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Recombinant drosophila cell line for expressing porcine atypical pestivirus fusion protein as well as preparation method and application of recombinant drosophila cell line

An atypical pestivirus and fusion protein technology, applied in the field of vaccines, to achieve the effects of high clinical application value, strong humoral and cellular immune responses, and efficient and stable expression

Pending Publication Date: 2021-12-03
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] There is currently no vaccine available to prevent and control APPV infection

Method used

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  • Recombinant drosophila cell line for expressing porcine atypical pestivirus fusion protein as well as preparation method and application of recombinant drosophila cell line
  • Recombinant drosophila cell line for expressing porcine atypical pestivirus fusion protein as well as preparation method and application of recombinant drosophila cell line
  • Recombinant drosophila cell line for expressing porcine atypical pestivirus fusion protein as well as preparation method and application of recombinant drosophila cell line

Examples

Experimental program
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Effect test

preparation example Construction

[0053] The invention provides a method for preparing a recombinant Drosophila cell line expressing porcine SARS virus E2Fc or E2ΔFc fusion protein, comprising the following steps:

[0054] 1) Using the primer pair pMT-E2-F / E2-Fc'-R, using the pEASY-Blunt-APPV E2 recombinant plasmid as a template, PCR amplifies the E2-Fc' fragment containing the partial sequence of the 5' end of IgG3Fc;

[0055] 2) artificially synthesizing the nucleotide sequence of the pig IgG3Fc protein, and retaining the dimer interaction site and receptor binding site of the pig IgG3Fc protein to obtain the IgG3ΔFc nucleotide sequence;

[0056] 3) Using the primer pair E2'-Fc-F / pMT-Fc-R, IgG3Fc or IgG3ΔFc in step 2) respectively as a template, PCR amplifies to obtain E2'-Fc or E2'-ΔFc fragments containing partial sequences at the 3' end of E2 ;

[0057] 4) Perform overlapping PCR using the E2-Fc' fragment described in step 1) and the E2'-Fc or E2'-ΔFc fragment described in step 3) as templates, and the ob...

Embodiment 1

[0085] Method for constructing recombinant plasmids expressing APPV E2, APPV E2Fc and APPV E2ΔFc proteins

[0086] 1. PCR amplification of APPV E2, APPV E2Fc and APPV E2ΔFc

[0087] (1) Amplification of APPV E2 sequence

[0088] Design the E2-specific primer pair pMT-E2-F / pMT-E2-R according to the genome sequence of APPV_GX-CH 2016 strain (GenBank accession no.: KY652092), using the pEASY-Blunt-APPV E2 recombinant plasmid stored in the laboratory as a template , to amplify and delete the sequence of the transmembrane region, and the primers were synthesized by Beijing Qingke Biotechnology Co., Ltd.

[0089] The specific primer sequences are shown in Table 1 (the underline represents the EcoR I / Xho I restriction site, the sequence in bold is 6×His, and the sequence in italics represents the GS Linker sequence).

[0090] Primer sequences for sequence amplification in Table 1

[0091]

[0092] The PCR reaction system is shown in Table 2.

[0093] Table 2 Amplification syst...

Embodiment 2

[0127] Construction method of recombinant Drosophila cell lines expressing APPV E2, E2Fc and E2ΔFc proteins

[0128] 1. Co-transfection test of pMT-Bip-aE2 / aE2Fc / aE2ΔFc and pCoHygro

[0129] (1) One day before transfection, S2 cells were seeded into 6-well plates (1.0×10 6 cells / mL), static culture at 27°C to allow it to fully adhere to the wall;

[0130] (2) Preparation of solution A: 4 μg of recombinant plasmid + 0.2 μg of hygromycin B resistance plasmid (pCoHygro, Miaoling Biotechnology), and adding anti-resistance medium (SF-SFM, Suzhou Womei Biotechnology) to 100 μL; preparation of solution B: Transfection reagent (Cellfectin II Reagent, ThermoFisher) 10 μL + anti-antibody-free medium 90 μL;

[0131] (3) Let it stand for 5 minutes, add B to A and mix well, let it stand for 30 minutes (flick and spin once to mix well), add 800 μL of antibiotic-free medium and mix well;

[0132] (4) Discard the old medium in the 6-well plate, add 1mL of the above mixed solution, and cult...

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Abstract

The invention provides a recombinant drosophila cell line for expressing porcine atypical pestivirus fusion protein as well as a preparation method and application of the recombinant drosophila cell line, and belongs to the technical field of vaccines. The recombinant drosophila cell line S2-aE2Fc or S2-aE2deltaFc for expressing the fusion protein of the atypical porcine pestivirus E2Fc and E2deltaFc provided by the invention can be used for preparing subunit vaccines, and after piglets are immunized, relatively strong specific immune response aiming at the atypical porcine pestivirus can be generated. The recombinant drosophila melanogaster cell lines S2-aE2Fc and S2-aE2deltaFc can be used for obtaining aE2 protein antigens in a dimer form, a prepared subunit vaccine can stimulate an organism to generate stronger humoral immunity and cellular immunity responses, and the recombinant drosophila melanogaster cell lines can be used as a promising candidate genetic engineering vaccine for preventing piglet congenital tremor caused by atypical porcine pestivirus.

Description

technical field [0001] The invention belongs to the technical field of vaccines, and in particular relates to a recombinant fruit fly cell line expressing porcine SARS virus fusion protein and a preparation method and application thereof. Background technique [0002] Piglet congenital tremor (CT), commonly known as "piglet trembling disease", is a neurological disease that occurs in newborn piglets. Due to the hypomyelination of the central nervous system (CNS), affected piglets show clinical symptoms such as trembling of the head and limbs, and paroxysmal convulsions. Severe cases may be accompanied by ataxia leading to reduced suckling ability of piglets and further death due to starvation and insufficient intake of colostrum. The syndrome was first described in the United States by Kinsley et al. in 1922 [1] . An outbreak of an unexplained disease in a herd of pigs caused the piglets to shake their heads, shake their limbs, and appear to be dancing, hence the name "da...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N5/10C07K19/00A61K39/12A61K39/385A61K39/39A61P31/14C12R1/91
CPCC12N15/85C07K14/005A61K39/12A61K39/385A61K39/39A61P31/14C12N2800/105C07K2319/30C12N2770/24322C12N2770/24334A61K2039/55516A61K2039/6056Y02A50/30
Inventor 钱平李祥敏任旭皎陈焕春
Owner HUAZHONG AGRI UNIV
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