本文選題：衣原體噬菌體 + VP1蛋白�。� 參考：《天津醫(yī)科大學(xué)》2010年博士論文

【摘要】： 沙眼衣原體(chlamydia trachomatis, C.t)可以導(dǎo)致身體多部位的感染,包括眼睛和泌尿生殖道。所導(dǎo)致的沙眼是世界首位的致盲因素,迄今仍肆虐在許多發(fā)展中國(guó)家。泌尿生殖道感染更為普遍,其并發(fā)癥有異位妊娠、不孕不育癥、嬰幼兒肺炎和Reiter's綜合癥等。 噬菌體是一類能感染細(xì)菌、真菌、放線菌和螺旋體等微生物的細(xì)菌病毒的總稱。根據(jù)噬菌體與宿主菌的相互關(guān)系,可分成兩種類型。一種能在宿主菌細(xì)胞內(nèi)復(fù)制增殖,產(chǎn)生許多子代噬菌體,并最終裂解細(xì)菌,稱為毒性噬菌體。另一種噬菌體基因與宿主菌染色體整合,不產(chǎn)生子代噬菌體,但隨細(xì)菌DNA的復(fù)制而復(fù)制,并隨細(xì)菌的分裂而傳代,稱為溫和噬菌體或溶原性噬菌體。噬菌體作為分子生物學(xué)研究工具及新型的治療方法正受到越來(lái)越多的關(guān)注。 衣原體也有自己的噬菌體,而且研究顯示衣原體噬菌體可能具有潛在的臨床治療價(jià)值。目前已發(fā)現(xiàn)了六種衣原體噬菌體Chp1、Chp2、Chp3、Chp4、ΦCPAR39和PhiCPGl,其宿主均為嗜衣原體屬的成員,而在衣原體屬的成員中尚未發(fā)現(xiàn)噬菌體。組成衣原體噬菌體殼的衣殼蛋白主要成分是Vp1、Vp2和Vp3,其中Vp1是最主要的結(jié)構(gòu)蛋白,在噬菌體對(duì)衣原體的黏附和植入中可能有重要作用。同時(shí)該蛋白高度保守而特異,因而是在其它衣原體種中尋找衣原體噬菌體的良好標(biāo)志物。 為了探索沙眼衣原體中是否存在噬菌體,本實(shí)驗(yàn)通過(guò)原核表達(dá)制備并純化了衣原體噬菌體PhiCPGl的衣殼蛋白Vpl,利用制備的蛋白為免疫原,通過(guò)雜交瘤技術(shù)獲得抗Vpl的單克隆抗體雜交瘤分泌株,利用ELISA、染色體分析、Western-blot、SDS-PAGE等方法對(duì)單克隆抗體進(jìn)行鑒定,采用動(dòng)物體內(nèi)誘生腹水的方法大量制備單克隆抗體并通過(guò)G-蛋白親和層析法純化。 通過(guò)免疫熒光法篩查沙眼衣原體噬菌體,首先采用McCoy細(xì)胞培養(yǎng)的方法分離制備待檢測(cè)的沙眼衣原體野生株。共20株,每株標(biāo)本均設(shè)復(fù)孔,孵育48h后,棄去孔中上清液,用PBS洗3次,每次5分鐘,冰甲醇固定15分鐘,自然干燥,沖洗3次。在復(fù)孔中加入0.5%的TritonX-100處理2分鐘。向標(biāo)本中加入制備的單克隆抗體,37℃孵育40分鐘,PBS沖洗3次,干燥后加入1：100稀釋的FITC標(biāo)記的羊抗鼠IgG25ul,稀釋液為含0.0025%Evans藍(lán)的PBS,避光反應(yīng)1小時(shí),熒光顯微鏡下觀察結(jié)果。 經(jīng)三次基礎(chǔ)免疫后小鼠血清中的抗體效價(jià)可達(dá)到1：6400。融合后一周左右,雜交瘤細(xì)胞可生長(zhǎng)至1／3-1／2孔,取上清液測(cè)抗體分泌情況,篩選出3個(gè)陽(yáng)性孔。將陽(yáng)性孔擴(kuò)大培養(yǎng)后進(jìn)行三次亞克隆化,獲得單克隆,擴(kuò)大培養(yǎng)后進(jìn)行雜交瘤細(xì)胞及單克隆抗體的鑒定。雜交瘤細(xì)胞染色體分析發(fā)現(xiàn)染色體數(shù)目平均為98。結(jié)構(gòu)上多數(shù)為端著絲點(diǎn)染色體,還有少數(shù)亞中部著絲點(diǎn)染色體。3株細(xì)胞分泌的單克隆抗體的免疫球蛋白類別均為IgG1。動(dòng)物體內(nèi)誘生的腹水中抗體經(jīng)純化后效價(jià)大于1：102400。利用得到的單克隆抗體檢測(cè)了20份臨床標(biāo)本,結(jié)果均為陰性。 綜上所述,本研究成功獲得了衣原體噬菌體PhiCPG1的衣殼蛋白Vpl的純品,制備了抗衣原體噬菌體衣殼蛋白Vp1的單克隆抗體,采用免疫熒光法將其用于臨床沙眼衣原體噬菌體的篩查,尚未發(fā)現(xiàn)陽(yáng)性標(biāo)本。
[Abstract]:Chlamydia Pneumoniae ( C . t ) can lead to infection of multiple parts of the body , including eye and urinary tract . The resulting trachoma is the leading cause of blindness in the world . So far , the infection of urinary genital tract is more common , and its complications include ectopic pregnancy , infertility , infantile pneumonia and Reiter ' s syndrome .



Bacteriophages are the general name of a class of bacteria , such as bacteria , fungi , actinomyces and spirosis . According to the relationship between phage and host bacteria , it can be divided into two types . One can replicate and proliferate in host bacteria cells , produce many progeny ages , and eventually lyse the bacteria , called virulent phage .



Chp1 , Chp2 , Chp3 , Chp4 , 桅CPAR39 and PhiCPG1 have been found .



In order to explore the presence of phage in Chlamydia trachoma , we prepared and purified the capsid protein Vpl of Chlamydia PhiCPG1 by prokaryotic expression . Using the prepared protein as immunogen , the monoclonal antibody against Vpl was obtained by hybridoma technique . The monoclonal antibody was prepared by ELISA , chromosome analysis , Western - blot , SDS - PAGE and other methods . The monoclonal antibody was prepared by using the method of inducing ascites in vivo and purified by G - protein affinity chromatography .



The samples were washed three times with PBS and washed three times with PBS . After drying , 0.5 % Triton X - 100 was added to the specimen . After drying , the diluted solution was added with PBS containing 0.0025 % Evans blue , and the light was allowed to react for 1 hour , and the results were observed under fluorescence microscope .



The antibody titer in serum of mice immunized with three basic immunization can be up to 1 : 64 . After fusion , hybridoma cells can grow to 1 / 3 - 1 / 2 wells . Three positive wells are screened . Three sub - clones are obtained after the positive wells are expanded and cultured . The number of chromosomes is 1 : 102400 . The titer of the monoclonal antibodies secreted by the cells is greater than 1 : 102400 .



In conclusion , this study successfully obtained the pure product of capsid protein Vpl of Chlamydia PhiCPG1 , prepared the monoclonal antibody against Chlamydia capsid protein Vp1 , and used the immunofluorescence method to screen the clinical trachoma chlamydia phage , and the positive specimen has not been found .

【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】：2010
【分類號(hào)】：R392

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