CN110407920A - 一种鲤疱疹病毒ⅱ型衣壳蛋白orf66的原核表达、蛋白纯化方法 - Google Patents
一种鲤疱疹病毒ⅱ型衣壳蛋白orf66的原核表达、蛋白纯化方法 Download PDFInfo
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Abstract
本发明首先公开了一种编码鲤疱疹病毒Ⅱ型ORF66蛋白的基因的核苷酸序列;然后公开了所述基因编码的鲤疱疹病毒Ⅱ型ORF66蛋白的氨基酸序列;接着公开了包含所述基因的重组载体,最后公开了一种鲤疱疹病毒Ⅱ型衣壳蛋白ORF66的原核表达、蛋白纯化方法。到目前为止,尚未有人体外表达过鲤疱疹病毒(CyHV‑2)ORF66蛋白,而本发明通过基因工程方法,首次成功表达纯化了鲤疱疹病毒(CyHV‑2)ORF66蛋白。
Description
技术领域
本发明属于生物工程技术领域,具体涉及一株鲤疱疹病毒Ⅱ型衣壳蛋白ORF66的基因克隆和原核表达、纯化方法。
背景技术
鲫鱼是我国淡水养殖的常见鱼种,属于鲤形目(Cypriniformes),鲤科(Cyprinidae),鲫属(Carassius)。鲫鱼(Crucian carp)饲养成本低,养殖地域广,在中国淡水养鱼上占有重要地位,中国鲫鱼养殖主要集中在东部、中部和南部地区,如江苏、湖北、江西、安徽、山东、广东、四川、辽宁、湖南、浙江等省。据统计,鲫鱼的养殖总产量已达到300多万吨,约占全国淡水鱼总养殖产量的12%。2013年中国鲫鱼年产量达259.44万吨,其中主要养殖品种为异育银鲫(Carassius auratus gibelio)。异育银鲫具有生长快速、抗逆性强、肉质好的优点,深受人们的喜爱。然而,随着养殖规模的逐渐扩大、集约化程度的不断提高,异育银鲫的病害问题日益突出。2012年,江苏省盐城地区养殖的异育银鲫爆发了一种以“鳃出血”为主要症状的疾病,称为鲫造血器官坏死病(俗称“鳃出血病”)。该病具有发病急、传播快、死亡率高的特点,严重威胁我国异育银鲫养殖业发展。研究发现,鲤疱疹病毒Ⅱ型(Cyprinid herpesvirus 2,CyHV-2)是造成鲫造血器官坏死病的病原。
鲤科疱疹病毒II型,又称为疱瘆造血器官坏死病毒(或金鱼造血器官坏死病毒)。该病毒隶属于疱疹病毒目(Herpesvirales)、鱼疱疹病毒科(Alloherpesviridae)、鲤疱疹病毒属 (Cyprinivirus),CyHV-2与分离自鲤科鱼类的另外两种病毒鲤疱疹毒1(Cyprinidherpesvirus 1, CyHV-1)和鲤疱疹病毒3(Cyprinid herpesvirus 3,CyHV-3)亲缘关系较近,与沟鲇疱疹病毒 (Ictalurid herpes-virus 1,IcHV-1)关系相对较远。在病原学方面,CyHV-2具有鱼类疱疹病毒的一般特征。病毒呈球形,具有二十面体立体对称的衣壳结构,内部包有长度约为290,304bp 的双股DNA,外面为皮质层和囊膜。现已确认,鱼类疱疹病毒基因组含有12个核心ORF,这些编码蛋白在CyHV-1和CyHV-3中均有分布,主要参与病毒复制、装配以及衣壳的形成等过程。Jung等在1995年对CyHV-2进行了第一次报道,发现CyHV-2是金鱼造血器官坏死病的致病病原。之后在美国、澳大利亚、中国台湾、英国养殖的金鱼也暴发该病。自从 2011年匈牙利首次在养殖的鲫鱼体内检测到CyHV-2后,近几年来关于CyHV-2感染鲫鱼的报道也逐渐增多,该病毒在中国、日本、美国、澳大利亚、英国、新西兰等地广泛传播,造成养殖鲫鱼和金鱼大量死亡。这些报道均表明CyHV-2是一种新生养殖鲫鱼病毒病,其发病区域在逐年扩大。CyHV-2病害已经引起我国水产养殖业管理部门及养殖户高度重视。
发明内容
针对现在技术的不足,本发明第一个目的是提供一株鲤疱疹病毒Ⅱ型ORF66蛋白的基因;本发明的第二个目的是提供鲤疱疹病毒Ⅱ型ORF66蛋白基因编码的蛋白质;本发明的第三个目的是提供一种鲤疱疹病毒Ⅱ型衣壳蛋白ORF66的原核表达、蛋白纯化方法。
本发明解决其技术问题采用的技术方案是:
一株鲤疱疹病毒Ⅱ型ORF66蛋白的基因,其核苷酸序列如SEQ IDNO:1所示。
上述鲤疱疹病毒Ⅱ型ORF66蛋白基因编码的蛋白质,其氨基酸序列如SEQID NO.2所示相应的,本发明提供包含上述CyHV-2蛋白ORF66基因的重组载体和表达方法。
具体地,所述重组载体的制备方法如下:提取异育银鲫感染鲤疱疹病毒Ⅱ型脾脏RNA,并反转录出cDNA;以cDNA为模板扩增ORF66基因;将ORF66扩增产物经BamH I和HindIII双酶切后,通过DNA连接酶***到经同样双酶切的pET-28a表达载体中,得到重组质粒pET28a-ORF66。
优选地,以cDNA为模板,利用以下引物扩增ORF66基因,引物序列如下:
引物序列如下:
上游引物:ORF66-F:AGGATCCATGACCTCCCTCCAGCAAGCTAC下划线为BamHI酶切位点;
下游引物:ORF66-R:TAAGCTTATAAATCATGAGATCGTCTAGGTC下划线为Hind III酶切位点;
上下游引物分别如SEQ ID NO:3和SEQ ID NO:4所示。
具体地,一种鲤疱疹病毒Ⅱ型衣壳蛋白ORF66的原核表达、蛋白纯化方法其特征在于,包括以下步骤:
(1)用上述重组载体转化大肠杆菌细胞,得到表达ORF66的重组原核表达菌株pET28a-ORF66-BL21;
(2)利用步骤(1)所述菌株pET28a-ORF66-BL21生产重组鲤疱疹病毒Ⅱ型ORF66蛋白的方法,具体为将菌株pET28a-ORF66-BL21接种在LB液体培养基中,37℃,180转/分培养14-16小时,按照1:100接种在LB液体培养基中,37℃,180转/分培养至OD600达到0.6-0.8,然后加入IPTG至终浓度为0.1-1.0mM,37℃,180转/分培养2小时以上离心并收集菌体,超声破碎后、离心、收集上清,经分离纯化得到重组鲤疱疹病毒Ⅱ型ORF66蛋白。
优选的,大量诱导表达的条件为37℃、0.9mM、过夜。
优选的,还包括如下步骤:将分离纯化得到的重组鲤疱疹病毒Ⅱ型ORF66蛋白加入到透析袋中,在20mMPB,0.5mM NaCl,0.5mM EDTA,pH8.0进行透析过夜;然后把透析后的蛋白溶液加入到超滤管中,4℃,6000g离心1-2小时,去掉滤出液,得到重组鲤疱疹病毒Ⅱ型ORF66蛋白。
有益效果
本发明的有益效果是:到目前为止,尚未有人体外表达过鲤疱疹病毒(CyHV-2)ORF66蛋白,而本发明通过基因工程方法,首次成功表达纯化了鲤疱疹病毒(CyHV-2)ORF66蛋白。本发明的目的在于提供一株鲤疱疹病毒Ⅱ型衣壳蛋白ORF66的基因克隆和原核表达、蛋白纯化方法。
基因的原核表达是研究基因功能的常用技术之一,但在原核表达中,蛋白通常以包涵体的形式存在于沉淀中,本发明得到的ORF66的蛋白包涵体的形式存在于沉淀中,并且针对蛋白表达条件进行一系列的优化,发现ORF66在BL21(DE3)菌株中当诱导温度为37℃时,其表达量最佳;随着IPTG浓度的增加,蛋白表达量逐渐增加,最佳诱导浓度为0.9mM;在37℃,IPTG浓度为0.9mM时,表达量为最佳。经SDS-PAGE电泳检测,得到分子量约为48kDa的目的条带,为ORF66蛋白预期大小。
附图说明
图1为酶切结果示意图,M为DNA ladder(1000,2000,3000,4000,5000,6000,7000,8000,10000);1为重组质粒未经EcoRⅠ和HindⅢ双酶切;2为重组质粒经EcoR Ⅰ和HindⅢ双酶切;
图2为ORF66蛋白可溶性分析的图,其中,M为蛋白质分子质量标准;1为未诱导菌裂解上清;2为未诱导菌裂解沉淀;3为诱导菌裂解上清;4为诱导菌裂解沉淀;
图3为pET-28a-ORF66在不同IPTG浓度下表达的SDS-PAGE分析:其中,M为蛋白质分子质量标准;1-6依次为0mM、0.1mM、0.3mM、0.5mM、0.7mM和0.9mM的IPTG诱导全菌;
图4为pET-28a-ORF66在不同诱导时间下表达的SDS-PAGE分析;其中,M为蛋白质分子质量标准;1-5依次为诱导过夜、6h、4h、2h、0h后的诱导全菌;
图5为SDS-PAGE电泳测试最佳诱导温度的结果图,其中,M为蛋白质分子质量标准;1-5依次为20℃、25℃、28℃、30℃、37℃后的诱导全菌;
图6为ORF66蛋白纯化后的电泳结果图;其中,M为蛋白质分子质量标准;1为未诱导菌裂解上清;2为未诱导菌裂解沉淀;3为诱导菌裂解上清;4为诱导菌裂解沉淀;5为变性蛋白;6为磁珠洗涤Ⅰ次;7为磁珠洗涤Ⅱ次;8为洗脱蛋白;
图7为western blot检测图:其中,M为蛋白质分子质量标准;1为纯化后样品。
具体实施方式
以下结合实施例对本发明做进一步详细说明。所用试剂或者仪器设备未注明生产厂商的,均视为可以通过市场购买的常规产品。
实施例1:
实施例1:鲤疱疹病毒Ⅱ型ORF66蛋白的基因大肠杆菌表达载体pET28a-ORF66的构建
1、其参照GenBank上公布的CyHV-2SY-C1株(Access No.KM200722.1)中ORF66基因全长序列独立自主设计引物;
2、提取异育银鲫感染鲤疱疹病毒Ⅱ型脾脏RNA,并反转录出cDNA;
3、以cDNA为模板,利用以下引物扩增ORF66基因,引物序列如下:
上游引物:ORF66-F:AGGATCCATGACCTCCCTCCAGCAAGCTAC;下划线为BamHI酶切位点;
下游引物:ORF66-R:TAAGCTTATAAATCATGAGATCGTCTAGGTC;下划线为Hind III酶切位点;
上下游引物分别如SEQ ID NO:3和SEQ ID NO:4所示。
用于载体构建的基因扩增所用的PCR扩增体系如下:5×buffer 10μL,FastPfuFlyDNA Polymerase 1μL(全式金),2.5mmol dNTPs 4μL,cDNA模板0.5μL,10μmol上下游引物各1μL,ddH2O 32.5μL。
PCR反应条件为:94℃预变性5min;94℃变性1min,退火1min,72℃延伸1 min 30s,共33个循环;然后72℃延伸10min。
4、构建OR66基因重组载体
合成的鲤疱疹病毒Ⅱ型ORF66基因和pET28a载体用限制性内切酶BamH I和Hind III37℃双酶切5h。体系如下:内切酶各1μL;重组质粒14μL;10×Buffer T 14μL;总体系 20μL。
酶切产物进行琼脂糖凝胶电泳,用天根切胶回收试剂盒进行回收,用TAKARAT4连接酶16℃连接过夜。
pET-28a(+)0.5μL;目的基因4.5μL;T4DNA Ligase 0.5μL;10×Ligase Buffer 1μL;ddH2O 3.5μL;总体系10μL。
将连接产物用标准氯化钙转化法转入大肠杆菌BL21中,用LB平板筛选转化子,以标准方法提取质粒,用限制性内切酶BamH I和Hind III双酶切重组质粒,得到大和小两个片段,大小分别与编码鲤疱疹病毒Ⅱ型ORF66蛋白的基因和表达载体pET28a(+)大小相同,再通过上海生工生物科技有限公司测序验证其序列正确性。测序结果表明,所获得的ORF66基因编码区序列与预计相符,证明编码鲤疱疹病毒Ⅱ型ORF66蛋白的基因已克隆入大肠杆菌表达载体pET28a(+)中,重组质粒命名为pET28a-ORF66。酶切分析图分别见图1。
实施例2:ORF66蛋白的诱导表达
1、获得ORF66的重组原核表达菌株
挑选实施例1中测序成功的单克隆接种到50ug/mL卡那霉素液体培养基中,37℃、200rpm 过夜培养,按照天根质粒小量提取试剂盒将pET28a-ORF66重组表达载体提取出来,取重组表达载体质粒转化大肠杆菌表达菌株BL21(DE3)检测ORF66蛋白的表达。
2、诱导表达
将制备好的重组质粒pET28a-ORF66用标准氯化钙转化法转入大肠杆菌BL21感受态细胞中,在LB平板上37℃进行培养。过夜后长出单克隆,挑取大肠杆菌工程菌pET28a-ORF66-BL21单菌落接种在10mL LB液体培养基中,37℃,180转/分培养过夜培养。将培养过夜的菌液按1:100的比例接种于10mL LB液体培养基中,37℃,180转/分培养至OD600到0.6- 0.8;添加IPTG至终浓度为1mM,30℃、180r/min振荡培养6h;获得ORF66的重组蛋白, SDS-PAGE电泳进行分析。
实施例3:鲤疱疹病毒Ⅱ型ORF66的大肠杆菌重组株pET28a-ORF66-BL21的构建及表达条件优化
1、ORF66基因诱导表达最佳IPTG的浓度的确定
挑取大肠杆菌工程菌pET28a-ORF66-BL21单菌落接种在10mL LB液体培养基中,37℃,180转/分培养过夜培养。将培养过夜的菌液按1:100的比例接种于10ml LB液体培养基中,37℃,180转/分培养至OD600到0.6-0.8;添加IPTG分别用终浓度0mM、0.1mM、0.3mM、 0.5mM、0.7mM和0.9mM的IPTG诱导表达菌6h。诱导完成后4℃,1,2000rpm离心2min,收集菌体用2mL预冷PBS buffer(用前加入1mM PMSF)悬浮,用超声破碎仪进行细胞破碎,每次10s,共10次,每次间隔30s;1,2000rpm离心10min,取上清,SDS-PAGE电泳检测,测试最佳IPTG诱导浓度,结果如图3所示,当IPTG浓度为0.9mM时,ORF66表达最多。
2、ORF66基因诱导表达最佳时间的确定
挑取大肠杆菌工程菌pET28a-ORF66-BL21单菌落接种在10mL LB液体培养基中,37℃,180转/分培养过夜培养。将培养过夜的菌液按1:100的比例接种于10ml LB液体培养基中,37℃,180转/分培养至OD600到0.6-0.8;在最佳诱导浓度条件下,于诱导后的第0h、2h、 4h、6h和过夜。诱导完成后4℃,1,2000rpm离心2min,收集菌体用2mL预冷PBS buffer(用前加入1mM PMSF)悬浮,用超声破碎仪进行细胞破碎,每次10s,共10次,每次间隔30s;1,2000rpm离心10min,取上清,SDS-PAGE电泳检测,测试最佳IPTG诱导浓度,结果如图4所示,当IPTG浓度为0.9mM时,诱导过夜时ORF66表达最多。
3、ORF66基因诱导表达最佳温度的确定
挑取大肠杆菌工程菌pET28a-ORF66-BL21单菌落接种在10mL LB液体培养基中,37℃,180转/分培养过夜培养。将培养过夜的菌液按1:100的比例接种于10ml LB液体培养基中,37℃,180转/分培养至OD600到0.6-0.8;在最佳诱导浓度条件加入0.9mM的IPTG,在 20℃、25℃、28℃、30℃、37℃诱导表达过夜。SDS-PAGE电泳分析表明,重组蛋白ORF66 在0.9mM最佳诱导的时间进行优化。诱导完成后4℃,1,2000rpm离心2min,收集菌体用 2mL预冷PBSbuffer(用前加入1mM PMSF)悬浮,用超声破碎仪进行细胞破碎,每次10sec,共10次,每次间隔30sec;1,2000rpm离心10min,取上清,SDS-PAGE电泳检测,测试最佳IPTG诱导浓度,结果如图5所示,当IPTG浓度为0.9mM时,诱导过夜时ORF66表达最多。从图中可以看出,在37℃诱导过夜时,重组蛋白ORF66的表达量达到最大。
因此,大量诱导表达的条件为37℃、0.9mM、过夜。
实施例4:ORF66重组蛋白的纯化
按照最佳诱导条件大量表达,冰浴超声破碎,沉淀经洗涤后,用含6mo1/L尿素的变性剂溶解,His磁珠纯化,蛋白定量。
(1)单菌落震荡培养过夜,次日按1%的比例接种于LB液体培养基(40μg/mL Kan)中,按最佳诱导条件诱导,5000rpm/min离心l0min,倾去上清;
(2)湿菌用PBS缓冲液重悬,冰浴超声破碎,超声条件为:300w功率,超5s停5s,共20min,10000rpm,4℃离心15min,弃上清;
(3)沉淀重悬于洗涤缓冲液(2M尿素、20mMPB,0.5mM NaCl、2%Triton X-100,PH8.0)缓冲液中,4℃涡旋搅拌30min,10000rpm/min离心10min,收集沉淀,重复洗涤一次;
沉淀用变性缓冲液(8ML尿素、20mMPB,0.5mM NaCl、2%Triton X-100、l0mmol/L咪唑、PH8.0)重悬。冰浴lh。4℃,16000g离心30min,上清采用0.45μm滤膜过滤后上柱。
按照磁珠试剂盒说明书的方法进行蛋白纯化。
①用移液枪取出5mL磁珠悬液于15mL离心管,进行磁性分离,弃上清;
②加5mL Binding Buffer到上述离心管中,上下翻转离心管数次,使磁珠重新悬浮;进行磁性分离,弃上清。
③重复洗涤2次。
④取10mL制备好的粗蛋白样品加入到装有预处理磁珠的离心管中,将离心管置于漩涡混匀器震荡15s。
⑤将离心管置于旋转混合仪上,试问旋转20-30min(可以在2-8℃的低温环境中,旋转1h,防止目标蛋白降解。)
⑥将离心管置于磁性分离器上进行分离,移出上清液至新的离心管以备后续检测,从此行分离器取下离心管进行后续洗涤步骤。
⑦加10mL Washing Buffer到装有磁珠的离心管,轻轻翻转数次,使磁珠重新悬浮,磁性分离,移出清洗液至新的离心管。以备取样检测。
⑧重复上述步骤2-3次。
⑨在装有磁珠的离心管中加入2-10mL Elution Buffer上下翻转离心管数次,使磁珠悬浮,磁性分离,收集洗脱液到新的离心管,即为纯化的目标蛋白。
⑩重复上述步骤1次。
按照蛋白定量测定试剂盒说明书,测定蛋白浓度,用Elution Buffer调整纯化产物浓度调整至1mg/mL。
2.2.2.4蛋白透析复性及纯度分析
将纯化的变性蛋白转移到透析袋中,尿素浓度梯度复性,取蛋白制样进行SDS-PAGE,分析蛋白纯度,蛋白分装后低温保存。
(1)新透析袋经2%NaHC03(m/v)中煮沸处理后,ddH20清洗后置于lmmol/L EDTA中煮沸l 0min,透析袋冷却后保存在EDTA中;
(2)将蛋白放入处理好的透析袋后,依次放入50倍体积的含4mo1/L,3mo1/L,2mo1/L,lmol/L,0.5mol/L尿素的复性缓冲液(20mMPB,0.5mM NaCl,0.5mM EDTA,10%甘油,1%精氨酸,0.1%PEG8000,PH8.0)的烧杯中,4℃磁力搅拌,复性缓冲液每6h更换一次,最终在PB缓冲液(PH8.0)中透析过夜;
(3)次日收集复性蛋白,4℃,10000rpm/min离心20min。取样进行SDS-PAGE,结果见图6。
实施例5:ORF66重组蛋白的western blot检测
(一)提取经过纯化后的ORF66蛋白:
1.培养含有pET28a-ORF66质粒的大肠杆菌表达菌株,IPTG诱导表达;
2.加入蛋白裂解液,用前加入1mM PMSF,1mM DTT,用超声波破碎细胞后,离心收集上清,冰上放置20min;
3. 15,000g,4℃离心5min,弃沉淀;
4.取200μL上清加入5×Loading Buffer,震荡混匀,剩余样品-80℃冻存;
5.沸水煮5min,冰浴,12,000rpm离心5min,吸取上清15μL进行蛋白电泳,剩余样品-20℃冻存;
6.制备SDS-PAGE胶,用100V电压进行电泳,至溴酚蓝跑出分离胶时停止电泳。
(二)转膜
用裁纸刀裁出比胶稍大一些的PVDF膜(对于PVDF膜,先用甲醇浸泡30秒,再在ddH2O 中浸泡3-5min,然后放入转膜buffer中)将胶板夹放入转移电泳槽。倒入转膜缓冲液,4℃,100V 90min。
(三)封闭
转膜完成后,取出PVDF膜,放于用5%脱脂奶粉的PBST溶液中,摇床上封闭2h。
(四)一抗孵育
弃去封闭液,加入Anti His抗体(1:5000),摇床孵育1h。
(五)二抗孵育
倒掉一抗,用1×PBST洗涤PVDF膜3次,每次5min;加入对应的二抗(羊抗小鼠),摇床孵育1h。
(六)显色
弃二抗,用1×PBST洗涤PVDF膜3次,每次5min;在PVDF膜上加入HRP化学显色底物液,从结果可以看出成功表达出ORF66蛋白。
本发明的保护内容不局限于以上实施例。在不背离发明构思的精神和范围下,本领域技术人员能够想到的变化和优点都被包括在本发明中,并且以所附的权利要求为保护范围。
序列表
<110> 盐城工学院
<120> 一种鲤疱疹病毒Ⅱ型衣壳蛋白ORF66的原核表达、蛋白纯化方法
<160> 4
<170> SIPOSequenceListing 1.0
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<211> 1197
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgacctccc tccagcaagc tacgggcgat ggtctgccgg ccatcggcta tgagcacgag 60
atcctcaacg tgcctctctg gtgctgcacg gcatccgaca agatcgaaca ctttgaggcc 120
ttgatcaaat gtaacctggg cgccttggac gaagtgtgcg aactcaagga tttcaggacg 180
ctcatcaaga cttgcatcga atccagaaac ctgaccgaca tcatctactc gggaggcggc 240
atcactacag tgggggctcc cctgtacgcc ttcatgaagc ggtggaacac cgccgtggat 300
aacgtcgcgg ccaaactgga attcatgagc gtcagacagc tggtggacga ctacaccaac 360
atgctagatc tggccgccaa aaagggcacc ctctttaacg agagcgacgt ggacatcatc 420
acacaggcca tcgtactatg cacgagggac ggagaggatt tttcaagact ctttaccttt 480
gccggcatag tgacctacca caactttcac acctgggaga acatggaatt cagtcacgac 540
agacccaggc agttcaacat catccgcagg ggaaccacca cgctcgaact caacaggcta 600
gagtttactc acaaggtacc ctacggtcag gtcaccaacc cttacatcat cctggcaacc 660
gtacccatga gagtctcttc ttcttcgtcc agccgaggaa gtctcaccaa actccaactc 720
ttttactggt cagatacaaa gattcacctc agcgccgctc tcacagcaga gtccgactgg 780
ggacacactt gcttcgattc gggcatcaga aacgcagaca tcaacctcat cgacaaatgg 840
aacaagtcag agactctcaa actgaacagc gatcagtacg tgggagccta ctacgtgatg 900
ggcaaacaaa tcacccagaa tccattctct ccgctcgaaa aacacagggc tcctcacgtc 960
aacatctgga gtaccgaaag ctggaccctc tactccaggc atcccatggt agagtacgaa 1020
cgaatcgaat ctcaactgga accagataca ggagccagtt ccttcgtaga caaactggtg 1080
gaaaagagcg atctctcagc ctgtctctca cacaccgatg ggaccatcga attacccagt 1140
agttgggtcg acccgagagc caaacagttt cacgacctag acgatctcat gatttat 1197
<210> 2
<211> 446
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
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Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
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Gly Ser Met Thr Ser Leu Gln Gln Ala Thr Gly Asp Gly Leu Pro Ala
35 40 45
Ile Gly Tyr Glu His Glu Ile Leu Asn Val Pro Leu Trp Cys Cys Thr
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Ala Ser Asp Lys Ile Glu His Phe Glu Ala Leu Ile Lys Cys Asn Leu
65 70 75 80
Gly Ala Leu Asp Glu Val Cys Glu Leu Lys Asp Phe Arg Thr Leu Ile
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Lys Thr Cys Ile Glu Ser Arg Asn Leu Thr Asp Ile Ile Tyr Ser Gly
100 105 110
Gly Gly Ile Thr Thr Val Gly Ala Pro Leu Tyr Ala Phe Met Lys Arg
115 120 125
Trp Asn Thr Ala Val Asp Asn Val Ala Ala Lys Leu Glu Phe Met Ser
130 135 140
Val Arg Gln Leu Val Asp Asp Tyr Thr Asn Met Leu Asp Leu Ala Ala
145 150 155 160
Lys Lys Gly Thr Leu Phe Asn Glu Ser Asp Val Asp Ile Ile Thr Gln
165 170 175
Ala Ile Val Leu Cys Thr Arg Asp Gly Glu Asp Phe Ser Arg Leu Phe
180 185 190
Thr Phe Ala Gly Ile Val Thr Tyr His Asn Phe His Thr Trp Glu Asn
195 200 205
Met Glu Phe Ser His Asp Arg Pro Arg Gln Phe Asn Ile Ile Arg Arg
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Gly Thr Thr Thr Leu Glu Leu Asn Arg Leu Glu Phe Thr His Lys Val
225 230 235 240
Pro Tyr Gly Gln Val Thr Asn Pro Tyr Ile Ile Leu Ala Thr Val Pro
245 250 255
Met Arg Val Ser Ser Ser Ser Ser Ser Arg Gly Ser Leu Thr Lys Leu
260 265 270
Gln Leu Phe Tyr Trp Ser Asp Thr Lys Ile His Leu Ser Ala Ala Leu
275 280 285
Thr Ala Glu Ser Asp Trp Gly His Thr Cys Phe Asp Ser Gly Ile Arg
290 295 300
Asn Ala Asp Ile Asn Leu Ile Asp Lys Trp Asn Lys Ser Glu Thr Leu
305 310 315 320
Lys Leu Asn Ser Asp Gln Tyr Val Gly Ala Tyr Tyr Val Met Gly Lys
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Gln Ile Thr Gln Asn Pro Phe Ser Pro Leu Glu Lys His Arg Ala Pro
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His Val Asn Ile Trp Ser Thr Glu Ser Trp Thr Leu Tyr Ser Arg His
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Pro Met Val Glu Tyr Glu Arg Ile Glu Ser Gln Leu Glu Pro Asp Thr
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Gly Ala Ser Ser Phe Val Asp Lys Leu Val Glu Lys Ser Asp Leu Ser
385 390 395 400
Ala Cys Leu Ser His Thr Asp Gly Thr Ile Glu Leu Pro Ser Ser Trp
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aggatccatg acctccctcc agcaagctac 30
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<212> DNA
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taagcttata aatcatgaga tcgtctaggt c 31
Claims (7)
1.一种编码鲤疱疹病毒Ⅱ型ORF66蛋白的基因,其特征在于,所述基因的核苷酸序列如SEQ ID NO:1所示。
2.权利要求1所述基因编码的鲤疱疹病毒Ⅱ型ORF66蛋白的氨基酸序列为SEQ ID NO:2。
3.包含权利要求1所述基因的重组载体。
4.根据权利要求3所述的重组载体,其特征在于,所述重组载体的制备方法如下:提取异育银鲫感染鲤疱疹病毒Ⅱ型脾脏RNA,并反转录出cDNA;以cDNA为模板扩增ORF66基因;将ORF66扩增产物经BamH I和Hind III双酶切后,通过DNA连接酶***到经同样双酶切的pET-28a表达载体中,得到重组质粒pET28a-ORF66。
5.根据权利要求4所述的重组载体,其特征在于,以cDNA为模板,利用引物扩增ORF66基因,上游引物和下游引物分别如SEQ ID NO:3和SEQ ID NO:4所示。
6.一种鲤疱疹病毒Ⅱ型衣壳蛋白ORF66的原核表达、蛋白纯化方法,其特征在于,包括以下步骤:
(1)用权利要求3或4所述的重组载体转化大肠杆菌细胞,得到表达ORF66的重组原核表达菌株pET28a-ORF66-BL21;
(2)利用步骤(1)所述菌株pET28a-ORF66-BL21生产重组鲤疱疹病毒Ⅱ型ORF66蛋白,具体为:将菌株pET28a-ORF66-BL21接种在LB液体培养基中,37℃,180转/分培养过夜,按照1:100接种在LB液体培养基中,37℃,180转/分培养至OD600达到0.6-0.8,然后加入IPTG至终浓度为0.1-1.0mM,20-37℃,180转/分培养2小时以上离心并收集菌体,超声破碎后、离心、收集上清,经分离纯化得到重组鲤疱疹病毒Ⅱ型ORF66蛋白。
7.根据权利要求6所述的方法,其特征在于,还包括如下步骤:将分离纯化得到的重组鲤疱疹病毒Ⅱ型ORF66蛋白加入到透析袋中,在20mMPB,0.5mM NaCl,0.5mM EDTA,pH8.0进行透析过夜;然后把透析后的蛋白溶液加入到超滤管中,4℃,6000g离心1-2小时,去掉滤出液,得到重组鲤疱疹病毒Ⅱ型ORF66蛋白。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110408655A (zh) * | 2019-07-31 | 2019-11-05 | 盐城工学院 | 一种鲤疱疹病毒ⅱ型orf66蛋白重组杆状病毒表达载体及其制备方法 |
CN112342221A (zh) * | 2020-12-02 | 2021-02-09 | 江苏麦莎实业有限公司 | 一种烟草生物碱合成负向调控NtARF6蛋白的原核表达、蛋白纯化方法 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107056898A (zh) * | 2017-02-13 | 2017-08-18 | 中国水产科学研究院珠江水产研究所 | 鲤疱疹病毒3型1301株orf136基因重组表达蛋白、抗体及其应用 |
-
2019
- 2019-07-29 CN CN201910687629.0A patent/CN110407920A/zh active Pending
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN107056898A (zh) * | 2017-02-13 | 2017-08-18 | 中国水产科学研究院珠江水产研究所 | 鲤疱疹病毒3型1301株orf136基因重组表达蛋白、抗体及其应用 |
Non-Patent Citations (8)
Title |
---|
LI,L等: "Characterization and Prevalence of A New Fatal Genotype CyHV-2 in Mainland China", 《GENBANK》 * |
于爱鸣编: "《生物化学》", 31 July 2011 * |
周勇等: "鲤疱疹病毒Ⅱ型ORF4基因的克隆、表达与免疫学检测方法", 《淡水渔业》 * |
孔善云等: "一种基于Ⅱ型鲤疱疹病毒衣壳蛋白72的免疫学检测方法", 《中国水产科学》 * |
廖红等: "鲤疱疹病毒2型ORF5截短基因的克隆表达及免疫原性研究", 《中国兽医科学》 * |
张建社等编著: "《蛋白质分离与纯化技术》", 30 September 2009 * |
李坤: "鲤疱疹病毒Ⅱ型的DNA疫苗、卵黄抗体的研制及鱼卵中鲤疱疹病毒Ⅱ型的检测技术", 《中国优秀硕士学位论文全文数据库(电子期刊)》 * |
李燕等主编: "《分子生物学实用实验技术》", 31 December 2011 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110408655A (zh) * | 2019-07-31 | 2019-11-05 | 盐城工学院 | 一种鲤疱疹病毒ⅱ型orf66蛋白重组杆状病毒表达载体及其制备方法 |
CN112342221A (zh) * | 2020-12-02 | 2021-02-09 | 江苏麦莎实业有限公司 | 一种烟草生物碱合成负向调控NtARF6蛋白的原核表达、蛋白纯化方法 |
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