Preparation method and application of classical swine fever virus recombinant subunit vaccine

A subunit vaccine and swine fever virus technology, applied in the field of biological vaccine preparation, can solve the problems of allergic reactions in animals, not easy to mass-produce, easy to contaminate foreign bacteria, etc., to achieve no BVDV contamination risk, low production cost, The effect of easy quality control

Inactive Publication Date: 2015-08-12
NOVO BIOTECH CORP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the immunogenicity of baby rabbit tissue vaccine and adult rabbit spleen lymphocytic vaccine is relatively good, and there is no risk of BVDV contamination, but because rabbits need to be used in the vaccine preparation process, this type of vaccine has the following shortcomings, such as: it is not easy to produce on a large scale Different breeds, batches, and different environments have different sensitivities in rabbits; it is easy to contaminate exogenous bacteria; individual animals may have allergic reactions after injection

Method used

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  • Preparation method and application of classical swine fever virus recombinant subunit vaccine
  • Preparation method and application of classical swine fever virus recombinant subunit vaccine
  • Preparation method and application of classical swine fever virus recombinant subunit vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] The construction of embodiment 1 expression vector

[0026] 1.1 Extraction of CSFV RNA

[0027] Trizol was used to extract RNA from the culture supernatant of CSFV as a template for reverse transcription.

[0028] 1.2 RNA reverse transcription into cDNA

[0029] Experiments were performed using takara's Primescript RT-PCR kit.

[0030] 1.3PCR amplification primers (restriction sites are underlined):

[0031] Upstream primer: 5'-CG GAATTC CTAGCCTGCAAGGAAGATTAC-3'

[0032] Downstream primer: 5'-CCC AAGCTT TTA GTGATGGTGATGGTGATGAACAAATTCTGCGAAGTAATC-3’

[0033] The sample loading system is (50μl):

[0034]

[0035] PCR amplification program:

[0036]

[0037] 1.4 Gel recovery of DNA fragments:

[0038] (1) Perform 1% agarose gel electrophoresis (98V 45min) on 50 μl of the system reaction solution in step 1.3;

[0039] (2) Under the ultraviolet light, cut the gel and recover the DNA fragments in a 1.5ml EP tube; use the DNA extraction kit of Tiangen Bio t...

Embodiment 2

[0078] Example 2 Recombinant pFastBac1-KSPTE2 plasmid transforms Escherichia coli DH 10Bac

[0079] (1) Take 100 μl of DH10Bac competent cells and place them on ice;

[0080] (2) Add at least 1 ng of recombinant pFastBac1-KSPTE2 plasmid to 100 μl DH10Bac competent cells, and ice-bath for 30 minutes;

[0081] (3) Heat shock in a water bath at 42°C for 45 sec, put the EP tube back on ice, and let stand for 2 min;

[0082] (4) Add 900 μl non-resistant LB culture solution in the ultra-clean bench;

[0083] (5) Shaking culture at 220 rpm at 37°C for 4 hours;

[0084] (6) After 4 hours, take 10 μl of bacterial liquid and apply KGT three-antibody plate (50 μg / ml Kanamycin, 7 μg / ml Gentamicin and 10 μg / ml Tetracycline, 100 μg / ml Bluo-gal, and 40 μg / ml IPTG), and incubate at 37°C in the dark 48h;

[0085] (7) Select the white spot and transfer it to the KGT third antibody plate to continue culturing at 37°C for about 24 hours. Afterwards, uniform white spots were selected, and eac...

Embodiment 3ba

[0087] Example 3 bacmid-KSPTE2 extraction

[0088] (1) Transfer the white spot bacterial liquid identified as positive by PCR to 5ml KGT tertiary antibody LB liquid medium and continue overnight culture at 37°C and 225rpm;

[0089] (2) Inoculate 1ml of overnight cultured bacterial solution into 100ml KGT triple antibody LB liquid medium to continue overnight culture at 37°C 225rpm, and extract the Bacmid plasmid according to the alkaline lysis method;

[0090] (3) Divide the cultured bacterial solution into two 50ml centrifuge tubes, and centrifuge at 12,000rpm at 4°C for 10min;

[0091] (4) Discard the supernatant, blot up the culture medium as much as possible, recover the bacteria, add 10ml of ice-cooled solution I (50mmol / L glucose, 25mmol / L Tris HCl, 10mmol / L EDTA, pH 8.0, add 40μg / ml RNase A) Tap gently with a pipette to completely suspend the bacteria;

[0092] (5) Add 20ml of freshly prepared solution II (1% SDS, 0.2M NaOH), cap the tube tightly, gently invert the c...

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Abstract

The invention discloses a preparation method and application of a classical swine fever virus recombinant subunit vaccine with the amino acid sequence shown as SEQ ID No.1. The preparation method of the classical swine fever virus recombinant subunit vaccine typically includes the following steps: classical swine fever E2 truncated protein (TE2) coding gene is cloned into baculovirus vector pFastBacTM1, and is then transfected into Sf9 insect cells to obtain recombinant baculovirus capable of expressing protein TE2. The high five insect cells in logarithmic growth phase are infected by the recombinant baculovirus, so that a large amount of the protein TE2 can be expressed in a cell culture supernatant. Finally, the cell culture supernatant is recovered and purified to obtain a large amount of the recombinant protein TE2 with the purity more than 90%. According to the method, the target protein can be harvested from the cell culture supernatant, the time of protein purification is reduced, consumption of a large amount of time can be avoided, and the vaccine production process can be simplified. Under the premise of simplification of the vaccine production process, the recombinant protein TE2 has the advantages of strong immunogenicity and high safety, and the animal experiments prove that the recombinant protein can effectively stimulate the body to produce a highly effective humoral immune response.

Description

technical field [0001] The invention relates to a preparation method and application of a swine fever virus recombinant subunit vaccine, belonging to the technical field of biological vaccine preparation. Background technique [0002] Classical swine fever (CSF) is called classical swine fever (Classical Swine Fever, CSF) in Europe and is an acute, febrile, contact infectious disease of pigs caused by classical swine fever virus (Classical Swine Fever Virus, CSFV). It is characterized by 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. Swine fever is one of the main diseases that endanger the development of my country's pig industry. [0003] CSFV is a single...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/187C12N15/40A61P31/14
Inventor 宣春玲钱泓吴有强吴素芳吕朋查银河张屹峰汪正亮
Owner NOVO BIOTECH CORP
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