沙门菌FimA、SefA、Ail和Pad四种表面抗原的原核表达及抗原性鉴定

发布时间：2018-06-26 18:39

本文选题：沙门菌 + 原核表达　；参考：《中国人民解放军军事医学科学院》2011年硕士论文

【摘要】：沙门菌(Salmonella)是一类兼性胞内致病菌,目前已鉴定的能感染人和动物的血清型已经超过2500种,主要引起肠炎、伤寒等一系列病症[1]。沙门菌分布广泛,可从家畜、家禽以及野生动物肠道内分离到,肉类、蔬菜和蛋奶类食品都可能被沙门菌污染,其中鼠伤寒沙门菌、肠炎沙门菌、猪霍乱沙门菌是最常见的污染菌。据报道由沙门菌污染而引起食物中毒约占细菌性食物中毒的42.6%~60.0%,非伤寒沙门菌是引起食源性腹泻的最主要原因,已对食品安全构成严重威胁[2]。沙门菌的检测在食品安全及应对公共卫生突发事件中非常重要,目前沙门菌的检测方法主要有传统培养法,分子检测法和免疫学检测法等。本研究为国家传染病重大专项资助课题,主要应用基因工程方法获得沙门菌特异性表面抗原,通过制备相应抗体,期望在沙门菌快速检测中得到应用。本研究的主要研究内容如下: 第一部分:FimA、SefA、Ail和Pad四种表面抗原的原核表达候选靶抗原的筛选:本研究选取沙门菌表面Ⅰ型菌毛、Sef14菌毛、外膜蛋白Ail和Pad作为检测靶抗原。Ⅰ型菌毛是沙门菌最常见也是研究得最为透彻的一种菌毛,在沙门菌中较为保守,fimA基因编码的FimA蛋白是Ⅰ型菌毛的主要结构亚单位[3];Sef14菌毛是D组沙门菌特有的菌毛,而最常见引起疾病的肠炎沙门菌即位于D组,Sef14菌毛的主要结构亚单位由sefA基因编码[4];Ail蛋白是沙门菌属特异性外膜蛋白,具有遗传保守性,在沙门菌各血清型中均有低水平表达[5,6];Pad蛋白是与沙门菌黏附宿主有关的一种黏附素蛋白[7,8],本研究利用NCBI数据库分别对pad基因和Pad蛋白进行序列比对,发现pad基因和Pad蛋白在沙门菌属内相对保守,属特异性较高,目前国内外尚无利用沙门菌Pad蛋白作为检测靶点的文献报道。重组蛋白的原核表达:通过聚合酶链式反应(polymerse chain reaction,PCR)扩增沙门菌Ⅰ型菌毛主要结构亚单位基因fimA,编码SEF14菌毛主要结构亚单位基因sefA、以及编码两种外膜蛋白的基因ail和pad。fimA基因从1~543bp共扩增543bp全序列,sefA基因从142~584bp共扩增432bp核心序列,ail基因从61~498bp共扩增438bp核心序列,pad基因从46~720bp共扩增675bp核心序列。将得到的四个目的片段插入原核表达载体,分别获得重组表达载体pET32a(+)-fimA、pET32a(+)-sefA、pET32a(+)-ail和pET32a(+)-pad。通过序列测定可以判断,目的序列均以正确的开放读码框(Open reading frame,ORF)插入表达载体。序列比对分析表明fimA、sefA基因、ail基因和pad基因与Genbank中标准序列的同源性分别为99.45%、99.77%、98.86%和100%。将重组阳性质粒pET32a(+)-fimA、pET32a(+)-sefA、pET32a(+)-ail和pET32a(+)-pad导入大肠杆菌BL21(DE3)中,经IPTG诱导,获得四种重组蛋白的表达。重组蛋白的SDS-PAGE分析表明,rFimA、rSefA、rAil和rPad蛋白均以包涵体形式表达,表达量分别占全菌总蛋白的50%、40%、60%和40%,其相对分子质量分别为39kD、35kD、34kD和42kD。重组蛋白抗原性分析:应用Western blotting实验对四种重组蛋白进行抗原性分析,分析结果表明,四种重组蛋白都能够与羊抗沙门菌免疫血清发生阳性反应,说明四种重组蛋白具有较好的抗原性。重组抗原免疫血清的制备:纯化四种重组蛋白后,用紫外分光法测定四种重组蛋白浓度。用四种纯化后的重组蛋白按照免疫方案免疫家兔,制备兔抗重组抗原免疫血清。第3次免疫后,用琼脂糖双向扩散法测定免疫血清效价,最终获得的免疫血清效价都在1: 32及以上。第二部分:四种重组抗原免疫抗体在胶体金法中的初步应用免疫胶体金技术(Immune colloidal gold technique)是以胶体金颗粒作为显色标记物结合于检测抗体的免疫层析法的一种。胶体金在弱碱pH下带负电荷,可与蛋白质的正电基团通过静电作用力紧密结合,这种静电结合并不影响蛋白的生物学特性。胶体金颗粒本身带有颜色,当免疫胶体金探针与待检样品结合并被NC膜上的致敏配对物捕获,从而出现肉眼可见的红色沉淀线。免疫胶体金法操作简便、无需专业人员操作和专业设备,仅需按照说明书操作即可获得初步检测结果,可提高应对公共卫生突发事件的能力,较为适合基层及家庭使用。应用氯金酸还原法制备胶体金颗粒,并分别标记四种重组抗原免疫抗体,FimA、SefA、Ail和Pad抗体胶体金溶液的最佳标记浓度分别为25μg/ml、25μg/ml、30μg/ml和30μg/ml。相应胶体金标记探针在520nm处的吸光值分别为2.77、2.91、3.32和3.44,都在2.5~10适宜吸光值范围内,四种探针的浓度符合要求。使用四硼酸钠(10%蔗糖)保存液稀释抗体至致敏浓度,可以避免非特异条带的出现。Ail抗体的最适致敏浓度为2mg/ml,SefA、FimA和Pad抗体最适致敏浓度均为4mg/ml。对四种胶体金标记抗体的实验室初步评价:四种胶体金抗体均可检出肠炎沙门菌、鼠伤寒沙门菌、猪霍乱沙门菌、伤寒沙门菌和亚利桑那沙门菌等实验所用的6株沙门菌;SefA、Ail和FimA三种金标抗体对沙门菌的的最低检出浓度均为1×108个/ml,Pad金标抗体对沙门菌的最低检出浓度为1×107个/ml;在13株非沙门菌的检测中,四种胶体金标记抗体均不与菌体浓度为1×108个/ml的粪肠球菌、蜂房哈夫尼氏菌、白色葡萄球菌、多杀巴斯德氏菌、霍乱弧菌小川型、霍乱弧菌稻叶型、副溶血性弧菌、草绿色链球菌、绿脓杆菌和白色念球菌发生反应,但可与菌体浓度为1×108个/ml的屎肠球菌,普罗威登斯氏菌,福氏志贺氏菌2a和枸橼酸杆菌反应。
[Abstract]:Salmonella (Salmonella) is a kind of facultative intracellular pathogenic bacteria. There are more than 2500 serotypes that have been identified to infect humans and animals, mainly causing enteritis, typhoid and other diseases, [1]. Salmonella is widely distributed, and can be separated from domestic animals, poultry and wild animals, and meat, vegetables and egg milk are likely to be covered by sand. Salmonella typhimurium, Salmonella enteritis, Salmonella enteritis and swine cholera Salmonella are the most common contaminating bacteria. It is reported that the food poisoning caused by Salmonella is about 42.6%~60.0% of bacterial food poisoning. Non typhoid Salmonella is the main cause of foodborne diarrhoea, which poses a serious threat to food safety, [2].
The detection of Salmonella is very important in food safety and public health emergencies. At present, the detection methods of Salmonella are mainly traditional culture method, molecular detection method and immunological detection method. This study is a major project of national infectious diseases, and the main application of genetic engineering method to obtain Salmonella specific surface antigen, The preparation of corresponding antibodies is expected to be applied in rapid detection of Salmonella.
The first part: selection of the candidate target antigens for the prokaryotic expression of four surface antigens: FimA, SefA, Ail and Pad. This study selected Salmonella on the surface of type I pilus, Sef14 pilus, outer membrane protein Ail and Pad as the target antigen. The encoded FimA protein is the main subunit [3] of the type I pilus, and the Sef14 pilus is the special pilus of the Salmonella of group D, and the most common Salmonella enteritis is located in the D group. The main subunit of the Sef14 pili is encoded by the sefA gene [4], and the Ail protein is a specific outer membrane protein of the Salmonella, which has the conservatism of heredity and is in the sand gate. A low level of [5,6] was expressed in the serotypes of the bacteria; Pad protein was a kind of adhesion protein [7,8] associated with the adhesion of Salmonella to the host. The pad gene and the Pad protein were compared by the NCBI database. The pad gene and Pad protein were kept in the genus Salmonella, and the specificity was higher. At present, there is no use at home and abroad. Salmonella Pad protein as a target for detection is reported in the literature.
Prokaryotic expression of recombinant protein: polymerse chain reaction (PCR) amplified the main subunit gene fimA of Salmonella type I pilus, encoded the main subunit gene sefA of the SEF14 pilus, and the gene ail and pad.fimA encoding two outer membrane proteins from 1~543bp. The sefA gene was from the 1~543bp. 142~584bp amplified the core sequence of 432bp, ail gene amplified 438bp core sequence from 61~498bp, and pad gene amplified 675bp core sequence from 46~720bp. The four target fragments were inserted into the prokaryotic expression vector to obtain the recombinant expression vector pET32a (+) -fimA, pET32a (+) -sefA. The sequence alignment analysis showed that the homology of fimA, sefA gene, ail gene, pad gene and the standard sequence in Genbank were 99.45%, 99.77%, 99.77%, 98.86% and 100%., respectively 99.45%, 99.77%, 98.86% and 100%., respectively. A (+) -pad was introduced into the Escherichia coli BL21 (DE3) and induced by IPTG to obtain the expression of four recombinant proteins. The SDS-PAGE analysis of the recombinant protein showed that the proteins of rFimA, rSefA, rAil and rPad were expressed in inclusion body form, and the expression amount accounted for 50%, 40%, 60% and 40% of the total total protein, respectively.
Analysis of antigenicity of recombinant protein: the antigenicity of the four recombinant proteins was analyzed by Western blotting experiment. The results showed that the four recombinant proteins could be positive with the immune sera of Salmonella resistant sheep, indicating that the four recombinant proteins have good antigenicity.
The preparation of recombinant antigen immunization serum: after purification of four recombinant proteins, the concentration of four recombinant proteins was determined by ultraviolet spectrophotometry. The Rabbit anti recombinant antigen immunized serum was prepared by immunizing the rabbit with four purified recombinant proteins. After third immunization, the immune serum titer was determined by agarose bi-directional diffusion method, and the final immunization was obtained. The titer of pestilence serum is at 1:32 and above.
The second part: the preliminary application of four kinds of recombinant antigen immunization antibodies in colloidal gold method (Immune colloidal gold technique) is a kind of immunochromatography with colloidal gold particles as chromogenic markers combined with the detection of antibodies. Colloidal gold is negatively charged under the weak alkali pH, and can be used with the positive group of protein through static electricity. The electrostatic binding does not affect the biological characteristics of the protein. The colloidal gold particles have their own color. When the colloid gold probe is combined with the samples to be detected and is captured by the sensitized pair on the NC film, the red precipitate line is visible to the naked eye. The preliminary test results can be obtained only in accordance with the manual operation, which can improve the ability to respond to public health emergencies. It is more suitable for grass-roots and family use. Colloidal gold particles are prepared by the chlorchloric acid reduction method, and four kinds of recombinant antigen immunization antibodies, the best labeling of FimA, SefA, Ail and Pad antibody colloidal gold solution, are marked respectively. The absorbance of the colloid gold mark probe at 520nm was 25 mu g/ml, 25 g/ml, 30 mu g/ml and 30 micron g/ml. respectively, which were 2.77,2.91,3.32 and 3.44 respectively. The concentration of the four probes was in accordance with the suitable absorption range of 2.5~10. The dilution antibody of four borate (10% sucrose) was used to reduce the dilution antibody to the sensitization concentration, and the non-specific bands could be avoided. The most sensitized concentration of.Ail antibody was 2mg/ml, SefA, FimA and Pad antibody. The best sensitization concentration was 4mg/ml..
The preliminary evaluation of four kinds of colloidal gold labeling antibodies: four kinds of colloidal gold antibodies can detect 6 Salmonella strains used in the experiments of Salmonella enteritis, Salmonella typhimurium, swine cholera Salmonella, Salmonella typhi and Arizona Salmonella, and the lowest detection concentration of three kinds of gold standard against Salmonella by SefA, Ail and FimA are 1 x 108 /m L, the lowest detection concentration of Pad gold antibody against Salmonella was 1 x 107 /ml; in the detection of 13 non Salmonella strains, four kinds of colloidal gold labeled antibodies were not associated with 1 x 108 /ml of Enterococcus, hive, Staphylococcus alba, Pasteur, Vibrio cholerae, Vibrio cholerae, and parahaemolyticus Vibrio, Streptococcus green, Pseudomonas aeruginosa and Candida albicans, but can react with 1 x 108 /ml strains of Enterococcus faecium, Plo Weedon J S bacteria, Shigella flexneri 2a and citrobacilli.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392.1

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