本文選題：衣原體噬菌體 + phiCPG1��； 參考：《天津醫(yī)科大學(xué)》2017年碩士論文

【摘要】：近年來(lái),沙眼衣原體(Chlamydia trachomatis,CT)所致的非淋病性尿道炎的發(fā)病率日益增長(zhǎng),在美國(guó),其發(fā)病率位列美國(guó)常見(jiàn)病的前五名;在中國(guó),其門(mén)診檢出率達(dá)9.9%,以中青年人群居多。然而隨著近年來(lái)耐藥菌株使得沙眼衣原體感染的治療變得困難而艱巨。值得欣慰的是,目前許多的文獻(xiàn)資料和臨床研究表明,衣原體噬菌體可以作為一種新型抗菌療法治療沙眼衣原體感染。前期課題成功誘導(dǎo)表達(dá)并純化了衣原體噬菌體phiCPG1最大最主要的衣殼蛋白Vp1,并發(fā)現(xiàn)其能抑制Ct的生長(zhǎng),對(duì)Ct E型標(biāo)準(zhǔn)株的抑制率為78%。Vp1,基因序列長(zhǎng)度1659 bp,高度保守,但在其序列上存在兩特殊區(qū)域,分別在其216-299位及462-467位這兩氨基酸序列間,分別稱為IN5環(huán)和INS環(huán)。前者在噬菌體衣殼蛋白表面形成“蘑菇狀”突起結(jié)構(gòu),且IN5環(huán)在衣原體噬菌體與衣原體結(jié)合的過(guò)程中起受體識(shí)別的關(guān)鍵作用。噬菌體螺原病毒SpV4和衣原體噬菌體同屬微小病毒科,在SpV4衣殼蛋白表面也存在蘑菇狀突起,在其蘑菇狀突起的末端有疏水性氨基酸殘基形成的疏水性凹陷腔,后者參與噬菌體與宿主的識(shí)別過(guò)程。本研究通過(guò)改變衣原體噬菌體phiCPG1衣殼蛋白Vp1氨基酸的疏水性和截取表達(dá)Vp1蛋白的IN5環(huán),并將獲得的純化蛋白作用于沙眼衣原體比較抑制率的差別,為后期進(jìn)行Vp1蛋白優(yōu)化設(shè)計(jì)的探索提供參考價(jià)值。[目的]旨在獲得對(duì)沙眼衣原體抑制率更高的Vp1蛋白,開(kāi)辟全新的臨床治療思路。[方法]以衣原體噬菌體phiCPG1的衣殼蛋白Vp1為模板設(shè)計(jì)引物對(duì)Vp1蛋白的IN5區(qū)域進(jìn)行克隆擴(kuò)增構(gòu)建表達(dá)質(zhì)粒;將噬菌體SpV4與phiCPG1的衣殼蛋白Vp1進(jìn)行BLAST比對(duì),找到SpV4衣殼蛋白Vp1關(guān)鍵疏水性氨基酸對(duì)應(yīng)phiCPG1 Vp1的核酸位置,設(shè)計(jì)改變氨基酸的親疏水性,構(gòu)建突變質(zhì)粒Vp1m1;通過(guò)生物信息軟件PredictProtein分析噬菌體與宿主結(jié)合的關(guān)鍵位點(diǎn),同上改變氨基酸的疏水性構(gòu)建突變Vp1m2;將以上IN5、Vp1m1及Vp1m2三種目的蛋白進(jìn)行誘導(dǎo)表達(dá),利用SDS-PAGE、Western-blot對(duì)蛋白表達(dá)結(jié)果的進(jìn)行鑒定,最后獲得純化的目的蛋白Vp1m1、Vp1m2及IN5蛋白。在沙眼衣原體E型標(biāo)準(zhǔn)株的培養(yǎng)過(guò)程中,加入以上獲得的三種蛋白及各對(duì)照組。48h后間接免疫熒光計(jì)數(shù)包涵體,比較三種蛋白對(duì)沙眼衣原體抑制率的區(qū)別。[結(jié)果]成功誘導(dǎo)表達(dá)并純化了衣原體噬菌體phiCPG1衣殼蛋白Vp1氨基酸疏水性改變后兩個(gè)蛋白Vp1m1、Vp1m2及Vp1蛋白的IN5部分。Vp1m1、Vp1m2及IN5蛋白對(duì)Ct生長(zhǎng)抑制率分別為69.59%,89.07%及54.50%。[結(jié)論]獲得了比原Vp1蛋白對(duì)沙眼衣原體抑制率更高的蛋白Vp1m2。為尋找Vp1蛋白的優(yōu)勢(shì)功能區(qū)域及噬菌體與衣原體宿主作用的機(jī)制提供線索,也為臨床治療沙眼衣原體感染提供了全新的治療思路。
[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.
【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R759

【參考文獻(xiàn)】

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