The large yellow croaker iridovirus(LYCIV)ATPase gene was cloned via PCR technique.Sequencing result of PCR product showed that it consisted of 720bp nuceleotides,encoding a protein of 240 amino acids in which a typic...The large yellow croaker iridovirus(LYCIV)ATPase gene was cloned via PCR technique.Sequencing result of PCR product showed that it consisted of 720bp nuceleotides,encoding a protein of 240 amino acids in which a typical ATP/GTP binding site motif was contained.Then the LYCIV ATPase gene was highly expressed in Hi5 insect cells using Baculovirus Bac To Bac expressioin system.SDS PAGE analysis indicated the expression product was about 31kD,and accounted for about 12% of the total cellular protein.Recombinant ATPase was purified by Ni NTA column.A good foundation had been laid down for studying of the functions of LYCIV ATPase.展开更多
文摘目的本研究旨在建立一种实时荧光定量PCR方法,用于检测猕猴三磷酸腺苷结合盒转运蛋白G2(adenosine triphosphate-binding cassette transporter protein G2,ABCG2)mRNA的基因转录水平。方法使用NCBI上GenBank数据库猕猴(Macaca mulatta)的ABCG2核苷酸序列号NM_001032919.1及内参GAPDH核苷酸序列号NM_001195426.1,借助Primer premier 5.0软件设计PCR引物。提取猕猴新鲜肾组织的总RNA,并反转录合成cDNA。接着,利用PCR引物进行实时荧光定量PCR扩增,并根据反应体系中荧光的变化情况定量分析ABCG2的mRNA相对表达水平。结果PCR产物测序结果显示,扩增的ABCG2和GAPDH核苷酸序列与NCBI上猕猴的序列同源性分别为90.91%和91.14%。ABCG2和GAPDH的扩增效率均达到80%~120%,实时荧光定量PCR标准曲线的熔解曲线为单峰,R2接近1。结论本研究建立的检测猕猴ABCG2 mRNA实时荧光定量检测方法,为研究高尿酸血症的发病机制以及新药开发奠定基础。
文摘The large yellow croaker iridovirus(LYCIV)ATPase gene was cloned via PCR technique.Sequencing result of PCR product showed that it consisted of 720bp nuceleotides,encoding a protein of 240 amino acids in which a typical ATP/GTP binding site motif was contained.Then the LYCIV ATPase gene was highly expressed in Hi5 insect cells using Baculovirus Bac To Bac expressioin system.SDS PAGE analysis indicated the expression product was about 31kD,and accounted for about 12% of the total cellular protein.Recombinant ATPase was purified by Ni NTA column.A good foundation had been laid down for studying of the functions of LYCIV ATPase.
