关键词:  亚细胞定位； PTEN基因； 质粒

Abstract:

Objective
To construct the recombinant plasmid that highly expressed h
uman subcellular PTEN,inorder to provide a basis for further study on its anti-tumor effect.
Methods PTEN cDNA was amplified by RT-PCR based on mRNA of placenta.The PCR product was ligated into T-vector,and transfected into E.coli;the obtained T-PTEN plasmid was identified with restrictive digestion and sequencing.PCR was  used to incorporte nuclear signal of localization(NSL) into PTEN when T-PTEN was used as template.Then the PCR product  was  ligated into T-vector,and transfected into E.coli,and T-NSL-PTEN plasmid was obtained.pcDNA3.1 and T-NSL-PTEN were ligated after digested with EcoRⅠand BamHⅠ,and transfected into E.coli,the recombinant vector pcDNA3.1-NSL-PTEN was obtained,and identified with digestion and sequcncing.Results The recombinant  expression vector DUM-PTEN and PUM-NSL PTEN were  identified by restrictive digestion and DNA sequencing.As expected,by EcoRⅠ and BamHⅠ digestion,it showed the band of 1 200 bp.The sequencing result showed the NSL was incorporated successfully.The recombinant pcDNA3.1-PTEN was obtained with 1 200 bp,the sequencing result showed that its sequence was same as target gene;the recombinant pcDNA3.1-NSL-PTEN was comfirmed by restrictive digestion and sequencing,and the NSL was incorporated successfully. Conclusion The  recombinant expression plasmid pcDNA3.1-NSL-PTEN is constructed successfully which can highly express human subcellular PTEN.

Key words:  subcellular, PTEN gene, plasmid