CN101016547B - 海洋双rna病毒mabv重组蛋白的制备方法与应用 - Google Patents
海洋双rna病毒mabv重组蛋白的制备方法与应用 Download PDFInfo
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Abstract
本发明公开了一种含有海洋双RNA病毒MABV结构蛋白基因的重组蛋白制备方法和应用,所述的重组蛋白包括VP2融合蛋白或VP3融合蛋白以及VP2或VP3蛋白,其中VP2融合蛋白编码序列具有序列表中SEQID NO.1所述的核苷酸序列,VP3融合蛋白编码序列具有序列表中SEQID NO.2所述的核苷酸序列。本发明用重组蛋白制备的多克隆抗体在检测海洋双RNA病毒MABV中增加了检测的可信度;利用ELISA和westernblot进行检测,比其它方法(RT-PCR、原位杂交等)简单易行。利用制备VP2和VP3重组蛋白作为疫苗,比减毒疫苗更经济更安全。
Description
技术领域
本发明涉及基因工程领域,尤其是含有海洋双RNA病毒外壳蛋白基因VP2或VP3基因的融合蛋白及制备方法和应用。
背景技术
水生双RNA病毒属(Aquabirnavirus)隶属双RNA病毒科(Birnaviridae),此科的一个明显特征是病毒基因组是由双节段的双链RNA组成。Aquabirnavirus属的代表种是能引起鲑鱼急性传染性病的传染性胰坏死病病毒(IPNV)。Sorimachi M、Hara T.1985年首次从一种鰤(Seriolaquinqueradiana)分离到海洋双RNA病毒(Sorimachi M,Hara T.“Characteristics and pathogenicity of a virus isolated from yellowtailfingerlings showing ascites[J]”《Fish Pathol》1985,19:231-238.),魳感病的典型症状为贫血性腮坏死,肝脏出血,腹水等。其后从褐牙鲆、香鱼(徊游性)、琥珀鱼、红稽东方鲀、海蛸、珍珠贝和其他的海洋鱼类和十几种贝类病体中都分离到各种MABV病毒。最近甚至在浮游动物中也发现了这种病毒
Hosono H,Suzuki S,Kusuda R.在“Genogrouping of birnavirusesisolated from marine fish:a comparison of VP2/NS junction regions ongenome segment A[J]”(《J Fish Dis》1996,19:295-306)中的研究发现这些从海洋生物分离的双RNA病毒在血清型和基因组型上与IPNV不同,并把这些从海洋生物分离到的水生双RNA病毒暂命名为海洋双RNA病毒(MABV)。组成双RNA病毒基因组的两条片段是3.1kb的A片段和2.8kb的B片段。B片段编码一个分子量为90kDa的依赖RNA的RNA聚合酶;而A片段编码一个分子量106kDa的聚蛋白,编码的蛋白顺序为NH2-VP2-NS-VP3-COOH。其中VP2和VP3为病毒的结构蛋白(Zhang,CX,Suzuki,S(2004)Aquabirnaviruses isolated from marine organisms form adistinct genogroup from other aquabirnaviruses[J].(《J Fish Dis》2004,27:633-643)。
由于海洋双RNA病毒其寄主广泛,对多种鱼苗生产造成了很大的危害,在许多未发病成鱼体内也检测到较高比例的MABV病毒携带者,从而造成病毒的传播。除鱼贝病害诊断需要外,亲鱼引进和繁育中也迫切需要有对病毒携带的亲鱼进行灵敏检测的手段。到目前为止,已报道用来检测该病毒方法,包括间接免疫荧光法(indirect immunofluorescence)、蛋白免疫印记法(immunodot blot)及RT-PCR法等,相关检测技术可详见Rodriguez Saint-Jean S,Borrego JJ,Perez-Prieto SI.在“Comparativeevaluation of five serological methods and RT-PCR assay for the detection ofIPNV in fish”(《J Virol Methods》2001 Sep;97(1-2):23-31)中的研究。但至今还未见利用基因工程方法制备病毒外壳蛋白,作为标准蛋白、作为免疫疫苗,以及作为抗原制备抗体的专利和报道。
发明内容
本发明提供一种海洋双RNA病毒MABV蛋白的其制备方法和应用。
一种含有海洋双RNA病毒MABV基因的GST/VP2融合蛋白的生产方法,包括提取病毒MABV的RNA,反转录成cDNA,以cDNA为模板,设计引物:
VP2正向引物(5’-ggatccatgtccctcaccacg-3’)
VP2反向引物(5’-ctcgagttaggccaccagggtg-3’)
经PCR扩增后,得到的DNA片段克隆入pGEM-T-easy载体后用BamH I和Xho I双酶切,克隆到大肠杆菌表达载体pGEX-4t-2中,并转化到表达菌株BL21进行诱导表达,表达条件为细菌OD600=1.0,IPTG终浓度为0.8mM,37℃,摇6h,采用包涵体分离结合割胶回收的方法纯化得到GST/VP2融合蛋白。
一种含有海洋双RNA病毒MABV基因VP2蛋白的生产方法,包括将所述的GST/VP2融合蛋白经凝血酶处理后,用GST亲和柱吸附除去GST,回收得到的VP2蛋白。
一种含有海洋双RNA病毒MABV基因的GST/VP3融合蛋白的生产方法,包括提取病毒MABV的RNA,反转录成cDNA,以cDNA为模板,设计引物:
vp3正向引物(5’-ggatcctcagggatggatgaagag-3’)
vp3反向引物(5’-ctcgagttacacttctccgttatctcc-3’)
经PCR扩增后,得到的DNA片段首先克隆入pGEM-T-easy载体;将重组pGEM-T-easy载体用BamH I和Xho I双酶切,将vp3进一步克隆到大肠杆菌表达载体pGEX-4t-2中,并转化表达菌株BL21,进行诱导表达。GST/VP3表达条件为细菌OD600=0.8,IPTG终浓度=0.8mM,30℃摇8h,通过谷胱甘肽亲和柱直接分离纯化得到GST/VP3融合蛋白。
一种含有海洋双RNA病毒MABV基因VP3蛋白的生产方法,包括将所述的GST/VP3融合蛋白经凝血酶处理后,用GST亲和柱吸附除去GST,回收得到的VP3蛋白。
本发明还提供了所述蛋白或融合蛋白,即VP2蛋白、VP3蛋白、GST/VP2融合蛋白或GST/VP3融合蛋白作为疫苗在诱导鱼产生免疫反应中的应用。
本发明还提供了所述蛋白或融合蛋白,即VP2蛋白、VP3蛋白、GST/VP2融合蛋白或GST/VP3融合蛋白制备的多克隆抗体。所述的多克隆抗体可以为抗VP2或VP3蛋白或抗VP2或VP3蛋白序列中各抗原蔟的各种多克隆抗体。
本发明还提供了所述的多克隆抗体在检测海洋双RNA病毒MABV中的应用。采用ELISA检测方法或Western blot检测方法检测海洋双RNA病毒MABV。
ELISA检测方法,检测的条件为用包被液包被感染海洋双RNA病毒MABV动物的肾脏(血液或其它组织)提取液,一抗为抗VP2或VP3融合蛋白多克隆抗体,二抗为过氧辣根偶联的羊抗兔血清,以OPD为底物进行显色。这里“二抗”可以为任何抗兔的抗体,显色也可以为任何可以检测的ELISA显色方法。
Western blot检测方法,检测的条件为将感染海洋双RNA病毒MABV动物的肾脏(血液或其它组织)提取液经变性凝胶电泳后,转移至PVDF膜进行杂交,一抗为抗VP3融合蛋白多克隆抗体,二抗为过氧辣根偶联的羊抗兔血清,以DAB为底物进行显色。这里“二抗”可以为任何抗兔的抗体,显色也可以为任何可以检测的Western blot显色方法。
在本发明中,术语“基因”指编码具有海洋双RNA病毒(或多肽)的核苷酸序列。该术语还包括能编码具有与海洋双RNA病毒外壳蛋白相同功能的蛋白的序列的变异形式。这些变异形式包括(但并不限于):若干个核苷酸的缺失、插入和或取代,以及在5’和/或3’端添加数个核苷酸。
在本发明中,术语“海洋双RNA病毒外壳蛋白”指具有海洋双RNA病毒外壳蛋白活性的多肽。该术语还包括具有与海洋双RNA病毒外壳蛋白相同功能的序列的变异形式。这些变异形式包括(但并不限于):若干个氨基酸的缺失、插入和/或取代,以及在C末端和/或N末端添加一个或数个氨基酸。
该多肽的变异形式包括:同源序列、等位变异体、天然突变体,诱导突变体,在高或低的严谨度条件下能与海洋双RNA病毒DNA杂交的DNA所编码的蛋白,以及利用抗海洋双RNA病毒外壳蛋白的抗血清获得的多肽或蛋白。
本发明中,可选用本领域已知的各种载体,如市售的载体。所用的宿主细胞是指原核细胞。可用的原核宿主细胞如大肠杆菌,枯草杆菌等。本发明的抗体制备可以通过本领域内技术人员已知的技术进行制备。
本发明所提供的海洋双RNA病毒MABV的检测方法同时利用外壳蛋白VP2和VP3抗体进行双重检测,增加了检测的可信度;利用ELISA和western blot进行检测海洋双RNA病毒MABV,比其它方法(RT-PCR、原位杂交等)简单易行;利用制备VP2和VP3融合蛋白作为疫苗,比减毒疫苗更经济更安全。
附图说明
图1 vp2基因片段在大肠杆菌中表达。泳道1,蛋白标准分子量;泳道2,在诱导条件OD600=1.0、IPTG=0.8mM、温度37℃下的诱导表达产物;泳道3,表达产物的沉淀不可溶蛋白;泳道4,表达产物的上清可溶性蛋白;泳道5,载体pGEX4t-2的诱导表达产物;泳道6,大肠杆菌E.coliBL21(DE3)的裂解液;泳道7,表达产物的Western blot验证。
图2vp3基因片段在大肠杆菌中表达。泳道1,蛋白标准分子量;泳道2,在诱导条件OD600=0.8、IPTG=0.8mM、温度37℃下的诱导表达产物;泳道3,表达产物的沉淀不可溶蛋白;泳道4,表达产物的上清可溶性蛋白;泳道5,载体pGEX4t-2的诱导表达产物;泳道6,大肠杆菌E.coliBL21(DE3)的裂解液;泳道7,回收纯化的VP3融合蛋白;泳道8,表达产物的Western blot验证。
图3在MABV感染的CHSE-214细胞中通过免疫印迹来检测MABVVP2(VP2前体)和VP3蛋白。
泳道1:标准蛋白分子量;
A.泳道2和3:用GST/VP3抗血清检测VP3蛋白(约27kDa);泳道4:未感染病毒的细胞作为阴性对照。
B.泳道5和6:用GST/VP2抗血清检测VP2前体(约56kDa)以及成熟的VP2蛋白(约54kDa);泳道7:未感染病毒的细胞作为阴性对照。
图4A鱼病毒的ELISA检测,利用抗VP2抗体进行ELISA检测,灰柱为GST/VP2或GST/VP3抗血清与不同滴度病毒的反应;黑柱(control 1)为免疫前兔血清与不同滴度病毒的反应;白柱(control 2)为只有二抗与不同滴度病毒的反应。每个柱体为三个重复的平均值。
图4B鱼病毒的ELISA检测,利用抗VP3抗体进行ELISA检测,灰柱为GST/VP2或GST/VP3抗血清与不同滴度病毒的反应;黑柱(control 1)为免疫前兔血清与不同滴度病毒的反应;白柱(control 2)为只有二抗与不同滴度病毒的反应。每个柱体为三个重复的平均值。
图5AB用GST/VP2抗血清进行MABV在香鱼ayu(Plecoglossusaltivelis)中的ELISA分析。
黑柱感染后不同天数的鱼肾样品。白柱(control 1)为无病毒的鱼肾。灰柱(control 2)为感染病毒的鱼肾与免疫前兔血清反应。每个柱体为三个重复的平均值。
图5B用GST/VP3抗血清进行MABV在香鱼ayu(Plecoglossus altivelis)中的ELISA分析。
黑柱感染后不同天数的鱼肾样品。白柱(control 1)为无病毒的鱼肾。灰柱(control 2)为感染病毒的鱼肾与免疫前兔血清反应。每个柱体为三个重复的平均值。
图6鱼病毒的Western blot检测。用GST/VP3抗血清对香鱼体中的MABV病毒进行免疫分析。用MABV-Y6病毒感染鱼后取感染后不同天数的鱼肾样品作分析,未感染病毒的鱼肾作为对照。
具体实施方式
MABV病毒的分离、VP2和VP3基因克隆
将从感病香鱼(Plecoglossus altivelis)中分离到的MABV在鲑鱼细胞系CHSE-214(Chinook salmon embryo cell line)中于20℃中培养,繁殖病毒,并进一步提取病毒RNA,反转录成cDNA。
以MABV Y-6病毒感染的鱼细胞总RNA反转录的cDNA为模板,设计引物。其中正向引物含有BamHI酶切位点(下划线碱基),反向引物含有XhoI酶切位点(下划线碱基)。
vp2正向引物(5’ggatccatgtccctcaccacg-3’)
vp2反向引物(5’-ctcgagttaggccaccagggtg-3)
vp3正向引物(5’-ggatcctcagggatggatgaagag-3’)
vp3反向引物(5’-ctcgagttacacttctccgttatctcc-3’)
PCR扩增条件:94℃1min,54℃40s,72℃1min,30个循环。经PCR扩增后,得到的DNA片段首先克隆入pGEM-T-easy载体。并经测序确定了已克隆到病毒外壳蛋白Vp2和核心蛋白VP3基因。其中,Vp2基因的编码区的核苷酸和推导的氨基酸序列对应GenBank登录号:AY283781。vp3基因的编码区的核苷酸和推导的氨基酸序列对应GenBank登录号AY283781。
VP2和VP3在大肠杆菌中与GST融合高效表达
将重组pGEM-T-easy载体用BamH I和Xho I双酶切,分别将vp2和vp3进一步克隆到大肠杆菌表达载体pGEX-4t-2中,并转化表达菌株BL21,进行诱导表达。GST/VP2最适合表达条件为细菌OD600=1.0,IPTG终浓度为0.8mM,37℃,摇6h。而GST/VP3最适合表达条件为细菌OD600=0.8,IPTG终浓度=0.8mM,30℃摇8h。薄层扫描显示在最适合表达条件下,GST/VP2最高表达水平占细菌总蛋白的23.6%,而GST/VP3最高表达水平达可达到细菌总蛋白的42.7%。SDS-PAGE和用抗GST抗体进行的Western blot结果显示表达的GST/VP2大小约46kDa,而GST/VP3为53 kDa与预计的分子量相符合。SDS-PAGE分析还显示GST/VP2表达产物几乎全是不可溶性的包涵体,而GST/VP3除不可溶性的包涵体外,还有部分是可溶性蛋白。
其中VP2融合蛋白具有序列表中SEQ ID NO.1所述的核苷酸序列,VP3融合蛋白具有序列表中SEQ ID NO.2所述的核苷酸序列。
在SEQ ID NO.1所述的核苷酸序列及SEQ ID NO.2所述的核苷酸序列中第664~678位置编码的氨基酸序列为凝血酶(thrombin)切割位点,能将融合蛋白的GST和VP2(或VP3)切开,后面为VP2或VP3序列。
大肠杆菌表达的重组VP2和VP3蛋白的纯化
由于大肠杆菌表达的是N端融合了谷胱甘肽的VP3,而且部分是可溶性的, 因此可以通过谷胱甘肽亲和柱直接分离纯化GST/VP3。将细菌用溶菌酶lysozyme和超声波处理后,通过亲和柱分离回收了产物。产物经SDS-PAGE和western blot分析,结果显示(见图2)GST/VP3已经成功被纯化回收。由于大肠杆菌表达的GST/VP2是以不可溶的包涵体形式存在,我们采用了包涵体分离结合割胶回收的方法纯化GST/VP2。包涵体分离使用Novagen公司的Protein Refolding Kit,用lysozyme(溶菌酶)将含GST/VP2的表达细菌裂解在冷的1×PBS的溶液中,并经超声波处理后,通过离心和1×包涵体洗液处理3次,回收包涵体。再用1×包涵体溶解液溶解包涵体,通过SDS-PAGE,将含目的条带的PAGE胶割下后透析,回收GST/VP2蛋白。将凝血酶加入溶解在1×PBS的溶液的GST/VP2和GST/VP3溶液中,在20℃酶切12hr,然后将酶切好的混合物通过亲和层析柱,吸附除去GST,回收得到VP2和VP3蛋白。
VP2和VP3多克隆抗体制备
用纯化的重组VP2和VP3蛋白免疫14-16周龄的健康新西兰大白兔,每次注射500μl含80μg左右纯化的GST/VP3和GST/VP2。第一次加500μl福氏完全佐剂溶液乳化,皮下多点注射免疫;3周后,同剂量GST/VP3和GST/VP2加福氏不完全佐剂溶液乳化,腿部肌肉多点注射免疫;2周后,直接用GST/VP3和GST/VP2溶于1ml生理盐水免疫;1周后,再次用抗原加强免疫。1周后,收集兔抗血清,-70℃保存备用。制备的多克隆抗体的效价测定结果表明,抗Vp2多克隆抗体和抗VP3多克隆抗体的效价结果均为:1∶512 000,达到高效价水平,可以用于进一步检测应用。GST-VP2融合蛋白和GST-VP3融合蛋白作为疫苗诱导香鱼产生抗体
将纯化的GST-VP2融合蛋白和GST-VP3融合蛋白溶于PBS,配制成1μg/1μl的溶液,每100μl溶液加上适量5μlGen+抗生素,作为免疫抗原,免疫香鱼。分别制备鱼抗GST-VP2融合蛋白的血清和鱼抗GST-VP3融合蛋白的血清。结合ELISA方法评价了这两种血清对感染病毒的细胞的保护作用,中和抗体的效价分别为1∶3,200和1∶800。VP2融合蛋白免疫鱼的血清对感染病毒的细胞的保护作用比VP3融合蛋白免疫鱼的血清对病毒感染细胞的保护作用更强,为抗病毒疫苗的筛选提供了依据。
用多克隆抗体检测MABV病毒
用感染指数(MOI)为0.1的MABV感染CHSE-214细胞,当90%细胞出现病理症状时,收获细胞,进行SDS-PAGE和Western blot分析,以确定多克隆抗体的特异性和检测有效性。当用兔抗GST/VP3抗血清进行蛋白印迹检测时,可以清楚地检测到1条27kDa的特异性蛋白条带(图3中泳道2和3),其与预测的VP3分子量一致。当用兔抗GST/VP2抗血清检测时,可以检测到3条蛋白质条带,大小分别为56、54和35kDa((图3中泳道5和6),其中56kDa蛋白与VP2前体蛋白proVP2大小一致,54kDa蛋白则与VP2蛋白大小一致,35kDa则是未知蛋白,有可能是VP2的降解形式。在没有病毒感染的细胞中,VP2和VP3的都没有检测到阳性条带。结果表明本实验制备的兔抗VP2和兔抗VP3抗血清可以用于该病毒的特异性检测。
为了确定两种多克隆抗体的检测MABV灵敏度,对不同稀释浓度的病毒粒子进行了ELISA检测。结果表明,当MABV病毒滴度从9.76×107TCID50/ml稀释到1.00×104TCID50/ml时,无论是兔抗GST/VP2抗血清还是兔抗GST/VP3抗血清,均可以检测到。这说明两种多克隆抗体检测MABV都达到了相当高的灵敏度。
用多克隆抗体检测感病香鱼体内MABV病毒
将体长10cm香鱼分为2组,每组22条。1组腹膜下每条注射0.01mlMABV(107TCID50),另1组每条注射0.01ml PBS作为对照。感染后不同天数取鱼肾脏,进行ELISA和western blot检测。
ELISA检测的具体操作步骤如下:肾脏匀浆上清液溶在Tris溶液中使最终浓度(50mM Tris.Cl pH6.8,100mM DTT,2%SDS),沸水浴变性后样品用包被液(15mM Na2CO3,35mM NaHCO3,pH 9.6)包被在96孔的ELISA板上;再用封闭液PBS-T(PBS pH 7.4,0.5%Tween-20)加3%脱脂奶粉封闭30分钟;抗VP2抗体和抗VP3抗体以1∶2000分别稀释在3%脱脂奶粉的PBS-T溶液中37℃温浴1小时;用PBS-T洗涤3次,每次5分钟;用HRP偶联的羊抗兔抗体作为二抗37℃反应1小时;用PBS-T洗涤3次,每次5分钟;最后,加入100μl OPD柠檬酸溶液(0.4mg/ml,pH 5.0)显示,以50ul 50mM H2SO4终止反应,在492nm处测定吸光值。
Western blot的操作方法如Towbin等(1979)(Towbin H,Staehelin T,Gordon J.Electrophoretic transfer of proteins from polyacrylamidae gels tonitrocellulose sheets:Procedure and some applications.Proc Natl Acad SciUSA,1979,76:4350-4354)所述。
ELISA检测结果表明,在MABV感染后第4天GST/VP3抗血清就可以明显地检测到香鱼体内MABV病毒(图5B),而用GST/VP2抗血清则在第8天才能很明显地检测到香鱼体内病毒(图5A).结果提示使用ELISA检测方法时,VP3抗血清比VP2抗血清能更灵敏地检测病鱼体内的MABV病毒。
进一步用GST/VP3抗血清对感染后不同天数鱼肾进行Western blot分析,结果也显示在MABV感染后第4天可以明显地检测特异性条带(图6),这进一步确定了ELISA检测的结果。
从以上检测方法得到了如下检测结果:(1)利用VP3抗体,在病毒感染后4天就能用ELISA方法检测到病毒,而用VP2抗体则需6天;(2)利用VP3抗体,在病毒感染后6天就能用Western blot方法检测到病毒。
(3)VP3抗体的灵敏性高于VP2抗体。
SEQUENCE LISTING
<110>浙江大学
<120>海洋双RNA病毒MABV重组蛋白的制备方法与应用
<130>
<160>4
<170>PatentIn version 3.3
<210>1
<211>1320
<212>DNA
<213>GST/VP2
<400>1
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ctggttccgc gtggatccat gtccctcacc acgaaccccc aggacaaagt gaacaaccag 720
ctagtcacca aaggagtgac agtcctaaac ctaccaacag ggttcgacaa gccatacgtc 780
cgcctggagg acgagacccc acaagggccc cagtcaatga acggcgcccg gatgaggtgc 840
acagctgcaa ttgcaccacg aaggtacgaa atcgacctcc catccgcacg cctccccaca 900
gtaccagcga ctgggaccct cacaacaatc tatgaaggga acgccgacat tgtgaactca 960
accacagtga ccggcgacat cagcttcaga ctggaacaag accccccgaa tgacacgaag 1020
tacgacttcc agctcgactt cctcggctta gacaacaacg tccccgtcgt gtcaataacc 1080
agctctacgc tggccacaac cgacaactac aggggggtct cagtcaaatt cacacagtca 1140
atcccaacag agacaatcac aaaacccatc accagggtca agctgtccta caaaatcaac 1200
cagcagacag ctatcggcaa tgcagcaacg cttggacccc tggggccctc atccgtctca 1260
ttctcatcag gaaacggcaa cgtacctgga gtgctcaggc caatcaccct ggtggcctaa 1320
<210>2
<211>1395
<212>DNA
<213>GST/VP3
<400>2
atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60
ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120
tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180
ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240
atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300
gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360
gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420
acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480
gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540
aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600
tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660
ctggttccgc gtggatcctc agggatggat gaagagctgc agaagctgct gcacgccacc 720
atggccagag caaaagaggt gaaagacgcc gaagtcttca aacttctcaa gctgatgtcc 780
tggaccagga agaacgggct caccgaccac atgtatgaat ggtcaaaaga ggaccctgaa 840
gcagtcaaat ttggcaaact catcagcaca ccaccaaaac accaagagaa gccaaaagga 900
cccgaccagc acacggcaca ggaggcaaaa gccgtccgga tctcactaga tgcagtgaaa 960
gcaggagcag actttgcctc cccagactgg atcgcggaga atggataccg cggtccatca 1020
ccaggccagt tcaagtacta cgtcatcaca gggcgcgtcc cagacccacg agacgagtac 1080
gaggactacg tgcgaaaacc aataacaaga cccacggaca tggacaaaat cagacgccta 1140
gccaacagtg tctacggact tccccaccaa gaacctgcac cagaggaatt ctaccaagcg 1200
gtagtcgaga tcttcgcaga aaatggagga cgaggaccag atcaagacca gatgcaagac 1260
ctgagggact tggcccggca gatgaaacga cgaccccgac cagctgagac acgcaggcaa 1320
aaccgagctc caccacgggc ggcacccagt ggaagctcac gttttacccc ctccggagat 1380
aacggagaag tgtaa 1395
<210>3
<211>439
<212>PRT
<213>GST/VP2
<400>3
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg
210 215 220
Gly Ser Met Ser Leu Thr Thr Asn Pro Gln Asp Lys Val Asn Asn Gln
225 230 235 240
Leu Val Thr Lys Gly Val Thr Val Leu Asn Leu Pro Thr Gly Phe Asp
245 250 255
Lys Pro Tyr Val Arg Leu Glu Asp Glu Thr Pro Gln Gly Pro Gln Ser
260 265 270
Met Asn Gly Ala Arg Met Arg Cys Thr Ala Ala Ile Ala Pro Arg Arg
275 280 285
Tyr Glu Ile Asp Leu Pro Ser Ala Arg Leu Pro Thr Val Pro Ala Thr
290 295 300
Gly Thr Leu Thr Thr Ile Tyr Glu Gly Asn Ala Asp Ile Val Asn Ser
305 310 315 320
Thr Thr Val Thr Gly Asp Ile Ser Phe Arg Leu Glu Gln Asp Pro Pro
325 330 335
Asn Asp Thr Lys Tyr Asp Phe Gln Leu Asp Phe Leu Gly Leu Asp Asn
340 345 350
Asn Val Pro Val Val Ser Ile Thr Ser Ser Thr Leu Ala Thr Thr Asp
355 360 365
Asn Tyr Arg Gly Val Ser Val Lys Phe Thr Gln Ser Ile Pro Thr Glu
370 375 380
Thr Ile Thr Lys Pro Ile Thr Arg Val Lys Leu Ser Tyr Lys Ile Asn
385 390 395 400
Gln Gln Thr Ala Ile Gly Asn Ala Ala Thr Leu Gly Pro Leu Gly Pro
405 410 415
Ser Ser Val Ser Phe Ser Ser Gly Asn Gly Asn Val Pro Gly Val Leu
420 425 430
Arg Pro Ile Thr Leu Val Ala
435
<210>4
<211>464
<212>PRT
<213>GST/VP3
<400>4
Met Ser Pro Ile Leu Gly Tyr Trp Lys Ile Lys Gly Leu Val Gln Pro
1 5 10 15
Thr Arg Leu Leu Leu Glu Tyr Leu Glu Glu Lys Tyr Glu Glu His Leu
20 25 30
Tyr Glu Arg Asp Glu Gly Asp Lys Trp Arg Asn Lys Lys Phe Glu Leu
35 40 45
Gly Leu Glu Phe Pro Asn Leu Pro Tyr Tyr Ile Asp Gly Asp Val Lys
50 55 60
Leu Thr Gln Ser Met Ala Ile Ile Arg Tyr Ile Ala Asp Lys His Asn
65 70 75 80
Met Leu Gly Gly Cys Pro Lys Glu Arg Ala Glu Ile Ser Met Leu Glu
85 90 95
Gly Ala Val Leu Asp Ile Arg Tyr Gly Val Ser Arg Ile Ala Tyr Ser
100 105 110
Lys Asp Phe Glu Thr Leu Lys Val Asp Phe Leu Ser Lys Leu Pro Glu
115 120 125
Met Leu Lys Met Phe Glu Asp Arg Leu Cys His Lys Thr Tyr Leu Asn
130 135 140
Gly Asp His Val Thr His Pro Asp Phe Met Leu Tyr Asp Ala Leu Asp
145 150 155 160
Val Val Leu Tyr Met Asp Pro Met Cys Leu Asp Ala Phe Pro Lys Leu
165 170 175
Val Cys Phe Lys Lys Arg Ile Glu Ala Ile Pro Gln Ile Asp Lys Tyr
180 185 190
Leu Lys Ser Ser Lys Tyr Ile Ala Trp Pro Leu Gln Gly Trp Gln Ala
195 200 205
Thr Phe Gly Gly Gly Asp His Pro Pro Lys Ser Asp Leu Val Pro Arg
210 215 220
Gly Ser Ser Gly Met Asp Glu Glu Leu Gln Lys Leu Leu His Ala Thr
225 230 235 240
Met Ala Arg Ala Lys Glu Val Lys Asp Ala Glu Val Phe Lys Leu Leu
245 250 255
Lys Leu Met Ser Trp Thr Arg Lys Asn Gly Leu Thr Asp His Met Tyr
260 265 270
Glu Trp Ser Lys Glu Asp Pro Glu Ala Val Lys Phe Gly Lys Leu Ile
275 280 285
Ser Thr Pro Pro Lys His Gln Glu Lys Pro Lys Gly Pro Asp Gln His
290 295 300
Thr Ala Gln Glu Ala Lys Ala Val Arg Ile Ser Leu Asp Ala Val Lys
305 310 315 320
Ala Gly Ala Asp Phe Ala Ser Pro Asp Trp Ile Ala Glu Asn Gly Tyr
325 330 335
Arg Gly Pro Ser Pro Gly Gln Phe Lys Tyr Tyr Val Ile Thr Gly Arg
340 345 350
Val Pro Asp Pro Arg Asp Glu Tyr Glu Asp Tyr Val Arg Lys Pro Ile
355 360 365
Thr Arg Pro Thr Asp Met Asp Lys Ile Arg Arg Leu Ala Asn Ser Val
370 375 380
Tyr Gly Leu Pro His Gln Glu Pro Ala Pro Glu Glu Phe Tyr Gln Ala
385 390 395 400
Val Val Glu Ile Phe Ala Glu Asn Gly Gly Arg Gly Pro Asp Gln Asp
405 410 415
Gln Met Gln Asp Leu Arg Asp Leu Ala Arg Gln Met Lys Arg Arg Pro
420 425 430
Arg Pro Ala Glu Thr Arg Arg Gln Asn Arg Ala Pro Pro Arg Ala Ala
435 440 445
Pro Ser Gly Ser Ser Arg Phe Thr Pro Ser Gly Asp Asn Gly Glu Val
450 455 460
Claims (7)
1.一种含有海洋双RNA病毒MABV基因的GST/VP2融合蛋白的生产方法,其特征在于:提取病毒MABV的RNA,反转录成cDNA,以cDNA为模板,设计引物:
VP2正向引物为5’-ggatccatgtccctcaccacg-3’
VP2反向引物为5’-ctcgagttaggccaccagggtg-3’
经PCR扩增后,得到的DNA片段克隆入pGEM-T-easy载体后用BamH I和Xho I双酶切,克隆到大肠杆菌表达载体pGEX-4t-2中,并转化到表达菌株BL21进行诱导表达,表达条件为细菌OD600=1.0,IPTG终浓度为0.8mM,37℃,摇6h,采用包涵体分离结合割胶回收的方法纯化得到GST/VP2融合蛋白。
2.如权利要求1所述的方法而得到的GST/VP2融合蛋白,其特征在于:由序列表中SEQ NO.3的氨基酸序列组成。
3.一种含有海洋双RNA病毒MABV基因的VP2蛋白的生产方法,其特征在于:将权利要求2所述的GST/VP2溶解在1×PBS的溶液中,将凝血酶thrombin加入溶液中,在20℃酶切12hr,然后将酶切好的混合物通过亲和层析柱,吸附除去GST,回收得到VP2蛋白。
4.一种含有海洋双RNA病毒MABV基因的GST/VP3融合蛋白的生产方法,其特征在于:提取病毒MABV的RNA,反转录成cDNA,以cDNA为模板,设计引物:
vp3正向引物为5’-ggatcctcagggatggatgaagag-3’
vp3反向引物为5’-ctcgagttacacttctccgttatctcc-3’
经PCR扩增后,得到的DNA片段首先克隆入pGEM-T-easy载体;将重组pGEM-T-easy载体用BamH I和Xho I双酶切,将vp3进一步克隆到大肠杆菌表达载体pGEX-4t-2中,并转化表达菌株BL21,进行诱导表达,GST/VP3表达条件为细菌OD600=0.8,IPTG终浓度=0.8mM,30℃摇8h,通过谷胱甘肽亲和柱直接分离纯化得到GST/VP3融合蛋白。
5.如权利要求4所述的方法而得到的GST/VP3融合蛋白,其特征在于:由序列表SEQ NO.4的氨基酸序列组成。
6.一种含有海洋双RNA病毒MABV基因的VP3蛋白的生产方法,其特征在于:将权利要求5所述的GST/VP3融合蛋白溶解在1×PBS的溶液中,将凝血酶thrombin加入溶液中,在20℃酶切12hr,然后将酶切好的混合物通过亲和层析柱,吸附除去GST,回收得到VP3蛋白。
7.如权利要求2或5所述的融合蛋白制备的多克隆抗体。
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CN1229358A (zh) * | 1996-09-05 | 1999-09-22 | 马里兰大学-生物技术研究所 | 从合成的rna转录物制备双rna病毒的方法 |
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EP1731159A1 (en) * | 2004-03-24 | 2006-12-13 | Japan Science and Technology Agency | Method of preventing nodavirus infection and therapeutic method |
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SHI WJ, et al.Expression and purification of the malittin gene from polisteshebraeus by the GST fusion system..Journal of Shanghai Jiaotong University (Science)E-10 S1.2005,E-10(S1),55-60. * |
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林建国.MABVY-6 VP2 VP3、VP3和VP5基因在昆虫细胞中的表达及VP2、VP3和VP5基因特性分析.浙江大学博士学位论文.2006,66-75、87-93. * |
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