尿激酶抗人交联纤维蛋白—D—二聚体单抗化学偶合体的制备

利用双功能试剂Ｎ－琥珀酰亚胺－３（２－二硫吡啶）丙酸酯（ＳＰＤＰ）作交联剂,合成了尿激酶（ＵＫ）－抗人交联纤维蛋白降解物Ｄ－二聚体单抗（ＭＡ－ＨＩＤ１）化学偶合体（ＵＫ．ＭＡ－ＨＩＤ１）并用苯甲脒－Ｓｅｐｈａｒｏｓｅ６Ｂ及人交联纤维蛋白降解物Ｄ－二聚体－Ｓｅｐｈａｒｏｓｅ４Ｂ亲和柱纯化,获得偶合体产物,ＳＤＳ－ＰＡＧＥ呈现一条带,其分子量约为２０００００,纤维蛋白平板法测活结果显示,偶合体中酶比活     

高分子量和低分子量尿激酶的分离纯化及动力学性质研究

人尿激酶粗品经苯甲脒亲和柱纯化和Ｐｒｏｔｅｉｎ－ＰａｋＳＰ柱分离后,得到两种分子量的尿激酶（ＵＫ）,即高分子量尿激酶（ＨＵＫ）和低分子量尿激酶（ＬＵＫ）,采用民斯亮蓝法测定蛋白质浓度,纤维蛋白板法测定活力,测得ＨＵＫ比活为２．９×１０＾５ＩＵ／ｍｇ蛋白,ＬＵＫ为３．５１０＾５ＩＵ／ｍｇ蛋白,活力回收为７０％以上,经ＳＤＳ－ＰＡＧＥ鉴定,ＨＵＫ和ＬＵＫ均呈单一条带,分子量分别为５４ｋＤ和３３ｋＤ,Ｈ     

人胎肺细胞的条件化培养液中两种类型纤溶酶原活化物的分离

用正常人胎肺细胞体外培养,从其培养液中分离纤溶酶原活化物（ＰＡ）,通过ＣＭ－ＳｅｐｈａｄｅｘＣ－５０层析,硫酸铵沉淀和Ｆｉｂｒｉｎ－Ｓｅｐｈａｒｏｓｅ,Ｌｙｓｉｎｅ－Ｓｅｐｈａｒｏｓｅ亲和层析及ＳｅｐｈａｄｅｘＧ－５０凝胶过滤等步骤,从１０．５ｌ条件培养液中分离纯化得到两种类型的纤溶酶原活化物,ｔ－ＰＡ９０μｇ,ｕ－ＰＡ８００μｇ．在还原条件下ＳＤＳ－ＰＡＧＥ均显示单带,分子量ｔ－ＰＡ为７２ｋＤ,ｕ－ＰＡ为５４ｋＤ,纤溶比活分别为１５６０００ＩＵ／ｍｇ蛋白和１０６０００ＩＵ／ｍｇ蛋白．     

亲和层析纯化HepG_2细胞分泌的PAI－1

本文报道用抗ＰＡＩ－１单克隆抗体（ＭｃＡｂ）亲和层析建立了纯化ＰＡＩ－１的简便方法。经免疫亲和层析,ＳｅｐｈａｃｒｙｌＳ２００凝胶过滤,从ＨｅｐＧ２细胞培液中分离到糖基化和非糖基化两种形式的ＰＡＩ－１,回收率为８４％,ＰＡＩ－１比活性６．１×１０４ＩＵ／ｍｇ。糖基化ＰＡＩ－１分子量为５０ｋＤ,比活性５．８×１０４ＩＵ／ｍｇ。非糖基化ＰＡＩ－１分子量４３ｋＤ,占总ＰＡＩ－１３０％,仍具有ＰＡＩ－１活性。用ＣｏｎＡ－Ｓｅｐｈａｒｏｓｅ亲和层析进一步纯化得到ＳＤＳ－ＰＡＧＥ纯的糖基化ＰＡＩ－１。     

大熊猫乳酸脱氢酶同工酶H4的分离纯化和某些性质的研究

采用８－（６－氨己基）－氨基－５－ＡＭＰＳｅｐｈａｒｏｓｅ亲和层分析法和ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ离子交换层析法从大熊猫心肌中分离纯化出乳酸脱氢酶同工酶Ｈ４,纯化的大熊猫ＬＤＨ－Ｈ４,比活为４４５Ｕ／ｍｇ蛋白,经ＳＤＳ－ＰＡＧＥ,ＡＰＧＥ,等电聚焦电泳鉴定均为一条带,其亚基分了量为３６０００,等电点为５．４５。经测定大熊猫ＬＤＨ－Ｈ亚基Ｎ端被封闭,Ｃ端氨基酸残基经测定为Ｌｅｕ。氨基酸组成分析表明     

大熊猫乳酸脱氢酶同工酶H_4的分离纯化和某些性质的研究

采用８－（６－氨己基）－氨基－５＇－ＡＭＰＳｅｐｈａｒｏｓｅ亲和层析法和ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ离子交换层析法从大熊猫心肌中分离纯化出了乳酸脱氢酶同工酶Ｈ４．纯化的大熊猫ＬＤＨ－Ｈ４,比活为４４５Ｕ／ｍｇ蛋白,经ＳＤＳ－ＰＡＧＥ,ＰＡＧＥ,等电聚焦电泳鉴定均为一条带,其亚基分子量为３６０００,等电点为５．４５．经测定大熊猫ＬＤＨ－Ｈ亚基Ｎ端被封闭,Ｃ端氨基酸残基经测定为Ｌｅｕ．氨基酸组成分析表明每个亚基含有５个Ｃｙｓ,９个Ｍｅｔ．     

重组人尿激酶原的分离纯化及性质研究

本文报道从人尿激酶原（ｐｒｏ－ｕｒｏｋｉｎａｓｅ,ｐｒｏ－ＵＫ）基因重组工程菌Ｅ．ｃｏｌｉＪＡ２２１表达产物的复性液中纯化尿激酶原的方法。复性产物经Ｚｎ＾２＋选择性沉淀,尿激酶抗体亲和柱层析,ｂｅｎｚａｍｉｄｉｎｅ－Ｓｅｐｈａｒｏｓｅ ＣＬ ４Ｂ亲和吸附,即得比活达１１００００ＩＵ／ｍｇ的纯化产品。样品经ＳＤＳ－ＰＡＧＥ鉴定,在还原及非还原条件下,均表现为分子量为４３ｋｄ的单一条带。动力学研究测得     

凝血酶激活的单链尿激酶型纤溶酶原激活剂的构建、表达、纯化和性质研究

用Ｋｕｎｋｅｌ突变法,将单链尿激酶型纤溶酶原激活剂（ｓｃｕ－ＰＡ）ｃＤＮＡ基因中编码Ｐｒｏ１５５—Ｌｙｓ１５８的片段定点突变,并将此突变的ｓｃｕ－ＰＡ（ｔｓｃｕ－ＰＡ）的ｃＤＮＡ克隆到表达载体ｐＣＭ－β－ｎｅｏ中,与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞．获得的稳定表达株在无血清培养基中２４ｈ的表达量为６２０ＩＵ／１０６细胞．经锌离子螯合Ｓｅｐｈａｒｏｓｅ亲和层析得到ｔｓｃｕ－ＰＡ纯品．ＳＤＳ－ＰＡＧＥ显示ｔｓｃｕ－ＰＡ分子量为５３ｋＤ左右,与预期的结果相符．ｔｓｃｕ－ＰＡ是由凝血酶激活而不是由纤溶酶激活,但激活后也能转变为双链分子（ｔｃｕ－ＰＡ）．ｔｓｃｕ－ＰＡ仍保持了ｓｃｕ－ＰＡ的血纤维蛋白亲和性．酶动力学研究表明,激活后的ｔｓｃｕ－ＰＡ水解Ｓ２４４４的Ｋｍ和Ｋｃａｔ值与高分子量尿激酶（ＨＵＫ）相似．体外溶栓实验结果表明,ｔｓｃｕ－ＰＡ可以选择性地溶解富含凝血酶的血凝块,对贫凝血酶的血凝块作用不大     

重组组织型纤溶酶原激活剂大规模纯化及部分性质的研究

收集产重组组织型纤溶酶原激活剂（ｒｔ－ＰＡ）的ＣＨＯ工程细胞灌流培养上清,经过Ｓｔｒｅａｍｌｉｎｅ扩张柱床和赖氨酸－Ｓｅｐｈａｒｏｓｅ４Ｂ柱亲和吸附色谱纯化后,最后产品的比性达到６０００００ＩＵ／ｍｇ蛋白,ｒｔ－ＰＡ总活性回收率在９８％左右,经还原ＳＤＳ－ＰＡＧＥ分析主要为高相对分了质量ｒｔ－ＰＡ蛋白,ＨＰＬＣ分析达到色谱纯,Ｎ端１５个氨基酸序列和ｐＩ与献报道的一致。蛋白质印迹实验证实具有ｔ－Ｐ     

u-PA/t-PA嵌合型纤溶酶原激活剂(ut-PA)的构建、表达、纯化和性质研究

将尿激酶原（ｐｒｏ－ＵＫ）ｃＤＮＡ和组织型纤溶酶原激活剂（ｔ－ＰＡ）Ａ链ｃＤＮＡ克隆到Ｍ１３ｍｐ１８中,经过二次寡核甘酸诱导的大片段定点删除和一次寡核苷酸诱导的多位点突变,得到ｕ－ＰＡ（Ｌｅｕ１４４－Ｇｌｙ４０８）／ｔ－ＰＡ（Ｓｅｒ１－Ｔｈｒ２６３）（ｕｔ－ＰＡ）融合基因．将ｕｔ－ＰＡ融合基因克隆到表达载体ｐＣＭ－βｎｅｏ中,与ｐＣＭ－ｄｈｆｒ共转染ＣＨＯ／ＤＨＦＲ－细胞,筛选稳定表达株．收集无血清表达上清,经苯甲脒柱纯化得到ｕｔ－ＰＡ纯品,ＳＤＳ－ＰＡＧＥ和纤维蛋白自显影显示ｕｔ－ＰＡ有两种分子量形式,分子量分别为６８ｋＤ和６１ｋＤ．纤维蛋白亲和性试验表明,ＬＵＫ（低分子量尿激酶）对纤维蛋白没有亲和性,而含有ＬＵＫ的ｕｔ－ＰＡ则对纤维蛋白表现出很强的亲和性,但ｕｔ－ＰＡ的亲和性略低于亲本ｔ－ＰＡ．     

Characterization of an antibody-urokinase conjugate. A plasminogen activator targeted to fibrin

The plasminogen activator urokinase was linked covalently to a monoclonal antibody specific for the amino terminus of the beta chain of human fibrin by means of the unidirectional cross-linking reagent N-succinimidyl-3-(2-pyridyldithio)propionate. N-Succinimidyl-3-(2-pyridyldithio)propionate allowed the amino groups on urokinase to be coupled to the sulfhydryl groups on iminothiolane (which had been introduced into the antibody before the coupling reaction). The inter-heavy chain sulfhydryl of the Fab' of this antibody was also linked to N-succinimidyl-3-(2-pyridyldithio)propionate-substituted urokinase. The antibody- or Fab'-urokinase complexes were purified by two affinity chromatography steps. In the first, benzamidine was used as ligand for urokinase, in the second, a heptapeptide consisting of the 7 amino-terminal residues of the beta chain of fibrin (beta peptide) was used as ligand for the antibody. The activity of the purified conjugates was compared with that of urokinase alone in an assay measuring lysis of 125I-fibrin monomer covalently linked to Sepharose CL-4B. For any concentration of either urokinase alone or urokinase-antifibrin antibody conjugate, an equivalent amount of lysis (release of labeled peptide from fibrin monomer-Sepharose) was obtained with 1/250 the concentration (with respect to urokinase content) of antifibrin antibody-urokinase conjugate. The antifibrin Fab'-urokinase conjugate exhibited a similar enhancement of activity in comparison with urokinase. Enhanced fibrinolysis was fully inhibited by beta peptide. These results suggest that antibody targeting enhances the concentration of urokinase in the vicinity of immobilized fibrin monomer, thereby also increasing the local conversion of plasminogen to plasmin, which in turn degrades its substrate, fibrin. Univalent antigen-antibody binding is sufficient for optimal efficiency.     

Biochemical properties of conjugates of urokinase-type plasminogen activator with a monoclonal antibody specific for cross-linked fibrin

Equimolar mixtures of recombinant single chain urokinase-type plasminogen activator (rscu-PA) and a murine monoclonal antibody (MA-15C5) directed against fragment-D dimer of human cross-linked fibrin were conjugated, using the cross-linking agent N-succinimidyl 3-(2-pyridyldithio)propionate (PySSProSu). The conjugate (rscu-PA/MA-15C5), purified by immunoadsorption on a urokinase antibody and affinity chromatography on fibrin fragment-D dimer with a yield of 42 +/- 15% (mean +/- SD, n = 3), contained an average of 1.2 +/- 0.3 IgG molecules/rscu-PA molecule. On non-reduced SDS/PAGE it migrated as a main band with apparent Mr of 200,000. Specific amidolytic activities expressed/mass of u-PA were less than 250 IU/mg for rscu-PA/MA-15C5 and rscu-PA, 140,000 +/- 13,000 IU/mg and 100,000 +/- 17,000 IU/mg for their plasmin-generated two chain derivatives rtcu-PA/MA-15C5 and rtcu-PA respectively. Specific activities on fibrin plates were 100,000 +/- 24,000 IU/mg and 130,000 +/- 49,000 IU/mg for rscu-PA/MA-15C5 and rtcu-PA/MA-15C5 respectively, as compared to 180,000 +/- 15,000 IU/mg for both rscu-PA and rtcu-PA. Activation of plasminogen with rscu-PA/MA-15C5 (Km = 0.37 +/- 0.16 microM, k2 = 0.0063 +/- 0.0030 s-1 or rtcu-PA/MA-15C5 (Km = 19 +/- 3.0 microM, k2 = 2.0 +/- 0.10 s-1) in purified systems followed Michaelis-Menten kinetics with Km and k2 values comparable to those of rscu-PA and rtcu-PA. In an in vitro system composed of a 125I-fibrin-labeled whole human plasma clot immersed in citrated human plasma, dose- and time-dependent lysis was obtained; 50% lysis in 2 h required 1.4 microgram/ml of rscu-PA or 0.33 microgram/ml of rtcu-PA, but only 0.22 microgram u-PA/ml of rscu-PA/MA-15C5 or 0.15 microgram u-PA/ml of rtcu-PA/MA-15C5. Addition of purified fragment-D dimer reversed the increased fibrinolytic potency of rscu-PA/MA-15C5 in a concentration-dependent way (50% inhibition at 7.2 micrograms fragment-D dimer/ml). Thus, conjugation of u-PA moieties with the fibrin-specific antibody MA-15C5 targets the plasminogen activator to the clot, resulting in a significant increase of their fibrinolytic potencies as compared to their unconjugated counterparts: 6.4-fold for rscu-PA and 2.2-fold for rtcu-PA.     

Isolation and purification of plasminogen activator from Yoshida ascites Sarcoma of rats

The plasminogen activator was purified to the extent of 150-fold from 20,000 x g supernatant of Yoshida ascites Sarcoma by ammonium sulphate precipitation at 33% saturation followed by affinity chromatography on p-aminobenzamidine-Sepharose 4B. The specific activity of the purified activator was 10,260 IU/mg expressed in terms of International units of urokinase, the known activator of plasminogen. The activator was homogeneous by polyacrylamide slab gel electrophoresis with an apparent molecular weight 75 kDa by gel filtration on Sephadex G-100. Analysis by SDS-polyacrylamide gel electrophoresis under reducing conditions, revealed the presence of two subunits of about 48 and 29 kDa. The activator displayed binding preference to fibrin and was immunologically distinguishable from urokinase, indicating that it could be of non-urokinase origin. The preparation further revealed similarity to standard tissue plasminogen activator with respect to fibrin binding and immunological cross reactivity.     

组织型纤溶酶原激活剂A链与尿激酶原B链嵌合分子的构建与表达

利用 Ｄ Ｎ Ａ 重组技术和定位删除技术,将组织型纤溶酶原激活剂（ｔ Ｐ Ａ）的 Ａ 链（ Ｓｅｒｌ Ｔｈｒ２６３）基因与尿激酶原（ｐｒｏ Ｕ Ｋ）的 Ｂ链（ Ｓｅｒ１３８ Ｌｅｕ４１１）基因相连,得到嵌合分子基因ｔｕ ｐａ,并在昆虫杆状病毒系统中进行表达,表达量可达 ５００ Ｉ Ｕ／ｍ ｌ．经单克隆抗体免疫亲和层析纯化细胞表达上清液,得到ｔｕ Ｐ Ａ 嵌合分子,其比活为 ２００ ０００ Ｉ Ｕ／ｍ ｇ 蛋白． Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 及 Ｗ ｅｓｔｅｒｎ ｂｌｏｔ 鉴定证明此表达产物分子量约为 ６０ ｋ Ｄ,与预期值相符．纤维蛋白平板测活及纤维蛋白亲和性分析初步证明,此嵌合分子的溶纤活性与ｐｒｏ Ｕ Ｋ 相近,而纤维蛋白亲和性高于 ｐｒｏ Ｕ Ｋ．     

鼠抗人纤维蛋白单链抗体-尿激酶杂合基因的构建及其在中国仓鼠卵巢细胞中的表达

采用 DNA重组技术构建了表达鼠抗人纤维蛋白单链抗体与低分子量尿激酶融合基因的真核表达载体。通过磷酸钙共沉淀法 ,将该表达载体转染到中国仓鼠卵巢细胞二氢叶酸还原酶基因缺陷株 ( CHO- dhfr-)中 ,利用选择培养基筛选出稳定表达的细胞株 ,溶解圈法测定融合蛋白的表达水平为每 1 0 6细胞每天 5 8IU。该融合蛋白保留了与纤维蛋白的结合活性和溶解纤维蛋白的溶纤活性。SDS- PAGE,Western印迹法分析证明融合蛋白的相对分子质量约为 70× 1 0 3     

高分子量和低分子量尿激酶的分离纯化及动力学性质研究

人尿激酶粗品经苯甲脒亲和柱纯化和Protein-PahSP柱分离后,得到两种分子量的尿激酶（UK）,即高分子量尿激酶（HUK）和低分子量尿激酶（LUK）．采用考马斯亮蓝法测定蛋白质浓度,纤维蛋白平板法测定活力,测得HUK比活为2.9×105IU／mg蛋白,LUK为3.5×105IU／mg蛋白,活力回收为70％以上．经SDS-PAGE鉴定,HUK和LUK均是单一条带,分子量分别为54kD和33kD．HUK和LUK水解显色底物S2444的动力学常数,分别测得HUK的Km为64μmol/L,Kcat为15s-1,LUK的Km为49μmol／L,kcat为13s-1,LUK的催化效率（Kcat／Km）稍高于HUK．     

人源化鼠抗人纤维蛋白单链抗体-尿激酶融合蛋白在大肠杆菌中的功能性表达

尿激酶是目前临床上广泛使用的一种有效的溶栓制剂 ,但由于尿激酶缺乏对血栓的特异亲和性 ,导致临床上尿激酶的使用剂量较大 ,容易出现全身性出血等副作用。因而开发对血栓特异的导向溶栓制剂是目前溶栓药研制的一个重要方向。本实验室在以前的工作中 ,利用噬菌体表面呈现技术 ,筛选到一种对人纤维蛋白特异的鼠单链抗体[1 ] ,在对该鼠抗体可变区进行结构模建的基础上[2 ] ,对其进行了人源化改造和体外亲和力成熟 ,得到亲和力和特异性较鼠抗体更好的人源化单链抗体[3 ] 。为研制导向溶栓制剂 ,本实验室构建了人源化单链抗体 低分子量尿激酶的…     

Covalent molecular weight approximately 92 000 hybrid plasminogen activator derived from human plasmin amino-terminal and urokinase carboxyl-terminal domains

The preparation of a new class of covalent hybrid plasminogen activators containing the fibrin-binding domains of human plasmin(ogen) and the catalytic active center of human urokinase will be described. Hybridization of the sulfhydryl form of the NH2-terminal plasmin-derived heavy (A) chain (PlnA) with the sulfhydryl form of the COOH-terminal urokinase-derived active heavy (B) chain (u-PAB) was carried out; a covalent PlnA-u-PAB hybrid plasminogen activator was prepared. The sulfhydryl form of PlnA (PlnA(SH)2) was isolated from reduced Lys-2-plasmin by L-lysine-substituted Sepharose column chromatography. For the isolation of the sulfhydryl form of u-PAB (u-PAB(SH], high molecular weight urokinase was adsorbed onto a benzamidine-Sepharose column and reduced with 100 mM 2-mercaptoethanol on the column. The urokinase NH2-terminal light (A) chain was washed off the column, and the u-PAB(SH) chain was eluted from the column. The specific activity of the isolated u-PAB(SH) chain was determined to be 242 000 IU/mg of protein. The PlnA(SH)2 and u-PAB(SH) chains were mixed at a molar ratio of PlnA(SH)2 to u-PAB(SH) of 3:2; the reducing agents were then removed by gel filtration. The hybridization (reoxidation) reaction was allowed to proceed for 48 h at 4 degrees C. The covalent hybrid activator, in 40% yield, was purified from the reaction mixture to homogeneity, by a sequential affinity chromatography method with L-lysine-substituted Sepharose followed by anti-low molecular weight urokinase IgG-Sepharose, and then gel filtration through Sephadex G-150.(ABSTRACT TRUNCATED AT 250 WORDS)     

Construction and characterization of a recombinant chimeric plasminogen activator consisting of a fibrin peptide and a low molecular mass single-chain urokinase

A recombinant chimeric plasminogen activator (f beta/scuPA-32k), with a fibrin beta-chain peptide (comprising Gly15 through Arg 42) linked to the N-terminal of a low molecular mass (32 kDa) single-chain urokinase (scuPA-32k, comprising Leu144 through Leu 411) via a 50 amino acid linker sequence, was produced by expression the corresponding chimeric cDNA in Escherichia coli cells. After refolding in vitro, the chimeric protein was purified to homogeneity by zinc chelate-Sepharose chromatography, Sephacryl S200 chromatography and benzamidine-Sepharose chromatography in sequence. The apparent molecular mass was 36 kDa shown by SDS-PAGE analysis. The special activity was 87,000 IU/mg detected by fibrin plate determination. F beta/scuPA-32k could directly activate plasminogen following Michaelis-Menten kinetics with K(m) = 0.52 microM and k(2) = 0.0024 s(-1). Mediated by plasmin, the single-chain molecule could be converted to the active two-chain molecule. The chimeric protein had 3.3 times higher fibrin affinity than scuPA-32k in the fibrin concentration of 3.2 mg/mL, while the chimeric protein inhibited the fibrin clotting and platelet aggregation. F beta/scuPA-32k showed a higher thrombolytic potency in vitro plasma clot lysis than scuPA-32k and depleted less fibrinogen in plasma. These results showed that the chimeric protein had not only higher fibrinolytic activity but also anti-thrombus activity. Further evaluation of the thrombolytic potential in appropriate animal models is required.