CN113073106B

CN113073106B - Novel coronavirus B.1.525 Nigeria mutant RBD gene and application thereof

Info

Publication number: CN113073106B
Application number: CN202110621618.XA
Authority: CN
Inventors: 翟俊辉; 杜红; 王轲珑
Original assignee: Beijing Huapeng Biotechnology Co ltd
Current assignee: Huaxi Biotechnology Qingdao Co ltd
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2021-09-07
Anticipated expiration: 2041-06-04
Also published as: CN113073106A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a gene of a novel coronavirus B.1.525 Nigeria mutant RBD and application thereof. The nucleotide sequence of the gene of the novel coronavirus B.1.525 Nigeria mutant RBD is shown as SEQ ID NO.1 or SEQ ID NO. 6. According to the invention, the gene sequence of the wild type novel coronavirus B.1.525 Nigeria mutant RBD is optimized, and the relative optimal sequence is determined by combining screening, and the expression efficiency of the clone generated by the optimized sequence is greatly improved compared with the expression efficiency of the wild type novel coronavirus B.1.525 Nigeria mutant RBD, so that the gene of the novel coronavirus B.1.525 Nigeria mutant RBD can be used for preparing novel coronavirus vaccines.

Description

Novel coronavirus B.1.525 Nigeria mutant RBD gene and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a gene of a novel coronavirus B.1.525 Nigeria mutant RBD and application thereof.

Background

2019 novel coronavirus SARS-CoV-2, causing novel coronavirus pneumonia COVID-19, is the 7 th coronavirus which is known to infect human, the other 6 are HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV (causing severe acute respiratory syndrome) and MERS-CoV (causing middle east respiratory syndrome), respectively.

Coronaviruses are a class of enveloped, linear, single-stranded, positive-stranded RNA viruses whose genomes are a large class of viruses that are widespread in nature. The coronavirus particles are irregular in shape and about 60-220nm in diameter; the genome is about 27-32kb in total length and is the largest virus among the currently known RNA viruses. The virion is surrounded by a fat membrane, the surface of which has three glycoproteins: spike glycoprotein (S, Spike Protein, which is the receptor binding site, cytolytic and major antigenic site); small Envelope glycoprotein (E, Envelope Protein, smaller, Envelope-bound Protein); membrane glycoproteins (M, Membrane proteins, responsible for transmembrane transport of nutrients, budding release of nascent viruses and formation of viral envelope).

The nucleic acid of coronavirus is non-segment single-stranded (+) RNA, the length of which is 27-31kb, and the nucleic acid is the longest RNA nucleic acid chain in RNA virus, and the mutation can be said to be the biggest characteristic of the RNA virus such as new coronavirus. After the virus invades the host cell, it can largely replicate itself to achieve infection transmission. During replication, the RNA virus has no correction mechanism, replication errors cannot be corrected by itself, and new variations are likely to occur in a large number of replications, and the new variations result in reduced effectiveness or failure of the vaccine.

Hundreds of variants of new coronaviruses have been discovered worldwide, and in 12 months of 2020, Nigeria first detected the B.1.525 strain in the viral genome sequence of a new coronaviruse patient, and later, the variants were also discovered soon in England, France, etc. After only 2 months, b.1.525 accounts for more than 20% of nigeria infected cases, and this virus is found in more than 200 cases worldwide. The major variants of the virus were amino acid substitutions in Q52R and A67 in the N-terminal domain of the S protein, and deletions at positions 69-70 and 144. The former is unique to the new variety, and the deletion of the latter 2 sites is also present in the previous british variety b.1.1.7. Because the N-terminal domain of the new coronavirus has high antigenicity, the region is mutated, and the phenomenon of antibody neutralization escape is easily caused. In addition, amino acid substitutions at positions 888 and 870 of the proximal stem region of the S protein membrane were also present in 1 antibody neutralization escape variant. B.1.525 carries a number of common new mutations of the coronavirus, such as D614G, this mutation that greatly enhances viral infectivity is present in almost all virus variants at this stage; also, the previous occurrence of the E484K mutation gave people approximately 3-fold resistance to several vaccines and 10-fold resistance to most convalescent antibodies. The amino acid substitution at position Q677H also occurs in the most recently discovered variant virus in the united states. This mutation, which occurs in the S1 portion of the S protein remote from the receptor binding domain, as with the D614G mutation, may increase the stability of the S1-S2 association, thereby enhancing transmission. The mutation may also facilitate open rather than closed binding of the receptor binding domain. B.1.525 also showed a number of mutations in genomic regions constituting other proteins, 3 of which occurred in genes constituting the large-scale replication complex, including Orf1a NSP3 (T1189I), NSP6 (3 amino acid 106-108 deletions), and 1 in the major RNA-dependent RNA polymerase Orf1b NSP12 (P323F). The genes for the viral structural proteins E (l21F), M (i82f) and N (A12G) and (T208I) also show 4 mutations. These mutations, which occur in large numbers in the transmembrane proteins NSP6, E, M, N, are highly antigenic in neocoronary patients. All of these mutations may lead to escape of antibody neutralization. The remaining mutations in orf1a may increase replication efficiency, or lead to escape of antibody neutralization, or both, leading to 2 consequences.

Research proves that most neutralizing antibodies are directed to the Receptor Binding Domain (RBD) of the spurt protein (S) of the novel coronavirus, the RBD is a key part for the virus to enter host cells, the RBD protein can interact with ACE2 receptor of the cells to open cell surface channels, so that virus particles can enter the cells to complete the virus invasion process, and therefore, the research and development of recombinant RBD vaccines directed to the novel coronavirus mutant strains are the key point for effectively preventing and controlling the virus mutant strains.

As a recombinant vaccine antigen composition, the preparation of a vaccine antigen by using a CHO, 293T and other cell eukaryotic expression systems is one of the best ways for ensuring the immunogenicity and the correct high-level structure of the vaccine antigen. However, mutant wild-type viral sequences are generally expressed at very low levels in eukaryotic expression systems, but for vaccine development, high expression levels are a prerequisite, which is influenced by several factors, such as antigen-encoded nucleotide sequences, mRNA secondary structure changes, screening for different signal peptides, vector selection, host cell selection, fermentation processes, culture process optimization, etc., but among these factors, protein gene coding sequences are the first to solve.

Therefore, the problem to be solved by those skilled in the art is how to provide a novel nucleotide sequence of Nigeria mutant RBD of coronavirus which is designed to be highly expressed and to express the nucleotide sequence.

Disclosure of Invention

The invention aims to provide a gene after design optimization of a novel coronavirus B.1.525 Nigeria mutant RBD.

It is still another object of the present invention to provide a recombinant expression vector containing the above gene.

The invention also aims to provide a method for preparing a novel coronavirus B.1.525 Nigeria mutant RBD by using the gene.

The invention also aims to provide the application of the gene in preparing a novel coronavirus vaccine.

The nucleotide sequence of the optimized gene of the novel coronavirus B.1.525 Nigeria mutant RBD according to the specific embodiment of the invention is shown as SEQ ID NO.1 or SEQ ID NO. 6.

Wherein, SEQ ID NO. 1:

AGAGTGCAGCCAACAGAGAGCATCGTGAGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCAACAAGGTTCGCCAGCGTGTACGCCTGGAACAGAAAAAGGATCAGCAACTGCGTGGCAGACTACAGTGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAATGCTATGGCGTGTCCCCCACCAAGCTGAACGATCTGTGTTTTACCAACGTGTACGCCGACTCCTTCGTGATTAGGGGCGACGAGGTGCGCCAGATCGCTCCTGGACAGACAGGAAAGATCGCCGACTATAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATTGCTTGGAACTCCAACAACCTGGACAGTAAAGTGGGCGGCAACTACAATTACCTGTACAGACTGTTCAGGAAGAGCAACCTGAAACCCTTCGAAAGAGACATCTCCACAGAGATCTACCAGGCCGGCAGCACCCCATGTAACGGAGTGAAGGGATTTAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCCAGCCAACAAACGGCGTGGGCTACCAGCCTTACAGGGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCCCCCGCTACAGTGTGTGGACCTAAGAAGTCCACCAACCTGGTGAAAAACAAATGTGTCAATTTC。

SEQ ID NO.6：

GCTAGC CCACCATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGAAGAGTGCAGCCAACAGAGAGCATCGTGAGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCAACAAGGTTCGCCAGCGTGTACGCCTGGAACAGAAAAAGGATCAGCAACTGCGTGGCAGACTACAGTGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAATGCTATGGCGTGTCCCCCACCAAGCTGAACGATCTGTGTTTTACCAACGTGTACGCCGACTCCTTCGTGATTAGGGGCGACGAGGTGCGCCAGATCGCTCCTGGACAGACAGGAAAGATCGCCGACTATAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATTGCTTGGAACTCCAACAACCTGGACAGTAAAGTGGGCGGCAACTACAATTACCTGTACAGACTGTTCAGGAAGAGCAACCTGAAACCCTTCGAAAGAGACATCTCCACAGAGATCTACCAGGCCGGCAGCACCCCATGTAACGGAGTGAAGGGATTTAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCCAGCCAACAAACGGCGTGGGCTACCAGCCTTACAGGGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCCCCCGCTACAGTGTGTGGACCTAAGAAGTCCACCAACCTGGTGAAAAACAAATGTGTCAATTTCtaaGCGGCCGC。

wherein the italic "GCTAGC" is the cleavage site and the underlined part "CCACC" is the KOZAK sequence.

In order to realize high expression in host cell specificity, the invention selects a signal peptide in a human serum albumin sequence, and the amino acid sequence of the signal peptide is as follows: MKWVTFISLLFLFSSAYSRGVFRR, the nucleotide sequence is: ATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGA are provided.

Therefore, the amino acid sequence of the RBD protein of the mature novel coronavirus B.1.525 Nigeria mutant strain is shown as SEQ ID NO. 9:

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF

in order to realize high expression in the specificity of the host cell, the invention can also select the signal peptide in the serum albumin sequence of the Chinese hamster, the amino acid sequence of the signal peptide is MKWVTFLLLLFVSDSAFS, and the nucleotide sequence of the optimized nucleotide of the signal peptide of the high expression secretion protein in the specificity of the host cell is as follows:

Atgaaatgggttactttcttattattattgtttgtatctgattctgctttttca。

the invention also provides a recombinant vector containing the novel coronavirus B.1.525 Nigeria mutant RBD, preferably pcDNA3.1 +. The novel coronavirus B.1.525 Nigeria mutant RBD of the invention is inserted between proper restriction enzyme cutting sites of an expression vector, so that the nucleotide sequence of the mutant RBD is operably connected with an expression regulation sequence. As a most preferred embodiment of the present invention, it is preferable that the novel coronavirus B.1.525 Nigeria mutant RBD is inserted between the Nhel/Notl double restriction sites of plasmid pcDNA3.1+, so that the nucleotide sequence is located at the downstream of the promoter and is controlled by the promoter, thereby obtaining a recombinant expression vector.

The invention also provides host cells of the novel coronavirus B.1.525 Nigeria mutant RBD, preferably Chinese hamster CHO cells and 293 cells. Chinese hamster CHO cells are easy to realize large-scale high-density culture and complete protein glycosylation modification. The signal peptide sequence of the host cell specific high expression secretory protein is a signal peptide in a human serum albumin sequence, and the amino acid sequence is as follows: MKWVTFISLLFLFSSAYSRGVFRR are provided.

The present invention also provides a method for preparing a novel coronavirus b.1.525 nigeria mutant RBD, the method comprising the steps of:

(1) transforming host cells with a recombinant expression vector containing the gene of the novel coronavirus B.1.525 Nigeria mutant RBD;

(2) culturing host cells, and inducing the expression of a novel coronavirus B.1.525 Nigeria mutant RBD;

(3) recovering and purifying the expressed novel coronavirus B.1.525 Nigeria mutant RBD.

The invention also provides the application of the gene with the nucleotide sequence shown as SEQ ID NO.1 or SEQ ID NO.6 in the preparation of a novel coronavirus vaccine. The novel coronavirus B.1.525 Nigeria mutant RBD protein is industrially produced by using a genetic engineering means, and is matched with a proper pharmaceutical adjuvant to be applied to novel coronavirus vaccines.

The invention has the beneficial effects that:

according to the invention, the gene sequence of the wild type novel coronavirus B.1.525 Nigeria mutant RBD is optimized, and the relative optimal sequence is determined by combining screening, and the cloning expression efficiency generated by the optimized sequence is improved by 6 times compared with the wild type novel coronavirus B.1.525 Nigeria mutant RBD sequence. The novel coronavirus B.1.525 Nigeria mutant RBD can induce a mouse to generate high-titer virus neutralizing antibodies. The gene of the novel coronavirus B.1.525 Nigeria mutant RBD can be used for producing novel coronavirus vaccines.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.

FIG. 1 shows a plasmid map of pcDNA3.1-RBD (B.1.525).

FIG. 2 shows the comparison of protein concentration secreted by clones constructed by eukaryotic expression of 4 sequences.

FIG. 3 is an SDS-PAGE pattern of purified RBD protein samples.

FIG. 4 shows serum anti-RBD antibody titers (RBD glycoproteins) in mice 14 days after the hyperimmunization.

FIG. 5 shows the results of a pseudovirus neutralization assay.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be described in detail below. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without any inventive step, are within the scope of the present invention.

Example 1 optimization of wild-type novel coronavirus B.1.525 Nigeria mutant RBD sequences

Based on the wild type novel coronavirus RBD amino acid sequence, the primarily optimized novel coronavirus B.1.525 Nigeria mutant RBD nucleotide sequence is obtained by the following optimization:

the invention optimizes the codon of the coding amino acid sequence in the coding sequence according to the genetic codon bias of Chinese hamster CHO cells;

wherein, according to the preference of codon, the previous protein high-efficiency expression experience in the laboratory, the secondary structure of mRNA and other factors, the nucleotide sequence of SEQ ID NO.1 is obtained by optimization:

the software optimized sequence provides SEQ ID No. 2:

AGAGTGCAGCCAACAGAGAGCATCGTGCGCTTCCCCAACATCACAAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCTACCAGGTTCGCTTCCGTGTACGCCTGGAACAGGAAGAGAATCTCCAACTGCGTGGCTGACTACTCCGTCCTCTACAACTCCGCTTCCTTCTCGACCTTCAAGTGCTACGGTGTGTCCCCTACCAAGCTGAACGACCTCTGCTTCACCAACGTCTACGCTGACTCCTTCGTGATCCGCGGCGACGAAGTCCGTCAAATCGCTCCTGGTCAGACCGGAAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGTTGCGTCATCGCTTGGAACTCCAACAACCTCGACAGTAAGGTGGGTGGTAACTACAACTACCTGTACCGCCTGTTCCGCAAGAGCAACCTGAAGCCCTTCGAAAGGGACATCTCCACCGAGATCTACCAGGCCGGCTCCACACCATGCAACGGAGTGAAGGGTTTCAACTGCTACTTCCCCCTGCAATCCTACGGTTTCCAGCCCACCAACGGTGTGGGATACCAGCCTTACCGCGTGGTGGTGCTCTCCTTCGAGCTCTTGCACGCCCCTGCTACCGTGTGTGGTCCTAAGAAGTCCACCAACCTCGTGAAAAACAAATGTGTCAATTTC。

the software optimized sequence provides SEQ ID No. 3:

CGTGTTCAGCCTACCGAATCTATTGTTCGTTTTCCTAATATTACCAACCTGTGTCCTTTTGGTGAAGTCTTTAATGCTACCCGTTTTGCTTCAGTTTATGCATGGAATCGTAAACGTATTAGTAACTGTGTTGCAGATTATAGCGTTCTGTATAACAGCGCCAGCTTTAGTACCTTTAAATGTTATGGTGTGAGTCCGACTAAACTGAATGATCTGTGTTTTACCAATGTTTATGCAGATAGCTTTGTTATTCGTGGTGATGAAGTTCGCCAGATTGCACCGGGTCAGACCGGTAAGATTGCCGATTATAATTATAAACTGCCGGATGATTTTACCGGTTGTGTGATTGCCTGGAATTCAAATAATCTGGATAGCAAAGTGGGTGGTAATTATAATTATCTGTATCGTCTGTTTCGCAAAAGCAATCTGAAACCGTTTGAACGTGATATTTCTACCGAAATTTATCAGGCGGGCAGCACACCGTGTAATGGTGTTAAAGGTTTTAACTGTTATTTTCCTCTGCAGTCTTATGGTTTTCAGCCGACCaacGGTGTTGGTTATCAGCCGTATCGCGTTGTGGTTCTGAGTTTTGAACTGCTGCATGCCCCGGCAACGGTTTGTGGTCCTAAAAAGTCAACCAATCTGGTTAAAAACAAATGTGTCAATTTC。

the wild type RBD nucleotide sequence of the novel coronavirus B.1.525 Nigeria mutant strain is shown as SEQ ID NO. 4:

AGAGTCCAACCAACAGAATCTATTGTTAGATTTCCTAATATTACAAACTTGTGCCCTTTTGGTGAAGTTTTTAACGCCACCAGATTTGCATCTGTTTATGCTTGGAACAGGAAGAGAATCAGCAACTGTGTTGCTGATTATTCTGTCCTATATAATTCCGCATCATTTTCCACTTTTAAGTGTTATGGAGTGTCTCCTACTAAATTAAATGATCTCTGCTTTACTAATGTCTATGCAGATTCATTTGTAATTAGAGGTGATGAAGTCAGACAAATCGCTCCAGGGCAAACTGGAAAGATTGCTGATTATAATTATAAATTACCAGATGATTTTACAGGCTGCGTTATAGCTTGGAATTCTAACAATCTTGATTCTAAGGTTGGTGGTAATTATAATTACCTGTATAGATTGTTTAGGAAGTCTAATCTCAAACCTTTTGAGAGAGATATTTCAACTGAAATCTATCAGGCCGGTAGCACACCTTGTAATGGTGTTAAAGGTTTTAATTGTTACTTTCCTTTACAATCATATGGTTTCCAACCCACTAATGGTGTTGGTTACCAACCATACAGAGTAGTAGTACTTTCTTTTGAACTTCTACATGCACCAGCAACTGTTTGTGGACCTAAAAAGTCTACTAATTTGGTTAAAAACAAATGTGTCAATTTC。

the invention further inserts a signal peptide sequence, a KOZAK sequence CCACC and a restriction enzyme cutting site GCTAGC in front of the optimized sequence, and adds a stop codon taa and a restriction enzyme cutting site GCGGCCGC at the tail end of the optimized sequence.

Selecting a host cell specificity high expression human serum albumin signal peptide sequence, wherein the nucleotide sequence is shown as SEQ ID NO. 5:

ATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGA。

the amino acid sequence of the signal peptide is as follows: MKWVTFISLLFLFSSAYSRGVFRR are provided.

The nucleotide sequence of the optimized novel coronavirus B.1.525 Nigeria mutant RBD containing signal peptide is shown as SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO. 8:

SEQ ID NO.6：

GCTAGCCCACCATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGAAGAGTGCAGCCAACAGAGAGCATCGTGAGGTTCCCCAACATCACCAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCAACAAGGTTCGCCAGCGTGTACGCCTGGAACAGAAAAAGGATCAGCAACTGCGTGGCAGACTACAGTGTGCTGTACAACTCCGCCTCCTTCTCCACCTTCAAATGCTATGGCGTGTCCCCCACCAAGCTGAACGATCTGTGTTTTACCAACGTGTACGCCGACTCCTTCGTGATTAGGGGCGACGAGGTGCGCCAGATCGCTCCTGGACAGACAGGAAAGATCGCCGACTATAACTACAAGCTGCCCGACGACTTCACCGGCTGCGTGATTGCTTGGAACTCCAACAACCTGGACAGTAAAGTGGGCGGCAACTACAATTACCTGTACAGACTGTTCAGGAAGAGCAACCTGAAACCCTTCGAAAGAGACATCTCCACAGAGATCTACCAGGCCGGCAGCACCCCATGTAACGGAGTGAAGGGATTTAACTGCTACTTCCCCCTGCAGTCCTACGGCTTCCAGCCAACAAACGGCGTGGGCTACCAGCCTTACAGGGTGGTGGTGCTGTCTTTTGAGCTGCTGCACGCCCCCGCTACAGTGTGTGGACCTAAGAAGTCCACCAACCTGGTGAAAAACAAATGTGTCAATTTCtaaGCGGCCGC。

SEQ ID NO. 7：

GCTAGCCCACCATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGAAGAGTGCAGCCAACAGAGAGCATCGTGCGCTTCCCCAACATCACAAACCTGTGCCCCTTCGGCGAGGTGTTCAACGCTACCAGGTTCGCTTCCGTGTACGCCTGGAACAGGAAGAGAATCTCCAACTGCGTGGCTGACTACTCCGTCCTCTACAACTCCGCTTCCTTCTCGACCTTCAAGTGCTACGGTGTGTCCCCTACCAAGCTGAACGACCTCTGCTTCACCAACGTCTACGCTGACTCCTTCGTGATCCGCGGCGACGAAGTCCGTCAAATCGCTCCTGGTCAGACCGGAAAGATCGCCGACTACAACTACAAGCTGCCCGACGACTTCACCGGTTGCGTCATCGCTTGGAACTCCAACAACCTCGACAGTAAGGTGGGTGGTAACTACAACTACCTGTACCGCCTGTTCCGCAAGAGCAACCTGAAGCCCTTCGAAAGGGACATCTCCACCGAGATCTACCAGGCCGGCTCCACACCATGCAACGGAGTGAAGGGTTTCAACTGCTACTTCCCCCTGCAATCCTACGGTTTCCAGCCCACCAACGGTGTGGGATACCAGCCTTACCGCGTGGTGGTGCTCTCCTTCGAGCTCTTGCACGCCCCTGCTACCGTGTGTGGTCCTAAGAAGTCCACCAACCTCGTGAAAAACAAATGTGTCAATTTCtaaGCGGCCGC。

SEQ ID NO. 8：

GCTAGCCCACCATGAAGTGGGTAACCTTTATTTCCCTTCTTTTTCTCTTTAGCTCGGCTTATTCCAGGGGTGTGTTTCGTCGACGTGTTCAGCCTACCGAATCTATTGTTCGTTTTCCTAATATTACCAACCTGTGTCCTTTTGGTGAAGTCTTTAATGCTACCCGTTTTGCTTCAGTTTATGCATGGAATCGTAAACGTATTAGTAACTGTGTTGCAGATTATAGCGTTCTGTATAACAGCGCCAGCTTTAGTACCTTTAAATGTTATGGTGTGAGTCCGACTAAACTGAATGATCTGTGTTTTACCAATGTTTATGCAGATAGCTTTGTTATTCGTGGTGATGAAGTTCGCCAGATTGCACCGGGTCAGACCGGTAAGATTGCCGATTATAATTATAAACTGCCGGATGATTTTACCGGTTGTGTGATTGCCTGGAATTCAAATAATCTGGATAGCAAAGTGGGTGGTAATTATAATTATCTGTATCGTCTGTTTCGCAAAAGCAATCTGAAACCGTTTGAACGTGATATTTCTACCGAAATTTATCAGGCGGGCAGCACACCGTGTAATGGTGTTAAAGGTTTTAACTGTTATTTTCCTCTGCAGTCTTATGGTTTTCAGCCGACCaacGGTGTTGGTTATCAGCCGTATCGCGTTGTGGTTCTGAGTTTTGAACTGCTGCATGCCCCGGCAACGGTTTGTGGTCCTAAAAAGTCAACCAATCTGGTTAAAAACAAATGTGTCAATTTCtaaGCGGCCGC。

example 2 expression and purification of recombinant RBD proteins

The complete target gene shown in SEQ ID NO.6 is subjected to Nhel/Notl double enzyme digestion, and then is connected to a pcDNA3.1+ eukaryotic expression vector subjected to the same enzyme digestion to obtain a recombinant vector, wherein the plasmid structure is shown in figure 1.

The recombinant vectors are respectively transformed into escherichia coli, plasmid amplification is carried out according to a conventional method, and then plasmids are extracted by using a reagent kit of Tiangen biology, Inc.

Transfection of chinese hamster CHO cells: preparing according to a Lipofectin kit manual to obtain a DNA-liposome mixture, adding the DNA-liposome mixture into Chinese hamster CHO cells cultured by a DMEM medium, and incubating for 2 hours at 37 ℃; the culture medium was changed to DMEM medium containing 10% FBS, and the culture was continued for 48 hours.

Selection of Neomycin resistant clones: the transfected cells were isolated from the flask at 1X 10⁵The cells/well were added to a 96-well plate, the transfected cells were further cultured in DMEM medium (with 10% FBS) containing 500. mu.g/mL Neomycin, and after 7 days, the cells forming the clone were selected and expanded and cultured in a 6-well plate.

Analysis of expression RBD clones: the NEO resistant clones were cultured at 1.5X 10⁵Cell density in/mL was inoculated into T25 flasks at 5% CO₂Culturing at 37 deg.C for 72h in incubator, and collecting supernatant to obtain RBD protein.

In the same way, the wild type novel coronavirus B.1.525 Nigeria mutant strain sequence containing signal peptide sequence and expression vectors of optimized strains SEQ ID NO.7 and SEQ ID NO.8 thereof are constructed, transformed and expressed.

The obtained supernatant was identified and analyzed for RBD protein content.

By identifying the sequence of a wild strain, and RBD proteins of novel coronavirus B.1.525 Nigeria mutant strains which can be expressed by SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, the concentration of the proteins secreted by clones constructed by eukaryotic expression of the four sequences is compared, and the results are shown in table 1 and figure 2.

TABLE 14 comparison of protein concentrations secreted by clones constructed by eukaryotic expression of the sequences (. mu.g/mL)

Results are shown in Table 1, the concentrations of the proteins expressed by SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8 are respectively 9.36. mu.g/mL, 3.72. mu.g/mL and 2.40. mu.g/mL, which are higher than the concentrations of the proteins expressed by the wild-type sequence, and meanwhile, the concentration of the protein expressed by SEQ ID NO.6 is significantly higher than the concentrations of the proteins expressed by SEQ ID NO.7 and SEQ ID NO. 8.

The amino acid sequence of RBD protein of mature novel coronavirus B.1.525 Nigeria mutant strain is shown as SEQ ID NO. 9:

RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVKGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNF。

the protein expressed by SEQ ID NO.6 was concentrated by a 10 kDa membrane pack while the medium therein was replaced with a low salt buffer, followed by further concentration using a 10 kDa ultrafiltration tube. After dilution by concentration, purification is carried out by ion exchange chromatography for use. As shown in FIG. 3, the molecular weight of the purified RBD protein is about 34kD, and SDS-PAGE shows that the RBD protein has uniform bands and better purity.

Example 3 mouse immunization experiment

20 female BALB/c mice aged 6-8 weeks are taken and randomly divided into the following groups:

immunization 1 group (10 μ g immunization group): on days 0 and 14, 100. mu.L of vaccine was injected intramuscularly. The vaccine used was 10. mu.g RBD + 100. mu.g Al (OH)₃Wherein RBD is the novel coronavirus B.1.525 Nigeria mutant RBD glycoprotein expressed by CHO cells prepared in example 2, and comprises 10. mu.g of RBD and 100. mu.g of Al (OH) in a volume of 100. mu.L₃The vaccine is prepared by using normal saline.

Immunization 2 group (5 μ g immunization group): on days 0 and 14, 100. mu.L of vaccine was injected intramuscularly. The vaccine used was 5. mu.g RBD + 100. mu.g Al (OH)₃Wherein, whereinRBD is the novel coronavirus B.1.525 Nigeria mutant RBD glycoprotein expressed by CHO cells prepared in example 2, and comprises 5. mu.g of RBD and 100. mu.g of Al (OH) in a volume of 100. mu.L₃The vaccine is prepared by using normal saline.

Adjuvant control group: injecting 100 μ L vaccine into muscle on day 0 and 14 respectively, and adjuvant is 100 μ g Al (OH)₃。

Saline control group: on days 0 and 14, 100. mu.L of physiological saline was injected intramuscularly.

Each of the above groups was bled on day 28.

The serum anti-RBD antibody titer of each group of mice was measured by ELISA. See the manual of molecular biology laboratory manual [ M ] science press 2008.

The results are shown in FIG. 4, and the specific antibody titer of 10. mu.g immunized mice in two-week two-immunization can reach 1X 10^6.22The specific antibody titer of 5 mu g of immunized mice can reach 1 multiplied by 10^5.24And the adjuvant group was 1X 10^1.58The normal saline solution group is 1 × 10^1.78. The antibody titer of the immunized group (10. mu.g or 5. mu.g group) was much higher than that of the saline control group or the adjuvant control group.

Example 4 pseudovirus neutralization assay

The pseudovirus was purchased from Nanjing Novozam Biotech GmbH, product number DD1451-02, and the procedure was as described in the product Specification. Neutralizing antibody neutralizes pseudovirus in vitro, so that pseudovirus loses the ability to infect cells, pseudovirus entering cells expresses fluc protein, luminescence value of pseudovirus is detected by a machine after reaction with luminescent substrate, inhibition percentage is calculated by comparing with luminescence value of pseudovirus control group, dilution multiple of antibody when pseudovirus is inhibited by 50% can be calculated by calculation formula, and ED50 is calculated, and ED50 represents the neutralizing activity of antibody to pseudovirus.

1) Sample preparation: after 4 groups of mice obtained in example 3 are immunized, the serum is inactivated in a water bath at 56 ℃ for 0.5 to 1 hour;

2) adding 260 mu L of high-pressure sterilized water into 36 holes on the periphery of the 96-hole plate, and sealing the edges, so as to reduce errors caused by the evaporation of a culture medium in the edge holes;

3) column 2 (cell control CC) was supplemented with DMEM complete medium 150. mu.L/well, column 3 (virus control VC) was supplemented with DMEM complete medium 100. mu.L/well, B4-B11 wells (i.e., columns 4-11 of a 96 well plate) were supplemented with medium 142.5. mu.L/well, and the remaining wells were supplemented with medium 100. mu.L/well, 6 wells per column;

4) a mouse immune serum sample to be detected is added into a B4-B11 hole (the first hole is added according to a ratio of 1:100 and diluted by times. ) 7.5 mu L;

5) repeatedly and gently blowing and sucking the liquid in the B4-B11 wells for 6-8 times, then transferring 50 mu L of liquid to the corresponding C4-C11 wells (namely, the C row 4-11 columns of the 96-well plate), and then diluting all the wells by 3 times;

6) pseudovirus was diluted to 1.3X 10 with DMEM complete medium⁴Adding 50 mu L of TCID50/mL to each hole of the 3 rd to 11 th rows;

7) the 96-well plate was placed in a cell incubator (37 ℃ C., 5% CO)₂) Incubating for 1 hour;

8) after incubation for 30min, digestion of Huh7 cells was started, diluting the cell concentration to 2 × 10⁵cells/mL；

9) After the incubation is finished, 100 mu L of cells are added into each hole, so that the cells in each hole are 2 multiplied by 104 cells;

10) put at 37 ℃ with 5% CO₂Culturing for 24 hours in a cell culture box;

11) after the culture is finished, 150 mu L of supernatant is sucked away, 100 mu L of Bright-GloTM luciferase detection reagent is added, reaction is carried out for 2min at room temperature in a dark place, blowing is carried out repeatedly, and 150 mu L of liquid is transferred to a white board;

12) reading luminescence values (RLU) by using a Perkinelmer EnSight multifunctional imaging microplate reader;

calculating the neutralization inhibition rate:

wherein, VC-virus contrast VC, CC-cell contrast CC.

The neutralizing antibody titer was expressed as the reciprocal of the serum dilution corresponding to an inhibition rate of 50% or the antibody concentration corresponding to an inhibition rate of 50%.

Positive judgment value: and a negative control and a positive control are required to be arranged in the neutralization experiment process as references to judge whether the experiment is established, wherein the negative control ED50 is less than 30, and the positive ED50 is greater than 30 as judgment values.

The results are shown in FIG. 5, and the virus-neutralizing antibody titer of the two-week two-immunization mouse with a high dose of 10. mu.g can reach 1 × 10^2.52The specific antibody titer of the low-dose 5 mu g immunization group mouse can reach 1 multiplied by 10^2.33And the adjuvant group was 1: no neutralizing antibodies were detected in the saline group 10. High dose 10 μ g immunization group (10 μ g RBD +100 μ g Al (OH)₃) And a low dose of 5. mu.g immunization group (5. mu.g RBD + 100. mu.g Al (OH)₃) Virus neutralizing antibody titer > 1:100, which is obviously higher than the agent control group and the normal saline control group.

The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the appended claims.

Sequence listing

<110> Beijing Hua Biotechnology Co., Ltd

<120> novel coronavirus B.1.525 Nigeria mutant RBD gene and application thereof

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

agagtgcagc caacagagag catcgtgagg ttccccaaca tcaccaacct gtgccccttc 60

ggcgaggtgt tcaacgcaac aaggttcgcc agcgtgtacg cctggaacag aaaaaggatc 120

agcaactgcg tggcagacta cagtgtgctg tacaactccg cctccttctc caccttcaaa 180

tgctatggcg tgtcccccac caagctgaac gatctgtgtt ttaccaacgt gtacgccgac 240

tccttcgtga ttaggggcga cgaggtgcgc cagatcgctc ctggacagac aggaaagatc 300

gccgactata actacaagct gcccgacgac ttcaccggct gcgtgattgc ttggaactcc 360

aacaacctgg acagtaaagt gggcggcaac tacaattacc tgtacagact gttcaggaag 420

agcaacctga aacccttcga aagagacatc tccacagaga tctaccaggc cggcagcacc 480

ccatgtaacg gagtgaaggg atttaactgc tacttccccc tgcagtccta cggcttccag 540

ccaacaaacg gcgtgggcta ccagccttac agggtggtgg tgctgtcttt tgagctgctg 600

cacgcccccg ctacagtgtg tggacctaag aagtccacca acctggtgaa aaacaaatgt 660

gtcaatttc 669

<210> 2

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

agagtgcagc caacagagag catcgtgcgc ttccccaaca tcacaaacct gtgccccttc 60

ggcgaggtgt tcaacgctac caggttcgct tccgtgtacg cctggaacag gaagagaatc 120

tccaactgcg tggctgacta ctccgtcctc tacaactccg cttccttctc gaccttcaag 180

tgctacggtg tgtcccctac caagctgaac gacctctgct tcaccaacgt ctacgctgac 240

tccttcgtga tccgcggcga cgaagtccgt caaatcgctc ctggtcagac cggaaagatc 300

gccgactaca actacaagct gcccgacgac ttcaccggtt gcgtcatcgc ttggaactcc 360

aacaacctcg acagtaaggt gggtggtaac tacaactacc tgtaccgcct gttccgcaag 420

agcaacctga agcccttcga aagggacatc tccaccgaga tctaccaggc cggctccaca 480

ccatgcaacg gagtgaaggg tttcaactgc tacttccccc tgcaatccta cggtttccag 540

cccaccaacg gtgtgggata ccagccttac cgcgtggtgg tgctctcctt cgagctcttg 600

cacgcccctg ctaccgtgtg tggtcctaag aagtccacca acctcgtgaa aaacaaatgt 660

gtcaatttc 669

<210> 3

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgtgttcagc ctaccgaatc tattgttcgt tttcctaata ttaccaacct gtgtcctttt 60

ggtgaagtct ttaatgctac ccgttttgct tcagtttatg catggaatcg taaacgtatt 120

agtaactgtg ttgcagatta tagcgttctg tataacagcg ccagctttag tacctttaaa 180

tgttatggtg tgagtccgac taaactgaat gatctgtgtt ttaccaatgt ttatgcagat 240

agctttgtta ttcgtggtga tgaagttcgc cagattgcac cgggtcagac cggtaagatt 300

gccgattata attataaact gccggatgat tttaccggtt gtgtgattgc ctggaattca 360

aataatctgg atagcaaagt gggtggtaat tataattatc tgtatcgtct gtttcgcaaa 420

agcaatctga aaccgtttga acgtgatatt tctaccgaaa tttatcaggc gggcagcaca 480

ccgtgtaatg gtgttaaagg ttttaactgt tattttcctc tgcagtctta tggttttcag 540

ccgaccaacg gtgttggtta tcagccgtat cgcgttgtgg ttctgagttt tgaactgctg 600

catgccccgg caacggtttg tggtcctaaa aagtcaacca atctggttaa aaacaaatgt 660

gtcaatttc 669

<210> 4

<211> 669

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

agagtccaac caacagaatc tattgttaga tttcctaata ttacaaactt gtgccctttt 60

ggtgaagttt ttaacgccac cagatttgca tctgtttatg cttggaacag gaagagaatc 120

agcaactgtg ttgctgatta ttctgtccta tataattccg catcattttc cacttttaag 180

tgttatggag tgtctcctac taaattaaat gatctctgct ttactaatgt ctatgcagat 240

tcatttgtaa ttagaggtga tgaagtcaga caaatcgctc cagggcaaac tggaaagatt 300

gctgattata attataaatt accagatgat tttacaggct gcgttatagc ttggaattct 360

aacaatcttg attctaaggt tggtggtaat tataattacc tgtatagatt gtttaggaag 420

tctaatctca aaccttttga gagagatatt tcaactgaaa tctatcaggc cggtagcaca 480

ccttgtaatg gtgttaaagg ttttaattgt tactttcctt tacaatcata tggtttccaa 540

cccactaatg gtgttggtta ccaaccatac agagtagtag tactttcttt tgaacttcta 600

catgcaccag caactgtttg tggacctaaa aagtctacta atttggttaa aaacaaatgt 660

gtcaatttc 669

<210> 5

<211> 72

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

atgaagtggg taacctttat ttcccttctt tttctcttta gctcggctta ttccaggggt 60

gtgtttcgtc ga 72

<210> 6

<211> 763

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gctagcccac catgaagtgg gtaaccttta tttcccttct ttttctcttt agctcggctt 60

attccagggg tgtgtttcgt cgaagagtgc agccaacaga gagcatcgtg aggttcccca 120

acatcaccaa cctgtgcccc ttcggcgagg tgttcaacgc aacaaggttc gccagcgtgt 180

acgcctggaa cagaaaaagg atcagcaact gcgtggcaga ctacagtgtg ctgtacaact 240

ccgcctcctt ctccaccttc aaatgctatg gcgtgtcccc caccaagctg aacgatctgt 300

gttttaccaa cgtgtacgcc gactccttcg tgattagggg cgacgaggtg cgccagatcg 360

ctcctggaca gacaggaaag atcgccgact ataactacaa gctgcccgac gacttcaccg 420

gctgcgtgat tgcttggaac tccaacaacc tggacagtaa agtgggcggc aactacaatt 480

acctgtacag actgttcagg aagagcaacc tgaaaccctt cgaaagagac atctccacag 540

agatctacca ggccggcagc accccatgta acggagtgaa gggatttaac tgctacttcc 600

ccctgcagtc ctacggcttc cagccaacaa acggcgtggg ctaccagcct tacagggtgg 660

tggtgctgtc ttttgagctg ctgcacgccc ccgctacagt gtgtggacct aagaagtcca 720

ccaacctggt gaaaaacaaa tgtgtcaatt tctaagcggc cgc 763

<210> 7

<211> 763

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gctagcccac catgaagtgg gtaaccttta tttcccttct ttttctcttt agctcggctt 60

attccagggg tgtgtttcgt cgaagagtgc agccaacaga gagcatcgtg cgcttcccca 120

acatcacaaa cctgtgcccc ttcggcgagg tgttcaacgc taccaggttc gcttccgtgt 180

acgcctggaa caggaagaga atctccaact gcgtggctga ctactccgtc ctctacaact 240

ccgcttcctt ctcgaccttc aagtgctacg gtgtgtcccc taccaagctg aacgacctct 300

gcttcaccaa cgtctacgct gactccttcg tgatccgcgg cgacgaagtc cgtcaaatcg 360

ctcctggtca gaccggaaag atcgccgact acaactacaa gctgcccgac gacttcaccg 420

gttgcgtcat cgcttggaac tccaacaacc tcgacagtaa ggtgggtggt aactacaact 480

acctgtaccg cctgttccgc aagagcaacc tgaagccctt cgaaagggac atctccaccg 540

agatctacca ggccggctcc acaccatgca acggagtgaa gggtttcaac tgctacttcc 600

ccctgcaatc ctacggtttc cagcccacca acggtgtggg ataccagcct taccgcgtgg 660

tggtgctctc cttcgagctc ttgcacgccc ctgctaccgt gtgtggtcct aagaagtcca 720

ccaacctcgt gaaaaacaaa tgtgtcaatt tctaagcggc cgc 763

<210> 8

<211> 763

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gctagcccac catgaagtgg gtaaccttta tttcccttct ttttctcttt agctcggctt 60

attccagggg tgtgtttcgt cgacgtgttc agcctaccga atctattgtt cgttttccta 120

atattaccaa cctgtgtcct tttggtgaag tctttaatgc tacccgtttt gcttcagttt 180

atgcatggaa tcgtaaacgt attagtaact gtgttgcaga ttatagcgtt ctgtataaca 240

gcgccagctt tagtaccttt aaatgttatg gtgtgagtcc gactaaactg aatgatctgt 300

gttttaccaa tgtttatgca gatagctttg ttattcgtgg tgatgaagtt cgccagattg 360

caccgggtca gaccggtaag attgccgatt ataattataa actgccggat gattttaccg 420

gttgtgtgat tgcctggaat tcaaataatc tggatagcaa agtgggtggt aattataatt 480

atctgtatcg tctgtttcgc aaaagcaatc tgaaaccgtt tgaacgtgat atttctaccg 540

aaatttatca ggcgggcagc acaccgtgta atggtgttaa aggttttaac tgttattttc 600

ctctgcagtc ttatggtttt cagccgacca acggtgttgg ttatcagccg tatcgcgttg 660

tggttctgag ttttgaactg ctgcatgccc cggcaacggt ttgtggtcct aaaaagtcaa 720

ccaatctggt taaaaacaaa tgtgtcaatt tctaagcggc cgc 763

<210> 9

<211> 223

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn

1 5 10 15

Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val

20 25 30

Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser

35 40 45

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

50 55 60

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

65 70 75 80

Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln

85 90 95

Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr

100 105 110

Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly

115 120 125

Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys

130 135 140

Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr

145 150 155 160

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

165 170 175

Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg Val

180 185 190

Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly

195 200 205

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

210 215 220

Claims

1. The gene of a novel coronavirus B.1.525 Nigeria mutant RBD is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 1.

2. A recombinant expression vector comprising a gene of the novel coronavirus B.1.525 Nigeria mutant RBD of claim 1.

3. A recombinant expression vector pcdna3.1+ comprising the gene of the novel coronavirus b.1.525 nigeria mutant RBD of claim 1.

4. A method for preparing a novel coronavirus b.1.525 nigeria mutant RBD, comprising the steps of:

(1) transforming a host cell with a recombinant expression vector containing a gene of the novel coronavirus b.1.525 nigeria mutant RBD of claim 1;

5. Use of the gene of the novel coronavirus B.1.525 Nigeria mutant RBD as claimed in claim 1 in the preparation of novel coronavirus vaccine.

6. The gene of a novel coronavirus B.1.525 Nigeria mutant RBD is characterized in that the nucleotide sequence of the gene is shown as SEQ ID NO. 6.

7. A recombinant expression vector comprising a gene of the novel coronavirus B.1.525 Nigeria mutant RBD of claim 6.

8. A recombinant expression vector pcDNA3.1+ comprising the gene of the novel coronavirus B.1.525 Nigeria mutant RBD of claim 6.

9. A method for preparing a novel coronavirus b.1.525 nigeria mutant RBD, comprising the steps of:

(1) transforming a host cell with a recombinant expression vector containing a gene of the novel coronavirus b.1.525 nigeria mutant RBD of claim 6;

10. Use of the gene of the novel coronavirus B.1.525 Nigeria mutant RBD as claimed in claim 6 in the preparation of novel coronavirus vaccine.