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Stable atypical porcine pestivirus subunit protein, and vaccine, preparation method and application thereof

A technology of subunit vaccine and classical swine fever virus, which is applied in the field of atypical swine fever subunit protein and its preparation, can solve the problems of not having a good vaccine for epidemic prevention

Active Publication Date: 2019-07-23
NOVO BIOTECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, so far, there is no good vaccine against this virus

Method used

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  • Stable atypical porcine pestivirus subunit protein, and vaccine, preparation method and application thereof
  • Stable atypical porcine pestivirus subunit protein, and vaccine, preparation method and application thereof
  • Stable atypical porcine pestivirus subunit protein, and vaccine, preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Selection of atypical swine fever virus subunit protein and optimization of gene sequence

[0045] By comparing the nucleotide sequence of atypical swine fever virus (GenBank: KY624591.1), swine fever virus sequence (see Chinese invention patent with application number 201710409930.6), and bovine viral diarrhea virus sequence (see Chinese invention with application number 201611239612.1) (Patent) comparison and analysis, it is found that the sequence 682L-941S is likely to be the main antigenic protein of atypical swine fever virus, temporarily named AE2 protein (Atypical porcine pestivirus E2, AE2), of which 682L-700G may be the secretion signal of AE2 protein Peptides, 701S-911H may be the extracellular domain of AE2 protein, 912L-934L may be the transmembrane domain of AE2 protein, and 935S-941S may be the intracellular domain of AE2 protein. Combined with our experience in the expression research of the E2 protein of classical swine fever virus and the E2 pr...

Embodiment 2

[0048] Example 2: Construction of pEE12.4-OPTI-AE2 recombinant plasmid

[0049] 2.1 PCR amplification target fragment OPTI-AE2

[0050] 2.1.1 PCR reaction

[0051] (1) Primer design and synthesis

[0052] Upstream primer: 5’-GCAAGCTT GCCGCCACCATGAAGGGCAACCTGATC-3’

[0053] Downstream primer: 5’-GCGAATTC TCAATGGTGATGGTGATGGTGGGTAGCGGC-3

[0054] (2) 50μL of sample addition system, as shown in the following table:

[0055]

[0056] PCR amplification program:

[0057]

[0058] 2.1.2 Gel recovery of PCR products

[0059] (1) Mark the sample collection EP tube, adsorption column and collection tube;

[0060] (2) Weigh the marked empty EP tube and record the value;

[0061] (3) Cut the single target DNA band from the agarose gel carefully with a scalpel on the gel cutter and put it into a clean 1.5mL centrifuge tube;

[0062] (4) Add 600μL of PC buffer to the 1.5mL centrifuge tube in step (3), and place it in a water bath at 50°C for about 5 minutes. During this time, gently flip the centrifuge tube...

Embodiment 3

[0127] Example 3: Establishment of pEE12.4-OPTI-AE2 recombinant plasmid transfection into CHO-K1 cells and monoclonal screening

[0128] 3.1 CHO-K1 cell transfection

[0129] (1) Preparation: UV sterilization in a biological safety cabinet for 30 minutes; DMEM / F12 (containing 10% serum, 1% double antibody), DMEM / F12 and PBS are placed in a 37°C water bath to preheat to 37°C.

[0130] (2) Take out the cells (10cm cell culture dish) from the 37°C incubator, discard the supernatant medium, wash the cells once with pre-warmed 8mL PBS, and discard the PBS.

[0131] (3) Add 1-2mL 0.25% trypsin-EDTA to each 10cm cell culture dish, digest for about 2min at room temperature, observe the cell shrinkage and round, and appear as a single cell under the microscope.

[0132] (4) Add 4 mL of DMEM / F12 (containing 10% serum, 1% double antibody) to terminate the digestion reaction, and blow the cells away with a pipette.

[0133] (5) Transfer the digested cells to a 15mL centrifuge tube, centrifuge at roo...

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Abstract

The invention discloses stable atypical porcine pestivirus subunit protein, and a vaccine, a preparation method and application thereof. The amino acid sequence of the atypical porcine pestivirus subunit protein is shown as SEQ ID NO.3, and the subunit protein has favorable immunogenicity. The subunit protein is prepared through the steps of (1) cloning the gene sequence shown as SEQ ID NO.1 to aeukaryotic expression carrier to obtain a recombinant plasmid containing the atypical porcine pestivirus subunit protein; (2) transfecting the recombinant plasmid containing the atypical porcine pestivirus subunit protein to CHO cells; (3)culturing, screening and domesticating the CHO cells in the step (2) to obtain cell strains high in expression; and (4) performing fermentation culturing on thecell strains in the step (3), and performing purification to obtain the atypical porcine pestivirus subunit protein. The method for preparing the subunit protein is simple, low in cost, and easy to amplify. An appropriate amount of the subunit protein and an adjuvant are mixed to obtain the subunit vaccine. The vaccine has favorable immunogenicity.

Description

Technical field [0001] The invention relates to an atypical swine fever subunit protein and a preparation method and application thereof, and belongs to the technical field of animal vaccines and veterinary biological products. Background technique [0002] From 2015 to 2017, European and American countries successively reported the phenomenon of congenital swine tremor (CT) in piglets, and the virus was isolated and identified as an atypical swine fever virus (APPV) of the Flaviviridae and Pestivirus genus. In May 2017, the virus was also isolated in Guangdong Province, my country. [0003] Atypical porcine pestivirus (APPV) mainly causes congenital tremor (CT), that is, piglets have local or systemic muscle twitches a few hours after birth. So far, only 2 laboratories have isolated the virus, and the virus titer is very low. For the virus, there is no clear analysis at present, nor is it clear which protein of the virus has good immunogenicity. Therefore, there is no good vacc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/18C12N15/85G01N33/68G01N33/569A61K39/187A61P31/14
CPCC07K14/005G01N33/68G01N33/56983A61K39/12C12N2770/24322G01N2333/183C12N2770/24334A61K2039/552
Inventor 钱泓吴有强卞广林张强徐玉兰吴素芳车影闻雪贾宝琴屠莉洁李泽峰查银河
Owner NOVO BIOTECH CORP