轮状病毒抗原蛋白VP4基因重组载体疫苗研究
本文关键词:轮状病毒抗原蛋白VP4基因重组载体疫苗研究 出处:《重庆医科大学》2009年硕士论文 论文类型:学位论文
更多相关文章: 轮状病毒抗原基因VP4 重叠PCR 穿梭载体pBEX/VP4 肠袢实验 口服疫苗
【摘要】: 轮状病毒(rotavirus,RV)感染是引起婴幼儿重症腹泻最重要的因素之一,也是造成发展中国家儿童死亡的重要原因。在世界各国,90%以上的婴儿在3岁之前均受到了RV的感染,在5岁以下腹泻住院患儿中,RV感染病例占20%--70%,5岁以上的儿童几乎全都感染过RV。美国每年与轮状病毒感染有关的医疗费用高达3—10亿元,可见轮状病毒腹泻不仅影响了全球儿童的健康,而且造成严重的经济负担。有效的疫苗接种可以显著减少由RV引起的儿童重症腹泻,也是唯一可行的预防控制RV较高发病率和死亡率的方法[1-2]。 轮状病毒颗粒由3层组成:外部衣壳、内部衣壳和核心。外部衣壳由VP7和VP4组成。VP7形成相对光滑的外表面,VP4组成钉状突起,二者都是中和抗原,可诱导机体产生中和抗体。VP4还在病毒感染过程中扮演重要的作用,作为一种细胞结合蛋白,VP4经胰酶切割后可增强轮状病毒对宿主的感染性。因此,以轮状病毒外壳蛋白基因VP4基因,将其重组到原核表达载体上,以研究病原基因在宿主菌中的表达情况,为新型口服疫苗的研究奠定基础[3-5]。 目的:构建携带轮状病毒外壳蛋白VP4基因的重组质粒(即pBEX/VP4),检测重组质粒的表达,并对重组载体表达蛋白的安全性进行验证。 方法:(1)根据Genbank中鼠源VP4的基因序列设计34对引物,通过重叠PCR合成VP4基因。以pGEX-5x-1为基础,构建穿梭表达载体pBEX,并将合成的VP4基因重组入pBEX,得到重组质粒pBEX/VP4。(2)将其转化大肠杆菌,SDS-PAGE验证蛋白的表达。通过肠袢实验验证表达蛋白的安全性。 结果:(1)测序表明,合成的VP4基因正确,并成功构建了穿梭载体pBEX/VP4。(2)VP4蛋白在大肠杆菌中成功表达,其表达蛋白经肠袢实验证实是无毒的。 结论:(1)在没有模板的情况下,重叠PCR法可以用来合成目的基因;(2)VP4蛋白在大肠杆菌中的表达,说明穿梭载体的构建是成功的,而肠袢实验验证了表达蛋白的安全性,为以后将重组穿梭载体转入宿主菌,作为新型口服疫苗的研究奠定了基础。
[Abstract]:Rotavirus (rotavirus, RV) infection is one of the most important factors of severe diarrhea in infants, but also an important cause of death in children in developing countries. In the world, more than 90% babies have been infected with RV before the age of 3, in the following 5 years old hospitalized children with diarrhea, RV infections accounted for 20%--70% 5 children over the age of almost all RV. infected each year in the United States with rotavirus infection related medical expenses up to 3 to 1 billion yuan, visible rotavirus diarrhea not only affects the global children's health, but also caused serious economic burden. A vaccine can significantly reduce the effect caused by RV in children with severe diarrhea. Is the only feasible method for the prevention and control of RV high morbidity and mortality [1-2].
Rotavirus particles consists of 3 layers: the outer capsid, internal and external capsid core. By VP7 and VP4.VP7 capsid formation of a relatively smooth surface, VP4 spikes, two are Neutralizing Antigen, can induce plays an important role in virus neutralizing antibody.VP4 during infection, as a cell binding protein VP4 of rotavirus infection was enhanced after cleavage. Therefore, in order to rotavirus coat protein gene VP4 gene was cloned into prokaryotic expression vector, to study the pathogenic gene expression in host strain, [3-5]. lay the foundation for the study of new oral vaccine
Objective: to construct a recombinant plasmid (pBEX/VP4) carrying the rotavirus coat protein VP4 gene, detect the expression of recombinant plasmid, and verify the safety of recombinant vector expressing protein.
Methods: (1) 34 pairs of primers were designed according to the gene sequence of mouse Genbank in VP4, VP4 gene was synthesized by overlapping PCR. On the basis of pGEX-5x-1, construct the shuttle expression vector pBEX, and recombinant VP4 into pBEX, the recombinant plasmid pBEX/VP4. (2) transformed Escherichia coli, the expression of SDS-PAGE protein to verify the expression of protein. The safety through intestinal loop experiments.
Results: (1) sequencing showed that the synthesized VP4 gene was correct and successfully constructed shuttle vector pBEX/VP4. (2) VP4 protein, which was successfully expressed in E.coli, and its expressed protein was proved to be non-toxic by intestinal loop test.
Conclusion: (1) in the absence of templates, overlapping PCR method can be used to synthesize the target gene; (2) the expression of VP4 protein in Escherichia coli, illustrate the construction of shuttle vector is successful, and the intestinal loop experiments verify the safety of protein expression, as will the recombinant shuttle vector into the host bacteria. As the research of new oral vaccine laid the foundation.
【学位授予单位】:重庆医科大学
【学位级别】:硕士
【学位授予年份】:2009
【分类号】:R392
【参考文献】
相关期刊论文 前10条
1 杨宇;吴元华;郑雅楠;;利用重叠延伸PCR技术进行DNA的人工合成[J];安徽农业科学;2006年09期
2 聂青和;罗新栋;;轮状病毒疫苗的种类及安全性问题[J];传染病信息;2006年01期
3 李晋涛,吴玉章,费蕾,唐艳,邹丽云;鼠源轮状病毒抗原基因的表达载体构建及农杆菌转化研究[J];第三军医大学学报;2002年08期
4 刘馨;孙茂盛;;轮状病毒VP4基因的原核表达、蛋白纯化及动物免疫试验[J];动物医学进展;2006年12期
5 曹阳;李冬田;尹冰楠;佟惠春;;重叠延伸PCR方法的建立与应用[J];河北医药;2005年11期
6 周鹏,董克家;利用套叠PCR技术进行基因突变和拼接[J];生命科学研究;2001年01期
7 陈波;;用重叠PCR合成植物甜蛋白brazzein基因[J];生物技术;2007年04期
8 李东;国泰;杨晓明;;轮状病毒疫苗的研究现状[J];中国生物制品学杂志;2007年11期
9 刘顺爱;浅野龙太郎;郭晶晶;刘志英;余康康;徐道振;;重叠延伸PCR法克隆重组复合干扰素[J];世界华人消化杂志;2008年05期
10 宋岩,李一经,魏丽丽,于小龙,樊琛,师东方;猪轮状病毒vp4基因在大肠杆菌中的表达[J];中国病毒学;2004年05期
,本文编号:1388440
本文链接:https://www.wllwen.com/yixuelunwen/shiyanyixue/1388440.html