CN113444155B

CN113444155B - MERS-CoV membrane protein receptor binding domain dimer, and coding gene and application thereof

Info

Publication number: CN113444155B
Application number: CN202010418710.1A
Authority: CN
Inventors: 张林琦; 周盼盼; 史宣玲
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2020-05-18
Filing date: 2020-05-18
Publication date: 2022-06-07
Anticipated expiration: 2040-05-18
Also published as: CN113444155A

Abstract

The invention discloses MERS-CoV membrane protein receptor binding domain dimer, and a coding gene and application thereof. The invention firstly protects the dimer of MERS-CoV RBD; MERS-CoV RBD is protein shown in a sequence 3 of a sequence table or protein shown in a sequence 7 of the sequence table. The invention determines the structure and immunogenicity of middle east respiratory syndrome coronavirus membrane protein receptor binding domain dimer (MERS-CoV RBD dimer). Furthermore, the invention discovers that the immunogenicity of MERS-CoV RBD dimer is better than that of MERS-CoV RBD monomer, and stronger neutralizing antibody reaction can be induced in animals. The discovery has important theoretical guidance value and wide application prospect for developing vaccines and the like for treating and preventing middle east respiratory syndrome.

Description

MERS-CoV membrane protein receptor binding domain dimer, and coding gene and application thereof

Technical Field

The invention relates to MERS-CoV membrane protein receptor binding domain dimer and a coding gene and application thereof.

Background

Middle East respiratory syndrome coronavirus (MERS-CoV) was first discovered in 2012 to infect humans in the Middle East region, and then the disease of such virus infection occurred in several countries and regions in Europe.

More than half of MERS-CoV infected patients develop severe respiratory disease with clinical symptoms very similar to SARS-CoV-induced atypical pneumonia in 2003. This virus has attracted a high degree of worldwide attention because it can be transmitted to humans.

Gene sequence alignment shows that MERS-CoV belongs to beta coronavirus, but the source and host thereof are not determined yet. Like other coronaviruses, MERS-CoV uses its surface membrane protein S glycoprotein to enter susceptible cells.

Disclosure of Invention

The invention aims to provide MERS-CoV membrane protein receptor binding domain dimer and a coding gene and application thereof.

The invention firstly protects the dimer of MERS-CoV RBD;

MERS-CoV RBD is (a1), (a2), (a3), (a4), (a5) or (a6) as follows:

(a1) protein shown in a sequence 3 in a sequence table;

(a2) a protein obtained by linking a signal peptide to the N-terminus of (a 1);

(a3) and (b) a fusion protein obtained by attaching a tag to the N-terminus or/and C-terminus of (a 1).

(a4) A fusion protein obtained by attaching a tag to the C-terminus of (a 2);

(a5) protein shown in a sequence 7 of a sequence table;

(a6) the protein shown as amino acid residues 39-287 in the sequence 7 of the sequence table.

Nucleic acid molecules encoding dimers of MERS-CoV RBD are also within the scope of the invention.

In particular, the nucleic acid molecule may be a DNA molecule.

The DNA molecule may be (c1) or (c2) or (c3) as follows:

(c1) a DNA molecule shown in a sequence 4 of a sequence table;

(c2) a DNA molecule shown as nucleotide No. 124-861 in the sequence 8 of the sequence table;

(c3) a DNA molecule shown in a sequence 8 of a sequence table.

Recombinant plasmids having the DNA molecules also belong to the scope of protection of the present invention. The recombinant plasmid can be specifically pFastBac^TMThe dual vector is a recombinant plasmid constructed by a starting vector.

The invention also provides a method for preparing the MERS-CoV RBD dimer, which comprises the following steps: the protein was prepared using the Bac-to-Bac system. The method specifically comprises the following steps: and preparing recombinant Bacmid by adopting the recombinant plasmid, preparing virus liquid by adopting Bacmid and insect cells, and preparing a dimer of MERS-CoV RBD by adopting the virus liquid and the insect cells. The insect cell may specifically be an Sf9 cell.

The invention also protects a kit for preparing the dimer of MERS-CoV RBD, which comprises the recombinant plasmid and insectsA cell. The recombinant plasmid is constructed by taking an expression vector of a Bac-to-Bac system as a starting vector and has the nucleic acid molecule; the nucleic acid molecule is a DNA molecule. The expression vector of the Bac-to-Bac system can be pFastBac^TMA dual vector. The insect cell may specifically be an Sf9 cell.

The MERS-CoV RBD dimer activity higher than the MERS-CoV RBD monomer also belongs to the protection scope of the invention. The activity is immunogenic. The activity is the neutralizing activity of the antibody obtained after immunizing animals as immunogen to MERS-CoV.

The invention also protects MERS-CoV RBD monomer, which is (b1), (b2), (b3), (b4), (b5) or (b 6):

(b1) protein shown in a sequence 1 in a sequence table;

(b2) a protein obtained by linking a signal peptide to the N-terminus of (b 1);

(b3) and (b1) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of (b 1).

(b4) A fusion protein obtained by attaching a tag to the C-terminus of (b 2);

(b5) protein shown in a sequence 5 in a sequence table;

(b6) the protein shown as 39 th-270 th amino acid residues in a sequence 5 of a sequence table.

Nucleic acid molecules encoding monomers of MERS-CoV RBD are also within the scope of the invention.

In particular, the nucleic acid molecule may be a DNA molecule.

The DNA molecule may be (d1) or (d2) or (d3) as follows:

(d1) a DNA molecule shown in a sequence 2 of a sequence table;

(d2) DNA molecule shown as 124-810 nucleotide in sequence 6 of the sequence table;

(d3) DNA molecule shown in sequence 6 of the sequence table.

Illustratively, the labels are shown in table 1. The respective labels and the relations of the sums in table 1 may also be relations of or.

TABLE 1

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Receptor binding region: receptor Binding Domain (RBD).

The invention also protects the application of the dimer of MERS-CoV RBD or the monomer of MERS-CoV RBD or any one of the nucleic acid molecules or the recombinant plasmid or the kit in the preparation of products; the application of the product is (e1) or (e 2):

(e1) as a middle east respiratory syndrome coronavirus vaccine;

(e2) as a medicament for the prevention and/or treatment of middle east respiratory syndrome.

The invention also protects a product, the active ingredient of which is the dimer of MERS-CoV RBD or the monomer of MERS-CoV RBD or any one of the nucleic acid molecules or the recombinant plasmid or the kit;

the application of the product is (e1) or (e 2):

(e1) as a middle east respiratory syndrome coronavirus vaccine;

The invention determines the structure and immunogenicity of a middle east respiratory syndrome coronavirus membrane protein receptor binding domain dimer (MERS-CoV RBD dimer). Furthermore, the invention discovers that the immunogenicity of MERS-CoV RBD dimer is better than that of MERS-CoV RBD monomer, and stronger neutralizing antibody reaction can be induced in animals. The discovery has important theoretical guidance value and wide application prospect for developing vaccines and the like for treating and preventing middle east respiratory syndrome.

Drawings

FIG. 1 is a diagram showing the superposition of a chromatogram (B) for molecular sieve chromatography in the case of performing step two using recombinant plasmid A and a chromatogram (A) for molecular sieve chromatography in the case of performing step two using recombinant plasmid B.

FIG. 2 is a non-reducing gel electrophoresis of MERS-CoV RBD monomer solution (B) and MERS-CoV RBD dimer solution (A).

FIG. 3 is a graph showing the results of an antigenicity analysis (ELISA test).

Fig. 4 is a diagram showing the results of the structure analysis.

Detailed Description

The following examples are intended to facilitate a better understanding of the invention, but are not intended to limit the invention thereto. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. Insect cellMedium (insert-Xpress Media): lonza Wokingham Ltd., cat # 12-730F. pFastBac^TMdual vector: ThermoFisher company, cat # 10712024. All PBS buffers used in the examples were 0.01M PBS buffer at pH7.4, unless otherwise specified.

Example 1 preparation of MERS-CoV RBD monomer and MERS-CoV RBD dimer

Construction of recombinant plasmid

Substituting double-stranded DNA molecule shown as sequence 6 in a sequence table for pFastBac^TMAnd a small fragment between BglII and SalI cleavage sites of the dual vector to obtain a recombinant plasmid A. In the sequence 6 of the sequence table, the 1 st to 114 th nucleotides encode gp67 signal peptide, the 124 th and 792 th nucleotides encode RBD monomer, and the 793 th and 810 th nucleotides encode His₆And (4) a label. The DNA molecule shown in sequence 6 of the sequence table encodes the protein shown in sequence 5 of the sequence table. The gp67 signal peptide is cut off during the expression and secretion process of insect cells to form a mature protein consisting of the 39 th-270 th amino acid residues in the sequence 5 of the sequence table.

Inserting a double-stranded DNA molecule shown as a sequence 8 in a sequence table into pFastBac^TMThe BglII and SalI sites of the dual vector yield recombinant plasmid B. In the sequence 8 of the sequence table, the 1 st to 114 th nucleotides encode gp67 signal peptide, the 124 th and 843 th nucleotides encode RBD dimer, and the 844 th and 861 th nucleotides encode His₆And (4) a label. The DNA molecule shown in the sequence 8 of the sequence table codes the protein shown in the sequence 7 of the sequence table. The gp67 signal peptide is cut off in the process of insect cell expression and secretion, and a mature protein consisting of amino acid residues 39-287 of the sequence 7 of the sequence table is formed.

Preparation and purification of bis, MERS-CoV RBD monomer and MERS-CoV RBD dimer

1. Preparation of recombinant Bacmid

(1) Adding the recombinant plasmid into a just thawed escherichia coli DH10 Bac competent cell, and placing for 30min on ice; then heat shocking for 75s at 42 ℃, and putting back on ice for 2 min; then adding 500 mul LB liquid culture medium, reviving for 5h at 37 ℃; then 10. mu.l of the suspension was applied to LB solid medium plates containing 50. mu.g/ml kanamycin, 7. mu.g/ml gentamicin, 10. mu.g/ml tetracycline, 40. mu.g/ml IPTG and 100. mu.g/ml X-gal, and cultured in the dark for three days until a clear blue-white spot was formed.

(2) White single colonies were picked, inoculated into 5mL of LB liquid medium containing 50. mu.g/mL kanamycin, 7. mu.g/mL gentamicin, 10. mu.g/mL tetracycline, and cultured at 37 ℃ for 12 hours with shaking at 220 rpm.

(3) Taking the culture system obtained in the step (2), and extracting plasmids by using a plasmid Miniprep Kit (QIAprep Spin Miniprep Kit, Qiagen company, product number 27106, which contains a P1 reagent, a P2 reagent and a P3 reagent), wherein the specific steps are as follows in sequence:

firstly, centrifuging the culture system at 13000rpm for 2min, collecting thalli precipitates, and resuspending thalli by using a P1 reagent;

adding a P2 reagent, slowly reversing and uniformly mixing for 6-8 times;

③ adding a P3 reagent, slowly reversing and uniformly mixing for 6-8 times (white precipitates can be seen), centrifuging at 13000rpm for 10min, and taking supernatant;

fourthly, taking 600 mul of supernatant, adding 800 mul of precooled isopropanol, standing for 10min at minus 20 ℃, then centrifuging for 15min at 13000rpm, and collecting the precipitate;

resuspending the precipitate with 500 μ l precooled 70% ethanol aqueous solution, centrifuging at 13000rpm for 5min, collecting the precipitate, drying the ethanol completely, and then using ddH preheated at 65 DEG C₂Dissolving the precipitate with O, centrifuging at 13000rpm for 5min, and sucking the supernatant, namely the solution of the recombinant Bacmid, which is called Bacmid solution for short.

2. Preparation and amplification of recombinant viruses

(1) And (3) adding the well-grown Sf9 cells into a 10cm culture dish, standing for 10min, adhering the cells to the wall, and observing under a microscope to ensure that about 70-80% of the bottom of the culture dish is covered by the cells.

(2) Cellffectin II Reagent 15. mu.l was taken and diluted with 100. mu.l of insect cell culture medium.

(3) 15-20. mu.l of Bacmid solution was taken and diluted with 100. mu.l of insect cell culture medium.

(4) Slowly adding the liquid phase obtained in the step (2) into the liquid phase obtained in the step (3), slowly and uniformly blowing, standing at room temperature for 30min, and diluting to 2ml with an insect cell culture medium.

(5) And (3) taking the culture dish which finishes the step (1), discarding the supernatant, slowly and uniformly dripping the liquid phase obtained in the step (4) into the culture dish, standing and culturing at 27 ℃ for 5 hours, then sucking away the supernatant, adding 7ml of fresh insect cell culture medium, sealing by using a sealing film, standing and culturing at 27 ℃ for 8 days, collecting the culture solution, centrifuging for 6min at 600g, taking the supernatant, adding fetal calf serum to ensure that the volume concentration of the fetal calf serum is 2-5%, and storing for a long time to obtain the virus solution of the P0 generation recombinant virus.

(6) Adding virus liquid of P0 generation recombinant virus into shake flask culture at a cell concentration of 2 × 10 according to a volume ratio of 1:1000⁶Culturing each cell/mL of Sf9 cell sap at 27 deg.C and 110rpm for 5 days, collecting culture solution, centrifuging for 6min at 600g, and collecting supernatant to obtain virus solution of P1 generation recombinant virus, P1 generation virus solution for short.

3. Expression and purification of proteins

(1) Taking P1-substituted virus solution, adding 1L of 2 × 10 cells at a volume ratio of 1:100⁶cells/mL of Sf9 cell solution were cultured at 27 ℃ for 72 hours at 125rpm, centrifuged at 4000rpm for 15min, and the supernatant was collected.

(2) And (2) carrying out suction filtration on the supernatant obtained in the step (1) by using a double-layer 0.45-micron glass fiber membrane, and collecting filtrate.

(3) The filtrate obtained in step (2) was concentrated by a tangential flow ultrafiltration system (Masterflex PharMed BPT Tubig system, Cole-Parmer Co., Ltd., product number 06508-24) while adding PBS buffer to dilute it continuously to displace the protein into the PBS buffer, and then centrifuged at 13000rpm for 30min to collect the supernatant.

(4) Affinity chromatography

Adding a Ni-NTA purification medium into the supernatant obtained in the step (3), incubating for 3 hours at 4 ℃, centrifuging for 5min at 400rpm, and taking a precipitate.

② the precipitate obtained in the first step was washed with 100mL of 20mM imidazole-containing PBS buffer to remove foreign proteins.

③ washing the precipitate obtained in the step (c) with 20mL of PBS containing 300mM imidazole, and collecting the solution.

(5) The solution obtained in step (4) was concentrated using a 10kD concentration tube to obtain 1.2ml of a protein concentrate.

(6) And (4) taking the protein concentrated solution obtained in the step (5), performing molecular sieve chromatography by adopting a Hiload superdex200 column, and adopting a PBS buffer solution as an eluent.

When the recombinant plasmid A is adopted to carry out the second step, the solution after passing through the column with the retention volume of 18.31-23.19ml is collected by molecular sieve chromatography, namely MERS-CoV RBD monomer solution.

And when the recombinant plasmid B is adopted to carry out the second step, collecting the solution which passes through the column and has the retention volume of 16.21.47-21.70ml by molecular sieve chromatography, namely the MERS-CoV RBD dimer solution.

The overlay of the chromatogram (B) of the molecular sieve chromatography in step two using recombinant plasmid A and the chromatogram (A) of the molecular sieve chromatography in step two using recombinant plasmid B is shown in FIG. 1.

The non-reducing gel electrophoresis patterns of MERS-CoV RBD monomer solution (B) and MERS-CoV RBD dimer solution (A) are shown in FIG. 2. MERS-CoV RBD dimer (dimer) has an expected molecular weight of 52.54 KD. The expected molecular weight of MERS-CoV RBD monomer (monomer) is 24.42 KD.

The MERS-CoV RBD dimer has a peak position earlier than that of MERS-CoV RBD monomer, and the molecular weight of the MERS-CoV RBD dimer is 2 times that of the MERS-CoV RBD monomer. The results show that MERS-CoV RBD dimer exists in the form of dimer. MERS-CoV RBD monomer exists in a monomer form.

Example 2 antigenic analysis (ELISA test)

Test antibody: MERS-4 or MERS-27 or 10E 8. MERS-4, namely monoclonal antibody MERS-4, and the preparation method is disclosed in the patent of monoclonal antibody MERS-4 and coding gene and application thereof (application No. 201310566227.8; grant No. CN 104628849B). MERS-27, namely monoclonal antibody MERS-27, and the preparation method is disclosed in the patent of monoclonal antibody MERS-27 and coding gene and application thereof (application number 201310565893. X; grant publication number CN 104628848B). MERS-4 and MERS-27 are both neutralizing antibodies against MERS-CoV RBD. 10E8 is a monoclonal antibody against HIV-1 membrane protein (used as negative reference).

Antigen solution: MERS-CoV RBD monomer solution or MERS-CoV RBD dimer solution prepared in example 1. Sealing liquid: PBS buffer containing 1% (by volume) fetal bovine serum. Test antibody solution: test antibody was taken and diluted with blocking solution to a protein concentration of 1 mg/ml. PBST solution: the content of Tween was 0.5% (by volume). Secondary antibody: HRP-labeled anti-mouse IgG antibody, Promega, cat # W4021.

1. Taking an antigen solution, and diluting the antigen solution with a PBS buffer solution until the protein concentration is 1 mu g/ml to obtain a coating solution; taking an enzyme label plate, adding coating solution (the adding amount of the antigen is respectively set to be 200 ng/hole, 100 ng/hole, 50 ng/hole, 25 ng/hole or 12.5 ng/hole; each coating amount is set to be 3 multiple holes), and coating overnight at 4 ℃.

2. After completion of step 1, the supernatant was discarded, blocking solution was added and incubated at 37 ℃ for 2 h.

3. After completion of step 2, the supernatant was discarded and washed three times with PBST solution.

4. After completion of step 3, test antibody solution (2000ng antibody/well) was added and incubated at 37 ℃ for 1 h.

5. After completion of step 4, the supernatant was discarded and washed 3 times with PBST solution.

6. After completion of step 5, the supernatant was discarded, and a secondary antibody working solution (100. mu.l/well) was added and incubated at 37 ℃ for 45 min.

7. After completion of step 6, the supernatant was discarded and washed 3 times with PBST solution.

8. And (4) after the step 7 is finished, adding TMB color development solution, reacting for 2-5 minutes, and detecting the absorbance at the wavelength of 450nm by using an enzyme-labeling instrument.

The results are shown in FIG. 3. In FIG. 3, each column corresponds to the antigen addition from high to low in the order from left to right. The binding capacity of the RBD dimer to the two neutralizing antibodies is superior to that of the RBD monomer.

Example 3 structural analysis

MERS-CoV RBD monomer solution and MERS-CoV RBD dimer solution prepared in example 1 were taken out separately, concentrated to 10mg/ml with a 10kD ultrafilter tube, and crystals were screened by sitting-drop method.

Spindle-shaped single crystals were grown under 25% PEG 1500.

Diffraction data collection was performed using a synchrotron radiation X-ray light source.

Data are processed by software such as HKL-2000, DPP4, Coot, phonix and the like, phases are solved by a molecular replacement method, and the structure is finally analyzed.

The results are shown in FIG. 4. A in FIG. 4 is the structure of MERS-CoV RBD monomer, and B in FIG. 4 is the structure of MERS-CoV RBD dimer. MERS-CoV RBD monomer contains 8 cysteines (Cys) and forms 4 pairs of intramolecular disulfide bonds. One monomer of MERS-CoV RBD dimer contains 9 Cys, wherein 8 Cys forms 4 pairs of intramolecular disulfide bonds, 1 extra Cys on each monomer forms 1 pair of intramolecular disulfide bonds, and two monomers form a dimer.

Example 3 neutralizing Activity of post-Immunity sera

One, group immunization

BalB/C mice (viton waals) were divided into 6 groups of 5 mice each, immunized separately as follows:

a first group: primary immunization on day 1, 2 immunization on

day

15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is intramuscular injection; for the primary immunization, the immunization volume of a single mouse is 40 mul, and the immune material is ' 20 mul of white emulsion formed by mixing 1mg/ml RBD monomer solution and 20 mul of Freund's complete adjuvant '; the boosting immunization, the single immunization volume of a single mouse is 40 mul, and the immune matter is ' 20 mul 1mg/ml RBD monomer solution and 20 mul Freund's incomplete adjuvant are mixed to form white emulsion ';

second group: primary immunization on day 1, 2 immunization on

day

15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is nasal mucosa immunization, the single immunization volume of a single mouse is 20 mu l, and the immune substance is a uniform mixture of 10 mu l of 2mg/ml RBD monomer solution and 10 mu l of CpGODN1826 solution;

third group: primary immunization on day 1, 2 immunization on

day

15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is nasal mucosa immunization, the single immunization volume of a single mouse is 20 mu l, and the immune substance is a uniform mixture of 10 mu l of 2mg/ml RBD monomer solution and 10 mu l C48/80 solution;

and a fourth group: primary immunization on day 1, 2 immunization on

day

15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is intramuscular injection; for the initial immunization, the immunization volume of a single mouse is 40 mul, and the immune substance is ' 20 mul of white emulsion formed by mixing 1mg/ml RBD dimer solution with 20 mul of Freund's complete adjuvant '; the boosting immunization, the volume of the single immunization of a single mouse is 40 mul, and the immune matter is '20 mul of white emulsion formed by mixing 1mg/ml RBD dimer solution with 20 mul of Freund incomplete adjuvant';

and a fifth group: primary immunization on day 1, 2 immunization on

day

15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is nasal mucosa immunization, the single immunization volume of a single mouse is 20 mu l, and the immune substance is a uniform mixture of 10 mu l of 2mg/ml RBD dimer solution and 10 mu l of CpGODN1826 solution;

a sixth group: primary immunization on day 1, 2 immunization on

day

15, 3 immunization on day 29, and 4 immunization on day 43; the immunization mode is nasal mucosa immunization, the single immunization volume of a single mouse is 20 mu l, and the immune substance is a uniform mixture of 10 mu l of 2mg/ml RBD dimer solution and 10 mu l C48/80 solution;

the MERS-CoV RBD dimer solution prepared in example 1 was used, and the concentration was adjusted with PBS buffer so that the protein concentration was 1mg/ml, i.e., a 1mg/ml RBD dimer solution. The MERS-CoV RBD dimer solution prepared in example 1 was taken and adjusted to a protein concentration of 2mg/ml with PBS buffer, thus obtaining a 2mg/ml RBD dimer solution. MERS-CoV RBD monomer solution prepared in example 1 was taken and adjusted to a protein concentration of 1mg/ml by PBS buffer, thus giving a 1mg/ml RBD monomer solution. MERS-CoV RBD monomer solution prepared in example 1 was taken and adjusted to a protein concentration of 2mg/ml with PBS buffer, thus giving a 2mg/ml RBD monomer solution. C48/80(Compound 48/80): sigma Co, lot C2313. C48/80 was dissolved in PBS buffer to a concentration of 2mg/ml, which was C48/80 solution. CpG ODN1826 (single-stranded DNA molecule, whole-strand thio-modified, sequence: TCCATGACGTTCCTGACGTT) is dissolved in PBS buffer solution to make the concentration 5mg/ml, namely CpGODN1826 solution.

The 2 nd, 3 rd and 4 th immunizations are collectively referred to as booster immunizations.

Blood is collected 7 days before immunization through the retroorbital venous plexus. After the primary immunization, blood was collected on day 36 and day 50, respectively, and blood was collected through the retroorbital venous plexus.

Preparation of MERS-CoV-2 pseudovirus

The plasmid expressing MERS-CoV full-length membrane protein (named MERS-CoV plasmid) and the skeleton plasmid pNL4-3R-E-luciferase are co-transfected into 293T cells, and after incubation, MERS-CoV pseudotyped virus with infectivity but no replication capacity can be obtained, and the infectivity of the virus is similar to that of live virus. Backbone plasmid pNL4-3R-E-Luciferase, i.e.a backbone plasmid pNL4-3R-E containing Luciferase (i.e.vector with the Luciferase conjugation backbone pNL4-3R-E in the literature): wang Q, Liu L, Ren W, Gettie a, Wang H, Liang Q, Shi X, Montefiori DC, Zhou T, Zhang l.cell rep.2019.

Inserting a double-stranded DNA molecule (protein shown in a sequence 9 of a coding sequence table) shown in a sequence 10 of a sequence table between BamHI and EcoRI enzyme cutting sites of a pcDNA3.1(+) vector to obtain MERS-CoV plasmid.

MERS-CoV plasmid and skeleton plasmid pNL4-3R-E-luciferase are co-transfected into 293T cells, standing incubation is carried out at 37 ℃ (DMEM culture medium containing 10% fetal calf serum is adopted), cell culture supernatant is collected after transfection is carried out for 48 hours, and virus liquid containing MERS-CoV pseudovirus (MERS-CoV virus liquid for short) is obtained.

Third, detection of neutralizing activity of immune serum

And (3) serum to be detected: and (4) taking the blood sample obtained in the step one, and separating to obtain serum.

1. And (3) adopting a DMEM medium containing 10% FBS to dilute the serum to be detected in a multiple ratio manner, and sequentially obtaining diluents with different serum concentrations.

2. 100 microliters of the dilution obtained in step 1 was mixed with 50 microliters of MERS-CoV virus solution (virus content: 100TCID50) prepared in step two, and incubated at 37 ℃ for 1 hour. A blank control was set up with 100 μ l DMEM medium containing 10% FBS instead of 100 μ l of diluent.

3. After completion of step 2, 50. mu.l of cell fluid of Huh7 cells (about 2X 10 cells) was added⁴Huh7 cells), and standing and incubating for 48 hours at 37 ℃ (in practical application, 48-72 hours can be used).

4. After completion of step 3, 100. mu.l of PBS buffer and 50. mu.l of cell lysate (Bright-GloTMLuciferase Assay System, Promega, E2650) were added, allowed to stand for 2min, and then luciferase activity was detected using a chemiluminescence apparatus.

Each treatment was set up with 3 replicates and the results averaged.

Neutralization activity ═ (fluorescence intensity of blank-fluorescence intensity of experimental group to which diluent was added)/fluorescence intensity of blank × 100%.

The serum dilution at 50% neutralization activity corresponds to position ID 50.

The ID50 values are shown in Table 2. The MERS-CoV RBD dimer induces the neutralizing antibody reaction generated by the mice to be stronger than the neutralizing antibody reaction induced by MERS-CoV RBD monomer by immunization through nasal mucosa, so that MERS-CoV infection cells can be more strongly inhibited.

TABLE 2

	Serum 7 days before immunization	Serum 36 days after primary immunization	Serum 50 days after primary immunization
				First group	<15	842	4089
Second group	<15	<15	45.6
				Third group	<15	52.2	339.6
Fourth group	<15	958.6	3780.2
				Fifth group	<15	<15	518.6
Sixth group	<15	564.8	1631

SEQUENCE LISTING

<110> Qinghua university

<120> MERS-CoV membrane protein receptor binding domain dimer, and coding gene and application thereof

<130> CGGNQAYX206035

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 223

<212> PRT

<213> Artificial sequence

<400> 1

Glu Ala Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu

1 5 10 15

Cys Asp Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn

20 25 30

Phe Lys Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu

35 40 45

Leu Ser Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro

50 55 60

Ala Ala Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe

65 70 75 80

Ser Tyr Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly

85 90 95

Pro Ile Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys

100 105 110

Leu Ile Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro

115 120 125

Leu Lys Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp

130 135 140

Arg Thr Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys

145 150 155 160

Val Ser Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg

165 170 175

Lys Gln Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly

180 185 190

Ser Thr Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr

195 200 205

Val Gln Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu

210 215 220

<210> 2

<211> 669

<212> DNA

<213> Artificial sequence

<400> 2

gaggctaagc catctggctc tgtggtggaa caggctgagg gagtggagtg tgacttcagc 60

ccactgctgt ctggcacacc tccacaggtc tacaacttca agagactggt gttcaccaac 120

tgtaactaca acctgaccaa actgctgtcc ctgttctctg tgaatgactt cacttgtagc 180

cagattagcc ctgctgccat tgccagcaac tgttactcct ccctgattct ggactacttc 240

tcctacccac tgagtatgaa gtctgacctg tctgtgtcct ctgctggacc aatcagccag 300

ttcaactaca agcagtcctt cagcaaccca acttgtctga ttctggctac agtgccacac 360

aacctgacca ccatcaccaa gccactgaaa tactcctaca tcaacaagtg tagcagactg 420

ctgtctgatg acaggacaga ggtgccacaa ctagtgaatg ccaaccaata cagcccatgt 480

gtgagcattg tgccaagcac agtgtgggag gatggagact actacaggaa gcaacttagc 540

ccattggagg gaggaggctg gctggtggca tctggcagca cagtggctat gacagaacaa 600

ctccaaatgg gctttggcat cacagtccaa tatggcacag acaccaactc tgtgtgtcca 660

aaattggag 669

<210> 3

<211> 240

<212> PRT

<213> Artificial sequence

<400> 3

Glu Ala Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu

1 5 10 15

Cys Asp Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn

20 25 30

Phe Lys Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu

35 40 45

Leu Ser Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro

50 55 60

Ala Ala Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe

65 70 75 80

Ser Tyr Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly

85 90 95

Pro Ile Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys

100 105 110

Leu Ile Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro

115 120 125

Leu Lys Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp

130 135 140

Arg Thr Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys

145 150 155 160

Val Ser Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg

165 170 175

Lys Gln Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly

180 185 190

Ser Thr Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr

195 200 205

Val Gln Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu Phe

210 215 220

Ala Asn Asp Thr Lys Ile Ala Ser Gln Leu Gly Asn Cys Val Glu Tyr

225 230 235 240

<210> 4

<211> 720

<212> DNA

<213> Artificial sequence

<400> 4

gaggctaagc catctggctc tgtggtggaa caggctgagg gagtggagtg tgacttcagc 60

ccactgctgt ctggcacacc tccacaggtc tacaacttca agagactggt gttcaccaac 120

tgtaactaca acctgaccaa actgctgtcc ctgttctctg tgaatgactt cacttgtagc 180

cagattagcc ctgctgccat tgccagcaac tgttactcct ccctgattct ggactacttc 240

tcctacccac tgagtatgaa gtctgacctg tctgtgtcct ctgctggacc aatcagccag 300

ttcaactaca agcagtcctt cagcaaccca acttgtctga ttctggctac agtgccacac 360

aacctgacca ccatcaccaa gccactgaaa tactcctaca tcaacaagtg tagcagactg 420

ctgtctgatg acaggacaga ggtgccacaa ctagtgaatg ccaaccaata cagcccatgt 480

gtgagcattg tgccaagcac agtgtgggag gatggagact actacaggaa gcaacttagc 540

ccattggagg gaggaggctg gctggtggca tctggcagca cagtggctat gacagaacaa 600

ctccaaatgg gctttggcat cacagtccaa tatggcacag acaccaactc tgtgtgtcca 660

aaattggagt ttgccaatga caccaagatt gccagccaac ttggcaactg tgtggaatac 720

<210> 5

<211> 270

<212> PRT

<213> Artificial sequence

<400> 5

Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr

1 5 10 15

Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala

20 25 30

Ala His Ser Ala Phe Ala Ala Asp Pro Glu Ala Lys Pro Ser Gly Ser

35 40 45

Val Val Glu Gln Ala Glu Gly Val Glu Cys Asp Phe Ser Pro Leu Leu

50 55 60

Ser Gly Thr Pro Pro Gln Val Tyr Asn Phe Lys Arg Leu Val Phe Thr

65 70 75 80

Asn Cys Asn Tyr Asn Leu Thr Lys Leu Leu Ser Leu Phe Ser Val Asn

85 90 95

Asp Phe Thr Cys Ser Gln Ile Ser Pro Ala Ala Ile Ala Ser Asn Cys

100 105 110

Tyr Ser Ser Leu Ile Leu Asp Tyr Phe Ser Tyr Pro Leu Ser Met Lys

115 120 125

Ser Asp Leu Ser Val Ser Ser Ala Gly Pro Ile Ser Gln Phe Asn Tyr

130 135 140

Lys Gln Ser Phe Ser Asn Pro Thr Cys Leu Ile Leu Ala Thr Val Pro

145 150 155 160

His Asn Leu Thr Thr Ile Thr Lys Pro Leu Lys Tyr Ser Tyr Ile Asn

165 170 175

Lys Cys Ser Arg Leu Leu Ser Asp Asp Arg Thr Glu Val Pro Gln Leu

180 185 190

Val Asn Ala Asn Gln Tyr Ser Pro Cys Val Ser Ile Val Pro Ser Thr

195 200 205

Val Trp Glu Asp Gly Asp Tyr Tyr Arg Lys Gln Leu Ser Pro Leu Glu

210 215 220

Gly Gly Gly Trp Leu Val Ala Ser Gly Ser Thr Val Ala Met Thr Glu

225 230 235 240

Gln Leu Gln Met Gly Phe Gly Ile Thr Val Gln Tyr Gly Thr Asp Thr

245 250 255

Asn Ser Val Cys Pro Lys Leu Glu His His His His His His

260 265 270

<210> 6

<211> 813

<212> DNA

<213> Artificial sequence

<400> 6

atgctactag taaatcagtc acaccaaggc ttcaataagg aacacacaag caagatggta 60

agcgctattg ttttatatgt gcttttggcg gcggcggcgc attctgcctt tgcggcggat 120

cccgaggcta agccatctgg ctctgtggtg gaacaggctg agggagtgga gtgtgacttc 180

agcccactgc tgtctggcac acctccacag gtctacaact tcaagagact ggtgttcacc 240

aactgtaact acaacctgac caaactgctg tccctgttct ctgtgaatga cttcacttgt 300

agccagatta gccctgctgc cattgccagc aactgttact cctccctgat tctggactac 360

ttctcctacc cactgagtat gaagtctgac ctgtctgtgt cctctgctgg accaatcagc 420

cagttcaact acaagcagtc cttcagcaac ccaacttgtc tgattctggc tacagtgcca 480

cacaacctga ccaccatcac caagccactg aaatactcct acatcaacaa gtgtagcaga 540

ctgctgtctg atgacaggac agaggtgcca caactagtga atgccaacca atacagccca 600

tgtgtgagca ttgtgccaag cacagtgtgg gaggatggag actactacag gaagcaactt 660

agcccattgg agggaggagg ctggctggtg gcatctggca gcacagtggc tatgacagaa 720

caactccaaa tgggctttgg catcacagtc caatatggca cagacaccaa ctctgtgtgt 780

ccaaaattgg agcaccacca tcaccatcat tag 813

<210> 7

<211> 287

<212> PRT

<213> Artificial sequence

<400> 7

Met Leu Leu Val Asn Gln Ser His Gln Gly Phe Asn Lys Glu His Thr

1 5 10 15

Ser Lys Met Val Ser Ala Ile Val Leu Tyr Val Leu Leu Ala Ala Ala

20 25 30

Ala His Ser Ala Phe Ala Ala Asp Pro Glu Ala Lys Pro Ser Gly Ser

35 40 45

Val Val Glu Gln Ala Glu Gly Val Glu Cys Asp Phe Ser Pro Leu Leu

50 55 60

Ser Gly Thr Pro Pro Gln Val Tyr Asn Phe Lys Arg Leu Val Phe Thr

65 70 75 80

Asn Cys Asn Tyr Asn Leu Thr Lys Leu Leu Ser Leu Phe Ser Val Asn

85 90 95

Asp Phe Thr Cys Ser Gln Ile Ser Pro Ala Ala Ile Ala Ser Asn Cys

100 105 110

Tyr Ser Ser Leu Ile Leu Asp Tyr Phe Ser Tyr Pro Leu Ser Met Lys

115 120 125

Ser Asp Leu Ser Val Ser Ser Ala Gly Pro Ile Ser Gln Phe Asn Tyr

130 135 140

Lys Gln Ser Phe Ser Asn Pro Thr Cys Leu Ile Leu Ala Thr Val Pro

145 150 155 160

His Asn Leu Thr Thr Ile Thr Lys Pro Leu Lys Tyr Ser Tyr Ile Asn

165 170 175

Lys Cys Ser Arg Leu Leu Ser Asp Asp Arg Thr Glu Val Pro Gln Leu

180 185 190

Val Asn Ala Asn Gln Tyr Ser Pro Cys Val Ser Ile Val Pro Ser Thr

195 200 205

Val Trp Glu Asp Gly Asp Tyr Tyr Arg Lys Gln Leu Ser Pro Leu Glu

210 215 220

Gly Gly Gly Trp Leu Val Ala Ser Gly Ser Thr Val Ala Met Thr Glu

225 230 235 240

Gln Leu Gln Met Gly Phe Gly Ile Thr Val Gln Tyr Gly Thr Asp Thr

245 250 255

Asn Ser Val Cys Pro Lys Leu Glu Phe Ala Asn Asp Thr Lys Ile Ala

260 265 270

Ser Gln Leu Gly Asn Cys Val Glu Tyr His His His His His His

275 280 285

<210> 8

<211> 864

<212> DNA

<213> Artificial sequence

<400> 8

atgctactag taaatcagtc acaccaaggc ttcaataagg aacacacaag caagatggta 60

agcgctattg ttttatatgt gcttttggcg gcggcggcgc attctgcctt tgcggcggat 120

cccgaggcta agccatctgg ctctgtggtg gaacaggctg agggagtgga gtgtgacttc 180

agcccactgc tgtctggcac acctccacag gtctacaact tcaagagact ggtgttcacc 240

aactgtaact acaacctgac caaactgctg tccctgttct ctgtgaatga cttcacttgt 300

agccagatta gccctgctgc cattgccagc aactgttact cctccctgat tctggactac 360

ttctcctacc cactgagtat gaagtctgac ctgtctgtgt cctctgctgg accaatcagc 420

cagttcaact acaagcagtc cttcagcaac ccaacttgtc tgattctggc tacagtgcca 480

cacaacctga ccaccatcac caagccactg aaatactcct acatcaacaa gtgtagcaga 540

ctgctgtctg atgacaggac agaggtgcca caactagtga atgccaacca atacagccca 600

tgtgtgagca ttgtgccaag cacagtgtgg gaggatggag actactacag gaagcaactt 660

agcccattgg agggaggagg ctggctggtg gcatctggca gcacagtggc tatgacagaa 720

caactccaaa tgggctttgg catcacagtc caatatggca cagacaccaa ctctgtgtgt 780

ccaaaattgg agtttgccaa tgacaccaag attgccagcc aacttggcaa ctgtgtggaa 840

taccaccacc atcaccatca ttag 864

<210> 9

<211> 1353

<212> PRT

<213> Artificial sequence

<400> 9

Met Ile His Ser Val Phe Leu Leu Met Phe Leu Leu Thr Pro Thr Glu

1 5 10 15

Ser Tyr Val Asp Val Gly Pro Asp Ser Val Lys Ser Ala Cys Ile Glu

20 25 30

Val Asp Ile Gln Gln Thr Phe Phe Asp Lys Thr Trp Pro Arg Pro Ile

35 40 45

Asp Val Ser Lys Ala Asp Gly Ile Ile Tyr Pro Gln Gly Arg Thr Tyr

50 55 60

Ser Asn Ile Thr Ile Thr Tyr Gln Gly Leu Phe Pro Tyr Gln Gly Asp

65 70 75 80

His Gly Asp Met Tyr Val Tyr Ser Ala Gly His Ala Thr Gly Thr Thr

85 90 95

Pro Gln Lys Leu Phe Val Ala Asn Tyr Ser Gln Asp Val Lys Gln Phe

100 105 110

Ala Asn Gly Phe Val Val Arg Ile Gly Ala Ala Ala Asn Ser Thr Gly

115 120 125

Thr Val Ile Ile Ser Pro Ser Thr Ser Ala Thr Ile Arg Lys Ile Tyr

130 135 140

Pro Ala Phe Met Leu Gly Ser Ser Val Gly Asn Phe Ser Asp Gly Lys

145 150 155 160

Met Gly Arg Phe Phe Asn His Thr Leu Val Leu Leu Pro Asp Gly Cys

165 170 175

Gly Thr Leu Leu Arg Ala Phe Tyr Cys Ile Leu Glu Pro Arg Ser Gly

180 185 190

Asn His Cys Pro Ala Gly Asn Ser Tyr Thr Ser Phe Ala Thr Tyr His

195 200 205

Thr Pro Ala Thr Asp Cys Ser Asp Gly Asn Tyr Asn Arg Asn Ala Ser

210 215 220

Leu Asn Ser Phe Lys Glu Tyr Phe Asn Leu Arg Asn Cys Thr Phe Met

225 230 235 240

Tyr Thr Tyr Asn Ile Thr Glu Asp Glu Ile Leu Glu Trp Phe Gly Ile

245 250 255

Thr Gln Thr Ala Gln Gly Val His Leu Phe Ser Ser Arg Tyr Val Asp

260 265 270

Leu Tyr Gly Gly Asn Met Phe Gln Phe Ala Thr Leu Pro Val Tyr Asp

275 280 285

Thr Ile Lys Tyr Tyr Ser Ile Ile Pro His Ser Ile Arg Ser Ile Gln

290 295 300

Ser Asp Arg Lys Ala Trp Ala Ala Phe Tyr Val Tyr Lys Leu Gln Pro

305 310 315 320

Leu Thr Phe Leu Leu Asp Phe Ser Val Asp Gly Tyr Ile Arg Arg Ala

325 330 335

Ile Asp Cys Gly Phe Asn Asp Leu Ser Gln Leu His Cys Ser Tyr Glu

340 345 350

Ser Phe Asp Val Glu Ser Gly Val Tyr Ser Val Ser Ser Phe Glu Ala

355 360 365

Lys Pro Ser Gly Ser Val Val Glu Gln Ala Glu Gly Val Glu Cys Asp

370 375 380

Phe Ser Pro Leu Leu Ser Gly Thr Pro Pro Gln Val Tyr Asn Phe Lys

385 390 395 400

Arg Leu Val Phe Thr Asn Cys Asn Tyr Asn Leu Thr Lys Leu Leu Ser

405 410 415

Leu Phe Ser Val Asn Asp Phe Thr Cys Ser Gln Ile Ser Pro Ala Ala

420 425 430

Ile Ala Ser Asn Cys Tyr Ser Ser Leu Ile Leu Asp Tyr Phe Ser Tyr

435 440 445

Pro Leu Ser Met Lys Ser Asp Leu Ser Val Ser Ser Ala Gly Pro Ile

450 455 460

Ser Gln Phe Asn Tyr Lys Gln Ser Phe Ser Asn Pro Thr Cys Leu Ile

465 470 475 480

Leu Ala Thr Val Pro His Asn Leu Thr Thr Ile Thr Lys Pro Leu Lys

485 490 495

Tyr Ser Tyr Ile Asn Lys Cys Ser Arg Leu Leu Ser Asp Asp Arg Thr

500 505 510

Glu Val Pro Gln Leu Val Asn Ala Asn Gln Tyr Ser Pro Cys Val Ser

515 520 525

Ile Val Pro Ser Thr Val Trp Glu Asp Gly Asp Tyr Tyr Arg Lys Gln

530 535 540

Leu Ser Pro Leu Glu Gly Gly Gly Trp Leu Val Ala Ser Gly Ser Thr

545 550 555 560

Val Ala Met Thr Glu Gln Leu Gln Met Gly Phe Gly Ile Thr Val Gln

565 570 575

Tyr Gly Thr Asp Thr Asn Ser Val Cys Pro Lys Leu Glu Phe Ala Asn

580 585 590

Asp Thr Lys Ile Ala Ser Gln Leu Gly Asn Cys Val Glu Tyr Ser Leu

595 600 605

Tyr Gly Val Ser Gly Arg Gly Val Phe Gln Asn Cys Thr Ala Val Gly

610 615 620

Val Arg Gln Gln Arg Phe Val Tyr Asp Ala Tyr Gln Asn Leu Val Gly

625 630 635 640

Tyr Tyr Ser Asp Asp Gly Asn Tyr Tyr Cys Leu Arg Ala Cys Val Ser

645 650 655

Val Pro Val Ser Val Ile Tyr Asp Lys Glu Thr Lys Thr His Ala Thr

660 665 670

Leu Phe Gly Ser Val Ala Cys Glu His Ile Ser Ser Thr Met Ser Gln

675 680 685

Tyr Ser Arg Ser Thr Arg Ser Met Leu Lys Arg Arg Asp Ser Thr Tyr

690 695 700

Gly Pro Leu Gln Thr Pro Val Gly Cys Val Leu Gly Leu Val Asn Ser

705 710 715 720

Ser Leu Phe Val Glu Asp Cys Lys Leu Pro Leu Gly Gln Ser Leu Cys

725 730 735

Ala Leu Pro Asp Thr Pro Ser Thr Leu Thr Pro Arg Ser Val Arg Ser

740 745 750

Val Pro Gly Glu Met Arg Leu Ala Ser Ile Ala Phe Asn His Pro Ile

755 760 765

Gln Val Asp Gln Leu Asn Ser Ser Tyr Phe Lys Leu Ser Ile Pro Thr

770 775 780

Asn Phe Ser Phe Gly Val Thr Gln Glu Tyr Ile Gln Thr Thr Ile Gln

785 790 795 800

Lys Val Thr Val Asp Cys Lys Gln Tyr Val Cys Asn Gly Phe Gln Lys

805 810 815

Cys Glu Gln Leu Leu Arg Glu Tyr Gly Gln Phe Cys Ser Lys Ile Asn

820 825 830

Gln Ala Leu His Gly Ala Asn Leu Arg Gln Asp Asp Ser Val Arg Asn

835 840 845

Leu Phe Ala Ser Val Lys Ser Ser Gln Ser Ser Pro Ile Ile Pro Gly

850 855 860

Phe Gly Gly Asp Phe Asn Leu Thr Leu Leu Glu Pro Val Ser Ile Ser

865 870 875 880

Thr Gly Ser Arg Ser Ala Arg Ser Ala Ile Glu Asp Leu Leu Phe Asp

885 890 895

Lys Val Thr Ile Ala Asp Pro Gly Tyr Met Gln Gly Tyr Asp Asp Cys

900 905 910

Met Gln Gln Gly Pro Ala Ser Ala Arg Asp Leu Ile Cys Ala Gln Tyr

915 920 925

Val Ala Gly Tyr Lys Val Leu Pro Pro Leu Met Asp Val Asn Met Glu

930 935 940

Ala Ala Tyr Thr Ser Ser Leu Leu Gly Ser Ile Ala Gly Val Gly Trp

945 950 955 960

Thr Ala Gly Leu Ser Ser Phe Ala Ala Ile Pro Phe Ala Gln Ser Ile

965 970 975

Phe Tyr Arg Leu Asn Gly Val Gly Ile Thr Gln Gln Val Leu Ser Glu

980 985 990

Asn Gln Lys Leu Ile Ala Asn Lys Phe Asn Gln Ala Leu Gly Ala Met

995 1000 1005

Gln Thr Gly Phe Thr Thr Thr Asn Glu Ala Phe Gln Lys Val Gln

1010 1015 1020

Asp Ala Val Asn Asn Asn Ala Gln Ala Leu Ser Lys Leu Ala Ser

1025 1030 1035

Glu Leu Ser Asn Thr Phe Gly Ala Ile Ser Ala Ser Ile Gly Asp

1040 1045 1050

Ile Ile Gln Arg Leu Asp Val Leu Glu Gln Asp Ala Gln Ile Asp

1055 1060 1065

Arg Leu Ile Asn Gly Arg Leu Thr Thr Leu Asn Ala Phe Val Ala

1070 1075 1080

Gln Gln Leu Val Arg Ser Glu Ser Ala Ala Leu Ser Ala Gln Leu

1085 1090 1095

Ala Lys Asp Lys Val Asn Glu Cys Val Lys Ala Gln Ser Lys Arg

1100 1105 1110

Ser Gly Phe Cys Gly Gln Gly Thr His Ile Val Ser Phe Val Val

1115 1120 1125

Asn Ala Pro Asn Gly Leu Tyr Phe Met His Val Gly Tyr Tyr Pro

1130 1135 1140

Ser Asn His Ile Glu Val Val Ser Ala Tyr Gly Leu Cys Asp Ala

1145 1150 1155

Ala Asn Pro Thr Asn Cys Ile Ala Pro Val Asn Gly Tyr Phe Ile

1160 1165 1170

Lys Thr Asn Asn Thr Arg Ile Val Asp Glu Trp Ser Tyr Thr Gly

1175 1180 1185

Ser Ser Phe Tyr Ala Pro Glu Pro Ile Thr Ser Leu Asn Thr Lys

1190 1195 1200

Tyr Val Ala Pro Gln Val Thr Tyr Gln Asn Ile Ser Thr Asn Leu

1205 1210 1215

Pro Pro Pro Leu Leu Gly Asn Ser Thr Gly Ile Asp Phe Gln Asp

1220 1225 1230

Glu Leu Asp Glu Phe Phe Lys Asn Val Ser Thr Ser Ile Pro Asn

1235 1240 1245

Phe Gly Ser Leu Thr Gln Ile Asn Thr Thr Leu Leu Asp Leu Thr

1250 1255 1260

Tyr Glu Met Leu Ser Leu Gln Gln Val Val Lys Ala Leu Asn Glu

1265 1270 1275

Ser Tyr Ile Asp Leu Lys Glu Leu Gly Asn Tyr Thr Tyr Tyr Asn

1280 1285 1290

Lys Trp Pro Trp Tyr Ile Trp Leu Gly Phe Ile Ala Gly Leu Val

1295 1300 1305

Ala Leu Ala Leu Cys Val Phe Phe Ile Leu Cys Cys Thr Gly Cys

1310 1315 1320

Gly Thr Asn Cys Met Gly Lys Leu Lys Cys Asn Arg Cys Cys Asp

1325 1330 1335

Arg Tyr Glu Glu Tyr Asp Leu Glu Pro His Lys Val His Val His

1340 1345 1350

<210> 10

<211> 4062

<212> DNA

<213> Artificial sequence

<400> 10

atgatccata gcgtcttcct gctgatgttc ctgctgacac ctactgaatc ttacgtcgat 60

gtgggacctg atagcgtgaa atccgcatgc atcgaggtgg atattcagca gactttcttt 120

gacaagacct ggcctcgacc aatcgatgtg agcaaagccg acggcatcat ctaccctcag 180

ggaaggacat attccaacat cacaattact taccagggcc tgttcccata tcagggcgac 240

cacggagata tgtacgtgta ttctgccgga catgctaccg ggaccacacc tcagaaactg 300

tttgtggcaa attatagcca ggacgtgaag cagttcgcca acgggtttgt ggtcagaatc 360

ggcgccgctg caaactctac cggcacagtg atcatttccc cttctacaag tgccactatc 420

cggaaaatct acccagcttt tatgctgggc agctccgtgg gaaacttcag cgatgggaag 480

atgggccgct tctttaatca cacactggtg ctgctgcctg acggatgcgg caccctgctg 540

agagccttct actgtatcct ggagcccaga tccggaaatc actgccctgc tgggaactct 600

tacaccagtt ttgccaccta tcatacacca gctactgact gttctgatgg caattataac 660

cggaatgcct cactgaacag cttcaaggaa tactttaatc tgcgcaactg cactttcatg 720

tacacctata atatcacaga ggatgaaatt ctggagtggt tcgggatcac tcagaccgct 780

cagggcgtgc acctgttttc tagtcgctac gtcgatctgt atggcggaaa catgttccag 840

tttgccacac tgcccgtgta cgacactatt aagtactata gcatcattcc tcattccatc 900

cgatctattc agagtgacag gaaggcttgg gccgctttct acgtgtataa actgcagcct 960

ctgaccttcc tgctggactt cagcgtggac ggatacatca ggagagccat tgattgcggg 1020

tttaacgacc tgtcccagct gcactgtagc tacgagagct tcgatgtgga gtcaggggtg 1080

tacagcgtct caagctttga ggctaagccc tcagggagcg tggtcgagca ggcagaaggc 1140

gtggagtgcg acttctcccc tctgctgtct ggcacacccc ctcaggtgta taatttcaaa 1200

agactggtct ttaccaactg taattacaac ctgacaaagc tgctgtccct gttctctgtg 1260

aacgacttta cctgcagtca gatcagccca gcagccattg ccagtaattg ttattcctct 1320

ctgatcctgg attacttctc atatcctctg agcatgaaat ccgacctgtc tgtgagttca 1380

gcaggcccaa tcagccagtt taattacaag cagtccttct ctaaccctac ctgcctgatt 1440

ctggccacag tgccacataa cctgactacc atcactaagc ccctgaaata ctcctacatc 1500

aataagtgca gtagactgct gtcagacgat cggaccgaag tgccacagct ggtcaatgcc 1560

aaccagtaca gcccatgcgt gagcatcgtc ccctctaccg tgtgggaaga cggagattac 1620

tatcggaagc agctgagccc actggaggga ggaggatggc tggtggcaag tgggtcaact 1680

gtcgccatga ccgagcagct gcagatgggc ttcggaatca cagtgcagta cggcacagat 1740

actaattctg tctgtccaaa gctggaattt gctaacgaca ctaaaattgc aagccagctg 1800

ggcaattgcg tggagtacag cctgtatgga gtgtccgggc gcggcgtctt ccagaactgt 1860

acagccgtgg gcgtccgaca gcagaggttc gtgtacgatg cttatcagaa cctggtcggc 1920

tactattccg acgatggaaa ttactattgc ctgagggcat gcgtgagcgt ccccgtgtca 1980

gtcatctacg acaaggaaac caaaacacac gcaaccctgt tcggctctgt ggcctgcgag 2040

catattagct ccacaatgag tcagtatagc agatccactc ggtcaatgct gaaacggcgc 2100

gacagcactt acggaccact gcagacccct gtgggatgcg tgctgggcct ggtgaactct 2160

agtctgttcg tcgaagattg caagctgcct ctgggacaga gcctgtgcgc actgccagac 2220

acaccctcca ctctgacccc acgcagtgtg cgatcagtcc caggagagat gcgactggca 2280

agcatcgcct tcaatcaccc aattcaggtg gatcagctga actcaagcta ctttaagctg 2340

tctatcccta ctaacttcag ttttggcgtg acccaggagt atatccagac aactattcag 2400

aaggtgacag tggactgcaa acagtacgtg tgcaatggat tccagaaatg cgaacagctg 2460

ctgagagagt atgggcagtt ttgttccaag atcaatcagg cactgcatgg cgccaacctg 2520

cgccaggacg attccgtgcg aaacctgttc gcctctgtca agtcctctca gagttcacct 2580

atcattccag ggttcggggg cgacttcaac ctgaccctgc tggaaccagt gtctatcagt 2640

accggcagca ggagcgccag atccgcaatc gaggatctgc tgtttgacaa agtgaccatt 2700

gccgaccccg gatacatgca ggggtatgac gattgcatgc agcagggacc tgccagcgcc 2760

agggacctga tctgcgctca gtacgtggca gggtataagg tcctgccacc cctgatggac 2820

gtgaacatgg aagctgcata taccagctcc ctgctgggga gcattgccgg agtggggtgg 2880

acagctggcc tgtctagttt cgccgctatc ccctttgctc agtccatttt ctaccggctg 2940

aacggcgtgg gaatcaccca gcaggtcctg tctgagaatc agaagctgat tgccaacaag 3000

ttcaaccagg ccctgggagc tatgcagaca gggtttacca caactaacga agctttccag 3060

aaagtgcagg atgcagtcaa caataacgca caggccctgt ccaagctggc tagcgagctg 3120

tccaatacct tcggagcaat ctccgcctct attggggata tcattcagag actggacgtg 3180

ctggaacagg atgcccagat cgaccggctg attaatggac gcctgaccac actgaacgct 3240

tttgtggcac agcagctggt ccgaagtgaa tcagcagccc tgagcgccca gctggctaag 3300

gacaaagtga acgagtgcgt caaggctcag tctaaacgga gtggcttttg tgggcagggc 3360

acccacatcg tgtccttcgt ggtcaatgca ccaaacggcc tgtactttat gcacgtggga 3420

tactatccca gtaaccatat cgaggtggtc tcagcttatg gcctgtgcga tgctgcaaat 3480

cctactaact gtattgcacc agtgaatgga tacttcatca aaaccaataa cacacggatt 3540

gtggacgaat ggtcttacac cggctcaagc ttttatgcac ccgagcctat cacaagtctg 3600

aacactaagt acgtggcccc ccaggtcaca tatcagaata tctcaactaa cctgcctcca 3660

cccctgctgg gcaatagcac cggaattgac ttccaggatg aactggacga gttctttaag 3720

aatgtgagca cttccatccc taactttggc agcctgaccc agattaacac taccctgctg 3780

gatctgacat acgagatgct gtccctgcag caggtggtca aggccctgaa tgaatcttac 3840

atcgacctga aagagctggg gaattatact tactataaca agtggccctg gtacatctgg 3900

ctggggttca ttgcaggcct ggtggctctg gcactgtgcg tcttctttat cctgtgctgt 3960

accggatgcg ggacaaattg tatgggcaag ctgaaatgta acaggtgttg tgatagatac 4020

gaagaatacg acctggagcc tcataaagtg catgtccatt ga 4062

Claims

Dimers of MERS-CoV RBD;

MERS-CoV RBD is (a1), (a2), (a3), (a4), (a5) or (a6) as follows:

(a1) protein shown in a sequence 3 in a sequence table;

(a2) a protein obtained by linking a signal peptide to the N-terminus of (a 1);

(a3) a fusion protein obtained by attaching a tag to the N-terminus or/and the C-terminus of (a 1);

(a4) a fusion protein obtained by attaching a tag to the C-terminus of (a 2);

(a5) protein shown in a sequence 7 of a sequence table;

(a6) the protein shown as amino acid residues 39-287 in the sequence 7 of the sequence table.
2. A nucleic acid molecule encoding a dimer of MERS-CoV RBD according to claim 1.
3. A recombinant plasmid having the nucleic acid molecule of claim 2, wherein said nucleic acid molecule is a DNA molecule.
4. A process for preparing a dimer of MERS-CoV RBD according to claim 1, comprising the steps of: the dimer of MERS-CoV RBD was prepared using the Bac-to-Bac system.
5. A kit for preparing dimers of MERS-CoV RBD of claim 1, comprising recombinant plasmids and insect cells; the recombinant plasmid is constructed by taking an expression vector of a Bac-to-Bac system as a starting vector and has the nucleic acid molecule of claim 2, and the nucleic acid molecule is a DNA molecule.
6. Use of a dimer of MERS-CoV RBD according to claim 1 or a nucleic acid molecule according to claim 2 or a recombinant plasmid according to claim 3 or a kit according to claim 5 for the preparation of a product for use as (e1) or (e 2):

(e1) as a middle east respiratory syndrome coronavirus vaccine;

(e2) as a medicament for the prevention and/or treatment of middle east respiratory syndrome.
7. A product, the active ingredient of which is a dimer of MERS-CoV RBD according to claim 1 or a nucleic acid molecule according to claim 2 or a recombinant plasmid according to claim 3 or a kit according to claim 5;

the application of the product is (e1) or (e 2):

(e1) as a middle east respiratory syndrome coronavirus vaccine;

(e2) as a medicament for the prevention and/or treatment of middle east respiratory syndrome.