猪白细胞介素-2基因在大肠杆菌中的表达及其生物学活性测定  

Expression of huainan porcine interleukin-2 in Escherichia coli and bioactivity analysis

作　　者：郭小参[1] 陈红英[1] 崔沛[2] 贾艳艳[1] 崔保安[1] 方剑玉[3] 王振亚[1] 

机构地区：[1]河南农业大学牧医工程学院,河南郑州450002 [2]河南省动物疫病预防控制中心,河南郑州450008 [3]南京农业大学动物医学院,江苏南京210095

出　　处：《西北农林科技大学学报（自然科学版）》2009年第11期1-6,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家农业科技成果转化资金项目(2007BAD07A06)

摘　　要：【目的】在大肠杆菌中表达猪白细胞介素-2(pIL2),并鉴定其生物学活性。【方法】以含猪IL-2基因的重组质粒pGEM-pIL2为模板,PCR扩增IL-2基因。将猪IL-2基因定向克隆到原核表达载体pGEX-6p-1中,转化大肠杆菌(Escherichia coli)BL21(DE3)感受态细胞,在IPTG诱导下表达融合蛋白GST-pIL2,并应用甲基噻唑基四唑(MTT)试验测定融合蛋白的生物学活性。【结果】以含有猪IL-2基因的重组质粒pGEM-pIL2为模板,PCR扩增出1条约420bp的带。所获重组质粒pGEX-pIL2经酶切、测序鉴定,证实含有目的片段,且连接、构建正确。SDS-PAGE电泳结果显示,可检测到相对分子质量约为42ku的GST-pIL2,Western-blot证实GST-pIL2能与兔抗猪IL-2单克隆抗体发生特异性反应。表达蛋白经薄层层析扫描分析,表达量约占菌体总蛋白的40%。MTT试验证实,表达产物经变性、复性后能极显著促进猪脾淋巴细胞增殖。【结论】猪IL-2基因在大肠杆菌中得到表达,表达的蛋白具有一定的生物学活性。[Objective] This study developed prokaryotic expression system which can express porcine interleukin-2 （pIL2） in Escherichia coli, and identified the bioactivity of the recombinant protein. [Method] Porcine IL-2 gene was amplified with recombination plasmid pGEM-pIL2 as a template, porcine IL-2 was cloned into the prokaryotic expression vector of pGEX-6p-1 and transformed into E. coli BL21 （DE3）competent cells,which can express fused protein GST-pIL2 in E. coli BL21（DE3） by IPTG inducing. [Result] A 420 bp fragment was amplified by PCR. DNA sequencing result showed that the sequence of the recombinant prokaryotic expression plasmid pGEX-pIL2 in the reading frame and the ligation part was correct. The expressed product was identified by SDS-PAGE and Western-blot. The expressed protein was about 42 ku and it could react with rabbit against porcine IL-2 monoclonal antibody. MTT essay confirmed that the expressed product can enhance lymphocyte proliferation. [Conclusion] Porcine IL-2 gene can express in E. coli,and the expressed protein has bioactivity.

关 键 词：猪白细胞介素-2 原核表达 生物学活性 

分 类 号：S851.31[农业科学—预防兽医学]