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【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XmaⅠ/EcoRⅠ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 0.5mg/L、温度(25±0.1)℃、光照70μmol/(m2·s)连续光照,继代培养条件为MS+NAA 1mg/L+6-BA 0.5mg/L、30μmol/(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 1mg/L+6-BA 0.5mg/L、接种量2.5g(50mL培养基)、蔗糖质量分数2%、水解酪蛋白800mg/L。pBastaCD20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。
【Objective】 The objective of this study was to investigate the conditions for establishing safflower suspension cell culture system with safflower callus and to express CD20 fragment antibody by safflower suspension cells through Agrobacterium-mediated transformation. 【Method】 Using the leaves of safflower as explants, the effects of different hormone combinations on the callus induction rate were studied. High-quality callus was screened for suspension culture, and the effects of initial inoculum size, sucrose content and hydrolyzed casein mass concentration Effect of suspension cell growth of safflower. The pEAST-T1-CD20 cloning vector and the pBasta-CD20 expression vector were constructed by introducing the CD20 fragment antibody gene into the XmaⅠ / EcoRⅠ restriction site at both ends and ligating it with pEASY-T1 and pBasta vectors. The constructed pBasta- The CD20 expression vector was transformed into Agrobacterium tumefaciens and the CD20 fragment was expressed by Agrobacterium tumefaciens-mediated safflower suspension cell transformation. 【Result】 The optimum conditions for the callus induction were as follows: MS + NAA 2mg / L + 6-BA 0.5mg / L, temperature 25 ± 0.1 ℃, light 70μmol / The conditions were as follows: MS + NAA 1mg / L + 6-BA 0.5mg / L, 30μmol / (m2 · s) continuous light and temperature (25 ± 0.1) ℃. The optimum condition for the culture of suspension cell system was MS + NAA 1 mg / L + 6-BA 0.5 mg / L without agar powder, inoculum 2.5 g (50 mL medium), sucrose 2%, hydrolyzed casein 800 mg / L . pBastaCD20 expression vector was successfully constructed, and the target protein was successfully expressed in safflower suspension cells. 【Conclusion】 The safflower suspension cell culture system was successfully constructed and the CD20 complex fragment antibody was successfully expressed.