CN108624602B - 一株具有阻断活性的抗尼帕病毒g蛋白的单克隆抗体及其应用 - Google Patents
一株具有阻断活性的抗尼帕病毒g蛋白的单克隆抗体及其应用 Download PDFInfo
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Abstract
本发明属于生物免疫分析技术领域,具体涉及一株具有阻断活性的抗尼帕病毒G蛋白的单克隆抗体及其应用。本发明经过密码子优化方法克隆得到尼帕病毒G蛋白基因,该蛋白基因的核苷酸序列如SEQ ID NO:1中第898‑1806位碱基位所述的序列,其蛋白质序列如SEQ ID NO:2所示。本发明提供了一种分泌抗尼帕病毒G蛋白且具有一定阻断活性的单克隆抗体的杂交瘤细胞株15‑D11(中国典型培养物保藏中心保藏编号为CCTCC NO.C2016207)。用原核表达并纯化的尼帕病毒G蛋白免疫BALB/c小鼠,筛选出一株能稳定分泌抗尼帕病毒G蛋白单克隆抗体的杂交瘤细胞。本发明制备的单抗可用于动物免疫分析技术领域。
Description
技术领域
本发明属于生物免疫分析技术领域,具体涉及一株具有阻断活性的抗尼帕病毒G蛋白的单克隆抗体及其应用。
背景技术
尼帕病毒(Nipah virus)是一种新发现的***共患病毒,属副粘病毒科RNA病毒。可引起人和猪以中枢神经***、呼吸***为主要病变的急性、高度致死性的传染性疾病。
1997年马来西亚Kinta地区爆发过发热性脑炎,造成1人死亡。1998年,马来西亚北部再度引起猪和人类致命性的脑炎,经由接触病猪组织及体液感染。而且两次的病患者均为养殖场工人、屠宰场工人以及从事生猪加工的人员。1999年3月新加坡某屠宰场发生发热性脑炎11例,造成1人死亡,其所屠宰的生猪80%以上来自马来西亚。此后,在印度和孟加拉国也有爆发此病,给这些国家的畜牧业发展和人民健康造成了严重的危害。
尼帕病毒在周边国家的流行态势对我国造成了潜在的威胁。我国东南沿海地区和云南地区具有适合尼帕病毒流行的生态坏境和宿主动物,且国内已从南方地区的蝙蝠体内检测到尼帕病毒的抗体,加上频繁的贸易往来使得该病传入我国的可能性不断增加,一旦爆发该病势必对我国的养殖业和人民的正常生活造成危害。
目前,该病诊断主要通过临床诊断结合病毒分离、免疫组化、PCR等技术。这些方法在病原检测或抗体检测中各有特点,在该病的诊断中发挥了重要的作用。但如何在此基础上研制开发纯度高、性能稳定且高效快速的检测试剂,更易在生产实践中推广应用的检测方法是尼帕病毒疾病诊断与防治工作的一个重点。
发明内容
本发明的第一个目的是提供一种尼帕病毒G基因编码的重组蛋白。
本发明的第二个目的是利用上述重组蛋白制备抗尼帕病毒G蛋白且具有一定阻断活性的单克隆抗体。
本发明的第三个目的是利用SEQ ID NO:2所示尼帕病毒G蛋白的重组蛋白作为抗原蛋白的应用。
为实现上述目的,本发明采取以下技术方案:
一种尼帕病毒G蛋白基因,该基因片段包含尼帕病毒的主要抗原表位,经密码子优化后核苷酸序列如SEQ ID NO:1中第898-1806位碱基所示(即CDS区),该基因编码303个氨基酸残基,其蛋白质序列如SEQ ID NO:2所示。
本发明还包括利用SEQ ID NO:2所示一种尼帕病毒G蛋白的重组蛋白作为抗原蛋白的应用。
申请人进一步制备了一种单克隆抗体,它是由序列表SEQ ID NO:2所述的尼帕病毒G蛋白的重组蛋白免疫动物后经融合实验得到的杂交瘤细胞株15-D11所分泌的,申请人将该株杂交瘤细胞株命名为杂交瘤细胞株15-D11,于2016年12月6日送交中国.武汉.武汉大学中国典型培养物保藏中心(CCTCC)保藏,保藏编号为CCTCC NO:C2016207。
上述杂交瘤细胞株15-D11能够分泌具有阻断活性的单克隆抗体,该单抗能特异性结合尼帕病毒G蛋白。
本发明的优点在于:本发明提供了抗尼帕病毒G蛋白的单克隆抗体和分泌该单克隆抗体的杂交瘤细胞株,该单克隆抗体特异性强,生物结合活性高,与转染表达尼帕病毒G蛋白的A549细胞发生反应,而与对照组的A549细胞不发生反应。
本发明为建立尼帕病毒快速检测方法奠定了技术基础,为该病毒的早期诊断以及流行病学调查提供有效的工具。
附图说明
序列表SEQ ID NO:1是本发明克隆的尼帕病毒基因,其中898-1806位碱基所示的序列是优化的尼帕病毒重组G蛋白的编码区,其编码303个氨基酸残基。
序列表SEQ ID NO:2是本发明克隆的尼帕病毒基因编码的蛋白质序列。
图1.通用的商业质粒PET-28a载体的图谱。
图2.通用的商业质粒PCAGGS载体的图谱。
图3.本发明构建的重组质粒PET-28a-NIV-G图谱。
图4.原核表达的尼帕病毒重组G蛋白,附图标记说明:图4中的1为PET-28a空载体对照;图4中的2为重组G蛋白。
图5.利用间接免疫荧光检测结果。附图标记说明:图5中的A图为已转染表达尼帕病毒G蛋白真核表达质粒的A549细胞;图5中的B图为转染空载体的A549细胞(对照)。
图6.杂交瘤细胞遗传稳定性的检测结果。附图标记说明:图6中的A图为瘤细胞(SP2/0)染色体数;图6中的B图为杂交瘤细胞染色体数。
具体实施方式
实施例1抗原的制备
1.尼帕病毒G蛋白基因片段的PCR扩增
从NCBI网站得到登录号Gene ID:920955的尼帕病毒G蛋白基因序列,其CDS区经密码子优化后,化学合成其编码序列并对该序列进行抗原表位分析,最终选取第898-1806位碱基进行PCR扩增。其中,模板为以上化学合成的G基因片段,上下游引物加入BamHI、HindIII酶切位点。扩增尼帕病毒G基因片段的上下游引物如下所示:
上游引物(P1):5'-CGGGATCCAACGCCGACAACATCAACAAG-3'
下游引物(P2):5'-CCCAAGCTTGGCGCGCCCGGTGGTCTG-3',
上述引物中划线部分为酶切位点。
PCR反应体系为50μl:
2.尼帕病毒G蛋白基因原核表达质粒的构建
尼帕病毒G蛋白基因片段和PET-28a载体(购自宝生物工程大连有限公司,原始载体图谱见图1)分别用BamHI、HindIII双酶切后,使用胶回收试剂盒(购自Magen公司)回收目的片段和载体,然后在16℃条件下用T4DNA连接酶进行连接,将所得到的阳性重组质粒NIV-G-28a送上海生工生物工程股份有限公司进行测序确认。得到重组质粒NIV-G-28a。
3.重组质粒NIV-G-28a的诱导表达
(1)将阳性重组质粒NIV-G-28a转化大肠杆菌感受态BL21(DE3)。
(2)将所得NIV-G-28a的阳性菌液按体积比为1:100接种到含有相应抗生素(例如卡那霉素)的LB液体培养基,在37℃摇床上与180r/min下培养。
(3)培养至OD600值在0.5-0.7,加入IPTG,使终浓度至1.0mmol/L,在37℃摇床上180r/min诱导4h。
(4)诱导结束后,于8000r/min离心5min,收集菌体,菌体用PBS(磷酸盐缓冲液,配制方法:8.0g NaCl,0.2g KCl,1.44g Na2HPO4,0.24g KH2PO4,溶于950mL蒸馏水中,调pH值至7.4,用ddH2O定容至1L,常规高温高压灭菌,室温下保存)洗涤一次,后用适量的PBS重悬,最后加入蛋白质电泳上样缓冲液,于95℃加热10min,处理后的样品放于冰上备用(以空载体质粒PET-28a做同样处理作为对照)。
(5)采用常规SDS-PAGE和Western-Blot免疫学检测方法对其进行表达鉴定。
4.重组蛋白的纯化
(1)诱导200mL菌体,于4℃,12000r/min下离心5min积菌,然后用PBS洗涤菌体2次,用20mL Buffer A重悬菌体,高压破碎至清亮。
(2)在4℃,12000rpm下离心30min,弃上清。
(3)用PBS洗涤沉淀2-3次。
(4)往沉淀中加入19.7mL Buffer A以及19.7uL的DTT及0.3mL的20%SKL(十二烷基肌氨酸钠),剧烈搅动,使其缓慢溶解,室温静置30min至2小时。
(5)在4℃,12000rpm下离心20min,取上清。
(6)加20%PEG4000 210uL至终浓度为0.2%,加50mM(0.03g/mL)的氧化型谷胱甘肽420uL至终浓度为1mM,加100mM(0.03g/mL)的还原型谷胱甘肽420uL至终浓度为2mM,静置30分钟至2小时(亦可4℃过夜)。
(7)以1×TE(pH 8.0)透析72小时,取出分装后-80℃保存备用。
实施例2单克隆抗体的制备
1.免疫试验
免疫试验用4周龄雌性的Balb/c小白鼠(购自华中农业大学兽医院,简称小鼠),用以上纯化的重组蛋白作为抗原,免疫程序如表1所示:
表1小鼠免疫程序
第三次免疫之后,尾静脉采血,采用Elisa方法检测小鼠血清效价,选择血清效价最高的小鼠做后续实验。
融合前三天对以上选取的血清效价最高的小鼠加强免疫(腹腔注射抗原0.20mg)。
2.细胞融合
(1)SP2/0瘤细胞的制备
从小鼠实体瘤中制备SP2/0细胞。具体步骤如下
1)复苏实验室冻存的SP2/0细胞于六孔板培养,待细胞状态良好时,用1640基础培养液(购买于武汉飞羿科技有限公司,品牌为HyClone)重悬,皮下注射BALB/c小鼠,13天后小鼠背部长出肿瘤。
2)将小鼠拉颈处死,用75%酒精浸泡5min。
3)在超净工作台上无菌状态下取瘤置无菌匀浆器中,加入5mL1640基础培养液充分研磨后补加10mL1640基础培养液,混匀后静置5min,待较大的组织块沉于管底后,吸取上层的细胞悬液于离心管中备用,再补加10mL1640基础培养液重悬组织,如此重复洗涤两次,将细胞悬液1000rpm离心10min后,取沉淀用15mL1640基础培养液重悬。
4)于另一50mL离心管中加入20mL淋巴细胞分离液,将细胞悬液轻轻加于淋巴细胞分离液之上,1000rpm离心10min,用吸管吸取位于界面致密的白色细胞层,用10mL1640基础培养液洗涤一次,计数后备用。
(2)免疫脾细胞的制备
取经过最后加强免疫的BALB/c小鼠一只,眼眶放血处死,收集血液并分离血清,即为阳性血清。
将小鼠拉颈处死,用75%酒精中浸泡5min后将小鼠移至超净台内放在解剖板上,前肢固定,后肢交叉(左后肢交叉)固定。先剪并撕开皮肤暴露腹膜,换一套剪刀镊子,剪开腹膜暴露脾脏,无菌条件下取出脾脏,放入匀浆器中,加入5mL 1640基础培养基研磨,再补加10mL 1640基础培养基,混匀后静置5min;轻轻吸取上层液体于离心管中,匀浆器中再补加10mL 1640基础培养基,混匀静置5min,如此重复洗涤2次,1000r/min离心10min,弃去上清,将脾细胞用适量1640基础培养基重悬后备用。
(3)饲养细胞的制备
取一只未经免疫的BALB/c小鼠,眼眶放血处死,收集血液并分离血清得到阴性血清。小鼠于75%酒精中浸泡5min后;按照上述步骤(2)中免疫脾细胞的制备方法制备饲养脾细胞,最后获得的饲养脾细胞用适量HAT培养基(购自sigma公司)重悬后均匀平铺在96孔细胞培养板中备用,100μL/孔。
(4)骨髓瘤细胞与免疫脾细胞体外融合
1)将SP2/0骨髓瘤细胞悬液(1×107cells)与免疫脾细胞悬液(1×108细胞)于50mL离心管中混匀,1000r/min离心10min;
2)倒空上清(可用灭菌的滤纸吸干),轻轻敲击离心管底使细胞松动;
3)将装有细胞混合物的离心管置于37℃水浴中,1min内缓慢加入预热至37℃的50%聚乙二醇(PEG)0.8mL,边加边轻轻用吸管尖搅拌;
4)继续搅拌1min;
5)5min内缓慢加入预热至37℃的1640基础培养基10mL,加入时需缓慢并不断轻轻搅拌;最后再缓慢加入30mL预热至37℃的1640基础培养基;
6)1000r/min离心10min弃去上清后,37℃放置7min;
7)用适量HAT培养基重悬,均匀平铺在已铺有饲养细胞的96孔细胞培养板,150μL/孔,于37℃,5%CO2培养箱中培养。
5)阳性杂交瘤细胞的筛选
融合后第4天补加50μL新鲜的HAT培养基,第8-10d后吸弃全部培养基,换为HT(购自sigma公司)培养基(即1640基础培养基+20%胎牛血清+1%青链霉素+2%HT(次黄嘌呤+胸腺嘧啶核苷)),待融合细胞形成的集落长至培养孔1/4大小,细胞上清变黄时,进行ELISA检测。
6)杂交瘤细胞上清ELISA检测
采用常规间接ELISA方法检测杂交瘤细胞上清,具体步骤为:
将纯化后的NIV-G重组蛋白和转化了空载体PET-28a的BL-21菌体蛋白(作为阴性对照)分别包被96孔酶标板,4℃过夜。包被后,用PBST缓冲液(PBS缓冲液+0.5‰吐温)洗涤三次,加1%封闭液(1g牛血清白蛋白溶于100mLPBST缓冲液中)于37℃温育1h;再用PBST洗涤三次,加入杂交瘤细胞培养上清,于37℃温育1h;PBST洗涤三次,加羊抗鼠IgG-HRP(武汉博士德生物工程有限公司),于37℃温育1h,PBST洗涤三次后,加入底物液(购自武汉科前动物生物制品有限责任公司)和显色液(购自武汉科前动物生物制品有限责任公司),10min后,观察显色反应并加终止液(购自武汉课前生物有限公司),在630nm波长下测OD值。同时测免疫小鼠阳性血清为阳性对照,设SP2/0细胞培养上清为阴性对照。OD630大于阴性对照两倍的样品为阳性。选取与抗原包被板反应阳性而与阴性对照板反应阴性且细胞生长旺盛形态良好的细胞生长孔作进一步亚克隆。
(7)阳性杂交瘤细胞的亚克隆
克隆前先制备饲养细胞【具体步骤同步骤(3)】,将Elisa检测为阳性的杂交瘤细胞进行有限稀释,使得每个细胞培养孔有1-2个杂交瘤细胞。之后定时观察孔内细胞生长情况并记录;待克隆8-10天,细胞长至约培养孔的1/3-1/2大小时,用间接ELISA方法【具体步骤同步骤(6)】检测;选单集落且检测呈阳性的孔用相同的方法再继续克隆,进一步纯化细胞株,直到所有克隆化细胞孔阳性率为100%即可建株。最后,扩大培养建株细胞以备用。
通过以上方法本发明获得一株稳定分泌抗尼帕病毒G蛋白且具有一定阻断活性的单克隆抗体的杂交瘤细胞株,申请人将该杂交瘤细胞株命名为杂交瘤细胞株15-D11,于2016年12月6日送交中国.武汉.武汉大学中国典型培养物保藏中心保藏,保藏编号为CCTCCNO:C2016207。
3.单克隆抗体的大量制备与纯化
取8周龄的BALB/c小鼠5只,腹腔注射不完全氟氏佐剂0.5mL/只;7-10d后收集扩大培养的杂交瘤细胞株15-D11,腹腔注射小鼠(105-106cells/只),7-10d后收集腹水,于2000r/min离心10min,取上清备用。
按照IgG抗体纯化试剂盒(购自Thermo公司)的操作说明,对以上离心粗提的腹水进行纯化,具体步骤如下:
1)去掉抗体纯化柱底部帽子,将纯化柱放在1.5ml Ep管上,在柱子中加入300μlBinding Buffer,盖上纯化柱盖子,颠倒混匀后,5000r/min,离心1min;
2)弃去下层离心管内液体,重复上述步骤2次;
3)在纯化柱内加入100μl待纯化的单克隆抗体腹水,上下颠倒混匀10min,5000r/min,离心1min,盛接在1.5ml Ep管内的液体为洗脱液,做好标记;
4)将纯化柱用Binding Buffer洗3次,每次5000r/min,离心1min;
5)在纯化柱中加入300μl洗脱液,上下颠倒混匀,并将柱子放在事先已加入40μl中和液的1.5ml Ep管上,5000r/min,离心1min,收集于1.5ml Ep管内的液体即为第一次纯化获得的单克隆抗体,并做好标记;
6)重复上述步骤,分别得到第二次和第三次纯化获得的抗体,做好标记;
7)将纯化柱用Binding Buffer洗3次,每次5000r/min,离心1min;
8)再将纯化柱用保存液(该试剂盒自带)洗3次,5000r/min,离心1min;
9)于纯化柱内加入300μl保存液,混匀后将试剂盒放回4℃保存。
实施例3单克隆抗体的生物活性检测
(1)腹水效价测定:用间接ELISA方法检测上述腹水中单克隆抗体的效价。用本发明制备的NIV-G-28a蛋白包被酶标板后,将腹水从体积比1:100倍比稀释至1:6553600作为一抗,以HRP标记的羊抗鼠IgG(购自武汉博士德生物有限公司)为二抗用于间接ELISA检测,并以未免疫小鼠血清作为阴性对照。
试验结果:本发明所述的单克隆抗体腹水效价为1:204800。
(2)利用间接免疫荧光检测
将A549细胞(购自中国典型培养物保藏中心,武汉大学)培养于24孔细胞培养板,待细胞长到70%~80%时,转染构建的真核表达质粒(PCAGGS-NIV-G),同时转染空载体PCAGGS(本实验室保存)作为阴性对照。
24h后,弃掉细胞培养上清,用100%甲醇(-20℃预冷30min)固定10min,然后用PBS洗涤三次,5min/次;
封闭:加5%牛血清白蛋白(BSA)封闭1h,再用PBS洗涤三次,每次5min;
一抗孵育:加入待检的单抗样品(用杂交瘤细胞15-D11制备的腹水稀释100倍),同时设置未免疫空白小鼠血清1:50稀释、SP2/O细胞制备的腹水1:100稀释作两个阴性对照。37℃温育1h,PBS洗三次,每次5min;
二抗孵育:加入异硫氰酸荧光素(fluorescein isocyanate,FITC)标记的羊抗鼠IgG(sigma 1:100),37℃温育30min,PBS洗三次,5min/次;
荧光显微镜下观察结果。
结果表明:筛选出的杂交瘤细胞15-D11分泌的抗体能与尼帕病毒G蛋白发生特异性结合反应。
试验结果如图5所示,实验组结果显示明显的绿色荧光,而对照组则无荧光反应,说明本发明制备的单克隆抗体能与尼帕病毒发生特异性结合。
实施例4单克隆抗体阻断活性的检测
(1)包被:用本发明中原核表达的尼帕病毒G蛋白包被酶标板,4℃过夜。次日,弃掉包被液,PBST洗3次,250μl/孔,3min/次;
(2)封闭:每孔加入100μl封闭液,37℃封闭1h后弃掉封闭液,用PBST洗3次,250μl/孔,3min/次;(3)加阻断剂:取免疫了尼帕病毒G蛋白的兔血清作为拟阻断剂,用封闭液将其按照2n*100倍比稀释加入已封闭的酶标板中,100μl/孔,最大稀释倍数为1:12800。同时,设封闭液代替兔血清为对照组;37℃孵育3h后,PBST洗涤三次;
(4)加待检测单抗:以上每孔加入杂交瘤细胞15-D11培养上清,100μl/孔,37℃孵育1h后,PBST洗3次;
(6)加二抗:每孔加入100μl HRP标记的羊抗小鼠二抗(购自博士德生物有限公司)(按体积比1:5000稀释)37℃孵育1h后PBST洗3次;
(5)显色:每孔加入50μl底物,50μl显色液,避光静置10min后加50μl终止液(底物液、显色液、终止液均购自武汉科前动物生物制品有限责任公司),使用酶标仪读取630nm波长下的吸光值。
表2本发明所制备的单克隆抗体的阻断活性检测
表2显示:当兔血清稀释200倍时,OD630值与对照组相比明显降低,说明本发明中的单克隆抗体与以上兔血清能够结合尼帕病毒G蛋白结构中相同的抗原表位,间接证明了该单克隆抗体具有阻断尼帕病毒G蛋白的能力。根据阻断率计算公式(PI)=100-OD实验组/OD对照组×100,本发明中杂交瘤细胞株15-D11分泌的单克隆抗体的阻断率为66.5%。
实施例5杂交瘤细胞15-D11遗传稳定性分析
1)利用秋水仙素处理生长状态良好的杂交瘤细胞15-D11,使细胞停留在***中期:取24孔细胞板培养的细胞,加入秋水仙素至终浓度为0.4μg/mL,37℃温箱培养3h。同时,骨髓瘤细胞做相同处理作为对照;
2)低渗处理,使细胞肿胀:用37℃预温的0.075mol/L的KCl(配制方法:将0.28gKCl加到50ml ddH2O中,混匀)低渗溶液处理细胞,37℃温箱培养35min;
3)收集秋水仙素处理后的细胞:倾出秋水仙素处理液,加入1640基础培养液后将细胞吹起。1600r/min离心5min收集细胞沉淀;
4)低渗处理,使细胞肿胀:将细胞沉淀用600μl 37℃预温的0.075mol/L的KCl低渗溶液悬浮,混匀后置37℃温育35min;
5)固定:加入新配制的固定液(甲醇∶冰乙酸)600μl,混匀,1600r/min离心5min,弃上清液。再加入固定液800μl,轻轻混匀,静置30min,1600r/min离心5min,弃上清液。再次加入固定液800μl,轻轻混匀,静置30min,1600r/min离心5min,弃上清液留细胞沉淀。
6)细胞重悬与浓度检测:用适量固定液重悬细胞,吸取一滴细胞悬液于4℃预冷的干净载玻片上,轻吹液滴,使细胞悬液铺展于玻片上,室温下自然干燥;
7)染色:用10%Giemsa染液染15min。流水冲洗,自然干燥;
8)观察与计数:将载玻片置显微镜油镜下观察计数。
结果显示,本发明制备的杂交瘤细胞15-D11的染色体数目约100条,瘤细胞的染色体数目约60条,而正常小鼠脾细胞的染色体条数约为40,本发明的杂交瘤细胞15-D11约为SP2/0细胞和正常脾细胞染色体数量之和(见图6)。说明本发明中杂交瘤细胞株15-D11为小鼠脾细胞和骨髓瘤细胞融合而成。
SEQUENCE LISTING
<110> 华中农业大学
<120> 一种具有阻断活性的抗尼帕病毒G蛋白的单克隆抗体及其应用
<130>
<141> 2017-03-20
<160> 2
<170> PatentIn version 3.1
<210> 1
<211> 1809
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<213> 尼帕病毒(Nipah virus)
<220>
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tcctgcccaa acccgctgcc cttccgcgag taccgccccc agaccgaggg cgtgtccaac 540
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ctgctggcca tggacgaggg ctacttcgcc tactcccacc tggagcgcat cggctcctgc 720
tcccgaggcg tgtccaagca gcgcatcatc ggcgtgggcg aggtgctgga ccgcggcgac 780
gaggtgccct ccctgttcat gaccaacgtg tggacccccc ccaaccccaa caccgtgtac 840
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Claims (2)
1.一种针对尼帕病毒G蛋白的单克隆抗体,其特征在于,所述的单克隆抗体是由保藏编号为CCTCC NO: C2016207杂交瘤细胞株15-D11所分泌的,所述的杂交瘤细胞株15-D11保藏在中国典型培养物保藏中心。
2.一株分泌针对尼帕病毒G蛋白的单克隆抗体的杂交瘤细胞株15-D11,其特征在于,所述的杂交瘤细胞株15-D11保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO:C2016207。
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Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1882704A (zh) * | 2003-09-22 | 2006-12-20 | 巴斯德研究院 | 尼帕病毒检测方法和提供抗汉尼巴病毒的免疫保护的方法 |
CN104244974A (zh) * | 2011-05-13 | 2014-12-24 | 硕腾有限公司 | 亨德拉和尼帕病毒g糖蛋白免疫原性组合物 |
CN105530956A (zh) * | 2013-09-05 | 2016-04-27 | 硕腾服务有限责任公司 | Hendra和Nipah病毒G糖蛋白免疫原性组合物 |
AU2016203298A1 (en) * | 2005-03-14 | 2016-06-09 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Human monoclonal antibodies against hendra and nipah viruses |
CN105828836A (zh) * | 2013-12-16 | 2016-08-03 | 硕腾服务有限责任公司 | 亨德拉病毒和尼帕病毒g糖蛋白免疫原性组合物 |
EP3381930A1 (en) * | 2004-07-09 | 2018-10-03 | The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. | Soluble forms of hendra and nipah virus g glycoprotein |
-
2017
- 2017-03-24 CN CN201710184873.6A patent/CN108624602B/zh not_active Expired - Fee Related
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN1882704A (zh) * | 2003-09-22 | 2006-12-20 | 巴斯德研究院 | 尼帕病毒检测方法和提供抗汉尼巴病毒的免疫保护的方法 |
EP3381930A1 (en) * | 2004-07-09 | 2018-10-03 | The Henry M. Jackson Foundation for the Advancement of Military Medicine, Inc. | Soluble forms of hendra and nipah virus g glycoprotein |
AU2016203298A1 (en) * | 2005-03-14 | 2016-06-09 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Human monoclonal antibodies against hendra and nipah viruses |
CN104244974A (zh) * | 2011-05-13 | 2014-12-24 | 硕腾有限公司 | 亨德拉和尼帕病毒g糖蛋白免疫原性组合物 |
CN105530956A (zh) * | 2013-09-05 | 2016-04-27 | 硕腾服务有限责任公司 | Hendra和Nipah病毒G糖蛋白免疫原性组合物 |
CN105828836A (zh) * | 2013-12-16 | 2016-08-03 | 硕腾服务有限责任公司 | 亨德拉病毒和尼帕病毒g糖蛋白免疫原性组合物 |
Non-Patent Citations (3)
Title |
---|
"中国西部地区WNV感染初步流行病学研究以及WNV和NiV主要蛋白的免疫信息学分析";陈晓;《中国博士学位论文全文数据库(电子期刊)医药卫生科技辑》;20100415(第4(2010)期);摘要,正文第94页第1.1.1小节,第101-103页第2.2小节 * |
"尼帕病毒G和F蛋白单克隆抗体的制备与鉴定";苏华等;《中国预防兽医学报》;20111115;第33卷(第11期);第898-901页 * |
"表达尼帕病毒G囊膜糖蛋白重组牛痘病毒的研究";王喜军等;《微生物学报》;20060804;第46卷(第4期);第647页左栏第3段,右栏最后1段 * |
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