梅毒螺旋体Gpd重组蛋白的表达、纯化及免疫活性研究

发布时间：2018-01-22 05:27

本文关键词： 梅毒螺旋体重组蛋白蛋白纯化 Gpd 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：构建含梅毒螺旋体(Treponema pallidum，Tp)外膜蛋白Gpd(Glycerophosphodiester Phosphodiesterase)1～353位氨基酸编码基因的重组表达载体，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免疫反应性分析，为探索Gpd重组蛋白在梅毒血清学诊断中的应用价值和其生物学功能提供一定的实验依据。方法：通过生物信息学分析，筛选并挑选Gpd基因优势抗原表位片段，以Tp Nichols株基因组DNA为模板，高保真聚合酶链反应扩增目的片断，将其亚克隆进原核表达载体pET28b(+)中、构建重组质粒pET28b(+)／Gpd，然后转化至表达宿主菌RIL中进行诱导表达，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。用纯化的Gpd重组蛋白包被微孔板，建立间接ELISA方法，检测梅毒参比血清和临床梅毒患者血清，同时与TPPA法进行比较，，根据重组蛋白与梅毒阴阳性血清的反应情况，评价重组抗原在梅毒血清学诊断中的应用价值。同时用纯化的Gpd重组蛋白免疫新西兰兔，间接ELISA方法检测免疫兔血清中Gpd多克隆抗体的效价，对Gpd重组蛋白的免疫原性进行分析。结果：软件分析Gpd基因的抗原表位，选择了Gpd基因的1～1059bp位碱基序列为目的基因片段(片段长度为1059bp，编码353个氨基酸)；PCR扩增得到以大小约为1059bp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Gpd目的基因，测序结果与Genbank上登录序列完全一致；
[Abstract]:Objective: to construct Treponema pallidum containing Treponema pallidum. TP) outer membrane protein Gpd(Glycerophosphodiester Phosphodiesterase. The recombinant expression vector of amino acid coding gene at position 1 ~ (353). The expression was induced in Escherichia coli, the expression product was purified and the immunogenicity and immunoreactivity were analyzed. To explore the application value and biological function of Gpd recombinant protein in the serological diagnosis of syphilis. Methods: the epitopes of Gpd gene were screened and selected by bioinformatics analysis. The genomic DNA of TP Nichols strain was used as template. The target fragment was amplified by high fidelity polymerase chain reaction and subcloned into prokaryotic expression vector pET28b. the recombinant plasmid pET28bwas constructed. Then it was transformed into the expression host strain RIL to induce the expression, and the expression product was analyzed and identified by SDS-PAGE and Western-Blot. The recombinant protein was purified by Ni-NTA affinity chromatography and the concentration of purified protein was determined by dilution dialysis renaturation. The purified Gpd recombinant protein was coated with micropore plate to establish an indirect ELISA method. To detect syphilis reference serum and clinical syphilis patient serum, and compare with TPPA method, according to the reaction of recombinant protein and syphilis yin-yang serum. To evaluate the application value of recombinant antigen in the serological diagnosis of syphilis and immunize New Zealand rabbits with purified Gpd recombinant protein. Indirect ELISA method was used to detect the titer of polyclonal antibody against Gpd in sera of immunized rabbits. The immunogenicity of Gpd recombinant protein was analyzed. Results: the antigenic epitopes of Gpd gene were analyzed by software, and the target gene fragment (1059bp) was selected as the target gene sequence of Gpd gene (1: 1059bp). Encoding 353 amino acids; The target fragment of 1059bp was obtained by PCR amplification. The recombinant plasmid was identified by restriction endonuclease digestion and sequencing. The result of sequencing was identical with that of Genbank.
【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】：R377

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