High-yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system

Recombinant hepatitis B surface antigen (HBsAg) constitutes currently used vaccines against hepatitis B virus, and has been successfully employed as a carrier for foreign epitopes. With the aim of developing an inexpensive, easily administered vaccine source for global immunization, several groups have expressed HBsAg in plant systems. Transgenic plant-derived HBsAg assembles into virus-like particles (VLPs) and is immunogenic in both mice and humans. However, HBsAg expression is relatively low in transgenic plant systems. The time-consuming and labour-intensive process of generating transgenic plants also significantly limits high-throughput analyses of various HBsAg fusion antigens. In this paper, the high-yield rapid production of HBsAg in plant leaf using a novel viral transient expression system is described. Nicotiana benthamiana leaves infiltrated with the MagnICON viral vectors produced HBsAg at high levels, averaging 295 µg/g leaf fresh weight at 10 days post-infection, as measured by a polyclonal enzyme-linked immunosorbent assay. Transiently expressed HBsAg accumulated as the full-length product, formed disulphide-linked dimers, displayed the conformational 'a' antigenic determinant and assembled into VLPs. Immunization of mice with partially purified HBsAg elicited HBsAg-specific antibodies. Furthermore, it was found that transient production of HBsAg using vacuum infiltration of whole plants, rather than syringe infiltration of leaves, was readily scalable, and greatly improved the accumulation of correctly folded HBsAg that displays the protective 'a' determinant.     

乙肝病毒表面抗原持续表达的新致病机制

乙型肝炎表面抗原(HBsAg)持续阳性是控制乙肝中难以解决的重大问题。本研究通过揭示HBsAg致病机制的基础研究,寻找抑制或清除HBsAg的新途径。通过建立有可比性的HBsAg转基因鼠和稳定表达细胞系及相应对照,进行比较转录组学和蛋白质组学研究,发现了HBsAg在HBV慢性感染中的一些新致病机制。其中包括:HBsAg促进肝细胞内CypA分泌,后者可趋化炎症细胞在HBsAg阳性灶周围浸润;在细胞模型中,HBsAg分泌可引起胞内GRP78蛋白下降,导致肝细胞抗凋亡能力减弱;发现HBsAg在细胞中可上调截短的LEF1基因的表达,缺乏活化全长LEF1促成瘤和增殖活性;而肝癌组织中LEF1则倾向于核内分布,并活化Wnt下游基因Cyclin D1与c-myc,有促肿瘤活性。在转基因鼠和细胞模型中都发现了物质和能量代谢相关的基因发生变化,并与临床慢性乙肝患者表现相符。研究中有关CypA的发现提供了抑制HBsAg的新途径;有关代谢的变化提出了改变饮食内容与习惯可能有利于HBsAg阳性感染者的预后。     

High-throughput protein localization in Arabidopsis using Agrobacterium-mediated transient expression of GFP-ORF fusions

We describe a streamlined and systematic method for cloning green fluorescent protein (GFP)-open reading frame (ORF) fusions and assessing their subcellular localization in Arabidopsis thaliana cells. The sequencing of the Arabidopsis genome has made it feasible to undertake genome-based approaches to determine the function of each protein and define its subcellular localization. This is an essential step towards full functional analysis. The approach described here allows the economical handling of hundreds of expressed plant proteins in a timely fashion. We have integrated recombinational cloning of full-length trimmed ORF clones (available from the SSP consortium) with high-efficiency transient transformation of Arabidopsis cell cultures by a hypervirulent strain of Agrobacterium. To demonstrate its utility, we have used a selection of trimmed ORFs, representing a variety of key cellular processes and have defined the localization patterns of 155 fusion proteins. These patterns have been classified into five main categories, including cytoplasmic, nuclear, nucleolar, organellar and endomembrane compartments. Several genes annotated in GenBank as unknown have been ascribed a protein localization pattern. We also demonstrate the application of flow cytometry to estimate the transformation efficiency and cell cycle phase of the GFP-positive cells. This approach can be extended to functional studies, including the precise cellular localization and the prediction of the role of unknown proteins, the confirmation of bioinformatic predictions and proteomic experiments, such as the determination of protein interactions in vivo, and therefore has numerous applications in the post-genomic analysis of protein function.     

Comparison of different elution conditions for the immunopurification of recombinant hepatitis B surface antigen

An immunoaffinity chromatographic method was developed using a mAb immunosorbent to purify recombinant hepatitis B surface antigen (r-HBsAg) from yeast. Elution conditions using a mAb-coated ELISA were improved to select the best conditions to purify r-HBsAg. The optimum results in terms of total quantitative recovery were obtained using 20 mM Tris pH 11.6. An increase in the CB.Hep-1 mAb (anti-HBsAg) useful immunosorbents half-life and in its yield per cycle was obtained when alkaline elution conditions were used. Moreover, the basic conditions do not affect either the antigenic characteristics or the purity or the molecular integrity of r-HBsAg.     

A modified hepatitis B surface antigen carrying both preS1 and preS2 epitopes

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分子筛层析法分析重组乙肝表面抗原聚集物的形成

分子筛层析作为分析蛋白质颗粒聚集物的一种有力工具,被用于研究重组乙肝表面抗原聚集物的形成。已去除聚集物的表面抗原放置在不同的理化条件下或经过不同的纯化方法处理后,应用HPLC分析其聚集物的形成。为研究发酵过程中是否形成表面抗原聚集物,酵母细胞破碎后立即用Sepharose 4 FF层析柱分离为不同的组分,并分别进行HPLC分析。结果发现,在纯化过程和酵母发酵阶段都有表面抗原聚集物的产生。     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

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     

Stable expression of hepatitis B surface antigen gene in Dunaliella salina (Chlorophyta)

A stable transformation system for theexpression of foreign genes in theunicellular marine green alga Dunaliella salina was established. Amongfive antibiotics, 60 g mL^-1chloramphenicol completely inhibitedgrowth. Of five promoters tested, theubiquitin-$Ohgr; promoter yielded thehighest -glucuronidase (GUS) activity.The hepatitis B surface antigen (HBsAg)gene was introduced into the cells by usingelectroporation. PCR and Southern blotanalysis amd it was shown that the gene wasintegrated into the genome. The stableexpression of HBsAg protein was confirmedby enzyme-linked immunosorbent assay(ELISA) and Western blot analysis. Theintroduced DNA and HBsAg expression weremaintained stable for at least 60generations in medium devoid ofchloramphenicol. This is an important steptoward the production of useful foreignproteins in the alga.     

血清乙肝病毒大蛋白与乙肝前S1抗原联合检测在HBeAg阴性乙肝患者诊断中的意义

目的 探讨乙型肝炎e抗原阴性[HBeAg（－）]乙肝患者血清乙肝病毒大蛋白（HBV-LP）和乙肝前S1抗原（PreS1-Ag）联合检测的临床意义。方法 采用酶联免疫吸附（ELISA）法测定300例慢性乙肝患者的血清HBV-LP和PreS1-Ag浓度;实时荧光定量PCR（qRT-PCR）法检测血清HBV-DNA表达量;比较不同HBV-M模式下HBV-LP、PreS1-Ag与HBV-DNA的阳性检出率;分析以HBV-DNA表达量作为HBV感染及复制的金标准时,血清HBV-LP和PreS1-Ag单独检测及联合检测对HBeAg阴性乙肝患者的阳性预测值和阴性预测值。结果 （1）115例HBeAg（+）血清中,HBV-LP和PreS1-Ag的阳性率均与HBV-DNA阳性率差异无统计学意义（Ps＞0.05）;120例HBeAg（－）HBeAb（+）血清中,HBV-LP阳性率（64.2%）明显高于HBV-DNA阳性率（P＜0.05）,而PreS1-Ag阳性率与HBV-DNA阳性率差异无统计学意义（P＞0.05）;65例HBeAg（－）HBeAb（－）血清中,HBV-LP阳性率（72.3%）和PreS1-Ag阳性率（67.7%）均明显高于HBV-DNA阳性率（Ps＜0.05）;（2）以185例HBeAg（－）乙肝患者的HBV-DNA表达量为参考标准,HBV-LP、PreS1-Ag的阳性预测值分别为72.6%、71.6%,阴性预测值分别为93.4%、84.1%;HBV-LP和PreS1-Ag联合检测,HBV-LP/PreS1-Ag双阳性中的HBV-DNA阳性率（66.0%）显著高于HBV-LP/PreS1-Ag双阴性（P＜0.05）。结论 血清HBV-LP和PreS1-Ag水平与HBV-DNA表达量有关,二者联合检测可灵敏地反映HBeAg（－）乙肝患者HBV的复制状态,预测HBV-DNA水平。     

Transient and stable expression of hepatitis B surface antigen in tomato (Lycopersicon esculentum L.)

Cotyledonary leaves of tomato cv. Megha were transformed with the hepatitis B virus ‘s’ gene, which encodes surface antigen. Six plant expression cassettes (pHBS, pHER, pEFEHBS, pEFEHER, pSHER and pEFESHER) were used to assay the possible expression levels by agroinfiltration. The maximum transient expression level of 489.5 ng/g D.W. was noted in pEFEHER-infiltrated cotyledonary leaves. Transgenic tomato plants with pEFEHBS and pEFEHER expression cassettes were regenerated and characterized by molecular analysis. The expression of the antigen in the fruits was confirmed by RT-PCR and ELISA analysis. This is the first report on the expression of hepatitis B surface antigen in tomato.     

待孕夫妇乙肝表面抗原及乙肝表面抗体阳性情况分析

目的 分析与探讨待孕夫妇乙肝表面抗原及乙肝表面抗体检测结果,并研究其对临床孕前检查的影响及评价。方法 随机选取2015‒2017年度在我院进行孕前检查的夫妇2440对（4 880例）为研究对象,按照年度将待孕夫妇分为两组,每组2 440例,两组均加强孕前检查中的乙肝表面抗原（HBsAg）及乙肝表面抗体（HBsAb）的检测。A组为2015年3月‒2016年2月在我院进行乙肝表面抗原及乙肝表面抗体检查的待孕夫妇;B组为2016年3月‒2017年2月在我院进行乙肝表面抗原及乙肝表面抗体检查的待孕夫妇。比较两组待孕夫妇乙肝表面抗原及乙肝表面抗体检测的阳性结果。结果 B组HBsAg阳性率、HBsAb阳性率明显高于A组（6.43% vs 4.63%;62.99% vs 58.44%）,差异有统计学意义（P＜0.05）。B组、A组男性HBsAg阳性率明显高于同组女性（59.87% vs 40.13%;60.18% vs 39.82%）,HBsAb阳性率低于同组女性（46.52% vs 53.48%;47.41% vs 52.59%）,差异均有统计学意义（P＜0.05）。B组、A组高中及以上学历HBsAg阳性率明显低于同组高中以下学历（38.85% vs 61.95%;38.05% vs 61.15%）,高中及以上学历HBsAb阳性率高于同组高中以下学历（53.15% vs 46.84%;51.75% vs 48.25%）,差异均有统计学意义（P＜0.05）结论 目前夫妇乙肝感染仍处于增高趋势,对于进行孕前检查的待孕夫妇加强乙肝表面抗原及乙肝表面抗体的检测,有助于疾病的早期诊断、干预及治疗,能够减少乙肝传播,可有效降低新生儿乙肝发病率,促进优生优育,提高出生人口整体素质。     

乙型肝炎病毒小表面抗原各拓扑区段对其表达和分泌的影响

乙型肝炎病毒小表面抗原(small hepatitis B virus surface antigen,SHB)在细胞内质网上表达,沿着细胞分泌途径分泌到胞外。为系统分析SHB拓扑结构对SHB表达和分泌的影响,首先通过生物信息学预测临床病毒株HBV C8和8种基因型(A~H)代表株的SHB拓扑结构,发现这些SHB均为四次跨膜蛋白,拥有基本相同的拓扑结构。相对内质网膜而言,SHB的拓扑结构拥有3个内质网腔内区段(Inside1~Inside3)、4个跨膜螺旋区(Tmhelix1~Tmhelix4)和2个内质网膜外区段(Outside1和Outside2)。6种基因型(基因型A、B、C、D、E和G)代表株与病毒株C8的SHB拓扑结构预测结果完全相同,而基因型F和H的SHB有4个区段与C8等不完全一致。通过对C8的SHB拓扑结构各区段进行缺失突变研究,发现Inside1区段不是SHB表达和分泌所必需的;Outside1、Tmhelix2和Inside2区段是SHB表达和分泌所必需的;Tmhelix1和Outside2不是SHB表达所必需的,但为SHB分泌所必需;Tmhelix3和Tmhelix4对SHB表达有重要影响,也是SHB分泌所必需的。进一步对Outside1和Outside2进行小片段(6个氨基酸)的缺失突变研究,发现小片段缺失基本不显著影响SHB的表达,但Outside1的氨基酸55~78及Outside2是SHB分泌所必需的。本研究首次系统性分析了SHB的拓扑结构各区段对SHB表达和分泌的影响,为深入探索SHB结构与功能的关系提供了线索。     

乙肝病毒表面抗原基因在胡萝卜中的克隆及表达

构建HBV表达抗原（HBsAg)植物表达载体并在胡萝卜植株中表达。采用自行构建adr亚型HBsAg基因克隆T-adr,再次酶切获得860bp含PreS2的HBsAg基因片段,将其插入到植物表达载体pBPC55,新质粒命名为pBPC91adr。将其与含除草剂抗性基因及GUS蛋白基因的筛选质粒pBPC93共同经基因枪（PDS-1000/He)转化胡萝卜悬浮细胞,经含除草剂（Biolaphos)的培养基筛选及植物激素诱导分化,获得除草剂抗性胡萝卜幼苗,结果为转化后8周,自胡萝卜细胞中分化出除草剂抗性胡萝卜幼苗,提取新分化幼苗总DNA,特异性引物PCR扩增后可见860bp扩增带;Southern-Blot证明有HBsAg基因整合,胡萝卜蛋白萃取物的Western-Blot及ELISA检测证实有HBsAg蛋白表达。利用基因枪转化使质粒pBPC91adr中HBsAg基因在胡萝卜幼苗内整合并表达,提示以植物生产疫苗具有可行性。     

乙肝病毒核心蛋白病毒样颗粒表面抗原密度对抗体应答水平的影响

【目的】为了探究乙肝病毒核心蛋白(HepatitisBviruscoreprotein,HBc)病毒样颗粒(Virus-like particles,VLPs)表面抗原密度对免疫后抗体应答水平的影响,制备了不同抗原密度的HBc VLPs疫苗,并检测了其在小鼠体内的抗体应答水平。【方法】首先制备了N端带有3个甘氨酸的人巨细胞病毒重组抗原域AD-4作为模式抗原,接着通过Sortase A的介导将AD-4连接到HBc VLPs表面上。将系列浓度梯度AD-4抗原在SortaseA介导下分别与相同浓度的HBcVLPs发生反应,制备不同抗原密度的HBc-AD-4 VLPs。将其分别免疫6–8周龄BALB/c小鼠3次,每次免疫间隔2 w,间接ELISA法检测被免疫小鼠血清的抗体应答水平。【结果】结果表明,当HBc VLPs表面抗原密度为44.4%时,即HBc反应浓度∶AD-4反应浓度为1:0.5时,不足以引起高滴度的抗体产生;当HBc VLPs表面抗原密度为64.2%时,即HBc反应浓度∶AD-4反应浓度为1:1时,HBc-AD-4 VLPs诱导的AD-4特异性抗体滴度与100%抗原密度的HBc-AD-4VLPs所引起的抗体滴度相当;当HBcVLPs表面抗原密度大于64.2%时,引起的抗体应答水平不因抗原密度增加而进一步增强。【结论】发现了HBcVLPs表面抗原密度与免疫后抗体滴度呈正相关,然而免疫64.2%抗原密度的HBc VLPs所产生的抗体滴度可达峰值,抗原密度进一步增加,抗体应答水平不会进一步加强。     

Aggregation of recombinant hepatitis B surface antigen in Pichia pastoris

The combination of immunoaffinity and size-exclusion chromatography (SEC) is a powerful tool to analyze multiprotein particle assembly. This approach was used to investigate the source of aggregation of recombinant hepatitis B surface antigen (HBsAg) detected in purified material. As HBsAg aggregation does not originate in the stresses, such as the concentration of HBsAg solutions, temperature and chaotropic agents, it is less probable that the HBsAg aggregate is produced during the process. To test whether aggregation takes place in vivo, crude yeast extract containing the expressed HBsAg was fractioned on a Sephacryl S-400 column just after cell disruption, and each fraction immunopurified individually. As a result, the HBsAg aggregate was isolated from a fraction corresponding to the elution of large particle aggregates only, not native HBsAg particles. It was biologically active, which demonstrates aggregate formation by specific assembly of partially or wholly folded HBsAg intermediates.     

Production of highly concentrated, heat-stable hepatitis B surface antigen in maize

Plant‐based oral vaccines are a promising emergent technology that could help alleviate disease burden worldwide by providing a low‐cost, heat‐stable, oral alternative to parenterally administered commercial vaccines. Here, we describe high‐level accumulation of the hepatitis B surface antigen (HBsAg) at a mean concentration of 0.51%TSP in maize T1 seeds using an improved version of the globulin1 promoter. This concentration is more than fourfold higher than any previously reported lines. HBsAg expressed in maize seeds was extremely heat stable, tolerating temperatures up to 55 °C for 1 month without degradation. Optimal heat stability was achieved after oil extraction of ground maize material, either by supercritical fluid extraction or hexane treatment. The contributions of this material towards the development of a practical oral vaccine delivery system are discussed.     

Efficient expression and characterization of hepatitis B virus preS2 antigen in Escherichia coli

The complete (encoding 55 amino acids, aa) or partial (encoding aa 1–26) preS2 region gene of hepatitis B virus (HBV) was fused to the 3-end of glutathion-S-transferase (GST) gene and expressed under the control of the inducible tac promoter in Escherichia coli at 37 °C. The fusion protein with the complete preS2 region was moderately expressed (8%) while the protein with the N-terminal 26 aa was expressed at a higher level, yielding about 20% of the total cellular proteins. The GST-preS2 (aa 1–26) protein, which contains the immunodominant epitope, was produced form the soluble protein fraction of the recombinant bacteria and purified by affinity chromatography using glutathione-agarose column. The purified preS2 fusion protein showed the antigenicity of preS2, as assessed by indirect and competitive ELISAs.     

Size-exclusion chromatographic study of the reduction of recombinant hepatitis B surface antigen

The reduction of the P. pastoris-derived hepatitis B surface antigen (HBsAg) has been investigated by size exclusion chromatography performed in a detergent solution containing 0.3% sodium dodecyl sulfate (SDS) and 0.1 M Tris–HCl, pH 7.0. The HBsAg, reduced under different conditions and passed through the TSK G4000 SW column (600×7.5 mm I.D.) at 0.9 ml min⁻¹, was resolved into two peaks corresponding to the reduced, monomeric, and non-reduced forms, respectively. Under these conditions, the antigen fraction corresponding to the HBsAg dimer can be separated and completely reduced to monomers by repeated reductive treatment with simultaneous lipid removal. The efficiency of reduction was maximal after sample treatment with an equal volume of a solution containing 417 mM dithiothreitol, 4.2% (w/v) SDS and 16% (v/v) 2-mercaptoethanol. In conclusion, complete reduction of recombinant HBsAg to monomer subunits is possible and depends on the efficiency of lipid removal during the reductive treatment.     

Sodium dodecylsulfate polyacrylamide gel electrophoresis of recombinant hepatitis B surface antigen particles

To investigate the factors leading to broadening of the recombinant hepatitis B surface antigen (HBsAg) peak in size-exclusion chromatography, the HBsAg particles eluting in different regions of the peak were subjected here to electrophoretic analysis. In nonreduced samples, the 24-kD band corresponding to the S monomer was detected when excessively large amounts of HBsAg were loaded onto the gel. Hence, some monomers are not disulfide-crosslinked in assembled particles. On the other hand, the results of alkylation experiments indicated the presence of free sulfhydryl group(s) in a little portion of freshly-purified HBsAg which was retarded on the size-exclusion chromatographic column and had significant antigenicity. This fraction of HBsAg was shown to be oligomeric and capable of spontaneous assembly into higher-order structures during aging.