利用红花悬浮细胞表达CD20抗体的研究
发布时间：2018-10-26 09:10
【摘要】：植物悬浮细胞具有良好的均一性、分散性、生长迅速、生物价格低廉等特点,因此利用植物悬浮系统作为植物反应器生产生物药,是极具价值与前景的项目。CD20分子为B淋巴细胞表面特有的抗原,CD20分子具有不易脱落、与抗体结合后不内化、在人体血清中无游离形式存在等特征,因此是作为治疗B细胞淋巴瘤的理想作用靶点而受到了人们的关注。本研究用红花愈伤组织建立红花悬浮细胞培养体系的条件,并通过农杆菌介导转化法利用红花悬浮细胞表达CD20复合片段抗体。以红花子叶为外植体,研究不同种类激素组合对愈伤诱导的影响,筛选优质愈伤组织进行悬浮培养,探索不同初始接种量、不同蔗糖质量浓度、不同水解酪蛋白质量浓度对红花悬浮细胞系建立的影响。将CD20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克隆载体与p Basta-CD20表达载体,并将构建的pBasta-CD20表达载体转入农杆菌,利用农杆菌介导红花悬浮细胞转化法表达CD20抗体。结果得出: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:不加琼脂粉的MS+NAA1mg/L+6-BA 0.5mg/L、接种量2.5g/50mL、蔗糖浓度20g/L、水解酪蛋白浓度800mg/L。结论:pBasta-CD20表达载体构建成功且目的蛋白在红花悬浮细胞成功表达,成功建立了红花悬浮细胞培养体系,并成功用该体系表达了CD20抗体,为利用悬浮细胞生产CD20抗体提供了新途径。
[Abstract]:Plant suspension cells have good uniformity, dispersity, rapid growth and low biological cost, so plant suspension system is used as plant reactor to produce biological drugs. CD20 molecule is a specific antigen on the surface of B lymphocytes, and CD20 molecule is not easy to fall off, does not internalize after binding with antibody, and has no free form in human serum. Therefore, as an ideal target for the treatment of B cell lymphoma, people pay more attention to it. In this study, the culture system of safflower suspension cells was established by callus of safflower, and CD20 antibody was expressed by Agrobacterium tumefaciens mediated transformation. The effects of different hormone combinations on callus induction were studied with the cotyledon of safflower as explants. The high quality callus was selected for suspension culture, and different initial inoculation amount and different sucrose concentration were explored. Effects of different concentration of hydrolyzed casein on the establishment of safflower suspension cell line. The two ends of CD20 antibody gene were introduced into the Xma I/EcoR I restriction site and connected with pEASY-T1 and pBasta vectors to construct pEASY-T1-CD20 clone vector and p Basta-CD20 expression vector. The constructed pBasta-CD20 expression vector was transformed into Agrobacterium tumefaciens. CD20 antibody was expressed by Agrobacterium tumefaciens mediated safflower suspension cell transformation. The results showed that the optimum conditions for callus induction in cotyledons of safflower were: MS NAA 2mg/L 6-BA 0.5 mg / L, temperature (25 卤0.1) 鈩，
本文编号：2295247
[Abstract]:Plant suspension cells have good uniformity, dispersity, rapid growth and low biological cost, so plant suspension system is used as plant reactor to produce biological drugs. CD20 molecule is a specific antigen on the surface of B lymphocytes, and CD20 molecule is not easy to fall off, does not internalize after binding with antibody, and has no free form in human serum. Therefore, as an ideal target for the treatment of B cell lymphoma, people pay more attention to it. In this study, the culture system of safflower suspension cells was established by callus of safflower, and CD20 antibody was expressed by Agrobacterium tumefaciens mediated transformation. The effects of different hormone combinations on callus induction were studied with the cotyledon of safflower as explants. The high quality callus was selected for suspension culture, and different initial inoculation amount and different sucrose concentration were explored. Effects of different concentration of hydrolyzed casein on the establishment of safflower suspension cell line. The two ends of CD20 antibody gene were introduced into the Xma I/EcoR I restriction site and connected with pEASY-T1 and pBasta vectors to construct pEASY-T1-CD20 clone vector and p Basta-CD20 expression vector. The constructed pBasta-CD20 expression vector was transformed into Agrobacterium tumefaciens. CD20 antibody was expressed by Agrobacterium tumefaciens mediated safflower suspension cell transformation. The results showed that the optimum conditions for callus induction in cotyledons of safflower were: MS NAA 2mg/L 6-BA 0.5 mg / L, temperature (25 卤0.1) 鈩，
本文编号：2295247
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