CN109734809A - 治疗性的人Plk1蛋白单克隆抗体及其制备方法 - Google Patents
治疗性的人Plk1蛋白单克隆抗体及其制备方法 Download PDFInfo
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Abstract
本发明提供了一种治疗性的人Plk1蛋白单克隆抗体及其制备方法,首先体外表达纯化了人Plk1蛋白,以重组Plk1蛋白为免疫原,免疫Balb/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤SP2/0细胞融合;通过间接ELISA法筛选分泌特异性McAb的杂交瘤细胞;用杂交瘤细胞株诱导小鼠产生腹水,再用蛋白G亲和层析法纯化抗体,最终获得5株治疗性的人Plk1蛋白单克隆抗体。相比现有技术,本发明的制备工艺简单、易行,试剂易得,一次性可提取5株针对癌症治疗靶点:人Plk1蛋白的鼠单克隆抗体,它们通过调控蛋白的活性调控细胞周期;可以通过进一步的抗体人源化改造使其成为具有治疗功能的抗癌药物前体。
Description
技术领域
本发明属于生物技术领域,尤其是治疗性人Plk1蛋白单克隆抗体及其制备方法。本发明涉及5株特异性针对癌症治疗靶点的人Plk1蛋白的鼠单克隆抗体,它们通过调控蛋白的活性调控细胞周期,可以通过进一步的抗体人源化改造使其成为具有治疗功能的抗癌药物前体。
背景技术
Polo样激酶1(Polo-like kinase 1,Plk1)是细胞周期中一种重要的调控蛋白,研究表明,Plk1和肿瘤发生发展有着密切关系,使其成为了肿瘤治疗的最具潜力的靶点之一。Plk1从其被发现开始就一直是肿瘤生物学领域的一个研究热点。无论是科研机构还是制药公司都竞相角逐,对其进行了广泛研究,希望以其为突破口深入研究肿瘤的发生机制以及找到合适的药物或治疗方案。目前对 Plk1的研究广泛,Plk1在细胞周期进程中对多种事件发挥重要调控作用,与肿瘤的发生发展密切相关。在肿瘤的治疗中,以Plk1为治疗靶点已表现出良好的应用前景。
抗体药物是一种由抗体物质组成的药物,从目前全球已经上市的抗体药物的治疗领域分布来看,主要用于肿瘤和自身免疫病的治疗。抗体的制备技术主要经历了三代:第一代是利用抗原免疫动物后获得抗体,成为多克隆抗体;第二代是通过杂交瘤技术制备出针对抗原中某一抗原决定簇的抗体,称为单克隆抗体;第三代是利用基因工程技术制备而来称为基因工程抗体。
现阶段针对Plk1的药物主要以合成的小分子药物为主,包括ATP竞争性抑制剂,非ATP竞争性抑制剂和其他抑制剂,在药物开发的过程中需要克服其造成的细胞毒性和脱靶效应。而抗体针对相应抗原具有高特异性和高亲和力的特性,使得其在疾病的诊断和治疗中显示出其他类型药物无可比拟的优势。
发明内容
为解决上述技术问题,本发明首先体外表达纯化了人Plk1蛋白,以重组Plk1 蛋白为免疫原,免疫Balb/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤SP2/0细胞融合;通过间接ELISA法筛选分泌特异性McAb的杂交瘤细胞;用杂交瘤细胞株诱导小鼠产生腹水,再用蛋白G亲和层析法纯化抗体,最终获得5株治疗性的人Plk1蛋白单克隆抗体。
本发明的技术方案如下:
治疗性的人Plk1蛋白单克隆抗体的制备方法,包括以下步骤:
S1,利用PCR技术体外扩增Plk1基因,Plk1基因的核酸序列为SEQ ID NO.1 所示;
S2,Plk1基因的表达和纯化:将Plk1基因连接进入pET-28a载体中,并将质粒转化进入大肠杆菌BL21(DE3)中,IPTG诱导后收集菌体;高压破碎菌体,离心后收集上清,利用Ni柱亲和层析纯化蛋白;纯化后的蛋白利用Superdex-200 柱子进行再次精细纯化,收集Plk1蛋白作为抗原备用,Plk1蛋白的氨基酸序列为 SEQ ID NO.2所示;
S3,小鼠的免疫:用纯化的Plk1蛋白作为抗原,对6-8周龄的雌性BALB/c 小鼠3只进行三次免疫,每次免疫间隔两周,第三次免疫后10天,尾部采血,并用间接ELISA检测小鼠产生的抗体滴度;在融合前的第3天尾静脉最后一次注射重组抗原,剂量为60ug/只,进行加强免疫一次;
S4,骨髓瘤细胞与免疫脾细胞的融合:分别收集免疫小鼠脾细胞和SP2/0细胞,DMEM培养基洗涤3次后,调整脾细胞数与SP2/0细胞数之比为1∶10,50% PEG1500作用1~2min后,加入含有HAT的完全培养基稀释,接种于96孔培养板,37℃,5%CO2培养;
S5,阳性杂交瘤细胞筛选与克隆化:经间接ELISA方法连续检测两次都为阳性的细胞孔,按有限稀释法进行亚克隆;如检出细胞孔有特异性抗Plk1蛋白抗体,可选择抗体效价高,呈单个克隆生长,形态良好的杂交瘤细胞孔,继续同法再克隆至少2次,克隆后阳性孔的杂交瘤细胞可移至24孔培养板,待24孔板中的杂交瘤细胞生长良好时,可冻存杂交瘤细胞;
S6,单克隆抗体的生产:对已经建株的杂交瘤细胞扩大培养同时在BALB/c 小鼠体内诱生小鼠腹水生产单克隆抗体;
S7,单克隆抗体的纯化:腹水用20mM PBS pH7.4稀释3倍以上,离心取上清,经HiTrap protein G亲和层析进行纯化,获得治疗性的人Plk1蛋白单克隆抗体。
作为该制备方法的优选方案,S3的免疫方案具体为:
第一阶段,小鼠选取:选取6-8周龄,健康活跃小鼠3只,编号;
第二阶段,抗原乳化:每只小鼠60ug,抗原+1×PBS总体积为350ul,与等体积福氏佐剂混合乳化3500次,200次/min,获得每0.2ml含60ug抗原的乳化液;
第三阶段,第一次免疫:用2ml注射器每只小鼠腹股沟皮下注射0.2ml乳化液,并记录注射时间和部位;
第四阶段,第二次免疫:间隔两周,用2ml注射器每只小鼠腹腔注射0.2ml 乳化液,并记录注射时间和部位;
第五阶段,第三次免疫:间隔两周,用2ml注射器每只小鼠腹股沟皮下注射注射0.2ml乳化液,并记录注射时间和部位;
第六阶段,测效价:间隔10±1天后取血检测进行Elisa检测。
作为该制备方法的优选方案,S4的亚克隆方案具体为:克隆前一天制备小鼠饲养细胞层;将要克隆的杂交瘤细胞从培养孔内轻轻地吹下,用血细胞计数板计数活细胞数;将细胞用完全培养基稀释到每毫升5、10、50个细胞;将上述三个浓度的细胞悬液分别加入已制备好的饲养细胞的96孔培养板中100μL/孔,使相应的每孔分别含0.5、1和5个细胞;培养第4天半量换液一次,第5-6天仔细观察各孔内细胞的生长情况,并记录;在克隆后第10-14天,细胞长满约1/4-1/2孔底时,进行全量换液,次日进行间接ELISA检测。
作为该制备方法的优选方案,S6的诱生小鼠腹水生产单克隆抗体方案具体为:取10周龄的BALB/c小鼠,腹腔注射液体石蜡0.5mL/只,7天后腹腔注射杂交瘤细胞5×105-1×106/只,7-10天后抽取小鼠腹水。
作为该制备方法的优选方案,获得的治疗性的人Plk1蛋白单克隆抗体,采用Ig类与亚类鉴定试剂盒鉴定人Plk1蛋白单克隆抗体的Ig亚型;通过间接ELISA测定人Plk1蛋白单克隆抗体的效价;利用Western Blotting鉴定人Plk1 蛋白单克隆抗体的特性。
作为该制备方法的优选方案,间接ELISA测定人Plk1蛋白单克隆抗体的效价的方法是:Plk1蛋白以100ng/孔包被,将单抗按1:10000、1:20000、1:40000、 1:80000、1:160000、1:320000、1:640000、1:1280000、1:2560000、1:5120000、1:10240000、1:20480000稀释,通过间接ELISA法测定其A450nm 值;以与免疫蛋白靶抗原发生反应的单克隆抗体的最大稀释度即为其效价,测定孔读数与阴性对照值之比大于2.1为阳性。
作为该制备方法的优选方案,利用Western Blotting鉴定人Plk1蛋白单克隆抗体的特性的方法是:将Plk1蛋白经蛋白质电泳后转移仪PVDF膜上,加入含3%BSA后4℃封闭过夜,分别加入5株Plk1单克隆抗体室温孵育2h,以HRP 标记的羊抗鼠IgG的抗体为二抗,室温孵育1h,用ECL显色。
本发明还保护如上所述的制备方法制备获得的5株治疗性的人Plk1蛋白单克隆抗体。
本发明首先体外表达纯化了人Plk1蛋白,以重组Plk1蛋白为免疫原,免疫 Balb/c小鼠,取免疫小鼠脾细胞与小鼠骨髓瘤SP2/0细胞融合。通过间接ELISA 法筛选分泌特异性McAb的杂交瘤细胞。用杂交瘤细胞株诱导小鼠产生腹水, 再用蛋白G亲和层析法纯化抗体。采用Ig类与亚类鉴定试剂盒鉴定该单克隆抗体的Ig亚型;通过间接ELISA测定McAbs的效价;利用Western Blotting鉴定McAbs的特性。
相比现有技术,本发明的有益效果为:本发明的制备工艺简单、易行,试剂易得,一次性可提取5株针对癌症治疗靶点:人Plk1蛋白的鼠单克隆抗体,它们通过调控蛋白的活性调控细胞周期;可以通过进一步的抗体人源化改造使其成为具有治疗功能的抗癌药物前体。
附图说明
图1是实施例1的S1中Plk1基因进行PCR扩增后的电泳图。
图2是实施例1的S2中Plk1蛋白纯化后抗原的电泳图。
图3是实施例1的S2中Plk1蛋白进行Superdex-200 10/300分子筛纯化后抗原的凝胶层析图谱。
图4是实施例1的S7中纯化后的单克隆抗体SDS-PAGE电泳图。
图5是实施例1的S8中对单克隆抗体Ig亚类鉴定结果图。
图6是实施例1的S9中对单克隆抗体的效价测定结果图。
图7是实施例1的S10中对单克隆抗体的免疫印迹鉴定结果图。
具体实施方式
为了更清楚地说明本申请实施例或现有技术中的技术方案,下面结合附图及具体实施例对本发明进一步说明。
实施例1
一、实验主要设备与试剂
低温冷冻高速离心机:2K15型,Sigma公司。
微量加样枪:Eppendorf公司。
恒温振荡仪:Eppendorf公司。
SDS-PAGE电泳系统:Mini型产品,Bio-Rad公司。
Western Blotting转印系统:Mini型产品,Bio-Rad公司。
酶标仪酶标分析仪:深圳雷杜。
福氏完全佐剂和不完全佐剂、Ig类与亚类二抗、二甲基亚砜(DMSO)、PEG SOLUTION均购于Sigma公司。
HPR标记羊抗鼠IgG抗体购于美国Southern Biotech公司。
DMEM、HAT、青链霉素、胎牛血清购于Gibco公司。
PVDF膜购于Millipore公司。
预染protein marker购于Thermo公司。
牛血清白蛋白(BSA)购于上海生工公司。
ECL显色液购于北京四正柏公司。
94孔、24孔细胞培养板、25cm2、75cm2细胞培养瓶、10ml、25ml移液管、 15ml、50ml离心管购于Corning公司。
二、制备步骤和测定方法:
S1.利用PCR技术(聚合酶链式反应)体外扩增Plk1基因,Plk1基因的核酸序列为SEQ ID NO.1所示。对体外扩增结果进行检测,结果如图1所示。
S2.Plk1基因的表达和纯化
将Plk1基因连接进入pET-28a载体中,并将质粒转化进入大肠杆菌BL21 (DE3)中,IPTG诱导后收集菌体。高压破碎菌体,离心后收集上清,利用Ni 柱亲和层析纯化蛋白。纯化后的蛋白利用Superdex-200柱子进行再次精细纯化,收集Plk1蛋白作为抗原备用。Plk1蛋白的氨基酸序列为SEQ ID NO.2所示。对纯化后的Plk1蛋白进行检测,结果如图2、3所示,蛋白纯度大于95%。
S3.小鼠的免疫
用纯化的Plk1蛋白作为抗原,免疫6-8周龄的雌性BALB/c小鼠3只,具体免疫方案请见表1,第三次常规免疫后10天,尾部采血,并用间接ELISA检测小鼠产生的抗体滴度。在融合前的第3天尾静脉最后一次注射重组抗原,剂量为60ug/只,进行加强免疫一次。
表1:小鼠的免疫方案
S4.骨髓瘤细胞与免疫脾细胞的融合
分别收集免疫小鼠脾细胞和SP2/0细胞,DMEM培养基洗涤3次后,调整脾细胞数与SP2/0细胞数之比约为1∶10,50%PEG1500作用1~2min后,加入含有HAT的完全培养基稀释,接种于96孔培养板,37℃,5%CO2培养。
S5.阳性杂交瘤细胞筛选与克隆化
经间接ELISA方法连续检测两次都为阳性的细胞孔,按有限稀释法进行亚克隆,克隆前一天制备小鼠饲养细胞层。将要克隆的杂交瘤细胞从培养孔内轻轻地吹下,用血细胞计数板计数活细胞数。将细胞用完全培养基稀释到每毫升5、 10、50个细胞。将上述三个浓度的细胞悬液分别加入已制备好的饲养细胞的96 孔培养板中100μL/孔。使相应的每孔分别含0.5、1和5个细胞。培养第4天半量换液一次,第5-6天仔细观察各孔内细胞的生长情况,并记录。在克隆后第 10-14天,细胞长满约1/4-1/2孔底时,进行全量换液,次日进行间接ELISA检测。如检出细胞生长孔有特异性抗Plk1蛋白抗体,可选择抗体效价高,呈单个克隆生长,形态良好的杂交瘤细胞孔,继续同法再克隆,一般克隆2次。2次后阳性孔的杂交瘤细胞可移至24孔培养板,待24孔板中的杂交瘤细胞生长良好时,可冻存杂交瘤细胞。
S6.单克隆抗体的生产
对已经建株的杂交瘤细胞扩大培养同时在BALB/c小鼠体内诱生小鼠腹水生产单克隆抗体。取10周龄的BALB/c小鼠,腹腔注射液体石蜡0.5mL/只,7 天后腹腔注射杂交瘤细胞5×105-1×106/只,7-10天后抽取小鼠腹水。
S7.单克隆抗体的纯化
腹水用20mM PBS pH7.4稀释3倍以上,离心取上清,经HiTrap protein G亲和层析进行纯化。
具体的,使用Protein G柱进行纯化,新柱子先用5ml超纯水过柱,再用5 ml 0.4MPB缓冲液(pH 7.0)平衡纯化小柱;抗体过柱,过程中要求缓慢过柱,以求抗体蛋白更好的结合在结合位点上;继续10ml 0.4M PB缓冲液(pH 7.0)平衡纯化小柱;5ml 0.1M甘氨酸-盐酸缓冲液(pH 2.7)洗脱结合位点上的抗体,并加入1MTris-HCl(pH 8.0)中和甘氨酸,使pH保持为适合抗体保存的中性。分别取5株纯化后的Plk1单克隆抗体进行蛋白质电泳,上样量为1ug,可清晰的看到抗体的重链和轻链(图4)。
S8.单克隆抗体的亚类鉴定
参考Sigma公司单克隆抗体亚类鉴定试剂盒操作说明,进行单克隆抗体亚类的鉴定。具体的,使用小鼠抗体亚类试剂盒鉴定抗体的亚型,其中1H7抗体为 IgG2b型,2A1、3A5、4A8、与5B12抗体均为IgG1型(图5)。
S9.单克隆抗体的效价测定
Plk1蛋白以100ng/孔包被,将单抗按1:10000、1:20000、1:40000、1: 80000、1:160000、1:320000、1:640000、1:1280000、1:2560000、1:5120000、1:10240000、1:20480000稀释,通过间接ELISA法测定其A450nm值。以与免疫蛋白靶抗原发生反应的单克隆抗体的最大稀释度即为其效价,测定孔读数与阴性对照值之比大于2.1为阳性。
通过间接ELISA方法检测抗体效价测定,具体的操作步骤为:调整抗原浓度为1ug/ml,4℃过夜包被。3%BSA封闭液,37℃封闭2h;以Plk1单克隆抗体为一抗,37℃孵育1h;HRP-羊抗小鼠IgG为二抗,37℃孵育1h。TMB显色液显色5min;2mM硫酸终止。读数A450nm读板,记录数据,测定孔读数与阴性对照值之比大于2.1为阳性。其中Plk1单克隆抗体以1:10000、1:20000、1: 40000、1:80000、1:160000、1:320000、1:640000、1:1280000、1:2560000、 1:5120000、1:10240000、1:20480000稀释,相对应的抗体使用浓度(ng/ml) 分别为100、50、25、12.5、6.25、3.125、1.5625、0.78125、0.390625、0.1953125、 0.09765625、0.048828125。五株抗体均能与Plk1蛋白结合,而且效价均能达到 500万,其中2A1抗体效价最高(图6)。
S10.单克隆抗体的特异性鉴定。
将Plk1蛋白经蛋白质电泳后转移仪PVDF膜上,加入含3%BSA后4℃封闭过夜,分别加入5株Plk1单克隆抗体室温孵育2h,以HRP标记的羊抗鼠 IgG的抗体为二抗,室温孵育1h,用ECL显色。
具体的,以Plk1蛋白为抗原使用Western-Blot检测抗体特异性。Plk1蛋白上样量为1ug,经蛋白电泳,电转至PVDF膜上,取下膜,TBS洗膜,5min×3。 3%BSA 4℃封闭过夜。按1:50000比例(抗体使用浓度0.02ug/ml)加入纯化的单克隆抗体,孵育1h。TBST洗膜,5min×3。加入1:10000稀释的HRP标记的羊抗鼠IgG抗体(二抗),孵育1h。TBST洗膜,5min×3。ECL显色液按A/B1:1 比例配制,发光仪上曝光。结果显示五株单抗均能与Plk1蛋白结合,与效价测定结果基本相符(图7)。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本发明的保护范围之内。
序列表
<110> 南方科技大学
<120> 一种治疗性的人Plk1蛋白单克隆抗体及其制备方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1809
<212> DNA
<213> 未知(Unknown)
<400> 1
atgagtgctg cagtgactgc agggaagctg gcacgggcac cggccgaccc tgggaaagcc 60
ggggtccccg gagttgcagc tcccggagct ccggcggcgg ctccaccggc gaaagagatc 120
ccggaggtcc tagtggaccc acgcagccgg cggcgctatg tgcggggccg ctttttgggc 180
aagggcggct ttgccaagtg cttcgagatc tcggacgcgg acaccaagga ggtgttcgcg 240
ggcaagattg tgcctaagtc tctgctgctc aagccgcacc agagggagaa gatgtccatg 300
gaaatatcca ttcaccgcag cctcgcccac cagcacgtcg taggattcca cggctttttc 360
gaggacaacg acttcgtgtt cgtggtgttg gagctctgcc gccggaggtc tctcctggag 420
ctgcacaaga ggaggaaagc cctgactgag cctgaggccc gatactacct acggcaaatt 480
gtgcttggct gccagtacct gcaccgaaac cgagttattc atcgagacct caagctgggc 540
aaccttttcc tgaatgaaga tctggaggtg aaaatagggg attttggact ggcaaccaaa 600
gtcgaatatg acggggagag gaagaagacc ctgtgtggga ctcctaatta catagctccc 660
gaggtgctga gcaagaaagg gcacagtttc gaggtggatg tgtggtccat tgggtgtatc 720
atgtatacct tgttagtggg caaaccacct tttgagactt cttgcctaaa agagacctac 780
ctccggatca agaagaatga atacagtatt cccaagcaca tcaaccccgt ggccgcctcc 840
ctcatccaga agatgcttca gacagatccc actgcccgcc caaccattaa cgagctgctt 900
aatgacgagt tctttacttc tggctatatc cctgcccgtc tccccatcac ctgcctgacc 960
attccaccaa ggttttcgat tgctcccagc agcctggacc ccagcaaccg gaagcccctc 1020
acagtcctca ataaaggctt ggagaacccc ctgcctgagc gtccccggga aaaagaagaa 1080
ccagtggttc gagagacagg tgaggtggtc gactgccacc tcagtgacat gctgcagcag 1140
ctgcacagtg tcaatgcctc caagccctcg gagcgtgggc tggtcaggca agaggaggct 1200
gaggatcctg cctgcatccc catcttctgg gtcagcaagt gggtggacta ttcggacaag 1260
tacggccttg ggtatcagct ctgtgataac agcgtggggg tgctcttcaa tgactcaaca 1320
cgcctcatcc tctacaatga tggtgacagc ctgcagtaca tagagcgtga cggcactgag 1380
tcctacctca ccgtgagttc ccatcccaac tccttgatga agaagatcac cctccttaaa 1440
tatttccgca attacatgag cgagcacttg ctgaaggcag gtgccaacat cacgccgcgc 1500
gaaggtgatg agctcgcccg gctgccctac ctacggacct ggttccgcac ccgcagcgcc 1560
atcatcctgc acctcagcaa cggcagcgtg cagatcaact tcttccagga tcacaccaag 1620
ctcatcttgt gcccactgat ggcagccgtg acctacatcg acgagaagcg ggacttccgc 1680
acataccgcc tgagtctcct ggaggagtac ggctgctgca aggagctggc cagccggctc 1740
cgctacgccc gcactatggt ggacaagctg ctgagctcac gctcggccag caaccgtctc 1800
aaggcctcc 1809
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Gly Lys Ile Val Pro Lys Ser Leu Leu Leu Lys Pro His Gln Arg Glu
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Lys Met Ser Met Glu Ile Ser Ile His Arg Ser Leu Ala His Gln His
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Val Val Gly Phe His Gly Phe Phe Glu Asp Asn Asp Phe Val Phe Val
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Val Leu Glu Leu Cys Arg Arg Arg Ser Leu Leu Glu Leu His Lys Arg
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Arg Lys Ala Leu Thr Glu Pro Glu Ala Arg Tyr Tyr Leu Arg Gln Ile
145 150 155 160
Val Leu Gly Cys Gln Tyr Leu His Arg Asn Arg Val Ile His Arg Asp
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Leu Lys Leu Gly Asn Leu Phe Leu Asn Glu Asp Leu Glu Val Lys Ile
180 185 190
Gly Asp Phe Gly Leu Ala Thr Lys Val Glu Tyr Asp Gly Glu Arg Lys
195 200 205
Lys Thr Leu Cys Gly Thr Pro Asn Tyr Ile Ala Pro Glu Val Leu Ser
210 215 220
Lys Lys Gly His Ser Phe Glu Val Asp Val Trp Ser Ile Gly Cys Ile
225 230 235 240
Met Tyr Thr Leu Leu Val Gly Lys Pro Pro Phe Glu Thr Ser Cys Leu
245 250 255
Lys Glu Thr Tyr Leu Arg Ile Lys Lys Asn Glu Tyr Ser Ile Pro Lys
260 265 270
His Ile Asn Pro Val Ala Ala Ser Leu Ile Gln Lys Met Leu Gln Thr
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Asp Pro Thr Ala Arg Pro Thr Ile Asn Glu Leu Leu Asn Asp Glu Phe
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Phe Thr Ser Gly Tyr Ile Pro Ala Arg Leu Pro Ile Thr Cys Leu Thr
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Ile Pro Pro Arg Phe Ser Ile Ala Pro Ser Ser Leu Asp Pro Ser Asn
325 330 335
Arg Lys Pro Leu Thr Val Leu Asn Lys Gly Leu Glu Asn Pro Leu Pro
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Glu Arg Pro Arg Glu Lys Glu Glu Pro Val Val Arg Glu Thr Gly Glu
355 360 365
Val Val Asp Cys His Leu Ser Asp Met Leu Gln Gln Leu His Ser Val
370 375 380
Asn Ala Ser Lys Pro Ser Glu Arg Gly Leu Val Arg Gln Glu Glu Ala
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Glu Asp Pro Ala Cys Ile Pro Ile Phe Trp Val Ser Lys Trp Val Asp
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Thr Trp Phe Arg Thr Arg Ser Ala Ile Ile Leu His Leu Ser Asn Gly
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Ser Val Gln Ile Asn Phe Phe Gln Asp His Thr Lys Leu Ile Leu Cys
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Pro Leu Met Ala Ala Val Thr Tyr Ile Asp Glu Lys Arg Asp Phe Arg
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Thr Tyr Arg Leu Ser Leu Leu Glu Glu Tyr Gly Cys Cys Lys Glu Leu
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Ala Ser Arg Leu Arg Tyr Ala Arg Thr Met Val Asp Lys Leu Leu Ser
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Claims (8)
1.治疗性的人Plk1蛋白单克隆抗体的制备方法,其特征在于,包括以下步骤:
S1,利用PCR技术体外扩增Plk1基因,Plk1基因的核酸序列为SEQ ID NO.1所示;
S2,Plk1基因的表达和纯化:将Plk1基因连接进入pET-28a载体中,并将质粒转化进入大肠杆菌BL21(DE3)中,IPTG诱导后收集菌体;高压破碎菌体,离心后收集上清,利用Ni柱亲和层析纯化蛋白;纯化后的蛋白利用Superdex-200柱子进行再次精细纯化,收集Plk1蛋白作为抗原备用,Plk1蛋白的氨基酸序列为SEQ ID NO.2所示;
S3,小鼠的免疫:用纯化的Plk1蛋白作为抗原,对6-8周龄的雌性BALB/c小鼠3只进行三次免疫,每次免疫间隔两周,第三次免疫后10天,尾部采血,并用间接ELISA检测小鼠产生的抗体滴度;在融合前的第3天尾静脉最后一次注射重组抗原,剂量为60ug/只,进行加强免疫一次;
S4,骨髓瘤细胞与免疫脾细胞的融合:分别收集免疫小鼠脾细胞和SP2/0细胞,DMEM培养基洗涤3次后,调整脾细胞数与SP2/0细胞数之比为1∶10,50%PEG1500作用1~2min后,加入含有HAT的完全培养基稀释,接种于96孔培养板,37℃,5%CO2培养;
S5,阳性杂交瘤细胞筛选与克隆化:经间接ELISA方法连续检测两次都为阳性的细胞孔,按有限稀释法进行亚克隆;如检出细胞孔有特异性抗Plk1蛋白抗体,可选择抗体效价高,呈单个克隆生长,形态良好的杂交瘤细胞孔,继续同法再克隆至少2次,克隆后阳性孔的杂交瘤细胞可移至24孔培养板,待24孔板中的杂交瘤细胞生长良好时,可冻存杂交瘤细胞;
S6,单克隆抗体的生产:对已经建株的杂交瘤细胞扩大培养同时在BALB/c小鼠体内诱生小鼠腹水生产单克隆抗体;
S7,单克隆抗体的纯化:腹水用20mM PBS pH7.4稀释3倍以上,离心取上清,经HiTrapprotein G亲和层析进行纯化,获得治疗性的人Plk1蛋白单克隆抗体。
2.根据权利要求1所述的治疗性的人Plk1蛋白单克隆抗体的制备方法,其特征在于,S3的免疫方案具体为:
第一阶段,小鼠选取:选取6-8周龄,健康活跃小鼠3只,编号;
第二阶段,抗原乳化:每只小鼠60ug,抗原+1×PBS总体积为350ul,与等体积福氏佐剂混合乳化3500次,200次/min,获得每0.2ml含60ug抗原的乳化液;
第三阶段,第一次免疫:用2ml注射器每只小鼠腹股沟皮下注射0.2ml乳化液,并记录注射时间和部位;
第四阶段,第二次免疫:间隔两周,用2ml注射器每只小鼠腹腔注射0.2ml乳化液,并记录注射时间和部位;
第五阶段,第三次免疫:间隔两周,用2ml注射器每只小鼠腹股沟皮下注射注射0.2ml乳化液,并记录注射时间和部位;
第六阶段,测效价:间隔10±1天后取血检测进行Elisa检测。
3.根据权利要求1所述的治疗性的人Plk1蛋白单克隆抗体的制备方法,其特征在于,S4的亚克隆方案具体为:克隆前一天制备小鼠饲养细胞层;将要克隆的杂交瘤细胞从培养孔内轻轻地吹下,用血细胞计数板计数活细胞数;将细胞用完全培养基稀释到每毫升5、10、50个细胞;将上述三个浓度的细胞悬液分别加入已制备好的饲养细胞的96孔培养板中100μL/孔,使相应的每孔分别含0.5、1和5个细胞;培养第4天半量换液一次,第5-6天仔细观察各孔内细胞的生长情况,并记录;在克隆后第10-14天,细胞长满约1/4-1/2孔底时,进行全量换液,次日进行间接ELISA检测。
4.根据权利要求1所述的治疗性的人Plk1蛋白单克隆抗体的制备方法,其特征在于,S6的诱生小鼠腹水生产单克隆抗体方案具体为:取10周龄的BALB/c小鼠,腹腔注射液体石蜡0.5mL/只,7天后腹腔注射杂交瘤细胞5×105-1×106/只,7-10天后抽取小鼠腹水。
5.根据权利要求1-4任一项所述的治疗性的人Plk1蛋白单克隆抗体的制备方法,其特征在于,获得的治疗性的人Plk1蛋白单克隆抗体,采用Ig类与亚类鉴定试剂盒鉴定人Plk1蛋白单克隆抗体的Ig亚型;通过间接ELISA测定人Plk1蛋白单克隆抗体的效价;利用Western Blotting鉴定人Plk1蛋白单克隆抗体的特性。
6.根据权利要求5所述的治疗性的人Plk1蛋白单克隆抗体的制备方法,其特征在于,间接ELISA测定人Plk1蛋白单克隆抗体的效价的方法是:Plk1蛋白以100ng/孔包被,将单抗按1:10000、1:20000、1:40000、1:80000、1:160000、1:320000、1:640000、1:1280000、1:2560000、1:5120000、1:10240000、1:20480000稀释,通过间接ELISA法测定其A450nm值;以与免疫蛋白靶抗原发生反应的单克隆抗体的最大稀释度即为其效价,测定孔读数与阴性对照值之比大于2.1为阳性。
7.根据权利要求5所述的治疗性的人Plk1蛋白单克隆抗体的制备方法,其特征在于,利用Western Blotting鉴定人Plk1蛋白单克隆抗体的特性的方法是:将Plk1蛋白经蛋白质电泳后转移仪PVDF膜上,加入含3%BSA后4℃封闭过夜,分别加入5株Plk1单克隆抗体室温孵育2h,以HRP标记的羊抗鼠IgG的抗体为二抗,室温孵育1h,用ECL显色。
8.治疗性的人Plk1蛋白单克隆抗体,其特征在于,采用如权利要求1-7任一项所述的制备方法制备获得,人Plk1蛋白单克隆抗体的株数为5。
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