本研究旨在对水牛固醇携带蛋白2(sterol carrier protein 2,SCP2)基因进行克隆及生物信息学分析,并检测其在水牛不同组织中的表达。以黄牛SCP2基因(登录号:NM_001033990.3)为种子序列成功克隆了水牛SCP2基因完整CDS区,该序列长1 632bp,...本研究旨在对水牛固醇携带蛋白2(sterol carrier protein 2,SCP2)基因进行克隆及生物信息学分析,并检测其在水牛不同组织中的表达。以黄牛SCP2基因(登录号:NM_001033990.3)为种子序列成功克隆了水牛SCP2基因完整CDS区,该序列长1 632bp,可编码543个氨基酸;其与黄牛、绵羊、山羊、白鲸、人、家犬和家猫的同源性分别为95.9%、93.4%、92.4%、89.4%、88.3%、86.3%和86.9%,说明SCP2基因CDS区在不同物种间具有较高的保守性。聚类分析则表明水牛与黄牛的分子进化关系最近;氨基酸序列分析表明,SCP2蛋白的分子式为C_(2602)H_(4131)N_(709)O_(774)S_(298),分子质量为58.66ku,理论等电点(pI)为8.59,不稳定系数为27.94,平均亲水性为-0.215,属于碱性、稳定、亲水蛋白质;二级结构分析表明水牛SCP2蛋白由α-螺旋、无规则卷曲和延伸链构成,其中α-螺旋占35.54%,无规则卷曲占48.99%,延伸链占15.47%,与三级结构预测结果一致;亚细胞定位分析表明,水牛SCP2蛋白分布在细胞质(43.5%)、过氧化物酶体(21.7%)、线粒体(17.4%)、细胞核(13.0%)和细胞骨架(4.4%);跨膜结构和信号肽预测分析表明,水牛SCP2蛋白不含跨膜结构和信号肽;磷酸化位点分析发现,水牛SCP2蛋白有13个Ser、3个Thr和2个Tyr可能成为蛋白激酶磷酸化位点;蛋白质结合位点预测结果显示,水牛SCP2蛋白含有12个蛋白质结合位点和1个多核苷酸结合位点;实时荧光定量PCR结果表明,水牛SCP2基因在肝脏中表达量最高,其他组织中表达量从高到低依次为乳腺、淋巴、肾脏、大肠、胃、肺脏、脾脏、卵巢、垂体、大脑和心脏。本试验为今后进一步探讨SCP2基因的功能奠定了基础。展开更多
目的:构建携带白蛋白启动子SCP2基因腺病毒载体,研究其与胆固醇结石形成的关系。方法:(1)利用RT-PCR技术克隆小鼠SCP2基因,在其上游接入白蛋白(ALB)启动子,下游连接绿色荧光报告基因(EGFP),构建穿梭质粒pDC312-ALB-SCP2-IRES2-EGFP;(2)...目的:构建携带白蛋白启动子SCP2基因腺病毒载体,研究其与胆固醇结石形成的关系。方法:(1)利用RT-PCR技术克隆小鼠SCP2基因,在其上游接入白蛋白(ALB)启动子,下游连接绿色荧光报告基因(EGFP),构建穿梭质粒pDC312-ALB-SCP2-IRES2-EGFP;(2)采用Ad Max TM Adenoviru5 Vector系统包装病毒,CsCl法纯化病毒、TCID50法测定滴度;(3)重组腺病毒感染小鼠hepa-1-6细胞,实时定量PCR检测mRNA的表达;Western印迹检测SCP2蛋白表达情况;结果:成功构建携带白蛋白启动子SCP2基因腺病毒载体;当SCP2基因过表达时,CYP7a1基因表达下调,(t=3.97,P<0.05)HMGCR基因基因表达上调,(t=3.23,P<0.05)。结论:当SCP2基因过表达时引起HMGCR、CYP7a1基因转录水平的变化,提示SCP2基因可能通过影响胆固醇和胆汁酸的代谢而参与胆固醇结石的形成。展开更多
BACKGROUND: Hypersecretion of biliary cholesterol is believed to be one of the important causes of lithogenic bile. Sterol carrier protein-2 ( SCP2 ) participates in choles- terol trafficking and metabolism and may pl...BACKGROUND: Hypersecretion of biliary cholesterol is believed to be one of the important causes of lithogenic bile. Sterol carrier protein-2 ( SCP2 ) participates in choles- terol trafficking and metabolism and may play a key role in cholesterol gallstone formation. This study was undertaken to investigate the expression of liver SCP2 mRNA in pa- tients with cholesterol gallstone and those patients with non-cholesterol gallstone. METHODS: The expression of liver SCP2 mRNA was studi- ed in 36 patients with cholesterol gallstone and 30 patients with non-cholesterol gallstone by reverse transcription-poly- merase chain reaction (RT-PCR). RESULT: The expression of SCP2 mRNA was increased more significantly in patients with cholesterol gallstone than in patients with non-cholesterol gallstone. CONCLUSION: The SCP2 gene was overexpressed in pa- tients with cholesterol gallstone, indicating that SCP2 may be one of the important causes of cholesterol gallstone.展开更多
文摘本研究旨在对水牛固醇携带蛋白2(sterol carrier protein 2,SCP2)基因进行克隆及生物信息学分析,并检测其在水牛不同组织中的表达。以黄牛SCP2基因(登录号:NM_001033990.3)为种子序列成功克隆了水牛SCP2基因完整CDS区,该序列长1 632bp,可编码543个氨基酸;其与黄牛、绵羊、山羊、白鲸、人、家犬和家猫的同源性分别为95.9%、93.4%、92.4%、89.4%、88.3%、86.3%和86.9%,说明SCP2基因CDS区在不同物种间具有较高的保守性。聚类分析则表明水牛与黄牛的分子进化关系最近;氨基酸序列分析表明,SCP2蛋白的分子式为C_(2602)H_(4131)N_(709)O_(774)S_(298),分子质量为58.66ku,理论等电点(pI)为8.59,不稳定系数为27.94,平均亲水性为-0.215,属于碱性、稳定、亲水蛋白质;二级结构分析表明水牛SCP2蛋白由α-螺旋、无规则卷曲和延伸链构成,其中α-螺旋占35.54%,无规则卷曲占48.99%,延伸链占15.47%,与三级结构预测结果一致;亚细胞定位分析表明,水牛SCP2蛋白分布在细胞质(43.5%)、过氧化物酶体(21.7%)、线粒体(17.4%)、细胞核(13.0%)和细胞骨架(4.4%);跨膜结构和信号肽预测分析表明,水牛SCP2蛋白不含跨膜结构和信号肽;磷酸化位点分析发现,水牛SCP2蛋白有13个Ser、3个Thr和2个Tyr可能成为蛋白激酶磷酸化位点;蛋白质结合位点预测结果显示,水牛SCP2蛋白含有12个蛋白质结合位点和1个多核苷酸结合位点;实时荧光定量PCR结果表明,水牛SCP2基因在肝脏中表达量最高,其他组织中表达量从高到低依次为乳腺、淋巴、肾脏、大肠、胃、肺脏、脾脏、卵巢、垂体、大脑和心脏。本试验为今后进一步探讨SCP2基因的功能奠定了基础。
文摘目的:构建携带白蛋白启动子SCP2基因腺病毒载体,研究其与胆固醇结石形成的关系。方法:(1)利用RT-PCR技术克隆小鼠SCP2基因,在其上游接入白蛋白(ALB)启动子,下游连接绿色荧光报告基因(EGFP),构建穿梭质粒pDC312-ALB-SCP2-IRES2-EGFP;(2)采用Ad Max TM Adenoviru5 Vector系统包装病毒,CsCl法纯化病毒、TCID50法测定滴度;(3)重组腺病毒感染小鼠hepa-1-6细胞,实时定量PCR检测mRNA的表达;Western印迹检测SCP2蛋白表达情况;结果:成功构建携带白蛋白启动子SCP2基因腺病毒载体;当SCP2基因过表达时,CYP7a1基因表达下调,(t=3.97,P<0.05)HMGCR基因基因表达上调,(t=3.23,P<0.05)。结论:当SCP2基因过表达时引起HMGCR、CYP7a1基因转录水平的变化,提示SCP2基因可能通过影响胆固醇和胆汁酸的代谢而参与胆固醇结石的形成。
基金This study was supported by a grant from the Health Bureau of Tianjin, China(No. 00kyzd8).
文摘BACKGROUND: Hypersecretion of biliary cholesterol is believed to be one of the important causes of lithogenic bile. Sterol carrier protein-2 ( SCP2 ) participates in choles- terol trafficking and metabolism and may play a key role in cholesterol gallstone formation. This study was undertaken to investigate the expression of liver SCP2 mRNA in pa- tients with cholesterol gallstone and those patients with non-cholesterol gallstone. METHODS: The expression of liver SCP2 mRNA was studi- ed in 36 patients with cholesterol gallstone and 30 patients with non-cholesterol gallstone by reverse transcription-poly- merase chain reaction (RT-PCR). RESULT: The expression of SCP2 mRNA was increased more significantly in patients with cholesterol gallstone than in patients with non-cholesterol gallstone. CONCLUSION: The SCP2 gene was overexpressed in pa- tients with cholesterol gallstone, indicating that SCP2 may be one of the important causes of cholesterol gallstone.
