人-防御素-3与人表皮生长因子融合蛋白的克隆及原核表达

    Cloning and prokaryotic expression of d-EGF and -defensin-3 fusion protein

    • 摘要: 目的:克隆融合蛋白d-EGF成熟链基因,构建原核表达载体,并在大肠埃希菌(E.coli)表达体系中进行有效表达、分离纯化并鉴定其特异性。方法:以蛋白基因序列为基础,RT-PCR扩增人防御素-和表皮生长因子基因,分别构建-防御素-3(-defensin-3)、EGF重组质粒,应用重组PCR方法,将EGF的DNA序列拼接到-defensin-3DNA序列的3端,将基因克隆入原核表达载体pET-SUMO,构建重组质粒pET-SUMO-d-EGF。采用PCR、测序等方法鉴定后转化E.coliBL21(DE3),经IPTG诱导表达,Ni-trap柱纯化,SDS-PAGE检测表达及纯化结果,最后以Westernblot对其进行特异性鉴定。结果:成功构建-defensin-3、EGF重组质粒,经重组PCR成功扩增出294bp的目的片段,重组体PCR结果与预期结果一致,测序正确。转入重组质粒的E.coliBL21(DE3)经IPTG诱导经SDS-PAGE分析得到28kDa左右的目的蛋白条带。Westernblot鉴定出融合蛋白含有抗EGF、-防御素-3两种抗原特异性的蛋白。结论:成功构建原核表达载体pET-SUMO-d-EGF,并获得纯化的融合蛋白,为进一步的活性分析奠定了实验基础。

       

      Abstract: To recombine the dGF and(3lefensin 3 gene in prokaryotic expression vector express fusion protein inProkaryotic system and purify and identify its specific. Methods:On the basis of gene sequence(3lefensin 3 gene and epidermalgrowth factor(EGF)gene were amplified by RTCR. Recombinant plasmid ofplefensin 3 and dGF plasmid were constructed andthe DNA sequences of dGF andplefensin 3 gene was cloned into the pET SUMO prokaryotic expression vector. The pET SUMOd-EGF vector was identified by PCR and sequenced and transformed into E. coli BL21(DE3),which was induced by IPTG purified usingNIrap column and identified with westernlot. Results:plefensin 3 and dGF plasmid were constructed. The fragment of 294 bywas successfully amplified by the recombinant PCR in accordance with the expected results and sequencing was correct. TwentyightkDa protein was obtained through tranforming into E. coli BL21 DE3)and IPTG Inducing. The fusion protein containing dGF andp-defensin 3 two proteins were identified with westernlot. Conclusions:Prokaryotic expression vector pET SUMOGF was successfully. onstructed and fusion protein purified was obtained. It provides a basis for analysing its activity

       

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