本研究旨在克隆小尾寒羊钙粘和蛋白1(Calcium and integrin binding protein 1,CIB1)基因cDNA全长,并在原核载体中表达。参照已发表的CIB1基因的核苷酸序列,设计了1对特异性引物,采用RT-PCR法,从小尾寒羊睾丸组织中扩增获得CIB1基因全长...本研究旨在克隆小尾寒羊钙粘和蛋白1(Calcium and integrin binding protein 1,CIB1)基因cDNA全长,并在原核载体中表达。参照已发表的CIB1基因的核苷酸序列,设计了1对特异性引物,采用RT-PCR法,从小尾寒羊睾丸组织中扩增获得CIB1基因全长cDNA。将其克隆到pMD19-T载体,并进行测序分析。将该基因编码区重组于融合表达质粒pET32a中,构建了重组原核表达载体(pET32a-CIB1)。将其转化到BL21(DE3)pLysS宿主菌中,用IPTG进行诱导表达。结果表明,克隆的CIB1基因cDNA与GenBank上登录的牛、猪、猕猴、人、小鼠、大鼠、黑猩猩等动物该基因序列的同源性达90%以上,编码氨基酸的同源性在93%以上,并且该序列包含有完整的开放阅读框,大小为576 bp。实现了高效特异性融合表达,表达产物的分子质量约为38 ku。本研究结果为进一步研究CIB1蛋白功能打下良好的基础。展开更多
Background: Uveal melanoma (UVM) is the most common primary intraocular tumor in adults. However, identification of the effective biomarker for the diagnosis and treatment of UVM remains to be explored. Calcium and in...Background: Uveal melanoma (UVM) is the most common primary intraocular tumor in adults. However, identification of the effective biomarker for the diagnosis and treatment of UVM remains to be explored. Calcium and integrin-binding protein 1 (CIB1) is emerging as an important factor in tumor progression. Purpose: To determine the contribution of CIB1 in the diagnosis of UVM. Method: Immunohistochemical staining is used to detect the CIB1 expression level, while Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and UALCAN online tools were used to analyze patient survival and CIB1 correlation genes in UVM. Integrative analysis using STRING and GeneMANIA predicted the correlated genes with CIB1 in UVM. Results: CIB1 expression level in UVM was significantly enhanced when compared with that in paracancerous tissues. A higher CIB1 expression level resulted in a significantly worse disease-free survival as well as overall survival. Moreover, the survival probability of patients was associated with body weight and gender of the patients with UVM. The correlated genes with CIB1 in UVM, and the similarity of the genes in UVM expression and survival heatmap were verified. Furthermore, Gene ontology enrichment analysis revealed that CIB1 and its correlated genes are significantly enriched in ITGA2B-ITGB3-CIB1 complex, regulation of intracellular protein transport and regulation of ion transport. Conclusions: Our novel findings suggested that CIB1 might be a potential diagnostic predictor for UVM, and might contribute to the potential strategy for UVM treatment by targeting CIB1.展开更多
Objective To construct effective RNA-interference plasmids targeting mouse HIF-la gene and testify their effects and specificities in interfering HIF-1α expression. Methods Three RNA-interference plasmids targeting m...Objective To construct effective RNA-interference plasmids targeting mouse HIF-la gene and testify their effects and specificities in interfering HIF-1α expression. Methods Three RNA-interference plasmids targeting mouse HIF- 1α gene, pBS/U6/HIF-1α-siRNAI-Ⅲ, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1α in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1αi-II in SH-SY5Y cell line was further investigated. Results All the three RNA-interference plasmids, especially pBS/U6/HIF1αi-Ⅱ, showed significant inhibition in HIF-1α expression in 293T cell line. pBS/U6/HIF1αi-Ⅱ could also inhibit HIF-1α expression in SH-SY5Y cell line, in a dosedependent way. Conclusion Plasmid pBS/U6/HIF1αi-Ⅱ constructed in our study can effectively and specifically inhibit HIF- 1α expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/ HIF1αi-Ⅱ plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and i schemia development.展开更多
文摘本研究旨在克隆小尾寒羊钙粘和蛋白1(Calcium and integrin binding protein 1,CIB1)基因cDNA全长,并在原核载体中表达。参照已发表的CIB1基因的核苷酸序列,设计了1对特异性引物,采用RT-PCR法,从小尾寒羊睾丸组织中扩增获得CIB1基因全长cDNA。将其克隆到pMD19-T载体,并进行测序分析。将该基因编码区重组于融合表达质粒pET32a中,构建了重组原核表达载体(pET32a-CIB1)。将其转化到BL21(DE3)pLysS宿主菌中,用IPTG进行诱导表达。结果表明,克隆的CIB1基因cDNA与GenBank上登录的牛、猪、猕猴、人、小鼠、大鼠、黑猩猩等动物该基因序列的同源性达90%以上,编码氨基酸的同源性在93%以上,并且该序列包含有完整的开放阅读框,大小为576 bp。实现了高效特异性融合表达,表达产物的分子质量约为38 ku。本研究结果为进一步研究CIB1蛋白功能打下良好的基础。
文摘Background: Uveal melanoma (UVM) is the most common primary intraocular tumor in adults. However, identification of the effective biomarker for the diagnosis and treatment of UVM remains to be explored. Calcium and integrin-binding protein 1 (CIB1) is emerging as an important factor in tumor progression. Purpose: To determine the contribution of CIB1 in the diagnosis of UVM. Method: Immunohistochemical staining is used to detect the CIB1 expression level, while Gene Expression Profiling Interactive Analysis 2 (GEPIA2) and UALCAN online tools were used to analyze patient survival and CIB1 correlation genes in UVM. Integrative analysis using STRING and GeneMANIA predicted the correlated genes with CIB1 in UVM. Results: CIB1 expression level in UVM was significantly enhanced when compared with that in paracancerous tissues. A higher CIB1 expression level resulted in a significantly worse disease-free survival as well as overall survival. Moreover, the survival probability of patients was associated with body weight and gender of the patients with UVM. The correlated genes with CIB1 in UVM, and the similarity of the genes in UVM expression and survival heatmap were verified. Furthermore, Gene ontology enrichment analysis revealed that CIB1 and its correlated genes are significantly enriched in ITGA2B-ITGB3-CIB1 complex, regulation of intracellular protein transport and regulation of ion transport. Conclusions: Our novel findings suggested that CIB1 might be a potential diagnostic predictor for UVM, and might contribute to the potential strategy for UVM treatment by targeting CIB1.
基金supported by the Innovative Foundation of the Third Military Medical University (N0.2007XG42)
文摘Objective To construct effective RNA-interference plasmids targeting mouse HIF-la gene and testify their effects and specificities in interfering HIF-1α expression. Methods Three RNA-interference plasmids targeting mouse HIF- 1α gene, pBS/U6/HIF-1α-siRNAI-Ⅲ, were constructed and identified using double digestion method in the present study. RT-PCR, immunostaining and western blotting were employed to detect the expression alterations of HIF-1α in 293T cells following transfections of the three plasmids, respectively. The interference effect of pBS/U6/HIF1αi-II in SH-SY5Y cell line was further investigated. Results All the three RNA-interference plasmids, especially pBS/U6/HIF1αi-Ⅱ, showed significant inhibition in HIF-1α expression in 293T cell line. pBS/U6/HIF1αi-Ⅱ could also inhibit HIF-1α expression in SH-SY5Y cell line, in a dosedependent way. Conclusion Plasmid pBS/U6/HIF1αi-Ⅱ constructed in our study can effectively and specifically inhibit HIF- 1α expression, and its role in neural tube development and dysfunction will be further investigated. Construct of pBS/U6/ HIF1αi-Ⅱ plasmid will provide a useful tool to study the role of HIF-1 pathway in embryogenesis, oncogenesis and i schemia development.
