CN112538461A - Glp1r报告基因稳转细胞株、构建方法、应用 - Google Patents
Glp1r报告基因稳转细胞株、构建方法、应用 Download PDFInfo
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- CN112538461A CN112538461A CN202011512406.XA CN202011512406A CN112538461A CN 112538461 A CN112538461 A CN 112538461A CN 202011512406 A CN202011512406 A CN 202011512406A CN 112538461 A CN112538461 A CN 112538461A
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Abstract
本申请涉及生物制药技术领域,具体公开了GLP1R报告基因稳转细胞株、构建方法、应用。GLP1R报告基因稳转细胞株为2020年11月18日保藏于武汉大学的中国典型培养物保藏中心的GLP1R报告基因稳转的人胚肾293T细胞DG1‑6,保藏编号为CCTCC No.C2020221;所述GLP1R报告基因稳转细胞株包括有GLP1R受体基因表达体系以及报告基因表达体系,所述报告基因表达体系包括CREB结合区域、minipromoter以及报告基因。GLP1R报告基因稳转细胞株的构建方法,包括以下步骤:S1、转染CREB报告基因质粒;S2、转染GLP1R表达质粒;S3、细胞株筛选。本申请的GLP1R报告基因稳转细胞株应用于GLP1R激动剂或者拮抗剂生物学活性测定、GLP1R激动剂或者拮抗剂定量生物分析检测、GLP1R激动剂或者拮抗剂的中和抗体检测。
Description
技术领域
本申请涉及生物医药技术领域,更具体地说,它涉及GLP1R报告基因稳转细胞株、构建方法、应用。
背景技术
GLP1R(UniProt ID:P43220)是胰高血糖素样肽1(GLP-1)的受体,属于G蛋白偶联受体家族的一员。GLP1R通过cAMP信号通路发挥生理功能,过程如下:GLP-1通过和细胞膜表面的GLP1R结合,激活胞内的信号,导致细胞内的腺苷酸环化酶活化和胞内cAMP浓度的上升,cAMP浓度的上升激活蛋白激酶A(cyclic-AMP dependent protein kinase A),磷酸化CREB转录因子,磷酸化CREB处于活化状态,进入细胞核激活胰岛素的转录翻译,最终分泌胰岛素(KEGG PATHWAY:map04024)。GLP1R是调节体内血糖水平的关键受体蛋白。
GLP1R的激动剂已被批准用于二型糖尿病的治疗,上市的GLP1R的激动剂药物有Dulaglutide(Trulicity),Liraglutide(Victoza),Albiglutide(Tanzeum)等。GLP1R激动剂药物在控制血糖及减轻受试者体重方面的效果,具有治疗糖尿病、肥胖及非酒精性脂肪性肝炎(NASH)的潜力。已上市的GLP1R激动剂是百亿美元市场,基于糖尿病、肥胖及非酒精性脂肪性肝炎(NASH)等慢性病领域庞大的市场,更加长效的GLP1R激动剂药物和有协同功能的双特异抗体GLP1R激动剂药物如IBI362,HM12525A的研发正在迅速推进,GLP1R的激动剂药物的研发竞争格局十分激烈。
生物治疗药物的研发主要包括药物发现,临床前和临床研究和商业化生产三个大的阶段。
在药物发现的过程中,药物相对于靶点体外的生物学活性测定和筛选是必不可少的过程。具体到GLP1R激动剂的药物发现过程,需要开发候选药物和GLP1R蛋白的结合测定方法,由于GLP1R为七次跨膜蛋白,基于现有的实验技术限制,市场上无提供有活性重组GLP1R的蛋白,构建GLP1R受体高峰度表达的稳转细胞株进行结合活性的测定是较优的选择;有结合活性的候选药物是否有细胞学激动剂的效果,需要在细胞上验证其活性,现有检测技术对候选药物活性测定均是建立在表达GLP1R受体的稳转细胞株基础之上,测定激动剂激活GLP1R稳转细胞后,采用试剂盒检测新增的cAMP浓度如(R&D KGE002B,Cisbio62AM4PEB)来测定活性,两个检测方法试剂成本较高,灵敏度相对较差,信号和检测的目的产物cAMP之间为非线性相关,数据的解读比较复杂,信号和背景的倍比较低区分度差,信号稳定性较差等不利影响。检测的变化是细胞激活后信号通路中间过程的产物cAMP,信号通路的激活最终效果是胰岛素的表达,该方法能是不能完全反应候选药物的激活效果。
在临床前和临床研究中,药物的药代动力学是评估药物安全性和有效性必不可少的检测项目,药物的药代动力学依赖定量分析方法,具体到GLP1R激动剂药物,采用受体配体结合实验(LBA)进行生物分析,由于市面上无活性的GLP1R重组蛋白,需要通过候选药物免疫动物通过杂交瘤技术获得抗候选药物特异性抗体进行方法开发,时间周期需要3-6个月时间周期较长,且每一种候选药物均需要制备特异性抗体,不具备通用性,且定量方法的稳定性和灵敏性受特异性抗体的特点影响较大。
在临床前和临床研究中,抗药抗体和中和抗体的检测是评估药物安全性和有效性必不可少的步骤,特别是对于需要长期用药的糖尿病治疗领域。在长期的用药过程中,机体在产生的抗药抗体和中和抗体会逐渐的增强,最终导致药物的失活和不良反应的产生,在用药初期微弱的中和抗体可能会在长期的药用过程中随着机体的免疫应答而逐渐增强,中和抗体的测定方法对方法的灵敏度度有很高的要求。目前针对中和抗体的检测,现有检测技术对候选药物中和抗体的测定均是建立在表达GLP1R受体的稳转细胞株基础之上,测定中和抗体和激动剂药物反应后,是否能够继续激活GLP1R稳转细胞,采用试剂盒检测新增的cAMP浓度如(R&D KGE002B,Cisbio 62AM4PEB)来测定中和抗体的,此类方法的灵敏度较差,且检测的是激活过程中中间信号cAMP的变化,容易受到以血清为基质的检测样品中其他细胞因子的干扰,特异性相对较差,灵敏度更好且抗干扰能力更强的中和抗体的检测方法有着迫切的需求。
发明内容
为了提升对GLP1R激动剂相关检测的抗干扰性和通用性,本申请提供一种GLP1R报告基因稳转细胞株、构建方法、应用。
第一方面,本申请提供GLP1R报告基因稳转细胞株,采用如下的技术方案:GLP1R报告基因稳转细胞株,所述GLP1R报告基因稳转细胞株为2020年11月18日保藏于武汉大学的中国典型培养物保藏中心的GLP1R报告基因稳转的人胚肾293T细胞DG1-6,保藏编号为CCTCC No.C2020221;所述GLP1R报告基因稳转细胞株包括有GLP1R受体基因表达体系以及报告基因表达体系,所述报告基因表达体系包括CREB结合区域、minipromoter以及报告基因。
通过采用上述技术方案,由于采用GLP1R受体基因表达体系,使细胞株的细胞膜可表达GLP1R受体,从而可结合GLP1R激动剂,激活稳转细胞株,产生第二信使cAMP;由于采用报告基因表达体系,且报告基因表达体系包括CREB结合区域、minipromoter以及报告基因,因此第二信使cAMP可进一步激活下游信号分子转录因子CREB,进一步通过minipromoter启动报告基因的转录,表达报告蛋白。
因此,本申请通过检测GLP1R下游信号分子CREB的活性,甚至更下游的报告蛋白的活性来表征GLP1R激动剂相关检测的结果,相比于检测中游信号cAMP,大大减少了其他细胞因子的干扰,从而大大提升了检测的特异性。
优选的,所述报告基因为荧光素酶基因。
通过采用上述技术方案,荧光素酶以ATP,氧气和荧光素luciferin为底物,催化化学反应,释放出可以被仪器检测到光,化学发光的强弱能够代表荧光素酶多少。
荧光素酶报告基因检测方法相对于其他荧光类报告基因方法有极低的背景信号,有极高的灵敏程度,检测到的荧光素酶的浓度下限能够达到10–11M数量级别,因此可大大提升GLP1R激动剂相关检测的灵敏度以及可靠度。
第二方面,本申请提供GLP1R报告基因稳转细胞株的构建方法,采用如下的技术方案:
GLP1R报告基因稳转细胞株的构建方法,包括以下步骤:
S1、转染CREB报告基因质粒
将CREB报告基因质粒转染293T细胞,对293T细胞进行Puromycin筛选,获得报告基因稳转细胞。
S2、转染GLP1R表达质粒
将GLP1R表达质粒转染报告基因稳转细胞,对报告基因稳转细胞进行puromycin/Hygromycin筛选,获得GLP1R报告基因稳转细胞。
S3、细胞株筛选
对GLP1R报告基因稳转细胞进行单克隆细胞株筛选,获得所述的GLP1R报告基因稳转细胞株。
通过采用上述技术方案,仅需依次转染CREB报告基因质粒以及GLP1R表达质粒,进一步通过Puromycin筛选和puromycin/Hygromycin筛选后,进一步通过单克隆细胞株筛选即可得到纯度较高的GLP1R报告基因稳转细胞株,操作简单便于生产。
优选的,S1中所述的Puromycin筛选包括:将转染CREB报告基因质粒的293T细胞转入T25细胞培养瓶中进行传代并且加入0.5ug/ml puromycin DMEM培养基中进行加压培养。
优选的,S1中所述的Puromycin筛选还包括:维持0.5ug/ml puromycin DMEM生长传代3次。
优选的,S2中所述的puromycin/Hygromycin筛选包括:将转染GLP1R表达质粒的293T细胞转入新的培养皿进行传代,并且加入1ug/ml puromycin和125ng/ml Hygromycin的DMEM培养基中进行加压培养。
优选的,S3中所述的单克隆细胞株筛选包括:待细胞长满后,维持抗性压力传代两次后,进行96孔板单克隆铺板;96孔板单克隆铺板12天后,挑选出单克隆进入24孔板,24孔板细胞经过2周培养后转入6孔板,然后经T25、T75、T175放大培养。
第三方面,本申请提供GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂生物学活性测定的应用。
第四方面,本申请提供GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂定量生物分析检测的应用。
第五方面,本申请提供GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂的中和抗体检测的应用。
综上所述,本申请具有以下有益效果:
1、由于采用报告基因表达体系,且报告基因表达体系包括CREB结合区域、minipromoter以及报告基因,因此第二信使cAMP可进一步激活下游信号分子转录因子CREB,进一步通过minipromoter启动报告基因的转录,表达报告蛋白。
因此,本申请通过检测GLP1R下游信号分子CREB的活性,甚至更下游的报告蛋白的活性来表征GLP1R激动剂相关检测的结果,相比于检测中游信号cAMP,大大减少了其他细胞因子的干扰,从而大大提升了检测的特异性。
2、荧光素酶报告基因检测方法相对于其他荧光类报告基因方法有极低的背景信号,有极高的灵敏程度,检测到的荧光素酶的浓度下限能够达到10–11M数量级别,因此可大大提升GLP1R激动剂相关检测的灵敏度以及可靠度。
3、仅需依次转染CREB报告基因质粒以及GLP1R表达质粒,进一步通过Puromycin筛选和puromycin/Hygromycin筛选后,进一步通过单克隆细胞株筛选即可得到纯度较高的GLP1R报告基因稳转细胞株,操作简单便于生产。
4、在单克隆细胞株筛选的过程中加入了活性测定评价的过程,更加直接的筛选出可用于后续应用的最优的单克隆细胞株。
5、构建的单克隆细胞株能够用于GLP1R激动剂或者拮抗剂药物从临床前活性测试,临床阶段生物分析测定和中和抗体测定等多方面的应用,在药物研发的全流程中多有应用,应用前景广泛。
附图说明
图1是本申请CREB报告基因的转染效率柱状图;
图2是本申请的GLP1R和CREB报告基因双转染的转染效率的柱状图;
图3是本申请GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂生物学活性测定的应用中GLP1R激动剂的剂量曲线;
图4是本申请GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂定量生物分析检测的应用中度拉糖肽在GLP1R报告基因细胞株中的标准曲线;
图5是本申请GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂的中和抗体检测的应用中独立血清相对于混合血清的比值分布图;
图6是本申请GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂的中和抗体检测的应用中抗度拉糖肽中和抗体剂量曲线。
具体实施方式
以下结合附图和实施例对本申请作进一步详细说明。
实施例
本实施例提供GLP1R报告基因稳转细胞株。
GLP1R报告基因稳转细胞株,所述GLP1R报告基因稳转细胞株为2020年11月18日保藏于武汉大学的中国典型培养物保藏中心的GLP1R报告基因稳转的人胚肾293T细胞DG1-6,保藏编号为CCTCC No.C2020221;所述GLP1R报告基因稳转细胞株包括有GLP1R受体基因表达体系以及报告基因表达体系,所述报告基因表达体系包括CREB结合区域、minipromoter以及报告基因。
本实施例进一步提供GLP1R报告基因稳转细胞株的构建方法,包括以下步骤:
S1、转染CREB报告基因质粒
将CREB报告基因质粒转染293T细胞,对293T细胞进行Puromycin筛选,获得报告基因稳转细胞。具体操作为:4ug CREB报告基因质粒(生工定制,含有CREB结合区域,minipromoter SEQ ID NO.:2和luciferase基因)和4ul X-tremeGENE HP DNA转染试剂(Roche-06366244001),使用opti-MEM培养基(Thermo 31985-070)100ul混合均匀,室温静置半小时后加入有1ml DMEM、60%汇合度的293T细胞的6孔板单孔中;第二天进行细胞换液;第三天转入T25细胞培养瓶中进行传代并且加入0.5ug/ml puromycin的DMEM培养基中进行加压培养,维持0.5ug/ml puromycin DMEM生长传代3次后,将细胞铺于10cm细胞培养皿中,获得报告基因稳转细胞。
S2、转染GLP1R表达质粒
将GLP1R表达质粒转染报告基因稳转细胞,对报告基因稳转细胞进行puromycin/Hygromycin筛选,获得GLP1R报告基因稳转细胞。具体操作为:取20ug GLP1R表达质粒(SinoBiological-HG13944,SEQ ID NO.:1)和20ul X-tremeGENE HP DNA转染试剂用使用opti-MEM培养基400ul混合均匀,室温静置半小时后加入有10ml 1ug/ml puromycin DMEM 60%汇合度的293T细胞的10cm培养皿。维持转染条件至转染第三天,转入新的10cm培养皿进行传代并且加入1ug/ml puromycin和125ng/ml Hygromycin的DMEM培养基中进行加压培养,待细胞长满后,维持抗性压力传代两次。
S3、细胞株筛选
单克隆细胞株筛选包括:待细胞长满后,维持抗性压力传代两次后,进行96孔板单克隆铺板;96孔板单克隆铺板12天后,挑选出单克隆进入24孔板,24孔板细胞经过2周培养后转入6孔板,然后经T25、T75、T175放大培养,获得GLP1R报告基因稳转细胞株。
检测评估
一、报告基因和GLP1R表达质粒转染效率评估
取CREB报告基因质粒转染过的293T细胞,经1ug/ml puromycin DMEM加压培养两代后,取80uL经消化后的细胞悬液到96孔板每孔中,加入20ul浓度为150uM的Forskolin或者20ul的培养基,混合均匀后,放入37℃、5%CO2的培养箱培养孵育5小时。加入100ulBright-GloTMLuciferase Assay试剂(Promega E2620),室温1000rpm混匀5分钟,放入化学发光检测仪检测。
检测结果见图1,实验结果表明转染过报告基因的293T细胞经Forskolin刺激后有明显的剂量响应,信噪比大于80倍,有很高的转染效率。
取GLP1R表达质粒和CREB报告基因质粒转染过的293T细胞,经1ug/ml puromycin,125ng/ml Hygromycin的DMEM加压培养两代后,取320uL消化后的细胞悬液到96孔板3个孔中,分别加入20ul浓度为5uM的Liraglutide,20ul浓度为150uM的Forskolin或者20ul的培养基,混合均匀后,放入37℃、5%CO2的培养箱培养孵育5小时。加入100ul Bright-GloTMLuciferase Assay试剂,室温1000rpm混匀5分钟,放入化学发光检测仪检测。
检测结果见图2,Forskolin刺激和Liraglutide刺激后,转染后的细胞均有很强的信号响应,信噪比大于60倍,实验结果表明报告基因和GLP1R质粒的293T细胞有很好的转染效率,CREB报告基因和GLP1R表达质粒均已插入293T细胞中。
二、单克隆细胞株功能评价。
从单克隆铺板的96孔板中选取单克隆转移到24孔板中,待24孔板中的克隆长满后,100ul胰酶消化细胞2-3分钟后,加入400ul培养基重悬细胞,取320uL消化后的细胞悬液到白色不底透的96孔板3个孔中,分别加入20ul浓度为5uM的Liraglutide,20ul浓度为150uM的Forskolin或者20ul的培养基,混合均匀后,放入37℃、5%CO2的培养箱培养孵育5小时。加入100ul Bright-GloTMLuciferase Assay试剂,室温1000rpm混匀5分钟,放入化学发光检测仪检测。
单克隆的检测结果见表1,实验结果表明不同细胞株单克隆在培养基Forskolin和Liraglutide产生的非常不一样的信号值,选取Forskolin/培养基,Liraglutide/培养基信噪比较优的克隆DG1-16进行进一步扩增培养和进行方法学开发。
表1不同细胞株克隆的信噪比和Z factor
本实施例进一步提供GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂生物学活性测定的应用。
选取GLP1R报告基因稳转细胞株,10万个/每孔,80ul/孔种入96孔板;将度拉糖肽进行梯度稀释,初始工作浓度为80ng/ml的度拉糖肽,2倍梯度稀释共12个点,加入孔中;设置加培养基的阴性对照组和加高浓度度拉糖肽的阳性对照组,混合均匀;96孔板放入37℃、5%CO2的培养箱培养孵育5小时,共孵育5小时,加入100ul Bright-GloTMLuciferase Assay试剂,室温1000rpm混匀5分钟,放入化学发光检测仪检测,检测结果参见图3。
本实施例进一步提供GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂定量生物分析检测的应用。
选取GLP1R报告基因稳转细胞株,10万个/每孔,80ul/孔种入96孔板;以度拉糖肽为标准品,将度拉糖肽进行梯度稀释,初始工作浓度为80ng/ml的度拉糖肽,2倍梯度稀释共12个点,加入有稳转细胞株克隆DG1-16的孔中,混合均匀;96孔板放入37℃、5%CO2的培养箱培养孵育5小时,加入100ul Bright-GloTMLuciferase Assay试剂,室温1000rpm混匀5分钟,放入化学发光检测仪检测,检测结果参见图4和表2。
本实施例进一步提供GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂的中和抗体检测的应用。
选取GLP1R报告基因稳转细胞株10万个/每孔,80ul/孔种入白色不底透的96孔板;抗度拉糖肽的中和抗体用Pool血清稀释到初始工作浓度9600ng/ml,2倍梯度稀释10个点;度拉糖肽用培养基稀释到工作浓度为12.5ng/ml,梯度稀释后的抗度拉糖肽的中和抗体和度拉糖肽稀释液对半稀释,并且独立血清和度拉糖肽稀释液对半稀释,对半稀释后的溶液以20ul/孔加入含80ul/孔细胞悬液中,混合均匀;96孔板放入37℃、5%CO2的培养箱培养孵育5小时,加入100ul Bright-GloTMLuciferase Assay试剂,室温1000rpm混匀5分钟,放入化学发光检测仪检测,检测结果参见图5和图6。
检测与分析
一、参见图3可得,GLP1R报告基因稳转细胞株能够十分灵敏地表征度拉糖肽的活性;
二、参见图4和表2,实验结果表明GLP1R报告基因稳转细胞株建立的度拉糖肽定量方法,标准曲线有非常好的回测准确性,灵敏度能够达到15.625pg/ml,有非常好的方法灵敏度;
表2度拉糖肽标准曲线的回测偏差表
Conc.(pg/ml) | Siganl | Back Cal. | Accurancy% |
1000 | 153515 | 1000.31 | 100 |
500 | 76587 | 499.86 | 100 |
250 | 31646 | 250.44 | 100 |
125 | 12535 | 124.41 | 100 |
62.5 | 5698 | 61.95 | 99 |
31.25 | 3402 | 30.89 | 99 |
15.625 | 2672 | 16.99 | 109 |
三、参见图5和图6,通过独立血清/混合血清(NC)的比值统计,可得测定中和抗体方法的cut point为0.82。
抗度拉糖肽的中和抗体的剂量曲线实验结果,实验结果表明GLP1R报告基因稳转细胞株中和抗体检测方法的灵敏度能够达到150ng/ml,相对行业标准1ug/ml,有极佳的方法灵敏度。
本具体实施例仅仅是对本申请的解释,其并不是对本申请的限制,本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改,但只要在本申请的权利要求范围内都受到专利法的保护。
序列表
<110> 上海精翰生物科技有限公司
<120> GLP1R报告基因稳转细胞株、构建方法、应用
<130> WZF006PTA01F1906702
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<170> SIPOSequenceListing 1.0
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atggccggcg cccccggccc gctgcgcctt gcgctgctgc tgctcgggat ggtgggcagg 60
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gaataccgac gccagtgcca gcgctccctg actgaggatc cacctcctgc cacagacttg 180
ttctgcaacc ggaccttcga tgaatacgcc tgctggccag atggggagcc aggctcgttc 240
gtgaatgtca gctgcccctg gtacctgccc tgggccagca gtgtgccgca gggccacgtg 300
taccggttct gcacagctga aggcctctgg ctgcagaagg acaactccag cctgccctgg 360
agggacttgt cggagtgcga ggagtccaag cgaggggaga gaagctcccc ggaggagcag 420
ctcctgttcc tctacatcat ctacacggtg ggctacgcac tctccttctc tgctctggtt 480
atcgcctctg cgatcctcct cggcttcaga cacctgcact gcacccggaa ctacatccac 540
ctgaacctgt ttgcatcctt catcctgcga gcattgtccg tcttcatcaa ggacgcagcc 600
ctgaagtgga tgtatagcac agccgcccag cagcaccagt gggatgggct cctctcctac 660
caggactctc tgagctgccg cctggtgttt ctgctcatgc agtactgtgt ggcggccaat 720
tactactggc tcttggtgga gggcgtgtac ctgtacacac tgctggcctt ctcggtcttc 780
tctgagcaat ggatcttcag gctctacgtg agcataggct ggggtgttcc cctgctgttt 840
gttgtcccct ggggcattgt caagtacctc tatgaggacg agggctgctg gaccaggaac 900
tccaacatga actactggct cattatccgg ctgcccattc tctttgccat tggggtgaac 960
ttcctcatct ttgttcgggt catctgcatc gtggtatcca aactgaaggc caatctcatg 1020
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ttatactgct ttgtcaacaa tgaggtccag ctggaatttc ggaagagctg ggagcgctgg 1260
cggcttgagc acttgcacat ccagagggac agcagcatga agcccctcaa gtgtcccacc 1320
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Claims (10)
1.GLP1R报告基因稳转细胞株,其特征在于,所述GLP1R报告基因稳转细胞株为2020年11月18日保藏于武汉大学的中国典型培养物保藏中心的GLP1R报告基因稳转的人胚肾293T细胞DG1-6,保藏编号为CCTCC No .C2020221;所述GLP1R报告基因稳转细胞株包括有GLP1R受体基因表达体系以及报告基因表达体系,所述报告基因表达体系包括CREB结合区域、minipromoter以及报告基因。
2.根据权利要求1所述的GLP1R报告基因稳转细胞株,其特征在于,所述报告基因为荧光素酶基因。
3.一种权利要求1-2任一项所述的GLP1R报告基因稳转细胞株的构建方法,其特征在于,包括以下步骤:
S1、转染CREB报告基因质粒
将CREB报告基因质粒转染293T细胞,对293T细胞进行Puromycin筛选,获得报告基因稳转细胞;
S2、转染GLP1R表达质粒
将GLP1R表达质粒转染报告基因稳转细胞,对报告基因稳转细胞进行puromycin/Hygromycin筛选,获得GLP1R报告基因稳转细胞;
S3、细胞株筛选
对GLP1R报告基因稳转细胞进行单克隆细胞株筛选,获得所述的GLP1R报告基因稳转细胞株。
4.根据权利要求3所述的GLP1R报告基因稳转细胞株的构建方法,其特征在于,S1中所述的Puromycin筛选包括:将转染CREB报告基因质粒的293T细胞转入T25细胞培养瓶中进行传代并且加入0.5ug/ml puromycin DMEM培养基中进行加压培养。
5.根据权利要求3所述的GLP1R报告基因稳转细胞株的构建方法,其特征在于,S1中所述的Puromycin筛选还包括:维持0.5ug/ml puromycin DMEM生长传代3次。
6.根据权利要求3所述的GLP1R报告基因稳转细胞株的构建方法,其特征在于,S2中所述的puromycin/ Hygromycin筛选包括:将转染GLP1R表达质粒的293T细胞转入新的培养皿进行传代,并且加入1ug/ml puromycin 和125ng/ml Hygromycin 的DMEM培养基中进行加压培养。
7.根据权利要求3所述的GLP1R报告基因稳转细胞株的构建方法,其特征在于,S3中所述的单克隆细胞株筛选包括:待细胞长满后,维持抗性压力传代两次后,进行96孔板单克隆铺板;96孔板单克隆铺板12天后,挑选出单克隆进入24孔板,24孔板细胞经过2周培养后转入6孔板,然后经T25、T75、T175放大培养。
8.权利要求1-2任一项所述的GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂生物学活性测定的应用。
9.权利要求1-2任一项所述的GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂定量生物分析检测的应用。
10.权利要求1-2任一项所述的GLP1R报告基因稳转细胞株于GLP1R激动剂或者拮抗剂的中和抗体检测的应用。
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