Preliminary Study On VP2 Vaccine Of EV71 C33 Strain In Inner Mongolia

Enterovirus virus type 71(enterovirus,EV71)infection can lead to HFMD(Hand,foot and mouth disease,HFMD),There is no effective treatment for the disease caused by EV71 virus,and whether the inactivated vaccine has certain preventive effects,need further study.VP2 is exposed to the surface with the main antigen sites of EV71,The VP2 not only contains antigen-determining cluster,and an important protein molecule involved in activating the cell immune responses for resisting EV71.Because there are many strains of EV71,the epidemiological characteristics of EV71 are generally different depend on the regions in which they spread.To check the detail variation of EV71 in Inner Mongolia and investigate the possibility of using VP2 as a vaccine to EV71,we selected the Inner Mongolia C33 strain of EV71 to clone its vp2 gene,and experssed recombinant VP2 protein in E.coli to test its immune-activating ability.The viral RNA was extracted and the 774 bp of vp2 cDNA was amplified and sequenced.The vp2 gene sequence of Inner Mongolia C33 strain has two SNPs compared to the GZ120-2010(HQ456307.1)of Guangzhou province,of which the G78?A and C288?G in the ORF will cause amino acid alterations of A?T and P?A,respectively.Then the cloned VP2 cDNA was inserted into pET3 a plasmid to construct expression vector of pET3a-VP2 and pET3a-HIS-VP2.The recombinant VP2 protein and HIS-VP2 protein(with His-tag)were expressed efficiently in E.coli BL21(DE3).Mice were immunized with purified VP2 and HIS-VP2 followed by injection of S180 cells to generate the ascites polyclonal antibodies against VP2 and HIS-VP2.Both the ascites antibody of VP2 and HIS-VP2 could recognize EV71 specificily without significant difference.When the diluted ascites antibody(1:16)were added into medium of Vero cells with EV71 virus,its CPE less than 50%,indicating the antibodies had efficiently protected the cells from EV71 infection.The survival rate of those one-day-old infant mice injected with a mixture of antibodies with EV71(200 ?L/each)is nearly 70%,also suggesting the antibodies have good protection efficiency.Taken together,the VP2 ascites antibodies prepared by this experiment have good immunogenicity,and certain protective effect on cells and animals,thus may be a candidate for EV71 vaccine.