Abstract:

Objective     To clone the human thioredoxin-1 gene (hTrx1) and construct the recombinant vector containing the target gene.  Methods   The cDNA gene of  human thioredoxin was amplified by RT-PCR from gastric cancer cells BCG823 and MNK45, using  specific primers containing double endonuclease digesting sites. The hTrx1 cDNA was then inserted into the EZ-T vector to construct the EZ-T-hTrx1 recombinant vector. The next step was to double digest the EZ-T-hTrx1 recombinant vector and insert the target gene into the pcDNA3.1myc-His to construct the pcDNA3.1myc-His-hTrx1 recombinant vector, which was  identified by double digestion and DNA sequencing.  Results    By double digestion, the hTrx1 cDNA was successfully inserted into the pcDNA3.1myc-His vector; and through DNA sequencing, the sequence of the target gene was inserted and was in accordance with GenBank(NM003329). The pcDNA3.1myc-His-hTrx1 recombinant vector was successfully gained.  Conclusion    The successful cloning of the hTrx1 gene and construction of its recombinant vector pcDNA3.1myc-His-hTrx1 lays the foundation for the study of the biological activity of hTrx1 and mechanism of its function in tumor genesis.

Key words: Human thioredoxin-1; Recombinant vector; Reverse transcription-PCR; Double endonuclease digestion