Cloning and Expression of hGAD65 Gene in E. Coli BL21  

Oleh:

Rohmah, Rista Nikmatu ; Widyasari, Soraya ; Aulanni’am ; Fatchiyah

Jenis: Article from Journal - ilmiah nasional - terakreditasi DIKTI
Dalam koleksi: Indonesian Journal of Biotechnology vol. 18 no. 1 (Jun. 2013), page 52-57.
Topik: hGAD65; PCR; pET-28a; RFLP
Fulltext: 169_Ros.pdf (232.06KB)

Isi artikel

The aim of this study is to construct the hGAD65 gene and to identify the hGAD65 clone by using PCR and RFLP. The samples were derived from normal person and DM patient’s blood. Blood DNA was isolated by salting out method and then amplifi ed by PCR with a pair of specifi c primer, GAD65-F-BamH1-807 and 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 confi rmed by PCR and RFLP by BamH1 and 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 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 specifi cally. These results indicated that the hGAD65 gene inserted into pET28a properly and provided the GAD65 protein expression.

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