Construction of recombinant adenovirus expression vector for Wistar rat heat shock protein 70 gene and preparation of recombinant adenovirus

Objective To construct a recombinant adenovirus expression vector for Wistar rat heat shock protein 70(HSP70) gene and prepare recombinant adenovirus.Methods HSP70 gene was amplified from spleen tissue of Wistar rats by RT-PCR and cloned into vector pMD18-T.The coding reading frame of cloned HSP70 gene was identified by sequencing and subcloned into shuttle plasmid pShuttle-CMV.The constructed recombinant shuttle plasmid pShuttle-CMV-HSP70 was digested withⅠ-CeuⅠandⅠ-SceⅠ,and the obtained expression frame of HSP70 gene was linked to vector pAdxsi carrying adenovirus backbone.The recombinant adenovirus backbone plasmid was linearized by digestion with PacⅠ,and transfected into 293(R)cells for packaging to obtain recombinant adenovirus pAd-CMV-HSP70.BRL cells were infected with the collected virus supernatant,in which the expression of HSP70 was determined by Western blot.Virus particles were purified by cesium chloride density gradient centrifugation,and determined for purity,titer and infectivity.Results Target gene band at a length of about 2 269 bp was observed in the cloning and shuttle plasmids as proved by PCR and restriction analysis,of which the sequence was consistent with that reported in GenBank.Digestion with XhoⅠ showed that HSP70 gene was cloned to adenovirus vector correctly.The expression level of HSP70 in BRL cells infected with recombinant adenovirus increased significantly(P 0.01).The recombinant adenovirus reached a purity of 1.29 after purification.The titer of virus particles was 5.31 × 1012 VP/ml,while the infectious titer was 1.0 × 1011 pfu/ml.Conclusion The recombinant adenovirus vector for Wistar rat HSP70 gene was constructed successfully,and high titer recombinant adenovirus particles were prepared,which laid a foundation of further study on biological activity and action mechanism of HSP70.