A transgenic mouse model of the chronic hepatitis B surface antigen carrier state

In an attempt to establish a model of the healthy carrier state in hepatitis B virus (HBV) infections, transgenic mice expressing HBV genes were produced. Fertilized one-cell eggs were microinjected with subgenomic fragments of HBV DNA containing the coding regions for the HBV surface antigen (HBsAg) and pre-S and X antigens. Either the normal (HBV) or metallothionein promoters were used to obtain expression of the HBV genes. There was no evidence of viral replication or tissue pathology. The integrated HBV DNA sequences were inherited in a normal Mendelian fashion. Three of 16 transgenic mice expressed HBV-encoded gene products to which they were immunologically tolerant. Expression was not tissue specific and may be influenced by the genomic integration site and cellular factors. Both HBsAg and pre-S antigen were detectable within the cytoplasm of hepatocytes and renal tubular epithelial cells. High serum concentrations of HBsAg were detectable and the secreted product appeared authentic as judged by mean density, morphology, mean particle diameter, polypeptide composition, and antigenicity. The absence of tissue pathology in these immunologically tolerant animals supports the hypothesis that cellular injury under these conditions is not a direct consequence of expression of the pre-S or HBs regions of the HBV genome.     

Antibodies to hepatitis B surface antigen potentiate the response of human T lymphocyte clones to the same antigen

Human t-helper lymphocyte clones specific for hepatitis B virus surface antigen (HBsAg) proliferate on stimulation with HBsAg in vitro. Antibodies specific for HBsAg, but no other antibodies, augment this proliferative response. In the presence of antibodies to HBsAg, the maximum response could be achieved at HBsAg concentrations that were 1 percent of those required in the absence of the antibodies. These findings suggest that antigen-specific antibodies exert regulatory controls on T cells that recognize the same antigens.     

A poliovirus neutralization epitope expressed on hybrid hepatitis B surface antigen particles

The hepatitis B virus (HBV) envelope protein carrying the surface antigen (HBsAg) is assembled with cellular lipids in mammalian cells into empty viral envelopes. In a study to evaluate the capacity of such particles to present foreign peptide sequences in a biologically active form, in-phase insertions were created in the S gene encoding the major envelope protein. One of the sequences inserted was a synthetic DNA fragment encoding a poliovirus neutralization epitope. Mammalian cells expressing the modified gene secreted hybrid particles closely resembling authentic 22-nanometer HBsAg particles. These particles reacted with a poliovirus-specific monoclonal antibody and induced neutralizing antibodies against poliovirus. The results indicate that empty viral envelopes of HBV may provide a means for the presentation of peptide sequences and for their export from mammalian cells.     

Inhibition of secretion of hepatitis B surface antigen by a related presurface polypeptide

The presurface (preS) proteins of hepatitis B virus are structural components of the viral envelope that may play important roles in virion assembly and infectivity. They are specified by a large open reading frame that includes the coding region for the major surface (S) protein in its 3' half. Translation of the preS proteins initiates upstream from the S region, giving rise to proteins that are composed of the S domain and an additional 163 (preS1) or 55 (preS2) amino acids. Little is known about the biosynthesis and assembly of these proteins. The expression of the S and preS1 proteins was examined by transfecting cultured mammalian cells with viral DNA and injecting synthetic messenger RNA's into Xenopus oocytes. In contrast to the proteins encoded by the S region, the preS1 proteins are not detectably secreted into the culture medium. Furthermore, when the S and preS1 proteins are synthesized together, secretion of the S proteins is specifically and strongly inhibited. The results suggest a unique molecular interaction during secretion of the S and preS proteins that may be important for virus assembly.     

乙肝病毒表面抗原基因转化番茄

将乙肝病毒表面抗原 (HBs Ag)基因与 Ca MV3 5 s启动子及 nos终止子构建植物表达载体 p1 3 0 1 HBs,直接法转入根癌农杆菌 EHA1 0 5 (Agrobacterium tumefaciens) ,以该菌株介导叶盘法转化番茄 ,得到抗潮霉素的再生植株 .抗性苗总 DNA经 PCR、Southern斑点杂交证实目的基因已整合到番茄基因组中 ,EILSA检测证明在番茄中正确表达了乙肝表面抗原蛋白 .     

Specific expression of hepatitis B surface antigen (HBsAg) in transgenic mice

Two transgenic mice were obtained that contain in their chromosomes the complete hepatitis B virus (HBV) genome except for the core gene. These mice secrete particles of HBV surface antigen (HBsAg) in the serum. In one mouse, HBV DNA sequences that had integrated at two different sites were shown to segregate independently in the first filial generation (F1) and only one of the sequences allowed expression of the surface antigen. Among these animals the males produced five to ten times more HBsAg than the females. A 2.1-kilobase messenger RNA species comigrating with the major surface gene messenger RNA is expressed specifically in the liver in the two original mice. The results suggest that the HBV sequences introduced into the mice are able to confer a tissue-specific expression to the S gene. In addition, the HBV transgenic mice represent a new model for the chronic carrier state of hepatitis B virus infection.     

Human hepatocellular carcinoma cell lines secrete the major plasma proteins and hepatitis B surface antigen

Analysis of the cell culture fluid from two new human hepatoma-derived cell lines reveals that 17 of the major human plasma proteins are synthesized and secreted by these cells. One of these cell lines, Hep 3B, also produces the two major polypeptides of the hepatitis B virus surface antigen. When Hep 3B in injected into athymic mice, metastatic hepatocellular carcinomas appear. These cell lines provide experimental models for investigation of plasma protein biosynthesis and the relation of the hepatitis B viru genome to tumorigenicity.     

应用昆虫杆状病毒载体在昆虫细胞中表达乙肝表面抗原

人乙型肝炎病毒（ａｄｒ亚型）表面抗原Ｓ基因克隆到ＰｕＡＣ－５苜蓿尺蠖核型多角体病毒转移载体上，所构建的重组转移载体质粒与野生型ＡｃＮＰＶＤＮＡ共转染Ｓｆ－２１细胞，经空斑法筛选和纯化，得到了含ＨＢＶＳ基因的重组病毒ＡｃＮＰＶＳ．用放射免疫法测定了ＡｃＮＰＶＳ感染的Ｓｆ－２１细胞及其培养液中的ＨＢｓＡｇ，总表达量为１．２２μｇ／１０￣６细胞．     

Location and chemical synthesis of a pre-S gene coded immunodominant epitope of hepatitis B virus

Immunodominant, disulfide-bond independent epitopes recognized by human antibodies to hepatitis B virus (HBV) are located within the 55-residue amino terminal portion (coded for by the pre-S region of HBV DNA) of minor HBV envelope components larger than the major protein constituents encoded by the S gene. A peptide having the sequence of the first 26 amino acids from the amino terminal methionine was synthesized and elicited antibodies (at dilutions of greater than or equal to 1 to 10(5) ) to the HBV envelope. These antibodies can be utilized for diagnostic tests. The immunogenicity of the peptide was substantially increased by covalent attachment to liposomes. The disulfide bond-independent determinants on sequences coded for by the pre-S gene may be more easily mimicked by peptide analogs than "conformational" determinants on the S-gene product.     

Australia antigen (hepatitis B antigen): a conformational antigen dependent on disulfide bonds

Reduction and alkylation of purified hepatitis-associated Australia antigen (hepatitis B antigen) resulted in a total loss of serologic activity. The reduced and alkylated protein formed a single band with a sedimentation coefficient of 31S on analytical ultracentrifugation, and no subunits were detected by Sephadex gel filtration. Although this preparation induced a delayed hypersensitivity response when injected into guinea pigs, it failed to stimulate humoral antibody formation. The data suggest that hepatitis B antigen is a conformational antigen critically dependent upon the disulfide bonds of the protein moiety.     

A liver-specific enhancer in the core promoter region of human hepatitis B virus

An 88-base pair fragment in the core promoter of the human hepatitis B virus (HBV) contains a functional promoter and a strong liver-specific enhancer. This enhancer functions in human hepatoma cells, where it is much more active than the previously described HBV enhancer in stimulating expression of the linked bacterial chloramphenicol acetyltransferase gene expressed from heterologous promoters. Studies of the role of this enhancer-promoter in HBV may help to clarify mechanisms of gene expression in cells infected with HBV and the role of the virus in the pathogenesis of hepatitis and hepatocellular carcinoma.     

Proteolytic self-cleavage of hepatitis B virus core protein may generate serum e antigen

A model is proposed to explain the presence of the e antigen (HBeAg) of hepatitis B virus (HBV) in the serum of individuals infected with this virus. The e antigen, which has only recently been characterized, is a fragment of the virus core, or nucleocapsid, protein. Serum HBeAg is a valuable clinical marker for active HBV infection because its appearance correlates both with virus replication in the liver and with the presence of circulating virions. In this study a protease-like amino acid sequence was identified at the amino terminus of the core protein sequence. Experimental evidence indicates that HBeAg may be produced by proteolytic self-cleavage of the core protein.     

The nucleocapsid of hepatitis B virus is both a T-cell-independent and a T-cell-dependent antigen

One characteristic of the immune response during hepatitis B virus (HBV) infection in humans is the vigorous production and subsequent persistence of antibodies of immunoglobulin (Ig) classes M and G to the nucleocapsid antigen (HBcAg). In this study HBcAg was shown to be similarly immunogenic in mice. When injected into athymic (nude) B10.BR and athymic BALB/c mice, HBcAg induced IgM and IgG class antibodies to HBc in spite of the absence of T cells in nude mice. In euthymic mice, HBcAg efficiently stimulated T-cell proliferation in vitro and helper T-cell function in vivo. The dual functions of HBcAg as a T-cell-independent and a T-cell-dependent antigen may explain its enhanced immunogenicity. Denaturation of HBcAg yields a nonparticulate antigen designated HBeAg; when HBeAg was used as the immunogen, antibody production required helper T-cell function. Although HBcAg and HBeAg are serologically distinct, they are structurally related, and in these experiments were highly cross-reactive at the T-cell level. These results suggest that the elevated levels of IgM antibodies to HBc and the enhanced immunogenicity of HBcAg during HBV infection in humans reflect the ability of HBcAg to directly activate B cells to produce antibodies to HBc in the presence or absence of HBcAg- or HBeAg-sensitized T cells.     

Antibodies to peptides detect new hepatitis B antigen: serological correlation with hepatocellular carcinoma

The expression of a previously unidentified gene product, encoded by the hepatitis B virus (HBV) genome, has been achieved with a recombinant SV40 expression vector. Antibodies against synthetic peptides representing defined regions of this protein were used to screen cells infected with recombinant virus as well as tissues naturally infected with HBV. A 24,000-dalton protein (p24) was detected in cells infected with recombinant virus and a 28,000-dalton protein (p28) was detected in tissues infected with HBV. The peptides or recombinant-derived protein were used as antigens to screen sera from individuals infected with HBV. Specific antibodies were detected predominantly in sera from patients with hepatocellular carcinoma. The presence of p28 in tissues infected with HBV and the appearance of specific antibodies in infectious sera establish the existence of an additional marker for HBV infection.     

急性乙型病毒性肝炎肝组织中Fas抗原和Fas配体的表达

探讨Ｆａｓ－ＦａｓＬ系统在急性病毒性肝炎发病中的作用。方法采用免疫组织化学技术对３８例急性乙型肝炎患者组织中Ｆａｓ和ＦａｓＬ表达进行检测,并与１０例正常肝组织作对照。结论：由ＣＴＬ－ＦａｓＬ系统介导的肝细胞凋亡在急性乙型肝炎的发病中可能起了较重要的作用。     

猪O型口蹄疫病毒抗原表位/VP1蛋白的融合表达及免疫原性研究

目的 获得可稳定表达猪O型口蹄疫病毒(Foot-and-mouth disease virus,FMDV)抗原表位融合结构蛋白VP1的中国仓鼠卵巢细胞株(CHO-K1),制备亚单位疫苗。方法 设计合成含FMDV抗原表位与VP1基因序列的重组基因RP1,将其克隆到表达载体pCDH-CMV-MCS-EF1-Puro中,将构建的重组质粒与辅助质粒PLP1、PLP2和PLP3共转染HEK-293T细胞,获得重组慢病毒HIV-RP1;将收获的病毒液感染CHO-K1细胞,经筛选获得单克隆细胞株,通过间接免疫荧光试验(Indirect immunofluorescence assay,IFA)和Western blot鉴定,获得可表达RP1的阳性细胞株,命名为CHO-K1-RP1;将CHO-K1-RP1连续传30代,每隔5代收获相同数量的细胞样品进行Western blot鉴定。结果 IFA结果显示,表达RP1的细胞发出绿色荧光,而空白对照无绿色荧光;Western blot结果显示,在约55 kU处能观察到清晰的条带;表明成功获得了融合蛋白。将获得的融合蛋白与佐剂等体积混合制备成亚单位疫苗免疫BALB/c雌鼠,抗体检测结果显示,二次免疫后,该亚单位疫苗组与口蹄疫(Foot-and-mouth disease,FMD)商品化灭活疫苗组小鼠之间抗体水平无显著差异,两者抗体水平均显著高于对照组(P<0.05)。结论 本研究构建的亚单位疫苗能有效刺激小鼠机体产生免疫应答反应,为猪口蹄疫新型疫苗的研制提供了参考。     

Expression of the major surface antigen of Plasmodium knowlesi sporozoites in yeast

The circumsporozite protein, a surface antigen of the sporozoite stage of the monkey malarial parasite Plasmodium knowlesi, was expressed in the yeast Saccharomyces cerevisiae by using an expression vector containing the 5' regulatory region of the yeast alcohol dehydrogenase I gene. It was necessary to eliminate the entire 5' upstream region of the parasite DNA to obtain the expression of this protein. Only the circumsporozoite precursor was produced by the yeast transformants, as detected by immunoblotting. About 55 and 20 percent of the circumsporozoite protein produced in yeast was associated wtih the 25,000 g and 150,000 g particulate fractions, respectively. The protein could be solubilized in Triton X-100 and was stable in solubilized extracts.