【作者】 林志鹏；

【导师】 万敏；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2017， 硕士

【摘要】 本文以口蹄疫病毒VP1的B细胞表位和T细胞表位作为研究对象,以表位串联的方式构建仅由B表位组成的12B1重组蛋白及由B表位和T表位组成的13B1T重组蛋白,分析二者的空间结构,并针对大肠杆菌表达的两种蛋白免疫活性进行了研究。研究结果表明12B1重组蛋白具有良好的免疫活性,但13B1T重组蛋白的免疫活性差。本研究对仅由表位组成的亚单位疫苗的抗原构建具有一定的参考价值。更多还原

【Abstract】 VP1 is one of foot-and-mouth virus capsid proteins,it exposure at the surface of the spatial structure of the virus and can stimulate the body to produce neutralizing antibodies against FMDV.There is a G-H loop structure of VP1 through space structure analysis.The G-H loop is a important neutralization epitope.of VP1,it is very easy mutative,but there is a highly conserved amino acid sequence in G-H loop-arginine-glycine-aspartic acid(RGD).It binds to the integrin receptors of cell surface.The paper selected a neutralizing epitope which contain RGD sequence through analysis of space structure and named it B1 epitope.Through series restructuring ways to get a recombinant protein which construct of 12 B1 epitopes and construct another protein which connected by B1 epitopes and T epitope of FMDV.Then analysis the activation of both of recombinants in E.coli and Baculovirus system.The 12B1 recombinant protein firstly expressed in BEVs-insect cell expression system.Baculovirus expression system is one kind of eukaryon expression system which can fuse exogenous gene and bacluvirus.It has post-translational modification and high safety adventages and now it is widely used in research and clinical applications.The expriment results show that 12B1 recombinant can express in insect cells,but the expression level is low.So we choos the E.coli expression system instead of BEVs.E.coli expression system is the most commonly expression system for foreign genes,it is clear genetic background,simple and convenient operation,short cultivation period,high conversion efficiency,low costs,high expression and easy to purification.But the prokaryotic expression system do not contain the function of post-translational modification,expression of recombinant protein activity may be lower.To express the 12B1 and 13B1 T proteins in E.coli expression system,sooptimized the codons.The expriments results show that 12B1 and 13B1 T recombinant proteins can express in E.coli.The proteins both express in soluble forms and get high concentrations recombinant proteins.ELisa and Western results show that 12B1 and13B1 T recombinant can be identified by anti-FMDV-positive serum.The 12B1 recombinant protein can stimulate mice producing anti-FMDV antibody.But the13B1 T recombinant protein can not stimulate mice producing anti-FMDV antibody and it can not stimulate the B cells proliferation and activation after 13B1 T recombinant protein stimulating spleen cells in vitro.Above all,12B1 and 13B1 T recombinant proteins are able to simulate space structure of FMDV VP1 and 12B1 recombinant protein can stimulate body to produce specific neutralizing antibody of FMDV,but13B1 T recombinant protein can not.So using the method of single epitope repeated concatenation to build the recombinant proteins may get better immunogenicity.This way provides a reference for building vaccine.But the activation of 12B1 and 13B1 T recombinant proteins are not very high.So we can choose other systems for proteins expression.更多还原