Construction and Identification of Novel Virus-like Particles of Swine influenza virus
ZHOU Yu-Tong1, ZHANG Meng-Jia2,3, QIAO Wen-Yue1, LANG Yi-Fei1, YAN Qi-Gui1, LI Wen-Tao2,3,*, ZHAO Shan1,*
1 College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China; 2 College of Veterinary Medicine, Huazhong Agricultural University, Wuhan 430070, China; 3 National Key Laboratory of Agricultural Microbial Resource Exploration and Utilization, Huazhong Agricultural University, Wuhan 430070, China
Abstract：Swine influenza (SI) is an acute infectious disease of pigs (Sus scrofa) caused by Swine influenza virus (SIV). In recent years, 3 SIV subtypes, namely H1N1, H1N2 and H3N2, are most prevalent in China. This study aimed to develop virus-like particles (VLP) of SIV H1 and H3 subtype and determine their morphological structure and biological characteristics. Genes encoding the SIV surface protein—hemagglutinin (HA) of H1 and H3 subtypes were synthesized and ligated unto the expression vector. The recombinant plasmids was obtained and transfected to HEK-293T cells for protein expression. Meanwhile, the gene encoding the 2,4-dihydroxyprotenine synthase (LS) derived from the super pyretic Aquifex aeolicus was synthesized, and both a signaling peptide and immunoglobulin-bin-binding domain sequence in streptococcal protein G (pG) were introduced at the N-terminus of the sequence. The pG-LS recombinant plasmids were constructed and expressed separately through Escherichia coli and HEK-293T cell expression systems. After the conjugation of the 2 proteins, the morphology and the formation of VLP could be observed by transmission electron microscopy. Average particle diameters of the conjugated proteins were measured with dynamic light scattering method while the biological properties of the complex protein were analyzed by SDS-PAGE, Western blot and hemagglutination assay. SDS-PAGE results indicated that both recombinant proteins (H1-Fc and H3-Fc) were correctly expressed; at the same time, Western blot showed that both prokaryotic and eukaryotic systems resulted in successful expression of pG-LS nanoparticles. Together with SDS-PAGE analysis, it was shown that pG-LS expressed in HEK-293T cells had higher purity, and henceforth used for the subsequent construction of pG-LS-HA virus-like particles. Electron microscopy analysis of the conjugated proteins showed that the pG-LS-HA forms spherical virus-like particles, with a diameter around 80 nm. Further immunological analysis and particle diameter measurement both showed the successful construction of VLPs. The results of hemagglutination assays indicated HA-Fc alone could not agglutinate erythrocytes, while the virus-like particles prepared from 2 conjugated proteins showed exponential affinity, which could efficiently agglutinate red blood cells of different species. This study successfully constructed pG-LS nanoparticles and 2 pG-LS-HA VLPs, where HA proteins successfully displayed at the VLP surface. Both developed pG-LS-HA had stable property and biological function, with a potential of large-scale production. In the meantime, this study found that both H1 and H3 HA could be effectively displayed to form VLP, which shed light on the future use of multi-subtype influenza virus HA protein co-presentation strategy to develop novel, multivalent VLP vaccine candidates.
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