猬迭宫绦虫27kDa半胱氨酸蛋白酶基因原核表达条件优化及组织定位研究

发布时间：2018-02-13 09:34

  本文关键词： 猬迭宫绦虫 裂头蚴 半胱氨酸蛋白酶 表达条件优化 组织定位　出处：《贵州医科大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:克隆、表达猬迭宫绦虫27kDa半胱氨酸蛋白酶(Cysteine Protease,CP)基因,优化原核表达条件,并应用免疫荧光技术检测27kDa-CP在成虫、裂头蚴不同组织中的分布情况。方法:(1)根据NCBI中公布的猬裂头蚴27kDa半胱氨酸蛋白酶基因序列(27kDa-CP,D63670),提取蛇源猬裂头蚴总RNA,逆转录合成cDNA,应用PCR技术对目的片段进行体外扩增,并克隆入原核表达载体pET-28a(+),构建pET-28a(+)-27kDa-CP重组质粒,转入大肠埃希菌Transetta(DE3)中,观察在不同培养时间、不同浓度异丙基硫代-β-D-半乳糖苷(IPTG)及不同培养温度条件下27kDa-CP重组蛋白的表达情况。用镍离子柱亲和层析法纯化目的蛋白,表达产物用SDS-PAGE及Western Blotting进行分析。(2)用纯化后的重组27kDa-CP蛋白免疫小鼠,制备多克隆抗体(一抗),用异硫氰酸荧光素(FITC)标记的羊抗小鼠IgG作为二抗,对27kDa-CP在猬迭宫绦虫成虫、裂头蚴组织中的位置分布进行免疫荧光定位。结果:(1)pET-28a(+)-27kDa-CP重组质粒经IPTG诱导后,蛋白在大肠埃希菌Transetta(DE3)中成功表达,最适表达条件为:培养温度30℃,IPTG浓度为0.3mmol/L,培养时间为18h。重组蛋白主要以包涵体形式表达,纯化后测得其浓度为0.2mg/mL。(2)纯化后的重组蛋白经Western Blotting检测,其相对分子质量大小(Mr)约为35kDa,与预期大小相符,且能被重组蛋白免疫小鼠血清识别。(3)免疫荧光定位显示27kDa-CP在裂头蚴皮层及排泄管中呈高度表达;在成虫中,以节片(孕节和成节)子宫内虫卵卵壳的表达最强,其次为排泄管道及皮层。结论:(1)成功构建了猬迭宫绦虫27kDa-CP基因原核表达体系,通过表达条件的优化,在大肠埃希菌Transetta(DE3)中27kDa-CP重组蛋白可获得高效表达。(2)通过免疫荧光技术组织定位初步提示27kDa-CP可能参与了虫体的入侵、移行、营养吸收、排泄、免疫逃避、生长发育及生殖等过程。
[Abstract]:Objective: to clone and express the 27kDa cysteine protease (CPN) gene of Taenia hedgehoi, optimize the prokaryotic expression conditions, and detect the expression of 27kDa-CP in adult by immunofluorescence technique. Methods according to the 27kDa cysteine protease gene sequence published in NCBI, we extracted the total RNAs of serpentine hydatid, synthesized cDNAs by reverse transcription, and amplified the target fragments by PCR in vitro. The recombinant plasmid pET-28a (-27kDa-CP) was cloned into the prokaryotic expression vector pET-28a (pET-28a). The recombinant plasmid pET-28a (-27kDa-CP) was transferred into E. coli TransettaDa-DE3. Expression of 27kDa-CP recombinant protein at different concentrations of isopropylthiothio- 尾 -Dgalactoside (IPTG) and under different culture temperature. The target protein was purified by nickel ion column affinity chromatography. The expressed product was analyzed by SDS-PAGE and Western Blotting.) mice were immunized with purified recombinant 27kDa-CP protein to prepare polyclonal antibody (first antibody) and sheep anti-mouse IgG labeled with fluorescein isothiocyanate (FITCC) as the second antibody. Results the protein was successfully expressed in Escherichia coli TranasettasettaDE3 after the recombinant plasmid was induced by IPTG. The optimal expression conditions were as follows: the culture temperature was 30 鈩，

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