斑点叉尾鮰病毒囊膜蛋白的鉴定及定位研究
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摘要
斑点叉尾鮰病毒(CCV)是最早被发现的鱼类疱疹病毒。因为通过SignaIP3.0(Bendtsen et al.2004) and SOSUI (Hirokawa et al.1998)计算程序,orf10被预测编码Ⅰ型囊膜蛋白。该囊膜蛋白具有一个潜在的裂解信号序列(amino acids, aa1-24)和一个跨膜区(amino acids, aa101-123),所以使用E. coli表达orf10,表达的产物作为抗原用来制备抗体。。本研究分为两部分：第一部分研究orf10基因编码的囊膜蛋白的功能特性和定位；第二部分是选择orf6基因研究了斑点叉尾鮰病毒PCR-ELISA检测方法以及该基因原核表达产物的免疫原性。主要取得以下结果：
1、成功地克隆了CCV的orf10基因。orf10有456bp,通过序列比对,和α疱疹病毒科其他已知的种类没有很大的同源性。通过斑点杂交分析,估计的蛋白分子质量接近35kDa,比理论的分子质量稍大(16kDa)。这个差异有两个原因引起,一是转译后修饰,二是和别的囊膜蛋白产生相互作用。同时,完整粒子和囊膜在斑点杂交中的两条带存在2kDa的差异,这可能是因为二硫键和/或糖基化作用,因为orf10被预测存在两个潜在的N-糖基化位点,分别在Asn64和Asn84。同样在原核表达系统中,orf10基因表达的蛋白也显示了同样分子质量,和预期的大小一致,同样缺少翻译后修饰。
2、成功地确定了orf10基因在病毒粒子的位置。基于斑点杂交试验和免疫电子显微实验,基因orf10编码的蛋白被证明是一个囊膜蛋白。
3、中和实验的结果表明CCV感染力降低,可能是因为位阻现象或者与抗体结合的封闭作用。这些结果都说明orf10和病毒侵入有直接的关系,虽然准确的机制还不是很清楚。因此,下一步的研究重点应该是抗原表位的研究。
4、通过斑点杂交试验表明使用新型表达载体表达的orf6囊膜蛋白具有很好的免疫原性。
5、根据CCV的orf6基因设计引物和捕获探针,其中一条引物5‘端标记生物素,而捕获探针的3‘端标记地高辛,利用链亲和素--生物素的亲和作用捕获样品,采用热变性的方法,使用PCR仪完成样品和检测探针的杂交,使用标记碱性磷酸酶的抗地高辛二抗进行显色反应,建立了CCV的PCR-ELISA检测方法。
6、建立的PCR-ELISA检测方法具有特异性强,重复性好和敏感度高的特点。重复性检测的批内变异系数小于10%；批间变异系数不大于15%。该方法的敏感度为5龟CCV DNA。
Channel catfish virus (CCV) was the first herpesvirus which was discovered in fish. Because orf10was predicted to encode a type I membrane protein with a potential, cleavable signal sequence (amino acids, aa1-24) and a transmembrane domain (amino acids, aa101-123), as determined by the computer program SignaIP3.0(Bendtsen et al.2004) and SOSUI (Hirokawa et al.1998), it was expressed in E. coli and used as an antigen to prepare an antibody. This study was disparted parts:first member studied function characteristic and location; second member studied the method of DNA probe detection of the channel catfish virus and immunity of production. the main results were as following.
1. In this study, we have successfully cloned the orf10gene of CCV. orf10is456bp and has no significant homology with other known viruses in the Alloherpesvirus family by sequence alignment. The estimated molecular mass of the protein by western blot analysis was approximately35kDa, which is much greater than the theoretical molecular mass (16kDa). This difference may be attributed to post-translational modifications or interactions with other envelope proteins. Also there were two bands that differed in size by about2kDa present in the intact virion and envelope lanes of the western blot, which might be due to disulfide bonding and/or glycosylation for ORF10was predicted to contain two potental N-glycosylted sites at Asn64and Asn84. As in prokaryotic expression system, the product of orf10gene displayed the equal molecular mass with expected size for lacking the posttranslational modification.
2. In this study, we have successfully identified its location in virions.The product of the orf10gene was identified to be an envelope protein, based on the results of western blotting assay and immuno-electron microscopy.
3. The results of the neutralization assay showed that CCV infection was reduced, due to the steric hindrance or the blocking of the binding with its cellar receptor by the primary antibody.These results suggested that orf10was also involved in the entry of CCV, although its exact mechanism was not clear. More interesting would be a later consideration of whether neutralizing this one epitope was likely to be sufficient to protect fish.
4. ORF6envelope protein using new expression carrier had good immunity.
5. A probe and a pair of primers were designed based on the orf6of CCV.5'end of primer was labeled by biotin,3'end of probe was labeled by digoxigenin, sample was captured by streptoavidin-biotin function. The hybridization with sample and probe was carried out on PCR. Then the hybridized product was detected with a AP-labeled anti-DIG antibody and PNPP substrate.
6. Established PCR-ELISA measure method had characteristic of strong special, good repetition and high sensitivity. Variance coefficient of repetition measure to a patch was less than10%; Variance coefficient of repetition measure among patchs was less than15%. Sensitivity of this method was5fg CCV DNA.
1、成功地克隆了CCV的orf10基因。orf10有456bp,通过序列比对,和α疱疹病毒科其他已知的种类没有很大的同源性。通过斑点杂交分析,估计的蛋白分子质量接近35kDa,比理论的分子质量稍大(16kDa)。这个差异有两个原因引起,一是转译后修饰,二是和别的囊膜蛋白产生相互作用。同时,完整粒子和囊膜在斑点杂交中的两条带存在2kDa的差异,这可能是因为二硫键和/或糖基化作用,因为orf10被预测存在两个潜在的N-糖基化位点,分别在Asn64和Asn84。同样在原核表达系统中,orf10基因表达的蛋白也显示了同样分子质量,和预期的大小一致,同样缺少翻译后修饰。
2、成功地确定了orf10基因在病毒粒子的位置。基于斑点杂交试验和免疫电子显微实验,基因orf10编码的蛋白被证明是一个囊膜蛋白。
3、中和实验的结果表明CCV感染力降低,可能是因为位阻现象或者与抗体结合的封闭作用。这些结果都说明orf10和病毒侵入有直接的关系,虽然准确的机制还不是很清楚。因此,下一步的研究重点应该是抗原表位的研究。
4、通过斑点杂交试验表明使用新型表达载体表达的orf6囊膜蛋白具有很好的免疫原性。
5、根据CCV的orf6基因设计引物和捕获探针,其中一条引物5‘端标记生物素,而捕获探针的3‘端标记地高辛,利用链亲和素--生物素的亲和作用捕获样品,采用热变性的方法,使用PCR仪完成样品和检测探针的杂交,使用标记碱性磷酸酶的抗地高辛二抗进行显色反应,建立了CCV的PCR-ELISA检测方法。
6、建立的PCR-ELISA检测方法具有特异性强,重复性好和敏感度高的特点。重复性检测的批内变异系数小于10%；批间变异系数不大于15%。该方法的敏感度为5龟CCV DNA。
Channel catfish virus (CCV) was the first herpesvirus which was discovered in fish. Because orf10was predicted to encode a type I membrane protein with a potential, cleavable signal sequence (amino acids, aa1-24) and a transmembrane domain (amino acids, aa101-123), as determined by the computer program SignaIP3.0(Bendtsen et al.2004) and SOSUI (Hirokawa et al.1998), it was expressed in E. coli and used as an antigen to prepare an antibody. This study was disparted parts:first member studied function characteristic and location; second member studied the method of DNA probe detection of the channel catfish virus and immunity of production. the main results were as following.
1. In this study, we have successfully cloned the orf10gene of CCV. orf10is456bp and has no significant homology with other known viruses in the Alloherpesvirus family by sequence alignment. The estimated molecular mass of the protein by western blot analysis was approximately35kDa, which is much greater than the theoretical molecular mass (16kDa). This difference may be attributed to post-translational modifications or interactions with other envelope proteins. Also there were two bands that differed in size by about2kDa present in the intact virion and envelope lanes of the western blot, which might be due to disulfide bonding and/or glycosylation for ORF10was predicted to contain two potental N-glycosylted sites at Asn64and Asn84. As in prokaryotic expression system, the product of orf10gene displayed the equal molecular mass with expected size for lacking the posttranslational modification.
2. In this study, we have successfully identified its location in virions.The product of the orf10gene was identified to be an envelope protein, based on the results of western blotting assay and immuno-electron microscopy.
3. The results of the neutralization assay showed that CCV infection was reduced, due to the steric hindrance or the blocking of the binding with its cellar receptor by the primary antibody.These results suggested that orf10was also involved in the entry of CCV, although its exact mechanism was not clear. More interesting would be a later consideration of whether neutralizing this one epitope was likely to be sufficient to protect fish.
4. ORF6envelope protein using new expression carrier had good immunity.
5. A probe and a pair of primers were designed based on the orf6of CCV.5'end of primer was labeled by biotin,3'end of probe was labeled by digoxigenin, sample was captured by streptoavidin-biotin function. The hybridization with sample and probe was carried out on PCR. Then the hybridized product was detected with a AP-labeled anti-DIG antibody and PNPP substrate.
6. Established PCR-ELISA measure method had characteristic of strong special, good repetition and high sensitivity. Variance coefficient of repetition measure to a patch was less than10%; Variance coefficient of repetition measure among patchs was less than15%. Sensitivity of this method was5fg CCV DNA.
引文
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