Volume 19, Issue 2 (2016)
mjms 2016, 19(2): 45-58 |
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Taheri M, Shenagari M, Nikoukar I, Khosravi M, Nemattalab M. Design, cloning and expression assay of NP gene in a bicistronic vector harboring mice IL-18 gene: potential implications for type A influenza vaccine investigations. mjms 2016; 19 (2) :45-58
URL: http://mjms.modares.ac.ir/article-30-11221-en.html
URL: http://mjms.modares.ac.ir/article-30-11221-en.html
1- Department of Biotechnology, Faculty of Para Medicine, Guilan University of Medical Sciences, langroud, Iran
2- Department of Microbiology, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran
2- Department of Microbiology, Faculty of Medicine, Guilan University of Medical Sciences, Rasht, Iran
Abstract: (9267 Views)
Background: Vaccination is the most effective tool for preventing influenza virus morbidity and mortality. Despite mutability of surface antigen, inner proteins of influenza virus are remarkably conserved among various strains. The high similarity of NP protein sequences among various strains of influenza type A from one side, and the potency of IL-18 molecular adjuvant in shifting immune response toward Th1 from other side, persuaded us to evaluate the possibility of cloning and expression of Influenza type A NP gene in a bicistronic vector harboring mice IL-18 gene in order to assess their immunogenic activities in a susceptible mouse model in the following study.
Materials and Methods: At the beginning, the genomic RNA of Influenza virus PR8 was extracted and after the process of cDNA synthesis, the target gene encoding NP was amplified by PCR. In the next step, NP gene was cloned in the pJET1.2/blunt TA vector. The accuracy of cloned gene was confirmed by PCR, enzymatic digestion and sequencing. The pJET1.2-NP plasmid and pIRES-Igk/mIL18/Fc plasmids were simultaneously digested by BstXI/NotI enzymes. Digested NP fragment was inserted into pIRES-Igk/mIL18/Fc plasmid using T4 ligase. Transformation into DH5α and colony selection was done. Henceforth, gene cloning was confirmed by PCR, enzymatic double-digestion and sequencing. Eventually, by transfection of the constructed mIL-18-pIRES2-NP plasmid into BHK-21 and RAW264.7 cell lines, the expression of NP and IL-18 was assessed by Indirect Immunofluorscence and ELISA respectively.
Results: Electrophoresis of PCR product, enzymatic digestion and sequencing showed that Influenza NP gene was successfully cloned into pIRES-Igk/mIL18/Fc to generate mIL-18-pIRES2-NP plasmid. The results of Indirect Immunofluorscence and ELISA indicated the successful expression of NP and mIL-18 from produced plasmid in eukaryotic cell lines.
Conclusion: The results of the present study shows that the expression of NP and IL-18 genes from mIL-18-pIRES2-NP is successful.
Materials and Methods: At the beginning, the genomic RNA of Influenza virus PR8 was extracted and after the process of cDNA synthesis, the target gene encoding NP was amplified by PCR. In the next step, NP gene was cloned in the pJET1.2/blunt TA vector. The accuracy of cloned gene was confirmed by PCR, enzymatic digestion and sequencing. The pJET1.2-NP plasmid and pIRES-Igk/mIL18/Fc plasmids were simultaneously digested by BstXI/NotI enzymes. Digested NP fragment was inserted into pIRES-Igk/mIL18/Fc plasmid using T4 ligase. Transformation into DH5α and colony selection was done. Henceforth, gene cloning was confirmed by PCR, enzymatic double-digestion and sequencing. Eventually, by transfection of the constructed mIL-18-pIRES2-NP plasmid into BHK-21 and RAW264.7 cell lines, the expression of NP and IL-18 was assessed by Indirect Immunofluorscence and ELISA respectively.
Results: Electrophoresis of PCR product, enzymatic digestion and sequencing showed that Influenza NP gene was successfully cloned into pIRES-Igk/mIL18/Fc to generate mIL-18-pIRES2-NP plasmid. The results of Indirect Immunofluorscence and ELISA indicated the successful expression of NP and mIL-18 from produced plasmid in eukaryotic cell lines.
Conclusion: The results of the present study shows that the expression of NP and IL-18 genes from mIL-18-pIRES2-NP is successful.
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