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Mycoplasma hyopneumoniae subunit vaccine and its preparation method and application

A technology for mycoplasma hyopneumoniae and subunit vaccines, applied in biochemical equipment and methods, vaccines, veterinary vaccines, etc., can solve problems such as difficult production quality control, difficult cultivation of mycoplasma zoonoticum, and endotoxin pollution, and achieve enhanced mucosal immune protection effect, solve the endotoxin problem, reduce the effect of steric hindrance

Active Publication Date: 2022-06-28
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Since Mycoplasma hyopneumoniae has very strict requirements on the nutritional conditions of the culture medium, it is difficult to grow in general culture medium, and it is a kind of difficult culture in animal mycoplasma, and the culture period is long, and it is very easy to contaminate Mycoplasma hyorhina during the culture process. Mycoplasma flocculus, etc., make the production cost of Mycoplasma pneumoniae whole-bacteria inactivated vaccine remain high
Although the protein expressed by the baculovirus expression system has a post-translational modification system, the use of baculovirus to express foreign genes in insect cells can carry out post-translational modifications, correct protein folding and glycosylation, and the expression of eukaryotic gene products It can better phosphorylate, amidate, cut signal peptides, and form tertiary or quaternary structures, but it has disadvantages such as high production costs, many steps, long cycle, and difficult control of production quality.
Although the Escherichia coli expression system is simple and easy to operate, there are problems such as endotoxin contamination and inability to secrete and express

Method used

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  • Mycoplasma hyopneumoniae subunit vaccine and its preparation method and application
  • Mycoplasma hyopneumoniae subunit vaccine and its preparation method and application
  • Mycoplasma hyopneumoniae subunit vaccine and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of Mycoplasma Antigen Chimeric Protein Expression Vector Based on Bacillus megaterium Expression System

[0049] (1) Construction and identification of expression vector

[0050] 1. Acquisition of Escherichia coli heat-labile enterotoxin LTB gene

[0051] Referring to the E. coli LTB gene sequence (WP_012846869.1), a 390 bp LTB gene sequence was artificially synthesized according to the codon preference of Bacillus megaterium.

[0052] 2. Mycoplasma chimeric protein LTB-P36-P65-P46-Dcpep and LTB-P97-LpptM-P146-Dcpep gene acquisition and vector construction.

[0053] Referring to the genome sequence of Mhp (J strain NC_007295.1), according to the amino acid sequences of immunogenic proteins such as P36, P46, P65, P97C, P146N, LpptM, etc., artificially synthesized LTB-P36-P65 according to the codon preference of Bacillus megaterium -P46-Dcpep and LTB-P97-LpptM-P146-Dcpep chimeric protein genes, and add the corresponding restriction sites at both e...

Embodiment 2

[0068] Example 2 Preparation and animal experiments of chimeric protein fusion1 and fusion2 subunit vaccines

[0069] 1. Subunit vaccine prepared by the chimeric proteins fusion1 and fusion2 of the present invention

[0070] After measuring the concentration of purified fusion1 and fusion2, they were mixed with Montanide TM Gel01 adjuvant (purchased from SEPPIC) was emulsified to prepare a subunit vaccine, so that the protein concentration in each milliliter was 150 micrograms, which was used in the following examples and stored at 4°C.

[0071] 2. Inspection of the technical indicators of subunit vaccines prepared by the chimeric proteins fusion1 and fusion2 of the present invention

[0072] (1) Physical properties

[0073] Appearance This product is a milky white homogeneous emulsion. The dosage form is a one-way dosage form (oil-in-water), take a clean straw to absorb a small amount of vaccine drop in cold water, both on the water surface and in the water. Viscosity Us...

Embodiment 3

[0096] Example 3 Subunit challenge protection effect prepared by chimeric proteins fusion1 and fusion2

[0097] 1. Preparation and immunization of fusion1 and fusion2 subunit vaccines

[0098] Fusion1 and fusion2 prepared in Example 1 were sequentially added to a sterile beaker. Then add 38ml of PBS solution with pH of 7.2, and finally add 10ml of Gel01 adjuvant (produced by SEPPIC, France), so that the content of the two antigen proteins is respectively 150μg / ml, 37℃, 500rpm stirring for 10 minutes, that is, the vaccine is obtained 100ml.

[0099] The immunization and challenge situation are shown in Table 2.

[0100] Table 2 Vaccine immunization and challenge grouping

[0101]

[0102]

[0103] 2. Mycoplasma hyopneumoniae challenge results

[0104] The results of lung lesion index after challenge with Mycoplasma hyopneumoniae are shown in Table 3. The results showed that the average lung lesion index of vaccine 1 and vaccine 2 were 4.0 and 4.2, respectively. The l...

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Abstract

The invention provides a mycoplasma hyopneumoniae subunit vaccine, a preparation method and application thereof. The invention connects multiple antigenic proteins of the mycoplasma through a flexible linker to express a chimeric protein, and makes each protein independent of each other and can retain its own immunogenicity. By adding Escherichia coli heat-labile enterotoxin B subunit and dendritic cell-inducing peptide to the chimeric protein, the mucosal immune protection effect of the vaccine is greatly enhanced. The invention also relates to a preparation method of the mycoplasma hyopneumoniae chimeric antigen and its application in a subunit vaccine for preventing mycoplasma hyopneumoniae infection. Expressing the mycoplasma chimeric antigen of the present invention in Bacillus megaterium can be used for industrial production of safe mycoplasma hyopneumoniae subunit vaccine.

Description

technical field [0001] The invention belongs to the field of veterinary vaccines, in particular to a Mycoplasma hyopneumoniae subunit vaccine and a preparation method and application thereof. Background technique [0002] Mycoplasma hyopneumoniae (Mycoplasma hyopneumoniae, Mhp) can cause Mycoplasma hyopneumoniae pneumonia, also known as endemic pneumonia, is a chronic wasting respiratory disease in pigs, the clinical infection rate is very high. Its main symptoms are cough and wheezing, and it is characterized by high morbidity and low mortality. Once a pig herd is infected with Mhp, it is difficult to clear it, which not only seriously affects the growth and development of the pig herd, and increases the dosage of drugs, but also is prone to secondary infection with PRRSV, PCV, etc., which leads to the failure of various vaccinations. The incidence of Mycoplasma suis pneumonia in my country is 50% to 80%, and the mortality rate in new epidemic areas is 5% to 15% (mostly su...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/75A61K39/02A61K39/39A61P11/00A61P31/04
CPCA61K39/0241A61K39/39A61P11/00A61P31/04C12N15/75C07K14/30C12N2800/22A61K2039/55544A61K2039/552A61K2039/55516C07K2319/55
Inventor 徐高原卢强王凤陈波郝根喜张华伟周明光
Owner WUHAN KEQIAN BIOLOGY CO LTD
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