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Porcine circovirus-mycoplasma pneumonia duplex subunit vaccine and preparation method thereof

A technology of porcine circovirus and mycoplasma pneumoniae, which is applied in the direction of virus/bacteriophage, virus antigen components, biochemical equipment and methods, etc., can solve the problem of high production cost, difficult growth, and high production cost of inactivated vaccines of whole bacteria of mycoplasma pneumoniae and other issues to achieve the effect of reducing production costs and shortening the growth cycle

Active Publication Date: 2018-12-21
WUHAN KEQIAN BIOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Since Mycoplasma hyopneumoniae has very strict requirements on the nutritional conditions of the culture medium, it is difficult to grow in general culture medium. 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 perform post-translational modifications, correct protein folding and glycosylation, and the expression of eukaryotic gene products It can better phosphorylate, lipase, phthalate, cleave signal peptide, and form tertiary or quaternary structures, etc., but it has the disadvantages of high production cost, many steps, long cycle, and difficult control of production quality.

Method used

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  • Porcine circovirus-mycoplasma pneumonia duplex subunit vaccine and preparation method thereof
  • Porcine circovirus-mycoplasma pneumonia duplex subunit vaccine and preparation method thereof
  • Porcine circovirus-mycoplasma pneumonia duplex subunit vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Construction of a mycoplasma antigen fusion protein and porcine circovirus Cap protein expression vector based on the Escherichia coli expression system

[0045] 1. Expression vector construction and identification

[0046] 1. Acquisition of Escherichia coli heat-labile enterotoxin LTB gene

[0047] 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 Escherichia coli.

[0048] 2. The acquisition and vector construction of the main immunogenic genes P36, P46, P65, P97, P146 and Lppt of Mycoplasma.

[0049] Referring to the genome sequence of Mhp (J strain NC_007295.1), respectively design primers for the whole gene sequence or partial gene sequence of immunogenic proteins such as P36, P46, P65, P97, P146, Lppt, etc. -lapping PCR方法,加上大肠杆菌不耐热性肠毒素LTB基因,将LTB(LTB-F:GCGGATCCATGgagatataccggatccatgccccagtctattacagaacta;LTB-R:tctaagcatttttttgttttccatactgattgcc...

Embodiment 2

[0072] Example 2 Subunit vaccines and animal experiments prepared by the fusion proteins Fusion1, Fusion2 and Cap protein of the present invention

[0073] 1. The subunit vaccine prepared by the fusion protein Fusion1, Fusion2 and Cap protein of the present invention

[0074] After measuring the concentration of the purified Fusion1, Fusion2 and Cap proteins, they were emulsified with MontanideTM Gel o1 adjuvant (purchased from SEPPIC) to prepare subunit vaccines, so that the concentration of each protein in each milliliter was 150 micrograms, which were used in the following examples and placed for 4 ℃ storage.

[0075] 2. Inspection of subunit vaccine technical indicators prepared by fusion proteins Fusion1, Fusion2 and Cap protein of the present invention

[0076] (1) Physical properties

[0077]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 dro...

Embodiment 3

[0106] Example 3 The protection effect of the subunit prepared by the fusion protein Fusion1, Fusion2 and Cap protein of the present invention against viruses

[0107] 1. Fusion1, Fusion2 and Cap protein subunit vaccine preparation and immunization

[0108] Fusion1, Fusion2 and Cap proteins prepared in Example 1 were sequentially added 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 three antigenic proteins are respectively 150 micrograms / ml, 37 ° C, 500 rpm, and stir for 10 In minutes, 100ml of vaccine is obtained.

[0109] The immunization and challenge conditions are shown in Table 2.

[0110] Table 2 Vaccine immunization and challenge groups

[0111]

[0112]

[0113] 2. Results of Mycoplasma hyopneumoniae challenge

[0114]Table 3 shows the results of lung lesion index after M. hyopneumoniae challenge. The results showed that the average lung ...

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Abstract

The invention belongs to the technical field of vaccine production, and specifically discloses a porcine circovirus-mycoplasma pneumonia duplex subunit vaccine and a preparation method thereof. The preparation method comprises the following steps: (1) dividing six coding genes of coded mycoplasma hyopneumoniae membrane proteins P36, P46, P65, P97, Lppt and P146 into two groups, constructing two mosaic genes which separately contain two groups of coding genes, and separately constructing the two mosaic genes in an expression vector; (2) constructing a coding gene of the porcine circovirus Cap protein into an expression vector; (3) constructing the expression vector comprising different genes into engineering bacteria, and expressing two mosaic genes to obtain two fused proteins and expressthe porcine circovirus Cap protein; and (4) utilizing the expressed protein to prepare the porcine circovirus-mycoplasma pneumonia duplex subunit vaccine. Multiple protens of the mycoplasma hyopneumoniae are fused, so that the growth period is greatly shortened, and production cost is greatly reduced.

Description

technical field [0001] The invention belongs to the technical field of vaccine production, and in particular relates to a porcine circovirus-mycoplasma pneumoniae dual subunit vaccine and a preparation method thereof. Background technique [0002] Porcine circovirus type 2 (PCV2) infection can cause post-weaning multisystemic wasting syndrome (PMWS), which mainly occurs in weaned piglets aged 6 to 15 weeks. Granulocyte infiltration etc. In addition, porcine circovirus type 2 infection can also cause porcine dermatitis and nephrotic syndrome, reproductive failure in sows, proliferative necrotizing pneumonia, and congenital tremor in piglets. PCV2 can also be co-infected with pathogens such as porcine parvovirus (PPV), porcine reproductive and respiratory syndrome virus (PRRSV), porcine pseudorabies virus (PRV), causing porcine respiratory disease syndrome, increasing the scope of disease diagnosis, prevention and control. difficulty. Therefore, how to effectively control p...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/70C12N15/81A61K39/295A61K39/12A61K39/02A61P31/20A61P31/04
CPCA61K39/0241A61K39/12A61P31/04A61P31/20C12N15/70C12N15/815C07K14/005C07K14/30C12N2750/10022C12N2750/10034A61K2039/552A61K2039/5256A61K2039/70Y02A50/30
Inventor 卢强徐高原周明光陈波张华伟郝根喜王凤
Owner WUHAN KEQIAN BIOLOGY CO LTD
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