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Aim: To clone prokaryotic expression vector of Cdc25C, purify the fusion protein of GST-Cdc25C, and identify its function preliminarily., Methods: Human Cdc25C coding region was amplified from human mammary cDNA library by PCR, and cloned into the prokaryotic expression vector pGEX-KG. The fusion protein GST-Cdc25C was expressed in E.coli Rossate and purified by GST-Sepharose 4B beads. The function of purified GST-Cdc25C was identified by GST pull-down assay., Results: The GST-Cdc25C recombinant plasmid was successfully obtained by double digestion identification. The inserted fragment was confirmed correctly by sequencing. SDS-PAGE and Western blot analysis showed that the fusion protein was expressed. The fusion protein of about M(r); 80 000 was successfully induced, and identified by SDS-PAGE and Western blot analysis. GST pull-down assay showed that GST-Cdc25C could interact with Chk2 which verified its known function., Conclusion: Cdc25C was successfully cloned and purified.

Aim: To prepare anti-PES1 monoclonal antibody (mAb) and detect the PES1 expression in several kinds of human cancer cell lines and its tissue distribution in the adult rat., Methods: pGEX-PES1(1-322aa) fusion protein was purified and inject-ed into mice for immunization. Anti-PES1 mAb was produced by cell fusion and screening after immunization. Anti-PES1 mAb was identified by Western blot. Several human cancer cell lines and different tissue samples of adult rat were detected the PES1 expressions with the mAb prepared., Results: Aanti-PES1 mAb was determined to be specific to PES1 with Western blot analysis. PES1 were expressed in all kinds of breast, ovary, liver and lung cancer cell lines detected in different levels with prepared mAb. Pescadillo was obviously expressed in the breast and ovary but not other tissues of adult rat using prepared mAb., Conclusion: Anti-PES1 mAb was successfully prepared. PES1 may play an important role in the tumorigenicity and may also play a role in the pathway of estrogen since breast and ovary, the most important estrogen target organ of adult rat, obviously express pesca-dillo.

Aim: To construct the prokaryotic expression vector of human histone-1, obtain the purified GST-H1 protein, and detect its activity., Methods: Human histone-1 coding region was amplified from human mammary cDNA library, and was inserted into prokaryotic expression vector pGEX-KG. The recombinant plasmid pGEX-KG-H1 was transformed into E.coli Rossate. The expressed product was purified by GST-Sepharose 4B beads and identified by SDS-PAGE and Western blot analysis., Results: The DNA fragment of about 650 bp was successfully amplified by PCR, cloned into pGEX-KG, and identified by sequencing. The recombinant protein of about M(r); 52 000 was successfully induced, purified and tested well by Kinase assay., Conclusion: The recombinant protein of GST-H1 is obtained successfully, which lay the foundation for further research on cell cycle control.

Aim: To construct a vector of ErbB2 small interfering RNA(siRNA), and to investigate its effect on ErbB2 expression and the growth of ZR75-1 breast cancer cells., Methods: Two ErbB2-siRNAs were designed and inserted into the pSliencer 2.1-U6 neo vector. After confirmed by restriction and DNA sequencing, siRNA vectors were co-transfected with the FLAG-ErbB2 expression vector into human embryonic kidney 293T cells, or transfected into SKBR3 and ZR75-1 breast cancer cells. The effects of siRNAs on the expression of ErbB2 were identified by Western blot and the proliferation of ZR75-1 cells was repressed., Results: Two siRNAs could effectively inhibit the expression of exogenous and endogenous ErbB2 proteins, and could repress the growth of ZR75-1 cells., Conclusion: ErbB2 siRNAs effectively inhibit the expression of ErbB2, thus repressing the growth of ZR75-1 cells.

Aim: To construct the eukaryotic expression vector of small interfering RNA (siRNA) targeting human FHL2 and transfect it into human embryo kidney 293T cells to investigate the silencing effect of FHL2 siRNA on the expression of FHL2 gene., Methods: Two FHL2 siRNAs were designed and inserted into pSliencer 2.1-U6 neo expression vector. Then human embryo kidney 293T cells were cotransfected with the recombinant plasmids and FLAG-tagged FHL2 expression vector. The silencing effect of FHL2 siRNAs on the expression of FHL2 gene was identified by Western blot., Results: The expression vectors of FHL2 siRNAs were constructed and confirmed by DNA sequencing. Western blot showed that FHL2 siRNAs effectively inhibited expression of FHL2., Conclusion: The eukaryotic expression vectors of FHL2 siRNAs are constructed successfully. The siRNAs effectively inhibit the expression of FHL2.

Aim: To construct human Egr-1 promoter luciferase reporter system and study its activity induced by ionizing radiation., Methods: Egr-1 promoter was obtained by human genomic PCR and cloned into pGL3-basic vector. After transfection of recombinant plasmid into human tumor cells, the Egr-1 promoter activity induced by ionizing radiation was detected by luciferase reporter assay., Results: The luciferase reporter system of Egr-1 promoter was successfully constructed. The activity of Egr-1 promoter was substantially increased after different doses of IR and reached to the peak at the time point of 48 h after IR., Conclusion: The Egr-1 promoter was constructed in this study showed IR inducible activity in tumor cells, laying foundation for the research of radiation. mediated gene therapy.

Aim: To prepare and characterize antibody against Memo protein and to detect the tissue distribution of Memo in mice., Methods: Fusion protein GST-Memo was expressed and purified, and polyclonal antibody against Memo was prepared by immunizing mice. A FLAG-tagged eukaryotic expression vector pcDNA3-FLAG-Memo was constructed. The specificity of the antibody was detected by Western blot., Results: An eukaryotic expression vector pcDNA3-FLAG-Memo was obtained. The polyclonal antibody was found to be specific to Memo. Memo protein was widely expressed in mouse tissues using the obtained antibody in Western blot., Conclusion: Antibody specific to Memo has been successfully obtained, which provides useful tool for investigation into Memo-associated mechanisms of tumor metastasis and invasiveness.