小鼠层粘连蛋白α_5 LG3组件的表达及纯化

Expression and Purification of the Recombinant Laminin α_5 LG3 Fragment in E.coli

目的:用原核表达的方法获得带有6×His标记的小鼠层粘连蛋白α_5 LG3组件的重组蛋白。方法与结果:从小鼠心肌组织中提取总RNA,利用RT-PCR扩增出小鼠层粘连蛋白α_5 LG3组件的cDNA片段,并与pET-28a载体连接,构建重组表达质粒pET-LG3,限制性酶切分析和DNA序列测定均证实该克隆插入片段为小鼠层粘连蛋白α_5 LG3组件基因的成熟肽编码序列。用IPTG诱导,LG3蛋白在大肠杆菌BL21(DE3)中得到了表达,并经Ni-NTA层析柱获得纯化。结论:层粘连蛋白α_5 LG3组件的重组蛋白得到表达和纯化,为后续相关研究奠定了基础。

Objective： To obtain the laminin α5 LG3 recombinant protein with 6xHis tag by prokaryotic expression. Methods and Results： Total RNA of heart cell was prepared from a mature mouse and used in RT-PCR to amplify the laminin α5 LG3 cDNA fragment. The resulted fragment with the expected size was cloned into the vector of pET-28a to construct the recombinant plasmid pET-LG3. The plasmid pET-LG3 was subjected to restriction enzyme analysis and sequenced, the results showed that the laminin α5 LG3 gene sequence was 549 bp long and completely matched with the laminin α LG3 sequence in GenBank. The recombinant plasmid was transformed into E.coli BL21（DE3）, and E.coli BL21 （DE3）/pET-LG3 was induced by IPTG. The fusion protein was effective expressed and pur/ficated. Conclusion： The target protein laminin α5 LG3 with 6xHis tag was obtained.