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  本文選題：大腸桿菌不耐熱腸毒素B亞單位　切入點：表達　出處：《蘭州大學》2006年碩士論文

【摘要】：目前所知道的最強的粘膜免疫抗原當屬霍亂毒素(cholera toxin CT)與大腸桿菌不耐熱腸毒素(heat-labile enterotoxin,LT),但是兩者分別能引起典型的霍亂樣腹瀉,及旅行者腹瀉。同時它具有很強的潛在的免疫原性及粘膜免疫佐劑效應,以他們作為佐劑能夠顯著刺激增強機體對抗原的免疫反應,但是其毒性限制了其的應用,隨著LT的三維結(jié)構(gòu)的獲悉,推動了人們對其B亞單位及其減毒突變體的研究,期望得到具有很強的粘膜佐劑活性,同時其毒性具有相對較低甚至沒有毒性的突變體。 本實驗的目的是為了研究大腸桿菌不耐熱腸毒素(Escherichia coli heat-labile enterotoxin(LT))B亞單位(LTB)的免疫佐劑活性。從產(chǎn)腸毒素大腸桿菌44815#菌株中提取其大質(zhì)粒DNA,根據(jù)GENE BANK的序列設(shè)計引物調(diào)出LTB的原始基因,并將該基因通過PCR的手段進行擴增。進而將該基因克隆入pMD 18-T vector,通過PCR及雙酶切進行篩選鑒定,陽性克隆測序。將正確的基因克隆到原核表達載體pET-21b(+)中,得到重組原核表達株(pET21b-LTB),經(jīng)氯化鈣法轉(zhuǎn)化大腸桿菌BL21(DE3),通過PCR及IPTG誘導表達經(jīng)過SDS-PAGE篩選陽性克隆,命名為pET21b-LTB。表達產(chǎn)物經(jīng)SDS-PAGE分析,分子量為14KD,與理論值相符。凝膠灰度掃描顯示pET21b-LTB表達量約占菌體總蛋白的16%。 將構(gòu)建好的重組菌株進行擴大培養(yǎng),離心收集細胞,經(jīng)超聲破碎后離心收集破碎上清、通過陽離子交換柱(CM-FF)對目的蛋白進行初步純化。以該純化蛋白為佐劑,將其分別與流感三價疫苗,Ⅱ型皰疹病毒(HSV-Ⅱ)gD糖蛋白混合后通過腹腔注射及鼻飼法免疫BALB/c小鼠,設(shè)單純鋁佐劑免疫為對照。免疫2,4,6周后尾靜脈采血,ELISA法檢測其抗體水平,檢測抗體水平升高后。將小鼠摘除眼球采血,全部處死后取其鼻、肺、腸及陰道洗液檢測sIgA抗體水平,取其血清檢測IgA、IgM、IgG抗體水平。通過與氫氧化鋁佐劑對比表明其滴度顯著高于氫氧化鋁佐劑對照組。 結(jié)果表明:重組大腸桿菌不耐熱腸毒素B亞單位具有粘膜免疫佐劑功效,該研究結(jié)果為今后LTB的研究及應用奠定了基礎(chǔ)。
[Abstract]:The strongest known mucosal immune antigens are cholera toxin cholera toxin and Escherichia coli heat-labile enterotoxin in LTL, but they can cause typical cholera diarrhea, respectively. And traveller diarrhea. It also has a strong potential immunogenicity and mucosal immune adjuvant effect, using them as adjuvants can significantly stimulate the body's immune response to antigens, but its toxicity limits its use. With the knowledge of the three-dimensional structure of LT, the study of its subunit B and its attenuated mutants is promoted, and it is expected to have strong mucosal adjuvant activity, and the mutants with relatively low toxicity or no toxicity are expected to be obtained. The aim of this study was to study the immunoadjuvant activity of Escherichia coli heat-labile enterotoxin(LT))B subunit (LTBs), to extract its large plasmid DNA from Enterotoxigenic Escherichia coli 44815# strain, and to design primers according to the sequence of GENE BANK to extract the original LTB gene. The gene was amplified by PCR, then cloned into pMD 18-T vector, screened by PCR and double enzyme digestion, and sequenced. The correct gene was cloned into the prokaryotic expression vector pET-21b (). The recombinant prokaryotic expression strain pET21b-LTBN was obtained and transformed into Escherichia coli BL21DE3 by calcium chloride method. The expression was induced by PCR and IPTG and the positive clone was screened by SDS-PAGE. The expression product was named pET21b-LTB.The expression product was analyzed by SDS-PAGE. The molecular weight was 14kD, which was consistent with the theoretical value. Gel gray-scale scanning showed that the expression of pET21b-LTB was about 16% of the total bacterial protein. The recombinant strain was expanded and cultured, the cells were collected by centrifugation, the supernatant was collected by centrifugation after ultrasonic crushing, and the target protein was preliminarily purified by cationic exchange column CM-FF.The purified protein was used as adjuvant. BALB/c mice were immunized with influenza trivalent vaccine and herpesvirus type 鈪，

本文編號：1667639