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Recombinant protein and porcine epidemic diarrhea vaccine composition

A porcine epidemic diarrhea and vaccine composition technology, applied in the biological field, can solve the problems of inapplicability of emergency vaccination, large vaccination, and inability to produce sufficient immunity, etc., and achieves convenience for large-scale production, low production cost, and good immune response Effect

Active Publication Date: 2022-06-17
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, inactivated vaccines require a large dose of vaccination, and a single vaccination cannot produce sufficient immunity, so emergency vaccination is not suitable
Attenuated vaccines have the risk of spreading the virus and returning to strength

Method used

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  • Recombinant protein and porcine epidemic diarrhea vaccine composition
  • Recombinant protein and porcine epidemic diarrhea vaccine composition
  • Recombinant protein and porcine epidemic diarrhea vaccine composition

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Construction of recombinant plasmids for eukaryotic expression of recombinant proteins

[0034] In this example, based on the S gene sequence of PEDV AH2012 / 12 strain (GenBank: KU646831.1), a fusion gene with the DNA sequence shown in SEQ ID NO. 6 was synthesized by Nanjing GenScript Biotechnology Co., Ltd., denoted as S1- Fc. The fusion gene S1-Fc contains three truncated fragments of the S protein gene, which are the N-terminal domain (NTD, aa19-233) with sialic acid binding activity in the S1 subunit, and the neutralizing antigen core region (COE, aa499~638) and the B cell recognition epitope (aa744~774) in the S2 subunit, these three truncated fragments are connected in series through the linker polypeptide (linker), and then fused with the Fc fragment of the pig IgG antibody.

[0035] The synthesized fusion gene was cloned into the plasmid vector pcDNA3.1 by conventional molecular biological means, and the recombinant plasmid pcDNA3.1-S1-Fc for expres...

Embodiment 2

[0040] Example 2: Preparation of recombinant protein for eukaryotic expression

[0041] This example is provided by Expi293 TM Expression systems to produce large quantities of recombinant proteins. ExpiFectamine used in this example TM 293 transfection kit was purchased from Gibco Company.

[0042] The specific operations are as follows:

[0043] (1) in 30mL Expi293 TM Inoculate 6 x 10 in expression medium 7 Expi293F TM Living cells;

[0044] (2) at 37℃, 8%CO 2 , Incubate cells in an orbital shaker at 125 rpm;

[0045] (3) Use an automated cell counter to determine cell number and viability, the cell density should be 3 × 10 6 ~5×10 6 cells / mL, cell viability should be > 95%;

[0046] (4) Use 25.5mL Expi293 in a 125mL flask TM Expression medium will be 7.5 x 10 7 cells were diluted to 2.9 × 10 6 pcs / mL;

[0047] (5) Dilute 30 μg of pcDNA3.1-S1-Fc eukaryotic expression plasmid to a total volume of 1.5 mL with Opti-MEM in tube A, and dilute 81 μL of ExpiFectami...

Embodiment 3

[0064] Example 3: Preparation of vaccine compositions

[0065] The vaccine composition was prepared by mixing the commercially available Freund's adjuvant and the layered double metal hydroxide (LDH) adjuvant prepared by the inventors with the purified recombinant protein prepared in Example 2, respectively.

[0066] The layered double metal hydroxide (LDH) adjuvant was prepared as follows: First, prepare 15 mL of a mixed salt solution containing 8.0 mmol of Mg(NO) 3 ) 2 and 4.0 mmol of Al(NO 3 ) 3 Next, prepare 20 mL of 4.0 mol / L NaOH solution, add 20 mmol of lactic acid (88%), and stir vigorously for 2 h to obtain a NaOH mixture; then, add 15 mL of mixed salt solution to 11 mL of NaOH mixture for reaction, and the reaction The resulting precipitate was sonicated in an ice bath for 10 minutes and centrifuged at 5000 rpm for 10 minutes to obtain pure LDH slurry, which was washed twice with water and then manually dispersed in 20 mL of water to obtain the prepared LDH adju...

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Abstract

The invention provides a recombinant protein and porcine epidemic diarrhea vaccine composition. The recombinant protein is a fusion protein that is tandemly formed by a truncated fragment from porcine epidemic diarrhea virus S protein (Spike, spike protein) and an Fc fragment of pig IgG. The truncated fragment of S protein is preferably selected from the S1 subunit of S protein. The N-terminal domain (NTD) with sialic acid binding activity in the base, the neutralizing epitope domain (COE) and multiple B cell recognition epitopes in the S2 subunit. The vaccine composition includes recombinant protein and adjuvant. After the recombinant protein of the present invention immunizes mice, it can produce higher levels of IgG antibodies and neutralizing antibody titers, CD 3+ cd 4+ 、CD 3+ cd 8+ The proportion of lymphocytes and the concentration of IFN-γ and IL-4 in lymphocytes showed a significant increase.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant protein and a porcine epidemic diarrhea vaccine composition. Background technique [0002] Porcine Epidemic Diarrhea Virus (PEDV) is a class I coronavirus that causes porcine epidemic diarrhea disease, a highly contagious intestinal disease characterized by severe watery diarrhea, vomiting and Dehydration, as well as some systemic symptoms such as vomiting, fever, anorexia, and lethargy. For suckling pigs, it is more sensitive to dehydration and manifests as more severe clinical symptoms, causing huge economic losses to the pig industry worldwide. [0003] At present, the porcine epidemic diarrhea vaccines on the market are mainly traditional PEDV inactivated vaccines and attenuated vaccines. However, inactivated vaccines require a large dose of inoculation, and a single inoculation cannot produce sufficient immunity, so emergency vaccination is not applic...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/85A61K39/215A61K39/39A61P31/14
CPCC07K14/005C12N15/85A61K39/12A61K39/39A61P31/14C07K2319/30C12N2770/20022C12N2770/20034C12N2770/20051C12N2800/107A61K2039/552A61K2039/55566A61K2039/55505A61K2039/575A61K39/215C07K2319/31C12N2770/20031C12N2770/20071
Inventor 李彬范宝超时丹怡周金柱郭容利何孔旺赵永祥朱雪蛟李澧汪伟王丹丹祝昊丹
Owner JIANGSU ACAD OF AGRI SCI
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