Reovirus major capsid protein expressed in Escherichia coli

A DNA copy of the open reading frame of the S4 gene of reovirus type 3 was cloned into a temperature-regulated bacterial expression vector. Induction at 42 degrees C resulted in the synthesis of a polypeptide that comigrated with virion capsid protein sigma 3 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reacted with sigma 3-specific antisera. The protein was expressed in bacteria as insoluble aggregates that accumulated in polar inclusion bodies. Aggregated product also resulted when the expression system was manipulated to induce bacterial sigma 3 (b sigma 3) synthesis at temperatures below 42 degrees C. Various methods used to solubilize b sigma 3 did not yield the monomeric protein. The results indicate that sigma 3, the major surface component of reovirions, is expressed in transfected Escherichia coli as an aggregated, disulfide cross-linked protein.     

Expression, purification, and characterization of VP2 capsid protein of canine parvovirus in Escherichia coli

The capsid protein VP2 of canine parvovirus (CPV) was expressed in Escherichia coli using pMAL-c2x vector. After codon optimization, the mutant resulted in high-level expression of fusion protein. A simple procedure was used to purify fusion protein and remove endotoxin from fusion protein preparations. The fusion protein was antigenical similar to the native capsid protein as Western-blot assay performed with polyclonal antibodies obtained from dogs vaccinated with CPV. The immunogenicity of fusion protein was demonstrated in a vaccination experiment conducted with rabbits subcutaneously immunized. Rabbits vaccinated with fusion protein developed high titers of virus-specific antibodies response that neutralized CPV in vitro.     

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.     

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。     

Production of FMDV virus-like particles by a SUMO fusion protein approach in Escherichia coli

Virus-like particles (VLPs) are formed by the self-assembly of envelope and/or capsid proteins from many viruses. Some VLPs have been proven successful as vaccines, and others have recently found applications as carriers for foreign antigens or as scaffolds in nanoparticle biotechnology. However, production of VLP was usually impeded due to low water-solubility of recombinant virus capsid proteins. Previous studies revealed that virus capsid and envelope proteins were often posttranslationally modified by SUMO in vivo, leading into a hypothesis that SUMO modification might be a common mechanism for virus proteins to retain water-solubility or prevent improper self-aggregation before virus assembly. We then propose a simple approach to produce VLPs of viruses, e.g., foot-and-mouth disease virus (FMDV). An improved SUMO fusion protein system we developed recently was applied to the simultaneous expression of three capsid proteins of FMDV in E. coli. The three SUMO fusion proteins formed a stable heterotrimeric complex. Proteolytic removal of SUMO moieties from the ternary complexes resulted in VLPs with size and shape resembling the authentic FMDV. The method described here can also apply to produce capsid/envelope protein complexes or VLPs of other disease-causing viruses.     

Self-assembly of sapovirus recombinant virus-like particles from polyprotein in mammalian cells

The SaV genome is a positive-sense, non-segmented single-strand RNA molecule of approximately 7.5 kb that is polyadenylated at its 3' terminus. The major capsid (VP1) of SaV is thought to be produced as the ORF1 polyprotein followed by cleavage, or translation from subgenomic RNA (3'-coterminal with the virus genome), or both. We have recently reported the formation of SaV VLP from subgenomic-like RNA in mammalian cells. In the present study, we demonstrated that the VP1 cleaved from a part of ORF1 polyprotein self-assembled into VLP in mammalian cells when a transient expression system using a recombinant vaccinia virus encoding T7 RNA polymerase was used.     

Characterization of monoclonal antibodies generated against Norwalk virus GII capsid protein expressed in Escherichia coli

The Norwalk virus (NV) causes outbreaks of acute non-bacterial gastroenteritis in humans. The virus capsid is composed of a single 60 kDa protein. The capsid protein of NV36 (genogroup II, Mexico virus type) was expressed in an Escherichia coli system and ten monoclonal antibodies (MAbs) were generated against it. The reactivity of these MAbs was characterized using enzyme-linked immunosorbent assay (ELISA) and Western blot (WB) analysis towards 20 overlapping fragments of the NV36 capsid protein expressed in E. coli. All of the MAbs recognized sequential (continuous) epitopes on the three antigenic regions. Six of the 10 MAbs recognized fragment 2 (equivalent residues 31-70), three MAbs recognized fragment 13 (residues 361-403) and one MAb recognized fragment 7 (residues 181-220), suggesting that the N-terminal domain (residues 1-220) may contain more antigenic epitopes than the C-terminal domain (residues 210-548). Furthermore, two MAbs (1B4 and 1F6) reacted in WB with three purified NV strains (genogroup II) derived from patients' stool samples. It was also found that genogroup I recombinant NV96-908 (genogroup I, KY89 type) could be detected as sensitively as recombinant NV36 (genogroup II) by ELISA with a set of the MAbs produced here.     

新型鸭细小病毒样颗粒的制备及鉴定

【背景】鸭短喙侏儒综合征(beak atrophy and dwarfism syndrome, BADS)是由新型鸭细小病毒(novel duck Parvovirus, NDPV)感染导致雏鸭生长发育迟缓、上下喙萎缩的疾病。BADS的暴发给我国养鸭业造成了巨大的经济损失。【目的】利用大肠杆菌表达系统制备NDPV病毒样颗粒(virus-like particles, VLPs),为研制NDPV相关疫苗奠定基础。【方法】对NDPV VP2序列全长进行密码子优化、合成,连接至pColdTF表达载体,获得pColdTF-NDPV-VP2重组质粒,酶切、测序鉴定正确后将重组质粒转化至大肠杆菌BL21(DE3)中进行诱导表达,利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulphate-polyacrylamide gel electrophoresis, SDS-PAGE)对蛋白表达进行可溶性分析;使用凝血酶(thrombin)切除trigger factor (TF)标签,利用镍柱(Ni-NTA)亲和层析方法纯化重组蛋白;利用Western blotting对纯化后的VP2蛋白进行反应原性分析;利用透射电镜、动态光散射观察重组蛋白形态以及能否形成VLPs。【结果】构建了pColdTF-NDPV-VP2重组质粒,在大肠杆菌中主要以可溶性形式表达,融合蛋白TF-VP2大小约为115 kDa,去除TF标签经镍柱纯化后得到67 kDa的VP2蛋白;Western blotting试验表明VP2蛋白能与NDPV鸭阳性血清发生特异性结合;通过透射电镜可以观察到形状规则、直径约为20−25 nm的病毒样颗粒。【结论】利用大肠杆菌表达系统制备了NDPV VLPs,为下一步研发BADS相关亚单位疫苗及生物相关制品提供了基础。     

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     

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...     

Immunogenicity of the capsid protein VP2 from porcine parvovirus expressed in low alkaloid transgenic tobacco

Porcine parvovirus is a widespread infectious viral disease with serious consequences to the reproductive health of swine. We have expressed the VP2 capsid protein of porcine parvovirus in the leaves of low alkaloid transgenic tobacco at approximately 0.3% of total soluble protein. Self-assembled virus-like particles were observed in planta by electron microscopy. Total soluble protein was extracted from the plant tissue and administered to mice by subcutaneous injection. An immune response was detected in these mice. The ability of serum antibodies to neutralize the infectivity of porcine parvovirus was further examined by a serum neutralization assay and was determined to be 1:2700–1:3900, a clear indication of the potential of VP2 expressed in plant material as a subunit vaccine against porcine parvovirus.     

细小病毒B19壳抗原VP2在大肠杆菌中的表达及血清学检测

为了进行B19感染临床的血清学诊断,利用原核表达载体PQE31克隆和表达B19壳蛋白VP2,酶切鉴定PCR产物及PQE31-VP2克隆的正确性,Western-blot证明表达蛋白的特异性,并对其表达条件和纯化条件进行了优选。在D600为0.7,诱导时间为5h时表达量最高。Ni2+亲和色谱,用0.5mol/L咪唑洗脱液洗脱,获得纯化蛋白。利用纯化蛋白检测100份人群血清,免疫斑点法结果为阳性94例,阴性6例;ELISA结果为阳性84例,阴性16例,两种方法结果一致(0.25>P>0.1)。     

Generation in yeast and antigenic characterization of hepatitis E virus capsid protein virus-like particles

Applied Microbiology and Biotechnology - Hepatitis E is a globally distributed human disease caused by hepatitis E virus (HEV). In Europe, it spreads through undercooked pork meat or other products...     

Identification of domains in canine parvovirus VP2 essential for the assembly of virus-like particles.

Canine parvovirus capsids are composed of 60 copies of VP2 and 6 to 10 copies of VPl. To locate essential sites of interaction between VP2 monomers, we have analyzed the effects of a number of VP2 deletion mutants representing the amino terminus and the four major loops of the surface, using as an assay the formation of virus-like particles (VLPs) expressed by recombinant baculoviruses. For the amino terminus we constructed three mutants with progressively larger deletions, i.e., 9, 14, and 24 amino acids. Deletions of 9 and 14 amino acids did not affect the morphology and assembly capabilities of the mutants. However, the mutant with the 24-amino-acid deletion did not show hemagglutination properties or correct VLP morphology, stressing again the relevance of the RNER domain in canine parvovirus functionality. Three of the four mutants with deletions in the loops failed to make correct VLPs, indicating that these regions are essential for correct capsid assembly and morphology. Only the mutant with the deletion in loop 2 was able to assemble in regular VLPs, suggesting that this loop has little or no effect in capsid morphogenesis. Further research has demonstrated that this region can tolerate the insertion of foreign epitopes that are correctly exposed in the surface of the capsid. This result opens the door to the use of these VLPs for antigen delivery.     

Production of human papillomavirus type 33 L1 major capsid protein and virus-like particles from Bacillus subtilis to develop a prophylactic vaccine against cervical cancer

We developed a bacterial expression system to produce human papillomavirus (HPV) type 33 L1 major capsid protein and virus-like particles from a recombinant Bacillus subtilis strain. For the first time, we have isolated self-assembled virus-like particles (VLPs) of HPV type 33 from B. subtilis, a strain generally recognized as safe (GRAS). The gene encoding the major capsid protein L1 of HPV type 33 was amplified from viral DNA isolated from a Korean patient and expressed in B. subtilis; a xylose-induction system was used to control gene activity. HPV33 L1 protein was partially purified by 40% (w/v) sucrose cushion centrifugation and strong cation exchange column chromatography. Eluted samples exhibited immunosignaling in fractions of 0.5-1.0 M NaCl. The HPV33 L1 protein was shown to be approximately 56 kDa in size by SDS-PAGE and Western blotting; recovery and purity were quantified by indirect immuno-ELISA assay. The final yield and purity were approximately 20.4% and 10.3%, respectively. Transmission electron microscopic analysis of fractions immunoactive by ELISA revealed that the L1 protein formed self-assembled VLPs with a diameter of approximately 20-40 nm. Humoral and cellular immune responses provoked by the B. subtilis/HPV33 L1 strain were approximately 100- and 3-fold higher than those of the empty B. subtilis strain as a negative control, respectively. Development of a VLP production and delivery system using B. subtilis will be helpful, in that the vaccine may be convenient production as an antigen delivery system. VLPs thus produced will be safer for human use than those purified from Gram-negative strains such as Escherichia coli. Also, use of B. subtilis as a host may aid in the development of either live or whole cell vaccines administered by antigen delivery system.     

Assembly of truncated HCV core antigen into virus-like particles in Escherichia coli

Core protein is one of the most conserved and immunogenic of the hepatitis C virus proteins. Several pieces of experimental evidence suggest its ability for formation of virus like particles alone or in association with other viral proteins in mammalian or yeast cells with great similarity to those detected in patient sera and liver extract. In this work we report an Escherichia coli-derived truncated hepatitis C core protein that is able to aggregate. SDS-PAGE and size exclusion chromatography patterns bring to mind the aggregation of monomers of recombinant protein Co.120. The Co.120 protein migrated with buoyant density of 1.28 g/cm(3) when analyzed using CsCl density gradient centrifugation. Spherical structures with an average diameter of 30 nm were observed using electron microscopy. We report here that VLPs are generated when the first 120 aa of HCV core protein are expressed in E. coli.     

乙肝病毒核心蛋白病毒样颗粒作为载体在表位疫苗中的应用

乙型肝炎病毒核心抗原(hepatitis B core antigen,HBcAg)是由乙型肝炎病毒(Hepatitis B virus,HBV) C基因编码的病毒蛋白,是HBV的衣壳蛋白,主要存在于HBV核衣壳表面。乙肝病毒核心蛋白(hepatitis B virus core protein,HBc)来源于HBcAg,由183或185个氨基酸组成。由于HBc本身具有高度的免疫原性,并且可携带自身或外源病原体的抗原/表位在体外自组装成病毒样颗粒,20世纪80年代开始HBc就被用于疫苗载体的研究。现对HBc近年来作为载体在表位疫苗中的应用作一概述。     

Purification of virus-like particles of recombinant human papillomavirus type 11 major capsid protein L1 from Saccharomyces cerevisiae

Recombinant major capsid protein, L1 (M(r) = 55,000), of human papillomavirus type 11 was expressed intracellularly at high levels in a galactose-inducible Saccharomyces cerevisiae expression system by an HPV6/11 hybrid gene. The capsid protein self-assembled into virus-like particles (VLPs) and accounted for 15% of the total soluble protein. A purification process was developed that consisted of two main steps: microfiltration and cation-exchange chromatography. The purified VLPs were 98% homogeneous, and the overall purification yield was 10%. The final product was characterized by several analytical methods and was highly immunogenic in mice.