解脲脲原体MB抗原保守区段的表达与抗体制备及初步应用

发布时间：2017-12-31 14:19

  本文关键词：解脲脲原体MB抗原保守区段的表达与抗体制备及初步应用　出处：《南华大学》2006年硕士论文　论文类型：学位论文

  更多相关文章： 解脲脲原体 重组蛋白 表达与纯化 MB抗原保守区段 多克隆抗体

【摘要】：目的：构建含解脲脲原体(Ureaplsama urealyticum，Uu)膜蛋白MB抗原保守区段(20～130aa)基因的重组表达载体，在大肠杆菌中进行诱导表达，纯化产物免疫新西兰兔获得特异性的多克隆抗体(PcAb)，检测Uu感染的临床标本，分析其敏感性与特异性，为制备Uu临床检测试剂盒奠定实验基础。 方法：通过生物信息学分析，筛选MB抗原保守区基因片段优势抗原表位，以Uu1型血清型标准株基因组DNA为模板，通过聚合酶链反应(PCR)扩增目的片段，将其克隆至原核表达载体pQE30，构建MB抗原基因保守区段重组表达质粒pQE30／partMBA，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对表达产物进行分析和鉴定；镍琼脂糖凝胶F．F．亲和层析柱纯化重组蛋白，BCA法测定纯化蛋白浓度。用纯化的含MB抗原保守区段的重组蛋白免疫新西兰兔，间接ELISA方法检测免疫兔血清中抗MB抗原保守区段PcAb的效价，对表达的MB抗原保守区段重组蛋白的免疫原性进行分析。然后用制备的特异性PcAb为一抗，将收集的临床尿液标本处理后包被微孔板，，建立间接ELISA方法检测临床标本中Uu，同时与培养法进行比较。根据PcAb检测临床样本中Uu的敏感性与特异性，评价重组蛋白制备的特异PcAb在临床标本检测中的应用价值。 结果：软件分析MB抗原的抗原性、疏水性和跨膜区，选取了MB抗原保守区段基因的第208～540bp位碱基序列为目的基因(片段长度为333bp，编码111
[Abstract]:Objective : To construct a recombinant expression vector containing a conserved segment ( 20 - 130aa ) of Ureaplaria ( Ureapl ) membrane protein MB antigen , induce expression in E . coli , purify the product immunized New Zealand rabbits to obtain the specific polyclonal antibody , and analyze its sensitivity and specificity , and lay a foundation for the preparation of the clinical detection kit . Methods : By bioinformatics analysis , the dominant epitope of MB antigen conserved region gene fragment was screened . The target fragment was amplified by polymerase chain reaction ( PCR ) . The recombinant expression plasmid pQE30 / partMBA was constructed by polymerase chain reaction ( PCR ) , then transformed into prokaryotic expression vector pQE30 . The expression product was analyzed and identified by SDS - PAGE and Western - Blot . F . The purified recombinant protein was purified by affinity chromatography column . The purified protein concentration was determined by BCA method . The titer of the conserved segment of anti - MB antigen in serum of immunized rabbits was analyzed by using purified recombinant protein containing MB antigen conserved segment , and the immunogenicity of the expressed MB antigen conserved segment recombinant protein was analyzed . Results : The antigenicity , hydrophobicity and transmembrane domains of MB antigen were analyzed by software . The nucleotide sequence of 208 锝

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