鸡白细胞介素-2原核表达与生物学活性初探

Study on the Expression and Bioactivity of Chick IL-2

根据pGEX-6p-1表达载体多克隆位点,设计带有限制性内切酶酶切位点的IL-2引物,RT-PCR克隆获得目的基因,连接pGEM-T载体,转化JM109感受态细胞,经蓝白斑筛选、测序和双酶切鉴定,与pGEX-6p-1表达载体连接,转化BL21感受态细胞,经双酶切和PCR鉴定,用IPTG诱导表达,通过菌体裂解、包涵体洗涤、溶解、复性、Sepharose 4B柱层析,SDS-PAGE和Western blot检测,证明表达并纯化了IL-2融合蛋白,而且纯化的IL-2融合蛋白具有促进淋巴细胞增殖的特性。

A pair of primers with restriction enzyme for prokaryotic expression were designed respectively according to the gene＇s CDs complete sequence.The gene was amplified by RT-PCR,then cloned into pGEM-T vector and transformed into the JM109 competent cells,the plasmid of white spot was identified by PCR and sequenced.The positive plasmid was digested by the restriction enzyme.The objective gene fragment was retrieved and cloned into expressing vector pGEX-6p-1 and transformed into BL21 competent cells.The plasmid of white spot was identified by PCR and restriction enzyme of endonucleases.The fusion proteins was expressed by different inducing conditions of IPTG,and purified by Sepharose 4B chromatography.The specific band was identified by Western blot analysis.The biological activity was tested by lymphocyte proliferation test.