[Expression and biological function analysis of chicken aminopeptidase N]

Area of Science:

• Molecular Biology
• Biochemistry
• Virology

Background:

• Aminopeptidase N (APN) plays crucial roles in various biological processes.
• Understanding the function of chicken APN (chAPN) is important for poultry health.
• Expression of chAPN in a prokaryotic system can facilitate functional studies.

Purpose of the Study:

• To clone and express the chicken aminopeptidase N (chAPN) gene in Escherichia coli (E. coli).
• To analyze the biological function of the expressed chAPN.
• To investigate the potential antiviral activity of chAPN.

Main Methods:

• RT-PCR amplification of chAPN gene from chicken embryo kidney cells.
• Cloning into prokaryotic expression vector pCOLD-TF and transformation into E. coli BL21(DE3).
• Optimization of expression conditions (induction time, concentration) and purification using Ni-NTA His Bind Resin.
• Identification via SDS-PAGE and Western blotting.
• Functional analysis using Leu-PNA substrate and ELISA for viral binding.

Main Results:

• Successfully cloned and expressed soluble recombinant chAPN in E. coli.
• Purified chAPN was confirmed by SDS-PAGE and Western blotting.
• The expressed chAPN exhibited dose-dependent binding to infectious bronchitis virus (IBV).

Conclusions:

• The chAPN gene was successfully cloned and expressed in E. coli.
• The recombinant chAPN is soluble and shows potential for antiviral applications.
• This study provides a foundation for further investigation into chAPN's enzymatic and antiviral properties.