Konstruksi Plasmid Pengekspresi Antigen Rekombinan HCV Berbasis Multiepitop untuk Deteksi Antibodi Anti-HCV
Abstract
Abstract
Hepatitis C virus (HCV) infection can cause chronic liver disease that develops into cirrhosis and liver cancer. It is estimated that are more than 170 million of th world’s population suffering from HCV. Accurate diagnosis is needed to provide appropriate early treatmen, including preventing further transmission of the virus. The purpose of this study was to construct plasmid expression of recombinant antigen for detection of anti-HCV antibodies. The antigen coding gene is designed so that is composed to epitopes that are immunodominant, sustainable and and represent HCV subtypes circulating in Indonesia and globally. Furthermore, the gene was made by synthetic DNA techniques by DNA synthesis service providers and accepted by the researchers in the form of blinding on the PUC57 plasmid to pQE80L plasmid with BamHI and HindIII cloning sites. Subcloned recombinant plasmids were then propagated on Top10 Escherichia coli cells and verified by PCR colony tecnique, restriction, and sequencing analysis. HCV recombinant antigen coding gene is 1200 bp. Cloning of these gene on the PUC57 vector produced a plasmid pUC57-HCV_ME (3910 bp) and subcloned in the pQE80L vector producing pQE80L-HCV_ME plasmid (5909bp). Based on verification results of pQE80L-HCV_ME plasmid the expression of recombinant antigen for detection of anti-HCV antibodies has been successfully constructed.
Abstrak
Infeksi hepatitis C virus (HCV) dapat menyebabkan penyakit hati kronis yang berkembang menjadi sirosis dan kanker hati. Diperkirakan terdapat lebih dari 170 juta penduduk dunia menderita HCV. Diagnosis yang akurat diperlukan untuk memberikan penanganan tepat secara dini, termasuk mencegah penularan virus tersebut lebih lanjut. Tujuan penelitian ini adalah mengonstruksi plasmid pengekspresi antigen rekombinan untuk deteksi antibodi anti-HCV. Gen pengode antigen tersebut dirancang sedemikian rupa sehingga tersusun atas epitop yang bersifat imunodominan, lestari, serta mewakili subtipe HCV yang bersirkulasi di Indonesia maupun global. Selanjutnya gen tersebut dibuat dengan teknik DNA sintetik oleh perusahaan penyedia jasa sintesis DNA dan diterima oleh peneliti dalam bentuk terklona pada plasmid pUC57. Untuk ekspresi pada sel Escherichia coli, gen penyandi antigen rekombinan disubklona dari plasmid pUC57 ke plasmid pQE80L dengan situs pengklonaan BamHI dan HindIII. Plasmid rekombinan hasil subklona kemudian dipropagasi pada sel Escherichia coli Top10 dan diverifikasi dengan teknik PCR koloni, analisis dengan enzim restriksi dan sekuensing. Gen penyandi antigen rekombinan HCV berbasis epitop multipel (HCV_ME) berukuran 1200 pb. Pengklonaan gen tersebut pada vektor pUC57 menghasilkan plasmid pUC57-HCV_ME (3910 pb) dan subklona pada vektor pQE80L menghasilkan plasmid pQE80L-HCV_ME (5909 pb). Berdasarkan pada hasil verifikasi plasmid pQE80L-HCV_ME pengekspresi antigen rekombinan untuk deteksi antibodi anti-HCV telah berhasil dikonstruksi.
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