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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 large inoculation, insufficient immunity, and inapplicability of emergency vaccination, and achieve low production cost, convenient large-scale production, and good immune response Effect

Active Publication Date: 2021-08-24
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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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, 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 sequentially connected through a linker polypeptide (linker), and then fused with the Fc fragment of the pig IgG antibody.

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

Embodiment 2

[0040] Embodiment 2: prepare the recombinant protein of eukaryotic expression

[0041] In this example, Expi293 TM expression systems to produce large quantities of recombinant proteins. The used ExpiFectamine of this embodiment TM 293 transfection kit was purchased from Gibco.

[0042] The specific operation is as follows:

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

[0044] (2) At 37°C, 8% CO 2 , Incubating the cells in an orbital shaker at 125rpm;

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

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

Embodiment 3

[0064] Embodiment 3: preparation vaccine composition

[0065] The commercially available Freund's adjuvant and the layered double metal hydroxide (LDH) adjuvant prepared by the inventors were mixed with the purified recombinant protein prepared in Example 2 to prepare vaccine compositions.

[0066] The layered double hydroxide (LDH) adjuvant was prepared as follows: First, prepare 15 mL of mixed salt solution containing 8.0 mmol of Mg(NO 3 ) 2 and 4.0mmol of Al(NO 3 ) 3 ; Next, prepare 20mL of 4.0mol / L NaOH solution, add 20mmol of lactic acid (88%), stir vigorously for 2h to obtain NaOH mixture; then, add 15mL of mixed salt solution to 11mL of NaOH mixture for reaction, and react The resulting precipitate was sonicated in an ice bath for 10 minutes, centrifuged at 5000 rpm for 10 minutes to obtain a pure LDH slurry, and the LDH slurry was washed twice with water, and then manually dispersed in 20 mL of water to obtain the prepared LDH adjuvant. Use Nicomp 380Z3000 nano p...

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Abstract

The invention provides a recombinant protein and porcine epidemic diarrhea vaccine composition. The recombinant protein is a fusion protein formed by connecting a truncated fragment from a porcine epidemic diarrhea virus S protein (Spike, spike protein) and an Fc fragment of porcine IgG in series, and the truncated fragment of the S protein is preferably selected from an N-terminal structural domain (NTD) with sialic acid binding activity in an S1 subunit of the S protein, a neutralizing epitope domain (COE) and a plurality of B cell recognition epitopes in an S2 subunit. The vaccine composition comprises a recombinant protein and an adjuvant. After a mouse is immunized with the recombinant protein, a high-level IgG antibody and a high-level neutralizing antibody titer can be generated, and the proportion of CD3 + CD4 + and CD3 + CD8 + lymphocytes and the concentration of IFN-gamma and IL-4 in the lymphocytes are remarkably increased.

Description

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

Claims

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Application Information

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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 ACADEMY OF AGRICULTURAL SCIENCES
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