1. Islamic azad university-central branch 2. Islamic azad university-central branch 3. Islamic azad university- Hamedan branch
Abstract
Introduction
Influenza virus, which causes very important respiratory disease, belongs to orthomyxoviridae family. vaccination is intended to control the disease as an effective solution. the factor that makes influenza vaccines inefficient is antigenic drift. attention to antigenic shift and antigenic drift is essential to design an effective vaccine against influenza virus. to design efficient vaccine, conserved regions in viral proteins are targeted. in influenza virus, external matrix protein (m2e), hemagglutinin (ha) and nucleoprotein (na) are the most conserved proteins. in this research, the recombinant construct of m2e and ha genes were designed by using bioinformatics software for cloning in bacillus subtilis host.
Methods
: the ha and m2e genes of h1n1 were amplified and separated by cultivation in hela cell lines and rt-pcr. m2e and ha genes were cloned in t&a vector firstly and finally into pht43 plasmid following digestion by bamh1, xba1 and xma1. after the verification of cloning process by pcr and enzymatic digestion analysis, the accuracy of m2e and ha gene’s orf in the t/a cloning vector were confirmed by sequencing .the chimeric construct pht43-m2e-ha was transformed into bacillus subtilis (wb600). expression of chimer protein m2e-ha was approved by sodiumdodecyl sulfate polyacrylamide gel electrophoresis (sds-page), bradford assay and western blot analysis.
Results
The results of clony pcr, restriction enzyme digestion and sequencing revealed that the construction of m2e-ha was correctly cloned in bacillus subtilis. in addition, antibodies animal test showed that the chimeric protein of m2e-ha stimulates the mice immune system properly.
Conclusion
Identification of antibodies against conserved epitopes of ha and m2e is an important step toward development of influenza vaccine, hence, m2e-ha chimeric protein prepared in this study could be an appropriate subunit vaccine candidate for preventing influenza virus infection.
Keywords
Chimeric protein, m2e gene, ha gene, subunit vaccine, influenza virus
