CN112834753A - 一种融合蛋白体外生物学活性的检测方法 - Google Patents
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Abstract
本发明提供了一种融合蛋白体外生物学活性的检测方法,该方法采用纤维蛋白或者明胶作为包被液,对细胞培养瓶进行包被,采用AlarmarBlue作为显色液,克服了原代细胞长势差、信噪比低、四参数曲线拟合不好等难点,有效提高了融合蛋白体外生物学活性的检测方法的准确度。
Description
技术领域
本发明涉及蛋白质医药生物工程与技术领域,涉及一种VEGFR-Fc融合蛋白体外生物学活性的检测方法。
背景技术
血管内皮生长因子(Vascular endothelial growth factor,VEGF)是目前已知的专属性最高、活性最强的血管生成因子,其可以促进血管内皮细胞的增殖和迁移,而肿瘤的生长依赖新生血管为肿瘤细胞提供所需的营养,研究表明,VEGF在肿瘤的发展过程中全程表达,因此VEGF可作为肿瘤治疗的重要靶点之一。VEGFR-Fc融合蛋白是一种由人VEGF受体的胞外区序列与人IgG1 Fc段融合形成的融合蛋白。VEGFR-Fc可以有效阻断新生血管的生成,从而使肿瘤组织由于缺乏血管的营养补给而不能正常生长。因此准确性好、灵敏度高的VEGFR-Fc融合蛋白类药物的生物学活性检测方法的建立是十分必要的。
发明内容
目前常用的VEGFR-Fc融合蛋白生物活性的检测方法有荧光素酶报告基因法以及HUVEC细胞法。报告基因法根据信号通路来模拟VEGF的作用原理进行实验设计,其不如HUVEC细胞法更为直观、更为全面的表现其对机体的生物学作用。此外,从现有文献报道来看,HUVEC细胞法测定VEGFR-Fc融合蛋白生物学活性目前存在一些问题。如HUVEC细胞属于原代细胞,培养条件不同直接影响细胞状态,进而影响实验结果;培养原代HUVEC细胞均使用市售的表面特殊处理的细胞瓶,但其对HUVEC的生长并没有明显的优势作用,细胞培养时间很长;显色体系采用的是四唑类化合物氧化还原体系进行显色,虽然显色操作方便,但造成实验本底较高,信噪比较低。有鉴于此,本发明的目的在于针对现有VEGFR-Fc融合蛋白生物活性测定方法存在的问题与不足,提供一种VEGFR-Fc融合蛋白生物活性的测定方法,为VEGFR-Fc融合蛋白生物活性检测提供了更准确,更高效的检测方法。
为实现本发明的目的,本发明采用如下技术方案:
一种VEGFR-Fc融合蛋白生物活性的检测方法,该方法中采用包被液对细胞培养瓶进行包被,使用AlarmarBlue进行显色,本发明所使用的包被溶液能够提高细胞粘附力,使其更好贴壁,且对细胞生长无副作用的粘附剂,例如纤维蛋白或者明胶,进一步优选明胶。
本发明所述的VEGFR-Fc融合蛋白生物活性的检测方法,其具体方法步骤如下:
(1)包被:吸取包被液至细胞培养瓶,使其完全覆盖细胞培养瓶底;
(2)细胞复苏、培养和传代:取HUVEC细胞进行复苏,次日换液,待细胞长满细胞瓶的70%以上时,进行传代培养;使用胰酶消化细胞,离心后重悬细胞并调整至合适的细胞密度,放入预先包被的细胞培养瓶中进行培养;
(3)细胞铺板、饥饿培养:将处于对数生长期的HUVEC细胞使用胰酶消化,离心后重悬细胞并计数调整至合适的细胞密度铺至96孔细胞培养板进行培养;
(4)参比品及供试品的制备:按照参比品和供试品溶液浓度进行稀释;
(5)rhVEGF165溶液的制备:将rhVEGF165稀释至一定的浓度;
(6)样品与rhVEGF165作用:吸取稀释后相应浓度的参比品和供试品分别与rhVEGF165等体积混匀,37℃±2℃,5%CO2培养箱中孵育;
(7)HUVEC细胞刺激:取上述作用后的样品与rhVEGF165混合物,分别吸取不同浓度梯度的混合溶液100μl加至饥饿培养的96孔细胞培养板进行孵育;
(8)显色:每孔加入显色液,孵育一段时间;
(9)读板:取出96孔板,根据显色液要求波长读数并根据得到的IC50值计算供试品的生物学活性。
其中所述步骤1中明胶作用时间进一步优化为37℃±2℃至少30min。
其中所述步骤2中细胞复苏、培养应使用含有生长因子和肝素钠的培养基,例如内皮细胞培养基(ECM)或者培养基199(M199),进一步优化为M199培养基。
其中所述步骤3中铺板HUVEC细胞为10代以内,进一步优化为3-7代。
其中所述步骤4中的参比品和供试品是人VEGF受体胞外区序列与人IgG1 Fc段融合的重组VEGFR-Fc蛋白。
其中所述步骤4中VEGFR-Fc参比品及样品稀释梯度为在1600-0ng/ml进行2倍系列稀释,进一步优化为初始浓度约400ng/ml,分别稀释200ng/ml、100ng/ml、50ng/ml、25ng/ml、12.5ng/ml、6.25ng/ml、0ng/ml,共8个稀释梯度。
其中所述步骤6中VEGF165稀释后与样品作用时间为1-3h,为进一步多短实验时间,进一步优化为37℃±2℃孵育1h。
其中所述步骤8中显色液还可以是CCK-8等四唑类化合物。
其中所述步骤7中HUVEC细胞刺激时间根据染色液性质不同可以是显色45h-79h,在优化显色液的基础上进一步优化为48±3h。
本发明通过增加细胞培养瓶明胶包被、优化培养基配方、驯化细胞、使用AlarmarBlue显色液等措施,完全克服了原代细胞长势差、信噪比低、四参数曲线拟合不好等难点,同时该方法也更能全面的从细胞整体水平表现VEGFR-Fc对机体的生物学作用,可以对VEGFR-Fc的活性进行准确测定,有利于对VEGFR-Fc的药效做出正确评价,更有助于指导工艺,达到获得高活性VEGFR-Fc的目的。
附图说明
图1:实施例1未添加明胶细胞铺板生物学活性检测拟合曲线。
图2:实施例1添加明胶细胞铺板生物学活性检测拟合曲线。
图3:实施例2CCK-8显色液生物学活性检测拟合曲线。
图4:实施例2AlarmarBlue显色液生物学活性检测拟合曲线。
图5:实施例3VEGFR-Fc融合蛋白体外生物学活性检测结果。
具体实施方式
为了进一步阐述本发明,下面将结合本发明的具体实施例,对本发明实施例中的技术方法进行清楚、完整地描述。如无特殊说明,本发明实施例中所涉及的试剂均为市售产品,均可以通过商业渠道购买获得。
实施例1 未添加明胶和添加明胶的活性检测
(1)包被
吸取明胶溶液至细胞瓶中,使明胶溶液完全覆盖细胞培养瓶底部,置于37℃,5%CO2培养箱中,至少30min;
(2)细胞复苏、培养和传代
取1支液氮冻存的HUVEC细胞进行复苏,次日换液,待细胞长满细胞瓶的70%以上时,进行传代培养。使用胰酶消化细胞,离心后重悬细胞并调整至合适的细胞密度,放入预先包被的细胞培养瓶中进行培养;
(3)细胞铺板、饥饿培养
取对数生长期的HUVEC细胞经胰酶消化后制成细胞悬液,计数,调整细胞密度至约1×105个/ml后,以100μl/孔加入96孔细胞培养板中,然后将细胞培养板置于37℃,5%二氧化碳培养箱中过夜饥饿培养;
(4)供试品稀释
取供试品,稀释至400ng/mL,200ng/ml、100ng/ml、50ng/ml、25ng/ml、12.5ng/ml、6.25ng/ml、0ng/ml共8个浓度梯度;
(5)rhVEGF165溶液的制备
将rhVEGF165稀释至100ng/ml;
(6)样品与rhVEGF165作用
吸取稀释后相应浓度的供试品分别与rhVEGF165等体积混匀,37℃,5%CO2培养箱中孵育1h;
(7)HUVEC细胞刺激
取上述作用后的样品与rhVEGF165混合物,分别吸取不同浓度梯度的混合溶液100μl加至饥饿培养的96孔细胞培养板进行孵育;
(8)显色
每孔加入30μl AlarmarBlue,孵育一段时间;
(9)读板
取出96孔板,室温避光振荡30min,酶标仪读数查看四参数曲线的拟合情况;
(10)结果分析
四参数拟合曲线见图1及图2,R2如下表所示:
四参数曲线R2未添加明胶比添加明胶差,图1与图2比较曲线S型较差,浓度点整体向下偏移,说明在相同种板浓度下未添加明胶培养的HUVEC状态较差;实验中使用的细胞培养瓶是市售的表面已经进行特殊处理的能增强细胞贴壁能力的培养瓶,但对于HUVEC细胞来说并无明显作用,因此HUVEC培养时要添加明胶。
实施例2 使用CCK-8及AlarmarBlue的活性检测
(1)包被
吸取明胶溶液至细胞瓶中,使明胶溶液完全覆盖细胞培养瓶底部,置于37℃,5%CO2培养箱中,至少30min;
(2)细胞复苏、培养和传代
取1支液氮冻存的HUVEC细胞进行复苏,次日换液,待细胞长满细胞瓶的70%以上时,进行传代培养。使用胰酶消化细胞,离心后重悬细胞并调整至合适的细胞密度,放入预先包被的细胞培养瓶中进行培养;
(3)细胞铺板、饥饿培养
取对数生长期的HUVEC细胞经胰酶消化后制成细胞悬液,计数,调整细胞密度至约1×105个/ml后,以100μl/孔加入96孔细胞培养板中,然后将细胞培养板置于37℃,5%二氧化碳培养箱中过夜饥饿培养;
(4)供试品稀释
取供试品,稀释至400ng/mL,200ng/ml、100ng/ml、50ng/ml、25ng/ml、12.5ng/ml、6.25ng/ml、0ng/ml共8个浓度梯度;
(5)rhVEGF165溶液的制备
将rhVEGF165稀释至100ng/ml;
(6)样品与rhVEGF165作用
吸取稀释后相应浓度的供试品分别与rhVEGF165等体积混匀,37℃,5%CO2培养箱中孵育1h;
(7)HUVEC细胞刺激
取上述作用后的样品与rhVEGF165混合物,分别吸取不同浓度梯度的混合溶液100μl加至饥饿培养的96孔细胞培养板进行孵育;
(8)显色
每孔加入相应显色液,孵育一段时间;
(9)读板
取出96孔板,室温避光振荡30min,酶标仪读数查看四参数曲线的拟合情况;
(10)结果分析
四参数拟合曲线见图1及图2,信噪比如下表所示:
四参数曲线信噪比CCK-8比AlarmarBlue小,AlarmarBlue曲线S型更明显,且使用CCK-8细胞刺激时间为72小时左右,而AlarmarBlue可以在孵育45小时加入即可,因此选择AlarmarBlue作为显色液更具优势。
实施例3 VEGFR-Fc融合蛋白生物活性检测
(1)包被
吸取明胶溶液至细胞瓶中,使明胶溶液完全覆盖细胞培养瓶底部,置于37℃,5%CO2培养箱中,至少30min;
(2)细胞复苏、培养和传代
取1支液氮冻存的HUVEC细胞进行复苏,次日换液,待细胞长满细胞瓶的70%以上时,进行传代培养。使用胰酶消化细胞,离心后重悬细胞并调整至合适的细胞密度,放入预先包被的细胞培养瓶中进行培养;
(3)细胞铺板、饥饿培养
取对数生长期的HUVEC细胞经胰酶消化后制成细胞悬液,计数,调整细胞密度至约1×105个/ml后,以100μl/孔加入96孔细胞培养板中,然后将细胞培养板置于37℃,5%二氧化碳培养箱中过夜饥饿培养;
(4)参比品及供试品稀释
取参比品及供试品,稀释至400ng/mL,200ng/ml、100ng/ml、50ng/ml、25ng/ml、12.5ng/ml、6.25ng/ml、0ng/ml共8个浓度梯度;
(5)rhVEGF165溶液的制备
将rhVEGF165稀释至100ng/ml;
(6)样品与rhVEGF165作用
吸取稀释后相应浓度的参比品和供试品分别与rhVEGF165等体积混匀,37℃,5%CO2培养箱中孵育1h;
(7)HUVEC细胞刺激
取上述作用后的样品与rhVEGF165混合物,分别吸取不同浓度梯度的混合溶液100μl加至饥饿培养的96孔细胞培养板进行孵育
(8)显色
每孔加入30μl AlarmarBlue,孵育一段时间;
(9)读板
从CO2培养箱中取出96孔板,100rpm室温避光振荡30min,在激发光波长530nm,发射光波长590nm进行检测,查看四参数拟合情况;
(10)结果分析
用参比品孔和供试品孔测定值与加样浓度的量效关系进行四参数曲线拟合,确定参比品与供试品的IC50值,曲线拟合常数R2。按照下列公式计算供试品的细胞增殖抑制生物学活性,四参数曲线拟合见图5:
供试品相对活性=参比品IC50值/供试品IC50值×100%
图5中供试品相对活性=64.05/65.41×100%=98%。
综上所述,本发明建立了一种VEGFR-Fc融合蛋白体外生物学活性的检测方法,并对该方法进行了方法验证,其精密度(6次重复性RSD=6.4%,2实验人员中间精密度RSD=8.7)、耐用性(不同细胞密度、不同孵育时间、不同细胞代次RSD均小于10%)、准确度等均满足要求,该方法实验周期较短且能够更灵敏、更准确且更稳定的进行VEGFR-Fc融合蛋白体外生物学活性的检测,对VEGFR-Fc药物研究开发具有重要的意义。
Claims (9)
1.一种VEGFR-Fc融合蛋白体外生物学活性的检测方法,该方法包括采用包被液对细胞培养瓶进行包被,使用AlarmarBlue进行显色,其中包被液是纤维蛋白或者明胶。
2.根据权利要求1所述的检测方法,其特征在于该方法包括以下步骤:
(1)包被:吸取包被液至细胞培养瓶,使其完全覆盖细胞培养瓶底;
(2)细胞复苏、培养和传代:取HUVEC细胞进行复苏,次日换液,进行传代培养,使用胰酶消化细胞,离心后重悬细胞并调整至合适的细胞密度,放入预先包被的细胞培养瓶中进行培养;
(3)细胞铺板、饥饿培养:将处于对数生长期的HUVEC细胞使用胰酶消化,离心后重悬细胞并计数调整至合适的细胞密度铺至96孔细胞培养板进行培养;
(4)参比品及供试品的制备:按照参比品和供试品溶液浓度进行稀释;
(5)rhVEGF165溶液的制备:将rhVEGF165稀释至一定的浓度;
(6)样品与rhVEGF165作用:吸取稀释后相应浓度的参比品和供试品分别与rhVEGF165等体积混匀,37℃±2℃,5%CO2培养箱中孵育;
(7)HUVEC细胞刺激:取上述作用后的样品与rhVEGF165混合物,分别吸取不同浓度梯度的混合溶液加至饥饿培养的96孔细胞培养板进行孵育;
(8)显色:每孔加入显色液,孵育一段时间;
(9)读板:取出96孔板,在适当波长下读数并根据得到的IC50值计算供试品的生物学活性。
3.根据权利要求1或2所述的检测方法,其特征在于包被时明胶作用时间为至少30min。
4.根据权利要求1所述的方法,其特征在于其中步骤(2)所述的细胞复苏、培养应使用含有生长因子和肝素钠的培养基,可以是ECM或者M199。
5.根据权利要求1所述的方法,其特征在于其中步骤(3)所述的细胞是通过胰酶消化、离心后使用不含生长因子的培养基重悬的原代HUVEC细胞悬液,计数并稀释至一定的浓度,100μl/孔铺至96孔细胞培养板中,37℃±2℃,5%CO2培养箱中过夜进行饥饿培养。
6.根据权利要求1所述方法,其特征在于,步骤(4)所述的参比品与供试品的浓度梯度是在1600-0ng/ml的浓度范围里进行2倍稀释。
7.根据权利要求1所述方法,其特征在于,步骤(6)所述样品与rhVEGF165作用时间为1-3小时。
8.根据权利要求1所述方法,其特征在于,步骤(7)所述对HUVEC细胞刺激时间为45h-79h。
9.根据权利要求1所述方法,其特征在于,步骤(9)所述供试品的生物学活性是通过以样品浓度为横坐标,以参比品孔和供试品孔测定值为纵坐标进行四参数拟合,得到参比品与供试品的IC50值,曲线拟合常数R2,并按照下列公式计算供试品的生物学活性,
供试品相对活性=参比品IC50值/供试品IC50值×100%。
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