CN113845577B - SARS-CoV-2 specific polypeptide and its application - Google Patents
SARS-CoV-2 specific polypeptide and its application Download PDFInfo
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Abstract
The invention discloses SARS-CoV-2 specific polypeptide and application thereof, belonging to the field of immunodetection. The invention provides two polypeptides specific to SARS-CoV-2, and prepares polypeptide-MHC tetramer with corresponding polypeptide, uses polypeptide-MHC tetramer to detect the T cells of SARS-CoV-2 infected convalescence person, the positive rate is 3.63% and 2.37% respectively, which is obviously higher than negative control group 0.061% and 0.096%, effectively increases the affinity of polypeptide-MHC and specific T cell surface TCR, can be used as an effective tool for T cell evaluation, and can also be used for separation and cloning of specific T cells, combining single cell sequencing technology to separate specific TCR, as T cell activating reagent and the like. Has higher application value in the aspect of T cell research of the crowd after SARS-CoV-2 infection or vaccination.
Description
Technical Field
The invention relates to SARS-CoV-2 specific polypeptide and its application, belonging to the field of immunodetection.
Background
The novel coronavirus SARS-CoV-2 was first discovered in 2019, and has spread rapidly worldwide since it was declared by the world health organization as a serious public health emergency of international interest (PHEIC). The current pandemic of SARS-CoV-2 has been sustained for one and a half years, resulting in 2.13 million cases of 2019 coronavirus disease (COVID-19), 440 thousands of deaths, which remains a serious challenge for global public health. The development of a safe and effective vaccine is one of the important means to control SARS-CoV-2 transmission. T cells play a very important role in viral clearance, and in the assessment of the immune effect of SARS-CoV-2 vaccine, the assessment of T cell immune response is particularly important. The evaluation of T cell immunity of SARS-CoV-2 infection convalescence people can provide basis for the research and development of vaccine. It is particularly important to develop efficient, rapid, and sensitive detection means for detecting T cell status in patients, rehabilitators, and vaccinators after SARS-CoV-2 infection.
The MHC-tetramer technology is a technology in which MHC monomer molecules are tetramerized to increase affinity with TCRs on T cells, thereby increasing detection sensitivity. The technology can be applied to detection of antigen-specific T lymphocytes, direct separation and cloning of T cells, separation of specific TCR, in-situ staining and the like, and provides a high-efficiency, rapid and sensitive detection means for researching a series of works related to cell immune reaction. However, there is no report on the level of T cell immunity in the recovered or vaccinated population of SARS-CoV-2 infection using tetramer technology.
Disclosure of Invention
A first object of the present invention is to provide SARS-CoV-2 specific polypeptide having the amino acid sequence as shown in SEQ ID NO. 1; or as shown in SEQ ID NO. 2.
The second object of the present invention is to provide a derivative of the specific polypeptide, which is a polypeptide derivative having the same antigenicity as the SARS-CoV-2 specific polypeptide by substituting or deleting or adding one or more amino acids to the amino acid sequence of the polypeptide.
It is a third object of the present invention to provide a polypeptide-MHC tetramer highly specific and highly sensitive to T cells of a population following infection or vaccination with SARS-CoV-2, said polypeptide-MHC tetramer being bound to said SARS-CoV-2 specific polypeptide by biotinylated MHC-I or to said specific polypeptide derivative by biotinylated MHC-I.
A fourth object of the present invention is to provide a method for preparing the polypeptide-MHC tetramer, comprising the steps of: (1) expressing an MHC light chain and an MHC heavy chain with escherichia coli; (2) dilution renaturation to prepare a polypeptide/MHC complex; (3) preparing a biotinylated polypeptide/MHC complex; (4) The biotinylated polypeptide/MHC complex reacts with labeled streptavidin.
In one embodiment of the invention, the MHC heavy chain C-terminal is linked to biotin.
In one embodiment of the invention, the C-terminal addition in the MHC is capable of ligating an amino acid sequence GGGLNDIFEAQKIEWHE of biotin.
In one embodiment of the invention, in step (3), the polypeptide/MHC complex is bound to D-biotin under the catalysis of the birA enzyme.
In one embodiment of the present invention, in the step (3), the concentration of the D-biotin is 400 to 600. Mu. Mol/L.
In one embodiment of the present invention, in the step (4), the molar ratio (4 to 6) is: (0.5-1.5) with a labeled streptavidin.
In one embodiment of the present invention, in the step (4), the molar ratio is 5:1, with marks is a chain avidin reaction.
In one embodiment of the invention, the method for preparing the polypeptide-MHC tetramer further comprises purification.
In one embodiment of the invention, the purification is a purification of the polypeptide/MHC complex prepared by molecular sieve.
In one embodiment of the invention, the method is specifically: the method comprises the steps of expressing an MHC light chain and a C-terminal connected biotin MHC heavy chain by using escherichia coli, preparing a polypeptide/MHC complex by using a dilution renaturation method, purifying by using superdex200, combining with D-biotin under the catalysis of birA enzyme to form a biotinylated polypeptide/MHC complex, and then combining with labeled streptavidin according to a molar ratio of 5:1 to give a polypeptide/MHC complex (polypeptide-MHC tetramer).
A fifth object of the present invention is to provide a polypeptide vaccine whose active ingredient contains the SARS-CoV-2 specific polypeptide and/or SARS-CoV-2 specific polypeptide derivative.
It is a sixth object of the present invention to provide a SARS-CoV-2 specific cellular immunoassay kit comprising said SARS-CoV-2 specific polypeptide and/or SARS-CoV-2 specific polypeptide derivative.
The invention also provides application of the polypeptide-MHC tetramer.
In one embodiment of the invention, the application comprises: preparing a vaccine; as an effective tool for T cell immunology evaluation, the T cell immune response of the human population after SARS-CoV-2 infection or vaccination was evaluated; flow cytometry detection of corresponding immune cells of SARS-CoV-2 infected or vaccinated population, in situ staining of tissue sections, isolation and cloning of specific T cells, isolation of specific TCR in combination with single cell sequencing technology, as T cell activating reagent.
The beneficial effects are that:
the tetramer prepared by using 2 polypeptides which are independently screened has high specificity and sensitivity, and can be used as an effective tool for T cell evaluation. The flow analysis shows that the positive rate of the polypeptide-MHC tetramer prepared by the method provided by the invention for detecting the T cells of a patient infected with SARS-CoV-2 is 3.63% and 2.37% respectively, which are obviously higher than that of a negative control group by 0.061% and 0.096%. The technology can also be used for isolation and cloning of specific T cells of SARS-CoV-2 infected or vaccinated people, isolation of specific TCR by combining single cell sequencing technology, as T cell activating reagent, etc.
Drawings
Fig. 1 is: HLA-A x 1101 and epitope polypeptide superdex200 molecular sieve bar graph;
Fig. 2 is: HLA-A x 1101 and epitope polypeptide forms a post-biotinylation superdex200 molecular sieve bar chart;
Fig. 3 is: M23-Tetramer infection SARS-CoV-2 infection convalescence person T cell flow analysis result represents a graph;
fig. 4 is: N25-Tetramer infection SARS-CoV-2 infection convalescence patient T cell flow analysis result represents a graph;
Detailed Description
The invention is further illustrated in the following drawings and specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Unless otherwise indicated, the reagents and materials used in the following examples are commercially available or may be prepared by known methods.
Example 1 SARS selection of CoV-2-specific Polypeptides
Predicting M and N proteins of SARS-CoV-2 with polypeptide prediction software to obtain 15-18 amino acid polypeptides, dissolving the polypeptides with DMSO, and mixing peptides of M and N proteins to obtain peptide library. PBMCs from rehabilitators were cultured with peptide libraries as stimuli and cells were harvested after 9 days of culture.
Numbering the synthesized polypeptides, arranging the polypeptides according to a matrix, crossing the polypeptides in a transverse N group, a vertical N group, and a transverse group and a vertical group, and respectively mixing the polypeptides in the transverse group and the vertical group into a secondary peptide library. The condition that the secondary peptide library generates IFN-gamma is detected by an Elispot technology, 2 functional peptides (shown as SEQ ID NO.1 and SEQ ID NO.2 in the amino acid sequence in the table 1) are obtained by positioning the intersection of the horizontal group and the vertical group of the secondary peptide library generating IFN-gamma, the CD8 + T epitope (shown as the table 1) is predicted, the detection by the Elispot technology has stronger specificity, the method can be used for preparing polypeptide vaccines, researching and developing diagnostic reagents, can be used as an effective tool for evaluating T cell immunology, and can be used for evaluating T cell immune response of a crowd after SARS-CoV-2 infection or vaccination.
Table 1 functional peptides obtained by screening and sequences thereof
EXAMPLE 2 preparation of polypeptide-MHC tetramers
(1) Preparation of polypeptide/MHC complex:
1) Adding an amino acid sequence GGGLNDIFEAQKIEWHE capable of being connected with biotin at the C end of a gene HLA-A 1101 (Genbank accession number MT 462157.1), constructing a recombinant plasmid pET28a-HLA-A 1101-Bio, connecting a B2m gene (Genbank accession number AAA 39668.1) with a vector, constructing a recombinant vector pET28a-B2m, and respectively transforming the plasmids into escherichia coli BL21 to obtain recombinant bacteria E.coli/pET28a-B2m and E.coli/pET28a-HLA-A 1101-Bio;
2) Culturing the escherichia coli BL21 carrying the plasmid in the step 1) at 37 ℃ and adding 1mmol/L IPTG to induce protein expression, collecting thalli, ultrasonically crushing the thalli, centrifuging at a high speed (12000 rpm,10 min), dissolving the precipitate in a dissolving buffer (6 mol/L guanidine hydrochloride, 10% glycerol, 50mmol/L Tris pH8.0, 100mmol/L NaCl,10mmol/L EDTA) to obtain heavy chain HLA-A 1101 and light chain B2m;
3) The polypeptide (named as polypeptide M23) with the sequence shown in SEQ ID NO.1 and the heavy chain and the light chain are subjected to dilution renaturation in renaturation Buffer (100 mmol/L Tris pH 8.0, 400mmol/L arginine, 2mmol/L EDTA) so as to form an MHC complex; preparing MHC complexes with the polypeptide shown in SEQ ID NO.2 (designated as polypeptide N25) and the heavy chain and the light chain, respectively, by the same method as described above;
4) Concentrating the renatured sample obtained in step 3) through a 10kDa filter membrane using a ultrafiltration cup and replacing the solution by concentration to Exchange Buffer (20 mmol/L Tris-HCl,50mmol/L NaCl, pH 8.0); after removal of the sample, the sample was centrifuged at 12000rpm for 10min at 4℃and the supernatant was transferred to an ultrafiltration tube and concentrated to about 0.5-1ml, and the sample was subjected to purification of polypeptide/MHC complexes by passing through a Superdex200 molecular sieve, the results of which are shown in FIG. 1.
(2) Biotinylation of polypeptides/MHC molecules
1) And (3) collecting the polypeptide/MHC complex protein sample purified by the molecular sieve obtained in the step (1) in an ultrafiltration concentration tube, concentrating to about 300 mu L, reacting with D-biotin under the catalysis of birA enzyme, and incubating overnight at 4 ℃ to obtain a biotinylated protein sample.
2) The biotinylated protein samples were centrifuged and passed through a superdex200 molecular sieve to perform biotinylated complex purification to remove excess biotin (fig. 2).
3) The purified polypeptide/MHC complex was concentrated to about 500. Mu.L using an ultrafiltration concentration tube, and sampled for Gel shift assay to verify the biotinylation effect.
Sample preparation:
2. Mu.L streptavidin+8. Mu.L molecular sieve Buffer.
B.8 μl of biotinylated polypeptide/MHC sample+2μl of 20mg/mL Streptavidin;
C.8. Mu.L of biotinylated polypeptide/MHC sample+2. Mu.L of molecular sieve buffer;
the three samples are placed on ice for incubation for 30min-2h and then are subjected to SDS-PAGE identification.
The biotinylated MHC can be combined with strepavidin to form macromolecules, so that the band hysteresis of the MHC in SDS-PAGE can be realized, the biotinylation effect can be judged by comparing the ratio of (C-B)/C MHC content, and the biotinylation effect of the technical scheme of the invention is about 70 percent.
(3) Biotinylation of MHC molecules:
Concentrating biotinylated MHC molecules, and tetramerizing the biotinylated MHC molecules according to the molar ratio of streptavidin to polypeptide/MHC complex of 1:5, wherein the streptavidin is the streptavidin with fluorescent marks, and incubating at 4 ℃ overnight to prepare the M23 tetramer.
N25 tetramer was prepared following the same procedure as described above.
EXAMPLE 3 application of polypeptide/MHC tetramer in T cell analysis
The characteristics of high affinity and high specificity of SARS-CoV-2 specific polypeptide/MHC tetramer are utilized to detect T cells of a crowd after SARS-CoV-2 infection and vaccine immunization, the cellular immune effect, separation and cloning of the T cells and the like of a person infected by SARS-CoV-2, a convalescence person and a crowd after vaccination are evaluated, and the T cell immune level of the person infected by SARS-CoV-2 is evaluated by adopting the following steps:
1) Selecting PBMC of SARS-CoV-2 infection convalescence person with HLA-A 1101 type, and culturing PBMCs by using synthetic peptide library as stimulus for 9 days;
2) The cultured cells were harvested and washed 2 times with FACS buffer/stabilizing buffer (PBS+0.5% BSA);
3) Cell surface molecule staining. Antibodies (e.g., FITC-CD8, APC-CD4, perCp-CD3, PE-Tetramer) were added.
Incubating on ice at 4 ℃ for 30min;
4) And (5) washing. Adding 200 mu L of FACS buffer for centrifugation, and washing for 2 times;
the results of the flow analysis by a cell flow meter are shown in fig. 3-4, and the positive rates of the T cells of the recovered patients infected by SARS-CoV-2 are 3.63% and 2.37% respectively, which are obviously higher than those of the negative control groups of 0.061% and 0.096% by using the polypeptide-MHC tetramer M23 and N25.
EXAMPLE 4 preparation of a vaccine from a polypeptide, polypeptide derivative
Dissolving the polypeptide or polypeptide derivative obtained by the preparation in an aqueous phase or oil phase adjuvant, diluting to a proper concentration, filtering and sterilizing. Optionally, emulsification is performed to prepare the vaccine.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> Chinese disease prevention control center viral disease prevention control institute
<120> SARS-CoV-2 specific polypeptide and use thereof
<130> BAA211318A
<160> 2
<170> PatentIn version 3.3
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<213> Artificial sequence
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Pro Lys Glu Ile Thr Val Ala Thr Ser Arg Thr Leu Ser Tyr Tyr Lys
1 5 10 15
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<213> Artificial sequence
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Leu Leu Asn Lys His Ile Asp Ala Tyr Lys Thr Phe Pro Pro Thr Glu
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Claims (7)
1. A polypeptide-MHC tetramer, wherein a biotinylated MHC-I with the amino acid sequence shown as SEQ ID No.1 and a SARS-CoV-2 specific polypeptide are combined, wherein the biotinylated MHC heavy chain of MHC-I is HLA-A x 1101 and the MHC light chain is B2m gene;
The HLA-A is 1101 Genbank accession number MT462157.1, and the Genbank accession number of the B2m gene is AAA39668.1.
2. The method for preparing the polypeptide-MHC tetramer according to claim 1, comprising the steps of: (1) expressing an MHC light chain and an MHC heavy chain with escherichia coli; (2) dilution renaturation to prepare a polypeptide/MHC complex; (3) preparing a biotinylated polypeptide/MHC complex; (4) The biotinylated polypeptide/MHC complex is reacted with labeled streptavidin.
3. The method of claim 2, wherein the MHC heavy chain C-terminal is linked to biotin.
4. The method of claim 2, wherein in step (3), the polypeptide/MHC complex is bound to D-biotin under the catalysis of BirA enzyme.
5. The method according to claim 2, wherein in the step (4), the molar ratio (4 to 6): (0.5 to 1.5) reacting the biotinylated polypeptide/MHC complex with a labeled streptavidin.
A SARS-CoV-2 specific cellular immunoassay kit comprising the polypeptide-MHC tetramer of claim 1.
7. Use of the polypeptide-MHC tetramer of claim 1 for the preparation of T cell immunoassays, flow cytometry detection of cells in vivo after infection or vaccination with SARS-CoV-2 and in situ staining of tissue sections of organisms, isolation and cloning of specific T cells, isolation of specific TCRs, as reagents for T cell activation.
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| CN106397549A (en) * | 2016-10-11 | 2017-02-15 | 中国疾病预防控制中心病毒病预防控制所 | MERS-CoV specific polypeptides and application thereof |
| CN109293739A (en) * | 2018-01-24 | 2019-02-01 | 中国疾病预防控制中心病毒病预防控制所 | A kind of A3 superfamily universal tumor antigen polypeptide and its application |
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| CN106397549A (en) * | 2016-10-11 | 2017-02-15 | 中国疾病预防控制中心病毒病预防控制所 | MERS-CoV specific polypeptides and application thereof |
| CN109293739A (en) * | 2018-01-24 | 2019-02-01 | 中国疾病预防控制中心病毒病预防控制所 | A kind of A3 superfamily universal tumor antigen polypeptide and its application |
Non-Patent Citations (3)
| Title |
|---|
| Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients;Yanchun Peng等;bioRxiv;1-36 * |
| Yanchun Peng等.Broad and strong memory CD4+ and CD8+ T cells induced by SARS-CoV-2 in UK convalescent COVID-19 patients.bioRxiv.2020,1-36. * |
| YANG,E.K.L.等.MT462157.1.Genbank.2020,FEATRUES,ORIGIN. * |
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