梅毒螺旋体Tp0453重组蛋白的表达、纯化及免疫活性研究

发布时间：2018-03-08 20:15

  本文选题：梅毒螺旋体　切入点：重组蛋白　出处：《南华大学》2005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Tp0453的优势表位(28-288aa)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免疫反应性分析,为探索Tp0453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实验依据。 方法:通过生物信息学分析,筛选并挑选Tp0453基因优势抗原表位,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pQE32中、构建重组质粒pQE32/Tp0453,然后转化至表达宿主菌M15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Tp0453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Tp0453重组蛋白免疫新西兰兔,间接ELISA方法检测免疫兔血清中Tp0453多克隆抗体的效价,对Tp0453重组蛋白的免疫原性进行分析。 结果:软件分析Tp0453基因的抗原表位选择了Tp0453基因的86-849bp位碱基序列为目的表位(片段长度为764bp,编码255个氨基酸);PCR扩增得到以大小约为800bp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Tp0453目的基因,测序结果与Genbank上登录序列完全一致;
[Abstract]:Objective: to construct the recombinant expression of the outer membrane protein Tp0453 containing Treponema pallidum TpP, and express it in Escherichia coli, purify the expressed product and analyze its immunogenicity and immunoreactivity. To explore the application value and biological function of Tp0453 recombinant protein in syphilis serological diagnosis. Methods: the dominant epitopes of Tp0453 gene were screened and selected by bioinformatics analysis. Using the genomic DNA of TP Nichols strain as template, the target fragment was amplified by high fidelity polymerase chain reaction and subcloned into prokaryotic expression vector pQE32. The recombinant plasmid pQE32 / Tp0453 was constructed and transformed into M15 to induce expression. The recombinant protein was purified by SDS-PAGE and Western-Blot. The concentration of purified protein was determined by dilution dialysis renaturation method. The purified Tp0453 recombinant protein was coated with micropore plate. Indirect ELISA method was established to detect syphilis reference serum and clinical syphilis serum, and compared with TPPA method. According to the reaction between recombinant protein and syphilis yin-positive serum, the application value of recombinant antigen in syphilis serological diagnosis was evaluated. New Zealand rabbits were immunized with purified recombinant Tp0453 protein. Indirect ELISA method was used to detect the titer of Tp0453 polyclonal antibody in serum of immunized rabbits and the immunogenicity of Tp0453 recombinant protein was analyzed. Results: the epitope of Tp0453 gene was analyzed by software. The 86-849bp base sequence of Tp0453 gene was selected as the target epitope (the fragment length was 764 BP, encoding 255 amino acids) to amplify the target fragment of 800 BP. 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】：2005
【分类号】：R377

【引证文献】

相关硕士学位论文 前1条

1 严加林;梅毒螺旋体Gpd重组蛋白的表达、纯化及免疫活性研究[D];南华大学;2006年

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