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.     

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.     

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.     

Fermentation of recombinant yeast producing hepatitis B surface antigen

Summary Fermentations were performed to determine parameters affecting the expression of hepatitis B surface antigen (HBsAg) in the yeastSaccharomyces cerevisiae containing the HBsAg gene. These studies emphasized inereasing both the relative abundance (HBsAg: cell mass) and total production of HBsAg. Specific activity was increased 70-fold when cells were grown in shake flasks containing nonselective rather than selective medium. The addition of adenine, ammonium sulfate or glucose to the complex medium reduced the production of antigen. Results similar to those achieved in shake flasks were obtained when the growth was performed in fermenters. A nutrient addition system was employed to increase the production of cells and HBsAg. The addition of glucose to the culture medium increased cell mass 6-fold but decreased the production of antigen. This imbalance was corrected by supplementing the glucose with complex nutrients.     

两种配方的氢氧化铝吸附基因工程乙肝疫苗的效果比较

氢氧化铝［Ａｌ（ＯＨ）３］作为ＷＨＯ 推荐的一种佐剂,已广泛应用于生物制品的各种菌疫苗制剂中。本文对两种不同配方的氢氧化铝吸附基因工程乙肝表面抗原后疫苗的效果进行比较,结果显示：三氯化铝加氢氧化钠配方制备的Ａｌ（ＯＨ）３ 配制工艺简捷,易控制工艺参数及佐剂质量,对疫苗效力的增强作用优于三氯化铝加氨水的配方。     

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.     

Patterns of circulating hepatitis B surface antigen variants among vaccinated children born to hepatitis B surface antigen carrier and non-carrier mothers

Hepatitis B virus (HBV) variants that possessed missense mutation within the neutralization epitope of the major S antigen as defined by amino acid residues (aa#) 124–147, termed the a determinant variants, were identified through a population-based serosurvey of 2,305 children of the vaccinated birth cohorts born after 1986. Data on the 678 nucleotides encoding the S antigen of HBV were available for 75 HBV strains that were collected from 63 vaccinated children and 12 unvaccinated or incompletely vaccinated children, and 21 HBV strains from 25 unvaccinated adults. Among the diverse patterns of one to three amino acid substitutions within the a determinant, 145-Arg occurred most frequently (5/14); other variants were: 126-Ala, 127-Thr, 126-Ser/131-Asn/133-Thr, 129-His, 129-Arg, 123-Asn/131-Ile, 133-Leu, 141-Glu, and 141-Arg/144-Ala. Only one of these variants occurred in the 16 hepatitis B surface antigen (HBsAg)-carrier children born to HBsAg-negative mothers, whereas 12 of these variants occurred in the 20 (50%) children born to HBsAg-positive mothers. In addition, early administration of HBV vaccine within the noenatal period increased the likelihood of the emergence of these variants to 64.7% (11/17). Five of the 21 (23.8%) unvaccinated HBsAg-carrier adults harbored the a determinant variants possessing mutations within aa# 125–136, i.e. the putative first loop formed by the cysteine disulfide bonds. Vaccinated children were likely to harbor HBV variants possessing mutations involving altered charge of side chains and/or its hydrophobicity of amino acid residues within the putative second loop between aa#140 and 146. Our data suggest that emergence of these HBV S gene mutants in the phase of HBV vaccination program would be most common among populations in whom perinatal/vertical transmission of HBV is most common, i.e. southeast Asian and the Taiwanese.     

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。     

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。     

Magnetic nanoparticle-based immunosensor for electrochemical detection of hepatitis B surface antigen

An electrochemical immunosensor was developed for the detection of hepatitis B surface antigen (HBsAg). The biotinylated hepatitis B surface antibody was immobilized on streptavidin magnetic nanoparticles and used for targeting the HBsAg. By the addition of horseradish peroxidase conjugated with secondary antibody (HRP–HBsAb), a sandwich-type immunoassay format was formed. Aminophenol as substrate for conjugated HRP was enzymatically changed into 3-aminophenoxazone (3-APZ). This electroactive enzymatic production (3-APZ) was transferred into an electrochemical cell and monitored by cyclic voltammetry. Under optimal conditions, the cathodic current response of 3-APZ, which was proportional to the HBsAg concentration, was measured by a glassy carbon electrode. The immunosensor response was linear toward HBsAg in the concentration range from 0.001 to 0.015 ng/ml with a detection limit of 0.9 pg/ml at a signal/noise ratio of 3.     

Secretion of hepatitis B surface antigen in transformed tobacco cell suspension cultures

Six different expression cassettes of hepatitis B surface antigen (HBsAg) were used to transform tobacco cell suspension cultures. The transgenic nature of the cells was confirmed by PCR. The secreted HBsAg was assayed by ELISA and analyzed by Western blotting. A maximum of 31 μg antigen/l was obtained in the spent medium from the transformed cells. The use of an ethylene-forming enzyme promoter and incorporation of C-terminal endoplasmic-reticulum-retention signal enhanced the secretion of HBsAg. Salicylic or jasmonic acid at 10 μM increased secretion of HBsAg by six fold.     

Expression of hepatitis B surface antigen major subtypes in Pichia pastoris and purification for in vitro diagnosis

This study describes the expression in Pichia pastoris of hepatitis B surface antigens (HBsAg) corresponding to the S region of the four major subtypes: adr, adw2, ayr and ayw3 and to the preS2-S region of the two subtypes adr and adw2. The recombinant yeast strains have been selected amongst methanol utilization positive (Mut⁺) or sensitive strains (Mut^s) and cultivated to high cell density in bioreactor using a short protocol. Our results prove the efficiency of P. pastoris to produce all the major HBsAg subtypes and confirm the ability of the methanol regulated promoter of alcohol oxidase I gene (AOX) to express heterologous protein through phenotype Mut⁺ or Mut^s strains.All these recombinant HBsAg proteins, including subtype ayr, whose production has never been presented, have been highly purified using a short original sequence of steps which includes high-pressure cell disruption associated with detergent treatment, ultrafiltration and immunopurification chromatography using a mAb anti-HBs. The whole process avoids possible alterations of antigenic properties and allows to obtain with high yield, high quality reagents for in vitro diagnosis.     

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.     

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。     

Therapeutic inhibition of hepatitis B virus surface antigen expression by RNA interference

RNA interference (RNAi) mediated inhibition of virus-specific genes has emerged as a potential therapeutic strategy against virus induced diseases. Human hepatitis B virus (HBV) surface antigen (HBsAg) has proven to be a significant risk factor in HBV induced liver diseases, and an increasing number of mutations in HBsAg are known to enhance the difficulty in therapeutic interventions. The key challenge for achieving effective gene silencing in particular for the purpose of the therapeutics is primarily based on the effectiveness and specificity of the RNAi targeting sequence. To explore the therapeutic potential of RNAi on HBV induced diseases in particular resulted from aberrant or persistent expression of HBsAg, we have especially screened and identified the most potent and specific RNAi targeting sequence that directly mediated inhibition of the HBsAg expression. Using an effective DNA vector-based shRNA expression system, we have screened 10 RNAi targeting sequences (HBsAg-1 to 10) that were chosen from HBsAg coding region, in particular the major S region, and have identified four targeting sequences that could mediate sequence specific inhibition of the HBsAg expression. Among these four shRNAs, an extremely potent and highly sequence specific HBsAg-3 shRNA was found to inhibit HBsAg expression in mouse HBV model. The inhibition was not only preventive in cotransfection experiments, but also had therapeutic effect as assessed by post-treatment protocols. Moreover, this HBsAg-3 shRNA also exhibited a great potency of inhibition in transgenic mice that constitutively expressed HBsAg. These results indicate that HBsAg-3 shRNA can be considered as a powerful therapeutic agent on HBsAg induced diseases.     

Vaccine- and hepatitis B immune globulin-induced escape mutations of hepatitis B virus surface antigen

Hepatitis B virus surface antigen (HBsAg) vaccination has been shown to be effective in preventing hepatitis B virus (HBV) infection. The protection is based on the induction of anti-HBs antibodies against a major cluster of antigenic epitopes of HBsAg, defined as the 'a' determinant region of small HBsAg. Prophylaxis of recurrent HBV infection in patients who have undergone liver transplantation for hepatitis B-related end-stage liver disease is achieved by the administration of hepatitis B immune globulins (HBIg) derived from HBsAg-vaccinated subjects. The anti-HBs-mediated immune pressure on HBV, however, seems to go along with the emergence and/or selection of immune escape HBV mutants that enable viral persistence in spite of adequate antibody titers. These HBsAg escape mutants harbor single or double point mutations that may significantly alter the immunological characteristics of HBsAg. Most escape mutations that influence HBsAg recognition by anti-HBs antibodies are located in the second 'a' determinant loop. Notably, HBsAg with an arginine replacement for glycine at amino acid 145 is considered the quintessential immune escape mutant because it has been isolated consistently in clinical samples of HBIg-treated individuals and vaccinated infants of chronically infected mothers. Direct binding studies with monoclonal antibodies demonstrated a more dramatic impact of this mutation on anti-HBs antibody recognition, compared with other point mutations in this antigenic domain. The clinical and epidemiological significance of these emerging HBsAg mutants will be a matter of research for years to come, especially as data available so far document that these mutants are viable and infectious strains. Strategies for vaccination programs and posttransplantation prophylaxis of recurrent hepatitis need to be developed that may prevent immune escape mutant HBV from spreading and to prevent these strains from becoming dominant during the next decennia.     

A highly efficient integrated chromatographic procedure for the purification of recombinant hepatitis B surface antigen from Hansenula polymorpha

The high expression level of recombinant hepatitis B surface antigen obtained from Hansenula polymorpha yeast cell (Hans-HBsAg) made it possible to produce HBsAg vaccine in a large scale and by cost-effective process. However, the present available purification process was somewhat tedious, time-consuming and difficult to scale up. To improve the purification efficiency and simplify the purification process, an integrated chromatographic process was developed and optimized. The downstream process included ion-exchange chromatography (IEC), hydrophobic interaction chromatography (HIC) and gel filtration chromatography (GFC). A series of chromatographic adsorbents were evaluated for their performances on the purification of Hans-HBsAg, and then the suitable adsorbents for IEC and HIC were screened out, respectively. After clarification by centrifugation, the supernatant of cell disruption (SCD) was purified by standard chromatographic steps, IEC on DEAE Sepharose FF, HIC on Butyl-S-QZT and GFC on Sepharose 4FF. Furthermore, HBsAg recovery, purification factor (PF) and purity during the downstream process were evaluated with enzyme-linked immunosorption assay (ELISA), sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and high-performance size-exclusion chromatography (HPSEC). The results demonstrated that in the scale of 550ml SCD, the total HBsAg recovery and PF of the whole procedure were about 21.0+/-0.9% and 80.7+/-8.4 (n=3) respectively, with the purity of above 99%. This new downstream process was efficient, reproducible and relatively easy to be scaled up.     

乙型肝炎病毒e抗原的生物学功能及血清学转换的免疫学基础

目前在临床乙型肝炎的治疗中,乙型肝炎病毒e抗原(HBeAg)消失及其抗体的出现已成为重要的疗效指标.本文回顾了HBeAg的发现及其生物学和医学意义,对HBeAg与乙型肝炎病毒核心抗原(HBcAg)的免疫原性进行了比较,阐述了HBeAg血清学转换的免疫学基础,并对出现HBeAg血清学转换的意义作了分析.