本文選題：弓形蟲 + P30�。� 參考：《青島大學》2007年碩士論文

【摘要】： 目的構建剛地弓形蟲(Toxoplasma gondii)表膜蛋白P30基因的表達載體，在大腸桿菌中誘導表達，純化表達產物P30并進行抗原性分析，研究P30在弓形蟲病血清學診斷中的應用價值。 方法(1)P30蛋白的表達、純化與復性巨噬細胞培養(yǎng)弓形蟲，提取弓形蟲DNA為模板，PCR擴增目的片斷，將目的片斷克隆至pUCm-T Vector，酶切后用PCR鑒定，再亞克隆到原核表達載體pET28b(+)中、構建重組質粒pET28b(+)／P30，經(jīng)酶切、PCR及測序鑒定后轉化至表達宿主菌BL21(DE3)中誘導表達，SDS-PAGE和Western-Blot鑒定表達產物；包涵體經(jīng)8M尿素變性，Ni-NTA親和層析柱純化重組蛋白，變性蛋白經(jīng)稀釋透析復性，SDS-PAGE和Western-Blot分析和鑒定復性P30蛋白，BCA法測定復性蛋白濃度，用于ELISA包板。(2)小鼠弓形蟲抗血清的制備及臨床弓形蟲病患者陽性血清的收集純化的弓形蟲速殖子經(jīng)～80℃反復冷凍3次，制備抗原，免疫昆明小鼠，制備小鼠弓形蟲陽性血清。(3)弓形蟲病人IgG抗體ELISA檢測方法的建立以純化復性后的P30為抗原，包板，用間接ELISA檢測臨床疑似弓形蟲病人，，用制備的小鼠弓形蟲陽性血清為對照，并同時與國外試劑盒檢測結果比較，以評價純化的P30在弓形蟲病診斷中的應用價值。 結果1．PCR擴增得到長800bp的目的片斷，測序結果與Genbank上登錄序列一致；2．含pET28b(+)／P30重組菌經(jīng)IPTG誘導，SDS-PAGE電泳結果顯示，得到一分子量(Mr)約30 kDa的重組蛋白，目的蛋白在菌體細胞內以包涵體形式存在；3．8M尿素溶解后經(jīng)Ni-NTA親和柱純化獲得了純度大于95％的重組蛋白；4．復性后，得到溶解狀態(tài)的復性蛋白，Western-Blot結果顯示，該蛋白能被抗6—His單抗、小鼠抗弓形蟲血清和部分臨床弓形蟲病患者陽性血清識別；5．以純化的P30重組蛋白為包被抗原建立間接ELISA法，檢測小鼠抗弓形蟲血清和臨床弓形蟲病患者陽性血清，與進口試劑盒檢測的結果比較，結果：靈敏度為93.3％，特異度為100％，ELISA法與進口試劑盒的總符合率為96.7％。 結論 1．獲得了高純度的弓形蟲P30復性蛋白； 2．復性的P30蛋白具有較好的抗原性，能與小鼠抗弓形蟲陽性血及部分臨床疑似弓形蟲病患者發(fā)生特異性反應，可用于弓形蟲病人血清學診斷。
[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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