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Updated: Mar 18, 2026
High-throughput Detection Method for Influenza Virus
Published on: February 4, 2012
[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 (PB 1, PB2 and PA) which plays an important role in the transcription and replication of the influenza A virus. Polymerase basic protein 1 (PB 1) 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 BL2 1. The expression of His tagged PB 1 protein was induced by IPTG, and His-PB 1 proteins were purified by Ni-NTA resin. For preparation of PB 1 protein antiserum, rabbits were immunized with His-PB 1 fusion protein 3 times. Then the titer of PB 1 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 PB 1 protein from WSN virus infected or pCMV FLAG-PB 1 transfected cells. Meanwhile, PB 1 antibody can also recognize specifically other subtype strains of influenza A virus such as H9N2 and H3N2. PB 1 polyclonal antibody we generated will be a useful tool to study the biological function of PB1.
Insights
Researchers developed a high-quality Polymerase Basic protein 1 (PB 1) antibody for Influenza A virus studies. This new PB 1 antibody specifically detects the virus in infected cells and across multiple strains, aiding future research.
Area of Science:
- Virology
- Immunology
- Molecular Biology
Background:
- Influenza A virus, an Orthomyxoviridae family member, utilizes 8 RNA segments encoding 16 proteins.
- The viral polymerase complex, comprising PB 1, PB 2, and PA proteins, is crucial for viral transcription and replication.
- Polymerase Basic protein 1 (PB 1) is a key component of this essential viral polymerase complex.
Purpose of the Study:
- To generate a high-quality antibody targeting the Polymerase Basic protein 1 (PB 1) of Influenza A virus.
- To facilitate further investigation into the biological functions of the PB 1 protein.
Main Methods:
- Amplification and cloning of the PB 1 conserved region (nt1648-2265) into a pET-30a vector for expression in Escherichia coli.
- Purification of His-tagged PB 1 protein using Ni-NTA resin following IPTG induction.
- Immunization of rabbits with purified His-PB 1 fusion protein, followed by antibody titer measurement via indirect ELISA, purification, and immunoblotting analysis.
Main Results:
- Successfully expressed and purified His-tagged PB 1 protein.
- Generated a PB 1 polyclonal antibody that specifically recognizes PB 1 protein in WSN virus-infected cells and cells transfected with pCMV FLAG-PB 1.
- Demonstrated that the antibody cross-reacts with other Influenza A virus subtypes, including H9N2 and H3N2.
Conclusions:
- A specific and effective PB 1 polyclonal antibody has been successfully generated.
- This antibody serves as a valuable tool for studying the role and function of PB 1 in Influenza A virus.
- The antibody's ability to detect multiple strains enhances its utility in influenza research.
