CN116096736A - Novel coronavirus RBD fusion proteins - Google Patents

Abstract

The invention relates to a novel coronavirus RBD fusion protein, an RBD protein dimer, a preparation method thereof and an application thereof in preventing SARS-CoV-2 infection of the novel coronavirus.

Description

Novel coronavirus RBD fusion proteins Technical Field

The invention relates to the field of biological medicine, in particular to a novel coronavirus RBD fusion protein, RBD protein dimer, and preparation and application thereof.

Background

Pneumonia caused by the novel coronavirus SARS-CoV-2 infection is a large-scale infection event in a plurality of countries worldwide, the coarse mortality rate is about 2.3%, the life health and life of human beings are greatly adversely affected, huge social panic is caused, and heavy loss is caused to economy. At present, no specific medicine is used for clinically curing the pneumonia caused by SARS-CoV-2 worldwide. Thus, there is an urgent need to develop effective and safe vaccines for protecting compromised populations.

By day 22 of 5 in 2020, 10 new crown vaccines have been developed in the world (5 from china) for clinical trials based on WHO statistics, and 114 have been developed for preclinical studies. The core principle of a vaccine is to let the immune system recognize the target virus in advance, in particular the structure specific to the target virus and functioning as a core, to generate a high quality immune response, thereby generating immunity thereto. The current strategies mainly include nucleic acid vaccines (RNA and DNA vaccines), adenovirus vector vaccines, recombinant protein vaccines, inactivated vaccines, attenuated virus vaccines and the like. Different types of vaccines have different characteristics and advantages and disadvantages. Nucleic acid vaccines, adenovirus vector vaccines rely on human expression of viral related proteins to generate an immune response, while recombinant protein vaccines, inactivated viral vaccines and attenuated viral vaccines use viral proteins directly. The recombinant protein vaccine is prepared through expressing partial functional gene of new coronavirus in cell or microbe and purifying. In principle, the recombinant protein vaccine has high safety, in particular to a related vaccine designed based on a novel coronavirus spike protein receptor binding domain RBD, the expression protein is controllable, and the risk of DNA and genome integration and the like is avoided theoretically, but the preparation process is complex, the technical difficulty is high, the immunogenicity is weak, and an adjuvant is needed to be added to improve the immunogenicity.

RBD derived from SARS-CoV-2 can induce highly potent antibody responses in immunized animals, including mice and horses (Xiaoyan Pan, pengfei Zhou, et al Immunoglobulin fragment F (ab') 2 against RBD potently neutralizes SARS-CoV-2 in vitro.Antiviral Research 2020). The induced antibody can neutralize novel coronavirus SARS-CoV-2 and inhibit virus infection of Vero-E6 cells. This suggests that RBD sequences in the raised protein of the novel coronavirus SARS-CoV-2 can induce highly potent neutralizing antibody responses and can be developed as an effective and safe subunit vaccine for the prevention of covd-19.

Disclosure of Invention

Novel coronavirus SARS-CoV-2 raised protein (S protein) contains two subunits S1 and S2. Wherein S1 mainly comprises a receptor binding domain (RBD, the sequence source is Genbank: QHR 63260.2), which can specifically bind to receptor-angiotensin converting enzyme ACE2 of target cells.

The present inventors found that the expression level and purity of the RBD fusion protein of the present invention were remarkably improved by using the cleavage site of RBD fusion protease of novel coronavirus SARS-CoV-2 and a tag that facilitates the formation of dimer. Further, the present inventors have found that, by fusing, for example, an Fc fragment containing NO cysteine in the hinge region or an Fc fragment containing NO hinge region (sequence shown as SEQ ID NO: 13), whether wild-type RBD or mutant RBD, the expression amount and purity of the RBD fusion protein are significantly improved, the proteolytic cleavage effect (e.g., improvement of the recovery rate of the target protein, reduction of the production of the hybrid protein) is improved, the proportion of the high polymer is reduced, and the obtained fusion protein can obtain a higher concentration of RBD dimer after proteolytic cleavage. The RBD protein dimer has better ACE2 binding activity. The fusion protein and the further formed RBD dimer can be used for preventing novel coronavirus infection vaccine or obtaining neutralizing antibodies for immunized animals and protecting people who are in initial contact with the novel coronavirus.

In particular, the invention relates to the following aspects:

1. the RBD fusion protein of the novel coronavirus SARS-CoV-2 comprises, in sequence, the RBD sequence of the novel coronavirus SARS-CoV-2 (preferably said RBD sequence comprises a cysteine positioned at position 220 according to the position numbering of the sequence set forth in SEQ ID NO:1 and having activity to bind to the human ACE2 receptor; more preferably the RBD sequence comprises an amino acid sequence set forth in any of SEQ ID NO: 1-8), a proteolytic cleavage site, and a tag facilitating dimer formation.

2. The novel coronavirus SARS-CoV-2 RBD fusion protein according to claim 1, wherein said tag facilitating dimer formation is selected from the group consisting of a leucine zipper (preferably a sequence not comprising HHHHHH in the sequence shown in SEQ ID NO:30 or 31) and an Fc fragment, wherein said Fc fragment does not comprise a cysteine of the hinge region (preferably said Fc fragment is an Fc fragment derived from human IgG, murine IgG or horse IgG, more preferably said Fc fragment does not comprise a hinge region, even more preferably said Fc fragment comprises an amino acid sequence as shown in any of SEQ ID NOs: 13-16, 29), preferably said leucine zipper is derived from a C-JUN or C-FOS protein, more preferably said leucine zipper is further attached at the C-terminus to a His, flag, C-myc or HA tag.

3. The novel coronavirus SARS-CoV-2 RBD fusion protein of claim 1 or 2, wherein said protease cleavage site and tag are linked by a linker peptide, preferably said linker peptide is a flexible peptide, more preferably said linker peptide is selected from the group consisting of SEQ ID NO:26 27 or 28.

4. The novel coronavirus SARS-CoV-2 RBD fusion protein of any of claims 1-3, wherein said protease is selected from the group consisting of thrombin, enterokinase, TEV protease, and HRC-3C protease; preferably, the amino acid sequence of the thrombin, enterokinase, TEV protease or HRC-3C protease cleavage site is as shown in SEQ ID NO: 17-20; more preferably, the amino acid sequence of the protease cleavage site is not duplicated by the amino acid sequence in the RBD sequence or Fc fragment.

5. The novel coronavirus SARS-CoV-2 RBD fusion protein of any of claims 1-4, wherein the N-terminus of said fusion protein further comprises a signal peptide, preferably the amino acid sequence of said signal peptide comprises the amino acid sequence as set forth in SEQ ID NO: 21-23.

6. Novel RBD dimer of coronavirus SARS-CoV-2, characterized by the amino acid sequence according to SEQ ID NO:1, the cysteines at positions 18 and 43, the cysteines at positions 61 and 114, the cysteines at positions 73 and 207, and the cysteines at positions 162 and 170, respectively, form an intra-chain disulfide bond, and an inter-chain disulfide bond is formed between the cysteines at position 220 of the two monomeric RBDs, preferably, the monomeric RBD sequence comprises the amino acid sequence as set forth in SEQ ID NO:1 and 5-8, more preferably, the two monomeric RBD sequences of the dimer are identical. Preferably, the RBD dimer is prepared by the method of 8 below.

7. A method for preparing the RBD fusion protein of novel coronavirus SARS-CoV-2 as claimed in any of claims 1-5 comprising sequentially ligating the RBD sequence of novel coronavirus SARS-CoV-2, a protease cleavage site and a tag facilitating dimer formation, preferably said protease cleavage site and said tag are linked by a linker peptide.

8. A method of preparing the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6, comprising cleaving the RBD fusion protein of the novel coronavirus SARS-CoV-2 of any of claims 1-5 with a protease, preferably the method further comprises purifying the RBD fusion protease cleavage product, preferably the purification comprises chromatography (e.g., affinity chromatography, ion exchange chromatography).

9. A polynucleotide encoding the RBD fusion protein of the novel coronavirus SARS-CoV-2 of any one of claims 1-5 or the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6.

10. A vector comprising the polynucleotide of claim 9.

11. A host cell comprising the polynucleotide of claim 9 or the vector of claim 10.

12. Vaccine, preferably for use in the prevention of infection by a novel coronavirus, characterized in that it comprises the RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any of claims 1-5, the RBD dimer of the novel coronavirus SARS-CoV-2 according to claim 6, or the polynucleotide according to claim 9.

13. The RBD fusion protein of novel coronavirus SARS-CoV-2 as claimed in any of claims 1-5, the RBD dimer of novel coronavirus SARS-CoV-2 as claimed in claim 6, or the polynucleotide as claimed in claim 9 for use in preventing infection of a human by novel coronavirus SARS-CoV-2, or for immunizing an animal, preferably a mammal, more preferably a horse, for obtaining an antiviral serum, preferably for use in preventing or treating infection of a human by novel coronavirus SARS-CoV-2.

14. A method for preventing or treating a novel coronavirus SARS-CoV-2 infection or obtaining neutralizing antibodies against the novel coronavirus SARS-CoV-2, comprising immunizing an animal, preferably a human or non-human mammal, more preferably a horse, with the RBD fusion protein of the novel coronavirus SARS-CoV-2 of any of claims 1-5, the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6, or the polynucleotide of claim 9 to obtain antiviral serum.

In some embodiments of the invention, the Fc fragment is an Fc fragment derived from human IgG, preferably SEQ ID NO: 13.

In some embodiments of the invention, the Fc fragment is an Fc fragment derived from horse IgG, preferably SEQ ID NO: 15.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

The terms referred to in the present invention have conventional meanings as understood by those skilled in the art. Where a term is used and/or is acceptable in the art, the definition of the term as used herein is intended to include all meanings as defined by two or more.

In determining the degree of sequence identity between two amino acid sequences, the skilled artisan may consider so-called "conservative" amino acid substitutions, which may generally be described as amino acid substitutions in which an amino acid residue is replaced by another amino acid residue having a similar chemical structure, and which have little or no effect on the function, activity, or other biological properties of the polypeptide. Such conservative amino acid substitutions are known in the art, for example, from WO 04/037999, GB-A-3357 768, WO 98/49185, WO 00/46383 and WO 01/09300; and such alternative (preferred) types and/or combinations may be selected based on the relevant teachings of WO 04/037999 and WO 98/49185 and other references cited therein.

A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, non-essential amino acid residues of an immunoglobulin polypeptide are preferably replaced with other amino acid residues from the same side chain family. In other embodiments, a series of amino acids may be replaced by a structurally similar series of amino acids, the latter differing in order and/or composition of the side chain family.

Non-limiting examples of conservative amino acid substitutions are provided in the following table, wherein a similarity score of 0 or higher indicates that there is a conservative substitution between the two amino acids.

	C	G	P	S	A	T	D	E	N	Q	H	K	R	V	M	I	L	F	Y	W
W	-8	-7	-6	-2	-6	-5	-7	-7	-4	-5	-3	-3	2	-6	-4	-5	-2	0	0	17
Y	0	-5	-5	-3	-3	-3	-4	-4	-2	-4	0	-4	-5	-2	-2	-1	-1	7	10
F	-4	-5	-5	-3	-4	-3	-6	-5	-4	-5	-2	-5	-4	-1	-0	1	2	9
L	-6	-4	-3	-3	-2	-2	-4	-3	-3	-2	-2	-3	-3	2	4	2	6
I	-2	-3	-2	-1	-1	0	-2	-2	-2	-2	-2	-2	-2	4	2	5

M	-5	-3	-2	-2	-1	-1	-3	-2	0	-1	-2	0	0	2	6
V	-2	-1	-1	-1	0	0	-2	-2	-2	-2	-2	-2	-2	4
R	-4	-3	0	0	-2	-1	-1	-1	0	1	2	3	6
K	-5	-2	-1	0	-1	0	0	0	1	1	0	5
H	-3	-2	0	-1	-1	-1	1	1	2	3	6
Q	-5	-1	0	-1	0	-1	2	2	1	4
N	-4	0	-1	1	0	0	2	1	2
E	-5	0	-1	0	0	0	3	4
D	-5	1	-1	0	0	0	4
T	-2	0	0	1	1	3
A	-2	1	1	1	2
S	0	1	1	1
P	-3	-1	6
G	-3	5
C	12

In some embodiments, the conservative substitution is preferably one in which one amino acid in the following groups (a) - (e) is replaced with another amino acid residue in the same group: (a) small aliphatic, nonpolar or weakly polar residues: ala, ser, thr, pro and Gly; (b) Polar, negatively charged residues and (uncharged) amides: asp, asn, glu and Gln; (c) polar, positively charged residues: his, arg and Lys; (d) large aliphatic, nonpolar residues: met, leu, ile, val and Cys; and (e) an aromatic residue: phe, tyr and Trp.

Particularly preferred conservative substitutions are as follows: substitution of Ala to Gly or Ser; arg is replaced by Lys; asn is replaced with gin or with His; asp is replaced by Glu; cys is replaced by Ser; gln is replaced by Asn; glu is replaced with Asp; gly to Ala or Pro; his is replaced with Asn or with Gln; lie is replaced with Leu or with Val; leu is replaced with Ile or with Val; lys is replaced with Arg, with gin or with Glu; met is replaced by Leu, tyr or Ile; phe to Met, leu to Tyr; substitution of Ser for Thr; thr to Ser; trp is replaced with Tyr; tyr is replaced with Trp; and/or Phe to Val, ile or Leu.

Drawings

FIG. 1 is a schematic representation of a recombinant fusion protein RBD-Fc.

FIG. 2 shows a non-reducing electrophoresis detection pattern of RBD-His SDS-PAGE. FIG. 2A, ni affinity chromatography sample, lanes 1-3, samples were collected from separate tubes for the same elution peak; FIG. 2B, cation exchange chromatography sample, lane 1, protein Marker, lanes 2-10, respectively, are different fractions eluting from low to high salt.

FIG. 3 shows an electrophoresis detection pattern of RBD-Fc SDS-PAGE. FIG. 3A, lane 1, RBD-TFc non-reduced; lane 2, rbd-YFc non-reduced; lane 3, RBD-TFc reduction; lane 4, rbd-YFc reduction; lane 5, rbd-eq igg1Fc non-reduced; lane 6, rbd-eq igg1Fc reduction; lane 7, rbd-eq igg4Fc non-reduced; lane 8, rbd-eq igg4Fc reduction; MK, protein Marker. FIG. 3B, lanes 1,RBD L455I F456V-YFc, non-reducing; lane 2, rbd g476s-YFc non-reducing; lane 3, rbd v483a-YFc non-reduced; lane 4, rbd s494p-YFc non-reduced; lanes 5,RBD L455I F456V-YFc reduced; lane 6, rbd g476s-YFc reduction; lane 7, rbd v483a-YFc reduction; lane 8, rbd s494p-YFc reduction; MK, protein Marker.

FIG. 4 shows a diagram of RBD-Fc HPLC-SEC detection.

FIG. 5 shows the detection of RBD-TFc and RBD-YFc by enzyme-cut SDS-PAGE non-reducing electrophoresis. Lane 1, before RBD-TFc cleavage; lane 2, after RBD-TFc cleavage; lane 3, RBD-YFc, before cleavage; lane 4, RBD-YFc after digestion.

FIG. 6 RBD of a non-labeled product _TFc And RBD _YFc Is to be identified. FIG. 6A, SDS-PAGE detection, lane 1, RBD _TFc Non-reduction; lane 2, RBD _YFc Non-reduction; lane 3, RBD _TFc Reducing; lane 4, RBD _YFc Reducing; FIG. 6B, HPLC-SEC detection diagram; FIG. 6C, mass Spectrometry molecular weight detection.

FIG. 7 shows the detection of the enzyme-cleaved SDS-PAGE of the RBD of the horse Fc tag. Lane 1, RBD-eq IgG1Fc before cleavage; lane 2, rbd-eq igg1Fc after cleavage; lane 3, rbd-eq igg4Fc pre-cleavage; lane 4, RBD-eq IgG4Fc after cleavage.

FIG. 8 RBD _eqG1Fc And (5) identification. FIG. 8A, SDS-PAGE, lane 1, RBD _eqG1Fc Non-reduction; lane 2, RBD _eqG1Fc Reducing; FIG. 8B, HPLC-SEC detection diagram; FIG. 8C, mass spectrum molecular weight detection plot.

FIG. 9 shows Biacore detection of binding of unlabeled products of different RBD-Fc to hACE2-Fc after cleavage and affinity chromatography.

FIG. 10 RBD _YFc Non-reducing SDS-PAGE and Coomassie brilliant blue staining patterns of the different fractions eluted by cation exchange chromatography.

FIG. 11 RBD _YFc Biacore assay of binding of dimer (A) and monomer (B) to hACE2-Fc, respectively.

FIG. 12 RBD _YFc Schematic of the structure of the dimer.

FIG. 13 RBD of unlabeled products of different RBD-Fc after cleavage and affinity chromatography _YFc9 ，RBD _YFc10 And RBD _YFc11 Is a non-reducing and reducing SDS-PAGE coomassie brilliant blue staining pattern.

Detailed Description

The following describes the embodiments of the present invention in detail with reference to the drawings. The present invention may be embodied in many other forms than those herein described and similarly modified by those skilled in the art without departing from the spirit of the invention, and it is therefore intended that the scope of the invention be limited only by the specific embodiments disclosed herein.

Example 1: fusion protein structural design

The RBD protein sequence is derived from Genbank: QHR63260.2 RBD gene fragments are obtained by total gene synthesis, constructed between the multiple cloning cleavage sites of eukaryotic expression vectors (such as pcDNA3.1 vector, invitrogen), and added with signal peptide (such as CD33 signal peptide, IL2 signal peptide, human albumin HSA signal peptide, etc.) at the N-terminal and protein tag (such as His, flag, HA, myc or Fc tag, etc.) at the C-terminal to facilitate purification. In addition, protease cleavage site encoding sequences are added between the RBD gene downstream and the tag gene upstream, proteases including, but not limited to, thrombin (Sigma), enterokinase (New England Biolabs), TEV protease (Invitrogen), or HRV3C protease (Novagen). Optionally, the protease cleavage site is linked to, for example, an Fc tag via a linker peptide. The fusion protein structure is specifically shown in figure 1. Specific sequence information for the constructs is shown in table 1.

RBD is a structural domain in S protein of SARS-CoV-2 novel coronavirus, and is positioned in 319-541 amino acid residue region (SEQ ID NO: 1) of whole S protein. There are studies reporting that RBDs of different regions are selected for expression alone or in fusion with Fc to obtain recombinant RBD proteins, such as selecting residues 319-545 of the S protein (SEQ ID NO: 2) for exogenous expression (literature: jingyun Yang, wei Wang, zimin Chen, et al A vaccine targeting the RBD of the S protein of SARS-CoV-2 induces protective immunity.Nature 2020.) or selecting residues 331-524 of the S protein (SEQ ID NO: 3) for exogenous expression (literature: hongjin Gu, qi Chen, guan Yang, et al adaptation of SARS-CoV-2 in BALB/c mice for testing vaccine efficacy.science 2020.), or selecting residues 319-537 of the S protein (SEQ ID NO: 4) for exogenous expression (literature: lianpan Dai, tianyi Zhen, kun Xu, et al A universal design of betacoronavirus vaccines against COVID-19,MERS and SARS.Cell 2020.). The construction of fusion Fc was performed for the RBD sequences described above for the different regions, see RBD-YFc, RBD-YFc7 and RBD-YFc8 of Table 1, wherein the RBD regions correspond to residues 319-545, 331-524 and 319-537, respectively, of the S protein.

Example 2: expression and purification of RBD proteins of different tags

Plasmid extraction was performed according to conventional plasmid extraction methods and used for chemical transfection of 293 (ATCC) or CHO-S (Gibco) cells. Transfected cells were incubated at 37℃with 5% CO ₂ And (3) suspending and shake culturing in a shaking table for 7-10 days. The supernatant was harvested by centrifugation at 3000 Xg and filtered through a 0.22 μm filter.

RBD-His feed liquid is harvested and subjected to Ni column affinity chromatography, the expression quantity is less than 1mg/L, and SDS-PAGE detection is carried out on the sample. As shown in FIG. 2A, the Ni column affinity chromatography is low in purity and impurity in sample, so that cation exchange chromatography is adopted for fine purification, low-salt (5 mM NaCl) hanging column high-salt (500 mM NaCl) linear elution is carried out on the sample, and different components eluted from low salt to high salt in the process are subjected to tube collection and SDS-PAGE detection, as shown in FIG. 2B, lane 2 is the highest-purity component, the band size is 50KD, the theoretical molecular weight of RBD-His is consistent, but the sample purification yield is extremely low and is not more than 20%. Similarly, the RBD-His1 expression liquid is subjected to the same Ni column and cation exchange chromatography purification process as described above, the purity of the obtained protein and the size of the band are the same, and the purification yield is not significantly different. The protein expression of RBD fusion His tag by using different signal peptides is not obviously different in expression level and purification yield, and the protein expression and yield are lower.

The C-terminal protein tag of RBD is replaced by other tags including but not limited to Flag tag, HA tag and C-Myc tag, corresponding RBD fusion tag proteins are RBD-Flag, RBD-HA and RBD-Myc respectively, and the constructed expression plasmid is transiently transfected to 293 or CHO-S cells and cultured in a suspension and vibration mode for 7-10 days, the expression level of the obtained feed liquid detection protein is similar to that of RBD-His, and the expression quantity is less than 1mg/L. After the corresponding affinity chromatography of the label is carried out, the yield of the obtained protein is not more than 20 percent. Thus, in the mammalian expression system, the C-terminal fusion of the short peptide protein tag of RBD has lower expression level and purification yield.

Protein expression feed solution of RBD fusion Fc tag was harvested and subjected to protein A affinity chromatography, and protein after one-step affinity purification was analyzed by SDS-PAGE, as shown in FIG. 3, with a band size of about 110KD, consistent with the theoretical molecular weight of RBD fusion Fc tag, and further tested for high polymer content by high performance size exclusion chromatography (HPLC-SEC), as shown in Table 2 and FIG. 4. It can be seen that the expression level and purity are remarkably improved after the RBD tag is replaced by Fc tag, and the expression level is remarkably improved after the horse IgG1Fc tag is adopted compared with that of human Fc tag. The RBD-Fc fusion protein is optimized, the HPLC-SEC purity of RBD-TFc before optimization is 89.43%, the purity of RBD-YFc after optimization is improved to 95.82%, and the proportion of high polymers is reduced. In addition, for the optimized RBD L455I F V-YFc, RBD G476S-YFc and RBD V483A-YFc, the high polymer proportion is obviously reduced, and the purity is improved by 5-10%; compared with RBD 494P-TFc before optimization, the optimized RBD 494P-YFc has obviously reduced high polymer proportion and improved purity by 5%. There was no significant difference between the expression levels and purities of RBD-YFc, RBD-YFc and RBD-YFc11 and RBD-YFc. There was no significant difference between the expression level and purity of RBD-epi 1Fc1 and RBD-epi 1 Fc. There was no significant difference between RBD-epi 4Fc1 and expression level and purity as compared to RBD-epi 4 Fc.

TABLE 2 expression levels and purities of different RBD-Fc

Construction article	Expression level	HPLC-SEC
RBD-TFc	80mg/L	89.43％
RBD-YFc	100mg/L	95.82％
RBD-YFc1	98mg/L	94.56％
RBD-YFc2	95mg/L	95.11％
RBD-YFc3	99mg/L	95.63％
RBD-YFc4	87mg/L	95.02％
RBD-YFc5	88mg/L	94.75％
RBD-YFc6	90mg/L	90.91％
RBD-YFc7	92mg/L	77.78％
RBD-YFc8	89mg/L	94.59％
RBD-YFc9	95mg/L	92.77％
RBD-YFc10	99mg/L	94.64％
RBD-YFc11	99mg/L	93.18％
RBD L455I F456V-YFc	108mg/L	60.70％
RBD G476S-YFc	94mg/L	94.41％
RBD V483A-YFc	97mg/L	95.28％
RBD S494P-TFc	43mg/L	55.00％
RBD S494P-YFc	54mg/L	60.66％
RBD-eqIgG1Fc	180mg/L	89.84％
RBD-eqIgG4Fc	92mg/L	78.13％

Example 3: label-free RBD preparation and authentication

In order to eliminate the nonspecific antibody induced by the Fc tag in vivo and the influence on RBD, the Fc tag is excised by protease to obtain unlabeled RBD protein, and the specific method is as follows: adding corresponding protease into RBD fusion Fc protein purified by affinity chromatography, incubating under specific conditions according to the requirements of the specification, and obtaining RBD protein by using a flow-through mode of protein A or protein G affinity chromatography, namely a label-free product. And detecting enzyme digestion effects of the pre-enzyme digestion and enzyme digestion samples of the RBD fusion Fc protein by SDS-PAGE, counting enzyme digestion purification recovery rate of the RBD protein, and detecting the final unlabeled RBD protein.

Preparation of ACE2 protein: a eukaryotic expression plasmid (the vector is pcDNA3.1) of a protein hACE2-Fc (SEQ ID NO: 24) fused with an Fc of human ACE2 extracellular domain is constructed, the plasmid is transiently transferred into 293 or CHO-S cells, the supernatant is obtained after 7-10 days of culture, and the hACE2-Fc protein is obtained by purification through protein A affinity chromatography.

As shown in lanes 2 and 4 of FIG. 5, the optimized construct RBD-YFc has significantly better cleavage efficiency than RBD-TFc prior to optimization. As shown in Table 3, the recovery rate of the optimized RBD-YFc, RBD-YFc1-RBD-YFc and RBD-eq IgG1Fc is obviously improved compared with that of RBD-TFc before optimization. And the enzyme digestion effect of the optimized RBD-YFc is better than that of RBD-TFc no matter the RBD is wild type or mutant type, and the recovery rate of the target protein can be improved by 20-30%. RBD for non-label products _TFc And RBD _YFc Identification was performed, both were identical in terms of purity (FIGS. 6A and 6B) and molecular weight (FIG. 6C), and the mass spectrum molecular weight detection RBD protein contained both monomer (31 KD) and dimer (62 KD) forms, which were identical to SDS-PAGE and SEC results. From the point of binding activity to hACE2-Fc (FIG. 9), the unlabeled product RBD _YFc Binding to hACE2-Fc is higher than RBD _TFc 。

TABLE 3 RBD recovery statistics

Construction article	RBD after Fc removal	Recovery rate of target protein
RBD-TFc	RBD TFc	58.6％
RBD-YFc	RBD YFc	82.3％
RBD-YFc1	RBD YFc1	78.8％
RBD-YFc2	RBD YFc2	79.3％
RBD-YFc3	RBD YFc3	77.5％
RBD-YFc4	RBD YFc4	77.4％
RBD-YFc5	RBD YFc5	78.9％
RBD-YFc6	RBD YFc6	79.2％
RBD-YFc7	RBD YFc7	78.9％
RBD-YFc8	RBD YFc8	77.36％
RBD-YFc9	RBD YFc9	76.9％
RBD-YFc10	RBD YFc10	80.1％
RBD-YFc11	RBD YFc11	79.3％
RBD-eqIgG1Fc	RBD eqG1Fc	70.7％

Note that: since each mg of RBD-Fc protein is composed of 0.5 mg of RBD and 0.5 mg of Fc tag, RBD target protein recovery = amount of RBD protein finally obtained/(amount of RBD-Fc protein x 50%).

RBD fusion Fc constructed using different protease cleavage sitesProteins such as RBD-YFc (thrombin), RBD-YFc1 (enterokinase), RBD-YFc2 (TEV protease) and RBD-YFc3 (HRV-3C protease), have better digestion effects than RBD-TFc (i.e., substitution of thrombin cleavage site in RBD-TFc with enterokinase cleavage site, TEV protease cleavage site, HRV-3C protease cleavage site) containing the corresponding cleavage site before optimization, and the recovery of RBD target protein is significantly improved, and the obtained label-free product RBD after cleavage, affinity chromatography and ion exchange chromatography _YFc 、RBD _YFc1 、RBD _YFc2 And RBD _YFc3 Has no significant difference in purity, molecular weight and activity of binding to hACE 2-Fc.

RBD fusion Fc proteins constructed by different regions of S protein, such as RBD-YFc, RBD-YFc7 and RBD-YFc8, are selected, and the unlabeled product RBD is obtained after enzyme digestion and affinity chromatography _YFc6 ，RBD _YFc7 And RBD _YFc8 Purity was determined by non-reducing SDS-PAGE as shown in Table 4. Wherein RBD _YFc6 Dimer ratio exceeding 80%, RBD _YFc7 Less than 30% dimer and more than 30% both high polymer (molecular weight greater than 90 kD) and monomer, RBD _YFc8 Only monomer. RBD-YFc 9-11 different from the connecting peptide of RBD-YFc, and obtaining a label-free product RBD after enzyme digestion and affinity chromatography _YFc9 、RBD _YFc10 And RBD _YFc11 All had approximately 90% dimer component (FIG. 13).

TABLE 4 non-reducing SDS-PAGE to check purity of RBD unlabeled products

RBD non-label product	High polymer ratio	Dimer ratio	Monomer ratio
RBD
TFc	0	65％	35％
RBD
YFc	0	90％	10％
RBD
YFc6	0	81％	19％
RBD YFc7	31％	29％	40％
RBD
YFc8	0	0	100％
RBD YFc9	O	87％	13％
RBD
YFc10	0	88％	12％
RBD
YFc11	0	89％	11％

Note that: RBD (radial basis function) _YFc7 And RBD _YFc8 None comprise a sequence according to SEQ ID NO;1 cysteine at position 220

As shown in lanes 2 and 4 of FIG. 7, the cleavage efficiency of the construct RBD-eqIgG1Fc was significantly better than that of RBD-eqIgG4Fc, and as shown in FIG. 8 and FIG. 9, RBD was obtained after RBD-eqIgG1Fc cleavage _eqG1Fc From the standpoint of purity, molecular weight and hACE2-Fc binding activity, it is bound to RBD _YFc And is highly uniform. RBD obtained after RBD-eq IgG4Fc cleavage _eqG4Fc Also having a dimer protein with a molecular weight of 62kD and a monomer with a molecular weight of 31kD, biacore assay and RBD _eqG1Fc Has uniform activity.

Example 4: detection of binding Activity of RBD protein and human ACE2

There are a large number of literature reports that the SARS-CoV-2 novel coronavirus RBD has high binding activity to human ACE2 receptor.

RBD (RBD) of label-free product _YFc (i.e., cleavage of the tag by protease) further by cation exchange chromatography (e.g., capto SP ImpRes, packing GE Co.) with a linear gradient of high salt (500 mM NaCl) and SDS-PAGE detection of the eluted fractions, as shown in FIG. 10, allows efficient separation of monomers from dimers. Pooled lanes 2-4 were collected as RBD _YFc Monomeric fractions were pooled and lanes 7-12 were collected as RBD _YFc The dimer component is subjected to HPLC-SEC purity detection, and the purity of the monomer and the dimer component respectively exceeds 97 percent.

RBD is respectively combined with _YFc Monomer and dimer Components undergo binding Activity with hACE2-Fc protein Biacore detection, as shown in FIG. 11, RBD _YFc Dimer has affinity of K with hACE2-Fc _D =0.192 nM, and RBD _YFc Affinity of monomer for hACE2-Fc was K _D ＝15.60nM。RBD _YFc The ability of the dimer to bind hACE2-Fc is significantly greater than RBD _YFc Ability of the monomer to bind hACE 2-Fc.

RBD _YFc The amino acid residue codes of the monomers are shown in Table 5.RBD (radial basis function) _YFc The dimer was analyzed by mass spectrometry after Chymotrypsin (Sigma) enzymatic hydrolysis and found that the cysteine at position 220 of both monomers formed a pair of interchain disulfide bonds. This indicates RBD _YFc Dimer is two RBDs _YFc The monomers are covalently linked by a pair of interchain disulfide bonds formed by cysteine 220.

TABLE 5 RBD _YFc Amino acid residue coding of monomer (SEQ ID NO: 1)

In summary, when the RBD region (such as residues 319-541) of the S protein of SARS-CoV-2 virus is selected, a C-terminal fusion is advantageous to the formation of dimer (such as Fc tag derived from IgG), preferably a protease cleavage site is added between RBD and tag and the connecting peptide and hinge region between protease cleavage site and tag are optimized, and the RBD-cleavage site-tag fusion protein is constructed, which has good expression level and purity in mammalian cells, and after protease cleavage, affinity chromatography and ion exchange chromatography, a RBD dimer component with high recovery rate can be obtained, and the dimer component has higher binding activity with hACE 2-Fc. The molecular weight of the RBD dimer is 62kD, which is formed by covalent linkage between two RBD monomers through a pair of interchain disulfide bonds (specifically disulfide bonds are formed between cysteines at position 220 shown in the code of table 5), and four pairs of interchain disulfide bonds exist for each RBD monomer (see fig. 12), respectively: (1) disulfide bonds between cysteine 18 and cysteine 43, (2) disulfide bonds between cysteine 61 and cysteine 114, (3) disulfide bonds between cysteine 73 and cysteine 207, and (4) disulfide bonds between cysteine 162 and cysteine 170, the sequence codes being shown in Table 5.

RBD dimer components obtained by purification, cleavage, affinity chromatography and ion exchange chromatography of RBD-mIgG2aFc, RBD-JUN and RBD-FOS (Table 2) have the same sequence and structure as the RBD dimer shown in FIG. 12 and binding activity to hACE 2-Fc.

Claims (14)

1. The RBD fusion protein of the novel coronavirus SARS-CoV-2 comprises, in sequence, the RBD sequence of the novel coronavirus SARS-CoV-2 (preferably said RBD sequence comprises a cysteine positioned at position 220 according to the position numbering of the sequence set forth in SEQ ID NO:1 and having activity to bind to the human ACE2 receptor; more preferably the RBD sequence comprises an amino acid sequence set forth in any of SEQ ID NO: 1-8), a proteolytic cleavage site, and a tag facilitating dimer formation.
2. The novel coronavirus SARS-CoV-2 RBD fusion protein according to claim 1, wherein said tag facilitating dimer formation is selected from the group consisting of a leucine zipper (preferably a sequence not comprising hhhhhhh in SEQ ID NO:30 or 31) and an Fc fragment, wherein said Fc fragment does not comprise a cysteine of a hinge region (preferably said Fc fragment is an Fc fragment derived from human IgG or horse IgG, more preferably said Fc fragment does not comprise a hinge region, even more preferably said Fc fragment comprises an amino acid sequence as set forth in any of SEQ ID NOs: 13-16, 29), preferably said leucine zipper is derived from a C-JUN or C-FOS protein, more preferably said leucine zipper is further attached at the C-terminus to a His, flag, C-myc or HA tag.
3. The novel coronavirus SARS-CoV-2 RBD fusion protein of claim 1 or 2, wherein said protease cleavage site and tag are linked by a linker peptide, preferably said linker peptide is selected from the group consisting of SEQ ID NO:26 27 or 28.
4. The novel coronavirus SARS-CoV-2 RBD fusion protein of any of claims 1-3, wherein said protease is selected from the group consisting of thrombin, enterokinase, TEV protease, and HRC-3C protease; preferably, the amino acid sequence of the thrombin, enterokinase, TEV protease or HRC-3C protease cleavage site is as shown in SEQ ID NO: 17-20; more preferably, the amino acid sequence of the protease cleavage site is not duplicated by the amino acid sequence in the RBD sequence or Fc fragment.
5. The novel coronavirus SARS-CoV-2 RBD fusion protein of any of claims 1-4, wherein the N-terminus of said fusion protein further comprises a signal peptide, preferably the amino acid sequence of said signal peptide comprises the amino acid sequence as set forth in SEQ ID NO: 21-23.
6. Novel RBD dimer of coronavirus SARS-CoV-2, characterized by the amino acid sequence according to SEQ ID NO:1, the cysteines at positions 18 and 43, the cysteines at positions 61 and 114, the cysteines at positions 73 and 207, and the cysteines at positions 162 and 170, respectively, form an intra-chain disulfide bond, and an inter-chain disulfide bond is formed between the cysteines at position 220 of the two monomeric RBDs, preferably, the monomeric RBD sequence comprises the amino acid sequence as set forth in SEQ ID NO:1 and 5-8, more preferably, the two monomeric RBD sequences of the dimer are identical.
7. A method for preparing the RBD fusion protein of novel coronavirus SARS-CoV-2 as claimed in any of claims 1-5 comprising sequentially ligating the RBD sequence of novel coronavirus SARS-CoV-2, a protease cleavage site and a tag facilitating dimer formation, preferably said protease cleavage site and said tag are linked by a linker peptide.
8. A method of preparing the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6, comprising cleaving the RBD fusion protein of the novel coronavirus SARS-CoV-2 of any of claims 1-5 with a protease, preferably the method further comprises purifying the RBD fusion protease cleavage product, preferably the purification comprises chromatography (e.g., affinity chromatography, ion exchange chromatography).
9. A polynucleotide encoding the RBD fusion protein of the novel coronavirus SARS-CoV-2 of any one of claims 1-5 or the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6.
10. A vector comprising the polynucleotide of claim 9.
11. A host cell comprising the polynucleotide of claim 9 or the vector of claim 10.
12. Vaccine, preferably for use in the prevention of infection by a novel coronavirus, characterized in that it comprises the RBD fusion protein of the novel coronavirus SARS-CoV-2 according to any of claims 1-5, the RBD dimer of the novel coronavirus SARS-CoV-2 according to claim 6, or the polynucleotide according to claim 9.
13. The RBD fusion protein of novel coronavirus SARS-CoV-2 as claimed in any of claims 1-5, the RBD dimer of novel coronavirus SARS-CoV-2 as claimed in claim 6, or the polynucleotide as claimed in claim 9 for use in preventing infection of a human by novel coronavirus SARS-CoV-2, or for immunizing an animal, preferably a mammal, more preferably a horse, for obtaining an antiviral serum, preferably for use in preventing or treating infection of a human by novel coronavirus SARS-CoV-2.
14. A method for preventing or treating a novel coronavirus SARS-CoV-2 infection or obtaining neutralizing antibodies against the novel coronavirus SARS-CoV-2, comprising immunizing an animal, preferably a human or non-human mammal, more preferably a horse, with the RBD fusion protein of the novel coronavirus SARS-CoV-2 of any of claims 1-5, the RBD dimer of the novel coronavirus SARS-CoV-2 of claim 6, or the polynucleotide of claim 9 to obtain antiviral serum.

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