The secretory core protein of human hepatitis B virus is expressed on the cell surface.

Human hepatitis B virus (HBV) causes acute and chronic liver disease, which can result in tumor formation. An as yet unexplained phenomenon is that virus elimination usually correlates with the development of antibodies directed against the HBeAg, a secretory HBV core gene product which can be detected in the serum of infected patients. Expression of HBeAg in a human hepatoma cell line by using recombinant vaccinia viruses revealed that the HBeAg is not only secreted from HBeAg-producing cells but also incorporated into the outer cell membrane. No membrane-expressed core gene product could be detected when the cytoplasmic core protein (HBcAg) was expressed. Immune sera from patients who developed anti-HBe antibodies efficiently recognized the membrane-bound HBeAg, suggesting that surface-expressed HBeAg can serve as a target for an antibody-mediated elimination of HBV-infected cells.     

Hepatitis B virus large surface protein is not secreted but is immunogenic when selectively expressed by recombinant vaccinia virus.

The envelope region of the hepatitis B virus (HBV) genome contains an open reading frame that begins upstream of the major surface protein gene. The two minor proteins that are initiated within this pre-s segment are immunogenic and may be involved in virus attachment to hepatocytes. We have constructed a recombinant vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus that contains the predicted coding segment for the large surface protein (LS) under control of a vaccinia virus promoter. Cells infected with the recombinant virus synthesized HBV polypeptides of 39 and 42 kilodaltons, corresponding to the unglycosylated and glycosylated forms of LS, respectively. The presence of pre-s epitopes in the 39- and 42-kilodalton polypeptides was demonstrated by binding of antibody prepared against a synthetic peptide. Synthesis of the 42-kilodalton species was specifically inhibited by tunicamycin, suggesting that it is N-glycosylated. Despite apparent glycosylation, LS was not secreted into the medium of infected cells. Nevertheless, rabbits vaccinated with the purified recombinant virus made antibodies that recognized s and pre-s epitopes. Antibody to the NH2 terminus of LS appeared before or simultaneously with antibody that bound to the major surface protein. The additional immunogenicity provided by expression of LS may be advantageous for the development of an HBV vaccine.     

Monoclonal antibody against non-dominant epitopes of HBV e Ag was raised by antigen-antibody co-immunization

Detection of hepatitis B e antigen (HBeAg) in the sera of individuals infected with hepatitis B virus (HBV) can indicate both a high infectivity of the disease and a poor prognosis of disease treatment. Most of monoclonal antibodies raised against HBV e proteins interact with immuno-dominant epitopes, such as HBeAg-beta. In order to raise antibodies against non-dominant epitopes of HBV e protein, in this study, mice were immunized with both recombinant HBeAg (rHBeAg) and an anti-HBeAg antibody (EWB) recognizing a dominant antigenic epitope of HBeAg (HBeAg-beta epitope). With this strategy, we successfully selected two monoclonal antibodies, S-29-3 and S-72-3. Both S-29-3 and S-72-3 bind to recombinant HBeAg with a high affinity. The epitope mapping assay determined that the S-73-2 recognizes the N-terminal of HBeAg (1-118 aa) and the S-29-3 recognizes the C-terminal of HBeAg (91-149 aa). Further experiment showed that these two antibodies could be formed a pair-Abs that is used in detecting native HBeAg from the sera of HBV patients. The conclusion is that the developed method is useful to raise mAb against non-dominant epitopes in given Ag.     

Inhibition of African swine fever virus binding and infectivity by purified recombinant virus attachment protein p12.

The African swine fever virus protein p12, involved in virus attachment to the host cell, has an apparent molecular mass of 17 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. We have also identified 12- and 10-kDa forms of the p12 protein in infected Vero cells and found that the mature 17-kDa protein is the only form present in virus particles. The p12 protein has been produced in large amounts in Spodoptera frugiperda insect cells infected with a recombinant baculovirus. A 17-kDa protein that possessed the biological properties of the viral protein was produced, since it bound to susceptible Vero cells and not to receptor-negative L cells, which do not support virus replication. The binding of the baculovirus-expressed protein p12 to Vero cells was specifically blocked by virus particles. In addition, the recombinant protein purified by immunoaffinity chromatography blocked the specific binding of virus particles to susceptible cells and prevented infection, demonstrating that the p12 protein mediates the attachment of virions to specific receptors and indicating that blocking the p12-mediated interaction between African swine fever virus and receptors in Vero cells can inhibit infection. However, although antibodies specific for protein p12 are induced in natural infections and in animals inoculated with inactivated virus or recombinant protein p12, these antisera did not inhibit virus binding to the host cell or neutralize virus infectivity.     

表达尼帕病毒G囊膜糖蛋白重组牛痘病毒的研究

采用牛痘病毒WR株,构建了表达哺乳动物密码子优化的NiV G蛋白基因的重组病毒rWR-NiV-G。Westernblot证实大小为66kDa的重组G蛋白在rWR-NiV-G感染的Hela细胞中获得表达;采用兔抗NiV高免血清间接免疫荧光检测重组痘病毒表达G蛋白显示出良好的特异免疫反应原性。rWR-NiV-G感染NiV敏感的BHK细胞系,并与NiV融合蛋白F共同表达,可形成强烈细胞融合现象。rWR-NiV-G感染免疫BALB/c小鼠,可诱导显著的NiV G蛋白特异体液免疫反应。以原核表达NiV G蛋白片段为包被抗原,间接ELISA检测rWR-NiV-G感染免疫小鼠血清中的G蛋白特异抗体,具有良好的敏感性和特异性。同时,rWR-NiV-G感染免疫小鼠血清中的G蛋白特异抗体可有效中和NiV囊膜蛋白F和G介导的伪型VSV重组病毒侵入NiV易感宿主细胞的感染性。结果表明,重组牛痘病毒表达的NiV G蛋白有良好的免疫原性和生物学活性功能,为进一步深入研究NiV G蛋白生物学功能、免疫原性及重组活载体疫苗研究奠定了重要基础。     

一种新的乙型肝炎病毒表面抗原结合蛋白的筛选、表达及其生物学活性的初步研究

为了研究乙肝病毒侵染肝细胞过程中的功能蛋白 ,通过印迹免疫分析技术从人肝cDNA噬菌体表达库中筛选出一株编码乙肝表面抗原结合蛋白 (hepatitisBsurfaceantigenbindingprotein ,HBsAg BP)的cDNA克隆 .基因测序结果表明 ,该cDNA具有独立的开放阅读框架 ,编码 1个由 344个氨基酸残基构成的可溶性蛋白分子 ,属于免疫球蛋白超家族成员 .将该基因克隆到原核表达载体pTriplEx后 ,在E .coliXL1 Blue菌株中获得 4 4kD的重组蛋白 .重组蛋白经Western印迹和ELISA实验证明具有与乙肝表面抗原特异性结合的能力 .进一步经流式细胞仪实验显示 ,在纯化的重组蛋白存在的情况下 ,天然的HBsAg与肝细胞株HepG2的亲和力显著增高 .结果显示 ,该乙肝表面抗原结合蛋白可能是介导乙肝病毒对肝细胞亲和侵染的可溶性辅助受体 .     

乙型肝炎病毒preC／C基因在杆状病毒载体系统中的表达

通过ＰＣＲ获得长度为６４０ｂｐ的ＨＢＶ ｐｒｅＣ／Ｃ基因片段,将其克隆入转移载体质粒ｐＳＸＩＶＶＩ＾＋Ｘ３／４,构建出重组质粒ｐＳＸＩＶＶＩ＾＝Ｘ３／４－ｐＣ＝Ｃ。利用共转染的方法,构建出既能形成多角体又能表达ｐｒｅＣ／Ｃ基因的重组毒株ＴｎＮＰＶ－Ｃ／Ｃ－ＯＣＣ＾＋。该重组毒株中ｐｒｅＣ／Ｃ基因受到串联的双启动子－合成启动子和含ＨｉｎｄⅢ接头的ＸＩＶ启动子的双重调控。     

Expression of the M gene of vesicular stomatitis virus cloned in various vaccinia virus vectors.

Initial attempts to clone the matrix (M) gene of vesicular stomatitis virus (VSV) in a vaccinia virus expression vector failed, apparently because the expressed M protein, and particularly a carboxy-terminus-distal two-thirds fragment, was lethal for the virus recombinant. Therefore, a transient eucaryotic expression system was used in which a cDNA clone of the VSV M protein mRNA was inserted into a region of plasmid pTF7 flanked by the promoter and terminator sequences for the T7 bacteriophage RNA polymerase. When CV-1 cells infected with recombinant vaccinia virus vTF1-6,2 expressing the T7 RNA polymerase were transfected with pTF7-M3, the cells produced considerable amounts of M protein reactive by Western blot (immunoblot) analysis with monoclonal antibodies directed to VSV M protein. Evidence for biological activity of the plasmid-expressed wild-type M protein was provided by marker rescue of the M gene temperature-sensitive mutant tsO23(III) at the restrictive temperature. Somewhat higher levels of M protein expression were obtained in CV-1 cells coinfected with a vaccinia virus-M gene recombinant under control of the T7 polymerase promoter along with T7 polymerase-expressing vaccinia virus vTF1-6,2.     

DNA-binding activity of hepatitis B e antigen polypeptide lacking the protaminelike sequence of nucleocapsid protein of human hepatitis B virus.

The characteristics of binding of hepatitis B core antigen (HBcAg) and hepatitis B e antigen (HBeAg) polypeptides to hepatitis B virus (HBV) DNA were analyzed. HBcAg polypeptide from recombinant HBV core particles and HBeAg polypeptide from partially purified serum HBeAg were prepared and verified to have molecular weights of 21,500 (P21.5) and of 17,000 (P17) and 18,000 (P18), respectively, by immunoblot analysis. By reaction of these proteins on a nitrocellulose membrane with cloned 32P-HBV DNA, it was revealed that the HBeAg polypeptide, which lacks the C-terminal 34 amino acids of P21.5, as well as the HBcAg polypeptide, bound to the DNA. The secondary structures of nucleocapsid proteins of HBV, woodchuck hepatitis virus, and ground squirrel hepatitis virus were predicted by the Garnier algorithm. Amino acid sequences which, in addition to those of the C-terminal regions, may contribute to binding were proposed to be the 21-amino-acid residues located at amino acids 100 to 120 of the nucleocapsid proteins of these hepadnaviruses.     

Distinctive properties of the hepatitis B virus envelope proteins.

Using recombinant adenoviral vectors, we expressed and characterized the large, middle, and major envelope proteins of hepatitis B virus (HBV). Cells infected with the recombinant adenovirus which contained the large envelope gene (HS1.HP) expressed predominantly large envelope and small but detectable quantities of middle (4%) and major (6%) envelope proteins in the cell lysate. No HBV envelope proteins were detected in the culture medium from HS1.HP-infected cells. Cells infected with recombinant adenovirus which contained the middle envelope gene (HS2.HP) expressed and secreted the middle and major envelope proteins in a molar ratio of 3:1. Cells infected with the recombinant adenovirus which contained the major envelope gene (HS.HP) expressed and secreted major envelope proteins. The HBV envelope proteins secreted by cells infected with either HS2.HP or HS.HP were assembled in 22-nm particles, as shown by velocity sedimentation rate determination, buoyant densities, and electron microscopy. Cells coinfected with a recombinant adenovirus which contained the large envelope gene and with either HS2.HP or HS.HP expressed similar quantities of the large, middle, and major envelope proteins in the cell lysates. Secretion of the major and middle envelope proteins was inhibited more than 95% by the presence of the large envelope proteins. These results suggest that differential biosynthesis, transport, and processing of the envelope proteins occur during HBV infection, allowing efficient assembly and secretion of virions and hepatitis B surface antigen particles.     

Recombinant expression of Garlic virus C (GARV-C) capsid protein in insect cells and its potential for the production of specific antibodies

Garlic cultivars in Brazil are infected by a complex of viruses and for some virus species, such as the allexivirus, purification of the virions is sometimes cumbersume. To overcome this problem, recombinant expression of viral proteins in heterologous systems is an alternative method for producing antibodies. The capsid gene from Garlic virus C (GarV-C), an Allexivirus, was inserted into the genome of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) generating the recombinant virus vSynGarV-C. The recombinant protein expression was confirmed by SDS-PAGE and western-blot of extracts from recombinant virus infected insect cells, where a protein band of approximately 32 KDa was observed only in extracts from recombinant infected cells. This protein corresponded to the predicted size of the capsid protein of the GarV-C. A rabbit polyclonal antibody was raised against this protein, shown to be specific for the GarV-C protein in western-blot and dot-Elisa, however with a low titer.     

甲3型流感病毒M2基因在痘苗病毒天坛株中的表达

为获得表达甲3型流感病毒(H3N2)M2蛋白的重组天坛株痘苗病毒RVJ1175M2,使用PCR方法扩增流感病毒全长M2基因,将其克隆到天坛株痘苗病毒同源重组质粒pJSC1175中,获得重组质粒pJSC1175M2,通过与痘苗病毒载体同源重组,构建了含流感病毒M2基因的重组痘苗病毒株RVJ1175M2。PCR检测结果证明,流感病毒(H3N2)M2蛋白基因准确插入到天坛株痘苗病毒TK区;Western blot、免疫荧光和流式细胞计数表明重组病毒RVJ1175M2可以有效地表达M2蛋白,表达的M2蛋白有两条带,分别为15kD和13kD,与相关文献报道一致;M2蛋白可有效分布在感染细胞的细胞膜上。这些结果表明重组痘苗病毒株RVJ1175M2可以有效地表达流感病毒M2蛋白,为使用表达M2蛋白的不同类型疫苗进行广谱流感疫苗效果的比较研究奠定了基础。     

Real-time analysis of the interaction of a multiple-epitope peptide with antibodies against classical swine fever virus using surface plasmon resonance

The E2 envelope glycoprotein is the major immunodominant protein of classical swine fever virus (CSFV), and can induce neutralizing antibodies and protective immune responses in infected swine. We developed a tandem-repeat multiple-epitope recombinant protein that contains two copies of each of the regions of E2 spanned by residues 693-704, 770-780, and 826-843, coupled by two copies of the region spanned by residues 1446-1460 of the CSFV nonstructural protein NS2-3. The chemically synthesized gene was expressed in Escherichia coli as a fusion with glutathione S-8 (GST), named GST-BT21. After it was purified with Glutathione Sepharose 4B, we used Western blotting to characterize the construct and surface plasmon resonance to analyze its affinity and specific interaction with CSFV-positive serum. Purified GST-BT21 protein displayed excellent immunoreactivity with antiserum against CSFV (Tian et al., 2012), and surface plasmon resonance confirmed the specific affinity between BT21, but not GST, and antibodies in serum from animals infected with CSFV. Surface plasmon resonance is a sensitive and precise method for epitope evaluation, and it can be used to characterize the immunogenicity and functions of recombinant proteins.     

Transient expression of hepatitis B virus surface antigen (HBsAg) gene in monkey cells by a SV40-based virus vector

Summary We have constructed a recombinant SV40-based vector carrying the S gene coding for the hepatitis B virus surface antigen (HBsAg). This vector replicates as an episome in monkey COS7 cells, producing high levels of hepatitis B virus surface antigen (HBsAg), which is liberated in the cell medium, probably as a membrane vesicule. The vector also carries the SV40-late genes and produces recombinant viruses. These viruses were used to infect fresh cell culture, with detection of HBsAg in the medium. Thus, this virus vector can efficiently transduce the gene for HBsAg.     

鸡传染性支气管炎病毒S1基因在昆虫细胞中表达水平初探

将含有鸡传染性支气管炎病毒 Ｓ１ 基因ｃ Ｄ Ｎ Ａ 的重组转移质粒ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３ Ｓ１ ． Ｈｏｌｔｅ 和ｐ Ｓ Ｘ Ｉ Ｖ Ｖ Ｉ＋ Ｘ３／４ Ｓ１ ． Ｈｏｌｔｅ 分别与粉纹夜蛾核型多角体病毒 Ｔｎ Ｎ Ｐ Ｖ Ｓ Ｖ Ｉ－ Ｇ Ｄ Ｎ Ａ（ Ｏ Ｃ Ｃ－ ,ｇａｌ＋ ） 共转染草地夜蛾（ Ｓｆ９） 细胞,经空斑纯化得到重组病毒 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 和 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 。将重组毒株分别感染 Ｔｎ５ Ｂ１ 细胞,并进行 Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 与 Ｗｅｓｔｅｒｎｂｌｏｔ 检测。结果表明, Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３／４） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 在感染的细胞中高效表达了 Ｓ１ 蛋白, Ｓ Ｄ Ｓ Ｐ Ａ Ｇ Ｅ 凝胶薄层色谱分析结果显示,感染病毒后７２ ｈ Ｓ１ 蛋白的表达量占细胞内总蛋白量的３５ ．８ ％ ,而 Ｔｎ Ｎ Ｐ Ｖ（ Ｘ３） Ｓ１ ． Ｈｏｌｔｅ Ｏ Ｃ Ｃ＋ 感染的细胞内检测不出 Ｓ１ 蛋白。经分析认为这一差异主要来自 Ｓ１ 基因翻译起始位点及其附近的周围环境。     

Characterization of humoral and CD4+ cellular responses after genetic immunization with retroviral vectors expressing different forms of the hepatitis B virus core and e antigens.

The humoral and CD4+ cellular immune responses in mice following genetic immunization with three retroviral vectors encoding different forms of hepatitis B virus core antigen (HBcAg) and e antigen (HBeAg) were analyzed. The retroviral vectors induced expression of intracellular HBcAg (HBc[3A4]), secreted HBeAg (HBe[5A2]), or an intracellular HBcAg-neomycin phosphoryltransferase fusion protein (HBc-NEO[6A3]). Specific antibody levels and immunoglobulin G isotype restriction were highly dependent on both the host major histocompatibility complex and the transferred gene. Humoral and CD4+ cellular HBcAg and/or HBeAg (HBc/eAg)-specific immune responses following retroviral vector immunization were of a lower magnitude but followed the same characteristics compared with those after immunization with HBc/eAg in adjuvant. Two factors influenced the humoral responses. First, in vivo depletion of CD8+ cells in HBc-NEO[6A3]-immunized H-2k mice abrogated both HBcAg-specific antibodies and in vitro-detectable cytotoxic T lymphocytes. Second, priming of H-2b mice with an HBc/eAg-derived T-helper (Th) peptide in adjuvant prior to retroviral vector immunization greatly enhanced the HBc/eAg-specific humoral responses to all three vectors, suggesting that insufficient HBc/eAg-specific CD4+ Th-cell priming limits the humoral responses. In conclusion, direct injection of retroviral vectors seems to be effective in priming HBc/eAg-specific CD8+ but comparatively inefficient in priming CD4+ Th cells and subsequently specific antibodies. However, the limited HBc/eAg-specific CD4+ cell priming can effectively be circumvented by prior administration of a recombinant or synthetic form of HBc/eAg in adjuvant.     

N端融合外源蛋白的兔出血症病毒衣壳蛋白自聚能力的研究

将兔出血症病毒衣壳蛋白VP6 0基因插入杆状病毒转移载体pBLUEBACHIS2_B的 6 HIS表达标签下游 ,与线性化野生型杆状病毒基因组DNA共转染Sf9昆虫细胞 ,经蚀斑纯化后获克隆化重组杆状病毒pBLUEBACHIS2B_VP6 0。以重组杆状病毒感染Sf9细胞 ,经SDS_PAGE和Westernblot检测显示高效表达一分子量为 6 9kD的重组蛋白 ,并且该蛋白可被兔抗RHDV高免血清识别。血凝试验表明 ,该重组蛋白可以凝集人“O”型红细胞 ,血凝价达 2 1 6 ,同时 ,该血凝性可被抗RHDV的高免血清所抑制。经电镜观察 ,重组病毒表达的融合有 6 HIS表达标签的衣壳蛋白仍可在昆虫细胞内自聚成不包裹核酸的、与天然RHDV病毒粒子在物理形态上相似的病毒样颗粒 (VLPs) ,并且该VLPs与兔抗RHDV高免血清作用后于电镜下可见凝集成团的现象 ,表明其与天然RHDV病毒粒子在抗原性上也极为相似