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1.
Zhong-hui Zhang Li-juan Du Di Xiang Shun-ying Zhu Ming-yuan Wu Hui-li Lu Yan Yu Wei Han 《Journal of Zhejiang University. Science. B》2009,10(2):79-86
Midkine is a heparin-binding growth factor, which plays important roles in the regulation of cell growth and differentiation.
The non-tagged recombinant human midkine (rhMK) is therefore required to facilitate its functional studies of this important
growth factor. In the present work, rhMK was expressed in Escherichia coli (E. coli) BL21 (DE3). The expression of midkine was efficiently induced by isopropyl-β-D-thiogalactopyranoside (IPTG). After sonication,
midkine was recovered in an insoluble form, and was dissolved in guanidine hydrochloride buffer. Renaturation of the denatured
protein was carried out in the defined protein refolding buffer, and the refolded protein was purified using S-Sepharose ion-exchange
chromatography. The final preparation of the rhMK was greater than 98% pure as measured by sodium dodecylsulfate-polyacrylamid
gel electrophoresis (SDS-PAGE) and reverse phase high performance liquid chromatography (RP-HPLC). The purified rhMK enhanced
the proliferation of NIH3T3 cells.
Project supported in part by the Hi-Tech Research and Development Program (863) of China (No. 2007AA02Z149) and the Science
and Technology Commission of Shanghai Municipality (No. 075407071), China 相似文献
2.
草鱼生长激素cDNA在大肠杆菌细胞中表达后，重组草鱼生长激素在胞内形成不溶性的包含体．这种包含体在洗涤、变性、复性和C12反相层析等步骤后，产物再经快速蛋白液相色谱，重组草鱼生长激素在SDS-聚丙烯酰凝胶电泳中显示均一性，纯度可达90％放射免疫分析表明：重组草鱼生长激素具有草鱼生长激素的免疫活住．分离纯化产率约为1L细菌培养液1～2m重组草鱼生长激素． 相似文献
3.
目的:在大肠杆菌(BL21)中构建可溶性表达的金黄色葡萄球菌B型肠毒素(SEB)受体拮抗剂。方法:首先确定SEB受体桔抗剂的基因序列,然后用含有SEB受体桔抗剂的基因序列重组质粒表达载体PGEX-4T-1转化大肠杆菌BL21(DE3),利用IPTG诱导表达获得蛋白,产物经GST柱纯化后,利用ELISA检测其与SEB的结合能力并进行其体内外药效学实验。结果:该质粒成功转化为可溶性表达,ELISA结果显示表达产物可与SEB特异性结合。结论:本研究成功对SEB受体拮抗剂GST可溶性表达并对其活性进行初步分析。 相似文献
4.
5.
研究用亲和融合谷胱甘肽 S 转移酶 (GST)的方法纯化重组人白细胞介素 6(IL 6)的发酵和纯化工艺 ,使用含有质粒pHZl818的E .coliJMl0 9在 2XYT培养基中进行发酵表达 ,IL 6表达为与谷胱甘肽 S 转移酶 (GST)融合的融合蛋白GST IL 6.融合蛋白存在可溶的活性蛋白和不可溶的包含体两种形式 ,此包含体无活性且无法复性 ,无法用亲和层析回收 ,通过实验优化摇床发酵的诱导温度和转速 ,以增加可溶融合蛋白的表达 .菌体超声破碎液后 ,上清液用作亲和柱层析 ,可将融合蛋白提纯至 80 00 ,每升发酵液可得 10mg融合蛋白 ,用凝血酶裂解处理 6h ,亲和标志物GST被特异性切除 ,裂解得到的IL 6用离子交换柱层析可纯化至 95 00 ,MTT法测得纯化的IL 6生物学活性为 1.0 2× 10 8IU/mg . 相似文献
6.
嗜水气单胞菌外分泌物的致病性 总被引：3，自引：0，他引：3
该菌分泌物有溶血性、细胞毒性和肠毒素毒性，毒性实验证实嗜水气单胞菌外分泌物对小白鼠和中华鳖有较高的致死毒性．层析纯化得到的1种蛋白质有上述3种毒性和致死性． 相似文献
7.
以pErl30a为载体构建的表达拟南芥热激因子AtHsfAla的大肠杆菌EcoliM15（pET30a／His6一AtHsfAla）为实验材料,以IPTG诱导6XHis融合蛋白的表达并经过Ni2＋柱分离纯化AtHsfAla,再通过SDS—PAGE鉴定表达蛋白和纯化蛋白．结果显示,AtHsfAla蛋白在在PET原核表达系统中能够有效表达,并能通过亲和层析获得纯化的AtHsfAla蛋白．研究结果为揭示拟南芥热激因子AtHsfAla的作用机理奠定基础． 相似文献
8.
以构建的表达拟南芥热激因子HsfA1a C端氨基酸331到氨基酸486（简称△HsfA1a）的大肠杆菌Ecoli M15为材料,用异丙基硫代-β-D-半乳糖苷（IVFG）诱导表达△HsfA1a,再通过Ni—NTA—Agarose亲和层析纯化表达的△HsfA1a,通过SDS—PAGE电泳分析表达蛋白和纯化蛋白,获得了表达纯化的△HsfA1a． 相似文献
9.
将人细胞周期蛋白D1基因克隆入原核表达载体pET-20b中获得重组质粒pET-20b-eyeD,经酶切鉴定正确后转化大肠杆菌BL21PlaysS后获得表达菌株．该菌株经IPTG诱导后表达的目的蛋白有部分分泌到培养基上清中,将培养基上清中的蛋白沉淀后用Ni^2＋螯合柱进行纯化,最后可得到纯度达到95％以上的目的蛋白．蛋白电泳显示纯化蛋白的分子量约为33KD,Westemblot分析表明,在电泳胶的相应分子量处出现特异性条带,说明已经成功表达和纯化了重组人细胞周期蛋白D1． 相似文献
10.
Jing LI Qian YANG Li-hua ZHAO Shu-mei ZHANG Yu-xia WANG Xiao-yu ZHAO 《Journal of Zhejiang University. Science. B》2009,10(4)
An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel P-100.The protein was absorbed on DEAE-cellulose and Bio-Gel P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pl value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited in-hibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia scle-rotiorum. The 50% inhibitory concentrations (IC50) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B291 also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germi-nated spores. 相似文献
11.
《天津大学学报(英文版)》2015,(1)
The Bacillus strain BH072 isolated from a honey sample showed strong antifungal activity against phytopathogen. Gene cloning test demonstrated that the strain had a tas A gene encoding an antifungal Tas A protein. Although the wild strain simultaneously produced various antifungal substances, only the physicochemical property and antifungal activity of Tas A protein were unclear due to the difficulty in extraction. In this study, tas A gene encoding the protein from Bacillus sp. BH072 was amplified by using the polymerase chain reaction(PCR) method and cloned into p ET 28a(+) vector, and then expressed in host cells Escherichia coli BL21(DE3). The expressed proteins were collected by centrifugation and ultrasonic treatment, and then purified by using nickel-nitrilotriacetic acid(Ni-NTA)metal affinity column and dialysis methods. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) test showed that an expected protein band appeared with a size of 31 k Da. The expressed products possessed antifungal activity against the phytopathogenic indicator strain Botrytis cinerea. A genetically engineered strain tas A of E. coli was established in this study which can efficiently express Tas A protein. 相似文献
12.
Objective: The aims of this research were to purify and identify the 130 kDa (CagA) protein of H. pylori clinical isolate HP97002 and evaluate the relationships between the purified 130 kDa (CagA) protein and gastric diseases. Methods: The procedure for isolating the protein included 6 mol/L guanidine extract, size exclusion chromatography and elusion from gel. Sera of 68 patients with gastric diseases (44 with chronic gastritis,15 with atrophic gastritis,7 with peptic ulcer disease,2 with gastric cancer ) were obtained, and the serological response to CagA was studied by Western-blot using the purified protein. Results: The purified protein was 130 kDa and preserved good antigenicity and revealed basic isoelectric point about of 8.1. Among 68 sera, 43 sera could recognize the purified protein associated with chronic gastritis 47.7% (21/44),atrophic gastritis 86.7% (13/15),peptic ulcer disease 100% (7/7),gastric cancer 100% (2/2). Compared with each other, the difference was significant (χ2=13.327, P=0.004), and 130 kDa (CagA) protein was associated with severe gastric diseases (rs=0.442, P=0.001). Conclusion: The 130 kDa (CagA) protein was associated with severe gastric diseases. 相似文献
13.
Jing Li Qian Yang Li-hua Zhao Shu-mei Zhang Yu-xia Wang Xiao-yu Zhao 《Journal of Zhejiang University. Science. B》2009,10(4):264-272
An antifungal protein was isolated from a culture of Bacillus subtilis strain B29. The isolation procedure comprised ion exchange chromatography on diethylaminoethyl (DEAE)-52 cellulose and gel filtration chromatography on Bio-Gel® P-100. The protein was absorbed on DEAE-cellulose and Bio-Gel® P-100. The purified antifungal fraction was designated as B29I, with a molecular mass of 42.3 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), pI value 5.69 by isoelectric focusing (IEF)-PAGE, and 97.81% purity by high performance liquid chromatography (HPLC). B29I exhibited inhibitory activity on mycelial growth in Fusarium oxysporum, Rhizoctonia solani, Fusarium moniliforme, and Sclerotinia sclerotiorum. The 50% inhibitory concentrations (IC50) of its antifungal activity toward Fusarium oxysporum and Rhizoctonia solani were 45 and 112 μmol/L, respectively. B29I also demonstrated an inhibitory effect on conidial spore germination of Fusarium oxysporum and suppression of germ-tube elongation, and induced distortion, tumescence, and rupture of a portion of the germinated spores. 相似文献
14.
绿僵菌是一类广泛应用于生物防治的昆虫病原真菌.研究表明小RNA能够调控基因的表达,其中Argonaute基因在整个小RNA通路中发挥重要的作用.本研究通过RT-PCR的方法从绿僵菌中获得了Argonaute基因的部分功能片段,构建了其重组原核表达载体,将重组载体转化至大肠杆菌进行诱导表达;采用镍柱亲和纯化重组的目的蛋白并通过Western blot技术鉴定.结果发现：通过RT-PCR的方法获得长度约为950bp的基因片段;重组原核表达载体经诱导表达后,SDS-PAGE检测发现分子量约为34kDa的目的蛋白条带;诱导5h后蛋白的表达量最高,采用镍柱亲和层析纯化重组蛋白,经Western blot技术检测,重组蛋白可与His-tag抗体发生特异性反应.该纯化重组蛋白的获得为将来绿僵菌Argonaute蛋白抗体的制备,并进一步通过该抗体获得绿僵菌体内的小RNA及其靶基因提供了基础. 相似文献
15.
Objective: The aims of this research were to purify and identify the 130 kDa (CagA) protein ofH. pylori clinical isolate HP97002 and evaluate the relationships between the purified 130 kDa (CagA) protein and gastric diseases.
Methods: The procedure for isolating the protein included 6 mol/L guanidine extract, size exclusion chromatography and elusion
from gel. Sera of 68 patients with gastric diseases (44 with chronic gastritis, 15 with atrophic gastritis, 7 with peptic
ulcer disease, 2 with gastric cancer) were obtained, and the serological response to CagA was studied by Western-blot using
the purified protein. Results: The purified protein was 130 kDa and preserved good antigenicity and revealed basic isoelectric
point about of 8.1. Among 68 sera, 43 sera could recognize the purified protein associated with chronic gastritis 47.7% (21/44),
atrophic gastritis 86.7% (13/15), peptic ulcer disease 100% (7/7), gastric cancer 100% (2/2). Compared with each other, the
difference was significant (χ2=13.327,P=0.004), and 130 kDa (CagA) protein was associated with severe gastric diseases (r
s=0.442,P=0.001). Conclusion: The 130 kDa (CagA) protein was associated with severe gastric diseases.
Project supported by the China Medical Board (96-628) and Zhejiang Province Hygiene Bureau (2000 A055) 相似文献
16.
余瑛 《重庆大学学报(英文版)》2003,2(2)
1. Introduction Acidic fibroblast growth factor (aFGF), a member of a family of structurally related polypeptides, is a kind of multifunctional protein that can stimulate cellular proliferation, migration and differentiation. In vivo, aFGF displays angiogenic activity promoting vascular endothelial cell mitogenesis as well as chemotaxis and induction of proteases involved in tissue regeneration [1]. Due to this wide range of biological activities, many potential therapeutic usages of aFGF … 相似文献
17.
骨形态发生蛋白-2（bone morphogenetic protein,BMP-2）具有多种生物活性,有潜在的药用价值.为探索利用基因工程技术重组BMP-2并在大肠杆菌表达系统表达的可行性.在大肠杆菌中重组和筛选BMp-2基因,SDS-PAGE电泳分析,显示外源蛋白带在相对分子量约12kD处,以离子交换层析DEAE和分子筛纯化蛋白,缓慢复性并在C2C12细胞内检测到其蛋白活性. 相似文献
18.
Due to their significant value in both economy and ecology, Daphnia had long been employed to investigate in vivo response of cholinesterase (ChE) in anticholinesterase exposures, whereas the type constitution and property of the enzyme remained unclear. A type of ChE was purified from Daphnia magna using a three-step procedure, i.e., Triton X-100 extraction, ammonium sulfate precipitation, and diethylaminoethyl (DEAE)-Sepharose?-Fast-Flow chromatography. According to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), molecular mass of the purified ChE was estimated to be 84 kDa. Based on substrate studies, the purified enzyme preferred butyrylthiocholine iodide (BTCh) [with maximum velocity (V max)/Michaelis constant (K m)=8.428 L/(min·mg protein)] to acetylthiocholine iodide (ATCh) [with V max/K m=5.346 L/(min·mg protein)] as its substrate. Activity of the purified enzyme was suppressed by high concentrations of either ATCh or BTCh. Inhibitor studies showed that the purified enzyme was more sensitive towards inhibition by tetraisopropylpyrophosphoramide (iso-OMPA) than by 1,5-bis(4-allyldimethylammoniumphenyl) pentan-3-one dibromide (BW284C51). Result of the study suggested that the purified ChE was more like a type of pseudocholinesterase, and it also suggested that Daphnia magna contained multiple types of ChE in their bodies. 相似文献
19.
以乌鳢鱼卵为材料,通过生物化学手段和研究方法,从乌鳢鱼卵匀浆液中分离得到了一种相对分子质量大约为42kDa的蛋白酶抑制剂．该蛋白对胰蛋白酶具有专一性的抑制活性,最低抑制浓度为130μg／mL,对胰蛋白酶的抑制常数为30．3nM．SDS—PAGE电泳检测表明,该抑制剂为不合二硫键的单链蛋白．热稳定性和酸碱稳定性研究表明,该蛋白酶抑制剂在30℃～100℃温度下能保持76％以上的活性,在pH值2～11的酸碱条件下,能保持90％以上的活性。 相似文献
20.
Objective:To purify Mannan-binding lectin(MBL)from human serum and detect its binding ability to several kindsof bacteria common in infections diseases of children.Methods:MBL was purifide from human serum by affinity chromatographyon mannan-Sepharose 4B column.Its binding ability to eight species,97 stratus of bacteria was detected by enzyme-linked lectinassay(ELLA).Results:MBL has different binding ability to bacteria and shows strong binding ability to Klebsiella ornithinolvticaand Escherichia coli,but shows relatively lower binding ability to Staphylococcus haemolyticus,Enterobacter cloacae andStaphylococcus epidermidis.To different isolates of Klebsiella pneumoniae,Haemophilus influenzae and Staphylococcus aureus,MBL shows quite different binding ability.Conclusions:MBL has different binding ability to different bacteria,and has relativelystronger binding ability to Gram-negative bacteria.Its binding ability to different isolates of certain kinds of bacteria is quitedifferent. 相似文献