血管内皮生长因子单链抗体在大肠埃希菌中的高效表达
|
摘要
以血管内皮生长因子单链抗体(pTA-anti-VEGF-scFv)为研究对象,考察不同培养基配方和培养条件对血管内皮生长因子单链抗体表达量的影响。经均匀设计试验,确定工程菌R22-4高效表达重组抗体的最佳培养基为以2×YT培养基为基础,添加质量分数为0.3%葡萄糖、1.0%酪蛋白胨、0.4%玉米浆、0.133%KH_2PO_4、0.159 6%K_2HPO_4、0.532%NaCl、0.01%微量元素、0.04%维生素B_1;诱导条件为30℃、0.1 mmol/L IPTG(异丙基硫代半乳糖苷,Isopropylβ-D-Thiogalactoside)诱导5 h。经ELISA (酶联免疫吸附测定,enzyme linked immunosorbent assay)检测,血管内皮生长因子单链抗体的表达量是原始对照组的2.27倍,实现了血管内皮生长因子单链抗体在大肠埃希菌中的高效表达。
A vascular endothelial growth factor(VEGF) was used as research object to investigate the influence of different media and culture conditions on the expression of pTA-anti-VEGF-scFv. Based on the uniform design experiment, the optimal growth media for high-level expression of recombinant antibodies in engineered strain R22-4 was found to be 2×YT, adding 0.3% glucose, 1.0% casein peptone, 0.4% corn starch, 0.133% KH_2PO_4, 0.159 6% K_2HPO_4, 0.532% NaCl, 0.01% trace elements, 0.04% vitamin B_1. Induction conditions was 30 ℃, 0.1 mmol/L IPTG to induce for 5 h. ELISA detected results demonstrated that the expression of pTA-anti-VEGF-scFv was 2.27 times higher than that of original control group,and the high expression of vascular endothlial growth factor single-chain achieved in Escherichia coli.
A vascular endothelial growth factor(VEGF) was used as research object to investigate the influence of different media and culture conditions on the expression of pTA-anti-VEGF-scFv. Based on the uniform design experiment, the optimal growth media for high-level expression of recombinant antibodies in engineered strain R22-4 was found to be 2×YT, adding 0.3% glucose, 1.0% casein peptone, 0.4% corn starch, 0.133% KH_2PO_4, 0.159 6% K_2HPO_4, 0.532% NaCl, 0.01% trace elements, 0.04% vitamin B_1. Induction conditions was 30 ℃, 0.1 mmol/L IPTG to induce for 5 h. ELISA detected results demonstrated that the expression of pTA-anti-VEGF-scFv was 2.27 times higher than that of original control group,and the high expression of vascular endothlial growth factor single-chain achieved in Escherichia coli.
引文
[1] 齐鹏鹏,于士洋,吴梓齐,等.血管内皮生长因子的研究进展[J].牙体牙髓牙周病学杂志,2016,12(6):381-384.
[2] 刘刚,李金源.血管内皮生长因子与肿瘤生长的关系[J].军医进修学院学报,2010,12(1):93-94.
[3] 郭艳风,韩芳毅,周红艳,等.L型氨基酸转运体1和血管内皮生长因子在甲状腺乳头状癌中的表达及意义[J].新乡医学院学报,2017,34(1):36-38.
[4] 杜明伟,方长青,王秀茹,等.血管内皮标记物及血管内皮生长因子在胸水细胞中的表达和意义[J].微生物学杂志,2012,32(4):47-52.
[5] 刘珊,罗义辉.利用基于单链抗体的BIAcore技术检测蔬菜中的杀螟硫磷残留[J].河南农业科学,2017,46(5):95-99.
[6] Boushey HA.Experiences with monoclonal antibody therapy for allergic asthma[J].J Allergy Clin Immunol,2001,108(2):77-83.
[7] Terpe K.Overview of bacterial expression systems for heterologous protein production:from molecular and biochemical fundamentals to commercial systems[J].Appl Microbiol Biotechnol,2006,72(2):211-222.
[8] Paola J,LorenzoV,Luis A,et al.Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of Escherichia coli:Evidence that chaperone activity is the prime effect of thioredoxin[J].Journal of Molecular Biology,2006,357(1) :49-61.
[9] 刘启刚,代云见,张勇侠,等.抗IgE单链抗体在大肠杆菌中可溶性高效表达条件的研究[J].中国生物工程杂志,2012,32(11):23-28.
[10] 刘野,邹婷婷,宋焕禄.响应曲面法优化西瓜籽蛋白提取工艺[J].食品工业科技,2013,34(3):250-254.
[11] 蒋冬花,冯青青,任浩,等.高产莫纳可林K低产桔霉素红曲霉菌株的筛选和发酵条件初步优化[J].微生物学杂志,2016,36(6):10-16.
[12] 肖玲玲,杨洁,张瑞,等.IPTG浓度对大肠杆菌中重组GroEL表达的影响[J].口腔医学研究,2017,33(2):140-144.
[13] Olaofea OA,Burtona SG,Cowan DA,et al.Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli[J].Biochem Eng J,2010,52(1):19-24.
[14] Jordan E,Hust M,Roth A,et al.Production of recombinant antibody fragments in Bacillus megaterium[J].Microbial Cell Factories,2007,6(1):1-11.
[15] Schirrmann T,Al-Halabi L,Dübel S,et al.Production systems for recombinant antibodies[J].Fronti Biosci,2008,13 (12):4576-4594.
[2] 刘刚,李金源.血管内皮生长因子与肿瘤生长的关系[J].军医进修学院学报,2010,12(1):93-94.
[3] 郭艳风,韩芳毅,周红艳,等.L型氨基酸转运体1和血管内皮生长因子在甲状腺乳头状癌中的表达及意义[J].新乡医学院学报,2017,34(1):36-38.
[4] 杜明伟,方长青,王秀茹,等.血管内皮标记物及血管内皮生长因子在胸水细胞中的表达和意义[J].微生物学杂志,2012,32(4):47-52.
[5] 刘珊,罗义辉.利用基于单链抗体的BIAcore技术检测蔬菜中的杀螟硫磷残留[J].河南农业科学,2017,46(5):95-99.
[6] Boushey HA.Experiences with monoclonal antibody therapy for allergic asthma[J].J Allergy Clin Immunol,2001,108(2):77-83.
[7] Terpe K.Overview of bacterial expression systems for heterologous protein production:from molecular and biochemical fundamentals to commercial systems[J].Appl Microbiol Biotechnol,2006,72(2):211-222.
[8] Paola J,LorenzoV,Luis A,et al.Thioredoxin fusions increase folding of single chain Fv antibodies in the cytoplasm of Escherichia coli:Evidence that chaperone activity is the prime effect of thioredoxin[J].Journal of Molecular Biology,2006,357(1) :49-61.
[9] 刘启刚,代云见,张勇侠,等.抗IgE单链抗体在大肠杆菌中可溶性高效表达条件的研究[J].中国生物工程杂志,2012,32(11):23-28.
[10] 刘野,邹婷婷,宋焕禄.响应曲面法优化西瓜籽蛋白提取工艺[J].食品工业科技,2013,34(3):250-254.
[11] 蒋冬花,冯青青,任浩,等.高产莫纳可林K低产桔霉素红曲霉菌株的筛选和发酵条件初步优化[J].微生物学杂志,2016,36(6):10-16.
[12] 肖玲玲,杨洁,张瑞,等.IPTG浓度对大肠杆菌中重组GroEL表达的影响[J].口腔医学研究,2017,33(2):140-144.
[13] Olaofea OA,Burtona SG,Cowan DA,et al.Improving the production of a thermostable amidase through optimising IPTG induction in a highly dense culture of recombinant Escherichia coli[J].Biochem Eng J,2010,52(1):19-24.
[14] Jordan E,Hust M,Roth A,et al.Production of recombinant antibody fragments in Bacillus megaterium[J].Microbial Cell Factories,2007,6(1):1-11.
[15] Schirrmann T,Al-Halabi L,Dübel S,et al.Production systems for recombinant antibodies[J].Fronti Biosci,2008,13 (12):4576-4594.
© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号 地址：北京市海淀区学院路29号 邮编：100083 电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700 |