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African swine fever virus recombinant protein containing intramolecular adjuvant, expression vector and application

A technology of African swine fever virus and recombinant protein, which is applied in the field of biotechnology and pharmaceuticals, can solve the problems of poor immunity of vaccines, achieve the effect of simple purification steps and maintain immunogenicity

Active Publication Date: 2021-07-23
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Aiming at the problem of poor immunity of existing African swine fever virus subunit vaccines, the first purpose of the present invention is to provide a recombinant protein OPM, the second purpose is to provide the expression vector of the gene encoding the recombinant protein, and the third purpose is to provide the recombinant protein OPM Application in the preparation of medicines or kits for diagnosis or prevention of African swine fever virus

Method used

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  • African swine fever virus recombinant protein containing intramolecular adjuvant, expression vector and application
  • African swine fever virus recombinant protein containing intramolecular adjuvant, expression vector and application
  • African swine fever virus recombinant protein containing intramolecular adjuvant, expression vector and application

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Construction and Identification of Recombinant Plasmids

[0027] 1. According to the sequence of the existing African swine fever virus ASFV-SY18 (accession number: MH766894.1) in GenBank, select the amino acid sequences of p30 and p54, wherein the transmembrane region of p54 His30-Phe52 replaces the connecting peptide (GGGGS) 3 To obtain mP54, pass mP54 through the connecting peptide (GGGGS) 3 Connected to the C-terminus of p30 and the C-terminus of OprI sequence (accession number: X13748.1) in sequence, and sent the concatenated gene sequence to Nanjing GenScript Biotechnology Co., Ltd. for synthesis, and directly synthesized into pET-30a(+) On the expression vector, the restriction sites at both ends are NdeI and XhoI, and the recombinant expression plasmid pET-30a(+)-OPM is obtained.

[0028] The amino acid sequence of the synthetic recombinant protein is SEQ ID NO: 2; the nucleotide sequence encoding the recombinant protein is SEQ ID NO: 1; the recombina...

Embodiment 2

[0035] Expression and purification of embodiment 2 OPM

[0036] Take 10mL of the bacterial solution containing the positive plasmid and transfer it to 1000mL of Kanal LB medium, and continue to culture on a shaker at 220rmp at 37°C until the OD of the bacterial solution is 600 When it reaches 0.6-0.8, add IPTG to induce it to a final concentration of 0.4mM, then culture it on a shaker at 37°C at 220rmp for 6h, centrifuge at 8000rmp for 7min to collect the bacteria, wash the collected bacteria with PBS once, add 50mLbinding buffer (300 mM NaCl, 20 mM NaH 2 PO 4 , 5 mM imidazole; pH 8.0) resuspended the cells, ultrasonically lysed the cells for 30 min, centrifuged at 12,000 rpm for 20 min to collect the inclusion bodies and supernatant, and performed SDS-PAGE electrophoresis detection. The results showed that the expression of OPM was in the form of inclusion bodies. Dissolve the inclusion body in the binding buffer containing 6M guanidine hydrochloride, and purify the recombi...

Embodiment 3

[0037] Example 3 In vitro stimulation test of dendritic cells (DCs)

[0038] Select SPF grade 6-8 week old C57BL / 6 mice, separate bone marrow primary cells from the femur and tibia of the mouse to make a single cell suspension, and the cells collected by centrifugation are lysed by red blood cells and washed with PBS, then resuspended to contain 20ng / mL rmGM-CSF, 10ng / mL rmIL-4 and 10% FBS in RPMI1640, adjust the cell density to 1×10 6 / mL, placed at 37 degrees 5% CO 2 1 / 2 volume was replaced with fresh medium every 2 days, and supplemented with 20ng / mL rmGM-CSF and 10ng / mL rmIL-4. The cells were cultured to differentiate into bone marrow-derived dendritic cells (BMDCs) on day 6. BMDCs were collected and resuspended in RPMI1640 containing 10% FBS to adjust the cell density to 1×10 6 / mL, add 1µg / mL, 5µg / mL and 10µg / mL of OPM and incubate at 37 degrees for 24h, and set the groups without stimulation and adding LPS (0.1µg / mL) as negative and positive controls. Collect the c...

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Abstract

The invention relates to an African swine fever virus recombinant protein containing an intramolecular adjuvant, an expression vector and application, and belongs to the field of biotechnology pharmacy. The recombinant protein is formed by fusing African swine fever virus p30 and mp54 (modified p54) and an outer membrane protein I of pseudomonas aeruginosa. The recombinant protein has a general formula of OprI-(Linker)n-p30- (Linker)n-mp54, wherein a Linker sequence of a connecting peptide is selected from GGGGS or GGSGG, and is preferably GGGGS; and n is 1, 2, 3 or 4, and is preferably 3. Experiments prove that the recombinant protein can significantly promote maturation and differentiation of dendritic cells and secretion of cytokines. A vaccine immune animal prepared from the recombinant protein can stimulate an organism to generate a high-level neutralizing antibody and a cellular immune response, and can be used for preparation of an African swine fever virus diagnostic reagent and a preventive or therapeutic drug.

Description

technical field [0001] The invention belongs to the field of biotechnology and pharmaceuticals, and in particular relates to an African swine fever virus recombinant protein containing an intramolecular adjuvant, an expression vector and application thereof. Background technique [0002] African swine fever (ASF) is an acute and severe infectious disease of pigs caused by African swine fever virus (ASFV), with a mortality rate of up to 100%. There is no commercial vaccine yet. The disease was introduced into my country in August 2018, and quickly spread to more than 30 provinces and regions in China, causing direct economic losses of more than tens of billions of yuan. At present, ASF is still prevalent in my country and poses a serious threat to the pig industry in my country. Safe and effective vaccines are urgently needed for the prevention and control of the disease. The early research experience of African swine fever vaccine showed that: (1) the inactivated African sw...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/62C12N15/70A61K39/12A61K39/39A61P31/20G01N33/68G01N33/569
CPCC07K14/005C07K14/21C12N15/70A61K39/12A61K39/39A61P31/20G01N33/6854G01N33/56983C12N2710/12022C12N2710/12034C07K2319/00C07K2319/21A61K2039/552A61K2039/55516G01N2333/01G01N2469/20Y02A50/30
Inventor 常惠芸张光磊邵军军刘伟常艳燕
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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