抗克百威单链抗体的可溶性表达、纯化与鉴定

詹屋强; 黄宇婷; 张瑾如; 李家冬; 刘姚; 沈兴; 王弘

doi:10.13386/j.issn1002-0306.2017.20.015

抗克百威单链抗体的可溶性表达、纯化与鉴定

Soluble expression, purification, and identification of scFv against carbofuran

摘要

摘要: 在前期获得了抗克百威单链抗体基因的基础上,为进一步研究克百威单链抗体的性质,本文构建了两种抗克百威单链抗体可溶表达载体,p ET22b（+）-C-6His-CBF-sc Fv和p ET22b（+）-N-6His-CBF-sc Fv。比较His标签处于不同位置时,目的蛋白与Ni结合作用,发现His标签处于N端的目的蛋白与Ni结合作用比His标签处于C端时要强。对影响N-6His-CBF-sc Fv可溶性表达的条件进行优化,进而对其进行纯化。结果表明,当选用LB培养基,表达温度18℃,表达时间为16 h,IPTG浓度为1 mmol/L时可溶表达效果最好,可溶表达的目的蛋白量占总目的蛋白量从优化前的10%增加到50%。优化表达后的N-6His-CBF-sc Fv破壁上清经Ni Sepharose HP金属螯合亲和层析及阳离子交换层析两步纯化,纯化后纯度达90%。经ic ELISA鉴定,抗克百威单链抗体保持抗原结合活性,IC₅₀值为63.80 ng/m L。获得的抗克百威单链抗体可用于后续特性研究。

Abstract: The lab constructed anti-carbofuran sc Fv gene, for further study of the nature of anti-carbofuran sc Fv, two kinds of anti-carbofuran sc Fv soluble expression vector p ET22b ( +) -C-6His-CBF-sc Fv and p ET22b ( +) -N-6His-CBF-sc Fv were constructed.N-6His-CBF-sc Fv was better than C-6His-CBF-sc Fv for separation. The conditions of soluble expression and purification effect factors about N-6His-CBF-sc Fv were optimized.The results showed that soluble expression was the best when LB medium was used, the expression temperature was 18 ℃, the expression time was about 16 hours and the concentration of IPTG was 1 mmol/L.Ni Sepharose HP and Iex Cap SP HP were used separate N-6His-CBF-sc Fv.The purity of the purified N-6His-CBF-sc Fv was 90%.Identified by ic ELISA, anti-carbofuran sc Fv maintains antigen binding activity, IC₅₀ value was 63.80 ng/m L, which could be used for the further study.

HTML全文

参考文献(23)

施引文献

资源附件(0)