Tesis

Pengembangan seed vaksin tuberkulosis: Konstruksi vaksin DNA resuscitation promoting factor B (rpfB) dan ekspresinya secara in vitro dan in vivo. = Development of tuberculosis vaccine seed: Construction of resuscitation promoting factor B (rpfB) DNA vaccine and its in vitro and in vivo expression.

Tuberkulosis (TB) merupakan penyakit infeksi kronis yang disebabkan oleh Mycobacterium tuberculosis dengan angka kematian yang tinggi diseluruh dunia. TB dapat dicegah salah satunya dengan vaksinasi. Vaksin yang tersedia saat ini adalah vaksin BCG yang berasal dari Mycobacterium bovis. Vaksin BCG memiliki beberapa kelemahan yakni efikasi yang berbeda pada setiap individu, tidak memberikan perlindungan TB paru pada individu dewasa serta reaktivasi subsekuen. Protein RpfB yang dikode oleh gen rpfB Mycobacterium tuberculosis memiliki karakteristik biologi dan imunologi tertinggi diantara protein lain dalam famili Rpf. Diduga imunitas host terhadap protein RpfB berperan dalam respon imun manusia terhadap Mycobacterium tuberculosis. Pada penelitian ini gen rpfB Mycobacterium tuberculosis strain Beijing diamplifikasi dengan PCR kemudian diklon kedalam plasmid pcDNA3.1. Plasmid rekombinan kemudian digunakan untuk imunisasi mencit Balb/C jantan dan transfeksi pada sel Chinese hamster ovary (CHO-K1). Pengklonan berhasil mendapatkan 2 klon plasmid rekombinan pcDNA3.1-rpfB yang membawa gen sisipan rpfB dan telah terkonfirmasi dengan PCR koloni, restriksi plasmid dan sekuensing. Hasil western blot menunjukkan bahwa gen rpfB berhasil menginduksi respon imun humoral mencit dengan adanya pita spesifik pada kisaran 66 kDa, sedangkan transfeksi pada sel CHO-K1 memperlihatkan protein RpfB berhasil terekspresi pada sel mamalia berdasarkan uji imunostaining.
Kata kunci: gen rpfB, Mycobacterium tuberculosis, sel CHO-K1, vaksin DNA


Tuberculosis (TB) is a chronic infection caused by Mycobacterium tuberculosis with a high mortality rate worldwide. TB can be prevented by vaccination. Current vaccine available is a BCG vaccine developed from Mycobacterium bovis. BCG vaccine has several weaknesses, different efficacy in each individual, BCG does not provide protection for lung TB in adult and not prevent subsequent reactivation. The RpfB protein encoded by the rpfB gene of Mycobacterium tuberculosis has the highest biological and immunological characteristics among other proteins in the Rpfs family. Immunity of the host to protein RpfB is suspected to play a role in the human immune response to Mycobacterium tuberculosis. In this study, the rpfB gene of Mycobacterium tuberculosis Beijing strain was amplified by PCR and cloned into a pcDNA3.1 plasmid. The recombinant plasmid was used for immunization of male Balb/C mice and transfection in Chinese hamster ovary (CHO-K1) cells. Cloning succesfully obtained 2 recombinant plasmid pcDNA3.1-rpfB carrying the rpfB inserted gene and confirmed with PCR colonies, plasmid restriction and sequencing. The western blot result indicated that rpfB gene succesfully induced the humoral immune responseof the mice in the presence of a specific band in range of 66 kDa, while transfection in CHO-K1 cells showed a protein of RpfB succesfully expressed in mammalian cells based on immunostaining assays.
Key words: CHO-KI cells, DNA vaccine, Mycobacterium tuberculosis, rpfB gene