构建pMD19-T-groEL重组质粒,经BamHⅠ和HindⅢ双酶切鉴定正确后,构建重组质粒pET-28a(+)-groEL。将鉴定正确的pET-28a(+)-groEL质粒转化至E.coli BL21(DE3)感受态细胞中,经IPTG诱导表达,运用SDS-PAGE和Western blotting方法进行蛋白质鉴定,利用DNAMAN和BioEdit等软件进行生物信息学分析。结果发现,试验成功克隆了类鼻疽伯克霍尔德菌groEL基因并进行了蛋白表达,表达的融合蛋白大小约为64 ku,GroEL蛋白的分子式为C4510H7381N1641O1840S521,原子总个数为15 893,消光系数为32 500,不稳定指数为40.31,亲水性平均值为0.901。GroEL蛋白二级结构中α-螺旋(Hh)、延伸链(Ee)、无规则卷曲(Cc)分别占48.71%、13.19%和38.10%。本试验结果为深入探究类鼻疽杆菌groEL基因的分子作用机理奠定了基础。

This study was aimed to clone and express groEL gene of Burkholderia pseudomallei,and perform the bioinformatics analysis of groEL protein.The genome DNA of the bacteria was extracted as the template,apair of primers were designed with reference to the groEL gene sequence of Burkholderia pseudomallei in GenBank.The groEL gene fragment of 1 641 bp was amplified by PCR,and ligated into pMD19-T vector to construct pMD19-T-groEL recombinant plasmid.The recombinant plasmid pET-28 a（＋）-groEL was constructed by digestion with BamHⅠand HindⅢ.The correct pET-28 a（＋）-groEL was transformed into E.coli BL21（DE3）competent cells and induced by IPTG.Protein was identified by SDS-PAGE and Western blotting.DNAMAN and BioEdit softwares were used to perform the bioinformatics analysis.The re-sults showed that groEL gene of Burkholderia pseudomallei was successfully cloned and expressed.The fusion protein was about 64 ku.The formular of GroEL protein was C4510 H7381 N1641 O1840 S521,the molecular mass was 15 893,the extinction coefficient was 32 500,the instability index was 40.31,and the average hydrophilicity was 0.901.The alpha helix（Hh）,extended chain（Ee）and random coil（Cc）accounted for 48.71%,13.19%and 38.10%,respectively.This results laid a foundation for the further study of molecular mechanism of Burkholderia pseudomallei groEL gene.