Localization, Construction,Identification And Expression Of Prokaryotic And Enkaryotic Expression Vector Of Human Tumor PRR11 Gene

PRR11 is a proline-rich protein and contains 360 amino acids .The gene located in 17q22 chromosome, this region is a high tumor areas of gene amplification, such as lung cancer.The early studies have shown that this gene may be a new tumor-related genes, the study of tumor-related genes will help to clarify the occurrence and development mechanism of tumor, and these may be as a possible target for an effective search of the new anticancer drugs and new diagnostic methods. By constructing eukaryotic and prokaryotic expression vector, We can observe the over-expression of PRR11 protein and the localization, This provides the basis for further research of PRR11 molecule function. Part one Construction,identification and expression of Prokaryotic expression vector of human tumor PRR11gene Objective:To construction and identification of prokaryotic expression vector of human PRR11. Methods:PRR11 gene was amplified by PCR from Hela cell cDNA,then the gene was cloned into prokaryotic expression vector PET-28a to construct PET28a-PRR11.The recombinant plasmid was transducted to E. coli BL21 and the strain was induced by(IPTG).The protein PRR11/His was examined by SDS-PAGE and Western blotting technique.Results:PRR11 gene was successfully amplified by PCR and cloned into PET-28a vector by restriction endonuclease analysis. SDS—PAGE and Western blotting demonstrated that the protein PRR11/His was expressed in E.coli BL21. Conclusion:The construction of recombinant plasmid is successful., which provides the basis for further research of PRR11 molecule.Part two Construction,identification and expression of enkaryotic expression vector of human tumor PRR11gene. Objective:To construction and identification of enkaryotic expression vector of human PRR11. Methods:PRR11 gene was amplified by PCR from PET-28a-PRR11,then the gene was cloned into enkaryotic expression vector pcDNA3.1 to construct recombinant plasmid pcDNA3.1-PRR11 . Results : PRR11 gene was successfully amplified by PCR and cloned into pcDNA3.1 vector by restriction endonuclease analysis. Western blotting demonstrated that the protein PRR11 was expressed. Conclusion:The construction of recombinant plasmid is successful.Part three The cellular localization of PRR11 protein and the research of function. Objective: To construct eukaryotic expression vector and to observe cellular localization of PRR11 protein, as well as the impaction of the cell cycle. Methods: PRR11 gene was amplified by PCR from PET-28a-PRR11,then the gene was cloned into enkaryotic expression vector pEGFP-C1 to construct pEGFP-C1-PRR11.Results:PRR11 gene was successfully amplified by PCR and cloned into pEGFP-C1 vector by restriction endonuclease analysis. Western blotting demonstrated that the protein PRR11 was expressed. GFP-PRR11 protein located Cytoplasm.