肺炎衣原体OmpA基因VD2-VD3区重组蛋白的表达、纯化及免疫活性研究

发布时间：2018-01-19 05:12

本文关键词： 肺炎衣原体主要外膜蛋白基因表达蛋白纯化免疫活性　出处：《南华大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的：构建含肺炎衣原体(Chlamydia pneumoniae，Cpn)主要外膜蛋白(MOMP)优势表位(147～297aa)基因的重组表达体，在大肠杆菌BL21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，为探索重组蛋白在肺炎衣原体血清学诊断中的应用奠定前期实验基础。方法：通过生物信息学分析，筛选OmpA(MOMP的编码基因)优势抗原表位的编码基因，以Cpn AR-39标准株DNA基因组为模板，Taq聚合酶链反应扩增目的片段，将此片段克隆于pUCm-T载体，，经酶切和PCR鉴定后，目的片段亚克隆于原核表达载体pET30a中，构建重组体pET30a-MOMP_(VD2-VD3)，然后转化至表达宿主菌E．coli BL21(DE3)，经酶切、PCR和测序筛选阳性克隆，挑取单个含重组质粒pET30a-MOMP_(VD2-VD3)的工程菌阳性克隆进行培养和IPTG诱导表达，利用SDS-PAGE和Western-Blot进行分析和鉴定表达产物；Ni-NTA亲和层析柱纯化重组蛋白，并进行稀释透析复性，BCA法测定纯化蛋白浓度。用纯化复性的MOMP_(VD2-VD3)重组蛋白免疫BALB／c小鼠，间接ELISA方法检测免疫鼠血清中MOMP_(VD2-VD3)多克隆抗体的效价，对MOMP_(VD2-VD3)重组蛋白的免疫原性进行分析；并以纯化的MOMP_(VD2-VD3)重组蛋白包被微孔板，建立间接ELISA方法，检测Cpn参比血清和临床冠心病患者血清，同时与晶美公司Cpn IgG ELISA诊断试剂盒检测结果进行比较，根据间接ELISA检测效果，评价重组抗原在Cpn血清学诊断中的应用价值。结果：软件分析OmpA的抗原表位编码基因并选择了OmpA的442～873bp位碱基序列为目的基因(片段长度为432bp，编码144个氨基酸)，PCR扩增
[Abstract]:Objective: to construct Chlamydia pneumoniae containing Chlamydia pneumoniae. The recombinant expression of the dominant epitope of CpN, the main outer membrane protein (MOMP), was induced and expressed in Escherichia coli (E. coli) BL21. The expression products were purified and immunogenicity and antigenicity analysis were carried out, which laid the experimental foundation for exploring the application of recombinant protein in the serological diagnosis of Chlamydia pneumoniae (Chlamydia pneumoniae). Methods: by bioinformatics analysis, the coding genes of dominant antigen epitopes of OmpA(MOMP were screened, and the DNA genome of Cpn AR-39 standard strain was used as template. The target fragment was amplified by Taq polymerase chain reaction and cloned into pUCm-T vector. After restriction endonuclease digestion and PCR identification, the target fragment was subcloned into prokaryotic expression vector pET30a. The recombinant pET30a-MOMPStace VD2-VD3 was constructed and then transformed into E. coli 21 (DE3), which was digested by enzyme. PCR and sequencing were used to screen the positive clones, and a single engineering strain containing recombinant plasmid pET30a-MOMPStace VD2-VD3 was selected for culture and IPTG induced expression. SDS-PAGE and Western-Blot were used to analyze and identify the expressed products. The recombinant protein was purified by Ni-NTA affinity chromatography and refolded by dilution and dialysis. BCA method was used to determine the concentration of purified protein. BALB/c mice were immunized with purified recombinant protein of MOMPS VD2-VD3. The titers of polyclonal antibodies against VD2-VD3 in the serum of immunized mice were detected by indirect ELISA method, and the immunogenicity of the recombinant protein was analyzed. The recombinant protein of MOMPD _ 2-VD3 was coated with micropore plate and indirect ELISA method was established to detect the reference serum of Cpn and the serum of clinical patients with coronary heart disease. At the same time, the results were compared with Cpn IgG ELISA diagnostic kit of Jingmei Company, according to indirect ELISA detection effect. To evaluate the value of recombinant antigen in the serological diagnosis of Cpn. Results: the antigenic epitope encoding gene of OmpA was analyzed by software and the target gene was selected as the target gene (the length of the fragment was 432bp). PCR Amplification of Encoding 144 Amino acids
【学位授予单位】：南华大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R374

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