PDCD5Gene Recombinant Plasmid Construction And Its Expression In Transfected KM3Cells

Objective:The recombinant plasmid pYr-ads-4-PDCD5was constructed and then transferred into multiple myeloma KM3cells by using liposomes for detecting the expression of PDCD5. The study in this dissertation lays the foundation and provides tools for the research in the cell apoptosis mechanism of multiple myeloma promoted by PDCD5gene in the future.Methods:The PDCD5genes coding segment (CDS) was Amplified by using pCMV-SPORT6-PDCD5as a template. After the success of identification of electrophoresis agarose gel, the CDS of PDCD5was connected to pYr-adshuttle-4plasmid by double digestion. The product of the connection was transformed into E. coli DH5a competent cells, cultured in containing kanamycin (Kan) medium. Then positive clones were picked for the identification of double restriction endonuclease digestion, The corrected pYr-ads-4-PDCD5were sent for sequencing mensuration. The sequencing report was compared with the desired target sequenceing in order to determine whether pYr-ads-4-PDCD5recombinant plasmid was successfully constructed. If pYr-ads-4-PDCD5was successfully constructed, a large number of plasmid was amplified and extracted. After setting the transfected target gene group, the transfected empty vector group, and the non-transfected group, liposome-mediated by LipofectamineTM2000pYr-ads-4-PDCD5was transfected to multiple myeloma KM3cell line, transfection efficiency was observed under fluorescence microscopy after48hours, and real-time fluorescent quantitative PCR (real-time by Quantitative PCR, qPCR) and Western blot technique (Western blot) were used separately to detect mRNA and protein expression of PDCD5in KM3cells.Results:With the template of pCMV-SPORT6-PDCD5, PDCD5gene coding segment (CDS) was successfully amplified, and agarose gel electrophoresis obtained about400bp of the target gene, which was consistent with PDCD5CDS. PDCD5gene fragments were ligated with pYr-adshuttle-4plasmid after double digestion, and after transformation and culture of the product of the ligation, about400bp PDCD5target gene segment and7000bp carrier segmet were cut out by double digestion. The gene sequencing mensuration result of pYr-ads-4-PDCD5was compared with the desired target sequenceing, the results showed that the sequnence of the amplified PDCD5gene fragments sequence was completely correct and the pYr-ads-4-PDCD5recombinant plasmid was successfully constructed. After48hours for LipofectamineTM2000transfected, the KM3cells of the target gene group and empty vector group were observed under the fluorescence microscope, the result showed the transfection efficiency was45%. The expression levels of PDCD5mRNA and PDCD5protein measured by qPCR and Western blot determination were significantly higher than transfected empty vector group and untransfected group, they increased to about7times and2times respectively. There was no significant difference between the transfected empty vector group and the untransfected group. Conclusions:1. The recombinant plasmid pYr-ads-4-PDCD5was constructed successfully.2. The multiple myeloma KM3cells transfected by pYr-ads-4-PDCD5recombinant plasmid could let PDCD5be successfully expressed...