大肠杆菌不耐热肠毒素B亚单位的克

发布时间：2018-03-26 11:08

  本文选题：大肠杆菌不耐热肠毒素B亚单位　切入点：表达　出处：《兰州大学》2006年硕士论文

【摘要】：目前所知道的最强的粘膜免疫抗原当属霍乱毒素(cholera toxin CT)与大肠杆菌不耐热肠毒素(heat-labile enterotoxin,LT),但是两者分别能引起典型的霍乱样腹泻,及旅行者腹泻。同时它具有很强的潜在的免疫原性及粘膜免疫佐剂效应,以他们作为佐剂能够显著刺激增强机体对抗原的免疫反应,但是其毒性限制了其的应用,随着LT的三维结构的获悉,推动了人们对其B亚单位及其减毒突变体的研究,期望得到具有很强的粘膜佐剂活性,同时其毒性具有相对较低甚至没有毒性的突变体。 本实验的目的是为了研究大肠杆菌不耐热肠毒素(Escherichia coli heat-labile enterotoxin(LT))B亚单位(LTB)的免疫佐剂活性。从产肠毒素大肠杆菌44815#菌株中提取其大质粒DNA,根据GENE BANK的序列设计引物调出LTB的原始基因,并将该基因通过PCR的手段进行扩增。进而将该基因克隆入pMD 18-T vector,通过PCR及双酶切进行筛选鉴定,阳性克隆测序。将正确的基因克隆到原核表达载体pET-21b(+)中,得到重组原核表达株(pET21b-LTB),经氯化钙法转化大肠杆菌BL21(DE3),通过PCR及IPTG诱导表达经过SDS-PAGE筛选阳性克隆,命名为pET21b-LTB。表达产物经SDS-PAGE分析,分子量为14KD,与理论值相符。凝胶灰度扫描显示pET21b-LTB表达量约占菌体总蛋白的16%。 将构建好的重组菌株进行扩大培养,离心收集细胞,经超声破碎后离心收集破碎上清、通过阳离子交换柱(CM-FF)对目的蛋白进行初步纯化。以该纯化蛋白为佐剂,将其分别与流感三价疫苗,Ⅱ型疱疹病毒(HSV-Ⅱ)gD糖蛋白混合后通过腹腔注射及鼻饲法免疫BALB/c小鼠,设单纯铝佐剂免疫为对照。免疫2,4,6周后尾静脉采血,ELISA法检测其抗体水平,检测抗体水平升高后。将小鼠摘除眼球采血,全部处死后取其鼻、肺、肠及阴道洗液检测sIgA抗体水平,取其血清检测IgA、IgM、IgG抗体水平。通过与氢氧化铝佐剂对比表明其滴度显著高于氢氧化铝佐剂对照组。 结果表明:重组大肠杆菌不耐热肠毒素B亚单位具有粘膜免疫佐剂功效,该研究结果为今后LTB的研究及应用奠定了基础。
[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，

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