CN113121680A - 一种抗h5亚型禽流感纳米抗体蛋白及其编码基因与应用 - Google Patents
一种抗h5亚型禽流感纳米抗体蛋白及其编码基因与应用 Download PDFInfo
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Abstract
本发明属于生物药物技术领域，具体涉及一种抗H5亚型禽流感纳米抗体蛋白及其编码基因与应用。所述的抗H5亚型禽流感病毒纳米抗体蛋白的氨基酸序列如SEQ ID NO.1所示。该纳米抗体蛋白特异性针对甲型H5亚型禽流感病毒可以解决现有H5亚型禽流感诊断试剂研发过程中抗体亲和力低、生产过程中纯度低、检测过程中灵敏度低的问题。本发明进一步将抗H5亚型禽流感病毒纳米抗体蛋白在大肠杆菌体内和麦芽糖结合蛋白标签融合表达，可实现快速检测抗原，并应用于甲型H5亚型禽流感快速检测试剂盒的开发，且该融合蛋白可用于防治甲型H5亚型禽流感产品的开发。
Description
技术领域
本发明属于生物药物技术领域，具体涉及一种抗H5亚型禽流感纳米抗体蛋白及其编码基因与应用。
背景技术
禽流感病毒(AIV)属于正黏病毒科正黏病毒属，是一类有包膜的单股负链分节段RNA病毒，根据流感病毒核衣壳蛋白(NP)和基质蛋白(M)的不同分为A、B和C型，国内又称甲、乙和丙型。根据血凝素(HA)和神经氨酸酶(NA)的抗原性差异，甲型流感病毒可分为不同的亚型。到目前为止，已有17种抗原上不同的甲型流感血凝素亚型，它们被进一步归类为第一组或第二组血凝素(第一组：H1、H2、H5、H6、H8、H9、H11、H12、H13、H16和H17病毒；第二组：H3、H4、H7、H10、H14、H15病毒)。
血凝素(HA)作为一种糖蛋白三聚体存在于流感病毒粒子的表面。每个HA单体最初表达为HA0，随后被宿主蛋白酶裂解成HA1和HA2亚基，通过二硫键连接。HA可分为球状头(HA1)和茎(部分HA1与全部HA2)两个区域，头部HA1相对茎部HA2较为容易发生突变，头部区域包含调节病毒与宿主底物结合能力的受体结合位点，针对该区域的抗体可以阻断受体结合，并被认为具有中和作用。而茎部相对保守，针对茎部的抗体更可能存在交叉反应，因此茎部是广谱中和抗体的潜在靶点。
H5是由甲型流感病毒引起的一种禽类烈性传染病，我国将其列为一类动物疫病，其潜伏期短，发病急剧，发病率和死亡率高。甲型流感病毒由8个基因节段构成，当不同的流感感染同一个细胞时会出现基因节段随机交换重组，也叫重配，导致流感抗原性转变，出现新的亚型和致病力的改变。近5年来，H5亚型AIV的基因分化速度快，出现多个亚分支，增加了与其他亚型发生重配的概率。而高致病性病毒源于低致病性病毒，病毒在不断进化和广泛传播，研制H5流感病毒检测设备和检测方法具有重要意义。在流感病毒发生快速、准确地诊断其亚型，大量研究报道了各种检测流感病毒的方法，比如病毒分离、免疫荧光、聚合酶链式反应(PCR)、酶染免疫吸附试验(ELISA)和血清学方法，然而这些方法费时费力，需要实验室设备以外花费还较高。例如，病毒分离被认为是诊断的金标准，也是常规的快速实验室确认人流感必不可少的，但它往往需要5-7天的测试与劳动密集型和长程序；而分子检测方法包括普通PCR、实时PCR等费时且技术要求高，而且样品间的交叉污染可能导致假阳性结果。
抗体介导的免疫检测因其简便而成为一种流行的检测方法，多克隆抗体容易产生交叉反应导致亚型判断错误，单克隆抗体开发周期长、稳定性差，价格不菲，若动物源的抗体在人体治疗上则很可能引起人体的免疫反应；核糖体展示技术的稳定性问题使得特异性和高亲和力、体积小的抗体难以获得。趋向于小型化的抗体——纳米抗体(Nbs)能避开上文提到的诸多不足，在食品、环境、化学检测上的运用逐年呈现上升趋势。纳米抗体是骆驼科动物(骆驼、大羊驼、羊驼及其近亲物种)缺失轻链的天然重链抗体的可变区(VHH)组成的单域抗体，该抗体只包含一个VHH和两个常规的重链恒定(CH2与CH3区)。具体地说，VHH是由不同的区域组成的，其中非CDR部分成为骨架区(FR)比较保守，其他区域负责特异性地识别抗原，称为互补决定区域(CDRs)，CDR3是识别程度最好的识别位点，VHH的高变区CDR3为16-18个氨基酸较任何小鼠的CDR3区平均长度要长，可变区的扩大能够形成更丰富的抗原结合构象，在一定程度上弥补了轻链缺失导致结合力下降的不足，从而使得纳米抗体本身具有较强的抗原结合能力，发生特异性结合时更容易且紧密和稳定。CDR3区还会形成一个大部分折叠在FR2上的凸环，凸环上的半胱氨酸和CDR1或者FR2上的半胱氨酸形成二硫键而增加其结构的稳定性，抗体基因序列多样性使得重链抗体可形成大量不同抗体结构形式的凸环；另外，人类VH3和骆驼VHH胚系基因具有高度相似性，实现抗体人源化降低免疫原性反应只需要较少的改变即可实现。VHH晶体为2.5nm，长4nm，分子量只有15KD。它具有分子量小，易表达，可溶性好，穿透性强，亲和性强、特异性高、稳定性高，能微生物表达，免疫原性低等优点，克服分子大、结构复杂、价格昂贵等缺点，与传统抗体相比较，在结构上存在较大差异，所以其特性也表现不一样，成为生物医药领域的新方向，具有广阔的应用前景。
发明内容
为了克服禽流感抗体在原核表达系统中以包涵体形式存在，产量低和变复性、生产成本高限制禽流感抗体在生产的运用等缺点和不足，本发明的第一个目的在于提供一种抗H5亚型禽流感病毒纳米抗体蛋白。
本发明的另一目的在于提供一种编码上述抗H5亚型禽流感纳米抗体蛋白的基因。
本发明的再一目的在于提供上述抗H5亚型禽流感病毒纳米抗体蛋白的应用。
本发明的第四个目的在于提供一种可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白，该融合蛋白可实现规模化生产。
本发明的第五个目的在于提供上述可溶性抗H5禽流感病毒纳米抗体融合蛋白的应用。
本发明的目的通过下述技术方案实现：
一种抗H5亚型禽流感病毒纳米抗体蛋白，其氨基酸序列如SEQ ID NO.1所示；
编码上述抗H5亚型禽流感病毒纳米抗体蛋白的基因，其核苷酸序列如SEQ IDNO.9所示：
所述的抗H5亚型禽流感病毒纳米抗体蛋白在制备抗禽流感病毒或检测抗原产品中的应用；
一种可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白，包含上述抗H5亚型禽流感病毒纳米抗体蛋白和麦芽糖结合蛋白(MBP)；
所述的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的氨基酸序列如SEQ IDNO.10所示，其中，抗H5亚型禽流感病毒纳米抗体蛋白和麦芽糖结合蛋白(MBP)通过连接肽(GGCGGCGGGTCA)连接；
所述的编码上述可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的基因的核苷酸序列如SEQ ID NO.11所示；
一种重组表达载体，为含有编码上述可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的基因的表达载体，是将编码上述可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的基因的核苷酸序列克隆进入表达载体得到；
所述的表达载体为PET系列载体，优选为pET-3a、pET-9a、pET-28a(+)、pET-22b(+)、pET-26b(+)或pET-31b(+)，进一步优选为pET28a(+)；
所述的编码上述可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的基因优选通过酶切位点Xho I和Nco I插入pET28a(+)中；
一种重组表达菌株，为含有上述重组表达载体的宿主菌株，是将上述重组表达载体转入宿主菌株得到；
所述的宿主菌株为细菌、酵母或真菌；优选为细菌，进一步优选为大肠杆菌(Escherichia coli)，最优选为大肠杆菌BL21(DE3)；
所述的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的制备方法，是将编码上述可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的基因克隆进入表达载体，得到重组表达载体，然后将重组表达载体转入宿主菌株，得到重组表达菌株；将重组表达菌株诱导表达，收集菌液，进行破碎、分离纯化，得到可溶性抗H5亚型禽流感纳米抗体融合蛋白；
所述的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的制备方法，优选包含如下步骤：
(1)将编码上述抗H5亚型禽流感病毒纳米抗体蛋白的基因和编码麦芽糖结合蛋白(MBP)的基因依次连接，得到融合基因序列；
(2)将步骤(1)得到的融合基因序列克隆入表达载体，得到重组表达载体；
(3)将步骤(2)得到的重组载体转入宿主菌株，得到重组表达菌株；
(4)培养步骤(3)得到的重组表达菌株并进行诱导表达，收集菌液，分离纯化，得到可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白；
步骤(2)中所述的表达载体为pET系列载体，优选为pET-3a、pET-9a、pET-28a(+)、pET-22b(+)、pET-26b(+)或pET-31b(+)，进一步优选为pET28a(+)；
步骤(3)中所述的宿主菌株为细菌、酵母和真菌，优选为细菌，进一步优选为大肠杆菌(Escherichia coli)，最优选为大肠杆菌BL21(DE3)；
所述的诱导表达的诱导剂优选为IPTG；
所述的IPTG的用量为按终浓度为0.1mmol/L；
所述的诱导表达的条件为28℃诱导16h；
步骤(4)中所述的重组表达菌株优选培养至菌液OD600为0.6～0.8时加入诱导剂进行诱导，进一步优选培养至菌液OD600为0.6时加入诱导剂进行诱导；
所述的分离纯化的方法优选为镍柱亲和层析法；
所述的可溶性抗H5亚型禽流感纳米抗体融合蛋白在制备抗禽流感或检测抗原产品中的应用；
本发明的原理：
(1)构建针对抗H5亚型禽流感病毒纳米抗体文库
本发明将禽流感病毒H5亚型Re-8株的血凝抑制实验抗原免疫驼科动物，抽取免疫后的供体外周淋巴血液，分离其中的外周淋巴细胞(PBMC)进行RNA抽提，进行反转录获得cDNA，然后构建对抗原有针对性的纳米抗体文库；
(2)利用噬菌体展示技术筛选抗H5亚型禽流感病毒纳米抗体蛋白
将禽流感病毒H5亚型Re-8株的血凝抑制实验抗原固定在ELISA板上，加入将编码目的蛋白基因融合到噬菌体衣壳蛋白基因中的噬菌体与抗原相结合，不含特异性配体的噬菌体克隆则会被洗掉，将筛选到的噬菌体克隆在大肠杆菌中扩增，经过3-5轮“吸附-洗脱-扩增”，最后挑选所得噬菌体的单菌落进行ELISA法筛选阳性克隆，当P/N(即阳性孔OD读数/对照孔OD读数的比值)大于等于3为阳性，扩增测序后得到抗H5亚型禽流感病毒纳米抗体蛋白的序列。
本发明提供了一种特异性针对甲型H5亚型禽流感病毒的抗H5亚型禽流感病毒纳米抗体蛋白及编码该纳米抗体蛋白的基因，也建立了该纳米抗体蛋白在诊断中的应用方法，以解决现有H5亚型禽流感诊断试剂研发过程中抗体亲和力低、生产过程中纯度低、检测过程中灵敏度低的问题。
本发明进一步将抗H5亚型禽流感病毒纳米抗体蛋白在大肠杆菌体内和麦芽糖结合蛋白标签融合表达，可实现快速检测抗原，并应用于甲型H5亚型禽流感快速检测试剂盒的开发，且该融合蛋白可用于防治甲型H5亚型禽流感产品的开发。
本发明相对于现有技术具有如下的优点及效果：
(1)本发明利用噬菌体展示技术筛选抗H5亚型禽流感病毒纳米抗体蛋白，该纳米抗体特异性针对甲型H5亚型禽流感病毒。
(2)本发明通过基因工程的方法，将编码上述抗H5亚型禽流感病毒纳米抗体蛋白的基因通过连接肽(GGCGGCGGGTCA)与麦芽糖结合蛋白(MBP)基因连接，构建表达载体，诱导融合蛋白表达，来促进抗H5禽流感病毒纳米抗体融合蛋白在表达系统中的可溶表达，从而解决了抗H5禽流感纳米抗体蛋白在原核系统呈不可溶的难题，提高了准确性和重复性。
(2)本发明提供的抗H5禽流病毒感纳米抗体融合蛋白的生产方法操作简单、成本低，可实现抗H5禽流感病毒纳米抗体规模化生产。
(3)本发明生产的抗H5禽流感病毒纳米抗体蛋白纯度较高、检测过程中灵敏度高，具有良好的稳定性和生物学活性，对鸡H5禽流感的监测具有重大的意义。
附图说明
图1是可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白质粒p6-mbp in pET-28a(+)的示意图，其中，VHH-6基因是指编码抗H5亚型禽流感病毒纳米抗体蛋白的基因。
图2是LC/Q-TOF检测抗H5亚型禽流感病毒纳米抗体的分子量和蛋白质谱鉴定分析图。
图3是p6-mbp in pET-28a(+)转化至受体菌DH5α后，对其抽质粒并进行酶切验证的结果图，其中，1：DL10000 Marker，2：载体PET28a(+)，3：对转化了p6-mbp in pET-28a(+)的受体菌DH5α进行质粒抽提后，并对质粒做Xho I和Nco I双酶切。
图4是SDS-PAGE电泳检测融合蛋白纯化结果分析结果图，其中，1：Thermo26616Marker，2：诱导剂IPTG 0.1mmol/L，28℃诱导16h的融合蛋白纯化结果。
图5是针对不同抗原的血凝抑制(HI)图，其中，第一行抗原为H5(Re-8)，第二行抗原为H7(H7-Re1株)，第三行抗原为H9(H9亚型)，第11列为阴对照，第12列为空白对照。
图6是可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白与不同的包被抗原的ELISA反应折线图，其中，横坐标为包被抗原的稀释倍数，纵坐标是加入显色液后在620nm的波长下的OD读数。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述，但本发明的实施方式不限于此。
实施例中，2×Taq Plus Master Mix购于南京诺唯赞生物公司，DNA胶回收试剂盒购于美国OMEGA公司，Not I内切酶、Sif I内切酶等购于英国NEB公司，引物均由上海上海生工生物有限公司进行合成，HRP-M13购于北京义翘神州，TMB底物显色液购于翊圣生物有限公司，
PCR Master Mix酶购于翊圣生物有限公司，兔源抗mbp多抗和羊抗兔-HRP抗体等购于以翊圣公司。
实施例1构建噬菌体展示文库
(1)将禽流感病毒H5亚型Re-8株的血凝抑制实验抗原(购于哈尔滨维科生物技术开发公司)免疫新疆双峰驼(甘肃兰州畜牧兽医研究所)，每三周免疫一次，一共免疫四次，抽取免疫后的供体外周淋巴血液，分离其中的外周血淋巴细胞(PBMC)进行RNA抽提并反转录获得cDNA；
(2)第一轮巢氏PCR反应：以步骤(1)获得的cDNA作为模板进行第一轮巢氏PCR反应，其中，反应体系为：cDNA模板2μL，2×Taq Plus Master Mix10μL，引物CALL001和CALL002各1μL，用ddH2O补足至20μL；扩增反应条件为：95℃5min；95℃30s，55℃30s，72℃45s，35个循环；对第一轮巢氏PCR反应产物进行凝胶电泳验证分析，扩增产物为约700bp的片段，然后按照DNA胶回收试剂盒进行PCR产物回收；其中，引物CALL001和CALL002的核苷酸序列如下所示：
引物CALL001：5’-GTCCTGGCTGCTCTTCTACAAAG-3’；
引物CALL002：5’-GGTACGTGCTGTTGAACTGTTCC-3’；
(3)第二轮巢氏PCR反应：以步骤(2)获得的第一轮PCR反应产物为模板进行第二轮巢氏PCR反应，其中，反应体系为：第一轮PCR反应产物(约700bp)2μL，2×Taq Plus MasterMix 25μL，引物VHH-Forward和VHH-Reverse各1μL，用ddH2O补足至50μL，第二轮PCR扩增反应条件为：94℃5min；94℃40s，64℃40s，72℃40s，5个循环；94℃40s，68℃45s，32个循环；72℃10min；对第二轮巢氏PCR反应产物进行凝胶电泳验证分析，扩增产物为约400bp的VHH片段，然后按照DNA胶回收试剂盒进行PCR产物回收，测定目的片段浓度并保存于-20℃；其中，引物VHH-Forward和VHH-Reverse的核苷酸序列如下所示(横线部分为酶切位点)：
引物VHH-Forward：
5’-TCGCGGCCCAGCCGGCCCAGGTCCAACTGCAGGAGTCTGGGG-3’；
引物VHH-Reverse：
5’-ATAAGAATGCGGCCGCTGAGGAGACGGTGACCTGGGTCCCC-3’；
(4)将步骤(3)扩增得到的VHH片段与pCANTAB 5E噬菌粒载体(购于GE公司)分别先用Not I 37℃酶切4h，其中，VHH片段酶切体系(50μL)为：步骤(3)扩增得到的VHH片段(约400bp)14μL，Not I 1μL，10×Buffer5μL，ddH2O 30μL；pCANTAB 5E噬菌粒载体酶切体系为：pCANTAB5E噬菌粒载体15μL，Not I 1μL，10×Buffer 5μL，ddH2O 29μL；
(5)Not I酶切后的VHH片段与pCANTAB 5E噬菌粒载体分别加入Sif I进行第二次酶切，反应条件为50℃酶切4h，其中，VHH片段酶切反应体系为：Not I酶切后的VHH片段14μL，Sif I 1μL，10×Buffer 5μL，ddH2O 30μL；pCANTAB 5E噬菌粒载体酶切体系为：Not I酶切后的pCANTAB 5E 15μL，Sif I1μL，10×Buffer 5μL，ddH2O 29μL；
(6)将Sif I酶切后的VHH片段与pCANTAB 5E噬菌粒载体参照T4 DNA连接酶的说明书，用T4 DNA连接酶进行连接，反应体系为：Sif I酶切后pCANTAB5E 20μL，Sif I酶切后的VHH片段6μL，Buffer 2μL，T4 DNA连接酶2μL；混合后16℃连接4h，4℃过夜连接；将10μL连接产物加入100μL TG1感受态(购于碧云天公司)中，以热击转化的方式转化；将转化后的单菌落PCR验证无误后全部加入至100mL 2×YT的无抗液体培养基过夜振荡12h，加入终浓度为25％(V/V)的甘油混匀，每支1mL文库菌分装后冻至-80℃，进而得到对抗原有针对性的纳米抗体文库；
(7)取1支步骤(6)获得的纳米抗体文库菌接种到50mL的2×YT/2％Glu/Amp液体中，37℃振荡培养1～2h，使OD值达到0.6左右，加入扩增后辅助噬菌体M13KO7(感染复数为20:1)，37℃静置15min，220rpm振荡培养45min，然后4000rpm离心10min，弃去上清；用200mL的2×YT/Amp/Kana重悬沉淀，30℃、200rpm震荡过夜；取出过夜摇菌的菌液，4℃、10000rpm离心20min，离心后取上清，加入1/5体积的PEG/NaCl(PEG6000含量为20％(W/V)，NaCl含量为2.5M)，冰浴4h以沉淀噬菌体，得到均匀浑浊的悬浮物；冰浴后将液体分装到5个50mL的离心管中，8000rpm离心20min，弃掉上清，得到噬菌粒沉淀；离心后，每管用10mL PEG/NaCl重悬，收集到1个50mL离心管中，8000rpm再次离心20min，弃去上清；将沉淀用4mL的PBS重悬，加入终浓度25％(V/V)的甘油，分装到-80℃保存，此为构建好的噬菌体展示文库；其中，辅助噬菌体M13KO7(购于NEB公司)的作用是为宿主细胞的噬菌粒DNA提供复制和包装所需要的蛋白酶和外壳蛋白；辅助噬菌体的扩增是将辅助噬菌体以10的N次方倍稀释法稀释，再感染OD600＝0.5的大肠杆菌TG1，37℃中静置30min侵染后加入到已融化好的上层琼脂中，然后倒入2×YT液体培养基中，放置于37℃培养箱中过夜；次日，观察培养板中噬斑形成情况，计算辅助噬菌体滴度；挑取单个噬菌斑加入到5mL OD600＝0.5的大肠杆菌TG1，并在37℃下200rpm摇菌2h；离心收集菌体至250mL的2×YT培养基中，室温下200rpm振荡培养1h；再加入Kana至终浓度为50μg/mL，30℃震荡过夜培养，此为扩增后的辅助噬菌体，加入终浓度为25％(V/V)的甘油，分装到-80℃保存。
实施例2噬菌体纳米文库的筛选
(1)用碳酸钠-碳酸氢钠缓冲液(0.05mol/L，pH＝9.6)将H5亚型Re-8株的血凝抑制实验抗原(购自哈尔滨维科生物技术开发公司)包被在酶标板上，每孔100μL，其中，H5亚型Re-8株的血凝抑制实验抗原占体积百分比为10％，放于37℃包被2h；弃去孔中液体，用300μL PBS洗一遍后加入300μL质量百分比为3％的BSA并置于室温封闭1h；弃去封闭液，加入100μL实施例1获得的噬菌体展示文库，在室温孵育1h；弃去孔中液体及未结合噬菌体，再用含体积百分比0.05％吐温20的PBS洗5次，5min/次；加入100μL含0.2M甘氨酸的洗脱液(pH＝2.7)混匀，将洗脱液收集起来，按照洗脱液和Tris体积比为1:0.2的比例加入1M Tris(pH＝9.1)，混合均匀，使得含有噬菌体的液体呈中性，获得第一轮富集筛选的噬菌体，此为“吸附-洗脱”的过程；
(2)将步骤(1)中和后的噬菌体加入到5mL OD600＝0.6的TG1大肠杆菌，室温孵育30min后，从侵染菌液中取出100μL，以10的N次方倍稀释后取100μL涂布于2×YT/Amp/Glu固体培养基中，放置于37℃中培养过夜，计算侵染的细菌文库滴度；取剩余侵染菌液加入到100mL的2×YT/Amp/Glu培养基中，37℃摇至OD600＝0.6，按照感染复数MOI＝20:1加入辅助噬菌体M13KO7，37℃静置15min，200rpm振荡培养30min，4000rpm离心15min，弃去上清，再加入到200mL的2×YT/Amp/Kana培养基，37℃、200rpm过夜振荡培养使筛选到的噬菌体进一步扩增；取出过夜摇菌的菌液，4℃、10000rpm离心20min，离心后取上清，加入1/5体积的PEG/NaCl(PEG6000含量为20％(W/V)，NaCl含量为2.5M)，冰浴4h以沉淀噬菌体，冰浴后8000rpm离心20min，弃掉上清得到噬菌粒沉淀；离心后再用10mL PEG/NaCl重悬，8000rpm再次离心20min，弃去上清；将沉淀用4mL的PBS重悬，加入终浓度25％(V/V)的甘油，分装到-80℃保存，此为第一轮筛选后的噬菌体扩增液，作为第二轮筛选的噬菌体文库，该过程为“扩增”的过程；
(3)三轮“吸附-洗脱-扩增”的筛选过程，最终富集筛选获得特异性较强的噬菌体克隆。
实施例3 ELISA法筛选阳性单克隆
(1)将实施例2第三轮筛选出来的噬菌体先与5mL的TG1菌(OD600＝0.5)，37℃温育30min后，从侵染菌液中取出100μL进行10的N次方梯度稀释，重新吸取菌液100μL分别划线于2×YT/Amp/Glu固体培养基，37℃过夜培养，再将单菌落接种在50mL的2×YT/Amp/Glu液体培养基中，220rpm 37℃过夜培养，第二天吸取1mL菌液至100mL 2×YT/Amp/Glu液体培养基中，220rpm37℃摇菌至OD600＝0.6左右，加入感染复数MOI＝20:1的辅助噬菌体M13KO7，37℃静置20min，200rpm振荡培养30min，进行辅助噬菌体侵染，3000g离心弃去上清包括培养基的葡萄糖，用新鲜的2×YT/Amp/Kana培养基重悬，37℃、250rpm过夜震荡培养，离心弃去上清，得到噬菌体阳性克隆；
(2)按照实施例2包被方法包被将H5亚型Re-8株的血凝抑制实验抗原(购自哈尔滨维科生物技术开发公司)4℃过夜包被至酶标板上后进行封闭处理，以空白酶标板(即不包被)为空白对照；加入100μL步骤(1)筛选扩增后的噬菌体阳性克隆并室温孵育2h，用含体积百分比0.05％吐温-20的PBS洗5次，5min/次，再加入100μL/孔HRP-M13，37℃孵育1h；弃去孔中液体及未结合噬菌体，用含体积百分比0.05％吐温-20的PBS洗5次，5min/次，加入100μL/孔TMB底物显色液，避光室温放置20min，酶标仪测波长620nm的OD数值，以P/N值(即阳性孔OD读数/对照孔OD读数的比值)大于等于3为阳性。将阳性克隆菌液送去上海生工生物股份有限公司测序，即可得到抗H5亚型禽流感病毒纳米抗体的核苷酸序列。
其中，抗H5亚型禽流感病毒纳米抗体蛋白的氨基酸序列(图2)如下所示(SEQ IDNO.1)：
MDNQVQLQESGGGLVQPGGSLRLSCAASGFTISSSSITWVRQPPGKGLEWVSSLYSVSSNTFYAESVKDRFTISGDYAKNTVYLQMNSLKSEDTAVYYCATDPRISVPDLVVAKTISRADFGTWGQGTLVTVSS
其中，抗H5亚型禽流感病毒纳米抗体蛋白包括框架区(FR)和抗体基因互补决定区(CDR)，所述框架区分为四部分，依次定义FR1、FR2、FR3、FR4，其氨基酸序列如SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.5所示；所述互补决定区分为三部分，依次可定义为CDR1、CDR2、CDR3，其氨基酸序列如SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8所示；
SEQ ID NO.2：MDNQVQLQESGGGLVQPGGSLRLSCAAS
SEQ ID NO.3：WVRQPPGKGLEWVSS
SEQ ID NO.4：FYAESVKDRFTISGDYAKNTVYLQMNSLKSEDTAVYYC
SEQ ID NO.5：WGQGTLVTVSS
SEQ ID NO.6：GFTISSSSIT
SEQ ID NO.7：LYSVSSNT
SEQ ID NO.8：ATDPRISVPDLVVAKTISRADFGT
编码上述抗H5亚型禽流感病毒纳米抗体蛋白的基因的核苷酸序列如下所示(SEQID NO.9)：
ATGGACAATCAAGTTCAGTTACAGGAATCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTAGCTCCAGCATTACCTGGGTCCGCCAGCCTCCAGGGAAGGGACTGGAGTGGGTGTCCAGTCTTTATAGTGTTAGTAGTAACACATTTTATGCAGAGTCCGTGAAGGACCGATTCACCATCTCCGGAGACTACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAATCTGAGGACACGGCCGTATATTACTGTGCCACTGACCCCCGAATCAGCGTCCCCGATTTGGTGGTAGCTAAAACAATCTCTCGCGCTGACTTTGGCACCTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCA
实施例4
(1)可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白质粒p6-mbp in pET-28a(+)的构建
根据编码抗H5亚型禽流感病毒纳米抗体蛋白(SEQ ID NO.1)的基因的序列，将其C末端与麦芽糖结合蛋白(MBP)通过连接肽(GGCGGCGGGTCA)进行连接构成可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的基因序列，通过基因合成对融合基因两端分别加上Xho I和Nco I酶切位点，与进行Xho I和Nco I双酶切的pET-28a(+)载体相连，命名为p6-mbp inpET-28a(+)，得到的连接片段由上海通用公司合成，经测序正确后，获得大小为6770bp的重组载体(图1)。
可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的氨基酸序列(SEQ ID NO.10)：
MDNQVQLQESGGGLVQPGGSLRLSCAASGFTISSSSITWVRQPPGKGLEWVSSLYSVSSNTFYAESVKDRFTISGDYAKNTVYLQMNSLKSEDTAVYYCATDPRISVPDLVVAKTISRADFGTWGQGTLVTVSSGGGSKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTHHHHHH.
可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的核苷酸序列(SEQ ID NO.11)：
ATGGACAATCAAGTTCAGTTACAGGAATCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCATCAGTAGCTCCAGCATTACCTGGGTCCGCCAGCCTCCAGGGAAGGGACTGGAGTGGGTGTCCAGTCTTTATAGTGTTAGTAGTAACACATTTTATGCAGAGTCCGTGAAGGACCGATTCACCATCTCCGGAGACTACGCCAAGAACACGGTGTATTTGCAAATGAACAGCCTGAAATCTGAGGACACGGCCGTATATTACTGTGCCACTGACCCCCGAATCAGCGTCCCCGATTTGGTGGTAGCTAAAACAATCTCTCGCGCTGACTTTGGCACCTGGGGCCAGGGGACCCTGGTCACCGTCTCCTCAGGCGGCGGGTCAAAAATCGAAGAAGGTAAACTGGTAATCTGGATTAACGGCGATAAAGGCTATAACGGTCTCGCTGAAGTCGGTAAGAAATTCGAGAAAGATACCGGAATTAAAGTCACCGTTGAGCATCCGGATAAACTGGAAGAGAAATTCCCACAGGTTGCGGCAACTGGCGATGGCCCTGACATTATCTTCTGGGCACACGACCGCTTTGGTGGCTACGCTCAATCTGGCCTGTTGGCTGAAATCACCCCGGACAAAGCGTTCCAGGACAAGCTGTATCCGTTTACCTGGGATGCCGTACGTTACAACGGCAAGCTGATTGCTTACCCGATCGCTGTTGAAGCGTTATCGCTGATTTATAACAAAGATCTGCTGCCGAACCCGCCAAAAACCTGGGAAGAGATCCCGGCGCTGGATAAAGAACTGAAAGCGAAAGGTAAGAGCGCGCTGATGTTCAACCTGCAAGAACCGTACTTCACCTGGCCGCTGATTGCTGCTGACGGGGGTTATGCGTTCAAGTATGAAAACGGCAAGTACGACATTAAAGACGTGGGCGTGGATAACGCTGGCGCGAAAGCGGGTCTGACCTTCCTGGTTGACCTGATTAAAAACAAACACATGAATGCAGACACCGATTACTCCATCGCAGAAGCTGCCTTTAATAAAGGCGAAACAGCGATGACCATCAACGGCCCGTGGGCATGGTCCAACATCGACACCAGCAAAGTGAATTATGGTGTAACGGTACTGCCGACCTTCAAGGGTCAACCATCCAAACCGTTCGTTGGCGTGCTGAGCGCAGGTATTAACGCCGCCAGTCCGAACAAAGAGCTGGCAAAAGAGTTCCTCGAAAACTATCTGCTGACTGATGAAGGTCTGGAAGCGGTTAATAAAGACAAACCGCTGGGTGCCGTAGCGCTGAAGTCTTACGAGGAAGAGTTGGTGAAAGATCCGCGTATTGCCGCCACTATGGAAAACGCCCAGAAAGGTGAAATCATGCCGAACATCCCGCAGATGTCCGCTTTCTGGTATGCCGTGCGTACTGCGGTGATCAACGCCGCCAGCGGTCGTCAGACTGTCGATGAAGCCCTGAAAGACGCGCAGACTCACCACCACCATCATCACTAA
(2)质粒抽提，双酶切确认质粒并送测序
将步骤(1)得到的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白质粒p6-mbp inpET-28a(+)转化至受体菌DH5α，菌液涂含卡那霉素的LB(含50mg/L的卡那霉素(Kan))板进行复苏活化，37℃培养16h后，挑取一部分单菌落分别转接至100mL的含终浓度为30mg/mL的Kana液体LB培养基中，37℃、200rpm的摇床上摇菌16h，先将部分菌液暂时分装放4℃保存；再取一部分菌液使用50mL离心管进行收集，8000rpm离心10min，弃去上清，进行质粒抽提，并对质粒做Xho I和Nco I双酶切(图3)，跑核酸电泳确认酶切片段大小，并将大小介于1500-2000bp的目的条带的克隆质粒进一步测序，将测序正确的阳性克隆扩大培养，抽取质粒保存至-20℃冰箱，将另外部分菌液用含终体积百分比为15～20％甘油的LB溶液放置-80℃进行保种。
实施例5可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的诱导表达
(1)将上述实施例4中保存的表达可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的质粒转化至受体大肠杆菌BL21(DE3)上，冰上放置30min，42℃水浴锅中热击90s后加入1mL LB肉汤，在37℃，220rpm的摇床上复苏活化之后，6000rpm离心1min，弃去90％的上清后将菌液涂含50mg/mL卡那霉素的LB板进行复苏活化，37℃培养16h后，挑取单个菌落利用T7引物(正向引物T7-F：TAATACGACTCACTATAGG；反向引物T7-R：TGCTAGTTATTGCTCAGCGG)进行菌落PCR确认，将大小介于1500-2000bp的目的条带的克隆菌落重新转接，一部分用于菌种保存，另一部用于下述步骤(2)；其中，扩增体系为：
PCR Master Mix酶25μL，正向引物(10pmol/μL)1μL，反向引物(10pmol/μL)1μL，基因模板2μL，ddH2O补足至50μL；扩增反应条件为：98℃3min；98℃15s、58℃15s、72℃60s，40个循环；72℃5min。PCR反应完成之后，使用质量体积比为1％的琼脂糖凝胶进行电泳，凝胶电泳显示大小介于1500-2000bp的目的条带。
(2)刮取转接后的部分菌转接至含终浓度为50mg/mL的Kana的100mL液体LB培养基中，37℃、200rpm的摇床上摇菌；待菌液OD600值为0.6时，用IPTG进行诱导，IPTG的终浓度为0.1mmol/L；28℃诱导16h后将菌液使用50mL离心管进行收集，8000rpm离心10min，弃去上清；
(3)用约30mL无咪唑的蛋白裂解液Lysis buffer(NaH2PO4·H2O(MW137.99g/mol)6.9g，即0.05M，NaCl(MW 58.44g/mol)17.54g即0.3M，加入约900mL去离子水，搅拌溶解后，加入NaOH调节溶液pH值至8.0，加入去离子水定容至1000mL)重悬步骤2中的菌体，使用超声破碎仪进行破碎，超声程序为破碎3s，间隔5s，超声破碎30min；
(4)将超声破碎后的产物8000rpm离心10min，收集上清，取出适量上清样品加入2×SDS上样缓冲液，混匀后，至沸水中煮10min，若加热后的样品有粘性产物，将样品进行瞬时离心后，取上清上样于购自南京金斯瑞的SDS-PAGE胶孔，同时加入等量的蛋白ThermoMarker 26616，以电泳电压调节至120V跑胶，跑胶30min。将凝胶块从玻璃板中取出，轻放于摇床上含有考马斯亮蓝溶液的染色槽中1h；然后将染色槽的考马斯亮蓝溶液倒出，加入清水冲洗干净后，凝胶块放于摇床中的染色槽中，用清水对凝胶块进行脱色30min即可见清洗条带。将脱色的凝胶放于成像系统中进行扫描，如图4所示，观察结果，目标条带大小在55KDa-70Kda之间，与预估蛋白大小约60Kda相符，且SDS-PAGE图条带清晰，证明成功表达该融合蛋白。
实施例6可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白功能的验证
抗H5亚型禽流感病毒纳米抗体有抑制血凝的功能，因此我们通过血凝抑制(HI)实验检验实施例5制得的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白，过程如下：先通过血凝(HA)试验测得病毒效价，配置四单位，再进行HI实验。
(1)HA实验：
①在96孔板1-11孔加入25μL PBS，第12孔加入50μL PBS；
②第1孔加H5亚型Re-8株的血凝抑制实验抗原(购自哈尔滨维科生物技术开发公司)25μL，混匀吸至第2孔，依次倍比稀释至第11孔，混匀后弃去25μL，第12孔不加；
③从1～11孔，每空加稀释好的25μL PBS；
④将1％鸡红细胞悬液轻轻摇动混匀，在1～12孔中每孔加入25μL；震荡，室温(24-25℃)静置40min后观察结果；其中，1％鸡红细胞悬液的制备：用肝素钠真空抗凝管抽取SPF鸡血，400g离心5min，用PBS清洗；再离心，吸取沉淀红细胞表面的白细胞，不断清洗直至红细胞表面无白细胞，PBS洗液呈透亮无颜色；以PBS：红细胞＝99：1的比例轻轻混匀，制得1％鸡红细胞悬液；
四单位配置：测得HA效价后，按照原液稀释2n-2倍的方法配置四单位抗原(4HAU)，配好后，需按测试HA的步骤进行四单位验证，当前两孔出现完全血凝现象，即为配好的四单位抗原。
(2)HI实验：
①在96孔板1-11孔加入25μL PBS，第12孔加入50μL PBS；
②第1孔加可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白(实施例5制得的上清液)25μL，混匀吸至第2孔，依次倍比稀释至第10孔，混匀后弃去25μLPBS，第11、12孔不加；
③从1～11孔，每空加稀释好的25μL AIV H5亚型Re-8株4单位抗原悬液，室温(24-25℃)静置至少30min，第12孔不加；
④将1％的红细胞轻轻摇动混匀，在1～12孔中每孔加入25μL；震荡，室温(24-25℃)静置40min后观察结果。
H7、H9血凝抑制实验步骤与H5亚型Re-8株的血凝抑制实验相同。H5、H7、H9禽流感血凝抑制试验抗原分别为Re-8株、H7-Re1株、H9亚型，均为购自哈尔滨维科生物技术有限公司的禽流感血凝抑制试验抗原。
HI结果显示，可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白对H5抗原效价为4log2，对H7、H9无血凝抑制，是特异性结合的抗H5禽流感纳米抗体融合蛋白抗体(图5)。
实施例7可溶性抗H5禽流感病毒纳米抗体融合蛋白对抗原的特异性检测应用
可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白是针对H5亚型禽流感病毒的特异性抗体，为此通过包被不同的抗原，用筛选表达的纳米抗体对包被的抗原进行检测。具体实验方案如下：
①抗原包被：分别取H5、H7、H9禽流感血凝抑制试验抗原(分别为Re-8株、H7-Re1株、H9亚型)10μL，按照第一孔抗原(抗原：碳酸盐包被缓冲液＝1:9)按照10倍稀释，第二孔开始4倍稀释，将抗原吸附在固相载体聚苯乙烯；即第一孔将抗原和包被液稀释均匀后，吸取25μL至第二孔，一次稀释至第七孔，第八孔只加包被液做为空白对照，每孔最终75μL，37℃孵育2h或者4℃过夜孵育后，弃去板内液体；
②封闭：加300μL质量百分比3％的BSA封闭1h；
③一抗：可溶性抗H5禽流感病毒纳米抗体融合蛋白(实施例5制得的上清液)即为一抗，按照1:1000的比例用PBS稀释加75μL稀释后抗体至每孔；
④洗涤：用含体积百分比0.5％Tween-20的PBS洗涤液洗涤3-5次，每次洗涤浸泡时间5min洗5次；
⑤二抗：兔源抗mbp多抗按照1:5000比例用PBS稀释，稀释加75μL稀释后抗体至每孔孵育1h；
⑥洗涤：用含体积百分比0.5％Tween-20的PBS洗涤液洗涤3-5次，每次洗涤浸泡时间5min，洗5次；
⑦三抗：羊抗兔-HRP抗体按照1:10000比例用PBS稀释，稀释加75μL稀释后抗体至每孔，孵育1h；
⑧洗涤：用含0.5％的Tween-20的PBS洗涤液洗涤3-5次，每次洗涤浸泡时间5min，洗5次；
⑨显色：加入HRP底物显色液进行显色20min，用酶标仪的波长620nm进行读数。
ELISA结果显示：随着H5抗原(Re-8)梯度递减的包被抗原(一定范围内)，P/N比值也梯度递减，其中空白对照数值为0.05，P/N最大比值大于3，证明原核表达的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白具有良好的生物活性；对比H7抗原、H9抗原，P/N最大比值均小于3，证明原核表达的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白具有良好的特异性(图6)。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
SEQUENCE LISTING
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<213> Artificial
<220>
<223> 可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的核苷酸序列
<400> 11
atggacaatc aagttcagtt acaggaatct gggggaggct tggtgcagcc tggggggtct 60
ctgagactct cctgtgcagc ctctggattc accatcagta gctccagcat tacctgggtc 120
cgccagcctc cagggaaggg actggagtgg gtgtccagtc tttatagtgt tagtagtaac 180
acattttatg cagagtccgt gaaggaccga ttcaccatct ccggagacta cgccaagaac 240
acggtgtatt tgcaaatgaa cagcctgaaa tctgaggaca cggccgtata ttactgtgcc 300
actgaccccc gaatcagcgt ccccgatttg gtggtagcta aaacaatctc tcgcgctgac 360
tttggcacct ggggccaggg gaccctggtc accgtctcct caggcggcgg gtcaaaaatc 420
gaagaaggta aactggtaat ctggattaac ggcgataaag gctataacgg tctcgctgaa 480
gtcggtaaga aattcgagaa agataccgga attaaagtca ccgttgagca tccggataaa 540
ctggaagaga aattcccaca ggttgcggca actggcgatg gccctgacat tatcttctgg 600
gcacacgacc gctttggtgg ctacgctcaa tctggcctgt tggctgaaat caccccggac 660
aaagcgttcc aggacaagct gtatccgttt acctgggatg ccgtacgtta caacggcaag 720
ctgattgctt acccgatcgc tgttgaagcg ttatcgctga tttataacaa agatctgctg 780
ccgaacccgc caaaaacctg ggaagagatc ccggcgctgg ataaagaact gaaagcgaaa 840
ggtaagagcg cgctgatgtt caacctgcaa gaaccgtact tcacctggcc gctgattgct 900
gctgacgggg gttatgcgtt caagtatgaa aacggcaagt acgacattaa agacgtgggc 960
gtggataacg ctggcgcgaa agcgggtctg accttcctgg ttgacctgat taaaaacaaa 1020
cacatgaatg cagacaccga ttactccatc gcagaagctg cctttaataa aggcgaaaca 1080
gcgatgacca tcaacggccc gtgggcatgg tccaacatcg acaccagcaa agtgaattat 1140
ggtgtaacgg tactgccgac cttcaagggt caaccatcca aaccgttcgt tggcgtgctg 1200
agcgcaggta ttaacgccgc cagtccgaac aaagagctgg caaaagagtt cctcgaaaac 1260
tatctgctga ctgatgaagg tctggaagcg gttaataaag acaaaccgct gggtgccgta 1320
gcgctgaagt cttacgagga agagttggtg aaagatccgc gtattgccgc cactatggaa 1380
aacgcccaga aaggtgaaat catgccgaac atcccgcaga tgtccgcttt ctggtatgcc 1440
gtgcgtactg cggtgatcaa cgccgccagc ggtcgtcaga ctgtcgatga agccctgaaa 1500
gacgcgcaga ctcaccacca ccatcatcac taa 1533
Claims (10)
1.一种抗H5亚型禽流感病毒纳米抗体蛋白，其特征在于其氨基酸序列如SEQ ID NO.1所示。
2.编码权利要求1所述的抗H5亚型禽流感病毒纳米抗体蛋白的基因，其特征在于其核苷酸序列如SEQ ID NO.9所示。
3.权利要求1所述的抗H5亚型禽流感病毒纳米抗体蛋白在制备抗禽流感病毒或检测抗原产品中的应用。
4.一种可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白，其特征在于包含权利要求1所述的抗H5亚型禽流感病毒纳米抗体蛋白和麦芽糖结合蛋白。
5.权利要求4所述的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的氨基酸序列如SEQ ID NO.10所示。
6.权利要求5所述的编码可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的基因的核苷酸序列如SEQ ID NO.11所示。
7.一种重组表达载体，其特征在于为含有编码权利要求4或5所述的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的基因的表达载体，是将编码权利要求4或5所述的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的基因的核苷酸序列克隆进入表达载体得到。
8.一种重组表达菌株，其特征在于为含有权利要求7所述的重组表达载体的宿主菌株，是将权利要求7所述的重组表达载体转入宿主菌株得到。
9.权利要求4或5所述的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的制备方法，其特征在于是将编码权利要求4或5所述的可溶性抗H5亚型禽流感病毒纳米抗体融合蛋白的基因克隆进入表达载体，得到重组表达载体，然后将重组表达载体转入宿主菌株，得到重组表达菌株；将重组表达菌株诱导表达，收集菌液，进行破碎、分离纯化，得到可溶性抗H5亚型禽流感纳米抗体融合蛋白。
10.权利要求4或5所述的可溶性抗H5亚型禽流感纳米抗体融合蛋白在制备抗禽流感或检测抗原产品中的应用。
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| CN116063465A (zh) * | 2022-08-09 | 2023-05-05 | 华南农业大学 | 针对h7亚型禽流感病毒的纳米抗体m111及其应用 |
| CN116063465B (zh) * | 2022-08-09 | 2023-08-04 | 华南农业大学 | 针对h7亚型禽流感病毒的纳米抗体m111及其应用 |
| CN116375851A (zh) * | 2023-03-03 | 2023-07-04 | 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 |
| 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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| CN117164705B (zh) * | 2023-08-31 | 2024-06-04 | 华南农业大学 | 一种靶向h5亚型禽流感病毒血凝素蛋白的纳米抗体 |
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