CD20片段抗体在红花悬浮细胞中的表达研究

摘要

[Objective] This study aimed to investigate the culture conditions for suspension cell system established by safflower callus and the expression of CD20 complex fragment antibody by agrobacteriummediated transformation in safflower suspension cells.[Method] The safflower cotyledon was used as explants for callus induction to research the effects of different hormone combinations on callus induction.High quality callus were selected to explore the effects of different initial inoculation amounts,sucrose concentrations and hydrolyzed casein concentrations on the establishment of safflower suspension cell.The Xma Ⅰ/EcoR Ⅰ restriction sites were introduced to both ends of the CD20 complex fragment antibody gene and it was connected with pEASY-T1 and pBasta carrier.The pEASY-T1-CD20 cloning vector and pBasta-CD20 expression vector were conducted and the pBasta-CD20 expression vector was transferred into agrobacterium to express CD20 complex antibody fragment in suspension cells by agrobacterium mediated transformation.[Result] The optimal conditions for safflower cotyledons callus induction were MS+NAA 2 mg/L+6-BA 0.5 mg/L,temperature (25±0.1) ℃ and continuous light at illumination 70 μmol/(m2 ·s).Subculturing conditions were MS+NAA 1 mg/L+6-BA 0.5 mg/L,continuous light at 30 μmol/(m2 ·s),and temperature (25±0.1) ℃.The optimal conditions for suspension cell culturing were no agar MS+NAA 1 mg/L+6-BA 0.5 mg/L,inoculation 2.5 g per 50 mL medium,sucrose 20 g/L and casein hydrolyzate concentration 800 mg/L.The expression vector of pBasta-CD20 was constructed successfully and the target protein was expressed in safflower suspension cells.[Conclusion] The culture system of safflower suspension cells was successfully constructed and the CD20 complex fragment antibody was successfully expressed by this system.%[目的]研究用红花愈伤组织建立红花悬浮细胞培养体系的条件,并利用红花悬浮细胞通过农杆菌介导转化法表达CD20复合片段抗体.[方法]以红花子叶为外植体,研究不同种类激素组合对愈伤诱导率的影响,筛选优质愈伤组织进行悬浮培养,探索不同初始接种量、蔗糖质量分数、水解酪蛋白质量浓度对红花悬浮细胞生长的影响.将CD20复合片段抗体基因两端引入Xma Ⅰ/EcoR Ⅰ酶切位点,并将其与pEASY-T1和pBasta载体相连,构建pEASY-T1-CD20克隆载体和pBasta-CD20表达载体,再将构建的pBasta-CD20表达载体转入根瘤农杆菌,利用根瘤农杆菌介导红花悬浮细胞转化法表达CD20复合片段抗体.[结果]红花子叶愈伤诱导最佳条件为MS+ NAA 2mg/L+6-BA 0.5 mg/L、温度(25±0.1)℃、光照70 μmol/(m2·s)连续光照,继代培养条件为MS+ NAA 1 mg/L+6-BA 0.5 mg/L、30 μmol/(m2·s)连续光照、温度(25±0.1)℃.悬浮细胞体系培养最佳条件为不加琼脂粉的MS+NAA 1 mg/L+6-BA 0.5 mg/L、接种量2.5g(50 mL培养基)、蔗糖质量分数2％、水解酪蛋白800 mg/L.pBasta-CD20表达载体构建成功,且目的蛋白在红花悬浮细胞中成功表达.[结论]成功构建了红花悬浮细胞培养体系,并用该体系成功表达了CD20复合片段抗体.