粘附结构蛋白migfilin基因N末端的克隆和抗体制备

Cloning of human migfilin N-terminal domain and preparation of anti-migfilin polyclonal antibody

目的克隆migfilin-N末端基因并使其在大肠杆菌中高效表达,纯化基因表达产物并进行多克隆抗体制备,为研究migfilin与大肠癌的关系奠定基础。方法根据人migfilin全长cDNA系列,设计PCR引物,以人大肠癌cDNA文库为模板克隆migfilin-N末端基因,将扩增产物克隆至大肠杆菌表达载体pGEX-4T-1中,经EcoRI/XhoI双酶切鉴定后,进行DNA序列测定。GST-migfilin-N融合蛋白在硫代半乳糖苷诱导下在大肠杆菌BL21中得到表达。利用谷胱甘肽亲和层析纯化融合蛋白,用纯化的GST-migfilin-N融合蛋白免疫家兔制备多克隆抗体。并用纯化的抗migfilin多克隆抗体在不同的细胞株中用Western blotting检测migfilin的表达。结果成功克隆了migfilin-N端基因片段,表达并纯化了GST-migfilin-N融合蛋白,制备了migfilin特异性抗体。Western blotting检测migfilin在不同细胞株中的表达,结果显示migfilin在肺癌及大肠癌细胞株中高表达。结论migfilin特异性抗体制备成功,为深入探讨migfilin在大肠癌中的作用奠定了基础。

Objective To clone migfilin-N terminal sequence into E.coli and obtain a fusion protein for preparing rabbit polyclonal antibody against migfilin, thereby facilitating the study of the role of migfilin in the biological behavior of colon cancer. Methods Based on human migfilin cDNA sequence, a pair of primers was designed to amplify migfilin-N terminal sequence by PCR. The PCR product was subcloned into the bacterial expression vector pGEX-4T-I with EcoRI/XhoI sites, and the target recombinant plasmids were identified with enzymatic cleavage followed by DNA sequence analysis. By transforming the expression vector into component E.coli BL2t cells, the GST-migfilin-N fusion protein was expressed with IPTG induction. Glutathione-sepharose beads were used to purify the fusion protein, and anti-migfilin polyclonal antibody was produced by immunization of rabbits with the purified GST-migfilin N-terminal fusion protein. The resultant anti-migfilin polyclonal antibody was purified by protein A beads and used for Western blotting for detecting migfilin expression in different cell lines. Results and Conclusion The migfilin-N terminal gene fragment was cloned successfully, and purified GST-migfilin N-terminal fusion protein and anti-rabbit migfilin polyclonal antibodies were obtained. Western blot analysis demonstrates that the antibodies specifically detected migfilin expression in the cell lines, which may facilitate further investigation of the role of migfilin in the biology of colon cancer.