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E.F Kaleta O Siegmann Rita Jank-Ladwig G Glünder 《Comparative immunology, microbiology and infectious diseases》1979,2(4):485-496
From each of two lentogenic Newcastle disease virus (NDV) strains of the type LaSota and Hitchner B1 a virulent subpopulation could be obtained. The two subpopulations were—in comparison to the two parent viruses—more resistant to the lipid solvent chloroform and more stable against thermal degradation. Also, the glycoproteins haemagglutinin and F (fusion) were more stable against thermal inactivation. Electron microscopic observations revealed in terms of size and morphology all of the characteristics of NDV. Both subpopulations possessed, however, the same elution kinetics as their respective parent strains. The intracerebral and intravenous pathogenicity indices as well as the mean death times of the two subpopulations allow to classify these viruses as virulent Newcastle disease viruses. 相似文献
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【目的】获得表达H9亚型禽流感病毒血凝素(HA)基因的重组禽痘病毒。【方法】将含禽痘病毒启动子LP2EP2驱动的HA基因,插入到禽痘病毒转移载体pSY681中,获得重组转移载体pSY681/HA。用脂质体将其转染已感染亲本禽痘病毒S-FPV-017株的鸡胚成纤维细胞,使其在鸡胚成纤维细胞内与禽痘病毒基因组发生同源重组,产生表达HA的重组禽痘病毒rFPV-HA。在含有X-gal的营养琼脂培养基上进行蓝斑筛选后,对重组病毒进行多次蚀斑克隆,并用间接免疫荧光法对感染重组病毒鸡胚成纤维细胞中HA的表达产物进行鉴定。【结果】以重组禽痘病毒DNA为模板,利用HA基因特异引物进行PCR,扩增出1条约1.7 kb左右的带。以间接免疫荧光法证实重组禽痘病毒能表达HA。【结论】成功构建了表达H9亚型禽流感病毒HA基因的重组禽痘病毒,且构建的重组禽痘病毒能表达具有生物学活性的HA。 相似文献
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为进一步了解福建省H9N2亚型禽流感病毒的基因遗传进化关系,本研究将福建省2011年分离的毒株FZ-04、FZ-11与GenBank上登录的2000～2011年福建省分离的H9N2毒株及国内外典型代表株进行HA、NA基因的序列比对和遗传进化分析。结果表明,分离株FZ-04和FZ-11的HA基因与CK/FJ/G9/09株核苷酸同源性最高,属于国内常见的CK/BJ/1/94亚系。HA裂解位点处的氨基酸序列为-PSRSSR/GL-,符合低致病性禽流感病毒的分子特征。NA基因在遗传进化关系上呈现独立的分支,与CK/FJ/10954/05毒株核苷酸序列同源性最高,属于CK/HK/G9/97亚系,且NA基因推导的469个氨基酸序列中没有缺失。同时,从HA和NA基因的遗传进化树上可知,2000～2011年福建省H9N2禽流感病毒进化相对比较稳定,可能有一个共同的起源。 相似文献
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犬瘟热病毒Guizhou株血凝素基因的克隆及序列分析 总被引：8，自引：0，他引：8
参照已发表的文献合成1对引物，以犬瘟热病毒Guizhou分离株感染Vero细胞收获病毒提取的RNA为模板，RT-PCR扩增获得血凝素蛋白基因（H）901～1661位大小为761bp的片段，并将PCR扩增产物进行克隆和测序。将Guizhou株与GenBank数据库中的31株CDV及国内NanjingOP株的H基因序列进行同源性和系统发生树分析，发现Guizhou株的H基因部分序列与疫苗毒株Convac和Onderstpoort的同源性最低，只有90％；与日本野毒株Tanu96、KDK-1、Hmanatsu、Yanaka、UENO的同源性较高，为96％～98％；且与国内从大熊猫和小熊猫分离的野毒株GP和LP的同源性也较高，为98％和96％。系统发生树分析显示，Guizhou株与国内野毒株GP和LP，及日本的5个野毒株应属同一类，其中和GP株基因型最近，推测这些毒株间有更近的共同祖先。因此怀疑该病毒较适应于野生动物，且在我国某些地区的野生动物中可能存在自然疫源性传播。 相似文献
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以临床疑似犬瘟热病犬外周血总RNA为模板,采用RT-PCR方法扩增犬瘟热病毒(CDV)血凝素(H)基因。序列测定和分析表明该病料犬瘟热病毒H基因序列与国内其他地区分离到的毒株同源性较高(94.6%~99.2%),而与Onderstepoort株、Convac株等疫苗毒株的同源性较低(90.4%~91.2%)。分子遗传进化分析显示所有毒株可分为7个大的分支,且各分支间具有一定的地域性,其中待检病料中CDV与大多数中国分离株一样处于Ⅰ型。进一步以H全基因为模板,截短表达其3′端816 bp、459 bp两个片段,并克隆入原核表达载体pET30 a进行表达。结果显示它们分别能表达大小约为36.4 ku和22.2 ku的融合蛋白。免疫转印表明该纯化蛋白均可与犬瘟热病毒抗血清发生阳性反应,说明重组蛋白具备抗原性,可用于进一步的血清学研究。 相似文献
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用灭菌棉拭共采集蛋鸭和肉鸭的泄殖腔样品 62只 ,鸡胚尿囊腔传代接种法分离到 1株病毒 ,该病毒可凝集鸡红细胞 ,且不能被 ND、EDS-76阳性血清抑制 ,用病变绒毛尿囊膜制备抗原与禽流感琼扩标准阳性血清作用 ,出现明显的白色沉淀线 ,证明该分离株为 A型流感病毒 ,血凝素亚型分析结果为 H9,电镜负染观察可见典型的禽流感病毒粒子 ,致病性试验结果表明该分离株对鸡表现为弱致病性。研究结果还表明 ,环境 (特别是水体 )贮毒可能是禽流感病毒得以长期存在和传播的重要因素和媒介 相似文献
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犬瘟热病毒H蛋白的原核表达及免疫原性的初步鉴定 总被引：1，自引：1，他引：0
本研究旨在对犬瘟热病毒(CDV)疫苗株H基因进行克隆及原核表达,并对产物的免疫原性做初步鉴定。根据犬瘟热病毒参考株Ondetstepoort的H基因序列,去除信号肽序列并选取其主要抗原表位设计引物,用反转录—聚合酶链式反应(RT-PCR)扩增目的片段;产物克隆至表达载体pET28b并转化宿主菌RosettaTM,优化诱导表达条件,纯化目的蛋白,并进行SDS-PAGE鉴定、Western blotting分析及间接酶联免疫吸附试验(ELISA);并将纯化的H蛋白免疫小鼠,进行中和试验检测抗体效价。结果显示,PCR扩增得到1113 bp DNA片段;在37 ℃、1.0 mmol/L IPTG诱导条件下可获得较高水平的表达;经SDS-PAGE鉴定,表达的H蛋白分子质量为42.38 ku,与预期值相符;Western blotting显示,在42.38 ku出现特异性目的条带;ELISA结果显示,表达的H蛋白能被抗CDV抗体识别,但与正常血清未发生非特异性反应;中和试验结果表明,血清中和抗体效价约为2－3.3。结果提示,H蛋白获得了正确表达,对CDV抗血清具有特异反应性,可作为实时检测动物机体免疫状况检测的候选抗原,为进一步研制犬瘟热抗体检测ELISA试剂盒和新型疫苗奠定基础。 相似文献
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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. 相似文献
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Roshan Pais Rekha Khushiramani Iddya Karunasagar & Indrani Karunasagar 《Aquaculture Research》2008,39(12):1339-1345
The levels of haemagglutinins in Penaeus monodon , following administration of immunostimulants, β-glucans and/or vibrio bacterin either orally or by immersion, were studied. The freshly drawn haemolymph was incubated with microbial materials like β-glucans/vibrio bacterin and serum obtained after coagulation was administered to naïve animals. The immunostimulant treatments either via immersion, feeding or injection were found to cause an increase ( P <0.006) in haemagglutination activity (HA) of the haemolymph against mouse erythrocytes. Injection of saline or heterologous haemolymph caused an increase in the HA, but injection of haemolymph serum obtained by clotting haemolymph in the presence of vibrio bacterin or glucan did not bring about an increase in HA. There was no change in the haemolymph protein profile of the groups receiving immunostimulants through immersion or feed. Two protein bands (27 and 30 kDa), which were present in the uninjected group, were found to be overexpressed in the haemolymph-injected groups. Three bands of 17, 21 and 23 kDa, which were absent in control or saline-injected groups, were present in all the haemolymph serum-injected groups. The study indicates that modulation of HA may partly account for the immunomodulatory activity of immunostimulants like β-glucan or vibrio bacterins. 相似文献