羊种布鲁氏菌dhbC基因的克隆、原核表达及生物信息学分析

Cloning,Prokaryotic Expression and Bioinformation Analysis of dhbC Gene of Brucella melitensis

试验旨在对羊种布鲁氏菌dhbC基因进行克隆及原核表达,并对其表达蛋白进行生物信息学分析。参照GenBank中布鲁氏菌M5-90株dhbC基因序列信息设计1对引物,通过PCR反应扩增获得dhbC基因片段。将得到的dhbC基因连接到pMD20-T载体,构建pMD20-T-dhbC重组质粒并转化大肠杆菌(E.coli)DH5α感受态细胞,提取质粒进行酶切鉴定。鉴定正确后构建pET28a-dhbC重组质粒,转化E.coli BL21(DE3)感受态细胞。经IPTG诱导表达,表达产物用SDS-PAGE和Western blotting进行分析。运用生物信息学软件DNAMAN及相关在线网站ProtParam、SOPMA及Protscale对dhbC基因编码的氨基酸序列进行生物信息学分析。结果表明,试验成功克隆了大小约为1 093bp的dhbC基因并进行了蛋白表达,表达的融合蛋白大小约为47ku,且主要以包涵体形式存在。dhbC蛋白的分子式为C1866H2968N544O562S15,分子质量为42 496.3u,理论等电点(pI)为5.81,消光系数为33 835,不稳定系数为36.76,疏水指数为86.19,总平均疏水性(GRAVY)为-0.215。预测在哺乳动物网织红细胞的半衰期为30h,其二级结构以α-螺旋(41.94%)和无规则卷曲(31.46%)为主。

This study was aimed to clone and express dhbCgene of Brucella melitensis,and analyze the bioinformatics of its expressed protein.A pair of primers were designed by referring to dhbCgene sequence information of Brucella melitensis M5-90 strain in GenBank,and the dhbC gene fragment was amplified by PCR method.The obtained dhbCgene was ligated into pMD20-T vector to construct pMD20-T-dhbC recombinant plasmid and transformed into E.coli DH5αcompetent cells.The plasmid was identified by restriction enzyme digestion.The recombinant plasmid pET28a-dhbC was constructed and transformed into E.coli BL21（DE3）competent cells.The expression was induced by IPTG.The expressed product was analyzed by SDS-PAGE and Western blotting.Bioinformatics analysis of the amino acid sequence encoded by dhbCgene was carried out using bioinformatics software DNAMAN and related online sites ProtParam,SOPMAand Protscale.The results showed that dhbCgene was cloned with the length of 1 093 bp,and protein expression was expressed.The expressed fusion protein was about 47 ku,and was mainly in the form of inclusion body.The molecular weight of the dhbC protein was C1866H2968N544O562S15,the molecular mass was 42 496.3u,the theoretical isoelectric point（pI）was 5.81,the extinction coefficient was 33 835,the instability coefficient was 36.76,the hydrophobic index was86.19,the total average hydrophobicity（GRAVY）was-0.215.The half-life of reticulocytes in mammals was predicted to be 30 h,and the secondary structure was dominated byα-helix（41.94%）and random coil（31.46%）.