Construction and Identification of Recombinant Adenovirus Containing Lipid Storage Droplet Protein 5

Objective:To construct mouse lipid storage droplet protein 5(LSDP5) gene recombinant adenovirus using AdEasy Adenoviral Vector System.Methods:The full-length LSDP5 gene cloned from mouse liver cDNA library was inserted to pMD18-T vec-tor and sequenced after enzyme digestion.Then LSDP5 gene was cloned into adenovirus shuttle plasmid pShuttle-CMV to construct a re-combinant plasmid pShuttle-CMV-LSDP5.The linearized plasmid by PmeI was transformed into E.coli strain BJ5183 with adenovirus backbone plasmid AdEasy-1.The recombinant plasmid extracting from the positive clones was linearized by PacI and transfected into adenovirus package cells AD293.The recombinant adenovirus was harvested by several freeze-thaw cycles.The recombinant adenovirus DNA was identified by PCR,and the titer of adenovirus was detected.Results:LSDP5 gene was cloned successfully.The specific frag-ment,about 1400bp,was obtained from recombinant pMD18-T vector cleavage.The recombinant plasmid pShuttle-CMV-LSDP5 was digested by KpnI and SalI to produce anticipated fragments.A bigger fragment of 30Kb and a smaller fragment of 4.5Kb were generated when the recombinant adenovirus vector was digested by PacI.The reombinat adenovirus was constructed after packaged in AD293 cells.The target DNA fragment was gained by PCR.The extracted virus was used to infect AD293 cells repeatedly for amplification.At last,the titer was about 2.5×109pfu /ml.Conclusion:The recombinant adnenovirus containing LSDP5 was successfully eastablished,and it may lay a foundation for the further functional study of LSDP5.