生殖支原体MgPa重组蛋白的表达、纯化及免疫活性研究

发布时间：2018-05-21 00:11

本文选题：生殖支原体 + MgPa　；参考：《南华大学》2006年硕士论文

【摘要】：目的：构建生殖支原体(Mycoplasma genitalium，Mg)黏附蛋白MgPa的优势表位(1075～1364 aa)基因(MgPa′)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MgPa重组蛋白在Mg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MgPa基因优势抗原表位，以Mg G-37标准株基因组DNA为模板，高保真聚合酶链反应扩增目的片段，将其亚克隆到原核表达载体pET-30a(+)中，构建重组质粒pET-30a(+)／MgPa′，然后转化至表达宿主菌E.coliRosett~(TM)2(DE3)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；用Ni-NTA亲和层析柱纯化重组蛋白，BCA法测定纯化蛋白浓度。用纯化的MgPa重组蛋白(rMgPa′)免疫新西兰兔，间接ELISA方法检测免疫兔血清中MgPa′多克隆抗体的效价，，对rMgPa′的免疫原性进行分析。同时用纯化的rMgPa′包被微孔板，建立间接ELISA方法，检测Mg感染者阳性血清及对照血清，根据重组蛋白与MS阴、阳性血清的反应情况，对rMgPa′的免疫反应性作出评价。结果：Expasy软件分析MgPa基因的抗原表位选择了MgPa′基因的3223～4092bp位碱基序列为目的基因(MgPa′，片段长度为870bp，编码290个aa)；PCR扩增得到大小约为870bp的目的片段；构建的重组质粒经酶切鉴定和测序鉴定证明其中插入片段为MgPa′目的基因，测序结果与Genbank上登录序列完全一
[Abstract]:Objective: to construct the recombinant expression of MgPa, a dominant epitope of Mycoplasma genitalium MgPa, 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 MgPa recombinant protein in mg serological diagnosis. Methods: the dominant epitopes of MgPa gene were screened and selected by bioinformatics analysis. The target fragment was amplified by high fidelity polymerase chain reaction using the genomic DNA of mg G-37 standard strain as template, and subcloned into prokaryotic expression vector pET-30a (). The recombinant plasmid pET-30a (pET-30a) was constructed, and then transformed into E. coli Rosettl TMT 2DE3 to induce the expression. The expression product was analyzed and identified by SDS-PAGE and Western-Blot, and the purified protein was determined by Ni-NTA affinity chromatography. New Zealand rabbits were immunized with purified recombinant MgPa protein rmg Pag. The immunogenicity of rMgPa' was analyzed by indirect ELISA method for detecting the titer of polyclonal antibody against MgPa' in the serum of immunized rabbits. At the same time, using purified rMgPa' coated with microporous plate, an indirect ELISA method was established to detect the positive serum of Mg-infected patients and the control serum. According to the reaction of recombinant protein with MS negative and positive serum, the immunoreactivity of rMgPa' was evaluated. Results the epitope of MgPa gene was analyzed by the: easy software. The 3223~4092bp base sequence of MgPa' gene was selected as the target gene. The length of the fragment was 870bp. the length of 870bp fragment was about 290 AAS. The recombinant plasmid was identified by restriction endonuclease digestion and sequencing. It was proved that the inserted fragment was a MgPa' target gene. The result of sequencing was identical to that of the Genbank login sequence.
【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】：R375

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