Fotouhi Fatemeh, Farahmand Behrokh, Heidarchi Behnaz, Esghaei Maryam, Rafati Sima, Tavassoti Kheiri Masoumeh
Influenza Research Lab, Department of Virology, Pasteur Institute of Iran, Tehran, IR Iran.
Virology Department, Iran University of Medical Sciences, Tehran, IR Iran.
Jundishapur J Microbiol. 2014 Sep;7(9):e11812. doi: 10.5812/jjm.11812. Epub 2014 Sep 1.
Permanent antigenic variation of influenza viruses causes a major concern to develop an effective human influenza vaccine. Conserved antigens are new vaccine candidates because it is not necessary to match the prepared vaccine with circulating strains. Ion channel M2 protein is conserved among all influenza A viruses, allowing the virus to enter host cells.
To prepare an effective vaccine against influenza A viruses, a chimerical DNA plasmid encoding Influenza virus M2 protein and Leishmania major HSP70 was constructed.
Influenza A/New Caledonia/20/99 (H1N1) was inoculated into MDCK cell line and total RNA was extracted. The full length M2 gene was amplified by RT-PCR using designed specific primers, cloned into pGEM-T Easy cloning vector and completely sequenced. The M2 gene was then subcloned into the pcDNA upstream of HSP70 gene. Recombinant plasmids were transfected into COS-7 cells to evaluate protein expression.
The recombinant plasmids were confirmed by PCR, restriction enzyme analysis and sequencing. Three dimensional structure of chimer protein was assessed using specific software. Transient protein expression in eukaryotic cells was confirmed by specific mRNA detection, indirect Immunofluorescence test and western blotting.
M2-HSP70 chimer protein was successfully expressed in eukaryotic cells. Computational studies of chimer peptide sequence revealed that fusing HSP to the C-terminal of M2 protein does not mask the predominant epitope of M2. HSP70 is a molecular chaperon and immunostimulatory component. Genetically fusing antigens to HSPs leads to the enrichment of DNA vaccine potency. The immunogenicity of this construct with different formulation would be evaluated in further investigations.
流感病毒的永久性抗原变异是开发有效人类流感疫苗的主要关注点。保守抗原是新的疫苗候选物，因为无需使制备的疫苗与流行毒株相匹配。离子通道M2蛋白在所有甲型流感病毒中保守，使病毒能够进入宿主细胞。
为制备一种有效的抗甲型流感病毒疫苗，构建了一种编码流感病毒M2蛋白和利什曼原虫主要热休克蛋白70（Leishmania major HSP70）的嵌合DNA质粒。
将甲型流感/新喀里多尼亚/20/99（H1N1）接种到MDCK细胞系中并提取总RNA。使用设计的特异性引物通过RT-PCR扩增全长M2基因，克隆到pGEM-T Easy克隆载体中并进行完全测序。然后将M2基因亚克隆到HSP70基因上游的pcDNA中。将重组质粒转染到COS-7细胞中以评估蛋白表达。
通过PCR、限制性内切酶分析和测序确认了重组质粒。使用特定软件评估嵌合蛋白的三维结构。通过特异性mRNA检测、间接免疫荧光试验和蛋白质印迹法确认了真核细胞中的瞬时蛋白表达。
M2-HSP70嵌合蛋白在真核细胞中成功表达。嵌合肽序列的计算研究表明，将HSP融合到M2蛋白的C末端不会掩盖M2的主要表位。HSP70是一种分子伴侣和免疫刺激成分。将抗原与HSP基因融合可提高DNA疫苗的效力。该构建体不同制剂的免疫原性将在进一步研究中评估。
