淋球菌外膜蛋白PI的克

发布时间：2018-04-10 05:13

本文选题：淋球菌　切入点：PI基因　出处：《四川大学》2005年硕士论文

【摘要】：目的构建淋球菌PI基因T-A克隆重组载体,对PI基因进行序列分析,以寻找PI基因抗原稳定性位点;构建PI基因表达重组子,诱导表达及纯化重组PI蛋白,研究重组蛋白的功能,为后续PI蛋白免疫学特性的研究、抗体制备,及淋病疫苗研制奠定基础。方法本文分为四个部分,分述如下: 第一部分淋球菌标准株PI基因克隆重组子的构建:提取淋球菌标准菌株29400和29403基因组DNA,PCR扩增淋球菌PI基因,产物经纯化后,在序列两端加A,并与pBS-T线性载体连接,连接产物转化DH5α感受态细胞,经氨卞青霉素抗性筛选和蓝白斑筛选,挑取白色菌落用于质粒提取,用FcoR I和Xho I双酶切所得质粒,初步鉴定阳性克隆。第二部分 PI基因序列分析:测定1中所得阳性重组子的PI基因序列,用blastn软件将所得序列与GenBank数据库中序列进行比较,寻找相似序列;用clustalW软件对所得2条标准株PI基因及本实验室之前所得4条临床分离株PI基因序列进行多序列同源性比较,分析标准株与临床株序列间差异;对6条序列的氨基酸序列进行多序列同源性比较,将本研究结果与他人的研究进行比较,预测PI蛋白的二级结构,并用蛋白质结构预测软件预测PI蛋白的三维构型。第三部分 PI基因表达重组子的构建:将1中所得pBS-PI克隆重组子经双酶切和胶回收得到有粘性末端的PI片段,同时用相同的限制性内切酶双酶切、去磷酸化及胶回收表达载体pET30b(+),将所得插入片段PI基因与载体进行连接,并转化DH5α感受态细胞,构建表达重组子的克隆重组菌;将所得表达重组质粒转
[Abstract]:Objective to construct the recombinant vector of Neisseria gonorrhoeae Pi gene T-A, to sequence the Pi gene, to find the stable site of Pi gene antigen, to construct the recombinant Pi gene expression plasmid, to induce and purify the recombinant Pi protein.The study of the function of recombinant protein will lay a foundation for the study of the immunological characteristics of Pi protein, the preparation of antibodies and the development of gonorrhea vaccine.Methods this paper is divided into four parts, as follows:The first part was the construction of clone recombinant of Pi gene of gonorrhoeae standard strain 29400 and 29403. The Pi gene of Neisseria gonorrhoeae was amplified by genomic DNA-PCR. After purification, A was added at both ends of the product and ligated with pBS-T linear vector.The ligation product was transformed into DH5 伪 competent cells. The plasmid was screened by ampicillin resistance screening and blue-white spot screening. The white colony was selected for plasmid extraction. The plasmids were digested by FcoR I and Xho I, and the positive clones were preliminarily identified.The second part is the sequence analysis of Pi gene: the Pi gene sequence of the positive recombinant in 1 was determined, and the sequence was compared with the sequence in GenBank database by blastn software to find the similar sequence.The sequence homology of Pi gene of two standard strains and four clinical isolates were compared by clustalW software, and the difference between standard and clinical strains was analyzed.The amino acid sequences of 6 sequences were compared with those of others. The secondary structure of Pi protein was predicted and the 3D configuration of Pi protein was predicted by protein structure prediction software.The third part of the construction of Pi gene expression recombinant: the recombinant pBS-PI cloned from 1 was digested with double enzyme and gelled to obtain the Pi fragment with sticky end, and was digested with the same restriction endonuclease, and the Pi fragment was digested with the same restriction endonuclease, and the Pi fragment was digested with the same restriction endonuclease.The expression vector pET30b (pET30b) was constructed by dephosphorylation and gel recovery. The inserted Pi gene was ligated with the vector and transformed into DH5 伪 receptive cells to construct the cloned recombinant bacteria expressing the recombinant plasmid, and the expressed recombinant plasmid was transformed into the recombinant plasmid.
【学位授予单位】：四川大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R346

【参考文献】