CN108728476A - 一种利用crispr系统产生多样性抗体文库的方法 - Google Patents
一种利用crispr系统产生多样性抗体文库的方法 Download PDFInfo
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Abstract
本发明属生物技术领域及基因工程技术领域,涉及抗体制备技术,具体涉及一种利用CRISPR系统产生多样性抗体文库从而筛选高亲和力抗体的方法。该方法利用CRISPR系统的剪切及后续NHEJ修复的途径,实现对抗体基因的可变区区域产生蛋白质序列的多样性,从而产生新抗体文库,为后续抗体筛选做准备。本发明方法比以往用易错pcr来形成抗体库及定向突变碱基替代等方法改进抗体库相比,产生的抗体库容大且多样化,打破抗体筛选时库容的限制,本方法通过调节CRISPR的表达,可循环产生新文库直至筛选出理想抗体,操作周期短,成本低,方便灵活。
Description
技术领域
本发明属生物技术领域及基因工程技术领域,涉及抗体制备技术,具体涉及一种利用CRISPR系统产生多样性抗体文库从而筛选高亲和力抗体的方法。
背景技术
现有技术公开了Kohler和Milstein发明了单克隆抗体技术(1975年),并在1984年获得了诺贝尔奖。因此,抗体对抗原的结合能力,使其在疾病诊断的免疫防治中具有重要意义。实践证实,抗体尤其是单克隆抗体已成为目前生物医药产业最具前景和应用价值的领域。现有技术公开了早年单抗的制备是B淋巴细胞和骨髓瘤细胞杂交形成的杂交瘤细胞产生的,其重链和轻链所形成的结构域可以识别和结合特异抗原。1998年,全球第一个人源化单克隆抗体帕利珠单抗(Palivizumab,商品名:Synagis),被美国食品药品管理局FDA批准上市,截至目前,FDA已批准上市了66个抗体药物。克隆抗体药物以其高特异性、有效性和安全性正在发展成为国际药品市场上一大类新型诊断和治疗药物,日益成为癌症及其他疾病治疗市场的新宠。因此,改进筛选和制备高效安全的单克隆抗体技术意义重大。
随着基因工程技术的迅速发展,治疗性单抗经历了早期100%的鼠源单抗,到嵌合抗体,人源化抗体,到近年的全人源性抗体几个时期,逐步消除了异源性抗体的免疫原性问题,在保持对抗原高亲和力的同时,改善了抗体的药代动力学。通过对抗体基因的改造,尤其是高变区基因,产生大量不同抗体文库是筛选高亲和力目标抗体的基础,如今多使用易错PCR技术扩大库容及定向突变碱基替代方法对可变区序列进行改进以提高亲和力,但这些方法相对局限并繁琐,周期长,成本高,限制了筛选高亲和力抗体的应用。
CRISPR系统是一种精确的万能基因武器,可以用来删除、添加、激活或抑制其他生物体的目标基因,这些目标基因包括人、老鼠、斑马鱼、细菌、果蝇、酵母、线虫和农作物细胞内的基因,这也意味着此基因编辑器是一种可以广泛使用的生物技术。
本领域公知,抗体文库的多样化及库容容量是后续筛选理想抗体的基础和关键。
基于现有技术的基础,本发明拟提供一种利用CRISPR系统产生多样性抗体文库从而筛选高亲和力抗体的方法。
发明内容
本发明的目的在于,基于现有技术的基础,旨在提供一种利用CRISPR系统产生多样性抗体文库从而筛选高亲和力抗体的方法。尤其涉及一种灵活简便的方法用于针对抗体基因高变区序列进行编辑并产生多样化新抗体文库,用于后续高亲和力抗体的筛选。该方法利用CRISPR系统的剪切及后续NHEJ修复的途径,实现对抗体基因的可变区区域产生蛋白质序列的多样性,从而产生新抗体文库,为后续抗体筛选做准备。
本发明方法比以往用易错pcr来形成抗体库及定向突变碱基替代等方法改进抗体库相比,产生的抗体库容大且多样化,打破抗体筛选时库容的限制,而且本方法通过调节CRISPR的表达,可循环产生新文库直至筛选出理想抗体,操作周期短,成本低,方便灵活。
为实现上述目的,本方法通过下述方法和步骤实现:
1、质粒的构建,包括选择性表达含有CRISPR系统的质粒及选择性表达抗体基因的展示质粒:
1)本发明所述的选择性表达含CRISPR系统的质粒(简称CRISPR质粒)质是指利用四环素系统或其它系统调节CAS9或sgRNA表达的质粒;可以根据初始抗体可变区特点设计多个sgRNA靶向序列;
2)本发明所述的选择性表达含有抗体基因的展示质粒是指利用半乳糖诱导或其他诱导抗体基因表达并展示在细胞表面的质粒,选自抗体展示质粒;
3)本发明所述的抗体基因是指Fab抗体、scFv抗体或其它用于筛选抗体的抗体基因序列,可选自非免疫抗体文库或诱变后的抗体文库;
2、质粒的转化以及阳性克隆的选择,得到含有上述两种质粒的细胞株:
1)本发明所述的细胞株是指与抗体展示质粒相配套的细胞株;
2)本发明所述的含有上述质粒的细胞株是指将CRISPR质粒及抗体展示质粒转化细胞株,可根据细胞株选择相应高效的转化方法;
3)本发明所述的阳性克隆的选择应根据质粒的筛选标记使用相应的选择性培养基,筛选得到的细胞株应同时含有CRISPR质粒及抗体展示质粒;
3、诱导CRISPR-CAS9对抗体序列的剪切及修复,获得新抗体文库:
本发明所述的新抗体文库的获得是指通过调控CRISPR系统的开启和关闭,从而对抗体可变区序列进行剪切,细胞通过NHEJ途径修复后随机形成多样化的突变,从而形成多样性新抗体文库;4、诱导新抗体文库的表达及展示:
本发明所述的诱导新抗体文库的表达及展示是指通过诱导抗体展示质粒的表达,使新的多样性抗体展示于细胞的表面,用于后续的筛选;
5、抗体的富集及高亲和力抗体的筛选:
(1)本发明所述的抗体的富集是指通过蛋白标签或抗原,运用流式或磁珠分选的方法,富集正确展示了抗体蛋白的细胞,去除因为随机修复造成移码突变或其它原因导致不能正确表达并展示特异性抗体的细胞;
(2)本发明所述的高亲和力抗体的筛选是指通过流式或其它抗体筛选方法筛选出高亲和力抗体;本发明通过调控CRISPR系统的开启和关闭,可循环性的产生新文库,直至筛选出理想抗体。
本发明提供了一种利用CRISPR系统产生多样性抗体文库从而筛选高亲和力抗体的方法。该方法能实现对抗体基因的可变区区域产生蛋白质序列的多样性,从而产生新抗体文库,为后续抗体筛选做准备。与现有技术的等方法相比,产生的抗体库容大且多样化,打破抗体筛选时库容的限制,本方法通过调节CRISPR的表达,可循环产生新文库直至筛选出理想抗体,操作周期短,成本低,方便灵活。
附图说明
图1是本发明的CRISPR质粒谱图。
图2是发明采用的展示质粒pYD-scFv-P2-G12图谱。
具体实施方式
下面的实例是对本发明提出的利用CRISPR系统产生并优化新抗体文库,从而进行高亲和力抗体筛选的进一步说明。
实施例1利用CRISPR系统产生新抗体文库并筛选高亲和力anti-P2-G12的抗体
(一)本实例所采用的CRISPR质粒及抗体展示质粒信息如下:
1)本实例所采用的CRISPR质粒为质粒pLM107CAS9-TET-ON-sgRNA,其为LEU筛选标记,多拷贝表达CAS9,由tet-on系统控制sgRNA表达,其谱图如图1所示:
2)本实例所采用的展示质粒为pYD-scFv-P2-G12:
a)实例所采用的酵母展示质粒来自初始质粒pYD1,Trp选择标记,Gal启动子调控下游抗体基因的表达,C端轻链和重链的可变区(VL和VH)序列由连接肽连接,C端带有myc标签和AGA2基因,其图谱信息如图2所示:
b)实例所采用的抗体基因为人程序死亡受体-1(PD1)的特异性抗体序列,其相关信息如下:
其中,
VL区碱基序列:高亮部分为VL3区
Gagctgactcagccaccctcggtgtcagtggccccaggaaagacggccaggattacctgtgggggaaacaacattggaagtaaaagtgtgcactggtaccagcagaagccaggccaggcccctgtgctggccatctattatgatagcgaccggccctcagggatccctgagcgattctctggctccaactctgggaacacggccaccctgaccatcagcagggtcgaagtcggggatgaggccgactattactgtttcggcggagggaccaagctgaccgtccta
VH区碱基序列:高亮部分为VH3区
gaggtgcagctggtgcagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagcctctggattcaccttcagtagctatagcatgaactgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccgattcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggctgtgtattactgt tggggccaagggacaatggtcaccgtctcctca
c)本实例针对人程序死亡受体-1(PD1)特异性抗体VL3及VH3可变区序列的sgRNA靶向序列信息如下:高亮区为PAM(NGG)
ccgactattactgtcaggtg
cgactattactgtcaggtgt
agtgttagtgatgttgtttt
tactgtgcgagagatcccta
tgcgagagatccctatggtt
gcgagagatccctatggttc
cgagagatccctatggttcg
tatggttcggggagttatta
atggttcggggagttattat
ggttcggggagttattatag
gttcggggagttattataga
ttcggggagttattatagag
gaggggatgcttttgatatc
aggggatgcttttgatatct
(二)本实例所采用的细胞株和相关试剂如下:
1)酵母菌株:EBY100,其基因型为MATa AGA1::GAL1-AGA1::URA3ura3-52trp1leu2-delta200his3-delta200pep4::HIS3prb11.6R can1GAL
2)相关培养基:
YPD完全培养基,
SD/-Trp选择培养基,
SD/-Trp-Leu选择培养基,
SD/-Trp-Leu+DOX诱导培养基,
SD/-Trp-Leu+Gal诱导培养基,
3)本实例采用LiAc法转化酵母,其相关试剂配方及转化步骤如下:
a)相关试剂配方LATE溶液:
0.1M LiAc,1×TE溶液(10mM Tris-HCl,1mM EDTA)PLATE转化液:40%PEG,0.1MLiAc,1×TE溶液50×TAE溶液:2M Tris-HCl,50mM EDTA单链鲑鱼精DNA
4)转化酵母EBY100及筛选步骤如下:
感受态细胞制备:
a)EBY100单克隆于YPD培养基,30℃,220rpm过夜培养;
b)将上述酵母培养物于100ml YPD培养基使OD600为0.2;
c)30℃,220rpm培养至OD600为1.0;
d)分装于50ml离心管,3000rpm离心2min,弃上清;
e)重悬于0.5ml LATE溶液中,并转移至1.5ml离心管
f)10,000rpm离心1min,弃上清;
g)重悬于300ul LATE溶液
h)加300ul 50%甘油,-80℃冻存转化:
a)单链鲑鱼精DNA(2.0mg/ml)与沸水中煮5min,立即置于冰上5min;
b)酵母感受态细胞室温10min;
c)将质粒及10ul单链鲑鱼精DNA及90ul酵母感受态细胞加入900ul PLATE转化液中,混匀;
d)30℃孵育30min;
e)42℃热激15min;
f)10,000rpm离心1min,吸去上清;
g)重悬于250ul YPD溶液,30℃孵育2.0h;
h)将上述悬液置于相应选择培养基平板中,30℃,48h得单克隆
5)本实例获得含有上述质粒的酵母菌株的转化及筛选过程如下:
a)质粒pYD-Fab转化EBY100菌株,用SD/-Trp选择培养基筛选,得EBY100/pYD-scFv-P2-G12酵母;
b)质粒pCM184CAS9-TET-ON-sgRNA转化EBY100/pYD-Fab酵母,用SD/-Trp-Leu选择培养基筛选,得到EBY100/pYD-scFv-P2-G12/pCM184-CAS9-TET-ON-sgRNA酵母菌株(简称P2-G12酵母菌株);
(三)本实例获得新文库过程如下:
1)P2-G12菌株酵母单克隆在SD/-Trp-Leu培养基中30℃过夜培养;
2)将上述培养物稀释于SD/-Trp-Leu+DOX诱导培养基至OD600=0.1,诱导培养12h得到新抗体文库;
3)新P2-G12抗体文库可于终浓度25%的甘油中-80℃冻存;
(四)本实例诱导新抗体文库的表达及展示过程如下:
1)冻存的新P2-G12抗体文库室温下解冻,在至少1L的SD/-Trp-Leu培养基中培养过夜(~20h)生长,使OD600至6~8;取1×1010个细胞在新的SD/-Trp-Leu的培养基重复孵育以去除死细胞;
2)取至少1×1010个细胞于50ml离心管中,2500,5min,去上清,细胞重悬于SD/-Trp-Leu+Gal诱导培养基使OD600至0.5~1.0,再在20℃,200rpm孵育过夜(>20h);
(五)本实例抗体的富集及高亲和力抗体的筛选过程如下:
1)取10倍于文库多样性数目细胞离心,14000g,离心30s,弃上清;
2)细胞重悬于1ml PBSM溶液(即含1g/L BSA及2mM EDTA的1×PBS溶液);
3)用生物素标记的抗原室温孵育细胞30min,冰上放置—分钟,(后续实验于冰上操作);
4)4℃,14000g,离心30s,用预冷的PBSM溶液洗一次;
5)将细胞重悬于缓冲液中,加入表面带有链霉亲和素的磁珠,冰上孵育10min,其间每分钟轻轻翻转混匀;
6)用磁力分选法富集抗原特异性的酵母;
7)将所得细胞重悬于SD/-Trp-Leu培养基中培养24h,再换至SD/-Trp-Leu+Gal培养基中诱导生长约12~16h后,进行第二轮磁珠分选,用表面带有抗生物素抗体的磁珠进行细胞分选,重复以上分选步骤,得到富集细胞;
8)将两轮分选后所得细胞重悬于500ul PBSF缓冲液(即含1g/L BSA的1×PBS溶液)中;
9)向酵母细胞悬液中加入myc抗体以及生物素标记的抗原,室温孵育细胞30min;
10)4℃,14000g,离心30s,用预冷的PBSM溶液洗一次;
11)加入被稀释的二抗,避光冰浴10min后离心,用冰冷缓冲液冲洗次;
12)重悬细胞于缓冲液中,于冰上避光放置直至流式细胞仪分选分选;
13)将分选后所得细胞加入SD/-Trp-Leu培养基中培养24h,再换至培养基中诱导生长约12~16h后,进行第二轮流式细胞仪分选,选择亲和素-APC做二抗,重复以上步骤;
14)将两轮分选后所得细胞重悬于缓冲液中,取菌液涂于SD/-Trp-Leu平板培养基中孵育;其余分选所得细胞进行扩大培养。
15)从生长有独立菌落的平板培养基上,挑选个克隆进行测序鉴定。
本方法与现有技术的等方法相比,产生的抗体库容大且多样化,打破抗体筛选时库容的限制,本方法通过调节CRISPR的表达,可循环产生新文库直至筛选出理想抗体,且操作周期短,成本低,方便灵活。
Claims (7)
1.一种利用CRISPR系统产生多样性抗体文库的方法,其特征在于,包括以下步骤:
(1)构建质粒,包括选择性表达含有CRISPR系统的质粒及选择性表达抗体基因的展示质粒;
(2)转化质粒以及阳性克隆的选择,得到含有上述质粒的细胞株;
(3)诱导CRISPR系统对抗体序列的剪切及修复,获得多样性新抗体文库;
(4)诱导新抗体文库的表达及展示;
(5)抗体的富集及高亲和力抗体的筛选。
2.根据权利要求1所述的方法,其特征在于,步骤(1)所述的选择性表达含CRISPR系统的质粒(简称CRISPR质粒)是指利用四环素系统或其它系统调节CAS9或sgRNA表达的质粒;sgRNA表达质粒根据初始抗体可变区特点设计多个sgRNA靶向序列;所述的选择性表达含有抗体基因的展示质粒是指利用半乳糖诱导或其他诱导抗体基因表达并展示在细胞表面的质粒,选自抗体展示质粒;所述的抗体基因是指Fab抗体、scFv抗体或其它用于筛选抗体的抗体基因序列,选自非免疫抗体文库或诱变后的抗体文库。
3.根据权利要求1所述的方法,其特征在于,步骤(2)所述的细胞株是指与抗体展示质粒相配套的细胞株;所述的质粒的转化是指将CRISPR质粒及抗体展示质粒转化细胞株,其中根据细胞株选择相应高效的转化方法;所述的阳性克隆的选择根据质粒的筛选标记使用相应的选择性培养基或培养液,筛选得到的细胞株同时含有CRISPR质粒及抗体展示质粒。
4.根据权利要求1所述的方法,其特征在于,步骤(3)所述的新抗体文库的获得是指通过诱导调控CRISPR系统的开启和关闭,对抗体可变区序列进行剪切,细胞通过NHEJ途径修复后随机形成多样化的突变,形成多样性新抗体文库。
5.根据权利要求1所述的方法,其特征在于,步骤(4)所述的诱导新抗体文库的表达及展示是指通过诱导抗体展示质粒的表达,使新的多样性抗体展示于细胞表面,用于后续的筛选。
6.根据权利要求1所述的方法,其特征在于,步骤(5)所述的抗体的富集是指通过蛋白标签或抗原,运用流式或磁珠分选的方法,富集正确展示了抗体蛋白的细胞,去除不能正确表达并展示特异性抗体的细胞。
7.根据权利要求1所述的方法,其特征在于,步骤(5)所述的高亲和力抗体的筛选是指通过流式或其它抗体筛选方法筛选出高亲和力抗体。
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