羊种布鲁氏菌dhbC基因的克

发布时间：2018-02-26 22:11

本文关键词： 布鲁氏菌 dhbC基因克隆原核表达生物信息学分析　出处：《中国畜牧兽医》2017年07期 　论文类型：期刊论文

【摘要】：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。
[Abstract]:The aim of the experiment was to clone and prokaryotic express the dhbC gene of Brucella ovis, and to analyze its expression protein by bioinformatics. According to the sequence information of dhbC gene of Brucella M5-90 strain in GenBank, a pair of primers were designed. The dhbC gene fragment was amplified by PCR reaction. The obtained dhbC gene was ligated into pMD20-T vector to construct the pMD20-T-dhbC recombinant plasmid and transform the E. coli E. coli DH5 伪 competent cell. The plasmid was extracted and identified by enzyme digestion. After the identification was correct, the pET28a-dhbC recombinant plasmid was constructed. Transformation of E. coli BL21 (DE3). Expression of E. coli BL21DDE3 was induced by IPTG. The expression products were analyzed by SDS-PAGE and Western blotting. The amino acid sequences of dhbC gene were analyzed by bioinformatics software DNAMAN and ProtParamie SOPMA and Protscale. The results showed that, The dhbC gene, which is about 1093 BP in size, was cloned and expressed successfully. The size of the expressed fusion protein is about 47ku.DhbC protein mainly exists in the form of inclusion body. The molecular formula is C1866H2968N544O562S15, the molecular weight is 42,496.3u, the theoretical isoelectric point (Ip I) is 5.81, the extinction coefficient is 33,835, the coefficient of instability is 36.76, the hydrophobic index is 86.19, and the total average hydrophobic index is 86.19. The hydrophobicity of GRAVY was -0.215.The half life of mammalian reticulocyte was predicted to be 30 h, the secondary structure of which was 伪 -helix 41.94 and irregular crimping 31.46).
【作者单位】：海南大学热带农林学院海南省热带动物繁育与疫病研究重点实验室海口市动物基因工程重点实验室;
【基金】：国家自然科学基金(31460670) 海南省重大科技计划项目(ZDKJ2016017-01)
【分类号】：S852.614

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