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Soraya Widyasari
Departement of Biology, Faculty of Mathematic and Natural Science, Universitas Brawijaya, Malang
Biochemistry, Genetics & Molecular Biology Immunology & microbiology Materials Science & Nanotechnology

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Cloning and Expression of hGAD65 Gene in E. Coli BL21 Rista Nikmatu Rohmah; Soraya Widyasari; A. Aulanni’am; F. Fatchiyah
Indonesian Journal of Biotechnology Vol 18, No 1 (2013)
Publisher : Universitas Gadjah Mada

Show Abstract | Download Original | Original Source | Check in Google Scholar | Full PDF (280.75 KB) | DOI: 10.22146/ijbiotech.7868

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
Co-Authors Aulanni'am, Aulanni'am fatchiyah . Rista Nikmatu Rohmah