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目的 构建人 p16基因第二外显子cDNA克隆 ,制备其cDNA探针 ,建立 p16基因第二外显子PCR SouthernBlot分析方法。研究原发性肝细胞癌 (HCC) p16基因第一、二外显子、p15基因第二外显子纯合性缺失 ,p53基因第七外显子纯合性缺失、点突变以及HBV前C区、S区、X区在HCC中整合情况。方法 用RT PCR获得p16基因第二外显子cDNA ,将其插入质粒 pGEM T中 ,构建为重组质粒pGEM pl6。经蓝白选择、限制性内切酶酶切图谱分析、DNA序列分析筛选获得重组质粒的阳性克隆 ,插入片段经限制性内切酶酶切、纯化后 ,用随机引物法标记地高辛 ,以此为探针 ,用SouthernBlot方法分析p16第二外显子PCR产物。应用PCR、PCR SSCP和PCR RFLP等方法 ,对临床 38例HCC手术后标本中的p16基因第一、二外显子、p15基因第二外显子、p53基因第七外显子纯合性缺失和点突变进行分析。应用PCR和 3’末端碱基特异性PCR分析肝细胞癌基因组HBV前C区野生型和突变型、PCR分析S区和X区整合的情况。同时检测了血清中HBV前C区的阳性率。结果 重组质粒的限制性内切酶酶切图谱分析表明其酶切位点与原设计相符 ,插入片段大小为 30 7bp ,与引物区间大小一致 ;地高辛标记探针可与 p16PCR产物杂交。发现在这 38例HCC患者中 ,p16基因第二外显子的缺失率为 34 2 %
Objective To construct the second exon cDNA clone of human p16 gene and prepare its cDNA probe, and to establish a PCR Southern Blot analysis method for the second exon of p16 gene. To investigate the homozygous deletion of exon 2 of p16 gene in p19 gene of primary hepatocellular carcinoma (HCC) and homozygous deletion of p15 gene, homozygous deletion of p53 gene in exon 7, point mutation and HBV prec Zone, S, and X zones are integrated in HCC. Methods The second exon cDNA of p16 gene was obtained by RT PCR and inserted into plasmid pGEM T to construct a recombinant plasmid pGEM pl6. The recombinant clones were screened by blue-white selection, restriction enzyme digestion analysis and DNA sequence analysis. The insert was digested with restriction enzyme and purified, and digoxin was labeled by random primer method. This is a probe and the p16 second exon PCR product was analyzed using the SouthernBlot method. Using PCR, PCR SSCP and PCR RFLP methods, the homozygosity of p16 gene exon 1, exon, p15 gene exon, and p53 gene exon 7 in 38 specimens of clinical specimens after HCC surgery And point mutations were analyzed. PCR and 3’-end base-specific PCR were used to analyze the integration of wild-type and mutant-type HBV pre-C regions, PCR analysis S-region and X-region. At the same time, the positive rate of HBV pre-C in serum was measured. RESULTS: Restriction endonuclease digestion analysis of the recombinant plasmid showed that the restriction site was consistent with the original design. The size of the insert was 30 7bp, which was the same as the size of the primer region. The digoxigenin-labeled probe could hybridize with the p16 PCR product. The missing rate of the second exon of p16 gene was 34 2 % in these 38 HCC patients.