本文選題：淋球菌　切入點：PI基因　出處：《四川大學(xué)》2005年碩士論文

【摘要】：目的 構(gòu)建淋球菌PI基因T-A克隆重組載體,對PI基因進行序列分析,以尋找PI基因抗原穩(wěn)定性位點;構(gòu)建PI基因表達重組子,誘導(dǎo)表達及純化重組PI蛋白,研究重組蛋白的功能,為后續(xù)PI蛋白免疫學(xué)特性的研究、抗體制備,及淋病疫苗研制奠定基礎(chǔ)。 方法 本文分為四個部分,分述如下: 第一部分 淋球菌標(biāo)準(zhǔn)株P(guān)I基因克隆重組子的構(gòu)建:提取淋球菌標(biāo)準(zhǔn)菌株29400和29403基因組DNA,PCR擴增淋球菌PI基因,產(chǎn)物經(jīng)純化后,在序列兩端加A,并與pBS-T線性載體連接,連接產(chǎn)物轉(zhuǎn)化DH5α感受態(tài)細胞,經(jīng)氨卞青霉素抗性篩選和藍白斑篩選,挑取白色菌落用于質(zhì)粒提取,用FcoR I和Xho I雙酶切所得質(zhì)粒,初步鑒定陽性克隆。 第二部分 PI基因序列分析:測定1中所得陽性重組子的PI基因序列,用blastn軟件將所得序列與GenBank數(shù)據(jù)庫中序列進行比較,尋找相似序列;用clustalW軟件對所得2條標(biāo)準(zhǔn)株P(guān)I基因及本實驗室之前所得4條臨床分離株P(guān)I基因序列進行多序列同源性比較,分析標(biāo)準(zhǔn)株與臨床株序列間差異;對6條序列的氨基酸序列進行多序列同源性比較,將本研究結(jié)果與他人的研究進行比較,預(yù)測PI蛋白的二級結(jié)構(gòu),并用蛋白質(zhì)結(jié)構(gòu)預(yù)測軟件預(yù)測PI蛋白的三維構(gòu)型。 第三部分 PI基因表達重組子的構(gòu)建:將1中所得pBS-PI克隆重組子經(jīng)雙酶切和膠回收得到有粘性末端的PI片段,同時用相同的限制性內(nèi)切酶雙酶切、去磷酸化及膠回收表達載體pET30b(+),將所得插入片段PI基因與載體進行連接,并轉(zhuǎn)化DH5α感受態(tài)細胞,構(gòu)建表達重組子的克隆重組菌;將所得表達重組質(zhì)粒轉(zhuǎn)
[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 偽 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 偽 receptive cells to construct the cloned recombinant bacteria expressing the recombinant plasmid, and the expressed recombinant plasmid was transformed into the recombinant plasmid.
【學(xué)位授予單位】：四川大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2005
【分類號】：R346

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