抗组织型纤溶酶原激活剂(tPA)单克隆抗体的制备和鉴定

用从人黑色素瘤细胞培液中提纯的tPA为抗原,通过杂交瘤技术获得TA₁,TA₂、TA₃和TA₄ 4株阳性杂交瘤细胞。经葡萄球菌A蛋白(SPA)亲和层析纯化抗tPA单克隆抗体(tPA．McAb)。固相酶联免疫吸附测定(ELISA)诱生含McAb的小鼠腹水,其效价达I：1O⁵。这些tPA McAb均属IgG₁亚型,特异地作用于tPA(包括基因工程生产的rtPA),与尿激酶(uK)无反应。用底物显色法测定表明所有tPA McAb均可抑制tPA的活性。     

医药其它

930930 .组织型血纤维蛋白溶酶原激活因子■组Kringlel区的表达、纯化及鉴定[英]/Deserrano,V.S.…,Arch.Bioehem.Biophys.·1992,294(1)..282"~290[译自DBA,1992,11(17),92.09686] 用新的融合蛋白表达质粒pST II(KZHPg)pfXa(KltPA)在大肠杆菌中表达纯的人组织血纤维蛋白溶酶原激活因子(tPA)kringle 1区(Ki tPA)残基C92~C173。该质粒顺序编码了人血纤维蛋白溶酶原的紧密赖氨酸结合kringle(K)1区(KIHPg),及含因子-Xa敏感键的肽(pfXa),在其下游插入K1tPA。经Sepharose-赖氨酸亲和层析从大肠杆菌DHs-a细胞的各种级分中纯化出…     

t-PA突变体真核表达载体的构建及其在COS-7细胞的瞬时表达

根据tPAcDNA的全长序列,设计了扩增tPA信号肽cDNA的引物,并进而获得了信号肽cDNA片段。将其回收纯化后,作为上游引物,结合原有的tPA下游引物,分别以tPA的缺失性原核表达载体pErA,以及后者的点突变体pErA(K)为模板,进行PCR反应,从而使二者各自增加了信号肽部分。进一步将其分别克隆至pcDNA30,构建了相应的真核表达载体pCSRA与pCSRK。酶切及测序结果均证明了构建的正确性。经LIPOFECTAMINE[TM]2000Reagent将其分别转染至COS7细胞,并同时设以pCDNA30为阴性对照。取转染后不同时间培养上清,以FAPA法进行检测。结果表明,上述构建载体的表达产物具有良好的溶圈活性。本实验为tPA突变体在真核细胞的稳定表达奠定了基础 。     

刺桐胰蛋白酶抑制剂亲和填料的制备及纯化瑞替普酶

刺桐属胰蛋白酶抑制剂(ETI)是一种丝氨酸蛋白酶抑制剂,能特异性的抑制胰蛋白酶、组织纤溶酶原激活剂(tPA)、瑞替普酶(rPA)等丝氨酸蛋白酶。实验利用ETI工程菌,经诱导表达、体外复性及纯化获得ETI蛋白,并将该蛋白键合到CNBr活化的琼脂糖凝胶,制备ETI亲和填料,纯化瑞替普酶 。     

tPA/gGH双基因转染山羊乳腺上皮细胞表达分析

人组织纤溶酶原激活剂(tissue-type plasminogen activator,tPA)是一种被广泛应用于临床的溶栓药物。双基因共整合入生物体内能够产生协同作用,从而提高目的基因的表达水平。但是目前,利用gGH基因与tPA基因共整合以期提高tPA表达水平的相关研究较少。为筛选获得tPA高表达的tPA/gGH双基因整合的单克隆转基因山羊乳腺上皮细胞株,本研究以β-casein基因作为调控序列,构建乳腺特异性表达载体PCL25/gGH,并通过电转染将tPA和gGH双基因共转染山羊乳腺上皮细胞;通过G418筛选获得抗性细胞株,经PCR检测获得转基因单克隆细胞株;利用催乳素诱导tPA表达,收集48 h后细胞诱导液进行ELISA()和Western blotting检测并分析其tPA表达水平。结果表明,共获得142株抗性单克隆细胞,其中有53株tPA单基因整合细胞株,34株tPA/gGH双基因整合细胞株,双基因整合率达23.9%(34/142)。共检测出29株细胞能够表达tPA,其中单基因表达细胞为12株,表达率为22.6%(12/53);双基因表达细胞为17株,表达率为50.0%(17/34);且单基因细胞表达tPA含量为7.5~52.0μg/mL,而双基因细胞表达tPA含量为40~360μg/mL,明显高于单基因表达水平。本研究通过电转染的方式成功获得了tPA/gGH双基因整合的单克隆山羊乳腺上皮细胞株,并证明双基因整合的细胞株表达tPA水平明显提高,为后期制备高表达转基因山羊奠定了基础。     

乳腺注射法表达人组织型纤溶酶原激活剂的研究

目的 :构建tPA乳腺定位表达载体 ,使其在牛乳汁中高效表达 ,观察目的基因表达的规律及其影响因素 ,为建立新型牛乳腺生物反应器提供理论基础。方法 :RT-PCR法克隆目的基因 ,通过酶切、连接、分离、纯化等方法构建含tPA-cDNA的乳腺定位表达载体 ;采用乳腺注射法将融合基因转入小鼠及牛的乳腺组织中。结果 :乳腺注射外源基因后 ,tPA可在小鼠和牛的乳汁中表达。结论 :乳腺注射法可使目的基因在乳腺组织中稳定地表达较长的时间 ,其表达量与显微注射法没有明显的差异 ,表明外源基因的表达不受转基因方法的影响。但tPA在牛乳汁中的表达量明显高于小鼠的表达量 ,提示不同动物的乳蛋白调控系统有一定的差异 ,可能受着不同的因素或调控系统的影响。     

恒河猴tPA基因的克隆、测序与真核表达

目的对恒河猴tPA编码区cDNA进行测序和表达.方法采用RT-PCR方法从恒河猴淋巴细胞中扩增tPA基因,将获得的cDNA克隆于T载体,序列确定后再克隆至真核表达载体.结果测序结果表明恒河猴tPAcDNA编码区与人tPAcDNA编码区的核苷酸序列同源性为96%,由此所推导的氨基酸序列的同源性为97.5%.随后将恒河猴tPAcDNA克隆于真核表达载体,转染CHO细胞后成功表达出了有活性的tPA.培养上清检测结果显示其活性约为50?U/ml,略低于人tPA在CHO细胞中表达产物的活性.结论本研究首次报道了恒河猴tPA基因编码区的全长cDNA序列并获得了有活性的恒河猴tPA真核表达产物.将为进一步比较灵长类动物间tPA的生物学特性奠定基础.     

人组织型纤溶酶原激活剂突变体微小基因的构建

tPA基因全长约36kb,至少由13个内含子分隔为14个外显子。根据tPA的第一、二外显子的编码情况,考虑建立从第二至第六外显子序列在内的tPA微小基因。即将tPA的部分基因组序列与LAtPA cDNA的序列在第六外显子的NarI位点处相连。     

卵泡闭锁与其tPA，LH受体及抑制素 α，βA亚基基因表达的关系

用DNA 3′-末端标记、免疫组化和原位杂交方法,通过连续切片比较研究了相同卵泡颗粒细胞抑制素亚基和LH受体（LHR）与卵细胞tPA表达和卵泡闭锁的关系.实验结果表明（1） 卵泡闭锁伴随卵细胞tPA活性明显增加;（2） 颗粒细胞抑制素的产生调节卵细胞tPA活性的表达并与卵泡发育状态密切相关;（3） 卵泡闭锁时,颗粒细胞几乎不表达LHR和抑制素亚基.上述结果提示卵细胞的tPA在闭锁卵泡中可能参与卵细胞的自我瓦解和清除过程;颗粒细胞表达的抑制素可能是tPA mRNA翻译的一种抑制因子,如其表达受阻,可导致卵细胞tPA蛋白活性增加引起卵泡闭锁.     

人组织性纤溶酶原激活物衍生物在大肠杆菌硫氧化还原蛋白融合表达系统中的表达

目的:克隆含tPA中355个氨基酸密码子(1-3和176-527氨基酸)的cDNA序列(tPA355),将其在大肠杆菌融合蛋白表达系统中表达,并在体外复性使其具有激活纤溶酶原的生物活性。方法:采用RT-PCR技术从人黑色素瘤细胞Bowes中克隆出tPA355cDNA,然后在pET32a(+)BL21(DE3)大肠杆菌表达系统中表达,将表达出的融合蛋白Trx-tPA355(Thioredoxin,Trx)包涵体在体外进行变性、复性和纯化以使其具有激活纤溶酶原的生物活性。结果:测序结果表明本研究克隆的编码tPA中355个氨基酸密码子的cDNA序列与美国专利(公开号:5,587,159)中对应的序列完全一致,将其在pET32a(+)/BL21(DE3)大肠杆菌表达系统中表达可获得稳定表达的融合蛋白Trx-tPA355包涵体,该包涵体占菌体总蛋白的30%左右,此融合蛋白经变性、复性后具有激活纤溶酶原的生物活性。结论:含tPA中355个氨基酸密码子(1-3和176-527氨基酸)的cDNA在大肠杆菌Trx融合蛋白表达系统中可获得稳定表达,表达的融合蛋白产物在体外经变性、复性后具有激活纤溶酶原的生物活性。     

Purification and characterization of a stimulator of plasmin generation from the antiangiogenic agent Neovastat: identification as immunoglobulin kappa light chain

We have recently shown that Neovastat, an antiangiogenic extract from shark cartilage, stimulates the in vitro activation of plasminogen by facilitating the tissue-type plasminogen activator (tPA)-dependent conversion of plasminogen to plasmin. In this report, we describe the purification and characterization of the stimulatory molecules. Neovastat was subjected to a three-step purification procedure including gel filtration, preparative isoelectric focusing, and preparative SDS-PAGE. Two 28-kDa proteins with pIs of approximately 4.5 and 6.5 were purified to apparent homogeneity and identified as immunoglobulin (Ig) kappa light chains by N-terminal microsequencing. Ig light chains do not directly stimulate the activity of tPA or plasmin, suggesting a mechanism of action involving an interaction with plasminogen. Kinetic analysis showed that both Ig light chains accelerate the in vitro tPA-dependent conversion of plasminogen in plasmin by increasing the affinity of tPA for plasminogen by 32- and 38-fold (Km decrease from 456 nM to 12-14 nM). Shark Ig light chains also stimulated the degradation of fibrin by the tPA/plasminogen system in an in vitro assay. A direct interaction between Ig light chains and plasminogen (KA=4.0-5.5 x 10(7) M(-1); KD=18-25 nM) and with tPA (KA=2.8 x 10(7) M(-1); KD=36 nM) was demonstrated using real time binding measured by surface plasmon resonance. Ig light chain is the first molecule associated with the antiangiogenic activity of Neovastat to be purified and identified.     

Expression, purification, and characterization of the recombinant kringle 1 domain from tissue-type plasminogen activator.

A novel fusion protein expression plasmid that allows ready purification and subsequent facile release of the target molecule has been constructed and employed to express in Escherichia coli and purify the tissue plasminogen activator kringle 1 domain ([K1tPA] residues C92-C173). The resulting plasmid encodes the tight lysine-binding kringle (K)1 domain of human plasminogen ([K1HPg]) followed by a peptide (PfXa) containing a factor Xa-sensitive bond, downstream of which [K1tPA] was inserted. The recombinant (r) [K1HPg]PfXa[K1tPA] fusion polypeptide was purified from various cell fractions in one step by Sepharose-lysine affinity chromatography. After cleavage with fXa, the mixture was repassaged over Sepharose-lysine, whereupon the r-[K1tPA]-containing polypeptide passed unretarded through the column. A homogeneous preparation of this material was then obtained after a simple step employing fast protein liquid chromatography. The purified r-[K1tPA], which contained the amino acid sequence SNAS[K1tPA]S, provided an amino-terminal amino acid sequence, through at least 20 amino acid residues, that was identical to that predicted from the cDNA sequence. The molecular mass of r-SNAS[K1tPA]S, determined by electrospray mass spectrometry, was 9621.9 +/- 4.0 (expected molecular mass, 9623.65). 1H-NMR spectroscopy and thermal stability studies of r-SNAS[K1tPA]S revealed that the purified material was properly folded and similar to other isolated kringle domains. Additionally, employment of this methodology revealed that only a very weak interaction between epsilon-aminocaproic acid and the isolated r-[K1tPA] domain occurred.     

Expression of Active Human Tissue-Type Plasminogen Activator in Escherichia coli

The formation of native disulfide bonds in complex eukaryotic proteins expressed in Escherichia coli is extremely inefficient. Tissue plasminogen activator (tPA) is a very important thrombolytic agent with 17 disulfides, and despite numerous attempts, its expression in an active form in bacteria has not been reported. To achieve the production of active tPA in E. coli, we have investigated the effect of cooverexpressing native (DsbA and DsbC) or heterologous (rat and yeast protein disulfide isomerases) cysteine oxidoreductases in the bacterial periplasm. Coexpression of DsbC, an enzyme which catalyzes disulfide bond isomerization in the periplasm, was found to dramatically increase the formation of active tPA both in shake flasks and in fermentors. The active protein was purified with an overall yield of 25% by using three affinity steps with, in sequence, lysine-Sepharose, immobilized Erythrina caffra inhibitor, and Zn-Sepharose resins. After purification, approximately 180 μg of tPA with a specific activity nearly identical to that of the authentic protein can be obtained per liter of culture in a high-cell-density fermentation. Thus, heterologous proteins as complex as tPA may be produced in an active form in bacteria in amounts suitable for structure-function studies. In addition, these results suggest the feasibility of commercial production of extremely complex proteins in E. coli without the need for in vitro refolding.     

重组刺桐胰蛋白酶抑制剂a在大肠杆菌中的表达和纯化

为了大量制备重组刺桐胰蛋白酶抑制剂a(rETIa) ,对构建的基因工程菌株E .coliBL2 1(DE3)pET2 2b mETIa进行了表达条件的优化 .用摇瓶培养 ,rETIa蛋白占菌体总蛋白 4 0 %以上 .经破碎菌体 洗涤包涵体 溶解包涵体 复性初步纯化后 ,再经二步柱层析纯化获得电泳纯的rETIa蛋白 .测定了rETIa对胰蛋白酶、胰凝乳蛋白酶、组织型纤溶酶原激活因子缺失突变体 (NTA)的抑制活性 .     

Transformed Lepidopteran insect cells: new sources of recombinant human tissue plasminogen activator.

Stable transformation was used to generate a cloned insect cell line (Bm5 silkmoth cells) over-expressing human tissue plasminogen activator (tPA). This cell line expressed 135 microg/mL single chain tPA in serum-free medium in static culture with a maximum specific activity of 120 IU/microg. In serum-containing medium, this line expressed 160 microg/mL of combined single-chain tPA, two-chain tPA, and a higher molecular weight SDS-stable tPA complex in suspension cultures with a maximum specific activity of 255 IU/microg. Approximately 100 copies of the tPA cDNA were randomly integrated into each Bm5 cell. For secretion of recombinant tPA from Bm5 cells, the native human tPA signal peptide is as effectively recognized as an insect specific signal peptide derived from a silkmoth chorion gene. Finally, stably transformed polyclonal populations of Bm5, High Five, and Sf21 cells expressing tPA were generated and compared for relative tPA expression.     

PC12 Cells Overexpressing Tissue Plasminogen Activator Regenerate Neurites to a Greater Extent and Migrate Faster than Control Cells in Complex Extracellular Matrix

Abstract: PC12 cells were stably transfected with expression vectors containing rat tissue plasminogen activator (tPA) under control of either a cytomegalovirus or rous sarcoma virus promoter. Cell lines were characterized using protease assays, ELISAs, immunoblots, northern blots, and Southern blots. Control PC12 cells or cells containing vectors alone released about 1 pg tPA/cell/24 h, whereas cells stably transfected with a tPA cDNA released 2–5 pg tPA/cell/24 h. A strong correlation existed between the amount of tPA released and the ability of cells to degrade extracellular matrix. Experiments with protease inhibitors and antibodies against tPA and plasminogen indicated that degradation of matrix involved tPA-generated plasmin and that the amount of matrix degraded was dependent on the amount of tPA released. Cells expressing high levels of tPA migrated on a three-dimensional matrix about twice as fast as control cells and regenerated neurites within three-dimensional gels of Matrigel to a greater extent than control cells. Antibodies that inhibited tPA and plasminogen decreased migration and neurite regeneration, indicating that tPA was involved in both events. PC12 cells overexpressing tPA should provide a useful model system for investigating neural functions of tPA including its role in migration and regeneration.     

A single-step separation of the one- and two-chain forms of tissue plasminogen activator

Preparations of one-chain tissue plasminogen activator (tPA), usually a mixture of 65- and 63-kDa differentially glycosylated forms, contain variable amounts of two-chain tPA. There is no effective procedure currently available for removal of the two-chain contaminant from one-chain tPA preparations. In this report, affinity chromatography on benzamidine-Sepharose was investigated for the separation of two-chain from one-chain tPA. Activase, a preparation of recombinant tPA containing 80% one-chain tPA, a mixture of 65- and 63-kDa variants, and 20% two-chain tPA, was applied to a column of benzamidine-Sepharose, equilibrated with 1 M ammonium bicarbonate. Under this condition, both one-chain and two-chain forms of tPA were adsorbed by the column. Addition of 0.1 M arginine to the equilibration buffer led to elution of two-peaks, corresponding to the 65- and 63-kDa variants of one-chain tPA. Two-chain tPA remained bound to the column, but could be eluted with sodium acetate buffer, pH 4.0, containing 0.1 M arginine. The present procedure allows rapid and effective removal of two-chain tPA with concomitant separation of 65- and 63-kDa one-chain glycoforms from preparations of one-chain tPA. Kinetic analysis for the hydrolysis of D-Ile-Pro-Arg-p-nitroanilide (S-2288) by the highly purified molecular forms of tPA suggests that 63-kDa one-chain tPA possesses 30% higher catalytic efficiency than the 65-kDa variant, while two-chain tPA is 9- or 12-fold more efficient than 63- or 65-kDa one-chain tPA, respectively.     

Allosteric regulation of tPA-mediated plasminogen activation by a modifier mechanism: evidence for a binding site for plasminogen on the tPA A-chain.

We studied the mechanism responsible for nonlinear double reciprocal plots for tissue type plasminogen activator (tPA)-mediated plasminogen activation reported previously by several groups. We found nonlinear Eadie-Scatchard plots for Glu-plasminogen activation by recombinant single-chain tPA confirming a non-Michaelis-Menten behavior of tPA. In order to characterize this mechanism, enzyme kinetic studies with truncated substrates (Lys- and miniplasminogen) and modified or truncated enzymes (two-chain tPA and tPA B-chain) were performed. Thereby it could be excluded that product-mediated modifications of the enzyme or the substrate are responsible for the nonlinear plots. Linear plots, i.e., Michaelis-Menten kinetics, were only found when tPA B-chain was used as a plasminogen activator, indicating that the tPA A-chain should be responsible for the non-Michaelis-Menten behavior. Binding studies of plasminogen to immobilized tPA A-chain in fact demonstrated a saturable binding of Glu- and miniplasminogen to the A-chain of tPA with a KD approximately 0.1 microM and one binding site per molecule of tPA A-chain. These data suggested a modifier mechanism responsible for the nonlinear plots whereby the substrate plasminogen itself could function as a modifier. When such a mechanism was included into a model for tPA-mediated plasminogen activation, the experimentally obtained data could be fitted into such a model by nonlinear regression analysis with resulting p-values of less than 0.001.     

Rat oocyte tissue plasminogen activator is a catalytically efficient enzyme in the absence of fibrin. Endogenous potentiation of enzyme activity

Rat oocytes synthesize tissue plasminogen activator (tPA) in response to stimuli which initiate meiotic maturation. Purified tPA exhibits optimal activity only in the presence of fibrin or fibrin substitutes. Because oocytes are not exposed to fibrin in situ, we investigated the possible stimulation of rat oocyte tPA activity by other endogenous factor(s). Oocytes were obtained from immature female rats which were induced to ovulate with gonadotropins. tPA activity was measured by the plasminogen-dependent cleavage of a chromogenic substrate. Measurements of kinetic parameters with Glu- or Lys-plasminogen revealed a Km for the rat oocyte enzyme of 1.3-2.1 microM compared with 23-24 microM for purified human tPA. Inclusion of the soluble fibrin substitute polylysine lowered the Km of human tPA by 30-fold (0.8 microM) but had no effect on the oocyte tPA Km. Polylysine had no significant effect on the Vmax values. The rate of plasminogen activation catalyzed by oocyte tPA was increased only 4.3-fold by fibrin while fibrin stimulated purified human tPA activity by 15.2-fold. After fractionation of oocyte extract by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, polylysine enhanced oocyte tPA activity as seen by casein zymography. tPA activity in the conditioned medium of a rat insulinoma cell line was also not stimulated with polylysine prior to fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These data suggest that extravascular cells which elaborate tPA may produce stimulatory factor(s) which allow for full tPA activity at physiological concentrations of plasminogen in the absence of fibrin.