| Objective: Interleukin-15 (IL-15) was found and cloned from monkey kidney epidermis cell (CV-1/EBNA) by Grabstein in 1994. Its mRNA was detectable in placenta> skeletal muscle, kidney, activated monocyte and macrophage. Although its sequences are not homologous with interleaukin-2(IL-2), it can interact with ?and V chains of IL-2 receptor and express bioactivity similar to IL-2. For example, it can induce proliferation of the PBMC, NKC and BC stimulated with PHA in vitro and generation of CTL and LAK cell. IL-15 also regulates macrophage to secrete cytokines by autocrine manner and actives neutrocyte and delays its apoptosis. IL-15 is also a the chemokine of T cell , so it is more important for primary immune response than IL-2. It could impel immune response from primary to secondary stage and strengthen the intensity of immune response.Coxsackievirus B3 (CVB3) is an important human pathogen which causes a wide spectrum of diseases, ranging from mild respiratory illness to severe myocarditis and neurological disorders. VP1 protein of CVB3 can stimulate host to produce protective neutralization antibodies. Theobject of this research is to construct a recombinant eukaryoticceu expression plasmid pcDNA3-VPl/IL-15 and observe the immune effect so that we can lay a foundation for developing an effective and safe vaccine against viral myocarditis.Methods: The mRNA of IL-15 was extracted from 2BS with RT-PCR and inserted into pGEM-T easy vector. E.coli DH5a were transfromed in Ampicillin 2YT culture and the recombinant clones were selected. The plasmid was extracted and identified by cutting with enzyme and sequensed. IL-15 gene fragment and plasmid pcDNA3 were cut with the same enzymes and Linked to construct a recombinant eukaryoticceu expression plasmid pcDNA3-IL-15. The VP1 gene was amplified from pcDNA3-VPl plasmid and inserted into pcDNA3-IL-15 plasmid. Then pcDNA3-VP1/IL-15 DNA was prepared and purified through transforming into the E.coli DH5a. Balb/c mice were immunized with this DNA vaccine by tibialis anterio muscle injection, lOOug per mice, three times altogether. Sera were collected from mice inoculated with pcDN A3-VP1/IL-15 in the sixth days after every immunization for measuring neutralization antibodies. The mice were infected with 800TCID50 CVB3 after three times of immunal injection and the protective effects of pcDNA3-VPl/IL-15 were observed.Results: The sequence of objective gene of recombinant eukaryoticceu expression plasmid pcDNA3-VPl/IL-15 is the same as the sequence of CVB3-VP1 and IL-15 ;Neutralization antibodies were produced after the mice were immunized with recombinant eukaryoticceu expression plasmid pcDNA3-VPl/IL-15. The liters of antibody were <1:5, 1:8.7 and 1:40 respectively; Infected mice with 800TCID50 CVB3,the survival rate of pcDNA3-VPl/IL-15 immunized group, pcDNA3-IL-15+pcDNA3-VPl, pcDNA3-IL-15, pcDNA3-VPl, pcDNA3 groups and saline control group were 35%,30%,10%,25%, 15%and 10% respectively. The survival rates of 6 groups had no evident difference (P>0.05).Conclutions: The eukaryoticceu expression plasmid pcDNA3-IL-15 has been successfully constructed; It is firstly successful in constructing the double expression plasmid pcDNA3-VPl/IL-15; The eukaryoticceu expression plasmid pcDNA3-VPl/IL-15, pcDNA3-IL-15+pcDNA3-VPl,pcDNA3-VPl could be expressed in mice and induce production of neutralization antibodies. Infected mice with 800TCID50 CVB3, the liters of antibody were increased along with the immunal times. There were no evident protective effects in mice immunized with pcDNA3-VPl/IL-15 for three weeks. We will try to lengthen the immunal time and give more times of immunal vaccination to build up mice resistance to CVB3.
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