Analysis of Protein Interactions:Probing the Function of Proteins with Yeast Two-Hybrid System

The yeast two\|hybrid system is a molecular genetic approach for protein interaction and it is widely used to screen for proteins that interact with a protein of interest in recent years.This process includes,construction and testing of the bait plasmid,screening a plasmid library for interacting fusion protein,elimination of false positives and delection analysis of true positives.This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with a protein of interest.More and more studies have demonstrated that the two\|hybrid system is a powerful and sensitive technique for the identification of genes that code for proteins that interact in a biologically significant fashion with a protein of interest in higher plants.This method has been used to identify new interaction protein in many laboratories.The recently reported yeast tri\|brid system,should allow the investigation of more complex protein\|protein interactions.The aim of this review is to outline the recent progress made in protein interactions by using yeast two\|hybrid system.

A Double-Blind, Randomized, Placebo-Controlled Nutritional Study Using an Insoluble Yeast Beta-Glucan to Improve the Immune Defense System

Purpose: In a placebo-controlled, double-blind, randomized clinical trial, the effect of an insoluble yeast beta-glucan preparation on the incidences of common colds and its effect on common cold symptoms were compared to placebo. Methods: 100 healthy participants with recurring infections were randomly assigned to receive either placebo or yeast beta-glucan (Yestimun?;n = 50 each group) over a period of 26 weeks. The subjects had to document each common cold episode in a diary, and rate 6 predefined infections symptoms on a 3-point rating scale during an infection period, resulting in an infection score. The common cold episodes were confirmed by the investigators. Results: A total of 171 common cold episodes were documented. Of these, 76 were experienced by 38 subjects in the beta-glucan group and 96 were experienced by 48 subjects in the placebo group (p = 0.406). The beta-glucan group had significantly more subjects without incidences of common cold than the placebo group (15.6% vs 2.0%;p = 0.019). During the most intense infection season (first 13 weeks of the study), the beta-glucan group had significantly less infections compared to placebo (p = 0.02). Beta-glucan significantly reduced the typical cold symptoms (“sore throat and/or difficulty swallowing”, “hoarseness and/or cough” and “runny nose”) as opposed to placebo. Conclusion: The present study demonstrates a prophylactic effect of yeast beta-glucan on the occurrence of common colds as opposed to placebo. In addition, when these episodes occurred, they were from the beginning less pronounced and subsided faster.

Screening of hepatocyte proteins binding to NS5ABP37 protein by yeast-two hybrid system

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.

Yeast One-hybrid System Used to Identify the Binding Proteins for Rat Glutathione S-transferase P Enhancer I

Objective To detect the trans-factors specifically binding to the strong enhancerelement (GPEI) in the upstream of rat glutathione S-transferase P (GST-P) gene. MethodsYeast one-hybrid system was used to screen rat lung MATCHMAKER cDNA library toidentify potential trans-factors that can interact with core sequence of GPEI(cGPEI).Electrophoresis mobility shift assay (EMSA) was used to analyze the binding of trans-factors to cGPEI. Results cDNA fragments coding for the C-terminal part of thetranscription factor c-Jun and rat adenine nucleotide translocator (ANT) were isolated. Thebinding of c-Jun and ANT to GPEI core sequence were confirmed. Conclusions Rat c-juntranscriptional factor and ANT may interact with cGPEI. They could play an important rolein the induced expression of GST-P gene.

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.

RapIDYeast Plus系统在鉴定常见酵母中的临床应用

目的：探讨RapIDYeast Plus（RYP）系统在鉴定临床常见酵母中的作用。方法：将所试菌株在沙氏培养基上传代2次，然后30℃孵育24-48h后，用RYP系统进行试验。结果：150株临床分离株中有139株被正确鉴定到种的水平；有8株得到了具有2种或2种以上可能性的鉴定结果，而在使用了附加试验后都得出了正确的鉴定结果；有3株鉴定错误。RYP系统在不用附加试验的情况下鉴定的准确率达到92％，采用附加试验后鉴定的准确率达到98％。结论：RYP系统适用于临床微生物实验室的常规鉴定，在不需要特殊仪器的条件下，就能准确地鉴定临床上40多种酵母菌和酵母样菌。

Isolation and Identification of Yeasts from Luzhou Flavor Daqu

A yeast strain had been isolated by dilution-plate from the Daqu samples in our study. The strain was identified as a strain of Rhodotorula aurantiaca through observation of its morphological features, micromorphological observation and biolog identification system.

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.

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...

利用酵母双杂交筛选菠萝AcSWEET11的互作蛋白

SWEET(sugars will eventually be exported transporter)基因在植物开花过程中具有重要的作用,但AcSWEET11在菠萝成花中的作用机制尚不清楚。通过鉴定成花过程中与AcSWEET11的互作蛋白,为菠萝成花机制的解析奠定基础。本研究利用共转化的方法在菠萝成花过程的cDNA膜文库中筛选AcSWEET11的互作蛋白,分析候选蛋白的表达量。结果表明,pBT3-STE-AcSWEET11+pPR3-N对NMY51酵母细胞无毒性,但有自激活活性。进一步研究结果显示,在TDO/3?AT培养基和QDO培养基上自激活受到抑制。利用该系统筛选到了81个阳性克隆,经测序鉴定出48个与AcSWEET11互作的候选蛋白,包括E3 ubiquitin-protein ligase RING1-like、Trehalose-phosphate synthase 7、Cytochrome P450、TranscriptionfactorLUX等。GO和KEGG分析结果显示,48个蛋白主要分布在细胞进程、代谢过程、刺激反应和催化活性等生物过程,参与脂类代谢、氨基酸代谢和碳水化合物代谢、信号转导和运输与分解代谢等新陈代谢途径。Trehalose-phosphate synthase 7(XP_020105459.1)、Protein TIFY 3-like(XP_020082835.1)、40S ribosomal protein S27(XP_020092770.1)、Heterogeneous nuclear ribonucleoprotein 1-like(XP_020112516.1)等4个基因与AcSWEET11表达趋势一致,在菠萝成花过程中下调表达;Dihydrolipoyl dehydrogenase 2(XP_020113798.1)、Putative lipid-transfer protein DIR1(XP_020086640.1)、clathrin assembly protein At4g32285(XP_020108161.1)等3个基因在菠萝成花过程中上调表达。这些结果表明,AcSWEET11可能通过与Trehalose-phosphatesynthase7等蛋白发生互作,参与菠萝成花过程。本研究进一步丰富了AcSWEET11的蛋白互作网络,为AcSWEET11在菠萝成花中的调控机制的解析奠定基础。

长足大竹象信息素结合蛋白CbuqPBP2互作蛋白的筛选与验证

【目的】筛选长足大竹象(Cyrtotrachelus buqueti)信息素结合蛋白CbuqPBP2的互作蛋白,为实现利用信息物质对长足大竹象种群持续控制提供参考。【方法】利用GST pull-down联合质谱技术,对长足大竹象信息素结合蛋白CbuqPBP2的互作蛋白进行筛选、鉴定和分析,并采用酵母双杂交试验验证了CbuqPBP2与信息素结合蛋白CbuqPBP1的特异性互作。【结果】GST pull-down共筛选出45个与长足大竹象信息素结合蛋白CbuqPBP2特异性结合的候选互作蛋白,包括CbuqPBP1、ND2、CYTB。这些互作蛋白主要参与细胞过程、定位、代谢过程、应激反应以及生物调控等多个生物学过程。使用酵母双杂交体系,构建了pGADT7-PBP1重组猎物质粒与pGBKT7-PBP2重组诱饵质粒,通过诱饵质粒毒性检测和自激活检测,表明重组诱饵质粒对Y2HGold酵母菌无毒性作用。共转化验证结果显示,诱饵质粒pGBKT7-PBP2共转化酵母菌株能够在TDO培养基上生长。【结论】CbuqPBP2和CbuqPBP1之间有相互作用,不同信息素结合蛋白间的互作对深入理解长足大竹象嗅觉感受机制提供了新的思路。

小麦γ-醇溶蛋白1基因克隆及其互作蛋白筛选

为进一步研究γ-醇溶蛋白1基因调控小麦面粉品质的分子机制,以郑麦158(低蛋白质含量、高面团强度)为研究材料,克隆小麦γ-醇溶蛋白1基因,构建诱饵载体pGBKT7-γ-醇溶蛋白1,利用酵母双杂交技术从郑麦158的cDNA文库中筛选γ-醇溶蛋白1的候选互作蛋白,并通过回转验证试验进一步验证其互作关系。结果表明,成功克隆了γ-醇溶蛋白1基因,其cDNA全长为1 020 bp。利用酵母双杂交技术共筛选到117个蓝色单克隆菌落,经菌液PCR检测、测序以及NCBI的BLAST比对分析,共获得10个与γ-醇溶蛋白1互作的候选蛋白,它们分别是富含半胱氨酸和跨膜结构域蛋白1(XM_044546254.1)、α-淀粉酶抑制剂(XM_044509330.1)、特异性内切-1,3:1,4-β-D-葡聚糖酶2(XM_044550285.1、XM_044594213.1)、蔗糖果糖基转移酶(XM_044554537.1)、支链淀粉酶1(XM_044577151.1)和半胱氨酸蛋白酶B-like(XM_044577994.1),还有与植物生殖生长尤其是果实或者籽粒生长有关的细胞数目调控因子CNR8-like(XM_044483337.1、XM_044491902.1)、CNR2-like(XM_044468782.1)以及参与蛋白质修饰的泛素结构域蛋白DSK2b-like(Dominant suppressor of KAR 2b like,XM_044546840.1)和CIP73(CCa MK-interacting protein of 73 ku,XM_044554473.1)。候选互作蛋白的回转验证试验结果表明,富含半胱氨酸和跨膜结构域蛋白1、α-淀粉酶抑制剂、特异性内切-1,3:1,4-β-D-葡聚糖酶2和半胱氨酸蛋白酶B-like等4个蛋白质可能与γ-醇溶蛋白1存在互作关系。推测γ-醇溶蛋白1可能与这些蛋白质相互作用参与小麦种子中贮藏蛋白(如半胱氨酸)和淀粉的合成与降解等过程。

红曲及相关食品的应用情况及其标准体系研究进展

红曲是一种由红曲霉发酵而成的天然产物,兼具食品和中药双重特性,广泛应用于保健食品、酿酒、腐乳制作、食品着色、调味剂添加等多个领域及中医药。然而,红曲发酵过程中产生的次级代谢产物可能会对肾脏、肝脏等造成损害。因此,即使是天然成分,也需要严格的质量控制和科学的风险评估,以确保其安全性。本文从红曲的实际应用出发,梳理分析红曲在食品和保健食品中的应用情况。同时对我国当前红曲及相关食品标准进行了总结,揭示了存在的问题,如洛伐他汀含量限定不明确、缺乏对桔青霉素和黄曲霉毒素的限量要求,以及发酵菌种选择、原料质量控制和发酵过程管理等方面的不足,为红曲及相关食品标准体系的建设及安全提供参考和建议。

常见酵母表达系统的菌株及其在猪用疫苗研制中的应用

近年来,猪类疫病病毒不断发生变异,给全球生猪养殖业造成了巨大的经济损失,疫苗作为最有效的防控手段,其基因工程疫苗研究工作备受关注。本文总结常见酿酒酵母、汉逊酵母和毕赤酵母表达系统菌株的优势及其在猪用疫苗研制中的应用,为今后酵母表达系统在研制猪用疫苗方面提供理论参考。

利用酵母双杂交系统筛选玉米ZmPRR73的互作蛋白

为了阐明 ZmPRR73基因的生物学功能,构建诱饵表达载体pGBKT7-ZmPRR73,利用酵母双杂交技术,从长日照光照环境诱导的热带玉米自交系的cDNA文库中筛选与ZmPRR73互作的蛋白。结果显示:构建的诱饵载体pGBKT7-ZmPRR73对酵母菌株无毒性,且对报告基因无自激活活性,共鉴定出12个与ZmPRR73互作的候选蛋白。生物信息学分析表明,这些候选互作蛋白的功能涉及植物的转录调控、离子跨膜转运的调节、信号转导、电子传递链等多个方面,推测ZmPRR73蛋白与以上蛋白互作参与多个信号转导和代谢途径,研究结果补充和完善了ZmPRR73蛋白参与的调控途径,为进一步研究生物钟核心元件ZmPRR73的分子功能提供了新的分子证据。

酵母多糖制备大鼠全身炎症反应综合征疾病动物模型

目的:通过研究使用酵母多糖来建立稳定的SIRS大鼠模型,为下一步探究全身炎症反应综合征(SIRS)发病机制及后续干预做准备。方法:Wistar大鼠均采用腹腔注射酵母多糖-石蜡混悬液制备SIRS动物模型,将模型组大鼠按照不同剂量分为400mg/kg、500mg/kg、600mg/kg、700mg/kg4组,通过对比观察,评估各组大鼠注射后的生存率(96h),给药后24h大鼠的一般情况、体温、白细胞(WBC)计数、外周血检测指标、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平以及肝肺脏器的病理苏木精-伊红(HE)染色。结果:腹腔注射酵母多糖-石蜡混悬液剂量为600mg/kg和700mg/kg的大鼠符合SIRS动物模型标准,600mg/kg组大鼠48h时生存率为40%,具有较长的生存周期;而700mg/kg组大鼠短期内的死亡率在24h后即可达到50%。结论:腹腔注射酵母多糖-石蜡混悬液是能成功制备较为稳定的SIRS模型大鼠的方法,600mg/kg剂量组的大鼠可存活的时间较长,故可用于SIRS发病机制研究,亦为SIRS的后续干预实验做好准备。

低温等离子体杀菌技术对泡菜源混菌体系杀菌效果的研究

该研究制备了泡菜源的3种乳酸菌和2种酵母组成的混菌体系,并考察不同低温等离子体杀菌(CPS)处理对泡菜源微生物模拟混菌体系的杀菌效果,并评估混菌体系的产膜、产气、产酸和耐酸特性。结果表明,当放电电压相同、处理时间相同时,5种微生物的存活率均随处理时间、放电电压的增加而降低。当处理时间≥9 min时,放电电压越高,膜璞积累量及产气能力下降;但产气效果差异不明显。最佳CPS处理条件为放电电压160 k V,处理时间9 min。在此优化条件下,当发酵31 h时,混菌体系中微生物生物量(OD_(600 nm)值)最低(1.35),有效减缓了乳酸菌和酵母菌的生长;pH值最高(4.12),抑制了微生物产酸,一定程度上缓解了后酸化。

基于核系统酵母双杂交技术的红螯螯虾IAG互作蛋白筛选

为探究胰岛素样促雄性腺激素(Insulin-like androgenic hormone,IAG)在甲壳动物性别分化过程作用的分子调控途径,挖掘与IAG蛋白存在互作关系的候选蛋白信息,研究使用红螯螯虾促雄性腺及输精管组织构建核体系酵母文库,利用酵母双杂交技术筛选与IAG互作的候选蛋白,并对关键候选蛋白的编码基因进行克隆与表达分析。获得初级文库容量为1.12×10^(7),次级文库容量为1.28×10^(7);成功筛选到25个阳性克隆,共注释到12个蛋白编码基因;克隆获得关键候选蛋白的编码基因muc5acl(Mucin-5AC-like)的CDS全长474bp;半定量与荧光定量表达结果表明,muc5acl在红螯螯虾的促雄性腺、精巢及输精管中特异表达,且在雄性个体促雄性腺中的表达水平显著高于间性,在输精管中的表达则相反,推测该基因可能参与雄性激素的分泌及精子运输过程,并与间性红螯螯虾的性腺发育有关。研究结果将为进一步解析IAG调控红螯螯虾间性性别形成的分子机制提供重要信息。

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。

酵母多糖对肉用仔鸡免疫系统的影响

目的 探讨酵母多糖对仔鸡免疫系统的影响。方法 在仔鸡常规育雏及育肥期间，用酵母多糖灌胃（20 mg/kg）或肌注（2 mg/kg），并观察其对仔鸡脾脏、腔上囊及T淋巴细胞的作用。结果 与对照组比较，无论是灌胃或是肌注，酵母多糖对仔鸡脾脏、腔上囊和T淋巴细胞都可产生明显影响：脾脏重量（mg/10 g体重）增加：淋巴细胞数量(万/cm3)上升，对MTT的吸收能力增强；腔上囊皱襞增生。结论 酵母多糖是一种既可促进鸡的细胞免疫能力，又可提高其体液免疫应答水平的免疫增强剂，因此将其开发应于家禽（畜）养殖业是有前途的。