口蹄疫病毒O型泛亚株VP1蛋白的二级结构及抗原表位预测

为获得口蹄疫病毒（FMDV）O/PanAsia毒株VPl蛋白的抗原信息,从而为制备多肽疫苗提供理论依据,运用生物信息学软件,对O/PanAsia毒株的结构蛋白VP1理化性质、结构功能以及细胞表位进行分析,预测抗原表位以选择合适肽段。应用ProParam、TMHMM Server、ProScale和SignaIP 等在线工具,分析VP1蛋白氨基酸序列,运用计算机技术和分子生物学软件,分析预测VP1蛋白的基本理化性质、结构功能以及可能的B细胞和T细胞抗原表位,筛选B细胞表位肽段并对VP1的两个主要T细胞表位CTL和Th细胞表位进行预测。结果显示：O/PanAsia毒株VP1 蛋白的等电点为9.49,相对分子质量为23 520.83;VP1蛋白的B细胞表位为8~23、135~149和193~205位氨基酸。预测该VP1有10个CTL和10个Th细胞抗原表位,表明其具有较好的免疫原性;最终筛选出VP1蛋白的5个CTL和5个Th优势表位。用生物信息学方法预测O/PanAsia毒株VPl蛋白为稳定性蛋白,含有T、B淋巴细胞抗原表位。本研究为FMDV的进一步研究和疫苗制备奠定了基础。     

非洲猪瘟病毒p72蛋白抗原表位预测分析及多表位疫苗的构建

为了预测非洲猪瘟病毒2018/AnhuiXCGQ株p72蛋白的T、B淋巴细胞抗原表位,并完成多表位疫苗的构建。试验采用生物信息学技术ExPASy、SOMPA、PSIPRED Server、DNASTAR及Phyre对该蛋白的理化性质、二级结构、三级结构进行初步预测分析,运用ABCpred Prediction、Scratch、NetCTL及IEDB预测其T、B淋巴细胞表位,最终设计构建得到多表位疫苗。依据各生物学方法分析预测,得到B淋巴细胞优势表位:110～120、77～88、45～55、12～18、27～37位置;T淋巴细胞优势表位298～307、520～531、203～212位置,并设计得到T、B淋巴细胞表位表达盒,ExPASy预测表示该表位盒为亲水性蛋白。以此多表位表达盒为目的基因,以pET-28a构建设计多表位疫苗。表明该蛋白有多个潜在抗原表位,并可依据预测得到的蛋白二级结构、三级结构等参数信息及多个预测抗原位点构建ASFV多表位疫苗,表达纯化用于免疫试验。     

1株非洲猪瘟病毒p30蛋白单克隆抗体识别的B细胞抗原表位的鉴定

旨在研究非洲猪瘟病毒(ASFV) p30蛋白的B细胞抗原表位,本研究制备了p30蛋白的单克隆抗体(mAb),并以该单克隆抗体为工具进行B细胞抗原表位定位。首先,通过原核表达及Ni柱亲和纯化获得p30蛋白,将纯化蛋白免疫BALB/c小鼠进行杂交瘤细胞制备,通过间接酶联免疫吸附试验(iELISA)筛选出阳性杂交瘤细胞,并以细胞表达的方法制备单克隆抗体;采用间接免疫荧光试验(IFA)和蛋白质免疫印迹(Western blot)对单克隆抗体的特异性进行鉴定。利用IEDB表位预测软件对p30蛋白B细胞抗原表位进行预测,根据预测结果对CP204L基因进行截短表达,利用IFA、Western blot和iELISA对其抗原表位进行鉴定。最后,利用噬菌体十二肽库对制备的p30单克隆抗体进行4轮生物淘选,筛选多肽表位,并与上述基因截短表达筛选方法进行比对。特异性鉴定结果显示,该单克隆抗体能成功识别感染猪肺泡巨噬细胞的ASFV;基因截短表达筛选结果表明,其识别的抗原表位区域为⁸⁴M～K¹⁴²;噬菌体淘选试验结果表明,¹¹⁶TSSFETLFE¹²⁴为本试验制备的单克隆抗体所识别的p30蛋白抗原表位核心序列,该结果进一步缩小了表位分布范围。本研究制备了p30蛋白的1株单克隆抗体,并对其抗原表位进行鉴定,为血清学诊断试剂的研发和p30蛋白功能研究奠定基础。     

猪嗜淋巴细胞疱疹病毒2型GD27株gB基因生物信息学分析

试验采用PCR方法扩增出猪嗜淋巴细胞疱疹病毒2型（porcine lymphotropic herpesviruses 2,PLHV-2）gB基因全序列,并进行测序,使用生物信息学软件对gB蛋白进行生物信息学分析和预测。结果显示,gB蛋白由876个氨基酸组成,分子式为C₄₅₀₀H₆₉₇₅N₁₁₉₉O₁₃₅₁S₄₀,分子质量为100.77 ku,等电点为6.45,不稳定系数为38.58;二级结构预测以无规则卷曲为主要结构,1-39位氨基酸为信号肽,761-780位氨基酸为跨膜区结构,具有多种功能位点,存在多处天然非规则结构蛋白;三级结构显示有一个具有溶解作用的环状构象。综合多种方法分析预测结果表明,gB基因存在多个B细胞线性抗原表位,可作为PLHV-2疫苗的候选抗原。     

鸭肠炎病毒gC糖蛋白胞外区优势抗原表位的筛选与鉴定

为筛选出鸭肠炎病毒(DEV)gC糖蛋白胞外区的优势抗原表位,本试验通过抗原表位作图法对DEV-gC基因进行分段克隆并构建重组表达载体,表达蛋白经纯化后进行Western blot分析。结果显示,经过4轮筛选,DEV-gC糖蛋白优势抗原表位为第73-88位氨基酸。该优势表位的发现为DEV-gC糖蛋白具体功能区的研究、诊断试剂及表位疫苗的研制奠定了物质基础。     

犬瘟热病毒N蛋白的B细胞抗原表位预测

将1段犬瘟热病毒N蛋白氨基酸序列（GenBank编号为：AEV77096．1）与GenBank登录的其他氨基酸序列进行比对,分析其同源性;通过DNAStar生物信息学分析软件中的Protean模块及The PredictProteinserver在线蛋白分析工具预测犬瘟热病毒N蛋白的理化性质、二级结构、亲水性、表面可及性、柔韧性、抗原指数、跨膜螺旋、蛋白相互作用位点、蛋白功能位点等特性,并预测其B细胞优势抗原表位。结果显示,犬瘟热病毒N蛋白具有规则的二级结构、亲水性、柔韧性片段多,多处于表面可及性大,抗原指数高,蛋白质相互作用位点区域。潜在的B细胞优势抗原表位为12～18、61～66、243～246、410～415、421～429、434～447、452～456、480～487氨基酸序列。结果表明,本试验预测了犬瘟热病毒N蛋白的B细胞优势抗原表位,为进一步设计犬瘟热病毒的诊断抗原多肽、免疫用抗原多肽和研发血清学检测试剂盒奠定了理论基础。     

马鼻肺炎病毒主要糖蛋白及其功能

马鼻肺炎(ER)是由亲缘关系密切的马疱疹病毒1型和马疱疹病毒4型(EHV-1/4)引起的马属动物几种高度接触传染性疾病的总称.其基因组包含约80个开放性阅读框,至少编码78种蛋白.目前发现EHV-1至少有12种糖蛋白,其中5个糖蛋白(gB、D、H、L、K)是病毒复制所必需的,另6个糖蛋白(gC、E、G、I、M、gp300)不是病毒在细胞培养物上生长所必需的,但存在于高毒力的野毒分离株.gB、C、D是重要的免疫原性抗原具有病毒中和表位,gM的功能是使病毒穿入和细胞间传播.论文就其中9种糖蛋白及其功能进行介绍,并对其应用前景进行了展望.     

羊口疮病毒B2L蛋白抗原表位预测及重组蛋白设计与验证

为得到羊口疮病毒（ORFV）B2L蛋白的可能抗原表位及截短后高效表达的表位蛋白，本试验采用DNAStar、Mega 7.0等软件对部分NCBI已登录的B2L基因序列进行分析，并应用不同在线服务器对其编码蛋白的信号肽、跨膜区、细胞毒性T细胞表位、辅助性T细胞表位、B细胞表位和二级结构进行预测，同时参考多模板进行三级结构预测，综合抗原表位、亲水性、表面可及性和抗原指数等主要预测数据进行抗原位点分析及融合His·tag抗原表位蛋白的设计，构建并原核表达截短后高效表达的表位蛋白。结果显示，得到了几个可能的优质抗原表位，分别为88-93、133-138、172-175、251-254、311-313和370-377位氨基酸；纯化并复性的蛋白以多聚体形式存在；Western blotting结果显示，其具有良好的反应原性。该研究确定了ORFV B2L蛋白抗原位点，构建并原核表达了截短后高效表达的表位蛋白，为羊口疮诊断制剂及亚单位疫苗的研究奠定了基础。     

猪δ冠状病毒RBD蛋白多肽抗体的制备与鉴定

【目的】设计合成猪δ冠状病毒(Porcine deltacoronavirus, PDCoV)受体结合结构域(receptor binding domain, RBD)的抗原表位,制备并鉴定多克隆抗体。【方法】为了特异性检测PDCoV RBD抗原,应用生物信息学技术预测其潜在抗原表位,将表位氨基酸序列与δ冠状病毒属中的其他15株病毒株以及14株其他猪冠状病毒株进行相似性比对,将筛选的优势抗原表位与载体蛋白血蓝蛋白(keyhole limpet hemocyanin, KLH)偶联,合成多肽并免疫小鼠,制备PDCoV RBD特异性抗体。通过Western blotting试验对不同系统表达的PDCoV RBD以及PDCoV、TGEV和PEDV感染ST细胞表达的S蛋白进行鉴定,通过间接ELISA方法测定抗体效价。【结果】经序列比对,利用生物信息学技术预测合成的表位抗原与δ冠状病毒属中的其他15株病毒株(0～14.28%)以及14株其他猪冠状病毒株(0～7.14%)序列相似性非常低,具有良好的保守性;Western blotting结果显示,制备的多肽抗体1∶500、1∶1 000、1∶2 ...     

狐源犬瘟热病毒F蛋白B细胞抗原表位基因的克隆表达

为了研究犬瘟热病毒的抗原表位区段,试验以犬瘟热病毒基因组序列为材料,根据预测的F蛋白N端含有抗原指数较高的区域,选择可能含有潜在的B淋巴细胞优势抗原表位的片段,将其连接到原核表达载体pGEX-6p-1上进行B淋巴细胞抗原表位基因的克隆表达。结果表明:该区段能够在原核载体上高效表达。     

Prediction and Identification of Linear B-cell Epitopes in gB Glycoprotein of Equine Herpesvirus Type 1

The study was aimed to predict B cell epitopes in gB glycoprotein of equine herpesvirus type 1 (EHV-1) with bioinformatics, and select epitopes which had potential diagnostic value. The DNA fragments of gB glycoprotein were predicted by protean of DNAStar software. Screening potential B cell epitopes after parameter comparison, the target B cell epitopes were selected, cloned and expressed. The expressed fusion proteins serviced as an antigen were used to react with equine herpesvirus positive serum to screen and identify antigenic epitopes. The results showed that according to predictive and analysis, the areas of amino acid from 6 to 10, 23 to 32, 53 to 65, 72 to 98, 111 to 120, 152 to 166, 173 to 180 might be gB glycoprotein B cell epitopes. Seven epitopes were successfully cloned into a prokaryotic expression vector, and confirmed by DNA sequencing. After expression and purification, Western blotting was performed to detect the antigen, which could be recognized by equine herpesvirus positive sera. Bioinformatics technology and molecular biology techniques were used to successfully screen five potential B cell epitopes, which provided the foundation for the diagnosis of EHV-1 and design of the epitope vaccine.     

Characteristics of glycoprotein B of equine herpesvirus 1 expressed by a recombinant baculovirus.

A recombinant baculovirus (Bac-EgB) containing the complete open reading frame of equine herpesvirus 1 glycoprotein B (EHV-1 gB) expressed recombinant products of 107-133 kDa, 58-75 kDa and 53-57 kDa, corresponding to EHV-1 gB precursor, large and small subunits respectively. High molecular mass products (>200 kDa) in the Bac-EgB infected insect cells were consistent with oligomerisation of the recombinant EHV-1 gB products, and analysis with tunicamycin and endoglycosidases indicated that the baculovirus-expressed gB contained N-linked sugars with high mannose and hybrid chains. N-terminal amino acid sequence analysis of the gB forms revealed identical signal and endoproteolytic cleavage sites to those of gB in EHV-1 infected mammalian cells, and authenticity of processing and transport was supported by the presence of EHV-1 gB antigen at the surface of infected insect cells. Immunogold labelling and electron microscopy of recombinant baculovirus particles indicated that the recombinant gB was also present in baculovirus envelopes. Bac-EgB infected insect cells were able to induce low levels of complement dependent virus neutralising antibody, and have been shown to evoke protective immune responses in murine models of respiratory disease and abortion.     

Lack of Detectable Equine Herpesviruses 1 and 2 in Paraffin-Embedded Specimens of Equine Sarcoidosis

Background: Equine sarcoidosis is a rare, multisystemic, noncaseating, granulomatous and lymphoplasmacytic disease of unknown etiology. A recent report described a horse with granulomatous skin disease displaying histologic, electron microscopic, and polymerase chain reaction (PCR) findings consistent with equine herpesvirus 2 (EHV-2).
Objective: To investigate the presence of EHV-2 and equine herpesvirus 1 (EHV-1) in 8 horses with sarcoidosis.
Animals: Eight horses with sarcoidosis, reported previously.
Methods: Retrospective study. PCR assays of the tissues were performed to detect DNA associated with EHV-1 and EHV-2. For both herpesviruses the target was their respective glycoprotein B gene. Positive controls consisted of DNA from viral cultures of culturettes from naturally occurring respiratory infections of EHV-1 and EHV-2.
Results: The PCR analyses for both equine herpesviruses' DNA were negative in all 8 horses.
Conclusion: The failure to detect DNA from EHV-1 and EHV-2 in paraffin-embedded skin of these 8 horses does not discount EHV-1 or EHV-2 as causing some cases of ES, but lends support to the presumably multifactorial etiologic nature of the disease.     

Prevalence of latent alpha-herpesviruses in Thoroughbred racing horses

The objective of this study was to detect and characterize latent equine herpes virus (EHV)-1 and -4 from the submandibular (SMLN) and bronchial lymph (BLN) nodes, as well as from the trigeminal ganglia (TG) of 70 racing Thoroughbred horses submitted for necropsy following sustaining serious musculoskeletal injuries while racing. A combination of nucleic acid precipitation and pre-amplification steps was used to increase analytical sensitivity. Tissues were deemed positive for latent EHV-1 and/or -4 infection when found PCR positive for the corresponding glycoprotein B (gB) gene in the absence of detectable late structural protein gene (gB gene) mRNA. The EHV-1 genotype was also determined using a discriminatory real-time PCR assay targeting the DNA polymerase gene (ORF 30). Eighteen (25.7%) and 58 (82.8%) horses were PCR positive for the gB gene of EHV-1 and -4, respectively, in at least one of the three tissues sampled. Twelve horses were dually infected with EHV-1 and -4, two carried a latent neurotropic strain of EHV-1, six carried a non-neurotropic genotype of EHV-1 and 10 were dually infected with neurotropic and non-neurotropic EHV-1. The distribution of latent EHV-1 and -4 infection varied in the samples, with the TG found to be most commonly infected. Overall, non-neurotropic strains were more frequently detected than neurotropic strains, supporting the general consensus that non-neurotropic strains are more prevalent in horse populations, and hence the uncommon occurrence of equine herpes myeloencephalopathy.     

Mutations in the US2 and glycoprotein B genes of the equine herpesvirus 1 vaccine strain RacH have no effects on its attenuation]

The equine herpesvirus 1 (EHV-1) modified live vaccine strain RacH is apathogenic for both laboratory animals and the natural host. The apathogenicity of RacH was caused by serial passages of the virus in heterologous cells. When compared to the virulent parental strain RacL11 several changes in the RacH genome occurred. Previous results have shown that the loss of the IR6 gene correlated with the loss of virulence. Additional important mutations were observed within the US2 gene which is directly adjacent to the IR6 gene and within the glycoprotein B (gB) gene. To answer the question whether these mutations contribute to the attenuation of RacH several recombinant EHV-1 were constructed: The mutated genes in RacH were replaced by the wild-type US2 gene or the wild-type gB gene, respectively. In addition, a RacL11 recombinant expressing the mutated (RacH) gB instead of the wild-type gene was generated. All recombinant viruses were tested for virulence using the EHV-1 mouse model. The results were as follows: i) The insertion of the RacL11 US2 gene into the RacH virus did not restore virulence and none of the infected mice showed typical signs of EHV-1-caused disease (symptoms and body weight loss). ii) Exchanging gB genes between RacL11 and RacH did not alter their virulence phenotypes remarkably either. Therefore, it is concluded that attenuation of the EHV-1 vaccine strain RacH is caused solely by the absence of the IR6 gene and protein.     

Potential of DNA-mediated vaccination for equine herpesvirus 1.

The potential of DNA-mediated immunisation to protect against equine herpesvirus 1 (EHV-1) disease was assessed in a murine model of EHV-1 respiratory infection. Intramuscular injection with DNA encoding the EHV-1 envelope glycoprotein D (gD) in a mammalian expression vector induced a specific antibody response detectable by two weeks and maintained through 23 weeks post injection. Immune responses were proportional to the dose of DNA and a second injection markedly enhanced the antibody response. EHV-1 gD DNA-injected mice developed neutralising antibodies, and a predominance of IgG2a antibodies after the DNA injection was consistent with the generation of a type 1 helper T-cell (Th1) response. Following intranasal challenge with EHV-1, mice immunised with 50 microg of EHV-1 gD DNA were able to clear virus more rapidly from lung tissue and showed reduced lung pathology in comparison with control mice. The data indicate that DNA-mediated immunisation may be a useful strategy for vaccination against EHV-1.     

Characterization of the glycoprotein D gene products of equine herpesvirus 1 using a prokaryotic cell expression vector

The gene encoding equine herpesvirus 1 (equine abortion virus; EHV-1) glycoprotein D was engineered into the prokaryotic vector pEX, and expressed as a β-galactosidase fusion product, which was recognized by pooled equine sera and anti-EHV-1 rabbit sera. Antibodies raised against the EHV-1 gD fusion product identified strong bands in infected cells at 66 and 68 K and at 138 K in purified virus, thus characterizing the several forms of this major envelope glycoprotein which is an important candidate for inclusion in subunit vaccines.     

Multiplex real-time PCR for the detection and differentiation of equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4)

A multiplex real-time PCR was designed to detect and differentiate equid herpesvirus 1 (EHV-1) and equid herpesvirus 4 (EHV-4). The PCR targets the glycoprotein B gene of EHV-1 and EHV-4. Primers and probes were specific to each equine herpesvirus type and can be used in monoplex or multiplex PCRs, allowing the differentiation of these two closely related members of the Alphaherpesvirinae. The two probes were minor-groove binding probes (MGB) labelled with 6-carboxy-fluorescein (FAM) and VIC for detection of EHV-1 and EHV-4, respectively. Ten EHV-1 isolates, six EHV-1 positive clinical samples, one EHV-1 reference strain (EHV-1.438/77), three EHV-4 positive clinical samples, two EHV-4 isolates and one EHV-4 reference strain (EHV-4 405/76) were included in this study. EHV-1 isolates, clinical samples and the reference strain reacted in the EHV-1 real-time PCR but not in the EHV-4 real-time PCR and similarly EHV-4 clinical samples, isolates and the reference strain were positive in the EHV-4 real-time PCR but not in the EHV-1 real-time PCR. Other herpesviruses, such as EHV-2, EHV-3 and EHV-5 were all negative when tested using the multiplex real-time PCR. When bacterial pathogens and opportunistic pathogens were tested in the multiplex real-time PCR they did not react with either system. The multiplex PCR was shown to be sensitive and specific and is a useful tool for detection and differentiation of EHV-1 and EHV-4 in a single reaction. A comprehensive equine herpesvirus disease investigation procedure used in our laboratory is also outlined. This procedure describes the combination of alphaherpesvirus multiplex real-time PCR along with existing gel-based PCRs described by other authors.     

Establishment of a novel equine cell line for isolation and propagation of equine herpesviruses

In the present study, an equine-derived cell line was established by transfecting primary fetal horse kidney (FHK) cells with expression plasmid encoding simian virus 40 (SV40) large T antigen and then cloning them by limiting dilution. The cloned cell line, named FHK-Tcl3, grew well and could be propagated over 30 times by splitting them 1:3. Equine herpesvirus (EHV)-1 and EHV-4 replicated well in FHK-Tcl3. EHV-2 and EHV-4 were isolated from samples collected from horses in the field using FHK-Tcl3, and EHV-3 also propagated in FHK-Tcl3. These results indicated that this novel cell line, FHK-Tcl3, can be used for isolation and propagation of equine herpesviruses.     

Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses, pregnant mares and young foals

The envelope glycoprotein D of equine herpesvirus 1 (EHV-1 gD) has been shown in laboratory animal models to elicit protective immune responses against EHV-1 challenge, and hence is a potential vaccine antigen. Here we report that intramuscular inoculation of EHV-1 gD produced by a recombinant baculovirus and formulated with the adjuvant Iscomatrix elicited virus-neutralizing antibody and gD-specific ELISA antibody in the serum of over 90% of adult mixed breed horses. The virus-neutralizing antibody responses to EHV-1 gD were similar to those observed after inoculation with a commercially available killed EHV-1/4 whole virus vaccine. Intramuscular inoculation of EHV-1 gD DNA encoded in a mammalian expression vector was less effective in inducing antibody responses when administered as the sole immunogen, but inoculation with EHV-1 gD DNA followed by recombinant EHV-1 gD induced increased gD ELISA and virus-neutralizing antibody titres in six out of seven horses. However, these titres were not higher than those induced by either EHV-1 gD or the whole virus vaccine. Isotype analysis revealed elevated gD-specific equine IgGa and IgGb relative to IgGc, IgG(T) and IgA in horses inoculated with EHV-1 gD or with the whole virus vaccine. Following inoculation of pregnant mares with EHV-1 gD, their foals had significantly higher levels of colostrally derived anti-gD antibody than foals out of uninoculated mares. The EHV-1 gD preparation did not induce a significant mean antibody response in neonatal foals following inoculation at 12 h post-partum and at 30 days of age, irrespective of the antibody status of the mare. The ability of EHV-1 gD to evoke comparable neutralizing antibody responses in horses to those of a whole virus vaccine confirms EHV-1 gD as a promising candidate for inclusion in subunit vaccines against EHV-1.