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Preparation methods and application of recombinant swine fever E2 protein and subunit vaccine of recombinant swine fever E2 protein

A subunit vaccine, swine fever technology, applied in biochemical equipment and methods, vaccines, veterinary vaccines, etc., can solve the problem that protein expression, folding and modification are not as good as mammalian cell expression systems, and it is difficult to achieve 500mg/L output, The differences in the structure of the natural antigen protein of the virus, etc., achieve the effect of no pollution risk, easy mass production, and good immunogenicity

Pending Publication Date: 2018-02-09
NOVO BIOTECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the vaccine is expressed through the insect baculovirus expression system. Compared with the prokaryotic expression system, the expression system can undergo post-translational processing and modification to a certain extent after protein expression, but there are still some differences in the structure of the natural antigenic protein of the virus. , protein folding and modification after expression is not as good as mammalian cell expression system
Moreover, when the system produces and prepares antigens, the cells will lyse and die after being infected by the virus, which will increase the difficulty of downstream purification and other production processes.
In addition, the yield of the target protein prepared by this system will not be very high, generally only 200-300mg / L, and it is difficult to achieve the yield of 500mg / L

Method used

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  • Preparation methods and application of recombinant swine fever E2 protein and subunit vaccine of recombinant swine fever E2 protein
  • Preparation methods and application of recombinant swine fever E2 protein and subunit vaccine of recombinant swine fever E2 protein
  • Preparation methods and application of recombinant swine fever E2 protein and subunit vaccine of recombinant swine fever E2 protein

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Codon optimization of classical swine fever E2 protein

[0042] Through codon optimization on the nucleotide sequence of the classical swine fever E2 protein, the sequence of OPTI-E2 was obtained, as shown in SEQ ID NO.2. This work was entrusted to Suzhou Jinweizhi Biotechnology Co., Ltd. to complete.

[0043] Comparing the sequences of SEQ ID NO.2 and SEQ ID NO.3, it is found that the nucleotide sequence of the classical swine fever E2 protein after codon optimization is 21.3% higher than the nucleotide sequence of the non-codon-optimized classical swine fever E2 protein , that is, 222 nucleotides out of 1044 nucleotides are different. see details Figure 8 shown.

Embodiment 2

[0044] Example 2: Construction of pEE12.4-OPTI-E2 recombinant plasmid

[0045] 2.1 PCR amplification of the target fragment OPTI-E2

[0046] 2.1.1 PCR reaction

[0047] (1) Primer design and synthesis

[0048] Upstream primer: 5'-CCAAGCTTGCCGCCACCATGAAAGTGCTGAGGGGCCAG-3'

[0049] Downstream primer: 5'-CCGGAATTCTTAGTGATGGTGATGGTGATGAGC-3'

[0050] (2) Add 50 μL of the sample system, as shown in the table below:

[0051]

[0052] PCR amplification program:

[0053]

[0054] 2.1.2 Gel recovery of PCR products

[0055] (1) Mark the sample collection EP tube, adsorption column and collection tube;

[0056] (2) Take the weight of the marked empty EP tube, and record the value;

[0057] (3) Carefully cut out a single target DNA band from the agarose gel with a scalpel on a gel cutter and put it into a clean 1.5mL centrifuge tube;

[0058] (4) Add 600 μL PC buffer to the 1.5mL centrifuge tube in step (3), place in a 50°C water bath for about 5 minutes, and gently turn th...

Embodiment 3

[0121] Embodiment 3: pCDNA3.1-OPTI-E2 recombinant plasmid construction

[0122] According to the steps in Example 2, the results of the double enzyme digestion identification experiment are shown in Figure 4 .

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Abstract

The invention discloses preparation methods and application of recombinant swine fever E2 protein and a subunit vaccine of the recombinant swine fever E2 protein. The preparation method of the recombinant swine fever E2 protein comprises the following steps that (1) a swine fever E2 protein coding gene is cloned into an eukaryotic expression vector to obtain recombinant plasmid containing the swine fever E2 protein coding gene; (2) then, the recombinant plasmid containing the swine fever E2 protein coding gene is transfected into a CHO cell strain; (3) the CHO cell strain obtained in the step(2) is cultured, screened and domesticated; and (4) the cell strain in the step (3) is fermented and cultured; and the recombinant swine fever E2 protein is obtained after purification. The methods provided by the invention have the advantages that the target protein can be obtained from cell culture supernatant; the yield reaches up to 1g / L; the protein purification time is shortened; the vaccineproduction steps are simplified; and the vaccine production cost is also greatly reduced.

Description

technical field [0001] The invention relates to a suspended CHO cell strain stably and efficiently expressing classical swine fever E2 protein, a method for constructing and screening the cell strain, a preparation method and application of a classical swine fever E2 protein subunit vaccine, and belongs to the technical field of animal vaccines and veterinary biological products . Background technique [0002] Classical swine fever (CSF), known in Europe as classical swine fever (CSF), is an acute, febrile, fatal disease caused by classical swine fever virus (CSFV). Classical swine fever is highly contagious, acute onset, persistent high fever, and degeneration of small blood vessel walls causing extensive hemorrhage, infarction, and necrosis. Domestic and wild pigs are its only natural hosts. The World Organization for Animal Health (OIE) defines it as a class A infectious disease, and my country's "Animal Epidemic Prevention Law" lists it as a class I infectious disease....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/85C07K14/18A61K39/187A61P31/14
CPCA61K39/12A61K2039/552C07K14/005C12N2770/24322C12N2770/24334
Inventor 钱泓吴有强车影吴素芳查银河
Owner NOVO BIOTECH CORP
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