人野生型P^(53)基因与逆转录病毒载体的构建及转染

CONSTRUCTION AND TRANSFECTION OF PLASMID OF HUMAN WILD TYPE P53 GENE IN RETROVIRAL VECTOR

应用逆转录病毒为载体构建了含人野生型P^(53)基因的重组质粒,并成功地转染了φcrip包装细胞.实验应用的为PLXSH(一种逆转录病毒载体),1.8kb野生型P^(53)作为插入片断,通过T4DNA连接酶进行连接.甲[α——^(32)P]dCTP标记野生型P^(53)基因片断(1.8kb)为探针进行原位杂交.筛选出重组体,将其命名为PLXSN—53,然后用该重组体对φcrip包装细胞进行转染.收集转染后的细胞,准备下一步感染P^(53)突变细胞株研究之用.该质粒的构建和细胞转染实验为进一步研究野生型P^(53)基因的抑癌作用及其表达与调控原理奠定了基础.

Retroviral vector was used to construct a plasmid containing human wild type p53 gene and the plasmid was transfected to ψ crip packaging cells. PLXSN as a vector and a 1. 8 kb fragment of wild type p53 as a target gene were used in this study, and ligated with T4DNA ligase. Posi-tive clones being recombinants were selected with 32P-labeled 1. 8 kb fragment of wild type p53 as a probe. We named the recombinants PLXSN-53, then transfected them to ψ crip packaging cells and , after collecting the surviving clones transfected them to p53 mutation cell lines. The con-struction of PLXSN-53 and the success in transfecting them to ψ crip have provided a foundation for studying the inhibition of tumor by wild type p53 as well as for the mechanism of regulation, control and expression of p53.