AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we ...AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.METHODS: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics. RESULTS: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4CD81 (TM4SF4).CONCLUSION: The pre-X gene exists in HBV genome.Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.展开更多
AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes.METHODS: We constructed p7 protein bait plasmid by doning the gene of p7 protein into pGBKT...AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes.METHODS: We constructed p7 protein bait plasmid by doning the gene of p7 protein into pGBKT7, then transformed it into yeast AH109 (a type). The transformed yeast was mated with yeast Y187 (α type) containing liver cDNA library plasmid, pACT2 in 2xYPDA 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 blue colonies, we performed sequence analysis by bioinformatics.RESULTS: Fifty colonies were selected and sequenced.Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nucleoporin 214 ku and two colonies were CLL-associated antigens.CONCLUSION: The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its associated protein.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 b...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 2xYPDA 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 calreticulin, one was human serum albumin (ALB)gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase,three were Homo sapiensNa+ 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 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
ELL2 (eleven-nineteen lysine-rich leukemia transcription elongation factor), a component of a larger complex with pTEFb (cyclin T and CDK9) and AF4, is up-regulated in plasma cells where it influences mRNA processing ...ELL2 (eleven-nineteen lysine-rich leukemia transcription elongation factor), a component of a larger complex with pTEFb (cyclin T and CDK9) and AF4, is up-regulated in plasma cells where it influences mRNA processing by increasing exon skipping and enhancing proximal poly (A) site use. ELL2 is needed to produce the secretory-specific Ig heavy chain mRNA while ELL1 mRNA does not change in abundance with B cell stages. To investigate the potential interactions of other proteins with the ELL1 and ELL2 proteins, we preformed yeast two-hybrid studies. HSP40 and Testin were found to bind to ELL2 in its amino-terminal half. PCNA binds to ELL2 in a region encompassing amino acids 186 - 344. The potent transcription factors HIF1 α and ZNF622 interact with both ELL1 and 2 in the central, proline rich region. Meanwhile, BBS2 and ING3 interact with ELL1 but not ELL2 in this central proline-rich region. Many of the ELL-interacting-proteins uncovered in the two-hybrid screen are tumour suppressors that may work through the ELL: pTEFb complex to suppress or activate sets of genes in plasma cells.展开更多
A specialized test of two-hybrid library type three-frame cDNA yeast for Muskmelon Fusarium oxysporum using the switching mechanism at the 5'end of RNA template(SMART)technology was constructed to screen for inter...A specialized test of two-hybrid library type three-frame cDNA yeast for Muskmelon Fusarium oxysporum using the switching mechanism at the 5'end of RNA template(SMART)technology was constructed to screen for interaction protein genes for wilt disease and to further research the molecular mechanisms of Fusarium oxysporum pathogenesis to explain the interactions between plant and pathogen.A 500-bp cDNA was purified and extracted using SMART and LD-PCR technology to synthesize ds cDNA and was then homogenized and purified to remove the fragments.After processing,the ds cDNA was connected to three types of reading frame pGADT7-SfiI carriers,and the three connection products in E.coli Electrocell were used to build the primary cDNA library.The titer of three ORF cDNA primary library storage capacities was 2.6×10^6,1.8×10^6 and 3×10^6 cfu;the PCR identification of the ORF 1 and 2 gene recombination rate was 94%,the ORF 3 gene recombination rate was 100%,and the insert length distribution was 0.5-4.0 kb as a single band.To reach the quality requirements for library construction,three kinds of reading frame cDNA primary libraries were mixed and amplified,and the plasmid was transformed into the Y187 yeast strain.The titer of the Y187 yeast library was determined to be 3.5×107 cfu?mL-1,and the base of the yeast library was approximately 1 600 000 cfu.The results showed that the construction of muskmelon Fusarium-specific two-hybrid library type three-frame cDNA yeast had a higher reservoir capacity and recombination rate and met the yeast two-hybrid screening requirements.展开更多
By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of re...By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...展开更多
The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which ...The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins (protein tyrosine-phosphatase non-receptor type 2, TBCl D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor a2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain), and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.展开更多
The yeast two-hybrid (Y2H) mating assay is a powerful method for detecting protein-protein interactions. Firstly, the gene of interest is cloned into specific Y2H vectors. Although multiple innovations in cloning meth...The yeast two-hybrid (Y2H) mating assay is a powerful method for detecting protein-protein interactions. Firstly, the gene of interest is cloned into specific Y2H vectors. Although multiple innovations in cloning methods were made in the past two decades, the conventional cloning method of restriction-enzyme (RE) digestion followed by ligation is still widely used. Unfortunately, many researchers, especially new-comers, often encounter difficulties in cloning a gene into a desired vector. Secondly, interaction between two proteins is commonly detected by growth of the diploids in specific media. This step takes about two weeks. Here, we describe improved cloning and detection procedures for the Y2H assay that accelerate the research progress. The changes in procedures involve running an agarose gel after the doubly digested vector and insert are ligated in the cloning step to determine the efficiency of RE digestion and ligation, and performing an additional replica-plating on plates for earlier assessment of interaction in the detection step. We show an example of Y2H interaction between Trs23 and Trs120 (respective subunits of TRAPP I and TRAPP II), as a proof of concept. By following the improved methods described here, the chances of successful cloning increased and the time for the whole Y2H experimental process is significantly shorter.展开更多
[Objective] The paper was to obtain host proteins interacting with porcine epidemic diarrhea virus (PEDV) N protein. [Method] The re-combinant vector pGBKT7-N of PEDV N gene was constructed and used as the bait plas...[Objective] The paper was to obtain host proteins interacting with porcine epidemic diarrhea virus (PEDV) N protein. [Method] The re-combinant vector pGBKT7-N of PEDV N gene was constructed and used as the bait plasmid to screen the proteins interacting with N protein ofPEDV from the cDNA library of porcine alveolar macrophage (PAM) by yeast two-hybrid method. [Result] There was no toxicity and self activationof bait protein in yeast hybridization system, and six proteins (FTH1, LGALS3, CORO1C, SNRPG, KRTAP5-3, ZNF598) interacting with N proteinwere indentified. It was confirmed that LGALS3 and SNRPG had specific interaction with N protein by return experiment and co-immunoprecipitation(CoIP) test. [Conclusion] The study lays a foundation for further studying the function of PEDV N protein and the pathogenic mechanism of PEDV.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 pGBKT7 vector, then the resulted pGBKT7-IFN-α vect...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 pGBKT7 vector, then the resulted pGBKT7-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) containing 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 self-activation 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 (ADH1B) have been identified as IFN-α-binding proteins.展开更多
基金Supported by the grants from the National Natural Science Foundation, No. C03011402, No. C30070690the 9.5 Research and Technique Foundation of PLA, No. 98D063+1 种基金 the Launching Foundation for Student Studying Abroad of PLA, No. 98H038 the 10.5 Youth Research and Technique Foundation of PLA, No. 01Q138andNo. 01MB135
文摘AIM: To identify the pre-X region in hepatitis B virus (HBV)genome and to study the relationship between the genotype and the pre-X region. To investigate the biological function of whole-X (pre-X plus X) protein, we performed yeast two-hybrid to screen proteins in liver interacting with whole-X protein.METHODS: The pre-X region of HBV was amplified by polymerase chain reaction (PCR) method, and was cloned to pGEM Teasy vector. After the target region was sequenced, Vector 8.0 software was used to analyze the sequences. The whole-X bait plasmid was constructed by using yeast two-hybrid system 3. Yeast strain AH109 was transformed. After expression of the whole-X protein in AH109 yeast strains was proved, yeast two-hybrid screening was performed by mating AH109 with Y187 containing liver cDNA library plasmid. The mated yeast was plated on quadruple dropout medium and assayed for α-gal activity. The interaction between whole-X protein and the protein obtained from positive colonies was further confirmed by repeating yeast two-hybrid. After extracting and sequencing of plasmid from blue colonies, we carried out analysis by bioinformatics. RESULTS: After sequencing, 27 of 45 clones (60%) were found encoding the pre-X peptide. Eighteen of twenty-seven clones (66.7%) of pre-X coding sequences were found from genotype C. Five positive colonies that interacted with whole-X protein were obtained and sequenced; namely, fetuin B, UDP glycosyltransferase 1 family-polypeptide A9, mannose-P-dolichol utilization defect 1, fibrinogen-B beta polypeptide, transmembrane 4 superfamily member 4CD81 (TM4SF4).CONCLUSION: The pre-X gene exists in HBV genome.Genes of proteins interacting with whole-X protein in hepatocytes were successfully cloned. These results brought some new clues for studying the biological functions of whole-X protein.
基金Supported by the National Natural Scientific Foundation, No. C03011402, No. C30070690the Research and Technique Foundation of PLA during the 9th-five year plan period, No. 98D063the Launching Foundation for Student Studying Abroad of PLA, No. 98H038and the Youth Research and Technique Foundation of PLA during the 10th-five year plan period, No. 01Q138the Research and Technique Foundation of PLA during the 10th-five year plan period, No. 01MB135
文摘AIM: To investigate the biological function of p7 protein and to look for proteins interacting with p7 protein in hepatocytes.METHODS: We constructed p7 protein bait plasmid by doning the gene of p7 protein into pGBKT7, then transformed it into yeast AH109 (a type). The transformed yeast was mated with yeast Y187 (α type) containing liver cDNA library plasmid, pACT2 in 2xYPDA 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 blue colonies, we performed sequence analysis by bioinformatics.RESULTS: Fifty colonies were selected and sequenced.Among them, one colony was Homo sapiens signal sequence receptor, seven colonies were Homo sapiens H19, seven colonies were immunoglobulin superfamily containing leucine-rich repeat, three colonies were spermatid peri-nuclear RNA binding proteins, two colonies were membrane-spanning 4-domains, 24 colonies were cancer-associated antigens, four colonies were nucleoporin 214 ku and two colonies were CLL-associated antigens.CONCLUSION: The successful cloning of gene of protein interacting with p7 protein paves a way for the study of the physiological function of p7 protein and its associated protein.
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 2xYPDA 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 calreticulin, one was human serum albumin (ALB)gene, one was Homo sapiens metallothionein 2A, two were Homo sapiens betaine-homocysteine methyltransferase,three were Homo sapiensNa+ 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 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.
文摘ELL2 (eleven-nineteen lysine-rich leukemia transcription elongation factor), a component of a larger complex with pTEFb (cyclin T and CDK9) and AF4, is up-regulated in plasma cells where it influences mRNA processing by increasing exon skipping and enhancing proximal poly (A) site use. ELL2 is needed to produce the secretory-specific Ig heavy chain mRNA while ELL1 mRNA does not change in abundance with B cell stages. To investigate the potential interactions of other proteins with the ELL1 and ELL2 proteins, we preformed yeast two-hybrid studies. HSP40 and Testin were found to bind to ELL2 in its amino-terminal half. PCNA binds to ELL2 in a region encompassing amino acids 186 - 344. The potent transcription factors HIF1 α and ZNF622 interact with both ELL1 and 2 in the central, proline rich region. Meanwhile, BBS2 and ING3 interact with ELL1 but not ELL2 in this central proline-rich region. Many of the ELL-interacting-proteins uncovered in the two-hybrid screen are tumour suppressors that may work through the ELL: pTEFb complex to suppress or activate sets of genes in plasma cells.
基金Supported by the National Nature Science Foundation of China(31372088)the "Academic Backbone" Project of Northeast Agricultural University(15XG05)China Agriculture Research System(CARS-26-02)
文摘A specialized test of two-hybrid library type three-frame cDNA yeast for Muskmelon Fusarium oxysporum using the switching mechanism at the 5'end of RNA template(SMART)technology was constructed to screen for interaction protein genes for wilt disease and to further research the molecular mechanisms of Fusarium oxysporum pathogenesis to explain the interactions between plant and pathogen.A 500-bp cDNA was purified and extracted using SMART and LD-PCR technology to synthesize ds cDNA and was then homogenized and purified to remove the fragments.After processing,the ds cDNA was connected to three types of reading frame pGADT7-SfiI carriers,and the three connection products in E.coli Electrocell were used to build the primary cDNA library.The titer of three ORF cDNA primary library storage capacities was 2.6×10^6,1.8×10^6 and 3×10^6 cfu;the PCR identification of the ORF 1 and 2 gene recombination rate was 94%,the ORF 3 gene recombination rate was 100%,and the insert length distribution was 0.5-4.0 kb as a single band.To reach the quality requirements for library construction,three kinds of reading frame cDNA primary libraries were mixed and amplified,and the plasmid was transformed into the Y187 yeast strain.The titer of the Y187 yeast library was determined to be 3.5×107 cfu?mL-1,and the base of the yeast library was approximately 1 600 000 cfu.The results showed that the construction of muskmelon Fusarium-specific two-hybrid library type three-frame cDNA yeast had a higher reservoir capacity and recombination rate and met the yeast two-hybrid screening requirements.
基金supported by grants from National Natural Sciences Foundation of China(No.30672227,30600668)"973"Program of China(No.2009CB521800)Joint Research Fund for Overseas Chinese,Hong Kong and Macao Young Scholars(No.30628029)
文摘By using a yeast two-hybrid system,a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins,and its effects on the growth of yeast cells and the activation of reporter genes were investigated.Total mRNA extracted from Hela cells was reversely transcribed into cDNA.Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector.The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing.Th...
基金the National Natural Science Foundation of China, No. 30300116
文摘The present study screened a human fetal brain cDNA library to find the proteins that interact with mutant superoxide dismutase 1 (SOD1) using a yeast two-hybrid system. Using BLAST software, 15 real proteins which interacted with mutant SOD1 were obtained, including 8 known proteins (protein tyrosine-phosphatase non-receptor type 2, TBCl D4, protein kinase family, splicing factor, arginine/serine-rich 2, SRC protein tyrosine kinase Fyn, β-sarcoglycan; glycine receptor a2, microtubule associated protein/microtubule affinity-regulating kinase 1, ferritin H chain), and 7 unknown proteins. Results demonstrated interaction of mutant SOD1 with microtubule associated protein/microtubule affinity-regulating kinase 1 and β-sarcoglycan.
文摘The yeast two-hybrid (Y2H) mating assay is a powerful method for detecting protein-protein interactions. Firstly, the gene of interest is cloned into specific Y2H vectors. Although multiple innovations in cloning methods were made in the past two decades, the conventional cloning method of restriction-enzyme (RE) digestion followed by ligation is still widely used. Unfortunately, many researchers, especially new-comers, often encounter difficulties in cloning a gene into a desired vector. Secondly, interaction between two proteins is commonly detected by growth of the diploids in specific media. This step takes about two weeks. Here, we describe improved cloning and detection procedures for the Y2H assay that accelerate the research progress. The changes in procedures involve running an agarose gel after the doubly digested vector and insert are ligated in the cloning step to determine the efficiency of RE digestion and ligation, and performing an additional replica-plating on plates for earlier assessment of interaction in the detection step. We show an example of Y2H interaction between Trs23 and Trs120 (respective subunits of TRAPP I and TRAPP II), as a proof of concept. By following the improved methods described here, the chances of successful cloning increased and the time for the whole Y2H experimental process is significantly shorter.
基金Supported by National Natural Science Foundation of China(31201915,31502071)Key Project of Science and Technology Promoting Agriculture in Shanghai City[HNKGZ(2013)No.3-6,No.5-5]
文摘[Objective] The paper was to obtain host proteins interacting with porcine epidemic diarrhea virus (PEDV) N protein. [Method] The re-combinant vector pGBKT7-N of PEDV N gene was constructed and used as the bait plasmid to screen the proteins interacting with N protein ofPEDV from the cDNA library of porcine alveolar macrophage (PAM) by yeast two-hybrid method. [Result] There was no toxicity and self activationof bait protein in yeast hybridization system, and six proteins (FTH1, LGALS3, CORO1C, SNRPG, KRTAP5-3, ZNF598) interacting with N proteinwere indentified. It was confirmed that LGALS3 and SNRPG had specific interaction with N protein by return experiment and co-immunoprecipitation(CoIP) test. [Conclusion] The study lays a foundation for further studying the function of PEDV N protein and the pathogenic mechanism of PEDV.
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 pGBKT7 vector, then the resulted pGBKT7-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) containing 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 self-activation 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 (ADH1B) have been identified as IFN-α-binding proteins.