CN108558993B - 一种抑制WSSV感染的Cq-Ns1abp基因及其蛋白抗病毒活性应用 - Google Patents

一种抑制WSSV感染的Cq-Ns1abp基因及其蛋白抗病毒活性应用 Download PDF

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CN108558993B
CN108558993B CN201810031865.2A CN201810031865A CN108558993B CN 108558993 B CN108558993 B CN 108558993B CN 201810031865 A CN201810031865 A CN 201810031865A CN 108558993 B CN108558993 B CN 108558993B
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刘海鹏
谢晓露
王克坚
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Abstract

一种抑制WSSV感染的Cq‑Ns1abp基因及其蛋白抗病毒活性应用,涉及白斑综合征病毒。将Cq‑Ns1abp基因连接到原核表达载体pMal‑C2x中,构建红螯螯虾抗WSSV感染调控因子pMal‑C2x‑Cq‑Ns1abp重组表达载体;Cq‑Ns1abp基因产物包含N端BTB结构域和C端Kelch结构域蛋白及全长蛋白;将所得重组表达载转化体分别导入宿主细胞,诱导表达得表达产物,再通过亲和层析获得较高纯度的rCq‑Ns1abp重组蛋白。由于Cq‑Ns1abp对WSSV感染具有抑制作用,对虾类抗病毒免疫具有重要作用,因此红螯螯虾抗WSSV感染调控因子在制备抗病毒感染药物和动物抗病饲料添加剂中应用。

Description

一种抑制WSSV感染的Cq-Ns1abp基因及其蛋白抗病毒活性 应用
技术领域
本发明涉及白斑综合征病毒,尤其是涉及一种抑制WSSV感染的Cq-Ns1abp基因及其蛋白抗病毒活性应用。
背景技术
白斑综合征病毒(White spot syndrome virus,WSSV)是目前虾类养殖中最严重的病毒性疫病病原。WSSV具有非常广泛的宿主范围和对甲壳类动物强烈的致病性,能够感染对虾、螯虾、蟹和龙虾等[1,2],已成为水产甲壳动物养殖的主要障碍,对水产甲壳动物养殖业造成严重的经济损失。迄今为止,依然缺乏治疗WSSV病害的有效药物。
甲型流感病毒非结构蛋白NS1-A结合蛋白(NS1ABP)在人类中被首次报道,因其能够结合甲型流感病毒的非结构性蛋白NS1-A而被命名[3]。Hela细胞中报道的Ns1abp蛋白含有N-末端BTB(bric-a-brac,tramtrack,broad complex)/POZ(poxviruses and zincfingers)结构域和约50个氨基酸的五个Kelch样串联重复元件。人Kelch重复结构域是最常见的底物结合结构域,人类基因组含有超过95个含BTB-Kelch结构域的蛋白质[4]。研究表明,Ns1abp还参与调控甲型流感病毒mRNA的选择性剪接,在甲型流感病毒基因表达中发挥重要作用[5]
哺乳动物细胞Ns1abp与剪接组装因子SC35共定位于细胞核中,病毒感染会破坏该定位,使之重新定位于整个核质中。果蝇和NIH3T3细胞的Ns1abp通过其Kelch结构域在细胞质中与肌动蛋白共定位并结合[6,7],并在细胞骨架重排中发挥重要作用。此外,A549细胞Ns1abp在流感病毒感染过程中具有抗凋亡作用[8]。对虾能够利用细胞凋亡途径防御WSSV的感染[9],然而,在甲壳类动物中关于Ns1abp的研究基本上还未见报道,Ns1abp是否存在于低等动物中,以及是否参与WSSV的感染均不清楚。
参考文献:
1.Sundar Raj N,Nathiga Nambi KS,Abdul Majeed S,Taju G,Vimal S,FarookMA,et al.High efficacy of white spot syndrome virus replication in tissues offreshwater rice-field crab,Paratelphusa hydrodomous(Herbst).J Fish Dis.201235:917-25.
2.Jiravanichpaisal P,Bangyeekhun E,Soderhall K,SoderhallI.Experimental infection of white spot syndrome virus in freshwater crayfishPacifastacus leniusculus.Dis Aquat Organ.200147:151-7.
3.Wolff T,O'Neill RE,Palese P.NS1-Binding protein(NS1-BP):a novelhuman protein that interacts with the influenza A virus nonstructuralNS1protein is relocalized in the nuclei of infected cells.J Virol.1998 72:7170-80.
4.Perez-Torrado R,Yamada D,Defossez PA.Born to bind:the BTB protein-protein interaction domain.Bioessays.2006 28:1194-202.
5.Tsai PL,Chiou NT,Kuss S,Garcia-Sastre A,Lynch KW,FontouraBM.Cellular RNA binding proteins NS1-BP and hnRNP K regulate influenza Avirus RNA splicing.PLoS Pathog.2013 9:e1003460.
6.Sasagawa K,Matsudo Y,Kang M,Fujimura L,Iitsuka Y,Okada S,etal.Identification of Nd1,a novel murine kelch family protein,involved instabilization of actin filaments.J Biol Chem.2002 277:44140-6.
7.Adams J,Kelso R,Cooley L.The kelch repeat superfamily of proteins:propellers of cell function.Trends Cell Biol.2000 10:17-24.
8.Othumpangat S,Noti JD,Blachere FM,Beezhold DH.Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus.Virology.2013 447:84-94.
9.Wang PH,Wan DH,Chen YG,Weng SP,Yu XQ,He JG.Characterization of fournovel caspases from Litopenaeus vannamei(Lvcaspase2-5)and their role in WSSVinfection through dsRNA-mediated gene silencing.PLoS One.2013 8:e80418.
发明内容
本发明的第一目的在于提供红螯螯虾抗WSSV感染调控因子(Cq-Ns1abp)的基因序列。
本发明的第二目的在于提供红螯螯虾抗WSSV感染调控因子(Cq-Ns1abp)的氨基酸序列。
本发明的第三目的在于提供红螯螯虾抗WSSV感染调控因子(Cq-Ns1abp)的应用。
所述红螯螯虾抗WSSV感染调控因子命名为Cq-Ns1abp。
所述红螯螯虾抗WSSV感染调控因子的基因序列为:
Figure BDA0001546739850000021
Figure BDA0001546739850000031
Figure BDA0001546739850000041
所述红螯螯虾抗WSSV感染调控因子的氨基酸序列为:
Figure BDA0001546739850000042
Figure BDA0001546739850000051
Figure BDA0001546739850000061
所述红螯螯虾(Cherax quadricarinatus)抗WSSV感染调控因子的制备方法包括以下步骤:
1)将Cq-Ns1abp基因连接到原核表达载体pMal-C2x中,构建红螯螯虾抗WSSV感染调控因子pMal-C2x-Cq-Ns1abp重组表达载体;所述Cq-Ns1abp基因产物包含N端BTB结构域和C端Kelch结构域蛋白及全长蛋白;
2)将步骤1)所得重组表达载转化体分别导入宿主细胞E.coli(BL21:DE3),并进行诱导表达,得表达产物;
在步骤2)中,所述诱导表达的条件可为0.1mM硫代半乳糖苷(IPTG)、诱导温度16℃、诱导时间20h。
3)分离纯化步骤2)得到的表达产物,通过亲和层析获得较高纯度的rCq-Ns1abp重组蛋白。
在步骤3)中,所述rCq-Ns1abp重组蛋白可包括rCq-Ns1abp-BTB、rCq-Ns1abp-Kelch及rCq-Ns1abp-Full-length等。
由于所述Cq-Ns1abp对WSSV感染具有抑制作用,对虾类抗病毒免疫具有重要作用,因此红螯螯虾抗WSSV感染调控因子(Cq-Ns1abp)可在制备抗病毒感染药物和动物抗病饲料添加剂中应用。
本发明根据Cq-Ns1abp基因序列特征成功构建重组表达载体,在大肠杆菌系统中诱导表达并纯化获得重组蛋白rCq-Ns1abp。该重组蛋白能够识别并结合WSSV的囊膜蛋白VP28,且具有较强的抗WSSV感染活性,能有效抑制WSSV在红螯螯虾Hpt细胞中的感染复制,是一种重要的抗WSSV感染调控因子。因此,重组基因工程产品rCq-Ns1abp在制备抗WSSV类新药开发应用中显示出非常诱人的应用前景。
构建的红螯螯虾转录组数据库显示,红螯螯虾中存在Ns1abp的序列。同时,目前在其他水产类动物中尚未见该基因功能活性的相关报道。红螯螯虾转录组数据显示Ns1abp表达能响应WSSV的感染而上调表达,说明红螯螯虾Ns1abp参与了WSSV的免疫防御过程,因此开展红螯螯虾Ns1abp的表达纯化及活性研究对WSSV的防治具有重要意义。
本发明源于红螯螯虾的Ns1abp基因及重组表达蛋白,该基因工程产品能够抑制WSSV在红螯螯虾造血组织(Hpt)干细胞中的增殖,是一种具有抗WSSV感染活性的基因工程产物,对于WSSV病害的有效防治具有重要应用前景。
附图说明
图1为SDS-PAGE分析pMal-C2x-Cq-Ns1abp重组载体克隆子诱导表达的电泳图谱。在图1中,M为SDS-PAGE标准蛋白质Marker,1、2泳道分别为导入重组载体pMal-C2x-Cq-Ns1abp-BTB的表达菌诱导前、后的菌液上清,3、4泳道分别为导入重组载体pMal-C2x-Cq-Ns1abp-Kelch的表达菌诱导前、后的菌液上清,5、6泳道分别为导入重组载体pMal-C2x-Cq-Ns1abp-Full-length的表达菌诱导前、后的菌液上清,2、4、6泳道中分别可见约55kDa、70kDa、130kDa的明显目的蛋白诱导条带。
图2为SDS-PAGE分析pMal-C2x-Cq-Ns1abp重组载体克隆子表达产物纯化的电泳图谱。在图2中,M为SDS-PAGE标准蛋白质Marker,1为表达纯化的rCq-Ns1abp-BTB重组蛋白,可见约55kDa的明显蛋白条带,2为表达纯化的rCq-Ns1abp-Kelch重组蛋白,可见约70kDa的明显蛋白条带,3为表达纯化的rCq-Ns1abp-Full-length重组蛋白,可见约为130kDa的明显蛋白条带。
图3为Pull down实验验证rCq-Ns1abp蛋白特异性识别结合WSSV主要囊膜结构蛋白VP28。在图3中,1为WSSV囊膜蛋白阳性对照,2为不加蛋白的阴性对照,3为加入MBP标签蛋白的阴性对照,4、5、6分别为加入rCq-Ns1abp重组蛋白的实验组。2-6中均加入了能特异性识别MBP标签蛋白的琼脂糖珠,用于亲和层析MBP标签蛋白及其互作蛋白。结果表明,阴性对照组(2、3泳道)与囊膜蛋白VP28不结合,而重组蛋白组(4、5、6泳道)与VP28有明显结合。
图4为Western Blot实验验证pulsin转染rCq-Ns1abp重组蛋白抑制WSSV感染复制的作用。在图4中,5个样品中均加入了转染试剂pulsin,1为不转染蛋白的阴性对照,2为转染MBP标签蛋白的阴性对照,3、4、5为转染rCq-Ns1abp重组蛋白的实验组。结果显示转染rCq-Ns1abp重组蛋白的实验组(3、4、5泳道),WSSV病毒的感染复制水平明显降低。
具体实施方式
以下通过实施例结合附图详细说明本发明的技术方案。
实施例1红螯螯虾抗WSSV感染调控因子Cq-Ns1abp原核表达载体的构建
分别设计扩增编码含Cq-Ns1abp(cDNA)基因两个重要结构域(BTB和Kelch)的蛋白及含ORF框的全长蛋白:Cq-Ns1abp-BTB的特异性上游引物BTB-F1和下游引物BTB-R1,Cq-Ns1abp-Kelch的特异性上游引物Kelch-F1和下游引物Kelch-R1,Cq-Ns1abp-Full-length的特异性上游引物Ns1abp-F1和下游引物Ns1abp-R1。分别在各上游引物的5′端添加EcoR I酶切位点;在下游引物的3′端添加Not I酶切位点及6*His标签,使重组蛋白同时带上MBP和6*His标签,便于进一步的纯化。然后对其相应的结构域区域进行PCR扩增。
Cq-Ns1abp-BTB上游引物BTB-F1:
5′-CCGGAATTCTGCGATGTCATCCTCCAGGT-3′,
Cq-Ns1abp-BTB下游引物BTB-R1:
5′-AATGCGGCCGCTTAGTGATGGTGATGGTGATGTAAATGAGCCACCAGATGCTCA-3′,
Cq-Ns1abp-Kelch上游引物Kelch-F1:
5′-CCGGAATTCCATCTTTTGGTCTGTGGAGG-3′
Cq-Ns1abp-Kelch下游引物Kelch-R1:
5′-AATGCGGCCGCTTAGTGATGGTGATGGTGATGTCTCTTCGTATAGTTATCAATGCC-3′
Cq-Ns1abp-Full-length上游引物Ns1abp-F1:
5′-CCGGAATTCATGACCTCCACATCTGCACAATC-3′
Cq-Ns1abp-Full-length下游引物Ns1abp-R1:
5′-AATGCGGCCGCTTAGTGATGGTGATGGTGATGATGGCCATTAGTGGCAATTGAA-3′
PCR反应条件为:98℃预变性3min;98℃变性10s,55℃退火15s,72℃延伸30s,重复35个循环;72℃延伸10min。
利用琼脂糖凝胶纯化试剂盒回收PCR产物,然后对PCR产物进行EcoR I/Not I双酶切,并与同样经EcoR I/Not I双酶切线性化的原核表达载体pMal-C2x连接,获得重组表达载体pMal-C2x-Cq-Ns1abp-BTB、pMal-C2x-Cq-Ns1abp-Kelch及pMal-C2x-Cq-Ns1abp-Full-length。测序鉴定读码框准确无误。
实施例2重组表达载体pMal-C2x-Cq-Ns1abp在大肠杆菌BL21(DE3)中的诱导表达
1、诱导表达:
1)将构建好的重组表达载体转化BL21(DE3),并涂布于含氨苄青霉素(Amp+)抗性的LB平板。
2)挑取单克隆菌落,接种于5ml含Amp+的LB培养基中,37℃,200rpm摇床培养12h。
3)以1︰100的比例接种至100ml含Amp+的LB培养基中,37℃,200rpm摇床培养至OD600为0.3至0.5。
4)加入IPTG至终浓度0.1mM,16℃下150rpm诱导20h。
结果显示,见图1(2、4、6泳道),IPTG诱导后在菌体中可检测到明显的诱导条带,大小依次为55kDa、70kDa、130kDa左右,说明该条件可以获得较高比例的表达产物。
2、纯化:
1)收集菌体,重悬于PBS,超声破碎,并于4℃,12000rpm离心5min。
2)如上菌体超声破碎上清可再次离心10min,以充分去除不溶菌体团块,并以0.22μm滤头过滤,即可用于亲和层析纯化。
3)用5~10个柱体积MilliQ水清洗层析柱。
4)平衡:用5~10个柱体积PBS缓冲液平衡层析柱。
5)挂柱:将过滤后的可溶性上清以0.8ml/min全部过柱。
6)洗柱:用5~10个柱体积的洗涤缓冲液(50mM磷酸盐[pH7.4],500mM NaCl,20mM咪唑)过柱,洗去非特异结合蛋白。
7)洗脱:利用洗脱液(50mM磷酸盐[pH7.4],500mM NaCl,150mM咪唑)进行洗脱。
8)收集:收集洗脱蛋白,取少量样品进行SDS-PAGE电泳鉴定。
结果显示,纯化洗脱峰对应较为单一的蛋白条带(图2),说明经亲和层析纯化后可以得到纯度较高的重组表达蛋白。
实施例3利用pull down实验证明rCq-Ns1abp重组蛋白与WSSV的识别结合
分别稀释2ug重组蛋白(MBP、rCq-Ns1abp-BTB、rCq-Ns1abp-Kelch、和rCq-Ns1abp-Full-length,其中control组不加任何蛋白)和5ug WSSV囊膜蛋白于1ml PBS溶液中,加入20ul能够特异性结合MBP标签蛋白的Dextrin beads,4℃旋转孵育4h。孵育后,500g离心3min,小心去除上清。再用1ml预冷的PBS重悬洗涤,500g离心3min,重复5次。最后用10mM麦芽糖洗脱beads上的结合蛋白,加入SDS上样缓冲液,煮沸10min变性,置于12%SDS-PAGE凝胶中电泳,免疫印迹法(Western Blot)检测重组蛋白与WSSV囊膜蛋白VP28的结合。结果如图3,不加蛋白和加入标签蛋白的对照组中(泳道2、3),未检测到VP28信号,而阳性对照Input(泳道1)和加入rCq-Ns1abp重组蛋白(rCq-Ns1abp-BTB、rCq-Ns1abp-Kelch、rCq-Ns1abp-Full-length)的实验组中(泳道4、5、6),能检测到VP28信号。该结果说明rCq-Ns1abp重组蛋白的N端、C端结构域及全长重组蛋白均能识别WSSV的囊膜蛋白,且全长蛋白的识别结合能力最强。
实施例4Western Blot实验验证pulsin转染rCq-Ns1abp重组蛋白抑制WSSV感染复制的作用
rCq-Ns1abp重组蛋白抗WSSV活性鉴定:分离螯虾Hpt细胞培养于96孔板中,分别稀释300ng重组蛋白(MBP、rCq-Ns1abp-BTB、rCq-Ns1abp-Kelch、rCq-Ns1abp-Full-length)于20ul HEPES溶液中,各加1ul pulsin蛋白转染试剂(control组中仅加pulsin,不加任何蛋白),室温孵育15min后加入到96孔板Hpt培养细胞中。26℃培养箱孵育4h后去除原培养基,更换新鲜培养基,并加入WSSV进行感染(MOI=1),24h后收集细胞,加10μl 1*SDSsamplebuffer裂解细胞,提取细胞总蛋白,进行Western Blot分析。
结果如图4,转染了rCq-Ns1abp重组蛋白的细胞(3、4、5泳道)中,WSSV的主要囊膜蛋白VP28的表达量较对照组(1、2泳道)显著减少,说明转染rCq-Ns1abp重组蛋白后WSSV的感染复制作用受到明显抑制,说明rCq-Ns1abp具有较强的抗WSSV感染活性。
本发明旨在获得红螯螯虾rCq-Ns1abp基因及氨基酸序列,并对其基因工程产品抗WSSV病毒活性进行了鉴定,以期用于高效抗WSSV新药物的开发。本发明成功构建了红螯螯虾抗WSSV感染调控因子rCq-Ns1abp基因工程表达重组质粒pMal-C2x-Cq-Ns1abp-BTB、pMal-C2x-Cq-Ns1abp-Kelch及pMal-C2x-Cq-Ns1abp-Full-length,在获得rCq-Ns1abp重组表达蛋白纯品后,进一步确认了rCq-Ns1abp识别及抗WSSV病毒活性,为其作为抗WSSV病害防治新药物的开发奠定了良好基础。
序列表
<110> 厦门大学
<120> 一种抑制WSSV感染的Cq-Ns1abp基因及其蛋白抗病毒活性应用
<141> 2017-12-15
<160> 2
<170> SIPOSequenceListing 1.0
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<211> 2232
<212> DNA
<213> 红螯螯虾(Cherax quadricarinatus)
<400> 1
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atggtgactg aaggcaagat gaataccagt cttgtgatgg aggacagtcc agaggtgctc 120
caccagcgcc tcggtcagct caaccagctc aggaagagca gacatttctg cgatgtcatc 180
ctccaggttg ggagctctga gatacatgct catcggtctg tcctggcttg tgcctctcca 240
tatttctttg agttgttcac gtctgatgat gaccgcaaga ctgcacatga aggaagactg 300
atgtacaagc tgagtagtgg cttcgagaga gactctgtgg agaggcttgt gaactatgtc 360
tatactggat gtatggaagt gcctgaccaa cttgtcaaga ctgtttttat tgctgccagg 420
aaacttaaga tggaaacggt ttctcgtgcc tgcggtgagc atctggtggc tcatttaacg 480
cccgaatcat gtctatcagt gagagccatt aatggtattg cttccaatgc tgcgcttgtc 540
aaccgtgttg atgagtacat tcagcaagtt agtgatctgg tgcaagtgac aagggatgcc 600
ttgggcatac caaagatcca ggtttcagtg atacacagga cacatgatga agctgccatt 660
actggtcgtg ctctttgtaa ccttgttttg gaatgggtca aaaagcaaat ggttgaggag 720
gaccttcatt tggacctaat gaaagaaaag aaacatatgc tgtatctgaa tattgataat 780
tcactgcatg actgttctga catccagtca ggtgatctca atgattctga tatggttcaa 840
gactacaaga agatgtccag gaaactctcc cagaccaata ttaagatgcg aagaaagtca 900
acaacgccac aacctgtcaa gccccgcctg atgctgtact ctcgtagcat ctctgataag 960
gatgattcag agcaagattc tgattggaaa ttgattgcct atgctcaagt ctcggagagt 1020
tcatgggtag ctgtggtgac attaaaggga tcagtcactg taatgtcagt gcaacaaaag 1080
atgggaagct catcaccgac tcacactcca atttcctcac gtccagcttc agtggagaag 1140
gttgattatt acacagttat tcctcatatg tcgtctccaa aatgtgcaac tggcacaggg 1200
aatttaaatg gtcatctttt ggtctgtgga ggatatgatc gtggtgagtg cctacggact 1260
gttgaagatt acaacccaga gatgaatgcc tggaccatac agcctcctat gcatcagggt 1320
cgtggacggt tcgatttgac agttctaaat ggaaaagcat atgctattgg tggctgtgat 1380
ggttctaagg agctcagtac tgtagaggtt ctagaggaaa acgccaagaa atggagcagt 1440
gtggcacctc tacccctggc tcgctctaac actggtgtgt gcagtttaga tgggaaagtc 1500
ttctgtatag gtggctggaa tggtcagtac ggcataaagc agtgtgacat gtatactcca 1560
gagactgata tatggcaaac cattgcttct cttcatattg gtcggtacca agctggagtg 1620
gctgcatata aaggaatggt gtatgccatt ggaggctgtg actcctggaa ctgcctcaac 1680
tccgttgagg tgtacgactc tagacttgac acatggcggt ttgctgctcc catgaccact 1740
ccaaggcgag gatgtggtgc tgaagttttc aaaggtaaac tgtatgtaat gggaggatca 1800
gatgggaccc atagcctgtg tacaacagag atctatgact tggagacaaa tacctggatg 1860
cctgggccat caatgactac atgcagagca aatgttggtg tagctgttgt gaatggaaag 1920
ctgtatgctg ttggaggatt ttcaggcaag aacttcctta acagcataga gtacctagat 1980
cctgcaactg atgaatggac caacttcact ccaaagccgg aagtgattag gaatggcatt 2040
gataactata cgaagagaag tggctataga aatgaagaaa atagccaggc tgttgaaagt 2100
gatggtacgc aaggcagtga tcatgaagat gctgttgagc atgaagctgt agcaaatggc 2160
catcatgtgg cagagaatgg ttgtaatggt ctcccaaatg gtgtacattc aattgccact 2220
aatggccatt aa 2232
<210> 3
<211> 743
<212> PRT
<213> 红螯螯虾(Cherax quadricarinatus)
<400> 3
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Asp Gln Leu Val Lys Thr Val Phe Ile Ala Ala Arg Lys Leu Lys Met
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Glu Thr Val Ser Arg Ala Cys Gly Glu His Leu Val Ala His Leu Thr
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Pro Glu Ser Cys Leu Ser Val Arg Ala Ile Asn Gly Ile Ala Ser Asn
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Leu Asn Asp Ser Asp Met Val Gln Asp Tyr Lys Lys Met Ser Arg Lys
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Leu Ser Gln Thr Asn Ile Lys Met Arg Arg Lys Ser Thr Thr Pro Gln
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Val Ser Glu Ser Ser Trp Val Ala Val Val Thr Leu Lys Gly Ser Val
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Thr Pro Ile Ser Ser Arg Pro Ala Ser Val Glu Lys Val Asp Tyr Tyr
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Asn Leu Asn Gly His Leu Leu Val Cys Gly Gly Tyr Asp Arg Gly Glu
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Cys Leu Arg Thr Val Glu Asp Tyr Asn Pro Glu Met Asn Ala Trp Thr
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Claims (6)

1.一种抑制WSSV感染的Cq-Ns1abp,其特征在于编码所述Cq-Ns1abp的基因序列为:
Figure FDA0002967708670000011
Figure FDA0002967708670000021
2.一种抑制WSSV感染的Cq-Ns1abp,其特征在于其氨基酸序列为:
Figure FDA0002967708670000022
Figure FDA0002967708670000031
Figure FDA0002967708670000041
3.如权利要求1~2任一项所述一种抑制WSSV感染的Cq-Ns1abp的制备方法,其特征在于包括以下步骤:
1)将Cq-Ns1abp基因连接到原核表达载体pMal-C2x中,构建红螯螯虾抗WSSV感染调控因子pMal-C2x-Cq-Ns1abp重组表达载体;所述Cq-Ns1abp基因产物包含N端BTB结构域和C端Kelch结构域蛋白及全长蛋白;所述Cq-Ns1abp的基因序列如SEQ ID NO.1所示;
2)将步骤1)所得重组表达载转化体分别导入宿主细胞E.coli,并进行诱导表达,得表达产物;
3)分离纯化步骤2)得到的表达产物,通过亲和层析获得较高纯度的rCq-Ns1abp重组蛋白。
4.如权利要求3所述一种抑制WSSV感染的Cq-Ns1abp的制备方法,其特征在于在步骤2)中,所述诱导表达的条件为0.1mM硫代半乳糖苷、诱导温度16℃、诱导时间20h。
5.如权利要求3所述一种抑制WSSV感染的Cq-Ns1abp的制备方法,其特征在于在步骤3)中,所述rCq-Ns1abp重组蛋白包括rCq-Ns1abp-BTB、rCq-Ns1abp-Kelch及rCq-Ns1abp-Full-length。
6.如权利要求3所述制备方法所制备的红螯螯虾抗WSSV感染调控因子在制备抗病毒感染药物和动物抗病饲料添加剂中应用。
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