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A foot-and-mouth disease vaccine mucosal immune enhancer, inactivated vaccine and preparation method thereof

A foot-and-mouth disease vaccine and Escherichia coli technology, applied in chemical instruments and methods, antiviral agents, pharmaceutical formulations, etc., can solve the problems of low antibody titer and non-detection, and achieve the effect of reducing labor costs and reducing the amount of virus carried

Active Publication Date: 2020-07-31
JIANGSU ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Bacterial flagellin (Flagella, F) and heat-labile enterotoxin (Labile toxin, LT) are adjuvants with good immunoenhancing effects. When used alone, they can increase the titer of serum neutralizing antibodies to a certain extent, but the induced IgA antibody titers are low or even undetectable

Method used

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  • A foot-and-mouth disease vaccine mucosal immune enhancer, inactivated vaccine and preparation method thereof
  • A foot-and-mouth disease vaccine mucosal immune enhancer, inactivated vaccine and preparation method thereof
  • A foot-and-mouth disease vaccine mucosal immune enhancer, inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Preparation of flagellin (F) and heat labile enterotoxin B subunit (LTB)

[0020] 1. Construction of recombinant plasmid

[0021] The complete amino acid sequence of the non-pathogenic E. coli flagellin (abbreviated as F) is shown in SEQ ID NO:1. The complete amino acid sequence of the heat labile enterotoxin B subunit (abbreviated as LTB) is shown in SEQ ID NO:2. The applicant used computer software to optimize the coding genes of F and LTB, and the optimized sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The codon optimized F and LTB coding genes were sent to GenScript for synthesis. The two synthetic sequences were cloned into the pCold I vector EcoR Ⅰ and Sal Between Ⅰ restriction sites, recombinant plasmids pCold I-F (with SEQ ID NO: 3 inserted) and pCold I-LTB (with SEQ ID NO: 4 inserted) were obtained.

[0022] 2. Construction and identification of recombinant bacteria

[0023] The obtained recombinant plasmids pCold I-F and pCold I-L...

Embodiment 2

[0034] Example 2 Preparation of Foot-and-Mouth Disease Vaccine Mucosal Immunity Enhancer and Control Immunity Enhancer

[0035] Recombinant proteins F and LTB were prepared according to the method in Example 1, FMDV inactivated virus solution (98 strains of porcine foot-and-mouth disease virus Burma, 4ug / ml) was provided by Lanzhou Veterinary Research Institute, and ISA206 was provided by Shanghai Seppic. PBS buffer (phosphate buffer, pH=7.4, 0.01mM) contains 0.27g / L potassium dihydrogen phosphate, 1.42g / L disodium hydrogen phosphate, 8g / L sodium chloride and 0.2g / L potassium chloride The aqueous solution was purchased from Nanjing Research Aid Biotechnology Co., Ltd.

[0036] 1. Foot-and-mouth disease vaccine mucosal immune enhancer and vaccine prepared by using the immune enhancer

[0037] (1) Preparation of mucosal immune enhancers A, B and C

[0038] Preparation of the recombinant protein F mother solution: The recombinant protein F purified in Example 1 was prepared into a solut...

Embodiment 3

[0050] Example 3: Immunity effects of foot-and-mouth disease vaccines A, B and C on pigs

[0051] Immunization method: 35 healthy FMDV-negative piglets aged 60 days were randomly divided into 7 groups with 5 pigs in each group. A group of piglets were immunized with foot-and-mouth disease vaccine A, foot-and-mouth disease vaccine B, foot-and-mouth disease vaccine C, control vaccine 1, control vaccine 2, control vaccine 3, and control vaccine 4, each at a dose of 2 ml / head. 14 days, 28 days and 56 days after immunization, blood was collected to determine the level of IgG antibodies, and nasal swabs were collected to detect the level of IgA antibodies.

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Abstract

The invention provides a foot-and-mouth disease vaccine mucosal immunopotentiator, an inactivated vaccine and a preparation method thereof, and belongs to the field of biological pharmacy. The mucosal immunopotentiator contains non-pathogenic escherichia coli flagellin and non-heatproof enterotoxin B subunit with a mass ratio of (0.2-4) to 1; the amino acid sequences of the non-pathogenic escherichia coli flagellin and the non-heatproof enterotoxin B subunit are shown by SEQ ID NO:1 and SEQ ID NO:2. The invention also provides a foot-and-mouth disease vaccine which contains inactivated foot-and-mouth disease virus, 25-80mu g / mL of the non-pathogenic escherichia coli flagellin and 25-80mu g / mL of the non-heatproof enterotoxin B subunit. The mucosal immunopotentiator provided by the invention can remarkably enhance immune response of an immune animal to antigen, particularly mucosal immune response; after immunization with the inactivated vaccine containing the immunopotentiator, high-titer IgG and sIgA can be induced, and the immune effect is good.

Description

Technical field [0001] The invention belongs to the field of biopharmaceuticals, and specifically relates to a foot-and-mouth disease vaccine mucosal immune enhancer, an inactivated vaccine and a preparation method thereof. Background technique [0002] Foot and mouth disease (Foot and mouth disease, FMD) is an acute, thermally highly contact infectious disease caused by foot-and-mouth disease virus (Foot-and-Mouth Disease Virus, FMDV) that causes cloven-hoofed animals to occur. Skin blisters and ulcers are the main clinical symptoms. my country lists FMD as the first type of animal disease. At present, vaccination is still the main means of preventing foot-and-mouth disease in my country. The foot-and-mouth disease vaccines currently in use are mainly inactivated vaccines, but the existing inactivated vaccines have obvious shortcomings, such as low antibody levels and inability to induce mucosal antibodies. FMDV is mainly infected through mucous membranes, and vaccination is ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/39A61P37/04A61K39/135A61P31/14C07K14/245
CPCA61K39/12A61K39/39A61K2039/5252A61K2039/55516A61K2039/55544C07K14/245A61K2300/00
Inventor 张雪寒孙小涵张碧成郭芸芸俞正玉祝昊丹汪伟何孔旺
Owner JIANGSU ACAD OF AGRI SCI
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