A kind of foot-and-mouth disease virus-like particle vaccine and preparation method thereof

A foot-and-mouth disease virus and particle technology, applied in the field of genetic engineering, can solve the problem of unable to express multiple capsid proteins at the same time, and achieve the effects of complete protective immunity, long immune protection period and lower production cost.

Active Publication Date: 2021-01-26
GPROAN BIOTECH (SUZHOU) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Prokaryotic expression system and yeast expression system, which are commonly used expression systems for recombinant vaccines, cannot simultaneously express multiple capsid proteins and self-assemble into virus-like particles. Insect cells and baculovirus systems can solve this problem well.

Method used

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  • A kind of foot-and-mouth disease virus-like particle vaccine and preparation method thereof
  • A kind of foot-and-mouth disease virus-like particle vaccine and preparation method thereof
  • A kind of foot-and-mouth disease virus-like particle vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] The gene sequence optimization of the P12A and 3C proteins of embodiment 1 encoding foot-and-mouth disease

[0050] According to GenBank serial number AET43040, (2332 aa linear VRL 15-FEB-2012; DEFINITIONpolyprotein[Foot-and-mouth disease virus-type O] ACCESSION AET43040, the main virus strain (serum type O) and Its gene sequence. On this basis, according to the preference of codons in the translation process of insect cells, the coding gene fragment required for FMD VLP was designed. The packaging of FMD VLP requires P12A and 3C proteins, so the chemically synthesized sequences correspond to P12A respectively Genes and 3C genes.

[0051] The nucleotide sequence of the P12A gene is shown in SEQ ID NO.1, and the sequence structure is: RsrII-Kozaksequence-initiation codon-HBS signal peptide-VP4-VP2-VP3-VP1-2A-stop codon-SV40PolyA Signal - EcoRI.

[0052] The nucleotide sequence of the 3C gene is shown in SEQ ID NO.2, and its sequence structure is: SphI-Polyhedrin promot...

Embodiment 2

[0053] Example 2 Construction of recombinant expression vector pFastBac HT-B-P12A-3C

[0054] 1. The P12A gene and 3C gene of foot-and-mouth disease virus were inserted in series into the transfer vector pFastBac HT-B

[0055] The 3C gene fragment and pFastBac HT-B were double digested with Sph I and Hind III, and the 3C gene fragment was ligated and transformed with pFastBac HT-B after digestion to obtain pFastBac HT-B-3C; The P12A gene fragment and pFastBac HT-B-3C were subjected to double enzyme digestion, followed by ligation transformation. For enzyme digestion results, see figure 1 and figure 2 .

[0056] The above reaction mixture was mixed and centrifuged, then ligated overnight at 16°C. Take out the E.coli DH5α competent cells frozen at -80°C and put them on ice until they melt, add the ligation product, flick the tube wall to mix, ice bath for 30min, heat shock in 42°C water bath for 90sec, immediately ice bath for 1min, add 800μL In LB medium without antibioti...

Embodiment 3

[0066] Example 3 Expression Identification of Recombinant Baculovirus Target Protein

[0067] 1. Sf9 cells transfected with rBacmid DNA

[0068] 1) Cultivation of Sf9 cells Sf9 suspension cells were cultured with SF900 III SFM, and cultured with shaking at 27° C. and 110 rpm. One day before transfection, Sf9 cells were subcultured to 6-well plates with a density of 8×10 per well. 5 .

[0069] 2) Transfection Take two Eppendorf tubes, add solution A and solution B respectively, and the system is as follows: Solution A: Mix 10 μL of recombinant Bacmid DNA and 90 μL of serum-free and antibiotic-free SF900 III medium.

[0070] Solution B: Mix 6 μL of liposome Cellfectin II transfection reagent (mix well by inverting 5-10 times before use) with 94 μL of serum-free and antibiotic-free SF900 III medium.

[0071] After liquid A and liquid B were allowed to stand at room temperature for 5 min, mix liquid A and liquid B, flick the tube wall to mix well, and place at room temperature ...

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Abstract

The invention provides a foot-and-mouth disease virus-like particle vaccine and a preparation method thereof. Insert the two ORFs of the codon-optimized FMDV P12A and 3C genes in series into the transfer vector to construct a recombinant expression plasmid; or clone a single ORF containing the P12A3C gene under the promoter SV40 into the vector to construct a recombinant expression plasmid, and transform competent cells to obtain recombinant expression Bacmid‑DNA was transfected into sf9 cells to obtain insect cells capable of expressing foot-and-mouth disease virus-like granule protein. After culturing the cells, the supernatant is collected, concentrated by ultrafiltration, centrifuged, and chromatographically purified to prepare the VLP vaccine.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a foot-and-mouth disease virus-like particle, a preparation method thereof, and a foot-and-mouth disease vaccine prepared by the same. Background technique [0002] Foot-and-mouth disease (FMD) is an acute, highly contagious animal disease. Mainly against artiodactyls, occasionally in humans and other animals. Susceptible animals mainly include more than 70 kinds of artiodactyl domestic and wild mammals in 20 families including cattle, buffalo, sheep, goats, camels and pigs. Due to its highly contagious nature and significant impact on the agricultural economy, the International Office of Epizootics (OIE) ranks foot-and-mouth disease at the top of Class A animal infectious diseases. [0003] The introduction of Asian type 1 foot-and-mouth disease in 2005, the introduction of type A foot-and-mouth disease in 2009, especially the widespread prevalence of type O neutra...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/42C12N15/63A61K39/135A61P31/14
CPCA61K39/135A61P31/14C12N15/63A61K39/125A61K2039/5258C07K14/005C12N15/66C12N2770/32123C12N2770/32134C12N2770/32151
Inventor 安海谦冉波赵荣茂方华明任玉珍王鹏
Owner GPROAN BIOTECH (SUZHOU) INC
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