LAPORAN PENELITIAN
HIBAH PENELITIAN STRATEGIS NASIONAL
TAHUN ANGGARAN 2009
IDENTIFIKASI PROTEIN ADHESI
Mycobacterium tuberculosis
DAN
RESEPTOR PADA MAKROFAG SEBAGAI BAHAN DIAGNOSTIK
PENDERITA TUBERCULOSIS
Oleh:
dr. Dwi Yuni Nur Hidayati, MKes (Ketua)
Dr. Uun Yanuhar (Anggota)
Husnul Khotimah SSi, MKes
(Anggota)Dibiayai oleh Direktorat Jenderal Pendidikan Tinggi, Departemen
Pendidikan Nasional, melalui DIPA Universitas Brawijaya No.
0174.0/023-04.2/XV//2009 tanggal 31 Desember 2008, dan berdasarkan SK Rektor
Nomor : 160/SK/2009, tanggal 7 Mei 2009
UNIVERSITAS BRAWIJAYA
NOVEMBER 2009
Ringkasan
Tuberkulosis merupakan masalah kesehatan yang utama di Indonesia dan juga merupakan penyumbang kasus Tuberkulosis (TB) terbesar ketiga didunia setelah Cina dan India. Diagnosis tuberkulosis umumnya berdasarkan gejala klinis dan gambaran radiologis disertai konfirmasi dengan pemeriksaan hapusan dan pemeriksaan sputum. Kurang sensitifnya pemeriksaan hapusan sputum dan kultur yang memerlukan waktu lama, merupakan kesulitan dalam mendiagnosa tuberkulosis paru, sehingga diperlukan pemeriksaan imunodiagnosis yang mudah dikerjakan, murah untuk dapat mendiagnosa tuberkulosis paru.
Adanya respon seluler terhadap M. tuberculosis akan memberikan dasar suatu bentuk model dimana protein adhesi dapat dipakai sebagai bahan diagnostik. Penelitian sebelumnya oleh Alteri (2007) telah ditemukan protein Mycobacterium tuberculosis
pili(Mtp). Secara lebih mendalam peran protein tersebut belum dikaji apakah protein Mtp
merupakan protein adhesi yang berhubungan dengan virulensi seperti kemampuan aglutinasi terhadap eritrosit manusia dan hewan. Penelitian ini mengarah pada penemuan protein adhesi M.tuberculosis dan reseptornya pada makrofag melalui eksplorasi interaksi antara protein adhesi M.tuberculosis dengan protein reseptor makrofag untuk mendapatkan protein spesifik dan bermanfaat sebagai tool diagnostik terhadap penderita tuberkulosis. Tujuan penelitian ini untuk mendeteksi adanya protein adhesi pada sputum dan serum penderita tuberkulosis.
Pada penelitian ini dilakukan karakterisasi molekul protein adhesi, dan reseptor makrofag dengan elektroforesis, elektroelusi serta dipreparasi lanjut sehingga mendapatkan karakter protein yang terukur. Tiap karakter protein yang terukur dilakukan uji haemaglutinin untuk menentukan jenis pita protein yang mempunyai titer haemaglutinin tertinggi berdasarkan konsentrasi pengenceran. Protein yang telah ditentukan, selanjutnya dilakukan uji respon imunogenitas protein adhesi maupun protein reseptornya dilakukan dengan SDS-Page dan ELISA, Western blotting dan dot blotting terhadap Ig A sputum dan Ig G penderita tuberkulosis .
Hasil penelitian menunjukkan bahwa identifikasi protein adhesi yang diisolasi dari M. tuberculosis mempunyai berat molekul 63 kDa menunjukkan protein haemaglutinin setelah dilakukan uji haemaglutinasi pada eritrosit darah O manusia dengan titer tertinggi yakni 1/64. Protein adhesin MTp 63 kDa yang telah dielusi dilakukan pengujian lebih lanjut terhadap respon imunogenitas pada IgA dan IgG penderita. Hasil respon menunjukkan adanya respon imun positif dengan teknik dot blott dan western blott. Hasil dot blotting memberikan spesifitas reaksi silang antara protein adhesin MTp 63 kDa dengan warna keunguan pada sampel penderita TB, sedangkan hasil western blotting menunjukkan bahwa protein adhesin MTp 63 kDa memberian respon imunogenitas terhadap IgG yang terukur. Hal ini dibuktikan dengan pemeriksaan sampel penderita (IgA) dengan protein MTp menggunakan ELISA. Hasil ELISA menunjukkan nilai kuantitatif pemeriksaan antara 0,49 hingga 0,688.
Berdasarkan penelitian ini disimpulkan bahwa protein adhesin MTp 63 kDa merupakan protein adhesin M. tuberculosis yang mempunyai pili dan juga merupakan protein haemaglutinin. Respon protein adhesin MTp 61 kDa mempunyai spesifitas terhadap IgA dan IgG penderita yang dibuktikan dengan pengujian pada makrofag.
Rekomendasi hasil penelitian untuk dilanjutkan pada produksi rekombinan protein ahdesin MTp 63 kDa baik rekombinan proteinnya maupun rekombinan antibodi.
Summary
Tuberculosis is main health problem in Indonesia and it is case contributor of Tuberculosis (TB) the biggest third in the world after Chinese and India. Generally, Tuberculosis diagnosis is based on clinical symptom and image of radiology accompanied by confirmation with inspection of smear and inspection of sputum. Less sensitively of inspection of smear sputum and culture required stripper time, be difficulty to diagnose a lung tuberculosis so it is required inspection of immunodiagnoses which is easy to be done, cheap to diagnose a lung tuberculosis.
Existence of response cellular to M. tuberculosis will give a base of model where adhesion protein able to be used as component of diagnostic. Recent research by Alteri (2007) has been found protein of Mycobacterium tuberculosis pili (Mtp). In more circumstantially, the role of the protein has not been studied what is protein of Mtp be an adhesion protein relating to virulence like ability of agglutination to man erythrocyte and animal. This research leads to invention of an adhesion protein M.tuberculosis and its receptor at Macrophage through exploration of interaction between adhesion proteins Mtuberculosis and Macrophage receptor protein to get specific protein and useful as diagnostic tool to tuberculosis patient. The purpose of this research is to detect existence of adhesion protein at sputum and tuberculosis patient serum.
At this research, it has be done characterization of an adhesion protein molecule, and Macrophage receptor using electrophoresis, electro elusion and further preparation until obtain measurable protein character. Every measurable protein character is done a haemaglutinin test to determine protein ribbon type having the highest haemaglutinin titer based on dilution concentration. Protein which has been determined, hereinafter it is done an immunogenity response test of adhesion protein and also receptor protein by using both SDS-Page and ELISA, Western blotting and dot blotting to Ig A sputum and Ig G tuberculosis patient.
Result of research indicates that identification of adhesion protein isolated from M. tuberculosis with weights molecule of 63 kDa shows a haemaglutinin protein after done by haemaglutination test on O blood erythrocyte of human with highest titer namely 1/64. Protein adhesin of MTp 61 kDa which has been elusion is done by further examination to response immunogenity of IgA and patient IgG. Result of response shows existence of positive immune response with technique of both dot blott and western blott. Result of dot blotting gives cross reaction specifity of proteins adhesin of MTp 63 kDa with purple colour at TB patient sample, while result of western blotting indicates that protein adhesin MTp 63 kDa gives immunogenity response to measurable IgG. This thing is proved with sampling inspection of patient (IgA) with protein MTp applies ELISA. Result of ELISA shows quantitative value of intermediate inspection have range 0,49 to 0,688.
Based on this research concluded that adhesin protein of MTp 61 kDa is adhesin protein of M. tuberculosis having pili as well as is a haemaglutinin protein. Protein response of adhesin MTp 63 kDa has specifity to IgA and IgG patient proved with examination at Macrophage.
Recommendation result of research to be continued on production of protein recombinant ahdesin MTp 63 kDa either its protein recombinant and also antibody recombinant.
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