CN113913389A - 杂交瘤细胞株、抗布鲁氏菌bab抗原的单克隆抗体及其制备和应用 - Google Patents
杂交瘤细胞株、抗布鲁氏菌bab抗原的单克隆抗体及其制备和应用 Download PDFInfo
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- CN113913389A CN113913389A CN202010661374.3A CN202010661374A CN113913389A CN 113913389 A CN113913389 A CN 113913389A CN 202010661374 A CN202010661374 A CN 202010661374A CN 113913389 A CN113913389 A CN 113913389A
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Abstract
本发明属于基因工程技术领域，特别是涉及一种杂交瘤细胞株、抗布鲁氏菌BAB抗原的单克隆抗体及其制备和应用。本发明针对具有良好的反应原性的BAB抗原制备、筛选得到杂交瘤细胞株，制备得到了抗布鲁氏菌BAB抗原的单克隆抗体，为布鲁氏菌诊断及新型疫苗的研制提供了基础。
Description
技术领域
本发明属于基因工程技术领域，特别是涉及一种杂交瘤细胞株、抗布鲁氏菌BAB抗原的单克隆抗体及其制备和应用。
背景技术
布鲁菌病(布病)是由布鲁氏菌引起的以流产和发热为特征的人兽共患病，严重地威胁着人和多种动物的生命健康，对农业生产和公共卫生造成了巨大的威胁。布鲁氏菌具有内寄生的特性,因此被感染的人或动物一般需要接受长时间的抗生素治疗,而且往往会留下严重后遗症。
反刍动物和猪对布鲁氏菌高度易感。布鲁氏菌特征性地侵害其生殖器官并导致母畜流产和不孕。当患病母畜分娩时，胎盘组织中布鲁氏菌达到极高数量，每克组织中细菌数可高达1014个，因此母畜流产是该病的传染性病症。在世界卫生组织(WHO)实验室生物安全手册中，布鲁氏菌被列为Ⅲ类危险级。布鲁氏菌病易传播给人，引起急性发热性疫病(波浪热)，可进一步发展成慢性型，引起肌肉-骨骼系统、心血管系统和中枢神经系统的严重并发症。本病流行区域应采取预防措施，防止人感染。职业性接触是常见的感染原因，口腔、呼吸道和结膜是基本感染途径。
布鲁菌是一种兼性胞内寄生菌，感染人畜后可在宿主体内建立慢性感染且难以被彻底清除，布鲁菌被认为缺乏病原菌所具有的经典毒力因子，因此对毒力基因的鉴定和研究对阐述其致病机制有着重要的意义。
鉴于此，特提出本发明。
发明内容
本发明的首要发明目的在于提供一种用于生产抗布鲁氏菌BAB抗原的单克隆隆抗体的杂交瘤细胞株。
本发明的第二发明目的在于提供一种抗布鲁氏菌BAB抗原的单克隆抗体。
本发明的第三发明目的在于提出上述杂交瘤细胞株、布鲁氏BAB抗原的单克隆抗体的应用。
本发明的第四发明目的在于提出上述杂交瘤细胞株、布鲁氏BAB抗原的单克隆抗体的制备方法。
为了完成上述发明目的，采用的技术方案为：
本发明提出一种杂交瘤细胞株，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.19941。
本发明还提出该杂交细胞株在用于产抗布鲁氏菌BAB抗原的单克隆隆抗体中的应用。
可选的，所述布鲁氏菌BAB抗原的表位如SEQ ID NO:1所示的氨基酸序列所示。
可选的，编码所述布鲁氏菌BAB抗原氨基酸序列的核苷酸序列如SEQ ID NO:2所示。
本发明还提出一种抗布鲁氏菌BAB抗原的单克隆抗体，其由保藏编号为CGMCCNo.19941的杂交瘤细胞株产生。
本发明还提出该单克隆抗体在制备布鲁氏菌BAB抗原检测试剂中的应用。
可选的，所述布鲁氏菌BAB抗原的表位如SEQ ID NO:1所示的氨基酸序列所示。
本发明还提出该杂交瘤细胞株的筛选方法，其特征在于，至少包括以下步骤：
以BAB蛋白作为免疫原，免疫注射BALB/c小鼠，取免疫小鼠脾脏细胞并将其与骨髓瘤细胞融合，获得了表达抗布鲁氏菌BAB抗原的杂交瘤细胞株；所述BAB蛋白的氨基酸序列如SEQ ID NO:1所示的氨基酸序列所示。
其中，编码所述BAB蛋白的氨基酸序列的核苷酸序列如SEQ ID NO:2所示。本发明还提出该单克隆抗体的制备方法，至少包括以下步骤：
以BAB蛋白作为免疫原，免疫注射BALB/c小鼠，取免疫小鼠脾脏细胞并将其与骨髓瘤细胞融合，获得了表达抗布鲁氏菌BAB抗原的杂交瘤细胞株；所述BAB蛋白的氨基酸序列如SEQ ID NO:1所示的氨基酸序列所示；
采用所述杂交瘤细胞株进行表达，获得所述单克隆抗体。
其中，编码所述BAB蛋白的氨基酸序列的核苷酸序列如SEQ ID NO:2所示。
本发明至少具有以下有益的效果：
本发明获得了具有良好的反应原性的BAB抗原，并针对BAB抗原制备、筛选得到杂交瘤细胞株，制备得到了抗布鲁氏菌BAB抗原的单克隆抗体，为布鲁氏菌诊断及新型疫苗的研制提供了基础。
附图说明
图1为免疫效价检测结果图；
图2 SDS-PAGE电泳鉴定图，2为本发明实施例保藏的杂交瘤细胞株，3为mark；
图3为Western-Blot检测结果图，左侧条带为mark，右侧条带为目的蛋白、12％SDS-PAGE。
保藏说明
杂交瘤细胞株保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.19941，分类命名：杂交瘤细胞株，保藏日期为2020年5月11日。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本申请而不用于限制本发明的范围。
具体实施方式
下面通过实施例和对比例进一步说明本发明，这些实施例只是用于说明本发明，本发明不限于以下实施例。凡是对本发明技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围，均应涵盖在本发明的保护范围中。
为了寻找布鲁菌的经典毒力因子，本发明实施例从布鲁氏菌Rev.I株基因组中扩增BAB-22915基因片段。布鲁菌的BAB22915基因为编码裂解酶基因，突变株表现出在RAW264.7细胞和小鼠体内存活率下降；突变株BAB22915在宿主细胞内不与溶酶体融合，不引起明显的病理损伤。本发明通过锐意研究发现，获得了具有良好的反应原性的BAB抗原，并针对BAB抗原制备、筛选得到杂交瘤细胞株，制备得到了抗布鲁氏菌BAB抗原的单克隆抗体，为布鲁氏菌诊断及新型疫苗的研制提供了基础。
本发明实施例的杂交瘤细胞株，保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，地址：北京市朝阳区北辰西路1号院3号，保藏编号为CGMCC No.19941，分类命名：杂交瘤细胞株，保藏日期为2020年5月11日。
本发明实施例的抗布鲁氏菌BAB抗原的单克隆抗体，其由保藏编号CGMCCNo.19941的杂交瘤细胞株产生。
本发明实施例还涉及该杂交细胞株在用于产抗布鲁氏菌BAB抗原的单克隆隆抗体中的应用。
具体的，布鲁氏菌BAB抗原的表位如SEQ ID NO:1所示的氨基酸序列所示。
具体的，SEQ ID No.1的氨基酸序列为：
GSMKIIASVLAAALFASTALSGAGAATTPAPAAATDNAVNMPKPECETDFGQWMENLTREAREAGVGEKGIAELHKASIDQKVLGRDRKQTVFNLTFTEFSKRLISEARLKKGQENLVKYADVFKKVEDAYGVPGPVLAAFWGLETDYGAIQGDFDTLNALVTLSYDCRRPDLFRPQLIALLKLFDQGVVDANTTGAWAGEIGMMQLLPKDYLERGVDGDGDGKVDLKNSVPDAMMTAGRMLSELGWKRGQPWLEEVRLTKDLPWEEAIRTNRKPHSWWAEHGVTGLNGPLGPDDGDASLLLPLGRKGPAFLSYLNFDIFVEWNKSIVYATTAAYFATRLAGAPAFDPGTPVPGLTQDQLKELQTKLQARGYEMGKIDGVFGVATRDAVRSEQLRLGMPADSWPTQELLDRLLE
可选的，编码布鲁氏菌BAB抗原氨基酸序列的核苷酸序列如SEQ ID NO:2所示。
SEQ ID NO:2所示的核苷酸序列为：
ggatccatgaagattatcgcctcagttctggctgcggccttgtttgcctccacggctctatcgggagccggggcagcgacaaccccggctcccgcagccgcgacagataatgccgtcaacatgccgaagccggaatgtgaaactgacttcggtcaatggatggaaaatctgaccagggaagcccgtgaagccggcgtcggtgaaaaaggcattgccgaattgcacaaggcttccatcgaccagaaagttctgggccgcgaccgcaagcagaccgttttcaatctcactttcaccgaattttccaagcgcctcatttccgaggcacgcctgaagaaaggccaggaaaatctcgtcaaatatgccgatgtcttcaagaaagttgaagatgcttatggcgttccgggtccggttcttgccgctttctgggggcttgaaaccgactatggcgcgattcagggcgatttcgacacgctcaatgcgctcgtcacgctctcctacgactgccgccgcccggacctgttccgtccgcagctcatcgccctgctcaagctcttcgatcagggcgtggtcgatgcaaatacgaccggcgcctgggcaggcgaaatcggcatgatgcagcttctgccgaaagactatctggaacgcggcgtcgatggcgacggcgatggcaaggtcgatctgaagaacagcgtgcccgatgccatgatgaccgcagggcgcatgctttcggaactgggctggaagcgcggccagccatggcttgaagaagtgcgcctcaccaaggacctcccctgggaagaggcgatccgcaccaaccgcaagccgcattcatggtgggctgagcatggcgtgacgggcctcaatggcccactcggccctgatgatggcgatgcatcccttctgctgccgcttggccgcaagggaccggctttcctgtcctatctgaattttgacatcttcgtcgaatggaacaaatccatagtctatgcgacgactgccgcctattttgcaacccgccttgcaggtgcccctgctttcgatccgggcaccccggttccggggctgacgcaggatcaactcaaggaattgcagaccaagcttcaggcgcgcggttatgaaatgggcaagatcgatggtgttttcggtgtcgcgacccgcgatgcggtacgctccgaacagttgcgccttggcatgcccgccgattcctggccgacacaggaacttctcgacaggcttctcgag
本发明实施例还涉及该单克隆抗体在制备布鲁氏菌BAB抗原检测试剂中的应用。
具体的，布鲁氏菌BAB抗原的表位如SEQ ID NO:1所示的氨基酸序列所示。
本发明实施例还涉及该杂交瘤细胞株的筛选方法，至少包括以下步骤：
以BAB蛋白作为免疫原，免疫注射BALB/c小鼠，取免疫小鼠脾脏细胞并将其与骨髓瘤细胞融合，获得了表达抗布鲁氏菌BAB抗原的杂交瘤细胞株；BAB蛋白的氨基酸序列如SEQID NO:1所示的氨基酸序列所示。
其中，编码BAB蛋白的氨基酸序列的核苷酸序列如SEQ ID NO:2所示。
本发明实施例还涉及该单克隆抗体的制备方法，至少包括以下步骤：
以BAB蛋白作为免疫原，免疫注射BALB/c小鼠，取免疫小鼠脾脏细胞并将其与骨髓瘤细胞融合，获得了表达抗布鲁氏菌BAB抗原的杂交瘤细胞株；BAB蛋白的氨基酸序列如SEQID NO:1所示的氨基酸序列所示；
采用杂交瘤细胞株进行表达，获得本发实施例的单克隆抗体。
其中，编码BAB蛋白的氨基酸序列的核苷酸序列如SEQ ID NO:2所示。
实施例1
1.免疫原
将氨基酸序列为SEQ ID No.1的BAB抗原，按60μg蛋白/只小鼠的量，皮下注射初次免疫4只SPF级BALB/c雌性小鼠，编号为：BAB-RS-1、BAB-RS-2、BAB-RS-3、BAB-RS-4。皮下第一次加强免疫，免疫量为30μg蛋白/只。皮下第二次加强免疫，免疫量为30μg蛋白/只。皮下第三次加强免疫，免疫量为30μg蛋白/只。眼眶采血，测血清效价。
2、免疫效价检测：
步骤：将氨基酸序列为SEQ ID No.1的BAB抗原，2μg/ml，4℃包被过夜；2％脱脂奶粉(PBS)，37℃封闭2h；血清从200倍开始2倍梯度稀释，空白对照(blank)为PBS，阴性对照(negative)为阴性血清200倍稀释。将稀释的血清分别加入包被BAB蛋白的ELISA板中，37℃孵育1h，加入PBST清洗，1:4000加入酶标抗体，37℃孵育1h，加入PBST清洗，加入TMB显色液显色，读取OD450值。
实验结果如表1和图1所示，效价为大于最大OD450/2的最小OD450读数所对应的稀释度。
表1：
效价为大于最大OD/2的最小OD读数所对应的稀释度。
结果：选取编号BAB-RS-3小鼠，腹腔注射免疫原50μg，进行细胞融合实验。
3、细胞融合实验
取小鼠脾细胞与SP2/0细胞，采用PEG法进行融合。使用半固体培养基(含HAT)进行细胞筛选培养。
实验步骤
1)将状态良好的sp2/0细胞轻柔的从培养瓶壁上吹打下来，吸入到50ml离心管中。
2)小鼠眼球采血，待小鼠死亡，放入75％的酒精中浸泡5min。
3)向平皿中倒入少量IMDM培养基，将细胞筛及注射器内芯放入平皿中。用剪刀和镊子取小鼠脾脏，放至细胞筛上。用注射器内芯轻轻地将脾充分碾碎，将碾好的细胞吸入到装sp2/0的离心管中，离心(1500rad/min，5min)。
4)用剪刀和镊子取小鼠的胸腺，碾碎。将碾好的胸腺细胞转移到15ml离心管中，加入2ml的HAT，2ml的HT放置孵箱备用。
5)离心细胞，弃掉细胞上清，使用无血清IMDM培养基将细胞轻柔地吹匀，离心(1500rad/min，5min)。
6)弃细胞上清。拍打离心管底充分悬浮细胞，将离心管放入37℃温水中，在1分钟内缓慢加入1ml的PEG，加完后，在温水中静置1min。然后2min内缓慢加入2ml的无血清的IMDM培养基，接着2min内缓慢加入8ml无血清的IMDM培养基。离心(1000rad/min，5min)。
7)弃细胞上清，加入10ml的血清，小心的将细胞吹匀，加入胸腺细胞及25ml半固体培养基，充分混匀。将其均匀倒入30个细胞培养皿。细胞培养皿放入湿盒，放入孵箱中培养。
4、挑克隆
挑10板×93个细胞单克隆，培养于96孔细胞培养板。
4.1单克隆细胞1筛
将氨基酸序列为SEQ ID No.1的BAB抗原包板，对挑选的克隆采用ELISA方法检测，进行第一次筛选，得到45株阳性杂交瘤细胞株。
4.1.1实验试剂
包被液：碳酸钠-碳酸氢钠缓冲液，pH9.6
PBS缓冲液：pH7.4
封闭液：2％脱脂奶粉(PBS)
洗液：PBS-T(0.05％吐温，PBS)
显色液：1％A液+10％B液(A液：1％TMB in DMSO；B液：0.1％H2O2in柠檬酸缓冲液)
终止液：2M硫酸
二抗：山羊抗小鼠IgG/HRP
4.1.2实验步骤
1)将包被液稀释氨基酸序列为SEQ ID No.1的BAB抗原，终浓度为2μg/ml，100μl/孔，4℃，过夜；用洗液洗涤3次。
2)封闭液封闭，200μl/孔，37℃温箱孵育，2h；后用洗液洗涤3次。
3)加入一抗(细胞培养上清)、阴性对照(SP2/0培养上清)、空白对照(PBS)、阳性对照(阳性血清PBBAB-RS0倍稀释)，100μl/孔，37℃作用1h；用洗液洗涤3次。
4)加入PBS稀释20000倍的二抗，100μl/孔，37℃作用1h；用洗液洗涤3次。
5)显色，显色液100μl/孔，显色时间为5min左右。
6)每孔加入50μl终止液终止。
7)双波长(450，630)测吸光值，记录保存数据。
4.2单克隆细胞2筛
将45株阳性的细胞株，用氨基酸序列为SEQ ID No.1的BAB抗原包板，采用ELISA方法检测，进行第二次筛选，得到阳性杂交瘤细胞株。将阳性细胞株进行亚类鉴定得到IgG类型阳性杂交瘤细胞株，保藏编号CGMCC No.19941。
实施例2单克隆细胞亚类鉴定
将阳性细胞株进行亚类阳性杂交瘤细胞株。具体实验步骤为：
1.1、实验试剂
包被抗体：(SBA Clonotyping System-HRP,Southern Biotech)
封闭液：2％BSA+3％蔗糖in PBS；
显色液：0.2ml A液+10μl 30％H2O2 in 10ml B液
(A液：15mg/ml ABTS in H2O；B液：柠檬酸缓冲液，pH4.0)
各型亚类二抗：(SBA Clonotyping System-HRP,Southern Biotech)
2、实验步骤
将100mM PBS(pH7.4)稀释包被抗体至0.5μg/ml，每孔加0.1ml，4℃，过夜。
PBS-T洗2次；每孔加入200μl封闭液，37℃孵育2h。
PBS-T洗3次；每孔加入100μl杂交瘤上清，37℃孵育1h。
PBS-T洗3次；将用封闭液1：2000稀释的HRP标记的抗体0.1ml每孔，分别加入适当的孔中，37℃孵育1h。
PBS-T洗3次；每孔加50μl底物溶液，10-20min内于双波长(450，630)测吸光值，记录保存数据。
实验结果如表2所示。
表2
本发明实施例制备得到的单克隆细胞亚类为G2b型。
实施例3
从得到的杂交瘤细胞株按每只0.5mL的剂量给小鼠腹腔注射弗氏不完全佐剂，免疫后第10天腹腔接种杂交瘤细胞，按细胞密度2.0×106～3.0×106/mL接种小鼠，每只小鼠的接种剂量为0.5mL，接种后第7～10天待小鼠腹腔变大时，收集腹水并离心。进行单抗腹水中IgG的纯化。
实验步骤为：
取1份腹水8000rpm离心5min，弃沉淀，加2份0.06mol/L(pH5.0)的醋酸缓冲液，用1.0M HCl调pH至4.8。按每毫升稀释腹水11μl辛酸的比例在室温搅拌下逐滴加入正辛酸，室温混匀30min，4℃静置至少2h，然后以12500rpm离心35min，弃沉淀。若离心后上清仍有细小颗粒悬浮，需要以12500rpm再离心15min，弃沉淀。
按照上清液体积的1/10加入0.01M PBS，用1.0M NaOH调pH至7.2。在冰水浴中按0.277g/ml加入硫酸铵至45％-50％饱和度，混匀30min后于4℃静置2h，12500rpm离心30min，弃上清。
沉淀溶于适量PBS，加入5％体积甘油，混匀后装入透析袋，于4℃冰箱中在2L0.01mol/L。PBS缓冲液中透析至少3h，期间更换缓冲液至少3次。
将透析物以10000rpm离心10min，除去不溶性沉渣，加入5％体积甘油，分装-20℃冻存。进行SDS-PAGE电泳鉴定。鉴定结果见图2。
实施例4
将保藏的杂交瘤细胞上清进行Western-Blot检测。
对纯化的BAB-RS蛋白进行SDS-PAGE后转移到PVDF膜上，14V电压电转50min；将膜放入预先用PBST缓冲液洗涤后的平皿，用1％的脱脂乳4℃过夜封闭，用PBST缓冲液洗涤PVDF膜3次，每次15min；加入1:200比例稀释的编号为14的杂交瘤细胞上清，37℃孵育4h；用PBST缓冲液洗涤PVDF膜3次，每次15min，然后加入1:5 000比例稀释的辣根过氧化氢酶标记的兔抗羊IgG抗体，37℃孵育2h；PBST缓冲液洗涤PVDF膜3次，每次15min，后用显色剂显色后拍照。
得到的实验结果如图3所示，其中，左侧条带为make，右侧条带为目的蛋白、12％SDS-PAGE。
本申请虽然以较佳实施例公开如上，但并不是用来限定权利要求，任何本领域技术人员在不脱离本申请构思的前提下，都可以做出若干可能的变动和修改，因此本申请的保护范围应当以本申请权利要求所界定的范围为准。
序列表
<110> 内蒙古农业大学
<120> 杂交瘤细胞株、抗布鲁氏菌BAB抗原的单克隆抗体及其制备和应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 414
<212> PRT
<213> 羊属布鲁氏菌(Brucella melitensis)
<400> 1
Gly Ser Met Lys Ile Ile Ala Ser Val Leu Ala Ala Ala Leu Phe Ala
1 5 10 15
Ser Thr Ala Leu Ser Gly Ala Gly Ala Ala Thr Thr Pro Ala Pro Ala
20 25 30
Ala Ala Thr Asp Asn Ala Val Asn Met Pro Lys Pro Glu Cys Glu Thr
35 40 45
Asp Phe Gly Gln Trp Met Glu Asn Leu Thr Arg Glu Ala Arg Glu Ala
50 55 60
Gly Val Gly Glu Lys Gly Ile Ala Glu Leu His Lys Ala Ser Ile Asp
65 70 75 80
Gln Lys Val Leu Gly Arg Asp Arg Lys Gln Thr Val Phe Asn Leu Thr
85 90 95
Phe Thr Glu Phe Ser Lys Arg Leu Ile Ser Glu Ala Arg Leu Lys Lys
100 105 110
Gly Gln Glu Asn Leu Val Lys Tyr Ala Asp Val Phe Lys Lys Val Glu
115 120 125
Asp Ala Tyr Gly Val Pro Gly Pro Val Leu Ala Ala Phe Trp Gly Leu
130 135 140
Glu Thr Asp Tyr Gly Ala Ile Gln Gly Asp Phe Asp Thr Leu Asn Ala
145 150 155 160
Leu Val Thr Leu Ser Tyr Asp Cys Arg Arg Pro Asp Leu Phe Arg Pro
165 170 175
Gln Leu Ile Ala Leu Leu Lys Leu Phe Asp Gln Gly Val Val Asp Ala
180 185 190
Asn Thr Thr Gly Ala Trp Ala Gly Glu Ile Gly Met Met Gln Leu Leu
195 200 205
Pro Lys Asp Tyr Leu Glu Arg Gly Val Asp Gly Asp Gly Asp Gly Lys
210 215 220
Val Asp Leu Lys Asn Ser Val Pro Asp Ala Met Met Thr Ala Gly Arg
225 230 235 240
Met Leu Ser Glu Leu Gly Trp Lys Arg Gly Gln Pro Trp Leu Glu Glu
245 250 255
Val Arg Leu Thr Lys Asp Leu Pro Trp Glu Glu Ala Ile Arg Thr Asn
260 265 270
Arg Lys Pro His Ser Trp Trp Ala Glu His Gly Val Thr Gly Leu Asn
275 280 285
Gly Pro Leu Gly Pro Asp Asp Gly Asp Ala Ser Leu Leu Leu Pro Leu
290 295 300
Gly Arg Lys Gly Pro Ala Phe Leu Ser Tyr Leu Asn Phe Asp Ile Phe
305 310 315 320
Val Glu Trp Asn Lys Ser Ile Val Tyr Ala Thr Thr Ala Ala Tyr Phe
325 330 335
Ala Thr Arg Leu Ala Gly Ala Pro Ala Phe Asp Pro Gly Thr Pro Val
340 345 350
Pro Gly Leu Thr Gln Asp Gln Leu Lys Glu Leu Gln Thr Lys Leu Gln
355 360 365
Ala Arg Gly Tyr Glu Met Gly Lys Ile Asp Gly Val Phe Gly Val Ala
370 375 380
Thr Arg Asp Ala Val Arg Ser Glu Gln Leu Arg Leu Gly Met Pro Ala
385 390 395 400
Asp Ser Trp Pro Thr Gln Glu Leu Leu Asp Arg Leu Leu Glu
405 410
<210> 2
<211> 1242
<212> DNA
<213> 羊属布鲁氏菌(Brucella melitensis)
<400> 2
ggatccatga agattatcgc ctcagttctg gctgcggcct tgtttgcctc cacggctcta 60
tcgggagccg gggcagcgac aaccccggct cccgcagccg cgacagataa tgccgtcaac 120
atgccgaagc cggaatgtga aactgacttc ggtcaatgga tggaaaatct gaccagggaa 180
gcccgtgaag ccggcgtcgg tgaaaaaggc attgccgaat tgcacaaggc ttccatcgac 240
cagaaagttc tgggccgcga ccgcaagcag accgttttca atctcacttt caccgaattt 300
tccaagcgcc tcatttccga ggcacgcctg aagaaaggcc aggaaaatct cgtcaaatat 360
gccgatgtct tcaagaaagt tgaagatgct tatggcgttc cgggtccggt tcttgccgct 420
ttctgggggc ttgaaaccga ctatggcgcg attcagggcg atttcgacac gctcaatgcg 480
ctcgtcacgc tctcctacga ctgccgccgc ccggacctgt tccgtccgca gctcatcgcc 540
ctgctcaagc tcttcgatca gggcgtggtc gatgcaaata cgaccggcgc ctgggcaggc 600
gaaatcggca tgatgcagct tctgccgaaa gactatctgg aacgcggcgt cgatggcgac 660
ggcgatggca aggtcgatct gaagaacagc gtgcccgatg ccatgatgac cgcagggcgc 720
atgctttcgg aactgggctg gaagcgcggc cagccatggc ttgaagaagt gcgcctcacc 780
aaggacctcc cctgggaaga ggcgatccgc accaaccgca agccgcattc atggtgggct 840
gagcatggcg tgacgggcct caatggccca ctcggccctg atgatggcga tgcatccctt 900
ctgctgccgc ttggccgcaa gggaccggct ttcctgtcct atctgaattt tgacatcttc 960
gtcgaatgga acaaatccat agtctatgcg acgactgccg cctattttgc aacccgcctt 1020
gcaggtgccc ctgctttcga tccgggcacc ccggttccgg ggctgacgca ggatcaactc 1080
aaggaattgc agaccaagct tcaggcgcgc ggttatgaaa tgggcaagat cgatggtgtt 1140
ttcggtgtcg cgacccgcga tgcggtacgc tccgaacagt tgcgccttgg catgcccgcc 1200
gattcctggc cgacacagga acttctcgac aggcttctcg ag 1242
Claims (10)
1.一种杂交瘤细胞株，保藏于中国微生物菌种保藏管理委员会普通微生物中心，保藏编号为CGMCC No.19941。
2.如权利要求1所述的杂交细胞株在用于产抗布鲁氏菌BAB抗原的单克隆隆抗体中的应用。
3.根据权利要求2所述的应用，其特征在于，所述布鲁氏菌BAB抗原的表位如SEQ IDNO:1所示的氨基酸序列所示。
4.根据权利要求3所述的应用，其特征在于，编码所述布鲁氏菌BAB抗原氨基酸序列的核苷酸序列如SEQ ID NO:2所示。
5.一种抗布鲁氏菌BAB抗原的单克隆抗体，其特征在于，其由保藏编号为CGMCCNo.19941的杂交瘤细胞株产生。
6.如权利要求5所述的单克隆抗体在制备布鲁氏菌BAB抗原检测试剂中的应用。
7.根据权利要求6所述的应用，其特征在于，所述布鲁氏菌BAB抗原的表位如SEQ IDNO:1所示的氨基酸序列所示。
8.一种如权利要求1所述的杂交瘤细胞株的筛选方法，其特征在于，至少包括以下步骤：
以BAB蛋白作为免疫原，免疫注射BALB/c小鼠，取免疫小鼠脾脏细胞并将其与骨髓瘤细胞融合，获得了表达抗布鲁氏菌BAB抗原的杂交瘤细胞株；所述BAB蛋白的氨基酸序列如SEQID NO:1所示的氨基酸序列所示。
9.一种如权利要求5所述的单克隆抗体的制备方法，其特征在于，至少包括以下步骤：
以BAB蛋白作为免疫原，免疫注射BALB/c小鼠，取免疫小鼠脾脏细胞并将其与骨髓瘤细胞融合，获得了表达抗布鲁氏菌BAB抗原的杂交瘤细胞株；所述BAB蛋白的氨基酸序列如SEQID NO:1所示的氨基酸序列所示；
采用所述杂交瘤细胞株进行表达，获得所述单克隆抗体。
10.根据权利要求8所述的筛选方法或权利要求9所述的制备方法，其特征在于，编码所述BAB蛋白的氨基酸序列的核苷酸序列如SEQ ID NO:2所示。
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