斑点叉尾鮰病毒ORF6和ORF10基因的原核表达及其功能的初步研究
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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,由Fijan等从驯养的斑点叉尾鮰病鱼体内分离得到,斑点叉尾鮰是其自然宿主。CCV是一种被膜的双链DNA病毒,给国内外的斑点叉尾鮰养殖业造成极其严重的经济损失。序列分析显示斑点叉尾鮰开放阅读框ORF6和ORF10为预测的膜蛋白基因,可能参与病毒的吸附、入侵以及传播过程,因此,本文选择ORF6和ORF10两个基因进行研究,主要取得以下结果：
1、将斑点叉尾鮰卵巢细胞CCO复苏,在28℃下,用含有10%胎牛血清的DMEM培养基传代培养,并以CCO来增殖斑点叉尾鮰病毒CCV,通过酚氯仿法从细胞培养液中提取CCV全基因组DNA。
2、根据CCV基因序列,设计并合成ORF6和ORF10的特异性引物,通过PCR扩增获得ORF6和ORF10全基因序列,大小分别为417 bp和456 bp；克隆至表达载体pET-32a,构建原核表达载体pET-32a-ORF6和pET-32a-ORF10;经IPTG诱导,ORF6和ORF10基因成功表达,目的条带显示的分子量大小和理论值35.4 KD和36.4KD稍有差异,目的蛋白均以包涵体形式存在。
3、纯化表达产物后免疫新西兰大白兔,制备ORF6和ORF10的多克隆抗体,Western Blot检测结果表明获得的ORF6和ORF10多克隆抗体特异性较好,且ORF10是CCV病毒粒子的结构蛋白。ORF6和ORF10多克隆抗体的中和实验表明两个多克隆抗体可以有效抑制CCV对CCO的感染,具有一定的保护作用；对于不同时相下的同一多克隆抗体浓度实验组而言,随着感染时间的加长,CCO细胞病变越来越严重,两个多克隆抗体的保护性越来越弱；对比同一时相同一浓度,ORF10多克隆抗体比ORF6多克隆抗体抑制细胞病变的能力强,推断ORF10多克隆抗体具有更好的保护性。
Channel catfish virus (CCV) is the first herpesvirus which was discovered in fish. It was separated by Fijan from farmed channel catfish affected by channel catfish virus, and channel catfish is the natural host. CCV is a double-strand DNA virus which owns envelope, cause acute hemorrhagic toxemia and led channel catfish farming to a huge economic loss. The open reading frame ORF6 and ORF10 of channel catfish virus was thought to be envelope protein, which can participate in adsorption, invasion and transmission of virus, so the two genes were chosen to be analyzed and the main results were as following.
1. CCO was recovered firstly and incubated in DMEM involved 10% FBS at 28℃.CCV was reproduced through infecting CCO, and then the genome of CCV was extracted.
2. Specific primers were designed according to gene ORF6 and ORF10, the full length of ORF6 and ORF10 sequences were amplified by PCR which was 417 bp and 456 bp respectively. ORF6 and ORF10 were cloned to expressed vector pET-32a and the recombinant plasmid pET-32a-ORF6 and pET-32a-ORF10 was constructed successfully. The expressed proteins with theoretical molecular weight of 35.4 KD and 36.4 KD respectively were analysed by SDS-PAGE and were in the inclusion.
3. The purified expressed proteins were used to immune rabbits separately and polyclonal antibodies were obtained. The results of Western Blot indicated that the polyclonal antibody of ORF10 could react specifically and was proved to be a structural protein of CCV, but the polyclonal antibody of ORF6 had no reaction. The results of neutralization experiment showed that ORF6 and ORF10 polyclonal antibodies can restrain the infection of CCV. It could be found the ability of protection of the two polyclonal antibodies became more and more weak with time lengthening, and the polyclonal antibody of ORF10 did better than the polyclonal antibody of ORF6.
1、将斑点叉尾鮰卵巢细胞CCO复苏,在28℃下,用含有10%胎牛血清的DMEM培养基传代培养,并以CCO来增殖斑点叉尾鮰病毒CCV,通过酚氯仿法从细胞培养液中提取CCV全基因组DNA。
2、根据CCV基因序列,设计并合成ORF6和ORF10的特异性引物,通过PCR扩增获得ORF6和ORF10全基因序列,大小分别为417 bp和456 bp；克隆至表达载体pET-32a,构建原核表达载体pET-32a-ORF6和pET-32a-ORF10;经IPTG诱导,ORF6和ORF10基因成功表达,目的条带显示的分子量大小和理论值35.4 KD和36.4KD稍有差异,目的蛋白均以包涵体形式存在。
3、纯化表达产物后免疫新西兰大白兔,制备ORF6和ORF10的多克隆抗体,Western Blot检测结果表明获得的ORF6和ORF10多克隆抗体特异性较好,且ORF10是CCV病毒粒子的结构蛋白。ORF6和ORF10多克隆抗体的中和实验表明两个多克隆抗体可以有效抑制CCV对CCO的感染,具有一定的保护作用；对于不同时相下的同一多克隆抗体浓度实验组而言,随着感染时间的加长,CCO细胞病变越来越严重,两个多克隆抗体的保护性越来越弱；对比同一时相同一浓度,ORF10多克隆抗体比ORF6多克隆抗体抑制细胞病变的能力强,推断ORF10多克隆抗体具有更好的保护性。
Channel catfish virus (CCV) is the first herpesvirus which was discovered in fish. It was separated by Fijan from farmed channel catfish affected by channel catfish virus, and channel catfish is the natural host. CCV is a double-strand DNA virus which owns envelope, cause acute hemorrhagic toxemia and led channel catfish farming to a huge economic loss. The open reading frame ORF6 and ORF10 of channel catfish virus was thought to be envelope protein, which can participate in adsorption, invasion and transmission of virus, so the two genes were chosen to be analyzed and the main results were as following.
1. CCO was recovered firstly and incubated in DMEM involved 10% FBS at 28℃.CCV was reproduced through infecting CCO, and then the genome of CCV was extracted.
2. Specific primers were designed according to gene ORF6 and ORF10, the full length of ORF6 and ORF10 sequences were amplified by PCR which was 417 bp and 456 bp respectively. ORF6 and ORF10 were cloned to expressed vector pET-32a and the recombinant plasmid pET-32a-ORF6 and pET-32a-ORF10 was constructed successfully. The expressed proteins with theoretical molecular weight of 35.4 KD and 36.4 KD respectively were analysed by SDS-PAGE and were in the inclusion.
3. The purified expressed proteins were used to immune rabbits separately and polyclonal antibodies were obtained. The results of Western Blot indicated that the polyclonal antibody of ORF10 could react specifically and was proved to be a structural protein of CCV, but the polyclonal antibody of ORF6 had no reaction. The results of neutralization experiment showed that ORF6 and ORF10 polyclonal antibodies can restrain the infection of CCV. It could be found the ability of protection of the two polyclonal antibodies became more and more weak with time lengthening, and the polyclonal antibody of ORF10 did better than the polyclonal antibody of ORF6.
引文
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3. 蔡铭升,程安春,汪铭书.疱疹病毒UL35基因及其编码蛋白的研究进展.病毒学报,2009,25(1)：73-76
4.柴玉波,大肠杆菌表达系统表达载体的类型及其研究进展.国外医学分子生物学分册,1997,19(4)：160-162
5.陈昌福.斑点叉尾鮰主要疾病与防治对策.科学养鱼,2004, (12)：12-13
6.陈建君,张毓金,杨增岐,等.鸭瘟病毒的分子生物学研究进展.动物医学进展,2003,24(6)：25-28
7.冯小黎.重组包涵体蛋白质的折叠复性.生物化学与生物物理进展,2001,28(4)：482-485
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