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猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swine fever virus,CSFV)引起的猪的高度接触性传染病,是严重危害养猪业的传染病之一。CSFV基因组为单股正链RNA分子,长约12.3kb,仅编码一个开放性阅读框。位于5’端的囊膜糖蛋白E^ms、E1和E2构成了CSFV的外壳, 相似文献
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猪瘟(Classical swine fever,CSF)是由猪瘟病毒(Classical swinefever virus,CSFV)引起的猪的高度接触性传染病,是严重危害养猪业的传染病之一.CSFV基因组为单股正链RNA分子,长约12.3kb,仅编码一个开放性阅读框.位于5'端的囊膜糖蛋白Erns、E1和E2构成了CSFV的外壳,其中Erns和E2参与病毒感染细胞的过程,并能诱导宿主产生保护性免疫应答[1].目前研究的CSFV基因工程疫苗主要以E2蛋白作为抗原,并通过检测Erns的抗体来区分E2标记疫苗免疫猪和野毒感染猪,有利于剔除猪群中潜在的传染源,达到最终消灭猪瘟的目的.氨基酸序列比较发现,CSFV的Erns氨基酸序列中有地衣类与植物核苷酸酶家族的特征序列,属于胞外RNase家族,具有RNase活性,Erns可降解病毒和细胞的RNA,在研究CSFV的致病机制方面具有重要意义[2].本研究利用RT-nPCR技术,克隆到了Erns基因,并利用大肠杆菌表达系统高效表达了Erns蛋白,纯化后的蛋白具有良好的生物学活性,为进一步建立Erns抗体的检测方法和探讨Erns蛋白在CSFV致病过程中的作用奠定了基础. 相似文献
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猪瘟(Classical swine fever,CSF)是严重危害养猪业的一种烈性传染病,常造成巨大的经济损失,是世界动物卫生组织要求必须申报的动物疫病之一。猪瘟的病原是猪瘟病毒(Classical swine fever virus,CSFV),CSFV的结构蛋白由衣壳蛋白(C)和囊膜糖蛋白(E~(rns)、E1、E2)构成。E2蛋白是CSFV主要的保护性抗原,可以诱导机体产生中和抗体,从而抵抗CSFV的感染。此前,本团队制备了一株针对CSFV E2蛋白的鼠源单克隆抗体HQ06。文中将HQ06抗体重链和轻链可变区基因与猪源恒定区基因嵌合后克隆至真核表达载体,利用中国仓鼠卵巢(CHO)细胞制备一株针对CSFV E2蛋白的嵌合猪源化单克隆抗体c HQ06。应用ELISA、Western blotting试验证实了c HQ06与CSFV E2蛋白具有良好的反应性;中和试验结果表明c HQ06可以中和CSFV。综上所述,本研究应用CHO细胞稳定表达了具有良好反应性和中和活性的针对CSFV E2蛋白的嵌合猪源化单克隆抗体c HQ06,为研究CSFV E2蛋白结构、功能以及开发新型的CSFV诊断和治疗制剂奠定基础。 相似文献
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猪瘟(Classical swine fever,CSF)是猪的一种急性、热性和致死性传染病。该病流行范围很广,而且致死率极高,给世界养猪业造成严重危害。目前,猪瘟流行地区或国家仍然采用接种弱毒疫苗的方法作为预防猪瘟的主要策略,但接种弱毒疫苗的传统预防控制方法无法区别猪瘟疫苗免疫抗体和野毒感染抗体。为了净化、消灭猪瘟,新型标记疫苗的研究已迫在眉睫。近些年,陆续有国内外研究者应用分子生物学和基因工程方法,对猪瘟野毒株或弱毒株进行基因修饰构建出新毒株,其中以Erns和E2为基础构建新毒株的方法占据着重要地位。部分候选疫苗具有较好的免疫效果,可用于区分免疫和自然感染动物,而且有望作为新一代疫苗来替代传统弱毒疫苗。 相似文献
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逆转录病毒载体介导的猪瘟病毒E2基因的真核表达 总被引:5,自引:0,他引:5
利用DNA重组技术将猪瘟病毒(Classical swine fever virus , CSFV)石门株囊膜蛋白E2基因插入逆转录病毒载体pBABE-puro 中构建成重组逆转录病毒载体pBABE-puro-E2,该重组逆转录病毒载体与pVSVg质粒经磷酸钙共转染法将其转入293GP细胞中包装逆转录病毒假病毒.用包装的假病毒感染PK-15细胞,经嘌呤霉素筛选阳性细胞后进行流式细胞技术(FACS)分析,结果表明CSFV E2基因在PK-15细胞膜上成功表达.将表达E2蛋白的PK-15细胞腹腔免疫Balb/c小鼠,成功诱导小鼠产生了抗E2蛋白的抗体. 相似文献
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猪瘟病毒E2基因真核表达质粒的构建及基因疫苗的研究 总被引:31,自引:1,他引:30
构建了猪瘟病毒(classical swine fever virus, CSFV)主要保护性抗原E2基因4种不同的真核表达质粒.小鼠免疫试验表明,E2基因上不同的功能区对基因疫苗的免疫应答有很大影响,有信号肽序列的E2基因可诱导产生特异性免疫反应,且无跨膜区序列的E2基因所诱导的免疫应答反应比有跨膜区序列的强,而无信号肽序列的E2基因则不能诱导产生CSFV特异性的免疫反应.攻毒保护试验表明,免疫家兔最少可抵抗10个最小感染剂量(MID)的猪瘟兔化弱毒苗(Hog cholera lap-inized virus, HCLV)的攻击;免疫猪可抵抗致死剂量的CSFV石门株强毒的攻击. 相似文献
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采用Bac-to-Bac表达系统构建重组杆状病毒rAcV-MBP-Erns,感染Sf9昆虫细胞,经免疫荧光及Western blot分析证实MBP-Erns融合蛋白在Sf9细胞中高效表达。表达的MBP-Erns以可溶和包涵体两种形式存在。在Sf9细胞中规模化增殖重组病毒,经Amylose Resin亲和层析纯化获得高纯度MBP-Erns,制备的MBP-Erns具有良好免疫原性,这些工作为研究该蛋白的生物学功能和免疫原性奠定基础。 相似文献
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猪瘟病毒囊膜糖蛋白E2中和表位的定位及其免疫反应性分析 总被引:1,自引:0,他引:1
猪瘟病毒(CSFV)囊膜结构糖蛋白E2(gp55)是激发保护性免疫应答的主要抗原蛋白。E^ms和E2与细胞表面受体的相互作用介导病毒对细胞的感染过程。采用抗CSFV中和性单克隆抗体c24/10,淘选噬菌体展示的12肽随机肽库,结合噬菌体拟位免疫反应性分析结果,对CSFV E2蛋白中和表位进行定位。结果表明:F2蛋白的SPTTLR基序(832~837位氨基酸)构成CSFV特异性线性中和表位,基序的第一、二、三位氨基酸是表位与单克隆抗体c24/10结合所必需的氨基酸,也是表位的关键性氨基酸。 相似文献
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Glycoprotein Erns of pestiviruses induces apoptosis in lymphocytes of several species. 总被引:10,自引:0,他引:10
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C J Bruschke M M Hulst R J Moormann P A van Rijn J T van Oirschot 《Journal of virology》1997,71(9):6692-6696
Classical swine fever virus and bovine virus diarrhea virus are members of the genus pestivirus, which belongs to the family of the Flaviviridae. Recently, envelope glycoprotein Erns was identified as an RNase. RNases can express different biological actions. They have been shown to be neurotoxic, antihelminthic, and immunosuppressive. We studied the immunosuppressive properties of Erns in vitro. The glycoprotein totally inhibited concanavalin A-induced proliferation of porcine, bovine, ovine, and human lymphocytes. We then studied the direct cytotoxic effects of Erns on lymphocytes and epithelial cells in protein synthesis assays. Erns strongly inhibited the protein synthesis of lymphocytes of different species, without cell membrane damage. This suggested an apoptotic process, and indeed apoptosis of lymphocytes was detected after incubation with Erns. Pestivirus infections are characterized by leukopenia and immunosuppression. Our results suggest that Erns plays an important role in the pathogenesis of pestiviruses. 相似文献
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猪瘟病毒E2基因的定点突变、在大肠杆菌中的高效表达及表达产物的免疫原性 总被引:9,自引:0,他引:9
用重组PCR技术对猪瘟病毒石门株E2基因进行了定点突变, 然后将突变后的基因克隆至表达载体质粒pET-28a(+)中,构建成重组质粒pETE2。将pETE2转入受体菌BL21(DE3)plysS中,在IPTG的诱导下, 重组转化菌可高效表达目的基因, 表达量平均可达菌体蛋白总量的28%。免疫印迹和间接ELISA表明所表达的蛋白是CSFV特异性的。此重组蛋白免疫的家兔可抵抗猪瘟兔化弱毒的攻击。 相似文献
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Hong TIAN Xiang-tao LIU Jing-yan WU You-jun SHANG Tao JIANG Hai-xue ZHENG Qing-ge XIE 《中国病毒学》2008,23(4)
E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD) of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15) by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain, and encourages further work towards the development of a vaccine against CSFV infection. 相似文献
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Hong Tian Xiang-tao Liu Jing-yan Wu You-jun Shang Tao Jiang Hai-xue Zheng Qing-ge Xie 《中国病毒学》2008,23(4):247-254
E2 is an envelope glycoprotein of Classical swine fever virus (CSFV) and contains sequential neutralizing epitopes to induce
virus-neutralizing antibodies and mount protective immunity in the natural host. In this study, four antigen domains (ABCD)
of the E2 gene was cloned from CSFV Shimen strain into the retroviral vector pBABE puro and expressed in eukaryotic cell (PK15)
by an retroviral gene expression system, and the activity of recombinant E2 protein to induce immune responses was evaluated
in rabbits. The results indicated that recombinant E2 protein can be recognized by fluorescence antibodies of CSFV and CSFV
positive serum (Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China) using Western
blot, indirect immunofluorescence antibody test (IFAT) and ELISA, Furthermore, anti-CSFV specific antibodies and lymphocyte
proliferation were elicited and increased by recombinant protein after vaccination. In the challenge test, all of rabbits
vaccinated with recombinant protein and Chinese vaccine strain (C-strain) were fully protected from a rabbit spleen virus
challenge. These results indicated that a retroviral-based epitope-vaccine carrying the major antigen domains of E2 is able
to induce high level of epitope-specific antibodies and exhibits similar protective capability with that induced by the C-strain,
and encourages further work towards the development of a vaccine against CSFV infection.
Foundation items: The National “973” (2005CB523201); Key Technology R&D Programme (2006BAD06A03). 相似文献
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含CSFV E2基因A/D区原核表达载体的构建、表达及其抗原性研究 总被引:1,自引:0,他引:1
应用RT PCR方法扩增了编码猪瘟病毒石门株 (CSFVshimenstrain)囊膜糖蛋白E2全基因 ,然后将其克隆到pMD 1 8T质粒中 ,获得重组质粒pMD E2。再以pMD E2为模板 ,另行设计两对引物 ,同时扩增其中一段适于在E .coli中表达且抗原反应性较好的基因片段 (E2蛋白A D抗原区基因序列 ) ,将扩增的两片段串联插入原核表达载体pET 32a中构建成重组质粒pET 2e。用酶切和序列分析鉴定插入目的基因的正确性。SDS PAGE和Western blot分析表明 ,经pET 2e转化、IPTG诱导的受体菌可表达目的蛋白 ,克隆在硫氧还蛋白 (thioredoxinprotein ,TrxA)基因下游的E2蛋白基因与TrxA基因获得了高效融合表达 ,并且具有免疫学反应活性 ,这为猪瘟的血清学诊断方法的建立打下了基础 。 相似文献