鸡白细胞介素2基因的克隆、原核表达及功能研究

以RT-PCR法从鸡脾脏细胞中扩增出鸡白细胞介素2编码基因,通过双酶切、连接步骤克隆进原核表达载体pQE30及pGEX-4T-3,构建了鸡白细胞介素2基因的重组原核表达质粒pQE30-chIL-2及pGEX-chIL-2.重组质粒转化大肠杆菌JM109后经诱导表达,分别得到两种表达产物.以SDS-PAGE和Westernblot检测确认表达产物为带6组氨酸标签及GST蛋白的融合鸡白细胞介素2,分子量分别为16 kDa及39.5 kDa.表达蛋白与抗鸡白细胞介素2单抗均有良好的免疫结合性,进一步证实为鸡白细胞介素2.表达产物经过纯化复性后,具有明显促进鸡脾淋巴细胞增殖的作用.

Study on the cloning, expression and function of chicken interleukin- 2

The gene encoding chicken interleukin-2(chIL-2) was amplified from chicken spleen cells by RT-PCR.Following double digestion by restriction endonucleases and ligation,chIL-2 was cloned into prokaryotic expression vectors of pQE30 and pGEX-4T-3 to construct recombinants of pQE30-chIL-2 and pGEX-chIL-2,which were consequently transformed into E.coli JM109.Two kinds of recombinant protein were obtained with IPTG induction.The molecular weights of the expressed proteins were 16?kDa and 39.5?kDa on SDS-PAGE and they were regarded as 6His and GST labeled protein(respectively) as checked by Western blot.Both proteins revealed specific reaction to anti-chIL-2 mAb,further confirming that recombinant proteins are chicken interleukin-2.After purification and(renaturation),the 6His-chIL-2 showed biological activity in stimulating chicken lymphocyte proliferation.