Chimeric empty viral-like particles derived from the infectious bursal disease virus (IDBV), process for their production and applications
一种法氏囊病、传染性的技术，应用在嵌合空心病毒样颗粒领域，能够解决未排除等问题
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Embodiment 1
[0102] Example 1 Obtaining IBDV CVLP-pVP2s in insect cells * and analysis of structural polymorphism
[0103] I. Materials and methods
[0104] Preparation of virus
[0105] IBDV Soroa strain, serotype I IBDV strain, was purified from quail muscle QM7 cells by standard procedures (Lombardo et al. 1999. VP1, the putative RNA-dependent RNA polymerase of infectious bursal disease virus, forms complexes with the capsid protein VP3, leading to Efficient encapsidation into virus-like particles. "VP1, the putative RNA-dependent RNA polymerase of infectious bursal disease virus, forms a complex with the capsid protein VP3, leading to efficient encapsidation into virus-like particles" J Virol 73, 6973-6983) and stored in 25mM PES buffer (piperazine-N-N'-bis(2-ethylsulfonic acid)[PIPES]pH=6.2, 150mM NaCl and 20mM CaCl 2 )middle.
[0106] Construction of recombinant baculovirus
[0107] The recombinant baculovirus (rBV) FB / VP2-456 has been reported before (Castón et al., 2001.C te...
Embodiment 2
[0159] IBDV CVLP-pVP2s * Characterization of Immunogenicity
[0160] In order to evaluate the immunogenicity of CVLP-pVP2-456 obtained in Example 1, immunoassays were carried out in 1-day-old chickens. Briefly, a group of 7 SPF (specific pathogen free) animals were immunized intramuscularly with a 200 μl single dose containing 10 μg CVLP-pVP2-456 / animal diluted in PBS. Similar groups were injected with PBS. Sera were drawn weekly from each animal in both groups. The sera from each group and time were pooled so that equal volumes of each individual in the group were represented by equal amounts of referenced homologous sera (pool). The sera were analyzed by means of ELISA. Therefore, 10 ng of CVLP-pVP2-456 was used to coat the wells. The assay was performed according to previously published methods (Current Protocols in Immunology "Prior Art in Immunology". Edited by: Bierer, Coligan, Margulies, Shevach, Strober, John Wiley & Sons http: / / www.interscience.wiley.com / c_p / in...
Embodiment 3
[0162] Obtaining CVLP-pVP2s in Yeast * (pVP2 * -BT)
[0163] IBDV CVLP-pVP2s obtained for studies in yeast (S. cerevisiae) cultures * For the purpose of possibility, a heterologous gene encoding FMVD chimeric peptide called BT (Zhang, Q. et al., 2002, Acta Virologica 46(1): 1-9) combined at the N-terminus of pVP2-456 was prepared. Expression plasmid pESCURA / pVP-456-BT. The chimeric BT peptide contains a B-cell epitope (located at positions 133-159 of the VP1 protein of FMDV serotype C Spanish isolate) and a T-cell epitope (located at positions 20-34 of the FMDV serotype C Spanish isolate VP4 protein). The amino acid sequence of the B cell epitope, expressed in single letter form, is SIINNYYMQQYQNSM (SEQ ID NO: 14), and the amino acid sequence of the T cell epitope, expressed in single letter form, is MTTTYTASARGDLAHLTTTHARHLP (SEQ ID NO: 15) .
[0164] The first step in the expression plasmid construct was achieved by cloning the coding region of the pVP2-456 protein into...
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