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Recombinant protein containing cap of classical swine fever e2 and circovirus and its preparation method and application

A protein and fusion protein technology, applied in the field of vaccines, can solve the problems of poor immune effect, immune death, short antibody time, etc., and achieve the effects of improving immunogenicity, improving protection effect, and improving the poor immune effect of expression products.

Active Publication Date: 2022-05-10
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

[0006] The technical problem to be solved by the present invention is: how to improve the poor immune effect of the expression product of the classical swine fever E2 protein in mammalian cells and / or how to overcome the short time for pigs to produce antibodies after the expression product of the prokaryotic expression system of the swine fever E2 protein is immunized to pigs and Deficiency of immune death induced by prokaryotic endotoxin residue

Method used

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  • Recombinant protein containing cap of classical swine fever e2 and circovirus and its preparation method and application
  • Recombinant protein containing cap of classical swine fever e2 and circovirus and its preparation method and application
  • Recombinant protein containing cap of classical swine fever e2 and circovirus and its preparation method and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Expression of embodiment 1, CSFVE2-SpyCatcher and Cap-SpyTag fusion protein

[0076] 1.1. Acquisition of coding genes

[0077] In order to improve the immunogenicity of wild-type E2, the protein sequence of classical swine fever E2 was fused with the SpyCatcher fragment to obtain the fusion protein CSFVE2-SpyCatcher, whose amino acid sequence is shown in SEQ ID No.2; at the same time, Cap was fused with SpyTag to obtain the fusion protein The amino acid sequence of Cap-SpyTag is shown in SEQ ID No.3. The gene encoding the fusion protein was synthesized according to the codon preference of mice, and was synthesized by Anhui General Biological Company. The gene encoding the fusion protein CSFVE2-SpyCatcher was named CSFVE2-SpyCatcher gene, and the CSFVE2-SpyCatcher gene was constructed into PMD18 to obtain the recombinant vector PMD18 -E2; wherein the gene encoding the fusion protein Cap-SpyTag is named Cap-SpyTag gene, and the Cap-SpyTag gene is constructed to PMD18 to ...

Embodiment 2

[0100] Embodiment 2, construction and protein expression of stably transfected cell lines

[0101] 2.1. Construction of stable transfected cell lines

[0102] Digest the recombinant expression vector PCHO1.0-E2-Cap with pvul and recover the digested product with a gel recovery kit. The recovered product is ready to be transfected into mammalian CHO cells / dhFr-, CHO / dhFr- was purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences (https: / / www.cellbank.org.cn / index.php), the catalog number is GNHa4 and the identification number is CSTR: 19375.09.3101HAMGNHa4.

[0103] Step 1: The day before transfection, inoculate a suitable cell density on a 6-well culture plate; when transfecting, the cells should reach 90-95% confluence;

[0104] Step 2: Prepare solution 1 and solution 2, mix solution 1 and solution 2, and place at room temperature for 20 minutes;

[0105]Solution 1: 240 μL serum-free medium (Gibco, catalog number: 1220...

Embodiment 3

[0128] Example 3, functional verification of classical swine fever E2-VLPs vaccine

[0129] 3.1. Experimental materials

[0130] (1) Experimental animals 4-6 week-old piglets were from non-immunized healthy pig herds. They were tested by classical swine fever antigen and antibody ELISA kits. Both antigen and antibody tests were negative, and the test was carried out under strict isolation conditions.

[0131] (2) Vaccine: the E2-VLPs vaccine prepared in 2.6 of Example 2, the batch numbers are 20160303c, 20160608a and 20160608b.

[0132] (3) Virus strain used in the test: AV1411 strain of classical swine fever Shimen blood virus (generation F115, production date: June 2011) was provided by the animal experiment company Jinyu Baoling Biological Medicine Co., Ltd., and the strain was provided by Jinyu Baoling Biological Medicine Co., Ltd The company purchased it from the Culture Collection Center of the China Veterinary Drug Administration.

[0133] (4) ELISA detection kit for ...

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Abstract

The invention discloses a recombinant protein containing hog fever E2 and Cap of circovirus, a preparation method and application thereof, and belongs to the field of vaccines. The active ingredient of the vaccine disclosed by the present invention is a recombinant protein, and the recombinant protein is formed by connecting the fusion protein CSFVE2-SpyCatcher and Cap-SpyTag, and the fusion protein CSFVE2-SpyCatcher is a protein whose amino acid sequence is SEQ ID No.2; The fusion protein Cap-SpyTag is a protein whose amino acid sequence is SEQ ID No.3. The present invention also provides a construction method for co-expressing classical swine fever E2 in a CHO cell line, using circular VLPs to display E2 to achieve E2 VLPsization, which can improve the immunogenicity of the E2 antigen, achieve mutual stimulation immunity, and improve classical swine fever and circular immunity Protective effect.

Description

technical field [0001] The invention belongs to the field of vaccines, in particular to a recombinant protein containing hog fever E2 and Cap of circovirus, a preparation method and application thereof. Background technique [0002] Classical swine fever, commonly known as "rotten intestinal fever", is an acute, febrile, contagious infectious disease caused by Classical swinefever virus (CSFV) of the family Flaviviridae, which is highly contagious and fatal. Pigs are the only natural host of the virus. CSFV is an enveloped virus, 40-60 nm, single-stranded positive-sense RNA. The CSFV genome is about 123kb long and contains only one large open reading frame (ORF). This ORF is translated into a polyprotein with 3898 amino acid residues and a molecular weight of about 438 kDa, and is further processed by the virus and host cell proteases. processed into mature protein. All structural and non-structural proteins of CSFV are encoded by this ORF, flanked by the 5' untranslated ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85C12N5/10A61K39/295A61K39/187A61K39/12A61P31/14A61P31/20
CPCC07K14/005C12N15/85C12N5/0682A61K39/12A61P31/14A61P31/20C12N2750/10022C12N2770/24322C07K2319/00C12N2750/10051C12N2770/24351C12N2800/22C12N2510/02A61K2039/70C12N2750/10034C12N2770/24334A61K2039/54A61K2039/552
Inventor 伏显华
Owner 北京中海生物科技有限公司