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应用PCR技术，对P16抑癌基因（CDKN2）进行体外定点突变．在P16cDNA中引入第48位密码子CCG（Pro）→CTG（Leu）和第74位密码子GAC（Asp）→AAC（Asn）突变，构建了p16－P48L和p16－D74N突变体，并把它们导入纯合缺失P16基因的人肺癌细胞株H460．经RNA点杂交、Northern印迹、Western印迹和细胞免疫化学染色，检测到P16表达．通过比较表达野生型和突变型P16的H460细胞在3H－TdR掺入及细胞所处周期的差异，证实P16表达抑制细胞进入S期，而P48L和D74N突变体对细胞进入S期没有影响．为了确证P48L和D74N突变体丧失抑制细胞增殖的功能是因为与CDK4结合功能下降，将野生型和突变型P16cDNA克隆于酵母表达载体plexA，用酵母双杂交筛选实验研究野生型和突变型p16蛋白与CDK4的结合．结果野生型P16和CDK4在酵母中表达并相互作用．而突变型P16cDNA和CDK4在酵母中相互作用受到影响．说明P48L和D74N突变影响了p16蛋白与CDK4的结合，从而影响了其调控细胞周期的功能．
Application of PCR technology, P16 tumor suppressor gene (CDKN2) in vitro site-directed mutagenesis. The p16-P48L and p16-D74N mutants were constructed by introducing the 48th codon CCG (Pro) → CTG (Leu) and the 74th codon GAC (Asp) → AAC (Asn) in P16 cDNA. A human lung cancer cell line H460 homozygous for deletion of the P16 gene was introduced. P16 expression was detected by RNA dot blot, Northern blot, Western blot and immunocytochemistry. Differences in 3H-TdR incorporation and cell cycle arrest of H460 cells expressing wild-type and mutant P16 confirmed that P16 expression inhibited cells entering S phase, whereas P48L and D74N mutants had no effect on cell entry into S phase. In order to confirm that the loss of P48L and D74N mutant cell proliferation inhibition is due to decreased binding to CDK4, the wild-type and mutant P16 cDNA was cloned into the yeast expression vector plexA, yeast two-hybrid screening experiment to study the wild-type and mutant p16 protein and CDK4 binding. Results Wild type P16 and CDK4 are expressed and interact in yeast. However, the interaction between mutant P16 cDNA and CDK4 in yeast is affected. These results suggest that P48L and D74N mutations affect the binding of p16 protein to CDK4 and thus affect its function of regulating the cell cycle.