籽鹅卵泡颗粒细胞α-烯醇化酶基因RNA干扰表达质粒的构建及鉴定

目的构建籽鹅卵泡颗粒细胞α-烯醇化酶(α-enolase,ENO1)基因RNA干扰表达质粒,并进行鉴定。方法利用RNAi Designer等网络在线RNA干扰设计软件设计3条可能会干扰籽鹅卵泡颗粒细胞ENO1基因表达的插入DNA序列:shRNA-ENO1-350、shRNA-ENO1-892、shRNA-ENO1-591,将体外合成的3条干扰序列分别与线性化的pGPU6/GFP/Neo载体连接,构建ENO1 RNA干扰表达质粒,在Lipofectamine 2000的介导下转染原代培养的籽鹅卵泡颗粒细胞,转染48 h后,荧光倒置显微镜下观察GFP的表达;实时荧光定量PCR和Western blot法检测转染细胞中ENO1基因mRNA的水平及蛋白的表达水平。结果 pGPU6/GFP/Neo-shDNA重组质粒经单酶切及测序证实构建正确;转染后48 h,转染重组质粒的颗粒细胞在荧光倒置显微镜下可见较强的绿色荧光,转染效率可达60%;与培养液组、转染试剂组和无关序列干扰组(shNC)相比,shRNA-ENO1-350组颗粒细胞中ENO1基因mRNA水平和蛋白表达水平均显著降低(P0.01),其他各组之间差异无统计学意义(P0.05)。结论成功构建了籽鹅卵泡颗粒细胞ENO1基因RNA干扰表达质粒,在原代培养的籽鹅卵泡颗粒细胞中,其表达量显著下降,为后续有关ENO1功能的研究奠定了基础。     

基于口蹄疫病毒2A片段的多基因共表达质粒的构建及表达

目的构建基于口蹄疫病毒(foot and mouth disease virus,FMDV)2A片段的多基因共表达质粒,并在293T细胞中共表达HBx、人Sox2及人c-Myc基因。方法以FMDV 2A自剪切片段连接HBx、Sox2和c-Myc基因编码序列,克隆至载体pEGFP-C1,构建多基因共表达质粒pEGFP-XSM;在脂质体LipofectamineTM2000的介导下,将重组质粒pEGFP-XSM转染293T细胞,同时设空载体组(转染空载体pEGFP-C1)和空白对照组(未转染),转染48 h后,荧光显微镜下观察细胞中绿色荧光蛋白(green fluorescent protein,GFP)的表达;Real-time PCR和Western blot法分别检测各组293T细胞中HBx、Sox2和c-Myc基因mRNA的转录水平和蛋白的表达水平。结果多基因共表达质粒pEGFP-XSM经酶切和测序鉴定构建正确;空载体组和pEGFP-XSM转染组细胞荧光镜下均可见GFP的表达;pEGFP-XSM转染组中HBx、Sox2和c-Myc基因mRNA的转录水平较空白对照组及空载体组均明显升高(P﹤0.001);pEGFP-XSM转染组可见相对分子质量约48 000的GFP-HBx-2A融合蛋白条带和约38 000的HA-Sox2-2A融合蛋白条带,各组均可见相对分子质量约49 000的c-Myc蛋白条带,pEGFP-XSM转染组c-Myc蛋白的表达水平明显高于空白对照组及空载体组(P﹤0.01)。结论成功构建了多基因共表达质粒pEGFP-XSM,并在293T细胞中实现了HBx、Sox2和c-Myc蛋白的共表达,为进一步深入研究该共表达蛋白在肝细胞肝癌(hepatocellular carcinoma,HCC)发生发展中的生物学机制奠定了基础。     

GINS2基因过表达对K562细胞增殖的影响

目的检测GINS2基因过表达对K562细胞增殖的影响。方法将Ad-GINS2感染K562细胞,扩增腺病毒,同时设空载体转染的空载组和未感染病毒的未处理组,PCR及Western blot法检测细胞中GINS2的转录及表达水平,MTT法检测细胞增殖水平。结果与空载组和未处理组相比,Ad-GINS2感染组细胞GINS2基因转录水平及蛋白表达水平显著升高(P0.05),Ad-GINS2能明显促进细胞增殖(P0.05)。结论 GINS2基因的重组腺病毒载体成功感染K562细胞并可促进K562细胞增殖,为进一步明确GINS2基因在白血病中的作用奠定了基础。     

α-烯醇化酶在籽鹅不同级别卵泡壁的定位及定量

目的对籽鹅不同级别卵泡壁中的α-烯醇化酶(α-enolase)进行定位及定量检测。方法分离产蛋期籽鹅等级卵泡(F1、F2、F3、F4、F5)、小黄卵泡(small yellow follicle,SYF)、大白卵泡(large white follicle,LWF)的卵泡壁;应用免疫组化方法检测不同级别卵泡壁的α-烯醇化酶的定位,采用Western blot及实时荧光定量PCR双标准曲线法检测α-烯醇化酶蛋白表达量及mRNA转录水平。结果不同级别卵泡壁均存在α-烯醇化酶表达,颗粒细胞层表达量总体高于膜层,SYF颗粒细胞层α-烯醇化酶表达量较高;F2级卵泡壁α-烯醇化酶mRNA转录水平显著高于LWF、F3及F4级别卵泡,差异有统计学意义(P 0. 01),F1、F5及SYF级卵泡壁α-烯醇化酶m RNA转录水平显著高于其他级别卵泡壁,差异有统计学意义(P 0. 01);SYF级卵泡壁α-烯醇化酶蛋白表达量显著高于其他级别卵泡,差异有统计学意义(P 0. 01)。结论α-烯醇化酶及糖酵解途径可能在卵泡发育中发挥重要功能,为α-烯醇化酶在禽类卵泡选择及生长发育中的功能研究奠定了基础。     

腺病毒介导的slit2 shRNA对缺氧诱导的人视网膜色素上皮细胞中血管内皮生长因子表达的影响

目的观察腺病毒介导的slit2 shRNA对缺氧诱导的人视网膜色素上皮细胞(retinal pigment epithelial cells,RPE)中血管内皮生长因子(vascular endothelial growth factor,VEGF)表达的影响,探讨slit2在脉络膜新生血管(chorodal neovascularation,CNV)中的可能作用,为CNV的治疗提供新的思路。方法分别采用RT-PCR和免疫组化法检测人视网膜色素上皮-19(ARPE-19)细胞中slit2及受体Robo1基因mRNA的转录和蛋白的表达;用200μmol/L氯化钴处理人ARPE-19细胞,建立化学缺氧模型,以未缺氧组作为对照,采用Real-time PCR和Western blot法检测在缺氧状态下细胞中slit2、Robo1、VEGF基因mRNA及蛋白水平表达的变化;将缺氧的人ARPE-19细胞随机分为shRNA处理组(加入Ad-slit2-shRNA)、空腺病毒组(加入空腺病毒)和缺氧组,24 h后收集各组细胞,Real-time PCR和Western blot法检测slit2 shRNA干扰后细胞中slit2、Robo1、VEGF基因mRNA和蛋白水平表达的变化,ELISA法检测细胞培养上清中VEGF蛋白水平的变化。结果在正常人ARPE-19细胞中有slit2和Robo1基因mRNA的转录及蛋白的表达;缺氧组人ARPE-19细胞中slit2、Robo1、VEGF基因mRNA和蛋白的表达水平均较未缺氧组明显升高(P0.05);与缺氧组相比,slit2-shRNA处理组人ARPE-19细胞中slit2、Robo1、VEGF基因mRNA和蛋白的表达水平均明显降低(P0.05),缺氧组与空腺病毒组slit2、Robo1、VEGF的变化差异无统计学意义(P0.05)。结论 slit2 shRNA沉默RPE细胞中的slit2后,可明显抑制缺氧诱导的RPE细胞中VEGF的表达,为临床CNV的治疗提供了新的思路。     

T-cadherin基因通过雄激素受体通路对前列腺癌LNCa P细胞增殖的抑制作用

目的分析前列腺癌组织中T-cadherin基因mRNA转录水平,探讨T-cadherin基因通过雄激素受体(androgen receptor,AR)通路抑制前列腺癌LNCaP细胞增殖的作用。方法收集前列腺癌及癌旁相应正常组织各60份,实时荧光定量PCR检测组织中T-cadherin mRNA转录水平;将培养24 h的前列腺癌LNCaP细胞分为腺病毒GFP组、腺病毒GFP-T-cadherin组及空白对照组(不含腺病毒),CCK-8法检测T-cadherin对前列腺癌LNCaP细胞增殖的影响,Western blot法检测过表达T-cadherin后前列腺癌LNCaP细胞中AR及前列腺特异性抗原(prostate specific antigen,PSA)蛋白表达水平,并检测不同组别LNCaP胞浆及胞核中AR蛋白表达水平。结果 T-cadherin基因在癌组织中较癌旁组织mRNA转录水平明显减少(P 0. 01),其转录水平与前列腺癌的分期、格里森评分及分化均有关(P均0. 05);与空白对照组比较,腺病毒GFP-T-cadherin组能抑制前列腺癌LNCaP细胞增殖(P 0. 05),而T-cadherin过表达能下调LNCaP细胞中AR及下游靶基因PSA的表达水平(P均0. 05)。结论前列腺癌组织中T-cadherin基因mRNA转录水平明显减少,其过表达能通过AR通路抑制前列腺癌LNCaP细胞的增殖,本研究为前列腺癌的治疗提供了新的靶点。     

热休克蛋白70过表达对过氧化氢诱导BRL细胞caspase 3表达的影响

目的探讨热休克蛋白(heat shock protein,Hsp)70过表达对过氧化氢(hydrogen peroxide,H_2O_2)诱导Buffalo大鼠肝细胞(BRL细胞)cleaved caspase 3表达的影响。方法将BRL细胞分为6组:空白对照组、Ad-CMV-Null组、Ad-CMV-Hsp70组、H_2O_2组、Ad-CMV-Null+H_2O_2组和Ad-CMV-Hsp70+H_2O_2组,Ad-CMV-Null、Ad-CMV-Hsp70、AdCMV-Null+H_2O_2、Ad-CMV-Hsp70+H_2O_2组分别加入1×10~7 PFU/ml的Ad-CMV-Null或Ad-CMV-Hsp70培养48 h后,H_2O_2、Ad-CMV-Null+H_2O_2和Ad-CMV-Hsp70+H_2O_2组分别加入150μmol/L H_2O_2处理2 h,空白对照、Ad-CMVNull和Ad-CMV-Hsp70组分别加入培养液培养。分别采用实时荧光定量PCR检测细胞中Hsp70基因mRNA的转录水平,Western blot法检测细胞中Hsp70及cleaved caspase 3蛋白的表达水平。结果与H_2O_2组相比,Ad-CMVHsp70+H_2O_2组细胞中Hsp70基因mRNA的转录水平和蛋白表达水平均显著升高(P0.01),cleaved caspase 3蛋白表达水平显著下降(P0.01)。结论Hsp70可抑制caspase 3的激活最终保护BRL细胞,减少凋亡损伤。     

乙型肝炎病毒X蛋白对小鼠胚胎肝干细胞凋亡及相关蛋白表达的影响

目的研究乙型肝炎病毒(hepatitis B virus,HBV)X蛋白(HBx)对小鼠胚胎肝干细胞(embryonic liver stemcell,ELSC)凋亡及相关蛋白Bcl2、Mcl1、Bax表达的影响。方法采用表达绿色荧光蛋白(green fluorescent protein,GFP)的腺病毒载体系统将HBx基因转入小鼠胚胎肝干细胞ELSC14.5中,采用RT-PCR和Western blot法检测细胞中HBx基因mRNA的转录和蛋白的表达;Hoechst33342染色法观察细胞核的改变;TUNEL法和流式细胞术检测细胞的凋亡情况;Real-time PCR和Western blot法检测抗凋亡因子Bcl2、Mcl1和促凋亡因子Bax基因mRNA的转录水平和蛋白的表达水平。结果重组腺病毒Ad-GFP-HBx能有效感染ELSC14.5细胞,HBx基因和蛋白均能特异性表达;感染的ELSC14.5细胞核呈现固缩,且边缘化的细胞数减少,细胞凋亡率降低;细胞中Bcl2和Mcl1基因mRNA的转录水平和蛋白的表达水平均增高,而Bax的表达降低。结论 HBx可通过调节Bcl2家族中抗凋亡因子和促凋亡因子的比例失衡,来抑制小鼠胚胎肝干细胞的凋亡,促进其存活。     

shRNA沉默P115基因对胃癌细胞增殖相关蛋白表达的影响及机制

目的探讨高尔基体囊泡转运蛋白(colgi-vesicular transport protein,P115)基因沉默对胃癌细胞株BGC-823中增殖相关因子细胞周期素D1(cyclinD1)、微小染色体维持缺陷蛋白2(mini chromosome maintenance protein 2,Mcm2)和增殖细胞核抗原(proliferating Cell Nuclear Antigen,PCNA)表达的影响及其可能的机制。方法采用脂质体介导法将重组表达质粒P115-shRNA2(P115-shRNA2组)和阴性对照质粒shNC(阴性对照组)转染至高表达P115的胃癌细胞株BGC-823中,并设未转染组,经G418筛选出稳定转染细胞,实时定量PCR(Real-time PCR)法检测细胞中P115、巨噬细胞移动抑制因子(macrophage migration inhibitory factor,MIF)、cyclinD1、Mcm2和PCNA基因mRNA的转录水平;Western blot法检测细胞中P115、MIF、pERK1/2、cyclinD1、Mcm2和PCNA蛋白表达水平;免疫共沉淀法检测BGC-823细胞中P115与MIF间的相互作用;ELISA法检测细胞上清液中MIF的分泌水平。结果与未转染组和阴性对照组相比,P115-shRNA2组细胞中P115、MIF、cyclinD1、Mcm2和PCNA的mRNA和蛋白表达水平均明显降低,ERK1/2的磷酸化水平明显下调,BGC-823细胞培养上清中MIF的含量明显降低;P115蛋白可在抗MIF的免疫沉淀物中检出,P115和MIF在BGC-823细胞中存在特异性的相互作用。结论 P115基因沉默下调胃癌细胞中cyclinD1、Mcm2和PCNA的表达,其分子机制可能与P115通过调控MIF的表达和分泌,进而启动下游的ERK1/2信号通路有关。P115可作为研究胃癌细胞增殖分子机制的新靶点。     

T-cadherin基因在前列腺癌组织中的表达及其对前列腺癌细胞DU145增殖的影响

目的探讨T-cadherin基因在前列腺癌组织中的表达及其对前列腺癌细胞DU145增殖的影响。方法采用RT-PCR法检测40份前列腺癌及相应癌旁组织中T-cadherin基因mRNA的表达。分别用10 ml滴度为1.6×10~(12)pdf/ml的GFP-T-cadherin腺病毒(Ad-GFP-T-cadherin)和GFP腺病毒(Ad-GFP)感染前列腺癌DU145细胞后,MTT法检测T-cadherin对前列腺癌DU145细胞增殖的影响,Western blot法检测T-cadherin对P21和cyclin D1蛋白表达水平的影响,同时设空白对照组(未感染病毒)。结果 T-cadherin在38/40(95%)的前列腺癌中表达下调(P0.01),其表达与前列腺癌的分期、格里森评分及分化有关。Ad-GFP-T-cadherin组DU145细胞中P21蛋白表达水平明显高于Ad-GFP组及空白对照组(P0.01),而cyclin D1蛋白表达水平及增殖活性明显低于Ad-GFP组及空白对照组(P0.01);空白对照组DU145细胞的增殖活性及细胞中P21、cyclin D1蛋白表达水平与Ad-GFP组比较,差异无统计学意义(P0.05)。结论 T-cadherin的表达与前列腺癌的发生密切相关,且可通过上调P21和下调cyclin D1蛋白的表达抑制前列腺癌DU145细胞的增殖。     

Lipid components that reduce protein solubility of soy protein isolates

A lipid fraction from a commercial soy protein isolate (SPI), previously found to be detrimental to SPI solubility, was analyzed by size-exclusion liquid chromatography, by high-performance liquid chromatography (HPLC), and for chemical composition. The molecular weight of most of this material was greater than 1,100 daltons. This lipid fraction was water-soluble yet required a strong nonpolar solvent mixture to elute it from a C₁₈ HPLC column. The lipid material was alkaline (pH 8.7) and composed of 3.0% nitrogen, 1.6% phosphorus, 17.5% nonvolatile crude fatty acids primarily hydroxylated), 10.4% long-chain bases, 9.9% hexuronic acid, 3.2% hexosamine, and 6.6% total sugar. The molecular weight, chemical composition, and physical characteristics (solubility characteristics, surfactant characteristics, and appearance) of this material were all similar to those reported for phytoglycolipid.     

Fish protein concentrate: A new source of dietary protein

The concept of fish protein concentrate (FPC) is based on the more efficient use of our fishery resource by converting under-utilized fish to acceptable products for human consumption. FPC is not a single product. It is rather a family of products produced by different processes. Each member in the family of products has different characteristics and can be used for different purposes. Although a variety of processes has been developed, most have been based on solvent extraction of whole fish to remove lipids and water. FPC produced by solvent extraction of fish of several different species contains between 75% and 95% protein, which is particularly high in quality. This type of product can be used in foods to improve markedly their nutritional quality without significantly changing their other characteristics. FPC produced by some solvent extraction methods, however, has limited functional properties. New processes are being developed which use enzymes, various solvents, or a combination of enzymes and solvents. Products from some of these processes have improved functional properties and these FPCs appear to be particularly promising for use in foods for their functional attributes. Several problems still remain to be solved and research is needed to determine how FPCs can be produced and utilized most efficiently and effectively. There is, however, a commercial industry emerging and indications are that FPCs will find a significant place in the market. One of 16 papers being published from the Symposium, “Oilseed Processors Challenged by World Protein Needs,” presented at the ISF-AOCS World Congress, Chicago, September 1970. National Marine Fisheries Service, National Oceanic and Atmospheric Administration, U.S. Department of Commerce.     

Development of grapeseed protein

The potential for grapeseed oil and protein in regions where grape production is significant is discussed. Extraction and concentration procedures which improve the nutritional value of grapeseed protein and problems related to protein digestibility are presented.     

Potential of genetic algorithms in protein folding and protein engineering simulations

Genetic algorithms are very efficient search mechanisms whichmutate, recombine and select amongst tentative solutions toa problem until a near optimal one is achieved. We introducethem as a new tool to study proteins. The identification andmotivation for different fitness functions is discussed. Theevolution of the zinc finger sequence motif from a random startis modelled. User specified changes of the repressor structurewere simulated and critical sites and exchanges for mutagenesisidentified. Vast conformational spaces are efficiently searchedas illustrated by the ab initio folding of a model protein ofa four ß strand bundle. The genetic algorithm simulationwhich mimicked important folding constraints as overall hydrophobicpackaging and a propensity of the betaphilic residues for transpositions achieved a unique fold. Cooperativity in the ßstrand regions and a length of 3–5 for the interconnectingloops was critical. Specific interaction sites were considerablyless effective in driving the fold.     

Development of safflower protein

Development of safflower protein and safflower protein isolate (SPI) containing as high as 95% protein (N×5.3) is described. SPI exhibits favorable nitrogen solubility, foaming, and bread-baking properties. Composition of SPI and select functional properties may be altered by the choice of pH used to precipitate the extracted protein (5 or 6). PER of SPI (1.26) was increased to as high as 2.13 by the addition of L-lysine at levels of 0.75% of the diet. Theoretical estimates of production costs for SPI are similar to estimates for soy protein isolate. SPI has been evaluated experimentally in pastas, baked products, and beverage systems. Nutritional and functional properties indicate that SPI has promising potential as either a protein fortificant and/or a functional ingredient in various foods.     

Expressed protein ligation for a large dimeric protein

Expressed protein ligation (EPL) is a protein engineering tool for post-translational ligation of protein or peptide fragments. This technique allows modification of specific parts of proteins, opening possibilities for incorporating probes for biophysical applications such as nuclear magnetic resonance (NMR) or fluorescence spectroscopy. The application for oligomeric proteins, however, is restricted by the need to obtain a large excess of active dimer over reactants and intermediates. Here, we explored the suitability of the EPL reaction for large dimeric proteins using the molecular chaperone Hsp90 as a model. We systematically varied the reaction conditions and the preparation protocols for the reactants. Modulation of the ligation site by shortening the flexible segment at the N-terminus of the C-terminal reactant increased the yield sufficiently to isolate the product by chromatography. Under those conditions, 41% of the used C-terminal fragment could be successfully ligated. We discuss possible up-scaling for segmental isotope labelling for NMR applications.     

马铃薯/乳清蛋白复合凝胶的性质和微观结构

为减少乳清蛋白用量，用马铃薯蛋白替代乳清蛋白质量的一半制备热诱导凝胶。通过色度、质构、水分分布、流变等考察了总蛋白浓度（40~80 g/L）对于复合凝胶物理性质的影响。结果表明，复合凝胶的最低成胶浓度为50 g/L，与乳清蛋白80 g/L的最低成胶浓度相比，乳清蛋白用量减少了2.2倍。总蛋白浓度从50 g/L提高到80 g/L时，复合凝胶偏黄，硬度和弹性分别增加了5.28倍和5.90%，但束缚水含量降低了3.63%。储能模量（G''）显示较弱的频率依赖性。CLSM和SEM观察表明，蛋白浓度80 g/L的复合凝胶孔隙尺寸小，具有由颗粒聚集体组成的均一致密网络结构，因而具有较高G''。红外光谱表征结果表明，复合凝胶中β-折叠和β-转角约占蛋白二级结构的70%。溶解度测试结果表明，维持复合凝胶结构的作用力中氢键和疏水相互作用的贡献高于二硫键。     

The ups and downs of protein topology; rapid comparison of protein structure

Protein topology can be described at different levels. At themost fundamental level, it is a sequence of secondary structureelements (a `primary topology string'). Searching predictedprimary topology strings against a library of strings from knownprotein structures is the basis of some protein fold recognitionmethods. Here a method known as TOPSCAN is presented for rapidcomparison of protein structures. Rather than a simple two-letteralphabet (encoding strand and helix), more complex alphabetsare used encoding direction, proximity, accessibility and lengthof secondary elements and loops in addition to secondary structure.Comparisons are made between the structural information contentof primary topology strings and encodings which contain additionalinformation (`secondary topology strings'). The algorithm isextremely fast, with a scan of a large domain against a libraryof more than 2000 secondary structure strings completing in     

蛋白质水力学半径的测定及用于蛋白质变性过程的实时监控

采用纳米粒度分析仪Zetasizer Nano测定了牛血清白蛋白（BSA）的水力学半径,考察了pH、离子强度、表面活性剂等的影响,并用于BSA热变性和脲变性过程的实时跟踪。随pH增大,BSA水力学半径呈“U”形变化趋势;酸性条件下,分子膨胀,随着盐浓度升高,BSA分子先小幅减小后显著增大;中性pH范围,离子强度对蛋白质分子尺寸影响很小,蛋白质性质相对稳定。离子型表面活性剂在蛋白质表面吸附,从而影响BSA水力学半径。通过水力学半径的测量实时跟踪蛋白质变性过程的分子变化,发现增加热变性中离子强度可加快变性速率,SDS加入增大了BSA变性温度Tm。脲在促使BSA分子扩张的同时,对链伸展有抑制作用;在DTT存在下,BSA的水力学半径随着时间变化逐渐增大。结果表明,简便的水力学半径测量可以用于蛋白质大小的表征,并可实时跟踪蛋白变性过程的分子尺度变化。