Preparation method of APP (Actinobacillus Pleuropneumoniae) OMVs (Outer Membrane Vesicles) and vaccine of APP OMVs

Abstract

The invention relates to a preparation method of APP (Actinobacillus Pleuropneumoniae) OMVs (Outer Membrane Vesicles) and a vaccine of the APP OMVs, and belongs to the technical field of biology. The preparation method comprises the steps of culturing APP shope strains in vitro by using a culture medium which is in iron ion limit, obtaining acellular cultural supernatant after carrying out centrifugation and 0.22-[mu]m filtration treatment, and preparing the OMVs released by germs through ultracentrifugation, wherein the observation through a transmission electron microscope shows that the diameter of most OMVs is 50 to 100 nm, the OMVs are used as subunit vaccines to carry out secondary intranasal immunization on a mouse, the weight of the OMV immune mouse is increased for a long time, and the visual forms of lungs have no obvious difference from a PBS (Phosphate Buffer Saline) immune group. Meanwhile, an experiment shows that the OMVs are efficient immune stimulants, not only can high-level IgG (Immunoglobulin G) be stimulated to be generated by mouse sera, but also high-level IgA (Immunoglobulin A) can be generated in mouse lungs, mucosal immunity of the mouse lungs is effectively stimulated, and a better application prospect of using the OMVs as the subunit vaccines is expressed.

Description

technical field [0001] The invention relates to a method for preparing outer membrane vesicles of Actinobacillus pleuropneumoniae and a vaccine thereof, in particular to in vitro culture of Actinobacillus pleuropneumoniae serotype 1 strains under iron ion-limited conditions to prepare outer membrane vesicles released by bacteria Bubbles are used as a subunit vaccine to evaluate the immune stimulating effect of the vaccine on mice, which belongs to the field of biotechnology. Background technique [0002] Actinobacillus (A.) pleuropneumoniae is a Gram-negative bacterium that can cause pneumonia in pigs. Acutely infected pigs show severe dyspnea, pulmonary hemorrhage and fibrous pleurisy, with high mortality , chronically resistant pigs grow retarded, and often become recessive pathogen carriers. This disease is recognized as one of the important infectious diseases that endanger the modern pig industry. A.pleuropneumoniae mainly invades the respiratory tract, and the importa...

AI Technical Summary

Problems solved by technology

Knocking out some virulence-related genes of A.pleuropneumoniae can artificially obtain attenuated virulence strains, which can be used as attenuated live vaccines to immunize animals with better cross-immune protection effects. However, considering safety issues, this type of vaccine has not yet been established. Carry out large-scale promotion and application

Compared with inactivated vaccines and live attenuated vaccines, subunit vaccines have better safety, but because subunit vaccines only contain one or several antigens, they often cannot induce high levels of Therefore, it is necessary to improve the immunogenicity of subunit vaccines by adding adjuvants, conjugated polysaccharides or controlled release of antigens

Through the treatment of antigen controlled release technology, the extremely small particles integrated with antigens can be obtained, which can better stimulate the immune system of the body and show attractive application prospects. However, this technology requires the purification and embedding of antigens, which greatly increases the production cost. The cost hinders the application of this technology in livestock and poultry vaccine preparations

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