目的分别构建绿色荧光蛋白(GFP)与氨基端或羧基端缺失突变乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)的重组表达载体,建立稳定表达GFP/HBxn、GFP/HBxc融合蛋白的HepG2细胞系,以进一步研究HBx缺失突变对其生物功能的影响。方法...目的分别构建绿色荧光蛋白(GFP)与氨基端或羧基端缺失突变乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)的重组表达载体,建立稳定表达GFP/HBxn、GFP/HBxc融合蛋白的HepG2细胞系,以进一步研究HBx缺失突变对其生物功能的影响。方法 PCR法分别扩增氨基端缺失50aa的HBx及羧基端缺失50aa的HBx基因,用HindⅢ和KpnⅠ双酶切定向插入pEGFP-C1相应酶切位点并转化宿主菌DH5α,双酶切鉴定pGFP/HBxn、pGFP/HBxc;脂质体转染法转染HepG2细胞,G418筛选出抗性细胞克隆,荧光显微镜下观察GFP的表达,挑选抗性克隆细胞扩大培养并传代。RT-PCR法、Western blot法检测转染细胞HBxn、HBxc基因、蛋白的表达。结果扩增的HBxn、Hbxc基因片段琼脂糖凝胶电泳显示符合预估大小。重组质粒pGFP/HBxn、pGFP/HBxc经HindⅢ和KpnⅠ双酶切后电泳,符合预估大小;转染pGFP/HBxn及pGFP/HBxc的HepG2细胞可见抗性细胞克隆形成,并可见阳性克隆细胞均有GFP表达。RT-PCR与Western blot法检测到HBxn、HBxc的表达。结论成功构建了GFP/HBxn、GFP/HBxc真核重组表达载体pGFP/HBXn、pGFP/HBxc,获得了稳定表达GFP/HBxn、GFP/HBxc融合蛋白的HepG2细胞系,为进一步研究HBx缺失突变对其生物功能的影响奠定了基础。展开更多
Objective To explore the relationship between a point mutation of codon 201 in deleted in colorectal carcinoma (DCC) gene and the biological behavior of colorectal carcinoma. Methods Tumor tissues and matched adjace...Objective To explore the relationship between a point mutation of codon 201 in deleted in colorectal carcinoma (DCC) gene and the biological behavior of colorectal carcinoma. Methods Tumor tissues and matched adjacent normal colon mucosa collected in 35 patients during surgical resection for colorectal carcinoma were analyzed. Forty normal colon mucosa tissues obtained by biopsy from patients who had neither colorectal tumor nor a family history of colorectal cancer during colonscopic examination were used as control. Codon 201 mutation was detected with allele specific PCR and a restriction enzyme digestion method. The tumors were reviewed as clinical data, tumor location, histology, metastasis, and pathological staging (Dukes classification). Results The frequency of mutation at codon 201 in tumor tissue and corresponding adjacent normal mucosa was 71.4% and 60%, respectively, and either of the rates was significantly higher than that of normal control(32.5%). The point mutation rate in tumor tissues did not differ from that in the corresponding normal adjacent tissues. Statistic analysis showed that the mutation rate had no relationship to the sex, age of the patients, the histological pattern, differentiation, and invasion depth of the tumors. However, 93.8% of the mutation rate in colorectal cancer with lymph node invasion and/or distant metastasis is significantly higher than 52.6% of mutant rate in colorectal cancer without lymph nodes invasion or metastasis (P<0 05). Conclusion The point mutation at codon 201 of DCC gene is an early genetic event in colorectal cancer, and play some role in invasion and metastasis of colorectal carcinoma. It may serve as a useful genetic marker for identifying higher risk patients with colorectal carcinoma.展开更多
Summary: Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots o...Summary: Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.展开更多
文摘目的分别构建绿色荧光蛋白(GFP)与氨基端或羧基端缺失突变乙型肝炎病毒X蛋白(hepatitis B virus X protein,HBx)的重组表达载体,建立稳定表达GFP/HBxn、GFP/HBxc融合蛋白的HepG2细胞系,以进一步研究HBx缺失突变对其生物功能的影响。方法 PCR法分别扩增氨基端缺失50aa的HBx及羧基端缺失50aa的HBx基因,用HindⅢ和KpnⅠ双酶切定向插入pEGFP-C1相应酶切位点并转化宿主菌DH5α,双酶切鉴定pGFP/HBxn、pGFP/HBxc;脂质体转染法转染HepG2细胞,G418筛选出抗性细胞克隆,荧光显微镜下观察GFP的表达,挑选抗性克隆细胞扩大培养并传代。RT-PCR法、Western blot法检测转染细胞HBxn、HBxc基因、蛋白的表达。结果扩增的HBxn、Hbxc基因片段琼脂糖凝胶电泳显示符合预估大小。重组质粒pGFP/HBxn、pGFP/HBxc经HindⅢ和KpnⅠ双酶切后电泳,符合预估大小;转染pGFP/HBxn及pGFP/HBxc的HepG2细胞可见抗性细胞克隆形成,并可见阳性克隆细胞均有GFP表达。RT-PCR与Western blot法检测到HBxn、HBxc的表达。结论成功构建了GFP/HBxn、GFP/HBxc真核重组表达载体pGFP/HBXn、pGFP/HBxc,获得了稳定表达GFP/HBxn、GFP/HBxc融合蛋白的HepG2细胞系,为进一步研究HBx缺失突变对其生物功能的影响奠定了基础。
文摘Objective To explore the relationship between a point mutation of codon 201 in deleted in colorectal carcinoma (DCC) gene and the biological behavior of colorectal carcinoma. Methods Tumor tissues and matched adjacent normal colon mucosa collected in 35 patients during surgical resection for colorectal carcinoma were analyzed. Forty normal colon mucosa tissues obtained by biopsy from patients who had neither colorectal tumor nor a family history of colorectal cancer during colonscopic examination were used as control. Codon 201 mutation was detected with allele specific PCR and a restriction enzyme digestion method. The tumors were reviewed as clinical data, tumor location, histology, metastasis, and pathological staging (Dukes classification). Results The frequency of mutation at codon 201 in tumor tissue and corresponding adjacent normal mucosa was 71.4% and 60%, respectively, and either of the rates was significantly higher than that of normal control(32.5%). The point mutation rate in tumor tissues did not differ from that in the corresponding normal adjacent tissues. Statistic analysis showed that the mutation rate had no relationship to the sex, age of the patients, the histological pattern, differentiation, and invasion depth of the tumors. However, 93.8% of the mutation rate in colorectal cancer with lymph node invasion and/or distant metastasis is significantly higher than 52.6% of mutant rate in colorectal cancer without lymph nodes invasion or metastasis (P<0 05). Conclusion The point mutation at codon 201 of DCC gene is an early genetic event in colorectal cancer, and play some role in invasion and metastasis of colorectal carcinoma. It may serve as a useful genetic marker for identifying higher risk patients with colorectal carcinoma.
文摘Summary: Mutations in the parkin gene have recently been identified in familial and isolated patients with early-onset Parkinson disease (PD) and that subregions between exon 2 and 4 of the parkin gene are hot spots of deletive mutations. To study the distribution of deletions in the parkin gene among variant subset patients with PD in China, and to explore the role of parkin gene in the pathogenesis of PD, 63 patients were divided into early onset and later onset groups. Exons 1-12 were amplified by PCR, templated by the genomic DNA of patients, and then the deletion distribution detected by agarose electrophoresis. Four patients were found to be carrier of exon deletions in 63 patients with PD. The location of the deletion was on exon 2 (1 case), exon 3 (2 cases) and exon 4 (1 case). All patients were belong to the group of early onset PD. The results showed that parkin gene deletion on exon 2, exon 3 and exon 4 found in Chinese population contributes partly to early onset PD.
