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PCV2 type baculovirus and mycoplasma hyopneumoniae bivalent inactivated vaccine and preparation method thereof

A technology of mycoplasma hyopneumoniae and double inactivated vaccine, which is applied in the direction of virus antigen components, virus/bacteriophage, botanical equipment and methods, etc. It can solve the problem that the long-term sustained release effect of water adjuvants is not as good as that of oil adjuvants and water-based adjuvants. Response is small and other problems, to achieve the effect of improving immunogenicity, small animal stress response, and improving stability

Inactive Publication Date: 2021-02-05
BEIJING KEMUFENG BIOLOGICAL PHARMA +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among the adjuvant materials in the prior art, water-based adjuvants have less side effects, early immune protection, and are favored for their convenience. Released immune booster is necessary

Method used

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  • PCV2 type baculovirus and mycoplasma hyopneumoniae bivalent inactivated vaccine and preparation method thereof
  • PCV2 type baculovirus and mycoplasma hyopneumoniae bivalent inactivated vaccine and preparation method thereof
  • PCV2 type baculovirus and mycoplasma hyopneumoniae bivalent inactivated vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] The construction of embodiment 1 recombinant baculovirus

[0065] Optimization and synthesis of target genes

[0066] The nucleotide sequence of porcine circovirus type 2 ORF2b gene was designed with reference to PCV2b subtype DBN-SX07 strain, and the original nucleotide sequence of ORF2b gene of PCV2b subtype strain was obtained as shown in SEQ ID No.5 (702bp), PCV2b subtype The original amino acid sequence of the strain Cap protein is shown in SEQ ID No.6 (233aa).

[0067] In this embodiment, taking the optimization of the ORF2b gene of PCV2b subtype strain as an example, the 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.

[0068] On 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 restrictio...

Embodiment 2

[0089] Example 2 Insect cell bioreactor serum-free suspension culture expresses CAP protein

[0090] Preparation of poisonous seeds for production

[0091] The basic virus seeds were inoculated at MOI=0.5-2.0 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.

[0092] Serum-free Suspension Culture of SF9 Cells in Bioreactor and Quantification of CAP Protein Expression

[0093] 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 until the cell density reached 2.0×10 6 -...

Embodiment 3

[0104] In this example, the nucleotide sequence of the porcine circovirus type 2 ORF2a gene was designed with reference to the PCV2a subtype strain, and the original nucleotide sequence of the ORF2a gene of the PCV2a subtype strain was obtained as shown in SEQ ID No.7 (702bp), The original amino acid sequence of the Cap protein of the PCV2a subtype strain is shown in SEQ ID No.8 (233aa).

[0105] 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 porcine circovirus type 2 baculovirus vector and mycoplasma hyopneumoniae bivalent inactivated vaccine, and furtherdiscloses a preparation method and application of the porcine circovirus type 2 baculovirus vector and mycoplasma hyopneumoniae bivalent inactivated vaccine. In the bivalent inactivated vaccine, theporcine circovirus type 2 baculovirus vector is a porcine circovirus type 2 isolate, and comprises two subtype strains of PCV2a and PCV2b, and CAP protein codons are subjected to insect cell baculovirus codon optimization, artificially synthesized codon-optimized PCV2 ORF2 sequences, cloning and transfection of SF9 cells to obtain recombinant baculoviruses. Then, SF9 or High five cells are infected to efficiently express CAP protein virus-like particles (VLPs), so that the immunogenicity of the protein is improved, and the minimum dosage of each pig is 4 [mu] g. The mycoplasma hyopneumoniae isa DJ-166 strain, the viable bacteria titer is not lower than 1010CCU / ml, the immunogenicity is good, and the mycoplasma hyopneumoniae content is not lower than 50[mu]g / pig. Two antigens of the bivalent inactivated vaccine do not interfere with each other, and two kinds of protection of one-injection immunization can be achieved.

Description

technical field [0001] The invention belongs to the field of veterinary vaccines, in particular to a porcine circovirus type 2 baculovirus vector and a dual inactivated vaccine of mycoplasma hyopneumoniae, and further discloses a preparation method and application thereof. Background technique [0002] Porcine circovirus type 2 (PCV2) can cause piglet weaning multisystemic wasting syndrome (PMWS), porcine respiratory disease syndrome (PRDC), porcine skin and nephrotic syndrome (PDNS), reproductive disorders in sows, etc. , causing great economic losses to the pig industry all over the world. The PCV2a subtype prevails in domestic pig farms, and there are some PCV2b subtype strains at the same time. There is a certain cross-protection between the two, but it cannot be completely protected. The full length of the PCV2 genome is 1767 / 1768bp, with a total of 11 open reading frames (Open reading frames, ORFs), of which ORF2 encodes the virus's only structural protein, the nucleo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/295A61K39/02A61K39/12A61K39/39A61P31/20A61P31/04A61P37/04C12N15/866C12N15/66C12N15/34C12N1/20C12R1/35
CPCA61K39/0241A61K39/12A61K39/39A61K2039/521A61K2039/55511A61K2039/55577A61K2039/70A61P31/04A61P31/20A61P37/04C07K14/005C12N1/20C12N15/66C12N15/86C12N2710/14043C12N2750/10022C12N2750/10034C12N2750/10051C12N2800/105C12N2800/22
Inventor 于萍萍刘奇张渊魁王敏刘昊徐慧敏吕帅阳汤波刘飞卢晓楠韩昱鹏
Owner BEIJING KEMUFENG BIOLOGICAL PHARMA
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