Sains Malaysiana 41(4)(2012): 423-430

Sistem Minigenom Virus Penyakit Newcastle (NDV) AF2240 Sebagai Sistem

Pengekspresan Gen Sel Mamalia

(Minigenome System for Newcastle Disease Virus (NDV) AF2240

as a Gene Expression System for Mammalian Cells)

Shahrul Hisham Zainal Ariffin*, Roslina Shamsudin,  Nurul Atikah

Ahmad & Zulkiflie Zamrod

Pusat Pengajian Biosains dan Bioteknologi, Fakulti Sains dan Teknologi

Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor D.E., Malaysia

Diserahkan: 7 Jun 2011 / Diterima: 19 September 2011

ABSTRAK

Sistem minigenom telah digunakan untuk mengkaji replikasi dan transkripsi virus RNA tidak bersegmen. Objektif kajian ini adalah untuk membina sistem minigenom bagi virus NDV strain tempatan, AF2240 serta bagi mengkaji mekanisme transkripsi dan replikasi virus ini. Bagi tujuan ini lima plasmid digunakan iaitu pMGNDV, pCITENP, pCITEP, pTriEX-T7, dan pGEML. Kesemua plasmid diekstrak secara berskala besar dan dimendakkan menggunakan polietilina glikol. Hasil

ekstrak ini digunakan untuk transfeksi ke dalam sel. Translasi in vitro dilakukan dengan menggunakan pCITENP, pCITEP dan pTriEX-T7 untuk memastikan kesemua konstruk ini berfungsi. Hasil pemblotan western menunjukkan protein bersaiz~100 kDa (T7), ~53 kDa (NP), ~53 dan 55 kDa (P) berjaya diekspreskan. Protein CAT diperoleh apabila plasmid yang mengekodkan minigenom NDV ditransfeksi bersama plasmid yang mengekodkan protein nukleokapsid (NP), fosfoprotein (P)

dan subunit besar polimerase (L) ke dalam sel BHK-21. Dianggarkan 55 pg protein CAT berjaya diperoleh menggunakan kit CAT ELISA. Hasil pemblotan western turut menunjukkan protein CAT bersaiz 25 kDa dihasilkan. Kesimpulannnya, system minigenom ini berupaya untuk berfungsi dan mampu mengekspreskan gen asing di dalam sel mamalia BHK-21.

Kata kunci: Pengekspresan gen; sistem minigenom; sistem pengekspresan sel mamalia; transfeksi; virus penyakit Newcastle

ABSTRACT

The minigenome system has been used as a model for studying transcription and replication for nonsegmented RNA viruses. The objective of this study was to develop a minigenome system for NDV local strain AF2240 and also to study the mechanism of replication and transcription of this virus. For this purpose, five recombinant plasmids; pMGNDV, pCITENP, pCITEP, pTriEX-T7 and pGEML were used. Transfection of all plasmids was carried out following large scale extraction and polyethylene glycol precipitation of plasmids. In vitro translational was performed by using pCITENP, pCITEP, and pTriEX-T7 to ensure the constructs are functional. Western blot analysis showed that protein with approximate molecular weights of 100 kDa (T7), 53 kDa (NP), 53 and 55 kDa (P) was successfully detected. CAT protein was detected when plasmid encoding the NDV minigenome was cotransfected into BHK-21 cells with plasmid encoding nucleocapsid (NP), phosphoprotein (P) and large polymerase subunit (L) protein. Approximately, 55 pg CAT protein was successfully quantified by CAT ELISA. Western blot analysis also showed that the CAT protein with the size of 25 kDa was successfully obtained. In conclusion, this study showed that the system was functional and able to express foreign gene in mammalian cells BHK-21.

Keywords: Gene expression; mammalian cell expression system; minigenome system; Newcastle disease virus; Transfection

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*Pengarang untuk surat-menyurat; email: hisham@cgat.ukm.my