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Recombinant PRRSV virus-like particle and preparation method thereof

A technology of recombinant baculovirus and virus, applied in the biological field, can solve the problems of decreased virus titer, late neutralizing antibody titer, etc., and achieve the effects of anti-virus prevention, good immune protection, and good immune protection effect

Active Publication Date: 2021-10-19
NORTHWEST A & F UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

And studies have shown (HP-PRRSV envelope protein 5 glycosylation site mutation and functional exploration), site-directed mutation of the amino acid of the A epitope has a certain impact on the replication of the virus, resulting in a decrease in the virus titer. Therefore, how to Reducing the antagonistic effect of epitope A on epitope B is an urgent technical problem to be solved in recombinant PRRSV virus-like particle vaccines. The inventors prepared immunogenic recombinant PRRSV virus-like particles (ZL201811114020.6) in 2018. However, in actual production, there is still the problem of late generation of neutralizing antibody titers, and there is still room for improvement

Method used

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  • Recombinant PRRSV virus-like particle and preparation method thereof
  • Recombinant PRRSV virus-like particle and preparation method thereof
  • Recombinant PRRSV virus-like particle and preparation method thereof

Examples

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Effect test

Embodiment 1

[0030] Embodiment 1: Construction of recombinant baculovirus Ac-PRRSVGP5-M

[0031] (1) Synthesis of PRRSV GP5 and M gene sequences:

[0032] Based on the sequence of PRRSV-SD16 strain (Genbank Accession No: JX087437.1), after artificial modification, the PRRSV GP5-M gene whose gene sequence is shown in SEQ ID No: 1 was obtained, and sent to Gene Synthesis Company for gene sequence synthesis , and set Bam H I and Hind III restriction sites at the 5' end and 3' end, respectively, to obtain insert fragments.

[0033] (2) Construction and identification of recombinant transfer vector:

[0034] The plasmid containing the target gene sequence GP5-M and the vector pBAC-5 (provided by Shaanxi Nuowei Lihua Biotechnology Co., Ltd., with a GFP marker gene on the vector, which can be used for fluorescence detection, see ZL201811114020.6) were subjected to Bam H I and Hind Ⅲ double enzyme digestion, after recovery and purification, use T4 ligase to perform ligation reaction, chemically ...

Embodiment 2

[0056] (1) Preparation of recombinant PRRSV virus-like particle vaccine:

[0057] The recombinant baculovirus Ac-PRRSV GP5-M prepared in Example 1 was inoculated into healthy sf9 cells at a ratio of 10%. After culturing at 27°C for 4-5 days, the cells and supernatant were collected by repeated freezing and thawing. After centrifuging for 20 minutes, collect the supernatant, then use the ammonium sulfate precipitation method to precipitate the target protein, resuspend the protein solution for 36-48 hours with binary ethyleneimine (BEI), and then use an equal amount of sodium thiosulfate Neutralize, and finally mix and emulsify the vaccine with Seppic ISA 206 adjuvant, and store it at 2-8°C for use.

[0058] In the vaccine preparation process, after the recombinant baculoviruses of Example 1 of the present invention, control group 1, control group 2 and control group 3 were cultured, they were all adjusted to a virus titer of 10 5 TCID 50 / ml, and then continue with subsequent ...

Embodiment 3

[0072] Example 3: Antibody ups and downs of vaccine immunized piglets

[0073] (1) The recombinant PRRSV virus-like particle vaccine and the vaccine of the control group 2 were prepared according to the method of Example 2, and the vaccine sample of the control group 3 (prepared by the invention patent ZL201811114020.6 was provided by Shaanxi Nuowei Lihua Biotechnology Co., Ltd. vaccine).

[0074] (2) Screen 10 one-month-old healthy piglets with double-negative PRRSV antigen and antibody, among which 1 piglet was randomly selected as the blank control group, and the remaining 9 piglets were randomly divided into 3 groups, 3 piglets in each group. The first group is immunized with the Ac-PRRSV GP5-M vaccine of the present invention (the amino acid sequence is shown in SEQ ID No: 2), the second group is immunized with the control group 2 vaccine (the amino acid sequence is shown in SEQ ID No: 6), and the third group Immune the vaccine of the control group 3 (the vaccine prepare...

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Abstract

A recombinant baculovirus capable of being efficiently replicated is obtained by modifying a GP5-M protein, the culture titer of the recombinant baculovirus is relatively high, the vaccine is further prepared by using prepared virus-like particles similar to PRRSV virions, the vaccine can generate a neutralizing antibody with higher titer, and the antibody swelling eliminating rule of immunized piglets shows that the vaccine provided by the invention can stimulate an organism to generate high-concentration antibodies earlier, and can stimulate the organism to generate higher average antibody titer after secondary immunization, so that better immune protection is realized.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and relates to a recombinant PRRSV virus-like particle and a preparation method thereof. Background technique: [0002] Porcine reproductive and respiratory syndrome (Porcine reproductive and respiratory syndrome, PRRS), also known as "blue ear disease", its pathogen is porcine reproductive and respiratory syndrome virus (Porcine reproductive and respiratory syndrome virus, PRRSV), which mainly causes reproductive problems in pregnant sows. Obstacles and respiratory disorders in piglets, usually manifested as coughing and dyspnea in piglets, premature birth, abortion or even stillbirth in sows. Since it was discovered in North America in the late 1980s, the worldwide prevalence and several outbreaks of the disease have led to huge economic losses in the swine industry around the world. [0003] PRRSV belongs to the Arteriviridae family, which includes Simian Hemorrhagic Fever Virus, Equine Arteriti...

Claims

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Application Information

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IPC IPC(8): C12N15/40C07K14/08C12N15/866C12N15/66A61K39/12A61P31/14
CPCC07K14/005C12N15/86A61K39/12A61P31/14C12N2770/10022C12N2770/10023C12N2770/10052C12N2770/10034C12N2710/14143C12N2800/105A61K2039/5258A61K2039/552
Inventor 陈瑞王晶钰南雨辰武春燕
Owner NORTHWEST A & F UNIV
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