Mycoplasma hyopneumoniae subunit vaccine and preparation method and application thereof

A technology for mycoplasma hyopneumoniae and subunit vaccines, applied in biochemical equipment and methods, vaccines, veterinary vaccines, etc., can solve the problems of inability to secrete and express, high production costs, and difficult control of production quality, so as to shorten the growth cycle and reduce Production cost, the effect of enhancing the protective effect of mucosal immunity

Active Publication Date: 2019-04-26
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 preparation method and application thereof
  • Mycoplasma hyopneumoniae subunit vaccine and preparation method and application thereof
  • Mycoplasma hyopneumoniae subunit vaccine and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Construction of the mycoplasma antigen chimeric protein expression vector based on the Bacillus megaterium expression system

[0049] (1) Expression vector construction and identification

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

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

[0052] 2. Acquisition of mycoplasma chimeric protein LTB-P36-P65-P46-Dcpep and LTB-P97-LpptM-P146-Dcpep genes 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., LTB-P36-P65 was artificially synthesized according to the codon preference of Bacillus megaterium - the gene of P46-Dcpep and LTB-P97-LpptM-P146-Dcpep chimeric protein, and corresponding enzyme ...

Embodiment 2

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

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

[0070] The concentration of purified fusion1 and fusion2 was measured with Montanide TM Gel01 adjuvant (purchased from SEPPIC) was emulsified to prepare a subunit vaccine with a protein concentration of 150 micrograms per milliliter, used in the following examples, and stored at 4°C.

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

[0072] (1) Physical properties

[0073] Appearance This product is milky white homogeneous emulsion. The dosage form is a one-way dosage form (oil-in-water). Take a clean straw to suck a small amount of vaccine and drop it in cold water, both on the water surface and in the water. Viscosity Use a pipette with an outlet diameter of 1.2mm to draw...

Embodiment 3

[0096] Example 3 The protective effect of subunits prepared by chimeric proteins fusion1 and fusion2 against viruses

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

[0098] Add fusion1 and fusion2 prepared in Example 1 in turn into a sterile beaker. Then add 38ml of PBS solution with a pH of 7.2, and finally add 10ml of Gel01 adjuvant (produced by SEPPIC, France), so that the contents of the two antigenic proteins are respectively 150μg / ml, stir at 37°C and 500rpm for 10 minutes, and the vaccine is obtained. 100ml.

[0099] See Table 2 for the status of immunization and challenge.

[0100] Table 2 Vaccine immunization and challenge groups

[0101]

[0102]

[0103] 2. Results of Mycoplasma hyopneumoniae challenge

[0104] Table 3 shows the results of lung lesion index after M. hyopneumoniae challenge. The results showed that the average lung lesion index of vaccine 1 and vaccine 2 were 4.0 and 4.2 respectively, and the lung lesion o...

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Abstract

The invention provides a mycoplasma hyopneumoniae subunit vaccine and a preparation method and application thereof. A plurality of antigenic proteins of mycoplasma are connected by a flexible Linker and expressed as a chimeric protein, and the proteins are mutually unaffected, and can retain respective immunogenicity. The mucosal immune protection effect of the vaccine is greatly enhanced by adding the escherichia coli heat-labile enterotoxin B subunit and the dendritic cell-inducing peptide to the chimeric protein. The invention also relates to a preparation method of a mycoplasma hyopneumoniae chimeric antigen and application of the mycoplasma hyopneumoniae chimeric antigen to the subunit vaccine for preventing mycoplasma hyopneumoniae infection. The mycoplasma chimeric antigen is expressed in bacillus megaterium and can be used for industrial preparation of safe mycoplasma hyopneumoniae subunit vaccines.

Description

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

Claims

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Application Information

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Patent Type & Authority Applications(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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