發(fā)布時(shí)間：2018-02-22 18:36

  本文關(guān)鍵詞： 馬流產(chǎn)沙門(mén)氏菌 鞭毛蛋白Flic 表達(dá)純化 SeM蛋白 gD蛋白 聯(lián)合免疫　出處：《新疆農(nóng)業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：馬流產(chǎn)沙門(mén)氏菌可引起妊娠母馬流產(chǎn),幼駒敗血癥,下痢,公馬睪丸炎,髯甲膿腫。該病可給馬養(yǎng)殖業(yè)造成嚴(yán)重經(jīng)濟(jì)損失。本研究首先對(duì)新疆地區(qū)養(yǎng)馬較為集中的昭蘇縣、富蘊(yùn)縣、特克斯縣、和靜縣、吉木乃縣的養(yǎng)殖場(chǎng)(戶)的2768份馬血清樣品進(jìn)行馬流產(chǎn)沙門(mén)氏菌血清學(xué)調(diào)查。間接ELISA法檢測(cè)結(jié)果顯示,這5縣馬養(yǎng)殖場(chǎng)(戶)中馬流產(chǎn)沙門(mén)氏菌病抗體平均陽(yáng)性率為28.68%；雌性馬匹的抗體陽(yáng)性率(36.9%)略高于雄性馬匹(29.7%),但差異不顯著；吉木乃和和靜縣馬匹抗體陽(yáng)性率相對(duì)較低,為3.24%-9.78%,特克斯縣和昭蘇縣抗體陽(yáng)性率相對(duì)較高,為27.17%～43.91%；從年齡統(tǒng)計(jì)結(jié)果來(lái)看,各年齡段的的馬匹均有一定的抗體陽(yáng)性率。本研究從流產(chǎn)馬匹的病料中分離純化,并經(jīng)生化和PCR鑒定得到馬流產(chǎn)沙門(mén)氏菌新疆分離株。本研究進(jìn)一步利用分離菌株的基因組,經(jīng)PCR擴(kuò)增得到了馬流產(chǎn)沙門(mén)氏菌鞭毛蛋白基因FliC2,大小為1427 bp,并構(gòu)建重組質(zhì)粒pET28a-FliC2,在大腸桿菌BL21(DE3)感受態(tài)細(xì)胞中經(jīng)IPTG進(jìn)行誘導(dǎo)表達(dá),經(jīng)SDS-PAGE電泳分析該重組蛋白大小約為52KDa,Western-blotting分析顯示該蛋白具有反應(yīng)原性。將純化后的融合蛋白F1iC2免疫小鼠,檢測(cè)結(jié)果顯示,免疫小鼠中產(chǎn)生了較高水平的抗體,抗體效價(jià)可達(dá)1：218 700,且以IgG1亞型為主。將純化后的融合蛋白免疫馬匹,免疫馬血清中同樣產(chǎn)生了較高水平的特異性抗體,抗體效價(jià)可達(dá)1：72 900。本研究進(jìn)一步利用PCR擴(kuò)增了截短的馬流產(chǎn)沙門(mén)氏菌鞭毛蛋白基因FliCl,大小為840 bp,并構(gòu)建重組質(zhì)粒pGEX-FliC1。誘導(dǎo)表達(dá)的融合蛋白FliCl-GST經(jīng)SDS-PAGE電泳分析大小為56 KDa。將純化后的FliC1蛋白和本實(shí)驗(yàn)室已表達(dá)的gD蛋白聯(lián)合免疫馬匹,結(jié)果表明,兩種蛋白聯(lián)合免疫組馬血清中抗體水平高于這兩種蛋白單獨(dú)免疫組,抗體效價(jià)可達(dá)1：218 700。檢測(cè)結(jié)果還表明,FliC1蛋白可促進(jìn)gD蛋白免疫水平的升高。且產(chǎn)生的抗體以亞型IgG1為主。對(duì)抗體持續(xù)期的檢測(cè)表明抗體維持較高水平可達(dá)170 d。為比較FliC兩個(gè)不同大小片段免疫效果的差異,本研究將FliC1和FliC2分別與SeM蛋白聯(lián)合免疫C57BL/6小鼠,對(duì)免疫小鼠抗體的效價(jià)、抗體亞型以及攻毒保護(hù)力評(píng)估不同大小鞭毛蛋白對(duì)SeM蛋白免疫效果的影響。結(jié)果顯示,FliC2與SeM聯(lián)合免疫組產(chǎn)生的抗體效價(jià)更高,抗體主要以IgG1為主；對(duì)小鼠的攻毒保護(hù)率最高,可達(dá)80%。該結(jié)果表明,鞭毛蛋白FliC2對(duì)SeM輔助免疫效果比FliC1更好。該結(jié)果對(duì)我們更好地利用FliC蛋白進(jìn)行相關(guān)研究奠定實(shí)驗(yàn)基礎(chǔ)。
[Abstract]:Salmonella equine abortion can cause miscarriage of pregnant mares, septicemia, dysentery, orchitis of male horses, and abscess of bearded armour. This disease can cause serious economic losses to horse breeding. In Fuyun County, Tex County, Hejing County, Jimonai County, 2768 horse serum samples were collected from farms (households) for serological investigation of Salmonella abortus. Indirect ELISA assay showed that, The average positive rate of salmonellosis antibodies in horse farms (households) in these five counties was 28.68; the positive rate of antibodies in female horses was 36.9%, which was slightly higher than that in male horses (29.7g), but the difference was not significant. The positive rate of horse antibodies in Jimenai and Hejing counties was relatively low. The positive rate of antibody was relatively high in Tex County and Zhaosu County (27.17%, 43.91%). According to the results of age statistics, there was a positive rate of antibody in horses of all ages. The isolates of Salmonella equi from Xinjiang were identified by biochemical and PCR methods. The flagellin gene FliC2 of Salmonella equina was amplified by PCR, and its size was 1427 bp.The recombinant plasmid pET28a-FliC2 was constructed and expressed by IPTG in E. coli BL21DE3. The recombinant protein was analyzed by SDS-PAGE electrophoresis to be about 52K Dawei Western-blotting. The purified fusion protein F1iC2 was immunized by immunizing mice. The results showed that a high level of antibody was produced in the immunized mice. The titer of the antibody reached 1: 218,700, and the IgG1 subtype was the main subtype. The purified fusion protein was used to immunize the horse, and a higher level of specific antibody was also produced in the serum of the immunized horse. The antibody titer can reach 1: 72 900.In this study, the flagellin gene of Salmonella equi was amplified by PCR, the size was 840 BP, and the recombinant plasmid pGEX-FliC1 was constructed. The induced expression of the fusion protein FliCl-GST was 56 by SDS-PAGE electrophoresis. The purified FliC1 protein was combined with the gd protein expressed in our laboratory to immunize horses. The results showed that the level of antibody in serum of the two kinds of protein combined immunized group was higher than that of the two kinds of protein alone immunization group. The titer of antibody reached 1: 218,700. The results also showed that FliC1 protein could promote the increase of immunological level of GD protein, and the antibody produced was mainly subtype IgG1. The detection of antibody duration showed that the antibody maintained a high level up to 170d. The difference of immune effect between two different size fragments of FliC, In this study, FliC1 and FliC2 were combined with SeM protein to immunize C57BL / 6 mice. The effects of flagellins of different sizes on the immune response of SeM protein were evaluated. The results showed that the antibody titers of FliC2 and SeM combined immunized group were higher, the main antibody was IgG1, and the protective rate of attacking virus to mice was the highest. The results showed that the flagellin FliC2 was more effective than FliC1 in assisting SeM immunization. The results laid an experimental foundation for us to make better use of FliC protein to carry on the related research.
【學(xué)位授予單位】：新疆農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：S852.61

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本文編號(hào)：1525025