摘要
目的:克隆人F3-Restin基因,表达有生物学活性的新型融合蛋白F3-Restin。方法:采用亚克隆技术构建融合表达载体PET32a(+)-F3-Restin,原核诱导表达,经Ni2+-NTA琼脂糖树脂亲和层析纯化,透析去盐。结果:在20℃、10h的条件下IPTG原核诱导表达、纯化,得到分子量约为40kD的高纯度的可溶性蛋白F3-Restin。结论:成功地克隆了人F3-Restin基因,构建融合表达载体PET32a(+)-F3-Restin,并表达出新型融合蛋白F3-Restin,为进一步研究F3-Restin蛋白抗肿瘤血管生长的活性及作用机理奠定了良好的基础。
Objective:To clone the DNA sequence coding the F3-Restin,and generate the fused protein F3-Restin.Methods:Prokaryotic expression vecter PET32a(+)-F3-Restin was constructed by subcloning technique,then the fusion protein of F3-Restin was expressed in E.coli and purified using Ni2+NTA agarose resin.Result:The expression of the fusion protein was at the highest level 10h in 20℃ induced by IPTG.After purification,the highly purified fusion protein(Mr 40000) was dissoluble.Conclusion:The F3-Restin prokaryotic expression vecter is successfully constructed,and the target protein is expressed and highly purified.A good foundation is established for the research of F3-Restin in future.
