本文選題：沙眼衣原體 + 噬菌體��； 參考：《天津醫(yī)科大學(xué)》2017年博士論文

【摘要】：[背景]沙眼衣原體(Chlamydia trachomatis,Ct)泌尿生殖道感染是國內(nèi)外最常見性傳播疾病(sexually transmitted disease,STD)之一,每年全世界范圍內(nèi)約超過1億人通過性傳播而被感染。其大多數(shù)患者臨床癥狀表現(xiàn)輕微而隱匿,但病程卻遷延且難以治愈。近年來,抗生素耐藥與衣原體持續(xù)感染的報(bào)道屢見不鮮,由于治療效果欠佳、缺乏有效疫苗,因此衣原體感染的治療是研究的熱點(diǎn)與難點(diǎn)。本課題組前期已發(fā)現(xiàn)豚鼠包涵體性結(jié)膜炎衣原體噬菌體phiCPG1中最大的衣殼蛋白VP1對(duì)沙眼衣原體有著抑制和破壞的效果。[目的]通過各類生物信息學(xué)手段分析衣原體噬菌體phiCPG1衣殼蛋白VP1的性質(zhì)與結(jié)構(gòu)。結(jié)合上述數(shù)據(jù)對(duì)VP1蛋白進(jìn)行截短表達(dá)并分別作用于沙眼衣原體,以期發(fā)現(xiàn)發(fā)揮VP1蛋白生物學(xué)功能的最佳區(qū)段并探究其功能結(jié)構(gòu)域,并通過蛋白相互作用檢測(cè)手段驗(yàn)證各截短VP1蛋白與沙眼衣原體的親和力。原核表達(dá)沙眼衣原體多形外膜蛋白I(PmpI)基因,并進(jìn)行免疫原性的鑒定,結(jié)合生物信息學(xué)分析探索其生物學(xué)特征。通過實(shí)驗(yàn)方法探究VP1蛋白在噬菌體對(duì)宿主衣原體侵襲過程中發(fā)揮作用的結(jié)構(gòu)位點(diǎn),為VP1蛋白結(jié)構(gòu)分析及噬菌體對(duì)衣原體的作用機(jī)制提供重要的依據(jù),為沙眼衣原體疾病的臨床治療開辟新的思路。[方法]用生物信息軟件分析PmpI的基因序列并預(yù)測(cè)此蛋白的B細(xì)胞抗原表位及二、三級(jí)結(jié)構(gòu),原核表達(dá)N端及全長PmpI蛋白并免疫白兔制備兔抗N-PmpI多克隆抗體,通過共聚焦顯微鏡觀測(cè)不同培養(yǎng)時(shí)相的PmpI蛋白在沙眼衣原體上的定位情況。用生物信息手段分析衣原體噬菌體phiCPG1衣殼蛋白VP1的基因序列并預(yù)測(cè)此蛋白的B細(xì)胞抗原表位及二、三級(jí)結(jié)構(gòu),通過密碼子優(yōu)化相關(guān)軟件設(shè)計(jì)并優(yōu)化VP1序列。結(jié)合計(jì)算機(jī)分析數(shù)據(jù)對(duì)VP1蛋白進(jìn)行截短表達(dá)并免疫小鼠制備鼠抗FL-VP1多克隆抗體。采用CCK-8試劑盒檢測(cè)VP1蛋白的細(xì)胞毒性作用并篩選出最適干預(yù)濃度;將VP1蛋白(終濃度50μg/mL)與沙眼衣原體E型標(biāo)準(zhǔn)株室溫孵育1小時(shí),并接種至單層致密的Hela細(xì)胞中,在Ct培養(yǎng)的過程中均加入VP1蛋白以模擬噬菌體感染其宿主衣原體的過程,通過碘染法來計(jì)數(shù)沙眼衣原體包涵體(inclusions)個(gè)數(shù)并計(jì)算生長抑制率,分析各截短VP1蛋白對(duì)沙眼衣原體生長發(fā)育過程的影響,探索發(fā)揮VP1蛋白生物學(xué)功能的最佳區(qū)段及其功能結(jié)構(gòu)域。以Far-Western blot及GST pull-down技術(shù)檢測(cè)不同截短長度VP1蛋白與沙眼衣原體多形外膜蛋白PmpI的結(jié)合力。通過最終實(shí)驗(yàn)結(jié)果探尋VP1蛋白的功能結(jié)構(gòu)域。[結(jié)果]對(duì)蛋白質(zhì)的二級(jí)結(jié)構(gòu)及氨基酸親水性、柔性區(qū)、表面可及性和抗原指數(shù)等預(yù)測(cè)結(jié)果進(jìn)行綜合分析,得出PmpI與VP1蛋白分別含有8個(gè)和12個(gè)優(yōu)勢(shì)B細(xì)胞表位,并得到了這兩個(gè)蛋白的三級(jí)結(jié)構(gòu)預(yù)測(cè)結(jié)果,獲得了VP1基因的密碼子優(yōu)化序列。成功重組N-PmpI及FL-PmpI原核表達(dá)質(zhì)粒,經(jīng)誘導(dǎo)表達(dá)、親和層析純化后獲得了活性蛋白并成功制備高效價(jià)多抗(1:51200)。成功構(gòu)建FL-VP1及各截短VP1蛋白原核表達(dá)重組體,經(jīng)表達(dá)、純化后獲得了活性蛋白并成功制備高效價(jià)多克隆抗體(1:102400)。經(jīng)過全基因組合成獲得了密碼子優(yōu)化后VP1序列(FL-oVP1),并成功獲得其誘導(dǎo)表達(dá)及純化產(chǎn)物。經(jīng)細(xì)胞毒性檢測(cè),選取干預(yù)實(shí)驗(yàn)中蛋白的濃度為50μg/mL,在此濃度下,Hela細(xì)胞的活力≥90%。干預(yù)后發(fā)現(xiàn)體外重組表達(dá)的各截短VP1蛋白對(duì)Ct E型標(biāo)準(zhǔn)株的生長有著不同程度的抑制作用,抑制率從83.2%到6.8%,而對(duì)照組低內(nèi)毒素BSA蛋白及PBS液并未表現(xiàn)出抑制作用。其中,表達(dá)18~476位氨基酸位點(diǎn)的VP1蛋白(VP1-C)抑制效果最好,為83.2%,抑制率高于全長表達(dá)的VP1蛋白(FL-VP1)。體外實(shí)驗(yàn)Far-Western blot及GST pull-down結(jié)果檢驗(yàn)了各截短VP1蛋白與D型沙眼衣原體標(biāo)準(zhǔn)株P(guān)mpI蛋白的不同結(jié)合力。綜合上述實(shí)驗(yàn)結(jié)果,推測(cè)VP1蛋白特異性的與Ct PmpI蛋白結(jié)合的區(qū)域可能主要在158~330氨基酸區(qū)域內(nèi),而330~502區(qū)域有較弱的輔助作用,推測(cè)VP1可能通過IN5環(huán)與Ins環(huán)共同作用來行使其生物學(xué)功能,不排除兩環(huán)附近區(qū)域有未知結(jié)構(gòu)輔助IN5及Ins環(huán)。而通過共聚焦熒光顯微鏡檢測(cè)E型Ct PmpI蛋白在不同培養(yǎng)時(shí)間段的定位情況,推測(cè)作為自轉(zhuǎn)運(yùn)蛋白的PmpI蛋白很可能有從膜內(nèi)到膜外的構(gòu)象變化。[結(jié)論]不同截短設(shè)計(jì)表達(dá)的衣殼蛋白VP1對(duì)沙眼衣原體(Ct)的生長具有不同程度的抑制作用,并結(jié)合蛋白互作檢測(cè)結(jié)果推測(cè)出了VP1蛋白可能的作用區(qū)域。為VP1蛋白在噬菌體對(duì)衣原體粘附作用機(jī)制的研究提供重要依據(jù),開辟了沙眼衣原體疾病臨床治療的新思路。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R374

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

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本文編號(hào)：1951858