衣原体噬菌体phiCPG1衣壳蛋白VP1的优化表达及其生物学效应的研究

发布时间：2018-05-29 17:57

本文选题：沙眼衣原体 + 噬菌体　；参考：《天津医科大学》2017年博士论文

【摘要】：[背景]沙眼衣原体(Chlamydia trachomatis,Ct)泌尿生殖道感染是国内外最常见性传播疾病(sexually transmitted disease,STD)之一,每年全世界范围内约超过1亿人通过性传播而被感染。其大多数患者临床症状表现轻微而隐匿,但病程却迁延且难以治愈。近年来,抗生素耐药与衣原体持续感染的报道屡见不鲜,由于治疗效果欠佳、缺乏有效疫苗,因此衣原体感染的治疗是研究的热点与难点。本课题组前期已发现豚鼠包涵体性结膜炎衣原体噬菌体phiCPG1中最大的衣壳蛋白VP1对沙眼衣原体有着抑制和破坏的效果。[目的]通过各类生物信息学手段分析衣原体噬菌体phiCPG1衣壳蛋白VP1的性质与结构。结合上述数据对VP1蛋白进行截短表达并分别作用于沙眼衣原体,以期发现发挥VP1蛋白生物学功能的最佳区段并探究其功能结构域,并通过蛋白相互作用检测手段验证各截短VP1蛋白与沙眼衣原体的亲和力。原核表达沙眼衣原体多形外膜蛋白I(PmpI)基因,并进行免疫原性的鉴定,结合生物信息学分析探索其生物学特征。通过实验方法探究VP1蛋白在噬菌体对宿主衣原体侵袭过程中发挥作用的结构位点,为VP1蛋白结构分析及噬菌体对衣原体的作用机制提供重要的依据,为沙眼衣原体疾病的临床治疗开辟新的思路。[方法]用生物信息软件分析PmpI的基因序列并预测此蛋白的B细胞抗原表位及二、三级结构,原核表达N端及全长PmpI蛋白并免疫白兔制备兔抗N-PmpI多克隆抗体,通过共聚焦显微镜观测不同培养时相的PmpI蛋白在沙眼衣原体上的定位情况。用生物信息手段分析衣原体噬菌体phiCPG1衣壳蛋白VP1的基因序列并预测此蛋白的B细胞抗原表位及二、三级结构,通过密码子优化相关软件设计并优化VP1序列。结合计算机分析数据对VP1蛋白进行截短表达并免疫小鼠制备鼠抗FL-VP1多克隆抗体。采用CCK-8试剂盒检测VP1蛋白的细胞毒性作用并筛选出最适干预浓度;将VP1蛋白(终浓度50μg/mL)与沙眼衣原体E型标准株室温孵育1小时,并接种至单层致密的Hela细胞中,在Ct培养的过程中均加入VP1蛋白以模拟噬菌体感染其宿主衣原体的过程,通过碘染法来计数沙眼衣原体包涵体(inclusions)个数并计算生长抑制率,分析各截短VP1蛋白对沙眼衣原体生长发育过程的影响,探索发挥VP1蛋白生物学功能的最佳区段及其功能结构域。以Far-Western blot及GST pull-down技术检测不同截短长度VP1蛋白与沙眼衣原体多形外膜蛋白PmpI的结合力。通过最终实验结果探寻VP1蛋白的功能结构域。[结果]对蛋白质的二级结构及氨基酸亲水性、柔性区、表面可及性和抗原指数等预测结果进行综合分析,得出PmpI与VP1蛋白分别含有8个和12个优势B细胞表位,并得到了这两个蛋白的三级结构预测结果,获得了VP1基因的密码子优化序列。成功重组N-PmpI及FL-PmpI原核表达质粒,经诱导表达、亲和层析纯化后获得了活性蛋白并成功制备高效价多抗(1:51200)。成功构建FL-VP1及各截短VP1蛋白原核表达重组体,经表达、纯化后获得了活性蛋白并成功制备高效价多克隆抗体(1:102400)。经过全基因组合成获得了密码子优化后VP1序列(FL-oVP1),并成功获得其诱导表达及纯化产物。经细胞毒性检测,选取干预实验中蛋白的浓度为50μg/mL,在此浓度下,Hela细胞的活力≥90%。干预后发现体外重组表达的各截短VP1蛋白对Ct E型标准株的生长有着不同程度的抑制作用,抑制率从83.2%到6.8%,而对照组低内毒素BSA蛋白及PBS液并未表现出抑制作用。其中,表达18~476位氨基酸位点的VP1蛋白(VP1-C)抑制效果最好,为83.2%,抑制率高于全长表达的VP1蛋白(FL-VP1)。体外实验Far-Western blot及GST pull-down结果检验了各截短VP1蛋白与D型沙眼衣原体标准株PmpI蛋白的不同结合力。综合上述实验结果,推测VP1蛋白特异性的与Ct PmpI蛋白结合的区域可能主要在158~330氨基酸区域内,而330~502区域有较弱的辅助作用,推测VP1可能通过IN5环与Ins环共同作用来行使其生物学功能,不排除两环附近区域有未知结构辅助IN5及Ins环。而通过共聚焦荧光显微镜检测E型Ct PmpI蛋白在不同培养时间段的定位情况,推测作为自转运蛋白的PmpI蛋白很可能有从膜内到膜外的构象变化。[结论]不同截短设计表达的衣壳蛋白VP1对沙眼衣原体(Ct)的生长具有不同程度的抑制作用,并结合蛋白互作检测结果推测出了VP1蛋白可能的作用区域。为VP1蛋白在噬菌体对衣原体粘附作用机制的研究提供重要依据,开辟了沙眼衣原体疾病临床治疗的新思路。
[Abstract]:[background] Chlamydia trachomatis (Ct) genitourinary tract infection is one of the most common sexual transmission diseases (sexually transmitted disease, STD) at home and abroad. More than 100 million people worldwide are infected through sexual transmission worldwide every year. Most of the patients are mild and occult in clinical symptoms, but the course of the disease is prolonged and difficult to cure. In recent years, reports of antibiotic resistance and Chlamydia persistent infection have been reported frequently. Due to poor therapeutic effect and lack of effective vaccines, the treatment of Chlamydia infection is a hot and difficult point in the study. The largest capsid protein VP1 of Chlamydia trachomatis phiCPG1 in guinea pig inclusion body conjunctivitis has been found in the previous year. The body has the effect of inhibition and destruction. [Objective] to analyze the properties and structure of Chlamydia phage phiCPG1 capsid protein VP1 by various bioinformatics methods. Combined with the above data, the VP1 protein was truncated and acted on Chlamydia trachomatis respectively, in order to find the best section and explore the function of VP1 protein bioactivity. The affinity of all truncated VP1 proteins to Chlamydia trachomatis was verified by protein interaction detection. The prokaryotic expression of I (PmpI) gene of Chlamydia trachomatis, and identification of immunogenicity, and bioinformatics analysis were carried out to explore the biological characteristics of VP1 protein in phage lodging. The structural sites that play a role in the process of Chlamydia invasion provide an important basis for the structural analysis of VP1 protein and the mechanism of phage on Chlamydia. It opens a new way for the clinical treatment of Chlamydia trachomatis. [method] analysis of the sequence of PmpI gene and the prediction of B cell epitopes and two of this protein by bioinformatics software. The three stage structure, prokaryotic expression N end and full length PmpI protein and immune white rabbit to prepare Rabbit anti N-PmpI polyclonal antibody. The location of PmpI protein in Chlamydia trachomatis in different culture phase was observed by confocal microscopy. The gene sequence of Chlamydia phage phiCPG1 coat protein VP1 was analyzed by biological information method and the B of the protein was predicted. The cell antigen epitope and the two, three level structure were designed and optimized by the codon optimization software. The VP1 protein was truncated by the computer analysis data and immunized with the mouse anti FL-VP1 polyclonal antibody. The cytotoxic effect of VP1 protein was detected by the CCK-8 kit and the optimum intervention concentration was screened; VP1 eggs were screened. White (final concentration 50 g/mL) and Chlamydia trachomatis E standard strain incubated at room temperature for 1 hours, and inoculated into single layer and dense Hela cells, VP1 protein was added to simulate the process of phage infection of the host chlamydia in the process of Ct culture. The number of inclusion bodies (inclusions) in the trachoma and the growth inhibition rate were counted by iodine staining. The effects of each truncated VP1 protein on the growth and development of Chlamydia trachomatis were analyzed, and the best section and functional domain of VP1 protein biological function were explored. The binding force of the different truncated VP1 proteins to the multiform outer membrane protein PmpI of Chlamydia trachomatis was detected by Far-Western blot and GST pull-down. The final experimental results were explored. The functional domain of VP1 protein was found. [results] the two stage structure of protein and amino acid hydrophilicity, flexible region, surface accessibility and antigen index were synthetically analyzed. The results showed that PmpI and VP1 protein contain 8 and 12 dominant B cell epitopes, and the prediction results of the three structure of these two proteins were obtained, and the VP1 base was obtained. The N-PmpI and FL-PmpI prokaryotic expression plasmids were successfully reorganized. After induction, the active protein was purified by affinity chromatography and high effective valence polyanti (1:51200) was successfully prepared. The recombinant FL-VP1 and the prokaryotic expression recombinant of each truncated VP1 protein were successfully constructed. After expression, the active protein was obtained and the high effective price was prepared. Polyclonal antibody (1:102400). The VP1 sequence (FL-oVP1) after codon optimization was obtained by whole genome synthesis, and its induced expression and purification products were successfully obtained. By cytotoxicity test, the concentration of protein in the intervention experiment was 50 u g/mL. Under this concentration, the activity of Hela cells was more than 90%., and the recombinant expression in vitro was found to be truncated in vitro. VP1 protein had different inhibitory effects on the growth of Ct E type standard strain, the inhibition rate was from 83.2% to 6.8%, while the low endotoxin BSA protein and PBS solution in the control group did not show inhibition. Among them, the inhibitory effect of VP1 protein (VP1-C), which expressed the 18~476 bit amino acid site, was 83.2%, and the inhibition rate was higher than the VP1 protein (FL-VP1). In vitro experiments, Far-Western blot and GST pull-down, tested the different binding forces of the truncated VP1 protein and the D type Chlamydia trachomatis standard strain PmpI protein. The results showed that the region of the binding of VP1 protein specifically to Ct PmpI protein may be mainly in the 158~330 amino acid region, while the 330~502 region has a weaker auxiliary activity. It is presumed that VP1 may exercise its biological function through the joint action of IN5 ring and Ins ring, and does not exclude the unknown structure auxiliary IN5 and Ins ring in the region near the two ring. And the localization of E type Ct PmpI protein in different culture time segments is detected by confocal fluorescence microscopy, and it is presumed that the PmpI protein as the transfer protein may be from the membrane. The conformation changes outside the membrane. [Conclusion] the capsid protein VP1 expressed in different truncated designs has different inhibitory effects on the growth of Chlamydia trachomatis (Ct), and conjectured the possible region of action of VP1 protein by binding protein interaction. It provides an important basis for the study of the adhesion mechanism of VP1 protein in the phage. The new idea of clinical treatment for Chlamydia trachomatis disease has been opened up.
【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R374

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