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Construction of eukaryotic expression vector of TSLC1 gene
Qi-Lian Liang1, Bi-Rong Wang, Zhou-Yu Li
1Department of Oncology, Affiliated Hospital of Guangdong Medical College, Zhanjiang, China.
Introduction:
To construct a eukaryotic expression vector of the tumour suppressor in lung cancer 1 (TSLC1) gene, so as to explore the mechanisms of tumour suppression of the gene theoretically.
Material And Methods:
The open reading frame (ORF) of TSLC1 gene was amplified with RT-PCR from normal human foreskin acrobystia, and cloned to pMD19-T simple vector (TA Clone method). The resultant plasmid was transformed into Escherichia coli JM109 for amplification. The TA Clone recombinant was digested by double restriction enzyme (Bgl II/EcoR I) and analysed with agarose gel electrophoresis. The positive one was sequenced. The inserted DNA fragment was recovered, and then it was mounted into the eukaryotic expression vector pIRES2-EGFP, transformed into E. coli JM109 for amplification. A positive recombinant plasmid named pIRES2-EGFP-TSLC1 was confirmed by Bgl II/EcoR I double-enzyme digestion analysis.
Results:
RT-PCR amplified the ORF of the TSLC1 gene. It was approximately 1400 base pairs. The obtained DNA was confirmed a high degree of homology with the sequence of TSLC1 cDNA sequence (AY358334) stored at GenBank.
Conclusions:
Construction of a TSLC1 eukaryotic expression vector was successful, and it has established a solid foundation for further study.
