Cloning And Expression Of Porcine Interleukin 4 Gene And Effect Of Vaccine Adjuvant

Acording to the sequence of porcine IL-4 gene in GeneBank,the primers of IL-4 gene was designed by using Primerselect and Oligo5.0 software .The total RNA was extracted from Lymphocytes stimulated by LPS and PHA.Then pig IL-4 gene was cloned from the total RNA using RT-PCR,the PCR products were cloned into PGEM-T vector and the connection productions were transformed into E.coli JM109.The recombinant plasmids were identified with PCR ,enzyme,digestion and sequencing. Then we designed a pairs of expression primers containing BamHI and XhoI , The products was amplified from the PGEM-T/IL-4 recombinant plasmid using expressions primers, the PCR products and the PGEX,pcDNA3.1 were ligated together after digestion with BamI and XhoI enzyme,and then transformed into E..coli cells BL21. The recombinant plasmid of PGEX-IL-4 was induced in E.coli using IPTG in different temperature , The biological activity of two protein were detect by the way of MTT, the pcDNA3.1-IL-4 recombinant plasmid as an adjuvant with TSO18 and Cysticercus cellulosae antigen to immunize pig. detecting antibodies using ELISA way resulting in a high level of protein expression. In the studies , IL-4 gene was cloning successfully ,The nucleotide acide sequence and predicated amino acid sequences were compared with IL-4 genes in GeneBank using DNAstar software.The homologies were 99 and 97.8,respectively. The recombinant plasmid of PGEX-IL-4 and pcDNA3.1-IL-4 was constructed successfully. The expressed products of PGEX-IL-4 were analyzed by SDS-PAGE and showing that the 38 kDa IL-4 GST fusion was expressed successfully. The fusion protein was mainly expressed in inclusion in 32 centigrade, the expressed protein making up 30% of total E. coli proteins; part dissolubility protein was expressed in 28 centigrade. The inclusion was purified using sephadex G-200 and the solulating protein was purified with GST kit. the results showed that the two proteins could improve proliferation of differentiation to Lymphocytes ; Antibodies were subsequently detected, showing that the pcDNA3.1IL-4 recombinant plasmid has a promising role as an adjuvant to Cysticercus cellulosae vaccine and much more better than 206 adjuvant.