Detection for Transcriptional Activity of Alternaria Tenuissim Protein Elicitor in Yeast Two-hybrid System

The peaT1 gene fragment was amplified from pGEM-6p-l-peaT1 by PCR, and recovered target gene was cloned into pLexA vector. After digestion and sequencing, the bait vector pLexA-peaT1 was transformed into yeast strain EGY48 [p8op-lacZ] by PEG/LiAC, and the transcriptional activity of bait vector was detected. The results showed that recombinant bait plasmid pLexA-PEMG1 was constructed, for the two bands of recombinant bait plasmid in agarose gel eleetrophoresis were expected after digesting by restriction endonuclease EcoR I and Xho I. Therefore, the recombinant bait plasmid could be used in yeast two-hybrid system to screen a cDNA library.

Screening of Extracellular Binding Proteins of Rice Receptor-like Kinase CR4 by the Yeast Two-hybrid

[Objective] The research aimed to find the extracellular binding proteins of CR4.[Method] The extracellular domain of OsCR4 was as the bait protein,and the yeast two-hybrid was used to screen cDNA library of seedling which was cultivated 14 d.[Result] A lot of proteins which included a peroxide B（D26484）,a methionine thioredoxin reductase（ABF96078）and an unknown function protein were gained.[Conclusion] It provided the theory basis for studying the signal transduction mechanism of CR4.

Molecular epidemiological study on pre-X region of hepatitis B virus and identification of hepatocyte proteins interacting with whole-X protein by yeast two-hybrid

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.

Screening of genes of proteins interacting with p7 protein of hepatitis C virus from human liver cDNA library by yeast two-hybrid system

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 cloning the gene of p7 protein into pGBKTT, 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 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 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 nudeoporin 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 assodated protein.

Shared and discrete interacting partners of ELL1 and ELL2 by yeast two-hybrid assay

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.

Analysis of Environmental Endocrine Disrupting Activities in Wastewater Treatment Plant Effluents Using Recombinant Yeast Assays Incorporated with Exogenous Metabolic Activation System

Objective To measure the endocrine disrupting chemicals （EDCs） in wastewater and evaluate the EDCs removal efficiencies in the municipal wastewater treatment plants （WWTP）. Methods A battery of in vitro recombinant yeast bioassays incorporated with exogenous metabolic activation system （rat liver preparation, S9 mix） was conducted to assess the estrogen receptor （ER）, androgen receptor （AR）, progesterone receptor （PR）, and thyroid receptor （TR） ant/agonistic activities of effluents collected from Datansha WWTP. Results The indirect estrogenic, anti‐androgenic, anti‐progesteronic, and anti‐thyroidic activities were observed in the influent. The removal efficiencies of EDCs were above 74%, suggesting that the present wastewater treatment processes were good enough to remove most of these indirect endocrine disrupting chemicals. Conclusion The incorporation of exogenous metabolic capacity into the test system was valid for the study of indirect effects on ER, AR, PR, and TR.

Construction of a Three-frame Yeast Two-hybrid cDNA Library of Fusarium oxysporum

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.

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

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...

Screening of FOXP3-interacted proteins by yeast two-hybrid technique

Objective： To screen the proteins interacting with the Treg specification factor forkhead box protein P3 （FOXP3） by yeast two-hybrid system, Methods： Human FOXP3 gene was amplified by nest RT-PCR from peripheral blood mononuclear cells （PBMC） and inserted into plasmid pGBKT7 to construct the bait vector, then the self-activation and toxicity of the bait vector in host yeast strain AH109 were observed. Thereafter, a human liver cDNA library was screened by the bait vector. The positive clones were selected out by nutrient-deficient culture and back-hybridizing. The sequences from the candidate positive clones were blasted and analyzed by bioinformatics methods. Results： The constructed bait vector encoding FOXP3 was found no self-activation and toxicity in yeast AH109. Three proteins which interacted with FOXP3, including tumor protein D52, splicing factor 3b subunit 1 and hypothetical protein, were identified. Conclusion： Three new candidate proteins interacting with FOXP3 are selected out by this yeast two-hybrid system and library, which may facilitate the further study of FOXP3 in Treg.

Screening proteins that interact with mutant superoxide dismutase 1 from familial amyotrophic lateral sclerosis using a yeast two-hybrid system

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.

Screening of Host Proteins Interacting with PorcineEpidemic Diarrhea Virus (PEDV) N Protein by YeastTwo-hybrid System

[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.

The Self-activated Experimental of T_(1083) Substitution Mutation Vector pGBKT7-TS in Yeast

[Objective] The research aimed to study the self-activation test of inverting T1083 substitution mutation BD fusion vector pGBKT7-TS into yeast,and discuss whether its expression product can be used as bait for further two-hybrid screening.[Method] T1083 substitution mutation BD fusion vector pGBKT7-TS was inverted into yeast to make the self-activation and protein expression toxic detection test.[Result] This expression product of the fragment was inactive and had no toxic to yeast cell.It could be used as bait for further two-hybrid screening.[Conclusion] The research laid the experimental foundation for further study of the effect of T1083 substitution mutation on the interaction of NtKrp and its target partner.

Screening and Identifying of Interaction Protein AtL5 in Arabidopsis thaliana

Research background: The Arabidopsis-resistance protein L5 (AT1G12290) can trigger cell death in Nicotiana benthamiana, which is a characteristic function of an NBS-LRR (Nucleotide-Binding Sites and Leucine-Rich Repeat) protein activation. Purpose: To explore the function and molecular regulatory network of L5. Method: We employed yeast two-hybrid technology to search for interacting proteins of L5, combined with laser confocal microscopy to observe the subcellular localization of these candidate proteins, and analyzed the impact of these proteins on L5 function using an Agrobacterium mediated transient expression system. Results: Seven candidate interacting proteins were identified from the Arabidopsis cDNA library, including PPA1 (AT1G01050), RIN4 (AT3G25070), LSU1 (AT3G49580), BZIP24 (AT3G51960), BOI (AT4G19700), RING/U (AT4G22250) and PPA3 (AT2G46860). Functional analysis of these candidate interacting proteins showed that they participated in multiple pathways, including biological and abiotic stress, programmed cell death, protein degradation, material metabolism and transcriptional regulation. The results of laser confocal microscopy manifested that RIN4 was only localized on the plasma membrane (PM), and RING/U was mainly associated with the PM. PPA1, PPA3, LSU1, BZIP24, and BOI all emerged nuclear and cytoplasmic localization. The results of the transient assay proclaimed that both BOI and RING/U can inhibit cell death caused by L5. Conclusions: These results indicate that L5 immune receptors may participate in various pathways, and their protein levels and activities are strictly regulated at multiple levels, providing a basis for elucidating the mechanism of L5 immune receptors in Arabidopsis resistance.

基于酵母双杂交技术对与纳尔逊海湾正呼肠孤病毒σNS相互作用宿主蛋白的筛选

目的:探讨小鼠成纤维L929细胞中与纳尔逊海湾正呼肠孤病毒(NBV)σNS蛋白相互作用(互作)的宿主蛋白,阐明宿主蛋白对病毒复制的影响。方法:构建表达σNS蛋白的诱饵质粒pGBKT7-S3,采用测序技术验证诱饵质粒在Y2H酵母细胞中的准确表达。将pGBKT7-S3和pGADT7质粒分别和共同转化至Y2HGold酵母细胞中,涂布于固体培养基进行培养,观察菌落生长情况,确认σNS蛋白对酵母细胞无毒性,不能自行激活报告基因。诱饵质粒pGBKT7-S3与小鼠成纤维L929细胞的cDNA文库进行杂交,对编码互作蛋白的阳性克隆进行质粒抽提,阳性测序结果通过Uniprot数据库检索筛选得到与NBVσNS互作的蛋白。对互作蛋白进行基因本体论(GO)功能富集分析、京都基因和基因组百科全书(KEGG)信号通路富集分析和STRING生物信息学分析。结果:成功筛选出61个阳性克隆。DNA测序分析和BLAST比对分析去除23个未匹配到数据库和测序基本相同的阳性克隆。阳性测序结果通过Uniprot数据库检索筛选得到38个与NBVσNS互作的蛋白,31个蛋白参与细胞生物过程,36个蛋白是细胞解剖成分,31个蛋白具有结合功能,5个蛋白是线粒体呼吸链组成部分,7个蛋白是核糖体蛋白和核糖体大小亚基的组成部分,2个蛋白参与铁代谢稳态。GO功能富集分析,互作蛋白参与的生物过程(BP)富集在细胞过程、代谢过程、生物学调节、定位和对刺激的反应等;细胞组分主要是细胞解剖成分和含蛋白复合物;分子功能集中在结合、催化活性、结构分子活性和转运活性等方面。KEGG信号通路富集分析,蛋白在遗传信息处理途径的翻译、折叠、分类和降解信号通路中高度富集,在机体系统中主要与消化系统有关联;与多种病毒性传染病和癌症有关联。STRING分析,互作蛋白涉及核糖体蛋白类、蛋白修饰类、代谢类和免疫蛋白类等功能蛋白。结结论论:成功筛选出与NBVσNS蛋白互作的蛋白,宿主蛋白在病毒复制中具有重要作用。

环境雌激素重组酵母测评系统的建立

目的通过检测重组酵母中报告基因的表达产物来反映环境雌激素通过雌激素受体介导而产生的拟雌激素作用。方法用乙酸锂法将本实验室已构建成功的全长人雌激素受体酵母表达载体pGAD_hER和雌激素效应元件(ERE)调控的酵母报告载体 pLacZi_2ERE导入酵母细胞中 ,得到重组酵母YM_ADER_ERE。结果与对照DMSO相比 ,加入17β_雌二醇(E2)后YM_ADER_ERE中报告基因lacZ的表达产物 β_半乳糖苷酶活性显著升高。用终浓度为10-15～10-7mol/L的E2 进行实验 ,E2 在YM_ADER_ERE中最大效应EAmax 为94 31U ,EC50 为0 34nmol/L。结论重组酵母中报告基因的表达是雌激素配体依赖的 ,符合环境雌激素测评系统的设计思想和要求 。

对酵母细胞酶法破壁的研究

研究了在重组酵母测评系统中,重组酵母细胞破壁条件的优化问题。通过酶法破壁酵母细胞得知,对于0.75mL酵母液来说,最佳反应时间为4h,最适反应温度为40℃,最佳酶量为10mg。同时,蜗牛酶破壁效果明显好于溶菌酶。

环境雌激素的生物检测与应用

环境雌激素问题已成为环境领域的研究热点。因此,建立简单快速有效的检测方法显得尤其重要。目前对污染物雌激素效应的测评主要依赖于生物学方法,文章主要介绍了子宫生长实验、肝细胞卵黄蛋白原生成实验、人乳腺癌细胞(MCF-7)增值实验、重组基因酵母实验、酵母双杂交实验五种主要的环境雌激素生物检测方法及其在国内外环境激素测评中的应用现状。

自来水及水源水有机提取物类雌激素活性研究

为了检测自来水及水源水有机提取物的类雌激素活性 ,应用环境雌激素基因重组酵母、细胞增殖和子宫增重等三项短期生物学实验进行测定。结果发现 :水样有机提取物在相当于原水量 50ml时两项体外实验均检出雌激素活性 ,原水量 2 50ml时细胞增殖效应达到最大 ,以后随原水量增大细胞增殖效应逐步减弱。原水量 12 50ml时酵母菌β -半乳糖苷酶活性最强 ,以后随原水量增大 β -半乳糖苷酶活性逐步减弱。水厂水源水有机提取物低、中剂量组受试小鼠子宫湿重增加 ,与对照组比较有统计学意义 ,高剂量组湿重减少。提示水源水有机提取物具有类雌激素活性。采用这三项实验组成的生物学检测程序具有一定的灵敏度 ,且较快速、方便。

北方某水厂的类雌激素物质变化规律

对北方某水厂春季和夏季源水和各处理单元出水中类雌激素物质的变化规律进行了研究.将水样用固相萃取方法富集后,按不同极性洗脱,得到从非极性到极性的3个组分,分别对总提取物和各分级富集组分进行重组雌激素受体基因的酵母检测.结果表明:源水和各处理单元出水中能够检测到极低水平的类雌激素物质,其中源水中的类雌激素效应仅相当于5.4～11.0pg/L雌二醇当量,主要由非极性与弱极性的类雌激素物质引起;春季和夏季源水中的类雌激素效应相差不大;水厂传统的处理工艺对类雌激素物质的去除效果不明显;重组基因酵母检测技术结合水样的固相萃取、三步纯化分级前处理方法可以快速、有效地筛选和定量分析水样中未知类雌激素物质,并对水处理效果进行评价.

再生水有机提取物雌激素效应研究

目的研究天津市某再生水厂各处理单元水体的雌激素效应。方法采用反相C-18固相萃取柱提取各处理单元水样中目标物质,采用人乳腺癌细胞(MCF-7细胞)增殖试验和基因重组酵母试验检测水样有机提取物的雌激素效应。结果2项试验中各处理单元水样有机提取物均表现出雌激素效应,且在一定范围内,呈现出明显的剂量-效应关系。二级出水、混凝后出水、连续膜过滤后出水和O3处理后出水均在相当于原水1.00ml的浓度水平时表现出对MCF-7细胞最强的增殖效应,最大相对增殖效应依次为33.87%,19.49%,20.42%和16.24%。而且上述水样有机提取物均在相当于原水20ml的浓度水平时具有最高β-半乳糖苷酶活力(8.15,7.50,6.07和5.33)。结论天津市某再生水厂现有处理单元对水体雌激素效应具有有限的去除作用,但最终出水仍具有一定的雌激素效应。