Study On The Expression And Mucosal Immunization Of Enterovirus 71 Structure Protein VP1

Hand-Foot-mouth disease (HFMD) caused by enterovirus, Picornaviridae:Coxsackie virus(CA) 2,4,5,7,9,10 and 16 type of group A,1,2,3,4,5 type of group B; some ECHO viruses and enterovirus 71. Among of them the most common pathogens are CA16 and EV71. It is a acute infectious diseases, the main cinical symptoms are fever with rash appears on the hand, foot, mouth and buttock. Most of the patients of HFMD will generally recovered in 5 to 7 days, it is a self limiting diseases. Viruses can also infect the respiratory system, central nervous system, cardiovascular system, could cause severe diseases such as encephalitis, myocarditis, pulmonary edema, symptoms as acute flabby paralysis. For many reasons, severe illed children could die. Most of the severe cases caused by EV71. The HFMD could occur all over the year, particularly in the summer and autumn, most commonly attacks the children under five years old. Main routes of transmission are fecal-oral root, may also spread through the respiratory tract. HFMD has a strong contagious ability, could easily cause outbreak.In recent years, with the Hand-Foot-mouth disease outbreak is on the rise, more concern is paying to the main pathogen EV71. Due to no specific treatments for HFMD, vaccine have become the most effective preventive measures. EV71 is mostly spread through fecal-oral and aerosol form, according to the vaccine designing principle "in situ immunization", it is reasonable to immunize through the mucosal surface. Compared with the traditional vaccine, EV71 mucosal vaccine could be more security, economic and easy to achieve mass vaccination of population and many other advantages.There are three major parts in the research:Part one:purification of EV71 virus and expression of the VP1 protein in prokaryotic cellBy epidemiology information, in China mainland, the most EV71 epidemic caused by C4 subtype, so we select C4 subtype HN-8 isolate as vaccine candidate, The VP1 gene was amplified, sequenced and cloned into the pET30a, the expression of VP1 was induced by IPTG in BL21(DE3) and purified. Up to 30-40mg purified recombinant VP1 was obtained from per liter of induced bacteria. At last SDS-PAGE, Western blot were performed to analysis the purified VP1 protein. We also cultivate the isolated EV71 strain HN-8 in Vero cell, through the continuous passage,the titer could up to 108TCID50/1ml and we successfully purified the EV71 particle through ultra-centrifugation.Part two:VP1 express in insect cell and its purification.Amplification the VP1 gene from pET30a-VP1 plasmid and cloned into pAcGP67-B vector. After sequencing the clone, choose the right one which has no base change in the VP1 gene, mixed the plasmid pAcGP67-B-VP1 with the baculovirus linear DNA and co-transfect SF9 cells, continuous passage to increased titer of baculovirus. The expression of Vp1 was comfirmed by indirect immunefluorescence assay, SDS-PAGE and Western blot analysises. In order to detect IgM antibody in mice serum, we also conjugated the VP1 protein with HRP, and work out the VP1-HRP working concentration. When coating the anti mouseμchain antibody with 200ng per well, the best working concentration of VP1-HRP is 1:160.Part three:the immune effect by oral root immunize the mice with VP1 plus chitosanTolerance could easily be induced by mucosal root immunization, so adjuvant is need to effectively stimulate the mucosal immune response in mice. We mix the recombinant VP1 and chitosan adjuvant to compose the candidate vaccine, with female Balb/C mice as animal models. Immunize the mice by oral root. The VP1 mucosal vaccine has two does groups:100μg and 1000μg. We also designed three positive control groups:inactivated EV71 virus mixed with Chitosan (mucosal way immune); inactivated EV71 viruses mixed with AI(OH)3 adjuvant and VP1 protein mixed with AI(OH)3 adjuvant (muscle injection way immune). The negative control is PBS solution mixed with Chitosan and AI(OH)3 adjuvant separately. The 0 day,7 day and 28day immune program is adopted.In this part, we finished the VP1 protein high and low does mucosal immunization and evaluate the immunize effect with the positive and the negative control. In our experiment, we find out that after immunize the Balb/C mice with VP1 mucosal vaccine, it could induce the IgM and IgG specific response in the serum. The mucosal vaccine could also induce strong and long specific slgA response on the mucosal surface of small intestinal, vagina and respiratory tract. The specific slgA play a quite important role in protecting the mucosal surface against the EV71 pathogen infection. The IFN-γsecreted Thl cell in the mice spleen is also counted by the ELISPOT experiment, the IFN-γsecreted Thl cell in the VP1 mucosal vaccine group is apparently more than the negative control. The result of the neutralization results in vitro show that the VP1 high does mucosal immunize group neutralize antibody titer could achieve 1:16.With the prokaryotic cell expressed VP1 protein mixed with Chitosan as research model. We studied in this research by the way of oral root immunization, with the help of Chitosan adjuvant, VP1 protein (100μg and 1000μg) could induce specific humoral and cell level immune response. Our results offer a substantial theory and method support for the further study of EV71 mucosal vaccine.