噬菌体展示技术筛选副猪嗜血杆菌外膜蛋白P5的抗原表位

为了研究副猪嗜血杆菌（HPS）外膜蛋白P5的抗原表位,应用噬菌体展示随机12肽库,以抗HPS外膜蛋白P5的单克隆抗体作为固相分子,进行了3轮筛选。对随机挑选出的10个分离间隔良好的噬菌斑进行ELISA和West—ernblot等检验,最终确定了4个各结果均为阳性的优势短肽。将4个短肽序列与GenBank中HPS外膜蛋白p5的序列进行比对分析,发现这些序列没有同源性或同源性较低,但竞争ELISA结果显示这些短肽与单抗的结合都能够被外膜蛋白P5有效的抑制。以上结果显示,通过噬菌体展示技术成功获得了4个HPS外膜蛋白P5的模拟表位,为后期开展以抗原表位为基础的诊断、表位疫苗等研究提供了依据。     

Protein display by bovine herpesvirus type 1 glycoprotein B

Glycoprotein B (gB) of bovine herpesvirus 1 (BHV-1), a major component of the viral envelope, is essential for membrane fusion during entry and cell-to-cell spread. It is cleaved in the trans-Golgi network by the proprotein convertase furin. Integration of the open reading frame (ORF) encoding a mutated gB with a second furin cleavage site and mature boIFN-α as intervening peptide between the amino-terminal (NH₂) and carboxy-terminal (COOH) gB subunits yielded recombinant BHV-1/gB2FuIFN-α which, unexpectedly, express gB with an enlarged NH₂-subunit of 90 kDa. Here we show that boIFN-α-specific antibodies bind to the 90 kDa gB subunit and efficiently neutralize BHV-1/gB2FuIN-α infectivity. We also show that inactivated BHV-1/gB2FuIN-α virions induce an antiviral state in cells incubated with UV-inactivated particles. These results demonstrate that the 90 kDa protein is a NH₂-subunit/boIFN-α fusion protein whose boIFN-α domain is biologically active. To verify that BHV-1 gB is suitable for the display of (glyco)proteins on the surface of virions we constructed BHV-1 recombinants expressing within gB the first 273 amino acids of the NH₂-subunit (HA1) of avian influenza haemagglutinin, either flanked by two furin cleavage sites or with only one cleavage site between a gB/NH₂_HA1 fusion protein and the COOH subunit. The resulting recombinant BHV-1/gB2FuHA1 expressed gB from which 55 kDa HA1 was excised and secreted. In contrast, gB from BHV-1/gB_NH₂HA1 infected cells retained HA1 as fusion protein with the NH₂-subunit. Immunoblotting and neutralization analyses revealed that HA1 is incorporated into the envelope BHV-1/gB/NH₂_HA1 particles and exposed to the exterior of virions. Thus, this novel approach enables display of polypeptides and (glyco)proteins of at least 273 amino acids on viral particles which is of particular interest for development of novel diagnostics and vaccines as well as for, e.g. gene therapy applications especially when biologically active ligands need to be presented.     

伪狂犬病病毒gE／gI双基因缺失株的生物学特性

为了解gE和gI双基因缺失株伪狂犬病病毒gE-／gI—PRVSA738和野毒株PRV—SA的生物学特性,对该病毒进行了理化特性和体外增殖曲线的研究。结果显示：该缺失株对氯仿、酸、热及冻融次数敏感,从双基因缺失毒株与野毒株在BHK-21细胞上的增殖曲线来看,在病毒培养50h之前野毒株的毒价均高于缺失株,而在50h以后缺失株的毒价又高于野毒株,并且在36h时2株病毒的毒价均达到最高峰。     

Establishment of latency associated with glycoprotein E (gE) seroconversion after bovine herpesvirus 1 infection in calves with high levels of passive antibodies lacking gE antibodies

This study was conducted to investigate the glycoprotein E (gE) antibody response raised after inoculation with a low infectious dose of bovine herpesvirus 1 (BHV-1) in six calves possessing high levels of passive immunity from cows repeatedly vaccinated with gE deleted marker vaccine. Four out of the six calves developed gE antibodies 3-5 weeks after infection, whereas the two other ones remained seronegative to gE. After 5 months of infection, the six calves were treated with dexamethasone. Virus was only re-excreted by the four calves which previously seroconverted against gE. The two other calves became seronegative against BHV-1, 30-32 weeks after infection. A second dexamethasone treatment performed 11 months after infection failed to demonstrate a latent infection in these two calves. Moreover, the lack of identification of a cell-mediated immune response, after the two dexamethasone treatments, and the failure to detect BHV-1 DNA sequences in trigeminal ganglia strongly suggest that these two calves were not latently infected. In conclusion, the presence of high levels of maternal immunity lacking gE antibodies does not prevent latency after infection with a low titre of BHV-1. Moreover, latency is associated with a serological response to gE. These results confirm that the gE deletion is a good marker to identify young calves latently infected with a field virus.     

Characterization of equine P-selectin glycoprotein ligand-1 by using a specific monoclonal antibody

The FIV regulatory protein Rev and accessory proteins Vif and ORF-A are essential for efficient viral replication and full-blown pathogenesis. Expressed at very low level during viral replication, they are nevertheless processed for recognition by cytotoxic T-lymphocytes (CTLs) and trigger cellular immune responses in FIV-infected cats. The observation that the accessory ORF-A protein of FIV is continuously expressed during viral replication and targeted by cellular immune responses in natural FIV infection, prompted us to investigate the protective potential of this protein. To this aim cats were immunized with three different strategies (protein alone in alum adjuvant, DNA alone, or DNA prime-protein boost) and generated clearly detectable immune responses. Upon challenge with ex vivo homologous FIV, ORF-A immunized cats showed distinct enhancement of acute-phase infection possibly due to an increased expression of the FIV receptor CD134. However, at subsequent sampling points plasma viremia was reduced and CD4+ T-lymphocytes in the circulation declined more slowly in ORF-A immunized than in control animals. These findings support the contention that a multicomponent vaccine, with the inclusion of both accessory and structural proteins, can not only improve the host's ability to control lentivirus replication and slow down disease progression but also draw attention to the fact that even simple immunogens that eventually contribute to protective activity can transiently exacerbate subsequent lentiviral infections.     

Identification and characterization of peptides binding to newcastle disease virus by phage display

Three individual peptide sequences, EVSHPKVG, WVTTSNQW, and SGGSNRSP, which have potentials to bind to Newcastle disease virus (NDV), were identified by the biopanning method using phage display technology. The binding specificities of these peptides presented on phages were confirmed by ELISA competition assay using chicken anti-NDV antiserum. The synthetic peptides designed based on these results partially neutralized the infection of NDV in vitro. The peptide-motives identified here have the potential to lead to the identification of novel molecules that inhibit the NDV infection independent of the immune system.     

Expression and immunohistochemical distribution of duck plague virus glycoprotein gE in infected ducks

To determine the distribution of duck plague virus (DPV) gE protein in paraformaldehyde-fixed, paraffin-embedded tissues of experimentally DPV-infected ducks, an indirect immunoperoxidase assay was established to detect glycoprotein E (gE) protein for the first time. The rabbit anti-His-gE serum, raised against the recombinant His-gE fusion protein expressed in Escherichia coli BL21 (DE3), was prepared and purified. Western blotting and indirect immunofluorescence analysis showed that the anti-His-gE serum had a high level of reactivity and specificity and could be used as the first antibody for further experiments to study the distribution of DPV gE protein in DPV-infected tissues. A number of DPV gE proteins were distributed in the bursa of Fabricius, thymus, spleen, liver, esophagus, duodenum, jejunum, ileum, and kidney of DPV-infected ducks and a few DPV gE were distributed in the Harders glands, myocardium, cerebrum, and lung, whereas the gE was not seen in the skin, muscle, and pancreas. Moreover, DPV gE was expressed abundantly in the cytoplasm of lymphocytes, reticulum cells, macrophages, epithelial cells, and hepatocytes. The present study may be useful not only for describing the characteristics of gE expression and distribution in infected ducks but also for understanding the pathogenesis of DPV.     

犬瘟热病毒中国分离株囊膜糖蛋白基因免疫小鼠诱发的抗体应答

将CDV中国分离株(YZ0101)的两囊膜糖蛋白基因F和H与pGEM—rreasy载体构建的重组质粒分别采用EcoRI和Kprd、BamHI和Kpnl双酶切后定向克隆进真核表达载体pcDNA3．1(-)中，DNA测序和限制性酶切分析筛选阳性克隆pcDNA-H和pcDNA-F，以重组质粒DNA和脂质体共转染COS-7细胞，用间接免疫荧光试验验证转染的COS-7细胞胞浆中分别表达了CDV的H和F蛋白。以聚乙烯胺(PEI)为佐剂，将犬瘟热病毒F和H基因重组质粒pcDNA-H和pcDNA-F的DNA分别或混合肌注6周龄BABI／c小鼠，同时设pcDNA原载体DNA对照。以2周为间隔共免疫4次。最后一次免疫后3周，采血分离血清，酶联免疫吸附试验(ELISA)和病毒中和试验(SN)分析基因免疫在小鼠体内诱导的免疫应答。结果显示：pcDNA-H和pcDNA-F试验组免疫4次后分别激发了10^2.99 0.134、10^2.93 0.164滴度的ELISA抗体；两种质粒DNA混合免疫后产生了1：32～1：128的抗CDV的中和抗体；而原载体DNA免疫后，用ELISA和SN未检测出特异的CDV抗体。表明犬瘟热病毒囊膜糖蛋白F和H基因免疫小鼠可诱发产生特异性的体液免疫。     

A preliminary map of epitopes on envelope glycoprotein E1 of HCV strain Brescia.

Monoclonal antibodies (MAbs) directed against envelope glycoprotein E1 (gp51-54) of hog cholera virus (HCV) strain Brescia have been shown to recognize four different antigenic domains A, B, C and D. Epitopes of within domain A have mainly been found conserved among HCV strains, whereas epitopes within domains B, C and D are not conserved. We used transiently expressed hybrid E1 genes of HCV strains Brescia and "C" to map the non-conserved epitopes on E1. Epitopes in domains B and C are located within the ultimate N-terminal 104 amino acids. The non-conserved subdomain A3 is most probably located between domains B/C and a hydrophobic region, which is highly conserved between HCV strains Brescia and "C". The conserved subdomains A1 and A2 are probably located in the vicinity and C-terminally of this conserved, hydrophobic region, which is near the centre of the E1 amino acid sequence.     

法氏囊活性五肽BP5特异性结合肽的筛选及鉴定

为鉴定与具有免疫调节功能和抗肿瘤潜能的法氏囊活性五肽(BP5)特异性结合的多肽,本研究利用噬菌体展示技术,以BP5-BSA为靶分子,对噬菌体随机12肽库进行4轮亲和筛选,结合ELISA鉴定和竞争抑制试验,筛选出3个能够特异性与BP5结合的噬菌体阳性克隆.测序结果显示,其编码的12肽序列分别为:PINMQTLNCMAA(P2-12)、GCTLNPMSDLLG(P6-12)和MSSTLNGMLNSL(P7-12),其核心基序为TLNXM.通过人工合成P2-12、P6-12、P7-12多肽,并采用MTT法检测其对BP5抗肿瘤细胞增殖能力的影响,结果显示,P2-12和P7-12肽在0.2 μg/mL～20 μg/mL浓度下,P6-12肽在2μg/mL～20 μg/mL浓度下均能够抑制BP5抗小鼠WEHI-231 B淋巴瘤细胞增殖作用,其中P2-12在20 μg/mL浓度下抑制作用最为显著(p＜0.01).此外,p53荧光素酶活性检测结果显示,这3种BP5结合肽在实验浓度下均能够下调BP5促p53基因转录的活性.以上结果表明,这3种BP5特异性结合肽具有下调BP5抗肿瘤活性.本研究为进一步研究BP5抗肿瘤细胞增殖作用的机制提供了相关的实验依据.     

Generation of recombinant antibody fragments that target canine dendritic cells by phage display technology

One of the main goals in cancer immunotherapy is the efficient activation of the host immune system against tumour cells. Dendritic cells (DCs) can induce specific anti-tumour immune responses in both experimental animal models and humans. However, most preclinical studies using small animal models show only limited correlation with studies carried out in clinical settings, whereas laboratory dogs naturally develop tumours that are biologically and histopathologically similar to their human counterparts. Here, we describe the generation and characterization of recombinant antibodies against canine DCs, isolated using the Tomlinson phage display system. We successfully isolated highly specific single-chain variable fragment (scFv) antibodies in a sequential three-step panning strategy involving depletion on canine peripheral blood mononuclear cells followed by positive selection on native canine DCs. This provides the basis for an antibody-based method for the immunological detection and manipulation of DCs and for monitoring antigen-specific immune responses.     

Comparative functional genomic analysis of Pasteurellaceae adhesins using phage display

The Pasteurellaceae contain a number of important animal pathogens. Although related, the various members of this family cause a diversity of pathology in a wide variety of organ systems. Adhesion is an important virulence factor in bacterial infections. Surprisingly little is known about the adhesins of the Pasteurellaceae. To attempt to identify the genes coding for adhesins to some key components of the hosts extracellular matrix molecules, phage display libraries of fragmented genomic DNA from Haemophilus influenzae, Actinobacillus pleuropneumoniae, Pasteurella multocida and Aggregatibacter actinomycetemcomitans, were prepared in the phage display vector pG8SAET. The libraries were screened against human or porcine fibronectin, serum albumin or a commercial extracellular matrix containing type IV collagen, laminin and heparin sulphate. Four genes encoding putative adhesins were identified. These genes code for: (i) a 34 kDa human serum albumin binding protein from Haemophilus influenzae; (ii) a 12.8 kDa fibronectin-binding protein from Pasteurella multocida; (iii) a 13.7 kDa fibronectin-binding protein from A. actinomycetemcomitans; (iv) a 9.5 kDa serum albumin-binding protein from A. pleuropneumoniae. None of these genes have previously been proposed to code for adhesins. The applications of phage display with whole bacterial genomes to identify genes encoding novel adhesins in this family of bacteria are discussed.     

Dynamics of natural bovine herpesvirus-1 (BoHV-1) infection in a dairy herd

In this study, natural cycling of BoHV-1 infection was investigated in two groups of dairy cattle containing 2120 head. Group 1 comprised 127 animals and they were monitored for BoHV-1 infection virologically and serologically in six consecutive sampling periods. It consisted of naive heifers between 6 and 8 months of age, while in group 2, age, sex and the BoHV-1 serostatus of the animals were disregarded. The animals in group 1 were found to have seroconverted at the second sampling. Results of the serological study showed slight antibody response after natural BoHV-1 infection in the herd and neutralizing titres fell below protective levels in the 6–8 months after the peak. During the 2-year study period, one recurrence was detected after primary infection. Virus isolation studies revealed a cytopathic effect indicative of BoHV-1 in two nasal swabs taken during the fifth sampling period from animals with mild upper respiratory tract symptoms. As the study was carried out under natural conditions, it is not known whether the viruses isolated were from recurrences or re-infections. Data from cross-neutralization tests with herd isolates showed higher antibody response than those with the reference virus. The dynamics of BoHV-1 in both groups were found to be statistically similar.     

Characterization of pseudorabies virus glycoprotein B expressed by canine herpesvirus

A recombinant canine herpesvirus (CHV) which expressed glycoprotein B (gB) of pseudorabies virus (PrV) was constructed. The antigenicity of the PrV gB expressed by the recombinant CHV is similar to that of the native PrV. The expressed PrV gB was shown to be transported to the surface of infected cells as judged by an indirected immunofluorescence test. Antibodies raised in mice immunized with the recombinant CHV neutralized the infectivity of PrV in vitro. It is known that the authentic PrV gB exists as a glycoprotein complex, which consists of gBa, gBb and gBc. In MDCK cells, PrV gB expressed by the recombinant CHV was processed like authentic PrV gB, suggesting that the cleavage mechanism of PrV gB depends on a functional cleavage domain from PrV gB gene and protease from infected cells.     

Characterization of monoclonal antibodies against canine P-selectin glycoprotein ligand-1 (PSGL-1)

Thirteen different monoclonal antibodies against canine P-selectin glycoprotein ligand-1 (cPSGL-1) were obtained by immunization of rats with cells of a canine lymphoma cell line (Ema). O-sialoglycoprotein endopeptidase treatment of Ema cells showed that all of these antibodies recognized O-glycosylated peptides of canine PSGL-1. Experiments using deletion or point mutants of cPSGL-1 indicated that these antibodies could be categorized into several groups based on their cPSGL-1 recognition characteristics. These anti-cPSGL-1 monoclonal antibodies will be useful for analysis of the canine P-selectin and PSGL-1 system.     

抗猪流感噬菌体单链抗体库的构建与筛选

利用噬菌体展示技术构建猪流感病毒噬菌体抗体库,并筛选出猪流感高特异性、高亲和力的单链抗体(scFv)。以猪流感病毒免疫BALB/c小鼠,提取脾细胞总RNA,反转录后以cDNA为模板扩增获得VH基因和VL基因,并采用重叠延伸PCR(SOE-PCR),用柔性多肽Linker接头(Gly4Ser)按VH-Linker-VL方式将VH基因和VL基因拼接成scFv基因片段。将scFv基因和pCANTAB5E载体分别双酶切(SfiⅠ/NotⅠ)后连接,转化宿主菌TG1,经过辅助噬菌体M13K07拯救,构建噬菌体单链抗体库。以猪流感病毒为抗原包被96孔酶标板,经过3轮的亲和富集筛选,用Phage-ELISA鉴定阳性重组抗体。本研究成功构建出库容约为4×106cfu/mL抗猪流感病毒的单链抗体库,并筛选出4株特异性抗猪流感病毒的scFv抗体,能够与鼠源阳性多抗进行竞争结合猪流感病毒,为抗猪流感病毒转基因猪的研究奠定基础。