表达人PAI-1的重组腺病毒的制备及检测

人纤溶酶原激活剂抑制物1（PAI-1）基因与复制缺陷型腺病毒载体AdCMVHSgD重组,与pJM17质粒共转染293细胞,采用不铺琼脂的方法产生重组病毒.PCR证实PAI-1基因重组进入腺病毒.进一步感染B16（F10）细胞,细胞表面洗提物和上清分别经纤维显示胶和反向纤维蛋白显示胶检测PAI-1抑制纤溶酶原激活剂（PA）的活性.     

单链尿激酶型纤溶酶原激活剂Kringle结构域催化功能探讨

 Kringle(K)结构域广泛存在于与凝血和纤溶相关的各种因子中 .虽然 K结构域在一级结构上是一种比较保守的结构域 (与其他结构域相比 ) ,但是 K结构域的功能在各种因子中的作用有很大的区别 .为探讨单链尿激酶型纤溶酶原激活剂 (scu- PA)的 K结构域功能 ,构建了在 scu- PA的 K结构域的 1 1 8位 Gly与 1 1 9位 Leu之间插入 PRGDWR序列的突变体 (称为 insert mutant B,In B) ,并测定了野生型 scu- PA与 In B的纤溶相关反应的动力学常数 . scu- PA与 In B水解 S- 2 4 4 4反应的 Km 值无明显变化 (分别为 60 .4与 56.8μmol·L-1) ,而 In B的 kcat值 (0 .33s-1)比 scu- PA的 kcat值 (7.31 s-1)降低很多 ;In B激活纤溶酶原反应的 Km 值 (0 .397μmol· L-1)比 scu- PA Km 值(0 .648μmol·L-1)降低 40 % ,但 kcat值 (0 .0 1 65s-1)比 scu- PA的 kcat值 (0 .0 62 6s-1)降低 74% .以上结果说明 :K结构域主要与反应活性相关 ,而与酶及底物的亲和性无关 .     

可调控小鼠尿激酶原激活剂真核表达载体的构建及其在Huh7细胞中的表达

目的：建立基于Tet-on系统的可调控真核细胞表达小鼠尿激酶原激活剂（uPA）的诱导表达系统。方法：提取C57小鼠肾组织总RNA,RT-PCR扩增uPAcDNA序列;提取基因组DNA,扩增uPA编码区后最后一个外显子序列,构建pTRE2-uPAcDNA-700载体,将其与pTet-on瞬时共转染Huh7细胞系,24h后用强力霉素诱导表达,诱导后36h分别收集细胞和培养上清（诱导组）,提取细胞总RNA并进行细胞uPA转录水平的检测;采用溶圈法对细胞分泌至培养上清中uPA的生物活性进行检测,同时以转染但未诱导Hun7（未诱导组）和正常培养Huh7（正常对照组）细胞及培养上清作为对照。结果：与未诱导组和正常对照组相比较,仅诱导组在转录水平上扩增出目的条带;溶圈法证实转染的细胞不仅表达uPA,而且表达的蛋白具有一定的生物活性。结论：构建了可调控的uPA真核诱导表达系统,为进一步制备可调控肝细胞表达uPA转基因小鼠及进一步揭示uPA肝损伤机理奠定了基础。     

重组人组织型纤溶酶原激活剂(rht-PA)及其突变体的纯化

稳定高效表达重组人组织型纤溶酶原激活剂 (rht PA)的CHO细胞株和表达组合突变体的细胞株进行了 3L转瓶培养 .将培养上清分别进行了Lys Sepharose 4B亲和层析和Zn2 + Sepharose 4B层析两步纯化 ,rht PA纯度提高了 5 34倍 ,比活达 2 5× 10 5IU mg ,产率为 73% ;突变体纯度提高了1119倍 ,比活达 5 9× 10 5IU mg ,产率为 6 9% .纯化产物SDS PAGE分析显示 ,rht PA和突变体基本都呈单一条带 ,扫描分析均达到 98%以上纯度 .rht PA和突变体在纯化系统中的行为作对照分析发现 ,突变体的构建思想在Lys Sepharose 4B亲和层析过程中有充分体现 .这两步层析组合是很好的纯化t PA及其突变体的方法 ,尤其是Lys Sepharose 4B纯化突变体效果更好     

丝氨酸蛋白酶的重组表达

丝氨酸蛋白酶是蛋白酶家族中的一大家族 ,广泛分布于细菌、病毒、真菌、植物、动物中 ,并参与生命的各种反应。随着丝氨酸蛋白酶研究的深入 ,重组表达获得大量的目的蛋白酶已成为研究该类蛋白酶的一大基础技术。本文重点综述了丝氨酸蛋白酶在不同表达系统中重组表达的优缺点及最新进展。     

用于重组蛋白生产的哺乳动物细胞表达新技术

近年来,用于重组蛋白生产的哺乳动物细胞表达领域涌现出一系列革命性的新技术。优化的工程细胞为表达重组蛋白提供了优良的宿主;基于荧光的筛选方法可以快捷地得到高表达细胞株;高通量的培养工艺能够预测适合外源蛋白表达的细胞培养条件;可抛弃式生物反应器为大规模细胞培养提供了更多的选择;大规模瞬时表达技术节省了重组蛋白的生产时间。这些新技术提高了重组蛋白的研发和生产效率,加快了蛋白药物的工业化进程。     

重组抗体—尿激酶导向溶栓剂的基因构建及表达

为了获得高效、高特异性溶栓药物,应用基因工程技术,成功的表达了由人源化抗人活化血小板单抗和单链尿酶组成的抗体导向溶栓剂（ＳＺ５１Ｈｕ－ｓｃｕＰＡ）。通过基因重组ＰＣＲ方法将ｓｃｕＰＡ全长ｃＤＮＡ的Ｎ末端连接在ＳＺ５１重链恒区ＣＨ３末端,构建了含有目的蛋白融合基因的真核表达载体αｌｙｓ３０－ＳＺ５１ＶＨ／Ｈｕ－ｓｃｕＰＡ。采用脂转染法将表达载体导入分泌ＳＺ５１ＶＫ／Ｈｕ轻链的小鼠骨髓瘤细胞中,筛选出     

蛇毒纤溶酶原激活剂TSV—PA在昆虫细胞中的表达

将蛇毒TSV－PA基因插入昆虫杆状病毒供体质粒pFastBacHTa中,在粉纹夜蛾Tn-5B1-4细胞中进行表达。SDS－PAGE分析结果表明,表达产物为分子量33KD的TSV－PA蛋白。Western印迹分析证实了此结果,酶活力测定结果表明,昆明细胞表达的TSV－PA蛋白具有较高活性。     

Expression of Active Recombinant Human Tissue-Type Plasminogen Activator by Using In Vivo Polyhydroxybutyrate Granule Display

Recombinant human tissue plasminogen activator (rPA) is a truncated version of tissue plasminogen activator (tPA), which contains nine disulfide bonds and is prone to forming inactive inclusion bodies when expressed in bacteria. To obtain functional rPA expression, we displayed the rPA on the surface of polyhydroxybutyrate (PHB) granules using phasin as the affinity tag. rPA was fused to the N terminus of the phasin protein with a thrombin cleavage site as the linker. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis showed that rPA fusion was successfully displayed on the surface of PHB granules. An activity assay indicated that the rPA fusion is active. The in vivo surface display strategy for functional rPA expression in Escherichia coli is distinct for its efficient folding and easier purification and may be expanded to the expression of other eukaryotic proteins with complex conformation.Tissue plasminogen activator (tPA) derives from a fibrinolytic system of blood vessel endothelial cells, activates plasminogen to form plasmin, and is an effective drug for thrombolytic therapy. Native tPA is composed of 527 amino acid residues with five structural domains and 17 disulfide bonds (19). Recombinant human tissue plasminogen activator (rPA) is a variant version of tPA with nine disulfide bonds, consisting of kringle 2 and serine protease domain (12). rPA was confirmed to possess enhanced capability for thrombolysis compared with that of tPA. Therefore, rPA is more beneficial for the treatment of acute myocardial infarction (17, 26, 28).Heterologous expression of tPA as well as rPA in Escherichia coli often results in the formation of the insoluble aggregates known as inclusion bodies due to the multidisulfide bonds (3). The refolding of the inclusion bodies in vitro is a long and difficult task, especially for proteins with complex conformation and multiple disulfide bonds. In order to obtain directly the functional rPA from recombinant E. coli, many approaches have been utilized: expressing the rPA gene in E. coli trxB gor ahpC* mutant strains, of which the cytoplasm is highly oxidized; fusing the rPA gene with gpIII of ΦM13 and linking to the OmpA signal sequence, through which rPA is secreted into the medium; exploiting the novel twin-arginine translocation (Tat) pathway to obtain active rPA in the periplasmic space based on its inherent properties; and cosecreting of rPA with chaperones and adding low-molecular-size medium additives to promote the formation of disulfide bonds (6, 11, 15, 25). However, the successful expression of rPA in its soluble or active form gives rise to another task: separation and purification of soluble active rPA from large amounts of other proteins in cytoplasm or medium.Normal protein purification typically involves several chromatographic steps. Each step can be costly and time-consuming (4). The development of simple and reliable methods for protein purification, which can be applied to arbitrary products, is therefore an important goal in bioseparation technology developments. One method that was recently developed is the addition of an affinity tag sequence to the target protein gene (13). It was demonstrated that heterologous proteins can be displayed actively on the surface of biopolyester granules in E. coli by fusing to the polyhydroxyalkanoate (PHA) synthase (PhaC), which serves as an affinity tag of PHA granules (21). PHA granules are carbon inclusions produced intracellularly by bacteria for coping with changing, often oligotrophic environments (1). These inclusions are composed of a hydrophobic polyester core and hydrophilic phospholipid membrane with many embedded proteins (24). Besides PhaC, phasins (namely PhaPs) are the main proteins tightly attached to the surface of polyhydroxybutyrate (PHB) granules, which can stabilize and prevent coalescence of separate PHB granules (22). Due to the inherent properties, PhaP has been used as the affinity tag in vivo to display recombinant proteins on the surface of PHB granules (5).In this study, we fused the rPA gene to the N terminus of phaP. A thrombin cleavage site was introduced between them to release rPA from PHB granules. The fusion gene was then expressed in engineered E. coli, which was conferred with the PHB production pathway by cloning the PHB biosynthesis genes. We confirmed that recombinant rPA fusion was able to be actively expressed in vivo on the surface of PHB granules.     

小剂量重组组织纤溶酶原激活剂治疗急性心肌梗死临床研究概况

1990年代中期以来,国内130多家医院入组9378例急性心肌梗死患者,其中用小剂量（50mg）重组组织纤溶酶厚激活剂（rt-PA）治疗6693例,阻塞相关血管开通5318俐,开通率为79.46％;死亡293倒,病死率为4.38％;出血550俐,出血率勾8.22％,其中重度出血7例,颅内出血21例（0.31％）,再次梗塞60例（0.90％）。超过40家医院对rt-PA（50mg）与尿激酶治疗急性心肌梗死疗效进行了比较,共计入组3449倒急性心肌梗死患者,rt-PA治疗1689例,先静脉推注8mg,其余42mg在30或60和90min滴注;尿激酶治疗1760例,150万U位滴注30min。结果显示,阻塞相关冠脉血管开通率分别为79.40％（1341例）和5733％（1009例）,相差非常显著（P〈0.001）。12家医院研究了rt-PA50-9100mg治疗急性心肌梗死的效果,共计入组1054例患者,其中50mg组487例,100mg组567例,阻塞相关血管开通率分别为78.85％和82.36％。另有22家医院入组1017倒病人,行rt-PA50mg30rain给药临味试验,冠脉开通率达80.53％;18家医院行rt-PA50mg 60min给药临床试验,入组942例病人,阻塞相关血管开通率为77.92％;50家医院用rt-PA50mg 90min给药方案治疗急性心肌梗死患者,入组2768例患者,冠脉开通率为77.89％。6家医院对用rt-PA（50mg）与链激酶治疗急性心肌梗死的疗效进行了对比,结果表明相关血管开通率分别为81.4％和65.2％22家医院比较了小剂量rt-PA对急性心肌梗死患者症状发作不同时间的治疗效果,表明症状发作时间越短,用药的溶栓效果越好。刘光对入院前和入院后用小剂量rt-PA溶栓进行了比较研究,证明入院前溶栓比入院后效果好。对冠脉内输注rt-PA（50mg）和2次静脉推注小剂量rt-PA治疗急性心肌梗死的效果也进行了探索。     

人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化

 本文报道了培养的人黑色素瘤细胞分泌的组织纤溶酶原激活剂(t-PA)的纯化方法。Bowes株人黑色素瘤细胞的分泌产物,经CM-Sephadex C--50层析,赖氨酸-Sepharose 4B,苯甲眯-sepharose 4B亲和层析后,即可得到纯化470倍的蛋白纯品。样品经聚丙烯酰胺凝胶电泳鉴定为均一单带,测得其分子量约为72kD。纯化的t-PA与尿激酶相比较,发现前者有更高亲和纤维蛋白的能力。     

人组织型纤溶酶原激活剂(t-PA)突变体在CHO细胞中的高效表达

构建了二氢叶酸还原酶(dhfr)选择基因启动子上游无增强子(cnhancer)的组织型纤溶酶原激活剂(t-PA)组合突变体FrGGI真核表达质粒pZLFrGGI。将pZLFrGGI酶切线性化,采用大剂量DNA电击介导法,转染dhfr基因缺陷型中国仓鼠卵巢细胞系(CHO-dhfr~-)。用氨甲喋呤(MTX)筛选转染细胞。混合加压后,挑选克隆,在1×10~(-7)mol/L MTX压力下,得到了表达水平达1500～2500IU/10~6细胞·24小时的细胞株。此细胞株表达水平稳定,形态良好,倍增时间为36小时,且有进一步提高表达水平的潜能,有望发展为工程细胞株。     

人组织型纤溶酶原激活剂（t－PA）突变体FrGGI的构建、表达及特性分析

为了获得半衰期延长,特异活性提高及具有ＰＡＬ－１抗性的新型ｔ－ＰＡ溶栓剂,利用基因重组及定位突变技术构建了ｔ－ＰＡ的Ｋ１、Ｋ２区糖基化位点消除,ＰＡＩ－１结合位点缺失,Ｆ与Ｅ区连接序列Ｈｉｓ４４～Ｓｅｒ５０置换为纤粘蛋白Ⅰ型Ｆ区间连接序列ＧｌｕＳｅｒＬｙｓＰｒｏＧｌｕＡｌａＧｌｕＧｌｕ的ｔ－ＰＡ组合突变体ＦｒＧＧＩ,并在中国仓鼠卵巢细胞中获得了高效表达。对表达产物的生物学特性分析表明,ＦｒＧＧＩ在大鼠血浆中的半衰期延长了１５倍,并获得了ＰＡＩ－１抗性,是一株很有希望的新型溶栓剂候选株。     

A Comparative Investigation on Different Refolding Strategies of Recombinant Human Tissue-type Plasminogen Activator Derivative

Recombinant human tissue-type plasminogen activator derivative (r-PA), fused with thioredoxin (Trx), was expressed in Escherichia coli. The resultant fusion protein, Trx-r-PA, was almost completely in the form of inclusion bodies and without activity. Different refolding strategies were investigated including different post-treatment of solubilized Trx-r-PA inclusion bodies, on-column refolding by size-exclusion chromatography (SEC) using three gel types (Sephacryl S-200, S-300 and S-400), refolding by Sephacryl S-200 with a urea gradient and two-stage temperature control in refolding. An optimized on-column refolding process for Trx-r-PA inclusion bodies was established. The collected Trx-r-PA inclusion bodies were dissolved in 6 m guanidine hydrochloride (Gdm·HCl), and the denatured protein was separated from dithiothreitol (DTT) and Gdm·HCl with a G25 column and simultaneously dissolved in 8 m urea containing oxidized glutathione (GSSG). Finally a refolding of Trx-r-PA protein on Sephacryl S-200 column with a decreasing urea gradient combined with two-stage temperature control was employed, and the activity recovery of refolded protein was increased from 3.6 to 13.8% in comparison with the usual dilution refolding. Revisions requested 31 October 2005; Revisions received 20 December 2005     

重组人MASP2蛋白在大肠杆菌中的表达和纯化

目的:获得酶原形式的重组人甘露聚糖结合凝集素相关丝氨酸蛋白酶2(MASP2)。方法:在大肠杆菌中诱导表达重组人MASP2全长蛋白,包涵体裂解后,经复性、透析、浓缩、考马斯亮蓝染色、SDS-PAGE及Western印迹,鉴定纯化结果及酶活性。结果:复性后的MASP2蛋白经考马斯亮蓝染色未见杂带。自激活实验表明,当MASP2浓度在1μmool/L以下时,无论在4℃还是37℃,都能较稳定地保持酶原形式;蛋白浓度为3.5μmool/L时只能在4℃保持稳定,37℃发生自激活;蛋白浓度达到12μmool/L后,在4℃时已不能稳定存在。结论:获得了较纯的重组人MASP2蛋白,且具有自激活活性。     

重组人纤溶酶原Kringle1-3的生物学活性鉴定

目的：检测重组人纤溶酶原Kringle1-3（K1-3）的生物学活性。方法：用含重组人纤溶酶原K1-3基因的表达载体pET21a-Angio（K1-3）转化表达宿主菌大肠杆菌BL21（DE3）后诱导表达,表达产物经溶解、复性和纯化后,进行SDS-PAGE,计算其相对分子质量;用BCA法测蛋白浓度,用细胞抑制实验（MTT法）和鸡胚绒毛膜尿囊膜（CAM）实验鉴定纯化产物对血管内皮细胞增殖和血管生成的抑制效果。结果：表达产物的相对分子质量为38000,与预期值一致;细胞抑制实验和CAM实验结果表明表达产物具有特异抑制血管内皮细胞增殖、血管生成的功能。结论：所纯化的重组人纤溶酶原K1-3具有抑制血管内皮细胞的生物学活性,为该蛋白在病理性血管疾病等方面的应用研究提供了材料。     

重组尿激酶型纤溶酶原激活物受体在酵母系统中的表达及鉴定

构建截断型可溶性尿激酶型纤溶酶原激活物受体（ｕ－ＰＡＲ）的原核及酵母表达质粒并分别在大肠杆菌和Ｐｉｃｈｉａｐａｓｔｏｒｉｓ酵母中高效表达．利用ＰＣＲ扩增截断型可溶性ｕ－ＰＡＲｃＤＮＡ片段,并分别连入酵母及原核表达载体中,构建成表达质粒．后者经诱导表达的蛋白被用于免疫家兔,获得抗ｕ－ＰＡＲ的抗血清,用于对酵母表达产物的鉴定．前者在甲醇的诱导下表达,产物分泌至培养液中．结果表明,原核表达的ｕ－ＰＡＲ蛋白免疫家兔后获得高效价的抗血清,利用此抗血清所作Ｗｅｓｔｅｒｎｂｌｏｔ证实酵母表达蛋白具有ｕ－ＰＡＲ的免疫原性,表观分子量４０ｋＤ左右．Ｍｕｔ－表型在第５ｄ为最高（２．５μｇ／ｍｌ）,Ｍｕｔ＋表型在第４ｄ为最高（７．５μｇ／ｍｌ）．因此,构建的可溶性ｕ－ＰＡＲ表达质粒在Ｐｉｃｈｉａｐａｓｔｏｒｉｓ酵母细胞获得表达,为进一步竞争拮抗受体功能的研究奠定了基础．     

人组织型纤溶酶原激活剂(t-PA)组合突变体FrGGl在CHO细胞中的高效表达

为了得到ｔＰＡ组合突变体ＦｒＧＧＩ在ＣＨＯ细胞中的高效表达,将表达质粒筛选基因启动子上游的增强子（ｅｎｈａｎｃｅｒ）去除,构建了ＦｒＧＧＩ真核表达质粒ｐＺＬＦｒＧＧＩ。酶切线性化后,采用大剂量ＤＮＡ电击介导法,转染ｄｈｆｒ基因缺陷型中国仓鼠卵巢细胞系（ＣＨＯｄｈｆｒ－）。氨甲喋呤（ＭＴＸ）筛选转染细胞,混合加压,挑选克隆,在１×１０－７ｍｏｌ／ＬＭＴＸ压力下,获得表达水平达１５００～２５００ＩＵ／１０６细胞·２４ｈ的细胞株。此细胞株表达水平稳定,形态良好,倍增时间约为３６ｈ,且有进一步提高表达水平的潜能,有望发展为工程细胞株     

人组织型纤溶酶原激活剂(t-PA)mRNA5′端非翻译区(UTR)对其表达的调控

运用反转录-PCR技术,从黑色素瘤细胞中扩增出t—PA cDNA 5′末端460bp的片段,再经重组获得含完整5′-UTR的t—PA cDNA克隆,在兔网织红细胞裂解物中翻译和COS-7细胞中表达发现,t—PA mRNA 5′—UTR对其表达有明显的抑制作用。将t—PA mRNA 5′—UTR用苜蓿病毒RNA 5′—UTR替换,使t—PA的表达水平提高3-7倍,mRNA翻译起始区二级结构分析结果表明,翻译起始区的二级结构与t-PA的表达水平有关。     

重组人组织型纤溶酶原激活剂突变体基因的克隆、表达与生物活性鉴定

目的：研究重组人组织型纤溶酶原激剂突变体(reteplase,r-PA)基因的克隆和表达,并对表达产物进行活性鉴定。方法：通过PCR的方法从t-PA基因上扩增得到r-PA基因的编码序列,并克隆到pUC19载体中,经DNA测序正确后,将该基因克隆到含有,T7启动子的高效表达质粒pJZ-100中,转化E．coli BL21(DE3),得到含有r-PA基因的工程菌。工程菌经IPTG诱导表达,SDS-PAGE检测表达情况。提取包涵体,经过体外稀释法复性,亲和色谱层析纯化,测定产物的活性。结果：表达的重组蛋白约占可溶性总蛋白的20％,复性并纯化后测定活性为58000IU/mg。结论：成功构建了重组r-PA的表达质粒,表达产物具有良好的生物活性。