Preparation and detection of anti-influenza A virus polymerase basic protein 1 polyclonal antibody

Abstract:

Influenza A virus is an enveloped virus that belongs to the Orthomyxoviridae family. It has 8 negative RNA segments that encode 16 viral proteins. The viral polymerase consists of 3 proteins (PB1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB1) is a critical member of viral polymerase complex. In order to further study the function of PB1, we need to prepare the PB1 antibody with good quality. Therefore, we amplified PB1 conserved region (nt1648–2265) by PCR and cloned it into pET-30a vector, and transformed into Escherichia coli BL21. The expression of His tagged PB1 protein was induced by IPTG, and His-PB1 proteins were purified by Ni-NTA resin. For preparation of PB1 protein antiserum, rabbits were immunized with His-PB1 fusion protein 3 times. Then the titer of PB1 polyclonal antibody was measured by indirect ELISA. The antibody was purified by membrane affinity purification and subjected to immunoblotting analysis. Data showed that PB1 antibody can recognize PB1 protein from WSN virus infected or pCMV FLAG-PB1 transfected cells. Meanwhile, PB1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.