Highly pathogenic avian influenza A virus H5N1 non-structural protein 1 is associated with apoptotic activation of the intrinsic mitochondrial pathway

Bian, Qian; Lu, Jing; Zhang, Li; Chi, Ying; Li, Yan; Guo, Hongxiong

Construction of an NS1-expressing plasmid. Highly pathogenic avian influenza A/Jiangsu/1/2007 (H5N1) viral RNA was extracted from supernatants of infected cell cultures for use as a polymerase chain reaction (PCR) template for amplifying the NS1 gene. Total RNA was extracted from cell lysate using the QIAamp Viral RNA Mini kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The full-length NS1 gene was amplified using the SuperScript III One-Step Reverse Transcription-PCR (RT-PCR) system with Platinum Taq High-Fidelity Polymerase (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) from H5N1 virus cDNA. The sense and antisense primers used for NS1 (EU434690) were 5'-GTG CTC GAG ATG GAT TCC AAC ACT GTG TCA-3' and 5'-CAC GGT ACC TCA AAC TTC TGA CTC AAT TGT-3', respectively. The PCR conditions were 95ËšC for 15 min, followed by 34 cycles of 94ËšC for 30 sec, 58ËšC for 30 sec, and 72ËšC for 30 sec. The cloning insert was ligated into the pMD18-T vector (D101A; Takara Biotechnology Co., Ltd., Dalian, China) by quick ligase (M2200S; NEB Beijing Ltd., Beijing, China) incubating for 30 min at room temperature. pMD18-T-NS1 was subcloned into the expression plasmid pXJ40-hemagglutinin (HA) (Invitrogen; Thermo Fisher Scientific, Inc.) using XhoI (D1094A) and KpnI (D1068A) (both from Takara Biotechnology Co., Ltd., Dalian, China) sites to produce the recombinant HA-tagged construct, pXJ40-HA-NS1. The construction of plasmid pXJ40-HA-NS1 followed standard cloning procedures. Competent Escherichia coli TOP10 cells (Invitrogen; Thermo Fisher Scientific, Inc.) were transformed using pXJ40-HA-NS1 plasmids, and the plasmids were amplified and purified using a high-purity plasmid purification kit (Invitrogen; Thermo Fischer Scientific, Inc.). Clones were then screened by restriction enzyme digestion and sequence analysis using the version 3.1 BigDye Terminator ready reaction cycle sequencing kit (Applied Biosystems; Thermo Fischer Scientific, Inc.) according to the manufacturer's instructions.