脓肿分枝杆菌非溶血性磷脂酶C的克隆表达及活性鉴定

发布时间：2018-06-11 21:43

本文选题：脓肿分枝杆菌 + 磷脂酶C　；参考：《重庆医科大学》2012年硕士论文

【摘要】：目的克隆、表达脓肿分枝杆菌磷脂酶C基因，鉴定其活性，为进一步探讨该蛋白质的生物学功能及其在脓肿分枝杆菌感染过程中的致病作用提供实验材料。方法以脓肿分枝杆菌基因组DNA为模板，PCR扩增脓肿分枝杆菌磷脂酶C基因片段，克隆入pQE-30质粒，构建重组原核表达载体pQE-30-MAB_0555，将其转化入大肠杆菌DH5a，进行序列测定及利用生物信息学软件DNAstar5.0等分析其生物学特性，同时IPTG诱导蛋白表达，采用SDS-PAGE分析表达蛋白的相对分子质量，应用Western-blotting检测蛋白表达，离子交换层析纯化重组蛋白，杯碟法检测其活性。结果 PCR扩增获得长1440bp的MAB_0555基因片段，编码479个氨基酸，经PCR、双酶切及测序证明重组质粒pQE-30-MAB_0555构建正确。DNAstar等软件预测其蛋白质相对分子量（Mr）约为53KD,并显示出具有良好的抗原性。SDS-PAGE显示表达蛋白相对分子量约为53KD，同生物信息学分析软件所得的估计值一致；Western-blotting检测蛋白得到表达，杯碟法观察到在琼脂卵黄平板上可见乳白色晕圈，而血平板上未见溶血环。结论成功的克隆了脓肿分枝杆菌磷脂酶C基因和构建重组质粒pQE-30-MAB_0555，应用生物信息学方法分析出该蛋白具有良好的抗原性；同时应用Western-blotting技术分析重组蛋白得到正确表达，，并通过卵黄琼脂平板法证实重组蛋白具有溶解蛋黄中卵磷脂的作用，通过血平板法证实重组蛋白不具有溶解红细胞膜的作用。为进一步探讨该蛋白质的生物学功能及其在脓肿分枝杆菌感染过程中的致病作用提供实验材料。
[Abstract]:Objective to clone, express and identify the phospholipase C gene of Mycobacterium abscess. In order to further study the biological function of the protein and its pathogenicity in the process of infection of Mycobacterium abscess, the genomic DNA of Mycobacterium abscess was used as template to amplify the fragment of phospholipase C gene of Mycobacterium abscess by PCR. The recombinant prokaryotic expression vector pQE-30-MAB0555 was cloned into pQE-30 plasmid, and transformed into Escherichia coli DH 5a. Its biological characteristics were analyzed by bioinformatics software DNAstar5.0, and the expression of protein was induced by IPTG. SDS-PAGE was used to analyze the relative molecular weight of the expressed protein, Western-blotting was used to detect the protein expression, ion exchange chromatography was used to purify the recombinant protein, and its activity was detected by cup / disc method. PCR, double enzyme digestion and sequencing proved that the recombinant plasmid pQE-30-MAB0555 was constructed correctly. DNAstar and other software predicted that the relative molecular weight of the protein was about 53KD.SDS-PAGE showed good antigenicity. SDS-PAGE showed that the relative molecular weight of the expressed protein was about 53KDand the same biological information. The estimated value of the software was consistent with that of Western-blotting for protein expression. The cream white halo circle was observed on the Agar yolk plate by the cup plate method. Conclusion the phospholipase C gene of Mycobacterium abscess was cloned and the recombinant plasmid pQE-30-MAB0555 was constructed. At the same time, Western-blotting technique was used to analyze the correct expression of recombinant protein. The recombinant protein was proved to be able to dissolve lecithin in egg yolk by using egg yolk Agar plate method, and the recombinant protein was not able to dissolve erythrocyte membrane by blood plate method. To further study the biological function of the protein and its pathogenicity in the process of mycobacterium abscess infection.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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