目的　克隆人WAF1基因 ,构建成WAF1 pcDNA3真核表达质粒 ,探索WAF1基因的生物调控功能。方法　采用PT PCR和DNA直接测序方法从Hela细胞中扩增人WAF1基因 ,通过克隆到 pcDNA3真核克隆载体上 ,用脂质体法转染Hct 8细胞 ;经免疫荧光和免疫组化方法鉴定WAF1外源基因的表达活性和对Rb基因、cyclinD1基因的调控作用。结果　从Hela细胞扩增的人WAF1基因克隆至真核克隆表达载体 pcDNA3中 ,获得了人WAF1 pcDNA3重组质粒和高效表达P2 1WAF1/CIP1的Hct 8细胞株 ;证实了WAF1具有促进Rb、抑制cyclinD1蛋白表达的调控作用。结论　成功地构建了人WAF1表达质粒WAF1 pcDNA3,外源基因WAF1的表达对下游基因具有生物调控效应 Objective To clone human WAF1 gene and construct WAF1 pcDNA3 eukaryotic expression plasmid to explore the biological regulatory function of WAF1 gene. Methods Human WAF1 gene was amplified from Hela cells by PT PCR and DNA sequencing. The recombinant plasmid was cloned into pcDNA3 eukaryotic vector and transfected into Hct 8 cells by lipofectamine 2000. The expression of WAF1 was identified by immunofluorescence and immunohistochemistry Exogenous gene expression activity and regulation of Rb gene and cyclinD1 gene. Results The human WAF1 gene amplified from Hela cells was cloned into the eukaryotic clone expression vector pcDNA3 and the recombinant plasmid of human WAF1 pcDNA3 and Hct 8 cell line highly expressing P2 1WAF1 / CIP1 were obtained. It was confirmed that WAF1 could promote Rb and inhibit cyclinD1 protein The role of regulation. Conclusion The human WAF1 expression plasmid WAF1 pcDNA3 was successfully constructed and the expression of the foreign gene WAF1 had a biological regulatory effect on the downstream genes