抗纤维蛋白β链N端七肽抗体的制备

编码纤维蛋白β链Ｎ末端七肽（β七肽）的寡核苷酸片段,通过基因重组技术插入到金葡核酸酶（Ｐ－１蛋白）基因的５′端。在Ｐ_ＲＰ_Ｌ启动子的调控下,β七肽以融合蛋白（β七肽·Ｐ－１）的形式在Ｅ．ｃｏｌｆｉ细胞中得到高效表达。以纯化的融合蛋白为免疫原,制备出抗β七肽抗体;纤维蛋白原（ＦＧ,４ｇ／Ｌ抑制实验证明,该抗体对纤维蛋白（ＦＰ）有特异性反应。     

一步加热法和溴化氰切割生产胸腺肽α1

胸腺肽α1是一种已经商业化的化学合成多肽,它具有广泛免疫增强和抗肿瘤作用.随着越来越多的临床治疗作用的发现,胸腺肽α1的大量和低价生产逐渐引起人们的重视.我们通过一种新的重组和纯化技术来生产多肽胸腺肽α1,首先通过基因重组的方法在大肠杆菌中表达了四串体的胸腺肽α1,然后使用一步加热的方法将四串体的胸腺肽α1纯化.随后,使用溴化氰在50%～70%三氟乙酸溶剂中将四串体的胸腺肽α1切割成单体.使用高效液相色谱法将胸腺肽α1单体提纯后,其纯度可达到98%以上.最后,利用胸腺肽α1能刺激淋巴细胞增殖的原理证实经我们的方法得到的重组胸腺肽α1,与商业化的胸腺肽α1 (Zadaxin?)具有相似的生物活性.我们通过基因重组、加热纯化、合适的切割成功获得了与商业化的胸腺肽α1相似生物活性的重组胸腺肽α1.     

胸腺肽α1-Spl DnaX内含肽融合蛋白的切割动力学研究

目的:研究胸腺肽α1(Tα1)与Spl DnaX内含肽融合蛋白AS的体外切割动力学。方法:构建Tα1和SplDnaX内含肽的融合表达载体pET-AS,转化大肠杆菌BL21(DE3),经乳糖诱导获得可溶性表达的融合蛋白AS,用镍亲和层析纯化该蛋白;综合评价温度、β-巯基乙醇(β-ME)浓度和诱导切割时间对Spl DnaX内含肽介导融合蛋白AS自切割释放Tα1的影响。结果:在大肠杆菌中诱导表达了融合蛋白AS,经镍柱纯化获得该蛋白;随着温度升高和β-ME浓度增加,诱导切割时间延长,Spl DnaX内含肽介导的切割率逐渐增大;最终采用300 mmol/Lβ-ME切割24 h,融合蛋白的硫解切割率大于90%。结论:通过对Spl DnaX内含肽的诱导切割条件进行摸索,确定了最适宜的切割条件,为利用该方法制备Tα1和其他小分子多肽提供了技术基础。     

一步加热法和溴化氰切割生产胸腺肽α1（英文）

胸腺肽α1是一种已经商业化的化学合成多肽,它具有广泛免疫增强和抗肿瘤作用.随着越来越多的临床治疗作用的发现,胸腺肽α1的大量和低价生产逐渐引起人们的重视.我们通过一种新的重组和纯化技术来生产多肽胸腺肽α1,首先通过基因重组的方法在大肠杆菌中表达了四串体的胸腺肽α1,然后使用一步加热的方法将四串体的胸腺肽α1纯化.随后,使用溴化氰在50%～70%三氟乙酸溶剂中将四串体的胸腺肽α1切割成单体.使用高效液相色谱法将胸腺肽α1单体提纯后,其纯度可达到98%以上.最后,利用胸腺肽α1能刺激淋巴细胞增殖的原理证实经我们的方法得到的重组胸腺肽α1,与商业化的胸腺肽α1 (Zadaxin?)具有相似的生物活性.我们通过基因重组、加热纯化、合适的切割成功获得了与商业化的胸腺肽α1相似生物活性的重组胸腺肽α1.     

人β-淀粉样肽Aβ_(42)的融合表达与纯化

[目的]建立一种快速可靠、获取足量和高纯度的人β-淀粉样肽(Aβ_(42))的方法。[方法]首先利用重叠PCR技术扩增获得Aβ_(42)基因全长。随后将基因连入p GEX-4T-1载体,利用GST系统表达融合蛋白。分别在16℃、25℃、30℃和37℃诱导表达,SDS-PAGE检测融合蛋白的表达情况,确定表达的最佳温度。根据优化条件进行目的蛋白的大量表达,利用Gstrap FF柱亲和纯化GST-Aβ_(42)融合蛋白。[结果]成功构建p GEX/Aβ_(42)表达载体,确定30℃为诱导表达的最佳温度。大量表达并经过纯化可获得分子量为30.7 k Da的融合蛋白。[结论]利用GST融合系统表达纯化可得到纯度超过90%的GST-Aβ_(42)融合蛋白,重组蛋白的产率约为1.2 mg/L培养基。当用凝血酶切除GST融合标签后,Aβ_(42)易聚集沉淀。     

抑前胸腺肽在家蚕体内的活性作用

家蚕Bombyx mori抑前胸腺肽是昆虫脑神经肽的一种,体外实验表明它能抑制处于活动时期的家蚕前胸腺合成蜕皮激素,因此抑前胸腺肽可能对昆虫的变态起着重要的作用。将抑前胸腺肽以不同的浓度分单一注射和加强注射导入家蚕体内,不同的时间间隔取样,利用蜕皮激素放射免疫分析方法,观察到了抑前胸腺肽在家蚕体内的活性作用以及引起家蚕体内血淋巴中蜕皮激素浓度的动态变化,首次证明了抑前胸腺肽在体内对家蚕前胸腺合成蜕皮激素有强烈的抑制作用。     

抑前胸腺肽在家蚕体内的活性作用

家蚕Bombyx mori抑前胸腺肽是昆虫脑神经肽的一种,体外实验表明它能抑制处于活动时期的家蚕前胸腺合成蜕皮激素,因此抑前胸腺肽可能对昆虫的变态起着重要的作用。将抑前胸腺肽以不同的浓度分单一注射和加强注射导入家蚕体内,不同的时间间隔取样,利用蜕皮激素放射免疫分析方法,观察到了抑前胸腺肽在家蚕体内的活性作用以及引起家蚕体内血淋巴中蜕皮激素浓度的动态变化,首次证明了抑前胸腺肽在体内对家蚕前胸腺合成蜕皮激素有强烈的抑制作用。     

胸腺肽β4研究进展

β胸腺肽是一个多肽家族,由高度保守的极性氨基酸组成,相对分子质量约5000.研究表明,在绝大多数哺乳动物组织中,胸腺肽β4都是该家族丰度最高的成员。1991年发现胸腺肽β4是人血小板中主要的肌动蛋白隐蔽肽,随后发现它在创伤愈合和血管生成、角膜修复、抑制炎症及肿瘤转移等方面具有重要作用。我们简要综述胸腺肽β4的上述作用及临床应用的研究进展。     

胸腺肽Tβ4

胸腺肽Tβ4是具有多重生物功能的小肽,是肌动蛋白的结合蛋白之一,该多肽的基因差异性表达与肿瘤恶变和转移、胚胎发育等关系密切,本综述了胸腺肽Tβ4的研究进展。     

发现血管紧张肽的新功能——抗阿片作用

自从在脑内发现阿片肽以来,有人曾在脑内寻找是否存在着内源性的抗阿片物质。目前知道,促黑激素释放抑制因子、ACTH、β-内啡肽片断及八肽胆囊收缩素等均具有对抗阿片物质的作用。但实际上,这些肽都不是以抗阿片物质的形式真正从脑组织中分离提取出来。本文作者用100只牛脑(40kg),先经丙酮提取得到7.5kg干粉。然后,经分子筛、离子交换以及高压液相层析等步骤多次分离纯化,最终得到两个活性峰A和B。活性峰B已纯化为单一肽,并经氨基酸成分分析与[缬~5]-血管紧张肽Ⅰ(十肽)一致。在高压液相中该肽与合成的[缬~5]-血管紧张肽Ⅰ具有相同的保留时间(Rt)。A峰尚未完全纯化,但在四种不同流洗系统高压液相上的Rt与合成的[缬~5]-血管紧张肽Ⅱ     

SEPARATION OF APPARENT MULTIPLE FORMS OF HUMAN BRAIN CHOLINE ACETYLTRANSFERASE BY ISOELECTRIC FOCUSING

Abstract— Choline acetyltransferase (acetyl-CoA: choline O -acetyl transferase; EC 2.3.1.6; ChAc) purified from human brain (basal ganglia) and sciatic nerve were separated into apparent multiple enzyme forms by the method of isoelectric focusing (pH gradient 3-10) on acrylamide gel. A preparative separation of enzyme forms of human brain was accomplished by the column method, by using a sucrose gradient. When each separated form was re-electrofocused, only a portion of the ChAc activity was observed in its original pH region while more than one-half of the recovered activity for each fraction appeared at pH 7.8-8. Gel filtration and kinetic studies of separated forms indicated that the more acidic forms might be aggregates, while more basic forms might be configurational isomers. Human ChAc of sciatic nerve did not exhibit acidic forms on electrofocusing, but otherwise yielded an electrofocusing profile similar to that of human brain. ChAc of rabbit brain and sciatic nerve each exhibited only a single form at pH 7.1 ± 0.2. Although ChAc differs among species, the enzyme of brain and sciatic nerve of the same species cannot be clearly distinguished by electrofocusing.     

Preparative isoelectric focusing in immobilized pH gradients. I. General principles and methodology

A new method for preparative protein purification is described, based on the use of Immobiline matrices. After electrofocusing, the protein zone of interest is recovered by electrophoretic transfer to a hydroxyapatite gel, from which it is eluted with 0.2 M phosphate buffer, pH 6.8, with yields for the proteins studied in the range 76-98%. For six different proteins, the focusing step gives a common upper limit of approximately 45 mg protein/ml gel as mean concentration in a focused protein zone. It is demonstrated that in practical preparative work, components with a pI difference of 0.007 pH units can be completely resolved, and that on a 5-mm-thick gel of dimensions 240 X 110 mm, samples containing as much as 400 mg of the major protein component can be applied. Focusing of large amounts of a salt-containing sample is demonstrated with the aid of human serum. A theoretical expression is given relating the concentration distribution and maximum protein concentration within a focused zone to the applied voltage, the pH slope used and the zone width. Based on this expression and the finding of an upper concentration limit for a protein we shown how to optimize the parameters in preparative work with immobilized pH gradients in relation to the separation power needed. Finally, it is shown that, in comparison with conventional preparative electrofocusing in polyacrylamide gels, immobilized pH gradients allow a ten-fold increase in load, whilst still giving a resolution comparable to that of analytical isoelectric focusing.     

S-adenosylmethionine: Protein-carboxyl methyltransferase from erythrocyte

Protein methylase II (S-adenosylmethionine:protein—carboxyl methyltrans-ferase), which modifies free carboxyl residues of protein, was purified from both rat and human blood, and properties of the enzymes were studied. The pH optima for the reaction were dependent on the substrate proteins used; pH 7.0 was found with endogenous substrate, 6.1 with plasma, 6.5 with γ-globulin, and 6.0 with fibrinogen. The molecular weight of the enzymes from both rat and human erythrocytes were identical (25,000 daltons) determined by Sephadex G-75 chromatography. Partially purified enzyme from rat erythrocytes showed three peaks on electrofocusing column at pH 4.9, 5.5 and 6.0. The K_m values of the enzymes from rat and human erythrocytes showed 3.1 × 10^?6m and 1.92 × 10^?6m at pH 6.0, 1.96 × 10^?6m and 1.78 × 10^?6m at pH 7.2, respectively, for S-adenosyl-l-methionine. It is also found that S-adenosyl-l-homocysteine is a competitive inhibitor for protein methylase II with K_i value of 1.6 × 10^?6m.     

Isolation of a Pure Dextranase from Penicillium funiculosum

A dextranase, produced by Penicillium funiculosum, was purified 1,000-fold to yield the enzyme which was demonstrated by gel electrophoresis and electrofocusing to be a homogeneous protein. The purification method included acetone partition, ammonium sulfate fractionation, gel filtration, iron defecation and precipitation, and diethylaminoethyl-cellulose chromatography. The pure enzyme was also obtained by preparative gel electrophoresis. Gel-permeation chromatography indicates a molecular weight of 41,000. An isoelectric pH of 4.6 was established by electrofocusing. A 1-mg amount of the enzyme hydrolyzes a dextran substrate to yield 27,000 isomaltose reducing units in 2 hr.     

Purification and some properties of γ-conglycinin in soybean seeds

A 7S globulin (γ-conglycinin) which was one of four major antigenic components in soybean globulins was purified and found to be homogeneous on ultracentrifugation, disc electrophoresis, immunoelectrophoresis and disc electrofocusing by gel filtration, preparative-scale disc electrophoresis and two kinds of affinity chromatography. Subsequently, some physico-chemical properties of the protein were determined. The sedimentation coefficient, isoelectric point, MW and diffusion constant were 6·55S, pH 5·80, 104000 and 5·80 × 10^?7 cm²/sec, respectively. The protein was a glycoprotein which contained 5·49% total carbohydrate per protein. The protein did not aggregate and dissociate with a change of ionic strength from 0·1 to 0·5.     

Isolation, purification, and characterization of glucosamine-6-phosphate-N-acetylase from pig liver

The procedure of isolation, purification, and characterization of glucosamine-6-phosphate acetylase from the pig liver is described. The steps of purification were as follows: adsorption on hydroxylapatite, fractionation with ammonium sulfate, chromatography on cellulose phosphate, electrofocusing, and preparative gel electrophoresis. A highly purified (about 3000-fold) preparation of GlcN-6-P acetylase, with a yield of 23%, was obtained. It was found that GlcN-6-P acetylase from pig liver is heterogeneous and exists in two active forms. The characteristic features of the preparation were established: Mr, about 24 kDa; temperature optimum at 37 degrees; pH optimum at 7.45; and Km (GlcN-6-P) 3.7 x 10(-3) M and Km (AcCoA) 1.4 x 10(-3) M. The ions K+, Na+, NH4+, Mg2+, Mn2+, and CH3COO- do not stimulate the acetylase activity. The product of acetylase reaction (GlcNAc-6-P) inhibits this reaction according to the feedback process. The highly purified preparation of GlcN-6-P acetylase is unstable during storage and it is protected by ampholine or glycine from enzyme inactivation, but it is not protected by 2-mercaptoethanol.     

Purification and Properties of Neutral-cyclodextrin Glycosyl-transferase of an Alkalophilic Bacillus sp.

Neutral-cyclodextrin glycosyltransferase (EC 3.2.1.19) of alkalophilic Bacillus sp. (ATCC 21783) was purified by starch adsorption, DEAE-cellulose chromatography and Sephadex G–150 gel filtration chromatography followed by preparative polyacrylamide gel electrophoresis. Molecular weight of the purified enzyme was 85,000-88,000 by SDS-disc gel electrophoresis. The enzyme was most active at pH 7 and 50°C, and stable up to 60°C at pH 7 and in the range of pH 6~8 at 60°C by 30 min incubation. The apparent V_max and Km values for α- and β-cyclodextrin at a constant concentration of sucrose were 417, 70 µmoles glucose/min · mg protein and 10, 0.83 nm, respectively. About 85~90% of amylose, 75~80% of potato starch, 65~70% of amylopectin, 55~60% of glycogen, 45~50% of amylopectin β-limit dextrin, 20~25% of maltotriose and 10~15% of maltose were converted to cyclodextrins with 0.5~1% (w/v) of each substrate.

Schardinger β-dextrin was preferentially produced from starch, and α- or γ-dextrin was gradually formed after prolonged incubation. After 20 min incubation, about 0.4, 14 and 2.5% of α-, β- and γ-dextrin were formed from starch, respectively.     

人心肌酸性铁蛋白的分离纯化及临床应用

人心肌匀浆经热变性、酸化、硫酸铵盐析、超离心、ＳｅｐｈａｒｏｓｅＣＬ－４Ｂ柱层析和制备等电聚焦分离,得到酸性铁蛋白。经鉴定,所得酸性铁蛋白ｐＩ为５．０,Ｈ亚基分子量为２１ｋＤ,Ｌ亚基为１９ｋＤ,ＰＡＧＥ分析呈单一区带。制备了兔抗人酸性铁蛋白抗血清,用该抗血清建立的人酸性铁蛋白放射免疫分析可检测出８０％甲胎蛋白阴性肝癌病人。     

Human carbamylphosphate synthetase I. Stabilization, purification, and partial characterization of the enzyme from human liver

Carbamylphosphate synthetase I from human liver was stabilized, purified, and partially characterized. The labile enzyme was stabilized in cell-free extracts by the presence of MgATP and dithiothreitol at pH 7.8. The stabilized enzyme was purified by a rapid procedure consisting of ion exchange chromatograhy and electrofocusing The native molecular weight of the enzyme was determined by gel filtration to be 190,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a monomeric molecular weight of 165,000. The isoelectric point of the purified enzyme was 6.05, and only one species of active enzyme was observed during electrofocusing of both purified enzyme preparations and crude liver homogenates. The enzyme exhibited a pH optimum of 7.8. The apparent Michaelis constants for NH4+, HCO3-, MgATP, and the activator, N-acetyl-L-glutamic acid, were 0.8, 6.7, 1.1, and 0.1 mM, respectively.     

Calf-ovary protein kinases dependent on adenosine 3':5' -monophosphate. Analysis by electrophoresis and electro-focusing on polyacrylamide get.

High resolving power and quantitative application polyacrylamide-gel electrophopresis at various pore sizes and electrofocusing provide resolution of a calf-ovarian protein-kinase system at an increased level of magnification, as well as optimal preparative routes. Three protein kinases dependent on adenosine 3':5' -monophosphate are distinguished by polyacrylamide gel electrophoresis in calf ovarian cytosol. These enzymes which are observed in the pH range 7.5--10.2, appear to be aggregates of a commonsubmit or monomer. The three kinases are, by the criteria of polyacylamide gel electrophoresis, distinct from three adenosine-3':5' -monophosphate-binding proteins found in the calf ovarian system. Analysis by electrofocusing on polyacrylamide gel shows that conventionally purified preparations of the major kinase of cytosol contain an overwhelming majority of contaminant proteins.