Cloning And Expression Of Chicken IL-2 CDNA And Its Effect On DNA Vaccine

The chicken interleukin-2(chIL-2) gene , a cDNA fragment about 450 bp, wasamplified from ConA-stimulated chicken spleen cells by RT-PCR using a pair ofprimers which were designed and synthesized according to the chIL-2 gene sequencein GeneBank published by Sundick. .The gene was inserted into pBluescriptⅡKS(+)and identified by endonuclease and DNA sequencing. The reconstructed vector wasnamed pBluescriptⅡ-cIL2. The result of sequence anlysis showed that the geneencoded a proprotein of 142 amino acids residues. An amino acids absence was foundat the region of 49 compared with all other published chicken IL-2 genes. The otherregions of amino acids were consistent with Broiler,SC,Chenren and Xiaoshanchicken and there are close relationships in them, but 1~4 amino acids different fromKestrel leghorn,leghorn SPF,Obese,Silky and Xianju chicken. The gene we clonedin this study was the only chicken IL-2 gene which there is an amino acid absence.Then the reconstructed expression vectors pGEX-IL2 were constructed byenzyme digestion and subcloned into pGEX-6p-1,a GST expression vector. Thereconstructed expression plasmid were transformed into E. coli DH5α. Therecomebinant GST-chIL2 fusion proteins were observed on SDS-PAGE after inducedby IPTG. The band with 40kD of molecular weight was consistent with whichexpected. Based on the result of DNA sequencing of pBluescriptⅡ-cIL2, a pair ofprimers was designed and synthesized. The chicken IL-2 genes was amplified frompBluescriptⅡ-cIL2, and then was cloned to the site of pcDNA3. The reconstructedeukaryotic expression vectors pcDNA-cIL2 were identified by endonuclease, PCRand DNA sequencing. The result showed that the reconstructed vectors were theespecial chichen IL-2 gene eukaryotic expression vectors. Then the 14-day-old SPFchickens were vaccinated intramuscularly with DNA vaccine pcDNA-HN which...