[Construction and identification of recombinant adenovirus vector containing thioredoxin reductase gene]

Jiang-ying Xu, Lin Xu, Guang-yong Peng, Chuan-lin Ding, Shi-yue Lu, Kun Yao
Xi Bao Yu Fen Zi Mian Yi Xue za Zhi, Chinese Journal of Cellular and Molecular Immunology 2003, 19 (5): 440-2

AIM: To construct recombinant adenovirus vector containing human thioredoxin reductase (TR) gene and to explore the correlation between antioxidant activity of TR and the degenerative neuropathy.

METHODS: Full length TR cDNA was obtained from recombinant plasmid pGEM-TR via digestion with Apa I and Not I and was cloned into pShuttle vector and pShuttle-TR was recut with I-Ceu I and PI-Sce I. Fragment containing TR gene and CMV promoter was inserted into E1 and E3 deficient adeno-X virus DNA, and then the recombinant adenovirus vector was transfected into HEK 293 cells through lipofectamine and identified by PCR. The TR expression on and in cell lysate of CV1 cells infected with recombinant adenovirus was by immuno fluorescence assay and Western blot analysis.

RESULTS: After replication of recombinant adenovirus Adeno-TR, the virus titer was about 4.4x10(11) pfu/L. The TR expression on CV1 cells was proved by fluorescent microscopy. Western blot analysis showed a band with relative molecular mass (M(r)) of 55,000.

CONCLUSION: A recombinant adenovirus vector has been successfully constructed and TR is expressed on CV1 cells. This result lays the foundation for further study on function of TR and its correlation with degenerative neuropathy.

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