Construction And Identification Of Eukaryotic Expression Vector Expressing Human Aspartyl-asparaginyl-B-hydroxylase

Objective: The aim of this study was to construct recombinantadenovirus carries(Ad-AAH-IRES2-EGFP)and plasmid pcDNA3.1-AAH，respectively.These two eukaryotic expression vectors amplified target geneexpressing ASPH protein to meet the needs of experimental and clinicalstudies.Method:1)Total mRNA was Extracted from human hepatocellularcarcinoma tissues, AAH cDNA were amplified by RT-PCR,and verified byTA cloning and sequencing.2) Then plasmid containing the whole-lengthAAH gene pMD18-T-AAH was constructed, from which AAH gene wasamplified and cloned into the pcDNA3.1(-) to create the recombinantplasmid pcDNA3.1(-)-AAH.3) Gateway technology was used,the obtained18T-ASPH-1as a substrate, to build Ad-ASPH-IRES2-EGFP.4) Bothrecombinant vectors were transfected into293T cells, the expression ofAAH protein was detected by western blot.Results: AAH cDNA was verified by sequence analysis. RecombinantpcDNA3.1(-)–AAH and Ad-AAH-IRES2-EGFP was successfullyconstructed. The titer of adenovirus was as high as to5×109ifu/ml. Bothvectors were transfected into293T cells, the AAH protein expression levelsin Ad-ASPH-IRES2-EGFP group was significantly higher than controlgroups, and also higher than that in pcDNA3.1(-)–AAH group. Conclusion: Gateway technology to Ad-AAH-IRES2-EGFP is betterthan traditional recombination, overcoming the repeated enzyme digestion,tedious work, time-consuming and other shortcomings, purified viruseswere obtained in high titers and with a high level of activity. Recombinantplasmid pcDNA3.1-AAH was also successfully constructed andidentified.The research has provided an experimental basis for the study ofgene function,immune therapy and the industrial production of recombinantadenovirus.