口蹄疫3D蛋白N端T细胞表位重组蛋白的表达、纯化及鉴定

Expression,Purification and Identification of 3D Protein N Terminal T Cell Epitope Recombinant Protein of Foot and Mouth Disease Virus

为获得具有抗原性的口蹄疫病毒(FMDV)3D蛋白N端T细胞表位的重组表达蛋白,根据FMDV3D蛋白前115位氨基酸序列,设计优化并人工合成相应的核酸序列,将其克隆至pET-28a中,构建原核表达质粒pET28a-115,并将重组质粒转化至BL21(DE3)细胞。经IPTG诱导后,收集菌液进行SDS-PAGE和Western-bolt鉴定,结果显示,在16kD处有一条明显的蛋白表达条带。对表达产物进行可溶性分析,结果表达蛋白50%为可溶性蛋白,经Ni-NTA亲和层析纯化后获得了较高浓度和纯度的目的蛋白。研究结果表明,含FMDV 3D蛋白前115位氨基酸的重组蛋白在大肠杆菌中得到了高效可溶性表达,并具有较好的抗原活性,为开发口蹄疫诊断试剂和基因工程疫苗奠定了基础。

In order to obtain the recombinant protein that contained N terminal T cell epitopes in 3D protein of FMDV,the gene was synthesized according to the sequences of N terminal 115 ami- no acids. The recombinant expression vector pET28a-l15 was constructed by cloning the gene of 115 amino acids into the prokaryotic expression plasmid pET-28a. The BL21 （DE3） was trans- formed with pET28a-115 and the protein corresponding to the gene of 115 amino acids was ex- pressed after induction with IPTG. SDS-PAGE and Western-blot showed that the molecular mass of the protein was approximately 16 kD. The soluble analysis showed that the recombinant protein was almost solubly expressed. The high purity and activity protein was obtained after purified by Ni-NTA resins. The results showed that the recombinant protein that contained N terminal 115 amino acids of 3D protein was expressed solubly and efficiently in E. coli. The purified protein hasgoc igenicity,which lays foundation for developing the diagnosis antigen and genetic engineer- ing cine of FMDV.