Cloning And Expression Of Canine IL-2 CDNA And Renaturation Of Its Expressional Products

Based on the published nucleotide sequence of Cants familiaris IL-2 gene, a pair of RT-PCR primers was designed and synthesized. Total RNA, isolated from ConA-stimulated peripheral blood T cells of canine, was used as template to generate complementary DNA by reverse transcription. The 600bp DNA fragment was amplified by polymerase chain reaction, and cloned into pMD18-T vector. DNA sequencing , restriction enzyme digestion and PCR amplification all confirmed the inserted fragment was canine IL-2 and its sequence was identical to that published in GenBank. Sequence analysis and comparative analysis of both amino acid sequence and antigenecity showed that the canine interleukin-2 has high similarity to the ones of other animals.By the technology of DNA recombination, the CaIL-2 cDNA was cloned into expression plasmids pBV220 vector, A39 vector and pET28 vector, and was transformed to E.coli. The recombinant plasmids were induced to expression through the changes of temperature or IPTG. The results of SDS-PAGE showed that all the expression products were about 20KD and all reached their expression peaks about 4-5 hours after inducing. For the recombination plasmids of pBV220-CaIL-2 and A39-CaIL-2. both the inclusion bodies and the supernatant have the target protein. The expression protein of the pET28-CaIL-2 only exists in the inclusion bodies. The inclusion body proteins were renatured after refolding. The biological activities of supernatant and renaturation proteins were detected with lymphocyte transform assays. The results indicated that all the recombinant canine IL-2 proteins have the high ability to supply the proliferation of canine peripheral blood T cells activated by ConABased on the results above, The inclusion bodies in E.coli with plasmid pET28-Ca-IL-2, which has the highest expression level, were prepared by sonication of the bacterial cells, washed by urea, NaCl and TritonX-100 and renatured followed by refolding in the way of dialyzation. The result of SDS-PAGE shows that washing by urea, NaCl and TritonX-100 can decrease the amounts of bacteria proteins in the inclusion body. The dissolving effect of guanidine hydrochloride on the inclusion body is better than urea's. Both renatured IL-2 proteins solved in guanidine hydrochloride or in urea have the same activity to supply the proliferation of peripheral blood T cells of canine activated by ConA.