【作者】 陈明；

【导师】 周光宏； 汤晓艳；

【作者基本信息】 南京农业大学 ， 食品工程， 2012， 硕士

【摘要】 沙门氏菌是一种人畜共患病病原菌,可导致食物中毒,对人类健康造成很大的威胁。本论文通过扩增沙门氏菌fliC基因,构建pET28a-fliC重组子,实现沙门氏菌fliC基因在大肠杆菌中的高效表达,将纯化的融合蛋白免疫BALB/c小鼠,获得多克隆抗体,并对抗血清的效价和特异性进行检测,为后续利用该多抗建立沙门氏菌ELISA快速检测方法奠定基础。本文分三部分进行研究阐述：1鼠伤寒沙门氏菌鞭毛蛋白基因fliC的克隆及其在E.coli中的表达通过PCR方法从鼠伤寒沙门氏菌中扩增出fliC基因,连接到原核表达载体pET28a(+)上,构建pET-28a(+)-fliC重组质粒,然后转化至大肠杆菌BL21(DE3)pLysS,通过酶切鉴定和测序验证后加入IPTG诱导蛋白的表达。实验结果表明,PCR产物与预期的目的基因大小相吻合；构建的重组质粒经酶切鉴定和测序鉴定表明重组质粒构建成功；经IPTG诱导的重组蛋白分子量为55KD,与预期目标蛋白大小相吻合。2鼠伤寒沙门氏菌Ⅰ相鞭毛蛋白的纯化及鉴定用镍金属离子亲和层析对鞭毛融合蛋白进行纯化,然后通过SDS-PAGE检测蛋白纯度和分子量,并通过BCA法和Western-blot分别检测蛋白浓度和蛋白的特异性。实验结果表明,经Ni-NTA纯化的蛋白纯度较高,分子量与目的蛋白相符,经Western-blot分析,表达产物可与抗His-tag单抗在55KD处产生特异性结合,表明表达产物中确实含有His-tag标签,进一步证明表达出的蛋白为目的蛋白。3FliC多克隆抗体的制备将纯化的鞭毛蛋白免疫小鼠,制备抗血清,并用间接ELISA法测定抗体的效价和特异性。实验结果表明,纯化的表达产物能刺激小鼠产生特异性抗体,抗体效价均大于1：32000,并具有较好的特异性和一定的属内广谱性,为后续利用该多抗建立沙门氏菌ELISA决速检测方法奠定了基础。更多还原

【Abstract】 Salmonella was one kind of person and livestock pathogens.It could cause food poisoning and pose the very big threat to the humanity.The main aim of the study was to express FliC fusion protein in.E.coli in high effieiene by cloning the.fliC gene of Salmone lla into an expression plasmid to generate pET-fliC recombinant and expressed. Then the recombinant FliC protein was purified by Ni-NTA affinity chromatography and the polyclonal antibodies were obtained via imunning BALA/c mice with the purified protein. The characteristics of the polyclonal antibodies such as titer and the specificity were detected. This research would be helpful for the further study on establishing immunologic detection of Salmonella.The paper included three parts:1Cloning and expression of flagellar gene fliC of Salmonella Typhimurium in Escherichia colithe flagellar gene fliC of Salmonella Typhimurium was amplified by PCR from the Salmonella genome, and cloned into the prokaryotic expression vector pET28a(+) to construct the recombinants pET28a-fliC,then the recombinants were transformed to the strain BL21(DE3)pLysS,after enzyme digested and sequenced, The recombinant strains were induced by IPTG to express the protein.The results showed that the PCR product was about1509bp as expected, the enzyme digestion and sequencing identification showed the prokaryotic expression system was constructed successfully. The molecular weight of the protein which was induced by IPTG was about54.6kD as expected.2Purification and identification of Phase I flagellar of Salmonella TyphimuriumThe fusion protein was purified by Ni-NTA affinity chromatography,then the purity and weight were detected by SDS-PAGE.The concentration and specificity were determined by BCA and Western-blot.The results showed that the purity of the protein purified by Ni-NTA was high, and the weight was as expected. Western-blot analysis showed that the expressed protein had a specific reaction with the monoclonal antibody anti-His-tag at55KD,indicating that the expressed protein had His-tag and was the target protein.3The production of anti-FliC Polyclonal antibodiesThe polyclonal antibodies were obtained by immunning BALA/c mice with the purified protein, and the titer and specificity were detected by indirect ELISA.The results showed that the purified protein could stimulate the mice to produce specific polyclonal antibodies, the antibody titers reached1:32000and had good specificity and broad spectrum in Salmonella. This reearch would be helpful for the further study on establishing immunologic detection of Salmonella.更多还原