Cloning And Expression Of Caprine Interleukin-2 Gene And Biological Activity Detection Of Recombinant IL-2

cloning and Expression of Caprine Interleukin-2 Gene and BiologicalActivity Detection of the productYing QihuaDirected by Dr.Li Xiangrui and Dr.Lu Cliengping(Nanjing Agricultural University,Nanj ing,2 10095)A sensitive and simple coiorimetric assay (MTT assay)for ~at interleukin-2 activity were built.The results showed that 3~g/mL Con A and 36h induction time in vitro were the best constitution to obtain the Con A-stimul~ed target cell,when the peripheral blood T lymphocytes were used. Optimum conditions of MTT assay were as follow: MTT 20g~tL(5Oj.tg/~iL),additive time of MIT 4h,dissolving time of formazan particles 4h,interactive time of target cell with IL-2 18 h.Based on the nucleotide sequence of Caprine lnterleukin-2 published ,a pair of RT-PCR primers were designed and synthesized .Total Caprine cell RNA, isolated from ConA-stimulated peripheral blood of Caprine, was usedas tenWlate to generate complementary DNA by reverse transcription .The 0.5 kb DNA fragments were amplified by polymerase chain reaction (PCR),and cloned into the pBV22O expression plasmid. DNA sequencing confirmed the fragment was Caprine Interleukin-2 and the sequence was identical to the sequence of [L-2 publised in Genbank;.Sequence analysis showed that the cloned sequence have vety high similarity with that of ovine and bovine IL-2, over 96%; C~mpar~ive analysis of both amino acid sequence and antigenecity showed that the interleukin-2 of Caprine have high similarity to the one of ovine and bovine.The prokaryotic expression plasmid pBV22O/Rcap IL-2 was constructed and transformed into DH5ct. The recombinant protein was found to be expressed with.M.W. 16 KD.Most of the recombinant protein existed in the supernatant .Biological activity of the recombinant protein was significant as MTT assay shown. Studies on cross-species reactivity revealed that buff~lo lymphocyte responds to the recombinant caprine IL-2 sigificantly while that of swine~s was negligible.