Expression and Purification of H5N1 Subtype Avian Influenza Virus HA Protein in Rice Endosperm
In order to prepare the HA protein of H5N1 subtype avian influenza virus and evaluate its immunogenicity,the recombinant plant expression vector pCAMBIA1300-HA was constructed to express the recombinant HA protein of H5N1 subtype avian influenza virus by using a rice expression system,which had a GT13-specific promoter,signal peptide SP and terminator,and was conducive to the expression of exogenous genes in rice.The recombinant plasmid pCAMBIA1300-HA was introduced into Agrobacterium tumefaciens EHA105 by electroconversion,and the positive colonies were screened to infect rice calli.After dark culture,hygromycin screening,differentiation,rooting,they grew into seedlings.The DNA of rice leaves was extracted by CTAB method,and the results of PCR identification showed that the HA gene had been inserted into the rice genome,and the size of the recombinant HA gene was 4 660 bp.The positive plants were transplanted into the field,and the rice seeds were harvested 4 months later,and then the rice endosperm protein was extracted.Western blot identification results showed that HA protein was successfully expressed in the rice endosperm.The recombinant HA protein was separated and purified by Q anion chromatography,hydrophobic chromatography and gel filtration chromatography,and its purity reached more than 90%.The purified recombinant HA protein was mixed with ISA 50V adjuvant and emulsified to make a vaccine,which was used to immunize mice.The mice serum had a high level of specific antibodies,indicating that the immunogenicity of the recombinant HA protein was better.In summary,a rice expression system for the recombinant HA protein of H5N1 subtype avian influenza virus was successfully constructed,and the recombinant HA protein with high purity and good immunogenicity was isolated and purified.
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