KAJIAN PENANDA GENETIK DAN KLONING GEN SHIGA LIKE TOXIN-2 Escherichia coli O157:H7

DRH. I WAYAN SUARDANA, M.SI., Prof. Dr. drh. Wayan Tunas Artama

2012 | Disertasi | S3 Bioteknologi

Ternak sapi dikenal sebagai reservoar utama dari serotipe Escherichia coli O157:H7. Bakteri ini bersifat zoonosis yang dicirikan dengan marker virulensi intimin yang disandi oleh gen eae sebagai protein yang berperanan dalam penempelan bakteri pada hospes, serta toksin Stx yang yang disandi oleh gen stx yang dikenal sebagai Shiga-like toksin (Stx). Toksin Stx khususnya dari jenis Stx2 diketahui terkait erat dengan munculnya kejadian Hemolytic Uremic Syndrome (HUS) yang berlanjut menjadi gagal ginjal pada manusia. Ekspresi gen stx2 diketahui terkait dengan variasi dari sekuen gen q, sekuen promoter, dan Ribosome Binding Site (RBS) yang terletak pada daerah upstream dari gen. Penelitian diawali dengan isolasi dan identifikasi E. coli yang berasal dari 344 sampel dengan rincian 80 sampel asal feses sapi, 78 sampel asal daging sapi, 80 sampel asal feses ayam, 30 sampel asal feses manusia sehat serta 76 sampel asal feses manusia sakit yang diambil dari penderita gagal ginjal yang mengalami hemodialisis. Analisis genomik dari seluruh E. coli O157:H7 hasil isolasi dan identifikasi dilakukan dengan PCR-RAPD, dilanjutkan dengan analisis molekular terhadap marker virulensi, uji ekspresi toksin Stx2 serta uji daya toksisitasnnya pada kultur sel Vero. Tahap akhir dari penelitian dilakukan kloning gen stx2 pada vektor kloning dan vektor ekspresi yang diakhiri dengan uji potensi protein rekombinan sebagai agen imunodiagnostik dan bahan dasar produksi vaksin. Hasil penelitian menunjukkan prevalensi infeksi E. coli O157:H7 pada seluruh sampel adalah 6,4%. Analisis genomik dengan metode PCR-RAPD menunjukkan seluruh isolat memiliki nilai similaritas antara 75,1% sampai 96,6%. Hasil analisis RAPD juga menemukan adanya 2 pita/band pada posisi 1721 dan 700 bp dapat digunakan sebagai petunjuk untuk membedakan antara isolat E. coli O157:H7 yang berasal dari kasus sakit dan sehat. Analisis terhadap marker virulensi menunjukkan 2 isolat yaitu KL-48(2) asal manusia dan SM25(1) asal sapi positif membawa gen eae, stx1 dan stx2 seperti halnya kontrol ATCC 43894. Hasil penelitian juga menunjukkan ke-2 isolat diketahui memiliki kesamaan susunan sekuen gen q, sekuen promoter dan Ribosome Binding Site (RBS) dengan kontrol ATCC 43894, namun ke-2 isolat diketahui memiliki perbedaan dalam hal ekspresi toksin Stx2. Disamping itu, melalui teknik rekayasa genetik, gen stx2 dapat diklon ke dalam vektor kloning dan vektor ekspresi, serta toksin rekombinan hasil rekayasa genetik diketahui berpotensi digunakan sebagai agen imunodiagnostik dan bahan dasar produksi vaksin.

Cattle are considered as a main reservoir of the Escherichia coli O157:H7 agent. These bacteria are commonly known as zoonotic agents that are characterized by virulence markers i.e. intimin that is encoded by eae gene as a protein to attach the bacteria to the host cell, and Stx toxin that is encoded by stx gene as familiarly as Shiga like toxin. Stx toxin especially Stx2 is closely associated to the Hemolytic Uremic Syndrome (HUS) which continues to be renal failure in human. The expression of stx2 gene is known closely related to variation of q gene sequences, promoter sequences, and Ribosome Binding Site (RBS) located in the upstream of the gene. The study began by isolating and identifying E. coli originated from 344 samples i.e. 80 samples of cattle feces, 78 samples of beef, 80 samples of chicken feces, 30 samples of healthy human feces, and 76 samples of unhealthy human feces taken from patients with renal failure. Genomic analysis of all E. coli O157:H7 isolates was performed by PCR-RAPD, continued by molecular analyzing of virulence markers, Stx2 toxin expression testing, and the toxicity potency test was carried out by culturing of Stx2 toxin on Vero cell line. The cloning of stx2 gene to cloning and expression vectors was performed at the end of the study before the potency test of recombinant protein as a immunodiagnostic agent and vaccine candidate was conducted. The results show that the prevalence of E. coli O157:H7 infections in all samples is 6.4%. Genomic analysis of all E. coli O157:H7 isolates by PCR-RAPD method shows that the isolates are known to have similar value ranging from 75.1 to 96.6% and it also shows two specific bands, 1721 and 700 bp, known as a marker to indentify the E. coli O157:H7 isolates originated from healthy and unhealthy cases. The analysis of virulence marker shows that KL48(2) isolate originated from human and SM25(1) isolate originated from cattle are known bearing eae, stx1 and stx2 genes as equal as E. coli ATCC 43894 control. Although the study of the variation of q gene sequences, promoter sequences, and Ribosome Binding Site (RBS) of KL-48(2) and SM25(1) indicates similar sequances, they have different level expression of Stx2. In addition, through the genetic engineering, the stx2 gene can be cloned in cloning vector and expression vector, and recombinant protein as a result of genetic engineering is known to have a good potency to use as an immunodiagnostic agent and vaccine candidate.

Kata Kunci : E. coli O157:H7, gen stx2 dan sekuen upstream