多型FMDV VP1表位嵌入型S-层蛋白SLP-MultiVP1的纯化及其免疫原性试验

发布时间：2018-04-27 09:49

  本文选题：各型FMDV + VP1基因　； 参考：《内蒙古农业大学》2015年硕士论文

【摘要】：口蹄疫(foot-and-mouth disease,FMD)是由口蹄疫病毒(foot-and-mouth disease virus,FMDV)感染引起的一种烈性传染病。病毒的衣壳是由VP1、VP2、VP3、VP4四种结构蛋白的60个亚单位组成,其中VP1是诱导动物产生中和抗体的主要成分。FMD一旦爆发,不仅对畜牧业带来极其严重的损失,也会影响社会经济以及政治的稳定。目前FMD的预防主要还是依赖全病毒灭活疫苗,但是疫苗实践当中发现该疫苗仍存在诸多瓶颈问题,例如病毒的纯化工艺繁琐、免疫效果不稳定等。本试验具体研究内容如下：1、本研究对已构建的含有VPl表位肽基因的原核表达载体pGEX-SLP-MultiVPl进行IPTG诱导表达,成功获得了分子量约为80 kDa的嵌入型蛋白。通过Pierce(?) GST Spin Purification Kit亲和层析和切胶相结合的方法,成功纯化出嵌入型蛋白GST-SLP-MultiVPl,并用Prescission Protease切掉嵌入型蛋白中的GST标签蛋白后,获得了目的蛋白SLP-MultiVP1,其大小约为54 kDa。经Western blotting试验结果显示：该蛋白与牛A、0、Asia Ⅰ型疫苗株阳性血清均有特异性的结合,表明本试验正确纯化了嵌入型蛋白SLP-MultiVP1。随后,用该蛋白(浓度约为1.12mg/ml)免疫昆明小鼠3次后第7d采外周血分离血清,经间接ELISA方法检测结果显示：嵌入型蛋白SLP-MultiVP1能刺激小鼠机体产生高滴度的特异性抗体(OD150nm值为2.0以上),为下一步进行该蛋白免疫原性试验奠定了基础。2、将用已纯化的嵌入型蛋白SLP-MultiVP1,免疫接种6周龄昆明小鼠,以重组蛋白SLP与PBS作为对照组。分别在第一次免疫、第二次免疫和第三次免疫后安乐死昆明小鼠,分离其脾脏提取了总RNA,立即反转录成cDNAⅠ采用荧光定量PCR方法,分别检测了IL-4与IFN-γ的表达水平并进行统计学分析。试验结果显示：嵌入型蛋白SLP-MultiVP1免疫组的2种细胞因子表达量高于与其他2个对照组(P0.05),而且两个对照组间比较,2种细胞因子表达量无显著性差异(P0.05)。3、将新一批6周龄昆明小鼠随机分组,同样蛋白在第二次免疫后7d,从心脏采取血液。血液样品按既定方法进行处理后,在流式细胞仪中上样分析嵌入型蛋白SLP-MultiVP1对CD4+T和CD4+T细胞的刺激情况并进行统计学分析。试验结果显示：嵌入型蛋白SLP-MultiVP1免疫组的CD4+细胞的单个细胞数量菏于SLP重组蛋白和PBS对照组,差异显著(P0.05)；而PBS对照组和SLP重组蛋白免疫组之间无显著差异(P0.05)。并且在外周血中,嵌入型蛋白SLP-MultiVP1免疫组的CD4+T细胞/CD8+T细胞比值均高于SLP重组蛋白免疫组和PBS对照组,差异显著(P0.05)。本试验为进一步各型FMDV VP1基因表位嵌入型蛋白SLP-MultiVP1的免疫保护试验研究奠定了基础。
[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，

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