摘要
目的 :提高从半合成噬菌体抗体库中克隆的抗地高辛 (Dig)人单链抗体 (ADAscFv)在大肠杆菌 (E .coli)的分泌表达水平。方法 :①通过PCR从表达质粒 pHEN2 ADAscFv扩增ADAscFv基因并重组于质粒 p3MH构建表达质粒p3MH ADAscFv后 ,转化到E .coliXL1 blue及HB2 15 1;②ADAscFv分别在带有上述 2种表达质粒的E .coliHB2 15 1以及带有表达质粒 p3MH ADAscFv的E .coliXL1 blue进行表达 ,通过ELISA分析培养基上清中的可溶性ADAscFv ;③在培养基中加入蔗糖、甘氨酸及TritonX 10 0 ,通过ELISA分析培养基上清中的可溶性ADAscFv。结果 :①ADAscFv在含表达质粒p3MH ADAscFv的E .coliHB2 15 1及XL1 blue的分泌型表达水平比pHEN2 E .coliHB2 15 1表达系统高约 5倍 ;②在培养基中加入蔗糖 ,可使ADAscFv在含有表达质粒 p3MH ADAscFv的E .coliHB2 15 1及XL1 blue表达系统的分泌型表达提高 5倍多。结论 :通过采用不同的载体 E .coli表达系统以及在培养基中加入不被代谢的蔗糖 ,可使ADAscFv的分泌型表达提高 2
Objective:To increase the secretory expression level of the human single chain variable fragment (scFv) of anti digoxin antibody(ADA) cloned from a hemi synthetic phage antibody library Methods: (1) The ADA scFv gene was amplified from the experssion plasimd pHEN2 ADA scFv with PCR ,recombinated into the plasmid p3MH to construct the expression plasmid p3MH ADA scFv,and transformed into E.coli strain HB2151 and XL1 blue (2) ADA scFv was secreted into the culture medium as soluble protein in E.coli srain HB2151 carrying the two expression plasmid mentioned above and E.coli strain XL1 blue carrying expression plasmid p3MH ADA scFv,respectively ELISA was used to analyzed the soluble ADA scFv in the supernant of the culture medium (3) ADA scFv was secreted into culture medium with addition of various concentrations of sucrose ,glycine, and Triton X 100 in the E.coli strains mentioned above The soluble ADA scFv in the supernant of the culture medium was analyzed with ELISA Results: (1) The level of the soluble ADA scFv in culture medium expressed in E.coli srains of HB2151 and XL1 blue carrying expression plasmid p3MH ADA scFv was 5 fold of that expressed in E.coli HB2151 carrying expression plasmid pHEN2 ADA scFv (2)Addition of sucrose to the medium gave more than 5 fold increase in the level of soluble ADA scFv expressed in E.coli HB2151 and XL1 blue carrying expression plasmid p3MH ADA scFv Conclusions:The level of the soluble ADA scFv in culture medium was increased more than 25 fold when expressed in E.coli srains of HB2151 and XL1 blue carrying expression plasmid p3MH ADA scFv combined with the addition of sucrose to the culture medium
出处
《军医进修学院学报》
CAS
2002年第1期25-27,共3页
Academic Journal of Pla Postgraduate Medical School
基金
"九五"军队医药科研基金项目 (98M 14 5
96Z0 0 4)
