Open AccessCloning and Expression of hGAD65 Gene in E. Coli BL21
Author(s) -
Rista Nikmatu Rohmah,
Soraya Widyasari,
A Aulanni’am,
F. Fatchiyah
Publication year - 2015
Publication title -
indonesian journal of biotechnology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.173
H-Index - 2
eISSN - 2089-2241
pISSN - 0853-8654
DOI - 10.22146/ijbiotech.7868
Subject(s) - clone (java method) , microbiology and biotechnology , cloning (programming) , restriction enzyme , xhoi , gene , biology , restriction fragment length polymorphism , polymerase chain reaction , genetics , ecori , computer science , programming language
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