人源抗狂犬病毒二硫键稳定抗体在大肠杆菌中的融合表达

目的在大肠杆菌pMAL-P2x系统中表达anti-rabiesMBP-ScdsFv融合蛋白,酶切获得目的蛋白ScdsFv,进行纯化、鉴定及简单活性分析。方法利用DNA重组技术,将全长的ScdsFv蛋白基因克隆至原核表达载体pMAL-p2x中,重组质粒转化大肠杆菌E.coliER2566,IPTG诱导表达。表达产物经Xa酶切后,经Ni柱、AmyloseResin亲和层析进行纯化,SDS-PAGE和Western blot对其进行鉴定,ELISA检测其结合活性。结果成功地构建了重组质粒pMAL-ScdsFv,经诱导表达、蛋白酶切、亲和纯化后获得目的蛋白。ELISA检测其具有抗原特异结合活性,其热稳定性有显著提高。结论抗狂犬病毒ScdsFv蛋白具有良好的结合活性和生物学活性稳定性,可作为候选分子用于狂犬病的防治研究。

Expression of human anti-rabies ScdsFv fusion protein in E.coli

AIM: To express the anti-rabies MBP-ScdsFv fusion protein in E.coli and identify it's activity assay. METHODS: A full length of ScdsFv gene was Cloned into prokaryotic vector pMAL-p2x by recombinant DNA, Then the recombinant plasmid pMAL-ScdsFv was transformed to E.coli ER2566 for expression and induced by IPTG. The fusion protein was digested by Factor Xa, purified by Ni(+) and amylose resin affinity chromatography and Identified by SDS-PAGE and Western blot. The binding activity of the protein was determined by ELISA. RESULTS: Recombinant plasmid pMAL-ScdsFv was successfully constructed. The anti-rabies ScdsFv protein which was digested by Factor Xa and purified by Ni-NTA and amylose resin affinity chromatography had good binding activity and high stability. CONCLUSION: The anti-rabies ScdsFv protein is of good binding activity and high stability, and it can be used for the immune therapy.