Molecular Cloning And Immune Effects Of DNA Vaccination Of The E2 Gene From Classical Swine Fever Virus

Classical swine fever virus (CSFV) is highly contagious and causes often a fatal disease of pigs that has been responsible for large economic losses in different parts of the world. It is classified as an OIE List A disease and under strict control, and it is classified as the 1st animal disease in China . In the 1960s, Chinese vaccine strain (C-strain) operates crucial funcation in the process of prevention against CSF, and arrests the prevalence of CSF in China. However, CSF appeared again in our country. Thus, it is essential that investigated to develop more safe and effective CSF vaccines. First, Our studies constructed of DNA vaccines of CSFV E2 gene, and detected their expression efficiencies in vitro and immune effects in vivo; then, investigated to the DNA prime –protein boost regimens for enhancing immune effect of CSFV E2 DNA vaccines. A fragment of E2 gene of CSFV Shimen strain, was amplified by RT-PCR from total RNA of attenuate CSFV vaccine(II), and cloned into pMD18-T vector. The nucleotide sequence of this fragmant was sequenced, and was analysed by Genebank software. The sequence is core sequence of E2 gene. Compared with the corresponding region of Shimen strain of CSFV, the nucleotide sequence homology is 93.4%. Respectively, we subcloned E2 gene cDNA into eukaryotic expression vectors proVAX and pVP22, and then transfected Hela cell by lipofectamine. After transfected into Hela cells, both expressed products in vitro were accessed by indirect ELISA and RT-PCR and shown that the proVAX-E2n expressed significantly higher E2 protein than that from the pVP22-E2n, the KunMing White mice were immunized with the plasmids, proVAX-E2n and pVP22-E2n intramuscularly. The results showed that expression efficiency of the proVAX-E2n for in vitro and immune effect in vivo was better than that of pVP22-E2n . DNA vaccination is a relatively recent development in vaccine methodology in the 1990s. It is useful for producing humour immune response and cell-mediated immune response, easy to deposit, safe , in a wide variety of species. It showed more better immune effect than other vaccines. But, DNA vaccination induced relatively weak responses. All kinds of approaches have been developed recently in order to improve its efficacy. Prime-boost regimens have exhibited enhancement of immune responses and demonstrated its potential for vaccine development. In order to improve immune effect of proVAX-E2n, We adopted co-immunization the DNA vaccine proVAX-E2n with inactivated CSFV vaccine. KunMing White mice and swine were immunized by vaccination regimen consisting of consecutive priming with DNA vaccine proVAX-E2n and boosting with inactivated CSFV vaccine. After the second immunization, the antibody titer and T cell proliferation which KunMing White mice and swine produced were detected by indirect ELISA and MTS,the neutralization titer which swine produced was detected by the neutralization experiment. The result showed that Co-immunization with DNA vaccine proVAX-E2n and inactivated CSFV vaccine in animal induced higher neutralization titer of anti-CSFV and stimulation index of T cell proliferation than those immunized only with DNA vaccine proVAX-E2n or inactivated CSFV vaccine.