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.

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.

应用酵母单杂交体系筛选与大鼠谷胱甘肽S-转移酶P增强子GPEⅠ相互作用的转录激活因子

目的 探讨谷胱甘肽 S-转移酶 (GST- P)基因表达调控机制的多样性及其与化学致癌的关系 ,筛选与大鼠 GST- P增强子元件 GPE 相互作用的调控因子。方法 采用酵母单杂交体系筛选与 GPE 核心序列相互作用的转录激活因子 ,应用 DNA序列测定及计算机分析等手段对所测的 DNA序列进行分析。结果 共获得两个阳性克隆 p YGPE1和 p YGPE2。 DNA测序分析表明 :p YGPE1的插入片段与大鼠原癌基因 c- jun c DNA具有 99%的同源性 ,其编码的氨基酸序列与大鼠 c- Jun蛋白具有 10 0 %的同源性。 p YGPE2的插入片段与大鼠线粒体腺苷酸转位酶 c DNA具有 99%的同源性 ,其编码的氨基酸序列与大鼠腺苷酸转位酶具有 10 0 %的同源性。结论 大鼠 c- Jun蛋白和线粒体腺苷酸转位酶在酵母细胞内与大鼠 GST- P增强子 GPE 核心序列结合 ,可能是作用于 GPE 的反式作用因子。

IQCE mRNA与FXR1P在酵母三杂交体系中的相互作用

脆性X相关基因1编码蛋白FXR1P是一种RNA结合蛋白,其所结合的靶RNA目前所知甚少。本研究应用酵母三杂交技术对本课题组从pRH3-cDNA人脑海马RNA表达文库中筛选到的一种可能与FXR1P存在相互作用的RNA IQCE进行研究,以验证该RNA与FXR1P的相互作用。方法为:提取利用酵母三杂交技术初步筛选得到的酵母阳性克隆的质粒,转化大肠杆菌Top10,利用其质粒不相容性分离插入了目的片段的pRH3′-cDNA质粒,将该质粒转化入含目的基因FXR1的酵母菌株L40-ura3/pHyb lex/Zeo-MS2/pYESTrp3/FXR1,进行一对一的酵母三杂交验证,最后将该片段进行测序。测序结果为IQCE的一段编码序列,而目前尚无研究报导FXR1与IQCE的相互关系。结论:提示FXR1P与IQCE mRNA存在相互作用,IQCE可能是FXR1P发育调控网络组成成员之一。

Two novel eukaryotic translation initiation factor 5A genes from Populus simonii×P.nigra confer tolerance to abiotic stresses in Saccharomyces cerevisiae

The role of plant eIF5A proteins in multiple biological processes, such as protein synthesis regulation, translation elongation, mRNA turnover, programmed cell death and stress tolerance is well known. Toward using these powerful proteins to increase stress tolerance in agricultural plants, in the present study, we cloned and characterized PsneIFSA2 and PsneIFSA4 from young poplar （P. simonii × P. nigra） leaves. The deduced amino acid sequences of PsneIF5A2 and PsneIF5A4 were 98 % similar to each other, and they are orthologs of eIF5A 1 in Arabidopsis. In a subcellular localization analysis, PsneIF5A2 and PsneIF5A4 proteins were localized in the nucleus and cytoplasm, qRT-PCR analysis showed that PsneIF5A2 and PsneIF5A4 were transcribed in poplar flowers, stem, leaves, and roots. In addition, they were also induced by abiotic stresses. Transgenic yeast expressing PsneIF5A2 and PsneIF5A4 had increased salt, heavy metal, osmotic, oxidative tolerance. Our results suggest that PsneIF5A2 and PsneIF5A4 are excellent candidates for genetic engineering to improve salt and heavy metal tolerance in agricultural plants.

Screening of the whole human cytochrome P450 complement(CYPome)with enzyme bag cocktails

We have previously introduced the use of permeabilized fission yeast cells(enzyme bags)that recombinantly express full-length CYPs for drug metabolism studies.Such enzyme bags are cells with pores that function as enzymes in situ.They can easily be prepared without a need for ultracentrifugation and may be used in similar protocols as microsomes.In this study we report the preparation of enzyme bag cocktails that permit the testing of multiple CYPs in a single enzyme bag reaction.Moreover,we established a convenient testing scheme that permits a rapid screen of all human CYPs for activity towards any given candidate substrate.An important aspect of this approach is the reduction of individual CYP test assays.If a cocktail containing many CYPs tests negative,it follows that all CYPs included in that cocktail need not be tested individually,thus saving time and resources.The new protocol was validated using two probe substrates.

加锗酿酒酵母发酵中药制剂对猪肉品质及N、P排泄率的影响

试验研究日粮中添加富锗发酵中药制剂对猪肉品质及N、P排泄率的影响。选取40头"杜×长×大"三元杂交肥阉猪,随机分为2组,每组5个重复,每个重复4只。对照组猪饲喂基础日粮,试验组在基础日粮中添加0.5%富锗发酵中药制剂。试验期49 d。结果显示,与对照组相比,试验组猪肉锗含量极显著提高(P<0.01),蒸煮损失和离心失水率显著降低(P<0.05)。贮藏第4、7、11 d的猪肉pH值均显著降低(P<0.05)。贮藏第11 d的挥发性盐基氮(TVBN)含量显著降低(P<0.05)。与对照组相比,试验组猪肉总抗氧化能力(T-AOC)极显著提高(P<0.01),丙二醛(MDA)含量极显著降低(P<0.01),粪便中氮(N)、磷(P)含量均显著降低(P<0.05)。研究表明,富锗发酵中药制剂可提高肉中锗的沉积,增强抗氧化性能,降低N、P排泄率。

Cell Surface Display of Red-Grouper Nervous Necrosis Virus Capsid Protein on <i>Pichia pastoris</i>

Nervous necrosis virus (NNV), the etiological agent of viral nervous necrosis, has a high mortality rate of 100% in hatchery-reared larvae and juveniles. At present, there are still no effective vaccines available for NNV. Pichia pastoris surface display of viral capsid proteins was generated in hopes of developing an oral vaccine against red-grouper-nervous-necrosis virus (RGNNV) in fish. Fingerlings or juveniles that showed clinical signs of NNV infection were proved by RT-PCR for the appearance of expected length of 198 bpcDNA and further analysis by DNA sequencing. The DNA fragment containing AGα1 linked to RG-NNVRNA2, 2100 bp in length, was inserted into pPIC9K vector. Linearlized plasmids were electroporated into P. pastoris GS115 (mut+His?) and yeast isolates that had Muts?His+ and resistance phenotype at 4 mg/mL geniticin were selected to determine the integration of the target gene by PCR reaction. The extracted cell walls from the yeasts cultured in buffered-methanol-complex medium (BMMY) through an induction of 0.5% methanol for 6 days, were investigated for the fusion proteins by western blot. A protein band of 73 kDa predicted to be the fusion protein and a non-specific one of 56 kDa were detected. Staining of the fusion proteins expressing cells with corresponding antibodies revealed their presence of NNVRNA2, but varied the intensity of detected signals from cell to cell by confocal laser scanning fluorescence microscopy. The predicted fusion proteins tertiary structure also confirmed exposed conformation of the fusion protein on the cell wall. In this study, the capsid proteins from the red-spotted grouper nervous necrosis virus were successfully expressed on the cell surface of P. pastoris but still low levels of fusion protein expression. Further studies are required to optimize fully surface protein expression prior to evaluate the possible use of the constructed recombinant yeast as an oral vaccine against RG-NNV infection.

Therapeutic effect of 5-FC on YCD modified murine P388 leukemia in vivo

Objective: To investigate the therapeutic effect of a novel suicide gene system, yeast cytosine deaminase (YCD)/5-Fluorocytosine(5-FC), on P388/DBA murine leukemia model. Methods: P388-YCD-eGFP clone was selected after retrovirus transduction by limited dilution. P388-eGFP and wild type (wt) P388 cells were used as controls. (1) Tumorigenesis study: DBA mice were inoculated intravenously with P388-YCD-eGFP, P388-eGFP or wt P388 cells at a dosage of 5×10~6 per mouse (n=5). (2) Therapeutic effect study of 5-FC: After inoculation with P388-YCD-eGFP, P388-eGFP or wt P388 cells (n=5), 5-FC was administered at a dosage of 5 μmol/d in each mouse for 2 weeks. 20 μmol/d 5-FC were administered on the 8~ th day in case 5 μmol/d did not work. The percentage of eGFP^+ cells was detected by flow cytometry, frozen section or HE section. Results: Mice in YCD, eGFP and wt P388 group developed leukemia and survived (8.0±1.0) d, (7.6±0.89) d and (7.8±1.64) d (P>0.05), respectively. After being treated with 5-FC for 2 weeks, mice in YCD and eGFP^+ groups survived (13.2±1.79) d and (8.4±0.58) d (P<0.05). When 20 μmol/d 5-FC was administered, the percentage of eGFP^+ cells dropped from 21.9%±16.09% to 5.4%±5.6%, as well detected by FACS within 24h after treatment. Conclusion: 5-FC can eliminate YCD gene modified cells in vivo when cells are inoculated intravenously. YCD is an efficient suicide gene system in vivo.

Design and engineering of logic genetic-enzymatic gates based on the activity of the human CYP2C9 enzyme in permeabilized Saccharomyces cerevisiae cells

Gene circuits allow cells to carry out complex functions such as the precise regulation of biological metabolic processes.In this study,we combined,in the yeast S.cerevisiae,genetic regulatory elements with the enzymatic reactions of the human CYP2C9 and its redox partner CPR on luciferin substrates and diclofenac.S.cerevisiae cells were permeabilized and used as enzyme bags in order to host these metabolic reactions.We engineered three different(genetic)-enzymatic basic Boolean gates(YES,NOT,and N-IMPLY).In the YES and N-IMPLY gates,human CYP2C9 was expressed under the galactose-inducible GAL1 promoter.The carbon monoxide releasing molecule CORM-401 was used as an input in the NOT and N-IMPLY gates to impair CYP2C9 activity through inhibition of the Fe+2-heme prosthetic group in the active site of the human enzyme.Our study provides a new approach in designing synthetic bio-circuits and optimizing experimental conditions to favor the heterologous expression of human drug metabolic enzymes over their endogenous counterparts.This new approach will help study precise metabolic attributes of human P450s.

SARS-CoV S1蛋白在甲醇营养型酵母中表达及鉴定

目的获得保持生物学活性、易于纯化、高产量的严重急性呼吸道综合征冠状病毒(severe acute resp iratorysyndrom coronavirus,SARS-CoV)S1蛋白,以便进一步研究S1蛋白及其抗体的功能。方法将SARS-CoV S1基因重组入酵母表达载体pMETαA,经过酶切和测序鉴定后,通过电穿孔法将重组质粒S1-pMETαA转化入宿主酵母菌株PMAD11,使其在甲醇的诱导下表达目的蛋白。最后用覆盖分析法对重组转化子初步分析,SDS-PAGE和W esntern b lotting法鉴定目的蛋白的表达和特异性。结果酶切鉴定和测序结果证实了S1基因成功地克隆到pMETαA载体中;覆盖分析、SDS-PAGE和免疫印迹法证实S1基因得到正确的表达。结论利用酵母表达系统,成功地克隆和表达了SARS-CoV S1蛋白,为进一步纯化该蛋白和研究其功能奠定了基础。

弓形虫SAG1成熟蛋白编码区基因在甲醇酵母中的初步表达

目的 分析弓形虫主要表膜蛋白SAG1在甲醇酵母高效表达系统表达的可行性。方法 在 5′端和3′端引物分别引入EcoRⅠ和SpeⅠ酶切位点 ,PCR扩增SAG1成熟肽编码区基因 ,定向克隆到甲醇酵母分泌型表达质粒pMETαA中 ,构建不带 6个组氨酸尾序列的重组质粒。重组质粒被PacⅠ酶切下表达盒 ,氯化锂化学法转化腺嘌呤营养缺陷型毕赤甲醇酵母株PMD11和PMD16 ,通过腺嘌呤营养缺陷型选择培养基YPD筛选酵母重组子 ,并利用MM/MD选择培养板分析外源基因表达盒整合到重组酵母染色体中的方式。筛选甲醇利用野生型的重组酵母 ,用甲醇诱导表达 ,并分析SAG1的表达水平 ,从中筛选高表达转化子。结果 获得了经非同源重组整合到酵母染色体上的能有效利用甲醇作为唯一碳源的PMD11和PMD16转化株。在甲醇诱导后第 3天 ,细胞裂解液SDS PAGE检测开始出现分子量与目的蛋白预测分子量相同的蛋白带 ,但PMD11重组株中的该蛋白随培养时间延长而减少 ,而PMD16重组株中的目的蛋白量未见减少。上清中有 4 0kDa和 2 7kDa的两种蛋白 ,后者的量大于前者 ,并与SAG1成熟肽的大小一致 ,PMD16株的表达量大于PMD11株 ,总蛋白量约 35 μg/ml。 结论 弓形虫SAG1基因可在甲醇酵母表达系统中表达 ,但PMD11宿主菌会降解外源蛋白 ,而缺失了蛋白酶的PMD16?

高压方波脉冲电场对微生物的致死作用

研究了高压方波脉冲电场对食品中常见的腐败菌枯草芽孢杆菌 ,灰绿青霉的致死作用 ,比较了几种微生物的灭活效果 ,并以酿酒酵母为材料 ,初步探讨了杀菌机理。研究表明 :微生物的致死率随电场强度、脉宽、脉冲个数的增加而增大 ,同一灭菌条件 ,酵母菌 >大肠杆菌 >枯草芽孢杆菌 >灰绿青霉。电场作用后死亡酵母细胞表面出现凹陷 ,局部有孔洞产生。

高产β-葡萄糖苷酶野生酵母的筛选及产酶能力差异性分析

为了比较分析野生酵母菌产β-葡萄糖苷酶的情况,筛选高产β-葡萄糖苷酶的酵母菌株,该研究采用七叶灵显色法和4-硝基苯基-β-D-吡喃葡萄糖苷(p-NPG)显色法对从宁夏贺兰山东麓分离的941株野生酵母的产β-葡萄糖苷酶能力进行筛选,通过WL培养基上的酵母菌落形态和26S r DNA D1/D2区的序列分析对所有酵母菌株进行分类鉴定,并且对酵母产β-葡萄糖苷酶能力进行差异性分析。结果表明,941株酵母被鉴定为14个种,且筛选出了4株酵母菌高产β-葡萄糖苷酶:菌株SLY-4(98.51 U/L)、F2-24(76.93 U/L)、F2-16(62.72 U/L)和HX-13(47.95 U/L)。产酶能力差异性分析表明β-葡萄糖苷酶广泛分布于14种酵母,但是产酶能力表现出明显的种间和种内差异性。

啤酒废酵母中还原型谷胱甘肽的抽提新方法探讨

采用对羟基苯甲酸酯提取啤酒废酵母菌细胞中的谷胱甘肽(GSH)。研究表明,按菌体与破壁液比例1∶2(WV)加入0.5%的对羟基苯甲酸丙酯,30℃,pH5-pH6,搅拌3h能有效地从啤酒废酵母中提取谷胱甘肽(GSH),溶液经离心后,上清液中谷胱甘肽(GSH)含量可达96.71mg100mL。和现有的几种抽提方法比较,对羟基苯甲酸酯提取由于其提取含量高(96.71mg100mL)、不需要复杂和贵重的仪器、易于放大、经济性强而明显优于其他抽提方法。

人β-防御素3融合细菌膜穿透增加蛋白在毕赤酵母中的表达

目的:探讨采用酵母表达系统进行人β-防御素3(hBD3)与细菌膜穿透增加蛋白(BPI)融合表达的可行性.方法:将hBD3成熟肽基因通过Linker蛋白与BPI基因串联,克隆于酵母表达载体pPICZαB中,电转导入X-33毕赤酵母菌,经重组酵母基因组PCR和表型鉴定获得阳性克隆,对阳性克隆进行甲醇诱导表达,上清进行目的蛋白纯化和Western Blot鉴定.结果:重组载体经酶切和测序证实序列正确,重组X-33-pICZαB-hBD3-BPI克隆经甲醇诱导24h后,上清SDS-PAGE电泳显示有目的蛋白表达,Western Blot分析表明重组蛋白抗人hBD3和BPI均阳性,该目的蛋白依次通过疏水色谱、离子交换色谱纯化,蛋白纯度达到89%.结论:采用毕赤酵母系统融合表达hBD3和BPI是可行的.

鲐鱼蛋白液脱苦脱腥的研究

分别采用活性炭吸附、β-CD包埋法、乙醚萃取法、酵母发酵法对鲐鱼蛋白水解液进行处理。经比较发现,酵母发酵法效果最佳,水解液中加入20％酵母粉进行35℃、1h发酵后,腥味基本脱除。

人工染色体研究进展

细菌人工染色体（ＢＡＣｓ）、噬菌体Ｐ１衍生的人工染色体（ＢＡＣｓ）和酵母人工染色体（ＹＡＣｓ）是近年发展起来的ＤＮＡ克隆新技术。着重介绍了ＹＡＣｓ作为转移大分子外源ＤＮＡ的载体，在生物基因组分析，基因的结构与功能、表达与调控、定位与分离以及遗传病的基固治疗等研究领域的应用。比较了ＢＡＣｓ、ＰＡＣｓ和ＹＡＣｓ的主要特点。对哺乳动物人工染色体的研究情况也作了简要介绍。

酵母饲料添加剂在对虾育苗中的应用

在对虾育苗生产中试验了啤酒酵母饲料添加剂对育苗效果和防治发光细菌的作用。结果表明,无论是作为对虾幼体饵料添加剂或是作为水质改良剂,啤酒酵母饲料添加剂具有抑制病菌生长繁殖,提高对虾育苗存活率的作用。

应用酵母双杂交技术研究人睾丸特异表达基因HSD-3.8的功能

目的探寻本实验室筛选出的与不孕症相关的人睾丸特异表达基因HSD-3.8(GenBank接收号为AF311312,国际上命名为精子相关抗原1)编码蛋白质的作用配体,揭示其参与受精过程的机制。方法采用酵母双杂交体系,构建含有HSD-3.8基因部分序列(HSD-0.7)的诱饵质粒,筛选人卵巢cDNA文库。在获得阳性克隆后,根据蛋白质结构分析并结合PCR方法重新构建一系列缺失体,在酵母体内验证它们与被捕获的蛋白因子之间的结合。结果酵母双杂交实验获得一阳性克隆,其编码氨基酸与人G蛋白β1亚基羧基端的144个氨基酸同源性为100%。所构建的几种诱饵蛋白缺失体在酵母体内均不能与该捕获的蛋白因子相互作用。结论HSD-3.8编码蛋白质片段HSD-0.7可能通过G蛋白信号转导途径在受精过程中发挥功能,与G蛋白β1亚基的结合有赖于其结构的完整。