共查询到20条相似文献，搜索用时 15 毫秒
1.
2.
Pratik U. Joshi Stephanie M. Kroger Silviya P. Zustiak Caryn L. Heldt 《Biotechnology progress》2023,39(4):e3338
Aqueous two-phase systems (ATPS) have found various applications in bioseparations and microencapsulation. The primary goal of this technique is to partition target biomolecules in a preferred phase, rich in one of the phase-forming components. However, there is a lack of understanding of biomolecule behavior at the interface between the two phases. Biomolecule partitioning behavior is studied using tie-lines (TL), where each TL is a group of systems at thermodynamic equilibrium. Across a TL, a system can either have a bulk PEG-rich phase with citrate-rich droplets, or the opposite can occur. We found that porcine parvovirus (PPV) was recovered at a higher amount when PEG was the bulk phase and citrate was in droplets and that the salt and PEG concentrations are high. To improve the recovery, A PEG 10 kDa-peptide conjugate was formed using the multimodal WRW ligand. When WRW was present, less PPV was caught at the interface of the two-phase system, and more was recovered in the PEG-rich phase. While WRW did not significantly increase the PPV recovery in the high TL system, which was found earlier to be optimal for PPV recovery, the peptide did greatly enhance recovery at a lower TL. This lower TL has a lower viscosity and overall system PEG and citrate concentration. The results provide both a method to increase virus recovery in a lower viscosity system, as well as provide interesting thoughts into the interfacial phenomenon and how to recover virus in a phase and not at the interface. 相似文献
3.
Recombinant structural proteins (VP1 and VP2) of the human parvovirus B19 have been expressed simultaneously using the baculovirus expression system to form virus-like particles (VLPs) that have potential use as vaccines. In this study, we report optimization of extraction conditions to recover these VLPs from cell paste. Under hypotonic conditions with neutral pH these VLPs were poorly extracted (up to 3% extraction). Addition of reducing agents, detergents, salts, and sonication did not improve the extractability. While screening for conditions to improve the extractability of the VLPs, we discovered that a combination of higher pH and elevated processing temperature significantly increased the extraction. Whereas increasing pH alone increased extractability from 3% to 6% (pH increased from 8.0 to 9.5), the effect of elevated temperature was much more substantial. At 50 degrees C, we observed the extraction to be more than fivefold higher than that at room temperature (up to 25% extracted at pH 9.0). The kinetics of extraction at elevated temperatures showed a rapid initial rate of extraction (on the order of minutes) followed by a plateau. In addition, we compared the extraction of VP1 expressed alone. VP1 expressed alone is incapable of forming VLPs. We observed that non-VLP VP1 was easily extractable (up to 60% extracted) under conditions in which the VP1 + VP2 VLPs were not extractable. From these studies we conclude that parvovirus B19 structural proteins expressed to form VLPs have a hindered extractability as compared with non-VLP protein. This hindrance to extraction can be significantly reduced by processing at elevated temperatures and an increased pH, possibly due to the enhanced rates of solubilization and diffusion. 相似文献
4.
Yuan Lu John P. Welsh Wei Chan James R. Swartz 《Biotechnology and bioengineering》2013,110(8):2073-2085
Bacterial flagellin has been explored as a potential vaccine adjuvant for enhancing immune responses. In this article, we describe Escherichia coli‐based cell‐free protein synthesis (CFPS) as a method to rapidly produce soluble phase 1 flagellin (FliC) protein from Salmonella typhimurium. The yield was about 300 µg/mL and the product had much higher affinity for the TLR5 receptor (EC50 = 2.4 ± 1.4 pM) than previously reported. The flagellin coding sequence was first optimized for cell‐free expression. We then found that the D0 domain at the C‐terminus of flagellin was susceptible to proteolytic degradation in the CFPS system. Proteolysis was reduced by protease inhibitors, the use of protease‐deficient cell extracts or deletion of the flagellin D0 domain. A human Toll‐Like Receptor 5 (hTLR5)‐specific bioactivity analysis of purified flagellin demonstrated that, although the D0 domain is far from the TLR5 recognition region, it is important for flagellin bioactivity. We next incorporated a non‐natural amino acid displaying an alkyne moiety into flagellin using the CFPS system and attached flagellin to hepatitis B core virus‐like particles (VLPs) using bioorthogonal azide‐alkyne cycloaddition reactions. The ordered and oriented VLP display of flagellin increased its specific TLR5 stimulation activity by approximately 10‐fold. Biotechnol. Bioeng. 2013; 110: 2073–2085. © 2013 Wiley Periodicals, Inc. 相似文献
5.
乙型肝炎病毒核心蛋白(Hepatitis B viruscore protein,HBc)可以形成二十面体对称的颗粒样结构,由于其N端、C端和主要免疫显性区域(Major immunodominant region,MIR)允许一定程度的缺失和外源插入,并且能够将外源序列重复且高密度地暴露在颗粒的表面,诱发强烈的外源序列特异的体液和细胞免疫反应,从上世纪80年代中期就开始被运用于表位疫苗的研究。以下主要从影响HBc作为表位疫苗载体的因素,包括HBc长度、外源插入位点和表位序列的性质等来介绍HBc作为表位疫苗载体的应用。 相似文献
6.
Masaki Kikuchi Shinichiro Iwabuchi Tatsuhiko Kikkou Keiichi Noguchi Masafumi Odaka Masafumi Yohda Masaaki Kawata Chikara Sato Osamu Matsumoto 《Acta Crystallographica. Section F, Structural Biology Communications》2013,69(2):165-169
Recombinant hepatitis B virus core proteins dimerize to form building blocks that are capable of self‐assembly into a capsid. A core capsid protein dimer (CPD) linked to a green fluorescent protein variant, EGFP, at the C‐terminus has been designed. The recombinant fusion CPD was expressed in Escherichia coli, assembled into virus‐like particles (VLPs), purified and crystallized. The single crystal diffracted to 2.15 Å resolution and belonged to the cubic space group F432, with unit‐cell parameters a = b = c = 219.7 Å. The fusion proteins assembled into icosahedral VLPs in aqueous solution, but were rearranged into octahedral symmetry through the crystal‐packing process under the crystallization conditions. 相似文献
7.
乙肝核心抗原由于其天然的颗粒组装能力和特异性激发针对外源表位的体液免疫和细胞免疫作用的特性,成为载体蛋白研究的热点.本简要综述乙肝核心抗原的结构特点、免疫学特性、作为免疫载体蛋白的研究进展及其应用研究. 相似文献
8.
乙型肝炎病毒核心蛋白HBc,可在体外自组装形成二十面体对称结构的病毒样微粒VLPs。VLPs可将外源序列重复且高密度地展示在表面,VLPs进入机体后能够快速诱导机体产生针对外源性抗原的特异性体液免疫及细胞免疫应答,具有极强的免疫原性与生物活性。因此,HBc-VLPs可以作为一种安全、有效的疫苗载体。文中设计了一种能够实现与抗原定点偶联的HBc-VLPs,并开发了一套高效制备HBc-VLPs的方法。通过定点突变技术,使翻译后的多肽序列第80位氨基酸由Ala变为Cys,在HBc-VLPs的主要免疫显性区域引入一个定点交联位点,构建了原核表达载体pET28a(+)-hbc,表达、纯化获得了高纯度的HBc(A80C) 单体蛋白;在PB缓冲体系中,HBc(A80C) 蛋白自组装形成HBc-VLPs纳米粒子。粒度仪的测定结果表明,HBc-VLPs纳米微粒的平均粒径为29.8 nm,透射电子显微镜观察到HBc-VLPs形成粒径约为30 nm的球形微粒,其形态与天然的HBV微粒相似。以流感病毒M2e抗原肽为模式抗原,通过Sulfo-SMCC氨基-巯基双功能交联剂,将M2e定点连接于HBc-VLPs通过突变引入的Cys残基处,制备了M2e-HBc-VLPs模式疫苗,通过细胞荧光示踪,验证了HBc-VLPs结构的完整性与M2e的正确交联。动物免疫实验表明该疫苗能够有效刺激小鼠产生抗原特异性的IgG抗体,验证了疫苗载体HBc-VLPs的有效性。研究结果为HBc-VLPs作为疫苗载体的研究奠定了基础,能够促进HBc-VLPs载体疫苗的研发以及HBc-VLPs在其他领域的应用。 相似文献
9.
Xiaowei Xu Sulin Ren Xiaoxiao Chen Jun Ge Zhenxing Xu Hongying Huang Honglin Sun Yue Gu Tong Zhou Jianqiang Li Hanmei Xu 《Virologica Sinica》2014,(6)
Despite the long availability of a traditional prophylactic vaccine containing the HBV surface antigen(HBsA g) and aluminum adjuvant, nearly 10% of the population remains unable to generate an effective immune response. Previous studies have indicated that hepatitis B virus(HBV) PreS 2-S is abundant in T/B cell epitopes, which induces a stronger immune response than HBsA g, particularly in terms of cytotoxic T lymphocyte(CTL) reaction. In the current study, the HBV PreS 2-S gene encoding an extra26 amino acids(PreS 2 C-terminus) located at the N-terminus of HBsA g was cloned into the pV CH1300 expression vector. Pre S2-S expressed in the methylotrophic yeast, Hansenula polymorpha, was produced at a yield of up to 250 mg/L. Subsequent purification steps involved hydrophobic adsorption to colloidal silica, ion-exchange chromatography and density ultracentrifugation. The final product was obtained with a total yield of ~15% and purity of ~99%. In keeping with previous studies, ~22 nm viruslike particles were detected using electron microscopy. The generated PreS 2-S antigen will be further studied for efficacy and safty in animals. 相似文献
10.
Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d 相似文献
11.
12.
13.
Neil Taylor Wanli Ma Adam Kristopeit Sheng‐Ching Wang Andrew L. Zydney 《Biotechnology and bioengineering》2021,118(1):106-115
There is growing interest in the development of new vaccines based on live‐attenuated viruses (LAVs) and virus‐like particles. The large size of these vaccines, typically 100–400 nm, significantly complicates the use of sterile filtration. The objectives of this study are to examine the performance of several commercial sterile filters for filtration of a cytomegalovirus vaccine candidate (referred to as the LAV) and to develop and evaluate the use of a model nanoparticle suspension to perform a more quantitative assessment. Data obtained with a mixture of 200‐ and 300‐nm fluorescent particles provided yield and pressure profiles that captured the behavior of the viral vaccine. This included the excellent performance of the Sartorius Sartobran P filter, which provided greater than 80% yield of both the vaccine and model particles even though the average particle size was more than 250 nm. The particle yield for the Sartobran P was independent of filtrate flux above 200 L/m2/h, but increased with increasing particle concentration, varying from less than 10% at concentrations around 107 particles/ml to more than 80% at concentrations above 1010 particles/ml due to saturation of particle capture/binding sites within the filter. These results provide important insights into the factors controlling transmission and fouling during sterile filtration of large vaccine products. 相似文献
14.
Che‐Yen Wang Naoyuki Miyazaki Tetsuo Yamashita Akifumi Higashiura Atsushi Nakagawa Tian‐Cheng Li Naokazu Takeda Li Xing Erik Hjalmarsson Claes Friberg Der‐Ming Liou Yen‐Jen Sung Tomitake Tsukihara Yoshiharu Matsuura Tatsuo Miyamura R. Holland Cheng 《Acta Crystallographica. Section F, Structural Biology Communications》2008,64(4):318-322
Hepatitis E virus (HEV) accounts for the majority of enterically transmitted hepatitis infections worldwide. Currently, there is no specific treatment for or vaccine against HEV. The major structural protein is derived from open reading frame (ORF) 2 of the viral genome. A potential oral vaccine is provided by the virus‐like particles formed by a protein construct of partial ORF3 protein (residue 70–123) fused to the N‐terminus of the ORF2 protein (residues 112–608). Single crystals obtained by the hanging‐drop vapour‐diffusion method at 293 K diffract X‐rays to 8.3 Å resolution. The crystals belong to space group P212121, with unit‐cell parameters a = 337, b = 343, c = 346 Å, α = β = γ = 90°, and contain one particle per asymmetric unit. 相似文献
15.
Bingyang Zhang Shuang Yin Yingli Wang Zhiguo Su Jingxiu Bi 《Engineering in Life Science》2021,21(6):438
Inserting foreign epitopes to hepatitis B core (HBc) virus‐like particles (VLPs) could influence the molecular conformation and therefore vary the purification process. In this study, a cost‐effective purification process was developed for two chimeric HBc VLPs displaying Epstein–Barr nuclear antigens 1 (EBNA1), and hepatitis C virus (HCV) core. Both chimeric VLPs were expressed in soluble form with high production yields in Escherichia coli. Molecular dynamic (MD) simulation was employed to predict the stability of chimeric VLPs. HCV core‐HBc was found to be less stable in water environment compared with EBNA1‐HBc, indicating its higher hydrophobicity. Assisting with MD simulation, ammonium sulfate precipitation was optimized to remove host cell proteins with high target protein recovery yields. Moreover, 99% DNA impurities were removed using POROS 50 HQ chromatography. In characterization measurement, we found that inserting HCV core epitope would reduce the ratio of α‐helix of HCV core‐HBc. This could be another reason on the top of its higher hydrophobicity predicted by MD simulation, causing its less stability. Tertiary structure, transmission electron microscopy, and immunogenicity results indicate that two chimeric VLPs maintained correct VLP structure ensuring its bioactivity after being processed by the developed cost‐effective purification approach. 相似文献
16.
Virus-like particles (VLPs) are empty particles consisting of virus capsid proteins that closely resemble native virus but are devoid of the native viral nucleic acids and therefore have attracted significant attention as noninfectious vaccines. A recombinant baculovirus, vIBD-7, which encodes the structural proteins (VP2, VP3, and VP4) of infectious bursal disease virus (IBDV), produces native IBD VLPs in infected Spodoptera frugiperda insect cells. Another baculovirus, vEDLH-22, encodes VP2 that is fused with a histidine affinity-tag (VP2H) at the C-terminus. By co-infection with these two baculoviruses, hybrid VLPs with histidine tags were formed and purified by immobilized metal affinity chromatography (Hu et al., 1999). Also, we demonstrated that varying the MOI ratio of these infecting viruses altered the extent of VP2H incorporated into the particles. A dynamic mathematical model that described baculovirus infection and VLP synthesis (Hu and Bentley, 2000) was adapted here for co-infection and validated by immunofluorescence labeling. It was shown to predict the VLP composition as a dynamic function of MOI. A constraint in the VP2H content incorporated into the particles was predicted and shown by experiments. Also, the MOI ratio of both infecting viruses was shown to be the major factor influencing the composition of the hybrid particles and an important factor in determining the overall yield. ELISA results confirmed that VP2H was exhibited to a varied extent on the outer surface of the particles. This model provides insight on the use of virus co-infection in virus-mediated recombinant protein expression systems and aids in the optimization of chimeric VLP synthesis. 相似文献
17.
乙型肝炎病毒DNA疫苗的研究进展 总被引：2，自引：0，他引：2
预防与控制乙型肝炎发病的乙型肝炎病毒(HBV)疫苗,是有重大的社会和经济意义。HBV的持续感染可引起慢性肝脏疾患,并逐步发展为肝硬化和肝细胞癌(HCC)。目前的乙肝重组亚单位疫苗可以使90％的接种产生保护性抗体;但是对慢性HBV携带,由于其机体对HBsAg蛋白产生耐受,不能产生体液和细胞免疫,因此它只能作为一种预防性的疫苗。DNA疫苗(基因疫苗)是一种新的疫苗技术,通过向体内递送编码抗原的细菌质粒,刺激产生特异的体液和细胞免疫反应。在小鼠和其他的肝炎病毒感染动物模型中,HBV DNA疫苗可以特异性地引起体液和细胞免疫,清除HBV转基因动物血循环中的HBsAg颗粒和HBV DNA。如果加入各种免疫调节细胞因子的基因,可以进一步提高HBV DNA疫苗的免疫效果,因此它不仅可作为预防性疫苗,也可作为治疗型疫苗。 相似文献
18.
19.
20.
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。 相似文献