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
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.
文摘核因子(Nuclear Factor-Y,NF-Y)是一类能够在植物应对非生物胁迫过程中发挥转录调控作用的转录因子。为探究GmNF-YB2在大豆应对非生物胁迫过程中的功能和分子调控机制,本研究从大豆中克隆GmNF-YB2基因并对其进行生物信息学分析,采用实时荧光定量PCR(qRT-PCR)方法对GmNF-YB2的表达量进行检测,通过酵母双杂交方法分析GmNF-YB2蛋白与GmNF-YC6蛋白和GmNF-YC15蛋白的互作情况。结果显示:以大豆叶片cDNA为模板,PCR扩增获得678 bp GmNF-YB2基因序列。生物信息学分析结果显示,GmNF-YB2基因编码1个含有225个氨基酸的蛋白质,预测分子量为24.66 kD,等电点为6.97,是不稳定的亲水性蛋白质,定位于细胞核中;GmNF-YB2蛋白序列中含有一段保守的组蛋白折叠基序HFM。蛋白系统进化分析表明,GmNF-YB2与杨树PdNF-YB7和拟南芥AtNF-YB2的亲缘关系最近。qRT-PCR结果显示,GmNF-YB2在大豆幼苗中能够被干旱、盐和低温胁迫诱导表达,GmNF-YB2在大豆茎中的表达量最高。酵母双杂交结果显示,GmNF-YB2蛋白与GmNF-YC6蛋白和GmNF-YC15蛋白均存在互作关系。结果表明GmNF-YB2可能参与大豆对非生物胁迫的应答。
