CN108728461B - H3n2型犬流感病毒穿梭胞内抗体tat-4f - Google Patents
H3n2型犬流感病毒穿梭胞内抗体tat-4f Download PDFInfo
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- CN108728461B CN108728461B CN201810538735.8A CN201810538735A CN108728461B CN 108728461 B CN108728461 B CN 108728461B CN 201810538735 A CN201810538735 A CN 201810538735A CN 108728461 B CN108728461 B CN 108728461B
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- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明公开了一种H3N2型犬流感病毒穿梭胞内抗体TAT‑4F,H3N2病毒为A型流感病毒,最主要的中和抗体来自于表面糖蛋白血凝素(HA),因此HA成为以往主要研究靶标;然而流感病毒具有高度变异性,而不同变异分支之间的免疫交叉反应较弱,M1的抗体可以通过与M1蛋白结合,抑制其活性,干扰流感病毒的复制、转录及释放,从而起到抗病毒的作用;为此我们选择M1蛋白来制备相应抗体以获得稳定效价、将其与能将生物大分子转导进细胞的TAT蛋白PTD偶联,表达融合蛋白TAT PTD‑M1 ScFv,制备出抗犬流感病毒的穿梭抗体,为犬流感的治疗提供新的途径。
Description
技术领域
本发明属于生物工程及疾病防治领域,具体地涉及制备针对H3N2型犬流感病毒保守抗原的穿梭胞内抗体TAT-4F,以及此抗体对H3N2型犬流感病毒的抗病毒作用。
背景技术
长久以来,人们普遍认为自然状态下犬类不易感染各种亚型的流感病毒,因此犬一直被排除在流感病毒的易感范围之外,但2004年病毒学家从佛罗里达赛狗体内分离出犬流感病毒(Canine influenze virus,CIV),并证实了CIV在各个犬种中的感染之后,这种认识才得以改变。此后研究表明,这种曾在美国赛犬业和宠物饲养者中引起恐慌的流感病毒,实为马流感病毒H3N8的变异株。犬流感病毒H3N8作为一种新出现的犬类病原微生物,致死率可能高达8%。这种H3N8犬流感是马流感病毒变异之后,形成了从马到狗之间传播的能力。作为一种新发生的疫病,几乎所有的犬都对此病毒缺乏免疫力。2008年韩国研究人员报道了H3N2亚型的犬流感发生,这种病毒基因组中包含了几种不同禽流感病毒的基因片段,与东南亚流行的禽流感病毒同源性达95.5%-98.9%,试验证明H3N2亚型流感病毒已经具备了犬传播的能力,研究人员认为此病毒有可能成为即将流行的第二种犬流感病毒。2006年在我国发现了具有流感症状的病犬,从病犬鼻拭子中分离到4株A型流感病毒,进化分析表明4个毒株的8个基因均与韩国2007年分离自犬的H3N2 CIV种系分支接近,人工感染实验证明分离株能感染犬并能在犬之间直接传播,表明禽源H3N2 CIV已进入中国。2009-2012年在中国多个省市地区病犬中分离到CIV,人工感染实验中发现,分离自犬的流感病毒不仅能感染犬,还能感染小鼠和猪。表明H3N2 CIV已经完全适应了哺乳动物,成功实现了跨宿主传播,同时存在新的哺乳动物之间跨宿主传播的风险。
犬流感是犬的一种新发传染病,不同品种、不同年龄犬都可感染,针对犬流感病毒的研究国外仅限于疫苗研究,而国内对于犬流感病毒的研究尚处于空白期,尚没有相应的预防和治疗药物的出现。犬作为伴侣动物在现在人类生活中具有特殊的地位。由于犬和人及野生动物的亲密接触,给流感病毒的中间传播提供了更多的机会。因此,一旦出现犬流感的大流行和暴发将会产生严重后果,必须加快CIV相关疫苗和治疗药物的研究和开发。
在病毒不断产生耐药性的情况下,新型抗体药物成为应对犬流感病毒引发潜在流感大流行的有效手段。随着基因工程技术的发展,基因工程抗体的发展非常迅速,单链抗体以其特异性高、分子量小,结构简单,较亲本抗体免疫原性低,在临床应用中能够最大减轻异种蛋白引起的变态反应的独特优势,吸引了众多科研者的目光,其制备技术已趋于成熟,特别是噬菌体展示技术更提高了抗体及抗体基因的筛选效率。单链抗体对病毒感染性疾病的治疗将发挥重要的作用。
H3N2病毒为A型流感病毒,最主要的中和抗体来自于表面糖蛋白血凝素(HA),因此HA成为以往主要研究靶标。然而流感病毒具有高度变异性,而不同变异分支之间的免疫交叉反应较弱。鉴于病毒变异的影响,基于保守抗原组分的抗体能够对不同亚型流感病毒产生抗病毒作用。流感病毒基质蛋白M1是流感病毒主要的结构蛋白,位于病毒囊膜内侧,通过与宿主细胞靶蛋白结合而参与和调控病毒的复制、转录、释放等过程。M1蛋白序列保守,因此针对M1的抗体可以通过与M1蛋白结合,抑制其活性,干扰流感病毒的复制、转录及释放,从而起到抗病毒的作用。为此我们选择M1蛋白来制备相应抗体以获得稳定效价、不受病毒变异影响的抗体制剂。
M1蛋白位于病毒囊膜内侧,抗体需要进入感染细胞才能发挥作用。蛋白转导域(Protein transduction domain,PTD)是能介导蛋白跨过细胞膜的小肽段,能够携带大分子有效通过生物膜进入细胞。PTD介导的蛋白转运不依赖于受体、通道、能量及胞吞作用,可以直接作用于所有类型细胞的脂质双分子层完成跨膜运动,而且其跨膜功能不具有物种特异性。自PTD被识别并鉴定以来,已经有数百种化合物和蛋白质被成功转导进入不同的细胞,并表现出了相应的生物活性。在已发现的PTD中,人类免疫缺陷病毒-1(HIV-1)TAT蛋白PTD是研究最多、功能确切的PTD,Tat PTD 能将与之连接的多肽、蛋白质及DNA 以一种浓度依赖的方式高效快速地导入细胞内,而细胞的正常结构和功能不受影响。尽管蛋白转导的机制目前尚在研究中,但其可以直接将具有治疗作用的生物大分子送入细胞发挥生物学效应这一特性为疾病的生物治疗提供了新的思路,因而在医学研究领域受到广泛关注。1997年,Vives 等发现,Tat PTD是位于47 ~ 57 位的11个氨基酸(YGRKKRRQRRR),是一个富含碱性氨基酸的多肽片段。见于TAT PTD的生物转导特性,将该片段与M1 ScFv融合表达后其可将M1 ScFv带入病毒感染细胞,靶向作用于胞内的M1蛋白,阻止其发挥生物功能,抑制流感病毒的装配和释放,从而起到抗病毒的作用。
综上所述,选择犬流感病毒保守序列M1蛋白为靶标抗原,利用犬噬菌体抗体库筛选出抗M1蛋白的高亲和力单链抗体,将其与能将生物大分子转导进细胞的TAT蛋白PTD偶联,表达融合蛋白TAT PTD-M1 ScFv,制备出抗犬流感病毒的穿梭抗体,为犬流感的治疗提供新的途径。
发明内容
本发明的目的是提供一种针对H3N2型犬流感病毒保守序列M1蛋白的H3N2型犬流感病毒穿梭胞内抗体TAT-4F,此抗体,可进入细胞作用于靶标,可用于H3N2型犬流感病的治疗。
H3N2型犬流感病毒穿梭胞内抗体TAT-4F,它的核苷酸序列如序列表SEQID NO.2;
H3N2型犬流感病毒穿梭胞内抗体TAT-4F,它的氨基酸序列如序列表SEQID NO. 4所示;
H3N2型犬流感病毒穿梭胞内抗体TAT-4F在制备治疗H3N2型犬流感病药物方面的应用;
H3N2型犬流感病毒的诊断试剂盒,它包括SEQID NO. 4所示的蛋白。
本发明提供了一种H3N2型犬流感病毒穿梭胞内抗体TAT-4F,H3N2病毒为A型流感病毒,最主要的中和抗体来自于表面糖蛋白血凝素(HA),因此HA成为以往主要研究靶标;然而流感病毒具有高度变异性,而不同变异分支之间的免疫交叉反应较弱,M1的抗体可以通过与M1蛋白结合,抑制其活性,干扰流感病毒的复制、转录及释放,从而起到抗病毒的作用;为此我们选择M1蛋白来制备相应抗体以获得稳定效价、将其与能将生物大分子转导进细胞的TAT蛋白PTD偶联,表达融合蛋白TAT PTD-M1 ScFv,制备出抗犬流感病毒的穿梭抗体,为犬流感的治疗提供新的途径。
附图说明
图1 为PET-SUMO-M1蛋白表达工程菌在诱导后的表达结果;M:蛋白质Marker;1:阴性对照;2:诱导菌体超声沉淀;3:诱导菌体超声上清;4:诱导全菌;
图2为纯化后的M1蛋白;M:Marker;1:SUMO酶切并纯化后样品;
图3为10株阳性菌株的PCR鉴定结果;
图4为纯化后的M1-scFv;M:Marker;1:10%-55%饱和度硫酸铵沉淀样品;2:流穿3:纯化后样品;
图5为M1-ScFv基因的扩增;M:Marker;1-2:TAT-ScFv PCR产物;
图6为重组表达质粒pET-28a-TAT-M1 ScFv的诱导表达结果;M:蛋白分子量Marker;1:阴性对照;2-3:重组表达质粒pET-28a-TAT-M1ScFv诱导;
图7为纯化的scFv;M:Marker;
1:TAT-scFv表达菌诱导超声上清;
2:Cu2+200咪唑洗脱样品; 3:纯化的TAT-scFv。
具体实施方式
实施例1:M1重组表达质粒pET-SUMO-M1的构建、表达及M1蛋白的纯化
设计并合成引物M1P1、M1P2,以犬H3N2 cDNA为模板PCR扩增M1蛋白基因,并克隆入PET-SUMO载体中,构建质粒pET-SUMO-M1,然后转入T-shot感受态细胞,含kan抗性的琼脂平板进行初步筛选。挑选单菌落于LB液体培养基中培养;用质粒回收试剂盒提取质粒,PCR鉴定,产物经1%琼脂糖凝胶电泳分析,获得750 bp左右的条带,大小与插入的目的基因相符,并进行序列的测定,证明目的片段正确插入载体中,成功构建了重组质粒pET-SUMO-M1。
将重组质粒pET-SUMO-M1转化表达菌大肠杆菌BL21(DE3),IPTG诱导表达后,SDS-PAGE结果显示:重组蛋白SUMO-M1在40KD左右处有一明显表达带,大小与理论值相符,超声后目的蛋白主要在超声上清中,证明目的蛋白以可溶形式表达(见图1)。
M1蛋白的纯化,诱导表达菌体超声裂解后,取其上清,以20-45% AS沉淀,沉淀以PB(pH 7.0)重悬后过离子交换层析(SP FF),以含0.5M NaCl的PB线性洗脱,收集目的蛋白洗脱峰,过Cu2+柱,缓冲系统为PB+0.5M NaCl 分别以50mM、150mM咪唑洗脱,目的蛋白在150 mM咪唑洗脱峰中。将150 mM咪唑洗脱液稀释至咪唑浓度为50 mM,加入SUMO蛋白酶,30度酶切2h。酶切产物过Cu2+柱,20mM咪唑洗脱目的蛋白,再以SP FF浓缩,得到纯度在90%以上的M1蛋白,满足噬菌体抗体库筛选的需要(见图2)。
实施例2:噬菌体单链抗体库的扩增
1、构建犬噬菌体抗体库构建
犬淋巴细胞分离:比格犬6只外周血淋巴细胞分离,提取总RNA ,反转录合成cDNA;扩增VH基因,上、下游分别加入NcoⅠ、XhoⅠ酶切位点;扩增VL基因,上、下游分别加入SalⅠ、NotⅠ酶切位点;VH酶切连接入PIT2载体;VL酶切连接入PIT2载体。
2、构建好的载体电转化大肠杆菌TG1,涂布TYE平板(含终浓度100μg/mL Amp+1%葡萄糖),37℃过夜培养,从平板上随机挑取单克隆进行菌液PCR鉴定,计算重组率,并估算库容(库容=克隆数×稀释倍数×重组率)并保存的犬噬菌体scFv抗体库(库容为2.5×107)接入预热的250mL 2×TY培养基中(含终浓度100μg/mL Amp+1%葡萄糖),37℃振荡培养至OD600为0.4(约2h)。取50mL菌液加入2.0×1011的辅助噬菌体KM13,37℃水浴30min。将菌液以4℃,3300g离心20min,沉淀用100mL2×TY培养基(含终浓度100μg/mLAmp+50μg/mL Kana+0.1%葡萄糖)重悬,250rpm 30℃振摇培养过夜。将过夜菌液4℃,3300g离心30min,收集上清约为80mL,加入20mL PEG/NaCl溶液(终浓度为20% PEG-6000,2.5mol/L NaCl),混匀后置于冰上lh。 4℃ 3300g离心30min,沉淀用4m1 PBS重悬,充分混匀。4℃ 11600g离心10min,取上清,4℃保存,用于抗体库筛选同时进行噬菌体滴度测定。
实施例3:抗M1-scFv的筛选
纯化的M1蛋白为抗原包被于96孔酶标板上,4℃过夜。次日弃上清,用2%的Milk-PBS 37℃封闭2h,加入制备的噬菌体抗体库(滴度为1.0×1013pfu),室温下剧烈晃动孵育60min,静置60min。后弃去液体,用含0.1%Twenn-20的PBS洗涤10次,洗涤后将每孔中残留的液体轻轻拍干,每孔加入50µL洗脱液(5mg/mL的胰酶-PBS),室温下剧烈晃动10min,洗脱噬菌体,收集4℃保存。
用洗脱下的噬菌体侵染E.coli TG1,并涂布于TYE平板上(含100µg/mLAmp和1%的葡萄糖)37℃过夜培养。利用辅助噬菌体KM13扩增噬菌体库,通过PEG/NaCl回收噬菌体。重复以上过程3次,共4轮筛选。
筛选后的噬菌体侵染E.Coli HB2151,诱导表达后,利用ELISA鉴定,置酶标仪测定OD值(波长为490nm),每个样品做双孔测定,取OD平均值。阳性克隆菌株确定标准为:OD值为阴性对照的3倍以上。
合成两条特异性PCR引物扩增scFv全基因片段:
P3: 5’—CAG GAA ACA GCT ATG AC—3’
P4: 5’ —CTA TGC GGC CCC ATT CA—3’
扩增出900bp左右的片段,证明获得完整的单链抗体(图3)。
实施例4:M1-scFv的表达及纯化
将ELISA阳性菌株转接于5mL 2×TY培养基(含100 µg/mLAmp和1%的葡萄糖),37℃培养过夜。次日,转接200µL过夜培养物于2×TY培养基(含100 µg/mLAmp和0.1%的葡萄糖),37℃培养至OD600至0.9(约4 h),再加入终浓度为1 mmol/L IPTG,30℃振荡培养过夜诱导。次日将诱导菌液4200rpm离心20 min,取上清,以10%-55%硫酸铵沉淀,沉淀用30 mmol/L PB(pH7.2)重悬,在PBS中透析过夜,透析样品进行rProtein-A FF亲和层析,洗脱样品用PBS透析过夜, 12%SDS-PAGE分析显示目的蛋白大小约为31000 Da,纯化后的scFv纯度满足进行抗病毒试验的要求。(图4)
实施例5:TAT-M1 ScFv的构建、表达及纯化
设计合成2条引物:P5、P6,分别引入EcoRⅠ、Hind Ⅲ 酶切位点,提取M1-ScFv菌株质粒(TAT-4F、TAT-2C菌株质粒),以此为模板进行PCR(图5)。
P5: 5` GTGAATTCATGAAATACCTATTGCCT 3`
P6: 5` GCAAGCTTCTATGCGGCCCCATTCAG 3`
采用凝胶电泳回收上述PCR反应的扩增产物,分别用EcoRⅠ和HindⅢ双酶切PCR扩增回收产物及载体pET28a-TAT-GFP,T4连接酶连接PCR产物及载体片段,转化感受态大肠杆菌DH5α,PCR鉴定阳性克隆,提取PCR鉴定正确的重组质粒测序,结果表明:TAT-M1 ScFv片段以正确的阅读框架克隆到表达载体pET-28a中。
将构建的PET28a-TAT-M1 ScFv氯化钙法转化到BL21(DE3)中,挑取单菌落,接种到LB液体培养基中振荡培养至菌液OD600≈0.5以上,加IPTG诱导表达。收集诱导菌体,进行12%SDS-PAGE检测,以未诱导工程菌做为阴性对照,结果显示,在约30KDa处出现一条明显表达带,与融合蛋白预期大小完全一致(图6)。
TAT-M1 ScFv的纯化,诱导表达菌体超声裂解后,取其上清,过金属螯合Cu2+柱,缓冲系统为PBS(pH7.2),分别以20mM、200mM咪唑洗脱,目的蛋白在200 mM咪唑洗脱峰中。200mM咪唑洗脱液过亲和层析柱(rProteinA FF),洗脱样品以PBS透析,即获得纯化的TAT-M1ScFv(图7)。
实施例6:TAT-M1 ScFv的生物活性检测
将消化后的MDCK细胞铺96孔细胞培养板(3×104个细胞/孔),待细胞长成单层吸弃培养基,以DMEM洗细胞3次,分别加入不同稀释度的H3N2病毒:50 TCID50、100 TCID50、150 TCID50、200 TCID50、250 TCID50、300 TCID50、350 TCID50、400 TCID50,阴性对照孔加入PBS,37℃孵育3.5h,弃细胞外液,PBS洗细胞2次,分别加入纯化的TAT-2C、TAT-4F(10.8μg/孔),阳性对照加PBS,37℃作用1.5h ,吸弃细胞外液,每孔加入DMEM(含2%FBS),37℃过夜培养。次日,以过夜培养物上清做血凝试验。
血凝试验:反应板内加入培养上清,50μL/孔,每个样品做复孔,再向每孔中加入0.85%鸡红细胞悬液50μL/孔,室温放置30min,将反应板直立观察结果。
结果显示穿梭胞内抗体可中和H3N2病毒活性,TAT-2C、TAT-4F中和H3N2病毒的效价分别为250TCID50、200TCID50(见表1)。
表1 血凝试验结果表
*H3N2的TCID50为10-4.5/0.1mL。
序列表
<110> 军事科学院军事医学研究院军事兽医研究所
<120> H3N2型犬流感病毒穿梭胞内抗体TAT-4F
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 906
<212> DNA
<213> 犬(Canis lupus familiaris)
<400> 1
tatggtcgta aaaaacgtcg tcagcgtcgt cgtgaattca tgaaatacct attgcctacg 60
gcagccgctg gattgttatt actcgcggcc cagccggcca tggccgaggt gcagctgttg 120
gagtctgggg gaggcttggt acagcctggg gggtccctga gactctcctg tgcagcctct 180
ggattcacct ttagcagcta tgccatgagc tgggtccgcc aggctccagg gaaggggctg 240
gagtgggtct cagatattag taagtctggt tctaagacat cgtacgcaga ctccgtgaag 300
ggccggttca ccatctccag agacaattcc aagaacacgc tgtatctgca aatgaacagc 360
ctgagagccg aggacacggc cgtatattac tgtgcggaaa tgccttctgt ttttgactac 420
tggggccagg gaaccctggt caccgtctcg agcggtggag gcggttcagg cggaggtggc 480
agcggcggtg gcgggtcgac ggacatccag atgacccagt ctccatcctc cctgtctgca 540
tctgtaggag acagagtcac catcacttgc cgggcaagtc agagcattag cagctattta 600
aattggtatc agcagaaacc agggaaagcc cctaagctcc tgatctatga ggcatccaag 660
ttgcaaagtg gggtcccatc aaggttcagt ggcagtggat ctgggacaga tttcactctc 720
accatcagca gtctgcaacc tgaagatttt gcaacttact actgtcaaca gctgaatcat 780
cggcctcaga cgttcggcca agggaccaag gtggaaatca aacgggcggc cgcacatcat 840
catcaccatc acggggccgc agaacaaaaa ctcatctcag aagaggatct gaatggggcc 900
gcatag 906
<210> 2
<211> 906
<212> DNA
<213> 犬(Canis lupus familiaris)
<400> 2
tatggtcgta aaaaacgtcg tcagcgtcgt cgtgaattca tgaaatacct attgcctacg 60
gcagccgctg gattgttatt actcgcggcc cagccggcca tggccgaggt gcagctgttg 120
gagtctgggg gaggcttggt acagcctggg gggtccctga gactctcctg tgcagcctct 180
ggattcacct ttagcagcta tgccatgagc tgggtccgcc aggctccagg gaaggggctg 240
gagtgggtct caggtattaa tagtacgggt aagctgacaa agtacgcaga ctccgtgaag 300
ggccggttca ccatctccag agacaattcc aagaacacgc tgtatctgca aatgaacagc 360
ctgagagccg aggacacggc cgtatattac tgtgcgaaaa ggaggcttct gtttgactac 420
tggggccagg gaaccctggt caccgtctcg agcggtggag gcggttcagg cggaggtggc 480
agcggcggtg gcgggtcgac ggacatccag atgacccagt ctccatcctc cctgtctgca 540
tctgtaggag acagagtcac catcacttgc cgggcaagtc agagcactag cagctattta 600
aattggtatc agcagaaacc agggaaagcc cctaagctcc tgatctataa ggcatcctac 660
ttgcaaagtg gggtcccatc aaggttcagt ggcagtggat ctgggacaga tttcactctc 720
accatcagca gtctgcaacc tgaagatttt gcaacttact actgtcaaca gcggtataat 780
tctcctgcta cgttcggcca aagggaccaa agtggaaatc aaacggcggc cgcacatcat 840
catcaccatc acggggccgc agaacaaaaa ctcatctcag aagaggatct gaatggggcc 900
gcatag 906
<210> 3
<211> 301
<212> PRT
<213> 犬(Canis lupus familiaris)
<400> 3
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Glu Phe Met Lys Tyr
1 5 10 15
Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro
20 25 30
Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
35 40 45
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
50 55 60
Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
65 70 75 80
Glu Trp Val Ser Asp Ile Ser Lys Ser Gly Ser Lys Thr Ser Tyr Ala
85 90 95
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
100 105 110
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
115 120 125
Tyr Tyr Cys Ala Glu Met Pro Ser Val Phe Asp Tyr Trp Gly Gln Gly
130 135 140
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser
165 170 175
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
180 185 190
Ser Gln Ser Ile Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly
195 200 205
Lys Ala Pro Lys Leu Leu Ile Tyr Glu Ala Ser Lys Leu Gln Ser Gly
210 215 220
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
225 230 235 240
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
245 250 255
Gln Leu Asn His Arg Pro Gln Thr Phe Gly Gln Gly Thr Lys Val Glu
260 265 270
Ile Lys Arg Ala Ala Ala His His His His His His Gly Ala Ala Glu
275 280 285
Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala
290 295 300
<210> 4
<211> 301
<212> PRT
<213> 犬(Canis lupus familiaris)
<400> 4
Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Glu Phe Met Lys Tyr
1 5 10 15
Leu Leu Pro Thr Ala Ala Ala Gly Leu Leu Leu Leu Ala Ala Gln Pro
20 25 30
Ala Met Ala Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
35 40 45
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
50 55 60
Ser Ser Tyr Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
65 70 75 80
Glu Trp Val Ser Gly Ile Asn Ser Thr Gly Lys Leu Thr Lys Tyr Ala
85 90 95
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn
100 105 110
Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
115 120 125
Tyr Tyr Cys Ala Lys Arg Arg Leu Leu Phe Asp Tyr Trp Gly Gln Gly
130 135 140
Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
145 150 155 160
Ser Gly Gly Gly Gly Ser Thr Asp Ile Gln Met Thr Gln Ser Pro Ser
165 170 175
Ser Leu Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala
180 185 190
Ser Gln Ser Thr Ser Ser Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly
195 200 205
Lys Ala Pro Lys Leu Leu Ile Tyr Lys Ala Ser Tyr Leu Gln Ser Gly
210 215 220
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
225 230 235 240
Thr Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln
245 250 255
Gln Arg Tyr Asn Ser Pro Ala Thr Phe Gly Gln Arg Asp Gln Ser Gly
260 265 270
Asn Gln Thr Ala Ala Ala His His His His His His Gly Ala Ala Glu
275 280 285
Gln Lys Leu Ile Ser Glu Glu Asp Leu Asn Gly Ala Ala
290 295 300
Claims (4)
1.H3N2型犬流感病毒穿梭胞内抗体TAT-4F,它的核苷酸序列如序列表SEQID NO.2所示。
2.H3N2型犬流感病毒穿梭胞内抗体TAT-4F,它的氨基酸序列如序列表SEQID NO. 4所示。
3.权利要求1所述的H3N2型犬流感病毒穿梭胞内抗体TAT-4F在制备治疗H3N2型犬流感病药物方面的应用。
4.H3N2型犬流感病毒的诊断试剂盒,它包括SEQID NO. 4所示的蛋白。
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