弓形虫P30重组蛋白的表达、纯化与复性

发布时间：2018-07-05 18:00

  本文选题：弓形虫 + P30　； 参考：《青岛大学》2007年硕士论文

【摘要】： 目的构建刚地弓形虫(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，酶切后用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纯化的弓形虫速殖子经～80℃反复冷冻3次，制备抗原，免疫昆明小鼠，制备小鼠弓形虫阳性血清。(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为抗原，包板，用间接ELISA检测临床疑似弓形虫病人，，用制备的小鼠弓形虫阳性血清为对照，并同时与国外试剂盒检测结果比较，以评价纯化的P30在弓形虫病诊断中的应用价值。 结果1．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；2．含pET28b(+)／P30重组菌经IPTG诱导，SDS-PAGE电泳结果显示，得到一分子量(Mr)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；3．8M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；4．复性后，得到溶解状态的复性蛋白，Western-Blot结果显示，该蛋白能被抗6—His单抗、小鼠抗弓形虫血清和部分临床弓形虫病患者阳性血清识别；5．以纯化的P30重组蛋白为包被抗原建立间接ELISA法，检测小鼠抗弓形虫血清和临床弓形虫病患者阳性血清，与进口试剂盒检测的结果比较，结果：灵敏度为93.3％，特异度为100％，ELISA法与进口试剂盒的总符合率为96.7％。 结论 1．获得了高纯度的弓形虫P30复性蛋白； 2．复性的P30蛋白具有较好的抗原性，能与小鼠抗弓形虫阳性血及部分临床疑似弓形虫病患者发生特异性反应，可用于弓形虫病人血清学诊断。
[Abstract]:Objective to construct the expression vector of surface membrane protein P30 gene of Toxoplasma gondii (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 toxoplasmosis. Methods (1) expression of P30 protein, purification and culture of Toxoplasma gondii by renaturation of macrophages, extraction of Toxoplasma gondii DNA as template for PCR amplification, cloning of the target fragment into pUCm-T Vector. after restriction endonuclease digestion, it was identified by PCR and subcloned into prokaryotic expression vector pET28b (). The recombinant plasmid pET28b () / P30 was constructed and transformed into the expression host strain BL21 (DE3) by restriction endonuclease polymerase chain reaction (PCR) and sequencing. The recombinant protein was purified by 8M urea denatured Ni-NTA affinity chromatography column, and the recombinant protein was identified by SDS-PAGE and Western-Blot. Denatured protein was determined by SDS-PAGE and Western-Blot method. It was used for Elisa plate. (2) preparation of Toxoplasma gondii antiserum in mice and collection and purification of Toxoplasma gondii Tachyzoites collected and purified from clinical patients with Toxoplasma gondii. Tachyzoites of Toxoplasma gondii were frozen repeatedly at 80 鈩

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