本文選題：沙門菌 + 原核表達(dá)　； 參考：《中國人民解放軍軍事醫(yī)學(xué)科學(xué)院》2011年碩士論文

【摘要】：沙門菌(Salmonella)是一類兼性胞內(nèi)致病菌,目前已鑒定的能感染人和動物的血清型已經(jīng)超過2500種,主要引起腸炎、傷寒等一系列病癥[1]。沙門菌分布廣泛,可從家畜、家禽以及野生動物腸道內(nèi)分離到,肉類、蔬菜和蛋奶類食品都可能被沙門菌污染,其中鼠傷寒沙門菌、腸炎沙門菌、豬霍亂沙門菌是最常見的污染菌。據(jù)報(bào)道由沙門菌污染而引起食物中毒約占細(xì)菌性食物中毒的42.6%~60.0%,非傷寒沙門菌是引起食源性腹瀉的最主要原因,已對食品安全構(gòu)成嚴(yán)重威脅[2]。 沙門菌的檢測在食品安全及應(yīng)對公共衛(wèi)生突發(fā)事件中非常重要,目前沙門菌的檢測方法主要有傳統(tǒng)培養(yǎng)法,分子檢測法和免疫學(xué)檢測法等。本研究為國家傳染病重大專項(xiàng)資助課題,主要應(yīng)用基因工程方法獲得沙門菌特異性表面抗原,通過制備相應(yīng)抗體,期望在沙門菌快速檢測中得到應(yīng)用。本研究的主要研究內(nèi)容如下: 第一部分:FimA、SefA、Ail和Pad四種表面抗原的原核表達(dá)候選靶抗原的篩選:本研究選取沙門菌表面Ⅰ型菌毛、Sef14菌毛、外膜蛋白Ail和Pad作為檢測靶抗原。Ⅰ型菌毛是沙門菌最常見也是研究得最為透徹的一種菌毛,在沙門菌中較為保守,fimA基因編碼的FimA蛋白是Ⅰ型菌毛的主要結(jié)構(gòu)亞單位[3];Sef14菌毛是D組沙門菌特有的菌毛,而最常見引起疾病的腸炎沙門菌即位于D組,Sef14菌毛的主要結(jié)構(gòu)亞單位由sefA基因編碼[4];Ail蛋白是沙門菌屬特異性外膜蛋白,具有遺傳保守性,在沙門菌各血清型中均有低水平表達(dá)[5,6];Pad蛋白是與沙門菌黏附宿主有關(guān)的一種黏附素蛋白[7,8],本研究利用NCBI數(shù)據(jù)庫分別對pad基因和Pad蛋白進(jìn)行序列比對,發(fā)現(xiàn)pad基因和Pad蛋白在沙門菌屬內(nèi)相對保守,屬特異性較高,目前國內(nèi)外尚無利用沙門菌Pad蛋白作為檢測靶點(diǎn)的文獻(xiàn)報(bào)道。 重組蛋白的原核表達(dá):通過聚合酶鏈?zhǔn)椒磻?yīng)(polymerse chain reaction,PCR)擴(kuò)增沙門菌Ⅰ型菌毛主要結(jié)構(gòu)亞單位基因fimA,編碼SEF14菌毛主要結(jié)構(gòu)亞單位基因sefA、以及編碼兩種外膜蛋白的基因ail和pad。fimA基因從1~543bp共擴(kuò)增543bp全序列,sefA基因從142~584bp共擴(kuò)增432bp核心序列,ail基因從61~498bp共擴(kuò)增438bp核心序列,pad基因從46~720bp共擴(kuò)增675bp核心序列。將得到的四個目的片段插入原核表達(dá)載體,分別獲得重組表達(dá)載體pET32a(+)-fimA、pET32a(+)-sefA、pET32a(+)-ail和pET32a(+)-pad。通過序列測定可以判斷,目的序列均以正確的開放讀碼框(Open reading frame,ORF)插入表達(dá)載體。序列比對分析表明fimA、sefA基因、ail基因和pad基因與Genbank中標(biāo)準(zhǔn)序列的同源性分別為99.45%、99.77%、98.86%和100%。將重組陽性質(zhì)粒pET32a(+)-fimA、pET32a(+)-sefA、pET32a(+)-ail和pET32a(+)-pad導(dǎo)入大腸桿菌BL21(DE3)中,經(jīng)IPTG誘導(dǎo),獲得四種重組蛋白的表達(dá)。重組蛋白的SDS-PAGE分析表明,rFimA、rSefA、rAil和rPad蛋白均以包涵體形式表達(dá),表達(dá)量分別占全菌總蛋白的50%、40%、60%和40%,其相對分子質(zhì)量分別為39kD、35kD、34kD和42kD。 重組蛋白抗原性分析:應(yīng)用Western blotting實(shí)驗(yàn)對四種重組蛋白進(jìn)行抗原性分析,分析結(jié)果表明,四種重組蛋白都能夠與羊抗沙門菌免疫血清發(fā)生陽性反應(yīng),說明四種重組蛋白具有較好的抗原性。 重組抗原免疫血清的制備:純化四種重組蛋白后,用紫外分光法測定四種重組蛋白濃度。用四種純化后的重組蛋白按照免疫方案免疫家兔,制備兔抗重組抗原免疫血清。第3次免疫后,用瓊脂糖雙向擴(kuò)散法測定免疫血清效價,最終獲得的免疫血清效價都在1: 32及以上。 第二部分:四種重組抗原免疫抗體在膠體金法中的初步應(yīng)用免疫膠體金技術(shù)(Immune colloidal gold technique)是以膠體金顆粒作為顯色標(biāo)記物結(jié)合于檢測抗體的免疫層析法的一種。膠體金在弱堿pH下帶負(fù)電荷,可與蛋白質(zhì)的正電基團(tuán)通過靜電作用力緊密結(jié)合,這種靜電結(jié)合并不影響蛋白的生物學(xué)特性。膠體金顆粒本身帶有顏色,當(dāng)免疫膠體金探針與待檢樣品結(jié)合并被NC膜上的致敏配對物捕獲,從而出現(xiàn)肉眼可見的紅色沉淀線。免疫膠體金法操作簡便、無需專業(yè)人員操作和專業(yè)設(shè)備,僅需按照說明書操作即可獲得初步檢測結(jié)果,可提高應(yīng)對公共衛(wèi)生突發(fā)事件的能力,較為適合基層及家庭使用。應(yīng)用氯金酸還原法制備膠體金顆粒,并分別標(biāo)記四種重組抗原免疫抗體,FimA、SefA、Ail和Pad抗體膠體金溶液的最佳標(biāo)記濃度分別為25μg/ml、25μg/ml、30μg/ml和30μg/ml。相應(yīng)膠體金標(biāo)記探針在520nm處的吸光值分別為2.77、2.91、3.32和3.44,都在2.5~10適宜吸光值范圍內(nèi),四種探針的濃度符合要求。使用四硼酸鈉(10%蔗糖)保存液稀釋抗體至致敏濃度,可以避免非特異條帶的出現(xiàn)。Ail抗體的最適致敏濃度為2mg/ml,SefA、FimA和Pad抗體最適致敏濃度均為4mg/ml。 對四種膠體金標(biāo)記抗體的實(shí)驗(yàn)室初步評價:四種膠體金抗體均可檢出腸炎沙門菌、鼠傷寒沙門菌、豬霍亂沙門菌、傷寒沙門菌和亞利桑那沙門菌等實(shí)驗(yàn)所用的6株沙門菌;SefA、Ail和FimA三種金標(biāo)抗體對沙門菌的的最低檢出濃度均為1×108個/ml,Pad金標(biāo)抗體對沙門菌的最低檢出濃度為1×107個/ml;在13株非沙門菌的檢測中,四種膠體金標(biāo)記抗體均不與菌體濃度為1×108個/ml的糞腸球菌、蜂房哈夫尼氏菌、白色葡萄球菌、多殺巴斯德氏菌、霍亂弧菌小川型、霍亂弧菌稻葉型、副溶血性弧菌、草綠色鏈球菌、綠膿桿菌和白色念球菌發(fā)生反應(yīng),但可與菌體濃度為1×108個/ml的屎腸球菌,普羅威登斯氏菌,福氏志賀氏菌2a和枸櫞酸桿菌反應(yīng)。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R392.1

【參考文獻(xiàn)】

相關(guān)期刊論文 前4條

1 寧云山,李妍,王小寧;包含體蛋白質(zhì)的復(fù)性研究進(jìn)展[J];生物技術(shù)通訊;2001年03期

2 楊U，

本文編號：2071027