前肽缺失体vWF改善FVIII基因断裂转移 细胞的重链和活性分泌

通过转von Willebrand因子(vWF)的前肽缺失突变体(vWF-ΔPro)基因,探讨了vWF-ΔPro对双载体转凝血VⅢ因子(FVⅢ)基因的影响。将vWF-ΔPro基因和B-区缺失型FVⅢ(BDD-FVⅢ)的重、轻链基因共转染HEK293细胞48 h后,ELISA检测重链的分泌量为(142±29)ng/ml,明显高于未转vWF-ΔPro基因细胞(87±15)ng/ml;未转vWF-ΔPro基因时单独转重链基因的重链分泌水平很低,vWF-ΔPro存在时重链分泌量明显提高,但不如共转基因时的重链分泌,提示轻链可反式促进重链分泌;单独转轻链或与重链共转基因时轻链分泌水平均较高,且不受vWF-ΔPro影响;Coatest法检测分泌的凝血活性显示,转vWF-ΔPro基因可使细胞分泌的凝血活性明显高于未转vWF-ΔPro基因细胞(0.80±0.15 IU/ml vs 0.41±0.08 IU/ml)。另外,转vWF-ΔPro基因条件下,合并培养转重链和转轻链基因细胞的培养上清中,也检测到FVIII凝血活性(0.23±0.09IU/ml),提示vWF-ΔPro有助于分泌的重、轻链形成功能性异源二聚体。表明转vWF-ΔPro基因可促进双链转FVⅢ基因,为进一步动物体内实验奠定了基础。     

大肠杆菌BL21(DE3)中DnaE intein介导ABCA1蛋白的连接

摘 要：【目的】利用Ssp DnaE intein的蛋白质反式剪接技术研究在大肠杆菌中对ABCA1基因表达产物的连接作用。【方法】将ABCA1的cDNA于满足剪接所需的保守性氨基酸Cys978密码子前断裂为N端和C端两部分,分别与天然存在的反式作用Ssp DnaE intein的123个氨基酸的N端和36个氨基酸的C端编码序列融合,构建到原核表达载体pET-28a(+)。转化感受态大肠杆菌BL21(DE3)细胞,诱导表达后观察重组蛋白的表达和ABCA1的连接。【结果】转化菌经IPTG诱导表达,SDS-PA     

Met662和Asp1828的Cys突变增强蛋白内含子对共表达的BDD-FVIII因子重链和轻链的剪接

本文旨在通过B区缺失型凝血因子8(BDD-FVⅢ)重、轻链间二硫键形成,改善蛋白质反式剪接效率,提高双载体转BDD FVⅢ基因功效. 将BDD-FVⅢ重链A2区的Met662和轻链A3区的Asp1828突变为Cys,用蛋白内含子融合的重链和轻链基因共转染HEK293细胞,Western印迹检测到细胞内BDD-FVⅢ剪接量的提高以及重、轻链间二硫键的形成,用ELISA和Coatest测得细胞分泌至培养上清的剪接BDD-FVⅢ的量(119±14 ng/mL)和活性(1.06±0.08 IU/mL),明显高于野生型BDD FVⅢ重链和轻链基因共转染细胞的量(81±12 ng/mL)和活性(0.70±0.15 IU/mL);混合培养的转突变重链和轻链基因细胞培养基中剪接BDD FVⅢ的量(17±5 ng/mL)和活性(0.15±0.03 IU/mL),与混合培养的转野生型重链和轻链基因细胞 (分别为21±9 ng/mL和0.18±0.05 IU/mL)相近,反映不依赖细胞机制的蛋白质反式剪接. 结果表明,重、轻链间二硫键形成通过增强蛋白质反式剪接提高双载体转BDD FVⅢ基因的功效. 为进一步运用双AAV载体动物体内转BDD-FVⅢ基因提供了实验依据.     

双载体转重链Tyr664Cys和轻链Thr1286Cys突变体BDD-FⅧ基因

用双载体转运凝血Ⅷ因子基因在甲型血友病基因治疗研究中可克服AAV毒载体容量限制,但存在重链分泌低效和链不均衡性问题。为探索重、轻链间二硫键形成对重链分泌的促进作用,该文用双载体转B结构域大部缺失型FⅧ(BDD-FVⅢ)的重链和轻链基因,将重链的Tyr664和轻链Thr1826突变为Cys,研究了HEK293细胞共转基因后的基因表达、分泌至培养上清的重链量和凝血生物活性。用Western blot检测细胞裂解液结果显示,非还原条件下有明显的二硫键交联的重、轻链蛋白;链特异性ELISA定量检测细胞分泌的重链为(125±29)ng/mL,明显高于共转野生型重链和轻链基因细胞的(75±23)ng/mL;Coatest法显示细胞分泌的凝血活性为(0.78±0.29)U/mL,也明显高于共转野生型重链和轻链基因细胞(0.34±0.12)U/mL。结果表明,重、轻链间的二硫键形成可提高双载体转FⅧ基因的功效,为进一步在动物体内转基因提供了实验依据。     

基于蛋白质反式剪接的双载体凝血Ⅷ因子基因转移

以intein的蛋白反式剪接为工具,研究了运用双载体的真核细胞凝血Ⅷ因子（FⅧ）基因转移,通过翻译后剪接得到完整的功能性FⅧ蛋白.将B结构域大部分缺失（Δ761~1639）的人功能性FⅧ（BDD-FⅧ）cDNA于剪接所需保守残基Ser1657前断裂为重链和轻链,分别与106和48个氨基酸的mini Ssp DnaB intein的N端（IntN）和C端（IntC）编码序列融合,构建一对在质粒pcDNA3.1的强启动子CMV驱动下的真核表达载体.用脂质体共转染至293细胞和COS-7细胞,培养48h后,收集细胞上清,用ELISA检测培养上清中剪接形成的BDD-FⅧ蛋白水平,用Coatest法检测上清的功能性FⅧ生物活性,并用Western blot观察细胞内的BDD-FⅧ蛋白质剪接.结果显示,两种细胞培养上清中有较高水平的剪接BDD-FⅧ蛋白形成,分别达到（137±23）和（109±22）ng/mL,由细胞内和细胞外（培养上清）的剪接共同组成 并检测到培养上清中较高水平的FⅧ生物活性,分别为（1.05±0.16）和（0.79±0.23）IU/mL,包括细胞内、外剪接产物BDD-FⅧ共同形成 细胞总蛋白的Western blot进一步显示共转染后细胞内高效剪接形成的BDD-FⅧ蛋白.表明intein可用于双载体系统真核细胞FⅧ基因转移,并不完全依赖细胞内的剪接产生具有高FⅧ生物活性的BDD-FⅧ蛋白,为进一步在甲型血友病基因治疗研究中应用双腺相关病毒载体（AAV）转运FⅧ基因,克服AAV载体的容量限制提供了依据.     

双载体转重链Tyr664Cys和轻链Thr1286Cys突变体BDD-FVⅢ基因

摘要用双载体转运凝血VⅢ因子基因在甲型血友病基因治疗研究中可克服AAV毒载体容量限制,但存在重链分泌低效和链不均衡性问题。为探索重、轻链间二硫键形成对重链分泌的促进作用,该丈用双载体转B结构域大部缺失型FVⅢ（BDD-FVⅢ）的重链和轻链基因,将重链的Tyr664和轻链Thr1826突变为Cys,研究了HEK293细胞共转基因后的基因表达、分泌至培养上清的重链量和凝血生物活性。用Western blot检测细胞裂解液结果显示,非还原条件下有明显的二硫键交联的重、轻链蛋白;链特异性ELISA定量检测细胞分泌的重链为（125＋29）ng／mL,明显高于共转野生型重链和轻链基因细胞的（75＋23）ng／mL;Coatest法显示细胞分泌的凝血活性为（0．784±0．29）U／mL．也明显高于共转野生型重链和轻链基因细胞（0．34＋0．12）U／mL。结果表明,重、轻链间的二硫键形成可提高双载体转FVⅢ基因的功效,为进一步在动物体内转基因提供了实验依据。     

酸性区3融合重链促进基于蛋白质内含子的双载体B域缺失型凝血因子Ⅷ转基因表达

最近的研究表明,蛋白质内含子(intein)介导的B区缺失型凝血因子Ⅷ (BDD-FⅧ)的轻链和重链剪接可顺式促进后者的分泌,而且剪接反应在细胞内、外均可发生．为进一步提高基于蛋白质内含子的双载体转BDD-FⅧ基因的功效,将具有促进重链分泌作用的位于Pro¹⁶⁴⁰～Ser¹⁶⁹⁰的酸性区3(acidic region-3,AR-3)引入重链,检验对蛋白质内含子剪接的BDD-FⅧ蛋白分泌和活性的影响．用融合蛋白内含子的附加ar-3重链(HCAR3IntN)基因和轻链(IntCLC)基因共转染培养的HEK293细胞,分别用ELISA和Coatest法定量分析分泌至培养上清中剪接BDD-FⅧ蛋白量和生物活性,并用免疫印迹观察了细胞内的BDD-FⅧ剪接．结果显示,共转HCAR3IntN和IntCLC基因细胞,分泌至上清的剪接BDD-FⅧ蛋白量和活性分别为(173±26) μg/L和(1.31±0.15) U/ml,明显高于未添加ar-3的蛋白质内含子融合重链(HCIntN)与轻链(IntCLC)基因共转染细胞[(102±12) μg/L和(0.79±0.09) U/ml],提示AR-3对蛋白质内含子剪接的BDD-FⅧ蛋白分泌和活性有明显改善作用．而且,分别转HCAR3IntN和IntCLC基因细胞混合培养后的上清中,亦检测到剪接的BDD-FⅧ蛋白和活性[(35±7) μg/L和(0.28±0.08) U/ml],表明蛋白质内含子可进行不依赖细胞机制的蛋白质剪接．另外,转基因细胞总蛋白呈现明显的可与FⅧ多克隆抗体进行反应的剪接BDD-FⅧ蛋白条带,直观地反映细胞内BDD-FⅧ的剪接．为动物模体内运用蛋白质反式剪接技术的双腺相关病毒载体(AAV)转BDD-FⅧ基因实验提供了依据．     

亮氨酸拉链提高小鼠血浆中剪接的凝血因子Ⅷ凝血活性

培养细胞实验表明,亮氨酸拉链通过改善内含肽(intein)的蛋白质剪接效率,提高双载体转B区缺失型凝血因子Ⅷ(BDD-FⅧ)基因细胞剪接FⅧ蛋白的分泌量和活性.本文从C57BL/6小鼠门静脉注射含亮氨酸拉链和Ssp DnaB内含肽融合的BDD-FⅧ的重链和轻链基因双表达载体,48 h后,检测到血浆的重链分泌量和FⅧ活性分别为(298±67)μg/L和(1.15±0.29)U/mL,明显高于不含亮氨酸拉链的双载体转BDD-FⅧ基因对照小鼠((179±59)μg/L和(0.58±0.19)U/mL).结果表明,亮氨酸拉链通过改善蛋白质反式剪接,提高基于蛋白质剪接的双载体转BDD-FⅧ基因小鼠血浆的凝血活性,为进一步双腺相关病毒(AAV)载体转BDD-FⅧ基因的甲型血友病基因治疗研究提供了依据.     

双Intein介导3片段vWF的反式剪接

von Willebrand因子(vWF)基因突变导致血管性血友病(VWD),由于其基因过大在基因治疗研究中难以为多数病毒载体携带.利用双内含肽(intein)的蛋白质反式剪接功能研究断裂成3段的vWF基因分别表达后在蛋白水平的连接,旨在为vWF基因的3载体联合转移应用于VWD基因治疗研究提供依据.将vWF cDNA于满足剪接所需的保守性氨基酸Cys1099、Ser2004的密码子前断裂为3段(N、M和C),分别与splitSspDnaE intein的N端(En)、C端(Ec)和splitSspDnaB intein的N端(Bn)、C端(Bc)编码序列融合,构建到原核表达载体pET-28a(+)中的His-Tag的下游,得到3种表达载体pET-NEn、pET-EcMBn和pET-BcC.分别转化感受态大肠杆菌BL21(DE3)细胞,经IPTG诱导表达后,以SDS-PAGE分析融合蛋白的表达,并进一步用His-Tag的特异性抗体进行分析;亲和层析纯化分别表达的带His-Tag标签的3段蛋白,复性后体外混合进行剪接实验以观察3片段vWF的连接.结果显示,3段预期大小的融合intein的vWF蛋白均有表达,用His-Tag抗体进行的Western印迹得到进一步证实;3段纯化的蛋白混合后可见明显的剪接条带形成,与vWF的预期分子量大小一致,表明双intein通过蛋白质反式剪接可有效连接3个片段的vWF,为进一步应用蛋白质剪接技术的3重载体真核细胞转vWF基因奠定了基础.     

内含肽介导三段vWF基因真核细胞转移的翻译后连接及其功能性多聚体形成

vWF(von Willebrand factor)是一种超大分子质量的血浆多聚体糖蛋白,在血栓形成和生理凝血过程中发挥重要作用,其质和/或量的缺陷导致血管性血友病(VWD),由于VWD为单基因病,且vWF为分泌性蛋白,基于基因转移的基因治疗无需特异的靶器官,因此VWD特别适合于基因治疗,但vWF基因过大(8.4 kb),难以为多数病毒载体特别是优点较多的腺相关病毒(AAV)载体承载.运用内含肽(intein)的蛋白质反式剪接功能,研究了三重载体真核细胞共转断裂3段的vWF基因,以期通过转基因翻译后的蛋白质剪接作用形成完整的功能性vWF蛋白.将vWF的cDNA于满足剪接所需的保守性氨基酸Cys1099、Ser2004的密码子前断裂为3段,分别与2种不同的内含肽即Ssp DnaE内含肽和Ssp DnaB内含肽编码序列融合,构建到真核表达载体pcDNA3.1(+),得到3个分别融合内含肽的vWF片段基因真核表达载体,共转染培养的293细胞,通过瞬时表达,电泳观察培养上清中的vWF多聚体形态,分析vWF蛋白量和凝血Ⅷ因子(FⅧ)结合力;通过共转FⅧ基因,分析了培养上清中的FⅧ蛋白量及生物活性.结果显示,通过内含肽的蛋白质反式剪接作用,共转内含肽融合的三片段vWF基因细胞上清,表现与正常人血浆和转vWF基因阳性对照细胞相似的vWF多聚体模式和FⅧ结合力,而且可明显提高转FⅧ基因后表达的FⅧ蛋白的分泌量和活性,提示剪接vWF蛋白的FⅧ载体功能的恢复.结果表明,内含肽可作为一种有效的技术手段进行三重载体共转断裂的vWF基因,为进一步基于内含肽的三重AAV转断裂vWF基因应用于VWD基因治疗研究、克服AAV的容量限制提供了依据.     

Protein <Emphasis Type="Italic">trans</Emphasis>-splicing based dual-vector delivery of the coagulation factor VIII gene

A dual-vector system was explored for the delivery of the coagulation factor VIII gene, using intein-mediated protein trans-splicing as a means to produce intact functional factor VIII post-translationally. A pair of eukaryotic expression vectors, expressing Ssp DnaB intein-fused heavy and light chain genes of B-domain deleted factor VIII (BDD-FVIII), was constructed. With transient co-transfection of the two vectors into 293 and COS-7 cells, the culture supernatants contained (137±23) and (109±22) ng mL⁻¹ spliced BDD-FVIII antigen with an activity of (1.05±0.16) and (0.79±0.23) IU mL⁻¹ for 293 and COS-7 cells, respectively. The spliced BDD-FVIII was also detected in supernatants from a mixture of cells transfected with inteinfused heavy and light chain genes. The spliced BDD-FVIII protein bands from cell lysates were visualized by Western blotting. The data demonstrated that intein could be used to transfer the split factor VIII gene and provided valuable information on factor VIII gene delivery by dual-adeno-associated virus in hemophilia A gene therapy.     

Blood coagulation factor VIII: An overview

Factor VIII (FVIII) functions as a co-factor in the blood coagulation cascade for the proteolytic activation of factor X by factor IXa. Deficiency of FVIII causes hemophilia A, the most commonly inherited bleeding disorder. This review highlights current knowledge on selected aspects of FVIII in which both the scientist and the clinician should be interested.     

Locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII (antihemophilic factor A).

The locations of disulfide bonds and free cysteines in the heavy and light chains of recombinant human factor VIII were determined by sequence analysis of fragments produced by chemical and enzymatic digestions. The A1 and A2 domains of the heavy chain and the A3 domain of the light chain contain one free cysteine and two disulfide bonds, whereas the C1 and C2 domains of the light chain have one disulfide bond and no free cysteine. The positions of these disulfide bonds are conserved in factor V and ceruloplasmin except that the second disulfide bond in the A3 domain is missing in both factor V and ceruloplasmin. The positions of the three free cysteines of factor VIII are the same as three of the four cysteines present in ceruloplasmin. However, the positions of the free cysteines in factor VIII and ceruloplasmin are not conserved in factor V.     

Purification of porcine plasma factor VIII using chromatographic methods

Factor VIII was purified from porcine plasma using adsorption on aluminium hydroxide with CM-cellulose as a filtration aid, cold ethanol precipitation, and two anion-exchange (Q-Sepharose fast flow) chromatographies. The final product was purified 264-fold and had a specific activity of 10 U mg^–1. The method is suitable to produce purified porcine FVIII by an easy process where all steps can be scaled up. The final product is free of von Willebrand factor that is responsible for the main side effects in patients. Finally, this method can be used to obtain purified porcine plasma FVIII for use in haemophilic patients with inhibitors.     

Binding of recombinant human coagulation factor VIII to lipid nanotubes

Cryo-electron microscopy has the power to visualise lipid membranes at the closest to in vivo conditions. The structure of the lipid bilayer can be well resolved and the interactions between lipid-protein and protein-protein molecules followed at the molecular level. We undertook an extended Cryo-electron microscopy study to follow the factor VIII binding to phosphatidylserine containing lipid nanotubes at different lipid composition. Obtaining well ordered tubes is required to define the factor VIII membrane-bound structure. The observed alterations in the arrangement of the protein molecules are indicative for the flexibility of the membrane-bound factor VIII. Understanding the significance of these conformational changes is essential to comprehend the function of factor VIII in coagulation and as a drug for Hemophilia A.     

Kinetic parameters of monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 on coagulation factor VIII and their influence on factor VIII activity

The murine monoclonal antibodies ESH2, ESH4, ESH5, and ESH8 specifically bind and inhibit the procoagulant activity of human coagulation factor VIII (FVIII). They are frequently used as a model of inhibitors which are raised against injected FVIII in about 25% of hemophiliacs as a serious side effect of substitution therapy. However, binding kinetics of the interaction of these antibodies with FVIII and their influence on FVIII activity (inhibition) have not yet been examined systematically. For this, we examined association and dissociation of protein:antibody interaction using surface plasmon resonance (SPR) and determined their ability to inhibit the FVIII activity in a one‐stage and a two‐stage assay. SPR‐analysis revealed that the equilibrium dissociation constants (K_D) of ESH8 and ESH4 are low and in a similar range (ESH8: K_D(ESH8) = 0.542 nM; ESH4: K_D(ESH4) = 0.761 nM). A 5.7 times higher K_D than for ESH4 was observed for ESH2 (4.33 nM), whereas ESH5 showed the highest K_D of 28.8 nM. In accordance with the lowest K_D, ESH8, and ESH4 reduced FVIII activity of normal human plasma almost completely in a one‐stage clot inhibition assay (ESH8: 91.9%; ESH4: 90.1%). However, ESH8 inhibited FVIII activity more efficiently as only 1.0 µg/ml ESH8 was sufficient to obtain maximum inhibition compared to up to 600 µg/ml of ESH4. Despite its attenuated K_D, ESH2 inhibits FVIII:C still efficiently, reducing 61.3% of FVIII activity at a concentration of 9 µg/ml in the one‐stage clotting assay. However, a discrepancy of inhibitory efficiency was found depending on the method used to measure FVIII activity. These effects seem to be mainly caused by differences of activation time of FVIII during both FVIII activity assays. The systematic assessment of these results should support FVIII interaction studies, and can provide data to rationally test peptides/mimotopes to remove or neutralize inhibitors of FVIII activity. Copyright © 2009 John Wiley & Sons, Ltd.     

TMD2前断裂CFTR翻译后的连接及氯离子通道功能

CFTR基因突变导致一种常染色体隐性遗传疾病——囊性纤维化(CF)。利用split Ssp DnaB intein的蛋白质反式剪接技术的真核细胞双载体转CFTR基因,旨在研究翻译后水平CFTR的连接,以及由其建立的氯离子通道功能。于CFTR膜内第2个跨膜结构域(TMD2)前的Glu838密码子后将其cDNA断裂为N端和C端两部分,与具有蛋白质反式剪接作用的split Ssp DnaB intein编码序列融合,分别插入到载体pEGFP-N1和pEYFP-N1,构建一对真核表达载体pEGFP-NInt和pEYFP-IntC。用脂质体将这对载体共转染至幼年仓鼠肾细胞(BHK),瞬时表达实验用Western blotting观察CFTR蛋白质的连接,并用膜片钳技术记录Cl-通道电流。结果显示,基因共转染细胞呈现完整的CFTR蛋白条带,膜片钳记录到全细胞Cl-电流和单个Cl-通道开放活性。结果表明split Ssp DnaB intein的蛋白质反式剪接技术可用于双载体共转移CFTR基因,为CF基因治疗应用双腺相关病毒载体(AAV)转运CFTR基因,克服AAV的容量限制提供了依据。     

Quantification of plasma-derived blood coagulation factor VIII by real-time biosensor measurements

Plasma-derived blood coagulation factor VIII was analyzed in real time using biosensor technology. Monoclonal antibodies directed against the heavy and against the light chain of factor VIII were immobilized on different carboxymethyl dextran surfaces. Different factor VIII concentrations were injected over the antibody surfaces in parallel and response levels were determined from the dissociation phase at a fixed time after sample injection. Serial dilutions of plasma-derived factor VIII with known concentrations determined by a commercial FVIIIC:Ag ELISA were used as standards. A quantification limit of 0.9 I.U./ml with antibody 530p and 1.5 I.U./ml with antibody 531p was calculated. Intra-assay precision expressed as percent coefficient of variation was below 10% for concentrations above 0.6 I.U./ml. Inter-assay precision for antibody 530p was below 20% for concentrations higher than 0.6 I.U./ml. For 531p, inter-assay precision was below 10% for concentrations higher than 2 I.U./ml. A sensor chip lifetime in respect to regeneration of at least 100 cycles for both antibodies was found. The small sample requirement of 35 μl allows fast analysis of different FVIII products and the use of two monoclonal antibodies directed against two different FVIII domains provides additional information about the integrity of the FVIII molecule.