Classical swine fever virus multimer vaccine and preparation method thereof

A technology of vaccines and recombinant vectors, applied in antiviral agents, chemical instruments and methods, medical preparations containing active ingredients, etc., can solve problems such as unsatisfactory immune effects, and achieve the effect of increasing stability and stabilizing the state

Active Publication Date: 2022-08-02
浙江洪晟生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese swine fever rabbit attenuated vaccine has played a decisive role in the prevention and treatment of swine fever, but in recent years, the immune effect has not been satisfactory

Method used

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  • Classical swine fever virus multimer vaccine and preparation method thereof
  • Classical swine fever virus multimer vaccine and preparation method thereof
  • Classical swine fever virus multimer vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1, fusion protein design and construction of recombinant expression vector

[0064] 1.1. Design of fusion protein

[0065] In order to improve the immune effect of CSFV-E2 protein, the gene encoding CSFV-E2 was fused with the gene encoding the constant region of the porcine IgG heavy chain, and the gene encoding CRM19L was fused with the gene encoding the constant region of the porcine IgG light chain. SpyTag is a polypeptide segment, and SpyCatcher is a corresponding protein. The two can recombine and spontaneously form iso-peptide bond coupling.

[0066] Entrusted Beijing Qingke Bio Co., Ltd. to synthesize fusion protein (CSFV-E2)-SpyTag-HIgG encoding gene (SEQ ID No. 1) and fusion protein CRM197-SpyCatcher-LIgG encoding gene (SEQ ID No. 2), wherein (CSFV -E2)-SpyTag-HIgG gene replaces the fragment between the HindIII and ECORI restriction sites of the PEE12.4 vector (a small fragment between the HindIII and ECORI restriction sites) to obtain a recombinant...

Embodiment 2

[0074] Example 2. Construction of stably transfected cell line and protein expression

[0075] 2.1. Construction of stably transfected cell line

[0076] The recombinant expression vector PEEGSE2197 of Example 1 was digested with Pvu I and recovered to obtain a linearized expression vector PEEGSE2197. The linearized expression vector PEEGSE2197 was transfected into CHO cells, and the transfection steps included:

[0077] Step 1: 24h before transfection, adjust the cell density to 2.5-3×10 6 cells / mL, cultured overnight.

[0078] Step 2: On the day of transfection, dilute the cell density to 3 x 10 with fresh medium 6 cells / mL, cell viability ≥95%.

[0079] Step 3: Prepare the DNA-PEI complex, dilute DNA and PEI with Opti-MEM respectively, the final DNA concentration during transfection is 1-2 μg / mL, the mass ratio of PEI to DNA is 5:1, and the diluted DNA is PEIs were allowed to stand for 5 minutes and then mixed 1:1, then gently inverted and mixed several times.

[0080...

Embodiment 3

[0100] Embodiment 3, E2 vaccine safety and effect verification

[0101] The E2 vaccine prepared in 2.4 in Example 2 was used for this step of the experiment, and the commercial vaccine was Kewenjing (a swine fever virus E2 protein recombinant baculovirus inactivated vaccine (WH-09 strain)).

[0102] 3.1. Safety experiment of recombinant fusion protein

[0103] Take 6-8 weeks old Balb / C mice (purchased from Shanghai Slack Laboratory Animal Co., Ltd.) with a body weight of 18-22 g, 20 (half male and half male), and randomly divided into groups, 5 mice in each group, a total of 4 groups . The first group was E2 vaccine 50μg group, the second group was E2 vaccine 100μg group, the third group was commercial vaccine group (positive control), and the fourth group was PBS group (negative control).

[0104] Group 1: Each mouse was first subcutaneously inoculated with 0.1 mL of the E2 vaccine solution (500 μg / mL vaccine A) in 2.4.1 of Example 2, so that the dose of fusion protein E2 w...

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Abstract

The invention discloses a multimer vaccine of hog cholera virus and a preparation method of the multimer vaccine, and belongs to the field of biological products. The active ingredient of the vaccine disclosed by the invention is a protein which is formed by connecting a fusion protein (CSFV-E2)-SpyTag-HIgG (Human Immunoglobulin G) and a fusion protein CRM197-SpyCatcher-LIGG. The invention also provides a preparation method of the protein, and the preparation method comprises the step of expressing coding genes of the fusion protein (CSFV-E2)-SpyTag-HIgG and the fusion protein CRM197-SpyCatcher-LIGG in biological cells to obtain the protein. According to the present invention, the SpyTag is fused in the E2 and Fc proteins by using the protein engineering method, the SpyTag and the diphtheria toxin mutant CRM197 fusion SpyCatcher are co-expressed, and finally the immune stimulant polymer E2 protein is formed, such that the state is stable, and the immune effect is good.

Description

technical field [0001] The invention belongs to the field of biological products, in particular to a multimeric vaccine for preventing swine fever and a preparation method thereof. Background technique [0002] Swine fever, commonly known as "rotten intestinal fever", is an acute, febrile and contact infectious disease caused by Hogcholera virus (Hogcholera virus, Swine fever virus, referred to as CSFV) of the Flaviviridae family and is highly contagious. and lethality. Pigs are the only natural host for the virus. The infectious disease was first discovered in Ohio in 1833, and its epidemic has spread all over the world for more than 100 years. It is classified as a Class A infectious disease by the International Bureau of Animal Epidemiology, and is listed as a Class I infectious disease in China's Animal Epidemic Prevention Law. The Chinese swine fever rabbit-attenuated vaccine has played a decisive role in the prevention and treatment of swine fever, but the immune ef...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/187A61K9/12A61P31/14
CPCC07K14/005C07K14/34C07K7/08C07K14/315C12N15/85A61K39/12A61K9/12A61P31/14C12N2770/24322C07K2319/30C07K2319/02C07K2319/00C07K2319/55C12N2800/107C12N2800/22C12N2770/24334A61K2039/552Y02A50/30
Inventor 查银河盖其静余晓玉张稳涛
Owner 浙江洪晟生物科技股份有限公司
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