CN116041496B - H7n9亚型禽流感病毒中和性单克隆抗体及应用 - Google Patents
H7n9亚型禽流感病毒中和性单克隆抗体及应用 Download PDFInfo
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Abstract
本发明公开了一种H7N9亚型禽流感病毒中和性单克隆抗体及应用,其中单克隆抗体为IgG1、κ型,单克隆抗体包括重链可变区氨基酸及轻链可变区氨基酸,重链可变区氨基酸序列包括SEQ ID No.5所示的HCDR1、SEQ ID No.6所示的HCDR2、SEQ ID No.7所示的HCDR3;轻链可变区氨基酸序列包括SEQ ID No.8所示的LCDR1、SEQ ID No.9所示的LCDR2、SEQ ID No.10所示的LCDR3。
Description
技术领域
本发明涉及一种H7N9亚型禽流感病毒中和性单克隆抗体及应用,属于生物技术技术领域。
背景技术
流感病毒属于正粘病毒科,可分为A、B、C、D四型,其中A型流感病毒宿主范围最广,是引起流感大流行的主要类型。根据病毒囊膜表面蛋白血凝素(HA)和神经氨酸酶(NA)抗原性不同,A型流感病毒可分为18个HA亚型和11个NA亚型。
H7N9亚型禽流感病毒(AIV)能够跨越宿主屏障感染人类,引起发病甚至死亡,其中感染病人的死亡率高达约40%。疫苗接种是预防流感病毒感染的最有效有段,但是现有技术中仍没有人用的H7N9亚型禽流感疫苗。临床上,主要采用抗病毒药物,如奥司他韦和金刚烷胺等,进行流感病毒感染的治疗。抗病毒药物的最大问题是病毒的耐药性,临床研究发现:流感病毒在药物暴露期间会出现耐药性突变,导致对奥司他韦出现耐药。此外,抗流感病毒药物必须在症状出现后48小时内用药,治疗时间窗较短,限制了药物的治疗效果。基于H7N9亚型AIV对公共卫生的潜在威胁,研制H7N9亚型禽流感的新型防治药物迫在眉睫。单克隆抗体具有高度的特异性和良好的安全性,在癌症、自身免疫性疾病和重大传染病的治疗方面得到了广泛应用,取得了良好的效果。一些单克隆抗体药物已被批准用于预防和治疗多种传染病,例如预防呼吸道合胞病毒感染的Palivizumab和防治埃博拉病毒感染的Ansuvimab。
单克隆抗体的接种途径对其保护效力具有重要影响。现有的单克隆抗体药物大多数经过静脉注射途径进行给药,可保证药物的全身性分布和较高的血药浓度。但是,静脉接种途径却难以将药物递送至呼吸道等部位。H7N9亚型AIV主要在人的呼吸系统,包括气管与肺,中复制,导致急性上呼吸道感染和肺炎。单克隆抗体药物经静脉接种,在呼吸道与肺脏中的药物浓度低,对H7N9亚型AIV的抑制效率低。为保证抗体对呼吸系统中病毒的有效抑制,单克隆抗体经静脉途径接种往往需要增大剂量,增加治疗成本。为解决单克隆抗体向呼吸系统靶向递送的问题,研究者利用基因工程技术将抗体转变为适合鼻腔接种的类型,如纳米抗体、IgM,或利用可在呼吸道复制的病毒为载体递送抗体分子。但是,基因工程技术表达的重组抗体产量较低,且病毒载体技术需要复杂的分子生物学与病毒学技术,不利于工业化制备。
发明内容
本发明的目的在于克服现有背景技术中的不足,提供一种H7N9亚型禽流感病毒中和性单克隆抗体,应用于鼻内接种,提高对H7N9亚型禽流感病毒的抵抗效力。
为达到上述目的,本发明所采用的技术方案是:
本发明提供一种H7N9亚型禽流感病毒中和性单克隆抗体,所述单克隆抗体为IgG1、κ型,所述单克隆抗体包括重链可变区氨基酸及轻链可变区氨基酸。
所述重链可变区氨基酸序列包括SEQ ID No.5所示的HCDR1、SEQ ID No.6所示的HCDR2、SEQ ID No.7所示的HCDR3;所述轻链可变区氨基酸序列包括SEQ ID No.8所示的LCDR1、SEQ ID No.9所示的LCDR2、SEQ ID No.10所示的LCDR3。
进一步的,所述重链可变区氨基酸序列如SEQ ID NO.2所示,轻链可变区氨基酸序列如SEQ ID NO. 4所示。
进一步的,所述单克隆抗体的重链可变区核苷酸序列如SEQ ID NO.1所示,轻链可变区的核苷酸序列如SEQ ID NO. 3所示,能够利用常规基因工程或蛋白质工程的方法表达抗体,避免了杂交瘤细胞长期冻存抗体基因的丢失,同时有利于在基因和蛋白水平上进行抗体优化,提高抗体的特异性和亲和力。
本发明提供一种表达载体,所述表达载体含有上述所述的重链可变区核苷酸序列及轻链可变区的核苷酸序列。
本发明还提供一种宿主细胞,所述宿主细胞含有上述所述的表达载体。
进一步的,所述单克隆抗体由杂交瘤细胞分泌产生。
进一步的,所述单克隆抗体能够与H7N9亚型禽流感病毒血凝素蛋白的抗原表位特异性结合,所述抗原表位为血凝素蛋白第151位甘氨酸,与不同H7亚型HA蛋白具有交叉反应性。
进一步的,所述单克隆抗对H7N9病毒具有血凝抑制与病毒中和活性,应用在鼻内接种药物的制备,显著提高保护效力。
进一步的,所述单克隆抗在制备H7N9亚型禽流感病毒检测产品中的应用。
与现有技术相比,本发明所达到的有益效果:
本发明提供的一种H7N9亚型禽流感病毒中和性单克隆抗体,对H7N9病毒具有血凝抑制与病毒中和活性,应用在鼻内接种,有效提高保护效力,为研制喷鼻抗体制剂提供参考;
本发明提供的抗体识别HA蛋白Antigenic Site A的高度保守的G151位点,与不同H7亚型HA蛋白具有交叉反应性,为研制广谱性抗体药物提供有利信息;
基于本发明提供的单克隆抗体基因序列,可以利用常规基因工程或蛋白质工程的方法表达抗体,避免了杂交瘤细胞长期冻存抗体基因的丢失,同时有利于在基因和蛋白水平上进行抗体优化,提高抗体的特异性和亲和力;
本发明提供的单克隆抗体能够特异性识别H7亚型禽流感病毒血凝素蛋白保守的抗原表位,且具有较强的病毒中和活性,为探究H7亚型禽流感病毒的免疫学特性及病毒治疗制剂的研制提供了新的生物材料。
附图说明
图1 为本发明实施例提供的H7N9亚型禽流感病毒中和性单克隆抗体的生物学活性鉴定图,其中图A表示抗体的HI与VN效价,图B为抗体的终点反应浓度;
图2 含有单克隆抗体重链与轻链可变区基因的质粒PCR鉴定图。每个基因各挑取两个质粒进行PCR鉴定。
图3 为本发明实施例提供的H7N9亚型禽流感病毒中和性单克隆抗体识别抗原表位的定位图,其中图A为抗体针对野生病毒与逃逸病毒的HI效价,图B为抗体识别表位G151在HA蛋白上的空间定位;
图4 为本发明实施例提供的H7N9亚型禽流感病毒中和性单克隆抗体识别抗原表位的验证结果图;
图5 为本发明实施例提供的H7N9亚型禽流感病毒中和性单克隆抗体的交叉反应性测定图;
图6 为本发明实施例提供的H7N9亚型禽流感病毒中和性单克隆抗体经腹腔接种小鼠的预防性保护效力图,其中图A为小鼠的体重曲线;图B为小鼠在H7N9病毒感染后的生存率;
图7为本发明实施例提供的H7N9亚型禽流感病毒中和性单克隆抗体经腹腔接种小鼠的治疗性保护效力图,其中图A为攻毒前12 h使用抗体的小鼠体重曲线;图B为攻毒前12h使用抗体的小鼠生存率;图C为攻毒前24 h使用抗体的小鼠体重曲线;图D为攻毒前24h使用抗体的小鼠生存率;
图8 为本发明实施例提供的H7N9亚型禽流感病毒中和性单克隆抗体经腹腔、鼻内接种的预防性与治疗性保护效力图,其中图A为预防性保护试验小鼠体重曲线;图B为预防性保护试验小鼠生存率;图C为治疗性保护试验(攻毒前48 h使用抗体)小鼠体重曲线;图D为治疗性保护试验(攻毒前48 h使用抗体)小鼠生存率;图E为治疗性保护试验(攻毒前72 h使用抗体)小鼠体重曲线;图F为治疗性保护试验(攻毒前72 h使用抗体)小鼠生存率。
具体实施方式
下面结合附图对本发明作进一步描述。以下实施例仅用于更加清楚地说明本发明的技术方案,而不能以此来限制本发明的保护范围。
实施例一
进行H7N9亚型禽流感病毒中和性单克隆抗体的制备与鉴定,本实施例使用杂交瘤细胞技术制备H7N9亚型禽流感病毒特异性的单克隆抗体,并使用血凝抑制试验筛选具有病毒中和活性的抗体。由于抗体的中和活性大部分是针对病毒的HA蛋白,但是大多数流感病毒单克隆抗体的制备使用经超速离心纯化的全病毒作为免疫原,这样可能会降低中和抗体的筛选几率。对于免疫原的选择在本技术领域中仍然属于需要技术人员经过一定程度的选择和研究才可以进行的程度,本发明和本实施例优选在昆虫细胞中表达的H7N9 HA蛋白作为单克隆抗体制备的免疫原,提高病毒中和活性抗体的筛选效率。
具体步骤如下:
使用10 μg 纯化的HA蛋白与弗氏佐剂配伍,免疫8只SPF级小鼠,并间隔2周进行加强免疫,共3次;
定期监测血清抗体效价并据此进行加强免疫;
按照常规的杂交瘤细胞技术进行单克隆抗体的制备;以H7N9病毒为检测抗原,用血凝抑制试验筛选阳性杂交瘤克隆。用阳性杂交瘤细胞腹腔注射小鼠,收集腹水,用Protein G亲和层析法纯化抗体;
使用微量中和试验测定抗体针对H7N9亚型AIV的病毒中和活性。
如图1的实验结果显示,筛选到一株具有血凝抑制活性的单克隆抗体H7N9-NeuAb,其HI效价为512,终点浓度为1.5 μg/mL。
该单克隆抗体为IgG1亚类、κ型的鼠源抗体,抗体对H7N9亚型AIV的中和效价为5120,终点浓度为0.15 μg/mL。
实施例二
进行单克隆抗体可变区的序列测定,具体步骤如下:
① 取状态良好的杂交瘤细胞(106个)加1mL RNAiso Plus处理,再加入三氯甲烷反应后取上清液,上清液用异丙醇沉淀,沉淀干燥后用加水溶解,琼脂糖电泳检测RNA条带。
②根据TaKaRa反转录试剂盒操作过程进行反转录。
③使用百英生物杂交瘤细胞测序PCR试剂盒进行PCR,扩增轻链与重链可变区,琼脂糖电泳检测条带。
④PCR产物连接至pUC-19载体,转化TOP10感受态细胞。
⑤挑取过夜平板中单菌落,用M13+/ -引物进行菌落PCR,电泳鉴定阳性克隆。
⑥随机选择6个阳性菌,阳性菌液用M13+引物测序。
⑦随机挑取含有轻链与重链可变区基因的阳性质粒各两个,用M13+/ -引物进行PCR,扩增抗体可变区基因。
本实施例成功构建了含有抗体轻链与重链可变区基因的质粒(图2),并测定了轻链与重链可变区的氨基酸序列。序列分析显示,重链可变区为IgG1亚型,轻链可变区为Kappa亚型。重链可变区的HCDR1序列为DFWIH,HCDR2序列为AIDTSDSYTTYNQNFRG,HCDR3序列为LDDGFYNYAMDY;轻链可变区的LCDR1序列为KSSQSVLNSSNQKNYLA,LCDR2序列为WASTREA,LCDR3序列为HQYLSSYT。
可实施的,本发明提供的一种H7N9亚型禽流感病毒中和性单克隆抗体,其重链可变区氨基酸序列如SEQ ID NO.2所示,轻链可变区氨基酸序列如SEQ ID NO. 4所示;单克隆抗体的重链可变区核苷酸序列如SEQ ID NO.1所示,轻链可变区的核苷酸序列如SEQ IDNO. 3所示。
本发明提供一种表达载体,其中表达载体含有如SEQ ID NO.1所示的重链可变区核苷酸序列,以及如SEQ ID NO. 3所示的轻链可变区的核苷酸序列。
本发明还提供一种宿主细胞,其中宿主细胞含有上述的表达载体。
实施例三
本实施例进行H7N9亚型AIV单克隆抗体识别抗原表位的鉴定及验证,步骤分别如下:
(1)抗原表位的鉴定
由于筛选的单克隆抗体具有很强的病毒中和活性,利用免疫逃逸株筛选的方法鉴定其识别的抗原表位,简要流程如下:
①通过连续倍比稀释法将H7N9病毒稀释到106 EID50/500 μL,再取等量的100倍及200倍稀释的抗体与病毒混合,置于室温作用1 h;
②把上述混合液分别接种10日龄SPF鸡胚,0.1 mL/胚,每种混合液接种4个鸡胚,于35℃孵化箱中培养,每隔12 h观察并记录,在48-72 h期间收集死亡鸡胚,放置4℃冰箱收缩血管,收取尿囊液,测定血凝效价;
③将血凝活性阳性的尿囊液再做连续倍比稀释,至105、106、107和108 EID50/500 μL,取0.5 mL 100倍及200倍稀释的抗体与稀释的尿囊液等量混合,置于室温作用1 h;
④按步骤②进行鸡胚接种与病毒检测;
⑤筛选出上述过程中满足血凝活性阳性且稀释度最大两个条件的尿囊液,与抗体进行HI试验。若尿囊液与抗体的HI效价比野生型病毒低3个滴度(3 log2) 时,则可判定该病毒为抗体的免疫逃逸株;
⑥将获得的免疫逃逸株在鸡胚传代1次,收集尿囊液,RT-PCR扩增HA基因,测序后与野生型病毒的HA基因进行序列比对。
(2)抗原表位的验证
为了验证鉴定的表位是单克隆抗体识别的抗原表位,利用点突变的方法将表位中的关键氨基酸进行突变,利用反向遗传技术拯救重组病毒,测定病毒与单克隆抗体的HI与中和效价,具体过程如下:
①将拯救出的表位突变的重组病毒与抗体进行HI试验。与野生型毒株相比,重组病毒与抗体的HI效价下降8倍以上,则表明位点突变能够显著影响抗体与病毒的HI反应性。
②用微量中和试验评价单克隆抗体与表位突变的重组H7N9病毒的中和活性。与野生型毒株相比,重组病毒与抗体的中和效价下降100倍以上,则表明突变的位点能够显著影响单抗针对病毒的中和活性。
如图3中图A所示,为抗体针对野生病毒与逃逸病毒的HI效价,结果显示,H7N9病毒在单克隆抗体H7N9-NeuAb的免疫压力下,逃逸毒株与抗体的HI效价下降8倍,测序显示逃逸毒株的HA基因发生G151E突变。
如图4所示,为H7N9亚型禽流感病毒中和性单克隆抗体识别抗原表位的验证结果图,用反向遗传技术拯救含有HA G151E突变的H7N9病毒,突变病毒与抗体的HI效价下降16倍,病毒中和效价下降16倍,证实抗体H7N9-NeuAb特异性识别HA蛋白第151位甘氨酸,如图3中的图B所示,为抗体识别表位G151在HA蛋白上的空间定位。
实施例四
本实施例进行单克隆抗体与不同亚型流感病毒HA蛋白的交叉反应性,为评价单克隆抗体与不同亚型流感病毒HA蛋白的交叉反应性,选用H1N1、H2N2、H5N1、H9N2、H3N2、H4N6、H7N7、H7N9、H15N8亚型病毒的HA蛋白,进行ELISA试验,步骤如下:
①取100 μL碳酸盐-碳酸氢盐缓冲液(0.05 M Na2CO3/NaHCO3, pH 9.6)稀释的HA蛋白(0.25 μg/mL)加入96孔酶标板,4℃包被过夜;
②用PBST(含0.05% Tween-20的PBS)洗涤3次,然后用100 μL含5 mg/mL BSA的PBST进行封闭,37℃孵育1 h;
③PBST洗3次,每孔加入倍比稀释的单克隆抗体,100 μL/孔,37℃孵育1 h;
④PBST洗涤3次,每孔加入100 μL HRP标记的羊抗鼠二抗(1: 5000),37℃孵育1h;
⑤PBST洗涤3次,每孔加入100 μL的TMB底物,室温避光孵育10 min 后,再加入50μL的2 M H2SO4终止反应;
⑥在450 nm波长下读取吸光值。计算样本吸光值与阴性对照吸光值的比值,比值≥ 2.1认定为阳性反应。
如图5所示,为H7N9亚型禽流感病毒中和性单克隆抗体的交叉反应性测定图,结果显示,单克隆抗体仅对H7亚型(H7N7与H7N9)HA蛋白具有结合性,对H1N1、H2N2、H5N1、H9N2、H3N2、H4N6、H15N8亚型的HA蛋白无结合性,说明该抗体对H7亚型病毒具有一定的交叉反应性。
实施例五
本实施例进行单克隆抗体经腹腔接种在小鼠模型中的保护效力评价,分为预防性保护效力及治疗性保护效力试验,步骤分别如下:
(1)抗体的预防性保护效力试验
为了评估单克隆抗体的预防保护效果,先在攻毒前2 h经腹腔注射对应剂量的抗体,每组15只小鼠,设置注射PBS的攻毒对照组及正常对照组。单克隆抗体使用5、10、20、30mg/kg的剂量。2 h后,以1%戊巴比妥钠进行麻醉,麻醉剂量为150 μL/只,然后用10 MLD50的H7N9病毒经滴鼻途径进行攻毒,每日观察体况并称量体重,合计14 天。对体重下降超过25%的小鼠进行安乐死。在攻毒后第1、3、5天每组各扑杀3只小鼠,采集肺组织,加入1 mL无菌四抗PBS,反复冻融3次后匀浆,离心取上清10倍倍比稀释,接种鸡胚进行病毒定量。
如图6所示,为H7N9亚型禽流感病毒中和性单克隆抗体经腹腔接种小鼠的预防性保护效力图,其中图A为小鼠的体重曲线;图B为小鼠在H7N9病毒感染后的生存率。结果显示,在H7N9病毒感染前,经腹腔途径接种5、10、20、30 mg/kg的抗体,均能够完全保护小鼠不发病与死亡,保护率为100%,说明单克隆抗体的预防性保护效力较高。
(2)抗体的治疗性保护效力试验
为了评估单克隆抗体是否具有治疗作用,先以H7N9病毒经滴鼻途径感染小鼠(n =5),于感染后第12和24 h施用抗体,以体重变化与生存率为标准评价抗体对小鼠的保护性。
如图7所示,为H7N9亚型禽流感病毒中和性单克隆抗体经腹腔接种小鼠的治疗性保护效力图,其中图A为攻毒前12 h使用抗体的小鼠体重曲线;图B为攻毒前12 h使用抗体的小鼠生存率;图C为攻毒前24 h使用抗体的小鼠体重曲线;图D为攻毒前24h使用抗体的小鼠生存率。结果显示,经腹腔接种,单克隆抗体在高剂量下(20、30mg/kg)能够提供针对H7N9 病毒感染的治疗性保护作用,但是抗体的治疗时间窗较短,为12 h。
实施例六
本实施例进行单克隆抗体经腹腔与鼻内接种的保护效力比较,为了评估单克隆抗体经鼻内接种是否比经腹腔接种具有优势,抗体以0.1、0.3 mg/kg的剂量分别经腹腔注射与鼻内两种途径接种小鼠;2 h后,用7.5 MLD50的H7N9病毒经滴鼻途径进行攻毒,每日观察体况并称量体重,合计14天。对体重下降超过25%的小鼠进行安乐死。在攻毒后第1、3、5天每组各扑杀3只小鼠,采集肺组织,加入1 mL无菌四抗PBS,反复冻融3次后匀浆,离心取上清10倍比稀释,接种MDCK细胞进行病毒定量。
此外,为了评价单克隆抗体经腹腔和鼻内途径接种的治疗性保护效力,先用H7N9病毒感染小鼠,于感染后的第24、48、72 h,抗体以1、3、10 mg/kg的剂量进行接种。以体重变化与生存率为标准评价抗体对小鼠的保护性。
图8为H7N9亚型禽流感病毒中和性单克隆抗体经腹腔、鼻内接种的预防性与治疗性保护效力图,其中图8中的图A为预防性保护试验小鼠体重曲线;图B为预防性保护试验小鼠生存率,结果显示,预防保护性试验中,0.3mg/kg的单克隆抗体经腹腔接种仅能提供针对H7N9病毒感染的20%的保护率,而相同剂量的抗体经鼻内接种则可提供100%的保护,且更低剂量(0.1mg/kg)经鼻内接种仍然能够提供60%的保护率。
图8中的图C为治疗性保护试验(攻毒前48 h使用抗体)小鼠体重曲线;图D为治疗性保护试验(攻毒前48 h使用抗体)小鼠生存率;图E为治疗性保护试验(攻毒前72 h使用抗体)小鼠体重曲线;图F为治疗性保护试验(攻毒前72 h使用抗体)小鼠生存率,治疗保护性试验中,在病毒感染后48 h,鼻内接种10mg/kg的抗体能够提供80%的保护率。综合以上结果说明,单克隆抗体经鼻内接种的保护效力强于腹腔接种途径。
本发明涉及到的核苷酸或氨基酸序列具体如下:
SEQ ID NO. 1(H7N9-NeuAb重链可变区的核苷酸序列)
CAGGTCCAACTGCAGCAGCCTAGGGCTGAGCTTGTGATGCCTGGGGCCTCAGTGAGGATGTCCTGTCAGGCTTCTGGCTACACATTCACTGACTTCTGGATACACTGGGTGAAGCAGAGGCCTGGGCAAGGCCTTGAGTGGATCGGAGCGATTGATACTTCTGATTCTTATACTACCTACAATCAAAACTTCAGGGGCAAGGCCTCATTGACTGTCGACGAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTGCAAGATTAGATGATGGTTTCTACAACTATGCTATGGACTACTGGGGTCAAGGCACCTCAGTCACCGTCTCCTCA;
SEQ ID NO. 2(H7N9-NeuAb重链可变区的氨基酸序列)
QVQLQQPRAELVMPGASVRMSCQASGYTFTDFWIHWVKQRPGQGLEWIGAIDTSDSYTTYNQNFRGKASLTVDESSSTAYMQLSSLTSEDSAVYYCARLDDGFYNYAMDYWGQGTSVTVSS;
SEQ ID NO. 3(H7N9-NeuAb轻链可变区的核苷酸序列)
AACATTATGATGACACAGTCGCCATCATCTCTGGCTGTGTCTGCAGGAGAAAAGGTCACTCTGAACTGTAAGTCCAGTCAAAGTGTTTTAAACAGTTCAAATCAGAAGAACTACTTGGCCTGGTACCAGCAGAGACCAGGACAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAAGCTGGTGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTTACTCTTACCATCAGCAGTGTAAAAGCTGAAGACCTGGCAGTTTATTACTGTCATCAATACCTCTCCTCGTACACGTTCGGAGGGGGGACCAAATTGAAAATAAAA;
SEQ ID NO. 4(H7N9-NeuAb轻链可变区的氨基酸序列)
NIMMTQSPSSLAVSAGEKVTLNCKSSQSVLNSSNQKNYLAWYQQRPGQSPKLLIYWASTREAGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCHQYLSSYTFGGGTKLKIK;
SEQ ID NO. 5(H7N9-NeuAb重链可变区的HCDR1序列)
DFWIH;
SEQ ID NO. 6(H7N9-NeuAb重链可变区的HCDR2序列)
AIDTSDSYTTYNQNFRG;
SEQ ID NO. 7(H7N9-NeuAb重链可变区的HCDR3序列)
LDDGFYNYAMDY;
SEQ ID NO. 8(H7N9-NeuAb轻链可变区的LCDR1序列)
KSSQSVLNSSNQKNYLA;
SEQ ID NO. 9(H7N9-NeuAb轻链可变区的LCDR2序列)
WASTREA;
SEQ ID NO. 10(H7N9-NeuAb轻链可变区的LCDR3序列)
HQYLSSYT。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明技术原理的前提下,还可以做出若干改进和变形,这些改进和变形也应视为本发明的保护范围。
Claims (8)
1.一种H7N9亚型禽流感病毒中和性单克隆抗体,其特征在于,所述单克隆抗体为IgG1、κ型,所述单克隆抗体包括重链可变区氨基酸及轻链可变区氨基酸,
所述重链可变区氨基酸序列包括SEQ ID No.5所示的HCDR1、SEQ ID No.6所示的HCDR2、SEQ ID No.7所示的HCDR3;所述轻链可变区氨基酸序列包括SEQ ID No.8所示的LCDR1、SEQ ID No.9所示的LCDR2、SEQ ID No.10所示的LCDR3。
2. 根据权利要求1所述的一种H7N9亚型禽流感病毒中和性单克隆抗体,其特征在于,所述重链可变区氨基酸序列如SEQ ID NO.2所示,轻链可变区氨基酸序列如SEQ ID NO. 4所示。
3. 根据权利要求1所述的一种H7N9亚型禽流感病毒中和性单克隆抗体,其特征在于,所述单克隆抗体的重链可变区核苷酸序列如SEQ ID NO.1所示,轻链可变区的核苷酸序列如SEQ ID NO. 3所示。
4.一种表达载体,其特征在于,所述表达载体含有根据权利要求3所述H7N9亚型禽流感病毒中和性单克隆抗体的重链可变区核苷酸序列及轻链可变区的核苷酸序列。
5.一种宿主细胞,其特征在于,所述宿主细胞含有权利要求4所述的表达载体。
6.根据权利要求1所述的一种H7N9亚型禽流感病毒中和性单克隆抗体,其特征在于,所述单克隆抗体由杂交瘤细胞分泌产生。
7.权利要求1-3任一所述的单克隆抗体在制备鼻内接种药物的应用,所述单克隆抗体对H7N9病毒具有血凝抑制与病毒中和活性。
8.权利要求1-3任一所述的单克隆抗体在制备H7N9亚型禽流感病毒检测产品中的应用。
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