Gong Wei, Li Jie, Wang Yun-Ling, Nan Qing-Zhen, Jiang Bo, Zhang Hong-Quan
Department of Gastroenterology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China. gwei203@ yahoo.com.cn
Nan Fang Yi Ke Da Xue Xue Bao. 2008 Jun;28(6):915-8.
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.
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-1 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 BL(21) 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.
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.
将migfilin的N端序列克隆至大肠杆菌中，获得融合蛋白以制备抗migfilin的兔多克隆抗体，从而有助于研究migfilin在结肠癌生物学行为中的作用。
根据人migfilin cDNA序列设计一对引物，通过PCR扩增migfilin的N端序列。将PCR产物亚克隆至带有EcoRI/XhoI酶切位点的细菌表达载体pGEX-4T-1中，通过酶切鉴定及DNA序列分析鉴定目标重组质粒。将表达载体转化至大肠杆菌BL(21)感受态细胞中，经IPTG诱导表达GST-migfilin-N融合蛋白。用谷胱甘肽琼脂糖珠纯化融合蛋白，并用纯化的GST-migfilin N端融合蛋白免疫家兔制备抗migfilin多克隆抗体。所得抗migfilin多克隆抗体经蛋白A珠纯化后用于Western印迹检测不同细胞系中migfilin的表达。
成功克隆了migfilin的N端基因片段，获得了纯化的GST-migfilin N端融合蛋白及抗migfilin兔多克隆抗体。Western印迹分析表明，该抗体可特异性检测细胞系中migfilin的表达，这可能有助于进一步研究migfilin在结肠癌生物学中的作用。
