人乳头瘤病毒18型病毒样颗粒在大肠杆菌中的表达及免疫原性分析

利用大肠杆菌表达系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,经过纯化和重组装过程获得HPV18病毒样颗粒(VLPs),研究其免疫原性和诱发中和抗体生成的水平。首先,提取HPV18的基因组DNA,通过PCR扩增获得HPV18 L1基因片段,将其插入pTrxFus表达载体,在大肠杆菌中可溶性表达HPV18 L1蛋白;其次,通过硫酸铵沉淀、离子交换层析和疏水相互作用层析获得高纯度的HPV18 L1蛋白,而后透析去除预先加入的还原剂DTT,使HPV18 L1蛋白自发组装成VLPs;最后,通过动态光散射技术和透射电子显微镜鉴定HPV18 VLPs的大小和形态,利用假病毒细胞中和实验评价HPV18 VLPs在实验动物体内的免疫原性和中和抗体生成水平。结果表明,HPV18L1蛋白可以在大肠杆菌表达系统中以可溶形式表达,经过纯化的HPV18 L1蛋白可以自发组装成为半径约为29.34nm、与HPV病毒外观相似的VLP。该VLPs在小鼠体内的中和抗体半数有效剂量为0.006μg,在兔及山羊体内诱导中和抗体滴度高达107。总之,本研究利用原核表达系统可简便高效地获得具有高度免疫原性的HPV18 VLPs,为HPV18...     

易发生聚集的重组HBcAg病毒样颗粒的纯化*

目的:探索针对易发生聚集的重组HBcAg病毒样颗粒(VLP)的有效纯化方案。方法:培养的大肠杆菌经IPTG诱导重组HBcAg蛋白的表达,菌体超声破碎后的离心沉淀用含有不同浓度尿素的PBS缓冲液重悬溶解,经密度梯度离心并结合电镜观察对VLPs行为进行分析鉴定。以Sepharose 4 FF凝胶过滤层析在选定的尿素条件下纯化沉淀溶解液,纯化获得的目的蛋白进一步在含30%山梨醇的PBS中脱盐去除尿素。整个过程以SDS-PAGE及电镜进行各步骤样品中目的蛋白的分析。结果:含有1mol/L尿素的PBS缓冲溶液重悬超声沉淀,可有效溶解聚集的VLPs,在蔗糖密度梯度离心中显示典型HBcAg VLPs的行为,且电镜观察颗粒形态结构完整。经1mol/L尿素下凝胶过滤,VLPs进一步获得纯化。在脱尿素过程中流动相采用含30%山梨醇的PBS,有效避免了VLPs在尿素去除后重新聚集。结论:尿素与山梨醇的联合应用,为具有聚集现象的VLPs纯化制备提供了一种有效解决方案。     

人乳头瘤病毒18型L1蛋白在大肠埃希菌中的可溶性表达及病毒样颗粒的形成

目的利用大肠埃希菌系统可溶性表达人乳头瘤病毒18型(HPV18)L1蛋白,纯化和重组装获得HPV18病毒样颗粒(VLPs),为进一步研制HPV18基因工程疫苗奠定基础。方法首先按大肠埃希菌密码子偏好进行HPV18L1全基因合成,经PCR扩增出截短的HPV18L1基因,构建重组表达载体PET30a-L1,通过优化表达在大肠埃希菌BL21中可溶性表达L1蛋白,其次采用硫酸铵沉淀、离子交换层析、疏水层析后,获得高纯度的的L1蛋白,再通过解聚和重聚获得VLPs。结果全基因优化并截短的HPV18L1蛋白在大肠埃希菌系统中以可溶形式表达,纯化后的蛋白纯度达到90%以上,电镜下观察到直径为60 nm的VLPs颗粒。结论利用大肠埃希菌系统可溶性表达非融合HPV18L1蛋白,并获得均一的VLPs颗粒,为疫苗的开发奠定基础。     

衣壳修饰对病毒样颗粒稳定性及其免疫原性的影响

全球现有疫苗种类中，病毒样颗粒（Virus-like particles, VLPs）疫苗以其安全性和高效性而被广泛关注。VLPs是一种不含核酸的病毒蛋白衣壳，目前可根据不同需求设计目标衣壳蛋白序列并在不同的表达系统中构建生产。在疫苗设计环节，对VLPs衣壳蛋白进行合理设计和修饰，可大大增强VLPs的稳定性，尤其是提高衣壳组装过程及组装体的稳定性，间接影响VLPs的免疫原性，对提升VLPs疫苗的有效性和安全性有重要作用。近年来国内外研究表明影响VLPs衣壳组装过程的稳定性的基本因素是组装环境中的离子强度和pH值，而影响VLPs组装体稳定性及其免疫原性的主要因素有疏水性质和静电相互作用及糖基化水平等。疏水性对VLPs蛋白质结构的动态平衡和生物功能的发挥起关键作用，静电相互作用通过改变VLPs分子表面或内部的静电势能，影响VLPs衣壳内分子共价键的形成及其与外界环境的相互作用，糖基化修饰则利用自身修饰基团的生物学结构或其他功能性质影响整体VLPs颗粒的结构稳定性和免疫学功能。本文根据现阶段对VLPs的生产应用和结构的研究，综述了影响VLPs衣壳组装过程和组装体稳定性的主要因素，以及VLPs...     

人乳头瘤病毒52型病毒样颗粒制备及其生物活性

[目的]利用大肠杆菌系统表达人乳头瘤病毒52型(HPV52) L1蛋白,并获得HPV52病毒样颗粒(VLPs)。[方法]优化并合成HPV52L1基因,构建p ET-30a-52L1表达载体粒,转化大肠杆菌E. coli BL21 StarTM(DE3)。经IPTG诱导表达,阳离子交换层析纯化,SDS-PAGE和Western Blotting鉴定,BALB/c小鼠免疫2次,初次后4w检测免疫血清中HPV52中和抗体滴度。[结果]获得纯度90%的HPV52L1蛋白,将获得的HPV52L1蛋白进行解组装和重组装形成VLPs,动态光散射观察到粒径约为70nm,透射电镜观察到50～60nm的均一VLPs,中和抗体滴度达25600。[结论]大肠杆菌系统表达人乳头瘤病毒52型(HPV52) L1蛋白,并经过一步柱层析纯化获得纯度达90%的L1蛋白,经解组装重组装获得粒径大小为50～60nm的VLPs,具有良好免疫原性。     

口蹄疫病毒氨基酸改造对其病毒样颗粒稳定性的影响

病毒样颗粒 (Virus like particles,VLPs) 的稳定性是目前影响口蹄疫VLPs疫苗质量的主要因素。为进一步提升口蹄疫VLPs疫苗的质量,基于口蹄疫病毒三维空间结构,通过动力学分析软件设计并筛选出3个氨基酸改造位点。经点突变试剂盒成功制备出上述3种突变型重组质粒,转化大肠杆菌Escherichia coli BL21菌株后经体外诱导表达,Ni离子层析柱纯化后,SDS-PAGE结果证明3种氨基酸突变不影响目标蛋白的表达。体外组装获得的3种突变型VLPs稳定性研究结果发现,内部疏水性侧链氨基酸的引入使VLPs的形态变得更加均一 (N4017W),且其稳定性比其他两种VLPs明显提高。结果表明,衣壳内部疏水性作用力有助于VLPs的形成且有助于维持衣壳的稳定性,为提高VLPs疫苗质量提供了新的研究思路,有助于推进VLPs疫苗的发展。     

大肠杆菌来源的人乳头瘤病毒11型病毒样颗粒的制备及其免疫原性

摘要：【目的】 利用大肠杆菌表达系统制备人乳头瘤病毒11型病毒样颗粒（HPV11 VLPs）,并对其免疫原性和所诱导中和抗体的型交叉反应性进行研究。 【方法】 在大肠杆菌ER2566中非融合表达HPV11-L1蛋白,并通过离子交换层析,疏水相互作用层析其进行纯化。纯化后的HPV11-L1经体外组装形成病毒样颗粒,通过动态光散射,透射电镜检测其形态,并通过多种HPV型别假病毒中和实验评价HPV11 VLPs的免疫原性及型交叉反应性。 【结果】 HPV11-L1蛋白在大肠杆菌中可以以可溶形式表达。经过硫酸铵沉     

条斑紫菜蛋白酶解产物中α-葡萄糖苷酶抑制剂纯化及特性

以α-葡萄糖苷酶抑制活性为指标,优选出1398中性蛋白酶、碱性蛋白酶和氨肽酶在一定条件下复配酶解条斑紫菜蛋白制备α-葡萄糖苷酶抑制剂。酶解液加入乙醇至终浓度为60%以沉淀去除多糖,上清液为α-葡萄糖苷酶抑制剂粗品。该粗品利用SP Sepharose High Performance阳离子交换层析、Sephadex G-10凝胶层析和Mono Q阴离子交换层析进行分离纯化,获得一种肽类α-葡萄糖苷酶抑制剂(LGI)。LGI经反向高效液相层析测定纯度为62.4%,基本特性分析显示其具较好的温度和pH稳定性,对α-葡萄糖苷酶半抑制浓度IC50值为97.36μg/mL,属于一种非竞争性抑制剂。     

乙肝核心抗原病毒样颗粒呈现HPV 16L1抗原表位及特异抗体诱导

目的:构建呈现HPV 16L1抗原表位的病毒样颗粒(VLPs),为新型HPV疫苗及特异抗体制备提供新的思路。方法:将编码HPV 16L1抗原表位QPLGVGISGHPLLNKLDDTE寡聚核苷酸片段克隆于HBc Ag基因编码第78、79位氨基酸序列之间,重组质粒转化大肠杆菌DH5α。重组蛋白经IPTG诱导后以SDS-PAGE分析表达情况,并以Western blot鉴定重组蛋白中HPV 16L1表位的免疫反应性。菌体超声破碎后经硫酸铵盐析法和蔗糖密度梯度离心进行纯化,并经凝胶层析Sepharose G25脱盐,最后以电子显微镜及高效液相(HPLC)凝胶过滤色谱鉴定VLPs的存在并分析纯度。纯化的病毒样颗粒分别于0周、2周、4周经皮下注射免疫BALB/c小鼠,以Western blot分析血清特异识别L1蛋白的能力。结果:HBc Ag/L1肽嵌合蛋白获得成功表达,能够被商业化L1抗体特异识别,经密度梯度超速离心及HPLC分析显示其与HBc Ag行为一致,电子显微镜观察进一步确定其以HBc Ag病毒样颗粒形式存在。VLPs免疫小鼠获得的抗血清能够特异识别酵母表达的重组L1蛋白。结论:HBc Ag VLPs成功呈现HPV16L1蛋白并有效激发特异抗体应答。     

疏水相互作用层析分离重组人干扰素α2b

目的采用疏水相互作用层析分离重组人干扰素α2b,去除干扰素样品中的二聚体,得到高纯度的干扰素用于进一步的研究。方法首先采用阳离子交换层析纯化复性重组人干扰素α2b,去除了大部分的杂蛋白,然后采用疏水相互作用层析纯化重组人干扰素α2b,去除复性过程中产生的错误折叠体和二聚体,并考察盐浓度、pH值、流速和洗脱液中尿素对疏水相互作用层析纯化效果的影响。结果硫酸铵初始浓度1.2 mol/L、缓冲液pH值6.0、流速2.5 mL/min、洗脱液中添加尿素浓度为2 mol/L时疏水相互作用层析纯化效果最佳。最终得到的重组人干扰素α2b非还原型SDS-PAGE电泳均呈单一条带。结论确定了疏水层析纯化重组人干扰素α2b的最优条件,成功提取到具有高活性、高纯度的重组人干扰素α2b纯品。     

一种安全高效的血红蛋白纯化方法

目的:建立一种适用于大量制备的,安全、高效的血红蛋白纯化方法。方法: 将压积红细胞装入透析袋,以含有还原剂的Tris缓冲液透析破碎,破碎的上清经两级硫酸铵沉淀后透析至上样缓冲体系,离心后取上清即得血红蛋白提取液;红细胞提取液通过阴离子交换柱层析进一步分离,计算回收率。纯化产物浓缩后以SDS-PAGE及HPLC鉴定纯度,进行紫外-可见光谱扫描并以ABL800血气分析仪分析血气指标,以鲎试剂测定内毒素含量,以磷测定法测定脂质含量。结果: 血红蛋白提取液中脂质去除率98%,容易通过0.45μm滤膜;经阴离子交换层析纯化的血红蛋白经SDS-PAGE(银染法)及WB分析没有杂蛋白条带,HPLC分析纯度>99%、总回收率>85%;内毒素含量<2 EU,高铁血红蛋白含量<5%。结论: 该血红蛋白纯化方法安全高效、成本低廉、易于放大生产,具有较好的应用前景。     

低分子量神经丝蛋白（NF—L）的体外组装

Neurofilaments were isolated from bovine spinal cords by ultra-speed centrifugation and examined by negative staining. The neurofilament triplet proteins: NF-L, NF-M and NF-H were purified by DE-52 anion exchange chromatography in the presence of 6 mol/L urea. The reassembly of NF-L under controlled conditions was studied. NF-L can reassemble into 10 nm width filaments within 60 minutes at physiological condition of around 0.15 mol/L NaCl, 2 mmol/L MgCl2, neutral pH(pH 6.8) and 37 degrees C. In 6 mol/L urea, NF-L was examined as 12 nm-diameter particle by low angle rotary shadowing. When dialyzed against reassembly buffer for 20 minutes, some irregular filaments were formed. Further dialyzed for another 40 minutes, the long smooth filaments appeared. Some filaments were unraveled at the end regions, where existed 2-4 subfilaments. Four subfilaments were more often observed. That is to say, the 10 nm-width filament was composed of 4 subfilaments. While dialyzed against the alkaline buffer containing 0.15 mol/L NaCl, NF-L reconstituted into 45-180 nm-long, 10 nm-width filaments, which were not able to elongate into long filaments.     

Quantitative Disassembly and Reassembly of Human Papillomavirus Type 11 Viruslike Particles In Vitro

The human papillomavirus (HPV) capsid is primarily composed of a structural protein denoted L1, which forms both pentameric capsomeres and capsids composed of 72 capsomeres. The L1 protein alone is capable of self-assembly in vivo into capsidlike structures referred to as viruslike particles (VLPs). We have determined conditions for the quantitative disassembly of purified HPV-11 L1 VLPs to the level of capsomeres, demonstrating that disulfide bonds alone are essential to maintaining long-term HPV-11 L1 VLP structure at physiological ionic strength. The ionic strength of the disassembly reaction was also important, as increased NaCl concentrations inhibited disassembly. Conversely, chelation of cations had no effect on disassembly. Quantitative reassembly to a homogeneous population of 55-nm, 150S VLPs was reliably achieved by the re-formation of disulfide linkages following removal of reducing agent at near-neutral pH and moderate NaCl concentration. HPV-11 L1 VLPs could also be dissociated by treatment with carbonate buffer at pH 9.6, but VLPs could not be regenerated following carbonate treatment. When probed with conformationally sensitive and/or neutralizing monoclonal antibodies, both capsomeres generated by disulfide reduction of purified VLPs and reassembled VLPs formed from capsomeres upon removal of reducing agents exhibited epitopes found on the surface of authentic HPV-11 virions. Antisera raised against either purified VLP starting material or reassembled VLPs similarly neutralized infectious HPV-11 virions. The ability to disassemble and reassemble VLPs in vitro and in bulk allows basic features of capsid assembly to be studied and also opens the possibility of packaging selected exogenous compounds within the reassembled VLPs.     

龙须菜中溴过氧化物酶的分离纯化及酶学性质分析

对中国北方海域江蓠属养殖龙须菜(Gracilaria lemaneiformis)进行了溴过氧化物酶分离纯化及性质的研究。粗提液中酶催化检测反应不稳定, 活力单位较低或无; 经DEAE cellulose 52离子交换层析, 去除了结构多糖及藻胆蛋白, 酶催化反应稳定, 得到比活力为2.8的电泳纯溴过氧化物酶。对纯化溴过氧化物酶性质研究表明: 该溴过氧化物酶为单体酶, 分子量约66 kD, 溴化单氯双甲酮时的最适pH值为6.0, 在40°C以下和pH 3.0~9.0之间有很好的稳定性。钒酸盐可提高该溴过氧化物酶的催化活性, 而Fe2+、Fe3+、Cu2+、Zn2+和EDTA等化合物对其有较显著的抑制作用。反应动力学实验表明, 该酶对Br-、H2O2的Km分别为53.5 mmol/L和38 mmol/L。     

龙须菜中溴过氧化物酶的分离纯化及酶学性质分析

对中国北方海域江蓠属养殖龙须菜(Gracilaria lemaneiformis)进行了溴过氧化物酶分离纯化及性质的研究。粗提液中酶催化检测反应不稳定, 活力单位较低或无; 经DEAE cellulose 52离子交换层析, 去除了结构多糖及藻胆蛋白, 酶催化反应稳定, 得到比活力为2.8的电泳纯溴过氧化物酶。对纯化溴过氧化物酶性质研究表明: 该溴过氧化物酶为单体酶, 分子量约66 kD, 溴化单氯双甲酮时的最适pH值为6.0, 在40°C以下和pH 3.0~9.0之间有很好的稳定性。钒酸盐可提高该溴过氧化物酶的催化活性, 而Fe2+、Fe3+、Cu2+、Zn2+和EDTA等化合物对其有较显著的抑制作用。反应动力学实验表明, 该酶对Br-、H2O2的Km分别为53.5 mmol/L和38 mmol/L。     

Characterization of porcine circovirus type 2 (PCV2) capsid particle assembly and its application to virus-like particle vaccine development

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in pigs. The sole structural capsid protein of PCV2, Cap, consists of major antigenic domains, but little is known about the assembly of capsid particles. The purpose of this study is to produce a large amount of Cap protein using Escherichia coli expression system for further studying the essential sequences contributing to formation of particles. By using codon optimization of rare arginine codons near the 5'-end of the cap gene for E. coli, a full-length Cap without any fusion tag recombinant protein (Cap1-233) was expressed and proceeded to form virus-like particles (VLPs) in normal Cap appearance that resembled the authentic PCV2 capsid. The N-terminal deletion mutant (Cap51-233) deleted the nuclear localization signal (NLS) domain, while the internal deletion mutant (CapΔ51-103) deleted a likely dimerization domain that failed to form VLPs. The unique Cys108 substitution mutant (CapC/S) exhibited most irregular aggregates, and only few VLPs were formed. These results suggest that the N-terminal region within the residues 1 to 103 possessing the NLS and dimerization domains are essential for self-assembly of stable Cap VLPs, and the unique Cys108 plays an important role in the integrity of VLPs. The immunogenicity of PCV2 VLPs was further evaluated by immunization of pigs followed by challenge infection. The Cap1-233-immunized pigs demonstrated specific antibody immune responses and are prevented from PCV2 challenge, thus implying its potential use for a VLP-based PCV2 vaccine.     

A quantitative description of in vitro assembly of human papillomavirus 16 virus-like particles

The full-length human papillomavirus 16 major capsid protein L1 is expressed in Saccharomyces cerevisiae as virus-like particles (VLPs). However, yeast-expressed human papillomavirus 16 particles are irregular in shape and are prone to aggregate. When disassembled and reassembled, the resulting particles have improved stability and solubility. We have examined VLP dissociation and reassembly to define the important features of the assembly mechanism. We found that the VLPs rapidly disassemble at pH 8.2 and low ionic strength in the presence of low concentrations of reducing agents. The pH dependence of assembly kinetics and extent of assembly under reducing conditions were differentially sensitive to ionic strength. Assembly at pH 5.2 was very fast and led to heavily aggregated particles. This sort of kinetic trap is expected for overinitiated assembly. We observed that reassembly at pH 6.2, 7.2, and 8.2 yielded regular particles over a broad range of ionic strength. At these three pH values, assembly was quantitative at 1 M NaCl. At pH 7.2, much more than at pH 6.2 or pH 8.2, assembly decreased monotonically with ionic strength. The free energy of association ranged from − 8 to − 10 kcal/mol per pentamer. The effect of pH on assembly was further investigated by examining dissociation of reassembled particles. Though indistinguishable by negative stain electron microscopy, particles assembled at pH 7.2 disassembled slower than pH 5.2, 6.2, or 8.2 VLPs. We hypothesize that pH 7.2 assembly reactions lead to formation of particles with conformationally different interactions.     

Purification and characterisation of tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase

Microbial tyrosine decarboxylase (EC 4.1.1.25) and mammalian aromatic-L-amino-acid decarboxylase (EC 4.1.1.28) catalyse the formation of tyramine from L-tyrosine. These enzymes were characterised after isolation to purity by methods including fast polymer liquid chromatography (FPLC). Tyrosine decarboxylase was isolated from Streptococcus faecalis by FPLC anion exchange chromatography (11-times purification; 72% recovery; 23.2 U/mg protein). FPLC on Phenyl-Superose resulted in purification to 115 U/mg protein. Aromatic-L-amino-acid decarboxylase was isolated from pig kidney by ammonium sulfate fractionation, DEAE chromatography, and FPLC anion exchange chromatography (21-times purification; 22% recovery; 0.71 U/mg protein). By FPLC chromatofocusing, tyrosine decarboxylase eluted at pH 4.3 and aromatic-L-amino-acid decarboxylase at pH 5.0. Isoelectric focusing of tyrosine decarboxylase gave two bands (pI 4.4 and 4.5). With pyridoxal 5'-phosphate removed by ultrafiltration, only one band (pI 4.4) appeared, and SDS polyacrylamide electrophoresis confirmed the purity. FPLC gel filtration resulted in molecular weights 143,000 and 86,000, respectively, for tyrosine decarboxylase and aromatic-L-amino-acid decarboxylase. In SDS electrophoresis, tyrosine decarboxylase had the monomer molecular weight 75,000, showing a dimer structure for the enzyme.     

赤豆子叶β-半乳糖苷酶的纯化及其性质的研究

β-半乳糖苷酶 ( EC3.2 .1 .2 )广泛存在于动植物的组织中 ,如在杏仁、桃子、大豆、咖啡豆等植物 ,蜗牛 ,哺乳动物的肠道中都有 β-半乳糖苷酶 .同样 ,微生物也能产生β-半乳糖苷酶 ,俗称乳糖酶 .乳糖操纵子学说的提出就是建立在对微生物β-半乳糖苷酶研究基础之上的 .在过去的研究中 ,关于微生物、动物来源的乳糖酶报道较多[1] ,而对于植物来源的β-半乳糖苷酶研究报道却相对较少[2 ] .它可能降解多糖中 β-构型半乳糖苷键 ,为种子生长发育提供必要的能量来源 .但目前对β-半乳糖苷酶在植物中确切的生理生化功能尚不清楚 .为了进一步阐明…