山羊痘病毒抗凋亡蛋白基因的表达与细胞凋亡的相关性

以贵州本地分离纯化的山羊痘病毒为试验材料,感染Vero细胞,通过台盼蓝、吖啶橙/溴乙锭荧光染色分析细胞的死亡及凋亡比例;经特异性PCR从山羊痘病毒基因组中分离出山羊痘病毒抗凋亡蛋白样基因,基因长531bp,含有完整的编码框,与绵羊痘病毒抗凋亡蛋白基因的相似性为97%,因此确定为山羊痘病毒抗凋亡蛋白基因。进一步研究山羊痘病毒抗凋亡蛋白基因在Vero细胞中的表达,结果显示,病毒感染Vero细胞24h时没有检测到凋亡现象,山羊痘病毒抗凋亡蛋白基因的mRNA水平最高;感染48h时,Vero细胞的凋亡率上升到13%,山羊痘病毒抗凋亡蛋白基因的表达量随之下降。结果表明,山羊痘病毒抗凋亡蛋白基因的表达量与Vero细胞的凋亡呈负相关,有助于病毒在细胞内的生存和侵染等过程。     

犬瘟热病毒研究概况

犬瘟热(CD)是由犬瘟热病毒(CDV)引起的一种急性、高度接触性传染病,流行范围广,发病率、致死率高,临床症状多样。CDV感染宿主广泛,所有日龄的犬都有可能感染。CDV属于副黏病毒科麻疹病毒属,有囊膜包裹的单股负链线性RNA病毒。CDV基因组编码6种蛋白:核衣壳(N)蛋白、磷(P)蛋白、基质膜(M)蛋白、融合(F)蛋白、血凝素(H)蛋白和大(L)蛋白。N、P和L蛋白与病毒复制有关;M蛋白与病毒的装配和出芽有关;F、H蛋白在病毒的侵染过程中起到关键作用。近年来,随着我国宠物业、毛皮经济养殖业的迅速发展,CD在我国的发病率有升高的趋势。论文对CDV分子生物学研究进展进行归纳总结。     

表达红色荧光蛋白的重组鸭肠炎病毒的构建及鉴定

鸭肠炎病毒(Duck enteritis virus, DEV),又称为鸭瘟病毒,是一种只感染雁形目禽类的疱疹病毒。DEV具有基因组大、非必需基因多、能插入外源基因的容量大、遗传稳定等优点,其庞大而复杂的基因组为外源基因提供了诸多可插入位点,以DEV为载体,成功表达了禽流感病毒HA蛋白_^[1]、鸭病毒性肝炎病毒VP0蛋白_^[2]、鸭坦布苏病毒E基因_^[3]以及鹅细小病毒VP2蛋白_^[4]。构建重组DEV的重要一步是将报告基因插入到基因组中,目前常用的报告基因有绿色荧光蛋白(GFP)、增强型绿色荧光蛋白(EGFP)、红色荧光蛋白(RFP)以及LacZ报告基团。本研究用RFP报告基团插入DEV UL2基因中,获得表达红色荧光的重组病毒,一步生长曲线表明,RFP对DEV的生长无影响;连续传代12代,RFP能够稳定表达,为DEV载体研究奠定基础。     

新城疫病毒基质蛋白第23位苯丙氨酸对病毒复制与细胞致病性的作用

副黏病毒科的副流感病毒5型(Parainfluenza virus5,SV5)和腮腺炎病毒(Mumps virus,MuV)的基质蛋白(Matrix protein,M)都编码FPIV-like晚期结构域(Late-domain,L-domain),L-domain中的苯丙氨酸(Phenylalanine,F)对病毒出芽与复制有很重要的作用。新城疫病毒(Newcastle disease virus,NDV)M蛋白也包含潜在的L-domain23FPIV26,但23F对NDV的出芽及复制的作用尚不明确。本试验应用反向遗传学技术,以NDV ZJ1株为母本病毒,拯救一株M蛋白F23A突变的重组病毒ZJ1F23A,并在DF1细胞上对ZJ1F23A的复制性能和细胞致病性进行评价。结果显示,ZJ1F23A在DF1上的复制水平显著低于母本病毒ZJ1,ZJ1F23A对DF1的致病性比ZJ1弱。试验结果说明NDV M蛋白23F对病毒复制及病毒的细胞致病性具有重要作用。     

携带GFP基因的重组狂犬病病毒的拯救及其生物学特性研究

本研究旨在构建一株携带绿色荧光蛋白（GFP）的重组病毒,以便更快速、准确地检测疫苗效价。利用基因重组技术构建含有GFP基因的重组质粒,并利用反向遗传学操作进行重组病毒的拯救,在Vero细胞进行病毒感染并收集蛋白样品进行Western blotting试验,检测外源蛋白GFP的表达情况,测定一步法生长曲线,比较与原始毒株的生物学特性差异。结果显示,本试验成功拯救出携带GFP基因的重组狂犬病病毒毒株SAD-GFP;Western blotting试验结果显示,重组病毒可表达GFP,且随着病毒感染时间的延长,蛋白表达增多;病毒结构蛋白G蛋白表达并未受外源蛋白表达的影响,重组毒株与原始毒株的生长并无明显差异;传代后GFP仍可在病毒中稳定表达。在连续传代过程中,本试验成功拯救出SAD-GFP重组毒株,GFP基因并未丢失,且蛋白表达量并未降低,证明该位点表达的外源蛋白在病毒传代过程中可持续稳定表达,且对病毒生长特性并未产生影响。     

A complement-dependent neutralizing monoclonal antibody against glycoprotein II of pseudorabies virus

A monoclonal antibody (MAb), 1.21, was produced against pseudorabies virus (PRV). It exhibited virus neutralization activity only in the presence of complement. Immunoblot analysis after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of virions revealed that MAb 1.21 bound with the 230 kilodalton (kD) virus protein only under non-reducing conditions. This protein was purified by immuno-affinity chromatography using MAb 1.21 and was found to be composed of three subunits, 60, 70 and 120 kD polypeptides when analyzed by SDS-PAGE under reducing conditions. This protein is probably glycoprotein II of PRV.     

狂犬病病毒BD-06株基质蛋白的原核表达、纯化及其多克隆抗体的制备

本研究旨在获得抗狂犬病病毒M蛋白的多克隆抗体,为进一步研究M蛋白功能提供材料。本试验以狂犬病病毒BD06毒株为模板克隆M基因序列,将其克隆至原核表达载体pET28a;经酶切和测序鉴定,M蛋白基因序列已正确插入表达载体pET28a;以表达载体pET28a-M转化E.coli Rosetta株,并以0.5 mmol/L IPTG诱导,结果表达出27 ku左右的M蛋白;将诱导表达的蛋白质回收纯化,以纯化的M蛋白多次免疫兔子制备多克隆抗体;Western blotting分析和间接免疫荧光分析结果表明,制得的抗体与病毒能够反应,运用制得的多克隆抗体定位了基质蛋白在感染狂犬病病毒的BHK-21细胞中的位置。结果表明成功制得了抗狂犬病病毒M蛋白的多克隆抗体,为研究狂犬病病毒M蛋白功能奠定了基础。     

4型禽腺病毒抗体间接hexon-ELISA检测方法的建立

以4型禽腺病毒(FAdV-4)的DNA为模板,扩增其六邻体(hexon)部分基因并进行重组表达,以重组hexon蛋白为包被抗原,优化ELISA检测条件,建立了FAdV-4的ELISA抗体检测方法。该方法对FAdV-4阳性血清检测为阳性,对于其他鸡常见病毒,如禽流感病毒5、7、9型,新城疫病毒以及鸡传染性支气管炎病毒阳性血清检测均为阴性;与PCR方法的阳性符合率为83.3%。试验表明,建立的以重组hexon蛋白为包被抗原检测FAdV-4血清抗体的ELISA方法可以用于检测FAdV-4的感染及相关流行病学调查。     

乙型脑炎病毒E蛋白N端片段在大肠杆菌中的表达

利用 PCR扩增乙型脑炎病毒 E蛋白基因 5′端片段 ,克隆入原核表达载体 p ET-2 8a,转化大肠杆菌 BL2 1 ( ED3 )。经 IPTG诱导表达后 ,SDS-PAGE分析表达产物。结果表明 ,所表达的E蛋白片段的分子量约为 3 .4万 ,表达量约占菌体总蛋白的 3 5%。 JEV E蛋白片段在大肠杆菌中的成功表达 ,为制备 JE实验室诊断抗原和分析 E蛋白基因结构与功能的关系提供条件     

Properties of monoclonal antibodies against Berne virus (Toroviridae)

Seven hybridomas that secreted monoclonal antibodies (MAB) against the peplomer protein and one that secreted MAB against the nucleocapsid protein of Berne virus (proposed family Toroviridae) were isolated. All MAB directed against the peplomer protein neutralized virus infectivity and, with the exception of MAB 6A7, inhibited each other's binding in competition assays. Neutralization of Berne virus infectivity was potentiated when some MAB were used in pairs. The antibodies have been used to localize toroviral proteins in infected cells; use of antipeplomer MAB 6B10 yielded a diffuse intracytoplasmic immunofluorescence, whereas the antinucleocapsid MAB 1F1 detected antigen in the intra- and perinuclear compartments. By use of radioimmune precipitation, protein A of Staphylococcus aureus was found to bind directly to the nucleocapsid polypeptide, without the requirement for specific antibody. Using fluorescein isothiocyanate-conjugated protein A, the intranuclear accumulation of the nucleoprotein of Berne virus was confirmed by results of immunofluorescence.     

Production and immunogenicity of chimeric virus-like particles containing the spike glycoprotein of infectious bronchitis virus

Infectious bronchitis virus (IBV) poses a severe threat to the poultry industry and causes heavy economic losses worldwide. Vaccination is the most effective method of preventing infection and controlling the spread of IBV, but currently available inactivated and attenuated virus vaccines have some disadvantages. We developed a chimeric virus-like particle (VLP)-based candidate vaccine for IBV protection. The chimeric VLP was composed of matrix 1 protein from avian influenza H5N1 virus and a fusion protein neuraminidase (NA)/spike 1 (S1) that was generated by fusing IBV S1 protein to the cytoplasmic and transmembrane domains of NA protein of avian influenza H5N1 virus. The chimeric VLPs elicited significantly higher S1-specific antibody responses in intramuscularly immunized mice and chickens than inactivated IBV viruses. Furthermore, the chimeric VLPs induced significantly higher neutralization antibody levels than inactivated H120 virus in SPF chickens. Finally, the chimeric VLPs induced significantly higher IL-4 production in mice. These results demonstrate that chimeric VLPs have the potential for use in vaccines against IBV infection.     

表达猪圆环2型的多拷贝P28-Cap重组猪痘病毒的构建及表达量分析

为探究猪痘病毒作为载体表达猪圆环病毒2型（PCV2）-Cap蛋白，插入外源基因P28-ORF2的拷贝数与PCV2-Cap蛋白表达量的关系。本试验以猪痘病毒为载体，通过双酶切连接及无缝克隆方法构建重组质粒，并纯化到了8株含不同拷贝数（1~8拷贝） P28-ORF2的重组猪痘病毒，基于荧光斑大小、电镜观察病毒粒子及Western blotting结果进行判断。纯化到的8株重组病毒的荧光斑直径为317.41~384.96 μm，大小无明显差异。重组猪痘病毒粒子形态与亲本病毒一致。Western blotting结果为插入1拷贝P28-ORF2的重组蛋白表达量最低；随着P28-ORF2拷贝数的增加，PCV2-Cap表达量也随之增加；至插入4拷贝P28-ORF2时，PCV2-Cap表达量达到峰值；随后减少。8株重组猪痘病毒荧光斑大小无明显差异，表明猪痘病毒基因组中插入不同拷贝数P28-ORF2对重组病毒在PK15细胞内的增殖无影响。重组猪痘病毒粒子的形态未发生变化说明多拷贝外源基因的插入并未对猪痘病毒的结构造成影响。本研究结果表明，猪痘病毒为载体表达外源蛋白时，单启动子启动多拷贝外源序列能明显提高外源蛋白的表达量，插入4拷贝的外源序列为较合适的拷贝数。     

Pig paramyxovirus of the blue eye disease binding to a 116 kDa glycoprotein expressed in pig neuronal membranes

Pig paramyxovirus of the blue eye disease (PPBED) is a novel member of the paramyxoviridac family which infects pigs. In neonatal pigs it causes neurological damage, whereas in adult pigs it affects the reproductive function. As PPBED damages the new-born pig central nervous system (CNS), it is important to study whether PPBED binds to the membrane proteins of all brain tissue, or selectively binds to neuronal tissue of the brain stem, olfactory bulb, hippocampus, cerebellum, frontal, temporal and parietal brain cortex. It is also important to establish whether it also infects neurones obtained from new-born, 60-day-old and adult pigs, and the role of carbohydrate residues in virus binding. The effect on virus binding of polyclonal antibodies against viral envelope proteins was also studied. Binding studies were performed using dot blot and virus overlay protein binding assays. PPBED was able to bind to membrane proteins from all brain regions, particularly to a protein band of approximately 116 kDa. Neuraminidase treatment of neuronal membrane proteins decreased virus binding; subsequent treatments with beta-galactosidase and manosidase did not increase virus binding inhibition. N-glycosidase F and trypsin also decreased virus binding, but not the O-glycanase. Antibodies against viral haemagglutinin-neuraminidase blocked virus binding more efficiently than antibodies against viral fusion protein. In conclusion: (1) PPBFD is able to bind to pig neurones of all brain regions studied and at all ages analysed; (2) a 116 kDa membrane protein containing sialic acid residues with an N-linked oligosaccharide chain was specifically recognized; (3) PPBED haemagglutinin-neuraminidase protein seems to play a central role in neural receptor recognition.     

Production and characterization of monoclonal antibodies to budgerigar fledgling disease virus major capsid protein VP.

Eleven hybridoma cell lines producing monoclonal antibodies (MAbs) against intact budgerigar fledgling disease (BFD) virions were produced and characterized. These antibodies were selected for their ability to react with BFD virions in an enzyme-linked immunosorbent assay. Each of these antibodies was reactive in the immunofluorescent detection of BFD virus-infected cells. These antibodies immunoprecipitated intact virions and specifically recognized the major capsid protein, VP1, of the dissociated virion. The MAbs were found to preferentially recognize native BFD virus capsid protein when compared with denatured virus protein. These MAbs were capable of detecting BFD virus protein in chicken embryonated cell-culture lysates by dot-blot analysis.     

表达猪圆环病毒2型衣壳蛋白的重组猪痘病毒的构建及鉴定

为探索猪痘病毒（swinepox virus,SWPV）作为猪圆环病毒2型（porcine circovirus type 2,PCV2）疫苗载体的可行性,本试验以痘苗病毒启动子P11启动绿色荧光蛋白筛选标记,猪痘病毒TK基因为外源基因的插入位点,P28、P7.5启动子启动PCV2 ORF2基因,以猪痘病毒JX20G株为亲本病毒,采用同源重组技术分别构建了两株表达PCV2衣壳蛋白的重组猪痘病毒rSWPV11-28C和rSWPV11-7.5C。结果显示,重组猪痘病毒rSWPV11-28C和rSWPV11-7.5C均成功表达了PCV2衣壳蛋白,表达的蛋白能与PCV2单克隆抗体6E12发生特异性反应;痘苗病毒启动子P28的启动效果明显优于P7.5,P28适合用于启动目的基因;利用重组猪痘病毒制备的PCV2灭活疫苗免疫小鼠后,rSWPV11-28C疫苗组的PCV2抗体水平与某PCV2商品疫苗相当,该重组病毒的成功构建为PCV2相关疾病及其他疫病在猪群中的防控提供了新的方向。     

猪繁殖与呼吸综合征病毒E蛋白研究进展

猪繁殖与呼吸综合征(PRRS)是由猪繁殖与呼吸综合征病毒(PRRSV)引起的高度接触性传染病,主要引起母猪繁殖障碍,表现为妊娠后期流产、死胎、木乃伊胎及弱仔。E蛋白是由ORF2b编码的一个非糖基化结构蛋白,分子质量约10 ku,具有影响病毒复制的作用,促使病毒粒子脱壳,但对病毒的装配不起作用,认为具有离子通道蛋白活性。作者就E蛋白的特性、亚细胞定位和拓扑结构、离子通道活性作一综述。     

猪流感病毒蛋白研究进展

猪流感（swine influenza,SI）是由猪流感病毒（swine influenza virus,SIV）引起的猪的一种传染病,其在世界各地的广泛存在和流行,给养猪业带来了巨大的经济损失。猪流感病毒属于正黏病毒科A型流感病毒属,作者就猪流感病毒蛋白,包括血凝素（HA）、神经氨酸酶（NA）、核蛋白（NP）、基质蛋白（M）、聚合酶蛋白（PA、PB1和PB2）和非结构蛋白（NS）进行简要概述,以期为猪流感病毒的致病机制、诊断、分子流行病学等方面的研究提供参考。     

Diagnostic potential of recombinant protein of hexahistidine tag and infectious bursal disease virus VPX expressed in Escherichia coli

The current method to detect antibody titre against infectious bursal disease virus (IBDV) in chickens is based on enzyme-linked immunosorbent assay (ELISA) using whole virus as coating antigen. Coating the ELISA plates requires a purified or at least semi-purified preparation of virus as antigen, which needs special skills and techniques. In this study, instead of using whole virus, recombinant protein of hexahistidine tag (His 6 tag) and VPX protein of IBDV expressed in E. coli was used as an alternative antigen to coat the ELISA plates. There was a good correlation coefficient (R2 = 0.972) between the results of the ELISA using plates coated with monoclonal antibody against His 6 tag and those of the commercial IBDV ELISA kit. Hence, His 6 tag and VPX recombinant protein expressed in E. coli has the potential for the development of ELISA for the measurement of IBDV-specific antibody.     

非洲猪瘟病毒p62蛋白单克隆抗体的制备及初步应用

为制备非洲猪瘟病毒（ASFV）p62蛋白的特异性单克隆抗体,并初步应用于感染组织样品中ASFV抗原的免疫组化（IHC）检测,本研究以杆状病毒表达的非洲猪瘟病毒重组p62蛋白免疫BALB/c小鼠,取其脾细胞与骨髓瘤细胞进行融合获得杂交瘤细胞。结果显示：基于纯化的p62蛋白建立的间接ELISA方法对杂交瘤细胞进行筛选和亚克隆,获得了18株可稳定分泌抗非洲猪瘟病毒p62蛋白单克隆抗体的杂交瘤细胞株。经IFA检测,制备的单克隆抗体均与非洲猪瘟病毒反应,且不与猪瘟病毒、猪繁殖与呼吸综合征病毒、猪伪狂犬病病毒、猪圆环病毒2型等猪源常见病毒反应,特异性良好。抗体识别蛋白的鉴定结果显示,3株MAbs识别p35蛋白,15株MAbs识别p15蛋白。14株MAbs重链亚类为IgG1型,4株MAbs重链亚类为IgG2a,轻链均为κ链。利用18株MAbs对ASFV感染猪的肺、扁桃体、淋巴结等组织进行IHC检测,结果显示5株MAbs均能够与感染ASFV的组织发生特异性的免疫反应。本研究获得的非洲猪瘟病毒p62蛋白单克隆抗体可为非洲猪瘟病毒免疫学检测方法的建立及p62蛋白的结构功能等基础研究提供重要的生物材料。