Construction And Identification Of A Recombinant Adenovirus Vector Containing Human Hsp70 Gene

Objective To construct recombinant adenovirus vector containning human Hsp70 and to express the gene efficiently in eukaryotic cells. To provide a base for the further investigating for gene therapy.Method The target gene Hsp70 was amplified from plasmid pOTB7/Hsp70 by PCR. The PCR products were identified correctly by digestion, electrophoresis and DNA sequencing. The target gene was connected with enzyme linearized shuttle plasmid pDC-315-EGFP, and then transformed into coli DH5a competent cells. Positive clone was identified that the recombinant plasmid pDC-315-EGFP-Hsp70. After foreign genes which was in recombinant plasmid were identified good expression, then the recombinant plasmid with the adenovirus Admax together transfected the human embryonic kidney 293 cells by liposome method. The recombinant adenovirus Ad-Hsp70 was packaged in HEK 293 cells by the transfection complex, and then the foreign genes were expressed.Results The PCR product of plasmid pOTB7/Hsp70 was identified as gene Hsp70 by digestion and electrophoresis. With three synonymous mutations, DNA sequencing results were 99% similarly as which was reported in Genebank.PCR products of target gene were connected with linearized plasmid pDC315-EGFP together and then recombinanted DH5αcompetent cells. Then the recombinant plasmid pDC315-EGFP-Hsp70 got and the insertion direction of Hsp70 was identified correctly by electrophoresis. With three synonymous mutations, DNA sequencing results were 99% similarly as which was reported in Genebank. The HEK293 cells were transfected by recombinant plasmid with liposome method after 48 hours, the green fluorescent protein and Hsp70 protein were expressed.HEK293 cells were transfected by recombinant plasmid and adenovirus genome with liposome method. The recombinant adenovirus Ad-Hsp70 were successfully paked in HEK293 cells.2 days after transfected, the green fluorescent protein can be observed; 9 days after transfected, the CPE can be observed. HEK293 cells were transfected again by the harvested recombinant adenovirus. After 48 hours, the green fluorescent protein can be observed and the Hsp70 protein can been tested by western blot. The titer of recombinant adenovirus was 1011pfu/ml.Conclusion Recombinant adenovirus Ad-Hsp70 was constructed successfully. The exogenous gene was expressed in eukaryotic cells successfully.