Recombinant PRRSV virus-like particle antigen-antibody complex and preparation method thereof

An antibody complex, particle antigen technology, applied in the directions of virus antigen components, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of unstable serum antibody content, complicated preparation process, pathogen pollution, etc. Immune protection effect, high culture titer, anti-virus prevention effect

Active Publication Date: 2021-09-24
NORTHWEST A & F UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are few reports on antigen-antibody complex vaccines for pigs
Antigen-antibody complex vaccines also have shortcomings. For example, the traditional method of using serum polyclonal antibodies mixed with pathogens to prepare vaccines is affected by the preparation of serum polyclonal antibodies. The preparation process is complicated, and the serum antibody content between different batches is unstable. Animal-derived serum is easy to introduce exogenous pathogen contamination and other shortcomings, so it has certain limitations, while the use of monoclonal antibodies does not have such problems

Method used

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  • Recombinant PRRSV virus-like particle antigen-antibody complex and preparation method thereof
  • Recombinant PRRSV virus-like particle antigen-antibody complex and preparation method thereof
  • Recombinant PRRSV virus-like particle antigen-antibody complex and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Embodiment 1: Construction of recombinant baculovirus Ac-PRRSVGP5-M

[0035] (1) Synthesis of PRRSV GP5 and M gene sequences:

[0036] 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.

[0037] (2) Construction and identification of recombinant transfer vector:

[0038] 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

[0060] (1) Preparation of recombinant PRRSV virus-like particle vaccine:

[0061] 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.

[0062] 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 subsequen...

Embodiment 3

[0076] Example 3: Preparation of "IgM-RPPSV VLPs" immune complex

[0077] (1) Preparation of RPPSV VLPs:

[0078] 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 then use a 100kD molecular weight filter membrane (EMD Millipore Company) to concentrate 50 times through a Labscale TFF cut-off filter, and then purify the RPPSV VLPs particles by liquid chromatography to obtain RPPSV VLPs.

[0079] (2) Preparation of IgM-RPPSV VLPs immune complex:

[0080] RPPSV VLPs were mixed with monoclonal antibody 5D9 (ca...

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Abstract

According to the invention, 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 prepared virus-like particles similar to PRRSV virions are further compounded with IgM to prepare an IgM-RPPSV VLPs immune complex, and the IgM-RPPSV VLPs immune complex can stimulate an organism to generate a high-concentration antibody earlier.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and relates to a recombinant PRRSV virus-like particle antigen-antibody complex 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 Fev...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/12A61K39/42A61P31/14C07K14/08C12N15/40C12N15/866
CPCA61K39/12A61K39/42A61P31/14C07K14/005C12N15/86C12N2770/10022C12N2770/10023C12N2770/10034C12N2710/14043
Inventor 陈瑞王晶钰南雨辰武春燕
Owner NORTHWEST A & F UNIV
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