CN111732664A - Novel coronavirus recombinant protein, rabbit-human chimeric antibody, preparation method and application thereof - Google Patents

Novel coronavirus recombinant protein, rabbit-human chimeric antibody, preparation method and application thereof Download PDF

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CN111732664A
CN111732664A CN202010637858.4A CN202010637858A CN111732664A CN 111732664 A CN111732664 A CN 111732664A CN 202010637858 A CN202010637858 A CN 202010637858A CN 111732664 A CN111732664 A CN 111732664A
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于在江
袁志波
赵荣茂
卢帅
朱琳
孙宇天
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Taizhou Leiling Baiao Biotechnology Co ltd
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Abstract

The invention provides a novel coronavirus recombinant protein, a rabbit-human chimeric antibody, a preparation method and application thereof, wherein the amino acid sequence of the novel coronavirus recombinant protein is expressed from N end to C end by SEQ ID NO: 1. SEQ ID NO: 2 are obtained by connecting polypeptide fragments in sequence. The novel coronavirus rabbit-human chimeric antibody is characterized in that F (ab') 2 of a rabbit anti-polyclonal antibody of a novel coronavirus recombinant protein is coupled with an Fc fragment of a human IgG antibody through biotin and streptavidin, the novel coronavirus rabbit-human chimeric antibody can provide safe and effective positive quality control substances for an existing novel coronavirus antibody detection kit, and the chimeric antibody is strong in specificity, good in stability, simple in preparation method, capable of realizing accurate replication and capable of being popularized in a large scale.

Description

Novel coronavirus recombinant protein, rabbit-human chimeric antibody, preparation method and application thereof
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a novel coronavirus recombinant protein, a rabbit-human chimeric antibody, and a preparation method and application thereof.
Background
In order to effectively control the further spread of the virus and enable infected persons to be treated in time, people in suspected cases or serious regions of epidemic situations need to be subjected to virus detection as soon as possible. The existing method for detecting the neocoronary pneumonia mainly comprises a nucleic acid detection method and an immunological detection method, wherein the nucleic acid detection method is used for detecting a virus RNA genome and comprises the technologies of gene sequencing, fluorescent quantitative PCR, micro-droplet digital PCR, gene chips, loop-mediated isothermal amplification and the like. The immunological detection method is used for detecting virus antigen or specific antibody produced by human body, and includes colloidal gold test paper, enzyme-linked immunosorbent assay, chemiluminescence immunoassay and other techniques. Previously, nucleic acid detection methods that react promptly and more rapidly were once considered the "gold standard" for clinical diagnosis and large-scale high-risk population screening. However, researches show that after the novel coronavirus invades a human body for 3-5 days, specific IgM antibodies of the novel coronavirus are more positive, then the specific IgG antibodies of the novel coronavirus begin to change from negative to positive, and the titer of the novel coronavirus in a recovery phase is greatly increased compared with that in an acute phase, so that an immunodetection method mainly for detecting IgM and IgG of a patient is an important supplement of a nucleic acid detection method. In the diagnosis and treatment plan for new coronary pneumonia (trial seventh edition) published by Wei Jian Wei Jian Wei in 3/4 days in China, a novel serum coronavirus specific antibody is newly added in the original diagnosis standard as one of the clinical diagnosis standards. The combination of the nucleic acid detection method and the immunological detection method can effectively improve the detection rate and the diagnosis confirmation rate of patients, particularly suspected cases.
For immunological detection, because many conditions in the experimental process affect the detection result and cause the deviation of the result, quality control products are required to be adopted to strictly control the detection quality during detection. The positive quality control substance can be a confirmed known positive sample or an antibody protein constructed in vitro, and the like, and the preferred principle is that the characteristics of the control sample are as close as possible to the clinical sample. The positive quality control products used by the currently developed diagnostic reagent for detecting the antibody mainly have 2 sources, one is to collect serum of a patient with a disease, and the positive quality control products are inactivated for use, generally IgM in the early stage of the disease is positive, middle and late stages are negative, IgG is negative in the early stage, and middle and late stages are positive, but titer of IgM and IgG cannot be guaranteed to be unchanged, the sources are limited, and the inactivated effect cannot be verified, so that the potential infectivity is prohibited by the state; in order to overcome the defects of the traditional quality control material, the genetic engineering chimeric antibody consisting of the variable region of the murine antibody and the constant region of the human IgM/IgG antibody is used as the quality control material of the IgM/IgG antibody of a specific pathogen, and the quality control material overcomes the defects of the traditional quality control material to a certain extent, but the preparation of the quality control material relates to a hybridoma technology and the like, is complex and needs a longer preparation period.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a novel coronavirus recombinant protein, a nucleotide sequence for coding the recombinant protein, an expression vector containing the nucleotide sequence, a host cell, a monoclonal antibody, a rabbit-human chimeric antibody, a preparation method and application thereof. Firstly, the dominant epitope fragment of the N protein of the novel coronavirus is connected with the receptor binding region fragment of the S protein to obtain recombinant protein with strong immunogenicity, a rabbit-derived novel coronavirus polyclonal antibody is obtained by immunizing a rabbit with the recombinant protein, and then F (ab') 2 obtained by enzyme digestion of the rabbit-derived novel coronavirus polyclonal antibody is coupled with a human IgG antibody Fc fragment through biotin and streptavidin to obtain a novel coronavirus rabbit-human chimeric antibody.
In order to solve the above problems, an aspect of the present invention provides a novel recombinant coronavirus protein whose amino acid sequence is represented by SEQ ID NO: 1. SEQ ID NO: 2 are obtained by connecting polypeptide fragments in sequence. The invention firstly uses software BepipPre to predict the antigen epitope of the N protein amino acid sequence of the novel coronavirus (SARS-CoV-2), designs 3 sections of antigen epitopes which are NP1(1-165aa), NP2(140-332aa) and NP3(245-419aa) of the N protein, namely the amino acid at 1-165 position of the N protein, the amino acid at 140-332 position of the N protein and the amino acid at 245-419 position of the N protein, and finds out the amino acid fragment with stronger affinity with the antibody through experiments, NP1 (1-165), wherein the amino acid sequence is shown as SEQ ID NO: 1, then connecting the amino acid segment with a receptor binding region of an S protein of the novel coronavirus (299-position 600 amino acid of the S protein, and the amino acid sequence is shown as SEQ ID NO: 2) through a polypeptide segment to obtain the novel coronavirus recombinant protein which has stronger affinity with an antibody and strong immunogenicity. Selecting dominant antigen epitopes of two structural antigens of the novel coronavirus N and S, and performing efficient recombinant expression to avoid the operation biological safety risk of directly immunizing animals by adopting the virus, and simultaneously generating antibodies by immunizing animals to reflect the humoral immune response level of the novel coronavirus during infection more truly; the dominant antigen epitope of the N protein and the S protein are connected and recombined to obtain recombinant protein, the recombinant protein is used as immunogen to immunize animals to obtain antibodies aiming at two proteins of the virus, and the variable region of the antibodies is used for preparing a positive quality control substance to reduce the probability of missed detection.
The novel coronavirus referred to in this application refers to the virus named SARS-CoV-2 by the world health organization.
The nucleotide sequence of SEQ ID NO: 1 (165 amino acids) as follows:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGT。
the nucleotide sequence of SEQ ID NO: 2 (302 amino acids) as follows:
TKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP。
the amino acid sequence of the polypeptide fragment connecting the NP1 and the RBD is not particularly limited, so long as the polypeptide fragment can connect the amino acid fragments at two ends, the polypeptide fragment can be obtained by the market, and products of different manufacturers and models do not influence the realization of the technical effect of the invention; preferably, the polypeptide fragment is a soluble polypeptide; further preferably, to facilitate correct folding of the two amino acid fragments, the amino acid sequence of the polypeptide fragment is optimized, and preferably: GSESGSLSGSDSGWGSGSGHGGKG are provided. Preferably, the amino acid sequence of the novel coronavirus recombinant protein is represented by SEQ ID NO: 3, respectively.
The nucleotide sequence of SEQ ID NO: 3 (491 amino acids):
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTGSESGSLSGSDSGWGSGSGHGGKGTKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP。
another aspect of the present invention provides a nucleotide sequence encoding the above-described novel recombinant coronavirus protein.
Preferably, the nucleotide sequence encoding the novel coronavirus recombinant protein is shown as SEQ ID NO: 4, and the sequence shown in SEQ ID NO: 4 has the following sequence structure:
atgagcgataacggtccgcaaaaccagcgtaacgcgccgcgtattaccttcggtggtccgagcgatagcaccggtagcaaccaaaacggcgaacgtagcggtgcgcgtagcaaacaacgtcgtccgcaaggtctgccgaacaacaccgcgagctggtttaccgcgctgacccagcacggtaaagaagacctgaagttcccgcgtggtcagggcgtgccgattaacaccaacagcagcccggacgatcaaattggttattaccgtcgtgcgacccgtcgtatccgtggcggtgatggcaaaatgaaagatctgagcccgcgttggtacttctattacctgggtaccggcccggaagcgggtctgccgtacggtgcgaacaaggatggcatcatctgggttgcgaccgaaggtgcgctgaacaccccgaaagaccacattggtacccgtaacccggcgaacaacgcggcgattgttctgcagctgccgcaaggtaccggttctgaatctggttctctgtctggttctgactctggttggggttctggttctggtcacggtggtaaaggtaccaagtgcactctgaagtctttcaccgtggagaagggcatctaccagactagcaacttcagggtgcagccaaccgaatctatcgtcagattccccaacatcactaacctgtgcccattcggagaggtcttcaacgccaccagattcgcttccgtgtacgcctggaacaggaagagaatcagcaactgcgtcgctgactactctgtgctgtacaacagcgcctctttctcaaccttcaagtgctacggcgtgagccctactaagctgaacgacctgtgcttcaccaacgtctacgccgactctttcgtgatcaggggagacgaggtcagacagatcgctcccggccagactggaaagatcgccgactacaactacaagctgccagacgacttcaccggctgcgtcatcgcttggaactcaaacaacctggactccaaagtgggtggcaactacaactacctgtaccgcctgttccgtaagagcaacctgaagcctttcgagagggacatctcaactgaaatctaccaggctggttccaccccctgcaacggtgtcgagggcttcaactgctacttcccactgcaatcttacggtttccagcctactaacggtgtgggctaccagccctacagagtggtcgtgctgtcattcgaactgctgcacgccccagctactgtgtgcggtcctaagaagtccaccaacctggtcaagaacaagtgcgtgaacttcaacttcaacggcctgaccggaactggtgtcctgaccgagtcaaacaagaagttcctgccattccagcagttcggaagggacatcgctgacaccactgacgctgtgcgcgaccctcagaccctggaaatcctggacatcactccttgcagcttcggaggtgtctctgtgatcacccct。
in another aspect, the present invention provides a recombinant vector comprising the above-mentioned nucleotide sequence encoding the above-mentioned novel coronavirus recombinant protein. In particular, the recombinant vector may be a plasmid, such as Pet32a, Pet28 a.
The invention also provides a novel coronavirus polyclonal antibody, which is obtained by taking the novel coronavirus recombinant protein as an immunogen to immunize an animal, and separating and purifying.
Wherein, the animal can be rabbit, mouse, pig, etc., preferably, the animal is rabbit.
In another aspect of the present invention, there is provided a method for preparing a novel rabbit-human chimeric antibody against coronavirus, comprising the steps of:
coupling an F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody with activated biotin to obtain a biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment compound, wherein the rabbit-derived novel coronavirus antibody is a polyclonal antibody obtained by immunizing a rabbit with the novel coronavirus recombinant protein;
coupling the Fc fragment of the human IgG antibody or the IgM antibody with avidin or streptavidin by adopting a covalent crosslinking method to obtain an avidin/streptavidin-human IgG/IgM antibody Fc fragment compound;
coupling biotin of the biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex with avidin or streptavidin of the avidin/streptavidin-human IgG/IgM antibody Fc fragment complex to obtain a novel coronavirus rabbit-human chimeric antibody;
or coupling the F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody with avidin or streptavidin by a covalent crosslinking method to obtain an avidin/streptomycin avidin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment compound, wherein the rabbit-derived novel coronavirus antibody is a polyclonal antibody obtained by immunizing rabbits with the novel coronavirus recombinant protein;
coupling the Fc fragment of the human IgG antibody or the IgM antibody with activated biotin to obtain a biotin-human IgG/IgM antibody Fc fragment compound;
and coupling the avidin or streptavidin of the avidin/streptavidin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex with the biotin of a biotin-human IgG/IgM antibody Fc fragment complex to obtain the novel coronavirus rabbit-human chimeric antibody.
Wherein, biotin is a kind of sulphureous water-soluble vitamin, also known as vitamin H or coenzyme R, have imidazole ketone ring suitable for binding with avidin and thiophene ring binding with protein macromolecules such as antibody, etc., biotin side chain end carboxyl can make activation biotin with various active groups after chemical modification, and activation biotin is apt to couple with corresponding groups in the molecule such as various antigens, antibodies, etc. and form the biotinylation marker, and does not influence the immunological competence of antigen, antibody, for example, after NHS (N-hydroxy succinimide ester) activation, activation biotin can couple with amino in the protein; after MPB activation, activated biotin can be coupled to sulfhydryl groups in proteins. Avidin is a glycoprotein that binds closely to biotin molecules; streptavidin, referred to as SA for short, is a protein with biological properties similar to those of avidin, and is secreted by streptomyces and can also be specifically bound to biotin. The avidin and the streptavidin can generate covalent crosslinking reaction with the protein or the amino acid fragment under the action of the crosslinking agent, so that the avidin or the streptavidin and the protein are crosslinked. Wherein the crosslinking agent can be selected from aldehydes, carbodiimides, diepoxides, cyanoiso-acid salts, etc. Biotin and avidin or streptavidin can carry out coupling, and the combination between the two has strong specificity and extremely strong affinity, the combination between the two is irreversible reaction, and the combination has extremely strong stability once combined, so that the chimeric component does not need to be activated or modified, and the steric hindrance interference can be avoided.
The F (ab') 2 fragment obtained by pepsin hydrolysis of the antibody consists of 2 Fab and hinge regions, retains the biological activity of binding with corresponding antigen, and can simultaneously bind with two antigen epitopes. While the Fc fragment has no antigen binding activity but species specificity. The rabbit-human chimeric antibody of the novel coronavirus is obtained by the coupling of biotin and an amino acid fragment, the covalent crosslinking of avidin, streptavidin and the amino acid fragment, and the specific coupling of biotin, avidin and streptavidin, wherein the rabbit-derived novel coronavirus antibody F (ab') 2 fragment is chimeric with a human IgG/IgM antibody Fc fragment to obtain the chimeric antibody which has the activity of the novel coronavirus and the species specificity of human Fc. The chimeric antibody is used for detecting a positive quality control substance of a novel coronavirus antibody, avoids potential infectivity caused by directly adopting the inactivation of the serum of a patient with disease as the positive quality control substance, and simultaneously is compared with a genetic engineering chimeric antibody which consists of a murine antibody variable region and a human IgG/IgM antibody constant region and is used as the quality control substance of a specific pathogen IgG/IgM antibody.
Preferably, the method for preparing a novel rabbit-human chimeric antibody against coronavirus comprises the following steps:
s1, carrying out enzyme digestion on the rabbit-derived novel coronavirus antibody by adopting pepsin to obtain an F (ab') 2 fragment of the rabbit-derived novel coronavirus antibody, wherein the rabbit-derived novel coronavirus antibody is a polyclonal antibody obtained by immunizing a rabbit by adopting the novel coronavirus recombinant protein;
s2, carrying out enzyme digestion on the human IgG antibody or the IgM antibody by using papain to obtain an Fc fragment of the human IgG antibody or the IgM antibody;
s3, NHS-Biotin is dissolved in solvent, F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody is added for coupling reaction, the concentration of the F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody is 2-10mg/mL, the ratio of the F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody to NHS-Biotin is 1mg (0.8-2)
Figure 100002_DEST_PATH_IMAGE001
10-7Obtaining a biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment compound;
and S4, mixing NHS and EDC serving as cross-linking agents with avidin or streptavidin, adding the Fc fragment of the human IgG or IgM antibody, mixing, and carrying out covalent cross-linking reaction, wherein the mass ratio of the Fc fragment of the human IgG or IgM antibody to the avidin or streptavidin is 3: (4-6), obtaining an avidin/streptavidin-human IgG/IgM antibody Fc fragment complex;
s5, the biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex is combined with the avidin/streptavidin-human IgG/IgM antibody Fc fragment complex to obtain a mixture of (0.5-2): 1, and carrying out coupling reaction to obtain the novel coronavirus rabbit-human chimeric antibody.
When the coupling reaction is carried out between the activated biotin and the amino acid fragments, the activated biotin and the amino acid fragments have the proportion, so that the quantity of biotin molecules marked on each amino acid fragment can be controlled within a proper range, the activated biotin and the amino acid fragments can be well coupled, and the activity of a coupled product can be prevented from being influenced; NHS is N-hydroxysuccinimide, EDC is 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, NHS and EDC are used as cross-linking agents, the cross-linking effect of the avidin/streptavidin and the human IgG/IgM antibody Fc fragment is best, and the two agents are non-toxic and good in biocompatibility.
Further preferably, the method for preparing the novel coronavirus rabbit-human chimeric antibody comprises the following steps:
s1, dialyzing the rabbit-derived novel coronavirus antibody in 0.2M sodium acetate buffer solution at the pH value of 4.0 for overnight, mixing pepsin with the purified rabbit-derived novel coronavirus antibody solution, carrying out enzyme digestion reaction at 37 ℃ for 6 hours, wherein the mass ratio of the pepsin to the rabbit-derived novel coronavirus antibody is 1:20, adding 3M Tris to adjust the pH value to 8.0, separating by a ProteinA/G column, carrying out ultrafiltration concentration on an effluent part, and passing through a S200 molecular sieve column to obtain a purified rabbit-derived novel coronavirus antibody F (ab') 2 fragment;
s2, dialyzing the human IgG antibody or IgM antibody in a buffer solution containing 0.2Mtris, 0.15Mnacl, 1mM EDTA and 1mM DTT at the pH value of 8.0 for overnight, mixing papain with the purified human IgG antibody or IgM antibody solution, carrying out enzyme digestion reaction at 37 ℃ for 6 hours, wherein the mass ratio of the papain to the human IgG antibody or IgM antibody is 1:20, separating the mixture by a ProteinA/G column, eluting a binding part by 0.1M glycine at the pH value of 2.7, carrying out ultrafiltration concentration, and passing the binding part by a S200 molecular sieve column to obtain the Fc fragment of the purified human IgG antibody or IgM antibody;
s3, dissolving and preparing 10mM NHS-Biotin by using dimethyl sulfoxide as a solvent, regulating the concentration of the rabbit-derived novel coronavirus antibody F (ab ') 2 fragment after PBS dialysis and purification to be 2mg/mL, mixing the rabbit-derived novel coronavirus antibody F (ab') 2 fragment and the NHS-Biotin according to the proportion of adding 27 mu L of 10mM NHS-Biotin into 1mL of the rabbit-derived novel coronavirus antibody F (ab ') 2 fragment, reacting for 1 hour at room temperature, and dialyzing overnight by using 2L PBS to obtain a Biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment compound;
s4, taking 10mg of streptavidin, adding NHS 8mg and EDC13.3mg, stirring for 24 hours at room temperature, then dropwise adding 6mg of Fc fragment of human IgG antibody or IgM antibody, stirring for 3 hours at room temperature, and dialyzing the product overnight at 4 ℃ with 0.01M PBS to obtain a streptavidin-human IgG/IgM antibody Fc fragment compound;
s5, mixing the biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex with the streptavidin-human IgG/IgM antibody Fc fragment complex and the like, and incubating for 2 hours at room temperature to obtain the novel coronavirus rabbit-human chimeric antibody.
In another aspect, the present invention provides a novel rabbit-human chimeric coronavirus antibody prepared by the above method for preparing a novel rabbit-human chimeric coronavirus antibody.
The invention also provides application of the novel coronavirus rabbit-human chimeric antibody in preparation of a positive quality control product of the novel coronavirus detection kit.
In another aspect, the invention provides a novel coronavirus detection kit, which comprises the novel coronavirus rabbit-human chimeric antibody.
Compared with the prior art, the invention has the following beneficial effects:
1. the novel coronavirus recombinant protein is obtained by connecting the dominant epitope of the novel coronavirus N protein with the receptor binding region of the S protein through water-soluble polypeptide which is favorable for correct folding of two sections of amino acid fragments, and has stronger affinity with an antibody and strong immunogenicity; selecting dominant antigen epitopes of two structural antigens of the novel coronavirus N and S, and performing efficient recombinant expression to avoid the operation biological safety risk of directly immunizing animals by adopting the virus, and simultaneously generating antibodies by immunizing animals to reflect the humoral immune response level of the novel coronavirus during infection more truly; connecting and recombining the dominant antigen epitopes of the N protein and the S protein to obtain a recombinant protein, immunizing an animal by taking the recombinant protein as an immunogen to obtain antibodies aiming at two proteins of the virus, and preparing a positive quality control substance by utilizing the variable region of the antibodies to reduce the probability of missed detection;
2. the rabbit-human chimeric antibody of the novel coronavirus is obtained by the coupling of biotin and an amino acid fragment, the covalent crosslinking of avidin, streptavidin and the amino acid fragment, and the specific coupling of biotin, avidin and streptavidin, wherein the rabbit-derived novel coronavirus antibody F (ab') 2 fragment is chimeric with a human IgG/IgM antibody Fc fragment to obtain the chimeric antibody which has the activity of the novel coronavirus and the species specificity of human Fc. The chimeric antibody is used for detecting a positive quality control substance of a novel coronavirus antibody, avoids potential infectivity caused by directly adopting the inactivation of the serum of a patient with disease as the positive quality control substance, and simultaneously is compared with a genetic engineering chimeric antibody which consists of a murine antibody variable region and a human IgG/IgM antibody constant region and is used as the quality control substance of a specific pathogen IgG/IgM antibody.
Drawings
FIG. 1 is a diagram showing the results of SDS-polyacrylamide gel electrophoresis detection of the amino acid fragments of NP1, NP2, and NP 33 which were prepared and purified in example 1 of the present invention;
FIG. 2 is a diagram showing the results of SDS-polyacrylamide gel electrophoresis detection of the purified recombinant protein of the novel coronavirus in example 2 of the present invention;
FIG. 3 is a diagram showing the results of SDS-polyacrylamide gel electrophoresis detection of a polyclonal antibody against a novel coronavirus recombinant protein obtained from rabbit in example 3 of the present invention;
FIG. 4 is a graph comparing the results of the rabbit-human chimeric antibody against positive and negative human sera, which is a novel coronavirus in example 5 of the present invention;
FIG. 5 is a graph showing the results of measurement of the titer of rabbit-human chimeric antibody against the novel coronavirus in example 5 of the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 selection of dominant epitopes of the N protein of a novel coronavirus
1.1 expression of novel coronavirus N protein epitope
According to the novel coronavirus (SARS-CoV-2) N protein gene sequence (Genbank: NC045512), the antigen epitope prediction is carried out by using software Bepipred, and 3 sections of amino acid fragments are designed in total, wherein the amino acid fragments are respectively amino acid NP1(1-165aa) at positions 1-165 of the N protein (the amino acid sequence is shown as SEQ ID NO: 1), amino acid NP2(140-332aa) at positions 140-419 of the N protein, amino acid NP3(245-419aa) at positions 245-419 of the N protein, and the amino acid sequence of the N protein is shown as SEQ ID NO: and 5, performing prokaryotic expression codon optimization on the 3 segments of amino acid fragments, synthesizing optimized codons, synthesizing genes by Anhui general biology company, cloning the optimized codons to Pet32A respectively, performing prokaryotic expression to obtain the 3 segments of amino acid fragments, and purifying, wherein the purification result is shown in figure 1, and the purification result shows that the amino acid fragments are well purified.
The nucleotide sequence of SEQ ID NO: 1 has the following sequence structure:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGT。
the nucleotide sequence of SEQ ID NO: 5 has the following sequence structure:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTTLPKGFYAEGSRGGSQASSRSSSRSRNSSRNSTPGSSRGTSPARMAGNGGDAALALLLLDRLNQLESKMSGKGQQQQGQTVTKKSAAEASKKPRQKRTATKAYNVTQAFGRRGPEQTQGNFGDQELIRQGTDYKHWPQIAQFAPSASAFFGMSRIGMEVTPSGTWLTYTGAIKLDDKDPNFKDQVILLNKHIDAYKTFPPTEPKKDKKKKADETQALPQRQKKQQTVTLLPAADLDDFSKQLQQSMSSADSTQA。
1.2 identification of epitopes of N protein of novel coronavirus
NP1, NP2, and NP3 were coated, and the affinity of the antibody against SARS-CoV-2 (purchased from Wanhai, Xiamen) was measured by ELISA, and the results are shown in Table 1 below, and the result of Table 1 shows that NP1 has the best affinity for the antibody.
TABLE 1
NP1(1ug/ml) NP2(1ug/ml) NP3(1ug/ml)
Quality control 1:1000 1.969 0.859 1.056
Quality control 1:2000 1.679 0.433 0.645
Quality control 1:4000 1.358 0.213 0.283
Quality control 1:8000 1.014 0.185 0.190
Negative control 0.184 0.188 0.177
EXAMPLE 2 preparation of novel coronavirus recombinant proteins
2.1 Gene optimization of novel coronavirus recombinant proteins
In the determination of NP1 as N protein amino acid fragment (amino acid sequence is shown as SEQ ID NO: 1), selecting receptor binding region RBD (i.e. 299-600 amino acids of S protein) to represent novel coronavirus S protein amino acid fragment (amino acid sequence is shown as SEQ ID NO: 2), inserting a soluble peptide segment which can help epitope folding: GSESGSLSGSDSGWGSGSGHGGKG, and the amino acid sequence after the tandem is shown as SEQ ID NO: 3, respectively.
The nucleotide sequence of SEQ ID NO: 2 has the following sequence structure:
TKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP。
the nucleotide sequence of SEQ ID NO: 3 has the following sequence structure:
MSDNGPQNQRNAPRITFGGPSDSTGSNQNGERSGARSKQRRPQGLPNNTASWFTALTQHGKEDLKFPRGQGVPINTNSSPDDQIGYYRRATRRIRGGDGKMKDLSPRWYFYYLGTGPEAGLPYGANKDGIIWVATEGALNTPKDHIGTRNPANNAAIVLQLPQGTGSESGSLSGSDSGWGSGSGHGGKGTKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP。
and (3) carrying out codon optimization on the amino acid sequence to obtain a nucleotide sequence for expressing the recombinant protein, such as SEQ ID NO: 4, SEQ ID NO: 4 has the following sequence structure:
atgagcgataacggtccgcaaaaccagcgtaacgcgccgcgtattaccttcggtggtccgagcgatagcaccggtagcaaccaaaacggcgaacgtagcggtgcgcgtagcaaacaacgtcgtccgcaaggtctgccgaacaacaccgcgagctggtttaccgcgctgacccagcacggtaaagaagacctgaagttcccgcgtggtcagggcgtgccgattaacaccaacagcagcccggacgatcaaattggttattaccgtcgtgcgacccgtcgtatccgtggcggtgatggcaaaatgaaagatctgagcccgcgttggtacttctattacctgggtaccggcccggaagcgggtctgccgtacggtgcgaacaaggatggcatcatctgggttgcgaccgaaggtgcgctgaacaccccgaaagaccacattggtacccgtaacccggcgaacaacgcggcgattgttctgcagctgccgcaaggtaccggttctgaatctggttctctgtctggttctgactctggttggggttctggttctggtcacggtggtaaaggacctaagtgcactctgaagtctttcaccgtggagaagggcatctaccagactagcaacttcagggtgcagccaaccgaatctatcgtcagattccccaacatcactaacctgtgcccattcggagaggtcttcaacgccaccagattcgcttccgtgtacgcctggaacaggaagagaatcagcaactgcgtcgctgactactctgtgctgtacaacagcgcctctttctcaaccttcaagtgctacggcgtgagccctactaagctgaacgacctgtgcttcaccaacgtctacgccgactctttcgtgatcaggggagacgaggtcagacagatcgctcccggccagactggaaagatcgccgactacaactacaagctgccagacgacttcaccggctgcgtcatcgcttggaactcaaacaacctggactccaaagtgggtggcaactacaactacctgtaccgcctgttccgtaagagcaacctgaagcctttcgagagggacatctcaactgaaatctaccaggctggttccaccccctgcaacggtgtcgagggcttcaactgctacttcccactgcaatcttacggtttccagcctactaacggtgtgggctaccagccctacagagtggtcgtgctgtcattcgaactgctgcacgccccagctactgtgtgcggtcctaagaagtccaccaacctggtcaagaacaagtgcgtgaacttcaacttcaacggcctgaccggaactggtgtcctgaccgagtcaaacaagaagttcctgccattccagcagttcggaagggacatcgctgacaccactgacgctgtgcgcgaccctcagaccctggaaatcctggacatcactccttgcagcttcggaggtgtctctgtgatcacccct。
2.2 culture of novel coronavirus recombinant protein prokaryotic expression engineering strains
The recombinant plasmid Pet28a-nCoV having the aforementioned nucleotide sequence (SEQ ID NO: 4) was synthesized by genetic engineering means, and the recombinant plasmid was synthesized by Anhui general Bio Inc. Pet28a-nCoV was transformed into BL21 (DE 3) competent cells, and the transformants were plated on LB agar plates (containing 100. mu.g/mL ampicillin) and cultured overnight at 37 ℃ to prepare primary seeds. The primary seeds (single colonies) were inoculated into 3 mL of LB medium (containing 100 ug/mL of ampicillin) and cultured at 37 ℃ for 10 hours to give production seeds. The strain for production is taken, inoculated into LB culture medium (containing 100 ug/mL ampicillin) according to the amount of 1 percent of the total volume of the culture medium, cultured for about 3 hours at 37 ℃ and 220 r/min by shaking until OD600 is 0.5, cooled for 1.5 hours at 16 ℃, added with 0.1 mM IPTG at the final concentration, induced for 16 hours at 16 ℃ and 220 r/min, and then the strain is collected.
2.3 prokaryotic expression of novel coronavirus recombinant proteins
The cells were centrifuged at 4000 rpm/min at 4 ℃ for 5 minutes and washed twice with PBS (0.01 mol/L, pH 7.2), the cells were collected and resuspended in 1/10 volumes of PBS (0.01 mol/L, pH 7.2), the cells were lysed by sonication on ice for 30 minutes (2 seconds with 2 second intervals), and the supernatant was collected at 12000 rpm/min at 4 ℃. Purifying the novel coronavirus recombinant protein by a nickel ion affinity chromatography method. Firstly, filling Ni-NTA His Bind Resin (Nanjing Kingsri) into an empty chromatographic column, naturally dripping liquid by the action of gravity, washing the chromatographic column by using a balance Buffer with the volume 10 times that of NTA, adding the supernatant sample into the chromatographic column, combining the supernatant sample on ice for 30 minutes and then allowing the supernatant sample to flow out; washing the chromatographic column with 5 times of balance Buffer, eluting with NTA-60 (5 times of NTA volume) elution Buffer, collecting eluate to obtain novel coronavirus recombinant protein, filtering for sterilization, measuring protein concentration with ultraviolet spectrophotometer to be not less than 1.0 mg/mL, and subpackaging and freezing for use.
2.4 purity identification of recombinant proteins of novel coronavirus
The purity of the purified protein was checked by protein SDS-polyacrylamide gel electrophoresis:
preparing 12% separation glue, carefully injecting into a gap of a glass plate until the height is 11cm, adding pure water on the separation glue, isolating air, polymerizing for 30-60 min at room temperature, pouring out the pure water, sucking residual liquid by absorbent paper, preparing concentrated glue, injecting the upper end of the separation glue, wherein the height is 1cm, carefully inserting a comb to avoid generating bubbles, and polymerizing for 30-45 min at room temperature. Mu.l of the sample was mixed with an equal volume of 2 Xloading buffer and heated at 100 ℃ for 5 min. And (3) loading 20 mu l of sample per hole, firstly, carrying out 80V, adjusting the sample to be 120V after the sample enters the separation gel, and till the bromophenol blue reaches the bottom of the separation gel. Coomassie brilliant blue staining: and (3) soaking the gel in Coomassie brilliant blue dye solution, oscillating and dyeing for 4h, decolorizing the decolorized solution until protein bands are clear, analyzing the protein bands, and separating the protein bands clearly without impurity bands as shown in figure 2 to meet the purification requirement.
EXAMPLE 3 preparation of polyclonal antibodies against the recombinant protein of the novel coronavirus
3.1 preparation of Freund's complete adjuvant antigen and incomplete adjuvant antigen
Freund's complete adjuvant antigen: freund's complete adjuvant (Sigma, USA) 3 mL plus the new coronavirus recombinant protein (pet 28a-nCOV NP recombinant protein) 1mg plus physiological saline to 6 mL.
Freund's incomplete adjuvant antigen: freund's incomplete adjuvant (Sigma, USA) 3 mL plus the new coronavirus recombinant protein (pet 28a-nCOV NP recombinant protein) 1mg plus physiological saline to 6 mL.
The adjuvant antigens are prepared before immunization, and are fully mixed and emulsified.
3.2 immunization procedure
Taking the fully emulsified Freund complete adjuvant antigen, inoculating the antigen under the skin of feet of two hind legs, the skin of popliteal lymph nodes and the skin of the back of each rabbit at multiple points, wherein the total inoculation amount is 2mL, and after 14 days, the antigen and the dosage are still inoculated from the skin of the back at multiple points for secondary immunization; after 11 days, two points of the shoulder muscle of each rabbit were inoculated with the Freund's incomplete adjuvant antigen subcutaneously at the back, and the total amount of inoculation was 2mL, which was used for the third immunization.
3.3 blood test
After 7-14 days, blood serum is separated from the ear marginal vein blood collection, blood is tested by ELISA test, and if the ELISA titer of the immune rabbit blood serum reaches 1:8000 or above, blood is collected formally. If the titer of the immune rabbit serum is low, the immune rabbit can be boosted once again according to the third dose and the immune way, and blood test is performed after 7-14 days, if the titer of the immune rabbit serum meets the requirement, the hyperimmune serum can be prepared according to the method. Otherwise, abandon it.
Protein coating: respectively coating recombinant proteins (coating buffer solution carbonate buffer solution sodium carbonate 1.59g and sodium bicarbonate 2.93g are fixed to volume in 1L of pure water) in a microporous plate, wherein the coating concentration is 1 mug/mL;
sealing the coated plate: sealing with 1% gelatin, 150 microliter per hole, washing the plate with washing solution for 1 time at 37 ℃ for 2 hours, and drying;
the resulting immune rabbit antiserum was diluted in PBS with a gradient of 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, and 50. mu.L was added to the antigen-coated plate and reacted at 37 ℃ for 30 min.
And (3) throwing liquid in the hole, washing the plate for 4 times by using PBST washing liquor (0.1%) with 250 mu L/hole, beating the plate to be dry, adding HRP enzyme-labeled goat anti-rabbit secondary antibody (diluted by PBS according to a ratio of 1: 10000) with 50 mu L/hole, reacting at 37 ℃ for 30min, washing the plate for 4 times again, beating the plate to be dry, adding TMB developing solution (commercialized) with 100 mu L/hole, developing at room temperature for 10min, finally adding 0.5M sulfuric acid with 50 mu L/hole, stopping the reaction, measuring an OD450nm value by using an enzyme-labeled instrument, and taking rabbit serum before immunization as a negative control.
3.4 ammonium sulfate precipitation method for purifying immune rabbit serum IgG of novel coronavirus recombinant protein
Saturated solution of ammonium sulfate: dissolving 800-850 g of ammonium sulfate in 1L H2O, heating until most of O is dissolved, filtering while hot, standing overnight at room temperature, 28% NH4OH to adjust the pH value to 7.0.
0.01mol/L pH 7.4PB solution: 19ml of the solution A and 81ml of the solution B are taken, water is added to the solution B to 1000ml, and the solution A and the solution B are filtered by a filter membrane with the diameter of 0.22 mu m for standby.
Liquid A (0.01 mol/L NaH)2PO4Liquid) NaH2PO4·2H2O15.6 g plus H2O to 1000 ml;
liquid B (0.01 mol/L Na)2HPO4Liquid) Na2HPO4·12H235.8g of O plus H2O to 1000 ml.
Nano liquid: HgI 115g, KI 80g, plus H2Dissolving O to 500ml, filtering, adding 20% NaOH 500ml, and mixing for use.
Eluent: 0.3 mol/L NaCl solution.
IgG extraction step: 20ml of serum is taken, 20ml of physiological saline is added, and then (NH) is added dropwise4)2SO4Saturated solution 10ml, make 20% (NH)4)2SO4Saturating the solution, stirring while adding, and standing for 30min at 4 ℃; centrifuging at 4000rpm and 4 deg.C for 20min, removing precipitate, and removing fibrin; adding (NH) into the supernatant4)2SO4Saturated solution 30ml, make 50% (NH)4)2SO4Mixing the saturated solution, and standing at 4 deg.C for 30 min; centrifuging at 4000rpm and 4 deg.C for 20min, and removing supernatant; adding 20ml of normal saline into the precipitate to dissolve the precipitate, and then adding (NH)4)2SO4Saturated solution 10ml, make 33% (NH)4)2SO4Mixing the saturated solution, and standing at 4 deg.C for 30 min; centrifuging at 4000rpm and 4 deg.C for 20min, discarding supernatant, and removing albumin. Repeating the step 5, 2-3 times; the precipitate was dissolved in 10ml of physiological saline and filled into a dialysis bag. Dialyzing in normal water at 4 deg.C overnight, dialyzing in physiological saline at 4 deg.C for 24 hr, and changing the medium for several times. With 1% BaCl2Checking the dialysate for SO4 2-Or checking NH with Nassner reagent4 +(3-4 ml of dialysate is taken and added1-2 drops of reagent, appearing brick red, it is considered that there is NH4 +Present) until no SO is present4 2-Or NH4 +Until it occurs. Centrifuging at 4000rpm and 4 deg.C for 20min to obtain supernatant as crude IgG.
Passing through DEAE-cellulose chromatographic column by conventional method, eluting with 0.01mol/L pH 7.4PB solution (0.3 mol/L NaCl solution), and collecting eluate. Loading the eluate into dialysis bag, embedding PEG3500, and dialyzing at 4 deg.C. Collecting the concentrated eluate to obtain rabbit-derived polyclonal antibody against the novel coronavirus, and subpackaging the rabbit-derived polyclonal antibody into small tubes to be stored at-70 ℃ for later use.
3.5 identifying the purity of the purified rabbit-derived novel coronavirus recombinant protein polyclonal antibody
The purity and the titer of the rabbit-derived novel coronavirus recombinant protein polyclonal antibody are measured, the purity is shown in figure 3, the result shows that the concentration of the purified antibody is 10mg/ml, and the SDS polyacrylamide gel electrophoresis analysis shows that clear immunoglobulin heavy chains and light chains are visible and no miscellaneous bands exist, which indicates that the purity of the antibody meets the expected requirements. The titer of polyclonal antibodies against the rabbit-derived novel coronavirus recombinant proteins is shown in Table 2 below.
TABLE 2
Dilution factor Eramio rabbit serum Sanimmune rabbit serum White rabbit
1:1000 2.135 2.27 0.201
1:2000 1.826 1.973 0.202
1:4000 1.697 1.659 0.183
1:8000 1.327 1.338 0.162
1:16000 0.829 1.007 0.155
EXAMPLE 4 preparation of novel Rabbit-human chimeric antibody against coronavirus
S1, carrying out pepsin digestion on the rabbit-derived novel coronavirus polyclonal antibody, dialyzing the rabbit-derived novel coronavirus polyclonal antibody obtained in the example 3 in 0.2M sodium acetate buffer solution at the pH value of 4.0 overnight, mixing pepsin with the purified rabbit-derived novel coronavirus polyclonal antibody solution, carrying out enzyme digestion reaction at 37 ℃ for 6 hours, wherein the mass ratio of the pepsin to the rabbit-derived novel coronavirus polyclonal antibody is 1:20, adding 3M Tris (Tris) to adjust the pH value to 8.0, separating by a ProteinA/G column, obtaining an F (ab ') 2 fragment as an effluent part, carrying out ultrafiltration concentration, and passing through an S200 molecular sieve column to obtain the F (ab') 2 fragment of the purified rabbit-derived novel coronavirus antibody;
s2, carrying out papain digestion on a human IgG antibody, dialyzing the human IgG antibody in a buffer solution containing 0.2Mtris, 0.15Mnacl, 1mM EDTA and 1mM DTT at the pH value of 8.0 overnight, mixing papain with the purified human IgG antibody solution, carrying out enzyme digestion reaction at 37 ℃ for 6 hours, wherein the mass ratio of the papain to the human IgG antibody is 1:20, separating the mixture by a ProteinA/G column, wherein the combined part is an Fc fragment, eluting the mixture by 0.1M glycine at the pH value of 2.7, carrying out ultrafiltration concentration, and carrying out S200 molecular sieve column chromatography to obtain the Fc fragment of the purified human IgG antibody;
s3, coupling a rabbit-derived novel coronavirus antibody F (ab ') 2 fragment with Biotin, dissolving the fragment with dimethyl sulfoxide to prepare 100 mu L of 10mM NHS-Biotin (the reagent is EZ-Link NHS-Biotin Reagents from Thermo company, cat # 21343), adjusting the concentration of the rabbit-derived novel coronavirus antibody F (ab ') 2 fragment after PBS dialysis and purification to 2mg/mL, mixing the rabbit-derived novel coronavirus antibody F (ab ') 2 fragment and 27 mu L of 10mM NHS-Biotin according to the proportion of 1mL of the rabbit-derived novel coronavirus antibody F (ab ') 2 fragment and 27 mu L of NHS-Biotin, reacting for 1 hour at room temperature, and dialyzing with 2L of PBS overnight to obtain the Biotin-rabbit-derived novel coronavirus antibody F (ab ') 2 fragment compound;
s4, coupling a human IgG antibody Fc fragment and streptavidin, taking 10mg of streptavidin, adding NHS 8mg and EDC13.3mg, stirring at room temperature for 24 hours, then slowly dropwise adding 6mg of the human IgG antibody Fc fragment, stirring at room temperature for 3 hours, and dialyzing the product with 0.01M PBS at 4 ℃ overnight to obtain the streptavidin-human IgG antibody Fc fragment compound;
s5, mixing the biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex with the streptavidin-human IgG antibody Fc fragment complex and the like, and incubating for 2 hours at room temperature to obtain the novel coronavirus rabbit-human chimeric antibody.
Example 5 Activity and titer determination of novel coronavirus rabbit-human chimeric antibody
5.1 commercial test paper strip for testing comparison of results of novel coronavirus rabbit-human chimeric antibody and negative and positive human serum
In order to ensure the activity of the chimeric antibody obtained after crosslinking, the chimeric antibody is detected by using a novel coronavirus IgM/IgG antibody detection reagent (colloidal gold method) of Spanish Bio-medomics, and the following steps are carried out:
(1) after all reagents are returned to the room temperature, taking out the test paper card and placing the test paper card on a dry plane;
(2) adding samples, namely adding 10 microliters of each of the negative control and the novel coronavirus rabbit-human chimeric antibody into a sample adding hole of the test paper card, and vertically dropwise adding 2-3 drops (about 100 microliters) of sample diluent;
(3) the results were checked within 10-15 minutes after loading.
FIG. 4 shows the comparison of the results of the novel coronavirus rabbit-human chimeric antibody and the positive and negative human serum, and it can be seen from the figure that the detection results of the novel coronavirus rabbit-human chimeric antibody and the positive control are consistent, which indicates that the novel coronavirus rabbit-human chimeric antibody has good activity and can be used as a positive quality control product for replacing serum.
5.2 novel coronavirus rabbit-human chimeric antibody titer assay
The detection is carried out by using a novel coronavirus IgM/IgG antibody detection reagent (colloidal gold method) of Spain Bio-medomics, and the steps are as follows:
the mixture of the rabbit-human chimeric antibody against the novel coronavirus and the negative serum was diluted 1:10, 1:20, 1:40, 1:80, 1:160, and 1:320 with PBS, and the dilution was performed in a gradient manner according to the procedure 5.1.
The test results are shown in fig. 5, from which it can be seen that the dilution factor of the antibody is 1: at 320, the rabbit-human chimeric antibody of the novel coronavirus still has detection titer, which indicates that the rabbit-human chimeric antibody of the novel coronavirus can be used as a positive quality control substance for detecting the novel coronavirus.
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
Sequence listing
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Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu ProAsn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Gly Ser Glu Ser Gly Ser Leu Ser Gly Ser Asp
165 170 175
Ser Gly Trp Gly Ser Gly Ser Gly His Gly Gly Lys Gly Thr Lys Cys
180 185 190
Thr Leu Lys Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn
195 200 205
Phe Arg Val Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr
210 215 220
Asn Leu Cys Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser
225 230 235 240
Val Tyr Ala Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr
245 250 255
Ser Val Leu Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly
260 265 270
Val Ser Pro Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala
275 280 285
Asp Ser Phe Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly
290 295 300
Gln Thr Gly Lys Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe
305 310 315 320
Thr Gly Cys Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val
325 330 335
Gly Gly Asn Tyr Asn Tyr Leu Tyr Arg Leu Phe Arg Lys Ser Asn Leu
340 345 350
Lys Pro Phe Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser
355 360 365
Thr Pro Cys Asn Gly Val Glu Gly Phe Asn Cys Tyr Phe Pro Leu Gln
370 375 380
Ser Tyr Gly Phe Gln Pro Thr Asn Gly Val Gly Tyr Gln Pro Tyr Arg
385 390 395 400
Val Val Val Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys
405 410 415
Gly Pro Lys Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe
420 425 430
Asn Phe Asn Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys
435 440 445
Lys Phe Leu Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr
450 455 460
Asp Ala Val Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro
465 470 475 480
Cys Ser Phe Gly Gly Val Ser Val Ile Thr Pro
485 490
<210>4
<211>1473
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<400>4
atgagcgataacggtccgca aaaccagcgt aacgcgccgc gtattacctt cggtggtccg 60
agcgatagca ccggtagcaa ccaaaacggc gaacgtagcg gtgcgcgtag caaacaacgt 120
cgtccgcaag gtctgccgaa caacaccgcg agctggttta ccgcgctgac ccagcacggt 180
aaagaagacc tgaagttccc gcgtggtcag ggcgtgccga ttaacaccaa cagcagcccg 240
gacgatcaaa ttggttatta ccgtcgtgcg acccgtcgta tccgtggcgg tgatggcaaa 300
atgaaagatc tgagcccgcg ttggtacttc tattacctgg gtaccggccc ggaagcgggt 360
ctgccgtacg gtgcgaacaa ggatggcatc atctgggttg cgaccgaagg tgcgctgaac 420
accccgaaag accacattgg tacccgtaac ccggcgaaca acgcggcgat tgttctgcag 480
ctgccgcaag gtaccggttc tgaatctggt tctctgtctg gttctgactc tggttggggt 540
tctggttctg gtcacggtgg taaaggtacc aagtgcactc tgaagtcttt caccgtggag 600
aagggcatct accagactag caacttcagg gtgcagccaa ccgaatctat cgtcagattc 660
cccaacatca ctaacctgtg cccattcgga gaggtcttca acgccaccag attcgcttcc 720
gtgtacgcct ggaacaggaa gagaatcagc aactgcgtcg ctgactactc tgtgctgtac 780
aacagcgcct ctttctcaac cttcaagtgc tacggcgtga gccctactaa gctgaacgac 840
ctgtgcttca ccaacgtcta cgccgactct ttcgtgatca ggggagacga ggtcagacag 900
atcgctcccg gccagactgg aaagatcgcc gactacaact acaagctgcc agacgacttc 960
accggctgcg tcatcgcttg gaactcaaac aacctggact ccaaagtggg tggcaactac 1020
aactacctgt accgcctgtt ccgtaagagc aacctgaagc ctttcgagag ggacatctca 1080
actgaaatct accaggctgg ttccaccccc tgcaacggtg tcgagggctt caactgctac 1140
ttcccactgc aatcttacgg tttccagcct actaacggtg tgggctacca gccctacaga 1200
gtggtcgtgc tgtcattcga actgctgcac gccccagcta ctgtgtgcgg tcctaagaag 1260
tccaccaacc tggtcaagaa caagtgcgtg aacttcaact tcaacggcct gaccggaact 1320
ggtgtcctga ccgagtcaaa caagaagttc ctgccattcc agcagttcgg aagggacatc 1380
gctgacacca ctgacgctgt gcgcgaccct cagaccctgg aaatcctgga catcactcct 1440
tgcagcttcg gaggtgtctc tgtgatcacc cct 1473
<210>5
<211>419
<212>PRT
<213> Artificial Sequence (Artificial Sequence)
<400>5
Met Ser Asp Asn Gly Pro Gln Asn Gln Arg Asn Ala Pro Arg Ile Thr
1 5 10 15
Phe Gly Gly Pro Ser Asp Ser Thr Gly Ser Asn Gln Asn Gly Glu Arg
20 25 30
Ser Gly Ala Arg Ser Lys Gln Arg Arg Pro Gln Gly Leu Pro Asn Asn
35 40 45
Thr Ala Ser Trp Phe Thr Ala Leu Thr Gln His Gly Lys Glu Asp Leu
50 55 60
Lys Phe Pro Arg Gly Gln Gly Val Pro Ile Asn Thr Asn Ser Ser Pro
65 70 75 80
Asp Asp Gln Ile Gly Tyr Tyr Arg Arg Ala Thr Arg Arg Ile Arg Gly
85 90 95
Gly Asp Gly Lys Met Lys Asp Leu Ser Pro Arg Trp Tyr Phe Tyr Tyr
100 105 110
Leu Gly Thr Gly Pro Glu Ala Gly Leu Pro Tyr Gly Ala Asn Lys Asp
115 120 125
Gly Ile Ile Trp Val Ala Thr Glu Gly Ala Leu Asn Thr Pro Lys Asp
130 135 140
His Ile Gly Thr Arg Asn Pro Ala Asn Asn Ala Ala Ile Val Leu Gln
145 150 155 160
Leu Pro Gln Gly Thr Thr Leu Pro Lys Gly Phe Tyr Ala Glu Gly Ser
165 170 175
Arg Gly Gly Ser Gln Ala Ser Ser Arg Ser Ser Ser Arg Ser Arg Asn
180 185 190
Ser Ser Arg Asn Ser Thr Pro Gly Ser Ser Arg Gly Thr Ser Pro Ala
195 200 205
Arg Met Ala Gly Asn Gly Gly Asp Ala Ala Leu Ala Leu Leu Leu Leu
210 215 220
Asp Arg Leu Asn Gln Leu Glu Ser Lys Met Ser Gly Lys Gly Gln Gln
225 230 235 240
Gln Gln Gly Gln Thr Val Thr Lys Lys Ser Ala Ala Glu Ala Ser Lys
245 250 255
Lys Pro Arg Gln Lys Arg Thr Ala Thr Lys Ala Tyr Asn Val Thr Gln
260 265 270
Ala Phe Gly Arg Arg Gly Pro Glu Gln Thr Gln Gly Asn Phe Gly Asp
275 280 285
Gln Glu Leu Ile Arg Gln Gly Thr Asp Tyr Lys His Trp Pro Gln Ile
290 295 300
Ala Gln Phe Ala Pro Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile
305 310 315 320
Gly Met Glu Val Thr Pro Ser Gly Thr Trp Leu Thr Tyr Thr Gly Ala
325 330 335
Ile Lys Leu Asp Asp Lys Asp Pro Asn Phe Lys Asp Gln Val Ile Leu
340 345 350
Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu Pro
355 360 365
Lys Lys Asp Lys Lys Lys Lys Ala Asp Glu Thr Gln Ala Leu Pro Gln
370 375 380
Arg Gln Lys Lys Gln Gln Thr Val Thr Leu Leu Pro Ala Ala Asp Leu
385 390 395 400
Asp Asp Phe Ser Lys Gln Leu Gln Gln Ser Met Ser Ser Ala Asp Ser
405 410 415
Thr Gln Ala

Claims (10)

1. A novel recombinant coronavirus protein, wherein the amino acid sequence of the novel recombinant coronavirus protein consists of SEQ ID NO: 1. SEQ ID NO: 2 are obtained by connecting polypeptide fragments in sequence.
2. A nucleotide sequence encoding the novel recombinant coronavirus protein of claim 1.
3. A recombinant vector comprising the nucleotide sequence of claim 2.
4. A novel coronavirus polyclonal antibody, which is obtained by immunizing an animal with the novel coronavirus recombinant protein as defined in claim 1 as an immunogen, and separating and purifying the animal.
5. A method for preparing a novel rabbit-human chimeric antibody against coronavirus, comprising the steps of:
coupling an F (ab ') 2 fragment of a rabbit-derived novel coronavirus antibody with activated biotin to obtain a biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex, wherein the rabbit-derived novel coronavirus antibody is a polyclonal antibody obtained by immunizing a rabbit with the novel coronavirus recombinant protein as defined in claim 1;
coupling the Fc fragment of the human IgG antibody or the IgM antibody with avidin or streptavidin by adopting a covalent crosslinking method to obtain an avidin/streptavidin-human IgG/IgM antibody Fc fragment compound;
coupling biotin of the biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex with avidin or streptavidin of the avidin/streptavidin-human IgG/IgM antibody Fc fragment complex to obtain a novel coronavirus rabbit-human chimeric antibody;
or, coupling the F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody with avidin or streptavidin by a covalent crosslinking method to obtain an avidin/streptomycin avidin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex, wherein the rabbit-derived novel coronavirus antibody is a polyclonal antibody obtained by immunizing a rabbit with the novel coronavirus recombinant protein as defined in claim 1;
coupling the Fc fragment of the human IgG antibody or the IgM antibody with activated biotin to obtain a biotin-human IgG/IgM antibody Fc fragment compound;
and coupling the avidin or streptavidin of the avidin/streptavidin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex with the biotin of a biotin-human IgG/IgM antibody Fc fragment complex to obtain the novel coronavirus rabbit-human chimeric antibody.
6. The method for preparing a rabbit-human chimeric antibody against coronavirus according to claim 5, comprising the following steps:
s1, carrying out enzyme digestion on a rabbit-derived novel coronavirus antibody by adopting pepsin to obtain an F (ab') 2 fragment of the rabbit-derived novel coronavirus antibody, wherein the rabbit-derived novel coronavirus antibody is a polyclonal antibody obtained by immunizing a rabbit by adopting the novel coronavirus recombinant protein as claimed in claim 1;
s2, carrying out enzyme digestion on the human IgG antibody or the IgM antibody by using papain to obtain an Fc fragment of the human IgG antibody or the IgM antibody;
s3 dissolving NHS-Biotin in solventAdding F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody to perform a coupling reaction, wherein the concentration of the F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody is 2-10mg/mL, and the ratio of the F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody to NHS-Biotin is 1mg (0.8-2)
Figure DEST_PATH_IMAGE001
10-7Obtaining a biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment compound;
and S4, mixing NHS and EDC serving as cross-linking agents with avidin or streptavidin, adding the Fc fragment of the human IgG antibody or the IgM antibody, mixing, and carrying out covalent cross-linking reaction, wherein the mass ratio of the Fc fragment of the human IgG antibody or the IgM antibody to the avidin or streptavidin is 3: (4-6), obtaining an avidin/streptavidin-human IgG/IgM antibody Fc fragment complex;
s5, the biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex is combined with the avidin/streptavidin-human IgG/IgM antibody Fc fragment complex to obtain a mixture of (0.5-2): 1, and carrying out coupling reaction to obtain the novel coronavirus rabbit-human chimeric antibody.
7. The method for preparing a rabbit-human chimeric antibody against coronavirus according to claim 6, comprising the following steps:
s1, dialyzing the rabbit-derived novel coronavirus antibody in 0.2M sodium acetate buffer solution at the pH value of 4.0 for overnight, mixing pepsin with the purified rabbit-derived novel coronavirus antibody solution, carrying out enzyme digestion reaction at 37 ℃ for 6 hours, wherein the mass ratio of the pepsin to the rabbit-derived novel coronavirus antibody is 1:20, adding 3M Tris to adjust the pH value to 8.0, separating by a ProteinA/G column, carrying out ultrafiltration concentration on an effluent part, and passing through a S200 molecular sieve column to obtain an F (ab') 2 fragment of the purified rabbit-derived novel coronavirus antibody;
s2, dialyzing the human IgG antibody or IgM antibody in a buffer solution containing 0.2Mtris, 0.15Mnacl, 1mM EDTA and 1mM DTT at the pH value of 8.0 for overnight, mixing papain with the purified human IgG antibody or IgM antibody solution, carrying out enzyme digestion reaction at 37 ℃ for 6 hours, wherein the mass ratio of the papain to the human IgG antibody or IgM antibody is 1:20, separating the mixture by a ProteinA/G column, eluting a binding part by 0.1M glycine at the pH value of 2.7, carrying out ultrafiltration concentration, and passing the binding part by a S200 molecular sieve column to obtain the Fc fragment of the purified human IgG antibody or IgM antibody;
s3, dissolving and preparing 10mM NHS-Biotin by using dimethyl sulfoxide as a solvent, adjusting the concentration of the F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody to 2mg/mL after PBS dialysis and purification, mixing the F (ab') 2 fragment of the rabbit-derived novel coronavirus antibody and 27 mu L of 10mM NHS-Biotin according to the proportion of adding the F (ab ') 2 fragment of the rabbit-derived novel coronavirus antibody to the NHS-Biotin, reacting for 1 hour at room temperature, and dialyzing overnight by using 2L PBS to obtain a Biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment compound;
s4, taking 10mg of streptavidin, adding NHS 8mg and EDC13.3mg, stirring for 24 hours at room temperature, then dropwise adding 6mg of Fc fragment of human IgG antibody or IgM antibody, stirring for 3 hours at room temperature, and dialyzing the product overnight at 4 ℃ with 0.01M PBS to obtain a streptavidin-human IgG/IgM antibody Fc fragment compound;
s5, mixing the biotin-rabbit-derived novel coronavirus antibody F (ab') 2 fragment complex with the streptavidin-human IgG/IgM antibody Fc fragment complex and the like, and incubating for 2 hours at room temperature to obtain the novel coronavirus rabbit-human chimeric antibody.
8. A novel rabbit-human chimeric antibody against coronavirus prepared by the method for preparing a novel rabbit-human chimeric antibody against coronavirus according to any one of claims 5 to 7.
9. Use of the rabbit-human chimeric antibody against coronavirus of claim 8 for preparing positive quality control substance of antibody detection kit against coronavirus.
10. A novel coronavirus antibody detection kit comprising the novel coronavirus rabbit-human chimeric antibody of claim 8.
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* Cited by examiner, † Cited by third party
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US11732030B2 (en) 2020-04-02 2023-08-22 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments
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