鹦鹉热嗜衣原体蛋白酶样活性因子(CPAF)原核表达与纯化

应用分子生物学技术,选择鹦鹉热嗜衣原体(Chlamydophila psittaci,C.psittaci,Cps)6BC株的CPAF蛋白的免疫优势区基因,进行构建pGEX6p-2/CPAFm重组质粒与重组菌,使用IPTG诱导重组蛋白的表达并分析诱导温度、诱导剂剂量及诱导时间对蛋白表达的影响.重组蛋白以GST琼脂糖凝胶...     

CPP32 cDNA的克隆及其表达和活性检测

CPP32在细胞编程死亡调控中起重要的作用.利用CPP32专一性引物,用RT-PCR方法从人鼻咽癌CNE细胞中扩增出约830 bp的片段,经序列分析证明与已发表的CPP32序列完全一致.将扩增出的编码人全长CPP32的cDNA片段克隆入pGEX-2T中,转化大肠杆菌DH5α.转化菌经诱导表达出较高含量的GST-CPP32融合蛋白.进一步研究显示,细菌中表达的CPP32蛋白能自我切割,而且能裂解体外翻译的PARP,从而证明其具有生物活性.     

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。     

家蝇半胱氨酸蛋白酶抑制剂基因MdCPI的克隆、表达及活性检测

半胱氨酸蛋白酶抑制剂是具有抑制半胱氨酸蛋白酶活性的一类蛋白超家族。本研究根据EST序列信息,通过RACE技术克隆得到1条家蝇Musca domestica半胱氨酸蛋白酶抑制剂基因MdCPI,该基因含有1个357 bp开放阅读框,编码118个氨基酸残基,推导的多肽N端17个氨基酸残基为信号肽序列。同源分析表明,MdCPI 氨基酸序列与红尾肉蝇Sarcophaga crassipalpis的CPI相似性最高（identity=51%）。以邻接法（NJ）构建的系统树表明,家蝇与其他双翅目昆虫CPI起源于共同的祖先,属于I25A型蛋白家族。为了解家蝇CPI对半胱氨酸蛋白酶的抑制活性,构建pET-17b-MdCPI表达载体,并转入大肠杆菌Escherichia coli BL21（DE3）进行重组表达。研究发现1 μg重组家蝇CPI能够抑制约14 μg木瓜蛋白酶的水解活性。结果表明MdCPI确属CPI家族,可能同其他家族成员具有相似的功能,参与免疫及生理调控。这些结果为研究MdCPI在家蝇体内作用机制奠定了基础。     

vWF和vWF-CP在紫癜性肾炎患者中的表达及意义

已知血管性血友病因子(vWF)参与介导血小板对血管和微血管的粘附作用,并且vWF在慢性肾病患者血浆中的水平明显升高。为了揭示vWF和血管性血友病因子裂解酶(vWF-CP)在紫癜性肾炎患者血浆中的表达,以及vWF和vWF-CP在发病机制中的作用。本研究采用酶联免疫吸附试验(ELISA)和残余胶原蛋白结合试验分别检测紫癜性肾炎患者(HSPN组, n=62)、微小病变性肾小球病患者(MCN组, n=16)和健康体检者(对照组, n=10)血浆vWF水平和vWF-CP活性。应用免疫组织化学法检测肾组织中vWF和vWF-CP的表达。另外,应用ELISA检测了紫癜性肾炎患者血浆肿瘤坏死因子-α(TNF-α)水平,并分析了各指标间的相关性。研究结果显示,HSPN组和MCN组的vWF水平均显著高于对照组,而vWF-CP活性均显著低于对照组。随着ISKDC分级的升高,紫癜性肾炎患者的vWF水平逐渐升高。而vWF-CP活性则随着ISKDC分级的升高而显著降低。大量蛋白尿组(≥2.0 g/24 h)的vWF水平((183.4±27.37)%)显著高于非大量蛋白尿组(2.0 g/24 h,(132.64±19.79)%),而大量蛋白尿组的vWF-CP活性((44.47±6.63)%)显著低于非大量蛋白尿组((74.86±11.17)%)。HSPN组和MCN组的TNF-α水平均显著高于对照组。在紫癜性肾炎患者和微小病变性肾小球病患者中,vWF的阳性表达率显著高于健康对照组(p0.05),而vWF-CP显著低于健康对照组(p0.05)。紫癜性肾炎患者的vWF水平与TNF-α水平显著正相关(r=0.452, p=0.010),而vWF-CP活性与TNF-α水平显著负相关(r=-0.496, p=0.006)。本研究初步结论表明vWF和vWF-CP参与紫癜性肾炎的发病机制,并且与病情严重程度密切相关,且vWF和vWF-CP的合成和分泌可能受到TNF-α等促炎症因子的调节。     

重组人丝氨酸蛋白酶抑制因子Hespintor Kazal结构域的原核表达、纯化及活性鉴定

Hespintor是应用抑制消减杂交技术 (SSH) 从肝母细胞瘤细胞系HepG2中筛选得到的一未知功能蛋白,序列分析表明该蛋白属于Kazal型丝氨酸蛋白酶抑制因子 (Serine proteinase inhibitor, Serpin) 家族中的一个分泌型新成员,具有与食管癌相关基因2 (Esophageal cancer related gene 2, ECRG2) 高度同源的Serpin基本结构。为了进一步阐明Hespintor的生物学功能,必须得到纯化的Hespintor蛋白。先将Hespintor Kazal结构域编码序列亚克隆至原核表达载体pET-40b(+),转化至Rosetta (DE3) 表达宿主菌中。经 0.25 mmol/L IPTG,30 ℃诱导5 h获得了分子量约为42 kDa的Hespintor-Kazal重组融合蛋白的优化表达,Western blotting证实了重组蛋白的特异性。Hespintor-Kazal重组融合蛋白以包涵体形式在宿主菌中表达,利用金属螯合亲和层析和阴离子交换层析柱对重组蛋白进行两步纯化。初步的活性鉴定表明,纯化的Hespintor-Kazal重组融合蛋白能特异性抑制胰蛋白酶的水解活性,提示Hespintor具有作为一种新型抗肿瘤药物的潜在开发价值。     

甲硫氨酸γ-裂解酶基因在大肠杆菌中的高效表达和活性

【背景】为了开发海洋蕴藏的新型微生物资源,本研究团队采用不依赖培养的宏基因组技术,构建了深海宏基因组文库,并对其中的重要基因进行后续研究。【目的】使用来自深海宏基因组文库中的甲硫氨酸γ-裂解酶基因(mgl)在大肠杆菌中高效表达并对其活性进行检测。【方法】将mgl基因克隆到表达载体pET-28a(+)并转化大肠杆菌BL21(DE3),经异丙基-β-D-硫代半乳糖苷(IPTG)诱导,并对表达条件进行优化,获得甲硫氨酸γ-裂解酶(Methionine-lyase,r MGL)的大量表达。亲和层析纯化重组蛋白后对酶的活性进行研究。【结果】亲和纯化后获得大量表达蛋白r MGL,大小与预测的46 kD相符合,并具有很高的裂解L-甲硫氨酸的活性。r MGL能催化L-甲硫氨酸和DL-同型半胱氨酸的裂解,但几乎不作用于L-半胱氨酸和L-胱氨酸,其中对DL-同型半胱氨酸的催化效率比对L-甲硫氨酸的催化效率高,相对活性约为对L-甲硫氨酸催化效率的1.4倍。【结论】来自深海宏基因组文库中的mgl基因能够利用p ET-28a(+)/BL21(DE3)高效表达r MGL。     

小RNA病毒3C蛋白酶及其裂解底物

小RNA病毒科是一类大的动物病毒科,小RNA病毒蛋白的合成需要自身蛋白酶裂解形成多个结构蛋白和功能蛋白,3C蛋白酶是一些小RNA病毒的自身蛋白水解酶之一,3C蛋白酶还可以裂解一些宿主的蛋白,利于病毒的复制。3C蛋白酶结构特点、活性中心、酶切位点如何,裂解的底物功能怎样,是进一步了解3C蛋白酶作用机制的关键。就这些问题进行综述。     

可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白的真核表达及生物学活性检测

本研究设计和构建了一种人肿瘤坏死因子受体II胞外区与人脂联素球部的融合基因sTNFRII-gAD,且相应的融合蛋白在哺乳动物细胞BHK-21S的无血清培养体系中实现了表达,并对该融合蛋白进行了初步鉴定。首先,用RT-PCR方法从人的外周血淋巴细胞总RNA中扩增人肿瘤坏死因子II型受体胞外区基因片段,与脂联素球部基因片段融合,克隆至pAAV2neo表达载体中,构建成pAAV2neo-sTNFRII-gAD。随后,用pAAV2neo-sTNFRII-gAD转染BHK-21S细胞获得G418抗性细胞BHK-21S/pAAV2neo-sTNFRII-gAD;然后,将原来含有血清的培养液换成无血清的化学成分限定的培养液,细胞从贴壁培养方式转换成悬浮培养方式;最后,收集BHK-21S/pAAV2neo-sTNFRII-gAD无血清悬浮培养24h后的培养上清,进行sTNFRII-gAD融合蛋白的鉴定分析。酶切鉴定和测序结果显示,所构建的pAAV2neo-sTNFRII-gAD质粒结构正确,sTNFRII-gAD序列与预期一致;分别用抗人肿瘤坏死因子受体II和抗人脂联素球部的单克隆抗体检测pAAV2neo-sTNFRII-gAD瞬时转染的BHK-21S细胞,免疫荧光呈现阳性;免疫印迹分析在pAAV2neo-sTNFRII-gAD稳定转染的BHK-21S细胞上清中检测到sTNFRII-gAD融合蛋白的表达,并以单体、三聚体和三聚体以上的多聚体形式存在。活性测定结果表明,sTNFRII-gAD融合蛋白具有显著抑制TNFα杀伤L929细胞的活性。因此,本研究为下一步大量制备sTNFRII-gAD融合蛋白用于体内外功能研究提供了良好基础。     

ATP依赖的人Lon蛋白酶的表达、纯化及其活性鉴定

ATP依赖的人Lon蛋白酶是一种同质寡聚、环状的蛋白酶,主要位于细胞线粒体基质中。许多研究表明,Lon蛋白酶对于维护细胞的内环境稳定起着重要作用,并参与线粒体蛋白质量控制和代谢调控。将pPROEX1 His6-Lon重组质粒在Escherichia coli Rosetta 2菌株中诱导表达用Ni2＋柱亲和层析法纯化,获得纯度较高的目的蛋白。经纯化后,Lon蛋白酶的比酶活达到0.17 U/mg。通过多肽底物Rhodamine 110、bis-（CBZ-L-alanyl-L-alanine amide）[（Z-AA）2 Rh110]的降解检测显示,Lon蛋白酶具有肽酶活性,并被ATP所刺激。Casein和线粒体转录因子A降解实验表明,纯化的Lon蛋白酶具有蛋白水解活性,而且蛋白水解活性依赖于ATP。     

Establishing reference intervals for von Willebrand factor multimers

Backgroundvon Willebrand factor (VWF) multimers (VWF:MM) methodologies are technically difficult, laborious, time consuming, non-standardized and results vary between laboratories. A new semi automated VWF:MM assay is available for routine use (Sebia). Due to lack of reference values for VWF:MM fractions, results interpretation can be challenging in some cases. The aim of this study was to determine reference intervals for low molecular weight (LMWM), intermediate molecular weight (IMWM) and high molecular weight (HMWM) multimers.MethodsBy the international cooperation initiated between 4 countries (Estonia, Latvia, France, and USA) 131 samples of relatively healthy individuals were analyzed for VWF:MM (in total 51 males and 80 non-pregnant females aged 17-69 years). Reference intervals were calculated according to CLSI C28-A3 standard.ResultsThe proposed reference intervals for VWF:MM were calculated for LMWM 10.4-22.5%, IMWM 22.6-37.6%, HMWM 45.6-66.6%. Age related differences were seen in IMWM and HMWM (p<0.001 and 0.038). There was no gender related difference observed. Geographically LMWM results of France were different from the other regions (p<0.05).ConclusionsQuantification of VWF:MM fractions, in addition to qualitative assessment of VWF:MM patterns, has the potential to aid in differential diagnosis of von Willebrand disease (VWD) subtypes. The reference values calculated in this study can be used in future research to establish clinical decision limits.     

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.     

Evaluation of a new semi-automated Hydragel 11 von Willebrand factor multimers assay kit for routine use

BackgroundAccurate diagnosis and classification of von Willebrand disease (VWD) are essential for optimal management. The von Willebrand factor multimers analysis (VWF:MM) is an integral part of the diagnostic process in the phenotypic classification, especially in discrepant cases. The aim of this study was to evaluate the performance of a new Hydragel 11VWF multimer assay (H11VW).MethodsAnalytical performance characteristics such as repeatability (intra-assay variability, in gel between track variation), reproducibility (inter-assay variability, between gel variation), sensitivity, EQA performance and differences between two commercially available VWF:MM kits (H5VW and H11VW) were analysed in healthy volunteers'' plasmas using in-house prepared reference plasma.ResultsRepeatability and reproducibility results of H11VW demonstrated acceptable and equivalent performance with previously verified H5VW. Participation in EQA was successful. No statistically significant difference was detected between H5VW and H11VW kits for different fractions of multimers: LMWM p=0.807; IMWM p=0.183; HMWM p=0.774.ConclusionsH11VW demonstrated acceptable analytical performance characteristics. H11VW kit conveniently offers a more significant number of samples on a single gel. H5VW and H11VW kits can be used in daily practice interchangeably.     

人vWF-A1多肽的融合表达及生物学活性鉴定

血管性血友病因子 (vWF)通过与血小板膜糖蛋白结合介导血小板的粘附和聚集 ,在血栓形成过程中发挥重要作用 .通过阻断血小板与vWF的结合可抑制血栓形成 .应用RT PCR方法从人脐带内皮细胞中克隆vWF A1区基因并在原核细胞内进行表达 ,经过纯化、复性 ,获得重组蛋白(rvWF A1) .用流式细胞术检测rvWF A1与转染了糖蛋白Ib(GPⅠb)的CHO K1细胞和血小板GPⅠb的结合能力 ,血小板聚集仪测定rvWF A1对瑞斯托霉素 (ristocetin)诱导的血小板聚集作用的影响 .重组表达载体pET 2 0b(+ ) vWF A1在大肠杆菌BL2 1(DE3)plus中得到有效表达 ,表达的重组蛋白量占菌体总蛋白 30 % .次氮基三乙酸镍琼脂糖 (Ni NTAagarose)柱纯化后 ,其纯度为 95 % .经复性的rvWF A1蛋白具有良好的生物学活性 ,它可与转染了GPⅠb的CHO K1细胞和血小板结合 ,阳性率分别为 96 90 %与 78 6 0 % ,且可以抑制ristocetin诱导的血小板聚集 ,其抑制效应呈剂量依赖性 .IC50 的rvWF A1浓度为 0 5 6 μmol L ,当浓度为 1 4 μmol L时抑制率最高达 84 70 % .结果表明 ,在原核细胞中表达人rvWF A1区蛋白可抑制血浆中野生型vWF与血小板的结合 ,具有抗血栓形成的潜在应用前景     

Structure determination of the major asparagine-linked sugar chain of human factor VIII--von Willebrand factor

N-glycosidically-linked glycans released by hydrazinolysis of human factor VIII/von Willebrand factor (FVIII/vWf) were separated by high-voltage electrophoresis. Five fractions were obtained, one of them representing 60% of the total amount of the N-glycosidically-linked glycans of FVIII/vWf. On the basis of the carbohydrate composition, methylation analysis and 500 MHz 1H-NMR spectroscopy, we describe the primary structure of this major glycan which is of the monosialylated and monofucosylated biantennary N-acetyllactosaminic type.     

Molecular modeling of the von Willebrand factor A2 Domain and the effects of associated type 2A von Willebrand disease mutations

A homology model for the A2 domain of von Willebrand factor (VWF) is presented. A large number of target–template alignments were combined into a consensus alignment and used for constructing the model from the structures of six template proteins. Molecular dynamics simulation was used to study the structural and dynamic effects of eight mutations introduced into the model, all associated with type 2A von Willebrand disease. It was found that the group I mutations G1505R, L1540P and S1506L cause significant deviations over multiple regions of the protein, coupled to significant thermal fluctuations for G1505R and L1540P. This suggests that protein instability may be responsible for their intracellular retention. The group II mutations R1597W, E1638K and G1505E caused single loop displacements near the physiologic VWF proteolysis site between Y1605–M1606. These modest structural changes may affect interactions between VWF and the ADAMTS13 protease. The group II mutations I1628T and L1503Q caused no significant structural change in the protein, suggesting that inclusion of the protease in this model is necessary for understanding their effect.Figure Homology model of the von Willebrand factor A2 domainElectronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00894-004-0194-9     

An engineered EBV vector expressing human factor VIII and von Willebrand factor in cultured B-cells

BACKGROUND: Hemophilia A is a congenital disorder caused by a deficiency of the blood-clotting factor VIII (FVIII) and is an attractive candidate for gene therapy. Most of the studies have only explored the potential of hepatocytes and muscle cells as the targets for gene transfer. Attempts to transfer the genes into hematopoietic cells have so far been mostly unsuccessful due to inefficiency of most viral vectors to transduce these cells and the supposed inability of the cells to express FVIII. METHODS: We demonstrate the generation of an engineered Epstein-Barr virus (EBV) vector with a BAC backbone that has the unique capacity to carry either the full-length FVIII cDNA or its B-domain-deleted form; a modified version of the vector that carries B-domain-deleted FVIII along with the von Willebrand factor (vWF) cDNA or the reporter gene DsRed2 was also used. All these vectors have been safety modified with viral thymidine kinase cDNA to transduce human B-cells in culture. RESULTS: Low-level expression of FVIII in the order of 5-8 ng FVIIIC/ml were observed in the cells stably transduced with full-length FVIII, while cells with the B-domain-deleted version expressed 8-16 ng FVIIIC/ml. Expression of vWF and B-domain-deleted FVIII resulted in a moderate expression of 18-30 ng FVIIIC/ml. Long-term expression for 12-16 weeks was observed in these cells regardless of selection pressure. CONCLUSIONS: These results support the development of an episomal engineered EBV vector for treatment of hemophilia A using the hematopoietic cells as a target for providing immediate secretion of functionally active product in the circulating bloodstream.     

Effects of Pro1266Leu mutation on structure and function of glycoprotein Ib binding domain of von Willebrand factor

Glycoprotein Ibα (GpIbα) binding ability of A1 domain of von Willebrand factor (vWF) facilitates platelet adhesion that plays a crucial role in maintaining hemostasis and thrombosis at the site of vascular damage. There are both “loss as well as gain of function” mutations observed in this domain. Naturally occurring “gain of function” mutations leave self-activating impacts on the A1 domain which turns the normal binding to characteristic constitutive binding with GPIbα. These “gain of function” mutations are associated with the von Willebrand disease type 2B. In recent years, studies focused on understanding the mechanism and conformational patterns attached to these phenomena have been conducted, but the conformational pathways leading to such binding patterns are poorly understood as of now. To obtain a microscopic picture of such events for the better understanding of pathways, we used molecular dynamics (MD) simulations along with principal component analysis and normal mode analysis to study the effects of Pro1266Leu (Pro503Leu in structural context) mutation on the structure and function of A1 domain of vWF. MD simulations have provided atomic-level details of intermolecular motions as a function of time to understand the dynamic behavior of A1 domain of vWF. Comparative analysis of the trajectories obtained from MD simulations of both the wild type and Pro503Leu mutant suggesting appreciable conformational changes in the structure of mutant which might provide a basis for assuming the “gain of function” effects of these mutations on the A1 domain of vWF, resulting in the constitutive binding with GpIbα.     

Differential localization of P-selectin and von Willebrand factor during megakaryocyte maturation

Abstract

An important step in megakaryocyte maturation is the appropriate assembly of at least two distinct subsets of α-granules. The mechanism that sorts the α-granule components into distinct structures and mediates their release in response to specific stimuli is now emerging. P-selectin and von Willebrand factor are two proteins present in the α-granules that recognize P-selectin glycoprotein ligand on neutrophils and collagen in the subendothelial matrix. These proteins may play an important role in determining the differential release of the α-granule contents in response to external stimuli. If P-selectin and von Willebrand factor are localized in the same or different α-granules is not known. To clarify this question, we analyzed by immunoelectron microscopy the localization of von Willebrand factor and P-selectin during the maturation of wild-type and Gata1^low megakaryocytes induced in vivo by treating animals with thrombopoietin. Gata1^low is a hypomorphic mutation that blocks megakaryocyte maturation, reduces the levels of von Willebrand factor expression and displaces P-selectin on the demarcation membrane system. The maturation block induced by this mutation is partially rescued by treatment in vivo with thrombopoietin. In immature megakaryocytes, both wild-type and Gata1^low, the two receptors were co-localized in the same cytoplasmic structures. By contrast, the two proteins were segregated to separate α-granule subsets as the megakaryocytes matured. These observations support the hypothesis that P-selectin and von Willebrand factor may ensure differential release of the α-granule content in response to external stimuli.