类弹性蛋白多肽-红色荧光蛋白融合蛋白的表达纯化及细胞相容性

Prokaryotic Expression,Purification and Cell Compatibility of ELP-mCherry Fusion Protein

目的构建携带红色荧光蛋白(mCherry)标签的类弹性蛋白(ELP)原核表达载体,并表达纯化ELPmCherry融合蛋白,探讨ELP-mCherry融合蛋白与细胞的相容性.方法对利用限制性内切酶Xba I和Xho I对mCherry质粒和pET28a-ELP载体同时进行双酶切,将酶切产物回收、连接并转化DH5α大肠杆菌感受态细胞,构建重组质粒pET28a-ELP-mCherry;将重组质粒转化到BL21(DE3)大肠杆菌感受态细胞中,诱导表达ELPmCherry融合蛋白;利用可逆转变循环(ITC)法对ELP-mCherry融合蛋白进行纯化;通过MTT法探讨ELPmCherry融合蛋白与人胚肾上皮细胞HEK293T和小鼠胚胎成纤维细胞NIH3T3的细胞相容性.结果DNA测序结果显示成功构建了ELP-mCherry原核表达载体;利用BL21(DE3)大肠杆菌表达系统成功表达ELP-mCherry融合蛋白,通过ITC法纯化得到了高纯度融合蛋白.MTT结果表明:在所有ELP-mCherry蛋白测试浓度下,HEK293T和NIH3T3细胞存活率接近或超过100%.结论成功构建ELP-mCherry原核表达载体,成功表达并纯化得到高纯度的ELP-mCherry融合蛋白,ELP-mCherry融合蛋白在HEK293T和NIH3T3细胞中具有良好的细胞相容性.

Objective To construct elastin-like protein(ELP)prokaryotic expression vector with red fluorescent protein(mCherry)tag,to purify the ELP-mCherry fusion protein and investigate the biocompatibility of ELPmCherry protein on cells.Method mCherry plasmids and pET28a-ELP plasmids were double digested by restriction endonucleases Xba I and Xho I,which were used to construct pET28a-ELP-mCherry plasmid.The recombinant plasmids were transformed into BL21(DE3)E.coli competent cells to express ELP-mCherry fusion protein.ELP-mCherry fusion was purified by reversible transformation cycle method(ITC).The biocompatibility of ELP-mCherry protein was evaluated on human renal epithelial cells and mouse embryonic fibroblasts by MTT method.Results DNA sequencing result indicated the successful construction of ELP-mCherry plasmid,and ELP-mCherry fusion protein was acquired and purified by using BL21(DE3)E.coli system via ITC method.MTT results showed that HEK293T and NIH3T3 treated with ELP-mCherry fusion protein under all tested concentration has a cell viability of 100%or more.Conclusion We successfully constructed ELP-mCherry prokaryotic expression vector,and got the high purity ELP-mCherry fusion protein,ELP-mCherry protein had good cell compatibility on HEK293T and NIH3T3.