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[Construction of recombinant plasmid vector pIRES-CD and its expression in ACC-2 cells]
Sheng-Yun Huang1, Dong-Sheng Zhang, Shi-Zhou Zhang
1Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Shandong University, Jinan 250021, Shandong Province, China. huangsy28@163.com
Purpose:
To clone CD gene, construct its eukaryotic expression vector pIRES-CD and obtain positive ACC-2 cells expressing E.coli CD gene stably.
Methods:
PCR amplification was performed using primers based on E.coli CD gene sequence from Genebank, E.coli genomic DNA as template. PCR product was inserted into pMD18-T. After sequence confirmation, the gene was subcloned to pIRES to construct recombinant eukaryotic expression vector pIRES-CD. Then the combinant plasmid was conducted into ACC-2 cell by electroporation. ACC-2 cells stably expressing CD was obtained by 10-day positive selection with 400 mug/mL G418. Total RNA was extracted and the expression of the CD gene in transfected ACC-2 cells was identified by RT-PCR.
Results:
PCR yielded a fragment of 1280bp and CD was verified by sequence analysis. A fragment of 6.1kb and inserted fragment of 1280bp were obtained by cutting positive recombinant plasmid of pIRES-CD with XbaI and NotI. RT-PCR analysis demonstrated that CD gene could be effectively expressed in ACC-2 cells.
Conclusions:
The CD gene is successfully amplified and the eukaryotic expression plasmid containing E.coli CD is successfully constructed.The positive ACC-2 cell clones expressing CD gene stably are obtained, which provide a basis for further study of adenoid cystic carcinoma gene therapy with CD/5-FC suicide gene system. Supported by Natural Science Foundation of Shandong Province(Grant No.Z2003C03).
