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Objective: Coxsackie virus group B (Coxsackievirus group B, CVB) is the the viral myocarditis main pathogen, more than 50% of myocarditis caused by the viral infection, some patients develop chronic myocarditis and dilated cardiomyopathy, CVB3 infection the most common. There is no effective methods of prevention. VP1 is CVB3 major neutralizing antigen, the plasmid vector construct CVB3 VP1 nucleic acid vaccine intramuscularly immunized mice, can only induce inefficient monovalent neutralizing antibody can not effectively prevent lethal virus infections. In order to enhance the effect of the vaccine, some researchers dendritic cell chemotactic factor gene target gene splicing, to build into the target nucleic acid vaccines. Targeted DNA vaccine is conducive to the uptake of the antigen processing and presentation, and dendritic cell expression of costimulatory molecules, non-immunogenic tumor antigens or immunogenic the weaker virus antigen immunogenicity significantly enhanced. MDC is CC chemokines, chemokine single nuclear / macrophages, dendritic cells and T cells. Our previous constructed by the appropriate adapter sequence with a signal peptide of the MDC and VP1 fusion gene the DNA vaccine pcDNA3/MDC-VP1, immune mice displayed significantly enhanced the protective effect of the lethal dose of virus infection, but still can not completely protect mice resistance lethal viral infection. Compared with the plasmid vector, adenovirus vector into the host cell's ability, a wide host range, upregulation of costimulatory molecules in infected cells, and induction of cytokine and chemokine production, exogenous gene expression and high viral titer characteristics, and does not integrate into the host chromosome, no insertion mutation. Demonstrate the advantages of the vaccine vector selection. Several studies have shown that the adenovirus vaccine vector construction satisfactory immune. The subjects of AdEasy adenovirus vector system build, packaging recombinant adenovirus Ad/MDC-VP1 alone or with plasmid pcDNA3/MDC-VP1 combined immune to enhance the VP1 antigen immune effect. Methods: a recombinant shuttle plasmid with the restriction endonucleases Hind III and Xba Ⅰ plasmid pcDNA3/MDC-VP1 double digested recycling purposes fragment MDC-VP1 and by the same digested adenovirus shuttle vectors pAdTrack-CMV is connected by transformation, screening, and restriction enzyme digestion, to construct recombinant shuttle plasmid pAdTrack-CMV/MDC-VP1. 2 adenovirus plasmids build pAdTrack-CMV/MDC-VP1 plasmid with endonuclease linearized with Pme Ⅰ to using AdEasy system by a two-step method in the E. coli BJ5183 with backbone vector pAdEasy-1, homologous recombination, recombinant adenovirus plasmid pAd/MDC-VP1. Pac Ⅰ restriction enzyme digestion, the positive plasmid to transform competent E. coli DH5α positive clone was picked. Extracting the recombinant plasmid, and to a large number of the alkaline lysis method was purified with polyethylene glycol (polyethylene glycol, PEG) precipitation. 3 recombinant adenovirus packaging adenovirus plasmids pAd/MDC-VP1 was amplified endonuclease Pac Ⅰ linearization HEK293 cells transfected with cationic liposomes and observed the green fluorescent protein (green fluorescent protein, GFP) expression. The freeze-thaw method to collect the first generation of virus solution. Taken early-generation virus fluid infection of HEK293 cells, a second round of amplification, the same method for the third, and four large-scale amplification, a large collection of the fourth-generation virus fluid, purified with adenovirus purification kit. 4 recombinant adenovirus titer was determined to be HEK293 cells grown in 96 well plates to 70 8 0% confluence, different dilution generation virus fluid and the purified virus was infected cells were cultured for 48 hours later, GFP-positive cell count method to the determination of virus titers. The virus titer = the number of fluorescent cells × dilution of the virus liquid / liquid amount of virus. 5 with amplified by purified Our previous build Ad, and viral titer was determined. 6, section 3-generation Ad/MDC-VP1 virus infected HEK293 cells after 48 hours, the cells were collected and the supernatant, the expression of the viral protein detected by Western blot. 7 using the alkaline lysis method from the DH5α E. coli transformed with large number of extracted plasmid pcDNA3/MDC-VP1, purified by precipitation with polyethylene glycol. 8 animal experiments: 6 8 -week-old male BALB / c mice were randomly divided into six groups, quadriceps injection of plasmid or recombinant adenovirus. Ad/MDC-VP1 group (A): Every time 1.2 107pfu/100μl, immune, interval 2w; pcDNA3/MDC-VP1 group (B): Every time 100μg/100μl immunized three times, the interval 3w; pcDNA3/MDC-VP1 Ad/MDC-VP1 group (C): immune, the initial inoculation plasmid after vaccination interval 3w Ad/MDC-VP1 strengthen 2 interval 2w dose ibid; Ad/MDC-VP1 pcDNA3 / MDC-VP1 group (D group): Immune three times first two vaccinations Ad/MDC-VP1, interval 2w, 3rd to vaccination pcDNA3/MDC-VP1, interval 3w, dose ibid; Ad group (E group): immune twice interval 2w, dose Ibid; 3 times PBS group (F group): each 100μl, immune interval 3w. Serum CVB3-specific neutralizing antibodies 14 days after each immunization, inner canthal vein blood serum was separated serum CVB3 VP1-specific IgG levels were determined by ELISA; microneutralization test (the fixed virus - the diluted serum law) level; three weeks after the last immunization, each group of three mice spleen lymphocyte suspension was prepared, using a cell counting kit (cell counting kit-8, CCK-8) lymphocyte proliferation and specific cytotoxic T-lymphoid cells (cytotoxic T lymphocyte, CTL) killing activity detection; each Another 3 mice with 3LD 50 CVB3 attack virus injection on day 7 after blood collection, and for the virus titer in blood determination; remaining 12 mice in each group injected intraperitoneally with a lethal dose of CVB3 (4LD 50 ), observed and recorded for 21 days after the survival of mice to infection. The result: a successful build to shuttle the plasmid pAdTrack-CMV/MDC-VP1 restriction enzyme analysis and found to comply with the expected design. 2 a successful build the adenovirus plasmids pAd/MDC-VP1, digested with Pac Ⅰ about 30kb fragment and a 4.5kb fragment. 3 48 hours after transfection, the fluorescence under a microscope can be observed to the gene of GFP expression in HEK293 cells reported the adenovirus Ad/MDC-VP1 packaging success. First-generation virus fluid again infection of HEK293 cells, can be observed under fluorescent microscope daily obvious cytopathic and fluorescence aggregation generations of the virus was infected cells have comet sample fluorescence plaque formation. The first generation to fourth-generation virus titer recombinant adenovirus Ad/MDC-VP1 were: 2.2 × 10 3 pfu / ml, 1.3 × 10 4 pfu / ml 4 × 10 6 pfu / ml and 5.8 × 10 8 PFU / ml. Recombinant adenovirus Ad/MDC-VP1 three generations of the virus was infected HEK293 cells after 48h, respectively, total protein extracted supernatant and cell supernatant Western Blot display fusion protein expression MDC-VP1 various times after immunization, blood CVB3 VP1-specific IgG average were: group A: 1:75.78,1:527.72; group B: 1:66.07,1:199.99,1:459.41; C group: 1:65.97,1:229.72,1:696.31; Group D: 1:65.98,1:459.41,1:1392.52; group E and group F were not detected. Single-factor analysis of variance showed that successive increase (P lt; 0.01) In addition to the E, F group, each group VP1-specific IgG; each group after the last immunization, the antibody titers descending order: D group gt; C group gt; A group gt; group B (P lt; 0.05). 7 after the third immunization blood in CVB3 average and antibody levels were: group A: 1:8.41,1:53.38; group B: 1:7.07,1:25.2,1:50.39; C group: 1:7.49,1 : 28.28,1:67.26; the D group: 1:8.41,1:50.39,1:71.26,; group E and group F was not detected. Single-factor analysis of variance showed that successive increase (P lt; 0.01) In addition to the E, F group, each group CVB3 neutralizing antibody levels; antibody titers after the second immunization in order were: A group gt; D group gt ; C group gt; B group (P lt; 0.05), after the last immunization was no significant difference among groups. The 8 lymphocyte proliferation test to inactivated of CVB3 and concanavalin egg A (concanavalin A, Con A) in vitro stimulation of lymphocytes to detect cell proliferation activity. Single-factor analysis of variance showed that CVB3 stimulation, no statistically significant among the three groups; ConA stimulation, C, D group was significantly higher than the other groups (P lt; 0.01). 9 mouse spleen-specific CTL killing activity by univariate analysis of variance showed that the killing rate descending order of C gt; D gt; A gt; gt B; E gt; the F twenty-two addition to C and D D and A difference was not statistically significant, other groups differences were statistically significant (P lt; 0.05). 10 3LD 50 after CVB3 mice challenged the 7d blood virus titer measurement results show that the A, B, C, D group was significantly lower, pairwise comparisons showed that among the groups C, D group was significantly lower than group B (P lt; 0.05). 11 4LD 50 21d survival rates in the After CVB3 attack mice were: D group: 41.7%, group C: 33.33%, A, group B: 25%, E, F group had no survival after Kaplan -Meier survival analysis showed that the survival difference (P lt; 0.01). Conclusion: a successful build and packaging recombinant adenovirus Ad/MDC-VP1 MDC-VP1 fusion protein secreted into the cell culture medium can be expressed in HEK293 cells. 2 the adenovirus vaccine Ad/MDC-VP1 immune 2 can improve the humoral immune response, enhanced nonspecific lymphocyte proliferation and CTL activity, and can reduce the titer of virus in the blood of mice after virus attack . 3 adenovirus vaccine DNA vaccine combination, the level of humoral immunity, non-specific lymphocyte proliferation and CTL activity was significantly higher than immunohistochemistry alone. 4 after the lethal dose of virus attack mice, DNA vaccine and adenovirus vector vaccine in combination can significantly prolong survival time.
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