Cloning and Expression of hGAD65 Gene in E. Coli BL21
<div>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 spe...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Universitas Gadjah Mada, Yogyakarta
2015-11-01
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| Series: | Indonesian Journal of Biotechnology |
| Online Access: | http://journal.ugm.ac.id/ijbiotech/article/view/7868 |
| Summary: | <div>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-</div><div>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 RFLP</div><div>with 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.</div><div><br /></div><div>Key words: hGAD65, PCR, pET-28a, RFLP</div> |
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| ISSN: | 0853-8654 2089-2241 |