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Fusion protein of chicken Newcastle disease virus and its preparation method, application and vaccine

A fusion protein, chicken Newcastle disease technology, applied in the fields of application and vaccine, fusion protein of chicken Newcastle disease virus and preparation method thereof, can solve the problems of low antibody titer of expression product, weak immune protection, complicated production process, etc., and shorten the empty window. long-term, good antigenicity and wide application range

Active Publication Date: 2021-10-15
天康制药股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, inactivated vaccines, attenuated vaccines, and genetically engineered vaccines are mainly used for Newcastle disease vaccines. Among them, inactivated vaccines and attenuated vaccines have the disadvantages of complex production process, weak immune protection and high production costs, and are gradually replaced by genetically engineered vaccines.
Now commonly used Newcastle disease genetically engineered vaccines mostly use baculovirus to express HN protein or F protein to prepare vaccines. Although the vaccine is safe and stable, the antibody titer of the expressed product is low, which affects its practical application.

Method used

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  • Fusion protein of chicken Newcastle disease virus and its preparation method, application and vaccine
  • Fusion protein of chicken Newcastle disease virus and its preparation method, application and vaccine
  • Fusion protein of chicken Newcastle disease virus and its preparation method, application and vaccine

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preparation example Construction

[0045]The present invention also provides a method for preparing the above-mentioned fusion protein, which can be performed by expressing the gene of the above-mentioned fusion protein in the host, for example, but not limited to, expression in E. coli expression system, yeast expression system, insect expression system, plant expression system, or mammalian expression system. Since the protein expressed by mammalian cells is translated and processed, its structure and biological characteristics are closer to natural proteins, so the present invention preferably uses a mammalian expression system to express the fusion The gene for the protein, more preferably the gene for expressing the fusion protein using a CHO cell expression system. CHO cells are Chinese hamster ovary cells. The CHO cell expression system has the following advantages:

[0046] (1) It has accurate post-translational folding and modification functions, and the expressed protein is closest to the natural prot...

Embodiment 1

[0061] Embodiment 1: F-HN gene design and synthesis

[0062] F-HN gene design and synthesis: The F gene sequence and NH gene sequence of chicken Newcastle disease virus La Sota strain were selected for analysis, and the codon optimization of the F gene and HN gene sequences was carried out, and the conserved sequence and antigenic epitope sequence were retained. Make F gene and HN gene have broad-spectrum antigenicity. After optimization, the F gene (GeneID:912271) is designed to be 1032bp in length and has the sequence shown in SEQ ID NO.1; the HN gene (GeneID:912270) is optimized to be 1229bp in length and has the sequence shown in SEQ ID NO.2 . The linker sequence is used to connect the F gene and the HN gene, and the linker sequence has the sequence shown in SEQ ID NO.3. The full length of the F-HN gene is 2291bp, and has the amino acid sequence shown in SEQ ID NO.4 and the nucleotide sequence shown in SEQ ID NO.5. F-HN gene synthesis was completed by Shanghai Sangon Bi...

Embodiment 2

[0063] Embodiment 2: pcDNA3-F-HN recombinant plasmid construction

[0064] 2.1 Add restriction sites: Add restriction sites respectively upstream and downstream of the F-HN gene sequence by PCR amplification: HindⅢ, XbaⅠ, PCR amplification upstream primers are shown in SEQ ID NO.6, PCR amplification downstream Primers are shown in SEQ ID NO.7. pcDNA3-F-HN plasmid map as figure 1 shown.

[0065] 2.2 F-HN gene and vector double enzyme digestion reaction

[0066] 2.2.1 F-HN tandem gene and vector double enzyme digestion reaction

[0067] 10×buffer 2.5μL DNA sample 1μg HindⅢ 1μL XbaI 1μL

[0068] Supplement ddH 2 0 to 25 μL, mix each component uniformly and then bathe in water at 37°C for 2h.

[0069] 2.2.2 Recovery of the DNA fragments of the enzyme-digested target: Perform agarose gel electrophoresis on the digested products, and recover the DNA fragments using a DNA gel recovery kit (purchased from Beijing Laibao Technology Co., Ltd.) ...

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Abstract

The invention provides a fusion protein of chicken Newcastle disease virus, its preparation method, application and vaccine, and relates to the field of biotechnology. The fusion protein connects chicken Newcastle disease virus F gene and HN gene sequences in series, and the expressed fusion protein has the advantages of good antigenicity and high expression level. The fusion protein can be applied to the preparation of vaccines, antibodies and kits for detecting chicken Newcastle disease virus. The vaccine containing the fusion protein has good immunogenicity, and the geometric mean titer (GMT) of the HI antibody can be measured 14 days after the vaccine is not lower than 1:64, while the conventional inactivated vaccine 21 days after immunization, the HI antibody The geometric mean titer (GMT) is not lower than 1:64, which shortens the window period of vaccine immunity and reduces the production cost.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a fusion protein of chicken Newcastle disease virus and its preparation method, application and vaccine. Background technique [0002] Newcastle disease (ND) is an acute and contagious disease of poultry caused by Newcastle disease virus. Newcastle disease virus (NDV) belongs to the genus Avulavirus of the family Paramyxoviridae, and its genome is single. Negative-strand unsegmented RNA. Newcastle disease virus can mainly cause chicken septicemia, prone to diarrhea, dyspnea, nervous disorders, serosal and mucous membrane hemorrhage. The fatality rate of the disease is high, which seriously threatens poultry breeding and is a severe infectious disease. In the prevention and control of Newcastle disease, a lot of manpower, material and financial resources have been invested to study the prevention measures of Newcastle disease. [0003] At present, inactivated vaccines, attenuated v...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10C07K16/10G01N33/569A61K39/17A61P31/14
CPCA61K39/12A61K2039/552A61P31/14C07K14/005C07K16/1027C12N15/85C12N2760/18122C12N2760/18134G01N33/56983
Inventor 贺笋任立松李延涛陈泽良任郭子君
Owner 天康制药股份有限公司
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