| 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420 bp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42 ku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。 |
| 关键词: 猪白细胞介素-2 原核表达 生物学活性 |
| DOI： |
| 分类号: |
| 基金项目:国家农业科技成果转化资金项目(2007BAD07A06) |
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| Expression of huainan porcine interleukin-2 in Escherichia coli and bioactivity analysis |
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GUO Xiao can1 CHEN Hong ying1 CUI Pei2 JIA Yan yan1 CUI Bao an1 FANG Jian yu3 WANG Zheng ya1
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| Abstract: |
| 【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.coliBL21(DE3)competent cells,which can express fused protein GST-pIL2 in E.coliBL21(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. |
| Key words: porcine interleukin-2 prokaryotic expression bioactivity |
