CN111171146B - 抗h9n2亚型禽流感病毒的纳米抗体、制备方法和应用 - Google Patents
抗h9n2亚型禽流感病毒的纳米抗体、制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种抗H9N2亚型禽流感病毒的纳米抗体、制备方法和应用，该纳米抗体氨基酸序列如SEQ ID NO:1所示。本发明通过原核表达技术表达了H9N2亚型禽流感病毒的核衣壳蛋白，然后将其作为免疫原免疫双峰驼，构建噬菌体库，再利用噬菌体展示技术筛选获得了1株抗H9N2核衣壳蛋白的纳米抗体，并将该纳米抗体与辣根过氧化物酶(HRP)进行融合表达，成功制备了纳米抗体与HRP的融合蛋白，将该融合蛋白应用到检测鸡血清中抗H9N2亚型禽流感病毒(AIV)抗体中，发现该融合蛋白构建的检测方法灵敏度高，并且具有操作简单、不需要使用二抗、检测样品耗时短等优点。
Description
技术领域
本发明涉及生物技术领域，特别是涉及一株抗H9N2亚型禽流病毒核衣壳蛋白的纳米抗体，该纳米抗体的制备方法，以及该纳米抗体与辣根过氧化物酶融合表达后在检测鸡血清中抗H9N2禽流感病毒抗体中的应用。
背景技术
禽流感(Avian influenza,AI)是由流感病毒引起的禽类全身性或呼吸系统性疾病，该病在全世界范围内广泛发生。在我国，近年来随着高致病性AI疫苗的强制免疫，低致病性的H9N2亚型禽流感病毒(AI virus,AIV)已成为危害我国养禽业健康发展的重要疫病之一。该病毒感染可引起蛋鸡产蛋下降、肉鸡生长发育迟缓、鸡只的免疫抑制，从而给养禽业造成严重的经济损失。对我国部分省市发病鸡只的AIV感染的分子流行病学监测发现,H9亚型AIV的阳性率最高，说明该病毒感染在我国鸡群中广泛存在。
AIV属于正黏病毒科流感病毒属单股负链RNA病毒，依据其核衣壳蛋白(NP)与基质蛋白(M1)的抗原性不同，可将其分为A、B、C三型。A型流感病毒的基因组由8个单股负链RNA片段组成，共编码10种蛋白(PA,PB1,PB2,HA,NA,NP,M1,M2,NS1,NS2)，其中核衣壳(NP)蛋白在不同型的AIV中较为保守，免疫原性较强，是流感病毒的血清学监测中主要使用的靶蛋白。目前，依托NP蛋白的抗原性，建立了多种AIV的血清学检测方法。
纳米抗体(Nanobody,Nb)是源自骆驼科重链抗体(sdAb)可变域的单域抗体(VHH)。由于纳米抗体的特殊结构，其往往更倾向识别抗原的构象表位。因此，纳米抗体具有较高的特异性。此外，纳米抗体分子量小，易于基因工程改造，耐极端环境，所以，纳米抗体被广泛地应用于免疫学检测及科学研究等领域。
目前，用于检测家禽H9N2 AIV感染的诊断方法主要包括血凝和血凝抑制试验、病毒分离鉴定、酶联免疫吸附试验、免疫荧光、乳胶凝集试验以及核酸探针检测技术等，然而每种方法都存在各自的缺陷。比如：用于检测家禽血清中抗H9N2 AIV抗体的金标方法为血凝抑制(HI)实验。然而，该方法操作繁琐，需要主观判断，易产生假阳性，特别是对血清中的抗体效价进行定量时，容易产生误差；血清学ELISA检测鸡血清中抗H9N2 AIV抗体的方法中包括间接和阻断ELISA，但已有商品化ELISA试剂盒均是依托传统抗体进行的开发和生产，该类ELISA试剂盒，需要标记抗体和使用酶标二抗，导致生产成本升高，阻碍了其大范围的推广使用。基于纳米抗体具有分子量小和易于基因工程改造的优点，因此，可以将纳米抗体与酶等报告基因进行融合表达，从而直接获得纳米抗体与酶的融合蛋白，避免了抗体的标记和使用二抗，从而简化了生产工艺，降低了生产成本，具有非常广阔的市场应用前景，并且对于解决目前存在的技术问题具有重要意义。
发明内容
本发明的目的是提供一种抗H9N2亚型禽流感病毒的纳米抗体、制备方法和应用，以解决上述现有技术存在的问题，该纳米抗体和HRP融合表达的蛋白可用于检测鸡血清中抗H9N2亚型禽流感病毒抗体，并且该检测方法具备操作简单，不需要二抗，检测样品耗时短，为后续开发该纳米抗体应用于H9N2亚型禽流感病毒抗体检测商品化试剂盒提供关键材料。
为实现上述目的，本发明提供了如下方案：
本发明提供一种抗H9N2亚型禽流感病毒的纳米抗体，其氨基酸序列如SEQ ID NO:1所示。
本发明还提供一种所述的抗H9N2亚型禽流感病毒的纳米抗体的制备方法，包括以下步骤：
步骤1：将编码H9N2亚型禽流感病毒的核衣壳蛋白(NP)的核苷酸序列连接到原核表达载体pET-28a，构建重组原核表达质粒pET-28a-NP；
步骤2：将pET-28a-NP转入感受态细胞，经诱导表达、纯化后，获取诱导表达的重组目的蛋白H9N2-NP；
步骤3：将所得H9N2-NP与等体积的佐剂乳化后，免疫双峰驼5次，采集外周血分离淋巴细胞，构建抗H9N2-NP蛋白的噬菌体展示文库；
步骤4：利用噬菌体展示技术，经过3轮筛淘，筛选获得纳米抗体H9N2-NP-Nb5。
优选的是，编码H9N2亚型禽流感病毒的核衣壳蛋白NP的核苷酸序列如SEQ ID NO：2所示；氨基酸序列如SEQ ID NO：3所示。
本发明还提供一种抗H9N2亚型禽流感病毒的融合蛋白，包括所述的纳米抗体。
优选的是，由所述纳米抗体和辣根过氧化物酶通过基因工程融合而成。
本发明还提供一种所述的抗H9N2亚型禽流感病毒的融合蛋白的构建方法，包括如下步骤：
步骤1：将编码纳米抗体的核苷酸序列连接至改造的真核表达载体pEGFP-N1-HRP中，转染大肠杆菌，获取H9N2-NP-Nb5-HRP阳性质粒；
步骤2：将H9N2-NP-Nb5-HRP阳性质粒与转染试剂混合均匀后，转染293T细胞，培养，收集上清液获取H9N2-NP-Nb5-HRP融合蛋白。
优选的是，步骤2中培养条件为：37℃培养48h。
本发明还提供一种所述的抗H9N2亚型禽流感病毒的融合蛋白在检测鸡血清中抗H9N2亚型禽流感病毒抗体的应用。
本发明公开了以下技术效果：
本发明首先通过原核表达技术，原核表达H9N2亚型AIV的NP蛋白，然后将其作为免疫原免疫双峰驼，构建噬菌体库，利用噬菌体展示技术筛选获得了1株抗H9N2 NP蛋白的纳米抗体，随后将该纳米抗体与辣根过氧化物酶(HRP)进行融合表达，成功制备了纳米抗体与HRP的融合蛋白，并将该融合蛋白应用到检测鸡血清中抗H9N2 AIV抗体中，发现利用该融合蛋白建立的检测方法灵敏度高，并且操作简单，不需要使用二抗，检测样品耗时短，可为后续该纳米抗体应用到H9N2 AIV抗体检测的商品化试剂盒的开发提供关键材料。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为PCR扩增编码H9N2-NP蛋白基因结果；1，2，3，4：扩增的目的片段；
图2为H9N2-NP蛋白的基因和pET-28a空载体双酶切结果；1：H9N2 NP蛋白的基因双酶切结果；2：pET-28a空载体双酶切结果；
图3为菌液PCR鉴定pET28a-H9N2-NP阳性单克隆；1，4，5为阳性克隆，2，3为阴性克隆；
图4为SDS-PAGE和Western blot分析原核表达的重组蛋白；a、b中1：pET28a空载对照；2：重组蛋白的菌体裂解液；3：包涵体蛋白；4：可溶性蛋白；5：镍柱亲和层析纯化后的蛋白；
图5为骆驼血清中抗H9N2-NP蛋白特异性抗体的ELISA效价；
图6为通过Ficoll淋巴细胞分离液分离获免疫骆驼外周血的淋巴细胞；
图7为琼脂糖凝胶电泳检测分析巢式PCR第一轮PCR扩增产物；
图8为琼脂糖凝胶电泳检测分析巢式PCR第二轮PCR扩增产物；
图9为琼脂糖凝胶电泳分析菌液PCR扩增构建的噬菌体文库插入的VHH基因；
图10为间接ELISA方法检测重组纳米抗体粗提物与H9N2-NP蛋白的反应性；
图11为琼脂糖凝胶电泳分析包含纳米抗体基因序列的重组质粒pMECS-H9N2-NP-Nbs和重组载体pCMV-N1-HRP双酶切产物结果；
图12为将构建的纳米抗体重组表达质粒转染293T细胞后，IFA分析H9N2-NP-Nb5-HRP融合蛋白在293T细胞中的表达情况；
图13为利用ELISA分析H9N2-NP-Nb5-HRP融合蛋白分泌表达情况；
图14为利用H9N2-NP-Nb5-HRP融合蛋白建立的竞争ELISA法检测H9N2攻毒SPF鸡不同日龄血清中抗H9N2 AIV的抗体的消长规律。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
应理解本发明中所述的术语仅仅是为描述特别的实施方式，并非用于限制本发明。另外，对于本发明中的数值范围，应理解为还具体公开了该范围的上限和下限之间的每个中间值。在任何陈述值或陈述范围内的中间值以及任何其他陈述值或在所述范围内的中间值之间的每个较小的范围也包括在本发明内。这些较小范围的上限和下限可独立地包括或排除在范围内。
除非另有说明，否则本文使用的所有技术和科学术语具有本发明所述领域的常规技术人员通常理解的相同含义。虽然本发明仅描述了优选的方法和材料，但是在本发明的实施或测试中也可以使用与本文所述相似或等同的任何方法和材料。本说明书中提到的所有文献通过引用并入，用以公开和描述与所述文献相关的方法和/或材料。在与任何并入的文献冲突时，以本说明书的内容为准。
在不背离本发明的范围或精神的情况下，可对本发明说明书的具体实施方式做多种改进和变化，这对本领域技术人员而言是显而易见的。由本发明的说明书得到的其他实施方式对技术人员而言是显而易见的。本申请说明书和实施例仅是示例性的。
关于本文中所使用的“包含”、“包括”、“具有”、“含有”等等，均为开放性的用语，即意指包含但不限于。
实施例1
H9N2亚型禽流感病毒NP蛋白原核表达载体的构建及其表达、纯化
1、H9N2亚型禽流感病毒NP蛋白原核表达载体的构建
取含H9N2 AIV的尿囊液1mL，按照
Viral DNA/RNA Kit(TransGen，ER201-01)说明书提取病毒RNA，以RNA为模板，反转录获得cDNA，反转录体系如表1所示。
表1
反应条件：25℃反应10min；50℃反转录30min；85℃反应10s终止反应。
依据编码H9N2 NP蛋白的基因序列(如SEQ ID NO：2)，设计特异性引物：H9N2-NP-F1：ATGGCGTCTCAAGGCACCAA；
H9N2-NP-R1：TCAATTGTCATATTCCTCTG。
以cDNA为模板，利用聚合酶
GXL DNA Polymerase(R050A,TaKaRa)，PCR扩增NP的基因序列。反应体系如表2所示：
表2
反应条件：98℃预变性2min；98℃变性2min，55℃退火15s，68℃延伸2min，共30个循环；循环完成后68℃继续延伸7min。
反应完成后，加入20μL聚合酶Premix TaqTM(RR901A,TaKaRa)，72℃反应10min加入poly A尾巴，将PCR产物经琼脂糖凝胶电泳，获得预期大小1600bp的片段(如图1所示)。利用凝胶回收试剂盒回收PCR产物，并连接至pMD19T载体(Code NO.6013，TaKaRa)。连接体系如表3所示。
表3
反应条件：16℃连接过夜，将连接产物转化入感受态细胞Trans(5α)中，次日挑取单克隆菌落，菌液PCR鉴定阳性单克隆送至西安擎科公司进行测序。
同时，设计带有酶切位点的扩增NP蛋白的特异性引物：
H9N2-NP-F2：TCCGAATTCATGGCGTCTCAAGGCACCAA；
H9N2-NP-R2：GTGCTCGAGATTGTCATACTCCTCTGCAT，其中下划线为EcoR I和Xho I酶切位点。以pMD19T-H9N2-NP重组质粒为模板，利用PCR扩增获得目的基因。PCR反应体系为如表4所示。
表4
反应条件：98℃预变性2min；98℃变性2min，55℃退火15s，68℃延伸2min，共30个循环；68℃延伸7min。
利用EasyPure Quick Gel Extraction Kit(全式金生物技术有限公司)凝胶回收试剂盒回收PCR扩增产物，然后将商品化载体pET-28a与回收产物同时用QuickCutTM EcoR I(1611,TaKaRa)和QuickCutTM Xho I(1635,TaKaRa)进行双酶切，反应体系如表5和表6所示。
经1％琼脂糖凝胶电泳，结果显示，获得了大小分别约为1500bp和5000bp的酶切片段(如图2所示)。T4连接酶连接(16℃过夜)后，将连接产物转化至Trans(5α)感受态细胞中，37℃培养12h。次日，挑取单个菌落，接种于5mL的LB液体培养基(胰蛋白胨10g，酵母提取物5g，NaCl 10g，去离子水补充至1000mL)，振荡培养12h后，经菌液PCR鉴定后，结果显示3个为阳性(如图3所示)，将阳性菌液送测序，阳性质粒命名为pET-28a-H9N2-NP。
2、H9N2亚型禽流感病毒NP蛋白的原核表达
将重组阳性质粒pET-28a-H9N2-NP转化至Transetta(DE3)感受态细胞中，37℃过夜培养；次日，挑取单克隆菌落接种于LB液体培养基中，37℃过夜培养；然后将过夜培养菌液接种于新鲜的含卡那霉素的LB液体培养基中，待菌液OD600值达到0.6-0.8后，加入终浓度为0.5mM的IPTG(异丙基-β-D-硫代半乳糖苷)，25℃诱导表达12h。SDS-PAGE分析重组蛋白H9N2-NP的表达，结果显示：成功获得了预期大小的H9N2-NP蛋白(如图4a所示)。
3、H9N2亚型禽流感病毒的NP重组蛋白(H9N2-NP)的纯化
将表达的重组H9N2-NP蛋白采用琼脂糖镍柱进行亲和层析纯化。将菌体超声裂解(20kHz频率，150W功率，工作时间3s，停3s，超声时间40min)和离心后，收集上清。取适量的Ni Resin装入柱子，然后将H9N2-NP蛋白加入柱中，4℃孵育过夜。用低密度咪唑(20mM)洗脱杂蛋白，然后用高密度咪唑(250mM)洗脱目的蛋白。
SDS-PAGE结果显示：H9N2-NP蛋白成功被纯化(如图4b所示)。
4、H9N2亚型禽流感病毒NP蛋白抗原性分析
将表达的H9N2-NP蛋白经SDS-PAGE后，采用湿转法将蛋白质电转至硝酸纤维素(PDVF)膜，利用封闭液[5％脱脂奶粉的洗涤液(含0.5％吐温-20的PBS缓冲液)]37℃封闭1h后，加入1:200稀释的阳性鸡血清，37℃孵育1h后，加入HRP标记的兔抗鸡二抗(1:5000稀释)，37℃孵育1h。最后，加入ECL发光显色液。
结果显示：原核表达的H9N2-NP蛋白，可以很好的与阳性鸡血清发生免疫反应(如图4b所示)。
实施例2
抗H9N2亚型禽流感病毒NP蛋白纳米抗体的筛选与制备
1、蛋白乳化
将1mL纯化的H9N2-NP蛋白与相同体积的弗氏佐剂乳化后，颈部皮下免疫阿拉善双峰驼，第1次用完全弗氏佐剂乳化，后4次用不完全弗氏佐剂乳化。
2、骆驼免疫
将乳化的免疫原，通过颈部皮下，免疫成年雄性阿拉善双峰驼。此后，每隔两周用弗氏不完全佐剂乳化的免疫原，采用相同方法加强免疫5次，最后一次免疫后4天采血；间接ELISA(以纯化的H9N2-NP重组蛋白为包被抗原，400ng/孔)检测5免后骆驼血清中的抗体效价。结果发现：血清中抗H9N2-NP重组蛋白的效价为1:128000(如图5所示)。
3、VHH噬菌体抗体文库的构建及淘选
3.1外周血淋巴细胞的分离
采集200mL免疫后骆驼的抗凝血，首先利用等体积的RPMI 1640(01-100-1ACS，BI)培养液进行稀释，然后利用Ficoll-Paque PLUS淋巴细胞分离液(Greiner bio-one公司)分离采集的外周血的淋巴细胞。离心后，血浆与白色透明淋巴细胞分离液之间的一层环状乳白色物质，即为淋巴细胞(如图6所示)。用血细胞计数板对分离的淋巴细胞进行计数，1×107个细胞/支，离心的细胞沉淀直接用于RNA提取。
3.2VHH基因片段的扩增
利用
Plus Mini RNA提取试剂盒(QIAGEN公司)抽提淋巴细胞总RNA。利用
III反转录酶，以提取的总RNA为模板，合成第一链cDNA，然后利用巢式PCR扩增VHH基因。反转录体系，首先配制RNA/primer混合物，其体系如表7所示。
表7
然后，将RNA/primer在65℃孵育5min后，立刻置于冰水浴中1min。随后，配制cDNA合成混合物，体系如表8所示。
表8
将上述cDNA合成混合物(10μL)加入RNA/primer混合物中，混匀，50℃孵育50min，85℃5min终止反应。
以反转录的cDNA为模板，巢式PCR扩增VHH基因，所用的引物序列如表9所示。
表9
首先，利用引物CALL001和CALL002进行第一轮PCR扩增，其反应体系如表10所示。
表10
反应条件：94℃预变性3min；94℃30s，55℃30s，72℃1min，28个循环；72℃延伸5min。
PCR产物用1.2％琼脂糖凝胶电泳鉴定，结果如预期在700bp和900bp位置各有一条条带(如图7所示)，利用胶回收试剂盒EasyPure Quick Gel Extraction Kit回收700bp的PCR产物。随后，以回收的产物为模板，利用VHH-FOR和VHH-REV引物进行第二轮PCR的扩增，其反应体系如表11所示。
表11
反应条件：94℃预变性3min；94℃30s，55℃30s，72℃1min，18个循环；72℃延伸5min。
PCR产物经1.5％琼脂糖凝胶电泳，结果显示扩增获得了目的条带(400bp左右)(如图8所示)。同样，采用胶回收试剂盒EasyPure Quick Gel Extraction Kit回收PCR产物。
3.3VHH噬菌体展示载体的构建
将上述回收的产物通过Pst I和Not I双酶切后，连入商品化pMECS噬菌体展示载体中，具体酶切体系如表12所示。
表12
酶切条件：37℃，16h。用EasyPure Quick Gel Extraction Kit回收酶切产物，然后，用T4 DNA连接酶将回收的产物连入噬菌体展示载体pMECS中，连接体系如表13所示。
表13
利用上述连接体系，于16℃连接16h。
3.4制备大肠杆菌TG1感受态细胞
将大肠杆菌TG1培养至OD600nm为0.4-0.6后，将其迅速冷却，离心后，用预冷的10％甘油反复洗三次，最后用10％甘油重悬TG1感受态细胞。
3.5连接产物转化TG1感受态细胞及噬菌体抗体文库的收获
将连接产物加入到感受态细胞中，轻轻混匀加入到电转杯中，用Eppendorf电穿孔仪，设置参数为1.8kV，25μF，200Ω，1mm，电转化感受态细胞。电转化完成后立即加入SOC培养基重悬细胞。37℃120r/min，震荡培养1h后，涂布于LB/AMP-GLU平板，37℃培养6-8h。用细胞刮刀收集菌苔，加入1/3体积的50％甘油，即为制备的噬菌体文库。
3.6噬菌体文库多样性和库容的测定
将电转后的重组菌进行10倍稀释，稀释至10-5cfu/mL，然后涂布于LB/AMP-GLU平板，37℃培养12h，计算转化子数量，最终得到库容为9.8×108CFU(colony-forming units)的噬菌体文库。随机挑取46个单克隆，利用VHH-FOR和VHH-REV引物进行PCR鉴定，琼脂糖凝胶电泳鉴定阳性克隆，目的大小400bp左右(如图9所示)。
4、抗H9N2-NP蛋白的特异性纳米抗体的筛选
4.1噬菌体文库的救援
将获得的噬菌体文库接种于2×YT/AMPGLU培养基中，37℃200r/min，培养至对数期后加入M13KO7辅助噬菌体，37℃静置30min；2800g离心10min，菌体用2×YT/AMP-KAN培养基重悬，37℃200r/min培养12h。3800g 4℃离心后，收集上清，并加入1/5体积预冷的PEG/NaCl溶液；3800g 4℃离心，加入PBS重悬噬菌体沉淀，使其充分溶解。
同时将噬菌体溶液10倍梯度稀释，然后取稀释度为10-2、10-4、10-6、10-8、10-10的样品，加入对数生长期的TG1细胞，37℃静置浸染15min。然后，涂布于LB/AMP-GLU平板，37℃培养8h，计算重组噬菌体滴度为1×1013噬菌体空斑形成单位(plaque forming unit,pfu)。
4.2抗H9N2-NP蛋白特异性重组噬菌体的淘选
将纯化的H9N2-NP蛋白包被ELISA板，PBS为无抗原对照。取上述制备的噬菌体溶液，加入ELISA板中，室温孵育2h，弃去噬菌体样品。然后，每孔中加入新鲜配制的0.1M三乙胺，室温静置10min，将洗脱液迅速用等体积1M Tris-HCl(pH 7.4)中和。
取洗脱液浸染对数期的TG1细胞，37℃静置30min，然后加入2×YT/AMP-GLU培养基，37℃200r/min培养至OD600达到0.6-0.8。重复上述4.1的操作，进行噬菌体文库的救援；得到救援的噬菌体文库后，利用上述相同的方法进行第二轮和第三轮的筛淘，每轮通过测定噬菌体滴度，结果显示：噬菌体文库得到了富集(如表14所示)。
表14筛选过程中H9N2-NP蛋白特异性噬菌体的富集情况
5、重组纳米抗体的诱导表达和粗提物的获取
第三轮洗脱后，从测得的噬菌体滴度的平板上随机挑选96个单菌落，接种于96孔板中，每孔加入LB/AMP-GLU培养基，37℃ 200r/min培养8h。每个克隆吸取培养液，转接到24孔培养板中，然后分别加入1mL TB培养基，37℃ 200r/min培养至对数期，每孔加入10mM的IPTG诱导表达。表达后的细菌离心沉淀后，反复冻融3次，3500g 4℃离心15min，收集上清，即为可溶性重组纳米抗体粗提物。
6、ELISA检测重组纳米抗体粗提物与H9N2-NP蛋白的反应
将纯化的H9N2-NP重组蛋白包被ELISA板，同时做PBS对照。取可溶性重组纳米抗体粗提物，用封闭液1:1稀释后加入酶标板中。用PBS’T洗酶标板后，加入用封闭液1:2000稀释的鼠抗HA-tag抗体，室温孵育2h；然后加入HRP标记的羊抗鼠二抗，室温孵育2h，加入TMB显色底物，室温避光显色30min。3M浓硫酸终止反应，自动酶标仪读数OD450nm的值。
结果显示：94个粗提物可以与H9N2-NP蛋白特异性结合(如图10所示)。将阳性得菌株进行测序，结果显示成功筛选获得了1株抗H9N2 NP蛋白的纳米抗体，命名为H9N2-NP-Nb5。
实施例3
一株抗H9N2-NP蛋白纳米抗体与HRP融合蛋白的表达和制备方法
1、H9N2-NP-Nb5-HRP重组真核表达载体的构建
基于改造的纳米抗体-HRP融合蛋白的真核表达载体pEGFP-N1-HRP(Sheng，Y.，etal.，Nanobody-horseradish peroxidase fusion protein as an ultrasensitive probeto detect antibodies against Newcastle disease virus in the immunoassay。JNanobiotechnology,2019.17(1)：p.35)，通过用Pst I和Not I双酶切，将获得的编码纳米抗体的VHH基因，连接至pCMV-N1-HRP载体中，菌液PCR和测序鉴定获得阳性质粒(如图11所示)。
2、H9N2-NP-Nb5-HRP融合蛋白的表达和制备
将构建成功的H9N2-NP-Nb5-HRP阳性质粒与
Medium和Lipo8000TM转染试剂混匀后，加入293T细胞，37℃培养72h，收集上清即为获得的H9N2-NP-Nb5-HRP纳米抗体。
转染48h后，选取部分转染的293T细胞，利用鼠抗His(1:2000)单抗，免疫荧光检测重组融合蛋白是否在293T细胞中表达。结果显示：H9N2-NP-Nb5-HRP融合蛋白正确表达，细胞发出绿色荧光(如图12所示)。
此外，将收集的细胞上清，利用间接ELISA检测表达的重组H9N2-NP-Nb5-HRP融合蛋白是否分泌到293T细胞上清中，结果显示：表达的纳米抗体与HRP融合蛋白分泌到细胞上清中(如图13所示)。
实施例4
H9N2-NP-Nb5-HRP融合蛋白在检测鸡血清中抗H9N2 AIV抗体中的应用
1、利用H9N2-NP-Nb5-HRP融合蛋白检测鸡血清中的抗H9N2 AIV抗体
将纯化的H9N2-NP重组蛋白包被ELISA板，封闭过夜后，将H9N2-NP-Nb5-HRP融合蛋白利用封闭液按照1:320稀释，该稀释液为临床鸡血清的稀释液。然后，将已知的抗H9N2AIV抗体的阳性和阴性鸡血清，分别用上述配制的血清稀释液按1:10稀释后加入酶标板中，37℃孵育20min，同时，设置未加血清的孔作为空白对照。随后，加入TMB显色底物，室温避光显色10min，显色完成后加入3M硫酸终止反应，观察ELISA板的颜色变化。结果显示：阳性血清无任何颜色变化，阴性血清为黄色。
2、利用H9N2-NP-Nb5-HRP融合蛋白检测鸡血清中的抗H9N2 AIV抗体与HI实验及商用ELISA试剂盒检测结果比较
选取H9N2 AIV攻毒SPF鸡后0，5,7,10,14,21,28天的血清，同时用上述方法，HI实验和商品化ELISA试剂盒进行检测，比较分析3种方法的检测结果。
结果发现：利用H9N2-NP-Nb5-HRP融合蛋白检测方法，在攻毒后第7天的所有血清中有12份为抗H9N2抗体阳性(如图14所示)，但HI测试只有4个样品呈阳性，而商用ELISA试剂盒则有2个样品呈阳性(如表15所示)。
表15不同检测方法比较结果
以上结果表明，与HI试验和商用ELISA试剂盒相比，利用H9N2-NP-Nb5-HRP融合蛋白建立的检测方法其灵敏度更高，说明制备的H9N2-NP-Nb5-HRP融合蛋白可以很好的应用于鸡血清中抗H9N2 AIV抗体的检测。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。
序列表
<110> 西北农林科技大学
<120> 抗H9N2亚型禽流感病毒的纳米抗体、制备方法和应用
<160> 3
<170> SIPOSequenceListing 1.0
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Gly Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
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Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Leu Asn Ser
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tggcgtctca aggcaccaaa cgatcctatg aacagatgga aactggtggg gagcgccaga 60
atgctactga gataagggca tctgttggaa gaatggttag cggcattggg agattctaca 120
tacagatgtg tacagaactc aaactcagtg acaatgaagg gaggctgatt cagaacagta 180
taacaataga gagaatggta ctctctgcat ttgatgaaag aaggaacaga tacctggaag 240
agcaccccag tgcaggaaag gaccctaaga aaactggagg tccaatttac aggagaagag 300
acggaaaatg ggtgagagag ttgatcctgt atgacaaaga ggaaatcagg agaatttggc 360
gacaagcgaa caatggagag gatgcaactg ctggtcttac ccatctgatg atatggcatt 420
ccaacctgaa tgatgctacc tatcagagga cgagagccct cgtgcgtact ggaatggatc 480
cccggatgtg ctctctgatg caaggatcaa ctctcccgag gagatcagga gctgcaggtg 540
cagcagtgaa ggggataggg acaatggtga tggaactgat tcggatgata aaacgaggga 600
tcaatgacag gaatttctgg agaggcgaaa atggaagaag gacaagaatt gcatatgaga 660
gaatgtgcaa catcctcaaa gggaaattcc aaacagcagc acaaagggca atggtggatc 720
aagtgcgaga gagcagaaat cctgggaatg ctgaaataga agatctcatt tttctggcaa 780
ggtctgcact catcctgaga ggatcagtgg ctcataaatc ctgcttgcct gcttgtgtgt 840
acggacttgc agtggctagt ggatatgact ttgagagaga agggtactcc ttggttggaa 900
tagatccttt ccgtctgctt caaaacagcc aggtctttag tctcattaga ccaaatgaga 960
acccagcaca taagagccaa ctagtgtgga tggcatgcca ctctgcagcg tttgaggacc 1020
ttagggtctc aagtttcatt agagggacaa gaatggtccc aagaggacag ctatccacta 1080
gaggggttca aattgcttca aatgagaaca tggaagcaat ggactccaat actcttgaac 1140
tgagaagtag atattgggct ataagaacca gaagcggagg gagcaccaac caacagagag 1200
catctgcagg acagatcagc gttcaaccca ctttctcagt acagagaaac cttcctttcg 1260
aaagatcaac cattatggca gcatttacag gaaatactga gggtagaacg tctgacatga 1320
ggactgaaat cataagaatg atggaaagtg ccagaccaga agatgtgtca ttccagggac 1380
ggggagtctt cgagctctcg gacgaaaagg caacgaaccc gatcgtgcct tcctttgaca 1440
tgaataatga aggatcttat ttcttcggag acaatgcaga ggagtatgac aattaa 1496
<210> 3
<211> 498
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Ala Ser Gln Gly Thr Lys Arg Ser Tyr Glu Gln Met Glu Thr Gly
1 5 10 15
Gly Glu Arg Gln Asn Ala Thr Glu Ile Arg Ala Ser Val Gly Arg Met
20 25 30
Val Ser Gly Ile Gly Arg Phe Tyr Ile Gln Met Cys Thr Glu Leu Lys
35 40 45
Leu Ser Asp Asn Glu Gly Arg Leu Ile Gln Asn Ser Ile Thr Ile Glu
50 55 60
Arg Met Val Leu Ser Ala Phe Asp Glu Arg Arg Asn Arg Tyr Leu Glu
65 70 75 80
Glu His Pro Ser Ala Gly Lys Asp Pro Lys Lys Thr Gly Gly Pro Ile
85 90 95
Tyr Arg Arg Arg Asp Gly Lys Trp Val Arg Glu Leu Ile Leu Tyr Asp
100 105 110
Lys Glu Glu Ile Arg Arg Ile Trp Arg Gln Ala Asn Asn Gly Glu Asp
115 120 125
Ala Thr Ala Gly Leu Thr His Leu Met Ile Trp His Ser Asn Leu Asn
130 135 140
Asp Ala Thr Tyr Gln Arg Thr Arg Ala Leu Val Arg Thr Gly Met Asp
145 150 155 160
Pro Arg Met Cys Ser Leu Met Gln Gly Ser Thr Leu Pro Arg Arg Ser
165 170 175
Gly Ala Ala Gly Ala Ala Val Lys Gly Ile Gly Thr Met Val Met Glu
180 185 190
Leu Ile Arg Met Ile Lys Arg Gly Ile Asn Asp Arg Asn Phe Trp Arg
195 200 205
Gly Glu Asn Gly Arg Arg Thr Arg Ile Ala Tyr Glu Arg Met Cys Asn
210 215 220
Ile Leu Lys Gly Lys Phe Gln Thr Ala Ala Gln Arg Ala Met Val Asp
225 230 235 240
Gln Val Arg Glu Ser Arg Asn Pro Gly Asn Ala Glu Ile Glu Asp Leu
245 250 255
Ile Phe Leu Ala Arg Ser Ala Leu Ile Leu Arg Gly Ser Val Ala His
260 265 270
Lys Ser Cys Leu Pro Ala Cys Val Tyr Gly Leu Ala Val Ala Ser Gly
275 280 285
Tyr Asp Phe Glu Arg Glu Gly Tyr Ser Leu Val Gly Ile Asp Pro Phe
290 295 300
Arg Leu Leu Gln Asn Ser Gln Val Phe Ser Leu Ile Arg Pro Asn Glu
305 310 315 320
Asn Pro Ala His Lys Ser Gln Leu Val Trp Met Ala Cys His Ser Ala
325 330 335
Ala Phe Glu Asp Leu Arg Val Ser Ser Phe Ile Arg Gly Thr Arg Met
340 345 350
Val Pro Arg Gly Gln Leu Ser Thr Arg Gly Val Gln Ile Ala Ser Asn
355 360 365
Glu Asn Met Glu Ala Met Asp Ser Asn Thr Leu Glu Leu Arg Ser Arg
370 375 380
Tyr Trp Ala Ile Arg Thr Arg Ser Gly Gly Ser Thr Asn Gln Gln Arg
385 390 395 400
Ala Ser Ala Gly Gln Ile Ser Val Gln Pro Thr Phe Ser Val Gln Arg
405 410 415
Asn Leu Pro Phe Glu Arg Ser Thr Ile Met Ala Ala Phe Thr Gly Asn
420 425 430
Thr Glu Gly Arg Thr Ser Asp Met Arg Thr Glu Ile Ile Arg Met Met
435 440 445
Glu Ser Ala Arg Pro Glu Asp Val Ser Phe Gln Gly Arg Gly Val Phe
450 455 460
Glu Leu Ser Asp Glu Lys Ala Thr Asn Pro Ile Val Pro Ser Phe Asp
465 470 475 480
Met Asn Asn Glu Gly Ser Tyr Phe Phe Gly Asp Asn Ala Glu Glu Tyr
485 490 495
Asp Asn
Claims (3)
1.一种抗H9N2亚型禽流感病毒的纳米抗体，其特征在于，其氨基酸序列如SEQ ID NO:1所示。
2.一种抗H9N2亚型禽流感病毒的融合蛋白，其特征在于，由权利要求1所述的纳米抗体和辣根过氧化物酶通过基因工程融合而成。
3.一种如权利要求2所述的抗H9N2亚型禽流感病毒的融合蛋白在制备检测鸡血清中抗H9N2亚型禽流感病毒抗体试剂盒中的应用。
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| CN111647610B (zh) * | 2020-06-02 | 2021-09-03 | 扬州大学 | 一种互换ha和ns1缺失基因包装信号的h9n2亚型禽流感病毒及其构建方法和应用 |
| CN111732661A (zh) * | 2020-06-11 | 2020-10-02 | 军事科学院军事医学研究院军事兽医研究所 | 抗h5n1病毒入胞抗体ptd-7b及其应用 |
| CN111944044A (zh) * | 2020-08-28 | 2020-11-17 | 西北农林科技大学 | 一种抗ASFV-p30蛋白的纳米抗体及其制备方法和应用 |
| CN113527476B (zh) * | 2021-06-25 | 2023-01-17 | 华南农业大学 | 抗h5亚型禽流感病毒新纳米抗体及其应用 |
| CN114907475B (zh) * | 2022-05-31 | 2023-06-02 | 合肥中科长木生物科技有限公司 | 一种抗甲型流感病毒m2抗原的纳米抗体及其应用 |
| CN116375851B (zh) * | 2023-03-03 | 2024-03-22 | 中国农业科学院兰州兽医研究所 | 一种抗禽流感病毒np蛋白的纳米抗体及其制备方法与应用 |
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