人β-防御素-3与人表皮生长因子融合蛋白的克隆及原核表达

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-β和表皮生长因子基因,分别构建β-防御素-3(β-defensin-3)、EGF重组质粒,应用重组PCR方法,将EGF的DNA序列拼接到β-defensin-3 DNA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.coli BL21(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Western blot对其进行特异性鉴定。结果:成功构建β-defensin-3、EGF重组质粒,经重组PCR成功扩增出294 bp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.coli BL21(DE3)经IPTG诱导经SDS-PAGE分析得到28 kDa左右的目的蛋白条带。Western blot鉴定出融合蛋白含有抗EGF、β-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。

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

Objective:To recombine the d-EGF and β-defensin-3 gene in prokaryotic expression vector,express fusion protein in Prokaryotic system,and purify and identify its specific. Methods:On the basis of gene sequence,β-defensin-3 gene and epidermal growth factor(EGF) gene were amplified by RT-PCR.Recombinant plasmid of β-defensin-3 and d-EGF plasmid were constructed and the DNA sequences of d-EGF and β-defensin-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 using NI-trap column and identified with western-blot. Results:β-defensin-3 and d-EGF plasmid were constructed.The fragment of 294 bp was successfully amplified by the recombinant PCR in accordance with the expected results and sequencing was correct.Twenty-eight kDa protein was obtained through tranforming into E.coli BL21 DE3) and IPTG Inducing.The fusion protein containing d-EGF and β-defensin-3 two proteins were identified with western-blot. Conclusions:Prokaryotic expression vector pET-SUMO-EGF was successfully constructed and fusion protein purified was obtained.It provides a basis for analysing its activity.