布鲁氏菌强启动子的筛选及重组口蹄疫病毒VP1基因的表达

发布时间：2018-04-20 17:46

本文选题：猪种布鲁氏菌 + 口蹄疫病毒VP1基因　；参考：《山东农业大学》2017年硕士论文

【摘要】：布鲁氏菌病(Brucellosis,简称布病)是由布鲁氏菌引起的一种重要的人兽共患病,2012年我国国务院印发了《国家中长期动物疫病防治规划(2011-2020年)》,将布病列为优先防治的国内16种动物疫病之一,并作为当前防控工作的重中之重。该病对畜牧业和人类健康均构成严重威胁,使用疫苗免疫是防控布氏菌病的重要措施之一。目前,我国用于控制布病的疫苗主要是S2株(猪型2号苗),其具有毒力较弱、免疫效果良好、安全性高等特点。布鲁氏菌能够引起机体强烈的Th1型免疫应答,这使得布鲁氏菌成为理想的异源性抗原载体,为了开发出能高效表达外源基因的布鲁氏菌活载体疫苗,在本课题中我们首先使用绿色荧光蛋白作为报告基因,将布鲁氏菌组成型启动子Psod、Pdnaj和Pdnak分别克隆入YGT-p BBR1mcs2,得到重组表达载体YGTp BBR1mcs2-Pdnaj、YGT-p BBR1mcs2-Psod和YGT-p BBR1mcs2-Pdnak并转化至布鲁氏菌。荧光显微镜检测GFP蛋白的表达情况。结果表明,3种不同强度的启动子均能在布鲁氏菌S2中启动GFP基因的表达且启动能力由强到弱依次为:Pdnak、Pdnaj、Psod。口蹄疫(Foot and mouth disease,FMD)是由口蹄疫病毒(Foot and mouth disease virus,FMDV)引起的偶蹄动物的一种急性、热性、高度接触性传染病,该病可引起偶蹄类动物的口、足等部位皮肤出现水泡而造成部份动物死亡,严重影响畜牧产业的发展。VP1蛋白是FMDV的主要结构蛋白,是病毒感染细胞的关键,具有与细胞受体结合的位点。口蹄疫VP1蛋白可作为研制口蹄疫基因工程疫苗的首选抗原。本研究以猪种布鲁氏菌S2株为亲本株,利用蔗糖自杀质粒载体,将FMDV的VP1基因表达框部分替代了布鲁氏菌wbo A基因,成功构建了重组口蹄疫病毒VP1基因的布鲁氏菌病活疫苗。使用荧光定量PCR的方法对重组菌的VP1基因的转录情况进行检测,发现VP1基因在宿主菌内有显著的转录变化,说明口蹄疫病毒VP1基因在m RNA水平上表达。将重组菌与原始菌灭活后进行超声破碎,行进Western Blot验证,结果显示,重组菌比原始菌多出一条分子量大小为30k Da左右的条带,与VP1蛋白理论大小一致,证明了口蹄疫病毒VP1在布鲁氏菌体内的表达。本研究初步筛选了布鲁氏菌的启动子,并对其强弱进行验证;初步建立了以猪种布鲁氏菌为载体表达外源基因的方法,为布鲁氏菌基因工程活载体疫苗的进一步研究奠定了基础。
[Abstract]:Brucellosis (brucellosis) is an important zoonosis caused by brucellosis. In 2012, the State Council of our country issued the National medium and long term Animal epidemic Prevention Program 2011-2020, which listed brucellosis as the priority control in China. One of the animal blight species, And as the top priority of the current prevention and control work. The disease poses a serious threat to animal husbandry and human health. Vaccine immunization is one of the important measures to prevent and control brucellosis. At present, the main vaccine used to control brucellosis in China is S2 strain, which has the characteristics of weak virulence, good immune effect and high safety. Brucella can induce a strong Th1 immune response, which makes Brucella become an ideal xenogenic antigen vector, in order to develop brucellosis live vector vaccine which can express foreign gene efficiently. In this study, we first used green fluorescent protein as reporter gene and cloned Psodnaj and Pdnak into YGT-p BBR1mcs2, respectively, to obtain the recombinant expression vectors YGTp BBR1mcs2-PdnajAYGT-p BBR1mcs2-Psod and YGT-p BBR1mcs2-Pdnak and transformed them into Brucella. The expression of GFP protein was detected by fluorescence microscope. The results showed that all three kinds of promoters with different intensities could initiate the expression of GFP gene in Brucella S2, and the sequence of promoter ability from strong to weak was: 1. Foot and mouth disease (FMD) is an acute, feverish, highly contact infectious disease of cloven-hoofed animals caused by foot and mouth disease virus, which can cause blisters in the mouth and foot of cloven-hoofed animals and cause some animals to die. VP1 protein is the main structural protein of FMDV, which is the key of virus infection, and has the site of cell receptor binding. Foot-and-mouth disease (FMD) VP1 protein can be used as the preferred antigen for the development of FMD genetic engineering vaccine. In this study, using S _ 2 strain of porcine brucella as parent strain, using sucrose suicide plasmid vector, the wbo A gene of brucella was partially replaced by VP1 gene expression frame of FMDV, and the brucellosis vaccine of recombinant VP1 gene of foot-and-mouth disease virus was successfully constructed. Fluorescence quantitative PCR was used to detect the transcription of the VP1 gene of the recombinant strain. It was found that the VP1 gene had significant transcriptional changes in the host bacteria, indicating that the VP1 gene of foot-and-mouth disease virus was expressed at m RNA level. After inactivation of recombinant bacteria and original bacteria, ultrasonic fragmentation was carried out, and Western Blot verification was carried out. The results showed that the recombinant bacteria had more bands with molecular weight of about 30kDa than the original bacteria, which was consistent with the theoretical size of VP1 protein. The expression of foot-and-mouth disease virus (FMDV) VP1 in brucella was confirmed. In this study, the promoter of brucella was screened, and its strength was verified, and a method of expressing foreign gene in porcine brucella was established. It lays a foundation for the further research of brucella genetic engineering live vector vaccine.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.6

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