Construction And Identification Of Recombinant Adenovirus Expressing Main Antigen Genes Of CSFV And IL2 Of Porcine

Main antigen genes, non-structure protein gene of Classical Swine Fever Virus (CSFV) and IL2 of porcine were cloned into non-replicative Adenoviurs vector, and then several recombinant adenovirus were constructed. It is the base to study live adenovirus vector-based vaccine.Coding genes E0,E2 , NS2.3 of CSFV and IL2 of porcine which were got by the method of RT-PCR were inserted into MCS A or MCS B of pIRES plasmid, which is an plasmid-mediated in order to use its internal ribosome entry sites(lRES). The recombinant pIRES plasmids were confirmed by PCR, restriction endonuclease analysis and sequencing. 10 recombinant adenoviral pShuttle-CMV(pSC) vectors were constructed by digesting plasmids-mediated to get special genes to insert into MCS of linearized pSC. These recombinant pSC vectors can be used to create recombinant adenovirus expressed main antigen genes of CSFV and cytokine of swine, and they were cotransformed into E-coli BJ5183 which contained Adenovirus backbone vector pAdEasy-I by electroporation. At the presence of recombinant of E-coli BJ5183, homologous recombination between linearied pSC-X plasmid(X stand for E2, E2E2,IL2, E2IL2, E2E2IL2) and pAdEasy-1 vector occurred. Consequently, the foreign gene fragments were transferred into adenovirus backbone vector pAdEasy-1 and selected with kanamycin. E2 and IL2 gene was screened with PCR and correct homologous recombination was further screened with Pacl digestion. Then plasmids were transfected into 293A cell with lipofectmine^TM2000 transfection reagent. pAdEasy-1 was encapsided a whole live virus in 293A when the CPE is obvious. The expression of E2 of CSFV was identified by RT-PCR, immunofluorescent assay (FA). The expression of IL2 of porcine was identified correctly by ELISA, and their concentration were 148.4, 120.4, 114.2pg/ml. TCID₅₀ of the recombinant adenovirus were: 10^6.76(rAd-E2),10^4.36(rAd-IL2), 10^4.56(rAd-E2IL2),10^3.70(rAd-E2E2), 10^4.16(rAd-E2E2IL2); The recombinant adenovirus was passaged 3 times and titer is stable. This result showed that CSFV E2 protein expressed in recombinanted adenovirus is alive stablly and recombinant adenovirus of CSFV had been constructed. It is the base to study live adenovirus vector-based vaccine and its further progress.