2.8 Western blot analysis
The perR gene was amplified by PCR with primers perR -F (CGGGATCC ATGGCTGCACAT -GAATTAAA) and perR -R (CCCTCGAG GTGGTTCTCTTTTTTGGAAC), subcloned into the pET28a vector, then the recombinant plasmid was transformed into Escherichia coli BL21. The expression of PerR was induced by IPTG (2 mM), purified by Ni-NAT column (Qiagen, German), then used for intraperitoneal immunization of mice at 50 μg/mouse for 3 dosages after emulsification with Freund’s adjuvant (Sigma, USA) [38]. After one week, the sera were collected for the following Western blot assay.
The cell pellets were prepared as the method described above for isolating mRNA. The cell pellets were lyzed by ultrasonification, then the total proteins concentration of each sample was adjusted to 10 μg/μL. After being mixed with sample buffer, 10 μL of each protein sample was separated by 15% SDS-PAGE gel, transferred onto the polyvinylidene fluoride (PVDF) membrane (Millipore, USA), then incubated with the antisera at a dilution of 400 x for 1 h. After extensive wash, the membrane was incubated with goat anti-mouse IgG labeled with Horseradish Peroxidase (HRP) (Boster Biological Technology, China) at a dilution of 5,000 x for 30 min. After that, the membrane was washed, reacted with BeyoECL Star (Beyotime Biotechnology, China), then scanned by Monad QuickChemi 5100 (Zhuhai, China) [38].