To Construct The Eukaryotic Expression Plasmid EX-Y2069-M29 And Test The Expression Of LRP16 Gene

Objective:Construction of LRP16 gene eukaryotic expression plasmid EX-Y2069-M29. was transfected into human endometrial carcinoma HEC-1-B cells, a stable cell line expression of LRP16 protein, and detection of LRP16 gene in human endometrial carcinoma HEC-1-B cells.Methods:Full-length LRP16 fragment was amplified by polymerase chain reaction(PCR), was subcloned into the eukaryotic expression vector pReceiver-M29 to construct recombinant plasmid EX-Y2069-M29. Analysis nucleotide sequence and DNA sequencing confirmed that the recombinant plasmid EX-Y2069-M29 insert the correct nucleotide sequence of LRP16. Liposome EX-Y2069-M29 transfected HEC-1-B cells and used Western blot to detect protein expression of LRP16 in human endometrial carcinoma HEC-1-B cells.Results:1. In the extraction process total RNA of LRP16 gene did not occurred significant degradation,the total degradation of RNA integrity and non-degradable.It is shows that total RNA and mRNA of LRP16 gene is integrated.Product of PCR showed a clear specific band at 980 bp and it is show that there is the correct size consistented with the length to the estimated cDNA.2. The eukaryotic expression plasmid of EX-Y2069-M29 is successfully constructed.LRP16 gene fragments in eukaryotic expression vector EX-Y2069-M29 is correct by digestion and PCR.3. To detect the expression of LRP16 protein in human endometrial carcinoma HEC-1-B cells by Western blot. Control group and the EX-NEG-M29 group relative expression of LRP16 protein, the difference was not significant (P> 0.05); EX-Y2069-M29 group and the control group, the relative expression of LRP16 protein, the difference was significant (P<0.05); EX-Y2069-M29 group and the EX-NEG-M29 group relative expression of LRP16 protein, the difference was statistically significant (P<0.05)Conclusion:LRP16 gene was successful-ly constructed and transformed into the eukaryotic expression vector EX-Y2069-M29 plasmid cloning site.2. Recombinant plasmid EX-Y2069-M29 constructed with LRP16 gene transfected HEC-1-B cells.The stablely expression in HEC-1-B cells make the further study of the foundation for LRP16.