Quad inactivated vaccine of PCV2-type baculovirus, swine mycoplasma hyopneumoniae, porcine influenza virus and haemophilus parasuis

A technology of mycoplasma hyopneumoniae and swine influenza virus, which is applied in the direction of virus antigen components, virus/bacteriophage, vaccines, etc., and can solve the problem of no porcine circovirus haemophilus parasuis

Inactive Publication Date: 2021-02-23
BEIJING KEMUFENG BIOLOGICAL PHARMA +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, there are no combined vaccines for porcine circovirus type 2, mycoplasma hyopneumoniae, swine influenza virus and Haemophilus parasuis domestically. Blank of virus (H1N1 subtype), Haemophilus parasuis (type 4, 5, 13) quadruple inactivated vaccine

Method used

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  • Quad inactivated vaccine of PCV2-type baculovirus, swine mycoplasma hyopneumoniae, porcine influenza virus and haemophilus parasuis
  • Quad inactivated vaccine of PCV2-type baculovirus, swine mycoplasma hyopneumoniae, porcine influenza virus and haemophilus parasuis
  • Quad inactivated vaccine of PCV2-type baculovirus, swine mycoplasma hyopneumoniae, porcine influenza virus and haemophilus parasuis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] The construction of embodiment 1 PCV2 type virus recombinant baculovirus

[0079] Optimization and synthesis of target genes

[0080] Design the PCV2 type ORF2b gene nucleotide sequence with reference to the PCV2b subtype DBN-SX07 strain, and obtain the original nucleotide sequence of the PCV2 subtype strain ORF2b gene as shown in SEQ ID No.5 (702bp), and the PCV2b subtype strain Cap protein The original amino acid sequence is shown in SEQ ID No.6 (233aa).

[0081] In this embodiment, the optimization of the ORF2b gene of the PCV2b subtype strain is taken as an example, and its codon optimization process is described in detail, and the optimization of the ORF2a gene of the corresponding PCV2a subtype strain is carried out with reference to this process.

[0082] Under the premise of keeping the amino acid of PCV2b subtype strain ORF2b unchanged, the codons were transformed into eccentric codons in the expression system of insect cell baculovirus, EcoRI and XhoI restric...

Embodiment 2

[0103] Example 2 Insect cell bioreactor serum-free suspension culture expresses CAP protein

[0104] Preparation of poisonous seeds for production

[0105] The basic virus seeds were inoculated at MOI=0.01-5.0 and grew well, with a cell density of 2.0×10 6 -2.5×10 6 / ml of Sf9 cells, cultivated at 27°C for 72-96 hours, harvested the virus liquid, quantitatively dispensed, indicated the name, harvest date, virus generation, etc., and carried out virus content detection, specificity detection, and purity detection. Content ≥ 1.0×10 8 PFU / ml, store at 2-8°C for later use.

[0106] Serum-free Suspension Culture of SF9 Cells in Bioreactor and Quantification of CAP Protein Expression

[0107] Suspension cell culture method was used. Take a 2000ml shake flask to culture 800ml of SF9 cells, the cell viability is 95%, and the cell density reaches 2.0×10 6 -2.5×10 6 cells / ml, according to 0.5×10 6 Cells / ml were transferred to a 5L bioreactor and continued to culture for 3-4 days...

Embodiment 3

[0119] In this example, the PCV2 type ORF2a gene nucleotide sequence was designed with reference to the PCV2a subtype strain, and the original nucleotide sequence of the PCV2a subtype strain ORF2a gene was obtained as shown in SEQ ID No.7 (702bp), and the PCV2a subtype strain The original amino acid sequence of the Cap protein is shown in SEQ ID No.8 (233aa).

[0120] Similarly, the optimization process of the above-mentioned PCV2a subtype strain ORF2a gene is carried out with reference to the method of the above-mentioned embodiment 1-2, and the obtained PCV2b type CAP protein has an amino acid sequence as shown in SEQ ID No.2, and its coding gene has a sequence as shown in SEQ ID No. The nucleotide sequence shown in 1.

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Abstract

The invention belongs to the field of veterinary vaccines, particularly relates to a quad inactivated vaccine of PCV2-type baculovirus, swine mycoplasma hyopneumoniae, a porcine influenza virus and haemophilus parasuis and further discloses a preparation method for the quad inactivated vaccine and application of the quad inactivated vaccine. The quad inactivated vaccine disclosed by the inventioncontains inactivated protein Cap expressed by the PCV2-type baculovirus, inactivated swine mycoplasma hyopneumoniae, an inactivated porcine influenza virus H1N1subtype virus, inactivated haemophilus parasuis types 4, 5 and 13 and an adjuvant of the vaccine; four kinds of antigens are free of interference to one another, four kinds of protection can be achieved by one injection, and four kinds of epidemic diseases can be prevented through one-time immunization; and meanwhile, in view of toxic substance counteracting protection and serum antibody level, the immunization effect reaches or exceedsthat of each single commodity vaccine, the immunization duration is long, the efficacy is durable, and the quad inactivated vaccine has the advantages that the safety is good, the preparation methodis simple, the immunization is convenient, the immunization cost is reduced, and the like.

Description

technical field [0001] The invention belongs to the field of veterinary vaccines, in particular to a quadruple inactivated vaccine of PCV2 type baculovirus, mycoplasma hyopneumoniae, swine influenza virus and haemophilus parasuis, and further discloses its preparation method and application. Background technique [0002] Porcine circovirus is also known as "AIDS" of pigs. The immune system of infected pigs will be completely destroyed, and the immune response to other vaccines will be lost, leading to immune failure and secondary infection of other pathogens. Among them, according to statistics, clinically, the secondary infection caused by porcine circovirus usually exceeds 50%, and the mortality rate reaches more than 40%. Among them, porcine circovirus type 2 (Porcine circovirus type 2, PCV2) can cause piglet weaning multisystemic wasting syndrome (PMWS), porcine respiratory disease syndrome (PRDC), porcine skin and nephrotic syndrome (PDNS), sow reproductive Obstacles, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/295A61K39/12A61K39/02A61K39/102A61K39/145A61K39/39A61P31/16A61P31/04A61P31/20C12N15/866C12N15/66C12N15/34C12N1/20C12N7/00C12R1/35C12R1/21C12R1/93
CPCA61K39/0241A61K39/102A61K39/12A61K39/39A61K2039/521A61K2039/5256A61K2039/55505A61K2039/55583A61K2039/572A61K2039/575A61K2039/70A61P31/04A61P31/16A61P31/20C07K14/005C12N1/20C12N7/00C12N15/66C12N15/86C12N2710/14043C12N2750/10022C12N2750/10034C12N2750/10051C12N2760/16134C12N2760/16151C12N2800/105C12N2800/22
Inventor 刘奇车艳杰于萍萍王亚玲汤波刘飞孙石静聂思静刘运平张渊魁王敏
Owner BEIJING KEMUFENG BIOLOGICAL PHARMA
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