CN-121737081-B - Recombinant FCV antigen, construction method and application thereof

Abstract

The invention discloses a recombinant FCV antigen, a construction method and application thereof. The construction method of the recombinant FCV antigen comprises the steps of fusing a T cell epitope coding sequence derived from a non-structural protein NS7 of FCV with a coding sequence of a SpyTag peptide through a coding sequence of a connecting peptide (linker), cloning the fused product onto a baculovirus transfer vector to obtain a recombinant plasmid, and integrating the T cell epitope coding sequence into the Bacmid through Tn7 transposition to finally obtain the recombinant FCV antigen. According to the invention, the NS7 is selected as a core immunogen to guide an immune system to generate a high-cell immune response, so that the defect of the existing vaccine in clearing intracellular viruses is overcome, and meanwhile, a T cell epitope with specificity is selected from a sequence of the NS7 as the immunogen, so that the mutation degree of antigens among different strains and the immune escape caused by virus antigen drift can be avoided, and the broad-spectrum protection is provided.

Inventors

• TENG XIAONUO
• FANG PENGFEI
• Xu Wanshi
• DONG YANPENG
• Chen Die

Assignees

• 苏州沃美生物有限公司

Dates

Publication Date

20260512

Application Date

20260225

Claims (11)

1. 1. A recombinant FCV antigen is characterized in that the amino acid sequence of the recombinant FCV antigen is shown as SEQ ID NO. 2.
2. 2. The construction method of the recombinant FCV antigen is characterized by comprising the steps of fusing a T cell epitope coding sequence derived from a non-structural protein NS7 of FCV with a coding sequence of a SpyTag peptide segment through a coding sequence of a connecting peptide Linker to construct a SpyTag-Linker-FCV-NS7 recombinant gene; Cloning SpyTag-Linker-FCV-NS7 recombinant genes onto a baculovirus transfer vector to obtain recombinant plasmids, then converting the recombinant plasmids into competent cells of escherichia coli, and integrating the T cell epitope coding sequences into Bacmid through Tn7 transposition to obtain recombinant baculovirus genome; transfecting insect cells with a recombinant baculovirus genome, screening to obtain recombinant baculovirus, and amplifying and purifying to obtain SpyTag-Linker-FCV-NS7 recombinant polypeptide, namely the recombinant FCV antigen; wherein the nonstructural protein NS7 is an RNA-dependent RNA polymerase RdRp; the amino acid sequence of the T cell epitope is shown as SEQ ID NO. 1; and the amino acid sequence of the recombinant FCV antigen is shown as SEQ ID NO. 2; and, the insect cell is an Sf9 insect cell.
3. 3. A recombinant FCV antigen prepared by the construction method of claim 2.
4. 4. A multivalent antigen system, comprising the directed covalent conjugate of a recombinant FCV antigen and a SpyCatcher-protein nanoparticle according to claim 1 or 3, wherein the molar ratio of the recombinant FCV antigen to the SpyCatcher-protein nanoparticle is 2:1; the protein in the Spycatcher-protein nanoparticle is ferritin Ferritin.
5. 5. The multivalent antigen system according to claim 4, wherein the preparation method of the SpyCatcher-protein nanoparticle comprises cloning the encoding gene of the SpyCatcher-Ferritin protein into a prokaryotic expression vector, then converting the prokaryotic expression vector into an expression system, performing IPTG induced expression, fusing the SpyCatcher-Ferritin protein into escherichia coli intracellular soluble expression, and obtaining the SpyCatcher-protein nanoparticle after self-assembly.
6. 6. The multivalent antigen system of claim 5, wherein the amino acid sequence of the SpyCatcher-protein is shown in SEQ ID NO. 3.
7. 7. An immune composition comprising a polypeptide of formula I, characterized by comprising the following steps: The recombinant FCV antigen of claim 1 or 3 or the multivalent antigen system of any one of claims 4-6; And, a pharmaceutically acceptable carrier.
8. 8. The composition of claim 7, wherein the pharmaceutically acceptable carrier is a combination of one or more of isotonic fluids; And/or the immune composition further comprises one or more of an oil-in-water emulsion adjuvant, a saponin adjuvant, a water-in-oil emulsion adjuvant and a white oil for enhancing cellular immunity.
9. 9. The composition of claim 7, wherein the pharmaceutically acceptable carrier is phosphate buffered saline.
10. 10. A feline calicivirus recombinant subunit vaccine comprising the recombinant FCV antigen of claim 1 or 3, the multivalent antigen system of any one of claims 4-6, or the immune composition of any one of claims 7-9.
11. 11. A feline viral antigen composition comprising a physical mixture of the feline calicivirus recombinant subunit vaccine of claim 10 and an FCV capsid protein antigen or a feline viral antigen.

Description

Recombinant FCV antigen, construction method and application thereof Technical Field The invention belongs to the technical field of vaccines, and particularly relates to a recombinant FCV antigen, and a construction method and application thereof. Background The feline calicivirus (Feline Calicivirus, FCV) is one of the main pathogens causing upper respiratory tract diseases (infectious rhinotracheitis) and canker sores in cats, and has the characteristics of high morbidity and high variability, which brings serious threat to the global cat industry and pet health. FCV is a single-stranded positive strand RNA virus of the genus herpesviridae, family caliciviridae, and is non-capsular and icosahedral in symmetry. The genome of the virus comprises three open reading frames, ORF1, ORF2 and ORF3, respectively, wherein ORF1 is located at the 5' end of the genome, encoding 6 nonstructural proteins p 5.6, p32, p39 (NTPase), p30, p13 (VPg) and p76 (Pro)Pol)。 Currently, commercial vaccines against FCV are mainly inactivated and live attenuated vaccines. The immunogens of these traditional vaccines are mainly focused on viral capsid proteins (such as VP 1), the protection mechanism of which depends on inducing the body to produce neutralizing antibodies, belonging to humoral immune-dominated immune responses. The prior art approaches with humoral immunity as a core suffer from the following inherent drawbacks and limitations, including: First, the protective spectrum is narrow, immune escape is easy to occur, and FCV capsid protein is one of regions with highest mutation rate in viral genome. The neutralizing antibody induced by the existing vaccine mainly aims at a specific antigen epitope on the protein. When epidemic strains undergo point mutation or antigen drift, the original vaccine can not effectively neutralize new strains, so that protection fails, and an immune escape phenomenon occurs, so that vaccine strains need to be frequently updated in the vaccine production process, and the production cost and epidemic prevention complexity are increased. Secondly, the immune response type is single, and the intracellular viruses cannot be effectively cleared, wherein the traditional vaccine is focused on inducing humoral immunity, and the generated antibodies mainly act on extracellular virus particles, but have limited virus clearing capacity for the viruses which enter the cells. The lack of an effective T cell (especially CTL) immune response may result in incomplete immune protection, and failure to completely prevent viral replication and detoxification. Thirdly, the attenuated live vaccine has biological safety risk that although the attenuated live vaccine can induce stronger immune response, the attenuated live vaccine has potential risk of virulence reversion, and can possibly cause harm to immunodeficiency individuals or kittens, and the safety is doubtful, and subunit vaccines in the prior art cannot fundamentally avoid the risk of virulence reversion due to the inclusion of virus genetic material, so that the risk of vaccine use is increased. Fourth, the immune potential of conserved nonstructural proteins is ignored, and prior art schemes generally ignore the value of FCV nonstructural proteins (Non-Structural Protein, NSP) as vaccine targets. Unlike the highly variant structural protein, the nonstructural protein (RNA-dependent RNA polymerase NS 7) plays a key role in viral replication, with sequences more conserved among different strains. Targeting the immune response of these conserved regions is expected to overcome the problem of protection failure due to viral variation, providing a broader spectrum of cross protection. The present invention systematically uses specific T cell epitopes of FCV nonstructural proteins for subunit vaccine design for the first time to induce cellular immunity against conserved regions. Therefore, the invention develops a novel FCV vaccine which takes the non-structural protein NS7 of the feline calicivirus as a core immunogen and stimulates powerful cellular immunity by designing a T cell epitope subunit vaccine so as to thoroughly eliminate infected cells aiming at the technical problems of narrow protection spectrum, easy occurrence of immune escape defect and the like caused by virus antigen drift of the vaccine in the prior art. Disclosure of Invention The invention mainly aims to provide a recombinant FCV antigen, a construction method and application thereof, so as to overcome the defects of the prior art. In order to achieve the above object, the present invention adopts the following technical solutions. As a first aspect of the invention, the invention provides a recombinant FCV antigen, the amino acid sequence of which is shown as SEQ ID NO. 2. As a second aspect of the invention, the invention provides a construction method of recombinant FCV antigen, comprising the steps of fusing a T cell epitope coding sequence derived from a non-structural protein NS7