Cloning and expression of influenza A virus recombinant Noraminidase antigen for use in diagnostic tests

Cloning and expression of influenza A virus recombinant Noraminidase antigen for use in diagnostic tests
Zahra Latif ,^1,* Farida Behzadian ,² Zahra Barghi ,³ Maryam Saheh ,⁴ Behrokh Farahmand,⁵
1. Research Center of Bioscience and Biotechnology, Malek- Ashtar University of Technology, Tehran, Iran and Department of Influenza Virus Research and Other Respiratory Viruses, Pasteur Institute of Iran, Tehran, Irann
2. Research Center of Bioscience and Biotechnology, Malek- Ashtar University of Technology, Tehran, Iran
3. Department of Influenza Virus Research and Other Respiratory Viruses, Pasteur Institute of Iran, Tehran, Iran
4. Department of Influenza Virus Research and Other Respiratory Viruses, Pasteur Institute of Iran, Tehran, Iran
5. Department of Influenza Virus Research and Other Respiratory Viruses, Pasteur Institute of Iran, Tehran, Iran
Introduction: During the last few years, Highly Pathogenic H5N1 Influenza (HPIV) Virus other than human mortality, has created losses in the poultry industry and causes seasonal epidemics, deaths and extensive economic damages annually. So we should have diagnostic tests to control influenza infections, research on diagnostic tests containing virus proteins that have conserved sequences is always needed to elicit effective and cross-immune responses against different virus subtypes. Neuraminidase Protein(NA) is one of the influenza virus conserved proteins that can trigger a sufficient host immune response.
Methods: Bioinformatics studies on the amino acid sequence of the protein have shown that NA protein, has physico-chemical feature and contains immunogenic epitopes that can be the targets of stimulate humoral immune system. So , NA protein can be an appropriate candidate for diagnostic tests development. In this study, cloning and expression of the influenza (A/Indonesia/5/2005(H5N1) virus’s NA protein in the prokaryotic host (Escherichia coli) was investigated for using in the diagnostic tests production. Thus, the NA gene fragment was amplified by polymerase chain reaction (PCR) process with designed primers. After enzymatic digestion, the gene fragment was cloned into pET21a; an expression vector, transferred to a replication host (Top10f′), and after confirmation of cloning accuracy, pET21-NA gene structure was transferred to an expression host (BL21). The NA protein expression with IPTG inducer was determined by SDS-PAGE and Western blotting using initial Anti-His6 antibody.
Results: The expression was optimized under conditions: 0.5 mM IPTG inducer at 37°C in over night. This method provides us a significant amount of NA protein (H5N1)in a short time for future purposes and demonstrates the efficient production of recombinant protein in the prokaryotic system for using in the diagnostic tests influenza infection.
Conclusion: Our data and western blot analyses showed that recombinant noraminidase has potential to be efficient production in the prokaryotic system for using in the diagnostic tests influenza infection.
Keywords: Nueraminidase , Prokaryote System, Diagnostic tests, Influenza A(H5N1)Virus