CN113355344B - 一种流感病毒ns1蛋白的表达质粒、重组蛋白和特异性单克隆抗体及应用 - Google Patents
一种流感病毒ns1蛋白的表达质粒、重组蛋白和特异性单克隆抗体及应用 Download PDFInfo
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Abstract
本发明提供了一种流感病毒NS1蛋白的表达质粒、重组蛋白和特异性单克隆抗体及应用，涉及抗体工程技术领域。本发明通过体外重组表达A型流感病毒的NS1蛋白，筛选出了对流感病毒感染的小鼠具有治疗效果的单克隆抗体。在本发明实施例中，所述单克隆抗体，对A型流感病毒感染的小鼠具有治疗效果，为预防和治疗流感病毒提供了一种策略，具有广阔的临床应用前景。
Description
技术领域
本发明属于抗体工程技术领域，具体涉及一种流感病毒NS1蛋白的表达质粒、重组蛋白和特异性单克隆抗体及应用。
背景技术
流感病毒是一种能够造成人类和动物患流行性感冒的RNA病毒，属正黏病毒科，为分节段的单负链RNA病毒。流感病毒易发生抗原变异，抗原漂移与抗原转变是主要的抗原变异形式。按核蛋白核基质蛋白抗原性的不同，流感病毒分为A、B、C、D四型，A型流感病毒最易发生变异，已引起多次大流行。B型流感病毒对人类致病性也比较强，但是人们还没有发现B型流感病毒引起过世界性大流行；C型流感病毒只引起人类不明显的或轻微的上呼吸道感染，很少造成流行。流感病毒在健康人群的感染多为自限性，尚无特效药物。接种疫苗是预防流感病毒的最有效的方法，但由于流感病毒容易发生抗原漂移和抗原转换，导致每年流行株的变化、疫苗免疫力的差异，在大流行流感发生时，生产用毒株选择的滞后导致疫苗不能够快速上市，难以在病毒流行初期为人群提供有效的免疫保护。因此，开发针对流感病毒有效的预防和治疗药物依然任重道远。
发明内容
有鉴于此，本发明的目的在于提供一种流感病毒NS1蛋白的表达质粒、重组蛋白和特异性单克隆抗体及应用，所述单克隆抗体对A型流感病毒具有治疗效果，为流感病毒的治疗提供一种参考。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种包含A型流感病毒NS1保守片段的重组质粒，所述NS1保守片段的编码基因包括SEQ ID NO.1所示的序列。
优选的，所述重组质粒的基础载体包括pET28a。
优选的，所述NS1保守片段的氨基酸序列包括SEQ ID NO.2所示的序列。
本发明还提供了一种表达A型流感病毒NS1保守片段的重组菌，所述重组菌的基因组包含上述重组质粒。
本发明还提供了一种与重组NS1蛋白发生特异性反应的单克隆抗体，所述单克隆抗体包括5F11和3E5，所述5F11的重链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.3~SEQ ID NO.5所示；所述5F11的轻链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.8~SEQ ID NO.10所示；
所述3E5的重链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.13~SEQ ID NO.15所示；所述3E5的轻链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.18~SEQ ID NO.20所示；
所述重组NS1蛋白由上述重组菌经IPTG诱导得到。
优选的，所述5F11的重链可变区的氨基酸序列如SEQ ID NO.6所示，所述5F11的轻链可变区的氨基酸序列如SEQ ID NO.11所示。
优选的，编码所述5F11的重链可变区的核苷酸序列如SEQ ID NO.7所示，编码所述5F11的轻链可变区的核苷酸序列如SEQ ID NO.12所示。
优选的，所述3E5的重链可变区的氨基酸序列如SEQ ID NO.16所示，所述3E5的轻链可变区的氨基酸序列如SEQ ID NO.21所示。
优选的，编码所述3E5的重链可变区的核苷酸序列如SEQ ID NO.17所示，编码所述3E5的轻链可变区的核苷酸序列如SEQ ID NO.22所示。
本发明还提供了上述单克隆抗体在制备预防和/或治疗A型流感病毒感染引发疾病的疫苗或药物中的应用。
有益效果：本发明通过体外重组表达A型流感病毒的NS1蛋白，筛选出了对流感病毒感染的小鼠具有治疗效果的单克隆抗体。在本发明实施例中，所述单克隆抗体，对A型流感病毒感染的小鼠具有治疗效果，这是除HA和NA保护性抗体之外的治疗流感病毒的又一方法。由于针对的是A型流感病毒NS1中的保守序列，既然该抗体能够治疗感染H1N1的小鼠，对其他的A型流感病毒也具有一定的理论保护作用，即为预防和治疗流感病毒提供了一种策略，具有广阔的临床应用前景。
附图说明
图1为pET28a-NS1蛋白的SDS-PAGE鉴定图；
图2为重组NS1蛋白与小鼠血清的反应性；
图3为杂交瘤细胞上清检测结果；
图4为单克隆抗体的SDS-PAGE鉴定图；
图5为纯化的单克隆抗体与NS1蛋白的结合力；
图6为单克隆抗体交叉反应分析；
图7为小鼠感染和给药后的体重变化。
具体实施方式
本发明提供了一种包含A型流感病毒NS1保守片段的重组质粒，所述NS1保守片段的编码基因包括SEQ ID NO.1所示的序列。
在本发明中，NS1蛋白（Nonstructral protein 1）是流感病毒第8条RNA编码的非结构蛋白，是一种RNA结合蛋白，具有重要的调节活性。NS1蛋白具有高度的保守性，仅存在于病毒感染的细胞内，并未被包装进病毒颗粒内；NS1蛋白在调节流感病毒的毒力和致病性方面发挥着重要作用，其可通过抑制宿主细胞蛋白的合成、诱导细胞凋亡及拮抗干扰素的产生等方面调节机体的抗病毒反应，从宿主和病毒两方面调节病毒的毒力，间接地发挥增强病毒致病性的作用。NS1蛋白虽然为流感病毒的非结构蛋白，却在病毒复制中具有不可替代的作用。在本发明中，所述NS1保守片段经过序列优化，形成SEQ ID NO.1所示的序列；且该片段的氨基酸序列优选如SEQ ID NO.2所示。
本发明所述重组质粒的基础载体优选包括pET28a，将所述NS1保守片段的编码基因插入pET28a的BamHI和SalI酶切位点之间。
本发明还提供了一种表达A型流感病毒NS1保守片段的重组菌，所述重组菌的基因组包含上述重组质粒。
本发明优选将上述重组质粒pET28a-NS1转化BL21（DE3）感受态细胞，转化菌涂布于LB琼脂平板（含50μg/mL卡那霉素），37℃过夜培养。本发明对所述转化的方法并没有特殊限定，利用本领域的常规转化方法即可。
本发明还提供了一种利用上述重组菌诱导产生重组NS1蛋白的方法，优选包括以下步骤：挑取上述LB琼脂平板过夜培养的单菌落接入5 mL LB培养基（含50μg/mL卡那霉素）中，37℃，220转/分振荡培养过夜。按培养基总体积的1%的量接种到LB培养基（含50μg/mL卡那霉素）中，37℃，220转/分振荡培养3小时左右，至OD600为0.5，于16℃降温1.5小时，加终浓度为0.1 mM IPTG，16℃，220转/分诱导16小时后收集菌体，菌体破碎后纯化。在本发明中，因表达的重组蛋白均带有组氨酸标签，因此用GE公司的AKTA Start和HisTrapTM HP层析柱进行纯化。
本发明还提供了一种与重组NS1蛋白发生特异性反应的单克隆抗体，所述单克隆抗体包括5F11和3E5，所述5F11的重链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.3~SEQ ID NO.5所示；所述5F11的轻链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.8~SEQ ID NO.10所示；
所述3E5的重链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.13~SEQ ID NO.15所示；所述3E5的轻链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.18~SEQ ID NO.20所示；
所述重组NS1蛋白由上述重组菌经IPTG诱导得到。
本发明所述5F11的重链可变区的氨基酸序列优选如SEQ ID NO.6所示，所述5F11的轻链可变区的氨基酸序列优选如SEQ ID NO.11所示。本发明编码所述5F11的重链可变区的核苷酸序列优选如SEQ ID NO.7所示，编码所述5F11的轻链可变区的核苷酸序列优选如SEQ ID NO.12所示。
本发明所述3E5的重链可变区的氨基酸序列优选如SEQ ID NO.16所示，所述3E5的轻链可变区的氨基酸序列优选如SEQ ID NO.21所示。本发明编码所述3E5的重链可变区的核苷酸序列优选如SEQ ID NO.17所示，编码所述3E5的轻链可变区的核苷酸序列优选如SEQID NO.22所示。
本发明对所述单克隆抗体的制备方法并没有特殊限定，优选通过杂交瘤结合腹水的方法制备。在本发明中，优选利用A型流感病毒鼠肺适应株PR8/H1N1对所述杂交瘤细胞的制备进行说明，但不能仅将其认定为本发明的保护范围。本发明所述杂交瘤细胞的制备方法，优选包括：（1）利用A型流感病毒连续滴鼻感染2次BALB/c雌性小鼠，14日后将纯化的重组NS1蛋白加等量的弗氏完全佐剂对所述BALB/c雌性小鼠进行免疫；
（2）取免疫小鼠的所有脾细胞与处于对数生长期的SP2/0骨髓瘤细胞进行融合，置于HAT培养基中进行筛选培养，得所述杂交瘤细胞。
本发明步骤（1）所述滴鼻感染优选包括A型流感病毒用无菌PBS稀释100倍，戊巴比妥钠溶液进行腹腔麻醉的BALB/c雌性小鼠，按25μL/只剂量连续滴鼻感染2次，间隔一周。本发明进行步骤（1）所述免疫时，NS1蛋白免疫总剂量为30μg/只，背部皮下注射。本发明在所述免疫后，于融合的前3~5日优选再次加强免疫，腹腔注射重组NS1蛋白，30μg/只。
本发明步骤（2）所述筛选培养，优选包括：待融合细胞长至孔底1/2时，以重组NS1蛋白和表达载体pET28a进行检测，筛选出能与重组NS1蛋白反应，但不与表达载体pET28a反应的细胞株，并通过2~3次连续的有限稀释克隆法达到阳性率为100%时，对细胞株进行扩大培养和冻存。
本发明还提供了一种单克隆抗体的制备方法，包括以下步骤：筛选上述杂交瘤细胞中与重组NS1蛋白反应阳性率100%的杂交瘤细胞进行扩大培养，腹腔注射雌性BALB/c小鼠，收集腹水，离心后纯化，得单克隆抗体。
本发明优选利用上述扩大培养和冻存的杂交瘤细胞进行腹腔注射，所述腹腔注射的量优选为0.2ml（含2.5×106个细胞）。本发明所述雌性BALB/c小鼠在腹腔注射前，优选还包括利用弗氏不完全佐剂100μL对小鼠进行腹腔注射预处理。本发明在所述腹腔注射后约10日，待小鼠腹部明显肿大时，使用无菌注射器针头采集腹水。
本发明还提供了从所述腹水中纯化出单克隆抗体的方法，优选包括：将腹水以12000r/min离心5分钟，取上清5ml，加入20ml醋酸缓冲液（68mmol/L，pH值4.5），混合均匀后，缓慢加入50μL正辛酸，边加边搅动，加完后继续搅拌30分钟，在2~8℃下，以12000r/min离心30分钟，取上清。将上清用脱脂棉过滤，并用NaOH（1mol/L）调节pH值至7.0~7.4，按终体积50%（V/V）的比例加入饱和硫酸铵，边加边搅拌，加完后继续搅拌30分钟，置2~8℃下沉淀4~5小时，在2~8℃下，以12000r/min离心30分钟，取沉淀。将沉淀用3ml Tris-HCl（10mmol/L，pH值9.0）重悬，装入透析袋（MW：8000~14000），在2~8℃下，置2L Tris-HCl（10mmol/L，pH值9.0）溶液中，透析14小时。将透析袋中的液体转移到离心管中，以12000r/min离心5分钟，上清即为纯化的单克隆抗体。
本发明还提供了上述单克隆抗体在制备预防和/或治疗A型流感病毒感染引发疾病的疫苗或药物中的应用。
在本发明实施例中，利用所述单克隆抗体，可治疗A型流感病毒感染的小鼠，所以可将其用于制备相应的疫苗或药物。
下面结合实施例对本发明提供的一种重组质粒、重组菌和与重组蛋白特异反应的杂交瘤细胞、单克隆抗体及应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
在本发明实施例中，所用的试剂和材料如非特殊说明，均为本领域的常规市售产品：
原核表达载体pET28a； A型流感病毒鼠肺适应株PR8/H1N1及其感染小鼠阳性血清由中国国家流感中心惠赠。Balb/C小鼠购自北京华阜康生物科技股份有限公司（SCXK（京）2019-0008）。
实施例1
1、A型流感病毒NS1原核表达
根据序列比对分析，选取A型流感病毒相对保守的NS1片段690nt，经密码子优化后合成（SEQ ID NO.1），克隆至原核表达载体pET28a，基因合成由安徽通用生物公司完成。
合成的重组质粒pET28a-NS1按常规方法转化BL21（DE3）感受态细胞（《分子克隆》第三版，科学出版社），转化菌涂布于LB琼脂平板（含50μg/mL卡那霉素），37℃过夜培养。挑取单菌落接入5 mL LB培养基（含50μg/mL卡那霉素）中，37℃，220转/分振荡培养过夜。按培养基总体积的1%的量接种到LB培养基（含50μg/mL卡那霉素）中，37℃，220转/分振荡培养3小时左右，至OD600为0.5，于16℃降温1.5小时，加终浓度为0.1 mM IPTG，16℃，220转/分诱导16小时后收集菌体。
2、重组蛋白的纯化
因表达的重组蛋白均带有组氨酸标签，用GE公司的AKTA Start和HisTrapTM HP层析柱进行纯化。A缓冲液为50mM Tris-Hcl pH 8.0，B缓冲液为50mM Tris-Hcl pH 8.0 0.5M咪唑，层析柱用A缓冲液平衡，然后将发酵好的菌液8000rpm离心20min，沉淀用A液重悬，在冰水中超声破碎30min，间隔5秒超声5秒，12000rpm离心30min，上清用0.22微米滤器过滤，上样，层析柱用A缓冲液洗涤，最后用B缓冲液梯度洗脱，通过SDS-PAGE蛋白凝胶电泳观察纯化情况。选择目的蛋白收集峰，用0.01M PBS透析换液，Nanodrop 测定蛋白浓度，分装成1ml/管，-20℃保存。
3、重组蛋白的鉴定
3.1用SDS-PAGE电泳鉴定蛋白纯度：
a) 蛋白样品前处理：分别留样加同等体积的2×SDS loading buffer，置于沸水浴中煮10min，12000rpm离心3min，取上清加至加样孔。
b) SDS-PAGE电泳胶的配制：垂直电泳玻璃板洗净晾干，垂直放于制胶架上，按照要求配好分离胶体系，加入玻璃板间隙，至胶面达到所要求的高度后，加入无水乙醇密封；室温凝固30min~1h后，弃去胶上层密封液体，并用吸水纸吸干，按比例配制浓缩胶，并插入适合的梳子，室温凝固30min~1h。
c) SDS-PAGE电泳：将5×甘氨酸缓冲液稀释至工作浓度，加入电泳槽至合适液面高度，将梳子从凝固凝胶中轻轻拔出。在加样孔内加入蛋白marker和10μL处理过的蛋白样品。接通电源，调整电压80V恒压电泳至分离胶，再改为120V直至溴酚蓝至凝胶底部。
d) 染色及脱色：将胶从玻璃板上切下，置于考马斯亮蓝染液，振荡染色4 h，后脱色液脱色至条带清晰（图1）。
3.2 ELISA 鉴定重组蛋白的活性
用纯化的重组NS1蛋白包被（1μg/ml），ELISA方法鉴定其与阳性血清的反应，阳性血清为HIN1鼠肺适应株感染的小鼠血清。先在微孔板中包被重组蛋白（包被缓冲液碳酸盐缓冲液：碳酸钠1.59g，碳酸氢钠2.93g，定容至1L纯水中），包被浓度为1μg/mL，50μL/孔，4℃过夜；1%BSA封闭，每孔100 μL，37℃ 2小时，用洗涤液（PBST，含0.05%的Tween-20的PBS）洗板1次，拍干；将阳性小鼠血清按1:100、1:1000、1:10000、1:10000的梯度进行（PBS）稀释，加入50μL到已包被抗原的微孔板中，同时用空白小鼠血清作阴性对照，37℃反应30min。甩出孔中液体， PBST洗液洗板4次，拍干后加入HRP标记的羊抗鼠二抗（以PBS按照1:5000稀释）50μL/孔，37℃反应30min，再洗板4次，拍干后加入TMB显色液50μL/孔室温显色10min，最后加入2M硫酸，终止反应，用酶标仪测定OD450值。
结果如图2所示，NS1重组蛋白可与PR8/H1N1感染小鼠阳性血清特异性反应，表明重组蛋白具有良好的生物学活性，蛋白表达成功，可以用于后续的免疫及筛选实验。
4、单克隆抗体的筛选与制备
4.1小鼠免疫：
A型流感病毒鼠肺适应株PR8/H1N1用无菌PBS稀释100倍，戊巴比妥钠溶液进行腹腔麻醉的BALB/c雌性小鼠按25ul/只剂量连续滴鼻感染2次，间隔一周。14日后，将纯化的重组NS1蛋白加等量的弗氏完全佐剂，背部皮下注射加强免疫，30μg/只；于融合的前3~5日再次加强免疫，腹腔注射重组NS1蛋白，30μg/只。
4.2杂交瘤细胞株的筛选
取免疫小鼠的所有脾细胞与处于对数生长期的SP2/0骨髓瘤细胞进行融合后，置于HAT培养基中进行筛选培养。待融合细胞长至孔底1/2时，，以重组NS1蛋白和表达载体pet28a进行检测，筛选出能与NS1重组蛋白反应，但不与表达载体pet28a反应的细胞株，，并通过2~3次连续的有限稀释克隆法达到阳性率为100%时，对细胞株进行扩大培养和冻存。
按3.2所述间接ELISA方法，取杂交瘤细胞培养上清50μL加入到已包被NS1抗原的微孔板中，同时用空白小鼠血清作阴性对照，37℃反应30min。甩出孔中液体， PBST洗液洗板4次，拍干后加入HRP标记的羊抗鼠二抗（以PBS按照1:5000稀释）50μL/孔，37℃反应30min，再洗板4次，拍干后加入TMB显色液50μL/孔室温显色10min，最后加入2M硫酸，终止反应，用酶标仪测定OD450值。筛选结果如图3所示，筛选出的9株杂交瘤细胞培养上清均与NS1蛋白反应，其中有5株OD450在1.5以上。
4.3单克隆抗体腹水的制备
将筛选到的杂交瘤细胞株进行扩大培养后，于腹腔注射0.2ml（含2.5×106个细胞）经弗氏不完全佐剂预处理的雌性BALB/c小鼠，约10日后，待小鼠腹部明显肿大时，使用无菌注射器针头采集腹水。
4.4单克隆抗体的纯化
将腹水以12000r/min离心5分钟，取上清5ml，加入20ml醋酸缓冲液（68mmol/L，pH值4.5），混合均匀后，缓慢加入50μL正辛酸，边加边搅动，加完后继续搅拌30分钟，在2~8℃下，以12000r/min离心30分钟，取上清。将上清用脱脂棉过滤，并用NaOH（1mol/L）调节pH值至7.0~7.4之间，按终体积50%（V/V）的比例加入饱和硫酸铵，边加边搅拌，加完后继续搅拌30分钟，置2~8℃下沉淀4~5小时，在2~8℃下，以12000r/min离心30分钟，取沉淀。将沉淀用3ml Tris-HCl（10mmol/L，pH值9.0）重悬，装入透析袋（MW：8000~14000），在2~8℃下，置2LTris-HCl（10mmol/L，pH值9.0）溶液中，透析14小时。将透析袋中的液体转移到离心管中，以12000r/min离心5分钟，上清即为纯化的单克隆抗体。纯化的单克隆抗体行SDS-PAGE电泳，微量紫外分光光度计进行浓度测定，分装1ml/管，-80℃以下保存，避免反复冻融。
5、纯化的单克隆抗体鉴定
5.1 ELISA检测单克隆抗体与NS1蛋白的结合力
包被纯化的NS1蛋白（1μg/ml）进行间接ELISA实验，检测9株单克隆抗体与NS1蛋白的结合能力。ELISA包被和检测方法同上，加入的单克隆抗体浓度为2μg/ml和0.5μg/ml，同时无关单克隆抗体（ADV）和PBS作为阴性对照。
结果如图4所示NS1蛋白与纯化后的9株单抗均反应，而不与无关单抗反应，其中5株单抗在0.5ug/ml与NS1蛋白的反应OD值在1.8以上，表明纯化的单抗具有较好的亲和力。
5.3 单克隆抗体交叉反应鉴定
用包被液（包被缓冲液碳酸盐缓冲液：碳酸钠1.59g，碳酸氢钠2.93g，定容至1L纯水中）将pET28a-NS1、A型流感病毒NP蛋白pET28a-ANP（购自义翘神州）、H1N1-Virus等稀释至约1μg/ml，包被酶联反应板，100μL/孔，在2~8℃下作用包被过夜。甩去孔内液体，用洗涤液洗涤酶联反应板。1%明胶封闭，每孔200μL，37℃ 2小时。加入待鉴定的单克隆抗体（0.5μg/ml），100μL/孔，同时无关单克隆抗体和PBS作为阴性对照，37℃反应30min。甩出孔中液体，PBST洗液洗板4次，拍干后加入HRP标记的羊抗鼠二抗（以PBS按照1:5000稀释）100μL/孔，37℃反应30min，再洗板4次，拍干后加入TMB显色液100μL/孔室温显色10min，最后加入0.5M硫酸，终止反应，用酶标仪测定OD450值。
结果如图5所示，9株单抗均只与NS1蛋白反应，而不与A型流感病毒和NP蛋白反应，表现出很好的特异性。
6、流感病毒小鼠半数致死量（LD50）确定
用无菌PBS对流感病毒鼠肺适应株进行10倍梯度稀释。取6~8周龄的雌性BALB/C小鼠使用戊巴比妥钠溶液进行腹腔麻醉，之后分别将各个稀释度的病毒经鼻腔给药，每组10只小鼠，每只小鼠25μL，每日观察小鼠的死亡情况，直至病毒攻击后第14天，使用Reed-Muench公式计算病毒的小鼠半数致死量。
7、单克隆抗体的流感病毒感染小鼠治疗实验
病毒攻击：取6~8周龄的雌性BALB/C小鼠使用戊巴比妥钠溶液进行腹腔麻醉, 鼻腔给药给以25μL含有10倍LD50的病毒。实验小鼠分为治疗组和对照组，其中治疗组：同时在病毒攻击当天，按50mg/KG小鼠体重比例经腹腔注射给以200μL不同的单克隆抗体，每株单抗组有6只小鼠。对照组有6只小鼠，同时腹腔注射200μL的无菌PBS。每日观察不同组小鼠的状态、活率和体重。
结果如图6和图7所示，病毒攻击后小鼠在第5-6天出现毛乱、精神萎靡等症状，其中PBS和ADV单抗对照组体重下降明显，感染后第8天全部死亡。单抗治疗组中3D9、3F5、4C5、1C10、4D3、4F4和2D6组小鼠均呈现体重持续下降趋势，症状严重，3D9组在第9天全部死亡，1C10和3F5组在第11天全部死亡，4C5和4F4组在第12天全部死亡，4D3和2D6组在第13天全部死亡。5F11和3E5组在感染初期有毛乱等轻微症状，但不严重，到第14天仍分别有4只和3只小鼠存活，且其平均体重基本未下降。综上5F11和3E5两株单抗在小鼠腹腔注射给药后，可以减轻流感病毒感染的症状，降低死亡率，这可以为流感病毒感染的治疗提供一种参考。
表1 小鼠存活率数据
表2 小鼠平均体重
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，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 北京溯本源和生物科技有限公司
<120> 一种流感病毒 NS1 蛋白的表达质粒、重组蛋白和特异性单克隆抗体及应用
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Claims (6)
1.一种与重组NS1蛋白发生特异性反应的单克隆抗体，其特征在于，所述单克隆抗体为5F11或3E5，所述5F11的重链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.3~SEQ ID NO.5所示；所述5F11的轻链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.8~SEQ ID NO.10所示；
所述3E5的重链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQID NO.13~SEQ ID NO.15所示；所述3E5的轻链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.18~SEQ ID NO.20所示；
所述重组NS1蛋白由表达A型流感病毒NS1保守片段的重组菌经IPTG诱导得到；所述重组菌的基因组包含重组质粒，所述重组质粒包含A型流感病毒NS1保守片段；
所述NS1保守片段的编码基因包括SEQ ID NO.1所示的序列；
所述重组质粒的基础载体包括pET28a；
所述NS1保守片段的氨基酸序列包括SEQ ID NO.2所示的序列。
2.根据权利要求1所述单克隆抗体，其特征在于，所述5F11的重链可变区的氨基酸序列如SEQ ID NO.6所示，所述5F11的轻链可变区的氨基酸序列如SEQ ID NO.11所示。
3.根据权利要求1或2所述单克隆抗体，其特征在于，编码所述5F11的重链可变区的核苷酸序列如SEQ ID NO.7所示，编码所述5F11的轻链可变区的核苷酸序列如SEQ ID NO.12所示。
4.根据权利要求1所述单克隆抗体，其特征在于，所述3E5的重链可变区的氨基酸序列如SEQ ID NO.16所示，所述3E5的轻链可变区的氨基酸序列如SEQ ID NO.21所示。
5.根据权利要求1或4所述单克隆抗体，其特征在于，编码所述3E5的重链可变区的核苷酸序列如SEQ ID NO.17所示，编码所述3E5的轻链可变区的核苷酸序列如SEQ ID NO.21所示。
6.权利要求1~5任一项所述单克隆抗体在制备预防和/或治疗A型流感病毒感染引发疾病的疫苗或药物中的应用。
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| CN110028578A (zh) * | 2019-03-12 | 2019-07-19 | 中国科学院武汉病毒研究所 | 广谱抗h7流感病毒的中和性人源单克隆抗体及其应用 |
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