1.Research On B-cell Peptide And Antigens Originated From Spike Protein In SARS-CoV Infection 2.Preparation And Identification Of Genetically Engeering Vaccines Of Enterovirus 71

Severe acute respiratory syndrome(SARS) is caused by SARS-CoV, it was designated as one of the most virulent contagious diseases in 21st century for fast spreading, broad scope and high mortality. SARS-CoV is a new number of coronavirus family, it is an unknown coronavirus neither a variant nor a recombinant of coronavirus. As a new type of coronavirus, its virulence exceeds other family numbers so that there are no effective antivirus medicines for SARS treatment. It is commonly recognized that the most fundamental method to prevent SARS prevailing again is to develope a safe and effective SARS vaccine and carrying out vaccination among people. There are many vaccine forms including SARS vaccine of virus inactivation, DNA vaccine originated SARS-CoV structural proteins and live-vector vaccine. Epitope vaccine is a new developing vaccine form with excellent security and strong pertinence. In our research, we demonstrated the functions of B-cell epitope peptides and their antigens originated from SARS-CoV Spike protein in the process of SARS-CoV infection, and discussed the mechanisms of their roles, these works will provide powerful scientific evidences for development of SARS-CoV vaccine.In this study, the monoclonal antibodies of four B-cell epitope peptides originated from Spike protein were used on Vero-E6 cell which was challenged with SARS-CoV. The neutralizing antibodies were screened out and at the same time, the Vero-E6 cell expressed infection enhancement with the increasing antibody concentration of S(597-625)43-3-14. Subsequently, different combinations of the four epitope pipetides were immunized in rhesus macaque, after stabilizing antibody level was detected, the monkeys were challenged with SARS-CoV and then euthanatized for detection of pathological changes and virus load of lung tissues. The results showed that monkeys immunized with peptide S(597-625) displayed more severe pulmonary interstitial inflammation and higher virus load than monkeys in control group and other test groups.Another test of antibody for making clear antibody dependent enhancement(ADE) was performed immediately. S(597-625)43-3-14 antibody with three gradually increasing concentrations were injected into monkeys intravenous followed by SARS-CoV infection. Euthanasia of monkeys were performed at the same days post infection for detection of pathological changes and SARS-CoV virus load. The results showed that pathological changes degree and virus load all increased positively correlated to concentration. Consequently, we can conclude that antibody S(597-625)43-3-14 can improved SARS-CoV infection both in vitro and in vvio.Our findings indicated that an FcyR-independent ADE can be mediated by antibodies to a specific major B-cell linear immunoantigen. It can reduce, countervail the function of the other neutralizing antibodies, even promote the virus infection. Although the exact mechanism is still unknown, this phenomenon is a big obstacle of the vaccine researching. As conclusion, our research will provide precise and powerful scientific basis for SARS-CoV vaccine evaluation research. Hand-foot-mouth disease(HFMD) is a kind of virus dermatopathy characterized by blisters on palm, sole and oral mucosa, the most common pathogens are coxsackievirus A16(CoxA16) and human enterovirus 71(EV71), Echo virus and other enteroviruses can also cause HFMD. Except severe complications of myocarditis, hydropneumonia, sterile meningitis even death in few clinical cases, the disease expressed selflimitation. The situation of the whole HFMD epidemiology in our country, area location of virus strain and variability are all needed investigated further. The epidemiological investigation of HFMD developed by Jin Qi during 2000-2005 showed 2/3 cases were caused by CA16, and 1/3 cases were caused by EV71. While,70%cases locally populated in Shan Dong were infected by EV71 virus and 20%were infected by CoxA16.The annual report demonstrated that 115618 infected and 50 dead. Minister Deng Haihua, The Information Office of Ministry of Health said 54713 infected and 31 dead at March, 2009.The infection expressed recessive, common infection cases developed into HFMD/herpangina, few patients can involved nervous system, respiratory system and circulatory system. Neurogenic pul-monary edema(NPE) has high mortality, it is the important complication and main death reason and also for death of children death in FuYang, is displayed paroxysmal tachycardia, dyspnea, cyanosis and shock.90% patients dead after 12h.HFMD affected physical and mental health of juvenile. People should strengthen body health, increase immunity and resist virus infection, the start-up of vaccine research should be given primary importance among prevention and cure of HFMD.We utilized EV71 strain, EU703812 rooted in Fu Yang, An Hui as template, and expressed 100-120 amino acid peptide according to it. The peptides were then immunized in ICR mouse, the specific antibodies were took into immunological test and neutralization test. We found the immunological effect is good in 1#,5#,6#,7#,10#,13#, 14#,18#,19#,20# peptides.1# locates at VP4 protein,5# nd 6# locates at VP3 protein, 7# locates at VP1 protein,10# locates at 2A protein,13# and 14# locate at 2C protein, 18# locates at 3C protein,19# and 20# locate at 3D protein. We decided to use antigen 1#,5#,6#,7#,10#,13#,14#,18#,19#,20# as basic units for vaccine combination, then the combinatied vaccine can improve immunocompetence.Our research will provide scientific data for EV71 vaccine preparation and evaluation.