本文選題：幽門螺桿菌 + ureA　； 參考：《山東理工大學》2010年碩士論文

【摘要】： 幽門螺桿菌是導致人類多種消化道疾病的致病原,有廣泛地傳染性,臨床難以進行徹底的治療,利用免疫保護是預(yù)防和治療幽門螺桿菌感染的重要方法。目的為研究幽門螺桿菌(Helicobacter pylori)尿素酶A亞單位的免疫原性和交叉免疫保護作用,本實驗克隆了幽門螺桿菌26695菌株尿素酶A亞單位編碼基因ure A;構(gòu)建原核表達載體,表達并純化了ureA蛋白；用純化蛋白免疫家兔,制備抗血清,研究抗體特異性和交叉免疫保護作用。為進一步探討ureA蛋白免疫保護作用及單克隆抗體用于治療或預(yù)防幽門螺桿菌引發(fā)的相關(guān)疾病奠定基礎(chǔ)。 方法(1)用PCR方法從幽門螺桿菌26695菌株基因組中擴增ureA基因片段；并將其克隆至pMD18-T Vector上,轉(zhuǎn)化大腸桿菌DH5α,進行鑒定；再將目的基因引入pBV220-ILl表達載體中,構(gòu)建pBV220-IL1-ureA表達重組載體,進行表達載體鑒定、基因序列測定和生物信息學比對。(2)溫度誘導含重組載體pBV220-ILl-ureA的工程菌DH5α,表達目的蛋白；SDS-PAGE分析ureA蛋白表達結(jié)果；Western-blotting進行目的蛋白的鑒定；膠制備法大量分離純化蛋白。(3)將純化蛋白與弗氏佐劑混合免疫家兔,制備抗血清；ELISA法檢測家兔抗血清效價及與免疫原蛋白UreA蛋白的結(jié)合特異性；利用競爭性結(jié)合實驗鑒定兔抗血清與病人陽性血清交叉保護作用。 結(jié)果(1)PCR特異性擴增獲得全長714bp目的片段,測序后與GenBank數(shù)據(jù)庫比對,確定目的片段為幽門螺桿菌26695菌株ureA基因序列,同源性高達99%。 (2)獲得pBV220-IL1-ureA原核重組表達質(zhì)粒,工程菌經(jīng)42℃熱誘導,獲得包涵體表達的ureA蛋白,融合蛋白分子量約為41kD,膠制備法獲得了大量純化蛋白,濃度約為1.Omg/mL。(3)將純化的ureA蛋白用于家兔免疫,獲得家兔抗血清,效價為1：1.28×104。ELISA法檢測家兔抗血清與病人抗血清具有良好的競爭性。 結(jié)論(1)成功構(gòu)建了pBV220-ILl-ureA重組質(zhì)粒,插入序列經(jīng)測序證實正確,無移碼現(xiàn)象,與GenBank公布的基因序列高度同源。(2)成功表達并純化了ureA蛋白,且其具有良好的免疫原性,為動物免疫奠定基礎(chǔ)。(3)所獲家兔抗血清與ureA蛋白具有良好的特異結(jié)合性。(4)競爭性實驗顯示,兔抗血清和人陽性血清具有交叉反應(yīng),能夠產(chǎn)生競爭性的免疫保護作用。這為利用ureA多抗或單抗進行免疫預(yù)防和治療幽門螺桿菌感染導致的疾病奠定了實驗基礎(chǔ)。
[Abstract]:Helicobacter pylori is the causative agent of many digestive tract diseases in human beings, which is widely contagious and difficult to be treated thoroughly. The use of immune protection is an important method to prevent and treat Helicobacter pylori infection. Objective to study the immunogenicity and cross-immune protection of urease A subunit of Helicobacter pylori (pylori), we cloned the urease A subunit encoding gene ure A from Helicobacter pylori 26695 strain and constructed a prokaryotic expression vector. The ureA protein was expressed and purified, the rabbit was immunized with the purified protein, and the antiserum was prepared to study the specificity of antibody and the protective effect of cross immunity. To further explore the immune protection of ureA protein and monoclonal antibodies used in the treatment or prevention of Helicobacter pylori related diseases. Methods (1) ureA gene fragment was amplified from the genome of Helicobacter pylori 26695 strain by PCR, cloned into pMD18-T vector, transformed into Escherichia coli DH5 偽 for identification, and then the target gene was introduced into pBV220-ILl expression vector to construct pBV220-IL1-ureA expression vector. Expression vector identification, gene sequencing and bioinformatics comparison were carried out. (2) temperature induced recombinant vector pBV220-ILl-ureA engineering strain DH5 偽, expressed target protein was analyzed by SDS-PAGE analysis ureA protein expression results were identified by Western-blotting. (3) immunized rabbits with purified protein and Freund's adjuvant. The antiserum was prepared to detect the titer of rabbit antiserum and its binding specificity to UreA protein. The cross protective effect of rabbit antiserum and patient positive serum was evaluated by competitive binding experiment. Results (1) the full-length 714bp fragment was amplified by PCR and compared with the GenBank database. The target fragment was confirmed to be the ureA gene sequence of Helicobacter pylori 26695 strain. (2) the prokaryotic expression plasmid pBV220-IL1-ureA was obtained. After heat induction at 42 鈩，

本文編號：2114508