Construction of bait plasmid containing SIGIRR cytoplasmic tail and detection of self-activation in yeast two hybrid system / 重庆医学

Objective To construct the bait plasmid of pSos-single immunoglobin IL-1 receptor related protein (SIGIRR) in Cy-toTrap yeast two hybrid system ,and to test its self-activation .Methods The cDNA fragments of SIGIRR(480 -1 230 bp) were amplified from pReceiver-LV19-SIGIRR and ligated into the bait plasmid pSos to generate the plasmid pSos-SIGIRR .The pSos-SI-GIRR was identified by DNA sequencing and dual-site endonuclease digestion .Then the recombinant plasmid and control plasmid were introduced into the yeast cell cdc25H .The transformants were inoculated on plates of 25 ℃ /SD/Glucose(-UL) ,25 ℃/SD/Ga-lactose(-UL) ,37 ℃ /SD/Glucose(-UL) and 37 ℃ /SD/Galactose ,respectively and the proliferation ability of transformant was ob-served for 6 d .The Western blot was adopted to detect the expression of target protein .Results The pSos-SIGIRR vector was cor-rectly constructed and proved of no self-activation and toxic action .The Western blot showed that the target protein was expressed in a form of fusion protein of 170KD .Conclusion The bait plasmid containing SIGIRR cytoplasmic tail can be applied to the yeast two-hybrid system and lays the important foundation for seeking the interacting protein with SIGRR from the human lung cDNA li-brary in .

Screening of proteins interacting with HSPC016 by yeast two-hybridization technique / 中华皮肤科杂志

Objective To screen and identify proteins interacting with hematopoietic stem/progenitor cell differentiation-related gene HSPC016, and to explore the molecular mechanism involved in the regulation by HSPC016 on the aggregative behavior of dermal papilla cells. Methods By using yeast two-hybridization,HSPC016 gene was sub-cloned into pGBKT7 to construct the bait plasmid (named as pGBKT7-HSPC016) in yeast AH109. The cDNA yeast expression library of human dermal papillae cells in yeast Y187 was screened with the bait plasmid and the proteins interacting with HSPC016 were identified. Yeast two-hybridization retransformation experiment was conducted to exclude the false positive clones and verify the interactions, then,the positive clones were sequenced and analyzed by using bioinformatic methods. Results The bait plasmid pGBKT7-HSPC016 was constructed successfully and there was no self-activation in or toxicity against yeast AH 109. Four proteins,including forkhead family of transcription factors (FOXO 1 ), mitogen-activated protein kinase 11 (MAPK 11 ), phosphoinositide-3-kinase (PIK3R3) and liver X receptor were screened and identified. Bioinformatic analysis revealed that these proteins had close relationship with intracellular energy metabolism and translational regulation. Conclusions HSPC016 may regulate the aggregative behavior of DPCs by regulating the levels of intracellular reactive oxygen species (ROS) and interacting with signaling molecules involved in intracellular energy metabolism and translational regulation.

Analysis of the molecular characterization of a Chinese pedigree with rare β thalassemia genotype / 中华检验医学杂志

Objective To investigate the molecular characterization of a Chinese pedigree with rare β thalassemia genotype.Methods Phenotypic analysis was performed using standard hematological tests to measure red blood cell parameters, including RBC,Hb,MCV,MCH,MCHC and RDW.SPIFE automatic Hb agarose gel electrophoresis instrument was used to measure hemoglobin fraction Hb A,Hb A2 and Hb F.The alleles of β thalassemia mutation were determined by RDB assay, and then cloning and sequencing were performed to define the mutation sites.Results The proband and his father had typical microcytic hypochromic anemia with low MCV and MCH(79.8, 63.1 fl and 19.9, 20.9 pg, respectively) and high level of Hb A2 (5.66% and 5.60%, respectively).The proband′s mother had normal MCV and MCH. β thalassemia mutation analysis with RDB assay showed that the proband had thalassemia minor resulting from double mutations on one globin gene.One showed codons 41/42 (-TTCT) mutation and the other was CAP mutation from positions +40 to +43 in the promoter region.These two mutations were inherited from his father.The genotype of the proband and his father was β41/42、CAP/βA ,and the genotype of his mother was βA/βA.Conclusions It′s rare that double mutations occur on single β globin gene, with one mutation on CD41/42(-TTCT) and the other mutation from positions +40 to +43 relative to the mRNA cap site in the promoter region.The findings enrich knowledge of the mutation spectrum of β thalassemia.

Verification of interaction between glutamate-ammonia ligase and nuclear localization signal-retinoic acid receptor α protein inside and outside cells / 第二军医大学学报

Objective: To verify the interaction between glutamate-ammonia ligase (GLUL) and nuclear localization signal-retinoic acid receptor α (NLS-RARα) protein by yeast two-hybrid and co-immunoprecipitation method. Methods: The two plasmids expressing NLS-RARα bait-protein and GLUL protein were co-transformed into yeast AH109 to investigate the interaction in vivo. Tagged fusion protein eukaryotic expression vectors were constructed and co-transfected into HEK 293 cells. Co-immunoprecipitation was used to investigate the interaction between NLS-RARα and GLUL in vitro. Results: Positive blue clones were found in the QDO/X-α-gal plate. Eukaryotic expression vectors were co-transfected into HEK 293 cells, then HA-NLS-RARα protein was immunoprecipitated by anti-HA polyclonal antibody, and GLUL-cMyc protein expression was confirmed by Western blotting analysis using anti c-Myc monoclonal antibody. Conclusion: The interaction between NLS-RARα and GLUL has been verified by both yeast two-hybrid and co-immunoprecipitation.

Construction and identification of yeast cDNA expression library of human dermal papillae cells in primary culture / 中华皮肤科杂志

Objective To construct yeast cDNA expression library of human dermal papillae cells (DPCs) in primary culture.Methods Human dermal papilla cells (DPCs) were isolated by two-step digestion method and cultured in DMEM medium.Total RNA was extracted from primary DPCs that exhibited an aggregative behavior in culture,then,cDNA was synthesized and amplified by using CloneMinerTM cDNA Library Construction kit to construct primary cDNA library and yeast cDNA expression libary.Results The average titer and total clones were 7.0×106 colony forming units(cfu)/ml and 1.4×107 cfu respectively in the primary library,5.5×106 cfu/ml and 1.1×107 cfu respectively in the yeast expression library.The average insert size Was 1.2 kb and the recombination rate was above 95%.Conclusions The yeast cDNA expression library of DPCs in primary culture has been successfully constructed.which will lay a foundation for screening proteins interacting with HSPC016 gene in DPCs with yeast two-hybrid system.

Langerhans cell protein 1 (LCP1) binds to PNUTS in the nucleus: implications for this complex in transcriptional regulation

Protein phosphatase-1 (PP1) nuclear targeting subunit (PNUTS), also called PP1R10, p99, or CAT 53 was originally isolated as a mammalian nuclear PP1-binding protein. In this study, we performed yeast two-hybrid screens to identify PNUTS-interacting proteins. Here, we report that LCP1 (epidermal Langerhans cell protein 1), a novel member of the HMG-box protein family, binds tightly to PNUTS. Co-immunoprecipitation of deletion constructs revealed that the C-terminus of LCP1 is sufficient for the interaction with an N-terminal region of PNUTS that is distinct from its PP1-binding domain. Furthermore, immunofluorescence studies showed that a subpopulation of LCP1 co-localizes with PNUTS in nuclear speckles. Importantly, we found that the N-terminus of LCP1 has a strong trans-activation activity in a GAL4-based heterologous transcription assay. The transcriptional activity of LCP1 is markedly suppressed by its interaction with PNUTS, in a PP1-independent manner. These findings suggest that the coordinated spatial and temporal regulation of LCP1 and PNUTS may be a novel mechanism to control the expression of genes that are critical for certain physiological and pathological processes.

Effect of interaction of human papillomavirus type 16 E6 protein and hDaxx on apoptosis of HeLa cells / 肿瘤

Objective: To study the interaction of human papillomavirus type 16 (HPV16 E6) protein and human death domain associated protein (hDaxx) and its effect on apoptosis of HeLa cells to provide the experimental basis for exploring the oncogenic mechanism of HPV16 protein. Methods: Recombinant vectors of pGADT7/E6 or pGBKT7/hDaxx were constructed. The interaction of E6 protein and hDaxx was detected by yeast two-hybrid system. Their expression in yeast was detected by Western blotting. The eukariotic plasmids of E6 and hDaxx were transfected into HeLa cells. Apoptosis was induced by 5-FU. The apoptotic ratio was measured by flow cytometry. Results: E6 protein had intracellular interaction with hDaxx. The apoptotic rate was rising with the increase in the transfection quantity of pcDNA3.1(-)/hDaxx in pcDNA3.1(-)/E6 and pcDNA3.1(-)/hDaxx co-transfected cells. The difference was significant (P < 0.01). Conclusion: There existed an intracellular interaction between HPV16 E6 protein and hDaxx. The over-expression of hDaxx could increase the sensitivity of E6 protein-positive HeLa cells to 5-FU. The effect was in a dose-dependent manner. HPV16 E6 protein inhibited the apoptosis of HeLa cells by interacting with hDaxx.

Reverse dot blot compared with hybrid capture 2 to detect human papillomavirus infection in cervix / 中国基层医药

Objective To evaluate the capacity of reverse dot blot(RDB)technique in screening Human papillomavirus infection in cervix.Methods 49 cases examined by hybrid capture 2(HC2)were collected for RDB technique testing.Results In the 49 cases,the positive of HR-HPV examined by HC2 was 89.8%(44/49),53.1%(26/49)by RDB,respectively.Sensitivity,specificity of RDB technique was 59.1%,100(5/5),conordance rate was 63.3%(31/49),positive predictive value and negative predictive value of RDB was 100%,21.7%,respectivdy.Kappa=0.23.Conclusion It is superior in the economy for screening human papillomavirus infection in cervix that RDB technique is developed and optimized,cheap RDB technique should be substituted for expensive HC2.

Development of a multiple PCR-based reverse line blot hybridization assay(mPCR-RLB)to detect seven sexually transmitted disease pathogens / 中华皮肤科杂志

Objective To develop a multiple PCR-based reverse line blot hydrization assay (mPCR-RLB)to simutaneously detect several STD pathogens:Neisserria gonorrhoeae,Chlamydia trachomatis,Ureaplasma urealyticum,U.parvum,Mycoplasma genitalium,M,hominis and Trichomonas vaginalis.Methods Seven pairs of biotin-labelled primer were designed and synthesized to target the 16S rRNA-23S rRNA intergenic spacer regions of Neisserria gonorrhoeae,Chlamydia trachomatis,Mycoplasma,and repetitive DNA sequence of Trichomonas to identify and subtype thesc pathogens.DNA was extracted from the referrence strains of seven pathogens and used as templates.mPCR was performed to simutaneously amplify the target regions of these pathogens.Then,the biotin-labelled amplicons were hybridized with membrane-bound specific oligonucleotide probes followed by the detection of bound amplicons with chemiluminescence assay.Serially diluted plasmids containing the target genes of pathogens were amplified with this method to detect its sensitivity.Two-hundred and eleven specimens,including 104 male urethral swabs and 107 female cervical swabs,were collected from the STD clinic of Wuhan First Hospital;mPCR-RLB and single-primer PCR were performed.For specimens with inconsistent results,nested PCR was performed to confirm the results.Results The assay sensitively and specifically identified referrence strains of the tested pathogens.The detection limit of mPCR-RLB was 100 copies for all the pathogens.Of the 211 clinical specimens,2.8%(6/21)were negative for single-primer PCR,but positive for mPCR-RLB,and nested-PCR results were consistent with those of mPCR-RLB.Conclusion mPCR-RLB is a sensitive,specific and rapid method for the detection of STD pathogens from clinical specimens.

Novel Protein Interactions of G Protein-Coupled Receptor Kinase 5 (GRK5) Searched with Yeast Two-Hybrid System

BACKGROUND AND OBJECTIVES: G protein-coupled receptors were considered to be the only natural substrates of G protein-coupled receptor kinases (GRKs). However, it was recently demonstrated that GRKs can also bind to other signal molecules. The purpose of this study was to investigate new molecules that might interact with the GRK5 using a yeast two-hybrid system to screen the cDNA library. MATERIALS AND MEDTHODS: For the yeast two-hybrid system, the "bait" was constructed to generate a LexA-GRK5 fusion protein in the EGY48 yeast strain. Rat library cDNA was inserted into the "prey". The first step in the library screening was performed by a galactose dependent leucine orthotrophism. For the second step screening, a beta-galactosidase dependent discoloration of colonies was used. Sequencing and searching of the gene bank was undertaken to characterize the clones. RESULTS: We screened a total of 1.3X10 6 clones from the cDNA library. On the first screening, 162 clones were identified by leucine orhotrophism. Another 54 clones were identified on the second screening by beta-galactosidase activation. Seven clones were selected by PCR and restriction patterns. Sequencing of seven molecules revealed that four of the clones were emerin fragments, with 2 of the remaining 3 clones being: an ID2 protein and a mitochondrial cytochrome c oxidase subunit II, with the last one remaining an unknown molecule. For the emerin fragments, their interactions with the GRK5 were confirmed by immunoprecipitation. CONCLUSION: We describe the novel protein-protein interactions of the GRK5, specifically, with three molecules. At first, these proteins may modulate the activation of the GRK5 through this specific protein-protein interaction desensitizing the beta-adrenergic receptors. Conversely, the localization of these molecules inside the cell indicates a potential new physiological role for the GRK5.

Reverse hybridization applied in detection on human papillomavirus infection of twenty-three subfamilies / 中华检验医学杂志

Objective To establish a method of reverse hybridization to detect five subfamilies of low risk Human Papillomaviruses(HPV6,11,42,43 and 44)and eighteen subfamilies of high risk HPV (HPV16,18,31,33,35,39,45,51,52,53,56,58,59,66,68,73,83 and MM4)in one reaction.Methods Special probes for twenty-three HPV subfamilies were fixed on nylon membrane bars,biotin labeled general primers mediated polymerase chain reaction(GP-PCR)were applied in HPV DNA amplification.PCR amplified DNA fragments were reversely hybridized with special probes that were fixed on the membranes. All samples(136)detected by reverse hybridization method were paralleled with the methods of Hybridization Capture Ⅱ(HC-Ⅱ)and sequencing.Results Positive rate of the 136 samples detected by reverse hybridization was 41.9%,while HC-Ⅱ 42.6% and sequencing 40.4%.Reverse hybridization detection indicated coherence with the other two methods(Kappa 0.8644 and 0.9089,respectively).While sequencing was lab standard for DNA test,the sensitivity was 96.36%,specificity was 95.06%,accuracy was 95.59%.Conclusions Method of reverse hybridization is adaptable to 23 kinds of HPV subfamilies, which can confirm the exactly subfamilies of HPV infection.This method is adaptable in clinical detection of HPV,with high sensitivity,high specificity,simply and convenient operation and the results are easily to be read.

Interaction between HCV core protein and HCBP6 in mammalian cells / 解放军医学杂志

Objective To study the interaction between HCV core protein and HCBP6 in mammlian cells using CheckMateTM Mammalian Two-Hybrid System.Methods cDNA fragments encoding HCV core protein and HCBP6 were amplified by PCR and subsequently cloned into pGEM-T vector.After verified by sequencing,the target fragments were subcloned into mammalian two-hybrid plasmids,pBIND and PACT,respectively.The recombinant plasmids,pBIND-core and pACT-HCBP6 were co-transfected into HepG2 cells with reporter plasmid pG5luc.pBIND+pACT were induced as background controls,pBIND-Myod+pACT-Id as positive controls,and pBIND-core+pACT,pBIND+pACT-HCBP6 as blank controls.The expression of G5luc,which indicated the interaction between HCV core protein and HCBP6 in mammlian HepG2 cells,was assayed through Dual-Luciferase Report Assay System and Turner Biosystems Veritas Microplate Lunimometer.Results The recombinant vectors pBIND-core and pACT-HCBP6 were successfully constructed.When co-transfected into HepG2 cells with reporter plasmid pG5luc,there were significant differences in the luciferase activity in the pBIND-core and pACT-HCBP6 groups compared with every control group.Conclusions HCBP6 can interact with HCV core protein in HepG2 cells,which provides clues for further study on the function of HCBP6 and core proteins,and on the mechanism of HCV core in cell apoptosis and cancer transformation.

Screening exonic genes of proteins in hepatocytes interacting with HBEBP2 by yeast two-hybrid technique / 解放军医学杂志

Objective To construct the eukaryotic expression vector of HBEBP2 gene,and screen the exonic genes of proteins in hepatocytes interacting with HBEBP2.Methods The DNA fragment of HBEBP2 was amplified by PCR.The eukaryotic expression vector pGBKT7-HBEBP2 was constructed successfully by yeast-two hybrid system 3 and then transformed into yeast cells AH109.The transformed AH109 were then mated with yeast cells Y187 containing hepatic cDNA library plasmid.The diploid yeast cells were plated on synthetic dropout nutrient medium(SD/-Trp/-Leu/-His/-Ade) containing X-?-gal for the first selection.Library plasmids pACT2-DNA were extracted and co-transformed into yeast cell AH109 together with pGBKT7-HBEBP2.Then the yeast cells were plated on synthetic dropout nutrient medium(SD/-Trp/-Leu /-His/-Ade) containing X-?-gal for the second screening to eliminate false positive clones.The real positive clones were sequenced and analyzed by bioinformatics.Results Six proteins binding to HBEBP2 were screened,including human sapiens lactate dehydrogenase D,human sapiens mitochondrion,human sapiens mannose,human sapiens aldehyde oxidase,human sapiens serpin peptidase inhibitor and other two unknown proteins.Conclusions A novel class of proteins in hepatocytes interacting with HBEBP2 has been obtained.It is presumed that HBEBP2 protein is correlated with glycosylation,lipid metabolism and cell proliferation,etc.

SCREENING AND IDENTIFICATION OF A NOVEL HEPATITIS B VIRUS E ANTIGEN BINDING PROTEIN E-36 FROM HEPATOCYTE cDNA LIBRARY / 解放军医学杂志

Objective To investigate the biological functions of hepatitis B virus e antigen (HBeAg) protein. Methods Yeast-two hybrid technique was performed to look for proteins interacting with HBeAg from hepatocytes. HBeAg bait plasmid was constructed by cloning the HBeAg gene into carrier plasmid pGBKT7, then the yeast AH109 (a type) was transformed. The transformed yeast cells was amplified and mated with yeast cells Y187 (?type) containing liver cDNA library plasmid pCAT2 in 2?YPDA medium. Diploid yeast cells were plated on synthetic dropout nutrient medium and were selected for two times. Results Plasmids of positive colonies were extracted and analyzed by DNA sequencing and BLAST in GenBank. Conclusions After the full sequence of new gene E-36 was amplified from the mRNA of HepG2 cells by reverse transcription polymerase chain reaction (RT-PCR) and cloned into pGADT7 vector, the recombinant plasmid was translated by reticulocyte lysate and analyzed by immuno-coprecipitation technique in vitro together with HBeAg. The result provided some new clues for the study of the biological functions of HBeAg.

Identification of differentially expressed genes in endometrium during the window of implantation using suppression substractive hybridization / 中华妇产科杂志

Objective To identify genes differentially expressed in the window of implantation and explore the molecular basis of the development of endometrial receptivity.Methods A subtracted cDNA library of the window of implantation was constructed by suppression subtractive hybridization(SSH) method.The screened clones of the subtracted library were sequenced and GenBank homology search was performed.The differential expression of ribosomal protein(RP)L7,RPL7 pseudogene(RPL7p),RPL19 and tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta polypeptide(YWHAZ) were further confirmed by RT-PCR.Results After sequencing and GenBank homology search of 50 clones, 35 differentially expressed genes were detected in the window of implantation,of which 23 were known genes,and 12 were unknown genes.Some of the known genes have been proved to be associated with implantation,while others were firstly screened out by us.The results of RT-PCR confirmed that RPL7, RPL7p,RPL19 and YWHAZ were highly expressed in the window of implantation,0.75?0.21,1.72? 0.30,1.23?0.31,and 1.28?0.08,respectively.Conclusions SSH is a useful technique to detect differential expression genes and an effective method to clone novel genes.It provides a new method to investigate the molecular basis of the development of endometrial receptivity.

Screening of differentially expressed genes in placentas with hepatitis B virus infection by suppression subtractive hybridization technique / 中华妇产科杂志

Objective To screen differentially expressed genes in placentas with hepatitis B virus (HBV)infection and to discuss the molecular mechanism of HBV intrauterine infection.Methods Thirty placenta tissue specimens from HBsAg and HBV DNA positive pregnant women were used as the study group and 30 placenta tissue specimens from normal pregnant women with HBsAg and HBV DNA negativity were served as the control group.The suppression subtractive hybridization(SSH)technique was used.Total RNAs of placenta tissue of the study group were mixed as the tester,and total RNAs of placenta tissue of the control group were mixed as the driver.A subtractive cDNA library was constructed by PCR-selective cDNA subtraction systems.Amplifications of the library were carried out with E.coil strain DH5? by reverse spot hybridization.RT-PCR confirmed that phosphatidylinositol 3-kinase(PI3K)was up-regulated in placenta tissue with HBV infection.Results Colony PCR showed that the clones contained 200-1000 bp inserts. Thirty five clones were confirmed by reverse spot hybridization and analyzed by sequencing and bioinformatics.Thirty three known genes and 2 genes with unknown function were obtained.RT-PCR preliminarily confirmed that PI3K gene was up-regulated in HBV infected placenta.Conclusions The differentially expressed genes in placentas with hepatitis B virus(HBV)infection using SSH technique has been screened out successfully.These differentially expressed genes encoding proteins participating in cell vital metabolism and malformation,and signal conduction-antiapoptosis pathway.This finding brings some new clues for studying the mechanisms of HBV intrauterine infection.

Screening and sero-immunoscreening of ovarian epithelial cancer associative antigens / 中华妇产科杂志

Objective To explore epithelial ovarian cancer(EOC)antigens that are potentially useful for cancer early detection and therapy.Methods A high quality cDNA library derived from ascites tumor cells of EOC patients(3 cases of serous EOC,1 case of mucinous EOC,and 1 case of endometrial carcinoma of ovary)was constructed,and the method of combining serological analysis of recombinant cDNA expression libraries(SEREX)and suppression subtractive hybridization(SSH)was used for screening cDNA library.All of the positive clones were sequenced and bioinformatics analysis with BLAST software in GenBank was performed.Serological mini-arrays of recombinant tumor antigens(SMARTA)was used to investigate the prevalence of autoantibodies to these antigens in both 96 ovarian cancer patients and 96 cancer-free controls.Results Fifty-five positive clones encoding different antigenic genes of EOC recognized by IgG and(or)IgM were obtained.It showed that these 55 clones derived from 45 distinct genes and these genes could be grouped into 6 classes as following according to homology with known expressed sequence tag(EST):(1)known ovarian carcinoma related genes:BARD1,et al;(2)homologous genes with other tumors:TM4SF1,et al;(3)homologous genes with special tissues:ILF3,FXR1,et al;(4) homologous genes with special function:TIZ,C1 D,et al;(5)embryo originating genes:PKHD1,et al; (6)novel genes:OV-189,et al.SMARTA results showed that the positive ratio of five EOC antigens TM4SF1(28% vs 9%),CID(21% vs 6%),BARD1(23% vs 5%),FXR1(23% vs 8%),OV-189 (31% vs 13%)which reacting with their IgG autoantibodies,three antigens TIZ(26% vs 8%),FXR1 (28% vs 11%),and OV-189(18% vs 7%)which reacting with their IgM autoantibodies in patients was higher than in controls(P

Screening of interferon?binding proteins by yeast-two hybrid technique / 中华传染病杂志

Objective To screen proteins binding with interferon?(IFN?)from human hepatic cDNA libraty by yeast-two hybrid technique.Methods The IFN?gene was amplified by polymerase chain reaction(PCR)and constructed into pGBKT7 vector as the bait plasmid in yeast-two hybrid system3,pGBKT7-IFN?was then transfected into yeast AH109.The transfected yeast were 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 synthetic dropout nutrient medium(SD/-Trp-Leu-His-Ade)containing X-?-gal for selecting.After plasmid extracting and en- zyme cutting analysis,the blue colonies were performed sequence analysis,the results were analyzed by bioinformatics.Results IFN?gene was successfully cloned and expressed in yeast cells.Thirty- four positive colonies were obtained using yeast-two hybrid technique.After sequence analysis,eight clones were found may have a binding effect with IFN protein.Conclusions IFN?genes was success- ful cloned and eight proteins that could bind with IFN?protein were also screened.

Interaction of novel gene AngRem104 and Bardet-Biedl syndrome 2 protein in mammalian cells / 中华肾脏病杂志

Objective To screen for proteins interacting with novel gene AngRem104 and to identify the putative interaction of novel gene AngRem104 and Bardet-Bied1 syndrome 2 (BBS2) protein in mammalian cells. Methods The yeast strain AH109 was transformed with AngRem104pGBKT7/c-myc and yeast-mating was utilized to screen for interacting proteins with AngRem104 in pretransformed human kidney cDNA library. The human embryonic kidney (HEK 293T) cells were transformed with two recombined plasmids,AngRem104-pcDNA3.1/V5-His and BBS2-pCMV/c-myc. Mouse anti-human V5 monoclonal antibody and mouse anti-human c-myc monoclonal antibody were used in immunoprecipitation and immunoblot analysis, respectively. Results Seven proteins that interact with AngRem104, including BBS2 were identified. The AngRem104-V5 and the BBS2-c-myc fusion protein were detected respectively in the immunoprecipitation by anti-c-myc and anti-V5 antibody. Conclusion The novel gene AngRem104 may interact with BBS2 protein in mammalian cells,which provides insights as to the function exploration of novel gene AngRem104 and the pathogenesis investigation of Bardet-Biedl syndrome.

Cloning of telomere-associated protein T-STAR and the interaction between T-STAR and hTERT / 解放军医学杂志

Objective To clone the telomere-associated protein T-STAR and study the relationship between T-STAR and the telomerase catalyzed subunit hTERT in mammalian cells. Method The expression vector pGBKT7-hTERT was constructed and acted as a decoy in cDNA library screened by yeast two-hybrid technology. Recombinant vectors pVP16-T-STAR and pM-hTERT were constructed and co-transfected with report gene CAT into SGC-7901 cells with liposome. pM-53+pVP16-T and pM3-VP16 were introduced as positive controls, pM-53+pVP16-CP as negative control, and pM-hTERT+pVP16, pM+pVP16-T-STAR and pM+pVP16 as crossing negative controls. The expression of CAT was assayed by ELISA. Results The eukaryotic expression vector pGBKT7-hTERT was successfully constructed. One positive clone achieved by cDNA library screening was sequenced and compared with the isogenous sequences in GenBank by Blast software via Internet. As a result, T-STAR, a recorded cDNA sequence was obtained. The recombinant vectors of pVP16-T-STAR and pM-hTERT were constructed successfully and co-transformed into gastric cancer cells SGC-7901. The OD value of reported gene CAT was 0.258, which was significantly higher than that of the negative and crossing negative controls (0.002-0.015). It revealed that T-STAR interacted with hTERT in the mammalian cells. Conclusions T-STAR interacts with hTERT in the gastric cancer cells. T-STAR may be a new member of telomere-associated protein, and it participates in the regulation of telomerase through hTERT.