Construction of a bicistronic eukaryotic vector expressing M1 and NP genes of influenza virus

Shenagari, Mohammad; Sabahi, Farzaneh; Tavasoti Kheiri, Masoumah; Forghan Parast, Kambiz; Jamali, Abbas; Hashemi, Hamidreza; Khodamoradi, Shadi

Construction of a bicistronic eukaryotic vector expressing M1 and NP genes of influenza virus

Authors

Mohammad Shenagari ¹

Farzaneh Sabahi ²

Masoumah Tavasoti Kheiri ³

Kambiz Forghan Parast ⁴

Abbas Jamali ⁵

Hamidreza Hashemi ⁶

Shadi Khodamoradi ⁷

¹ Ph.D. Student, Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University

² Associated Professor, Department of Virology, Faculty of Medical Sciences, Tarbiat Modares University

³ Assistant Professor, Department of Influenza, Pasteur Institute of Iran

⁴ Associated Professor, Department of Bacteriology, Faculty of Medicine, Gilan University of Medical Sciences, Iran

⁵ Post-Doctoral fellow, Department of Influenza, Pasteur Institute of Iran, Iran

⁶ Ph.D. Student, Department of Virology, School of Public Health, Tehran University of Medical Sciences, Iran

⁷ M.Sc. Student, Department of Influenza, Pasteur Institute of Iran, Iran

Abstract

Objective: In this study, two conserved genes (M1 and NP) of influenza virus were expressed in a bicistronic vector in order to develop a universal gene based vaccine.
Materials and Methods: Plasmids M1-pIRES2-EGFP, pIRES2-NP were constructed by cloning the PCR products of M1 and NP genes which were amplified from the A/Peurto Rico/8/34 (H1N1) influenza virus strain into the plasmid expression vector pIRES2-EGFP, respectively. For construction of M1-pIRES2-NP bicistronic plasmid, M1 gene was extracted from M1-pIRES-EGFP plasmid and sub-cloned into pIRES2-NP construct. Finally, simultaneous expression of both genes was assessed by transient transfection of bicistronic plasmid into BHK-21 cell lines and subsequent immunofluorescence staining.
Results: The results of enzymatic double digestions on the constructed plasmids and sequencing demonstrated the success of cloning processes of above mentioned genes. Correct expression of these genes was confirmed by M1-pIRES2-NP plasmid expression in BHK-21 cell lines confirmed by immunofluoresence microscopy.
Conclusion: Simultaneous expression of influenza M1 and NP genes from a bicistronic plasmid containing “IRES” sequence is achievable.

Keywords

Influenza Virus

bicistronic vector

Simultaneous Expression