UNGGULAN PERGURUAN TINGGI- HIBAH BERSAING
LAPORAN AKHIR HASIL PENELITIAN UNGGULAN PERGURUAN TINGGI – HIBAH BERSAING (TAHUN KE- 1 ) TAHUN ANGGARAN 2012
Judul
: Pengaruh Pemberian Outer Membran Protein Mycobacterium tuberculosis terhadap ekspresi sel T CD4 dan Interferon – γ
Ketua
:dr. Dwi Yuni Nur Hidayati, Mkes (Ketua)
Anggota
: 1. Husnul Khotimah SSi,Mkes (Anggota ) 2. dr. Dewi Santosaningsih, Mkes (Anggota)
Dibiayai oleh Direktorat Jenderal Pendidikan Tinggi, Kementerian Pendidikan dan Kebudayaan, Melalui DIPA Universitas Brawijaya nomor: 0636/02304.2.16/15/2012, tanggal 9 Desember 2011, dan berdasarkan SK Rektor Universitas Brawijaya Nomor: 058/SK/2012 tanggal 8 Pebruari 2012
Lembaga Penelitian dan Pengabdian kepada Masyarakat UNIVERSITAS BRAWIJAYA 2012
ABSTRAK
Pengaruh Pemberian Outer Membrane Protein Mycobacterium tuberculosis terhadap ekspresi sel T CD4 dan Interferon γ Pencegahan tuberkulosis sampai saat ini dilakukan dengan pemberian vaksin BCG (Bacille Calmette- Guerine). Tampaknya pemberian vaksin BCG, belum dapat melindungi terjadinya infeksi tuberkulosis pada orang dewasa. Oleh karena itu, masih diperlukan pengembangan terhadap vaksin tuberkulosis, mengingat efek proteksi BCG pada tuberkulosis paru orang dewasa bervariasi sekitar 0-80%. Pengembangan vaksin berdasarkan materi permukaan bakteri yang disebut adhesin sebagai target untuk pengembangan vaksin mulai banyak diteliti. Proses adhesi merupakan salah satu sifat virulensi dari bakteri yang berperan penting untuk terjadinya kolonisasi sampai timbulnya infeksi. Mycobacterium tuberculosis memiliki Outer membran protein (OMP) yang merupakan bagian bakteri yang berperan pada pelekatan dengan sel epitel host dan OMP dapat menginduksi respons imun, dengan dasar mekanisme ini OMP dapat dipakai sebagai kandidat vaksin Tujuan penelitian ini adalah untuk mengungkap OMP Mycobacterium tuberculosis mempunyai protein hemaglutinin yang merupakan protein adhesin dan pemberiannya dapat menginduksi respons imun seluler sehingga dapat menghambat kolonisasi bakteri Mycobacterium tuberculosis pada jaringan paru. Pada penelitian ini, dilakukan SDS-PAGE dan uji hemaglutinasi. Didapatkan protein pili berat molekul 38 kDa merupakan protein hemaglutinin dan selanjutnya dilakukan uji adhesi, protein OMP 38 kDa merupakan protein adhesin. Kesimpulan OMP 38 kDa Mycobacterium tuberculosis merupakan protein adhesin Kata kunci: OMP, adhesin, Mycobacterium tuberculosis.
ABSTRACT The Effect Outer Membrane Protein (OMP) Mycobacterium tuberculosis at expression T cell CD4 and Interferon γ
Prevention of tuberculosis has so far been done with the BCG vaccine (Bacille Calmette-Guerine). Apparently BCG immunization has failed to provide protection against tuberculosis infection in adults. Therefore, development of the vaccine against tuberculosis, is still requires considering that protective effect of BCG given to adult pulmonary tuberculosis varies around 0-80%. The development of vaccines based on bacterial surface material called adhesin as a target for vaccine development is beginning to be widely studied. The process of adhesion is one of the virulence properties of bacteria playing an important role for the occurrence of colonization until the onset of infection. Mycobacterium tuberculosis has outer membrane (OMP), part of bacteria that play a role in adhesion to host epithelial cells, and OMP can induce an immune response, on the basis of this mechanism OMP can be used as vaccine candidate. The purpose of this study is to reveal that OMP Mycobacterium tuberculosis has the hemagglutinin protein which is an adhesin protein, and adhesin protein delivery can induce cellular immune responses that can inhibit the growth and colonization of the bacteria Mycobacterium tuberculosis in lung tissues. In this study, SDS-PAGE and hemagglutination testing were performed. OMP with molecular weight of 38 kDa, which is a protein of hemagglutinin was obtsined, and adhesion testing was conducted, the OMP of 38 kDa was adhesin protein. The conclusion is that protein of 38 kDa OMP Mycobacterium tuberculosis is an adhesin Keywords :OMP, adhesin, Mycobacterium tuberculosis,
RINGKASAN Pengaruh Pemberian Outer Membrane Protein Mycobacterium tuberculosis terhadap ekspresi sel T CD4 dan Interferon γ Tuberkulosis (TB) merupakan penyakit menular yang disebabkan oleh bakteri Mycobacterium tuberculosis. Diperkirakan sepertiga penduduk dunia telah terinfeksi oleh M.tuberculosis, 5-10% akan berkembang menjadi tuberkulosis dengan manifestasi klinis. Pencegahan tuberkulosis sampai saat ini dilakukan dengan pemberian vaksin BCG (Bacille Calmette- Guerine). Tampaknya pemberian imunisasi BCG, belum dapat melindungi terjadinya infeksi TB pada orang dewasa. Oleh karena itu, masih diperlukan pengembangan terhadap vaksin tuberkulosis, mengingat efek proteksi BCG yang diberikan pada kejadian tuberkulosis paru orang dewasa bervariasi sekitar 0-80%. Pengembangan vaksin berdasarkan materi permukaan bakteri yang disebut adhesin sebagai target untuk pengembangan vaksin mulai banyak diteliti. Proses adhesi merupakan salah satu sifat virulensi dari bakteri yang berperan penting untuk terjadinya kolonisasi sampai timbulnya infeksi. Mycobacterium tuberculosis memiliki outer membran protein (OMP) yang merupakan bagian bakteri yang berperan pada pelekatan dengan sel epitel host, dengan dasar mekanisme ini OMP dapat dipakai sebagai kandidat vaksin Manfaat penelitian ini secara teoritis diharapkan temuan penelitian ini dapat menjadi dasar pengembangan Ilmu Pengetahuan dan Teknologi, mengenai protein adhesin OMP Mycobacterium tuberculosis dalam menginduksi respons imun seluler. Manfaat Praktis diharapkan temuan penelitian ini dapat dilanjutkan dan digunakan sebagai pengembangan bahan vaksin yang dapat digunakan untuk membantu menurunkan terjadinya infeksi tuberkulosis. Metode penelitian dilakukan dalam 2 tahap, yaitu: isolasi outer membrane protein Mycobacterium tuberculosis strain H37Rv dan SDS-PAGE (Tahap-1); Uji Hemaglutinasi dan uji adhesi outer membrane protein Mycobacterium tuberculosis strain H37Rv(Tahap-2). Hasil penelitian tahap-1 menunjukkan bahwa outer membrane protein Mycobacterium tuberculosis strain H37Rv 38 kDa mampu mengaglutinasi eritrosit pada pengenceran titer 1/32 , uji hemaglutinasi bertujuan untuk mendapatkan protein hemaglutinin; karena pada umumnya protein hemaglutinin juga merupakan protein adhesin. Setelah didapatkan protein hemaglutinin Mycobacterium tuberculosis strain H37Rv 38 kDa selanjutnya dilakukan uji adhesi untuk membuktikan bahwa protein tersebut merupakan protein adhesin. Pada uji adhesi ini menggunakan 6 perlakuan pengenceran protein adhesin 38 kDa (0,1,1/2,1/4,1/8,1/16) pada enterosit mencit. Dari hasil Anova menunjukkan protein hemaglutinin omp 38 kDa pada berbagai pengenceran berpengaruh sangat bermakna terhadap indeks adhesi(p<0.05), dimana makin tinggi pengenceran protein hemaglutinin OMP Mycobacterium tuberculosis strain H37Rv 38 kDa yang ditambahkan makin tinggi indeks adhesinya. Dengan demikian terbukti protein hemaglutinin OMP Mycobacterium tuberculosis strain H37Rv 38 kDa berperan sebagai protein adhesin.
SUMMARY
The Effect Outer Membrane Protein (OMP) Mycobacterium tuberculosis at expression T cell CD4 and Interferon γ Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis bacteria. It is estimated that one-third of the world's population has been infected with Mycobacterium tuberculosis, 5-10% will develop into clinical manifestations of tuberculosis. Prevention of tuberculosis has so far been done with the BCG vaccine (Bacille Calmette-Guerine). Apparently BCG immunization has failed to provide protection against tuberculosis infection to adults. Therefore, the development of a vaccine against tuberculosis, is still requires considering that the protective effect of BCG given on adult pulmonary tuberculosis incidence varies around 0-80%. The development of vaccines based on bacterial surface material called adhesin as a target for vaccine development is beginning to be widely studied. The process of adhesion is one of the virulence properties of bacteria playing an important role for the occurrence of colonization until the onset of infection. Mycobacterium tuberculosis has outer membrane protein that are part of bacteria that play a role in adhesion to host epithelial cells and OMP can induce an immune response, on the basis of this mechanism it can be used as a vaccine candidate. The significance of this research is that theoretically it is expected that the findings of this study can be a basis for the development of Science and Technology, regarding the pili adhesin protein of Mycobacterium tuberculosis in inducing protective cellular immune responses. The practical benefit is that it is expected that the findings of this study can be further persued and used as vaccine development materials that can be used to help reduce the occurrence of tuberculosis infection. Method of research covered 2 phases: isolation of proteins of Mycobacterium tuberculosis strain H37Rv OMP and SDS-PAGE (Phase-1); hemagglutination and OMP adhesion protein testing of Mycobacterium tuberculosis strain H37Rv (Phase-2). ; pili adhesin protein immunogenicity testing of Mycobacterium tuberculosis H37Rv strain in inducing cellular immune response (Phase-3); research to prove the induction of adhesin protein of 11kDa Mycobacterium tuberculosis strain H37Rv in inhibiting bacterial colonization in mice lung tissues. The phase 1 research results showed that the Outer membrane protein of 38 kDa Mycobacterium tuberculosis strain H37Rv was able to agglutinate erythrocytes in dilution titer 1/32, hemagglutination testing was aimed to obtain hemagglutinin protein, because in general the hemagglutinin protein is also a protein of adhesin. Having obtained the hemagglutinin protein of 38 kDa Mycobacterium tuberculosis strain H37Rv adhesion test is then performed to demonstrate that the protein is an adhesin protein. In this adhesion testing using 6 dilution treatments of adhesin protein of 11kDa (0,1,1 / 2.1 / 4.1 / 8.1 / 16) in mice enterocytes. The result of ANOVA showed 38 kDa protein of hemagglutinin OMP at various dilutions had highly significant effect on adhesion index (p <0.05), where the higher the dilution of the hemagglutinin protein of 38 kDa Mycobacterium tuberculosis strain H37Rv OMP added to enterocytes, the higher adhesion index gained. Thus it proving that the hemagglutinin protein of 38 kDa Mycobacterium tuberculosis strain H37Rv OMP is an adhesin protein.
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