CN104788572A - 用于检测pcv2的融合蛋白、制备方法及应用 - Google Patents
用于检测pcv2的融合蛋白、制备方法及应用 Download PDFInfo
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- CN104788572A CN104788572A CN201510197838.9A CN201510197838A CN104788572A CN 104788572 A CN104788572 A CN 104788572A CN 201510197838 A CN201510197838 A CN 201510197838A CN 104788572 A CN104788572 A CN 104788572A
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Abstract
本发明提供用于检测PCV2的融合蛋白、制备方法及应用,涉及PCV2病毒防治诊断领域。所述融合蛋白,是将抗人红细胞H抗原纳米抗体和抗PCV2纳米抗体通过Linker序列连接而成。本发明还要求保护所述融合蛋白的编码基因,含有该基因的重组载体和重组菌以及上述融合蛋白在制备检测PCV2试剂盒方面的应用。本发明融合蛋白,在与人O型血红细胞和PCV2病毒同时作用时,会发生肉眼可观察到的凝集现象,因此可以用于快速检测PCV2,操作简单、灵敏度高且特异性强,能够满足基层现场使用快速、简便、成本低的要求。
Description
技术领域
本发明涉及PCV2病毒防治诊断领域,具体涉及用于检测PCV2的融合蛋白、制备方法及应用。
背景技术
猪圆环病毒(Porcine circovirus,PCV)是作为一种猪肾细胞系PK-15培养污染物而被发现的,其基因组为共价闭合的单股环状DNA分子,基因组全长约1.7kb。PCV是迄今为止人们发现的最小动物病毒,病毒粒子表面无囊膜,呈二十面体对称,粒子直径在17-20nm之间。根据PCV的致病性、血清型和基因型特点,将其分为非致病性的PCV1和致病性的PCV2(猪圆环病毒2型)两种类型。其中,PCV2是引起猪圆环病毒病(PCVD)的主要病原。PCV2主要侵害免疫系统,降低机体的抵抗力和免疫应答反应,导致被感染猪产生免疫抑制和其他病原微生物的继发感染,从而使死亡率明显上升。该病除能引起断奶仔猪多系统衰竭综合征(Postweaning multisystemic wasting syndrome,PMWS)之外,还是猪皮炎与肾病综合征(porcine dermatitis nephropathy syndrome,PDNS)、猪呼吸道综合征(porcine respiratory disease complex,PRDC)、增生性坏死性肺炎(proliferative necrotizing pneumonia,PRDC)、新生仔猪先天性震颤、怀孕母猪繁殖障碍等疾病的重要病原。
猪圆环病毒病一年四季均可发生,无严格的季节性,猪是PCV2的天然宿主,各种品种的猪,不论大小均可发病,主要感染5-12周龄猪。病猪和带毒猪是猪圆环病毒病的主要传染源,可以水平传播,也可通过感染的怀孕母猪通过垂直传播的方式传染仔猪。自1991年在加拿大首次报道猪群中存在PMWS以来,相继在美国、欧洲、东南亚等地发现猪群中有PMWS存在,并分离到PCV2。我国为养猪大国,PCV2感染也相当严重,自2000年郎洪武等报道在国内猪群中存在PCV2感染以来,PCV2引起的疾病在我国流行一直处于上升趋势。有研究显示,从2008年以来中国大部分省市PCV2抗体阳性率在25.9%到63.38%之间。血清学调查结果说明PCV2在我国猪群中广泛的流行。正因为PCV2能使感染猪机体免疫功能受到损害,机体抵抗力明显下降,易造成并发或继发感染而使病情加重,造成极大的经济损失。PCV2已成为严重威胁我国及世界其他国家和地区猪群健康的重要致病因素之一,阻碍了养猪业的健康发展,严重影响了世界各国的养猪生产水平。
及时、准确的对PCV2进行检测,是诊断、预防和控制PCVD的前提。目前PCV2的检测技术包括病毒的分离、电子显微镜观察、聚合酶链式反应(PCR)、免疫过氧化物单层培养法、酶联免疫法、间接免疫荧光法、原位杂交及核酸探针杂交实验等方法。迄今为止,尽管有多种检测PCV2的方法,但是这些检测方法要求较高,需要配备专门的仪器和专业技术人员,检测成本昂贵,而且耗时相对较长,这极大地限制了其在基层单位的应用。
发明内容
本发明的目的是提供用于检测PCV2的融合蛋白,该融合蛋白在与人O型血红细胞和PCV2病毒同时作用时,会发生肉眼可观察到的凝集现象,因此可以用于快速检测PCV2。
本发明的另一目的是提供所述融合蛋白的制备方法,由于该融合蛋白分子量较小、且易于原核系统的表达,所以产量较高。
本发明的再一目的是提供上述融合蛋白在制备检测PCV2试剂盒方面的应用,操作简单、灵敏度高且特异性强,能够满足基层现场使用快速、简便的要求。
本发明的目的采用如下技术方案实现。
一种用于检测PCV2的融合蛋白,是将抗人红细胞H抗原纳米抗体和抗PCV2纳米抗体通过Linker序列连接而成。
优选的技术方案中,所述融合蛋白的氨基酸序列如SEQIDNo:2所示。
本发明还要求保护所述融合蛋白的编码基因。
优选的技术方案中,所述编码基因的核苷酸序列如SEQIDNo:1所示。
本发明还要求保护含有所述基因的重组载体;优选的技术方案中,所述重组载体是将权利要求3或4所述基因插入表达载体所得;所述表达载体优选为pET-His。
另外,本发明还要求保护含有所述基因的重组菌,是将含有所述基因的重组载体导入宿主菌中得到的重组菌;所述重组载体是将所述基因插入表达载体pET-His所得。
本发明还要求保护制备所述融合蛋白的方法,诱导所述重组菌表达融合蛋白,收集所述融合蛋白的包涵体,复性所述融合蛋白。
最后,本发明还要求保护所述融合蛋白在制备检测PCV2试剂盒方面的应用。
本发明用于检测PCV2的融合蛋白,在与人O型血红细胞和PCV2病毒同时作用时,会发生肉眼可观察到的凝集现象,因此可以用于快速检测PCV2。本发明用于检测PCV2的融合蛋白可以单独与人O型血红细胞和PCV2病毒结合,但不会发生凝集现象。
在本发明所述融合蛋白的制备方法中,由于该融合蛋白分子量较小、且易于原核系统的表达,所以产量较高。
上述融合蛋白可以应用于制备检测PCV2的试剂盒。采用该试剂盒检测PCV2,操作简单、灵敏度高且特异性强,能够满足基层现场使用快速、简便、成本低的要求。
附图说明
下面结合附图所示实施例,作进一步说明,但本发明保护范围不限于此实施例。
图1融合蛋白A的诱导表达验证SDS-PAGE电泳图,M-Mark,泳道1-未诱导BL21-A全菌;泳道2-诱导后BL21-A的全菌。
图2重组融合蛋白A的双功能特性验证。
图3重组融合蛋白A的特异性验证。
图4重组融合蛋白A用于检测PCV2的灵敏性验证,其中第一、二排中,各列孔上方数字表示该孔内病毒的稀释度;第三排中均为对照。
图5重组融合蛋白B的诱导表达验证SDS-PAGE电泳图,M-Mark,泳道1-未诱导BL21-B全菌;泳道2-诱导后BL21-B的全菌。
具体实施方式
溶液Ⅰ:1MTris-HCl(pH8.5-9.0)缓冲液中含有1mM EDTA、50mM NaCl和0.5%(质量百分浓度)Triton X-100。
溶液Ⅱ:1M Tris-HCl(pH8.5-9.0)缓冲液中含有1mM EDTA、50mM NaCl、3M尿素和0.5%(质量百分浓度)Triton X-100。
溶液Ⅲ:1M Tris-HCl(pH8.5-9.0)缓冲液中含有1mM EDTA、50mM NaCl、6M盐酸胍、5%(质量百分浓度)甘油和5mMDTT(二硫苏糖醇)。
TGE Buffer:50mM Tris-HCl(pH7.9)缓冲液中含有0.5mM EDTA、50mMNaCl和5%(质量百分浓度)甘油。
0.1M、pH7.4的PBS缓冲液:称取11.65g Na2HPO4,1.65g KH2PO4,9g NaCl,去离子水溶解后用0.1M氢氧化钠溶液或盐酸调pH值到7.4,定容至1000ml。
实施例1用于检测PCV2的融合蛋白的制备
1.融合蛋白基因的设计与合成
人工设计分子量较小的用于检测PCV2的融合蛋白A。该融合蛋白由抗人红细胞H抗原纳米抗体和抗PCV2纳米抗体通过Linker序列连接而成。融合蛋白A的氨基酸序列如SEQ ID No:2所示。设计融合蛋白A的编码基因,核苷酸序列如SEQ ID No:1所示。融合蛋白A的编码基因由南京金斯瑞生物科技有限公司合成。
另选抗人红细胞H抗原抗体和抗PCV2抗体通过Linker序列连接,形成分子量较大的融合蛋白B。融合蛋白B的氨基酸序列如SEQ ID No:4所示,编码基因如SEQ ID No:3所示。融合蛋白B的编码基因由南京金斯瑞生物科技有限公司合成。
2.融合蛋白原核表达载体的构建及诱导表达
将融合蛋白A的基因片段(核苷酸序列如SEQ ID No:1所示)采用Nde Ⅰ和Hind Ⅲ进行双酶切,酶切体系:Nde Ⅰ 1μl、Hind Ⅲ 1μl、10×K buffer 2μl、融合蛋白A的基因片段6μl、ddH2O 10μl。酶切结束后进行核酸电泳,将大小约为790bp的目的基因胶回收,克隆至原核表达载体pET-His中,转化DH5α感受态细胞。提取质粒,经Nde Ⅰ和Hind Ⅲ双酶切鉴定正确后,大量增殖阳性克隆菌,提取质粒后再转化入宿主菌BL21中,得到重组菌BL21-A。
采用相同方法,将融合蛋白B的基因片段插入pET-His中,然后转化入宿主菌BL21中,得到重组菌BL21-B。
采用液体LB培养基培养重组菌BL21-A,当OD值达到0.6-0.8时,添加终浓度为1mM IPTG,在37℃条件下进行诱导培养4小时。将重组菌BL21-A诱导后的培养物离心,取菌泥,采用PBS缓冲液洗涤2次。取诱导后BL21-A全菌进行SDS-PAGE电泳,并以未诱导的BL21-A全菌作为对照。电泳结果如图1所示。从图1可以看出,诱导后重组菌BL21-A中有大量大小约为30kDa的目的蛋白,说明融合蛋白A成功表达。
取200mL诱导表达后的重组菌BL21-A培养物,离心取沉淀,采用超声波裂解重组菌。将裂解液离心,沉淀用溶液Ⅰ进行重悬;重悬后室温作用20min,离心收集沉淀,采用溶液Ⅱ重悬后室温作用20min,再次离心,收集沉淀,采用溶液Ⅲ溶解,4℃条件下放置12h,12000rpm离心10min,得到50ml上清液,该上清液中含有融合蛋白A包涵体。
采用相同方法诱导培养重组菌BL21-B,SDS-PAGE电泳结果如图5所示。采用相同方法,制备融合蛋白B的包涵体。
从图1和图5的SDS-PAGE结果来看,在相同的培养条件及诱导浓度下,融合蛋白A的表达量要显著高于融合蛋白B。
3.融合蛋白的复性
采用透析复性的方法,取本实施例标题2中含有融合蛋白A包涵体的上清液依次用浓度为4M、2M和1M的盐酸胍溶液透析,逐步去除纯化过程中引入的变性剂,每隔6h半量换液,最后用TGE Buffer过夜平衡;用PEG20000浓缩复性蛋白至30ml,得到融合蛋白A。用BCA蛋白定量试剂盒测定融合蛋白A的浓度,浓度为0.1mg/ml。
采用相同方法对融合蛋白B的包涵体进行复性,得到浓度为0.05mg/ml融合蛋白B。
上述结果,证明本发明融合蛋白分子量较小、且易于原核系统表达,产量较高。
实施例2融合蛋白A的双功能特性验证
红细胞凝集实验验证复性后的融合蛋白A的双功能特性。具体步骤如下:
(1)将新鲜的O型人红细胞用0.1M、pH7.4的PBS缓冲液洗涤两次后重悬制成10%(体积百分浓度)悬液,取此悬液缓慢加入到等体积1%(体积百分浓度)的戊二醛(25%戊二醛用0.1M、pH7.4的PBS缓冲液稀释制备)溶液中,50rpm、37℃条件下作用1h,然后再用0.1M、pH7.4的PBS缓冲液洗涤3次,最后用0.1M、pH7.4的PBS缓冲液重悬配成20%悬液,加入终浓度为0.1%(质量百分浓度)的NaN3防腐,得到醛化O型人红细胞母液,4℃冰箱保存备用。将醛化O型人红细胞母液离心取沉淀,用0.1M、pH7.4的PBS缓冲液稀释至浓度为2%后,与复性后的融合蛋白A(0.1mg/ml)等体积混合,37℃作用1h,其间每隔10min轻轻摇晃,保持红细胞悬浮,最终得到初步致敏人O型血红细胞。将初步致敏人O型血红细胞在0.1M、pH7.4的PBS缓冲液中洗涤3次,每次1500rpm离心5min,最后用0.1M,pH7.4的PBS重悬,得到致敏人O型血红细胞,浓度为1%。
(2)设置实验组、对照1组、对照2组和对照3组,每组3个重复。实验组中滴加50μl PCV2抗原(PCV2-DBN-SX07,Genbank NO.:FJ660968)和50μl致敏人O型血红细胞(浓度为1%);对照1组滴加50μl PCV2抗原(PCV2-DBN-SX07,Genbank NO.:FJ660968)和50μl醛化O型人血红细胞;对照2组,滴加50μl致敏人O型血红细胞和50μl PBS缓冲液;对照3组,滴加50μl醛化O型人血红细胞和50μl PBS。室温静置30min后观察各孔状态。
结果如图2所示,实验组出现强烈凝集现象,而其它三组对照均未观察到凝集现象。经红细胞凝集试验证实,融合蛋白A既能够与人O型血红细胞结合,又能够与PCV2抗原反应,具有双功能性,且同时与人O型血红细胞和PCV2抗原作用时,会发生肉眼可见的、强烈的凝集现象。
实施例3融合蛋白A的特异性验证
分别将猪流行性腹泻病毒(PEDV CV777,Genbank NO.:AF353511)、猪乙型脑炎病毒(JEV RP9,Genbank NO.:DQ648597.1)、猪繁殖与呼吸综合征病毒(PRRSV JX-1,Genbank NO.:AF331831)、PCV2病毒(PCV2-DBN-SX07,GenbankNO.:FJ660968)各50μl滴加于血凝板中,再滴加50μl致敏人O型血红细胞进行红细胞凝集试验(同实施例2),对照中加入PBS缓冲液替代病毒。结果如图3所示,红细胞凝集试验中,仅加入PCV2病毒的孔出现强凝集现象,加入其余病毒的孔均未观察到凝集现象。结果表明:融合蛋白A特异性强,只有同时与人O型血红细胞和PCV2抗原作用时,才会发生肉眼可见的、强烈的凝集现象;融合蛋白A与其它引起猪繁殖障碍性疾病的病毒无交叉反应性。
实施例4融合蛋白A的灵敏性验证
在血凝板的每孔中将滴加0.1M、pH7.4的PBS缓冲液50μl,在第一个孔中混入PCV2病毒原液(滴度为1×107TCID50/ml)50μl,然后依次倍比稀释至第八孔,最后一孔弃去50ul液体,混匀后每孔滴加50μl致敏人O型血红细胞(浓度为1%)进行红细胞凝集试验,同时做两个平行重复。结果显示如图4所示,前6列孔均出现了肉眼可见的、强凝集现象。由于PCV2病毒原液的滴度为1×107TCID50/ml,经过计算得出,融合蛋白A能检测出的最低PCV2病毒量约为8×104TCID50/ml,可以理解为100μl反应体系中,在融合蛋白A的作用下,8000个PCV2病毒粒子就能引起肉眼可见的凝集现象,表明该重组融合蛋白具有较高的灵敏度。
以上所述仅为本发明的较佳实施方式,并不用以限制本发明,凡在本发明的精神和原则之内,所做的任何修改、等同替换、改进等,均应包含在本发明的保护范围内。
SEQUENCE LISTING
<110> 江苏省农业科学院
<120> 用于检测PCV2的融合蛋白、制备方法及应用
<130> 20150423
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 783
<212> DNA
<213> artificial
<220>
<223> 融合蛋白A的基因序列
<400> 1
ggtgaaatca accagcaaag ctctaccatt aattatagcc cgccgctgaa agataaattt 60
attatctcgc gtgacaacgc taaaagcacg ctgtacctgc agatgaataa agttcgctcg 120
gaagataccg cgctgtatta ctgcgcacgt ctgtctctga cggcagcagg tttcgcatat 180
tggggtcagg gtaccctggt gacggttgca tcgggcggtg gcggtagcgg cggtggcggt 240
tctggcggtg gcggtagtga catcgtcatg tcacagtcgc cgagttccct ggccgtctct 300
gtgggcgaaa aagtgaccat gagttgtcgc tcatcgcaaa gtctgtttaa ctcccgtacc 360
cgcaaaaatt atctgacgtg gtaccagcaa aaaccgggtc agagcccgaa accgctgatt 420
tattgggcat caacccgtga atcgggcgtt ccggatcgtt ttaccggcag cggttctggc 480
acggacttca ccctgacgat ccgccagacc ccgggtaaag aacgtgaagg cgttgcagct 540
atctaccgtg gtggcctgcg cggtggcggt cgcacctatt acgcggattc cgtgaaaggt 600
cgcttcacca tttcacgtga caacgccgaa aatacggttt atctggaaaa caatggcctg 660
atcccggaag ataccgcaat gtattactgc gcagcaagca cgggtcgtct gtgggctggc 720
tacgattggt atcgcccgga accgtataac ttctggggtc aaggcacgca ggtcaccgtt 780
tca 783
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<212> PRT
<213> artificial
<220>
<223> 融合蛋白A的氨基酸序列
<400> 2
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Ser Gly Gly Gly Gly Ser Asp Ile Val Met Ser Gln Ser Pro Ser Ser
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Leu Ala Val Ser Val Gly Glu Lys Val Thr Met Ser Cys Arg Ser Ser
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Gln Ser Leu Phe Asn Ser Arg Thr Arg Lys Asn Tyr Leu Thr Trp Tyr
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Gln Gln Lys Pro Gly Gln Ser Pro Lys Pro Leu Ile Tyr Trp Ala Ser
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Thr Asp Phe Thr Leu Thr Ile Arg Gln Thr Pro Gly Lys Glu Arg Glu
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Tyr Asp Trp Tyr Arg Pro Glu Pro Tyr Asn Phe Trp Gly Gln Gly Thr
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Gln Val Thr Val Ser
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<210> 3
<211> 1182
<212> DNA
<213> artificial
<220>
<223> 融合蛋白B的基因序列
<400> 3
gaagtgcgtc tgctggaatc gggtggtggt ccggtgcaac cgggcggctc gctgaaactg 60
tcctgtgcgg catcgggctt tgattttagt cgctattgga tgaactgggt gcgtcgcgca 120
ccgggtaaag gtctggaatg gattggtgaa atcaaccagc aaagctctac cattaattat 180
agcccgccgc tgaaagataa atttattatc tcgcgtgaca acgctaaaag cacgcttctg 240
tttaactccc gtacccgcaa aaattatctg acgtgggtac ctgcagatga ataaagttcg 300
ctcggaagat accgcgctgt attactgcgc acgtctgtct ctgacggcag caggtttcgc 360
atattggggt cagggtaccc tggtgacggt tgcatcgggc ggtggcggta gcggcggtgg 420
cggttctggc ggtggcggta gtgacatcgt catgtcacag tcgccgagtt ccctggccgt 480
ctctgtgggc gaaaaagtga ccatgagttg tcgctcatcg caaagtacca gcaaaaaccg 540
ggtcagagcc cgaaaccgct gatttattgg gcatcaaccc gtgaatcggg cgttccggat 600
cgttttaccg gcagcggttc tggcacggac ttcaccctga cgatcagctc tgtccaggca 660
gaagatctgg ctgactatta ctgcaaacaa tcctacaacc tgcgcacctt cggcggtggc 720
acgaaactgg aaattaaacg tggtggcggt ggctccggtg gcggtggctc aggtggcggt 780
ggcggatccc aggttcaact ggtcgaaagc ggcggtggct ctgtgcaagc aggtggcagt 840
ctgcgcctgt cctgtaccgc ttcaggttat acgtacagtt ccaattatct gggctggttt 900
cgccagaccc cgggtaaaga acgtgaaggc gttgcagcta tctaccgtgg tggcctgcgc 960
ggtggcggtc gcacctatta cgcggattcc gtgaaaggtc gcttcaccat ttcacgtgac 1020
aacgccgaaa atacggttta tctggaaaac aatggcctga tcccggaaga taccgcaatg 1080
tattactgcg cagcaagcac gggtcgtctg tgggctggct acgattggta tcgcccggaa 1140
ccgtataact tctggggtca aggcacgcag gtcaccgttt ca 1182
<210> 4
<211> 388
<212> PRT
<213> artificial
<220>
<223> 融合蛋白B的氨基酸序列
<400> 4
Val Gln Leu Gln Glu Ser Gly Gly Gly Asn Thr Asp Tyr Lys Ser Ala
1 5 10 15
Leu Lys Ser Arg Leu Thr Ile Ser Lys Asp Asn Ser Arg Ser Gln Val
20 25 30
Leu Leu Lys Met Asn Ser Leu Gln Ile Asp Asp Thr Ala Ile Tyr Tyr
35 40 45
Cys Ala Arg Asn Tyr Gly Tyr Ser Pro Phe Val His Trp Gly Gln Gly
50 55 60
Thr Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Ile Thr Cys Thr
65 70 75 80
Val Ser Gly Phe Ser Leu Ser Gly Tyr Ser Val His Trp Val Arg Gln
85 90 95
Pro Pro Gly Lys Gly Leu Glu Trp Leu Gly Met Ile Trp Gly Thr Val
100 105 110
Thr Val Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Thr Leu Thr Ile
130 135 140
Gly Ser Val Gln Ser Glu Asp Leu Ala Tyr Tyr Phe Cys Gln Gln Leu
145 150 155 160
Tyr Arg Thr Pro Phe Thr Phe Gly Ser Gly Thr Ser Leu Ala Met Ser
165 170 175
Val Gly Gln Lys Val Thr Met Ser Cys Lys Ser Arg Gln Ser Leu Leu
180 185 190
Asn Ser Asp Asn Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro
195 200 205
Gly Gln Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Ser Arg Glu Ser
210 215 220
Gly Val Ser Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Lys Leu
225 230 235 240
Glu Ile Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
245 250 255
Ser Gln Val Gln Leu Val Glu Ser Arg Gly Gly Leu Arg Gly Gly Gly
260 265 270
Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
275 280 285
Asp Asn Ala Glu Asn Thr Val Tyr Leu Glu Asn Asn Gly Leu Ile Pro
290 295 300
Glu Asp Thr Ala Met Tyr Tyr Cys Ala Ala Ser Thr Gly Arg Leu Trp
305 310 315 320
Ala Gly Tyr Gly Gly Gly Ser Val Gln Ala Gly Gly Ser Leu Arg Leu
325 330 335
Ser Cys Thr Ala Ser Gly Tyr Thr Tyr Ser Ser Asn Tyr Leu Gly Trp
340 345 350
Phe Arg Gln Thr Pro Gly Lys Glu Arg Glu Gly Val Ala Ala Ile Tyr
355 360 365
Asp Trp Tyr Arg Pro Glu Pro Tyr Asn Phe Trp Gly Gln Gly Thr Gln
370 375 380
Val Thr Val Ser
385
Claims (8)
1.一种用于检测PCV2的融合蛋白,是将抗人红细胞H抗原纳米抗体和抗PCV2纳米抗体通过Linker序列连接而成。
2.根据权利要求1所述用于检测PCV2的融合蛋白,其特征在于所述融合蛋白的氨基酸序列如SEQ ID No :2所示。
3.权利要求1或2所述融合蛋白的编码基因。
4.根据权利要求3所述融合蛋白的编码基因,其特征在于所述编码基因的核苷酸序列如SEQ ID No:1所示。
5.含有权利要求3或4所述基因的重组载体;优选的技术方案中,所述重组载体是将权利要求3或4所述基因插入表达载体所得;所述表达载体优选为pET-His。
6.含有权利要求3或4所述基因的重组菌,其特征在于:是将含有权利要求3或4所述基因的重组载体导入宿主菌中得到的重组菌;所述重组载体是将所述基因插入表达载体pET-His所得。
7.一种制备权利要求1或2所述融合蛋白的方法,其特征在于诱导权利要求6所述重组菌表达融合蛋白,收集所述融合蛋白的包涵体,复性所述融合蛋白。
8.权利要求1或2所述融合蛋白在制备检测PCV2试剂盒方面的应用。
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CN109503711A (zh) * | 2018-11-30 | 2019-03-22 | 江苏省农业科学院 | 一种用于血凝方法检测pcv2病毒的双功能纳米抗体、编码基因及其应用 |
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CN109503711B (zh) * | 2018-11-30 | 2021-09-10 | 江苏省农业科学院 | 一种用于血凝方法检测pcv2病毒的双功能纳米抗体、编码基因及其应用 |
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