TGEV和PEDV融合S蛋白在乳酸乳球菌食品级细胞壁锚定高效诱导表达与免疫原性分析  

The Food-grade Cell Wall Anchored High Efficient Induction Expression the Fusion Protein S of TGEV and PEDV in Lactococcus lactis and the Immunogenicity Analysis

作　　者：徐波[1] 熊维娜[1] 李烨青 周通[1] 刘志文[1] 应碧 郭晓燕[1] 

机构地区：[1]江西农业大学生物科学与工程学院,南昌330045

出　　处：《中国食品学报》2015年第6期32-38,共7页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家自然科学基金项目(31360372);江西省科技支撑计划项目(2009BNB05704)

摘　　要：近年来猪传染性胃肠炎和猪流行性腹泻在我国广泛流行,给养殖业带来了巨大的经济损失。目前对此两种疾病的防治手段还停留在使用常规疫苗。现有的疫苗免疫效果不够理想,急需寻求一种安全高效的防治手段。本文首先构建了猪传染性胃肠炎病毒(TGEV)和猪流行性腹泻病毒(PEDV)融合S基因,以乳酸乳球菌食品级细胞壁锚定高效诱导表达载体p RNV48为基础,构建了TGEV和PEDV融合S基因的表达质粒p RNV48-TPs。将得到的重组质粒用电穿孔方法转入乳酸乳球菌NZ9000感受态细胞中,获得了重组菌乳酸乳球菌Z9000/p RNV48-TPs。再利用nisin对重组菌进行诱导,提取细胞壁蛋白后采用聚丙烯酰胺凝胶电泳(SDS-PAGE)分析目的蛋白的表达情况,然后分别对兔子进行注射免疫和口服免疫,并用Western Blot分析。通过乳酸乳球菌食品级细胞壁锚定高效诱导表达载体,诱导表达TGEV与PEDV融合S蛋白产生中和抗体,以期探讨口服疫苗的免疫反应机理,为由TGEV与PEDV研发的安全、高效的疫苗奠定良好的基础。Porcine transmissible gastroenteritis and porcine epidemic diarrhea are widely spread in China, which has brought huge economic losses to Chinese breeding industry. At present, the prevention and treatments of the two diseases are still using conventional vaccine. The existing vaccine is not effective enough and there is an urgent need for a safe and efficient means of prevention. Firstly, the fusion gene S of TGEV and PEDV was successfully constructed. The pRNV48-TPs were successfully constructed by using the food-grade cell wall anchored high efficient induced expression vector pRNV48 of Lactococcus lactis. To acquire the recombinant bacteria L. lactis Nzg000/pRNV48-TPs, the recombinant plasmid was transferred into the L. lactis NZ9000 competent cells by electroporation method. Then the recombinant strain was induced by nisin and the SDS-PAGE was used to analyse the expression of the target protein after the cell wall protein was extracted. And then rabbits were administrated by injection immunization and oral immunization respectively and then analyzed by Western Blot. In order to discuss the immune response mechanism of the oral vaccine and lay a good foundation for TGEV and PEDV research and development of a safe and efficient vaccine, the Lactococcus lactis food-grade cell wall anchored high efficient induced expression vector carrier was used to induce the fusion protein S of TGEV and PEDV to produce neutralizing antibodies.

关 键 词：TGEV和PEDV融合S蛋白 乳酸乳球菌 食品级载体 细胞壁锚定高效诱导表达 免疫原性 

分 类 号：S852.4[农业科学—基础兽医学]