RGD-葡激酶突变体(K130T,K135R)的制备与活性分析

以葡激酶突变体质粒mSAK(K130T ,K135R)-pBV220为模板,PCR重叠引物延伸法引入突变位点,并将该片段克隆至载体pBV220 ,构建了RGD-mSAK-pBV220质粒,转化大肠杆菌后热激诱导获得了高效表达,表达产物占菌体总蛋白的50%以上,且主要以可溶性形式存在,所获蛋白依次用Q SepharoseHP柱、SephaycrylS200HR柱和SP柱进行纯化,纯化的蛋白的纯度可达98%以上,纤维蛋白溶圈法体外溶栓活性测定结果表明,所获RGD-mSAK蛋白溶栓活性与野生型葡激酶相当,豚鼠体内免疫试验证明突变体的免疫原性也有所降低,血小板聚集试验分析突变体蛋白的抗血小板聚集能力,RGD 葡激酶突变体具有一定的抗血小板聚集能力。     

具有抗血小板聚集功能的新型葡激酶突变体的表达、纯化及性质

[目的]为解决溶栓后再栓塞问题,构建N-端含RGD(Arg-Gly-Asp)序列的葡激酶双功能突变体.研究突变体的表达和纯化,并进行性质分析.[方法]将突变后的葡激酶突变体序列连入pBV220质粒,转化大肠杆菌BL21进行表达.阳离子交换、凝胶过滤和阴离子交换三步层析法纯化表达产物,采用溶圈法对纯化产物进行生物学活性测定,并测定纯化产物对血小板聚集的抑制效应.[结果]PAGE扫描结果显示,葡激酶突变体蛋白在大肠杆菌BL21中的表达量约占菌体蛋白总量的40%～50%;三步层析纯化后,HPLC测定其纯度可达95%.酪蛋白凝胶板溶圈法测得其比活性分别为10.8×104和11.0×104HU/mg,与野生型葡激酶活性相当;且具有明显的抗血小板聚集活性,血小板聚集仪测定其血小板聚集抑制率分别为10.72%和19.71%,明显高于野生型葡激酶血小板聚集抑制率.本实验利用pBV220载体高效表达了葡激酶突变体基因,得到了高纯度、高活性的突变体蛋白,为葡激酶生产产业化和临床应用奠定了良好的基础.     

RGD肽与尿激酶B链融合蛋白原核重组表达质粒的构建、表达和功能的初步分析

将化学合成的RGD肽(Arg-Gly-Asp)编码寡核苷酸与尿激酶B链cDNA相连成为融合基因后,克隆至原核表达质粒pBV220中,在P-RP-L启动子的作用下,经42℃热诱导,在大肠杆菌DH5α中获得了融合基因的表达,其表达量占菌体总蛋白的9.2%,表达产物以无活性的包含体形式存在。经变复性处理得到纯化的融合基因的表达产物,经Western blotting分析表明产物具有与天然尿激酶相似的抗原性,体外活性分析显示其具有一定的纤溶和抗血小板聚集的活性。     

尿激酶原-RGDS双功能分子——构建、表达及性质研究

利用定点突变及DNA重组技术 ,构建了在尿激酶原K区C 端的β 发夹区插入了精氨酸 甘氨酸 天冬氨酸 丝氨酸 (RGDS)片段的尿激酶原嵌合体基因 ,并利用昆虫杆状病毒表达系统通过感染Sf9细胞对嵌合体尿激酶原进行了高效表达 .用尿激酶原单抗亲和柱纯化表达产物 ,获得初步纯化的嵌合体蛋白 .对嵌合体蛋白进行血小板膜结合实验表明 ,此嵌合体具有依赖于钙离子的结合活化血小板膜的活性 .用生色底物Chromozym U测定嵌合体的酰胺解活性 ,结果显示 ,纤溶酶激活后的嵌合体比活为 62 0 0 0IU/mg,与文献报道的纤溶酶激活的尿激酶原比活 65 3 5 5IU/mg相近 .纤溶酶激活后的嵌合体激活纤溶酶原的反应符合米氏方程 ,其Km 值为 0 .97μmol/L ,与天然尿激酶的Km 值 1.64μmol/L相近 .嵌合体还显示了较强的体外抑制血小板聚集活性 .这些结果表明 ,此尿激酶原 RGDS嵌合体有可能成为一种新的双功能溶栓药物 .     

尿激酶前体的纯化及其性质的研究

尿激酶前体是与尿激酶具有共同抗原性的一种新的纤溶酶原激活物。我们从人胎肾细胞条件培养液中提纯该物质。首先用抗UKIgG-Sepharose亲和层析得到尿激酶抗原相关蛋白,然后利用苯甲脒-Sepharose柱去除尿激酶获纯化的尿激酶前体。纯化倍数达930倍,得率为18％,所得尿激酶前体为单肽链结构蛋白质,分子量55kD,比活(11389IU/mg)低于尿激酶,二异丙基氟磷酸酯(DFP)不能抑制其活性。体外¹²⁵I-血凝块溶解试验表明尿激酶前体可特异地诱导血凝块溶解,对血浆纤溶系统无明显激活作用,血凝块溶解的时间曲线呈特征性“S”型。所得尿激酶前体是一种新的有别于尿激酶的纤溶酶原激活物。     

含RGD模体的人白细胞介素18(IL-18)突变体的酵母表达及其抗血小板聚集活性

利用定点突变及DNA重组技术,在人白细胞介素18(IL18)cDNA序列中插入GGC序列,使IL18第39位精氨酸残基和第40位天冬氨酸残基之间插入一个甘氨酸残基,从而构建了RGD模体.此重组的cDNA序列构建入表达质粒pPIC9K,并转化Pichiapastoris酵母GS115,利用表达系统进行了高效表达.用SephadexG100凝胶过滤纯化表达产物,获得初步纯化的蛋白.对该蛋白进行了血小板聚集抑制实验和对GPⅡbⅢa与Fn结合的抑制实验.含RGD模体的重组IL18(IL18RGD)显示了较强的体外抑制血小板聚集活性,IC50=8.8μmolL;并具有与GPⅡbⅢa的竞争结合活性,IC50=8.0μmolL.该含有RGD模体的重组IL18仍保存对PBMC诱导产生IFNγ能力.结果表明,此IL18RGD嵌合体在具有抗炎,抗感染的同时增添了新的抑制血小板聚集功能.     

日本七鳃鳗Arg-Gly-Asp毒素蛋白Lj-RGD3野生型与RGD全缺失突变体Lj-112的抗血管新生作用

七鳃鳗Arg-Gly-Asp (RGD) 毒素肽Lj-RGD3与富含组氨酸糖蛋白HRG具有序列同源性,而RGD毒素蛋白及HRG都具有抑制血管新生的活性,但作用靶点不同。为研究Lj-RGD3结构与功能的关系,对野生型Lj-RGD3及其RGD全缺失突变体Lj-112进行了抗血管新生功能研究。将3个RGD模体的全缺失突变体基因Lj-112全序列合成后构建于pET-23b载体,对野生型Lj-RGD3及突变体 Lj-112蛋白进行IPTG诱导表达,重组蛋白经组氨酸亲和层析纯化;采用MTT法测定野生型和突变体蛋白对人     

PRGDWR-尿激酶原双功能嵌合分子的性质研究

为赋予单链尿激酶型纤溶酶原激活剂(single chain urokinase-type plasminogen activator, scu-PA, 尿激酶原)以抗血小板聚集的功能,在scu-PA的kringle区118位Gly与119位Leu之间插入PRGDWR序列（insert mutant B,InB）.利用甲醇酵母（Pichia pastoris）进行分泌表达,经金属离子螯合亲和层析与S强阳离子交换层析,得到纯蛋白.实验测定InB对人工合成底物S-2444的酰胺解活性为5 900 IU/mg,动力学常数为：K_m,S-2444^InB=56.8 μmol·L^-1,k_cat,S-2444^InB=0.33 s^-1;水解天然底物plasminogen的动力学常数为：K_m,plg^InB=0.397 μmol·L^-1,k_cat,plg^InB=0.0164 s^-1.InB激活plasminogen的反应在有fibrin存在条件下InB的活性为无fibrin条件下的46.3％.该突变体在体外激活plasminogen的活性与同一系统表达的野生型scu-PA基本相同.该突变体表现出较强的抗血小板聚集活性,IC₅₀＝12.7 μmol·L^-1,而野生型scu-PA无此功能.实验表明scu-PA的K区插入突变体InB是一种极具潜力的双功能溶栓分子.     

大肠杆菌K1致病株外膜蛋白T体外功能

纤溶酶原激活及水解抗菌肽利于致病菌侵袭及体内存活。【目的】构建大肠杆菌K1致病株E44的ompT基因缺失突变株,证实E44外膜蛋白T(OmpT)体外激活纤溶酶原及水解抗菌肽鱼精蛋白的活性。【方法】采用基因同源重组技术敲除大肠杆菌K1株E44中的ompT基因,构建ompT缺失突变株;二步柱层析纯化E44外膜组分,S-2251发色底物法测定其纤溶酶原激活活性;考察野生株E44、ompT基因敲除株E44ompT及转化带有ompT完整阅读框的质粒pUCT的E44ompT/pUCT三者对0.1mg/mL阳离子抗菌肽鱼精蛋白的敏感程度。【结果】利用自杀性载体pCVD442和同源重组的原理构建E44的ompT基因敲除株E44ompT;纯化得到约37kDa的E44外膜组分,S-2251发色底物法证实其具有纤溶酶原激活活性,纤溶酶原激活与膜组分的加入量呈一定量效关系;与野生株E44相比,ompT敲除株E44ompT对0.1mg/mL鱼精蛋白敏感,转化入带有ompT完整序列的质粒pUCT有一定的回补作用,E44ompT部分恢复抗鱼精蛋白能力。【结论】外膜蛋白T在致病株E44中有表达,并具有激活纤溶酶原及水解鱼精蛋白的活性。     

血纤蛋白粘附肽与低分子量单链尿激酶融合基因在大肠杆菌中的表达

人工合成了血纤蛋白粘附肽基因,构建了粘附肽与低分子量单链尿激酶cDNA的融合基因,在大肠杆菌中表达了融合基因。融合基因表达产物的抗原性和天然尿激酶相同,并具有尿激酶的溶纤活性和粘附肽的抗纤维蛋白单体聚合的功能。     

SA-hirudin-RGD融合蛋白的原核表达及其抗凝血酶与抗血小板聚集功能的研究

目的:构建SA-hirudin-RGD重组载体,表达和纯化融合蛋白,并对其抗凝血酶、抗血小板聚集功能进行初步验证。方法:利用基因重组技术将链霉亲和素(SA)核心区与hirudin-RGD序列连接,并克隆到原核表达载体PET-44b中,Westernblotting鉴定经IPTG诱导后纯化的融合蛋白。抗凝血酶和抗血小板聚集作用分析证明该融合蛋白既有抗凝血酶又有抗血小板聚集的功能。结果:重组载体p ET44b-SA-hirudin-RGD经限制性酶切鉴定和基因测序证实构建成功;经IPTG诱导后SA-hirudin-RGD融合蛋白在大肠杆菌中高效表达;纯化得到该目的蛋白,相对分子质量经Westernblotting鉴定约为70000。抗凝血酶和抗血小板聚集的实验证明,融合蛋白SA-hirudin-RGD既有抗凝血酶又有抗血小板聚集的功能。结论:具有抗凝血酶和抗血小板聚集双重功能的SA-hirudin-RGD融合蛋白被成功表达和纯化,为下一步SA-hirudin-RGD的功能研究及临床应用确立了基础。     

RGD肽与尿激酶B链融合蛋白原核重组表达质粒的构建,表达和？…

将化学合成的ＲＧＤ肽（Ａｒｇ－Ｇｌｙ－Ａｓｐ）编码寡核苷酸与尿激酶Ｂ链ｃＤＮＡ相连成为融合基因后,克隆至原核表达质粒ｐＢＶ２２０中,在ＰＲＰＬ自动子的作用下,经４２℃热诱导,在大肠杆菌ＤＨ５α中获得了融合基因的表达,其表达量占菌体总蛋白的９．２％,表达产物以无活性的包含体形式存在。经变复性处理得到纯化的融合基因的表达产物,经Ｗｅｓｔｅｒｎ－ｂｌｏｔｔｉｎｇ分析表明产物具有与天然尿激酶相似的抗原性,     

Recombinant human, active site-blocked factor VIIa reduces infarct size and no-reflow phenomenon in rabbits

Oxygen free radicals induce de novo synthesis of tissue factor (TF), the initiator of the extrinsic pathway of coagulation, within the coronary vasculature during postischemic reperfusion. In the present study we wanted to assess whether TF expression might cause myocardial injury during postischemic reperfusion. Anesthetized rabbits underwent 30 min of coronary occlusion followed by 5.5 h of reperfusion. At reperfusion the animals received 1) saline (n = 8), 2) human recombinant, active site-blocked activated factor VII (FVIIai, 1 mg/kg, n = 8), or 3) human recombinant activated FVII (FVIIa, 1 mg/kg, n = 8). FVIIai binds to TF as native FVII, but with the active site blocked it inhibits TF procoagulant activity. The area at risk of infarction (AR), the infarct size (IS), and the no-reflow area (NR) were determined at the end of the experiment. FVIIai resulted in a significant reduction in IS and NR with respect to control animals (28.1 +/- 11.3 and 11.1 +/- 6.1% of AR vs. 59.8 +/- 12.8 and 24.4 +/- 2.7% of AR, respectively, P < 0.01), whereas FVIIa resulted in a significant increase in IS and NR to 80.1 +/- 13. 1 and 61.9 +/- 13.8% of AR, respectively (P < 0.01). In conclusion, TF-mediated activation of the extrinsic coagulation pathway makes an important contribution to myocardial injury during postischemic reperfusion.     

Use of whey protein beads as a new carrier system for recombinant yeasts in human digestive tract

A new immobilizing protocol using whey protein isolates was developed to entrap recombinant Saccharomyces cerevisiae. The model yeast strain expresses the heterologous P45073A1 that converts trans-cinnamic acid into p-coumaric acid. Beads resulted from a cold-induced gelation of a whey protein solution (10%) containing yeasts (7.5 x 10(7)cells ml(-1)) into 0.1M CaCl(2). The viability and growth capability of yeasts were not altered by our entrapment process. The release and activity of immobilized yeasts were studied in simulated human gastric conditions. During the first 60 min of digestion, 2.2+/-0.9% (n=3) of initial entrapped yeasts were recovered in the gastric medium suggesting that beads should cross the gastric barrier in human. The P45073A1 activity of entrapped yeasts remained significantly higher (p<0.05) than that of free ones throughout digestion (trans-cinnamic acid conversion rate of 63.4+/-1.6% versus 51.5+/-1.8% (n=3) at 120 min). The protein matrix seemed to create a microenvironment favoring the activity of yeasts in the stringent gastric conditions. These results open up new opportunities for the development of drug delivery system using recombinant yeasts entrapped in whey protein beads. The main potential medical applications include biodetoxication or the correction of digestive enzyme deficiencies.     

Artificial insemination with frozen semen in dogs: a retrospective study of 10 years using a non-surgical approach

From 1994 to 2003, a total of 526 bitches of 99 different breeds were artificially inseminated in 685 estrus cycles with domestic (n = 353) or imported (n = 332) frozen-thawed semen from 368 males. The overall whelping rate was 73.1% and mean (+/- S.E.M.) litter size 5.7 +/- 0.1 pups. The whelping rate was higher after intrauterine insemination (75.0%; n = 665) than after intravaginal insemination (10.0%, n = 20; P < 0.05). Insemination at the optimal time resulted in a higher whelping rate (78%, n = 559; P < 0.01) and larger litter size (5.8 +/- 0.2; P < 0.05) than inseminations performed late or too late (55.7% and 4.5 +/- 0.5, n = 61). Two inseminations (n = 384) yielded a higher whelping rate (P < 0.05) and mean litter size (P < 0.01) than one insemination (n = 241), 78.1% and 6.0 +/- 0.2 and 70.5% and 5.1 +/- 0.2, respectively. For inseminations performed at the optimal time, however, the whelping rate was not significantly different for bitches inseminated twice (79.3%, n = 358) versus once (76.8%, n = 168), but the litter size was larger (6.0 +/- 0.2 and 5.3 +/- 0.3). Semen classified as of poor quality (progressive motility < 50% or percentage abnormal sperm > 20%) resulted in a lower whelping rate (P < 0.01) than semen classified as of good quality (progressive motility > or = 50% and percentage abnormal sperm < or = 20%), 61 and 77%, respectively. Small breeds (n = 50) had a smaller litter size (3.9 +/- 0.3; P < 0.01) than larger breeds (medium [5.7 +/- 0.3, n = 94], large [5.9 +/- 0.2, n = 295] or giant breeds [6.1 +/- 0.5, n = 62] [P < 0.01]). Bitches older than 6 years had a lower whelping rate (68.2%) than younger ones (77.0%; P < 0.05). The duration of pregnancy was longer (P < 0.01) for bitches with a litter size of < 3 pups (61.7 +/- 0. 4 days, n = 30) than for bitches with larger litters (60.5 +/- 0.1 days, n = 177). These results show the potential of transcervical intrauterine insemination for routine artificial insemination in dogs. The results with frozen semen inseminations were optimised by inseminating bitches < or = 6 years old 2 and 3 days after ovulation with semen of good quality from males < or = 8 years old.     

Recombinant Saccharomyces cerevisiae expressing P450 in artificial digestive systems: a model for biodetoxication in the human digestive environment

The use of genetically engineered microorganisms such as bacteria or yeasts as live vehicles to carry out bioconversion directly in the digestive environment is an important challenge for the development of innovative biodrugs. A system that mimics the human gastrointestinal tract was combined with a computer simulation to evaluate the survival rate and cinnamate 4-hydroxylase activity of a recombinant model of Saccharomyces cerevisiae expressing the plant P450 73A1. The yeasts showed a high level of resistance to gastric and small intestinal secretions (survival rate after 4 h of digestion, 95.6% +/- 10.1% [n = 4]) but were more sensitive to the colonic conditions (survival rate after 4 h of incubation, 35.9% +/- 2.7% [n = 3]). For the first time, the ability of recombinant S. cerevisiae to carry out a bioconversion reaction has been demonstrated throughout the gastrointestinal tract. In the gastric-small intestinal system, 41.0% +/- 5.8% (n = 3) of the ingested trans-cinnamic acid was converted into p-coumaric acid after 4 h of digestion, as well as 8.9% +/- 1.6% (n = 3) in the stomach, 13.8% +/- 3.3% (n = 3) in the duodenum, 11.8% +/- 3.4% (n = 3) in the jejunum, and 6.5% +/- 1.0% (n = 3) in the ileum. In the large intestinal system, cinnamate 4-hydroxylase activity was detected but was too weak to be quantified. These results suggest that S. cerevisiae may afford a useful host for the development of biodrugs and may provide an innovative system for the prevention or treatment of diseases that escape classical drug action. In particular, yeasts may provide a suitable vector for biodetoxication in the digestive environment.     

Mechanism of thrombin-induced vasodilation in human coronary arterioles

Thrombin (Thromb), activated as part of the clotting cascade, dilates conduit arteries through an endothelial pertussis toxin (PTX)-sensitive G-protein receptor and releases nitric oxide (NO). Thromb also acts on downstream microvessels. Therefore, we examined whether Thromb dilates human coronary arterioles (HCA). HCA from right atrial appendages were constricted by 30-50% with endothelin-1. Dilation to Thromb (10(-4)-1 U/ml) was assessed before and after inhibitors with videomicroscopy. There was no tachyphylaxis to Thromb dilation (maximum dilation = 87.0%, ED(50) = 1.49 x 10(-2)). Dilation to Thromb was abolished with either hirudin or denudation but was not affected by PTX. Neither N(omega)-nitro-l-arginine methyl ester (n = 7), indomethacin (n = 9), (1)H-[1,2,4] oxadiazolo-[4,3-a]quinoxalin-1-one (n = 6), tetraethylammonium chloride (n = 5), nor iberiotoxin (n = 4) reduced dilation to Thromb. However, KCl (maximum dilation = 89 +/- 5 vs. 20 +/- 10%; P < 0.05; n = 7), tetrabutylammonium chloride (maximum dilation = 79 +/- 7 vs. 21 +/- 4%; P < 0.05; n = 5), and charybdotoxin (maximum dilation = 89 +/- 4 vs. 10 +/- 2%; P < 0.05; n = 4) attenuated dilation to Thromb. In contrast to animal models, Thromb-induced dilation in human arterioles is independent of G(i)-protein activation and NO release. However, Thromb dilation is endothelium dependent, is maintained on consecutive applications, and involves activation of K(+) channels. We speculate that an endothelium-derived hyperpolarizing factor contributes to Thromb-induced dilation in HCA.     

Responses of cardiac sympathetic nerve activity to changes in circulating volume differ in normal and heart failure sheep

Factors controlling cardiac sympathetic nerve activity (CSNA) in the normal state and those causing the large increase in activity in heart failure (HF) remain unclear. We hypothesized from previous clinical findings that activation of cardiac mechanoreceptors by the increased blood volume in HF may stimulate sympathetic nerve activity (SNA), particularly to the heart via cardiocardiac reflexes. To investigate the effect of volume expansion and depletion on CSNA we have made multiunit recordings of CSNA in conscious normal sheep and sheep paced into HF. In HF sheep (n = 9) compared with normal sheep (n = 9), resting levels of CSNA were significantly higher (34 +/- 5 vs. 93 +/- 2 bursts/100 heart beats, P < 0.05), mean arterial pressure was lower (76 +/- 3 vs. 87 +/- 2 mmHg; P < 0.05), and central venous pressure (CVP) was greater (3.0 +/- 1.0 vs. 0.0 +/- 1.0 mmHg; P < 0.05). In normal sheep (n = 6), hemorrhage (400 ml over 30 min) was associated with a significant increase in CSNA (179 +/- 16%) with a decrease in CVP (2.7 +/- 0.7 mmHg). Volume expansion (400 ml Gelofusine over 30 min) significantly decreased CSNA (35 +/- 12%) and increased CVP (4.7 +/- 1.0 mmHg). In HF sheep (n = 6) the responses of CSNA to both volume expansion and hemorrhage were severely blunted with no significant changes in CSNA or heart rate with either stimulus. In summary, these studies in a large conscious mammal demonstrate that in the normal state directly recorded CSNA increased with volume depletion and decreased with volume loading. In contrast, both of these responses were severely blunted in HF with no significant changes in CSNA during either hemorrhage or volume expansion.     

Plasminogen activation by single-chain urokinase in functional isolation. A kinetic study

The kinetics of the activation of Glu- and Lys-plasminogen by single-chain urokinase (sc urokinase) derived from the transformed human kidney cell line TCL-598 have been studied and compared with two-chain urokinase (tc urokinase). Plasminogen activation was determined by the increase in fluorescence polarization of fluorescein-labeled aprotinin, a high affinity inhibitor of plasmin. This methodology allows plasmin generation by sc urokinase to be measured in functional isolation, with no interfering generation of tc urokinase, sc urokinase was found to activate plasminogen to plasmin with apparent Michaelis-Menten-type kinetics. The Km for Glu-plasminogen activation was 47.7 microM, with a catalytic constant of 2.91 min-1. Lys-plasminogen activation by sc urokinase was characterized by a Km of 11.7 microM and a kcat of 5.60 min-1. The Km values for the activation of Glu- and Lys-plasminogen by tc urokinase were found to be similar to those for activation by sc urokinase (36.8 and 9.0 microM, respectively), but the catalytic constants were higher at 36.0 and 118 min-1, respectively. Therefore, on the basis of the catalytic efficiency kcat/Km, sc urokinase seems to have 16-27-fold lower activity than tc urokinase. This activity of sc urokinase is in contrast to its lack of activity against a low molecular weight peptide substrate (less than 0.2% of the activity of sc urokinase). The activation of sc urokinase to tc urokinase by plasmin was also characterized (Km = 3.0 microM, kcat = 105 min-1). Using these data, it was possible to calculate the theoretical rate of plasminogen activation by sc urokinase in the absence of aprotinin, when tc urokinase is generated by the action of plasmin. The calculated rate was in good agreement with that determined experimentally using the chromogenic substrate D-Val-Leu-Lys-p-nitroanilide. These data demonstrate that sc urokinase has properties which distinguish it from conventional serine protease zymogens. The lack of activity against low molecular weight peptide substrates demonstrates the inaccessibility of the substrate-binding pocket. However, there is a moderate activity against plasminogen, suggesting that plasminogen may be acting as both an effector and a substrate for sc urokinase.