表皮生长因子与前列腺增生及前列腺癌的关系

195 9年Ｃｏｈｅｎ[1] 在纯化神经生长因子 (ＮＧＦ)的过程中 ,首先从小鼠颌下腺中发现了一种对外胚层、中胚层和内胚层细胞具有促分裂活性的多肽生长因子 ,并能明显地促进表皮细胞的生长与角化 ,命名为表皮生长因子 (ＥｐｉｄｅｒｍａｌＧｒｏｕｔｈＦａｃｔｏｒＥＧＦ)。人的表皮生长因子以ｈＥＧＦ - β和ｈＥＧＦ -γ两种形式存在。由于它对胃酸具有很强的抑制作用 ,故又命名为尿抑胃素 (ｕｒｏｇａｓｔｒｏｎｅ)。 1975年Ｓｔａｒｋｅｙ等人[2 ] 从尿中纯化获得了ＥＧＦ的纯品 ,并确定其结构。ｈＥＧＦ - β和ｈＥＧＦ -γ具有高度的同…     

hEGF和hTGF—αN结构域与C结构域的功能差异

用ＰＣＲ的方法将人表皮生长因子和人转化生长因子－α（ｈＴＧＦ－α）的Ｎ结构域和Ｃ结构域互换,构造了两个嵌合分子Ｅ－ＴＧＦ（ＥＧＦ１－３２－ＴＧＦ－α３４－５０）和Ｔ－ＥＧＦ（ＴＧＦ－α１－３３－ＥＧＦ３３－５３）。野生型和嵌合分子基因在大肠杆菌ｐｈｏＡ系统表达并纯化定量。各重组体的受体竞争结合活性大小为ｈＥＧＦ〉ｈＴＧＦ－α和Ｅ－ＴＧＦ〉Ｔ０－ＥＧＦ,它们的促细胞生长活性的大小为ｈＴＧＦ－α和Ｅ－     

人工合成表皮生长因子基因在甲基营养型酵母中的高效表达

选用酵母菌偏爱密码子人工合成了编码５１个氨基酸的人表皮生长因子（ｈＥＧＦ）基因．将合成基因与编码酵母α因子前导肽８５个氨基酸的ＤＮＡ片段融合后克隆到醇氧化酶基因启动子下游,并构建出多拷贝表达载体．此载体转化甲基营养型酵母株ＧＳ１１５后筛选出整合型ＭｕｔＳＨｉｓ＋基因型菌株．高密度培养及诱导表达后该株可分泌具完好生物活性和正确物理化学性质的人表皮生长因子,产量达１００ｍｇ／Ｌ,经３次柱层析纯度达９５％以上,为观察其生物学作用打下了良好基础     

表皮生长因子，生长抑素对人早孕胎盘hCG分泌及基因表达的影响

观察了表皮生长因子（ＥＧＦ）,生长抑素（ＳＳ）对体外培养的人早孕绒毛膜促性腺激素（ｈＣＧ）分泌及ｈＣＧβ－ｍＲＮＡ含量的影响。发现ＥＧＦ可明显刺激绒毛分泌ｈＣＧ,显著增加ｈＣＧβ－ｍＲＮＡ含量,生长抑素虽然对绒毛ｈＣＧ分泌及ｈＣＧ　β－ｍＲＮＡ含量无明显影响,但可抑制ＥＧＦ,ＧｎＲＨ刺激的ｈＣＧ分泌及ｈＣＧβ－ｍＲＮＡ水平。提示ＥＧＦ,ＳＳ在妊娠早期参与了ｈＣＧ分泌的调节。     

表皮生长因子,生长抑素对人早孕胎盘hCG分泌及其因表达的影响

观察了表皮生长因子,生长抑素对体外培养的人早孕绒毛膜促性腺激素分泌及ｈＣＧβ－ｍＲＮＡ含量的影响,发现ＥＧＦ可明显刺激绒毛分泌ｈＣＧ,显著增加ｈＣＧ β－ｍＲＮＡ含量。生长抑素虽然对绒毛ｈＣＧ分泌及ｈＣＧβｍ－ＲＮＡ含量无明显影响,但可抑制ＥＧＦ,ＧｎＲＨ的ｈＣＧ分泌及ｈＣＧβ－ｍＲＮＡ。提示ＥＧＦ,ＳＳ在妊娠早期参与了ｈＣＧ分泌的调节。     

高活性重组hG-CSF的制备

我们构建了新的硫氧还蛋白（Ｔｈｉｏｒｅｄｏｘｉｎ）融合表达载体ｐＥＴＴｒｘＬ和ｐＥＴＴｒｘ－ＨｉｓＬ,它们可使功能蛋白在大肠杆菌胞质中以可溶性形式高效表达。利用此表达系统成功地获得的ｈＧ－ＣＳＦ－硫氧还蛋白融合蛋白的高效可溶性表达,表达水平达总细胞可溶蛋白的４１％以上。所表达的ｈＧ－ＣＳＦ－硫氧还蛋白融合蛋白可通过Ｃｕ２＋－ＩＤＡＳｅｐｈａｒｏｓｅＦＦ固相金属螯合层析柱,方便地从细胞破碎可溶上清中直接纯化。所获得的融合蛋白具有ｈＧ－ＣＳＦ特异的生物活性,其比活性达到０．５－１．３３×１０７ｕ／ｍｇ融合蛋白。这样表达的ｈＧ－ＣＳＦ融合蛋白能被ＩｇＡ蛋白酶特异地切割,将ｈＧ－ＣＳＦ从融合蛋白上切下获得与天然蛋白一级结构完全一致的重组ｈＧ－ＣＳＦ 。     

hTGF—α的C结构域半保守残基对其结构与功能的影响

ＥＧＦ家族Ｃ结构域有较高的同源性,其中有一些氨基酸表现为半保守性质。我们应用定点突变法对ｈＴ－ＧＦ－αＣ结构域的半保守残基进行突变,代之以ｈＥＧＦ的相应残基,构建３个突变分子ｈＴＧＦ－αＶ３５、ｈＴＧＦ－αＱ４４和ｈＴＧＦ－αＹ４５Ｒ４６。实验发现ｈＥＧＦ与ＥＧＦ受体的亲和力为ｈＴＧＦ－α的２倍,而３个突变体与ＥＧＦ受体的亲和力分别为ｈＴＧＦ－α的２２％,１３．４％和２５％;ｈＥＧＦ促ＮＲＫ－４９     

人主动脉硫酸乙酰肝素蛋白聚糖对培养的人血管壁细胞生长的作用

通过培养的人主动脉平滑肌细胞（ｈＡＳＭＣ）及脐静脉内皮细胞（ｈＵＶＥＣ）,应用３Ｈ－ＴｄＲ参入、Ｎｏｒｔｈｅｒｎｂｌｏｔ分析、逆转录多聚酶链反应（ＲＴ－ＰＣＲ）、放射免疫分析（ＲＩＡ）、和紫外比色法等技术观察了人主动脉中硫酸乙酰肝素蛋白聚糖（ＨＳＰＧ）对ｈＡＳＭＣ和ｈＵＶＥＣＤＮＡ合成的作用及对血小板源生长因子（ＰＤＧＦ）、ＰＤＧＦ受体、转化生长因子β（ＴＧＦ－β）、内皮素－１（ＥＴ－１）或碱性成纤维细胞生长因子（ｂＦＧＦ）基因表达和肾素－血管紧张系统（ＲＡＳ）的影响,结果显示,ＨＳＰＧ明显抑制培养的ｈＡＳＭＣ基础的ＤＮＡ合成（ｃｐｍ值为：１０３８５±３２６３ｖｓ,２５５４１±６４２１,Ｐ＜０．０１）及外源性ＰＤＧＦ诱导的ＤＮＡ合成（ｃｐｍ值为：９８７８±１９４７ｖｓ．１３４８１±４４ｌ０,Ｐ＜０．０５）;抑制ＰＤＧＦＡ链、ＴＧＦ－Ｂｐ和ＥＴ－１ｍＲＮＡ表达,提高ＰＤＧＦａ和β受体ｍＲＮＡ的表达;显著降低ｈＡＳＭＣ培养液中血管紧张素Ⅱ（ＡｎｇⅡ）的浓度和血管紧张素转换酶（ＡＣＥ）的活性,推测ＨＳＰＧ抑制ＰＤＧＦＡ链、ＴＧＦ－β及ＥＴ－１ｍＲＮＡ表达,降低ＡＣＥ活性及ＡｎｇⅡ浓度是其抑制ｈＡＳＭＣ增殖的重要机     

细支气管肺泡细胞增生和细支气管肺泡细胞癌内生长因子和蛋白激酶C的表达

表皮生长因子（ＥＧＦ）、转化生长因子－α（ＴＧＦα）、表皮生长因子受体（ＥＧＦＲ）和蛋白激酶Ｃ（ＰＫＣ）与细胞生长、增殖分化调节和细胞癌变有密切关系。作者用免疫组织化学方法检测了细支气管肺泡细胞增生（ＢＡＨ）和细支气管肺泡细胞癌（ＢＡＣ）的ＥＧＦ、ＴＧＦα、ＥＧＦＲ和ＰＫＣ表达。结果表明：ＢＡＣ中的ＥＧＦ、ＴＧＦα阳性率和阳性强度以及ＥＧＦＲ、ＰＫＣ阳性强度均明显高于ＢＡＨ。ＢＡＨ的重度不典型增生病例,其ＥＧＦ、ＴＧＦα、ＥＧＦＲ和ＰＫＣ均呈高表达。ＴＧＦα、ＥＧＦＲ和ＰＫＣ三者在ＢＡＣ和ＢＡＨ中的表达存在明显相关性。提示：ＴＧＦα及其受体ＥＧＦＲ和ＰＫＣ是细支气管肺泡细胞增生、恶性转化和肺泡癌细胞失控生长的重要因素。     

气道上皮细胞的抗氧化损伤保护及微环境调控

本工作建立了臭氧对原代培养的兔支气管上皮细胞（ＢＥＣ）损伤模型,观察到血管活性肠肽（ＶＩＰ）、表皮生长因子（ＥＧＦ）、热应激等微环境理化因子可减轻细胞损伤,具有细胞保护作用,其保护机制与提高还原谷胱甘肽（ＧＳＨ）含量有关,并依赖于蛋白激酶的磷酸化调节及基因转录。ＢＥＣ细胞在基础情况下有ｂｃｌ－２基因的低水平表达。ＶＩＰ和ＥＧＦ可促进ｂｃｌ－２基因转录,增强ＢＥＣ的抗氧化损伤能力。ＥＧＦ或热应激促进     

Batch and fed-batch cultivation for excretive production of human epidermal growth factor (hEGF) with recombinant E. Coli K12 system

Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32 degrees C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system.     

Batch and Fed-Batch Cultivation for Excretive Production of Human Epidermal Growth Factor (hEGF) with Recombinant E. Coli K12 System

Abstract

Batch and fed-batch production of recombinant human epidermal growth factor (hEGF) was studied in an E. coli secretary expression system. By using MMBL medium containing 5 g/L glucose, controlling the temperature at 32°C and maintaining the dissolved oxgen level over 20% saturation, a high yield of hEGF (32 mg/L) was obtained after an 18 hr batch cultivation with 0.2 mM IPTG induction at mid-log phase. Three different glucose feeding strategies were employed to further improve hEGF productivity in a bench top fermentor. Compared with the batch results, hEGF yield was improved up to 25.5% or 28.1%, respectively by intermittent or pH-stat glucose feeding, and up to 150% improvement of hEGF production was achieved by constant feeding of 200 g/L glucose solution at a rate of 0.11 mL/min. The effects of further combined feeding with other medium components and inducer on hEGF yield were also examined in the benchtop fermentor. This work is very helpful to further improve the productivity of extracellular hEGF in the recombinant E. coli system.     

Fed-batch cultivation ofEscherichia coli YK537 (pAET-8) for production ofphoA promoter-controlled human epidermal growth factor

Secretion of the expressed heterologous proteins can reduce the stress to the host cells and is beneficial to their recovery and purification. In this study, fed-batch cultures ofEscherichia coliYK537 (pAET-8) were conducted in a 5-L fermentor for the secretory production of human epidermal growth factor (hEGF) whose expression, was under the control of alkaline phosphatase promoter. The effects of feeding of glucose and complex nitrogen sources on hEGF production were investigated. When the fed-batch culture was conducted in a chemically defined medium, the cell density was 9.68 g/L and the secreted hEGF was 44.7 mg/L in a period of 60 h. When a complex medium was used and glucose was added in pH-stat mode, the secreted hEGF was improved to 345 mg/L. When the culture was fed with glucose at a constant specific rate of 0.25 gg⁻¹h⁻¹, hEGF reached 514 mg/L. The effects of adding a solution containing yeast extract and tryptone were further studied. Different rate of the nitrogen source feeding resulted in different levels of phosphate and acetic acid formation, thus affected hEGF expression. At the optimal feeding rate, hEGF production achieved 686 mg/L.     

Factors influencing excretive production of human epidermal growth factor (hEGF) with recombinant Escherichia coli K12 system

Human epidermal growth factor (hEGF) was expressed and excreted into culture medium in an excretory recombinant Escherichia coli system. Conditions for the production of hEGF in this system were investigated. Results showed that the slight reduction of promoter strength improved the stability of plasmid and the production of hEGF in this recombinant system. One favorable MMBL medium for hEGF production was formulated by evaluating the effects of medium components, ampicilin addition and pH. hEGF production was affected obviously by culture conditions, especially fermentation temperature. High temperature (32 °C) was very beneficial for culture process by increasing productivity and reducing the quantity of isopropyl-#-d-thiogalactopyranoside (IPTG) for sufficient induction. High cell density and hEGF productivity could be accomplished concomitantly by inducing the culture at middle or late log phase of cell growth. In comparison with the batch process, fed-batch cultivation could improve plasmid stability from ca. 72% to ca. 83%, and increased hEGF productivity by 24.4%. Under all these circumstances, almost all expressed hEGF (⃋%) was fully excreted into the culture medium.     

Improvement of hEGF production with enhanced cell division ability using dissolved oxygen responses to pulse addition of tryptone

Tryptone has multiple and complex effects on cell physiology and process performance in pulse fed-batch cultivation of recombinant Escherichia coli. By applying feedback control of dissolved oxygen signal responding to pulse in the feed rate, the production of acetate was avoided and the optimization of production of recombinant human epidermal growth factor (hEGF) was successfully achieved. With the addition of an optimum amount of tryptone along with glucose in the pulse fedbatch cultivation of E. coli, the ability of the cell to divide and the stability of the plasmid within the bacteria were improved. Consequently, segregation of the cells into a viable but non-culturable physiological state was alleviated. Addition of tryptone also enhanced cell respiration before and after hEGF expression and thus further benefited the production of recombinant hEGF. Excessive addition of tryptone resulted in low sensitivity of the oscillation of dissolved oxygen signal and poor operability of pulse fed-batch cultivation as this led to an accumulation of acetate, which weakened the dissolved oxygen signal responses. Consequently, the production of recombinant protein was considerably reduced. By combining the process performance and the positive effect of complex media pulse addition on bacterial metabolism, the optimal production conditions of hEGF were successfully determined. A high cell density of 91 g/L dry cell weight was obtained under these optimal production conditions. Furthermore, a high level of 0.24 g/L hEGF was attained leading to a 32.6% increase in product yield as compared to the controls.     

An improved procedure for production of human epidermal growth factor from recombinant E. coli

An improved procedure for the fermentation and purification of human epidermal growth factor (hEGF) was developed. Recombinant Escherichia coli HB-101 [lacUV5omp08hEGF] harboring plasmid lacUV5omp08hEGF encoding hEGF was used in fermentation to increase levels of hEGF. Medium composition, and the levels of inoculum, inducer (isopropyl-beta-D-thiogalactoside) and ampicillin were optimized with respect to volumetric fermentation of hEGF. As a result, the hEGF concentration reached a high value of 242 mg l(-1) and the amount of heterogeneous protein decreased by 62% compared with that before optimization in batch fermentation. High-quality hEGF was purified from the fermentation culture by centrifugation, salting-out, resuspension, recentrifugation and finally gel chromatography on a Grad-iFrac System using Sephadex G-50 superfine. The purity of hEGF and the total yield were more than 94% and higher than 36%, respectively, and SDS-PAGE of the purified hEGF demonstrated a single band corresponding to an hEGF standard. In particular, a very important phenomenon was found, i.e. that the amount of heterogenous protein in fermentation broths cultured in media with high concentrations of lactose is far less than that cultured in media with high concentrations of glucose.     

金黄色破囊壶菌发酵生产DHA的研究

顺 4,7,1 0 ,1 3 ,1 6 ,1 9 二十二碳六烯酸 (ｄｏｃｏｓａｈｅｘａｅｎｏｉｃａｃｉｄ ,简称ＤＨＡ)是ω 3系列多不饱和脂肪酸。近年的研究表明 ,ＤＨＡ是组成大脑和视网膜的重要结构物质 ,如大脑灰质结构脂质中 6 0 %的脂肪酸均为ＤＨＡ[1] 。ＤＨＡ对人体健康有益的生理功能主要表现在[2 ] :调节中枢神经系统功能 ;预防和治疗心血管疾病 ;治疗气喘、关节炎等 ;预防和治疗乳腺癌、结肠癌等。由于ＤＨＡ具有上述生理功能 ,已在医药、食品、保健品等领域得到广泛应用。目前 ,商品ＤＨＡ主要来源于深海鱼油 ,如沙丁鱼、金枪鱼等鱼油 ,由于鱼…     

一种来源于链霉菌的纤溶酶的纯化及其基因的克隆

链霉菌C3662的发酵液上清经 80 %硫酸铵沉淀 ,DEAE Sepharose和CM Sepharose层析分离后纯化出一种纤溶酶。SDS PAGE显示为单一的条带 ,分子量约为 30kD。以 pIJ699为载体 ,S .lividansTK2 4为宿主菌 ,鸟枪法克隆纤溶酶基因 ,从 30 0 0个转化子中挑选到 1个具活性转化子 ,经亚克隆 ,序列测定得到一个 90 3bp的完整ORF ,其GC %为 68.33% ,密码子第三位GC %为 95.6% ,符合链霉菌基因的典型特征。与多种蛋白酶具有较高的同源性     

基于pH的反馈补料方法在谷氨酸发酵中的应用

为简化谷氨酸发酵补料工艺,提出了一种新型的基于pH的补料方式。考察谷氨酸发酵过程中氨消耗量 (x) 和糖消耗量 (y) 发现,两者之间存在较好的线性关系 (y=7.4744x,R2=0.9989),以此为pH反馈补料工艺中补料液中葡萄糖与氨的混合比例,能较好地将谷氨酸发酵过程中葡萄糖浓度稳定在12~21 g/L。比较恒定葡萄糖浓度补料工艺与pH反馈补料工艺发现,采用pH反馈补料工艺进行发酵,葡萄糖转化率、谷氨酸产酸速率分别提高了9.06％和17.5％左右,同时发酵周期缩短2 h以上。     

人肝细胞生长因子(hdHGF)基因在毕赤酵母中的分泌表达

研究了hdHGF基因在毕赤酵母中的表达 .以人胎盘mRNA为模板 ,经逆转录、重叠PCR获得hdHGF全长和成熟基因片段 .将该基因片段克隆到pPIC9载体上 ,将重组表达质粒转化巴斯德毕赤酵母 (Pichiapastoris)GS115,筛选mut+ 表型 ,经甲醇诱导可实现rhdHGF的分泌表达 .经摇瓶培养筛选出 4株表达水平较高的酵母工程菌株 ,SDS PAGE分析和Western印迹试验表明 ,产物分子量约为80kD ,5L发酵罐高密度培养已使生物量达 13 5g L(干重 ) ,发酵液上清总蛋白量为 8 0g L ,电泳结果表明rhdHGF表达水平为总蛋白的 12 3 % .