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Classical swine fever virus E2-E0 fusion protein as well as preparation method and application thereof

An E2-E0, swine fever virus technology, applied in the field of preparation of swine fever virus E2-E0 fusion protein, can solve the problems of lack of glycosylation modification, lack of expression of target protein, affecting the immunogenicity of antigenic protein, etc. To achieve the effect of increasing protein expression and promoting expression

Pending Publication Date: 2022-07-29
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

When E0 is prepared by prokaryotic Escherichia coli, the product exists in the form of insoluble inclusion bodies and lacks glycosylation modification, which seriously affects the immunogenicity of the antigenic protein; through eukaryotic CHO cells with complete glycosylation modification function When preparing E0, the target protein is not expressed or the expression level is extremely low, which seriously hinders the development of CSF subunit vaccines

Method used

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  • Classical swine fever virus E2-E0 fusion protein as well as preparation method and application thereof
  • Classical swine fever virus E2-E0 fusion protein as well as preparation method and application thereof
  • Classical swine fever virus E2-E0 fusion protein as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Example 1 Preparation of swine fever virus E2-E0 truncated fusion protein and E2-E0 truncated mutant fusion protein

[0044] 1. Gene Fragment Synthesis

[0045] Gene fragments encoding swine fever virus E0 protein, E0 truncated protein, E0 truncated mutant protein, E2 protein, E2-E0 truncated fusion protein and E2-E0 truncated mutant fusion protein were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. , and was correctly identified by nucleic acid gel electrophoresis. The gene fragment encoding swine fever virus E0 protein is shown in SEQ ID NO.12, and the amino acid sequence is shown in SEQ ID NO.11; the gene fragment encoding swine fever virus E0 truncated protein is shown in SEQ ID NO.2, The amino acid sequence is shown in SEQ ID NO.1; the gene fragment encoding the swine fever virus E0 truncation mutation is shown in SEQ ID NO.8, and the amino acid sequence is shown in SEQ ID NO.7. The E0 truncated mutant protein refers to the The amino acids at positi...

Embodiment 2

[0056] Example 2 Immunogenicity detection

[0057]Indirect ELISA was used to detect mE0 protein, E2 protein, E2-E0 truncated fusion protein and E2-E0 truncated mutant fusion protein immunized rabbit serum to analyze their immunogenicity. The purified CSFV-E0 truncation mutant protein, CSFV-E2 protein, CSFV-E2-E0 truncation mutant fusion protein and CSFV-E2-E0 truncation mutant fusion protein (50ng / well) were packaged with 50 mM carbonate buffer, respectively. Incubate overnight at 4°C in an ELISA plate. Block with 1% bovine serum albumin (BSA) at 37°C for 2h. The serum to be tested was diluted with serum diluent at 1:2000 and added to the reaction well, 100 μL / well, each sample was set to 3 duplicate wells, and incubated at 37°C for 1 h. The HRP-labeled goat anti-rabbit IgG antibody was diluted with serum diluent at 1:5000 and added to the reaction well, 100 μL / well, and incubated at 37°C for 1 h. TMB color development, 50 μL per well, after 10 min of color development, add...

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Abstract

The invention belongs to the technical field of biology, and particularly relates to hog cholera virus E2-E0 fusion protein, a preparation method and application. The invention firstly provides the E2-E0 fusion protein of the classical swine fever virus E0 truncated protein and the classical swine fever virus E2 protein, which not only can promote the expression of the E0 protein, but also does not influence the immunogenicity of the E0 protein and the E2 protein, and can be used for preparing a classical swine fever subunit vaccine; on the basis of the classical swine fever virus E0 truncated protein, it is found that the 160th, 162th, 165 and 166th amino acids of the classical swine fever virus E0 truncated protein are mutated into glycine G, the expression quantity of the E2-E0 fusion protein can be further increased by 20% or above, and the immunogenicity of the E2-E0 fusion protein is not affected.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a swine fever virus E2-EO fusion protein, a preparation method and an application. Background technique [0002] Swine fever (Classicalswinefever, CSF), also known as hog cholera (Hogcholera, HC), is an acute febrile lethal disease caused by swine fever virus (HogCholeravirus, HCV or Classical swinefevervirus, CSFV). Swine fever is highly contagious and popular. Widespread, high morbidity and mortality, great harm. The International Bureau of Animal Epidemiology (OIE) used to classify it as a Class A infectious disease, and now it is listed as a notified epidemic disease, and my country has classified it as a Class I animal disease. [0003] CSFV is an enveloped virus with a virion size of about 40-60 nm. The viral genome is a single-stranded positive-strand RNA, about 12.3kb in length, containing a large open reading frame (ORF), encoding a polyprotein with 3898 amino ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/85A61K39/12A61P31/14
CPCC07K14/005C12N15/85A61K39/12A61P31/14C07K2319/00C12N2800/107C12N2770/24022C12N2770/24034A61K2039/552Y02A50/30
Inventor 郑海学茹毅李亚军郝荣增杨洋刘华南李丹卢炳州张贵财秦晓东张越陈娇吴秀萍赵东梅任蕊芳
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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