Khanh Tan Nguyen2,5, Hung Manh Tran2,5,Anh Tuan Trieu3,5, Van Thi Huynh Nguyen2,5, Hoang M.Nguyen4, Phu Tran Vinh Pham1,5

*Address(es):Phu Tran Vinh Pham, Ph.D.

• 1 Faculty of Medicine, Dong A University, 33 Xo Viet Nghe Tinh street, Da Nang 550000, Vietnam, phone number:(+84)-978-595-013.
• 2 Scientific Management Department, Dong A University, 33 Xo Viet Nghe Tinh street, Da Nang550000, Vietnam.
• 3 Faculty of Food Technology, Dong A University, 33 Xo Viet Nghe Tinh street, Da Nang 550000, Vietnam.
• 4 Faculty of Chemical Engineering, the University of Da Nang-University of Science and Technology, Da Nang 550000, Vietnam.
• 5 UDA-Institute of Applied Life Sciences (UDA-IALS), Dong AUniversity, 33 Xo Viet Nghe Tinh street, Da Nang 550000, Vietnam

Polymerase of influenza virus is made upof three subunits PB1, PB2, and PA, which are involved in viral genometranscription and replication. Purification of sufficient amounts of viral polymerase is essential to understand the catalytic function of viral polymerase. In this study, we generatedaviral polymerase expression system in human embryonic kidney 293T cells (293T cells). The cDNAs for RNA segments1, 2, and 3, which encode for PB2, PB1, and PA proteins, respectively, were integrated into the mammalian expression plasmids pCAGGS to simultaneously express all viral polymerase proteins in 293T cells. We purified the recombinant polymerases of human influenza virus A/PR/8/34 (H1N1) (PR8) and avian influenza virus A/Turkey/England/1969 (H3N2) (TE) using anti-FLAG M2 affinity resin. After confirming trimeric complexes, enzymatic properties of recombinant polymerases were characterized, including model viral RNA binding, in vitro transcription assays, and cap-snatching activity upon addition of cap1-70 mer vRNA as substrate. Taken together we conclude that 293T cells are a suitable expression system for sufficient amountisolation of functional recombinant influenza viral polymerases with 95% of purity.

Keywords:Cap-snatching, 293T cells, transcription, purification, viral polymerase

Tham khảo thêm thông tin tại: PURIFICATION AND ANALYSIS CATALYTIC FUNCTIONS OF RECOMBINANT INFLUENZA VIRAL POLYMERASES | Journal of microbiology, biotechnology and food sciences (jmbfs.org)