AMPLIFIKASI, SUBKLONING DAN SEKUENSING GEN PENYANDI PROTEIN INTERFERON GAMMA (IFN-γ) UNTUK PENGEMBANGAN DIAGNOSIS TUBERKULOSIS PADA ANJING

• Main Authors: , Mei Ita Riyanti, , Prof. Dr. drh. Ida Tjahajati, M.P.
• Format: Theses and Dissertations NonPeerReviewed
• Published: [Yogyakarta] : Universitas Gadjah Mada 2012
• Subjects: ETD
• Online Access: https://repository.ugm.ac.id/98064/; http://etd.ugm.ac.id/index.php?mod=penelitian_detail&sub=PenelitianDetail&act=view&typ=html&buku_id=54352
• Institution: Universitas Gadjah Mada

Tuberculosis is a disease caused by close related Mycobacterium strains such as M.tuberculosis, M.bovis, M.africanum, and M.microti which are known as M.tuberculosis complex (MTC). Tuberculosis (TB) is mainly described by the presence of tubercle or granulomatous inflammation which could happen in human as well as many species of animals. Tuberculosis in small animal gains more attention because it can endanger human health. This research aims to develop a new diagnostic tool in identification of tuberculosis infection in dogs by subcloning of interferon gamma protein coding gene. Obtained interferon gamma protein can be used to produce primary and secondary antibodies which can be used in tuberculosis diagnosis in dogs by ELISA method. The research started by amplification of IFN-γ from mRNA obtained from Tjahajati�s research (2008) to DNA by RT-PCR. Obtained DNA were ligated to plasmid vector pET SUMO and transformed to One Shot® Chemically Competent E. coli Mach 1 (One Shot® Mach1�-T1). Growing colonies were recultured to liquid Luria Bertani (LB) media containing kanamycin. The next step is isolating plasmid using High Pure Plasmid Isolation Kit (Roche). Isolated plasmids were tested using PCR with canine IFN-γ primer. Product of PCR than being sequenced and analysed with Mega version 4.0. The results showed that bacteria colonies successfully grown on LB plat media and grown in re-cultured liquid LB media. After plasmid isolation and PCR testing, positive results were marked by 541 bp band in electrophoresis equal to IFN-γ gene width. The sequence�s result show 91% similarity between gene canine IFN-γ from cloning and gene canine IFN-γ from Gene bank.