为了建立快速定量检测鸡Caspase-1基因的方法,本研究根据鸡Caspase-1基因序列设计特异性检测引物,PCR扩增Caspase-1基因片段,构建pMD-Caspase-1重组质粒。以该重组质粒为标准品进行荧光定量PCR扩增,建立标准曲线,并通过反应条件优化、...为了建立快速定量检测鸡Caspase-1基因的方法,本研究根据鸡Caspase-1基因序列设计特异性检测引物,PCR扩增Caspase-1基因片段,构建pMD-Caspase-1重组质粒。以该重组质粒为标准品进行荧光定量PCR扩增,建立标准曲线,并通过反应条件优化、特异性、敏感性及重复性等试验,建立了鸡Caspase-1基因SYBR Green I荧光定量PCR检测方法。结果显示,本研究建立的该荧光定量PCR方法在重组质粒标准品为1.0×10^(3)拷贝/μL~1.0×10^(8)拷贝/μL范围内具有良好的线性关系;该荧光定量PCR方法对Caspase-1基因的扩增具有较强的特异性;对重组质粒标准品的检测下限为1.0×10^(2)拷贝/μL,比常规PCR敏感100倍;批内和批间变异系数分别在0.51%~4.41%和0.37%~0.88%,重复性好。利用所建立的方法可以定量检测致病性禽腺病毒血清4型感染鸡不同组织中Caspase-1的转录水平。本研究方法的建立将为研究鸡Caspase-1在免疫反应和疫病发展过程中的作用提供技术支持。展开更多
Carboxylesterase 1(CES1), one of the most abundant serine hydrolases in mammals, has drawn much attentions in recent years, owing to this enzyme involves in many physiological processes via hydrolysis of both endogeno...Carboxylesterase 1(CES1), one of the most abundant serine hydrolases in mammals, has drawn much attentions in recent years, owing to this enzyme involves in many physiological processes via hydrolysis of both endogenous esters and xenobiotic esters. Herein, to real-time monitor the activities of CES1 in various biological systems, a practical and iso form-specific fluorescent probe was developed on the basis of the substrate preference of CES1, as well as the structural and optical properties of BODIPY dyes. After screening of a panel of BODIPY ester derivatives, probe 1 displayed the best combination of specificity,sensitivity, enzymatic kinetics and applicability for monitoring CES1 activities in real samples. This probe was successfully used to detect CESl activities in several biological systems including tissue preparations,living cells, tissue slices and zebrafish. Furthermore, the biomedical applications of probe 1 for screening of CES1 inhibitors were also demonstrated using tissue preparations or living cells as enzyme sources. In summary, a practical and broadly applicable tool for real-time monitoring CES1 in biological systems was developed and well-characterized, which held great promise for further investigations on CES1-associated drug discovery, clinical practice and fundamental research.展开更多
目的:建立红色荧光蛋白(red fluorescent prote in,RFP)标记的单纯疱疹病毒1型TK基因(Herpessimp lex virus thym id ine k inase,HSV-TK)的基因转移系统,并验证其有效性。方法:以pBLuescript-TK为模板,设计引物,将聚合酶链反应(PCR)扩...目的:建立红色荧光蛋白(red fluorescent prote in,RFP)标记的单纯疱疹病毒1型TK基因(Herpessimp lex virus thym id ine k inase,HSV-TK)的基因转移系统,并验证其有效性。方法:以pBLuescript-TK为模板,设计引物,将聚合酶链反应(PCR)扩增到的TK基因克隆到真核表达载体pD sRed2-N1中,获得以红色荧光蛋白为报告基因的重组质粒pD sRed2-N1-TK,利用脂质体转染体外培养的HepG2细胞,在活细胞状态下用荧光显微镜直接观察pD sRed2-N1-TK在细胞中的分布和定位,用RT-PCR方法验证其mRNA和蛋白质的表达。结果:空载体pD sRed2-N1转染组中,HepG2细胞内红色荧光呈弥散分布;重组质粒pD sRed2-N1-TK转染组中,红色荧光集中在细胞核中,随着表达量的增加,红色荧光在细胞核中聚集成团块状,2周后稳定表达于细胞浆中;RT-PCR的结果表明,HSV-TK基因在重组质粒转染组中有表达;丙氧鸟苷(GCV)杀伤效应确证了TK基因表达产物胸苷激酶的活性。结论:pD sRed2-N1-TK融合基因真核表达载体在真核细胞HepG2中获得了表达,细胞所表达的融合蛋白具有TK和RFP的双重活性,可用荧光显微镜直接观察其表达情况及亚细胞定位,为HSV-TK/GCV治疗肿瘤研究提供了实用而又方便的工具。展开更多
目的构建含有金属蛋白酶组织抑制因子-1(TIMP-1)的重组质粒并以其为模板建立荧光定量聚合酶链反应(PCR)技术检测TIMP-1的标准曲线,为荧光定量PCR准确检测TIMP-1奠定基础.方法提取大鼠星状细胞系HSC-T6的mRNA,经RT-PCR扩增TIMP-1基因后与...目的构建含有金属蛋白酶组织抑制因子-1(TIMP-1)的重组质粒并以其为模板建立荧光定量聚合酶链反应(PCR)技术检测TIMP-1的标准曲线,为荧光定量PCR准确检测TIMP-1奠定基础.方法提取大鼠星状细胞系HSC-T6的mRNA,经RT-PCR扩增TIMP-1基因后与pGMT-Vector 连接,转化到大肠杆菌DH5α,以筛选得到的阳性克隆质粒作为标准品进行梯度稀释,分别用Taqman荧光探针技术和SYBR Green I荧光染料技术进行荧光定量PCR检测,建立标准曲线,并对两者结果进行了对比.同时进行普通PCR检测,与荧光定量PCR方法检测结果进行比较.结果重组质粒经PCR扩增及序列测定,表明pGMT-TIMP-1基因已成功克隆.以不同稀释水平的标准品质粒进行荧光定量PCR扩增, 应用Taqman荧光探针技术建立的标准曲线的线性检测范围为7×104～7×108拷贝,检测灵敏度为7×104拷贝;应用SYBR Green I荧光染料技术的线性检测范围为7×106～7×108拷贝,检测灵敏度为7×106拷贝,且熔解曲线显示出现非特异性扩增;两种技术相对于普通PCR技术均具有定量功能.结论所构建的pGMT-TIMP-1基因荧光定量PCR检测标准品应用Taqman荧光探针技术建立的标准曲线线性关系好, 灵敏度和特异性高,准确可靠,此方法可作为荧光定量PCR检测TIMP-1基因的标准方法.展开更多
文摘为了建立快速定量检测鸡Caspase-1基因的方法,本研究根据鸡Caspase-1基因序列设计特异性检测引物,PCR扩增Caspase-1基因片段,构建pMD-Caspase-1重组质粒。以该重组质粒为标准品进行荧光定量PCR扩增,建立标准曲线,并通过反应条件优化、特异性、敏感性及重复性等试验,建立了鸡Caspase-1基因SYBR Green I荧光定量PCR检测方法。结果显示,本研究建立的该荧光定量PCR方法在重组质粒标准品为1.0×10^(3)拷贝/μL~1.0×10^(8)拷贝/μL范围内具有良好的线性关系;该荧光定量PCR方法对Caspase-1基因的扩增具有较强的特异性;对重组质粒标准品的检测下限为1.0×10^(2)拷贝/μL,比常规PCR敏感100倍;批内和批间变异系数分别在0.51%~4.41%和0.37%~0.88%,重复性好。利用所建立的方法可以定量检测致病性禽腺病毒血清4型感染鸡不同组织中Caspase-1的转录水平。本研究方法的建立将为研究鸡Caspase-1在免疫反应和疫病发展过程中的作用提供技术支持。
基金supported by the National Natural Science Foundation of China(Nos. 21572029, 31600641,81703604, 81773687, 81672961 and 81573501)the National Key Research and Development Program of China (Nos. 2017YFC1700200 and 2017YFC1702000)+2 种基金Program of Shanghai Academic/Technology Research Leader(No. 18XD1403600)Shuguang Program (No. 18SG40)supported by Shanghai Education Development Foundation and Shanghai Municipal Education Commission and the Innovative Entrepreneurship Program of High-level Talents in Dalian (Nos. 2016RQ025 and 2017RQ121)
文摘Carboxylesterase 1(CES1), one of the most abundant serine hydrolases in mammals, has drawn much attentions in recent years, owing to this enzyme involves in many physiological processes via hydrolysis of both endogenous esters and xenobiotic esters. Herein, to real-time monitor the activities of CES1 in various biological systems, a practical and iso form-specific fluorescent probe was developed on the basis of the substrate preference of CES1, as well as the structural and optical properties of BODIPY dyes. After screening of a panel of BODIPY ester derivatives, probe 1 displayed the best combination of specificity,sensitivity, enzymatic kinetics and applicability for monitoring CES1 activities in real samples. This probe was successfully used to detect CESl activities in several biological systems including tissue preparations,living cells, tissue slices and zebrafish. Furthermore, the biomedical applications of probe 1 for screening of CES1 inhibitors were also demonstrated using tissue preparations or living cells as enzyme sources. In summary, a practical and broadly applicable tool for real-time monitoring CES1 in biological systems was developed and well-characterized, which held great promise for further investigations on CES1-associated drug discovery, clinical practice and fundamental research.
文摘目的构建含有金属蛋白酶组织抑制因子-1(TIMP-1)的重组质粒并以其为模板建立荧光定量聚合酶链反应(PCR)技术检测TIMP-1的标准曲线,为荧光定量PCR准确检测TIMP-1奠定基础.方法提取大鼠星状细胞系HSC-T6的mRNA,经RT-PCR扩增TIMP-1基因后与pGMT-Vector 连接,转化到大肠杆菌DH5α,以筛选得到的阳性克隆质粒作为标准品进行梯度稀释,分别用Taqman荧光探针技术和SYBR Green I荧光染料技术进行荧光定量PCR检测,建立标准曲线,并对两者结果进行了对比.同时进行普通PCR检测,与荧光定量PCR方法检测结果进行比较.结果重组质粒经PCR扩增及序列测定,表明pGMT-TIMP-1基因已成功克隆.以不同稀释水平的标准品质粒进行荧光定量PCR扩增, 应用Taqman荧光探针技术建立的标准曲线的线性检测范围为7×104～7×108拷贝,检测灵敏度为7×104拷贝;应用SYBR Green I荧光染料技术的线性检测范围为7×106～7×108拷贝,检测灵敏度为7×106拷贝,且熔解曲线显示出现非特异性扩增;两种技术相对于普通PCR技术均具有定量功能.结论所构建的pGMT-TIMP-1基因荧光定量PCR检测标准品应用Taqman荧光探针技术建立的标准曲线线性关系好, 灵敏度和特异性高,准确可靠,此方法可作为荧光定量PCR检测TIMP-1基因的标准方法.
