發(fā)布時(shí)間：2018-02-13 09:34

  本文關(guān)鍵詞： 猬迭宮絳蟲 裂頭蚴 半胱氨酸蛋白酶 表達(dá)條件優(yōu)化 組織定位　出處：《貴州醫(yī)科大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：目的:克隆、表達(dá)猬迭宮絳蟲27kDa半胱氨酸蛋白酶(Cysteine Protease,CP)基因,優(yōu)化原核表達(dá)條件,并應(yīng)用免疫熒光技術(shù)檢測27kDa-CP在成蟲、裂頭蚴不同組織中的分布情況。方法:(1)根據(jù)NCBI中公布的猬裂頭蚴27kDa半胱氨酸蛋白酶基因序列(27kDa-CP,D63670),提取蛇源猬裂頭蚴總RNA,逆轉(zhuǎn)錄合成cDNA,應(yīng)用PCR技術(shù)對目的片段進(jìn)行體外擴(kuò)增,并克隆入原核表達(dá)載體pET-28a(+),構(gòu)建pET-28a(+)-27kDa-CP重組質(zhì)粒,轉(zhuǎn)入大腸埃希菌Transetta(DE3)中,觀察在不同培養(yǎng)時(shí)間、不同濃度異丙基硫代-β-D-半乳糖苷(IPTG)及不同培養(yǎng)溫度條件下27kDa-CP重組蛋白的表達(dá)情況。用鎳離子柱親和層析法純化目的蛋白,表達(dá)產(chǎn)物用SDS-PAGE及Western Blotting進(jìn)行分析。(2)用純化后的重組27kDa-CP蛋白免疫小鼠,制備多克隆抗體(一抗),用異硫氰酸熒光素(FITC)標(biāo)記的羊抗小鼠IgG作為二抗,對27kDa-CP在猬迭宮絳蟲成蟲、裂頭蚴組織中的位置分布進(jìn)行免疫熒光定位。結(jié)果:(1)pET-28a(+)-27kDa-CP重組質(zhì)粒經(jīng)IPTG誘導(dǎo)后,蛋白在大腸埃希菌Transetta(DE3)中成功表達(dá),最適表達(dá)條件為:培養(yǎng)溫度30℃,IPTG濃度為0.3mmol/L,培養(yǎng)時(shí)間為18h。重組蛋白主要以包涵體形式表達(dá),純化后測得其濃度為0.2mg/mL。(2)純化后的重組蛋白經(jīng)Western Blotting檢測,其相對分子質(zhì)量大小(Mr)約為35kDa,與預(yù)期大小相符,且能被重組蛋白免疫小鼠血清識(shí)別。(3)免疫熒光定位顯示27kDa-CP在裂頭蚴皮層及排泄管中呈高度表達(dá);在成蟲中,以節(jié)片(孕節(jié)和成節(jié))子宮內(nèi)蟲卵卵殼的表達(dá)最強(qiáng),其次為排泄管道及皮層。結(jié)論:(1)成功構(gòu)建了猬迭宮絳蟲27kDa-CP基因原核表達(dá)體系,通過表達(dá)條件的優(yōu)化,在大腸埃希菌Transetta(DE3)中27kDa-CP重組蛋白可獲得高效表達(dá)。(2)通過免疫熒光技術(shù)組織定位初步提示27kDa-CP可能參與了蟲體的入侵、移行、營養(yǎng)吸收、排泄、免疫逃避、生長發(fā)育及生殖等過程。
[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，

本文編號(hào)：1507851