METHOD FOR IMPROVING ANTIGEN IMMUNOGENICITY, CORONAVIRUS ANTIGEN, USE THEREOF, RECOMBINANT VECTOR, EXPRESSION KIT, TRANSGENIC CELL LINE, RECOMBINANT BACTERIUM, CORONAVIRUS VACCINE, PREPARATION METHOD OF ANTIGEN AND NUCLEOTIDE SEQUENCE

Abstract

Provided is a Helicobacter pylori ferritin-based novel coronavirus S protein single-region subunit nano-vaccine. According to the present invention, a receptor binding domain (RBD) of a virus is used as an antigen and is connected with a Helicobacter pylori multimeric protein (HP_Ferritin) to form a fusion protein RBD-HP_Ferritin, such that antigen multimerization is realized; and an eukaryotic cell expression system is then utilized for expression, so as to form a 24-mer nano-antigen by means of the self-assembly action of the HP_Ferritin. According to the solution, the defect that RBD monomers are insufficient in immunogenicity can be overcome; the obtained vaccine can remarkably improve the level of neutralizing antibodies of a host to viruses; and the generated antibodies have the capacity to strongly prevent the viruses from invading target cells.

Claims

1. A method for improving antigen immunogenicity, comprising combining a receptor binding domain (RBD) of a virus with Helicobacter pylori multimeric protein (Helicobacter pylori_Ferritin, Ferritin (HP)) to form a new fusion protein RBD-HP_Ferritin, which is then used as an antigen.

2. The method according to claim 1, wherein the antigen is a coronavirus antigen, and the receptor binding domain RBD of the virus are a receptor binding domain RBD of a coronavirus.

3. The method according to claim 2, wherein the coronavirus antigen is a novel coronavirus SARS-CoV-2 antigen, and the receptor binding domain RBD of the coronavirus are a receptor binding domain RBD of a novel coronavirus SARS-CoV-2.

4. The method according to claim 3, wherein the novel coronavirus SARS-CoV-2 antigen is a novel coronavirus SARS-CoV-2 surface spike protein (S protein) antigen.

5. The method according to claim 4, wherein a sequence of the receptor binding domain RBD of the novel coronavirus SARS-CoV-2 is shown in SEQ ID NO: 1, an amino acid sequence of the Ferritin(HP) is shown in SEQ ID NO: 2, SEQ ID NO: 1 and SEQ ID NO: 2 can be directly linked, or the two can be linked by a hinge region Linker to form a new fusion protein RBD-HP_Ferritin; preferably, when the Linker is GSG, an amino acid sequence of the resulting fusion protein RBD-HP_Ferritin is shown in SEQ ID NO: 3.

6. The method according to claim 1, wherein after the fusion protein is added with a signal peptide and a purification tag, an eukaryotic expression system is utilized to express antigen; preferably, the signal peptide is a secretory signal peptide (SP); preferably, the purification tag is a His tag (His-tag); preferably, an amino acid sequence of fusion of the SP, the His-tag, the receptor binding domain RBD of the novel coronavirus SARS-CoV-2 and HP_Ferritin is as shown in SEQ ID NO: 4.

7. A coronavirus antigen, wherein a fusion protein RBD-HP_Ferritin is constructed and obtained according to the method in claim 1.

8. Use of the coronavirus antigen in claim 7 in preparation of anti-coronavirus medicament.

9. The use according to claim 8, wherein the use is to combine the coronavirus antigen and a SAS adjuvant.

10. The use according to claim 8, wherein the use is for preparation of a kit; the kit contains the antigen, or a DNA molecule encoding the antigen, or a recombinant vector/ expression kit/ transgenic cell line/recombinant bacterium expressing the antigen.

11. A recombinant vector, expression kit, transgenic cell line or recombinant bacterium expressing the coronavirus antigen of claim 7.

12. A coronavirus vaccine, wherein the coronavirus vaccine is prepared by the coronavirus antigen of claim 7 as an antigen.

13. A preparation method of the antigen of claim 7, comprising: at a 3′ end of a nucleotide sequence corresponding to amino acids as shown in direct linking or hinge linking of SEQ ID NO: 1 and SEQ ID NO: 2, or a nucleotide sequence corresponding to amino acids as shown in SEQ ID NO: 3 or SEQ ID NO: 4, adding a translation terminator codon, performing clone expression, screening for a correct recombinant, then transfecting an eukaryotic expression system for expression, collecting a cell supernatant after expression, and purifying to obtain the novel coronavirus antigen.

14. A nucleotide sequence encoding and expressing the coronavirus antigen of claim 7, or a vector or transgenic cell line comprising the sequence.

15. A coronavirus antigen, wherein a fusion protein RBD-HP_Ferritin is constructed and obtained according to the method in claim 3.

16. A coronavirus antigen, wherein a fusion protein RBD-HP_Ferritin is constructed and obtained according to the method in claim 5.

17. A coronavirus antigen, wherein a fusion protein RBD-HP_Ferritin is constructed and obtained according to the method in claim 6.

18. A coronavirus vaccine, wherein the coronavirus vaccine is prepared by the coronavirus antigen of claim 15 as an antigen.

19. A coronavirus vaccine, wherein the coronavirus vaccine is prepared by the coronavirus antigen of claim 16 as an antigen.

20. A coronavirus vaccine, wherein the coronavirus vaccine is prepared by the coronavirus antigen of claim 17 as an antigen.

Description

DESCRIPTION OF DRAWINGS

[0051] FIG. 1 is a schematic diagram of self-assembly of RBD-HP_Ferritin fusion proteins into a nanoparticle.

[0052] FIG. 2 is a schematic diagram of structure of the RBD-HP_Ferritin fusion protein.

[0053] FIG. 3 is a schematic diagram of structure of plasmid expressing RBD-HP_Ferritin.

[0054] FIG. 4 is an enzyme cleavage verification of RBD-HP_Ferritin fusion.

[0055] FIG. 5 is an immunofluorescence image of 293F cells transfected with RBD-HP_Ferritin fusion protein.

[0056] FIG. 6 is a molecular sieve diagram for purification of RBD-HP_Ferritin fusion protein.

[0057] FIG. 7 is a SDS-PAGE image for purification of RBD-HP_Ferritin fusion protein (about 48 KD).

[0058] FIG. 8 is an immunization strategy for mice immunized with RBD-HP _Ferritin nano-vaccine.

[0059] FIG. 9 is a detection strategy for neutralizing antibody titer in mouse serum.

[0060] FIG. 10 shows that mice immunized with RBD-HP _Ferritin nano-vaccine produce neutralizing antibodies that block SARS-CoV-2 from invading into target cells.

[0061] FIG. 11 shows experimental results of the fusion protein RBD-PF_Ferritin in Comparative Example 1.

[0062] FIG. 12 shows experimental results of the fusion protein LS-RBD in Comparative Example 2.

DETAILED DESCRIPTION

[0063] The present invention is further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any form.

[0064] Unless otherwise specified, reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

[0065] Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

Embodiment 1 Construction of Novel Coronavirus SARS-CoV-2 Antigen (Fusion Protein RBD-HP _Ferritin)

[0066] The schematic diagram of self-assembly of RBD-HP_Ferritin fusion proteins into a nanoparticle, and the schematic diagram of structure is as shown in FIG. 1 and FIG. 2, respectively..

[0067] Specifically, construction and preparation method of fusion protein RBD-HP_Ferritin is as follows:

1. Preparation of Vector Expressing RBD-Ferritin Antigen

[0068] A translation terminator codon was added at a 3′ end of a nucleotide sequence of RBD-HP_Ferritin, which was then cloned and added between Xho I and Xba I enzyme cleavage sites in an Intron and WPRE expression-enhanced expression vector (pcDNA3.1-Intron-WPRE), and an expression vector pcDNA3.1-Intron-WPRE-RBD-Ferritin(HP)-IRES-GFP (as shown in FIG. 3) was constructed.

[0069] The recombinant plasmid was transformed into DH5α competent cells, cultured at 37° C. overnight, and positive clones were screened and identified by PCR. An endotoxin-depleted plasmid was extracted, then after enzyme cleavage and verification by sequencing, it was used for expression of nano-antigen protein (as shown in FIG. 4). The plasmid was transfected into HEK293F cells through a lipofection protocol, and a cell supernatant was harvested by centrifugation 3 days after transfection (the immunofluorescence image of 293F cells transfected with RBD-HP_Ferritin fusion protein is shown in FIG. 5), and a purification of target protein RBD-HP_Ferritin was carried out.

2. Purification of RBD-HP _Ferritin Nano-antigen

[0070] The supernatant of cells expressing RBD-HP_Ferritin was filtered through a .Math.m filter to remove cell debris. After ultrafiltration through a 10 K ultrafiltration tube, the filtered cell supernatant was combined with Histrap-excel at 4° C. for 30 minutes, and a HisTrap excel nickel column was used for crude purification.

[0071] Afterwards, firstly 50 ml was washed with PBS (pH 7.4) buffer and low-concentration imidazole buffer (PBS, 50 mM Imidazole, pH 7.4) to remove flow-through impurity protein. Thereafter, target protein was eluted by high imidazole-containing buffer (PBS, 500 mM Imidazole, pH 7.4). Subsequently, the target protein was enriched using a Lectin Agarose column (GE) packed with Con A and WGA at a ratio of 1:1.

[0072] Elution peaks of RBD-HP_Ferritin 24-mer were collected and combined, and finally purified by molecular sieve chromatography using a Siperose6 Increase 10/300 GL column (GE) to obtain a 24-multimerized RBD-HP_Ferritin protein with a purity greater than 99% (as shown in FIGS. 6-7), a buffer for molecular sieve chromatography was: PBS, pH 7.4. After the target protein was concentrated, it was divided into small portions, quickly frozen in liquid nitrogen and stored at -80° C.

Embodiment 2 Mouse Immunization Experiment

[0073] The RBD-HP _Ferritin antigen obtained in Embodiment 1 was diluted with physiological saline to 100 .Math.g/ml according to Table 1, and emulsified in groups with an equal volume of adjuvant SAS. 6-8 week-old Balb/C mice were then immunized in groups. The immunization strategy was as shown in FIG. 8, that is, by intraperitoneal injection, each mouse received 3 times of vaccine immunization on Day 0, Week 3 (Day 21), and Week 14 (Day 108), with an inoculation volume of 200 .Math.l (10 .Math.g) each time. On Day 10, Day 31, and Day 108, the mice were bled from the orbit. Mouse blood serum was obtained by centrifugation at 4° C. and 2800 rpm for 15 minutes after standing for a period of time until the blood serum was precipitated, and was immediately used for SARS-CoV-2 pseudotyped viruspseudotyped virus neutralization detection experiment.

TABLE-US-00005 Antigen/control Antigen content Adjuvant Number of animals RBD-HP_Ferritin 10 .Math.g SAS 4 PBS 0 SAS 4

Embodiment 3 Pseudotyped Virus Neutralization Test

1. Preparation of Pseudotyped Virus

[0074] According to a sequence published by NCBI, Spike protein of SARS-CoV-2 was synthesized and inserted into a pcDNA3.1 expression vector. 293T cells were co-transfected by the expression vector of SARS-CoV-2 Spike protein with pHIV-luciferase and psPAX2 plasmid. After 5 hours of transfection, cells were washed twice with PBS, and then continued to culture with replaced serum-free DMEM medium. After 48 hours, a supernatant was collected and centrifuged to remove cell debris. After dissolving with a small volume of serum-free DMEM, HIV-luc/SARS-CoV-2-S pseudotyped virus was obtained.

[0075] The pseudotyped virus can effectively simulate a process of wild-type SARS-CoV-2 invading cells. When it infects production cells or target cells, expression of luciferase reporter gene carried by SARS-CoV-2 pseudotyped virus can accurately reflect results of virus infection, so that results of the experimental system can be read accurately and quickly, which can be used as an excellent antibody neutralization titer monitoring system (as shown in FIG. 9).

2. Pseudotyped Virus TCID 50 Assay

[0076] The virus solution collected in the previous step was diluted 5-fold and added to HEK293T cells in a 96-well plate. After 4 hours of infection, the virus solution was discarded, cells were washed twice with PBS, replaced with DMEM complete medium containing 10% serum. After 48 hours, the medium was discarded, washed twice with PBS, added with a cell lysis buffer, and lysed by shaking for 30 minutes. After freeze-thawing once at -80° C., 30 .Math.l of each well was taken to detect a luciferase activity value using GloMax 96 (Promega). TCID 50 was calculated by Reed-Muech method.

3. Neutralization Test

[0077] The purified antibody was diluted 2-fold, mixed with pseudotyped virus of TCID 50 final concentration, and co-incubated at 37° C. for 1 hour. The mixture was added to HEK293T cells with a density of about 70% in a 96-well plate. After 48 hours, culture medium was discarded, cells were washed twice with PBS, cell lysis buffer was added, and the luciferase activity value was detected.

4. Result Analysis

[0078] Results are shown in FIG. 10. A neutralizing activity against SARS-CoV-2 pseudotyped virus was detected in serum of Balb/c mice 10 days after immunization of RBD-HP_Ferritin nano-antigen. The t-test shows that there is a significant difference between an experimental group and a control group. At a significance level of 0.05, a two-tailed probability level is less than 0.05.

[0079] The results show that combination of RBD-HP_Ferritin of the present invention and SAS adjuvant can stimulate humoral immunity of mice 10 days after once immunization, which is less than neutralizing antibody titer stimulated by 24-mer group as a parallel control, and there is a significant difference.

Comparative Example 1

[0080] Referring to the method of Embodiment 1, Helicobacter pylori multimeric protein (HP_Ferritin)was replaced by Pyrococcus furiosus multimeric protein (PF_Ferritin), and a fusion protein RBD-PF_Ferritin was constructed as an antigen.

[0081] And according to the method of Embodiments 2-3, mouse immunization experiment and pseudotyped virus neutralization test were carried out.

[0082] Results show (FIG. 11) that there was no significant difference in ability of serum of mice immunized with RBD-PF_Ferritin as an antigen to neutralize the SARS-CoV-2 pseudotyped virus compared to the control group.

Comparative Example 2

[0083] Referring to the method of Embodiment 1, Helicobacter pylori multimer protein (HP_Ferritin)was replaced with dioxotetrahydropyridine synthase multimer protein (Lumazine Synthase, LS) derived from Aquifex aeolicus strain to construct a fusion protein LS- RBD as an antigen.

[0084] And according to the method of Embodiments 2-3, mouse immunization experiment and pseudotyped virus neutralization test were carried out.

[0085] Results show (FIG. 12) that there was no significant difference in ability of serum of mice immunized with LS-RBD as an antigen to neutralize the SARS-CoV-2 pseudotyped virus compared to the control group.

[0086] The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, and simplifications shall be equivalent replacement modes, which are all included in the protection scope of the present invention.