鹿源BVDV E2的优化基因与布氏杆菌主要保护性抗原的融合基因在BCG中的共表达及rBCG免疫原性研究

Epidemiological investigation showed that at present our country the farmed deer of TB positive rate as high as 31-42%; Deer viral diarrhea - mucosal disease positive rate of 19.4-44.4%, the green deer is as high as 60-86.7%; Brucellosis were 8.87 -26.3%.Deer tuberculosis, brucellosis, viral diarrhea - mucosal disease had become the main seriously infectious diseases to threat the farmed deer breeding. Regretfully,no effective vaccines are currently available. To end that situation, on the basis of the early researches, based on collecting published BVDV E2 epitopes,we predicted and analyzeCTL,Th,and B-cell epitopes of BVDV E2 by bioinformatics methods.Based the above epitopes,CTL Th and B box were designed and the codons were optimized.Optimized BVDV E2 and L7/L12,Omp31 gene fusion by gene splicing by overlap extension PCR.Then constructed pVAX- E2- L7/L12-Omp31and pMV361L- E2- L7/L12-Omp31. pMV361L- E2- L7/L12-Omp31was transformed in BCG.RT-PCR,indirect immunofluorescence and Westen-Blotting was used for detection of each component antigen antigen.On the basis of analysis of constructed DNA vaccine and recombinant BCG, the Prime-Boostimmunization Strategy applied for analyzing the immunogenicity of DNA vaccine and recombinant BCG, the first using DNA vaccine then immuned with the recombinant BCG.The immunogenicity was research by indirect ELISA, IFN,ELISPO,WST-8 viable cell count,the Tlymphocyte subsets and serum cytokine.It is beneficial attempt and exploration to control and put out transmission and popular of viral diarrhea mucosal disease,brucellosis and tuberculosis in farmed deers.It would have play a profound inpact on the promotion of the great development of deer farming.

流行病学调查表明，目前我国鹿群的牛病毒性腹泻-黏膜病的阳性率达19.4-44.4%，布氏杆菌病的阳性率达8.87-26.3%，结核病的阳性率达31-42%。由于目前尚未研制出有效预防这些传染病的鹿防疫用专属疫苗，致使牛病毒性腹泻-黏膜病、布氏杆菌病、结核病在鹿群中的流行日益严重，已成为严重危害养鹿业发展的主要传染病。为了控制和扑灭这些鹿主要传染病，课题组拟在前期工作的基础上，系统的分析BVDV 主要保护性抗原E2基因的CTL、Th和B细胞表位，将各表位用柔性氨基酸优化串联后与布氏杆菌的主要保护性抗原L7/L12、Omp31的融合基因共同插入改造的pMV361L高效表达载体，转化至卡介苗中实现共同高效表达，引入prime-boost策略对构建的重组卡介苗的免疫原性进行测定分析。这将为研制开发出有效预防鹿主要传染病的多价基因工程疫苗做出有益的探索和尝试，对促进养鹿业的健康发展将产生深远的影响。

目前我国鹿群的牛病毒性腹泻-黏膜病的阳性率达19.4-44.4%，布氏杆菌病的阳性率达8.87-26.3%，结核病的阳性率达31-42%。由于目前尚未研制出有效预防这些传染病的鹿防疫用专属疫苗，致使牛病毒性腹泻-黏膜病、布氏杆菌病、结核病在鹿群中的流行日益严重，已成为严重危害养鹿业发展的主要传染病。本课题通过将BVDV 主要保护性抗原E2基因、布鲁氏菌保护性抗原L7/L12和Omp31基因融合表达，构建了单价、双价和三价重组卡介苗。同时构建了核酸疫苗。分别采用重组卡介苗免疫以及重组卡介苗初免和核酸疫苗加强免疫的方法，通过体液免疫和细胞免疫对重组卡介苗的免疫原性进行综合分析。研究表明BVDV E2-布鲁氏菌L7/L12-Omp31 rBCG可诱导实验动物产生良好的体液免疫和细胞免疫，重组卡介苗初免和核酸疫苗加强免疫的免疫效果优于重组卡介苗免疫。本研究对研发鹿主要传染病的多价基因工程疫苗做出有益的探索和尝试，对促进养鹿业的健康发展将产生深远的影响。