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人IL-23R胞内区基因的克隆与原核表达
引用本文:车昌燕,张国华,刘力.人IL-23R胞内区基因的克隆与原核表达[J].粉末涂料与涂装,2009,22(6).
作者姓名:车昌燕  张国华  刘力
作者单位:车昌燕,张国华,CHE Chang-yan,ZHANG Guo-hua(山西医科大学汾阳学院检验系,山西汾阳,032200);刘力,LIU Li(北京协和医学院基础学院病原生物学系,北京,100005) 
摘    要:目的克隆人IL-23R(hIL-23R)胞内区基因,并在大肠杆菌中表达融合蛋白。方法通过PCR获得hIL-23R基因的胞内区片段,克隆至载体pGEX-4T-1中,构建重组原核表达质粒pGEX-4T-1-hIL-23R(I),转化感受态大肠杆菌BL21(DE3),IPTG诱导表达,表达产物经SDS-PAGE及Westernblot进行鉴定。结果hIL-23R胞内区基因的PCR扩增产物经琼脂糖凝胶电泳分析可见约760bp的目的片段。重组原核表达质粒经双酶切及测序证明构建正确。表达的融合蛋白相对分子质量约为55000,在低温(20℃)、低IPTG浓度(0.5mmol/L)诱导条件下能以可溶形式表达,可溶性蛋白的表达量约占菌体可溶性蛋白总量的16.1%,且可被兔抗GST多克隆抗体识别。结论已成功克隆了hIL-23R胞内区基因,并在大肠杆菌中表达了可溶性的GST融合蛋白。

关 键 词:人IL-23受体  胞内区  基因克隆  原核表达

Cloning and Prokaryotic Expression of Gene Encoding Cytoplasmic Domain of Human IL-23 Receptor
CHE Chang-yan,ZHANG Guo-hua,LIU Li.Cloning and Prokaryotic Expression of Gene Encoding Cytoplasmic Domain of Human IL-23 Receptor[J].Chinese Journal of Biologicals,2009,22(6).
Authors:CHE Chang-yan  ZHANG Guo-hua  LIU Li
Abstract:Objective To clone the gene encoding cytoplasmic domain of human IL-23 receptor(IL-23R)and express fusion protein in E.coli.Methods The gene encoding cytoplasmic domain of human IL-23R was amplified by PCR and cloned into vector pGEX-4T-1.The constructed recombinant plasmid pGEX-4T-1-hIL-23R(I)was transformed to E.coli BL21(DE3)for expression under induction of IPTG.The expressed product was identified by SDS-PAGE and Western blot.Results Agarose gel electrophoresis proved that the target gene fragment at a length of about 760 bp was amplified by PCR.Both restriction analysis and sequencing proved that recombinant plasmid pGEX-4T-1-hIL-23R(I)was constructed correctly.The fusion protein with a relative molecular mass of about 55 000 was expressed in a soluble form under induction of 0.5 mmol / L IPTG at 20℃.The expressed product contained about 16.1% of total soluble somatic protein and was recognized with rabbit anti-GST polyclonal antibody.Conclusion The gene encoding cytoplasmic domain of human IL-23R was successfully cloned, and soluble GST fusion protein was expressed in E.coli.
Keywords:Human IL-23 receptor  Cytoplasmic domain  Gene cloning  Prokaryotic expression
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