CN111748034A - 一种滑液囊支原体单克隆抗体的制备方法 - Google Patents
一种滑液囊支原体单克隆抗体的制备方法 Download PDFInfo
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- CN111748034A CN111748034A CN201910230426.9A CN201910230426A CN111748034A CN 111748034 A CN111748034 A CN 111748034A CN 201910230426 A CN201910230426 A CN 201910230426A CN 111748034 A CN111748034 A CN 111748034A
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Abstract
本发明公开一种滑液囊支原体单克隆抗体的制备方法,针对支原体膜表面蛋白质大小为20Kda和13Kda两个假想膜蛋白进行了保守性分析,重组质粒合成、蛋白质诱导表达、亲和层析纯化,以纯化的重组蛋白作为免疫原免疫BALB/c小鼠,经抗体检测合格后进行细胞融合,融合细胞经过亚克隆后,获得单一化单克隆抗体细胞株,制备的单克隆抗体能够较好的特异性识别滑液囊支原体抗原。
Description
技术领域
本发明属于涉及单克隆抗体制备技术领域,具体涉及一种滑囊液支原体单克隆抗体的 制备方法。
背景技术
支原体是一类介于细菌和病毒之间的可在无生命培养基中生长繁殖的最小原核细胞 型微生物。滑液囊支原体被认为是一种能够特定引起鸡关节滑膜和关节囊膜疾病的病原。 滑液囊支原体无细胞壁结构,形态呈多形性特点。其基因组和蛋白质组非常小。滑液囊支 原体主要通过膜表面蛋白与宿主进行互相作用,完成粘附、定殖等过程。与宿主之间的免 疫应答反应也主要以膜表面蛋白为主。近些年来,滑液囊支原体(Mycoplasmasynoviae,MS) 在鸡群中的发病率不断升高,其可造成家禽全身性的损伤,并可引起免疫系统抑制。虽不 直接造成死亡损伤,但间接引起产蛋率下降和肉鸡胴体废弃物增加,影响鸡群的正常生长。 养殖户对MS的认识、检测及防控远远不足,导致MS感染正在从非典型的上呼吸道亚临 床症状转向严重的呼吸系统疾病,如气囊炎;当合并其他致病因子,如鸡新城疫病毒(NDV)、 传染性支气管炎病毒(IBV)、低致病性禽流感病毒或大肠杆菌感染时,会使病情恶化;此外, MS引起的传染性滑膜囊炎、蛋壳形状畸形及自身免疫性疾病同样威胁着全球家禽养殖业 的健康发展。在18届世界禽病大会上,就有专家提出“现在以及不远的将来MS的危害会 超过MG”的观点。最新的流行病学调查显示,在东欧、荷兰、墨西哥、阿根廷,MS感 染可引起严重的呼吸系统疾病和关节滑膜炎;在我国,MS的发病率亦呈明显上升趋势, 加上环境及鸡群状态不良等不利因素,导致MS感染在鸡群中爆发的现象日趋严重。
鸡滑液囊支原体感染是由滑液囊支原体(Mycoplasma synoviae,MS)引起的关节渗 出性滑膜炎、滑液囊和腱鞘发炎为主要特征的急性或慢性传染性疾病,也可引起气囊炎, 甚至导致全身感染,常常与新城疫病毒、传染性支气管炎病毒、大肠杆菌等致病微生物或 混合感染。其膜上含有丰富的脂蛋白,称为脂质相关膜蛋白(Lipid associatedmembrane proteins,LAMPs),具有很强的免疫原性。同时脂蛋白易发生相位、抗原变异,造成支原 体表面种类丰富多样化。虽然人们对支原体致病机理尚不明确,但越来越多的研究表明脂 蛋白在其致病过程中扮演重要的角色。单克隆抗体在疾病诊断中的作用不言而喻。目前针 对滑液囊支原体诊断性单抗的研究和开发甚少。研制效价高,特异性强的单克隆抗体是兽 医诊断行业的需求,在滑液囊支原体中,共有近100种膜表面假象蛋白,目前针对假想蛋 白的研究较少,而现有技术制备滑液囊支原体单克隆抗体大多采用其膜表面已知序列的蛋 白质作为抗原,仅仅针对一种特定的蛋白,无法针对支原体全菌起免疫反应。
如论文“温政.鸡滑液囊支原体LP78蛋白单克隆抗体的制备及鉴定[D].山东.山东农业 大学2018.06中利用滑液囊支原体上的LP78蛋白作为抗原制备单克隆抗体,但是其免疫反 应仅仅针对滑液囊支原体膜表面已知基因序列的LP78蛋白。
发明内容
本发明的目的是针对现有技术存在的问题,提供一种滑液囊支原体单克隆抗体的制备 方法。
为实现上述目的,本发明采用的技术方案是:
一种滑液囊支原体单克隆抗体的制备方法,包括以下步骤:
(1)制备免疫原:将P20基因和P13基因插入到表达载体PET-32a中,获得重组表达载体,用重组表达载体转化大肠杆菌感受态细胞DH5α,经酶切、PCR测序鉴定,筛选出 阳性克隆重组质粒PET-32a-P20-P13;
(2)重组蛋白表达:将步骤(1)筛选出的阳性克隆重组质粒PET-32a-P20-P13经过诱 导表达、纯化,获得PET-32a-P20-P13重组蛋白;
(3)小鼠免疫及筛选:使用步骤(2)中获得的PET-32a-P20-P13重组蛋白进行小鼠免 疫,获取免疫脾细胞;
(4)细胞融合、筛选及克隆化:筛选抗血清效价最高的免疫脾细胞与骨髓瘤细胞体外 融合获取融合细胞,筛选阳性杂交瘤细胞株,将所述阳性杂交瘤细胞进行亚克隆,获得能 够稳定分泌抗滑液囊支原体单克隆抗体的杂交瘤细胞株;
其中,步骤(1)中的P20基因序列如序列表SEQ ID NO:1所示,所述P13基因如序列表SEQ ID NO:2所示;所述PET-32a-P20-P13重组蛋白的基因序列如序列表SEQ ID NO:3 所示。
所述P20基因所编码的蛋白质氨基酸序列如序列表SEQ ID NO:4所示,所述P13基因 所编码的蛋白质氨基酸序列如序列表SEQ ID NO:5所示,经NCBI数据库中Blast工具与数据库中的数据进行比对,结果证明上述两个蛋白均和滑液囊支原体的相似性在86.76%以 上,与鸡毒支原体的相似性小于50%。证明上述两个蛋白质均具有较好的特异性和保守性。
进一步的,所述步骤(2)的具体步骤为:挑选转化后生长良好的单菌落,接种至含氨苄抗性的LB液体培养基中,培养过夜,次日取菌液扩大培养,待菌液OD600>0.8时, 加入阿拉伯糖诱导剂诱导表达过夜,收集菌体,离心,重悬洗涤,将重悬的菌体破碎离心, 取上清液进行His-镍柱进行亲和层析纯化。
进一步的,步骤(4)体外融合的具体步骤为:
A、饲养细胞的制备:取成熟健康的小鼠,腹部注射HAT培养基,抽回腹腔液体,计数并稀释后加入96孔细胞培养板中;
B、将免疫脾细胞与骨髓瘤细胞混合,离心,弃上清,混合均匀后,水浴静置,离心,弃上清,用HAT培养基悬浮沉淀细胞,分装于上述96孔细胞培养板中培养。
进一步的,步骤B中的培养条件为37℃,6%CO2。
进一步的,步骤(4)中亚克隆采用的方法为有限稀释法。
进一步的,步骤(4)中筛选阳性杂交瘤细胞株的方法为:使用滑液囊支原体全菌灭活抗原、PET-32a-P20-P13重组蛋白及PET-32a空载表达的蛋白分别包被ELISA板,采用 间接ELISA来筛选阳性克隆。
与现有技术相比,本发明的有益效果是:
本发明针对支原体膜表面蛋白质大小为20Kda和13Kda两个假想膜蛋白进行了保守性 分析,重组质粒合成、蛋白质诱导表达、亲和层析纯化,以纯化的重组蛋白作为免疫原免 疫BALB/c小鼠,经抗体检测合格后进行细胞融合,融合细胞经过亚克隆后,获得单一化单克隆抗体细胞株,制备的单克隆抗体能够较好的特异性识别滑液囊支原体全菌抗原。
附图说明
图1为本发明中制备的重组蛋白酶切电泳分析图,图中M:DNA marker,1: PET-32a-P20-P13连接产物,2:PET-32a-P20-P13经BamHI//XhoI酶切;
图2为本发明中制备的单克隆抗体细胞株与滑液囊支原体的反应原性检测图,图中M: 蛋白质Marker,1:阴性对照;2-6分别为6A11、5B9、4F2、1C8、6C2细胞上清。
具体实施方式
下面将结合本发明中的附图,对本发明的技术方案进行清楚、完整地描述,显然,所 描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例, 本领域普通技术人员在没有做出创造性劳动条件下所获得的所有其它实施例,都属于本发 明保护的范围。
实施例1:抗原的制备
(1)将P20基因和P13基因插入到表达载体PET-32a中,获得重组表达载体,用重组表达载体转化大肠杆菌感受态细胞DH5α,经酶切、PCR鉴定和测序,筛选出阳性克隆重 组质粒PET-32a-P20-P13;经BamHI//XhoI酶切、及重组质粒PET-32a-P20-P13的电泳检测 如图1所示,P20基因序列如序列表SEQ ID NO:1所示,所述P13基因如序列表SEQ ID NO:2 所示;所述PET-32a-P20-P13重组蛋白的基因序列如序列表SEQ ID NO:3所示。
所述P20基因所编码的蛋白质氨基酸序列如序列表SEQ ID NO:4所示,所述P13基因 所编码的蛋白质氨基酸序列如序列表SEQ ID NO:5所示,经NCBI数据库中Blast工具与数据库中的数据进行比对,结果证明上述两个蛋白均和滑液囊支原体的相似性在86.76%以 上,与鸡毒支原体的相似性小于50%。证明上述两个蛋白质均具有较好的特异性和保守性。
(2)重组蛋白的表达:
挑选转化后生长良好的单菌落,接种至10ml含氨苄抗性的LB液体培养基中,培养过 夜,次日取5ml上述菌液扩大培养至500ml,待菌液OD600在0.8以上时,加入阿拉伯糖诱导剂诱导25℃诱导表达过夜。然后收集菌体,离心后使用磷酸盐缓冲液重悬洗涤两遍,将重悬的菌体在高压破碎仪下进行破碎,破碎后再次离心分离破碎后上清和细胞碎片。破碎后上清液经过His-镍柱进行亲和层析纯化。纯化后蛋白经过SDS-PAGE电泳观察表达情况,并采用核酸蛋白浓度测定仪对所纯化的蛋白进行浓度测定。结果:重组蛋白为可溶性表达,纯化后蛋白浓度为3.2mg/mL。
实施例2:小鼠免疫、细胞融合及亚克隆
1)第一次免疫使用弗氏完全佐剂与等量重组蛋白进行乳化,乳化后皮下接种5只6-8 周龄Balb/c雌性小鼠,每只接种剂量200ug重组蛋白。之后每间隔两周免疫一次,第二次 免疫以后佐剂换为弗氏不完全佐剂,经过三次免疫后,间隔一周尾静脉采血检测抗体,使 用滑液囊支原体灭活全菌作为包被抗原进行抗体检测。
2)选择产生的抗体效价最高的小鼠作为细胞融合的小鼠,在融合前腹腔接种重组蛋 白加强免疫一次。
3)骨髓瘤细胞的准备:选择生长状态良好的SP2/0细胞,密度长至培养瓶底75%时弃上清,用10mL DMEM培养基将细胞轻轻吹下。
4)脾淋巴细胞的准备:取加强免疫后3天的小鼠,摘除眼球采血作为阳性血清;颈椎脱臼致死小鼠,置75%酒精中5min,腹部朝上用无菌打开腹腔取出脾脏,用DMEM培 养基洗涤,去除多余结缔组织和脂肪;随后将脾脏转移到另一个盛有DMEM培养基的平 皿中。用研磨棒在细胞筛网上将脾脏磨碎,收集脾细胞悬液。
5)饲养细胞的制备:取一只成熟健康的KM雌鼠,颈椎脱臼致死小鼠;置75%酒精中10min,然后剪开腹腔部位肚皮表皮层,用10mL注射器,12#针头,吸取10mL HAT 培养基小心注射到腹腔,持酒精棉球轻轻按摩腹部,抽回腹腔内液体,注入己准备好的容 器中,计数并稀释后加入96孔细胞培养板中。
6)融合:将上述制备的脾细胞与骨髓瘤细胞按5:1的比例混合于一支50mL的融合管 中,1000g离心10min,弃上清。将融合管置于手掌中,轻轻摩擦底部,使两种细胞充分 混匀;在37℃水浴中45s内缓慢加入1mL预热的PEG1500到融合管中,边加边轻轻摇匀; 随即在90s内先慢后快滴加30mL 37℃预热的DMEM培养基终止反应,37℃水浴中静置 10min,1000g离心10min;弃上清,用60mL HAT培养基轻轻悬浮沉淀细胞,分装于已 铺好饲养细胞的96孔细胞培养板,然后将培养板置37℃6%CO2培养箱内培养;5d后用 新鲜的HAT培养基进行半换液;10d后用预热的HT培养基换出HAT培养基;观察杂交 瘤细胞生长情况,待其细胞克隆分布至孔底面积的1/8以上时,吸取100μL细胞上清进 行抗体检测。
7)杂交瘤细胞的筛选:使用滑液囊支原体全菌灭活抗原、重组蛋白及PET-32a空载表 达的蛋白分别包被ELISA板,采用间接ELISA来筛选阳性克隆。选择滑液囊支原体全菌灭活抗原和重组蛋白检测均为阳性,且PET-32a空载表达蛋白检测为阴性的细胞孔定义为阳性孔,并对阳性孔进行有限稀释亚克隆,直至获得的阳性细胞株均为单一化的细胞为止。最后筛选出5株效果显著的单一化细胞株。
实施例3:抗体的制备与验证
1)抗体生产:分别将上述获得的5株细胞株先后经过放大培养,逐步实现抗体的大规模生产。过程如下,首先在24孔细胞板中复苏,复苏时加入饲养细胞促进杂交瘤细胞 株的快速生长,长至孔底75%面积时,将细胞转移至T25细胞培养瓶中培养,同时添加饲 养细胞。待T25细胞培养瓶长至瓶底75%面积时,将杂交瘤细胞转移至T75细胞培养瓶, 然后转至T225细胞培养瓶,随后转至10升转瓶上培养。每个阶段留样检测抗体效价,以 测定细胞的遗传稳定性,效价测定结果如下表1:
表1抗体效价测定
细胞株名称 | 24孔板 | T25瓶 | T75瓶 | T225瓶 | 10升转瓶 |
6A11 | 1:6400 | 1:6400 | 1:6400 | 1:6400 | 1:12800 |
5B9 | 1:3200 | 1:3200 | 1:3200 | 1:6400 | 1:6400 |
4F2 | 1:3200 | 1:3200 | 1:3200 | 1:6400 | 1:6400 |
1C8 | 1:6400 | 1:6400 | 1:6400 | 1:6400 | 1:12800 |
6C2 | 1:3200 | 1:3200 | 1:3200 | 1:6400 | 1:6400 |
结果表明,经过不同培养瓶条件下培养的杂交瘤细胞系均能够稳定分泌效价,而且在 大容量10L转瓶中培养的抗体效价是其他条件下的两倍。
2)亚类鉴定:使用单克隆抗体亚类鉴定试剂盒进行鉴定,5株细胞株均为IgG1,结果见表2。
表2单克隆抗体亚类鉴定结果
名称 | 6A11 | 5B9 | 4F2 | 1C8 | 6C2 |
亚类 | IgG1 | IgG1 | IgG1 | IgG1 | IgG1 |
与滑液囊支原体的反应原性:使用滑液囊支原体全菌灭活抗原进行SDS-PAGE,并转 印至PVDF膜上,经过5%脱脂乳封闭2h后,分别使用上述5种抗体与膜一起孵育过夜, 经PBST洗涤后,加入HRP标记的酶标二抗进行孵育2h,最后使用ECL化学发光增强液 进行显影,检测结果如图1,表明五种抗体均与滑液囊支原体具有较好的反应原性。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解 在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变 型,本发明的范围由所附权利要求及其等同物限定。
序列表
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tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg 60
cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc 120
ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg 180
gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc 240
acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt 300
ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc 360
ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta 420
acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaatttcag gtggcacttt 480
tcggggaaat gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 540
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 600
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 660
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 720
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 780
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 840
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 900
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 960
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 1020
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1080
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1140
tgcagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1200
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1260
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1320
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1380
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1440
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1500
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1560
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1620
aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 1680
accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 1740
aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 1800
ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 1860
agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 1920
accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 1980
gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 2040
tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 2100
cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 2160
cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 2220
cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 2280
ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga 2340
taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 2400
gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatatgg 2460
tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat 2520
cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct 2580
gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct 2640
gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct 2700
catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt 2760
tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg 2820
ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa 2880
tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc 2940
ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa 3000
aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta 3060
gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg 3120
tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag 3180
acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac 3240
cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca 3300
cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg 3360
gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca agcgacaggc 3420
cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag agcgctgccg 3480
gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg acgatagtca 3540
tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc atcggtcgag 3600
atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca ctgcccgctt 3660
tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 3720
gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac gggcaacagc 3780
tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac gctggtttgc 3840
cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca tgagctgtct 3900
tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc ggactcggta 3960
atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc agtgggaacg 4020
atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact ccagtcgcct 4080
tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca gccagccaga 4140
cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg ctggtgaccc 4200
aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa aataatactg 4260
ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt gcaggcagct 4320
tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc actgacgcgt 4380
tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg ttctaccatc 4440
gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc gacaatttgc 4500
gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga ctgtttgccc 4560
gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc cgcttccact 4620
ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga aacggtctga 4680
taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac attcaccacc 4740
ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt gcgccattcg 4800
atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga agcagcccag 4860
tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca aggagatggc 4920
gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac aagcgctcat 4980
gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat aggcgccagc 5040
aaccgcacct gtggcgccgg tgatgccggc cacgatgcgt ccggcgtaga ggatcgagat 5100
cgatctcgat cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa 5160
ttcccctcta gaaataattt tgtttaactt taagaaggag atatacatat gagcgataaa 5220
attattcacc tgactgacga cagttttgac acggatgtac tcaaagcgga cggggcgatc 5280
ctcgtcgatt tctgggcaga gtggtgcggt ccgtgcaaaa tgatcgcccc gattctggat 5340
gaaatcgctg acgaatatca gggcaaactg accgttgcaa aactgaacat cgatcaaaac 5400
cctggcactg cgccgaaata tggcatccgt ggtatcccga ctctgctgct gttcaaaaac 5460
ggtgaagtgg cggcaaccaa agtgggtgca ctgtctaaag gtcagttgaa agagttcctc 5520
gacgctaacc tggccggttc tggttctggc catatgcacc atcatcatca tcattcttct 5580
ggtctggtgc cacgcggttc tggtatgaaa gaaaccgctg ctgctaaatt cgaacgccag 5640
cacatggaca gcccagatct gggtaccgac gacgacgaca aggccatggc tgatatcata 5700
tcagctatta ctccttatta ttacaattat agtttcgaag aactttacaa ttactatcaa 5760
gaaattacaa atgcagctca aattcctatg ctaatttatt acttaccaca attagcaggt 5820
aaaaaagtat cagttgaaga atttcaaaaa ttacttgaaa ttccaaatgt tattggttca 5880
aaatatggat caacagattt atttaccttt gaaagattaa tggctaaatt ccctgataaa 5940
gtttttatgt ttgcccatga tgaagcttta gcacctggat taacatttgg agcaaaaggt 6000
tttattggtt cagcatataa tgttaacgca aaagcaacta gagaaataat tacagccgat 6060
aaagaaaaag ttgctaaatt aacccaccaa tataatgact acattcaagc tttaatttca 6120
aaaggtttaa tgcaatcact taaagctatt atacgtctta aaaatgtaaa tgctggttat 6180
acaagaaaac catttttaca ttatgaagac tcagtaattg aaaaacacgc acaagaagta 6240
ataaaaaaat acattaaagt ggaatttatc aagcgtagct atagcattaa atgtccttta 6300
attaaaacga tttacaattg aattaattca aaaatttgaa aaataaatcg caaaaattta 6360
cttagaagca attatgtatt tggcggcaaa aggcaaaaat ctgtccaaga gcgactagta 6420
agaaaacgct gagttaggcc ttttaaaatc ggattaagaa aagaattcgg ccactgggaa 6480
gcaaatttaa tcaaaggcaa aagagcaact aaggttcatt tactagtttt ccaagaaaga 6540
ctcaccagat ttgttttaat taaaaaaata atgactaaaa atccgtgaaa cattaatcta 6600
gaacttttaa atttagttaa aggctattga attcaccacc accaccacca ctgagatccg 6660
gctgctaaca aagcccgaaa ggaagctgag ttggctgctg ccaccgctga gcaataacta 6720
gcataacccc ttggggcctc taaacgggtc ttgaggggtt ttttgctgaa aggaggaact 6780
atatccggat 6790
<210> 4
<211> 187
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 4
Met Ser Ala Ile Thr Pro Tyr Tyr Tyr Asn Tyr Ser Phe Glu Glu Leu
1 5 10 15
Tyr Asn Tyr Tyr Gln Glu Ile Thr Asn Ala Ala Gln Ile Pro Met Leu
20 25 30
Ile Tyr Tyr Leu Pro Gln Leu Ala Gly Lys Lys Val Ser Val Glu Glu
35 40 45
Phe Gln Lys Leu Leu Glu Ile Pro Asn Val Ile Gly Ser Lys Tyr Gly
50 55 60
Ser Thr Asp Leu Phe Thr Phe Glu Arg Leu Met Ala Lys Phe Pro Asp
65 70 75 80
Lys Val Phe Met Phe Ala His Asp Glu Ala Leu Ala Pro Gly Leu Thr
85 90 95
Phe Gly Ala Lys Gly Phe Ile Gly Ser Ala Tyr Asn Val Asn Ala Lys
100 105 110
Ala Thr Arg Glu Ile Ile Thr Ala Asp Lys Glu Lys Val Ala Lys Leu
115 120 125
Thr His Gln Tyr Asn Asp Tyr Ile Gln Ala Leu Ile Ser Lys Gly Leu
130 135 140
Met Gln Ser Leu Lys Ala Ile Ile Arg Leu Lys Asn Val Asn Ala Gly
145 150 155 160
Tyr Thr Arg Lys Pro Phe Leu His Tyr Glu Asp Ser Val Ile Glu Lys
165 170 175
His Ala Gln Glu Val Ile Lys Lys Tyr Ile Lys
180 185
<210> 5
<211> 125
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 5
Met Glu Phe Ile Lys Arg Ser Tyr Ser Ile Lys Cys Pro Leu Ile Lys
1 5 10 15
Thr Ile Tyr Asn Trp Ile Asn Ser Lys Ile Trp Lys Ile Asn Arg Lys
20 25 30
Asn Leu Leu Arg Ser Asn Tyr Val Phe Gly Gly Lys Arg Gln Lys Ser
35 40 45
Val Gln Glu Arg Leu Val Arg Lys Arg Trp Val Arg Pro Phe Lys Ile
50 55 60
Gly Leu Arg Lys Glu Phe Gly His Trp Glu Ala Asn Leu Ile Lys Gly
65 70 75 80
Lys Arg Ala Thr Lys Val His Leu Leu Val Phe Gln Glu Arg Leu Thr
85 90 95
Arg Phe Val Leu Ile Lys Lys Ile Met Thr Lys Asn Pro Trp Asn Ile
100 105 110
Asn Leu Glu Leu Leu Asn Leu Val Lys Gly Tyr Trp Ile
115 120 125
Claims (6)
1.一种滑液囊支原体单克隆抗体的制备方法,其特征在于,包括以下步骤:
(1)制备免疫原:将P20基因和P13基因插入到表达载体PET-32a中,获得重组表达载体,用重组表达载体转化大肠杆菌感受态细胞DH5α,经酶切、PCR测序鉴定,筛选出阳性克隆重组质粒PET-32a-P20-P13;
(2)重组蛋白表达:将步骤(1)筛选出的阳性克隆重组质粒PET-32a-P20-P13经过诱导表达、纯化,获得PET-32a-P20-P13重组蛋白;
(3)小鼠免疫及筛选:使用步骤(2)中获得的PET-32a-P20-P13重组蛋白进行小鼠免疫,获取免疫脾细胞;
(4)细胞融合、筛选及克隆化:筛选抗血清效价最高的免疫脾细胞与骨髓瘤细胞体外融合获取融合细胞,筛选阳性杂交瘤细胞株,将所述阳性杂交瘤细胞进行亚克隆,获得能够稳定分泌抗滑液囊支原体单克隆抗体的杂交瘤细胞株;
其中,步骤(1)中的P20基因序列如序列表SEQ ID NO:1所示,所述P13基因如序列表SEQID NO:2所示;所述PET-32a-P20-P13重组蛋白的基因序列如序列表SEQ ID NO:3所示。
2.如权利要求1所述的一种滑液囊支原体单克隆抗体的制备方法,其特征在于,所述步骤(2)的具体步骤为:挑选转化后生长良好的单菌落,接种至含氨苄抗性的LB液体培养基中,培养过夜,次日取菌液扩大培养,待菌液OD600>0.8时,加入阿拉伯糖诱导剂诱导表达过夜,收集菌体,离心,重悬洗涤,将重悬的菌体破碎离心,取上清液进行His-镍柱进行亲和层析纯化。
3.如权利要求1所述的一种滑液囊支原体单克隆抗体的制备方法,其特征在于,步骤(4)体外融合的具体步骤为:
A、饲养细胞的制备:取成熟健康的小鼠,腹部注射HAT培养基,抽回腹腔液体,计数并稀释后加入96孔细胞培养板中;
B、将免疫脾细胞与骨髓瘤细胞混合,离心,弃上清,混合均匀后,水浴静置,离心,弃上清,用HAT培养基悬浮沉淀细胞,分装于上述96孔细胞培养板中培养。
4.如权利要求3所述的一种滑囊液支原体单克隆抗体的制备方法,其特征在于,步骤B中的培养条件为37℃,6%CO2。
5.如权利要求1所述的一种滑囊液支原体单克隆抗体的制备方法,其特征在于,步骤(4)中亚克隆采用的方法为有限稀释法。
6.如权利要求1所述的一种滑囊液支原体单克隆抗体的制备方法,其特征在于,步骤(4)中筛选阳性杂交瘤细胞株的方法为:使用滑液囊支原体全菌灭活抗原、PET-32a-P20-P13重组蛋白及PET-32a空载表达的蛋白分别包被ELISA板,采用间接ELISA来筛选阳性克隆。
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CN108484758A (zh) * | 2018-02-11 | 2018-09-04 | 浙江大学 | 抗埃博拉病毒vp40蛋白单克隆抗体a2g7及其应用 |
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CN108484758A (zh) * | 2018-02-11 | 2018-09-04 | 浙江大学 | 抗埃博拉病毒vp40蛋白单克隆抗体a2g7及其应用 |
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CN115141810A (zh) * | 2022-09-05 | 2022-10-04 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | 分泌抗鸡滑液囊支原体单克隆抗体的杂交瘤细胞株及应用 |
CN115141810B (zh) * | 2022-09-05 | 2022-11-08 | 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心) | 分泌抗鸡滑液囊支原体单克隆抗体的杂交瘤细胞株及应用 |
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