斑马鱼PKR的dsRBM1和dsRBM3重组及体外功能分析

IN VITRO FUNCTIONAL ANALYSIS OF THE RECOMBINANT DSRBM1 AND DSRBM3 FROM ZEBRA FISH(DANIO RERIO)PKR

为了重组斑马鱼(Daniorerio)双链RNA依赖的蛋白激酶(DrPKR)的双链RNA结合模体1(dsRBM1)和双链RNA结合模体3(dsRBM3)基因,并对重组基因表达的蛋白进行鉴定和功能分析,设计定向删除引物,应用PCR技术扩增DrPKR的ds RBM1和dsRBM3的基因片段,通过重叠PCR技术将dsRBM1和dsRBM3基因重组在一起得到dsRBM13基因,再将dsRBM13构建到原核表达质粒pET32a上并表达和纯化蛋白;通过表达蛋白的二聚化实验和pull down实验对重组蛋白dsRBM13进行体外功能分析。结果:DrPKR的ds RBM1和dsRBM3重组的蛋白dsRBM13可以进行二聚化和多聚化,且能结合Poly I:C(人工合成的dsRNA)。因此,重组DrPKR蛋白模体dsRBM13仍然具有二聚化和dsRNA结合活性,提示串联了三个dsRBM的Dr PKR激活过程中,串联两个dsRBM对DrPKR激活是必需的,并且在DrPKR结合dsRNA的激活过程中,因dsRNA的长短和空间结构的多样性,含有三个dsRBM的DrPKR可能存在更强和更多的结合方式,因此具有更强的抗病毒免疫反应功能。

To recombine the gene of dsRBM1 and ds RBM3 of double-stranded RNA dependent PKR(DrPKR)in zebra fish(Danio rerio),identify and analyze the function of the expressed protein in vitro,directional deletion primers were designed to amplify the gene segment of ds RBM1 and dsRBM3 of DrPKR,which were recombined to obtain dsRBM13 by overlapping PCR,then ds RBM13 was transferred to p ET32 a to express and purify the recombined protein;the dimerization assay and Poly I:C pull-down assay were introduced to test the function of the protein in vitro.The results showed that dsRBM13 could dimerize and multimerize,which also bind to poly I:C(synthetical dsRNA).Thus,the recombinant ds RBM13 could dimerize and bind to poly I:C,which implied that it was essential for two dsRBM in tandem to activate DrPKR;Furthermore,because of the diversity of length and spatial structure of dsRNA,DrPKR with three dsRBM could bind to dsRNA more powerful and with more binding pattern,hence,it might have a stronger function for antiviral immunity.