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appv-e2 fusion protein and its preparation method, application and vaccine

A technology of APPV-E2 and fusion protein, which is applied in the field of application and vaccine, APPV-E2 fusion protein and its preparation method, can solve the problems of farmers’ losses and achieve good immunogenicity and good protection effect

Active Publication Date: 2020-07-03
天康生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Cause huge losses to farmers, there is currently no effective way to prevent

Method used

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  • appv-e2 fusion protein and its preparation method, application and vaccine
  • appv-e2 fusion protein and its preparation method, application and vaccine
  • appv-e2 fusion protein and its preparation method, application and vaccine

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preparation example Construction

[0064] The application of the APPV-E2 fusion protein provided by the present invention, the above-mentioned preparation method or the protein prepared by the above-mentioned preparation method includes at least one of the following (x1)-(x5): (x1) preparing atypical swine fever vaccine; ( x2) Preparation of antibodies against atypical swine fever virus; (x3) preparation of diagnostic antigens for atypical swine fever virus; (x4) preparation of reagents and / or kits for detection of atypical swine fever virus; (x5) preparation of detection of atypical swine fever virus Reagents and / or kits for virus antibodies.

[0065] Since the APPV-E2 fusion protein provided by the invention has good immunogenicity, it can be used to prepare atypical swine fever vaccine, preferably APPV-E2 subunit vaccine, and can produce higher antibody titers after immunization of animals. The antibody against atypical swine fever virus prepared using the APPV-E2 fusion protein can be applied to the prepara...

Embodiment 1

[0070] Embodiment 1: APPV-E2 fusion protein gene design and synthesis

[0071] APPV-E2 gene (GenBank: LT594521.1) is optimized and divided into segment A and segment B; segment A is expressed by the nucleotide sequence shown in SEQ ID NO.1, and segment B is expressed by the nucleotide sequence shown in SEQ ID NO.2 The nucleotide sequence shown is expressed; segment A and segment B are connected by a Linker having the nucleotide sequence shown in SEQ ID NO.3. The linked APPV-E2 gene has a nucleotide sequence as shown in SEQ ID NO.4, with a length of 567bp. APPV-E2 gene synthesis was completed by Shanghai Sangon Biotech Co., Ltd.

Embodiment 2

[0072] Embodiment 2: Construction of PEE6.4-APPV-E2 recombinant plasmid

[0073] 2.1 Add restriction sites: Add restriction sites respectively upstream and downstream of the APPV-E2 gene sequence by PCR amplification: HindⅢ, XmaI, PCR amplification upstream primers are shown in SEQ ID NO.5, PCR amplification downstream The primers are shown in SEQ ID NO.6, and the PCR results are shown in figure 1 Shown, where lane 1 is the APPV-E2 gene, and lane M is the marker.

[0074] 2.2 APPV-E2 gene and vector double enzyme digestion reaction

[0075] 2.2.1 Construct a 50 μL reaction system, mix the components according to the table below, and then bathe in water at 37°C for 2 hours.

[0076] 10×buffer 5μL DNA sample 2μg HindⅢ 2.5μL wxya 2.5μL dd H 2 0

38μL

[0077] 2.2.2 Recovery of target DNA fragments: DNA gel recovery kit (purchased from Beijing Laibao Technology Co., Ltd.) was used to recover the enzyme-cut target fragments. The ste...

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Abstract

The invention provides APPV-E2 merged protein, as well as a preparation method and application thereof, and a vaccine, and relates to the field of a biological technology. According to the APPV-E2 merged protein, regions being good in antigenicity and easy to express highly in E2 genes of non-classical hog cholera viruses are connected in series, and comprise a segment A mainly expressed by a nucleotide sequence shown as SEQID NO.1 and a segment B mainly expressed by a nucleotide sequence shown as SEQID NO.2. The APPV-E2 merged protein has the advantages of being good in antigenicity and highin expression quantity. The APPV-E2 merged protein can be applied to preparation of the vaccine, antibodies and a reagent kit for detecting the non-classical hog cholera viruses. The vaccine containing the APPV-E2 merged protein has good immunogen properties, and can generate high titer antibodies after immunization of animals, so that the animals can be well protected.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an APPV-E2 fusion protein and its preparation method, application and vaccine. Background technique [0002] Classical swine fever (hog cholera, HC), also known as classical swine fever (Classical Swine Fever, CSF), is a highly contagious disease caused by swine fever virus (HCV, CSFV). It is one of the infectious diseases concerned by countries all over the world, and it is still one of the pig infectious diseases with the most incidence, the greatest harm and the widest prevalence in my country. [0003] With the development of immune defense against classical swine fever, especially in large-scale pig farms, the vaccination against classical swine fever has achieved obvious results, and the typical classical swine fever inventions are decreasing day by day. However, in recent years, atypical swine fever has appeared in my country due to the existence of sows with swine fever virus...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C07K16/10C12N15/62C12N15/85A61K39/12A61P31/14G01N33/569
CPCA61K39/12A61K2039/552A61P31/14C07K14/005C07K16/1081C07K2319/00C12N15/85C12N2770/24322C12N2770/24334G01N33/56983G01N2333/183
Inventor 贺笋任立松李延涛任郭子君
Owner 天康生物制药有限公司