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Celine parvovirus VP2 protein and obtained self-assembled virus-like particles

A feline parvovirus, virus-like technology, applied in the field of feline parvovirus VP2 protein and the obtained self-assembled virus-like particles, can solve the problems of high production cost, poor application effect, general protein expression in prokaryotic expression system, etc. Effects of immune activity

Pending Publication Date: 2022-07-05
深圳赫兹生命科学技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, insect cell expression systems are mainly used to express virus-like particles in research reports. The production cost is high, and the protein expression of prokaryotic expression systems is average, and the application effect is not good.

Method used

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  • Celine parvovirus VP2 protein and obtained self-assembled virus-like particles
  • Celine parvovirus VP2 protein and obtained self-assembled virus-like particles
  • Celine parvovirus VP2 protein and obtained self-assembled virus-like particles

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Construction of recombinant plasmid and prokaryotic expression

[0028] Referring to the FPV gene sequence, the sequence was optimized according to the codon preference of Escherichia coli, and the FPV VP2 gene fragment with a size of 1752 bp was designed. The nucleotide sequence is shown in SEQ ID No. 1. The FPV VP2 gene fragment with his tag, TrxA lytic tag, NcoI and xhoI restriction sites, TEV restriction site, G4S linker and myc tag was directly synthesized into the pET28a plasmid to obtain the correct recombinant plasmid pET28a -FPV VP2 as figure 1 shown.

[0029] Then, the collected recombinant plasmid pET28a-FPV VP2 was transformed into E.coli BL21 competent cells, and a single colony was picked to inoculate 20 mL of LB medium containing kanamycin (final concentration of 100 μg / mL), and cultured at 37°C for 2 h on a shaker. According to the volume ratio of 1:100, the culture was inoculated with 2 L of ampicillin LB medium, and 1 mM IPTG was added when...

Embodiment 2

[0030] Example 2 FPV VP2 protein purification and expression

[0031] The supernatant protein sample collected in Example 1 was added to the nickel column equilibrated with 50 mM Tris-HCl in advance, and incubated for 1 h to fully bind the protein to the nickel column. . Elute with 5 times the column volume of 50 mM and 250 mM imidazole and collect the protein; transfer the collected protein to an ultrafiltration tube, concentrate by ultrafiltration at 4°C, 3000 r / min, take 8 μl of the concentrated protein and add 2 μL of 5 ×The protein loading buffer was mixed and denatured for 10 min, and 8 μl samples were taken for SDS-PAGE identification.

[0032] SDS-PAGE electrophoresis showed that the target band appeared at about 70kDa in inclusion bodies, 50 and 250mM imidazole elution concentrates ( figure 2 A, B). And, purer FPV VP2 protein was obtained after 250mM elution concentrate. In addition, Western blot results showed that the FPV VP2 protein eluted with 250 mM imidazol...

Embodiment 3

[0033] Example 3 Preparation of Feline Parvovirus-Like Particles

[0034] The purified protein was loaded into a dialysis bag to remove the imidazole eluate. Dialysis assembly was performed at 4°C (50 mM Tris, 250 mM NaCl, pH=8.0), and VLPs were 20-30 nm in diameter, the closest to native feline parvovirus particles. In order to further determine the formation state of VLPs, transmission electron microscopy (TEM) detection was carried out, that is, the VLPs obtained by the above purification were negatively stained with 2% phosphotungstic acid, and the morphology of VLPs was observed by transmission electron microscopy. uniform, showing a hollow shape, the results are as follows image 3 As shown, the target protein FPV VP2 can spontaneously assemble into particles with a diameter of about 25 nm, with clear boundaries and high particle uniformity, similar to the natural virus-like particle morphology of FPV. Therefore, when the assembly conditions are 50 mM Tris, 250 mM NaCl...

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Abstract

The invention provides a feline parvovirus VP2 protein and an obtained self-assembled virus-like particle, and belongs to the field of VLP vaccines. The cat parvovirus VP2 gene provided by the invention has a nucleotide sequence as shown in SEQ ID NO. 1. The DNA sequence of the cat parvovirus VP2 gene is artificially modified and synthesized according to the preference of codons of escherichia coli, and the VP2 protein expressed by using the sequence has good immunocompetence and can effectively protect pet cats and felines from being invaded by cat plague viruses.

Description

technical field [0001] The invention belongs to the field of VLP vaccines, and in particular relates to a feline parvovirus VP2 protein and the obtained self-assembled virus-like particles. Background technique [0002] Feline parvovirus (feline parvovirus, FPV) is also known as feline leukopenia virus, feline distemper virus, feline infectious enteritis virus. It mainly occurs in kittens under the age of 12 months, especially kittens under the age of 36 months. Since FPV has caused great harm to my country's pet cat industry and wildlife protection, it is the top priority to develop new vaccines. [0003] FPV is a single-stranded non-enveloped DNA virus with a diameter of 20nm24nm and icosahedral symmetry. The VP2 protein is the main component of the parvovirus coat, and the VP2 protein is an immunogenic protein that can induce the body to produce neutralizing antibodies. Virus-like particles (VLPs), also known as core-like particles, are hollow particles ranging from 15...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/35C12N15/70C12N15/66C07K14/015
CPCC07K14/005C12N15/70C12N2750/14322C12N2750/14351C12N2750/14323C12N2800/22
Inventor 查丽莎周宇杭郑琪傅舟宇
Owner 深圳赫兹生命科学技术有限公司
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