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Study on the Expression and Mucosal Immunization of Enterovirus 71 Structure Protein VP1

Author: ZhangFuShun
Tutor: LiDeXin
School: Disease Control and Prevention Center
Course: Immunology
Keywords: EV71 virus Mucosal immune Vaccine VP1 protein
CLC: R392
Type: Master's thesis
Year: 2011
Downloads: 83
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Abstract


Hand, foot and mouth disease (Hand-Foot-Mouth Disease, HFMD) aka exanthematous vesicular stomatitis caused HFMD pathogens enterovirus Coxsackie virus (Coxsackie virus) A small RNA virus family (Picornaviridae) 2,4,5,7,9,10,16-type group, Coxsackie virus group B, 1, 2, 3, 4, 5 type; partially echovirus (ECHO-viruses) and enterovirus 71 (EV71) . The most common Coxsackie virus group A type 16 (CA16) and enterovirus 71. The clinical manifestations of fever and hand, foot, mouth and buttocks and other parts of a rash as the main clinical features of acute infectious diseases. The vast majority of HFMD patients with a good prognosis, generally heal in 5-7 days, is a self-limiting disease. The virus can also be violations of the respiratory system, central nervous system, cardiovascular system, causing encephalitis, myocarditis, pulmonary edema, flaccid paralysis and other symptoms, individual critically ill children can lead to death due to a variety of reasons. The incidence of the disease all year round, occur in the summer and autumn, and is common in children under 5 years of age. Severe HFMD caused by EV71. HFMD main route of transmission is fecal - oral route, can also be spread through the respiratory tract. HFMD is contagious, easily lead to epidemic or outbreak. In recent years, the HFMD epidemic trend grim increased concern of of HFMD major pathogens EV7. HFMD is no effective drug treatment, vaccine development become a top priority. EV71 mainly through fecal - oral route or dissemination in aerosol form, mucosal vaccine immune logical. Development EV71 mucosal vaccine compared with traditional vaccination with safe, economical and easy to mass populations inoculated many advantages, thus causing more and more attention of researchers. This work consists of three parts: the first part: of EV71 purified VP1 protein prokaryotic expression and purification according to recent epidemiological data, our mainland popular EV71 C4 subtypes, so this study selected C4 subtypes HN -8 isolates as the research object, and its gene sequence alignment analysis, confirmation the its main in, and the epitope: PDSRESLAWQTATNP and YPTFGEHKQEKDLEY, no occurrence of the change in the amino acid level. Extract the viral RNA, obtained by reverse transcription of the viral genome cDNA, in vitro amplification of the viral VP1 gene sequences, and cloned into the pET30a vector plasmid pET30a-VP1. VP1 protein VP1 protein expression was induced by IPTG, and purified the protein yield of up to 30-40mg per liter broth. By SDS-PAGE, Western blot analysis of the VP1 protein purified. This part of the HN-8 isolates were cultured in Vero cells, and titration of the virus and purified. Viral titer by serial passage 108TCID50. And relatively pure virus particles. The second part: VP1 protein in insect cell expression vector construction and purification. PET30a-VP1 plasmid sources of gene sequences amplified VP1 gene sequences cloned to pAcGP67-B carrier, restriction endonuclease digestion, the right cloning and sequencing, select VPl gene sequence base changes did not happen clone with rod virus linearized DNA co-transfected SF9 cells continuously enhance baculovirus virulence by serial passage. SF9 cells expressing VP1 protein by indirect immunofluorescence by SDS-PAGE, Western blot detection. To capture assay in mouse serum IgM, the present part of the VP1 protein with horseradish peroxidase conjugated and the microtiter the VP1 protein concentration determination. When anti-IgM μ chain capture antibody concentration 200ng, HRP-VP1 working concentration of 1:160. Part III: mucosal immunological effect of the prokaryotic expression of VP1 protein of prokaryotic expression of VP1 subunit vaccines require adjuvants assisted, in order to efficiently induce humoral immune responses. Part original nuclear expression of recombinant VP1 protein with mucosal adjuvant chitosan compatibility constitute vaccine as an animal model of female Balb / C mice were immunized by gavage route. The VP1 protein mucosal immune design the 100μg and the 1000μg two dose groups, and the establishment of a positive control group: inactivated EV71 virus with chitosan compatibility group (mucosal route immunity); inactivated EV71 whole virus with aluminum hydroxide compatibility group VPl protein and aluminum hydroxide compatibility group (intramuscularly immunized). The negative control group: PBS with chitosan and aluminum hydroxide respectively compatibility. For 0 days, 7 days, 28 days the three needle immune. The partially completed prokaryotic VP1 protein, low-dose group, mucosal immunity and its comparison with the positive effect of the control group and the the intramuscular group of immune. Found the VP1 protein chitosan compatibility immune Balb / C mice, can be induced not only serum specific IgM and IgG antibody responses can also be induced mouse intestinal, vaginal and respiratory mucosal surface is stronger and longer-lasting specific sIgA responses. This experiment also counted in IFN-γ-secreting cells of the mouse spleen found mucosal immune group of IFN-γ secreting Th1-type cells was significantly higher than the negative control group. Vitro neutralization experiments showed that: VPl protein high dose mucosal immune serum and titers up to 1:16. Preliminary evidence of mucosal immune effects of the VP1 protein. The vaccine research model for this study to the the prokaryotic expression VPl protein with chitosan compatibility by gavage ways of mice mucosal immunity, initially clarified the aid of chitosan adjuvant, VP1 protein can induce specific humoral and cellular immune response for the EV71 virus mucosal vaccine research provides a theoretical basis and method.

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