沙眼衣原体CPAF免疫活性区重组蛋白的表达及在临床诊断中的应用

发布时间：2018-12-18 19:57

【摘要】： 目的:构建含沙眼衣原体(Chlamydia trachomatis,Ct)蛋白酶样活性因子(chlamydial protease-like activity factor, CPAF)免疫活性区(200～338 aa)基因的重组表达体,在大肠杆菌中进行诱导表达,纯化表达产物并进行免疫原性和抗原性分析,建立间接ELISA法和IIF法检测Ct感染的临床标本,分析其敏感性和特异性,为制备Ct临床检测试剂盒奠定基础。方法:通过生物信息学分析和查阅资料,筛选并挑选Ct CPAF基因优势抗原表位,以D型Ct基因组DNA为模板,高保真聚合酶链反应扩增目的片断,将其亚克隆至原核表达载体pGEX-6p-2中,构建重组质粒pGEX-6p-2/CPAF,然后转化至表达宿主菌E.coli BL21(DE3)中进行诱导表达,利用SDS-PAGE和Western-Blot分析和鉴定表达产物;重组蛋白经稀释透析复性后,用GST琼脂糖凝胶FF纯化柱进行纯化,紫外吸收法测定纯化蛋白浓度。用纯化的Ct CPAF重组蛋白包被微孔板,建立间接ELISA方法,检测Ct参比血清和临床Ct阳性血清,同时与晶美公司Ct ELISA试剂盒检测结果进行对比分析,根据重组蛋白与Ct阴、阳性血清的反应情况,评价该重组抗原在Ct感染血清学诊断中的应用价值。同时用纯化的Ct CPAF重组蛋白免疫新西兰兔,间接ELISA方法检测免疫兔血清中Ct CPAF多克隆抗体的效价;并用溴化氰活化的琼脂糖凝胶4B对抗血清进行纯化,纯化后的抗血清作为一抗建立间接ELISA法和IIF法测定临床分泌物标本中的Ct CPAF抗原,与PCR法检测结果进行对比分析,评价该抗血清在临床标本抗原检测中的应用价值。结果:软件分析Ct CPAF基因的抗原表位选择了Ct CPAF基因的674～1 090bp位碱基序列为目的基因(片段长度为417bp,编码139个氨基酸);PCR扩增得到大小约为400bp的目的片断;构建的重组质粒经酶切鉴定和测序鉴定证明其中插入片断为Ct CPAF目的基因,测序结果与Genbank上登录序列完全一致;SDS-PAGE分析显示,在IPTG诱导下,重组表达菌表达了一相对分子量(Mr)约为43×103的目的蛋白条带,目的蛋白在菌体细胞内主要以包涵体形式存在;经GST琼脂糖凝胶FF纯化柱纯化获得了纯度在95%以上的重组蛋白。Western-blot检测其能与Ct阳性血清发生特异性反应;重组蛋白为包被抗原建立间接ELISA法检测Ct参考血清,其阴、阳性符合率均为100%;用自建的间接ELISA法和晶美公司Ct ELISA试剂盒分别检测250份阴、阳性血清,两种方法的符合率IgM为96.8%,IgG为98.4%。利用纯化的Ct CPAF重组蛋白免疫新西兰兔,间接ELISA法测定兔免疫血清特异性抗体效价在1:16 000以上;纯化后的抗血清作为一抗建立间接ELISA法和IIF法检测临床分泌物标本中的Ct CPAF抗原,建立的间接ELISA法及IIF法与上海复星公司Ct PCR试剂盒检测结果符合率分别为91.90%和90.00%。结论: 1、成功构建了pGEX-6p-2/ CPAF原核表达体,将其转化至E.coli BL21(DE3)后表达出了一相对分子量(Mr)约为43×103的重组蛋白; 2、Ct CPAF重组蛋白具有较好的免疫原性,能刺激新西兰兔产生高效价的特异性抗体; 3、Ct CPAF重组蛋白具有良好的抗原性,能与Ct感染者阳性血清特异性结合,可应用于Ct血清学诊断; 4、以Ct CPAF重组蛋白免疫新西兰兔制备的多克隆抗体为一抗建立的间接ELISA法和IIF法具有良好的敏感性和特异性,可望应用于Ct感染的抗原检测。
[Abstract]:Objective: To construct a recombinant expression of the immunoactive region (200-338aa) of the immunoactive region (200-338aa) of Chlamydia trachomatis (Ct) protease-like activity factor (CPAF), to induce expression in E. coli, to purify and express the product and to analyze the immunogenicity and antigenicity. To establish the indirect ELISA and the IIF method to detect the clinical specimens of Ct infection, the sensitivity and specificity were analyzed, and the basis for preparing the Ct clinical test kit was established. Methods: Through the analysis of bioinformatics and the data, screening and selection of the positive antigen epitope of the Ct CPAF gene, using the D-type Ct genomic DNA as the template and the high-fidelity polymerase chain reaction to amplify the target fragment, subcloning it into the prokaryotic expression vector pGEX-6p-2, and constructing the recombinant plasmid pGEX-6p-2. 2/ CPAF, and then transformed into E. coli BL21 (DE3) to be expressed, analyzed and identified by SDS-PAGE and Western-Blot, and the recombinant protein was subjected to dilution and renaturation, and purified by using a GST agarose gel FF purification column, and the ultraviolet absorption method purifying the protein concentration, coating the microporous plate with the purified Ct CPAF recombinant protein, establishing an indirect ELISA method, detecting the Ct reference ratio serum and the clinical Ct positive serum, and carrying out contrast analysis with the detection result of the Ct ELISA kit of the crystal beauty company, and according to the recombinant protein and the Ct negative and the positive blood, A clear response to evaluate the serologic diagnosis of the recombinant antigen in the Ct infection Use of purified Ct CPAF recombinant protein to immunize New Zealand rabbit, indirect ELISA method to detect the titer of Ct CPAF polyclonal antibody in immune rabbit serum, and use cyanogen bromide to activate agarose gel 4B. The serum was purified, the purified antiserum was used as an anti-establishing indirect ELISA method and the IIF method to measure the Ct CPAF antigen in the clinical secretion specimen, and compared with the detection result of the PCR method, the antiserum was evaluated for clinical specimen antigen detection. The results are as follows: The software analyzes the epitope of the Ct CPAF gene, and the target gene (the length of the fragment is 417bp, encodes 139 amino acids) is selected by the antigen table of the Ct CPAF gene, and the PCR amplification is large. The results showed that the inserted fragment is the target gene of Ct CPAF, and the sequencing result is completely consistent with the log-in sequence on Genbank; and the SDS-PAGE The analysis showed that, under the induction of IPTG, the recombinant expression bacteria expressed a target protein band with a relative molecular weight (Mr) of about 43-103, and the target protein was mainly in the form of inclusion bodies in the bacterial cells; and the purified column was purified by the GST agarose gel FF purification column. The purity of the recombinant protein was over 95%. Western-blot was used to detect the specific reaction with the positive serum of Ct. The recombinant protein was an indirect ELISA method for the detection of Ct reference serum. The positive rate of negative and positive was 100%. The coincidence rate of the two methods was 96% and the rate of the positive serum was 96. 8% and 98. 4% of IgG. The purified Ct CPAF recombinant protein was used to immunize New Zealand rabbits, and the indirect ELISA method was used to determine the specific antibody titer of the rabbit immune serum at 1: 16,000. The purified antiserum was used as an anti-establishment indirect ELISA method and the IIF method to detect the clinical secretion. The coincidence rate of Ct CPAF antigen, the established indirect ELISA method and the IIF method and the detection result of the Ct PCR kit of Shanghai Fuxing Co., Ltd. 91. Conclusion: 1. The pGEX-6p-2/ CPAF prokaryotic expression was successfully constructed and transformed into E. coli BL21 (DE3). The expression of a relative molecular weight (Mr) is about 43-103 recombinant protein; 2, Ct CP The AF recombinant protein has better immunogenicity and can stimulate the high-efficiency specific antibody of the New Zealand rabbit; and 3, the Ct CPAF Reorganization The protein has good antigenicity, can be specifically combined with the positive serum of a Ct-infected person, can be applied to the serological diagnosis of Ct,
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2007
【分类号】：R374;R446.6

【相似文献】