目的克隆TNF家族B细胞激活因子（B cell activating factor to the TNF family,BAFF）胞外区134～285（sBAFF134-285）氨基酸残基段缺失突变体（s△BAFF）的cDNA,并进行表达及纯化.方法以构建的重组质粒pUC19/sBAFF为模板,采用一步反向...目的克隆TNF家族B细胞激活因子（B cell activating factor to the TNF family,BAFF）胞外区134～285（sBAFF134-285）氨基酸残基段缺失突变体（s△BAFF）的cDNA,并进行表达及纯化.方法以构建的重组质粒pUC19/sBAFF为模板,采用一步反向PCR法,扩增缺失编码sBAFF的142～160位氨基酸的核苷酸序列.经测序证实后,克隆入原核表达载体pQE-80L.经IPTG诱导表达,SDS-PAGE和Western blot检测表达产物,Ni2＋-NTA柱层析纯化目的蛋白.结果经一步反向PCR扩增后得到401 bp的DNA片段,该片段序列与GenBank报道的编码人△BAFF的胞外区（s△BAFF）cDNA序列一致.含s△BAFF的表达载体在大肠杆菌中可表达出相对分子质量为18 000的蛋白质并以包涵体的形式存在,经Ni2＋-NTA柱层析纯化后得到高纯度的目的蛋白.结论已成功地制备出人s△BAFF蛋白,为其功能的研究创造了条件.展开更多
Endothelin(ET)is the most potent mammalian vasoconstrictor identified to data.As a pathogenic factor,ET is involved in the genesis of many diseases.In this study,a pair of primers was designed and synthesized accordin...Endothelin(ET)is the most potent mammalian vasoconstrictor identified to data.As a pathogenic factor,ET is involved in the genesis of many diseases.In this study,a pair of primers was designed and synthesized according to the human ETB receptor gene (hETBR)sequence.A 394bp of DNA fragment was amplified by polymerase chain reaction(PCR) and labeled with α 32 P CTP using Random Primer Labeling method.With this probe,rabbit lung cDNA library was screened by in situ hybridization and 11 positive clones were identified.Sequencing result showed that a complete reading frame of rabbit ETB receptor(rETBR)cDNA could be produced from three positive clones of eleven.By a series of subcloning,a recombinant plasmid including the 1326 bp of rETBR coding sequences,named pBlu Script rETBR,was constructed.The deduced amino acid sequence indicated that the rETBR is 441 residues in length,with an expected molecular mass of approximately 49.44 kD.N terminal 18 residues is the potential signal peptide (Score=11.11)and therefore the molecular mass of mature rETBR is 47.65 kD with 423 amino acid residues.Analysis of the rETBR hydropathy profile indicates the presence of seven hydrophobic regions,putative transmembrane domains.Potential N glycosylation sites are the 60th and the 118 th.The structure exhibits a significant sequence and topographical similarity with G protein coupled receptors.展开更多
Protein kinase B (PKB) is a member of the second messenger-regulated subfamily of protein kinases, and plays a key role in cell-cycle regulation, glucose uptake and promotion of cell differentiation. Evidence shows th...Protein kinase B (PKB) is a member of the second messenger-regulated subfamily of protein kinases, and plays a key role in cell-cycle regulation, glucose uptake and promotion of cell differentiation. Evidence shows that PKB undergoes activation in some human tumors and is involved in Ras pathway, which implies that PKB can trigger a pathway to induce oncogenic transformation. A nucleotide sequence of mouse Pkbywas used as a probe to screen homolog in a human liver cDNA library. A fragment of 1998 bp containing a 1440 bp ORF encoding 479 amino acid residues was obtained. Then the 3’-terminal of this fragment was extended to 2788 bp by ’electronic walking’ screening, and the extended fragment was confirmed by PCR amplification. The protein deduced by the gene had a high identity of 83% and 78% to the human PKBα and β, respectively, and was designated as human PKBγ. Northern hybridization detected two equally expressed transcripts of 8.5 and 6.5 kb in length in all 16 human tissues tested, with the展开更多
文摘Endothelin(ET)is the most potent mammalian vasoconstrictor identified to data.As a pathogenic factor,ET is involved in the genesis of many diseases.In this study,a pair of primers was designed and synthesized according to the human ETB receptor gene (hETBR)sequence.A 394bp of DNA fragment was amplified by polymerase chain reaction(PCR) and labeled with α 32 P CTP using Random Primer Labeling method.With this probe,rabbit lung cDNA library was screened by in situ hybridization and 11 positive clones were identified.Sequencing result showed that a complete reading frame of rabbit ETB receptor(rETBR)cDNA could be produced from three positive clones of eleven.By a series of subcloning,a recombinant plasmid including the 1326 bp of rETBR coding sequences,named pBlu Script rETBR,was constructed.The deduced amino acid sequence indicated that the rETBR is 441 residues in length,with an expected molecular mass of approximately 49.44 kD.N terminal 18 residues is the potential signal peptide (Score=11.11)and therefore the molecular mass of mature rETBR is 47.65 kD with 423 amino acid residues.Analysis of the rETBR hydropathy profile indicates the presence of seven hydrophobic regions,putative transmembrane domains.Potential N glycosylation sites are the 60th and the 118 th.The structure exhibits a significant sequence and topographical similarity with G protein coupled receptors.
文摘Protein kinase B (PKB) is a member of the second messenger-regulated subfamily of protein kinases, and plays a key role in cell-cycle regulation, glucose uptake and promotion of cell differentiation. Evidence shows that PKB undergoes activation in some human tumors and is involved in Ras pathway, which implies that PKB can trigger a pathway to induce oncogenic transformation. A nucleotide sequence of mouse Pkbywas used as a probe to screen homolog in a human liver cDNA library. A fragment of 1998 bp containing a 1440 bp ORF encoding 479 amino acid residues was obtained. Then the 3’-terminal of this fragment was extended to 2788 bp by ’electronic walking’ screening, and the extended fragment was confirmed by PCR amplification. The protein deduced by the gene had a high identity of 83% and 78% to the human PKBα and β, respectively, and was designated as human PKBγ. Northern hybridization detected two equally expressed transcripts of 8.5 and 6.5 kb in length in all 16 human tissues tested, with the
