Construction and expression of recombinant adenovirus containing human catalase gene in vitro / 浙江大学学报·医学版

ABSTRACT

<p><b>OBJECTIVE</b>To construct the adenovirus vector containing recombinant human catalase (CAT) and to express the recombinant gene in vitro.</p><p><b>METHODS</b>Total RNA was extracted from human leukocytes and full-length human CAT cDNA was obtained with RT-PCR method. The CAT gene was cloned into pcDNA3.1(+) vector and pcDNA3.1(+)CAT was constructed. The positive clones were confirmed by the restriction enzyme digestion and gene sequencing. The CAT gene was cloned into the entry vector pENTR1A, and pENTR1A-CAT vector was constructed. By LR reaction pENTR1A-CAT and pAd/CMV/V5-DEST was recombined in vitro, and the recombinant adenovirus pAd/CMV/V5-DEST-CAT was obtained. The positive pAd/CMV/V5-DEST-CAT was confirmed by sequencing and transfected into 293A cells with Pac I linearization and Lipofectamine 2 000, and the recombinant virus particles were packaged and amplified in the cells. The expression of CAT protein and CAT enzyme activities of the recombinant virus were determined by Western blot and 240 nm UV absorption methods.</p><p><b>RESULT</b>High expression of recombinant adenovirus was obtained and the expressed human catalase had high enzyme activity.</p><p><b>CONCLUSION</b>Ad/CMV/V5-DEST-CAT vector containing human catalase gene has been constructed successfully; and the expressed enzyme in 293A cells has high activity.</p>