目的构建组氨酸(Histidine)位点突变的丙型肝炎病毒(Hepatitis C virus,HCV)突变体,并检测其感染性。方法利用定点突变技术,分别将HCV包膜蛋白E2第490位和第621位的组氨酸突变为丙氨酸(Alanine),构建突变质粒H490A和H621A,采用T7体外转录法制备病毒RNA,通过电穿孔法导入Huh7.5.1细胞,免疫荧光法检测病毒蛋白的表达、电穿孔效率及细胞培养上清的感染性。结果 H490A和H621A突变型质粒经酶切及测序鉴定构建正确;体外转录获得的野生型与突变型病毒RNA均能在Huh7.5.1细胞中表达病毒蛋白,电穿孔效率高达90%以上;野生型病毒感染细胞培养上清中可检测到HCV阳性细胞,H490A病毒感染细胞培养上清中的阳性细胞数量比野生型明显减少,H621A病毒感染细胞培养上清中未检测到阳性细胞。结论成功构建了组氨酸位点突变的全长表达质粒H490A和H621A,两种突变体病毒的感染性均显著降低。
The p53 signaling pathway works as a potent barrier to tumor progression.Two single nucleotide polymor-phisms(SNPs)in the gene loci of p53 pathway,p53 codon 72 Arg72Pro and MDM2 SNP309(T>G),have been shown to cause perturbation of p53 function,but the effect of the two SNPs on the risk of hepatocellular carcinoma(HCC)remains inconsistent.This study in-vestigated the influence of combined p53 Arg72Pro and MDM2 SNP309 on the risk of developing HCC in pa-tients with chronic hepatitis B virus infection,and evaluated the significance of the two combined SNPs on patient prognosis.In total,350 HCC patients,230 non-HCC patients,and 96 healthy controls were geno-typed for the p53 Arg72Pro and MDM2 SNP309.The combined p53 Pro/Pro and MDM2 G/G genotype was significantly associated with HCC risk(P=0.047).Mul-tivariate analysis indicated that combined p53 Pro/Pro and MDM2 G/G genotype was an independent factor affecting recurrence and survival(P<0.05).Patients with combined p53 Pro/Pro and MDM2 G/G genotypes had a poorer prognosis than other genotypes,P<0.01 for both disease-free survival(DFS)and overall survival(OS).DFS and OS rates also differed significantly be-tween Barcelona Clinic Liver Cancer(BCLC)stage A patients with combined p53 Pro/Pro and MDM2 G/G and other genotypes(P<0.05).Thus,the combined p53 Pro/Pro and MDM2 G/G genotype is associated with increased risk of developing HCC and is an independ-ent adverse prognostic indicator in early stage HCC.
