重组人FOXL2基因的原核表达和纯化研究

目的通过pET32a（＋）原核表达载体，表达重组人叉头框蛋白L2（human forkhead box12，FOXL21）。并且进行纯化和鉴定。方法从正常人血液中提取基因组DNA，利用PCR扩增FOXL21目的基因片段，构建FOXL21原核表达重组质粒pET32a（＋）-FOXL21]并转化E．coli的BL21（DE3）菌株，IPTG诱导重组蛋白表达，经HisTrap FF亲和层析柱纯化，再通过SDS—PAGE和Western印迹鉴定。结果成功克隆到大小为1131bp的人源FOXL21基因片段并准确插入表达载体pET32a（＋），0．1mmol／LIPTG诱导转化菌8h可表达大量的FOXL21蛋白，并可经HisTrap FF柱亲和层析得到高度纯化。结论成功获得纯化的66kD重组人FOXL21蛋白，为后续进行FOXL21蛋白的功能研究奠定了基础。

Study on FOXL2 Gene Expression and Antibody Cloning

Objective To clone, express and purify human recombinant Forkhead BoxL2- FOXL2. Methods FOXL2 gene was amplified from total genomic DNA of human blood by PCR, and cloned into protein fusion expression vector pET32a （ ＋ ） . This recombinant plasmid was in- troduced into E. coli BL21 （DE3） for expression which was induced by IPTG. Resulted protein was purified by His TrapTM FF. S DS-PAGE and Western blotting were used to identify the expres- sion and purification of recombinant FOXL2. Results human FOXL2 fragment of l134bp was ob- tained, and was cloned into pET32a （ ＋ ） . A 66kDa recombinant FOXL2 was successfully in- duced to express in E. coli and further purified by His TrapTM FF chromatograph. Conclusion A method for recombinant expression and purification of FOXL2 was successfully established. This method laid a foundation for the subsequent function research of FOXL2.