衣原体噬菌体phiCPG1衣壳蛋白Vp1单克隆抗体的制备及初步应用

发布时间：2018-05-02 17:13

本文选题：衣原体噬菌体 + VP1蛋白　；参考：《天津医科大学》2010年博士论文

【摘要】： 沙眼衣原体(chlamydia trachomatis, C.t)可以导致身体多部位的感染,包括眼睛和泌尿生殖道。所导致的沙眼是世界首位的致盲因素,迄今仍肆虐在许多发展中国家。泌尿生殖道感染更为普遍,其并发症有异位妊娠、不孕不育症、婴幼儿肺炎和Reiter's综合症等。噬菌体是一类能感染细菌、真菌、放线菌和螺旋体等微生物的细菌病毒的总称。根据噬菌体与宿主菌的相互关系,可分成两种类型。一种能在宿主菌细胞内复制增殖,产生许多子代噬菌体,并最终裂解细菌,称为毒性噬菌体。另一种噬菌体基因与宿主菌染色体整合,不产生子代噬菌体,但随细菌DNA的复制而复制,并随细菌的分裂而传代,称为温和噬菌体或溶原性噬菌体。噬菌体作为分子生物学研究工具及新型的治疗方法正受到越来越多的关注。衣原体也有自己的噬菌体,而且研究显示衣原体噬菌体可能具有潜在的临床治疗价值。目前已发现了六种衣原体噬菌体Chp1、Chp2、Chp3、Chp4、ΦCPAR39和PhiCPGl,其宿主均为嗜衣原体属的成员,而在衣原体属的成员中尚未发现噬菌体。组成衣原体噬菌体壳的衣壳蛋白主要成分是Vp1、Vp2和Vp3,其中Vp1是最主要的结构蛋白,在噬菌体对衣原体的黏附和植入中可能有重要作用。同时该蛋白高度保守而特异,因而是在其它衣原体种中寻找衣原体噬菌体的良好标志物。为了探索沙眼衣原体中是否存在噬菌体,本实验通过原核表达制备并纯化了衣原体噬菌体PhiCPGl的衣壳蛋白Vpl,利用制备的蛋白为免疫原,通过杂交瘤技术获得抗Vpl的单克隆抗体杂交瘤分泌株,利用ELISA、染色体分析、Western-blot、SDS-PAGE等方法对单克隆抗体进行鉴定,采用动物体内诱生腹水的方法大量制备单克隆抗体并通过G-蛋白亲和层析法纯化。通过免疫荧光法筛查沙眼衣原体噬菌体,首先采用McCoy细胞培养的方法分离制备待检测的沙眼衣原体野生株。共20株,每株标本均设复孔,孵育48h后,弃去孔中上清液,用PBS洗3次,每次5分钟,冰甲醇固定15分钟,自然干燥,冲洗3次。在复孔中加入0.5%的TritonX-100处理2分钟。向标本中加入制备的单克隆抗体,37℃孵育40分钟,PBS冲洗3次,干燥后加入1：100稀释的FITC标记的羊抗鼠IgG25ul,稀释液为含0.0025%Evans蓝的PBS,避光反应1小时,荧光显微镜下观察结果。经三次基础免疫后小鼠血清中的抗体效价可达到1：6400。融合后一周左右,杂交瘤细胞可生长至1／3-1／2孔,取上清液测抗体分泌情况,筛选出3个阳性孔。将阳性孔扩大培养后进行三次亚克隆化,获得单克隆,扩大培养后进行杂交瘤细胞及单克隆抗体的鉴定。杂交瘤细胞染色体分析发现染色体数目平均为98。结构上多数为端着丝点染色体,还有少数亚中部着丝点染色体。3株细胞分泌的单克隆抗体的免疫球蛋白类别均为IgG1。动物体内诱生的腹水中抗体经纯化后效价大于1：102400。利用得到的单克隆抗体检测了20份临床标本,结果均为阴性。综上所述,本研究成功获得了衣原体噬菌体PhiCPG1的衣壳蛋白Vpl的纯品,制备了抗衣原体噬菌体衣壳蛋白Vp1的单克隆抗体,采用免疫荧光法将其用于临床沙眼衣原体噬菌体的筛查,尚未发现阳性标本。
[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 .

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2010
【分类号】：R392

【参考文献】