Construction and analysis of the recombined adenovirus carrying mouse estrogen receptor a gene
AIM: To construct recombinant adenovirus vector carrying C57BL/6 mouse estrogen receptora ( Esr1 ) gene, and further to propagate and purify the virus particles. METHODS: The CDS fragment of Esrl gene was amplified by RT-PCR from C57BL/6 mouse ovary cells and put into pMD19-T Simple vector, then verified by sequencing. The fragment was subcloned into the adenovirus shuttle plasmid pDNR-CMV. The recombinated shuttle plasmid was homogenously recombined with pLP-Adeno-X-CMV in ElectroMAX~(TM) DH10B~(TM) Cells and the recombinant adenoviral plasmid Adeno-Esr1 was generated. Then plasmid Adeno-Esr1 was linearizated by Pacl and transfected into HEK293 cells for packaging, amplifying and purifying to obtain recombined adenovirus Ad-Esr1 and its liter measured. HEK293 cell was infected by recombinated adenovirus Ad-Esrl and Esrl protein was detected by Western Blot. RESULTS: It was confirmed by sequencing that the extracted CDS fragment of Esrl gene had no mutation. Restriction endonuclease analysis and sequencing confirmed the successful cloning of the gene into the recombined adenovirus. Ad-Esr1 was successfully constructed with a titer of 6. 4 × 10~(13) ( pfu/L). Western blot showed Esrl protein expression in infected HEK293 cells and didn't express in uninfected HEK293 cells. CONCLUSION: The recombinant adenovirus of Esrl gene has been successfully constructed, which provides a basis for the research into the role of Esrl about the effect of biological function in nervous system and gene therapy in nervous system disease.
NETL
NSTL
万方数据
维普
