一种快速添加或替换蛋白标签的新方法及应用

A new method of rapid protein tag addition or replacement and its application

[目的]本文旨在建立一种快速添加或替换蛋白标签的新方法——多标签重组连接酶法,为进一步研究目的基因的功能奠定基础。[方法]利用同源重组连接酶反应,将目的基因、蛋白标签和酶切的表达载体同时进行连接,建立一种快速添加或替换目的基因蛋白标签的新方法。[结果]多标签重组连接酶法在用时和步骤上比融合PCR法和T4 DNA连接酶法具有一定的优势。以梨S7-RNase为目的基因,基于同源重组连接酶反应,成功将S7-RNase和GFP蛋白标签构建到pCold-TF载体上,并在15℃和IPTG终浓度为0.5 mmol·L^(-1)时,对重组质粒S7-RNase-GFP-pCold-TF进行原核表达并成功表达出了重组蛋白。同时,将p1300-35S-GFP载体中的GFP蛋白标签成功替换为mCherry蛋白标签,并将重组质粒p1300-35S-S7-RNase-mCherry在烟草细胞中成功表达。[结论]多标签重组连接酶法具有快速、简单和高效等特点,并且适用于构建多个蛋白标签或替换某个蛋白标签的表达载体,是一种可靠的高效获得目的基因所需蛋白标签的新方法。

[Objectives]In order to quickly add or replace the protein tag of the target gene,we established the multi-tag recombination ligase method,which could be applied as a useful tool in exploring gene functions.[Methods]Utilizing homologous recombinase reaction,the target gene and protein tag along wit digested vector were simultaneously connected,and the rapid protein tag addition or replacement of the target gene was subsequently accomplished.[Results]The multi-tag recombinant ligase method had certain advantages over the fusion PCR method and T4 DNA ligase method in terms of time and steps.S7-RNase in pear was applied as the target gene for assay certification.Utilizing the homologous recombinase reaction,the S7-RNase and GFP protein tags were successfully linked and constructed on the pCold-TF vector.At temperature of 15℃,the recombinant plasmid S7-RNase-GFP-pCold-TF was prokaryotically expressed and the recombinant protein was successfully expressed when the final concentration of IPTG was 0.5 mmol·L^(-1).Simultaneously,the GFP protein tag in the p1300-35S-GFP vector was successfully replaced with the mCherry protein tag,and the recombinant plasmid p1300-35S-S7-RNase-mCherry was successfully expressed in tobacco cells.[Conclusions]Multi-tag recombination ligase method is fast,simple and efficient,and it is suitable for constructing a variety of multiple protein tags or replacing a certain protein tag expression vector,so this method is a reliable and efficient new method to obtain the protein tags required by the target gene.