本文選題：無(wú)乳鏈球菌 + 表面蛋白��； 參考：《天津科技大學(xué)》2012年碩士論文

【摘要】：奶牛乳腺炎是世界奶牛業(yè)的主要危害因素之一,它不僅影響產(chǎn)奶量,造成經(jīng)濟(jì)損失,而且影響牛奶的品質(zhì),危害人類的健康。而無(wú)乳鏈球菌是引起奶牛乳腺炎的常見病原微生物之一,因而是乳業(yè)關(guān)心的主要問(wèn)題。建立無(wú)乳鏈球菌快速檢測(cè)方法,對(duì)于提高奶制品質(zhì)量與安全,推進(jìn)奶牛業(yè)發(fā)展,促進(jìn)人類健康意義重大。本研究克隆表達(dá)了三種無(wú)乳鏈球菌表面蛋白(Rib,Sip, Alpha),并分別進(jìn)行了抗體制備,為無(wú)乳鏈球菌快速檢測(cè)試紙條和多價(jià)疫苗的研制奠定了良好的基礎(chǔ)。 本實(shí)驗(yàn)首先將6種血清型的ATCC無(wú)乳鏈球菌標(biāo)準(zhǔn)菌進(jìn)行了活化,并分別提取了基因組DNA。根據(jù)Rib, Sip, Alpha三種表面蛋白基因序列設(shè)計(jì)特異性引物,以不同標(biāo)準(zhǔn)菌株基因組DNA為模板,分別擴(kuò)增得到長(zhǎng)度為453bp、1302bp和699bp的片段,將片段克隆連接至pMD18-T載體進(jìn)行序列測(cè)定,結(jié)果表明獲得的片段為正確的目的基因。將rib和sip基因片段亞克隆至重組表達(dá)載體pET26b、alpha基因片段亞克隆至重組表達(dá)載體pGEX-4T-1,并利用IPTG進(jìn)行誘導(dǎo)表達(dá),SDS-PAGE結(jié)果顯示3種基因均能夠重組表達(dá),外源蛋白的分子量分別為18kDa、61kDa和52kDa,與預(yù)期大小相符,但是其中Rib和Alpha形成包涵體。將3種基因進(jìn)行大規(guī)模重組表達(dá)并對(duì)形成的包涵體進(jìn)行變性、復(fù)性后,利用親和純化獲得了純度較高的目的蛋白。經(jīng)過(guò)質(zhì)譜鑒定,3種外源蛋白與與無(wú)乳鏈球菌Rib,Sip以及Alpha的相似度分別達(dá)到99.99%,100%以及100%。將三種蛋白分別免疫新西蘭白兔,制備多克隆抗體,經(jīng)測(cè)定抗體效價(jià)分別為480000：1,640000：1以及320000：1。利用3種抗血清,分別采用間接ELISA方法對(duì)ATCC標(biāo)準(zhǔn)菌進(jìn)行檢測(cè),結(jié)果顯示3種多克隆抗體均體現(xiàn)出一定親和性和特異性。
[Abstract]:Dairy cow mastitis is one of the main harmful factors of dairy industry in the world. It not only affects milk production, causes economic loss, but also affects the quality of milk and human health. Streptococcus actinomycetes is one of the common pathogenic microorganisms that cause dairy cow mastitis, so it is the main concern of dairy industry. It is of great significance to establish a rapid detection method for streptococcus lactococcus to improve the quality and safety of dairy products, promote the development of dairy industry and promote human health. In this study, three surface proteins of Streptococcus lactobacillus were cloned and expressed, and the antibodies were prepared, which laid a good foundation for the rapid detection of streptococcus lactobacillus and the development of multivalent vaccine. In this study, six serotypes of ATCC were first activated and genomic DNAA were extracted. According to the three surface protein gene sequences of Rib, Sip, Alpha, specific primers were designed and amplified by using genomic DNA of different standard strains as templates. The fragments of 453bpC1302bp and 699bp were amplified and cloned into pMD18-T vector for sequencing. The results showed that the obtained fragment was the correct target gene. The rib and sip gene fragments were subcloned into the recombinant expression vector pET26bGN alpha gene and subcloned into the recombinant expression vector pGEX-4T-1. The results of SDS-PAGE showed that all the three genes could be expressed by SDS-PAGE. The molecular weights of exogenous proteins were 18kDa 61kDa and 52kDa respectively, which were consistent with the expected size, but Rib and Alpha formed inclusion bodies. The three genes were expressed in large scale and the inclusion bodies were denatured. After renaturation, the purified target protein was obtained by affinity purification. The similarity of three exogenous proteins with Streptococcus lactis Ribsip and Alpha was 99.9999% and 100%, respectively. The polyclonal antibodies were prepared by immunizing New Zealand white rabbits with three proteins. The titers of the antibodies were 480000: 1, 6400: 1 and 320 000: 1, respectively. Three kinds of antiserum were used to detect ATCC standard bacteria by indirect ELISA method. The results showed that the three polyclonal antibodies showed certain affinity and specificity.
【學(xué)位授予單位】：天津科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2012
【分類號(hào)】：R155.57

【參考文獻(xiàn)】

相關(guān)期刊論文 前10條

1 夏啟玉;肖蘇生;鄧柳紅;易小平;張春發(fā);;包涵體蛋白的復(fù)性研究進(jìn)展[J];安徽農(nóng)業(yè)科學(xué);2008年14期

2 楊永弘,朱蔭芝;B族鏈球菌感染的研究進(jìn)展[J];北京醫(yī)學(xué);1997年03期

3 吳潤(rùn);郝保青;農(nóng)向;嚴(yán)丹紅;;奶牛隱性乳房炎的主要病原菌的PCR鑒定[J];中國(guó)牛業(yè)科學(xué);2006年02期

4 李春生;;免疫檢測(cè)技術(shù)在農(nóng)畜產(chǎn)品安全檢測(cè)中的應(yīng)用[J];河北省科學(xué)院學(xué)報(bào);2009年01期

5 解庭波;;大腸桿菌表達(dá)系統(tǒng)的研究進(jìn)展[J];長(zhǎng)江大學(xué)學(xué)報(bào)(自科版)醫(yī)學(xué)卷;2008年03期

6 王洪海;張曉平;梁平;黃蓉蓉;;免疫學(xué)新技術(shù)介紹:Western Blot Analysis[J];中國(guó)人民解放軍軍醫(yī)進(jìn)修學(xué)院學(xué)報(bào);1990年03期

7 賈玉萍,周東順,萬(wàn)仁忠,劉文強(qiáng),胡敬東,趙宏坤;巢式PCR檢測(cè)無(wú)乳鏈球菌16S rRNA方法的建立及應(yīng)用[J];農(nóng)業(yè)生物技術(shù)學(xué)報(bào);2005年05期

8 朱宇;;酶聯(lián)免疫檢測(cè)技術(shù)在食品安全檢測(cè)中的應(yīng)用[J];農(nóng)業(yè)質(zhì)量標(biāo)準(zhǔn);2006年01期

9 王桂玲;王孝會(huì);都田趙;陳薇;姜佩家;;原核表達(dá)載體GST-RUNX3的構(gòu)建及其在大腸桿菌表達(dá)[J];解剖科學(xué)進(jìn)展;2011年04期

10 陳來(lái)同,茹炳根;在大腸桿菌表達(dá)體系中以包涵體形式存在的真核生物蛋白質(zhì)的分離、復(fù)性和二硫鍵的形成[J];中國(guó)生化藥物雜志;1997年01期

，

本文編號(hào)：1831078