The construction and identification of prokaryotic expression vector pQE30-EWS-FLI1 / 重庆医科大学学报

ABSTRACT

Objective:To construct Ewing's sarcoma EWS-FLI1 gene prokaryotic expression vector.Methods:The target gene of EWS-FLI1 was obtained by RT-PCR method after the total RNA was extracted from Ewing's sarcoma A673 cells.The site sequences of restrictive endonuclease SacⅠand Hind Ⅲ were introduced into the upstream and downstream of target gene respectively.The target gene fragment were cloned into pMD18-T and transformed into E.Coli JM109.Screened positive clones were confirmed by PCR,restrictive endonuclease digestion and DNA sequencing.The EWS-FLI1 gene was sequentially subcloned into prokaryotic expression vector pQE30,and the recombinant plasmid pQE30-EWS-FLI1 was confirmed by restrictive endonuclease digestion and DNA sequencing.The proteins,expressed in E.coli JM109 transformed with EWS-FLI1recombinant plasmid under IPTG induction,were characterized by SDS-PAGE and Western-blot.Results:PCR result indicated that an amplified DNA fragment was in size of 1.5 kb.Restrictive endonuclease digestion analysis indicated that the target gene was in size of 1.5 kb.DNA sequencing analysis demonstrated that sequence of target gene accorded with anticipated one.The EWS-FLI1 with a molecular weight of 54 kD was highly expressed in pQE30-EWS-FLI1.Western blot proved that the expressed product had the antigenicity of EWS-FLI1.Conclusion:The recombinant prokaryotic expression vector pQE30EWS-FLI1 is constructed successfully,which will contribute to the further research of EWS-FLI1.