Generation Of Mouse Antiserum Against VP1 Of The Inner Mongolia C008 EV71 Virus Strain And Test Of Its Neutralizing Effect

Enterovirus71(EV71)is one kind of the Enterovirus in the Picornavirus family.It is a symmetrical icosahedron of 30 nm diameter capsid consisting of RNA but without outer envelope and neurite.Around in microsphere form,EV71 virus was initially reported in 1974.In the past decade,it has become the major pathogen of hand,foot and mouth disease(HFMD)in Asian-Pacific region.What’s worse,once being infected by EV71 virus,people may have a high fever,suffer from neurological complications,fatal encephalitis and pulmonary edema.The capsid particle of EV71 virus is made of four kinds of structural proteins(VP1～4),surrounding a 7.4 kb single strand RNA.Only VP4 protein embeds inside virus and closely connected with the RNA core,other three structural proteins all expose on surface of virus capsid,thus possess main antigenic determinant of EV71 virus.According to researches,the ORF(Open Reading Frame)region of VP1 gene is liable for variation which provides the virus with genetic diversity,and becomes the major classification criterion.The antigenic determinants on the VP1 protein is the determinant of EV71 virus antigenicity,consisting most of the epitope for antibody neutral reaction.Therefore,VP1 protein may be used as a vaccine to generate antibodies against EV71 virus.In this paper,in order to address the possibility to use VP1 as a vaccine,and genotyping of VP1 protein in EV71 virus in Inner Mongolia,we cloned VP1 gene from an EV71 virus strain on the pharynx test paper of child patient in Wuhai,Inner Mongolia.We extract the total RNA of EV71,and designed the primers according to the gene sequence of EV71 VP1 in NCBI,and amplified the cDNA of VP1 by one-step RT-PCR.Then,we inserted the VP1 cDNA into pET3a vector to express the HIS tagged-recombinant VP1 protein and VP1 without HIS tag in E.coli BL21.The recombinant proteins VP1 and HIS-VP1 were obtained and purified.Western blot using the serum of BALB/c mouse which were immunized with both purified VP1 protein and HIS-VP1 protein as well as the EV71 virus as the primary antibody confirmed that the recombinant VP1 could induce anti-VP 1 serum.Finally,we tested the ability of the anti serum against VP1 to protect cells from EV71 infection.The results of neutralization test indicated that both anti-VP1 and anti-HIS-VP1 of 1:65 536 serum dilution could protect the cell from EV71,while the anti-EV71 virus serum of 1:81 920 dilution could protect the cell.Although anti-HIS-VP1 serum is a little inferior to the anti-EV71 serum,our experiments demonstrated that the efficacy as antigen and liability to express in bacteria make the VP 1 and 6His-VP 1 good candidates to be safe vaccines to EV71.