表达狂犬病病毒糖蛋白的重组鸡痘病毒的构建和鉴定   总被引：4，自引：0，他引：4  

赵怀龙  金宁一  郭志儒  张茂林  夏志平  李萍 《中国兽医学报》2004,24(1):37-39

将 RVG基因插入到鸡痘病毒表达载体 p UTA- 2 Sm a 位点获得重组转移载体 p U TA- RVG,利用脂质体转染已感染中国鸡痘病毒疫苗株 2 82 E4株 2～ 3h的鸡胚成纤维 (CEF)细胞 ,收获病毒后 ,用 Brd U法进行加压筛选重组病毒 ,PCR检测到重组病毒 RVG基因 ,Western blot检测出 RVG,从而证实已成功构建了表达 RVG的重组鸡痘病毒  相似文献   

Re-emergence of rabies in Mazowieckie Voivodeship,Poland, 2021     

Marcin Smreczak  Anna Orłowska  Paweł Trębas  Agnieszka Stolarek  Conrad Freuling  Thomas Müller 《Zoonoses and public health》2023,70(1):111-116

Due to the oral vaccination of foxes against rabies most of the territory of Poland was freed from rabies of non-flying mammals. In January 2021, rabies was diagnosed in fox in the central part of Mazowieckie Voivodeship where rabies has not been detected since last 17 years. Subsequently, in the following months the rabies virus infection spread southward reaching the voivodeship of Świętokrzyskie in November 2021. Emergency actions were implemented aiming at rapid rabies elimination.  相似文献   

嵌合犬瘟热病毒H基因的重组狂犬病病毒的构建及免疫原性研究     

赵静  褚颖  罗均  郭霄峰 《中国畜牧兽医》2022,49(12):4776-4785

【目的】构建表达犬瘟热病毒（CDV）血凝素蛋白H基因的重组狂犬病病毒（RABV）,并研究其生物学特性及免疫原性。【方法】在RABV HEP-dG株基因组G与L基因之间插入CDV H基因,构建重组全长cDNA质粒pHEP-dG （H）。将pHEP-dG （H）与辅助质粒共同转染BHK细胞,拯救携带CDV H基因的重组RABV HEP-dG （H）。将重组病毒接种BHK细胞,分析病毒的扩散能力;将重组病毒接种小鼠,分析病毒的致病性和免疫原性。【结果】RT-PCR及直接免疫荧光显示,传至第3代的病毒仍能检测到H基因,表明CDV H基因已成功插入RABV基因组中,并在HEP-dG （H）中稳定遗传和正确表达。HEP-dG （H）在BHK细胞中的生长曲线与HEP-dG毒株相似,病毒滴度在96 h达到峰值,但HEP-dG （H）的滴度在每个时间点略低于HEP-dG。以感染复数（MOI）为0.005感染BHK细胞,HEP-dG （H）的扩散能力比HEP-dG毒株低。HEP-dG （H）与HEP-dG对6周龄成年小鼠均不致死,但HEP-dG （H）对成年小鼠体重的影响弱于HEP-dG。HEP-dG （H）与HEP-dG均能诱导小鼠产生抗RABV的中和抗体,免疫后7 d抗体已达到保护水平（0.5 EU/mL）;此外,HEP-dG （H）可诱导产生CDV中和抗体。HEP-dG （H）与HEP-dG免疫小鼠3周后,均能抵御RABV标准攻毒毒株CVS-24的攻击。【结论】本研究成功构建重组RABV HEP-dG （H）,其具有良好的免疫原性和安全性,可作为RABV-CDV新型二联基因工程候选疫苗。  相似文献   

应用半嵌套PCR扩增鹿源狂犬病野毒核蛋白基因     

李影  段锐  钱爱东 《经济动物学报》2004,8(4):202-204

应用一对外引物 (RNP1 ,RNP2 )和一个套式引物 (RNP3) ,对鹿源狂犬病野毒 82 0 2株核蛋白基因 (约135 0bp)进行 2次PCR扩增。第 1次仅扩增出一条约 75 0bp的片段 ,第 2次扩增时 ,将第一次扩增产物进行10 3倍稀释后能较好地扩增出目的片段。结果表明 ,半套式PCR引物的非特异性对目的片段的干扰 ,具有较高的敏感性。  相似文献   

Immunohistochemical detection of virus antigen in the nasal planum of rabid dogs     

Chun-Ho PARK  Sayaka KUBONIWA  Ryo MURAKAMI  Nozomi SHIWA  Satoshi INOUE  Kazunori KIMITSUKI  Ma. Ricci R. GOMEZ  Mark Joseph M. ESPINO  Alpha Grace B. CABIC  Sheila Marie C. ESPOSO  Daria L. MANALO 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2021,83(10):1563

The rabies virus is one of the most neurotropic of all viruses infecting mammals. During the terminal phases of infection, the virus spreads to peripheral tissues, including the skin. The external skin of the nose, called the nasal planum, is a sensory organ where numerous nerve bundles and terminal nerves are distributed. Therefore, the nasal planum is expected to serve as a postmortem diagnostic material. However, the distribution of rabies virus antigens in the nasal planum in rabid animals has not yet been studied. In this study, the nasal planum was obtained from 45 rabid dogs. In all rabid dogs, the viral antigen was detected in the peripheral nerve tissues, Merkel cells, and squamous cells. The viral antigen in the epidermis exhibited three patterns: first, a diffuse positive pattern from the basal layer to the squamous layer; second, a reticular positive pattern along the cell membrane in the squamous layer; and third, a basal layer pattern of the epidermis. In the dermis, viral antigens were detected more often in lamellated corpuscles just beneath the rete pegs. These results suggest that the nasal planum could serve as a useful alternative source for postmortem diagnosis in rabies endemic countries.  相似文献   

我国RV野毒株G基因主要功能区核酸序列的分子差异     

栾维民  钱爱东  侯世宽 《中国兽医学报》2000,20(6):532-534

对来源于鹿、牛和鼠的狂犬病病毒（RV）野毒株8202、BRV、MRV的G基因405～1 146位核苷酸序列进行了RT-PCR扩增、克隆和序列测定,并应用计算机软件对这3个毒株的测定序列和人源株CGX89的相应序列进行了分析比较。结果表明,这4个不同来源RV的G基因主要功能区的分子差异较大,4个野毒株的糖基化位点和主要诱导中和抗体产生的抗原决定簇内某些氨基酸亦不同,这些差异将影响它们的抗原性和毒性。研究结果证明,在我国流行的RV野毒株中存在亲缘关系较远的毒株,其野毒株G基因较大的分子差异可能影响疫苗的免疫保护效果。  相似文献   

两株狂犬病病毒强毒株糖蛋白基因的克隆及潜在抗原表位分析   总被引：2，自引：2，他引：2  

崔艳  李忠  王雷  张守峰  扈荣良 《中国预防兽医学报》2007,29(6):479-482

通过RT-PCR分别获得了狂犬病病毒强毒CVS株、DRV82株糖蛋白基因,进行克隆及测序,并推导出氨基酸序列,与犬用疫苗弱毒株ERA、SRV9、犬源性街毒株CGX及人用疫苗株PG的糖蛋白序列进行比较。结果表明,以上狂犬病病毒毒株间的核苷酸同源性为83.1%～99.2%,氨基酸序列同源性为87.0%～98.5%。经Jameson-Wolf抗原表位优势图分析,CVS株与其他各株相比发现在304位、372位抗原表位优势升高;而DRV82株与其它各株差异不明显。抗原优势变化可能导致狂犬病病毒糖蛋白出现新的潜在抗原位点,为下一步构建不同毒株的狂犬病病毒糖蛋白重组疫苗奠定了基础。  相似文献   

狂犬病病毒糖蛋白基因在原核细胞中的表达   总被引：2，自引：0，他引：2  

秦翠丽  金宁一 《中国兽医学报》1996,16(1):33-37

将狂犬病病毒糖蛋白（ＲＶｇｐ）基因ＢｇｌⅡ片段（１６７５ｂｐ）分别正向插入到原核高效表达载体ｐＥＴ－１７ｂ和ｐＥＴ－１７ｂ２（用ＳａｃⅠ－ＮｄｅⅠ缺失掉ｐＥＴ－１７ｂ６０ｂｐ含起始密码子ＡＴＧ小片段）的ＢａｍＨⅠ切点，构建重组质粒ｐＥＴ－１７ｂＲＶｇｐ和ｐＥＴ－１７ｂ２ＲＶｇｐ。将其分别转化表达受体菌Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）和Ｅ，ｃｏｌｉＢＬ２１（ＤＥ３）ｐｌｙｓＳ．ＩＰＴＧ诱导表达，菌体经超声波裂解处理后ＳＤＳ－ｐＡＧＥ，染色，在分子量约６００００处可见重组质粒表达的较宽的蛋白带，以抗ＲＶｇｐＭｃＡｂ进行Ｗｅｓｔｅｒｎ－ｂｌｏｔ检测，表明该表达蛋白为ＲＶｇｐ。通过扫描显示，表达的ＲＶｇｐ占菌体总蛋白的１０％～１４％，其中ｐＥＴ－１７ｂ２ＲＶｇｐ在Ｅ。ｃｏｌｉＢＬ２１（ＤＥ３）中的表达量最高。  相似文献   

2007年北京市丰台区犬只狂犬病病毒抗体效价调查分析     

魏德友  刘军  朱锡  宋继东  刘国胜  吴长福  刘娥娥  周媛媛  陈振文 《中国兽医学报》2008,28(12)

采集北京市丰台区城区和4个乡镇家养犬血清样品1 299份,采用免疫荧光中和试验法检测血清狂犬病病毒抗体效价。结果显示,被调查犬中有59.7%接种过狂犬疫苗,城区和乡镇接种率分别为75.9%和30.1%;有32.1%未接种过狂犬疫苗,城区和乡镇分别占16.1%和61.4%;有8.2%不清楚是否接种过狂犬疫苗,城区和乡镇分别占8%和8.5%。免疫犬狂犬病中和抗体效价d 0.5的占疫苗接种犬的57.1%,城区和乡镇分别为60.7%和40.6%。未免疫犬狂犬病中和抗体效价d>0的占未接种疫苗犬的27.6%,城区和乡镇分别为42.6%和20.3%。106只免疫不详犬均有狂犬病中和抗体。本次调查发现,犬群中仅有39%的犬只具有有效的狂犬病保护性抗体,疫苗接种率和狂犬病毒抗体效价城区均好于乡镇,但总体免疫水平仍不能有效控制群体中狂犬病的爆发和流行。  相似文献   

Preparation of Anti-Rabies Virus N Protein IgYs by DNA Immunization of Hens Using Different Types of Adjuvants     

Nanase Kubo  Mari Nishii  Satoshi Inoue  Akira Noguchi  Hajime Hatta 《The Journal of Poultry Science》2022,59(2):191

DNA immunization has been used to study vaccination methods and for production of specific antibodies. The present study aimed to apply DNA immunization to prepare specific IgYs, which react against rabies virus N protein (RV-N) and can be used to research and diagnose rabies virus. The DNA sequence of RV-N was ligated into a pcDNA 3.1 plasmid for constructing pcDNA-N. Eight hens were divided into four groups. Group 1 comprised the control group (non-immunized). In Groups 2, 3, and 4, hens were injected intramuscularly with pcDNA-N (400 µg/hen). Eight injections were administered every other week. From the 4th week, an adjuvant was injected in addition to pcDNA-N. Freund''s complete adjuvant (FCA) and λ-carrageenan were administered to Groups 3 and 4, respectively. Eggs were collected daily, and the specific antibody activities of egg yolks were measured by ELISA. IgYs were purified from pooled egg yolks at 16–19 weeks post-administration in each group. The detection sensitivities of the RV-N were compared using purified IgY as the primary antibody for ELISA, dot blotting, and western blotting. Egg yolks from one of the two hens in Group 2 (pcDNA-N alone) and all hens in Groups 3 (pcDNA-N + FCA) and 4 (pcDNA-N + λCarra) had increased ELISA values. The combined use of λ-carrageen in DNA immunization resulted in an adjuvant effect comparable to that of FCA. Each purified specific IgY detected RV-N in the ELISA, western blotting, and dot blotting; however, the detection sensitivity differed. Higher detection sensitivity of the +λCarra IgY was observed by ELISA, whereas there was higher detection sensitivity of +FCA IgY in western blotting and dot blotting. In summary, anti-rabies virus N protein IgY was prepared through DNA immunization of hens using FCA or λ-carrageenan as adjuvants and can be used as a primary antibody to detect rabies viruses.  相似文献