弓形虫P30重组蛋白的表达、纯化及诊断应用研究

发布时间：2019-01-05 00:16

【摘要】：目的 构建含刚地弓形虫(Toxoplasma gondii)表膜蛋白P30基因的表达载体，，在大肠杆菌中诱导表达，纯化表达产物P30并进行抗原性分析，研究P30在弓形虫病血清学诊断中的应用价值。 方法 (1)P30蛋白的表达、纯化与复性 巨噬细胞培养弓形虫，提取弓形虫DNA为模板，PCR扩增目的片断，将目的片断克隆至pUCm-T Vector，酶切和PCR鉴定，再亚克隆到原核表达载体pET28b(+)中、构建重组质粒pET28b(+)／P30，经酶切、PCR及测序鉴定后转化至表达宿主菌BL21(DE3)中诱导表达，SDS-PAGE和Western-Blot鉴定表达产物；包涵体经8M尿素变性，Ni-NTA亲和层析柱纯化重组蛋白，变性蛋白经稀释透析复性，SDS-PAGE和Western-Blot分析和鉴定复性P30蛋白，BCA法测定复性蛋白浓度，用于ELISA包板。(2)小鼠弓形虫抗血清的制备及临床弓形虫病患者阳性血清的收集 弓形虫速殖子经-80℃反复冷冻3次，经减毒后感染昆明小鼠，制备小鼠弓形虫阳性血清，收集临床弓形虫病人血清。(3)弓形虫病人IgG抗体ELISA检测方法的建立以纯化复性后的P30包板，间接ELISA检测临床弓形虫病人血清中抗弓形虫的抗体，以小鼠弓形虫阳性血清为对照，同时与国外进口试剂盒的检测结果比较，评价纯化的P30在弓形虫病血清学诊断中的应用价值。 结果 PCR扩增得到长800bp的目的片断，测序结果与Genbank上登录序列一致；含pET28b(+)／P30重组菌经IPTG诱导，SDS-PAGE电泳结果显示，得到一分子量(M_r)约30 kDa的重组蛋白，目的蛋白在菌体细胞内以包涵体形式存在；8M尿素溶解后经Ni-NTA亲和柱纯化获得了纯度大于95％的重组蛋白；
[Abstract]:Objective to construct the expression vector containing P30 gene of (Toxoplasma gondii) surface membrane protein of Toxoplasma gondii, express it in Escherichia coli, purify the expression product P30 and analyze its antigenicity, and study the application value of P30 in the serological diagnosis of Toxoplasma gondii. Methods (1) expression of P30 protein, culture of Toxoplasma gondii by purification and renaturation of macrophages, extraction of Toxoplasma gondii DNA as template, amplification of PCR fragment, cloning of the target fragment into pUCm-T Vector, digestion and PCR identification. The recombinant plasmid pET28b () / P30 was constructed by subcloning into prokaryotic expression vector pET28b (). The recombinant plasmid pET28b () / P30 was transformed into BL21 (DE3) by restriction endonuclease digestion, PCR and sequencing. The expression products were identified by SDS-PAGE and Western-Blot. The inclusion bodies were denatured with 8m urea and purified by Ni-NTA affinity chromatography. The denatured proteins were renatured by dilution and dialysis. The P30 protein was analyzed and identified by SDS-PAGE and Western-Blot. The concentration of renatured proteins was determined by BCA method. It was used for ELISA plate. (2) preparation of Toxoplasma gondii antiserum in mice and collection of Toxoplasma gondii positive sera from clinical patients with Toxoplasma gondii. Toxoplasma gondii Tachyzoites were frozen for 3 times at -80 鈩

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