目的:克隆人CXCR3B基因,并构建含有该目的基因的真核表达载体,获得稳定表达人CXCR3B分子的基因转染细胞株L929-huCXCR3B。方法:采用PCR方法从pMD19-T/huCXCR3A质粒中扩增人CXCR3B基因,通过双酶切装入真核表达载体pIRES2-EGFP中;脂质体...目的:克隆人CXCR3B基因,并构建含有该目的基因的真核表达载体,获得稳定表达人CXCR3B分子的基因转染细胞株L929-huCXCR3B。方法:采用PCR方法从pMD19-T/huCXCR3A质粒中扩增人CXCR3B基因,通过双酶切装入真核表达载体pIRES2-EGFP中;脂质体法转染L929细胞,G418加压筛选阳性克隆;分别用RT-PCR方法与免疫荧光技术分析阳性克隆中人CXCR3B在mRNA和蛋白水平的表达。MTT分析基因转染细胞株L929-huCXCR3B在Mig(monokineinduced by IFN-γ,IFN-γ诱导的单核因子)作用下的增殖能力。结果:成功克隆了人CXCR3B基因并构建了真核表达载体pIRES2-EGFP/huCXCR3B,转染该载体后获得了稳定表达人CXCR3B的基因转染细胞株L929-huCXCR3B,膜表面CX-CR3B分子的阳性表达率为93%。该基因转染细胞与其配体Mig共培养24、48及72 h,抑制率分别为41.44%、44.01%和24.80%。结论:L929-huCXCR3B细胞株的建立为研究CXCR3B信号转导及制备相应的单克隆抗体(mAb)奠定了基础。展开更多
从口蹄疫病毒(foot-and-m ou th d isease v irus,FM DV)细胞培养物中提取总RNA,经一步法RT-PCR获得了长约230 bp的3B基因片段,将PCR产物连接到pM D-18T载体上并测序,用B amHⅠ与H indⅢ双酶切后,将3B基因融合到pGEX-KG载体上形成表达质...从口蹄疫病毒(foot-and-m ou th d isease v irus,FM DV)细胞培养物中提取总RNA,经一步法RT-PCR获得了长约230 bp的3B基因片段,将PCR产物连接到pM D-18T载体上并测序,用B amHⅠ与H indⅢ双酶切后,将3B基因融合到pGEX-KG载体上形成表达质粒pGEX-KG-3B,转化到BL 21(DE 3)中在27℃诱导表达,将表达产物进行SDS-PAGE和蛋白质斑点EL ISA。结果表明3B基因主要以可溶形式高效表达,表达产物具有免疫原性。以纯化的表达产物为抗原建立了间接酶联免疫吸附试验(I-EL ISA)。方阵滴定其最佳包被浓度是6.1μg/孔,最佳血清稀释倍数为1∶80,通过测定36份FM DV阴性血清,确定了该方法的阳性判定标准。结果表明,该方法特异性强、重复性好;用3B-EL ISA方法与美国联合生物医学公司(U B I)生产的合成肽检测试剂盒U B I○RFM DV N S-EL ISA对比检测44份血清样品,符合率为93.1%。证明3B-EL ISA方法可用于口蹄疫的鉴别诊断。展开更多
We investigated the expression of TIMP1, TIMP2, SPARC, VCAN, and CLEC3B genes, encoded matricellular proteins with pleiotropic functions, and glucose intolerance in obese male subjects with normal and impaired glucose...We investigated the expression of TIMP1, TIMP2, SPARC, VCAN, and CLEC3B genes, encoded matricellular proteins with pleiotropic functions, and glucose intolerance in obese male subjects with normal and impaired glucose tolerance. The purpose of this study was to examine the association between the gene expressions and glucose intolerance in obesity. The results indicate that obesity leads to significant increase of TIMP1, TIMP2, E2F1 and CLEC3B gene expressions in subcutaneous adipose tissue, especially TIMP2 gene. However, more significant increase of the expression of TIMP1 and TIMP2 was found in adipose tissue of obese patients with glucose intolerance. No significant changes were found in the expression of VCAN and SPARC genes in adipose tissue of obese subjects with normal glucose tolerance but increased in the group of obese subjects with glucose intolerance. At the same time, the E2F1 and CLEC3B gene expressions were decreased in adipose tissue of obese patients with glucose intolerance. Results of this study provide evidence that changes in the expression of genes encoded TIMP1, TIMP2, VCAN, SPARC, E2F1 and CLEC3B in subcutaneous adipose tissue of obese individuals associate with glucose intolerance.展开更多
文摘目的:克隆人CXCR3B基因,并构建含有该目的基因的真核表达载体,获得稳定表达人CXCR3B分子的基因转染细胞株L929-huCXCR3B。方法:采用PCR方法从pMD19-T/huCXCR3A质粒中扩增人CXCR3B基因,通过双酶切装入真核表达载体pIRES2-EGFP中;脂质体法转染L929细胞,G418加压筛选阳性克隆;分别用RT-PCR方法与免疫荧光技术分析阳性克隆中人CXCR3B在mRNA和蛋白水平的表达。MTT分析基因转染细胞株L929-huCXCR3B在Mig(monokineinduced by IFN-γ,IFN-γ诱导的单核因子)作用下的增殖能力。结果:成功克隆了人CXCR3B基因并构建了真核表达载体pIRES2-EGFP/huCXCR3B,转染该载体后获得了稳定表达人CXCR3B的基因转染细胞株L929-huCXCR3B,膜表面CX-CR3B分子的阳性表达率为93%。该基因转染细胞与其配体Mig共培养24、48及72 h,抑制率分别为41.44%、44.01%和24.80%。结论:L929-huCXCR3B细胞株的建立为研究CXCR3B信号转导及制备相应的单克隆抗体(mAb)奠定了基础。
文摘从口蹄疫病毒(foot-and-m ou th d isease v irus,FM DV)细胞培养物中提取总RNA,经一步法RT-PCR获得了长约230 bp的3B基因片段,将PCR产物连接到pM D-18T载体上并测序,用B amHⅠ与H indⅢ双酶切后,将3B基因融合到pGEX-KG载体上形成表达质粒pGEX-KG-3B,转化到BL 21(DE 3)中在27℃诱导表达,将表达产物进行SDS-PAGE和蛋白质斑点EL ISA。结果表明3B基因主要以可溶形式高效表达,表达产物具有免疫原性。以纯化的表达产物为抗原建立了间接酶联免疫吸附试验(I-EL ISA)。方阵滴定其最佳包被浓度是6.1μg/孔,最佳血清稀释倍数为1∶80,通过测定36份FM DV阴性血清,确定了该方法的阳性判定标准。结果表明,该方法特异性强、重复性好;用3B-EL ISA方法与美国联合生物医学公司(U B I)生产的合成肽检测试剂盒U B I○RFM DV N S-EL ISA对比检测44份血清样品,符合率为93.1%。证明3B-EL ISA方法可用于口蹄疫的鉴别诊断。
文摘We investigated the expression of TIMP1, TIMP2, SPARC, VCAN, and CLEC3B genes, encoded matricellular proteins with pleiotropic functions, and glucose intolerance in obese male subjects with normal and impaired glucose tolerance. The purpose of this study was to examine the association between the gene expressions and glucose intolerance in obesity. The results indicate that obesity leads to significant increase of TIMP1, TIMP2, E2F1 and CLEC3B gene expressions in subcutaneous adipose tissue, especially TIMP2 gene. However, more significant increase of the expression of TIMP1 and TIMP2 was found in adipose tissue of obese patients with glucose intolerance. No significant changes were found in the expression of VCAN and SPARC genes in adipose tissue of obese subjects with normal glucose tolerance but increased in the group of obese subjects with glucose intolerance. At the same time, the E2F1 and CLEC3B gene expressions were decreased in adipose tissue of obese patients with glucose intolerance. Results of this study provide evidence that changes in the expression of genes encoded TIMP1, TIMP2, VCAN, SPARC, E2F1 and CLEC3B in subcutaneous adipose tissue of obese individuals associate with glucose intolerance.
