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Preparation and application of virus-like particle based on African swine fever virus P30 and P72 proteins

The technology of P72-SC and P30-SC is applied in the field of preparation of virus-like particles, which can solve the problems such as failure to develop a safe and effective vaccine, and achieve the effect of good application prospect, outstanding immune protection effect and high antigen purity.

Pending Publication Date: 2021-12-07
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] In order to solve the problems in the prior art, the present invention provides a preparation and application of virus-like particles based on African swine fever virus P30 and P72 proteins, so as to solve the problem that ASFV has not been able to develop a safe and effective vaccine so far in the prior art

Method used

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  • Preparation and application of virus-like particle based on African swine fever virus P30 and P72 proteins
  • Preparation and application of virus-like particle based on African swine fever virus P30 and P72 proteins
  • Preparation and application of virus-like particle based on African swine fever virus P30 and P72 proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1. Construction of engineered phage displaying SpyTag on the surface

[0039] (1) Obtaining of SpyTag fragments

[0040] The SpyTag fragment with a flexible peptide and EcoR I / Hind III restriction site sequence (with protective bases) is designed with a single-stranded complementary primer, annealed to synthesize a double-stranded gene fragment, and the sequence feature is SEQ No.1.

[0041] The target gene fragment required for the ligation reaction is 0.06pmol, and the total amount of the synthesized gene is 5nmol / tube, so after adding 50μL of ddH2O to dissolve, the concentration is 0.1nmol, which is 100pmol, so the fragment needs to be diluted 100 times to 1pmol, Take 1 μL for double enzyme digestion. The above-mentioned synthetic gene fragment was digested by EcoR I / Hind III, and the enzyme digestion system was as follows:

[0042]

[0043] After digesting for 2 hours at 37°C, inactivate the enzyme at 65°C for 10 minutes, so as to obtain the inserted ge...

Embodiment 2

[0081] Example 2. Prokaryotic expression and purification of P30-SC fusion protein

[0082] (1) Amplification of P30 and P72 genes

[0083] The P30 gene sequence refers to the ASFV virus P30 gene sequence (GenBank No.: JQ764967.1), and the existing plasmid in the laboratory is used as a template to design seamless cloning primers P30-F (SEQ ID NO.11: cagcaaatgggtcgcggatccATGAAAATGGAGGTCATCTTCAAAA) and P30-R (SEQ ID NO.12: accggaattcgagtcggatccTTTTTTTTTTTAAAAGTTTAATAACCATGAG) Amplified ASFV P30 protein coding gene P30 for prokaryotic vector ligation.

[0084] The P72 gene sequence refers to the ASFV virus P72 gene sequence (GenBank No.: AY578708.1), and the existing plasmid in the laboratory is used as a template to design seamless cloning primers P72-F (SEQ ID NO.13: cagcaaatgggtcgcggatccATGGCATCAGGAGGAGCTTTTTGTC) and P72-R (SEQ ID NO.14: accggaattcgagtcggatccGGTATGGCTACACGTTCGCTGCGTA) ASFV P72 protein coding gene P72 was amplified for prokaryotic vector ligation.

[0085] T...

Embodiment 3

[0118] Example 3. Preparation of T7 phage VLP particles displaying P30-SC and P72-SC on the surface

[0119] The protein obtained using the purification method in Example 2 is soluble in high-concentration imidazole, so dialysis is required to replace the protein buffer with 1×PBS. After concentrating the dialyzed protein through a concentrator tube, take 20 μL of the protein and incubate with the same volume of previously purified T7-ST phage, and overnight at 4°C to obtain the prepared VLP particles (P30-SC::T7-ST, referred to as P30 ::T7; P72-SC::T7-ST, referred to as P72::T7). The VLP structure obtained after combination is as follows Figure 10 As shown, in principle, up to 415 copies of the fusion protein can be displayed on the surface of each T7-ST phage.

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Abstract

The invention discloses preparation and application of a recombinant virus-like particle for surface display of African swine fever virus P30 and P72 proteins, a SpyTag tag protein is displayed on the surface of T7 bacteriophage through a T7 bacteriophage display technology, and therefore, engineered bacteriophage (T7-ST) is constructed. Meanwhile, through an escherichia coli prokaryotic expression system, a P30-Spycatcher fusion protein and a P72-Spycatcher fusion protein can be expressed and purified. In a proper buffer system, the T7-ST, the P30-SC fusion protein and the P72-SC fusion protein are mixed and incubated respectively, and finally, the recombinant VLP particle which carries out the surface play of the P30-SC protein and the P72-SC protein is obtained. The novel genetic engineering vaccine prepared by VLP has the characteristics of an outstanding immune protection effect, good safety, high antigen purity and the like, and multiple protection can be provided for the organism.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to the preparation and application of a virus-like particle (VLP) particle displaying African swine fever virus P30 and P72 proteins on the surface. Background technique [0002] African swine fever (Infection with African swine fever virus, ASF) is an acute, hemorrhagic and severe infectious disease caused by African swine fever virus (ASFV) infecting domestic pigs and wild boars. Acute African swine fever has a rapid onset and a short course, characterized by high fever, loss of appetite, purple, visceral hemorrhage, and a case fatality rate of nearly 100%. [1] . The World Organization for Animal Health (OIE) lists it as a legally notifiable animal disease, and my country lists it as one of the first-class animal diseases and one of the foreign animal diseases that must be guarded against. Since the first ASF outbreak in my country in August 2018, as of June 6, 2019, a total of 137 ou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/64C12N15/62C07K19/00C12N7/01A61K39/12A61P31/20C12R1/92
CPCC12N15/70C07K14/005C12N7/00A61K39/12A61P31/20C12N2710/12022C07K2319/20C07K2319/00C12N2710/12023C12N2710/12052C12N2710/12034C12N2795/10221C12N2795/10251A61K2039/5258A61K2039/552
Inventor 黄金海谭政郭艳余
Owner TIANJIN UNIV
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