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Bovine parainfluenza virus recombinant antigen and application thereof

A bovine parainfluenza virus, recombinant antigen technology, applied in the direction of virus antigen components, viruses, virus peptides, etc., can solve the problems of poor immune effect, reversion, short immune period, etc., to increase immunogenicity, long immune period, Strong immunogenic effect

Active Publication Date: 2021-01-22
苏州沃美生物有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the vaccines for preventing BPIV3 are mainly attenuated vaccines and inactivated vaccines. Although the manufacturing process of general inactivated vaccines is simple, the immunity period is short and the immune effect is poor. Attenuated vaccines can induce humoral immunity and cellular immunity, but there is a risk of reversion. Likely, making immunized cattle a potential source of infection

Method used

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  • Bovine parainfluenza virus recombinant antigen and application thereof
  • Bovine parainfluenza virus recombinant antigen and application thereof
  • Bovine parainfluenza virus recombinant antigen and application thereof

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preparation example Construction

[0043] The preparation method of the bovine parainfluenza virus recombinant antigen according to one embodiment of the present invention comprises the following steps: cultivating the above-mentioned host cells under suitable conditions, collecting the culture medium and / or the lysate of the host cells, and then separating and purifying to obtain bovine parainfluenza virus Viral Recombinant Antigen.

[0044] In a specific example, the separation and purification method includes one or more of ion exchange chromatography, hydrophobic chromatography, affinity chromatography and molecular sieve, which are not limited thereto and can be selected according to needs.

[0045] The bovine parainfluenza vaccine according to an embodiment of the present invention comprises the above-mentioned bovine parainfluenza virus recombinant antigen and a pharmaceutically acceptable adjuvant.

[0046] In a specific example, the adjuvant can be one or a combination of two or more of MONTANIDE ISA 2...

Embodiment 1

[0049] Embodiment 1 Construction of recombinant eukaryotic expression vector pCI-F-GS

[0050] 1. F gene amplification and purification

[0051] The codon-optimized F gene (SEQ ID NO: 3) was synthesized in Nanjing GenScript Biotechnology Co., Ltd. and cloned into the pUC-57 vector to obtain the pUC-F plasmid vector. Use pUC-F as a template and use F-F and F-R shown below as primers for PCR amplification. The amplification system is shown in Table 1. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0052] F-F: 5'-atactcgagatgttttttggcgaaattatt-3'

[0053] F-R: 5'-ataggtaccttatttgcccgcgctttt-3'

[0054] Table 1 F gene amplification system

[0055] reactive component Unit (μL) pUC-F plasmid 1 F-F primer 1 F-R primer 1 2×PCR MasterMix 12.5 wxya ...

Embodiment 2

[0072] Example 2 Construction of recombinant eukaryotic expression vector pCI-F-HN-GS

[0073] 1. Amplification and purification of HN gene expression cassette

[0074] The codon-optimized HN gene expression cassette (SEQ ID NO: 4) was synthesized in Nanjing GenScript Biotechnology Co., Ltd. and cloned into the pUC-57 vector to obtain the pUC-HN plasmid vector. Using pUC-HN as a template and HN-F and HN-R shown below as primers for PCR amplification, the amplification system is shown in Table 5. The reaction conditions were: pre-denaturation at 94°C for 5 minutes; denaturation at 95°C for 45 seconds, renaturation at 60°C for 45 seconds, extension at 72°C for 2 minutes, 30 cycles; extension at 72°C for 10 minutes, and storage at 4°C.

[0075] HN-F: 5'-ataggtaccatgcagctgtgcagcaccccg-3'

[0076] HN-R: 5'-atactcgagttatttgcccgcgcttttgctggt-3'

[0077] Table 5 HN gene expression cassette amplification system

[0078]

[0079]

[0080] Perform gel electrophoresis on the PCR...

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Abstract

The invention relates to a bovine parainfluenza virus recombinant antigen and application thereof. The bovine parainfluenza virus recombinant antigen comprises a bovine parainfluenza virus recombinantF protein with an amino acid sequence as shown in SEQ ID NO:1 and a bovine parainfluenza virus recombinant HN protein with an amino acid sequence as shown in SEQ ID NO:2, and the bovine parainfluenzavirus recombinant F protein and the bovine parainfluenza virus recombinant HN protein form a heterodimer. According to the bovine parainfluenza virus recombinant antigen, the local fragment of the screened F protein and the local fragment of the screened HN protein are taken as the basis rather than full-length protein, and a bovine antibody Fc fragment is added to a C end of an F protein fragment and a C end of an HN protein fragment, so that more stable heterodimer can be formed when the two protein fragments are expressed in one cell simultaneously, and the immunogenicity of the protein isremarkably improved.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a bovine parainfluenza virus recombinant antigen and its application. Background technique [0002] Bovine parainfluenza (BP) is an acute contagious disease of cattle caused by bovine parainfluenza virus type 3 (BPIV3). The disease has a worldwide distribution and can infect a variety of animals, including cattle, sheep, goats, and wild ruminants, as well as humans and non-primate humans. BPIV3 mainly causes pneumonia, bronchopneumonia, and substantive lesions of the infected lung lobes in adult cattle, calves, lambs, and young goats. . BPIV3 infection can gradually cause lung tissue damage and immunosuppression in cattle, and it can be secondary to bacterial or mycoplasma infection under stress conditions such as transportation, cold and starvation, such as Pasteurella multocida, Mannella hemolytica, Bovine Mycoplasma, etc., thereby causing severe bronchopneumonia, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/155A61P31/14
CPCA61K39/12A61K2039/53A61K2039/552A61P31/14C07K14/005C07K2319/00C12N5/0682C12N15/85C12N2510/02C12N2760/18622C12N2760/18634C12N2800/107C12N2800/22
Inventor 曹文龙孔迪滕小锘张大鹤
Owner 苏州沃美生物有限公司
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