CN109503720A - 甘薯spvd病毒重组双cp融合蛋白、由其制备的多克隆抗体、其制备方法及其应用 - Google Patents
甘薯spvd病毒重组双cp融合蛋白、由其制备的多克隆抗体、其制备方法及其应用 Download PDFInfo
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- CN109503720A CN109503720A CN201811454909.9A CN201811454909A CN109503720A CN 109503720 A CN109503720 A CN 109503720A CN 201811454909 A CN201811454909 A CN 201811454909A CN 109503720 A CN109503720 A CN 109503720A
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Abstract
本发明公开了一种甘薯SPVD病毒重组双CP融合蛋白、由其制备的多克隆抗体、其制备方法及其应用,属于生物技术领域。本发明的甘薯SPVD病毒重组双CP融合蛋白,是由甘薯褪绿矮化病毒和甘薯羽状斑驳病毒CP基因的融合片段构建到重组表达载体,转化表达菌株,诱导表达得到。并将制备的甘薯SPVD病毒重组双CP融合蛋白免疫兔,获得的抗血清即为甘薯SPVD病毒重组双CP多克隆抗体。本发明通过制备甘薯SPVD病毒重组双CP多克隆抗体,可同时检测甘薯褪绿矮化病毒和羽状斑驳病毒,提高了检测效率,降低了检测成本。
Description
技术领域
本发明属于生物技术领域,具体涉及一种甘薯SPVD病毒重组双CP融合蛋白、由其制备的多克隆抗体、其制备方法及其应用。
背景技术
甘薯是我国重要的粮食、饲料、工业原料及新型能源作物。病毒病是甘薯上的重要病害,是导致甘薯产量降低、品质下降和种性退化的主要原因之一。由于长期的无性繁殖,病毒可通过薯块和秧苗传播,同时对甘薯病毒缺乏有效的化学防治方法,甘薯病毒病成为困扰甘薯产业发展的难题。
目前已报道侵染甘薯的病毒有30余种,其中由甘薯褪绿矮化病毒(SPCSV)和甘薯羽状斑驳病毒(SPFMV)协生共侵染引起的甘薯病毒病(Sweetpotato virus disease,SPVD)是甘薯上最严重的病毒病之一。感染SPVD后,甘薯表现叶片扭曲、畸形、褪绿、明脉以及植株矮化等症状,产量损失可达70%以上,甚至绝收。2009年,我国首次发现SPVD病毒,短短几年时间在我国三大薯区均有SPVD发生,并且迅速蔓延。2014年SPVD和卷叶病毒在甘薯生产上大爆发,山东、河南、江西、湖北、重庆、安徽等地的上万亩甘薯田发病严重,发病田块产量损失达60%~80%。我国甘薯上的SPCSV存在东非和西非2个株系,其中西非株系分布范围更广,为我国的优势株系。
目前对甘薯病毒尚无有效的化学防治方法,采用茎尖组织培养,结合病毒检测技术以及利用无病毒种植材料,能有效地恢复品种优良种性、增强抗性、提高产量和改善品质、延长种薯的服务期限,增产幅度达20%~40%。但培养的脱毒苗需要进行病毒检测,才能服务于农户。目前,脱毒苗病毒检测的常用方法是ELISA法,有些研究组也制备了单种病毒重组 CP多克隆抗体,这种方法只用于单种病毒的检测,成本高,效率低。
发明内容
针对现有技术中存在的问题,本发明的目的在于提供一种甘薯SPVD病毒重组双CP融合蛋白以及由其制备的多克隆抗体,同时用于两种病毒的ELISA检测,提高检测效率,降低检验成本。
为了达到上述目的,本发明的技术方案为:
一种甘薯SPVD病毒重组双CP融合蛋白,是由甘薯褪绿矮化病毒和甘薯羽状斑驳病毒 CP基因的融合片段构建到重组表达载体,转化表达菌株,诱导表达得到。
在上述方案的基础上,所述甘薯褪绿矮化病毒和甘薯羽状斑驳病毒CP基因的融合片段的核酸序列如SEQ ID No:1所示。
在上述方案的基础上,所述融合蛋白序列如SEQ ID No:2所示。
在上述方案的基础上,所述甘薯SPVD病毒重组双CP融合蛋白的制备方法,步骤如下:
(1)重组表达载体的构建:在甘薯褪绿矮化病毒和甘薯羽状斑驳病毒CP基因的融合片段两端分别加上Nde Ⅰ和HindⅢ的酶切接头,酶切后,连接到原核表达载体pET22b,得到重组表达载体;
(2)转化表达菌株:将重组表达载体转化Rosetta(DE3)感受态细胞;
(3)诱导表达:挑取(2)中阳性克隆,诱导表达融合蛋白;
(4)融合蛋白纯化:采用镍离子亲和层析纯化融合蛋白。
在上述方案的基础上,所述诱导表达条件为:IPTG添加终浓度为0.5mM,诱导温度20℃,转速220rpm,诱导过夜。
上述方法制备的甘薯SPVD病毒重组双CP融合蛋白的应用。
一种甘薯SPVD病毒重组双CP多克隆抗体的制备方法,采用上述方法制备的甘薯SPVD 病毒重组双CP融合蛋白免疫兔,获得的抗血清即为甘薯SPVD病毒重组双CP多克隆抗体。
上述方法制备的甘薯SPVD病毒重组双CP多克隆抗体。
上述多克隆抗体在制备检测甘薯SPVD病毒的试剂中的应用。
一种检测甘薯SPVD病毒方法,步骤如下:
(1)取待测甘薯叶,用液氮研磨破碎后,按10mL/g加入病毒提取缓冲液并进一步研磨, 4℃、12000r离心5min后取上清液;
(2)每反应孔加150μL(1)中的上清液,4℃包被过夜后洗涤;加200μL 3%的BSA进行封闭;
(3)每孔加150μL稀释的上述多克隆抗体、150μL的酶标二抗稀释液及150μL的底物液,最后加100μL的1.5M NaOH终止反应;用酶标仪在450nm波长下测定OD值。
本发明技术方案的优点:
目前检测甘薯SPVD的方法是只能分别单独检测甘薯褪绿矮化病毒和甘薯羽状斑驳病毒,导致检测效率低、成本高。本发明通过制备甘薯SPVD病毒重组双CP多克隆抗体,可同时检测甘薯褪绿矮化病毒和羽状斑驳病毒,提高了检测效率,降低了检测成本。
附图说明
图1融合蛋白小试SDS-PAGE分析图(M:Protein Marker;1:诱导前总蛋白;2:20℃上清;3:20℃沉淀;4:37℃上清;5:37℃沉淀);
图2镍离子亲和层析纯化SDS-PAGE分析图(M:Protein marker;1:上样;2:流出;3:20mM Imidazole洗脱组分;4:50mM Imidazole洗脱组分;5:500mM Imidazole洗脱组分);
图3抗血清效价检测(第一排为重复1,第二排为重复2,孔1为:阴性对照;孔2为空白对照;孔3-12分别为抗体稀释度1:1K、2K、4K、8K、16K、32K、64K、128K、256K、 512K)
具体实施方式
在本发明中所使用的术语,除非有另外说明,一般具有本领域普通技术人员通常理解的含义。
下面结合具体实施例,并参照数据进一步详细的描述本发明。以下实施例只是为了举例说明本发明,而非以任何方式限制本发明的范围。
实施例1
1、甘薯褪绿矮化病毒和甘薯羽状斑驳病毒CP基因融合片段的获得
根据公布的甘薯褪绿矮化病毒CP基因(西非株系,GenBank登录号:FJ807785中的CP 基因,4356-5126bp)和甘薯羽状斑驳病毒CP基因(GenBank登录号:HQ844114)序列,采用化学合成法合成两个CP基因的融合片段,去掉终止密码子,在不改变编码的氨基酸的前提下将基因内部的Nde Ⅰ和HindⅢ酶切位点突变(原始序列在268-273bp,938-943bp, 1127-1132bp处各有1个Nde Ⅰ酶切位点,在合成基因序列时分别将270bp处、942bp、1131bp 处的碱基由T变成C,从而去除基因内部的Nde Ⅰ酶切位点,且不改变编码的氨基酸序列;在1415-1420bp处有1个HindⅢ酶切位点,在合成基因序列时分别将1419bp处的碱基由T 变成A,从而去除基因内部的HindⅢ酶切位点,且不改变编码的氨基酸序列),合成的基因融合片段如SEQ ID No:1所示,分别序列两端加上Nde Ⅰ和HindⅢ的酶切接头,连接到pMD18 载体。
2、含有甘薯褪绿矮化病毒和甘薯羽状斑驳病毒CP基因融合片段的原核表达载体的构建
将1中的甘薯褪绿矮化病毒和甘薯羽状斑驳病毒CP基因融合片段(SEQ ID No:1),经鉴定后,酶切,连接到原核表达载体pET22b,进行PCR、酶切和测序鉴定。
3、诱导表达融合蛋白
(1)转化
取1ul重组的pET22b载体转化Rosetta(DE3),42℃热击90s后冰上静置2min后涂布含有终浓度为50μg/mL氨苄青霉素和34μg/mL氯霉素的LB固体平板上,使用时4g 粉末用100mL dd water溶解,37℃培养过夜。
(2)小试培养选择最佳的诱导条件
挑取表达菌株Rosetta(DE3)的单菌落于10mL LB培养基。添加终浓度为50μg/mL氨苄青霉素和34μg/mL氯霉素)37℃,220rpm过夜培养。
将过夜培养的菌液按1:100比例分别接种于10mL LB培养基中,添加终浓度为50μg/mL 氨苄青霉素和34μg/mL氯霉素,37℃,220rpm培养。当OD值达到0.6时,添加终浓度为0.5mM 的IPTG,220rpm,分别20℃诱导过夜;37℃诱导4h,未加IPTG诱导剂的作为阴性对照。4000rpm离心10min收集菌体,弃上清,菌体用500μLPBS(PH7.4)缓冲液悬浮,超声破碎6min,超0.5s停1.5s,分别离心收集上清和沉淀,沉淀用500μL包涵体溶解液(8M Urea,50mM Tris-HCl,300mM NaCl,pH8.0)溶解,分别取40μL样品和10μL 5×protein loadingbuffer 混匀,沸水浴10min。
SDS-PAGE检测:准备12%的SDS-PAGE,Tris-Gly电泳缓冲液(Tris 3.0g,甘氨酸14.4g, SDS 1.0g,定容至1L),上样量10μL,浓缩胶80V 20min,分离胶120V 60min,凝胶电泳结束,考马斯亮蓝染色20min,脱色,获得约65KDa的目的蛋白(蛋白序列如SEQ ID No:2所示),结果与预期相符(图1)。
(3)大量诱导表达融合蛋白
将培养的菌液按1:100比例接种于3L的LB液体培养基中,添加终浓度为50μg/mL氨苄青霉素和34μg/mL氯霉素,37℃,220rpm培养,当OD值达到0.6时,添加终浓度为0.5mMIPTG,20℃,220rpm,诱导过夜,离心收集细胞菌体。
4、蛋白的纯化
(1)超声破碎菌体
将收集的细菌菌体用破碎Buffer(50mM Tris,300mM NaCl,0.2%Triton X-100,0.2mM PMSF,pH8.0)溶解,冰浴中超声破碎菌体,功率400W,20min。12000rpm,4℃,离心20min,收集上清进行下一步纯化。
(2)镍离子亲和层析
取5mL Ni-NTA,用5倍柱床体积的Binding buffer(50mM Tris,300mM NaCl,pH8.0) 清洗平衡柱子,流速5mL/min。将(1)中收集的上清液上柱,流速为2mL/min,收集穿透液。 5倍柱床体积的Binding buffer清洗柱子,流速5mL/min。先用Wash buffer(50mMTris,300mM NaCl,20/50mM Imidazole,pH8.0)洗杂,流速5mL/min,收集洗脱液;然后用Elution buffer (50mM Tris,300mM NaCl,500mM Imidazole,pH8.0)洗脱,流速2mL/min,收集洗脱液。
由图2可以看出,先用含20mM和50mM Imidazole的洗脱液洗脱,可去除绝大多数的杂质,再用含高浓度Imidazole(500mM)的Elution buffer洗脱,可大量富集目的蛋白。
将收集到的组分进行SDS-PAGE检测,将纯化后的蛋白,透析到:1×PBS,pH8.0中,4℃透析过夜,透析结束后,12000rpm,4℃,离心20min,保留上清液。0.45μm滤膜过滤后分装1mL/管,-80℃保存。采用Bradford法对蛋白进行定量。
5、抗原处理:
纯化蛋白总量为3mg,初免剂量为0.5mg/只,二免、三免、四免剂量为0.25mg/只。初免抗原为蛋白抗原与等体积弗氏完全佐剂混匀乳化,二免、三免、四免抗原为蛋白抗原与等体积弗氏不完全佐剂混匀乳化。
6、动物免疫:
动物选用健康雌性新西兰大白兔,4月龄,2.1kg。
免疫
一免:第1天,免疫用抗原为弗氏完全佐剂+蛋白抗原;
二免:第21天,免疫用抗原为弗氏不完全佐剂+蛋白抗原;
三免:第35天,免疫用抗原为弗氏不完全佐剂+蛋白抗原;
三免后采血:第42天,耳静脉采血1ml,ELISA检测抗血清效价;
四免:第49天,免疫用抗原为弗氏不完全佐剂+蛋白抗原;
四免后采血:第56天,耳静脉采血1ml,ELISA检测抗血清效价;
终放血:第57天,ELISA检测抗血清效价达到要求,颈动脉采全血。
7、重组CP多克隆抗体活性的间接ELISA检测
包被抗原:将纯化的蛋白用0.05mol/L碳酸盐(PH=9.6)按0.2ug/孔包板,100ul/孔,4℃孵育过夜。
洗板:取出用0.05%Tween-20(PBST)洗涤三次,3分钟/次。
封闭:每孔加入5%脱脂奶粉150ul封闭液,37℃封闭60分钟。
洗板:取出用0.05%Tween-20(PBST)洗涤三次,3分钟/次。
加一抗:将兔子的血清分别按照1:1000稀释,然后倍比稀释,37℃孵育1小时。
洗板:取出用0.05%Tween-20(PBST)洗涤三次,3分钟/次。
加二抗:辣根酶标记山羊抗兔IgG(H+L),货号:D110058,抗体公司:生工生物工程(上海)有限公司。1:8000稀释,37℃孵育45min。
洗板:取出用0.05%Tween-20(PBST)洗涤五次,3分钟/次。
显色:加入底物溶液(TMB)100ul/孔,反应15min,最后加入100ul 2mol/L硫酸终止反应。
测OD值:用酶标仪(科华ST-360)在450nm波长下测定OD值。
以重组CP为抗原包被反应孔,在间接ELISA测定中,阴性对照与空白对照都不显色,含纯化抗体的反应孔显色,目测结果反应为阳性,但稀释度增加后反应孔颜色变浅(图3)。
OD值测定结果显示抗体稀释度在1∶512k的范围内,多克隆抗体与抗原(重组CP)具有较强的结合活性,反应孔与阴性对照OD值之比≥2.5(表1),判断为阳性。
表1重组CP多克隆抗体活性的间接ELISA检测(OD450)
孔号 | 阴性 | 空白 | 1K | 2K | 4K | 8K | 16K | 32K | 64K | 128K | 256K | 512K |
重复1 | 0.046 | 0.029 | 1.653 | 1.071 | 0.928 | 0.808 | 0.724 | 0.645 | 0.552 | 0.462 | 0.365 | 0.257 |
重复2 | 0.038 | 0.026 | 1.968 | 1.212 | 0.936 | 0.817 | 0.761 | 0.633 | 0.531 | 0.463 | 0.327 | 0.271 |
8、SPVD病毒的间接ELISA检测
取感染SPVD的甘薯病叶,用液氮研磨破碎后,按1︰10(g︰mL)加入病毒提取缓冲液(含2%PVP-40000、1%BSA的PBST)并进一步研磨,4℃、12000r离心5min后取上清液用于检测。每反应孔加150μL上清液,4℃过夜后洗涤,包被结束后加200μL 3%的BSA进行封闭。随后每孔加150μL稀释的抗血清、150μL的酶标二抗稀释液及150μL的底物液 (0.6mg/mLpNPP,11.7%二乙醇胺),最后加100μL的1.5M NaOH终止反应。用酶标仪在 450nm波长下测定OD值。
利用制备的抗血清对感病叶片进行间接ELISA检测,反应孔颜色变黄,病叶汁液有较强的阳性反应,在抗血清稀释度小于1︰1000时,A/C大于2.0(表2)。健康叶片的汁液没有颜色变化,呈阴性反应。结果表明利用重组CP制备的抗血清可用于SPVD病毒的检测。
表2甘薯病叶的间接ELISA检测
注:A为重组CP抗血清反应孔的平均值,C为脱毒甘薯叶片(阴性对照)反应孔的平均值(0.065)。
以上所述,仅是本发明的较佳实施例而已,并非是对本发明作其它形式的限制,任何熟悉本专业的技术人员可能利用上述揭示的技术内容加以变更或改型为等同变化的等效实施例。但是凡是未脱离本发明技术方案内容,依据本发明的技术实质对以上实施例所作的任何简单修改、等同变化与改型,仍属于本发明技术方案的保护范围。
序列表
<110> 青岛农业大学
<120> 一种甘薯SPVD病毒重组双CP融合蛋白、由其制备的多克隆抗体、其制备方法及其应用
<130> 2018
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1716
<212> DNA
<213> 人工序列(Sweet potato chlorotic stunt virus and Sweet potatofeathery mottle virus)
<400> 1
atggctgata gcactaaagt cgaagagaag aactttgatt ccgactcaga tttggaaact 60
aacccaaaga ataaactaca gtccgagtat gcgtctcgtc gtgacgtcca gactgaagga 120
acggctggtt tgggaagacg agatatggag ttaactagtg gtattcttac ctcagaccag 180
ttagctcttg caagattggg taagatccaa gtatattcca attctccaga tattatgtca 240
aagagtcaag aggatgagtt taagcgtcac atggagaact tcgcaaaagt cgtgactggt 300
gaggctacaa taacgccaga aatttttgca gcgttttatg catcgttaat tcaggcatgg 360
gcaaatcaga gtacgtccga aaagaatgct tcgaacgtaa atcttgagaa tatgtttatg 420
gtcgacggga aagagtatag ttggaagact cacaacttta taaaccatat tcaatctaac 480
atgccagatg ttaagaacgc cataaggaag tgggcaaggg ctcatgcaaa tgattacaag 540
gtccttgttg gtatgggtat tgttaagccc gattaccatt tacaggcgaa acagggtgtc 600
ttacctgaat attggcattt ggccactgat ttcatgagag gtaatgattt ggcgacaacc 660
gcagatgggc tggctgcgac tttgatgatg aaaaggaacg ctctttgtaa caaggatagt 720
aagaattctg tctacaacgt tactcagttg actggaacag gtcttcactg ttctagtgaa 780
cgtactgaat tcaaagatgc gggagcgaac cctccagccc ctaagcctca gaatatccct 840
ccaccaccca caataactga ggttactgat ccagaagacc caaagcaggc agctttgaga 900
gctgcacgag ctaagcaacc cgcaaccatt ccagaatcat acggacgaga cactagcaag 960
gagaaggaat caatagtggg ggcatcatca aagggtgcga gggataaaga tgtaaacgtt 1020
ggaacagttg gtacgtttgt cgtgccacgt gttaagatga atgcaaacaa gaaaaggcaa 1080
ccaatggtaa atggaagggc cattataaat ttccaacact tgtcaacata cgagccagaa 1140
cagtttgagg ttgcaaacac ccggtcgact caagaacagt ttcaagcatg gtatgaggga 1200
gtgaaagggg actatggtgt tgacgataca ggaatgggga tcttattgaa tggattaatg 1260
gtttggtgca ttgaaaatgg cacatcccca aatataaatg gcgtgtggac tatgatggat 1320
ggtgatgagc aagtgacata tccaattaaa ccattgttgg accatgcagt gcctactttt 1380
aggcagatta tgacgcactt cagtgacgtt gctgaagcat acatagaaat gcgaaaccgt 1440
acaaaggcgt acatgccgag gtatggtcta caacgtaatt tgactgatat gagtcttgcg 1500
cgatatgcat ttgatttcta cgagctgcat tcaaccaccc ctgcacgtgc aaaagaagca 1560
catttacaga tgaaggcagc cgcgcttaag aatgcgaaaa atcggttgtt tggtttggac 1620
ggaaacgtct ccacgcaaga agaagatacg gagaggcaca cgacaactga tgttactaga 1680
aatatacata acctcttagg aatgaggggt gtgcaa 1716
<210> 2
<211> 585
<212> PRT
<213> 人工序列(Sweet potato chlorotic stunt virus and Sweet potatofeathery mottle virus)
<400> 2
Met Ala Asp Ser Thr Lys Val Glu Glu Lys Asn Phe Asp Ser Asp Ser
1 5 10 15
Asp Leu Glu Thr Asn Pro Lys Asn Lys Leu Gln Ser Glu Tyr Ala Ser
20 25 30
Arg Arg Asp Val Gln Thr Glu Gly Thr Ala Gly Leu Gly Arg Arg Asp
35 40 45
Met Glu Leu Thr Ser Gly Ile Leu Thr Ser Asp Gln Leu Ala Leu Ala
50 55 60
Arg Leu Gly Lys Ile Gln Val Tyr Ser Asn Ser Pro Asp Ile Met Ser
65 70 75 80
Lys Ser Gln Glu Asp Glu Phe Lys Arg His Met Glu Asn Phe Ala Lys
85 90 95
Val Val Thr Gly Glu Ala Thr Ile Thr Pro Glu Ile Phe Ala Ala Phe
100 105 110
Tyr Ala Ser Leu Ile Gln Ala Trp Ala Asn Gln Ser Thr Ser Glu Lys
115 120 125
Asn Ala Ser Asn Val Asn Leu Glu Asn Met Phe Met Val Asp Gly Lys
130 135 140
Glu Tyr Ser Trp Lys Thr His Asn Phe Ile Asn His Ile Gln Ser Asn
145 150 155 160
Met Pro Asp Val Lys Asn Ala Ile Arg Lys Trp Ala Arg Ala His Ala
165 170 175
Asn Asp Tyr Lys Val Leu Val Gly Met Gly Ile Val Lys Pro Asp Tyr
180 185 190
His Leu Gln Ala Lys Gln Gly Val Leu Pro Glu Tyr Trp His Leu Ala
195 200 205
Thr Asp Phe Met Arg Gly Asn Asp Leu Ala Thr Thr Ala Asp Gly Leu
210 215 220
Ala Ala Thr Leu Met Met Lys Arg Asn Ala Leu Cys Asn Lys Asp Ser
225 230 235 240
Lys Asn Ser Val Tyr Asn Val Thr Gln Leu Thr Gly Thr Gly Leu His
245 250 255
Cys Ser Ser Glu Arg Thr Glu Phe Lys Asp Ala Gly Ala Asn Pro Pro
260 265 270
Ala Pro Lys Pro Gln Asn Ile Pro Pro Pro Pro Thr Ile Thr Glu Val
275 280 285
Thr Asp Pro Glu Asp Pro Lys Gln Ala Ala Leu Arg Ala Ala Arg Ala
290 295 300
Lys Gln Pro Ala Thr Ile Pro Glu Ser Tyr Gly Arg Asp Thr Ser Lys
305 310 315 320
Glu Lys Glu Ser Ile Val Gly Ala Ser Ser Lys Gly Ala Arg Asp Lys
325 330 335
Asp Val Asn Val Gly Thr Val Gly Thr Phe Val Val Pro Arg Val Lys
340 345 350
Met Asn Ala Asn Lys Lys Arg Gln Pro Met Val Asn Gly Arg Ala Ile
355 360 365
Ile Asn Phe Gln His Leu Ser Thr Tyr Glu Pro Glu Gln Phe Glu Val
370 375 380
Ala Asn Thr Arg Ser Thr Gln Glu Gln Phe Gln Ala Trp Tyr Glu Gly
385 390 395 400
Val Lys Gly Asp Tyr Gly Val Asp Asp Thr Gly Met Gly Ile Leu Leu
405 410 415
Asn Gly Leu Met Val Trp Cys Ile Glu Asn Gly Thr Ser Pro Asn Ile
420 425 430
Asn Gly Val Trp Thr Met Met Asp Gly Asp Glu Gln Val Thr Tyr Pro
435 440 445
Ile Lys Pro Leu Leu Asp His Ala Val Pro Thr Phe Arg Gln Ile Met
450 455 460
Thr His Phe Ser Asp Val Ala Glu Ala Tyr Ile Glu Met Arg Asn Arg
465 470 475 480
Thr Lys Ala Tyr Met Pro Arg Tyr Gly Leu Gln Arg Asn Leu Thr Asp
485 490 495
Met Ser Leu Ala Arg Tyr Ala Phe Asp Phe Tyr Glu Leu His Ser Thr
500 505 510
Thr Pro Ala Arg Ala Lys Glu Ala His Leu Gln Met Lys Ala Ala Ala
515 520 525
Leu Lys Asn Ala Lys Asn Arg Leu Phe Gly Leu Asp Gly Asn Val Ser
530 535 540
Thr Gln Glu Glu Asp Thr Glu Arg His Thr Thr Thr Asp Val Thr Arg
545 550 555 560
Asn Ile His Asn Leu Leu Gly Met Arg Gly Val Gln Lys Leu Ala Ala
565 570 575
Ala Leu Glu His His His His His His
580 585
Claims (10)
1.一种甘薯SPVD病毒重组双CP融合蛋白,其特征在于:是由甘薯褪绿矮化病毒和甘薯羽状斑驳病毒CP基因的融合片段构建到重组表达载体,转化表达菌株,诱导表达得到。
2.根据权利要求1所述甘薯SPVD病毒重组双CP融合蛋白,其特征在于:所述甘薯褪绿矮化病毒和甘薯羽状斑驳病毒CP基因的融合片段的核酸序列如SEQ ID No:1所示。
3.根据权利要求1或2所述甘薯SPVD病毒重组双CP融合蛋白,其特征在于:所述融合蛋白序列如SEQ ID No:2所示。
4.根据权利要求3所述甘薯SPVD病毒重组双CP融合蛋白的制备方法,其特征在于:步骤如下:
(1)重组表达载体的构建:在甘薯褪绿矮化病毒和甘薯羽状斑驳病毒CP基因的融合片段两端分别加上NdeⅠ和HindⅢ的酶切接头,酶切后,连接到原核表达载体pET22b,得到重组表达载体;
(2)转化表达菌株:将重组表达载体转化Rosetta(DE3)感受态细胞;
(3)诱导表达:挑取(2)中阳性克隆,诱导表达融合蛋白;
(4)融合蛋白纯化:采用镍离子亲和层析纯化融合蛋白。
5.根据权利要求4所述甘薯SPVD病毒重组双CP融合蛋白的制备方法,其特征在于:所述诱导表达条件为:IPTG添加终浓度为0.5mM,诱导温度20℃,转速220rpm,诱导过夜。
6.权利要求4或5所述方法制备的甘薯SPVD病毒重组双CP融合蛋白的应用。
7.一种甘薯SPVD病毒重组双CP多克隆抗体的制备方法,其特征在于:采用权利要求4或5所述方法制备的甘薯SPVD病毒重组双CP融合蛋白免疫兔,获得的抗血清即为甘薯SPVD病毒重组双CP多克隆抗体。
8.权利要求7方法制备的甘薯SPVD病毒重组双CP多克隆抗体。
9.权利要求8所述多克隆抗体在制备检测甘薯SPVD病毒的试剂中的应用。
10.一种检测甘薯SPVD病毒方法,其特征在于:步骤如下:
(1)取待测甘薯叶,用液氮研磨破碎后,按10mL/g加入病毒提取缓冲液并进一步研磨,4℃、12000r离心5min后取上清液;
(2)每反应孔加150μL(1)中的上清液,4℃包被过夜后洗涤;加200μL 3%的BSA进行封闭;
(3)每孔加150μL稀释的权利要求7的多克隆抗体、150μL的酶标二抗稀释液及150μL的底物液,最后加100μL的1.5M NaOH终止反应;用酶标仪在450nm波长下测定OD值。
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