PENGEMBANGAN DETEKSI INFEKSI Streptococcus suis SEROTIPE 2 MELALUI KLONING GEN PENYANDI MURAMIDASE RELEASED PROTEIN (GEN mrp 864 bp)

ENDAH SUPRIYATI, Prof. Dr. drh. Siti Isrina Oktavia Salasia

2015 | Tesis | S2 Bioteknologi

Streptococcus suis merupakan agen penyebab berbagai penyakit pada babi yang ditandai dengan meningitis, bronkopneumonia, perikarditis, poliserositis dan septisemia. Infeksi Streptococcus suis terutama serotype 2 bersifat zoonosis pada manusia dengan gejala khas meningitis. Gen mrp 864 bp diduga merupakan protein transmembran yang dapat memicu respon imun pada hospes. Tujuan penelitian ini adalah untuk melakukan klonasi gen mrp 864 bp untuk mendapatkan protein rekombinan yang dapat digunakan untuk sarana deteksi infeksi Streptococcus suis yang imunogenik dan spesifik. Dalam penelitian ini digunakan Streptococcus suis strain R 735 yang diketahui sebagai S.suis serotipe 2 dan menghasilkan muramidase released protein (MRP+). Gen mrp 864 bp diamplifikasi dari genom S.suis, kemudian diligasi pada vektor pET SUMO. Hasil ligasi kemudian ditransformasikan pada sel kompeten E.coli Mach1 dan ditanam pada media Luria Bertani (LB) agar yang mengandung kanamycin. Koloni hasil transformasi yang tumbuh kemudian ditanam pada LB cair yang mengandung kanamycin lalu dilakukan isolasi plasmid. Analisis plasmid rekombinan dilakukan menggunakan primer mrpF, mrpR, SUMO forward dan T7 terminator untuk memastikan bahwa hasil rekombinan berada pada posisi yang tepat. Hasil rekombinan juga dilakukan sekuensing untuk memastikan urutan nukleotida mrp 864 bp. Plasmid yang postitif mengandung mrp 864 bp kemudian dimasukkan ke dalam vektor ekspresi E.coli BL21(DE3). Hasil penelitian menunjukkan bahwa gen mrp 864 bp berhasil diamplifikasi dari genom S.suis dan dapat direkayasa untuk menghasilkan protein rekombinan MRP. Hasil prediksi epitop terhadap protein rekombinan menggunakan software Ellipro menunjukkan bahwa protein bersifat imunogenik. Imunisasi terhadap mencit BALB/c menggunakan protein rekombinan dilakukan untuk memperoleh antibodi poliklonal. Respon imun mencit dievaluasi dengan enzyme linkage immunoabsorbant assay (ELISA). Antibodi terhadap MRP rekombinan berhasil dikembangkan pada mencit BALB/c. Antigen rekombinan dan antibodi terhadap MRP rekombinan mampu mendeteksi infeksi S.suis secara spesifik.

Streptococcus suis is a bacterial pathogen that cause various diseases of pigs, indicating meningitis, bronchopneumonia, arthritis, perycarditis, polyserositis and septicaemia. Streptococcus suis especially serotype 2 can infect human (zoonotic) with special symptom were meningitis. mrp 864 gene predicted gen encode transmembran protein, as one of the structural proein of S.suis that can stimulate immune responses of the host. The aim of the study was to cloned 864 bp gene of mrp to develop the MRP recombinant protein that can be used as an immunogenic and sensitive antigen to detect S.suis infection. Streptococcus suis strain R 735 as known as serotype 2 and MRP+ were used in this study. The 864 bp mrp gene was amplified from S.suis genome, then ligated with pET SUMO plasmid vector. The ligation product was transformed into the competence cell E.coli Mach1 and cultured on Luria Bertani (LB) agar containing canamycin. Cultured the colonies that inserted with 864 bp mrp gene into liquid LB containing canamycin and isolated the plasmid then. Plasmid inserting with 864 bp mrp gene were analysed by using primer of mrpF, mrpR, SUMO forward and T7 Terminator to ensure that the mrp recombinants has the right position in th genome. Recombinant plasmids were also sequenced to know the exact sequence of nucleotide of mrp gene in compare to the existing data in gene bank. Positive plasmid that inserted with were cloned into E.coli BL21(DE3) for protein expression. The result of the study showed that the 864 bp mrp gene was successfully amplified from the genom of S.suis and could be engineered to produce recombinant MRP protein. Prediction for recombinant MRP protein using Ellipro software showed that this protein could be immunogenic. BALB/c were immunized by recombinant MRP protein to produce polyclonal antibody against MRP recombinant. Antibody were evaluated by enzyme linkage immunoabsorbant assay (ELISA). The antibody against MRP can produced in BALB/c. Recombinant MRP protein and antibody against Recombinant MRP protein can detect S.suis infection specifically.

Kata Kunci : Streptococcus suis, muramidase released protein, mrp 864 bp, cloning, recombinant