SKRIPSI Jurusan Biologi - Fakultas MIPA UM, 2018

Ukuran Huruf:  Kecil  Sedang  Besar

IDENTIFIKASI PROFIL PROTEIN MEMBRAN LUAR Pseudomonas fluorescens SEBAGAI KANDIDAT VAKSIN NOSOKOMIAL

Damara Haris Ang Della

Abstrak


ABSTRAK

 

Della, Damara Haris Ang. 2018. Identifikasi Profil Protein Membran Luar Pseudomonas fluorescens sebagai Kandidat Vaksin Nosokomial. Skripsi, Jurusan Biologi, Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Negeri Malang. Pembimbing: (I) Dr. Umie Lestari, M. Si, (II) Dr. Endang Suarsini, M. Ked.

 

Kata Kunci: protein membran luar, Pseudomonas fluorescens, aktivitas fosforilasi, pewarnaan glikoprotein, kandidat vaksin nosokomial

Bakteri Pseudomonas fluorescens adalah bakteri Gram negatif yang dapat menyebabkan infeksi saluran kemih. Infeksi saluran kemih merupakan jenis infeksi nosokomial yang sering terjadi. Infeksi nosokomial adalah infeksi yang terjadi pada pasien selamaproses perawatan di rumah sakit atau fasilitas kesehatan lainnya yang tidak menunjukkan gejala infeksi saat masuk rumah sakit. P. fluorescens dilaporkan telah mengalami resistensi terhadap beberapa macam antibiotik. Vaksin menjadi alternatif terbaik untuk melawan resistensi bakteri dan beberapa jenis vaksin terhadap P. fluorecens telah dikembangkan seperti whole cell, protein membran luar, lipopolisakarida, dan biofilm. Tujuan penelitian ini untuk mengidentifikasi profil protein membran luar bakteri P. fluorescens dan menentukan potensi protein membran luar bakteri P. fluorescens melalui uji fosforilasi dan uji pewarnaan glikoprotein sehingga dapat dijadikan sebagai kandidat vaksin.

Penelitian ini merupakan penelitan deskriptif eksploratif dengan pendekatan eksperimen laboratorik yang dilakukan dengan analisis pengukuran uji fosforilasi dan uji pewarnaan glikoprotein protein membran luar bakteri P. fluorescens. Obyek penelitian adalah protein membran luar bakteri P. fluorescensyang berasal dari Laboratorium Mikrobiologi Jurusan Biologi FMIPA Universitas Negeri Malang. Penelitian ini bertempat diLaboratorium Biologi Molekular Gedung O5 Biologi Ruang 314, Laboratorium Mikrobiologi Gedung O5 Ruang 310 Fakultas Matematika dan Ilmu Pengetahuan Alam, Universitas Negeri Malang, dan Laboratorium Biomedik Universitas Brawijaya Malang pada bulan Desember 2017 hingga bulan April 2018. Tahapan penelitian meliputi beberapa tahap, yakni (1) kultur dan pertumbuhan bakteri, (2) pemisahan membran luar bakteri dengan pemotongan pili, (3) isolasi protein membran luar bakteri P. fluorescens, (4) pengukuran kadar protein membran luar bakteri P. fluorescens, (5) elektroforesis SDS-PAGE crude dan penentuan berat molekul protein membran luar bakteri P. fluorescens, (6) elektroelusi protein membran luar bakteri P. fluorescens, (7) pengukuran aktivitas fosforilasi protein membran luar bakteri P. fluorescens, dan (8) pewarnaan glikoprotein protein membran luar bakteri P. fluorescens yang dilakukan secara manual.

Hasil penelitian menunjukkan bahwa crude protein membran luar bakteri P. fluorescens hasil isolasi menggunakan NOG yang dilanjutkan dengan SDS-PAGE membentuk 6 pita (band) protein dengan berat molekul 69 kDa, 55 kDa, 38 kDa, 34 kDa, 10 kDa, dan 7 kDa. Pengukuran aktivitas fosforilasi protein membran luar bakteri P. fluorescens 34 kDa sebesar 9,239x10-2 µmol/mL.menit, sedangkan aktivitas fosforilasi protein membran luar bakteri P. fluorescens 55 kDa sebesar 15,384x10-2 µmol/mL.menit yang keduanya termasuk dalam kategori relatif rendah yang berarti bahwa kedua protein tidak mengganggu aktivitas sel target. Hasil uji pewarnaan glikoprotein menunjukkan hasil negatif pada protein membran luar bakteri P. fluorescens 34 kDa dan 55 kDa. Berdasarkan studi literasi diketahui bahwa reseptor bakteri tidak hanya berupa glikoprotein, namun dapat berupa protein. Dengan demikian, protein membran luar bakteri P. fluorescens 34 kDa dan 55 kDa dapat digunakan sebagai kandidat vaksin nosokomial.

 

SUMMARY

 

Della, Damara Haris Ang. 2018. Identification of Outer Membrane Protein Pseudomonasfluorescensas Vaccine Nosocomial Candidate. Thesis, Department of Biology, Faculty of Mathematics and Natural Sciences, Universitas Negeri Malang. Advisor: (I) Dr. Umie Lestari, M. Si, (II) Dr. Endang Suarsini, M. Ked.

 

Keywords:outer membrane protein, Pseudomonas fluorescens, phosphorylation activity, glycoprotein staining, vaccine nosocomial candidate

Pseudomonas fluorescens is a Gram-negative bacteria that can cause urinary tract infection. Urinary tract infection is a common type of nosocomial infection. Nosocomial infection is an infection that occurs in patients during the treatment process at a hospital or other health facility that does not show symptoms of infection when admitted to hospital. P. fluorescens has been reported to be resistant to determined antibiotics. Vaccines are the best alternative to resist bacterial resistance and some types of vaccines against P. fluorecens have been developed such as whole cells, outer membrane proteins, lipopolysaccharides, and biofilms. The aim of this study is to identify the profile of outer membrane protein of P. fluorescens bacteria and to determine the potential of outer membrane protein of P. fluorescens bacteria through phosphorylation test and glycoprotein staining test so that it can be used as vaccine candidate.

This research is aexplorative descriptive research with laboratory experimental approach which is done by analysis on the measurement of phosphorylation and glycoprotein staining test on outer membrane protein of P. fluorescens bacteria. The research object is the outer membrane protein of bacteria P. fluorescens taken from Microbiology Laboratory Department of Biology FMIPA UniversitasNegeri Malang. This research took place in Molecular Biology Laboratory Building O5 Room 314, Laboratory of Microbiology Building O5 Room 310 Faculty of Mathematics and Natural Sciences, UniversitasNegeri Malang, and Biomedical Laboratory UniversitasBrawijaya Malang on December 2017 until April 2018. This research was conducted through several stages, (1) bacterial culture and growth, (2) separation of outer membrane of bacteria by pili cutting, (3) isolation of outer membrane protein of P. fluorescens bacteria, (4) measurement of protein content in outer membrane of P. fluorescens bacteria, (5) electrophoresis SDS-PAGE crude and determining the molecular weight of outer membrane protein of P. fluorescens, (6) electro elution of outer membrane protein of bacteria P. fluorescens, (7) measurement of phosphorylation activity of outer membrane protein P. fluorescens, and (8) glycoprotein staining of the outer membrane protein of P. fluorescens which was done manually.

The findings showed that the crude of outer membrane protein of P. fluorescens as a result of isolation using NOG followed by SDS-PAGE formed 6 bands of protein with molecular weight of 69 kDa, 55 kDa, 38 kDa, 34 kDa, 10 kDa, and 7 kDa. The measurement of phosphorylation activity of outer membrane protein of P. fluorescens 34 kDa bacteria was 9,239x10-2 μmol / mL.min, while phosphorylation activity of outer membrane protein of P. fluorescens55 kDa bacteria was 15,384x10-2 μmol / mL.min which both fall into the category of relatively low which means that the two proteins do not interfere with the activity of the target cell. The results of the glycoprotein staining test showed negative results on the outer membrane protein of P. fluorescens 34 kDa and 55 kDa. Based on literacy studies it is known that bacterial receptors not only in the form glycoproteins, but also proteins. Thus, the outer membrane protein of P. fluorescens 34 kDa and 55 kDa can be used as a vaccine nosocomial candidate.