【摘要】 目的　构建编码肿瘤抑制基因 PTEN的正义与反义真核表达重组载体并进行其序列测定。　方法　新鲜胎盘组织抽提基因组 RNA;根据基因库 PTEN基因序列分别设计合成三对反义引物及一对正义引物 ,采用 RT-PCR法扩增编码 PTEN的基因片段 ;将 PTEN基因定向克隆到 pc DNA3.1/ Hygro(- )真核表达载体 ,筛选阳性重组子并鉴定 ;对重组子进行序列测定。　结果　RT- PCR所扩增的 PTEN基因片段为 2 4 1,2 39,2 2 7bp(反义 )及 12 0 9bp(正义 ) ,表明所克隆的基因为编码 PTEN的基因片段。　结论　成功构建编码肿瘤抑制基因 PTEN的 pc DNA3.1/Hygro(- )真核表达质粒 pc DNA3.1/ Hygro(- ) PTEN。更多还原

【Abstract】 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.更多还原