口蹄疫乳酸杆菌活载体疫苗的构建及其诱导的黏膜免疫效果
发布时间：2018-11-06 11:37
【摘要】：口蹄疫(Foot-and-Mouth Disease,FMD)是由口蹄疫病毒(Foot-and-Mouth Disease Virus,FMDV)引起的一种烈性、急性家畜传染病,传播速度快,影响范围大,给畜禽养殖业造成重大的经济损失。随着规模化养殖程度的提高,FMD等传染性疫病对家畜养殖业的危害越来越严重,防治难度不断加大,传统防治措施已突显其不足,对新的防控技术、特别是安全有效的新型疫苗的需求日益迫切。VP1是编码FMDV-VP1蛋白的免疫原性基因,表达的蛋白带有诱导FMD免疫反应的关键表位。本研究将FMDV VP1基因克隆并进行优化,构建重组表达质粒并检测其在大肠杆菌和乳酸杆菌中的表达,最后将重组乳酸杆菌免疫豚鼠检测其诱导的黏膜免疫保护效果。具体研究方法和结果如下:1.FMDV VP1基因的原核表达载体的构建及重组乳酸杆菌的制备从A型FMDV中克隆出VP1基因后,根据乳酸杆菌密码子偏好性对VP1进行优化,与大肠杆菌-乳酸杆菌穿梭表达载体p SIP411进行连接,并将连接产物转化到DH5α中筛选出阳性克隆菌,重组质粒酶切和PCR鉴定成功后再转化到BL21中进行诱导表达,表达产物经SDS-PAGE和western blot检测可见在24k Da处有明显的条带,证明VP1-p SIP411大肠杆菌-乳酸杆菌穿梭重组表达载体构建成功并且在BL21中得到表达的蛋白具有反应原性。然后将重组质粒p SIP411-VP1电转化到乳酸杆菌NC8和WCFS1中,通过质粒提取、双酶切鉴定和PCR鉴定等方法筛选出的阳性菌进行Spp IP诱导表达,表达产物经western blot检测可见明显的目的条带,证明VP1在NC8和WCFS1中得到表达并且具有反应原性。2.重组乳酸杆菌免疫豚鼠以及免疫效果的检测动物免疫试验中将豚鼠分为5组(p SIP411-VP1-NC8,p SIP411-NC8,p SIP411-VP1-WCFS1,p SIP411-WCFS1和阴性对照组),免疫期间采集豚鼠唾液、粪便和血清样品进行ELISA检测,用流式细胞仪检测外周血中CD4+、CD8+淋巴细胞表达水平,CFSE法测定脾脏淋巴细胞增殖情况,细胞中和试验检测中和抗体含量。结果显示,两组重组乳酸杆菌免疫组抗体水平明显高于其他三组,差异显著,CD4+、CD8+淋巴细胞含量明显增多,脾淋巴细胞在抗原刺激后增殖明显,可在血清中检测到中和抗体。以上结果表明,VP1在重组乳酸杆菌NC8和WCFS1中获得了表达,并且免疫动物后增强了动物的免疫功能,抵抗了FMDV的感染,为研制预防FMDV的重组乳酸杆菌疫苗奠定了实验基础。
[Abstract]:Foot-and-mouth disease (Foot-and-Mouth Disease,FMD) is a kind of acute infectious disease caused by foot-and-mouth disease virus (Foot-and-Mouth Disease Virus,FMDV). With the development of large-scale aquaculture, FMD and other infectious diseases are becoming more and more serious to livestock breeding, and the difficulty of prevention and control is increasing. The traditional prevention and control measures have highlighted its shortcomings, and the new technology of prevention and control has been brought to light. VP1 is the immunogenicity gene encoding FMDV-VP1 protein, and the expressed protein has the key epitope to induce FMD immune response. In this study, FMDV VP1 gene was cloned and optimized to construct recombinant expression plasmid and detect its expression in Escherichia coli and Lactobacillus coli. The specific research methods and results are as follows: construction of prokaryotic expression vector of 1.FMDV VP1 gene and preparation of recombinant Lactobacillus coli VP1 gene were cloned from type A FMDV and VP1 was optimized according to codon preference of Lactobacillus. The recombinant plasmid was ligated with Escherichia coli-Lactobacillus shuttle expression vector p SIP411, and the product was transformed into DH5 伪 to screen positive clones. The recombinant plasmid was digested and identified by PCR and then transformed into BL21 to induce expression. SDS-PAGE and western blot analysis showed that there were obvious bands at 24k Da, which proved that the recombinant expression vector of VP1-p SIP411 Escherichia coli-Lactobacillus shuttle was successfully constructed and expressed in BL21. Then the recombinant plasmid p SIP411-VP1 was electrotransformed into NC8 and WCFS1 of Lactobacillus. The positive bacteria screened by plasmid extraction, double enzyme digestion and PCR identification were induced by Spp IP expression. The expression of VP1 in NC8 and WCFS1 showed obvious target bands by western blot, which showed that VP1 was expressed in NC8 and WCFS1. 2. 2. Guinea pigs immunized with recombinant lactobacillus and their immunological effects the guinea pigs were divided into 5 groups (p SIP411-VP1-NC8,p SIP411-VP1-WCFS1,p SIP411-WCFS1 and negative control group) in the animal immune test. Saliva was collected from guinea pigs during immunization. The expression of CD4 and CD8 in peripheral blood was detected by flow cytometry, the proliferation of splenic lymphocytes by CFSE, and the content of neutralizing antibody by neutralization test. The results showed that the antibody levels of the two groups were significantly higher than those of the other three groups. The levels of CD4 and CD8 lymphocytes were significantly increased and the spleen lymphocytes proliferated after antigen stimulation. Neutralizing antibodies could be detected in serum. The results showed that VP1 was expressed in recombinant Lactobacillus NC8 and WCFS1, and the immune function of the animals was enhanced after immunization, and the infection of FMDV was resisted, which laid an experimental foundation for the development of recombinant Lactobacillus vaccine for the prevention of FMDV.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4
本文编号：2314156
[Abstract]:Foot-and-mouth disease (Foot-and-Mouth Disease,FMD) is a kind of acute infectious disease caused by foot-and-mouth disease virus (Foot-and-Mouth Disease Virus,FMDV). With the development of large-scale aquaculture, FMD and other infectious diseases are becoming more and more serious to livestock breeding, and the difficulty of prevention and control is increasing. The traditional prevention and control measures have highlighted its shortcomings, and the new technology of prevention and control has been brought to light. VP1 is the immunogenicity gene encoding FMDV-VP1 protein, and the expressed protein has the key epitope to induce FMD immune response. In this study, FMDV VP1 gene was cloned and optimized to construct recombinant expression plasmid and detect its expression in Escherichia coli and Lactobacillus coli. The specific research methods and results are as follows: construction of prokaryotic expression vector of 1.FMDV VP1 gene and preparation of recombinant Lactobacillus coli VP1 gene were cloned from type A FMDV and VP1 was optimized according to codon preference of Lactobacillus. The recombinant plasmid was ligated with Escherichia coli-Lactobacillus shuttle expression vector p SIP411, and the product was transformed into DH5 伪 to screen positive clones. The recombinant plasmid was digested and identified by PCR and then transformed into BL21 to induce expression. SDS-PAGE and western blot analysis showed that there were obvious bands at 24k Da, which proved that the recombinant expression vector of VP1-p SIP411 Escherichia coli-Lactobacillus shuttle was successfully constructed and expressed in BL21. Then the recombinant plasmid p SIP411-VP1 was electrotransformed into NC8 and WCFS1 of Lactobacillus. The positive bacteria screened by plasmid extraction, double enzyme digestion and PCR identification were induced by Spp IP expression. The expression of VP1 in NC8 and WCFS1 showed obvious target bands by western blot, which showed that VP1 was expressed in NC8 and WCFS1. 2. 2. Guinea pigs immunized with recombinant lactobacillus and their immunological effects the guinea pigs were divided into 5 groups (p SIP411-VP1-NC8,p SIP411-VP1-WCFS1,p SIP411-WCFS1 and negative control group) in the animal immune test. Saliva was collected from guinea pigs during immunization. The expression of CD4 and CD8 in peripheral blood was detected by flow cytometry, the proliferation of splenic lymphocytes by CFSE, and the content of neutralizing antibody by neutralization test. The results showed that the antibody levels of the two groups were significantly higher than those of the other three groups. The levels of CD4 and CD8 lymphocytes were significantly increased and the spleen lymphocytes proliferated after antigen stimulation. Neutralizing antibodies could be detected in serum. The results showed that VP1 was expressed in recombinant Lactobacillus NC8 and WCFS1, and the immune function of the animals was enhanced after immunization, and the infection of FMDV was resisted, which laid an experimental foundation for the development of recombinant Lactobacillus vaccine for the prevention of FMDV.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S852.4
【参考文献】
相关硕士学位论文 前2条
1 王吉玮;口蹄疫干粉制剂对动物鼻黏膜免疫效果研究[D];中国农业科学院;2011年
2 王景锋;羊口蹄疫病毒Asia 1型多表位疫苗的研制[D];中国农业科学院;2010年
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