CN106318971B - 一种人抗体基因重组质粒的转染方法 - Google Patents
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Abstract
一种抗体基因重组质粒的转染方法，是采用脂质体转染方法，将Lipofectamine3000作为转染试剂，HEK293细胞作为转染的宿主细胞，边传代边转染，将人抗体重链基因重组质粒和人抗体轻链基因重组质粒体外瞬时共转染至HEK293细胞，培养48h后，检测细胞培养上清IgG分泌量，从而将人抗体基因重组质粒成功转染至HEK293细胞。本发明所述转染方法操作简便，安全，重复性好，可自然降解，低细胞毒性，快速，高效，经济，适应性广等优点。
Description
技术领域
本发明涉及一种人抗体基因重组质粒的转染方法。
背景技术
细胞转染是真核细胞由于外源DNA或RNA掺入而获得新遗传标志的过程。它是基因功能研究、基因免疫的理论和技术基础，也是研究基因表达调控、突变分析等的常规工具。同时也是基因治疗、疫苗接种、药物开发等应用的重要工具。
细胞转染常用的方法有：生物学方法(即以病毒为载体的转染法)、化学方法(磷酸钙共沉淀法、DEAE-葡聚糖法、阳离子脂质体法、阳离子聚合物法)、物理方法(显微注射法、基因枪法、电穿孔法、激光照射法、声孔效应法、磁性纳米颗粒等)。病毒载体转染效率高，但其目的基因容量小，制备复杂，成本高，不能体内反复使用；另外，其可能在体内发生基因的重组或互补，存在致癌、致畸的风险，安全性差，适用性窄。磷酸钙法操作简单实用，但对细胞有一定的选择性，且重复性差，不能广泛应用。显微注射法需要特殊的技术，对细胞的损伤较大，不能广泛应用。基因枪法需要特殊的设备，价格昂贵，在转染时对细胞具有物理损伤而不能被普及应用。
影响基因转染效率的因素很多，包括转染试剂、转染方法、细胞种类、细胞状态，转染时间、质粒结构和纯度、转染试剂与DNA的比例等。理想的细胞转染方法应该具有较好的生物降解性、稳定性、靶向性强、有助于基因的表达并能应用于基因方面疾病的治疗。针对目前转染方法的不足，我们利用脂质体法将人抗体基因重组质粒体外瞬时转染至HEK293细胞，为单克隆抗体药物筛选提供研究基础。
发明内容
本发明的目的在于针对现有转染方法的不足，提供一种人抗体基因重组质粒的转染方法。本发明是利用脂质体转染方法，将Lipofectamine3000作为转染试剂，HEK293细胞作为转染的宿主细胞，边传代边转染，将人抗体重链基因重组质粒和人抗体轻链基因重组质粒体外瞬时共转染至HEK293细胞，培养48h后，检测细胞培养上清IgG分泌量，从而将人抗体基因重组质粒成功转染至HEK293细胞。
本发明所述转染方法为：
(1)人抗体基因重组质粒转染HEK293细胞，包括步骤：
(a)HEK293细胞传代、接种：使用含10％FBS的高糖DMEM培养基培养HEK293细胞，待细胞密度达到80％左右，将0.25％的胰酶-EDTA消化终止后的细胞，按每孔10000个细胞接种96孔板，再补加培养液至190μL。同时设置不同的对照组，对照组1为只加单个重链的质粒组；对照组2为只加单个轻链的质粒组；对照组3只加转染试剂和细胞，不加质粒；对照组4只加细胞，不加转染试剂和质粒；
(b)用opti-MEM分别稀释质粒和转染试剂，使得加入每孔的转染试剂Lipofectamine3000为0.15μL，每孔的P3000为0.2μL，每孔的人抗体重链基因重组质粒和人抗体轻链基因重组质粒各为50ng；
(c)按1：1的体积比例将稀释的质粒和稀释的转染试剂混匀，室温孵育5min后，将其加入到接种有细胞的孔中，37℃、5％的CO2培养箱中培养48h。
(2)Elisa检测细胞培养上清IgG分泌量：收集步骤(1)中的转染后细胞培养上清，使用Goat Anti-Human Kappa蛋白和Goat Anti-Human Lambda蛋白包被酶标板，采用人IgG作为标准品，标准品对应的二抗为1:5000稀释的HRP标记的Goat Anti-Human IgG，待检测的转染后细胞培养上清对应的二抗为1:10000稀释的Goat x-human IgG-Fc Fragment HRPConjugated，采用酶联免疫吸附测定方法检测转染后细胞培养上清特异性，看其是否分泌IgG；具体操作如下：
(A)用包被液(50μL/孔)将Goat Anti-Human Kappa蛋白和Goat Anti-HumanLambda蛋白(各100ng/孔)包被酶标板，4℃过夜。洗板机洗板5遍。洗液为含0.05％Tween的PBS；
(B)用封闭液200μL/孔，37℃封闭2h，洗板5遍；
(C)分别在对应孔中加入细胞培养上清，或者梯度稀释的IgG标准品，同时用水和PBS作为阴性对照，50μL/孔，37℃放置1h。洗板5遍；
(D)分别在对应孔中加入1:5000稀释的HRP标记的Goat Anti-Human IgG，以及1:10000稀释的Goat x-human IgG-Fc Fragment HRP Conjugated 50μL/孔，37℃放置45min，洗板5遍；
(E)加入显色液100μL/孔，室温避光显色10min；
(F)加入2mol/L硫酸50μL/孔，终止显色反应，检测OD450nm，参考波长为630nm。
所述步骤(1)采用的转染方法是HEK293细胞传代与人抗体基因重组质粒转染同时进行；
所述抗体基因重组质粒种属来源不仅限于人，抗体基因重组质粒种属来源可以是任一动物。
本发明的有益效果：采用脂质体转染方法，将Lipofectamine3000作为转染试剂，HEK293细胞作为转染的宿主细胞，边传代边转染，将人抗体重链基因重组质粒和人抗体轻链基因重组质粒体外瞬时共转染至HEK293细胞，培养48h后，检测细胞培养上清IgG分泌量，从而将人抗体基因重组质粒成功转染至HEK293细胞。本发明的优点是：一种人抗体基因重组质粒的转染方法操作简便，安全，重复性好，可自然降解，低细胞毒性，快速，高效，经济，适应性广等优点。
附图说明
图1为Elisa检测标准曲线。
具体实施方式
1、人抗体基因重组质粒转染HEK293细胞，包括步骤：
(a)HEK293细胞传代、接种：使用含10％FBS的高糖DMEM培养基培养HEK293细胞，待细胞密度达到80％左右，将0.25％的胰酶-EDTA消化终止后的细胞，按每孔10000个细胞接种96孔板，再补加培养液至190μL。同时设置不同的对照组，对照组1为只加单个重链的质粒组；对照组2为只加单个轻链的质粒组；对照组3只加转染试剂和细胞，不加质粒；对照组4只加细胞，不加转染试剂和质粒；其中表1为Elisa检测的96孔板整体布局；
(b)用opti-MEM分别稀释质粒和转染试剂，使得加入每孔的转染试剂Lipofectamine3000为0.15μL，每孔的P3000为0.2μL，每孔的人抗体重链基因重组质粒和人抗体轻链基因重组质粒各为50ng；
(c)按1：1的体积比例将稀释的质粒和稀释的转染试剂混匀，室温孵育5min后，将其加入到接种有细胞的孔中，在37℃及5％的CO2培养箱中培养48h。
2、Elisa检测细胞培养上清IgG分泌量
使用酶联免疫吸附测定方法检测转染效果，其中表2为Elisa检测细胞培养上清IgG的OD值，表3为Elisa检测样品的IgG浓度。具体操作如下：
(A)用包被液(50μL/孔)将Goat Anti-Human Kappa蛋白和Goat Anti-HumanLambda蛋白(各100ng/孔)包被酶标板，4℃过夜。洗板机洗板5遍。洗液为含0.05％Tween的PBS；
(B)用封闭液200μL/孔，37℃封闭2h，洗板5遍；
(C)分别在对应孔中加入细胞培养上清，或者梯度稀释的IgG标准品，同时用水和PBS作为阴性对照，50μL/孔，37℃放置1h。洗板5遍；
(D)分别在对应孔中加入1:5000稀释的HRP标记的Goat Anti-Human IgG，以及1:10000稀释的Goat x-human IgG-Fc Fragment HRP Conjugated 50μL/孔，37℃放置45min，洗板5遍；
(E)加入显色液100μL/孔，室温避光显色10min；
(F)加入2mol/L硫酸50μL/孔，终止显色反应，检测OD450nm，参考波长为630nm。
表1Elisa检测的96孔板整体布局
对照组1为只加单个重链的质粒组；对照组2为只加单个轻链的质粒组；
对照组3只加转染试剂和细胞，不加质粒；对照组4只加细胞，不加转染试剂和质粒。
表2
| 值 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | 1.126 | 1.142 | 1.132 | 0.065 | 0.067 | 0.064 | 0.067 | 0.066 | 0.063 | 0.061 | 0.062 | 0.063 |
| B | 0.976 | 1.022 | 0.997 | 1.069 | 1.086 | 1.095 | 1.034 | 1.016 | 1.017 | 1.076 | 1.072 | 1.081 |
| C | 0.752 | 0.898 | 0.831 | 1.073 | 1.064 | 1.068 | 1.126 | 1.109 | 1.105 | 1.06 | 1.076 | 1.066 |
| D | 0.601 | 0.622 | 0.608 | 1.112 | 1.115 | 1.118 | 1.026 | 1.009 | 1.029 | 0.063 | 0.061 | 0.161 |
| E | 0.405 | 0.389 | 0.395 | 1.052 | 1.057 | 1.054 | 1.102 | 1.106 | 1.107 | 0.072 | 0.069 | 0.068 |
| F | 0.211 | 0.206 | 0.209 | 1.063 | 1.081 | 1.092 | 1.063 | 1.079 | 1.065 | 0.069 | 0.088 | 0.079 |
| G | 0.143 | 0.134 | 0.141 | 1.059 | 1.062 | 1.069 | 1.083 | 1.097 | 1.101 | 0.055 | 0.057 | 0.056 |
| H | 0.114 | 0.106 | 0.109 | 0.065 | 0.065 | 0.062 | 0.061 | 0.061 | 0.062 | 0.064 | 0.061 | 0.062 |
表3
Claims (3)
1.一种人抗体基因重组质粒的转染方法，其特征在于，包括如下步骤：
（1）人抗体基因重组质粒转染HEK293细胞，包括步骤：
(a) HEK293细胞传代、接种：使用含10%FBS的高糖DMEM培养基培养HEK293细胞，待细胞密度达到80%左右，将0.25%的胰酶-EDTA消化终止后的细胞，按每孔10000个细胞接种96孔板，再补加培养液至190µL;
同时设置不同的对照组，对照组1为只加单个重链的质粒组；对照组2为只加单个轻链的质粒组；对照组3只加转染试剂和细胞，不加质粒；对照组4只加细胞，不加转染试剂和质粒；
(b)用opti-MEM分别稀释质粒和转染试剂，使得加入每孔的转染试剂Lipofectamine3000为0.15µL，每孔的P3000为0.2µL，每孔的人抗体重链基因重组质粒和人抗体轻链基因重组质粒各为50ng；
(c) 按1：1的体积比例将稀释的质粒和稀释的转染试剂混匀，室温孵育5min后，将其加入到接种有细胞的孔中，在37℃及5%的CO2培养箱中培养48h;
（2）Elisa检测细胞培养上清IgG分泌量：收集步骤（1）中的转染后细胞培养上清，使用Goat Anti-Human Kappa 蛋白和Goat Anti-Human Lambda蛋白包被酶标板，采用人IgG作为标准品，标准品对应的二抗为1:5000稀释的HRP标记的Goat Anti-Human IgG，待检测的转染后细胞培养上清对应的二抗为1:10000稀释的Goat x-human IgG-Fc Fragment HRPConjugated，采用酶联免疫吸附测定方法检测转染后细胞培养上清特异性，看其是否分泌IgG;
具体操作如下：
（A）用50µL/孔包被液将Goat Anti-Human Kappa 蛋白和Goat Anti-Human Lambda蛋白，各100ng/孔，包被酶标板，4℃过夜，洗板机洗板5遍，洗液为含0.05% Tween的PBS；
（B）用封闭液200µL /孔，37℃封闭2h，洗板5遍；
（C）分别在对应孔中加入细胞培养上清，或者梯度稀释的IgG标准品，同时用水和PBS作为阴性对照，50µL/孔，37℃放置1h，洗板5遍；
（D）分别在对应孔中加入1:5000稀释的HRP标记的Goat Anti-Human IgG，以及1:10000稀释的Goat x-human IgG-Fc Fragment HRP Conjugated 50µL/孔，37℃放置45min，洗板5遍；
（E）加入显色液100µL/孔，室温避光显色10min；
（F）加入2mol/L硫酸50µL/孔，终止显色反应，检测OD450nm，参考波长为630nm。
2.根据权利要求1所述的一种人抗体基因重组质粒的转染方法，其特征在于：所述步骤（1）采用的转染方法是HEK293细胞传代与人抗体基因重组质粒转染同时进行。
3.根据权利要求1所述的一种人抗体基因重组质粒的转染方法，其特征在于：所述抗体基因重组质粒种属来源是人或任一动物。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2014018572A2 (en) * | 2012-07-23 | 2014-01-30 | Zymeworks Inc. | Immunoglobulin constructs comprising selective pairing of the light and heavy chains |
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| WO2014018572A2 (en) * | 2012-07-23 | 2014-01-30 | Zymeworks Inc. | Immunoglobulin constructs comprising selective pairing of the light and heavy chains |
Non-Patent Citations (2)
| Title |
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| HEK293 PEAK瞬转快速表达抗体蛋白平台的建立;颜为海等;《安徽大学学报(自然科学版)》;20140331;第38卷(第2期);103-108 * |
| 双载体转重链Tyr664Cys和轻链Thr1286Cys突变体BDD-FVIII基因;朱甫祥等;《中国细胞生物学学报》;20120115;第34卷(第1期);18-24 * |
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