Cloning and Expression of hGAD65 Gene in E. Coli BL21

Abstract

The aim of this study is to construct the hGAD65 gene and to identify the hGAD65 clone by using PCR & RFLP. The samples were derived from normal person & DM patient’s blood. Blood DNA was isolated by salting out method and then amplified by PCR with a pair of specific primer, GAD65-F-BamH1-807 & GAD65-R-Xho1-945. The PCR-product was cloned into vector pET-28a and the pET28a-hGAD65-clone was transformed into E.coli BL21 competent cells. The pET28a-hGAD65-clone was confirmed by PCR and RFLP by BamH1 & XhoI. The PCR product of pET28a-hGAD65-clone was one band of 159bp and has two bands 5.3 kb and 159 bp by RFLPwith both restriction enzymes. The GAD65 protein is expressed in 65kD of pET28a-hGAD65-clone. PET28a-hGAD65-clone was able to recognize by gold standard monoclonal antibody specifically. These results indicated that the hGAD65 gene inserted into pET28a properly and provided the GAD65 protein expression. Key words: hGAD65, PCR, pET-28a, RFLP