Construction Of DisA Gene Expression Vector Induced With Propionate And Its Prokaryotic Expression

DisA is DNA integrity scanning protein A,it can detect the damaged DNA and repair the lesions,thus maintaining the integrity of DNA.The study found the DisA has diadenylate cyclase activity.It can convert two ATP molecules into one c-di-AMP.c-di-AMP is a second messenger in bacteria.It has a close relationship with the synthesis of cell wall and the regulation of cell morphology.It aslo has relationship with the pathogenicity of the pathogen and antibiotic resistance in bacteria.In order to get the soluble DisA for the synthesis of c-di-AMP,research the function and regulation mechanism of c-di-AMP.DisA gene was amplified by PCR and cloned to the ten expression vectors carries different soluble tags.The recombinant plamids were identified by bacterial PCR,digestion with restriction enzyme and sequencing.Ten expression vectors were induced with propionate.Choose the recombinant plamids which can express DisA to express lots of recombinant protein.Crush the bacterias by ultrasonic and detect the supernatant of the lysate of the recombinant proteins by analysis of SDS-PAGE and Western blotting.The results of bacterial PCR,digestion with restriction enzyme and sequencing showed that DisA gene was amplified and the ten recombinant plasmids containing DisA gene were constructed successfully.The target proteins were detected in total proteins by using the six kinds of recombinant plasmids.They are 6×His-Grifin-TEV-LIC?6×His-MBP-TEV-LIC?6×His-NusA-TEV-LIC?6×His-SUMO-TEV-LIC?6×His-Thioredoxin-TEV-LIC?6×His-?-crystallin-TEV-LIC,then crush the bacterias by ultrasonic.We detected the supernatant of the lysate of the six kinds of recombinant proteins by analysis of SDS-PAGE and Western blotting.The sesults showed that we got the soluble DisA.