Isolation And Identification Of Local Strains Of Enterovirus 71, Construction Of Eukaryotic Expression Vector Carring VP1 And Study Of Its Immunological

Objective Enterovirus 71 (EV71) is a major causative viral agent responsible for hand,foot and mouth disease (HFMD) and often causes a variety of nervous system complications. There is no effective antiviral treatment for severe EV71 infection and no vaccine is available. In this study, we isolated EV71 from specimens of patients with HFMD in Luan. Full-length VP1 gene was amplified and cloned into eukaryotic expression vector pcDNA3.1(+).To observe the humoral immune response and cellular immune response in mice induced by intramuscular injection in BALB/c mice. Find a new and effective way to prevent EV71 infection .Methods⑴First, in this study,we collected 51 specimens from 33 cases of children with HFMD (33 throat swab, 12 herpes fluid, 6 cerebrospinal fluid) and isolated virus using of Vero cell culture. All positive specimens were observed using transmission electron microscope and identified for EV71 by neutralization test using EV71 standard serum and by RT-PCR using EV71 specific primers;⑵Subsequently, the RNA were extracted from one EV71 local strains.And then the VP1 gene were amplified using RT-PCR and cloned into the pMD18-T simple vector. The constructs were transformed into competent E.coli cells and the positive clones were selected using restriction digestion, RT-PCR and DNA sequencing analysis. The correct sequencing of the VP1 gene was inserted into eukaryotic expression vector, pcDNA3.1(+). The recombinant plasmid were test for protein expression in COS-7 cells;⑶Eventually, in the in vivo studies, 18 female BALB/c mice aged between 6-8 weeks were randomly divided in 3 groups of 6 and were immunised by intramuscular injection with the recombinant plasmid., in which group A, the mice were injected with 100μg/100μl the recombinant plasmid pcDNA3.1(+)/VP1 per mouse. The two negative control groups were injected with the backbone vector pcDNA3.1(+) (100μg/100μl per mouse) and PBS (100μl per mouse). The mice were given booster injections at 2 and 4 weeks post-immunisation. Blood samples were collected from the mice via the tail vein before immunization and via the orbital artery and vein after the last immunization 2 weeks, meanwhiles, prepared T lymphocytes from the mouse. The indirect Enzyme Linked Immunosorbent Assay(ELISA) were performed to detect the presence of anti-VP1 IgG in the sera of immunised mice and the virus neutralizing assay were performed to assess the ability of the mice serum to neutralise the live EV71. The proliferation of T lymphocytes by MTT assay in order to observe the cell immunity.Results⑴In this study, 14 suspicimens enterovirus positive throat swab specimens (42.4%) were found from 14 patients with HFMD, in which 2 herpes fluid specimens (16.7%) at the same time.We confirmed they were enterovirus virus by transmission electron microscope.In the end,6 EV71 positive specimens(42.9%) were found by micro-neutralization test using EV71 standard serum and by RT-PCR using EV71 specific primers;⑵Sequencing results showed that the homology of nucleotide and amino acid between the VP1 of local strain and EV71 BrCr strain were 98.4% and 96.3%. Identified by restriction digestion analysis and RT-PCR analysis, the recombinant eukaryotic expression plasmid pcDNA3.1(+)/VP1 was successfully constructed. By RT-PCR,mRNA of VP1 gene fragment could be detected in transfected COS-7 cells.The result of Western blot showed that there was a protein band about 34KD in cell lysis of transfected COS-7 cells,and it could be specifically recognized by EV71 polyclonal antibody;⑶The recombinant eukaryotic expression plasmid pcDNA3.1 (+)/VP1 was used to immune BALB/c mice.The serum-specific IgG antibody levels significantly increased after the initial immunization, OD450 was 0.218, and then increased along with the number of immune increased. OD450 was 0.559 in week 2 after the last immunization. A significantly difference was test between A group and B group or C group.(p <0.01). The result of neutralizing assay showed that the anti-VP1 IgG had neutralising activity against EV71. The activity of T lymphocytes proliferation increases and the stimulation index (SI) were: A group 2.410±0.12, B group 1.400±0.21, C group 1.241±0.19, The SI of A group were significantly higher than B group and C group (p <0.01).Conclusions⑴EV71 is one of the main pathogens for the hand, foot and mouth disease occored in Liuan region.⑵The recombinant eukaryotic expression plasmid pcDNA3.1(+)/VP1 was successfully constructed, and VP1 protein express in COS-7 cells.⑶The female mice injected recombinant eukaryotic expression plasmid pcDNA3.1(+)/VP1 were available for a certain degree of humoral immunity and cellular immune responses.This study has given to a solid foundation for the nucleic acid vaccine against EV71.