CN-121987772-A - Application of activity-regulating cytoskeletal-related protein in REV nanoparticle vaccine and vaccine

Abstract

The invention relates to an application of an activity-regulating cytoskeletal related protein (ARC) in REV nanoparticle vaccines and vaccines, and belongs to the technical field of animal vaccine preparation, wherein the amino acid sequence of the activity-regulating cytoskeletal related protein (ARC) is shown in SEQ ID NO.3, the nucleotide sequence is shown in SEQ ID NO.4, the activity-regulating cytoskeletal related protein is used as carrier protein in the nanoparticle vaccines, and the nanoparticle vaccines are REV nanoparticle vaccines. The invention uses ARC as a carrier in nanoparticle vaccine for the first time, self-assembles into nanoparticles, can serve as an antigen delivery platform, plays a role in protein-antigen delivery, and stimulates the organism to generate immune response.

Inventors

• WANG GUIHUA
• WEI DIANFENG
• CHENG ZIQIANG
• ZHOU JIE
• SUN LIFENG
• Jia Ruping
• ZHAO YUXIN

Assignees

• 山东农业大学

Dates

Publication Date

20260508

Application Date

20260205

Claims (8)

1. 1. The application of the activity-regulating cytoskeletal related protein in REV nanoparticle vaccines is characterized in that the amino acid sequence of the activity-regulating cytoskeletal related protein is shown as SEQ ID NO.3, the nucleotide sequence is shown as SEQ ID NO.4, the activity-regulating cytoskeletal related protein is used as carrier protein in nanoparticle vaccines, the nanoparticle vaccines are REV nanoparticle vaccines, and the activity-regulating cytoskeletal related protein is ARC for short.
2. 2. The use according to claim 1, wherein the env gp90 antigen is formed by covalent coupling with an ARC in a nanoparticle vaccine, wherein the env gp90 antigen has the amino acid sequence shown in SEQ ID No.9 and the nucleotide sequence shown in SEQ ID No. 10.
3. 3. The use according to claim 1, wherein the carrier protein ARC has a SpyCatcher sequence attached to the N-terminus and a Flag sequence attached to the C-terminus.
4. 4. The use according to claim 1, wherein the antigen protein gp90 protein is linked at the N-terminus to a6 xhis sequence and at its C-terminus to a SpyTag tag sequence.
5. 5. The ARC nanoparticle vaccine is characterized by being formed by assembling env gp90 glycoprotein of REV and carrier protein ARC, wherein the N end of the ARC nanoparticle is connected with a SpyCatcher sequence and a Flag sequence to obtain an SC-ARC plasmid, the amino acid sequence of the SC-ARC plasmid is shown as SEQ ID N0.5, the nucleotide sequence of the SC-ARC plasmid is shown as SEQ ID N0.6, the env gp90 glycoprotein is connected with a6 xHis sequence at the N end and with a SpyTag tag sequence at the C end to obtain the ST-ARC plasmid, the amino acid sequence of the ST-ARC plasmid is shown as SEQ ID N0.11, and the nucleotide sequence of the ST-ARC plasmid is shown as SEQ ID N0.12.
6. 6. The preparation method of the ARC nanoparticle vaccine according to claim 5 is characterized by comprising the following specific steps of (1) obtaining an SC-ARC recombinant protein, wherein the SC-ARC recombinant protein is formed by connecting an SpyCatcher sequence to the N end of the ARC protein and a Flag tag to the C end of the ARC protein, and the amino acid sequence of the SC-ARC recombinant protein is shown as SEQ No. 5; (2) The SC-gp 90 recombinant protein is obtained, wherein the SC-gp 90 recombinant protein is formed by connecting a6 xHis sequence at the N end of p90 glycoprotein and a SpyTag tag sequence at the C end, and the amino acid sequence of the SC-gp 90 recombinant protein is shown as SEQ No. 11; (3) And (3) co-incubating and assembling the SC-ARC and ST-gp90 recombinant protein according to a molar ratio of 1:3 at 4 ℃ and purifying to obtain the ARC nanoparticle vaccine.
7. 7. The method according to claim 6, wherein in (1), when the pET21a (+) plasmid is recombinantly expressed as an expression vector, a recombinant plasmid pET-21a-SC-ARC is constructed by inserting an SC-ARC encoding nucleotide sequence, to which a Flag tag is attached, into the pET21a (+) plasmid between EcoRI and XhoI sites.
8. 8. The method according to claim 6, wherein in (2), the pET-30a (+) plasmid is used as a vector, and the nucleotide sequence of ST-gp90 is cloned and recombined into the pET-30a (+) plasmid to construct a recombinant plasmid pET-30a-ST-gp90.

Description

Application of activity-regulating cytoskeletal-related protein in REV nanoparticle vaccine and vaccine Technical Field The invention belongs to the technical field of animal vaccine preparation, and particularly relates to application of an activity-regulating cytoskeletal related protein in REV nanoparticle vaccines and the vaccines. Background Avian reticuloendotheliosis (Reticuloendotheliosis, RE) is an immunosuppression-based avian disease caused by avian reticuloendotheliosis virus (Reticuloendotheliosis Virus, REV), belonging to the genus C retrovirus of the family retroviridae, typically gamma retrovirus. REV mainly causes a group of syndromes characterized by reticuloendothelial hyperplasia, including acute reticuloendothelial hyperplasia, short syndrome, and chronic neoplastic diseases of lymphoid and other tissues. At present, commercial vaccines against REV are not available, but in the conventional vaccines such as inactivated vaccines, attenuated live vaccines and the like, the inactivated vaccines cannot effectively induce cellular immune responses and neutralizing antibody responses, and the attenuated live vaccines are worry about potential recombination and toxicity anti-strong risks. REV vaccine research has focused mainly on env proteins, envelope proteins (env) are glycoproteins comprising two peptide chains, gp20 and gp90, smaller gp20 is a transmembrane protein (TM) that can pass through the envelope of the virus, and studies have shown that larger gp90 is exposed outside the envelope, belonging to envelope proteins (SU), which are the major immunogenic proteins, contributing to the production of antibodies by the body. The activity-modulating cytoskeletal associated protein (activity regulated cytoskeleton associated protein, ARC) is an early gene product present at the end of the nerve synapses and is derived from the Ty3/gypsy retrotransposon lineage. As a protein having homology with retroviral gag protein, the main function of the gene is to regulate dynamic changes in the cytoskeleton, especially when the cell is subjected to external stimuli or changes in activity. The ARC protein acts as a bridge in the cell, linking the cytoskeleton and other structures within the cell, helping the cell maintain its morphology and function. Signal sensing ARC proteins are capable of sensing signals outside of cells, such as mechanical forces, chemicals, etc. In recent years, nanoparticle vaccines have received increasing attention for targeted delivery of antigen-carrying substances to immune organs, and for their preparation by means of self-assembly, etc. Compared with the traditional vaccine, the nano particles are easier to be absorbed by antigen presenting cells, and can reduce the degradation of antigen and improve the utilization rate of antigen, thereby improving the action effect of the vaccine. Disclosure of Invention The invention aims to provide an application of an activity-regulating cytoskeletal related protein in a nanoparticle vaccine and the vaccine, wherein ARC is selected as a REV nanoparticle vaccine core, and simultaneously a SpyTag/SpyCatcher system derived from streptococcus pyogenes is introduced to covalently couple ARC-based nanoparticles so as to improve immunogenicity. The invention is realized by the following technical scheme: The application of the activity-regulating cytoskeletal related protein in the REV nanoparticle vaccine is characterized in that the amino acid sequence of the activity-regulating cytoskeletal related protein (ARC) is shown as SEQ ID NO.3, the nucleotide sequence is shown as SEQ ID NO.4, the activity-regulating cytoskeletal related protein is used as carrier protein in the nanoparticle vaccine, and the nanoparticle vaccine is the REV nanoparticle vaccine. Further, the use, in nanoparticle vaccines, of env gp90 antigen is formed by covalent coupling with ARC. The ARC acts as an antigen delivery platform, exerting protein-antigen delivery effects, stimulating the immune response in the body. The env gp90 antigen has an amino acid sequence shown in SEQ ID NO.9 and a nucleotide sequence shown in SEQ ID NO. 10. Further, the N-terminal of the carrier protein ARC is connected with the SpyCatcher sequence and the C-terminal thereof is connected with the Flag sequence. Further, the antigen protein gp90 protein is linked to a 6 XHis sequence at the N-terminus and to a SpyTag tag sequence at the C-terminus. The invention also provides an ARC nanoparticle vaccine, which is formed by assembling env gp90 glycoprotein of REV and carrier protein ARC, wherein the N end of the ARC nanoparticle is connected with a SpyCatcher sequence and a Flag sequence to obtain an SC-ARC plasmid, the amino acid sequence of the SC-ARC plasmid is shown as SEQ ID N0.5, the nucleotide sequence of the SC-ARC plasmid is shown as SEQ ID N0.6, the env gp90 glycoprotein is connected with a 6 xHis sequence at the N end and with a SpyTag tag sequence at the C end to obtain the ST-ARC