Recombinant avian influenza trivalent vaccine as well as preparation method and application thereof

A flu vaccine, avian flu technology, applied in recombinant DNA technology, chemical instruments and methods, pharmaceutical formulations, etc., can solve problems such as inapplicability of emergency prevention, biosafety risks, affecting secondary immunization, etc., and achieve less antigen immunization dosage , The effect of reducing stress response and long half-life in vivo

Active Publication Date: 2022-04-26
北京中海生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the main method to prevent and control the occurrence of avian influenza is vaccination. The commonly used H5 subtype influenza vaccines are whole virus inactivated vaccines, such as Re-1, Re-4, Re-5, Re-6 and Re-7 commercially available in my country. , Re-8 and Re-10 strains and other vaccines, but this type of vaccine involves the operation of live viruses in the production process, so there are biosafety risks
In addition, there are recombinant live vector vaccines, DNA vaccines, etc. Since the presence of recombinant vector vaccines affects secondary immunization, DNA vaccines require multiple immunizations and are not suitable for emergency prevention, so there are no large numbers of applications in the market, so the developed A new and highly effective universal influenza subunit vaccine is imminent

Method used

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  • Recombinant avian influenza trivalent vaccine as well as preparation method and application thereof
  • Recombinant avian influenza trivalent vaccine as well as preparation method and application thereof
  • Recombinant avian influenza trivalent vaccine as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0106] Embodiment 1, the design of recombinant fusion protein and its coding gene sequence

[0107] The recombinant fusion protein of the present invention includes influenza protein (HA fusion protein) and influenza skeleton protein (M2-NA fusion protein). The specific protein sites are shown in Table 1.

[0108] Influenza protein: the present invention fuses the HA1 fragment of the avian influenza H5N1 subtype, the HA1 fragment of the avian influenza H9N2 subtype with the HA1 fragment and the HA2 fragment of the avian influenza H7N9 subtype to obtain the gene fragment shown in sequence 1, and its coding sequence 2 The protein shown (positions 19-3360 of Sequence 1 is the coding gene sequence of Sequence 2). Among them, the 19th-90th position of the sequence 1 is the coding gene sequence of the signal peptide, the 91-912th position of the sequence 1 is the coding gene sequence of the avian influenza H5N1 subtype HA1 fragment, and the 913-957th position of the sequence 1 is t...

Embodiment 2

[0113] Embodiment 2, preparation of recombinant fusion protein

[0114] 1. Construction of recombinant fusion protein expression vector

[0115] 1. Construction of recombinant vector pCHHA3372

[0116] The fragment between the AVRII recognition sites of the pCHO1.0 vector was replaced with the DNA molecule shown in Sequence 1 to obtain the recombinant vector pCHHA3372. The recombinant vector pCHHA3372 was sequenced, and the sequenced correct vector was verified by enzyme digestion ( figure 1 ).

[0117] 2. Construction of recombinant vector pCHO1.0HA-M2-NA

[0118] The fragment between the ECORV recognition sites of the recombinant vector pCHHA3372 was replaced with the DNA molecule shown in Sequence 3 to obtain the recombinant vector pCHO1.0HA-M2-NA. The recombinant vector pCHO1.0HA-M2-NA was sequenced, and the correct sequenced vector was digested and verified ( figure 2 ).

[0119] 2. Obtaining recombinant cell CHO

[0120] 1. One day before transfection, inoculate ...

Embodiment 3

[0152] Embodiment 3, the preparation of avian influenza fusion protein trivalent vaccine

[0153] 1. The water-based adjuvant SummitA004 (Luohe Summit Biotechnology Co., Ltd.) was sterilized by autoclaving at 115°C for 30-40 minutes in a seedling mixing tank, and then cooled to room temperature for later use.

[0154] 2. Properly dilute the stock solution of influenza fusion protein virus-like particles (obtained in step 1 of step 6 of Example 2) that has passed the test and mix well to obtain an antigen solution.

[0155] 3. Mix the antigen solution and the sterilized aqueous adjuvant according to the mass ratio of 4:1 to obtain a mixed solution, stir the mixed solution at 6000r / min for 30min, and add thimerosal with a final concentration of 0.005% before stopping the stirring and fully mixed to obtain the avian influenza fusion protein trivalent vaccine (H5 subtype + H7 subtype + H9 subtype) of the present invention, wherein the concentration of the fusion protein is 100 μg / ...

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Abstract

The invention discloses a recombinant avian influenza trivalent vaccine as well as a preparation method and application thereof. According to the invention, a cloned cell strain capable of secreting and expressing an HA1 neck ring part protein antigen in an H5N1 subtype, an H7N9 subtype and an H9N2 subtype and an H7N9 skeleton recombinant fusion protein is constructed, and the recombinant avian influenza trivalent vaccine disclosed by the invention is obtained through fermentation culture. Experiments show that the recombinant avian influenza trivalent vaccine immunizes SPF (specific pathogen free) chickens to generate neutralizing antibodies which are equal to those generated by products in the market at present, can be used for preventing avian influenza of H5N1 subtype, H7N9 subtype and H9N2 subtype, and has the advantages of low production cost, no need of chicken embryo culture viruses, environmental safety, no risk of virus dispersion and capability of preventing three types of influenza through one-time fermentation production.

Description

technical field [0001] The invention relates to a recombinant avian influenza trivalent vaccine and its preparation method and application, belonging to the field of veterinary products. Background technique [0002] Influenza virus belongs to the Orthomyxoviridae family and is a segmented RNA virus with a single negative strand. According to the different antigenic determinants on nucleoprotein (NP) and matrix protein (M1), influenza viruses can be divided into four types: A, B, C, and D. Types A and B can pose threats to human health, especially It is type A, which causes approximately 300,000-500,000 deaths each year due to global seasonal influenza and sporadic influenza, with a mortality rate as high as 60%. In addition to infecting humans, influenza A viruses can also cause influenza in poultry, swine, and other mammals. Type A influenza viruses circulating in chickens include subtypes such as H5N1, H7N9 and H9N2, among which, H5N1 subtype and H7N9 subtype have attra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/295A61K39/145A61P31/16
CPCC07K14/005C12N15/85C12N5/0682A61K39/12A61P31/16C12N2760/16122C12N2760/16151C12N2760/16123C12N2760/16134C07K2319/00C12N2800/107C12N2510/02A61K2039/552A61K2039/70A61K2039/5258
Inventor 伏显华
Owner 北京中海生物科技有限公司
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