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Screening of binding proteins interact with phosphotyrosine-interacting domain of DOC-2 by yeast two hybrid system
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Abstract:
Objective: To screen for proteins which can interact with phosphotyrosine-interacting domain (PID) of differentially expressed gene in human ovarian cancer cell line DOC-2 by yeast-two hybrid technique, so as to provide evidence for the signal pathway of DOC-2. Methods: The cDNA sequence of human DOC-2 gene was amplified and its PID domain (nDOC-2) was subcloned into the bait vector pGBKT7 of yeast two-hybrid system; the product was then used to screen an embryo brain cDNA library and the proteins interacting with nDOC-2 were identified. Quadrople dropout(QDO) medium and X-α-gal were used for selecting the positive clones. PCA was used to analyze the amplified sequence. After elimination of the false positive clones, the positive clones were sequenced and analyzed by bioinformatic methods. Results:Twenty-one candidate positive clones were obtained and 3 of them were plasmids encoding Homo sapiens partial mRNA for betaglycan (TBR III gene), Homo sapiens protocadherin gamma subfamily C 3 (PCDHGC3), and APLP1（amyloid beta precursor-like protein 1）.Conclusion: The proteins obtained in this study may play important roles in the signal pathway of DOC-2, which provides a new orientation for DOC-2 gene therapy of ovarian cancers
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