The Experimental Study On Recombinant Expression And Anti-tumor Effect Of ING4

Objective: (1)To construct pcDNA3.0-ING4 recombiant eukaryotic expression plasmid and explore the effect of ING4 on some kinds of tumour cells(;2)To construct pET-21a(+) recombiant prokaryotic expression plasmid and prepare the polyclonal antibody. Methods: Using RT-PCR method,the cDNA encoding the mouse ING4 was isolated with total RNA extracted from mouse liver tissue. (1) The cDNA fragment was subcloned into the eukaryotic expression plasmid pcDNA3.0-ING4, Analysis of PCR ,restricting enzyme digestion and DNA sequencing were carried out to demonstrate the sequence of the plasmid, Then we transfected it into some kinds of tumour cells by Lipofectamine to study the expression and the function of ING4;(2) The cDNA fragment was subcloned into the prokaryotic expression plasmid pET-21a(+).Analysis of PCR ,restricting enzyme digestion and DNA sequencing were carried out to demonstrate the sequence of the plasmid , Then the pET-21a(+)-ING4 was transformed into E.coli BL21(DE3), Recombiant protein was expressed in E.coli host strain BL21(DE3) in the form of inclusion body and primarily purified, the purified recombiant protein was used to immune the rabbit for preparing polyclonal antibody . Results: RT-PCR product is about 750bp specific fragment , Analysis by restricting enzyme digestion and PCR of pcDNA3.0-ING4 recombiant plasmid showed results were about 750bp, DNA sequencing revealed that ING4 cloning were successful.Cell cycle were examined by flow cytometry after transfection with pcDNA3.0-ING4, apoptosis was deteced by fluorescence microscope with Hoechst33258 staining and lazer scanning confocal microscope(LSCM).we found that ING4 could enhance apoptosis in some kinds of tumour cells. ING4 or IL-24 could enhance apoptosis in SMMC7721 cells. ING4 and IL-24 have no difference in results of apoptosis in SMMC7721 cells.The construction of pET-21a(+)-ING4 prokaryotic expression vector were successfully obtained and the polyclonal antibody was prepared. Conclusion: This is the first time to get ING4 cDNA clone identified in China. And It lays the foundation for further research on molecular mechanism on anti-tumors and the development of new gene therapy method .