Cloning And Prokaryotic Expression Of Bovine Interleukin-2 Gene

A pair of RT-PCR primers were designed and synthesized based on the published gene sequence of Bovine interleukin-2 in GenBank.About 500bp target DNA sequence were amplified by reverse transcription polymerase chain reaction depending on the template of total RNA isolated from ConA-stimulated peripheral blood lymphocytes of healthy Chinese HolsteinCattle,and cloned to pMD18-T Simple vector,extracted plasmid and sequenced.The result showed that the length of the IL-2 gene was 501bp,contain a complete Open Reading Frame of 465bp which encoding a protein of 155 amino acids.Sequence analysis showed that the gene sequence was 99.8% identical to BoIL-2 gene that published in GenBank.And confirmed it was Interleukin 2 of Chinese HolsteinCattle.By the technology of DNA recombination,the sequence coded BoIL-2 was subcloned to EcoRI/BamHI sites of the expression plasmid pGEX-2T vector.The recombined plasmid,named pGEX-BoIL2,was transformed to E.coli BL21 and induced with IPTG at 37℃for 4 hours.SDS-PAGE illuminated that about 43kD fusion protein was expressed in E.coli BL21,and the Chinese HolsteinCattle IL-2 protein was 17 kD among the rest.In conclusion,we successfully cloned Chinese HolsteinCattle IL-2 gene and constructed the recombinant palsmids pGEX-BoIL2 which were expressed in prokaryotic expression cells.All should provide effective experimental materials to further research on anti-infection,anti-virus and vaccine of Chinese HolsteinCattle.