Expression Of Recombinent Glutamic Acid Decarboxylase In BL-21 Cell

Glutamic acid decarboxylase(GAD) is a major target antigen of humoral and cellular autoimmunity in type 1 diabetes mellitus. The autoantibodies against GADes protein is the most early and specific marker for type 1 diabetes mellitus, so it has been applied to the clinical practices widely. In our experiment, the GADes gene from rat brain was cloned and expressed in E.coli, so as to make the recombinant GADes produced by gene technology for clinical application and further study on the relation between GAD and type 1 diabetes mellitus.Firstly, with the pUClS/GADes recombinant plasmid as template, the full-length encoding sequence of GADes was amplified by PCR, and cloned into pGEM-T vector. Secondly, the target gene was cut by EcoRI and inserted into restriction sites of pET42a vector for expression. After the sequence and open reading frame were confirmed by DNA sequencing, the GADes gene was expressed in E.coli. SDS-PAGE analysis suggested that the bacteria BL21(DE) produce a kind of fusion protein of 92KD and the expressed GADes fusion protein shows expected immunoreactivity by Western Blot. Optimization of condition and segregation > denaturation and renaturation of inclusion body was done in order to enhance the quantity of solvable fusion protein.This research will lay a foundation for GAD gene expression by genie engineering and application in clinical and experimental study.