Expression and secretion of preS containing hepatitis B surface antigen in vaccinia virus system

The expression and secretion of preS containing hepatitis B surface antigen in vaccinia virus system was investigated. The human TK- 143 cells were infected with the recombinant vaccinia viruses vTMS-1 or vTLS-1. Cells infected with vTMS-1, which contains the preS2 + S gene, produced preS2 containing middle HBsAg proteins. Similarly, cells produced preS1 containing large HBsAg proteins upon infection with vTLS-1, which carries the preS1 + preS2 + S gene. The expression products could be secreted and form 22 nm particles. They reacted specifically with anti-preS1 and/or anti-preS2 monoclonal antibodies, and exhibited pHSA-receptor (for polymerized human serum albumin) activity. In addition, the major S components of hepatitis B surface antigen were also present in the products expressed by vTMS-1 and vTLS-1.     

The Properties of an HBV Surface Antigen Protein Carrying the Binding Site for the Receptor of Hepatocytes——Its Formation of Surface Antigen Particles and Secretion From Discrete Cell Lines

A human hepatitis B virus (HBV) gene, which encodes the major surface antigen protein(S protein) carrying the hepatocyte receptor-binding site, was constructed with site-directed mutagenesis and in vitro recombination. When expressed in monkey kidney cell line COS-M6, this gene product (S309 protein) formed surface antigen (HBsAg) particles and secreted from the cells. It was stable within the cells and in the culture medium and could be immunoprecipitated with antisera directed against plasma-derived HBsAg or synthetic preS1 polypeptide. Isopycnic CsCl gradient centrifugation showed that the density of S309 protein particles (1.25 g/ml) was slightly higher than that of S protein particles. The S309 protein was readily secretable from hepatoma cell lines, and the amount secreted was comparable to that of the S protein. By contrast, only about 10% of the S309 protein was secreted from COS-M6 cells, and its appearance in culture medium was delayed. The efficiency of the secretion of the S309 protein can b     

An HBV large surface antigen protein which can be secreted from mammalian cells.

The N-terminal 54 base pairs (encoding amino acid residues 2-19) within the preS1 region of the human hepatitis B virus surface antigen gene were deleted by site-directed mutagenesis. Unlike the wild type large surface antigen protein, when this mutated gene was expressed in monkey kidney cell line COS-M6, the protein product (S301 protein) could be secreted from the cells. Moreover, the inhibition of the secretion of the major surface antigen protein by this altered large surface antigen protein was greatly reduced, suggesting that the deleted region contained a retention sequence which prevented the secretion of the large surface antigen. However, the coexpression of the major S protein was essential for the secretion of the S301 protein. When coexpressed, the secretion of these two proteins was synchronous. Like the wild type large surface antigen protein, the S301 protein could be translocated into endoplasmic reticulum and glycosylated after its synthesis in COS cells. The S301 protein was thermostable and proteinase-resistant. It also retained the antigenicity of the large S and major S proteins. Given the fact that the S301 protein is readily secretable, stable and identical to the large S protein in terms of their antigenicity, it may be developed into a new generation of recombinant vaccine for the prevention of viral hepatitis.     

Isolation and Purification of Recombinant HBsAg of Human Hepatitis B Virus from Silkworm Larvae

Recombinant HBsAg coded by preS1-preS2-S regions of hepatitis B virus was expressed in Bombyx mori silkworm larvae. Recombinant HBsAg was expressed (30–40 μg/mL, 0.1% of the total amount of extracted protein) by larvae infected with recombinant baculovirus rBmNPV-Hep-preS1-S containing cDNA of HBsAg strictly by the polyhedrin gene promoter. Recombinant HBsAg consisting of a polypeptide of molecular weight ∼36 kDa (p36) was purified by gel filtration and affinity chromatography to 92% purity. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 5, pp. 477–480, September–October, 2005.     

Selection of recombinant vaccinia viruses (Tian Tan strain) expressing hepatitis B virus surface antigen by using beta-galactosidase as a marker

We constructed a plasmid that contains a small piece of DNA with two vaccinia promoters running in opposite directions--a promoter from a late gene encoding an 11 K polypeptide (P11) and a promoter from an early gene encoding 25K (P25). These promoters were isolated from the Tian Tan strain of vaccinia virus and were flanked by the thymidine kinase (TK) sequence of the same virus. Genes encoding the hepatitis B virus surface antigen (HBsAg) and the Escherichia coli beta-galactosidase (LacZ) were inserted downstream of the 11 K and 25 K promoters respectively so that coexpression plasmids were constructed. Recombinant vaccinia viruses were selected directly by picking blue plaques formed under overlaying agarose medium containing X-gal. HBsAg was expressed to high level by these recombinant viruses. These recombinant viruses showed reduced virulence on rabbit skin and induced anti-HBs after intradermal inoculation of rabbits.     

Ultrasensitive potentiometric immunosensor based on SA and OCA techniques for immobilization of HBsAb with colloidal Au and polyvinyl butyral as matrixes

A novel potentiometric immunosensor for detection of hepatitis B surface antigen (HBsAg) has been developed by means of self-assembly (SA) and opposite-charged adsorption (OCA) techniques to immobilize hepatitis B surface antibody (HBsAb) on a platinum electrode. A cleaned platinum electrode was first pretreated in the presence of 10% HNO3 and 2.5% K2CrO4 solution and held at -1.5 V (vs SCE) for 1 min to make it negatively charged and then immersed in a mixing solution containing hepatitis B surface antibody, colloidal gold (Au), and polyvinyl butyral (PVB). Finally, HBsAb was successfully immobilized onto the surface of the negatively charged platinum electrode modified nanosized gold and PVB sol-gel matrixes. The modified procedure was characterized by electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV). The immobilized hepatitis B surface antibody exhibited direct electrochemical behavior toward hepatitis B surface antigen (HBsAg). The performance and factors influencing the performance of the resulting immunosensor were studied in detail. More than 95.7% of the results of the human serum samples obtained by this method were in agreement with those obtained by enzyme-linked immunosorbent assays (ELISAs). The resulting immunosensor exhibited fast potentiometric response (<3 min) to HBsAg. The detection limit of the immunosensor was 2.3 ng.mL(-1), and the linear range was from 8 to 1280 ng.mL(-1). Moreover, the studied immunosensor exhibited high sensitivity, good reproducibility, and long-term stability (>6 months).     

Translational Enhancer of Tobacco mosaic virus Enhancing Expression of Hepatitis B Surface Antigen in Transgenic Panax ginseng C. A. Meyer Callus

The 5＇-nontranslated leader（omega sequence） of Tobacco mosaic virus（TMV） was used as a translational enhancer sequence in the expression of the hepatitis B surface antigen（HBsAg） gene in transgenic ginseng callus cultures. The adr subtype HBsAg gene was placed under the control of the Cauliflower mosaic virus（CaMV） 35S promoter linking to the TMV leader sequence. The antisense omega sequence was used in a control construct. The resulting constructs cloned in the binary vector pBI121 were used to transform the ginseng callus tissue via the Agrobacterium-mediated procedure. The integration and expression of the HBsAg gene were evaluated by PCR and western blot, respectively. Enzyme-linked immunoassays（ELISA） using a monoclonal antibody directed against human serum-derived HBsAg revealed a three to four-fold enhanced expression of HBsAg in ginseng cells conferred by the TMV omega element.     

红细胞标记抗体的化学发光免疫分析

用红细胞代替辣根过氧化物酶作为双抗体夹心免疫分析中第二抗体的标记物, 建立了一种红细胞标记抗体的免疫化学发光测定乙型肝炎病毒表面抗原的新方法. 在免疫反应完成后, 结合了抗原-抗体免疫复合物的致敏红细胞在低渗溶液中溶血, 释放出血红蛋白. 基于血红蛋白对鲁米诺-H2O2体系化学发光具有催化作用的原理, 采用化学发光法测定血红蛋白含量. 测得的血红蛋白发光强度与待测抗原浓度呈线性关系. 采用这种方法可检测出0.5 ng/mL的乙型肝炎病毒表面抗原. 将该方法与酶联免疫吸附分析(ELISA)结合起来对乙型肝炎患者血清乙肝病毒表面抗原(HBsAg)进行检测, 两者符合率均为97%, 表明本法具有良好的灵敏度和特异性, 可用于临床标本测试.     

Synthesis of Novel Hydrophobic Media and Purification of Recombinant HBsAg

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Polydiacetylene/Anti‐HBs Complexes for Visible and Fluorescent Detection of Hepatitis B Surface Antigen on a Nitrocellulose Membrane

The immunochromatographic assay (ICA) using a nitrocellulose (NC) membrane offers several advantages. This technique is a rapid and straightforward method in contrast to other immunoassays. Polydiacetylene (PDA) vesicles have unique optical properties, displaying red color and red fluorescence at the same time. In this system, red‐phase PDA vesicles are used as a fluorescent dye as well as a surface for immobilized hepatitis B surface antibody (HBsAb). PDA has a remarkable stability compared with other fluorescent dyes. In this study, the most suitable PDA/HBsAb complexes are introduced for detecting hepatitis B surface antigen (HBsAg). Then, the PDA/HBsAb complexes affixed antibody is attached to NC membrane, which has two lines to confirm detection of HBsAg. The main advantage of this system is that the detection of HBsAg can be observed in both visible and fluorescent images due to the optical properties of polydiacetylene. Detection of HBsAg is observed up to 0.1 ng mL⁻¹ by fluorescent analysis and confirmed by red line on the NC membrane up to 1 ng mL⁻¹ (HBsAg) using the naked eye. Consequently, these results show that PDA/HBsAb complexes were successfully applied to ICA for the diagnosis of hepatitis B.     

乙肝疫苗注入后婴儿血液吞噬细胞吞噬功能变化的临床观察

为探讨乙开型肝炎表面抗原（ｈｅｐａｔｉｔｉｓ Ｂ ｓｕｒｆａｃｅ ａｎｔｉｇｅｎ,ＨＢｓＡｇ）阳性母亲的婴儿乙肝疫苗注入后早期机体免疫反应的特点,采用化我法测定了吞噬细胞的吞噬功能;用双抗原夹心法测定了抗－ＨＢｓ抗体。结果表明,（１）乙肝疫苗注入前两组间吞噬细胞的吞噬功能无明显差异,疫苗注入后３个月和６个月时实组外周血吞噬细胞的吞噬功能明显下降（Ｐ〈０．０５）。两组间同时间点相比,１个月和３个月都     

Label-free optical diagnosis of hepatitis B virus with genetically engineered fusion proteins

A simple biosensing strategy for the diagnosis of patients with hepatitis B virus (HBV) was developed. This study can be divided into two themes, both of which utilized gold-binding polypeptide (GBP) fusion proteins: HBV surface antigen PreS2 (HBsAg) detection with GBP-fused single chain antibody (GBP-ScFv) and anti-HBsAg detection with GBP-HBsAg. These GBP-fusion proteins can directly bind onto the gold surface via the high binding affinity between the GBP and the gold surface, while at the same time, orient the recognition sites toward the sample for target binding. This one-step immobilization strategy, which greatly simplifies a fabrication process as well as maintaining biological activity of the recognition elements, can be applied to optical analytical methods, such as surface plasmon resonance (SPR) and localized surface plasmon resonance (LSPR).     

Increased in vivo immunological potency of HB-110, a novel therapeutic HBV DNA vaccine, by electroporation

Pulse-induced permeabilization of cellular membranes, generally referred to as electroporation (EP), has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids, for gene therapeutic applications, and this technique has been shown to result in improved immunogenicity. In this study, we assessed the utility of EP as a tool to improve the efficacy of HB-110, a novel therapeutic DNA vaccine against chronic hepatitis B, now in phase 1 of clinical study in South Korea. The potency of HB-110 in mice was shown to be improved by EP. The rapid onset of antigen expression and higher magnitude of humoral and cellular responses in electric pulse-treated mice revealed that EP may enable a substantial reduction in the dosage of DNA vaccine required to elicit a response similar in magnitude to that achievable via conventional administration. This study also showed that EP-based vaccination at 4-week-intervals elicited a cellular immune response which was about two-fold higher than the response elicited by conventional vaccination at 2-week intervals. These results may provide a rationale to reduce the clinical dose and increase the interval between the doses in the multidose vaccination schedule. Electric pulsing also elicited a more balanced immune response against four antigens expressed by HB-110: S, preS, Core, and Pol.     

氯化血红素作为模拟酶荧光免疫分析乙肝表面抗原

乙肝表面抗原的测定在临床诊断上是一项很重要的指标．现在一般采用酶联吸附免疫分析技术测定，但是酶本身性质不稳定且价格昂贵、操作繁琐；更重要的是大分子的酶作为标记物，由于空间位阻效应而阻碍抗原-抗体的免疫反应．所以，用小分子催化剂代替大分子酶的研究显得日益重要[1，2]．近年来有关模拟酶在免疫分析中的应用已有报道[3，4]．本文提出了以氯化血红素作为辣根过氧化物酶（HRP）的模拟酶来标记抗体，以盐酸硫胺素（维生素B1）作为供氢体，成功地实现了乙肝表面抗原（HBsAg）的夹心法荧光免疫分析．测定范围是2．5～500ng／wel…     

Dot-immunobinding assay using indirect photoacoustic measurement

Dot-immunobinding assay of hepatitis B surface antigen (HBsAg) was performed by a conventionally dot-ELISA technique with 4-chloro-1-naphthol staining, and the quantitative results were measured by an indirect photoacoustic method. In this method, a 2-mW helium-neon laser was applied to provide the excitation beam, the blue spots on the membrane were detected in a piezoelectric transducer-based photoacoustic cell. The operational conditions of measurement were optimized. A significant difference from the negative human serum was obtained for 50 pg in HBsAg detection. The method proposed provides a sensitive quantitative technique for dot-immunobinding assay.     

Dot-immunobinding assay using indirect photoacoustic measurement

Dot-immunobinding assay of hepatitis B surface antigen (HBsAg) was performed by a conventionally dot-ELISA technique with 4-chloro-1-naphthol staining, and the quantitative results were measured by an indirect photoacoustic method. In this method, a 2-mW helium-neon laser was applied to provide the excitation beam, the blue spots on the membrane were detected in a piezoelectric transducer-based photoacoustic cell. The operational conditions of measurement were optimized. A significant difference from the negative human serum was obtained for 50 pg in HBsAg detection. The method proposed provides a sensitive quantitative technique for dot-immunobinding assay. Received: 2 November 1999 / Revised: 27 January 2000 / Accepted: 1 February 2000     

Behavior of Wild-type and Transfected S2 Cells Cultured in Two Different Media

An animal protein-free medium composed of IPL-41 containing 6 g L^?1 yeastolate ultrafiltrate, 10 g L^?1 glucose, 2 g L^?1 lactose, 5 g L^?1 glutamine, 1% lipid emulsion, and 0.1% Pluronic F-68 was used for producing recombinant proteins in batch mode employing two cell lines, S2AcRVGP2k expressing the G glycoprotein from rabies virus (RVGP) and S2AcHBsAgHy-9C expressing the surface antigen of hepatitis B virus (HBsAg), both obtained from Drosophila melanogaster S2 cells. Growth of wild-type S2 cells was also evaluated in the same medium. Cell behavior in the tested medium was compared to that verified in Sf900 II®. The results show that in shake flasks, S2AcRVGP2k and S2AcHBsAgHy-9C cells reached around 2?×?10⁷ cells mL^?1 in both media. In supplemented IPL-41 and Sf900 II® media, S2AcRVGP2k cells produced 367 ng RVGP mL^?1 and 638 ng RVGP mL^?1, respectively, while S2AcHBsAgHy-9C cells correspondently produced 573 ng HBsAg mL^?1 and 322 ng HBsAg mL^?1 in the mentioned media. In stirred tanks, S2AcRVGP2k cells reached 3?×?10⁷ cells mL^?1 and produced up to 758 ng RVGP mL^?1. In general, glucose was consumed by cells, while lactate and ammonia were produced.     

Highly sensitive potentiometric immunosensor for hepatitis B surface antigen diagnosis

Immunosenors are of great interest because oftheir potential utility as specific, simple, label-free anddirect detection techniques and reductions in size, costand time of analysis compared with conventional im-munoassay techniques. The immunoassays with …     

Application of high-speed counter-current chromatography for the isolation of antiviral eremophilenolides from Ligularia atroviolacea

Ligularia is mainly distributed in the western regions of China. Most of the species have been traditionally used in folk medicine for the treatment of hepatitis B, asthma, hemoptysis and pulmonary tuberculosis. In our continuation of research on antiviral components from traditional Chinese medicine, Ligularia atroviolacea was tested for inhibitory effects on hepatitis B virus (HBV). A bioassay-guided phytochemical examination of L. atroviolacea disclosed that its ethyl acetate extract, which was made up of two eremophilenolides, showed suppressive activity on the expression of HBV surface antigen (HBsAg) in the HepG2.2.15 cell line. Then a simple and effective preparative high-speed counter-current chromatography method was successfully developed for the isolation and purification of two main active metabolites, 8beta-hydroxyeremophil-3,7(11)-dien-12,8alpha;15,6alpha-diolide and 8beta-methoxyeremophil-3,7(11)-dien-12,8alpha;15,6alpha-diolide from the ethyl acetate extract of L. atroviolacea by a one-step separation using a two-phase solvent system composed of light petroleum (60-90 degrees C)-ethyl acetate-methanol-water (9:1:8:2, v/v/v). The chemical structures of the two eremophilenolides were identified by ESI-MS, (1)H-NMR and (13)C-NMR analysis. The anti-HBV activity of the two purified compounds was measured; both of them showed suppressive activity on the expression of HBsAg in the HepG2.2.15 cell line. The results support the continued and expanded exploitation and utilization of L. atroviolacea. Copyright (c) 2008 John Wiley & Sons, Ltd.