本文選題：各型FMDV + VP1基因�。� 參考：《內(nèi)蒙古農(nóng)業(yè)大學(xué)》2015年碩士論文

【摘要】：口蹄疫(foot-and-mouth disease,FMD)是由口蹄疫病毒(foot-and-mouth disease virus,FMDV)感染引起的一種烈性傳染病。病毒的衣殼是由VP1、VP2、VP3、VP4四種結(jié)構(gòu)蛋白的60個亞單位組成,其中VP1是誘導(dǎo)動物產(chǎn)生中和抗體的主要成分。FMD一旦爆發(fā),不僅對畜牧業(yè)帶來極其嚴(yán)重的損失,也會影響社會經(jīng)濟(jì)以及政治的穩(wěn)定。目前FMD的預(yù)防主要還是依賴全病毒滅活疫苗,但是疫苗實(shí)踐當(dāng)中發(fā)現(xiàn)該疫苗仍存在諸多瓶頸問題,例如病毒的純化工藝繁瑣、免疫效果不穩(wěn)定等。本試驗(yàn)具體研究內(nèi)容如下：1、本研究對已構(gòu)建的含有VPl表位肽基因的原核表達(dá)載體pGEX-SLP-MultiVPl進(jìn)行IPTG誘導(dǎo)表達(dá),成功獲得了分子量約為80 kDa的嵌入型蛋白。通過Pierce(?) GST Spin Purification Kit親和層析和切膠相結(jié)合的方法,成功純化出嵌入型蛋白GST-SLP-MultiVPl,并用Prescission Protease切掉嵌入型蛋白中的GST標(biāo)簽蛋白后,獲得了目的蛋白SLP-MultiVP1,其大小約為54 kDa。經(jīng)Western blotting試驗(yàn)結(jié)果顯示：該蛋白與牛A、0、Asia Ⅰ型疫苗株陽性血清均有特異性的結(jié)合,表明本試驗(yàn)正確純化了嵌入型蛋白SLP-MultiVP1。隨后,用該蛋白(濃度約為1.12mg/ml)免疫昆明小鼠3次后第7d采外周血分離血清,經(jīng)間接ELISA方法檢測結(jié)果顯示：嵌入型蛋白SLP-MultiVP1能刺激小鼠機(jī)體產(chǎn)生高滴度的特異性抗體(OD150nm值為2.0以上),為下一步進(jìn)行該蛋白免疫原性試驗(yàn)奠定了基礎(chǔ)。2、將用已純化的嵌入型蛋白SLP-MultiVP1,免疫接種6周齡昆明小鼠,以重組蛋白SLP與PBS作為對照組。分別在第一次免疫、第二次免疫和第三次免疫后安樂死昆明小鼠,分離其脾臟提取了總RNA,立即反轉(zhuǎn)錄成cDNAⅠ采用熒光定量PCR方法,分別檢測了IL-4與IFN-γ的表達(dá)水平并進(jìn)行統(tǒng)計(jì)學(xué)分析。試驗(yàn)結(jié)果顯示：嵌入型蛋白SLP-MultiVP1免疫組的2種細(xì)胞因子表達(dá)量高于與其他2個對照組(P0.05),而且兩個對照組間比較,2種細(xì)胞因子表達(dá)量無顯著性差異(P0.05)。3、將新一批6周齡昆明小鼠隨機(jī)分組,同樣蛋白在第二次免疫后7d,從心臟采取血液。血液樣品按既定方法進(jìn)行處理后,在流式細(xì)胞儀中上樣分析嵌入型蛋白SLP-MultiVP1對CD4+T和CD4+T細(xì)胞的刺激情況并進(jìn)行統(tǒng)計(jì)學(xué)分析。試驗(yàn)結(jié)果顯示：嵌入型蛋白SLP-MultiVP1免疫組的CD4+細(xì)胞的單個細(xì)胞數(shù)量菏于SLP重組蛋白和PBS對照組,差異顯著(P0.05)；而PBS對照組和SLP重組蛋白免疫組之間無顯著差異(P0.05)。并且在外周血中,嵌入型蛋白SLP-MultiVP1免疫組的CD4+T細(xì)胞/CD8+T細(xì)胞比值均高于SLP重組蛋白免疫組和PBS對照組,差異顯著(P0.05)。本試驗(yàn)為進(jìn)一步各型FMDV VP1基因表位嵌入型蛋白SLP-MultiVP1的免疫保護(hù)試驗(yàn)研究奠定了基礎(chǔ)。
[Abstract]:Foot-and-mouth disease (FMDV) is a severe infectious disease caused by foot-and-mouth disease virus (FMDV) infection. The capsid of the virus is composed of 60 subunits of four structural proteins of VP1VP2VP3VP4, in which VP1 is the main component that induces the production of neutralizing antibodies in animals. Once the virus erupts, it will not only cause extremely serious losses to animal husbandry. It also affects social, economic and political stability. At present, the prevention of FMD mainly depends on the whole virus inactivated vaccine, but in the vaccine practice, there are still many bottleneck problems, such as the complicated purification process of the virus, the unstable immune effect and so on. The specific contents of this study are as follows: 1. The constructed prokaryotic expression vector pGEX-SLP-MultiVPl containing VPl epitope peptide gene was induced by IPTG and the embedded protein with molecular weight of about 80 kDa was successfully obtained. Through Pierceau) The intercalated protein GST-SLP-MultiVPlwas successfully purified by GST Spin Purification Kit affinity chromatography and gel-cutting method. The target protein SLP-multiVP1 was obtained by Prescission Protease after the GST tag protein was cut off from the embedded protein, and the size of SLP-multiVP1 was about 54 kDa. The results of Western blotting test showed that the protein was specifically bound to the positive serum of bovine Agno Asia 鈪，

本文編號：1810238