羊源多杀性巴氏杆菌sodA基因的克隆、表达及其生物信息学分析

Cloning,Expression and Bioinformatics Analysis of sodA Gene of Pasteurella multocida

为了解羊源多杀性巴氏杆菌超氧化物歧化酶(SOD)的生物学功能,本试验对该菌sodA基因进行克隆及原核表达,并对克隆的sodA基因进行遗传进化树分析,对其表达的SOD蛋白进行生物信息学分析。参照GenBank中多杀性巴氏杆菌HN06株基因组中sodA基因序列信息设计引物进行PCR扩增,将产物与pET-28a(+)载体相连,构建pET-28a(+)-sodA重组质粒,将该质粒转化E.coli DH5α感受态细胞进行克隆,再转化E.coli BL21(DE3)感受态细胞进行表达,经IPTG诱导后对表达蛋白进行SDS-PAGE和Western blotting鉴定分析。结果显示,本试验成功扩增出大小为645bp的目的片段,并表达出大小约28ku的目的蛋白。遗传进化树分析表明,该基因与HN07(GenBank登录号:CP007040.1)和Pm70(GenBank登录号:AE004439.1)株亲缘关系较近,重组蛋白生物信息学分析显示,该融合蛋白为稳定的酸性亲水可溶性蛋白,分子式为C1085H1651N293O309S9,分子质量为24 032.36u,理论等电点为6.19,消光系数为45 170,不稳定系数为26.87(<40),在哺乳动物网织红细胞的半衰期预计为30h,疏水指数为82.15,总平均疏水性(GRAVY)为-0.282,二级结构以α-螺旋和无规则卷曲为主。以上研究结果为后续深入研究多杀性巴氏杆菌在羊体内的存活机制及研发预防巴氏杆菌病的疫苗提供了参考。

To investigate the biological functions of superoxide dismutase（SOD）of Pasteurella multocida,the sodAgene was cloned,expressed and analyzed by phylogenetic and bioinformatics,respectively.A pair of primers was designed based on the sodAgene sequence of Pasteurella multocida HN06 strain in GenBank.The PCR products were ligated with pET-28 a（＋）vector to construct pET-28 a（＋）-sodA recombinant plasmids.The recombinant plasmids were transformed into E.coli DH5αcompetent cells for cloning and then transformed them into E.coli BL21（DE3）competent cells for expression.The expressed proteins were induced by IPTG and identified by SDS-PAGE and Western blotting.The results showed that the target fragment with the length of645 bp was successfully amplified and the target protein which was 28 ku was successfully expressed.Phylogenetic analysis showed that the gene was closely related to the HN07（GenBank accession No.：CP〈007040.1）and Pm70（GenBank accession No.：AE004439.1）strains.Bioinformatics analysis results of the recombinant protein showed that it was a stable acidic hydrophilic soluble fusion protein.The molecular formula and molecular mass were C1085 H1651 N293 O309 S9 and24 032.36 u;The theoretical isoelectric point,extinction coefficient and instability coefficient were6.19,45 170 and 26.87（40）,repectively;The half-life of reticulocytes in mammals was estimated to be 30 h;The aliphatic index and total average hydrophobicity（GRAVY）were 82.15 and-0.282,respectively;And the secondary structure was mainly composed ofα-helix and random coil.The above results provided references for the further study of the survival mechanism of Pasteurella multocida and the development of a vaccine against pasteurellosis.