衣原体噬菌体phiCPG1衣壳蛋白Vp1优化设计的初步探索

发布时间：2018-04-30 18:46

  本文选题：衣原体噬菌体 + phiCPG1　； 参考：《天津医科大学》2017年硕士论文

【摘要】：近年来,沙眼衣原体(Chlamydia trachomatis,CT)所致的非淋病性尿道炎的发病率日益增长,在美国,其发病率位列美国常见病的前五名;在中国,其门诊检出率达9.9%,以中青年人群居多。然而随着近年来耐药菌株使得沙眼衣原体感染的治疗变得困难而艰巨。值得欣慰的是,目前许多的文献资料和临床研究表明,衣原体噬菌体可以作为一种新型抗菌疗法治疗沙眼衣原体感染。前期课题成功诱导表达并纯化了衣原体噬菌体phiCPG1最大最主要的衣壳蛋白Vp1,并发现其能抑制Ct的生长,对Ct E型标准株的抑制率为78%。Vp1,基因序列长度1659 bp,高度保守,但在其序列上存在两特殊区域,分别在其216-299位及462-467位这两氨基酸序列间,分别称为IN5环和INS环。前者在噬菌体衣壳蛋白表面形成“蘑菇状”突起结构,且IN5环在衣原体噬菌体与衣原体结合的过程中起受体识别的关键作用。噬菌体螺原病毒SpV4和衣原体噬菌体同属微小病毒科,在SpV4衣壳蛋白表面也存在蘑菇状突起,在其蘑菇状突起的末端有疏水性氨基酸残基形成的疏水性凹陷腔,后者参与噬菌体与宿主的识别过程。本研究通过改变衣原体噬菌体phiCPG1衣壳蛋白Vp1氨基酸的疏水性和截取表达Vp1蛋白的IN5环,并将获得的纯化蛋白作用于沙眼衣原体比较抑制率的差别,为后期进行Vp1蛋白优化设计的探索提供参考价值。[目的]旨在获得对沙眼衣原体抑制率更高的Vp1蛋白,开辟全新的临床治疗思路。[方法]以衣原体噬菌体phiCPG1的衣壳蛋白Vp1为模板设计引物对Vp1蛋白的IN5区域进行克隆扩增构建表达质粒;将噬菌体SpV4与phiCPG1的衣壳蛋白Vp1进行BLAST比对,找到SpV4衣壳蛋白Vp1关键疏水性氨基酸对应phiCPG1 Vp1的核酸位置,设计改变氨基酸的亲疏水性,构建突变质粒Vp1m1;通过生物信息软件PredictProtein分析噬菌体与宿主结合的关键位点,同上改变氨基酸的疏水性构建突变Vp1m2;将以上IN5、Vp1m1及Vp1m2三种目的蛋白进行诱导表达,利用SDS-PAGE、Western-blot对蛋白表达结果的进行鉴定,最后获得纯化的目的蛋白Vp1m1、Vp1m2及IN5蛋白。在沙眼衣原体E型标准株的培养过程中,加入以上获得的三种蛋白及各对照组。48h后间接免疫荧光计数包涵体,比较三种蛋白对沙眼衣原体抑制率的区别。[结果]成功诱导表达并纯化了衣原体噬菌体phiCPG1衣壳蛋白Vp1氨基酸疏水性改变后两个蛋白Vp1m1、Vp1m2及Vp1蛋白的IN5部分。Vp1m1、Vp1m2及IN5蛋白对Ct生长抑制率分别为69.59%,89.07%及54.50%。[结论]获得了比原Vp1蛋白对沙眼衣原体抑制率更高的蛋白Vp1m2。为寻找Vp1蛋白的优势功能区域及噬菌体与衣原体宿主作用的机制提供线索,也为临床治疗沙眼衣原体感染提供了全新的治疗思路。
[Abstract]:In recent years, the incidence of nongonococcal urethritis caused by Chlamydia trachomatistitis (CTL) has been increasing day by day. In the United States, the incidence of nongonococcal urethritis is among the top five common diseases in the United States. However, the treatment of chlamydia trachomatis infection has become difficult and arduous with drug resistant strains in recent years. It is gratifying to note that chlamydia phage can be used as a new antimicrobial therapy for the treatment of chlamydia trachomatis infection. Vp1, the largest and most important capsid protein of Chlamydia chlamydia phage phiCPG1, was successfully induced and purified, and it was found that Vp1 could inhibit the growth of Ct. The inhibition rate of CPE strain was 78. Vp1, the length of gene sequence was 1659 BP, and the gene sequence was highly conserved. However, there are two special regions in its sequence, namely, IN5 ring and INS ring, between the two amino acid sequences of 216-299 and 462-467, respectively. The former forms a "mushroom shaped" protruding structure on the surface of phage capsid protein, and the IN5 loop plays a key role in receptor recognition during the binding of chlamydia phage to chlamydia. Bacteriophage SpV4 and Chlamydia phage belong to the parvovirus family. There are also mushroom protuberances on the surface of SpV4 capsid protein and hydrophobic cavities formed by hydrophobic amino acid residues at the end of the mushroom shaped protuberances. The latter is involved in the recognition of phage and host. The aim of this study was to change the hydrophobicity of Vp1 amino acids of phiCPG1 capsid protein of Chlamydia chlamydia and to intercept the IN5 loop expressing Vp1 protein, and to apply the purified protein to Chlamydia trachomatis to compare the inhibition rate of Chlamydia trachomatis (Chlamydia trachomatis). It provides reference value for the optimization design of Vp1 protein in the later stage. Objective] to obtain Vp1 protein with higher inhibitory rate on chlamydia trachomatis and to develop a new clinical treatment. [methods] using the capsid protein Vp1 of chlamydia phiCPG1 as template, the IN5 region of Vp1 protein was cloned and amplified, and the SpV4 was compared with phiCPG1 capsid protein Vp1 by BLAST. The key hydrophobic amino acids of SpV4 capsid protein Vp1 were found to correspond to the nucleic acid position of phiCPG1 Vp1, the hydrophobicity of amino acids was changed, the mutant plasmid Vp1m1 was constructed, and the key site of phage binding to host was analyzed by PredictProtein. The protein was induced to express by SDS-PAGEG Western-blot, and the purified protein Vp1m1m2 and IN5 protein were obtained by SDS-PAGEG Western-blot. The results showed that Vp1m1m1m1m2and Vp1m1m2were purified by SDS-PAGEG Western-blot. The purified Vp1m1m1m1m2and Vp1m1m2protein were obtained by SDS-PAGEG Western-blot. In the culture of Chlamydia trachomatis E strain, three kinds of proteins were added and the inclusion bodies were counted by indirect immunofluorescence after 48 hours in each control group. The difference of the inhibition rate of the three proteins on chlamydia trachomatis was compared. [results] after the amino acid hydrophobicity of Chlamydia phage phiCPG1 capsid protein Vp1 was changed, the inhibition rates of two proteins, Vp1m1Vp1m2 and Vp1m1Vp1m2 and IN5 protein, on the growth of Ct were 89.07% and 54.50%, respectively. [conclusion] the protein Vp1 m2 was obtained with higher inhibition rate of Chlamydia trachomatis than proto Vp1 protein. It provides clues for finding the dominant functional region of Vp1 protein and the mechanism of host interaction between bacteriophage and chlamydia. It also provides a new therapeutic idea for clinical treatment of chlamydia trachomatis infection.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R759

【参考文献】

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