CN111647624A - Tsab人源单克隆抗体重组载体、重组抗体及其制备方法 - Google Patents
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Abstract
本发明属于生物技术领域,具体涉及一种甲状腺疾病Graves’病分型抗体TSAB人源单克隆抗体重组载体、重组抗体及其制备方法。针对TSAB抗体稳定性差、产量低等缺陷,本发明提供了一种重组TSAB人源单克隆抗体重组载体、重组抗体及其制备方法。本发明重组抗体制备时采用了CHO表达系统,构建稳定表达的细胞株,稳定表达量≥870mg/L,同时该抗体活性高,完全满足科研和检测的需求,实现该分型抗体的国产化,有助于提高疾病检出率,应用前景广阔。
Description
技术领域
本发明属于生物技术领域,具体涉及一种甲状腺疾病Graves’病分型抗体 TSAB人源单克隆抗体重组载体、重组抗体及其制备方法。
背景技术
自身免疫性甲状腺疾病(Autoimmune thyroid disorders,AITD)是机体对甲状腺组织发生自身免疫反应所导致的一组自身免疫性疾病。它主要由两个病组成:Graves’病(Graves’disease,GD)和桥本氏甲状腺炎(Hashimoto's thyroiditis, HT)。AITD共同的特点是患者血清中存在针对甲状腺组织的多种自身抗体,其中包括促甲状腺素受体抗体(thyrotropin receptor antibody,TRAb)、甲状腺球蛋白抗体(TGAb)、甲状腺过氧化物酶抗体(TPOAb)等。在HT患者中,TGAb 的流行率为25-50%,TPOAb流行率为90%,TRAb分类中的一种-TBAb(TSHR blocking autoantibodies)流行率为17%。GD患者血清中,TRAb在90%以上均升高,理论上,TRAb应该为100%阳性,才符合TRAb是GD的主要致病因素这一理论。但实际上,由于受到检测方法灵敏性的影响,TRAb无法达到100%阳性。TRAb是一组异质性抗体,可进一步分类为甲状腺刺激抗体(TSAb)、甲状腺阻断抗体(TBAb)和中性抗体,取决于它们各自对于TSHR所发挥的作用。这些抗体的产生是由于免疫系统失去对甲状腺抗原的耐受所致,目前认为这一失去耐受机制是多因素作用的结果,包括遗传易感性和环境因素等。TSHR抗体的确切结合位点被描述为TSHR亚基富含亮氨酸的区域。这也是甲状腺刺激激素(TSH) 结合的部位。TRAb的刺激性类型导致与TSH和TSHR结合的下游效应相同,包括腺苷酸环化酶的激活导致环状腺苷单磷酸生成(AMP),促进成纤维细胞透明质酸合成及下游信号通路的活化。
目前,市面上还没有TSAB分型抗体检测试剂盒,同时很多学者都在积极研究TSAB抗体对Graves’病的作用及其作用机制,但分型抗体TSAB的稳定性差,产量低,需从国外购买,成本较高也极为不便利,这严重阻碍了TSAB抗体对 Graves’病的作用相关方面的研究,影响技术进步。
发明内容
针对上述TSAB抗体稳定性差、产量低等缺陷,本发明提供了一种重组TSAB 人源单克隆抗体重组载体、重组抗体及其制备方法,可实现抗体的量产,有助于提高该疾病的诊出率。
本发明提供了一种TSAB人源单克隆抗体的重组载体,其包含表达TSAB 人源单克隆抗体的表达框架,将所述表达框架插入表达载体中构建而成。
其中,上述TSAB人源单克隆抗体的重组载体中,所述的TSAB人源单克隆抗体包括一条重链VH和一条轻链VL。
进一步的,所述重链VH的编码核苷酸序列如SEQ ID NO:1所示,所述轻链VL的编码核苷酸序列如SEQ ID NO:2所示。
SEQ ID NO:1重链VH的编码核苷酸序列
caaatgcagctggtgcagtctggagcagaggtgaaaaagcccggggagtctctgaagatctcctgtaggggttctg gatacaggtttaccagctactggatcaactgggtgcgccagctgcccgggaaaggcctagagtggatgggcaggattgat cctactgactcttataccaactacagtccatccttcaaaggccacgtcaccgtctcagctgacaagtccatcaacactgccta cctgcagtggagcagcctgaaggcctcggacaccggcatgtattactgtgcgaggctcgaaccgggctatagcagcacct ggtccgtaaattggggccagggaaccctggtcaccgtctcctca。
SEQ ID NO:2轻链VL的编码核苷酸序列
ctgcctgtgctgactcagccaccctcggtgtctggagcccccaggcagagggtcaccatctcctgttctggaaaca gctccaacatcggaaataatgctgtaaactggtaccagcagctcccaggaaaggctcccaaactcctcatttattatgatgat caactgccctcaggggtctctgaccgattctctggctccaggtctggcacctccgcctccctggccatccgtgggctccagt ctgaggatgaggctgattattactgtacatcatgggatgacagcctggatagtcaactgttcggcggagggaccaggctga ccgtccta。
其中,上述TSAB人源单克隆抗体的重组载体中,所述的表达载体为 pCaHO1.0。
本发明还提供了一种重组TSAB人源单克隆抗体,由TSAB人源单克隆抗体的重组载体导入宿主细胞中表达后纯化而得。
其中,所述的宿主细胞为CHO细胞株。所述细胞株购自ATCC,ATCC CCL-61。
本发明还提供了一种重组TSAB人源单克隆抗体的制备方法,包括以下步骤:
a、构建TSAB人源单克隆抗体重组载体;
b、将步骤a得到的重组载体采用电转的方法导入受体细胞CHO中,经终浓度10μg/mL嘌呤霉素、200nM/L甲氨蝶呤筛选稳定表达细胞株;
c、将稳定表达细胞株在CD011完全培养基中培养14天,待细胞存活率<75%时,停止培养,收集培养上清,将上清经蛋白亲和纯化,得到重组TSAB人源单克隆抗体。
其中,步骤c所述的CD011完全培养基的组成包括:CD011无血清培养基,含浓度为1mM/L的L-谷氨酰胺。
进一步的,所述CD011无血清培养基购自甘肃健顺科技有限公司, 88011-317;所述L-谷氨酰胺购自Sigma-Aldrich,G7513。
其中,步骤c所述培养时保持葡萄糖浓度恒定为3~8g/L。
与现有技术相比,本发明的有益效果为:
本发明提供了一种重组TSAB人源单克隆抗体重组载体、重组抗体及其制备方法,通过构建合理的重组载体,采用CHO表达系统,构建稳定表达细胞株,稳定表达量≥870mg/L,同时该抗体活性高,完全满足科研和检测的需求,实现该分型抗体的国产化,有助于提高疾病检出率,为疾病的治疗提供帮助。本发明能够简便获得大量的重组TSAB人源单克隆抗体,解决了国内科研工作与快速检测的需求,应用前景广阔。
附图说明
图1所示为实施例3中的随着时间变化,TSAB抗体蛋白浓度变化图;
图2所示为实施例4中的SDS-PAGE检测TSAB抗体蛋白分子量;
图3所示为实施例5中的经不同浓度TSAB刺激后,甲状腺上皮细胞cAM 活性变化;
图4所示为实施例5中的经不同浓度TSAB刺激后,皮肤成纤维细胞分泌透明质酸含量变化。
具体实施方式
下面结合附图通过具体实施方式的描述对本发明作进一步说明,但这并非对本发明的限制,本领域技术人员根据本发明的基本思想,可以作出各种变型或改进,只要不脱离本发明的基本思想,均在本发明的范围之内。
实施例中所用各试剂均为普通市售产品。
实施例1 构建pCHO1.0-TSAB表达质粒
稳定表达细胞株构建。
方法如下:
1.分离浆细胞:收集Graves’病患者血液,采用密度梯度离心方法分离浆细胞,提取RNA,采用RT-PCR实验技术,获得cDNA。
如SEQ ID NO:1和SEQ ID NO:2所示。
利用特定设计的上下游引物PCR克隆该杂交瘤细胞的重链可变区(VH)和轻链可变区(VL)。
其中重链(VH)引物序列为SEQ ID NO:3和SEQ ID NO:4所示:
上游引物:5'cctaggcaaatgcagctggtgcag3'(SEQ ID NO:3);
下游引物:5'gtatactgaggagacggtgaccag 3'(SEQ ID NO:4)。
轻链(VL)引物序列为SEQ ID NO:5和SEQ ID NO:6所示::
上游引物:5'gatatcctgcctgtgctgactcag 3'(SEQ ID NO:5);
下游引物:5'ttaattaactgcctgtgctgactcag 3'(SEQ ID NO:6)。
(1)将所述重链经内切酶AvrII、BstZ17处理,所述轻链经EcoRV、PacI 酶切处理,处理后的重链、轻链与克隆载体(pCHO1.0)连接,连接产物转化感受态细菌DH5a,由于pCHO1.0载体带有卡纳(Kana+)抗性基因,可将转化菌液涂在Kana抗性的LB固体培养基上,37℃过夜培养;
所述的内切酶的编码核苷酸如SEQ ID NO:7~SEQ ID NO:10所示。
AvrII:cctagg(SEQ ID NO:7);BstZ17:gtatac(SEQ ID NO:8);EcoRV:gatatc(SEQ ID NO:9);PacI:ttaattaa(SEQ ID NO:10)。
(2)待涂板细菌长出分散菌落,选择边缘清晰、生长良好的菌落,进一步测序鉴定;
(3)根据测序结果保留候选的重组质粒,再次PCR扩增出与表达载体匹配的重链(VH)和轻链(VL)序列,将PCR产物与双酶切预先处理的线性表达载体(pCHO1.0)连接,连接产物转化感受态细菌DH5a,由于表达载体带有卡那霉素 (Kana+)抗性基因,可将转化菌液涂在Kana抗性的LB固体培养基上,37°过夜培养;
(4)测序方法参照(2);对比两次测序结果,挑选正确序列的转化菌,扩大培养后进行质粒抽提。
实施例2 pCHO1.0-TSAB表达质粒导入受体细胞获得重组抗体
具体操作步骤如下:
(1)将实施例1得到的连接有目的单抗重链(VH)和轻链(VL)基因的表达载体共转染真核表达细胞株CHO;
其中,步骤(1)中,所述真核表达细胞株CHO为悬浮培养,用CD011完全培养基培养,培养条件为37℃,5%CO2,120rpm;取CHO细胞,用台盼蓝计数,细胞活率在98%以上,以0.5-1×106/mL浓度接种细胞于CD011完全培养基中,37℃,120rmp,5%CO2培养。
(2)接种当天记为第0天,约day3、day4时计数细胞密度,若细胞密度大于 4×106/mL,加入终浓度为1mM的丁酸钠(购自生工生物工程(上海)股份有限公司,A510838-0005),补加补料CD feed 002(购自甘肃健顺科技有限公司, 99014-008)为培养体积的5%,控制葡萄糖浓度在3-8g/L之间。将细胞转入32℃, 120rmp,5%CO2培养。
(3)此后,隔天测定葡萄糖浓度,控制葡萄糖浓度在3-8g/L,并于day4、6、 8、10补加5%CD feed 002。
(4)检测细胞存活率,当细胞存活率低于75%时,停止培养,收集培养液。通过7天连续培养后收获上清,4000g离心30min,去除上清中细胞等杂质,并用0.46um滤器过滤除菌;
(5)收获的上清含有目的抗体,经常规的蛋白亲和纯化分离,获得高纯度抗体蛋白。
实施例3 对重组抗体蛋白的含量测定
每隔1天,取培养基1mL,经BCA定量方法,测定抗体蛋白表达量。
具体步骤如下:
(1)蛋白标准品的准备
a.取0.8mL蛋白标准配制液加入到一管蛋白标准(20mg BSA)中,充分溶解后配制成25mg/mL的蛋白标准溶液。配制后可立即使用,也可以-20℃长期保存。
b.取适量25mg/mL蛋白标准,用培养基稀释至终浓度为0.5mg/mL。
(2)BCA工作液配制
根据样品数量,按50体积BCA试剂A加1体积BCA试剂B(50:1)配制;
(3)蛋白浓度测定
a.将标准品按0、1、2、4、8、12、16、20μL加到96孔板的标准品孔中,加标准品稀释液补足到20μL,相当于标准品浓度分别为0、0.025、0.05、0.1、 0.2、0.3、0.4、0.5mg/mL。
b.加10μL样品到96孔板的样品孔中。加标准品稀释液补足到20μL。
c.各孔加入200μl BCA工作液,37℃放置20~30分钟。
d.用酶标仪测定A562,或540~595nm之间的其他波长的吸光度。
e.根据标准曲线和使用的样品体积计算出样品的蛋白浓度。
经14天的连续检测,以0.5~1×106/mL浓度接种细胞于CD011完全培养基中,14天时,细胞活性低于75%,同时抗体蛋白浓度高达870mg/L(如图1所示)。
实施例4 对重组抗体蛋白的分子量测定
将抗体蛋白经SDS-PAGE电泳分离,检测其分子量。具体操作步骤如下:
(1)制12%分离胶。
(2)将分离胶灌入,胶面距顶面保留2cm,加入RO水封,室温待胶凝固后,将RO水倒出来,并用滤纸吸干,再灌入5%浓缩胶并插入梳子,静置待胶凝固。
(3)取50μL蛋白样品,加入5×Lodding Buffer 15μL,煮沸处理10min, 13000rpm离心10min,取上清加入样品槽。
(4)进行SDS-PAGE电泳(80V/30min,120V/70min)。
(5)当溴酚蓝指示剂跑到分离胶底部位置时,终止电泳。
(6)将含有目的片段的胶放在R250染色液中震荡染色30min。
(7)将胶放入脱色液中震荡脱色后取出拍照。
实验结果显示,经SDS-PAGE电泳检测,抗体蛋白分子量为30kd和50kd 左右,与理论一致(如图2所示)。
实施例5 对重组抗体蛋白的活性测定
1、采用50ng/mL、100ng/mL的TSAB抗体浓度,处理人正常甲状腺上皮细胞48h,并以Graves’病患血清纯化的IgG为阳性对照,测定cAMP活性。
具体操作如下:
(1)将经抗体处理后的细胞,置于冰上,经裂解液裂解处理30min,13000rpm 离心处理15min,取上清,备测。
(2)在测试孔板中,加入标准品或细胞裂解液100μL/孔,室温孵育10min。
(3)加入25μL/孔HRP-cAMP工作液,置于摇床上,室温孵育3h。
(4)取测试板中液体,加入200μL/孔洗涤工作液,洗涤5次。
(5)加入100μL/孔绿色荧光探针,室温避光孵育3h。即刻用酶标仪650nm 波长下测量OD值。
2、采用50ng/mL、100ng/mL的TSAB抗体浓度,处理人正常皮肤成纤维细胞48h,并以Graves’病患血清纯化的IgG为阳性对照,测定透明质酸的含量。具体操作如下:
(1)细胞培养上清准备:将细胞培养基移至无菌离心管,在4℃条件下 1000×g离心10min,搜集上清。
(2)标准品稀释:标准品梯度稀释:加入标准品/样本稀释液(SR1)1mL 至冻干标准品中,静置15分钟待其完全溶解后轻轻混匀(浓度为2000pg/mL),然后按照以下浓度:2000、1000、500、250、125、62.5、31.25、0pg/mL 进行稀释。
(3)加入100μL标准品及检测样本至反应孔中,封板后于37℃孵箱孵育 90min后,经洗涤液洗涤5次。
(4)加入100μL生物素化抗体工作液至反应孔中,封板后于37℃孵箱孵育60min后,经洗涤液洗涤5次。
(5)加入100μL酶结合物工作液至反应孔中,封板后于37℃孵箱孵育30 min后,经洗涤液洗涤5次。
(6)加入100μL显色底物至反应孔中,封板后于37℃避光显色15min后,加入50μL终止液,即刻用酶标仪450nm波长下测量OD值。
实验结果显示,经TSAB抗体刺激后,甲状腺上皮细胞中,cAMP活性明显减低(如图3所示);经抗体处理后,皮肤成纤维细胞分泌的透明质酸明显升高,并高于从病人血清中提取的IgG处理组(如图4所示)。
由实施例可知,通过本发明方法能够高效制备得到重组的TSAB抗体蛋白,其表达量高达870mg/mL,同时具有生物活性,可满足科研工作及临床检测的需求,有助于Graves’病患的诊断及治疗。
序列表
<110> 成都和同易创生物科技有限公司
<120> TSAB人源单克隆抗体重组载体、重组抗体及其制备方法
<141> 2020-06-10
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 363
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
caaatgcagc tggtgcagtc tggagcagag gtgaaaaagc ccggggagtc tctgaagatc 60
tcctgtaggg gttctggata caggtttacc agctactgga tcaactgggt gcgccagctg 120
cccgggaaag gcctagagtg gatgggcagg attgatccta ctgactctta taccaactac 180
agtccatcct tcaaaggcca cgtcaccgtc tcagctgaca agtccatcaa cactgcctac 240
ctgcagtgga gcagcctgaa ggcctcggac accggcatgt attactgtgc gaggctcgaa 300
ccgggctata gcagcacctg gtccgtaaat tggggccagg gaaccctggt caccgtctcc 360
tca 363
<210> 2
<211> 330
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ctgcctgtgc tgactcagcc accctcggtg tctggagccc ccaggcagag ggtcaccatc 60
tcctgttctg gaaacagctc caacatcgga aataatgctg taaactggta ccagcagctc 120
ccaggaaagg ctcccaaact cctcatttat tatgatgatc aactgccctc aggggtctct 180
gaccgattct ctggctccag gtctggcacc tccgcctccc tggccatccg tgggctccag 240
tctgaggatg aggctgatta ttactgtaca tcatgggatg acagcctgga tagtcaactg 300
ttcggcggag ggaccaggct gaccgtccta 330
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
cctaggcaaa tgcagctggt gcag 24
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
gtatactgag gagacggtga ccag 24
<210> 5
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gatatcctgc ctgtgctgac tcag 24
<210> 6
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
ttaattaact gcctgtgctg actcag 26
<210> 7
<211> 6
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
cctagg 6
<210> 8
<211> 6
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
gtatac 6
<210> 9
<211> 6
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
gatatc 6
<210> 10
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<212> DNA
<213> 人工序列(Artificial Sequence)
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ttaattaa 8
Claims (9)
1.TSAB人源单克隆抗体的重组载体,其特征在于:包含表达TSAB人源单克隆抗体的表达框架,将所述表达框架插入表达载体中构建而成。
2.根据权利要求1所述的TSAB人源单克隆抗体的重组载体,其特征在于:所述的TSAB人源单克隆抗体包括一条重链VH和一条轻链VL,所述重链VH的编码核苷酸序列如SEQ ID NO:1所示,所述轻链VL的编码核苷酸序列如SEQ ID NO:2所示。
3.根据权利要求1所述的TSAB人源单克隆抗体的重组载体,其特征在于:所述的表达载体为pCaHO1.0。
4.重组TSAB人源单克隆抗体,其特征在于:由权利要求1-3任一项所述的TSAB人源单克隆抗体的重组载体导入宿主细胞中表达后纯化而得。
5.根据权利要求4所述的重组TSAB人源单克隆抗体,其特征在于:所述的宿主细胞为CHO细胞株。
6.表达权利要求4或5所述的重组TSAB人源单克隆抗体的宿主细胞。
7.权利要求4或5所述的重组TSAB人源单克隆抗体的制备方法,其特征在于,包括以下步骤:
a、构建TSAB人源单克隆抗体重组载体;
b、将步骤a得到的重组载体采用电转的方法导入受体细胞CHO中,经终浓度10μg/mL嘌呤霉素、200nM/L甲氨蝶呤筛选稳定表达细胞株;
c、将稳定表达细胞株在CD011完全培养基中培养14天,待细胞存活率<75%时,停止培养,收集培养上清,将上清经蛋白亲和纯化,得到重组TSAB人源单克隆抗体。
8.根据权利要求7所述的重组TSAB人源单克隆抗体的制备方法,其特征在于:步骤c所述的CD011完全培养基的组成包括:CD011无血清培养基,含浓度为1mM/L的L-谷氨酰胺。
9.根据权利要求7所述的重组TSAB人源单克隆抗体的制备方法,其特征在于:步骤c所述培养时保持葡萄糖浓度恒定为3~8g/L。
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