Screening for Novel Binding Proteins Interacting with Human Papillomavirus Type 18 E6 Oncogene in the Hela cDNA Library by Yeast Two-Hybrid System

To screen for novel binding proteins interacting with high-risk HPV 18 E6 oncogene, the strain AH 109 was transformed with pGBKT7-HPV 18 E6 plasmid, and subsequent transference was utilized to screen for interacting proteins with HPV 18 E6 in human Hela cDNA library. HPV 18 E6 mRNA was expressed in yeast and there was no self-activation and toxicity in AH109. Seven proteins that interacted with HPV18 E6, including transmembrane protein 87B, phosphonoformate immuno-associated protein 5, vimentin, KM-HN-1 protein, dedicator of cytokinesis 7, vaccinia related kinase 2 and a hypothetical protein, were identified. It was suggested that yeast two-hybrid system is an efficient for screening interacting proteins. The high-risk HPV 18 E6 oncogene may interact with the proteins, which may be associated with signal transduction and transcriptional control, epithelial cell invasion and migration, as well as humoral and cellular immune etc. This investigation provides functional clues for further exploration of potential oncogenesis targets for cancer biotherapy.

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 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.

Screening of hepatocyte proteins binding to complete S protein of hepatitis B virus by yeast-two hybrid system

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.

Electrophoretic Analysis of Isozymes and Proteins in F_1 Hybrids of Ctenopharyngodon idellus ♀ × Elopichthys bambusa ♂

[ Objective] This study aimed to investigate the biochemical and genetic characteristics of F1 hybrids of Ctenopharyngodon idellus ♀× Elopichthys bambusa ♂ - [ Method ] By using vertical polyacrylamide gel electrophoresis technology, three isozymes （LDH, EST, MDH） in nine tissues （heart, brain, eyes, liver, kidney, spleen, fin, muscle, plasma） of F1 hybrids of C. ideUus and parents were compared. [Result] Three isozymes in F1 hybrids of ♀× E. bambusa ♀ were analyzed; differences in isozyme and proteins between F1 hybrids idellus ♀× E. bambusa ♀ exhibited varying degrees of tissue-specificity; isozymes and proteins in F, hybrids were signiticandy different from the parents. [ Conclusion] These nine differences could be used as indicators to identify F1 hybrids and parents.

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 development of a recombinant hybrid protein of Plasmodium falciparum and analysis of its antigenicity and protectivity

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Screening and cloning for proteins transactivated by the PS1TP5 protein of hepatitis B virus:A suppression subtractive hybridization study

AIM:To clone and identify human genes transactivated by PS1TP5 by constructing a cDNA subtractive library with suppression subtractive hybridization(SSH)technique.METHODS:SSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by PS1TP5 protein.The mRNA was isolated from HepG2 cells transfected with pcDNA3.1(-)-myc-his(A)-PS1TP5 and pcDNA3.1(-)-myc-his(A)empty vector,respectively,and SSH technique was employed to analyze the differentially expressed DNA sequence between the two groups.After digestion with restriction enzyme RsaⅠ,small size cDNAs were obtained.Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2,respectively.The tester cDNA was hybridized with driver cDNA twice and subjected to nested PCR for two times,and then subcloned into T/A plasmid vectors to set up the subtractive library.Amplification of the library was carried out with E.coli strain DH5α.The cDNA was sequenced and analyzed in GenBank with Vector NTI 9.1 and NCBI BLAST software after PCR amplification.RESULTS:The subtractive library of genes transactivated by PS1TP5 was constructed successfully.The amplified library contained 90 positive clones.Colony PCR showed that 70 clones contained 200-1000-bp inserts.Sequence analysis was performed in 30 clones randomly,and the full-length sequences were obtained by bioinformatics technique.Altogether 24 coding sequences were obtained,which consisted of 23 known and 1 unknown.One novel gene with unknown functions was found and named as PS1TP5TP1 after being electronically spliced,and deposited in GenBank(accession number:DQ487761).CONCLUSION:PS1TP5 is closely correlated with immunoregulation,carbohydrate metabolism,signal transduction,formation mechanism of hepatic fibrosis,and occurrence and development of tumor.Understanding PS1TP5 transactive proteins may help to bring some new clues for further studying the biological functions of pre-S1 protein.

大豆GmNF-YA13互作蛋白的筛选及鉴定

大豆GmNF-YA13蛋白是一个核转录因子Y(NF-Y),在干旱和高盐响应过程中均发挥重要作用。为研究其抗旱和耐盐的作用机理,寻找GmNF-YA13的互作蛋白,构建pGBKT7-GmNF-YA13诱饵载体,采用酵母双杂交筛选大豆酵母文库,并进行X-α-gal染色验证。结果显示:酵母双杂交获得85个阳性克隆,测序分析后得到36个候选的互作蛋白。功能预测显示互作蛋白主要参与生长发育、胁迫响应、能量代谢、转录调控和信号转导等生物过程。选择GmUVR8、GmCML41、GmFbox13和GmFBA与诱饵pGBKT7-GmNF-YA13进行一对一验证,只有GmFBA能与GmNF-YA13发生相互作用,预示GmNF-YA13功能的发挥需要GmFBA的参与。该结果可为NF-YA抗逆分子网络的研究提供基础。

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.

苏丹草和高丹草转录组测序及其差异基因表达分析

为了初步探明苏丹草[Sorghum sudanense(Piper) Stapf.]和高丹草[Sorghum bicolor(Linn.) Moench.×Sorghum sudanense(Piper) Stapf.]相关差异基因的表达,本研究对两者进行转录组测序,并对分蘖调控以及粗蛋白和木质素合成关联基因的差异表达进行分析。结果表明:与苏丹草相比,高丹草的单株分蘖数显著降低,粗蛋白、木质素和独角金内酯含量以及果糖激酶、苯丙氨酸解氨酶、多酚氧化酶、反肉桂酸4-单加氧酶活性显著提高,己糖激酶-3(HXK3)、果糖激酶2(FRK2)、天冬氨酸转氨酶(ASPAT)、S-腺苷甲硫氨酸合成酶1(SAMS1)、多酚氧化酶II(PPOII)、双功能天冬氨酸激酶/高丝氨酸脱氢酶2(AKI/DHI2)、苯丙氨酸解氨酶(PAL)、反-肉桂酸4-单加氧酶(C4H)基因表达上调,独角金内酯酯酶D14(SLsE D14)基因表达下调。本研究为苏丹草和高丹草相关功能基因的克隆、分子标记开发等工作奠定了基础。

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。

陆地棉抗黄萎病基因GhENODL6互作蛋白筛选

研究发现陆地棉ENODL6基因具有抗黄萎病功能。为进一步揭示其在棉花抗黄萎病过程中发挥的作用,通过Nimble Cloning技术将GhENODL6插入酵母表达载体pNC-GBKT7,构建了重组质粒pNC-GBKT7-ENODL6。重组质粒转入酵母菌Y2HGold后可在DDO培养基上正常生长,但不能生长于QDO/X/A培养基,表明GhENODL6蛋白对酵母宿主无毒害作用,没有自激活活性。将携带pNC-GBKT7-ENODL6诱饵载体的Y2HGold酵母菌与cDNA文库进行杂交筛选,结果获得一个能够显示蓝色的菌落。通过PCR扩增,在蓝色酵母菌中获得一段526 bp的非载体插入片段,与陆地棉基因组内基因WRKY47序列高度一致。通过同源扩增,从陆地棉中克隆到WRKY47开放读码框,全长1 587 bp,编码528个氨基酸残基。再次利用酵母双杂交技术确认了WRKY47与GhENODL6之间存在互作关系。综上,构建了重组载体pNC-GBKT7-ENODL6,并鉴定到与GhENODL6互作的蛋白WRKY47。

玉米大斑病菌cDNA文库的构建及转录因子StMR1互作蛋白的筛选

【目的】筛选玉米大斑病菌(Setosphaeria turcica)转录因子的互作蛋白,解析黑色素调控转录因子StMR1调控玉米大斑病菌致病性的分子机制。为解析玉米大斑病菌侵染过程中转录因子的调控网络,阐明病菌的致病机理提供参考。【方法】收集玉米大斑病菌菌丝和孢子不同萌发阶段作为试验材料,采用Gateway方法构建玉米大斑病菌cDNA文库,使用同源重组的方法构建转录因子StMR1的诱饵载体,采用酵母双杂交技术筛选其互作蛋白并进行一对一验证。【结果】构建的玉米大斑病菌文库插入的平均片段长度大于1000 bp,初级文库及次级文库的库容量为1.2×107和1.04×107CFU,重组率为100%,可以用于酵母双杂交筛选。成功构建可以用于筛库的诱饵载体pGBKT7-StMR1,经初筛与复筛得到3个互作蛋白,一对一验证短链脱氢酶、糖基转移酶、富含亮氨酸重复序列蛋白均与转录因子StMR1存在互作。【结论】成功构建了丰富度高且质量好的玉米大斑病菌cDNA文库并筛选到了与转录因子StMR1互作的蛋白。

白菜种子cDNA酵母文库的构建及BrTTG1互作蛋白的筛选及分析

【目的】通过构建白菜种子的cDNA文库,筛选WDR 40蛋白TRANSPARENT TESTA GLABRA 1(TTG1)的互作蛋白,探究TTG1参与MBW三元复合体调控种皮原花青素形成的分子机制。【方法】以棕籽白菜自交系‘B147’的种子为材料,提取总RNA并建立cDNA文库,通过gateway技术构建诱饵载体pGBKT7-TTG1并进行酵母双杂交筛库。【结果】酵母文库库容为1.2×10^(7)CFU,文库滴度是5.0×10^(7)CFU/mL,插入片段平均长度大于1000 bp,诱饵载体在酵母中无自激活活性。通过构建的诱饵载体pGBKT7-TTG1与构建的cDNA文库杂交,共获得了38个阳性互作蛋白,功能预测显示其中一个蛋白注释为MYB转录因子,注释为MYB73,序列分析结果显示该基因含有R2R3-MYB型抑制子保守基序C1和C2,推测该基因为白菜中参与种皮颜色形成的R2R3-MYB型抑制子,暗示着白菜中可能存在不同MYB转录因子参与的调控网络,影响着原花青素的形成。【结论】本研究构建了白菜种子组织的酵母双杂交cDNA文库,获得了38个TTG1阳性互作蛋白,首次挖掘到了可能影响白菜种皮颜色原花青素形成的R2R3-MYB型抑制子MYB73,为后期探究白菜种皮原花青素的调控网络奠定良好的基础。

巴西橡胶树HbPSKR2基因克隆及互作蛋白鉴定

磺肽素是一种高等植物特有的小肽类激素,广泛参与植物的生长发育、分裂分化、生物或非生物胁迫等生物学过程。磺肽素受体蛋白(phytosulfokine receptor,PSKR)是磺肽素的直接受体,对于磺肽素信号传导至关重要。本研究采用RT-PCR(reverse transcription-polymerase chain reaction)技术克隆了橡胶树的HbPSKR2基因,并对其进行生物信息学、基因表达模式、互作蛋白筛选及鉴定分析。结果显示HbPSKR2基因的开放阅读框全长3159 bp,编码1052个氨基酸,理论分子量为114.84 kDa,理论等电点为6.34。结构域分析显示HbPSKR2属于典型的跨膜蛋白,前640个氨基酸是由亮氨酸重复组成的天线结构,第692~714个氨基酸是跨膜结构域,第765~1052个氨基酸是膜内激酶结构域。对HbPSKR2膜内激酶结构域以及拟南芥和水稻的PSKR同源序列进行多重比对,结果显示均存在ATP binding site、CaM binding site、Activation segment、GC Centre等保守性位点。表达模式分析显示,HbPSKR2基因在橡胶树形成层区高丰度表达,其表达量在冠菌素处理前期显著上升。通过酵母双杂交技术筛选到12个与HbPSKR2互作的候选蛋白,并对其中的2个蛋白激酶(HbPBL8和HbPIX13)与HbPSKR2的互作关系进行荧光素酶互补成像验证。结果显示,在烟草中共转化HbPSKR2-nLUC/HbPBL8-cLUC和HbPSKR2-nLUC/HbPIX13-cLUC可以观察到强烈的荧光信号,进一步证明了HbPSKR2在体内可与HbPBL8激酶和HbPIX13激酶互作。橡胶树HbPSKR2基因克隆及互作蛋白鉴定将为深入揭示橡胶树乳管分化分子机制提供新的思路。

蜡梅CpBEAT3启动子克隆及其互作蛋白的初步验证

苯甲醇乙酰基转移酶(benzyl alcohol acetyltransferase,BEAT)能够以苯甲醇和乙酰辅酶A(acetyl-CoA)为底物催化合成乙酸苄酯,是植物乙酸苄酯生物合成代谢途径下游的关键酶。CpBEAT3是蜡梅叶中调控乙酸苄酯生物合成的关键基因,为了解析其在蜡梅乙酸苄酯生物合成中的转录调控机制,本研究克隆得到了CpBEAT3基因启动子区序列,使用生物信息学软件预测分析了该基因核心启动子区、顺式作用元件、CpG岛等结构特征,并通过酵母单杂交技术筛选可能与CpBEAT3结合的上游调控因子。结果表明,2115 bp的CpBEAT3核心启动子区可能位于-1844~-1794 bp处,有一个位于-1869~-1459 bp处且长度为410 bp的CpG岛,含有多种与光响应、激素调节、胁迫响应相关的顺式作用元件及多个转录因子结合位点。通过酵母单杂交技术筛选获得1个MYB类的转录因子,可以与CpBEAT3启动子结合。

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。

尿液基细胞荧光原位杂交检测对膀胱尿路上皮癌的诊断价值

目的探讨尿液基细胞学(LBC)靶向的荧光原位杂交(FISH)对膀胱尿路上皮癌(BUC)的诊断价值。方法回顾性收集2020年10月—2022年10月于首都医科大学附属北京天坛医院泌尿外科行膀胱镜检查的128例患者的临床资料。所有患者在行膀胱镜检查前进行尿核基质蛋白22(NMP22)检测、尿LBC检测与尿LBC靶向的FISH检测,以术后病理结果为标准,分析3种检查方法的灵敏度和特异度。结果NMP22、尿LBC与LBC靶向的FISH的灵敏度分别为61.11%、79.17%、82.46%,特异度分别为57.14%、73.21%、86.67%;NMP22、尿LBC的灵敏度在检测高级别BUC时优于低级别,差异有统计学意义(P=0.01,P=0.03);3种方法对肌层浸润性膀胱癌与非肌层浸润性膀胱癌检测的灵敏度比较,差异无统计学意义(P≥0.05)。结论尿LBC靶向的FISH对BUC诊断的灵敏度和特异度较高,尤其是对于低级别BUC,可以作为BUC早期筛查、诊断的重要方法。

致病疫霉菌效应蛋白Pi05440毒性功能验证及寄主候选靶标筛选

致病疫霉菌(Phytophthora infestans)侵染时会分泌大量效应蛋白进入寄主细胞,通过操纵寄主靶标抑制植物免疫反应。为研究致病疫霉菌效应蛋白Pi05440的功能,本研究重点分析了Pi05440的蛋白特征、毒性功能及鉴定其寄主靶标;利用生物信息学数据库,预测Pi05440的保守结构域和信号肽。构建pRI101-GFP-Pi05440表达载体用于Pi05440的亚细胞定位和毒性功能分析;同时,利用酵母双杂交技术对Pi05440的寄主靶标蛋白进行了筛选和鉴定。结果表明,Pi05440基因全长969 bp,编码322个氨基酸。结构预测结果显示Pi05440含有3个典型的KAZAL功能域。亚细胞定位结果表明Pi05440定位在质膜和细胞间隙。在本氏烟中瞬时表达该基因显著促进致病疫霉菌扩展。通过酵母双杂交筛选,初步鉴定到3个Pi05440的候选靶标蛋白,分别为马铃薯过氧化氢酶12、马铃薯几丁质酶以及马铃薯MYB-like A蛋白。本研究为探究致病疫霉菌效应蛋白Pi05440及其靶标蛋白如何调控植物免疫提供了重要线索。