抗凋亡蛋白Fortilin基因的克隆、表达与鉴定

目的在大肠杆菌中表达一种新近发现的抗凋亡蛋白Fortilin。方法用RT-PCR方法,从肺腺癌细胞中扩增出编码Fortilin蛋白的基因(GenBank中编码该蛋白的基因名为TPT1),克隆入pGEX-4T-1表达载体;重组质粒转入E.coliBL21(DE3),诱导表达Fortilin蛋白,并对表达产物进行免疫印迹鉴定。结果经双酶切及核酸序列分析鉴定,重组质粒pGEX-4T-1/TPT1成功构建,诱导后能高效表达可溶性Fortilin融合蛋白,SDS-PAGE及Western-blot验证该蛋白,表达的融合蛋白分子量为45000,与预期理论值相符。结论Fortilin蛋白在大肠杆菌中以融合蛋白形式进行表达,可提高其在宿主细胞中的稳定性和可溶性,且在大肠杆菌中获得高效表达,获得了充足的活性蛋白,这为进一步了解该蛋白的生物学功能,研究其抗凋亡机制奠定了基础。

Cloning and Expression of An Anti-apoptosis Protein Fortilin Gene

Objective To clone TPT1 gene and express the Fortilin protein in bacteria. Method The TPT1 gene was amplified from cell line A549(human adenocarcinoma of lung)by RT-PCR,and then was cloned into vector pGEX-4T-1 to construct the recombinant pGEX-4T-1/TPT1 plasmid and transfected into the E.coli BL21. IPTG was used to induced the expression of Fortilin fusion protein. SDS-PAGE and Western-blot was used to detect the expressed protein. Result The recombinant plasmid pGEX-4T-1/TPT1 was identified and confirmed by enzyme digestion and sequencing. A high level of Fortilin fusion protein was expressed in E.coli. SDS-PAGE and Western-blot analysis suggested that the molecular weight of recombinant fusion protein was 45 000 Dalton. Conclusion Successful amplification, cloning and expression of Fortilin fusion protein facilitates further studies on the structure, functions and protein-protein interaction of Fortilin,especially on the anti-apoptosis mechanism.