Construction and self-activation detection of yeast two-hybrid bait plasmid of human programmed cell death ligand 1 immunoglobulin variable region domain gene / 中国生物制品学杂志

@#Objective To construct a yeast two-hybrid recombinant bait plasmid of human programmed cell death ligand 1(PD-L1)immunoglobulin variable region(IgV)domain gene,detect its expression in yeast and detect the cytotoxicity and self-activation of PD-L1 IgV protein as well as the interaction between PD-L1 IgV and human thioredoxin(hTrx).Methods Human PD-L1 was analyzed by bioinformatics method,and primers were designed to amplify PD-L1 IgV domain based on the coding region of PD-L1 gene registered in NCBI GenBank database. PCR amplification was carried out with pENTERPD-L1 plasmid as template,and then cloned into yeast two-hybrid bait vector pGBKT7. The recombinant bait plasmid and pGBKT7 empty vector were transformed into Y2HGold yeast cells respectively,and the PD-L1 IgV gene and its expression were detected by PCR and Western blot;Meanwhile,the protein toxicity and self-activation of PD-L1 IgV were detected,and the interaction between PD-L1 IgV and hTrx was detected by drip plate method.Results The bioinformatics analysis results of PD-L1 were consistent with related reports. The recombinant bait plasmid pGBKT7-PD-L1 IgV was correctly constructed,and Y2HGold positive clone was obtained,in which PD-L1 IgV was stably expressed. The empty vector pGBKT7 and recombinant bait plasmid pGBKT7-PD-L1 IgV grew well on SD/-Trp and SD/-Trp/X-α-Gal plates with the same colony size and number and white colony,but they did not grow on SD/-Trp/X-α-Gal/AbA plates,which indicated that PD-L1 IgV protein had no toxicity and no self-activation effect on yeast. The results of drip plates test showed that all experimental groups grew well on SD/-Trp/-Leu plate,while only positive control group grew on SD/-Trp/-Leu/X-α-Gal/AbA plate and showed blue color,which indicated that bait protein PD-L1 IgV and hTrx did not self-activate,and there was no interaction between them.Conclusion Recombinant human PD-L1 IgV bait plasmid was successfully constructed. PD-L1 IgV protein showed no toxicity and self-activation effect on yeast cells,and there was no interaction between PD-L1 IgV and hTrx. Subsequently,hTrx can be used to construct a peptide aptamer library,from which peptide aptamers that specifically bind to PD-L1 IgV can be screened.

Light-driven CO2 conversion system: construction, optimization and application / 生物工程学报

The use of light energy to drive carbon dioxide (CO2) reduction for production of chemicals is of great significance for relieving environmental pressure and solving energy crisis. Photocapture, photoelectricity conversion and CO2 fixation are the key factors affecting the efficiency of photosynthesis, and thus also affect the efficiency of CO2 utilization. To solve the above problems, this review systematically summarizes the construction, optimization and application of light-driven hybrid system from the perspective of combining biochemistry and metabolic engineering. We introduce the latest research progress of light-driven CO2 reduction for biosynthesis of chemicals from three aspects: enzyme hybrid system, biological hybrid system and application of these hybrid system. In the aspect of enzyme hybrid system, many strategies were adopted such as improving enzyme catalytic activity and enhancing enzyme stability. In the aspect of biological hybrid system, many methods were used including enhancing biological light harvesting capacity, optimizing reducing power supply and improving energy regeneration. In terms of the applications, hybrid systems have been used in the production of one-carbon compounds, biofuels and biofoods. Finally, the future development direction of artificial photosynthetic system is prospected from the aspects of nanomaterials (including organic and inorganic materials) and biocatalysts (including enzymes and microorganisms).

Synergistic integration of metal nanoclusters and biomolecules as hybrid systems for therapeutic applications

Therapeutic nanoparticles are designed to enhance efficacy, real-time monitoring, targeting accuracy, biocompatibility, biodegradability, safety, and the synergy of diagnosis and treatment of diseases by leveraging the unique physicochemical and biological properties of well-developed bio-nanomaterials. Recently, bio-inspired metal nanoclusters (NCs) consisting of several to roughly dozens of atoms (<2 nm) have attracted increasing research interest, owing to their ultrafine size, tunable fluorescent capability, good biocompatibility, variable metallic composition, and extensive surface bio-functionalization. Hybrid core-shell nanostructures that effectively incorporate unique fluorescent inorganic moieties with various biomolecules, such as proteins (enzymes, antigens, and antibodies), DNA, and specific cells, create fluorescently visualized molecular nanoparticle. The resultant nanoparticles possess combinatorial properties and synergistic efficacy, such as simplicity, active bio-responsiveness, improved applicability, and low cost, for combination therapy, such as accurate targeting, bioimaging, and enhanced therapeutic and biocatalytic effects. In contrast to larger nanoparticles, bio-inspired metal NCs allow rapid renal clearance and better pharmacokinetics in biological systems. Notably, advances in nanoscience, interfacial chemistry, and biotechnologies have further spurred researchers to explore bio-inspired metal NCs for therapeutic purposes. The current review presents a comprehensive and timely overview of various metal NCs for various therapeutic applications, with a special emphasis on the design rationale behind the use of biomolecules/cells as the main scaffolds. In the different hybrid platform, we summarize the current challenges and emerging perspectives, which are expected to offer in-depth insight into the rational design of bio-inspired metal NCs for personalized treatment and clinical translation.

Screening and Analysis of Proteins Interacting with HCMV UL145 from a Human Fetal Brain cDNA Library / 中国医科大学学报

Objective To screen a human fetal brain cDNA library for proteins that can interact with HCMV UL145 using a yeast two-hybrid system.Methods A bait plasmid (pGBKT7-UL145) was constructed.Using HCMV UL145 as bait,a human fetal brain cDNA library was screened and proteins interacting with UL145 were identified using bioinformatic methods to sequence and analyze the positive clones.Results Three clones interacting with HCMV UL145 were found,and identified as FOXG1.Conclusion Several proteins interacting with HCMV UL145 in the human fetal brain cDNA library were identified as FOXG1,indicating that this protein may play an important role in the course of HCMV infection.

Screening and identification of proteins interacting with HCMV UL132 protein / 吉林大学学报(医学版)

Objective:To screen the proteins interacting with the human cytomegalovirus(HCMV)UL132 protein from the human fetus brain cDNA library by using Yeast Two-Hybrid System, and to elucidate the possible mechanism of UL132 protein in congenital cytomegalovirus infection.Methods:The HCMV UL132 fragment was amplified by polymerase chain reaction,the amplified HCMV UL132 fragment and expression vector pGBKT7 were digested and purified,and the HCMV UL132 fragment was linked to the vector pGBKT7.The pGBKT7-UL132 was constructed and transformed to yeast AH109, then the Human Fetal Brain DNA Library DNA was transformed into AH109 yeast.Using HCMV UL132 as abait, a human fetus brain cDNA was screened and the proteins interacting with UL132 protein were searched, the positive clone was sequenced and analyzed by bioinformatics methods.Results:The bait expression vector pGBKT7-UL132 was successfully constructed.The results of double enzyme digestion showed that there were two visible bands of 800 and 7 000 bp, respectively.After transformation of library plasmid, the transformation efficiency was calculated, and the transformation efficiency was 6.6×103 cfu· μg-1.There were 95 blue clones by X-gal coloration reactionsequencing and there were 10 clones interacting with the protein encoded by UL141 protein.The BLAST analysis showed that 7 of them were highly homologous with CAML.Conclusion:CAML might be one interaction protein with HCMV UL132 in Human Fetus Brain cDNA Library,suggesting that the interaction may be associated with the invasion and proliferation of the HCMV.

New ligands of the Cellular Retinoic Acid-Binding Protein 2 (CRABP2) suggest a role for this protein in chromatin remodeling

Retinoic acid (RA) regulates the transcription of a series of genes involved in cell proliferation, differentiation and apoptosis by binding to the RA Receptor (RAR) and Retinoid X Receptor (RXR) heterodimers. The cellular retinoic acid-binding protein 2 (CRABP2) is involved in the transport of RA from the cytosol to specific RA receptors in the nucleus, acting as a coactivator of nuclear retinoid receptors. In order to have a better understanding of the role of CRABP2 in RA signaling, we used the yeast two-hybrid system as a tool for the identification of physical protein-protein interactions. Twenty-three putative CRABP2-interacting proteins were identified by screening in the presence of RA, five of which are related to transcription regulation or, more specifically, to the process of chromatin remodeling: t-complex 1 (TCP1); H3 histone, family 3A (H3F3A); H3 histone, family 3B (H3F3B); β-tubulin (TUBB) and SR-related CTD-associated factor 1 (SCAF1). These results suggest a more direct role for CRABP2 in chromatin remodeling and may be a starting point for the elucidation of the fine-tuning control of transcription by RA receptors...

Function analysis of outer membrane protein VirB4 in Brucella infecting embryo trophoblastic cells / 中国人兽共患病学报

Potential target proteins binding to VirB4 of type Ⅳ secretion system were screened during Brucella infected bovine embryonic trophoblast cells .Brucella VirB4 genes were amplified by PCR with species-specific primers .Expression vector pGBKT7-virB4 was constructed and analysed by sequencing and restriction enzymes ,transforming to the yeast strain Y187 and testing self-activation and toxicity .The cells model and cDNA library of bovine embryonic trophoblast cells infected with Brucella abortus strain were constructed respectively .Utilizing yeast two-hybrid system was employed to screen the target proteins of bovine embryo trophoblastic cells which was conjunctive with virB4 .These proteins were detected by real-time fluo-rescence quantitative PCR .The results suggested that bait plasmid pGBKT7-virB4 was successfully transformed into the Y187 and there was no toxicity and self-activation;the cDNA library of bovine embryonic trophoblast cells infected with Brucella abortus strain was constructed .There screened 13 positive plasmids in which Q10 and SLC3A2 were up-regulated at the mRNA level .In this paper ,we reported the interactions between the VirB4 protein of Brucella and the bovine embryo trophoblastic cells ,which provide an upstream work for further elucidating the pathogenesis of Brucella infection of the host cell .

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 .

Characterization of microbial community structure in a hybrid biofilm-activated sludge reactor for simultaneous nitrogen and phosphorus removal.

The microbial communities in a hybrid biofilm-activated sludge reactor (HY) for nitrogen and phosphorus removal were characterized by 16S rRNA-based clone libraries and phylogenetic analysis. The hybrid reactor removed over 90% of COD, 92% of total nitrogen (TN) and 95% of total phosphorus (TP) from the municipal wastewater, respectively. The mean removal rates of COD, TN, and TP in the conventional suspended activated sludge reactor were above 80%, 80% and 94%, respectively. Community structures were determined by phylogenetic analyses of six clone libraries (each nearly 100 clones). The dominant bacterial group with which clones were affiliated to the b subclass of the Proteobacteria (31%~77%), following the Bacteroidetes group (10%~34%). In addition, several clone groups affiliated with unknown bacterial assemblages were identified in the clone libraries. Acinetobacter sp., which was thought to had played an important role in phosphate removal systems, was scarcely represented by clone sequences in both libraries. Differences in community structure were observed between the hybrid reactor and activated sludge reactors. Such differences may account for the differing wastewater treating capabilities of the two different systems.

Murine Cytomegalovirus IE3 Protein Interacts with Ankrd17 / 华中科技大学学报（医学）（英德文版）

Murine cytomegalovirus (MCMV) IE3 protein is a multifunctional viral protein that interacts with several target proteins of both viral and host cellular origin.To investigate the biological function of IE3 in the pathogenesis of the brain disorders caused by CMV,a screening for host cellular proteins that could interact with IE3 was performed.By yeast two-hybrid screening,ankyrin repeats domain 17 (Ankrd17,also known as Gtar) was identified as a host factor that could interact with IE3.This interaction was verified by yeast two-hybrid assay and chemiluminescent co-immunoprecipitaion.Mapping analysis suggested that the 1-148 residues of IE3 were responsible for the interaction.These results suggested that the interaction between Ankrd17 and IE3 may play a key role in the pathogenesis of MCMV-associated disease.

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.

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.

Screening of protein interacting with the transcript of UL128 gene showed two protein patterns by yeast two-hybrid from human fetus brain cDNA library / 中华微生物学和免疫学杂志

Objective Using yeast two-hybrid system to screen the proteins which can interact with the human cytomegalovirus (HCMV) UL128 which have two difference transcription structure from human fetus brain cDNA library, and compare the difference with structure and function of interacting proteins. Methods Two fragments of UL128 were amplified by 3'RACE and 5'RACE technology, the length are 519 bp and 642 bp, respectively. The "bait plasmid" (named as pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp) was constructed successfully. Using pGBKT7-UL128-519 bp and pGBKT7-UL128-642 bp as a bait, a human fetus brain cDNA was screened and the proteins interacting with UL128-519 bp and UL128-642 bp encoded protein were searched, and the positive clones were sequenced and analyzed by bioinformatic methods. Results EFEMP2 interacting with HCMV UL128-519 bp were identified, THY-1 interacting with HCMV UL128-642 bp were identified. Conclusion EFEMP2 and THY-1 proteins interacting with HCMV UL128-519 bp and UL128-642 bp in human fetus brain cDNA library were successfully screened, but same proteins weren't found from the proteins interacting with UL128-519 bp and UL128-642 bp protein, UL128-519 bp and UL128-642 bp protein may be play an different effect in the process of infect by HCMV.

Screening of proteins binding to mouse cytomegalovirus M122 protein from mouse brain cDNA library by yeast two-hybrid system / 中华微生物学和免疫学杂志

Objective To investigate the biological function of M122 in pathogenesis of MCMV in developmental brain disorders and brain damage, screening for mouse brain cDNA library interacting with M122 was performed by a yeast two-hybrid system. Methods The reconstructed bait plasmid pGBKT7-M122 was transformed into yeast cells AH109 and screened on the nutrient deficiency medium SD/-Trp. After express of the bait protein in AH109 yeast strains was detected by Western blot analysis, yeast-two hybrid screening was performed by mating AH109 with Y187 containing mouse brain cDNA library plasmid. The diploid yeast cells were plated on the nutrient deficiency medium SD/-Trp/-Leu/-His/-Ade. The second screening was performed with SD/-Trp/-Leu/-His/-Ade containing X-α-gal. The plasmids in positive colonies were extracted and transformed into E. coli JM109 cells. After plasmid DNA in JM109 cells were extracted form positive colonies and sequenced, the results were analyzed by bioinformatic methods. The interactions between M122 protein and the protein obtained from positive colonies were further confirmed by repeating yeast-two hybrid. Then, autoactivations of the proteins obtained from positive colonies were detected.Results The reconstructed bait plasmid was transformed into yeast cells AH109 successfully. The bait protein expressed in the yeast cells AH109 stably. 24 proteins interacting with MCMV M122 were screened, including syntaxin 8 ( Stx8 ), phosphoglucomutase 2 ( Pgm2 ), potassium voltage-gated channel, shaker-related subfamily, beta member 1 ( Kcnab1 ), collagen, type ⅪⅩ, alpha 1 ( Col19a1 ), archain 1 ( Arcn1 ), cytidylate kinase( Cmpk), DnaJ(Hsp40) homolog, subfamily A, member 1 (Dnaja1), ATPase, Na+/K + transporting, beta 3 polypeptide( Atp1b3 ), SH3-domain GRB2-like ( endophilin ) interacting protein 1 ( Sgip1 ),ankyrin repeat domain 17 (Ankrd17), Smg-7 homolog, nonsense mediated mRNA decay factor(Smg7),sperm associated antigen 9 ( Spag9 ), FK506 binding protein 1a ( Fkbp1a), MYST histone acetyltransferase monocytic leukemia 4 ( Myst4), hyaluronan and proteoglycan link protein 1 ( Hapln1), autophagy-related 3 (Atg3), splicing factor, arginine/serine-rich 5 ( Sfrs5 ), zinc finger, C3HC-type containing 1 ( Zc3hc1 ),thioredoxin-related transmembrane protein 1 ( Txndc1 ), adaptor protein complex AP-1, gamma 1 subunit (Ap1g1), Cullin 1 ( Cul1 ), and so on. Three of them were formerly unknown proteins. M122 protein could interact with the proteins obtained from positive colonies in the yeast cells AH109. Ap1g1 and Cul1 were proved to have autoactivation. Conclusion A class of proteins in brain interacting with M122 has been obtained. It is presumed that these proteins are correlated with neuropathogenesis of the brain disorders caused by CMV, but the candidates still need further confirmation for the interaction.

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 a Yeast Two-Hybrid Bait Vector and Its Effect on the Growth of Yeast Cells and the Self-Activating Function of Reporter Genes for Screening of HPV18 E6-Interacting Protein / 华中科技大学学报（医学）（英德文版）

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.The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector were transformed into the yeast cell AH109,respectively.After they were cultured respectively in YPDA liquid medium and nutrition-deficient culture medium,their toxicity and transcriptional activation were tested by both the phenotype assay and the color assay.The bait plasmid HPV18 E6 was successfully obtained.After being cultured in YPDA liquid medium for 16h,the A600 nm values of two yeast fluids were 0.98±0.03 and 0.99±0.02,respectively.The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector could grow to white colonies on SD/-Trp/X-α-gal plates,while no colony could survive on SD/-His/-Trp/X-α-gal,SD/-Ade/-Trp/X-α-gal plates,indicating that the bait plasmid pGBKT7-HPV18 E6 was constructed successfully and expressed correctly,and could not activate the transcription of reporter gene alone.The yeast two-hybrid GAL4 system 3 can be utilized to find HPV18 E6 interacting proteins.

Screening of Protein Interacting with HCMV UL130 Protein by Yeast Two Hybrid from Human Fetus Brain cDNA Library / 中国医科大学学报

Objective To screen the human proteins interacting with human cytomegalovirus（HCMV）UL130 from human fetus brain cDNA library by GAL4 two-hybrid system 3 technique and analyze the corresponding coding sequences.Methods The ＂bait plasmid＂（named as pGBKT7-UL130）was constructed.By using HCMV UL130 as the bait,a human fetus brain cDNA library was screened and the proteins interacting with UL130 protein were searched.The positive clones were sequenced and analyzed by bioinformatic methods.Results Nine clones interacting with HCMV UL130 were identified,two of them were synaptosome-associated protein（SNAP）.Conclusion Some proteins interacting with HCMV UL130 in human fetus brain cDNA library were successfully screened.SNAP might play an important role in HCMV infection pathogenesis.

High-throughput screening of telomerase inhibitors in actinomycetes fermentation broth / 中国药理学通报

Aim To discover specific telomerase inhibitors by high-throughput screening in the more than 2 000 strains of actinomycetes fermentation broth.Methods After verifying the three kinds of screening models for telomerase inhibitor by yeast PCR,the three yeast strains were inoculated overnight into yeast liquid culture which was lacking in histidine and contained 3-AT.Then concentration of yeast strains in culture liquid and dosage of screening sample were regulated for better adjustment to the high-throughput screening.At last the adjusted liquid culture containing yeast strain piped to 96-well plates,drug samples and control article were also added,then the yeast was incubated in the thermostat oscillator.At the same time OD_(595nm) of the yeast liquid was measured every 12 h by using Multifunctional Microplate Analyzer,and the samples whose inhibitory efficiency was over 0.45 were selected for secondary screening.Results The high-throughput screening method related to yeast three-hybrid system was effective in screening samples that had potential telomerase inhibitory ability.The initial concentration of yeast liquid OD_(595nm) was 0.04 and the best dosage of samples was 5 ml.14 active samples,whose inhibitory efficiency was over 0.45,were founded after primary and secondary screening.Conclusions High-throughput screening method related to yeast three-hybrid system is simple and stable in discovering telomerase inhibitors,and 14 new antitumor compounds are identified,which lays the foundation in the development of new anti-tumor agents.

Klotho: a protein molecule interacting with FPC protein / 中国病理生理杂志

AIM: To investigate the role of fibrocystin/polycystin (FPC) in autosomal recessive polycystic kidney disease (ARPKD) development by means of screening the protein interaction using yeast two-hybrid approach. METHODS: The constructed pGBKT7-FPC was used as the bait to screen the pre-transformed human fetal kidney cDNA expression library by yeast two-hybrid assay to obtain the host cell protein which interacted with C-terminal region of FPC. The sequence transformation screening experiment was applied to confirm the protein interactions in yeast. RESULTS: After yeast mating and co-transformation screening analysis, Klotho (KL) was selected from the host cells and the interaction between KL and FPC was further confirmed. CONCLUSION: C-terminal region of FPC can interact with KL, which probably provide the approach for further studying the role and biochemistry mechanism of FPC protein in ARPKD.