Construction and Sequence Determination of Recombinant Plasmid of PTEN Gene Fragment

Objective To construct a recombinant plasmid containing PTEN gene of gliblastoma. Methods Genomic RNA was extracted from fresh placenta with Trizol. Four pairs of primers were designed according to the gene sequence of PTEN. The gene PTEN fragment was amplified by means of reverse\|transcription polymerase chain reaction(RT\|PCR). The purified RT\|PCR products and \{pcDNA3 1\}/Hygro(-) plasmid were digested and ligated to form a recombinant expression plasmid which introduced into Escherichia coli(E.coli) Top 10. Sequence determination analysis was performed to identify the cloned gene. Results The PTEN gene fragment with the length about 241, 239, 227 bp(antisense) and 1209 bp(sense) were specifically amplified by RT\|PCR. Sequence determination analysis showed that the cloned gene was identical to the expected gene. Conclusion The recombinant \{pcDNA3 1\}/Hygro(-) containing PTEN gene from gliblastoma was constructed successfully.