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Foot-and-mouth disease virus-like particle, preparation method, vaccine composition and application

A foot-and-mouth disease virus and vaccine composition technology, applied in the field of veterinary biological products, can solve the problems of easy mutation of foot-and-mouth disease virus, decreased vaccine protection, weak immunogenicity, etc.

Active Publication Date: 2020-09-29
PULIKE BIOLOGICAL ENG INC +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The FMD virus-like particles that have been studied more at present are composed of the FMD structural proteins VP0, VP3 and VP1. However, the structure of the FMD virus-like particles prepared by this method is unstable, the activity is reduced, and the body cannot be well induced to produce an effective immune response.
[0004] In addition, foot-and-mouth disease virus is prone to mutation, especially the O-type foot-and-mouth disease virus CATHAY variant that is popular in my country. body develops sufficient immunity

Method used

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  • Foot-and-mouth disease virus-like particle, preparation method, vaccine composition and application
  • Foot-and-mouth disease virus-like particle, preparation method, vaccine composition and application
  • Foot-and-mouth disease virus-like particle, preparation method, vaccine composition and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Preparation of Type O Foot-and-Mouth Disease Virus Ⅰ Virus-like Particles (VP4, VP2, VP3, VP1)

[0094] 1. Preparation of foot-and-mouth disease gene fragments used as templates

[0095] The full length of the gene shown in the amino acid sequences of Type O foot-and-mouth disease virus I VP4, VP2, VP3, and VP1 shown in SEQ ID NO.1, 2, 3, and 4 was synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The full lengths of the synthesized gene fragments were 255bp, 654bp, 660bp, 639bp, respectively. The foot-and-mouth disease gene template of the present invention is prepared on the basis of the artificially synthesized foot-and-mouth disease gene fragment.

[0096] 2. Construction of foot-and-mouth disease gene expression vector

[0097] For the foot-and-mouth disease gene template synthesized in the previous step, primers were designed respectively (see Table 1), and the O-type foot-and-mouth disease virus I VP4, VP2, VP3, and VP1 genes were amplified.

[0098] T...

Embodiment 2

[0112] Preparation of Type O Foot-and-Mouth Disease Virus Ⅰ Virus-like Particles (VPO, VP3, VP1)

[0113] Referring to the method in Example 1, primers were respectively designed according to the gene sequences of type O foot-and-mouth disease virus I structural proteins VPO, VP3, and VP1 for tandem expression to prepare virus-like particles. The collected bacteria were resuspended according to the ratio of 1 g of bacteria to 10 ml of lysate, and the bacteria were crushed 4 times with a homogenizer at a pressure of 800 bar. Centrifuge at 13500rpm for 40min, save the supernatant, and detect by 15% SDS-PAGE electrophoresis. At this time, the expression level of the three serially expressed proteins in the supernatant is about 20%. Ammonium sulfate fractional precipitation was used for crude protein purification, followed by chromatographic purification. The purified protein was subjected to SDS-PAGE electrophoresis, which showed that the target protein was purified and enriched....

Embodiment 3

[0115] Comparative Test of Stability of Type O Foot-and-Mouth Disease Virus Ⅰ Virus-like Particles

[0116] The virus-like particles prepared in Example 1 and Example 2 were respectively placed in a water bath at 50° C. for 1 hour and treated at pH 6.0 for 30 minutes, and then observed under an electron microscope. The results show that the virus-like particles expressed in four segments in tandem have uniform size and hollow shape no matter in hot environment or in acidic conditions; while the virus-like particles expressed in three segments in tandem appear to aggregate and have different particle sizes . The specific results are shown in Table 3.

[0117] Table 3 Stability test of FMD virus-like particles

[0118] category VP4, VP2, VP3, VP1 VP0, VP3, VP1 50℃ water bath for 1h Stablize unstable pH6.0 treatment 30min Stablize unstable

[0119] It has been proved that the stability of the virus-like particle structure provided by the pre...

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Abstract

The invention discloses a foot-and-mouth disease virus-like particle, a preparation method, a vaccine composition and an application. The foot-and-mouth disease virus-like particle of the present invention is composed of the structural proteins VP4, VP2, VP3 and VP1 of the foot-and-mouth disease virus, and has a stable structure, which not only increases the temperature stability and acid environment stability, but also the prepared vaccine composition can quickly form specific antibodies, The immune duration is significantly increased, and the immune protection can be maintained for a longer time.

Description

technical field [0001] The invention relates to the field of veterinary biological products, more specifically, the invention relates to foot-and-mouth disease virus-like particles, a preparation method, a vaccine composition prepared from the virus-like particles and applications. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, highly contagious animal disease that can spread quickly and over long distances. It is the most contagious disease among mammals, and the infection of artiodactyls will cause significant economic losses worldwide. Animals affected by FMD include cattle, sheep, goats and pigs. The causative agent, foot-and-mouth disease virus (FMDV), is an aphthous sore virus of the picornavirus family. The virus is divided into 7 serotypes (A, O, C, Asia1, SAT1, SAT2, and SAT3), among which type O, Asia1 and A are mainly prevalent in my country. Vaccine immunization is an effective measure to control the disease and protect livestock from h...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N7/04A61K39/135A61K39/39A61P31/14C12R1/93
CPCY02A50/30Y02A40/70
Inventor 张许科袁于人孙进忠陈红英肖燕田克恭
Owner PULIKE BIOLOGICAL ENG INC
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