[Construction of recombinant plasmid vector pIRES-CD and its expression in ACC-2 cells].
OBJECTIVE
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).
Huang
Sheng-Yun
SY
Department of Oral and Maxillofacial Surgery, Shandong Provincial Hospital, Shandong University, Jinan 250021, Shandong Province, China. huangsy28@163.com
Zhang
Dong-Sheng
DS
Zhang
Shi-Zhou
SZ
Liu
Gui-Jun
GJ
Mu
Wen-Li
WL
Zhang
Jie
J
D015703:Antigens, CD / Q000235:genetics*; D045744:Cell Line, Tumor; D004926:Escherichia coli; D041722:Genes, Transgenic, Suicide; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids; D014162:Transfection
D015703:Antigens, CD