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.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
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)
