Construction and identification of eukaryotic expression vector of antisense MBD1 gene fragment / 中国普通外科杂志

ABSTRACT

Objective To construct eukaryotic expression vector of antisense MBD1 gene fragment and to provide a tool for studying MBD1 gene function. Methods PCR primers were designed according to the coding sequence of MBD1 gene. Xba I and Kpn I recognition sequences and cutting sites were added to the 5' end of the sense and antisense primer respectively. The 342 bp specific PCR fragment was obtained from the cDNA of biliary tract carcinoma cell line QBC-939 using RT-PCR, the purified PCR fragment was then inserted reversely into the multiple cloning site of eukaryotic expression vector pcDNA3. 1 ( + ). The constructed recombinant plasmid was identified by PCR confirmation, Xba I and Kpn I double enzyme digestion and DNA sequencing. Results The 322 bp specific DNA band was obtained by PCR, Xba I and Kpn I double digestion produced a 327 bp and a 5. 4 kb DNA band which represent the inserted target gene fragment and the vector respectively. The sequencing result confirmed that the sequence of inserted fragment was correct. Conclusion The eukaryotic expression vector of antisense MBD1 gene fragment was constructed successfully by using gene cloning technique. It will be a useful tool for studying MBD1 gene function in DNA methylation and tumorigenesis.