Objective To investigate the biological function of NS5ABP37 and to look for proteins interacting with NS5ABP37 protein in hepatocytes.Methods We constructed bait plasmid expressing NS5ABP37 protein of hepatitis C vir...Objective To investigate the biological function of NS5ABP37 and to look for proteins interacting with NS5ABP37 protein in hepatocytes.Methods We constructed bait plasmid expressing NS5ABP37 protein of hepatitis C virus(HCV)by cloning the gene of NS5ABP37 protein into pGBKT7,then the recombinant plasmid DNA was transformed into yeast AH109(α type).The transformed yeast AH109 was mated with yeast Y187(α type)containing liver cDNA library plasmid in 2×YPDA medium.Diploid yeast was plated on synthetic dropout nutrient medium(SD/-Trp-Leu-His-Ade)containing X-α-gal for selection and screening.After extracting and sequencing of plasmids from positive(blue)colonies,we made a sequence analysis by bioinformatics.Results We screened twenty-five proteins binding to NS5ABP37,including Homo sapiens cyclin I(CCNI)gene,Homo sapiens matrix metallopeptidase 25(MMP25)and Homo sapiens talin 1.Conclusion The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with NS5ABP37 of HCV.And the biological function of NS5ABP37 may be associated with glycometabolism,lipid metabolism and apoptosis.展开更多
AIM: To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes. METHODS: We constructed bait plasmid expressing complete S protein of HBV ...AIM: To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes. METHODS: We constructed bait plasmid expressing complete S protein of HBV by cloning the gene of complete S protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics. RESULTS: Nineteen colonies were selected and sequenced. Among them, five colonies were Homo sapiens solute carrier family 25, member 23 (SLC25A23), one was Homo sapiens calrer.iculin, one was human serum albumin (ALB) gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase, three were Homo sapiens Na+ and H+coupled amino acid transport system N, one was Homo sapiens CD81 antigen (target of anti-proliferative antibody 1) (CD81), three were Homo sapiens diazepam binding inhibitor, two colonies were new genes with unknown function. CONCLUSION: The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with complete S protein of HBV. The complete S protein may bind to different proteins i.e., its multiple functions in vivo.展开更多
OBJECTIVE: To investigate the biological function of augmenter of liver regeneration (ALR), we usedyeast-two hybrid technique to detect proteins in hepatocytes interacting with ALR.METHODS: ALR bait plasmid was constr...OBJECTIVE: To investigate the biological function of augmenter of liver regeneration (ALR), we usedyeast-two hybrid technique to detect proteins in hepatocytes interacting with ALR.METHODS: ALR bait plasmid was constructed by using yeast-two hybrid system 3, then transformedinto yeast AH109. The transformed yeast was mated with yeast Y187 containing liver cDNA libraryplasmid in a 2×YPDA medium. Diploid yeast was plated on a synthetic dropout nutrient medium(SD/-Trp-Leu-His-Ade) containing x-α-gal for selection and screening. After extracting and sequencingof the plasmid from blue colonies. Analysis was performed by bioinformatics.RESULTS: Of 36 colonies sequenced, 14 are metallothionein, 12 albumin, and 3 selenoprotein P. Onecolony is a new gene with unknown function.CONCLUSION: The successful cloning of gene of ALR interacting protein has paved the way forstudying the physiological function of ALR and associated proteins.展开更多
The aim of this study is to screen proteins interacting with interferon-α （IFN-α）. The IFN-α gene was amplified by polymerase chain reaction （PCR） and cloned into pGBKT-/vector, then the resulted pGBKT-/-IFN-α...The aim of this study is to screen proteins interacting with interferon-α （IFN-α）. The IFN-α gene was amplified by polymerase chain reaction （PCR） and cloned into pGBKT-/vector, then the resulted pGBKT-/-IFN-α vector was transformed into yeast strain AH109. The transformed yeast AH109 was mated with yeast Y187 containing liver cDNA library plasmid in 2 × YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade and SD/-Trp-Leu-His） con- taining X-α-gal for selection. After plasmid extraction and enzyme cutting analysis, the blue colonies were subjected to sequence analysis and the results were analyzed by bioinformatics. The results showed that IFN-α was successful cloned into the pGBKT7 vector. IFN-α was expressed and there was no selfactivation and toxicity in AH109. Thirty-four positive colonies were obtained after yeast-two hybrid technique screening. After sequence analysis, eight clones were found to have a binding effect with IFN-α protein. IFN-α was successfully cloned into the pGBKT7 vector. IFN-α protein was expressed and there was no self-activation and toxicity in AH109. Eight proteins that interacted with IFN-α, including vitronectin, fibrinogen A alpha polypeptide, HIV-1 Tat interactive protein 2, arginase, NADH dehydrogenase 1 beta subcomplex, transferrin receptor 2 alpha （TFR2）, HCC-1, alcohol dehydrogenase IB （ADHIB） have been identified as IFN-α-binding proteins.展开更多
SWEET(sugars will eventually be exported transporter)基因在植物开花过程中具有重要的作用,但AcSWEET11在菠萝成花中的作用机制尚不清楚。通过鉴定成花过程中与AcSWEET11的互作蛋白,为菠萝成花机制的解析奠定基础。本研究利用共转...SWEET(sugars will eventually be exported transporter)基因在植物开花过程中具有重要的作用,但AcSWEET11在菠萝成花中的作用机制尚不清楚。通过鉴定成花过程中与AcSWEET11的互作蛋白,为菠萝成花机制的解析奠定基础。本研究利用共转化的方法在菠萝成花过程的cDNA膜文库中筛选AcSWEET11的互作蛋白,分析候选蛋白的表达量。结果表明,pBT3-STE-AcSWEET11+pPR3-N对NMY51酵母细胞无毒性,但有自激活活性。进一步研究结果显示,在TDO/3?AT培养基和QDO培养基上自激活受到抑制。利用该系统筛选到了81个阳性克隆,经测序鉴定出48个与AcSWEET11互作的候选蛋白,包括E3 ubiquitin-protein ligase RING1-like、Trehalose-phosphate synthase 7、Cytochrome P450、TranscriptionfactorLUX等。GO和KEGG分析结果显示,48个蛋白主要分布在细胞进程、代谢过程、刺激反应和催化活性等生物过程,参与脂类代谢、氨基酸代谢和碳水化合物代谢、信号转导和运输与分解代谢等新陈代谢途径。Trehalose-phosphate synthase 7(XP_020105459.1)、Protein TIFY 3-like(XP_020082835.1)、40S ribosomal protein S27(XP_020092770.1)、Heterogeneous nuclear ribonucleoprotein 1-like(XP_020112516.1)等4个基因与AcSWEET11表达趋势一致,在菠萝成花过程中下调表达;Dihydrolipoyl dehydrogenase 2(XP_020113798.1)、Putative lipid-transfer protein DIR1(XP_020086640.1)、clathrin assembly protein At4g32285(XP_020108161.1)等3个基因在菠萝成花过程中上调表达。这些结果表明,AcSWEET11可能通过与Trehalose-phosphatesynthase7等蛋白发生互作,参与菠萝成花过程。本研究进一步丰富了AcSWEET11的蛋白互作网络,为AcSWEET11在菠萝成花中的调控机制的解析奠定基础。展开更多
文摘Objective To investigate the biological function of NS5ABP37 and to look for proteins interacting with NS5ABP37 protein in hepatocytes.Methods We constructed bait plasmid expressing NS5ABP37 protein of hepatitis C virus(HCV)by cloning the gene of NS5ABP37 protein into pGBKT7,then the recombinant plasmid DNA was transformed into yeast AH109(α type).The transformed yeast AH109 was mated with yeast Y187(α type)containing liver cDNA library plasmid in 2×YPDA medium.Diploid yeast was plated on synthetic dropout nutrient medium(SD/-Trp-Leu-His-Ade)containing X-α-gal for selection and screening.After extracting and sequencing of plasmids from positive(blue)colonies,we made a sequence analysis by bioinformatics.Results We screened twenty-five proteins binding to NS5ABP37,including Homo sapiens cyclin I(CCNI)gene,Homo sapiens matrix metallopeptidase 25(MMP25)and Homo sapiens talin 1.Conclusion The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with NS5ABP37 of HCV.And the biological function of NS5ABP37 may be associated with glycometabolism,lipid metabolism and apoptosis.
基金Supported by the National Natural Science Foundation of China, No. C03011402, No. C30070690the Science and Technique Foundation of PLA during the 9th Five-year plan period, No. 98D063 the Launching Foundation for Students Studying Abroad of PLA, No. 98H038 the Youth Science and Technique Foundation of PLA during the 10lh Five-year plan period, No. 01Q138and the Science & Technique Foundation of PLA during the 10th Five-year plan period, No. 01MB135
文摘AIM: To investigate the biological function of complete S protein and to look for proteins interacting with complete S protein in hepatocytes. METHODS: We constructed bait plasmid expressing complete S protein of HBV by cloning the gene of complete S protein into pGBKT7, then the recombinant plasmid DNA was transformed into yeast AH109 (a type). The transformed yeast AH109 was mated with yeast Y187 (α type) containing liver cDNA library plasmid in 2×YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-α-gal for selection and screening. After extracting and sequencing of plasmids from positive (blue) colonies, we underwent sequence analysis by bioinformatics. RESULTS: Nineteen colonies were selected and sequenced. Among them, five colonies were Homo sapiens solute carrier family 25, member 23 (SLC25A23), one was Homo sapiens calrer.iculin, one was human serum albumin (ALB) gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase, three were Homo sapiens Na+ and H+coupled amino acid transport system N, one was Homo sapiens CD81 antigen (target of anti-proliferative antibody 1) (CD81), three were Homo sapiens diazepam binding inhibitor, two colonies were new genes with unknown function. CONCLUSION: The yeast-two hybrid system is an effective method for identifying hepatocyte proteins interacting with complete S protein of HBV. The complete S protein may bind to different proteins i.e., its multiple functions in vivo.
文摘OBJECTIVE: To investigate the biological function of augmenter of liver regeneration (ALR), we usedyeast-two hybrid technique to detect proteins in hepatocytes interacting with ALR.METHODS: ALR bait plasmid was constructed by using yeast-two hybrid system 3, then transformedinto yeast AH109. The transformed yeast was mated with yeast Y187 containing liver cDNA libraryplasmid in a 2×YPDA medium. Diploid yeast was plated on a synthetic dropout nutrient medium(SD/-Trp-Leu-His-Ade) containing x-α-gal for selection and screening. After extracting and sequencingof the plasmid from blue colonies. Analysis was performed by bioinformatics.RESULTS: Of 36 colonies sequenced, 14 are metallothionein, 12 albumin, and 3 selenoprotein P. Onecolony is a new gene with unknown function.CONCLUSION: The successful cloning of gene of ALR interacting protein has paved the way forstudying the physiological function of ALR and associated proteins.
文摘The aim of this study is to screen proteins interacting with interferon-α （IFN-α）. The IFN-α gene was amplified by polymerase chain reaction （PCR） and cloned into pGBKT-/vector, then the resulted pGBKT-/-IFN-α vector was transformed into yeast strain AH109. The transformed yeast AH109 was mated with yeast Y187 containing liver cDNA library plasmid in 2 × YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium （SD/-Trp-Leu-His-Ade and SD/-Trp-Leu-His） con- taining X-α-gal for selection. After plasmid extraction and enzyme cutting analysis, the blue colonies were subjected to sequence analysis and the results were analyzed by bioinformatics. The results showed that IFN-α was successful cloned into the pGBKT7 vector. IFN-α was expressed and there was no selfactivation and toxicity in AH109. Thirty-four positive colonies were obtained after yeast-two hybrid technique screening. After sequence analysis, eight clones were found to have a binding effect with IFN-α protein. IFN-α was successfully cloned into the pGBKT7 vector. IFN-α protein was expressed and there was no self-activation and toxicity in AH109. Eight proteins that interacted with IFN-α, including vitronectin, fibrinogen A alpha polypeptide, HIV-1 Tat interactive protein 2, arginase, NADH dehydrogenase 1 beta subcomplex, transferrin receptor 2 alpha （TFR2）, HCC-1, alcohol dehydrogenase IB （ADHIB） have been identified as IFN-α-binding proteins.
文摘SWEET(sugars will eventually be exported transporter)基因在植物开花过程中具有重要的作用,但AcSWEET11在菠萝成花中的作用机制尚不清楚。通过鉴定成花过程中与AcSWEET11的互作蛋白,为菠萝成花机制的解析奠定基础。本研究利用共转化的方法在菠萝成花过程的cDNA膜文库中筛选AcSWEET11的互作蛋白,分析候选蛋白的表达量。结果表明,pBT3-STE-AcSWEET11+pPR3-N对NMY51酵母细胞无毒性,但有自激活活性。进一步研究结果显示,在TDO/3?AT培养基和QDO培养基上自激活受到抑制。利用该系统筛选到了81个阳性克隆,经测序鉴定出48个与AcSWEET11互作的候选蛋白,包括E3 ubiquitin-protein ligase RING1-like、Trehalose-phosphate synthase 7、Cytochrome P450、TranscriptionfactorLUX等。GO和KEGG分析结果显示,48个蛋白主要分布在细胞进程、代谢过程、刺激反应和催化活性等生物过程,参与脂类代谢、氨基酸代谢和碳水化合物代谢、信号转导和运输与分解代谢等新陈代谢途径。Trehalose-phosphate synthase 7(XP_020105459.1)、Protein TIFY 3-like(XP_020082835.1)、40S ribosomal protein S27(XP_020092770.1)、Heterogeneous nuclear ribonucleoprotein 1-like(XP_020112516.1)等4个基因与AcSWEET11表达趋势一致,在菠萝成花过程中下调表达;Dihydrolipoyl dehydrogenase 2(XP_020113798.1)、Putative lipid-transfer protein DIR1(XP_020086640.1)、clathrin assembly protein At4g32285(XP_020108161.1)等3个基因在菠萝成花过程中上调表达。这些结果表明,AcSWEET11可能通过与Trehalose-phosphatesynthase7等蛋白发生互作,参与菠萝成花过程。本研究进一步丰富了AcSWEET11的蛋白互作网络,为AcSWEET11在菠萝成花中的调控机制的解析奠定基础。
