Molecular cloning of eps8 gene from chicken embryonic cell

• Main Authors: Tsung-Kun Chen, 陳宗坤
• Other Authors: Tzeng-Horng Leu
• Format: Others
• Language: zh-TW
• Published: 1999
• Online Access: http://ndltd.ncl.edu.tw/handle/51481172423952507360

碩士 === 國立成功大學 === 藥理學研究所 === 87 === Previous studies have indicated that eps8 is one of the EGF receptor substrates and its overexpression can potentiate EGF-induced mitogenic effects. In v-Src transformed murine fibroblasts, two eps8 isoforms ( p97eps8 and p68eps8 ) can be recognized by anti-murine eps8 antibody generated in our laboratory previously. However, in chicken embryonic cells, a 200 kDa protein was detected by this antibody in the Western immunoblot analysis. In an attempt to confirm this 200 kDa protein was a chicken eps8 homologue, We screened a chicken embryonic cDNA library with 32P-labeled murine eps8 cDNA and got several positive clones. Sequence analysis of these chicken eps8-positive cDNA clones demonstrated the absence of the sequences at the 5' region. Utilizing 32P-labeled chicken eps8 cDNA as a probe, we demonstrated the molecular size of chicken eps8 mRNA was 4.7 Kb. And through 5'-RACE, we attempted to obtain the whole sequence of 4.7 kb chicken eps8 cDNA. Sequence comparison showed that eps8 cDNA from these two different species shared strong homology and both encoded 97 KDa protein. Thus, the detected 200 kDa protein in CE cell should represent a protein that shared the same antigen epitope present in mouse eps8. In addition, a rabbit polyclonal antibody specifically recognize chicken eps8 was attempted. Hopefully, with this antibody, we could study the interaction between v-Src and eps8 in CE cell.