Cloning And Expression Of Bovine Interleukin-2 Gene And Preparation Of Its Polyclonal Antibody

IVBovine Interleukin-2 (BoIL-2), which has the ability to regulate the immune response through immune network, is mainly secreted by T helper cell. BoIL-2 is an usful adjuvant and therapeutics in control of diseases. As these features, it was extensively used in treatment of malignant tumors and infectious diseases. Naturally, the production of BoIL-2 in animal body is limited in amount and thus confined its study and application. With modern genetic engineering technique, BoIL-2 gene was cloned using reverse transcription polymerase chain reaction (RT-PCR) method, expressed with prokaryotic plasmid and prepared its polyclonal antibody. This would provide solid basis for the studying of its biological activities and application.Specific primers for bovine IL-2 cDNA were designed and synthesized according to the previously published sequence in Genebank. The total cell RNA, isolated from ConA-stimulated peripheral blood lymphocyte of Bovine, was used as template to generate complementary DNA by reverse transcription. The 477bp DNA fragments were amplified by polymerase chain reaction (PCR), and cloned into the pMD-18-T vector. The fragment was confirmed bovine interleukin-2 by DNA sequencing. The sequence analysis showed that it was similar to the sequence of bovine IL-2 publised in Genbank, the homology was 98.7% in nucleotide acid and 99.4% in amino acid.Then the prokaryotic expression plasmid pET30a/Rcap IL-2 was constructed and transformed into BL21 cell. The recombinant rBoIL-2 was expressed efficiently in forms of inclusion body induced by IPTG. SDS-PAGE and western blot analysis showed that the recombinant fusion protein had a molecular weight 23ku. The inclusion body was solubilized by 8M Urea and purified by QIAGEN Ni-NTA resin. After annealing treatment of the recombinant protein with urea, its biological activity was tested with MTT assay. The results demonstrated that it had the biological activity to sustain the proliferation of bovine and goat peripheral blood lymphocytes stimulated with ConA preliminary, however no effect to that of swine. The protein was purified and used to immunize rabbit, and polyconal antibody was prepared and identificated by AGP and western blot.In general, the cloning, expression of bovine interleukin 2 gene and the preparation of its polyclonal antibody will provide solid basis for the biological activity study and application of bovine interleukin 2.