【摘要】：目的 構(gòu)建含剛地弓形蟲(chóng)(Toxoplasma gondii)表膜蛋白P30基因的表達(dá)載體，，在大腸桿菌中誘導(dǎo)表達(dá)，純化表達(dá)產(chǎn)物P30并進(jìn)行抗原性分析，研究P30在弓形蟲(chóng)病血清學(xué)診斷中的應(yīng)用價(jià)值。 方法 (1)P30蛋白的表達(dá)、純化與復(fù)性 巨噬細(xì)胞培養(yǎng)弓形蟲(chóng)，提取弓形蟲(chóng)DNA為模板，PCR擴(kuò)增目的片斷，將目的片斷克隆至pUCm-T Vector，酶切和PCR鑒定，再亞克隆到原核表達(dá)載體pET28b(+)中、構(gòu)建重組質(zhì)粒pET28b(+)／P30，經(jīng)酶切、PCR及測(cè)序鑒定后轉(zhuǎn)化至表達(dá)宿主菌BL21(DE3)中誘導(dǎo)表達(dá)，SDS-PAGE和Western-Blot鑒定表達(dá)產(chǎn)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(fù)性，SDS-PAGE和Western-Blot分析和鑒定復(fù)性P30蛋白，BCA法測(cè)定復(fù)性蛋白濃度，用于ELISA包板。(2)小鼠弓形蟲(chóng)抗血清的制備及臨床弓形蟲(chóng)病患者陽(yáng)性血清的收集 弓形蟲(chóng)速殖子經(jīng)-80℃反復(fù)冷凍3次，經(jīng)減毒后感染昆明小鼠，制備小鼠弓形蟲(chóng)陽(yáng)性血清，收集臨床弓形蟲(chóng)病人血清。(3)弓形蟲(chóng)病人IgG抗體ELISA檢測(cè)方法的建立以純化復(fù)性后的P30包板，間接ELISA檢測(cè)臨床弓形蟲(chóng)病人血清中抗弓形蟲(chóng)的抗體，以小鼠弓形蟲(chóng)陽(yáng)性血清為對(duì)照，同時(shí)與國(guó)外進(jìn)口試劑盒的檢測(cè)結(jié)果比較，評(píng)價(jià)純化的P30在弓形蟲(chóng)病血清學(xué)診斷中的應(yīng)用價(jià)值。 結(jié)果 PCR擴(kuò)增得到長(zhǎng)800bp的目的片斷，測(cè)序結(jié)果與Genbank上登錄序列一致；含pET28b(+)／P30重組菌經(jīng)IPTG誘導(dǎo)，SDS-PAGE電泳結(jié)果顯示，得到一分子量(M_r)約30 kDa的重組蛋白，目的蛋白在菌體細(xì)胞內(nèi)以包涵體形式存在；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；
[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

本文編號(hào)：2400997