CN113896774B - Recombinant protein K-S and preparation method and application thereof - Google Patents
Recombinant protein K-S and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a recombinant protein K-S and a preparation method and application thereof, belonging to the technical field of biology, wherein the scheme comprises that 6 key sites are mutated on a nucleotide sequence of the S1 protein, and the mutation sites and bases are respectively: G1251T, T1355G, G1450C, A1501T, A1841G and C2042G, the corresponding sites after encoding the proteins are: K417N, L452R, E484Q, N501Y, D614G and P681R; is connected to eukaryotic expression vector pcDNA3.1, expressed by CHO-S cells (thermoFisher) and purified to obtain recombinant protein K-S, and the recombinant protein K-S can be sequentially immunized with inactivated vaccine through the skin with novel coronavirus variant.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant protein K-S, a preparation method and application thereof.
Background
The global pandemic of covd-19 caused by SARS-Cov-2 has caused more than 2 hundred million infections and 430 more than one case of death. This trend is further exacerbated by the pandemic of variant strains that occur from time to time worldwide, which is a great concern and concern. With global pandemic, various variants continue to appear, including alpha, beta, gamma, delta, lambda, etc., and it is not anticipated what new variants will appear later. All the variants that have appeared have two common characteristics, both infectivity and immune escape are markedly enhanced. Vaccine is the most effective way to prevent and control infectious diseases, and vaccination is still the most effective prevention and control means in the face of the complex and changeable trend of global variant pandemics and the like. In order to cope with severe epidemic situation such as anti-flutter, many countries are ready to vaccinate 'booster needles' on the basis of the original two-needle vaccine. The so-called "booster" may be the original vaccine, or may be replaced by a new variant strain or a new gene sequence of interest. However, all of these vaccines are based on a variant of the prior art and have some hysteresis and limitations. It is desirable to produce a vaccine that can cope with the current variety of variants and also predict the potential epidemic strains in the future, so-called second generation vaccines, which can fundamentally solve the problem of immune escape of variants.
Disclosure of Invention
In order to solve the problems of infectivity and immune escape enhancement of a variant strain of the COVID-19, the invention provides a recombinant protein K-S, and a preparation method and application thereof.
The technical scheme of the invention is as follows:
a recombinant protein K-S, the nucleotide sequence of which is shown as SEQ ID NO:1, or a protein sequence such as seq id no:2.
the invention also provides a preparation method of the recombinant protein K-S, which comprises the following steps:
(1) Selecting the nucleotide sequence of the S1 protein according to the published gene sequence (MT 226610.1);
(2) On the nucleotide sequence of the S1 protein, 6 key sites are mutated to construct a new sequence, and the mutation sites and bases are respectively: G1251T, T1355G, G1450C, A1501T, A1841G and C2042G, the corresponding sites after encoding the proteins are: K417N, L452R, E484Q, N501Y, D614G and P681R;
(3) The two ends of the new sequence are added with cleavage sites NheI (GCTAGC) and KpnI (GGTACC) and are connected to eukaryotic expression vector pcDNA3.1, and CHO-S cells (ThermoFisher) are utilized for expression;
(4) Obtaining the supernatant after expression, and purifying the protein to obtain recombinant protein K-S;
the recombinant protein K-S nucleotide sequence or protein sequence is as claimed in claim 1.
The invention also provides application of the recombinant protein K-S, and the recombinant protein K-S according to the technical scheme is applied to novel coronavirus immunization.
Further, the immunization mode is intradermal immunization.
Further, the recombinant protein K-S is sequentially immunized with an inactivated vaccine.
The inactivated vaccine used in the invention is disclosed and prepared by the medical biology research institute of the national academy of medical science. The clinical separated SARS-CoV-2 virus is inoculated in African green monkey kidney cell line (Vero) for 3-5 days, the typical lesions appear on the cells, the virus culture solution is obtained and filtered, firstly 200 mug/ml formaldehyde (HCHO) is used for inactivating for 48 hours, then Beta Propiolactone (BPL) is used for inactivating for 24 hours, and then the vaccine stock solution is obtained through ultrafiltration concentration, purification and filtration, and then aluminium hydroxide is added to prepare the virus.
The beneficial effects are that: the invention establishes a vaccine antigen preparation technical system aiming at the virus variant strains which are already and will appear, adopts a sequential immunization strategy and creatively applies intradermal immunization mode for research, and the intradermal immunization is different from the traditional subcutaneous and intramuscular immunization, can quickly activate a large amount of immune cells in skin in a short time, provides theoretical and technical basis for quickly constructing a novel coronavirus immune system, and has specific experimental data, and beneficial effects and principles are shown in specific implementation modes.
Drawings
FIG. 1 is a diagram showing the immunogenicity verification of recombinant protein K-S and inactivated vaccine;
FIG. 2 is a graph comparing the immune response caused by intradermal immunity to muscle immunity;
FIG. 3 is a diagram of neutralizing antibodies and ELISA antibodies of immunized mice;
FIG. 4 is a diagram of an immunized mouse cellular immunity ELISPot assay (IFN-. Gamma.);
FIG. 5 is a diagram showing an analysis of the toxicity-counteracting protective effect of immunized mice;
FIG. 6 is a graph showing neutralization of three strains by mouse immune serum;
wherein FIG. 1 (a) is a diagram of the S1 recombinant protein K-S pattern containing 6 mutation sites; (b) The figure shows Western immunoblotting, conv: convalescent patient serum, immu: inoculator serum. 1: s1 protein, 2: K-S recombinant proteins; (c) The figure shows western blotting, 1:recombinant protein (K417N) 2:recombinant protein (L452R), 3: recombinant protein (E484Q), 4: recombinant protein (N501Y), 5: recombinant protein (D614G), 6: recombinant protein (P681R), 7: the RBD protein (d) is shown in a virus antigen immune electron microscope, S antigen (left), N antigen (right);
FIG. 2, (a) is a graph showing comparison of expression levels of mRNA of immune related molecules in skin at various time points, IM-V: a muscle immune inactivated vaccine; IM-K: muscle immune recombinant protein S1; ID-V: an intradermal immune inactivated vaccine; ID-K: intradermal immune recombinant protein S1; (b) Is a graph of the co-localization of antigen (red) and dendritic cells (green) in intradermal and intramuscular immune skin. (c) neutralizing antibodies and positive turnover plots for immunized mice.
FIG. 3, (a) a graph of a mouse sequential immune inactivated vaccine and a K-S recombinant protein neutralizing antibody; (b) Sequential immunization of mice with inactivated vaccine and ELISA antibody patterns of K-S recombinant protein. MD-1: a single dose of recombinant protein K-S group; MD-2: an adjuvant group; MD-3: influenza control group; MC-1: single low dose inactivated vaccine + recombinant protein K-S group; MC-2: single high dose inactivated vaccine + recombinant protein K-S group; MC-3: two doses of low dose inactivated vaccine + recombinant protein K-S group; MC-4: two doses of high dose inactivated vaccine + recombinant protein K-S group.
FIG. 4, (a) is a graph showing IFN-gamma levels expressed by specific T cells under stimulation with recombinant proteins (K417N), (L452R), (E484K), (N501Y) and (L452 R+E484Q) as antigens, respectively; (b) To map the IFN-gamma levels expressed by specific T cells under respective stimulation with protein N as antigen. Wherein, MD-1: a single dose of recombinant protein K-S group; MD-2: an adjuvant group; MD-3: influenza control group; MC-1: single low dose inactivated vaccine + recombinant protein K-S group; MC-2: single high dose inactivated vaccine + recombinant protein K-S group; MC-3: two doses of low dose inactivated vaccine + recombinant protein K-S group; MC-4: two doses of high dose inactivated vaccine + recombinant protein K-S group;
FIG. 5, (a) graph of body weight change after challenge of immunized mice. (b) mortality plot. (c) a map of viral load of each tissue after challenge. (d) diagram of nose and pharynx toxin expelling condition. MB-1: a single dose of recombinant protein K-S group; MB-2: an adjuvant group; MA-1: single low dose inactivated vaccine + recombinant protein K-S group; MA-2: single high dose inactivated vaccine + recombinant protein K-S group; MA-3: two doses of low dose inactivated vaccine + recombinant protein K-S group; MA-4: two doses of high dose inactivated vaccine + recombinant protein K-S group.
FIG. 6, MB-1: a single dose of recombinant protein K-S group; MB-2: an adjuvant group; MA-1: single low dose inactivated vaccine + recombinant protein K-S group; MA-2: single high dose inactivated vaccine + recombinant protein K-S group; MA-3: two doses of low dose inactivated vaccine + recombinant protein K-S group; MA-4: two doses of high dose inactivated vaccine + recombinant protein K-S group.
Detailed Description
The invention is further described below with reference to examples and figures.
Example 1
Recombinant protein K-S construction and expression
According to published gene sequences (MT 226610.1), the nucleotide sequence of the S1 protein is selected, and sequences (G1251T, T1355G, G1450C, A1501T, A1841G and C2042G) containing 6 mutation sites are constructed aiming at the current epidemic strains, wherein the corresponding protein mutation sites are K417N, L452R, E484Q, N501Y, D614G and P681R. The modified sequence is added with cleavage sites NheI (GCTAGC) and KpnI (GGTACC) at two ends, a entrusted company carries out artificial synthesis of the sequence, two ends of the sequence are connected into a eukaryotic expression vector pcDNA3.1, CHO-S cells (thermoFisher) are used for expression, and supernatant fluid is obtained for protein purification to obtain recombinant protein K-S. Protein electrophoresis and protein immunogenicity identification of recombinant proteins were performed as in example 2.
Example 2
Recombinant protein K-S and inactivated vaccine immunogenicity identification and analysis
Based on the currently reported epidemic variants and mutation sites thereof, as shown in the diagram of FIG. 1 (a), the invention designs S1 protein K-S (N501Y, K417N, E484Q, L452R, P681R and D614G) with 6 mutation sites. As shown in the graph (b) of fig. 1, the Western blotting result shows that the recombinant protein K-S can well identify the convalescent serum of a patient and the serum of a novel crown inactivated vaccine inoculator. To further solve the immunogenicity of recombinant protein K-S, serum was prepared by immunization of rabbits and immunoblotted with mutant S proteins (K417N), (L452R), (E484Q), (N501Y), (N439K), (A520V) and RBD proteins, respectively, as shown in FIG. 1 (c), which were well recognized by rabbit immune serum.
Preparation of inactivated vaccine
The inactivated vaccine used in the invention is prepared by medical biology research institute of Chinese medical science academy. The clinical separated SARS-CoV-2 virus is inoculated in African green monkey kidney cell line (Vero) for 3-5 days, the typical lesions appear on the cells, the virus culture solution is obtained and filtered, firstly 200 mug/ml formaldehyde (HCHO) is used for inactivating for 48 hours, then Beta Propiolactone (BPL) is used for inactivating for 24 hours, and then the vaccine stock solution is obtained through ultrafiltration concentration, purification and filtration, and then aluminium hydroxide is added to prepare the virus.
The inactivated vaccine used in the invention is prepared by respectively inactivating formaldehyde and beta propiolactone, as shown in a graph of fig. 1 (d), the virus envelope can be effectively cracked by adopting a two-step inactivation process, the main antigens of S and N are fully exposed, and the immunoelectron microscope research also proves the scientificity of the inactivation process. In large-scale clinical trials, higher antibody levels can be produced following inactivated vaccination of the population.
Example 3
Preparation of female C57 and C57BL/6hACE +/+ Mice, no specific pathogen grade (SpecificPathogenFree, SPF), 4 weeks old, 10-15g, purchased from Shanghai, hainan model biotechnology Co., ltd, were bred according to the standard breeding protocol of experimental animals. After 1 dose or 2 doses of inactivated vaccine, 28 days after the primary immunization, blood was taken to determine neutralizing antibodies and ELISA antibodies. Then, recombinant protein K-S was boosted, and after 14 days, blood was taken to measure neutralizing antibodies (India strain (B.1.617.2), british strain (B.1.351) and SARS-CoV-2) and ELISA antibodies, and virus challenge infection was performed 28 days after the boost, and nasal and pharyngeal swabs were collected daily for detoxification monitoring. Tissue viral load detection was performed on 3 dead per fraction on days 3, 7 and 11 post infection.
(1) Neutralizing antibody assay
0.5mL of non-anticoagulated blood is collected at different time periods after infection according to the experimental design time, and neutralizing antibody detection is carried out after serum separation. Diseases of known titerThe toxin samples were serially diluted 10-fold to 2X103CCID50/mL and the dilutions were added to 96-well plates at 50. Mu.L/well, while the antisera prepared from the mice were double diluted starting at 1:4 (1:4, 1:8,1:16,1:32,1:64 … …). Serial dilutions of serum were added to 96-well plates containing virus at 50. Mu.L/well, neutralized at 37℃for 2h, and digested Vero cell suspension was added at a cell concentration of about (2-3) x10 5 /mL. At 37 ℃,5% CO 2 And (5) standing and culturing in a incubator for 5-7 days, and observing cytopathic conditions. Antibody titers were calculated as the highest dilution of serum that could neutralize the corresponding virus and prevent cell damage.
(2) Elisa assay
Elisa assay kit was purchased from Beijing Yiqiao Shenzhou technologies Co. The enzyme-labeled instrument was purchased from chinese gene company. The ELISA plates pre-coated with the antibodies were awakened according to the kit instructions, and then standard and test samples were added to the plates while negative controls were established. The plates were then developed according to the kit instructions and absorbance values were read at 450nm using a microplate reader.
(3) Specific CTL detection
Peripheral anticoagulants were collected from mice at the corresponding time points, and after isolation of PBMC, they were temporarily stored at 4℃in RPMI1640 medium containing 10% fetal bovine serum. Antibody pre-coated ELISPOT plates were awakened according to the kit instructions, after which a concentration of PBMC cells was added to the plates. The antigenic peptide was then added to the culture broth followed by 5% CO at 37℃ 2 Culturing for 24 hr under the condition of avoiding shaking the plate. Then, the cells and the culture medium were removed, and the plates were developed according to the kit instructions, and recorded, counted and analyzed using an ELISPOT automatic analyzer (CTL company in the united states).
Example 4
The intradermal immune recombinant protein K-S and the inactivated vaccine can effectively activate the innate immune response of the mice.
Early studies showed that intradermal immunization not only effectively reduced the dose of antigen at the time of vaccination, but also enhanced the antiviral immune response of the body through innate and acquired immune responses.
In the invention, the recombinant protein K-S and the inactivated vaccine are respectively used for intradermal immunization and intramuscular immunization of the C57 mice, and the skin and the muscle at the injection site are respectively taken for detecting the expression level of immune-related cytokines 12 hours, 24 hours and 48 hours after immunization, as shown in figure 2, the detection methods are respectively different groups in figure 2, and fluorescent quantitative PCR is adopted, so that compared with the traditional intramuscular immunization, the immunoregulation signal molecules IFN-alpha, TNF-alpha, RANKL, BTLA, LIGHT, IKK beta, 4IBBL and interleukin IL-5, IL-9, IL-13, IL-25 and IL-33 are greatly improved. The results of immune confocal also indicate that the number of Dendritic Cells (DCs) induced by intradermal immunization is 2-3 times greater than that of the muscle immune pathway. These results indicate that the intradermal immune protein K-S and the inactivated vaccine can effectively improve the innate immune response in the epidermal tissue, thereby activating the acquired immune response and improving the whole antiviral immune response of the organism. The results of neutralizing antibodies after immunization of mice also showed (detection method see example 3 for neutralizing antibody assay), 100% for intradermal immune protein K-S (10 ug/dose) and inactivated vaccine (30U/dose) antibodies, 20-30% for GMT, and 50% for muscle immunization with the same dose of protein K-S and inactivated vaccine, 3-4% for GMT. The results show that the intradermal immunization of the novel coronatine or the inactivated vaccine has obvious advantages compared with the traditional muscle immunization.
Example 5
Intradermal sequential immune inactivated vaccine and recombinant protein K-S for effectively activating antibody level of organism
The invention uses C57BL/6hACE +/+ The transgenic mice were used as models for analysis of immune effects. At C57BL/6hACE +/+ In mice, after 1 dose or 2 doses of the low dose inactivated vaccine (30U/dose), recombinant protein K-S boosting was performed 28 days after the last vaccination, and neutralizing antibodies were measured and GMT was calculated for each group before and 14 days after boosting, as shown in FIG. 3, and the results showed 88.4 for GMT before boosting and 322.5 for 14 days after boosting, which was 3.2-fold higher. 1 dose or 2 doses of inactivated vaccine (50U/dose) are inoculated, recombinant protein K-S boosting is carried out 28 days after the last inoculation, neutralizing antibodies of each group are respectively measured and GMT is calculated before boosting and 14 days after boosting, and the result shows that the GMT before boosting is 70.2 and boosting is carried out222.9 after 14 days of immunization was raised 3.6-fold, and specific detection methods are described in example 3 for the neutralizing antibody assay.
For the current epidemic strain, whether the original inactivated vaccine is effective or not, and no clear theories exist at present. However, it is certainly not possible to prevent infection of several epidemic strains simultaneously by using an inactivated vaccine prepared from one strain. On the basis of inactivated vaccine inoculation, the invention uses the recombinant S1 protein containing a plurality of mutation sites to strengthen immunity, and can effectively prevent infection of a plurality of epidemic strains in theory.
Example 6
Intradermal sequential immune inactivated vaccine and protein K-S effective for activating cellular immunity level of organism
In the previous studies of the present invention, whether immunized animals or subjects, the memory T cell response was able to rapidly develop a cellular immune response upon stimulation with the novel coronal inactivated viral antigens S and N antigen 180 days after immunization, and specific detection methods are described in example 3 for specific CTL assays. However, it is unclear whether the organism can maintain an immunological memory response to various mutants after booster immunization with the recombinant protein.
C57BL/6hACE in example 5 +/+ In the mouse model, by isolating mouse PBMC, the recombinant S1 proteins at the different mutation sites (K417N, L452R, E484Q, N501Y and L452 R+E484Q) were stimulated, respectively, for specific detection methods as described in Elisa in example 3. As shown in FIG. 4, the number of times of immunization and the dose were not related, although the number of times of immunization was significantly increased in the body memory cell immunization compared with the negative control group (MD-1) and the adjuvant control group (MD-1). The induced cellular immune response was consistent with the trend of the cellular immune response generated by recombinant protein S1 under N antigen stimulation.
Example 7
Intradermal sequential immunization of C57BL/6hACE +/+ Protection analysis of mice against toxicity
By sequentially immunizing mice intradermally with neutralizing antibodies and cellular immunoassays, this strategy is capable of rapidly inducing an immune response in the body, and an immune memory response to various variants (417 n,452r, 284 q,501y,452r, 704 q) can be rapidly activated in a short time.
To further verify this immune effect, the present invention selected a variant B.1.617.2, C57BL/6hACE for the immunization described in example 5 above +/+ The mice were subjected to toxicity counteracting protection effect analysis. After challenge, clinical symptoms were observed for 11 consecutive days, as shown in FIG. 5, the single dose protein group (MB-1) and adjuvant group (MB-2) all had the appearance of bow back, hair fall and weight loss, all died 5-8 days after challenge, while the low dose groups (MA-1 and MA-3) only had clinical symptoms for individual mice, with mortality of 20-25%. The single high dose group (MA-2) showed clinical symptoms and death in 1 mouse 3 days after challenge, and the mortality was 10%. The mice in the group with 2 doses and high dose are free from abnormality and death. From the detoxification situation, the high dose group had substantially cleared the virus on day 7 post-infection, while the low dose group had substantially no detectable virus on day 9 post-infection. At days 3, 7 and 11 post-infection, 3 virus load assays were sacrificed, and essentially no virus was detected in each tissue of the high dose group. The results show that the high-dose group has good protection effect on immunized mice.
Example 8
Intradermal sequential immune serum neutralization epidemic and variant assays
The virus infects the body, and must first bind to the corresponding receptor on the body to successfully invade the cells and infect them. The neutralizing antibody is a soluble protein produced by B lymphocytes of adaptive immune cells, can be combined with viruses invading the body, and can prevent the viruses from invading and infecting cells. The neutralizing antibody can neutralize the virus to some extent, and reflect the protective effect on the body to some extent. By comparison with C57BL/6hACE +/+ The toxicity attack protection effect analysis of mice is carried out, and the infection attack of B.1.617.2 strains to the mice can be well protected by an intradermal sequential immunization mode.
The immune mouse serum and representative British strain B.1.351 and domestic SARS-CoV-2 are subjected to cross neutralization analysis, and as shown in figure 6, the immune serum can well neutralize B.1.617.2 strain, B.1.351 strain and SARS-CoV-2 strain, and has high antibody titer, and each group is more than 1:128. This also indirectly demonstrated that mice immunized sequentially intradermally were also resistant to infection by strain B.1.351 and SARS-CoV-2. In theory, the infection of various variants can be resisted by the intradermal sequential immunization mode.
Sequence listing
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<120> recombinant protein K-S, preparation method and application thereof
<130> 2021.09.09
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<400> 1
atgtttgttt ttcttgtttt attgccacta gtctctagtc agtgtgttaa tcttacaacc 60
agaactcaat taccccctgc atacactaat tctttcacac gtggtgttta ttaccctgac 120
aaagttttca gatcctcagt tttacattca actcaggact tgttcttacc tttcttttcc 180
aatgttactt ggttccatgc tatacatgtc tctgggacca aaggtactaa gaggtttgat 240
aaccctgtcc taccatttaa tgatggtgtt tattttgctt ccactgagaa gtctaacata 300
ataagaggct ggatttttgg tactacttta gattcgaaga cccagtccct acttattgtt 360
aataacgcta ctaatgttgt tattaaagtc tgtgaatttc aattttgtaa tgatccattt 420
ttgggtgttt attaccacaa aaacaacaaa agttggatgg aaagtgagtt cagagtttat 480
tctagtgcga ataattgcac ttttgaatat gtctctcagc cttttcttat ggaccttgaa 540
ggaaaacagg gtaatttcaa aaatcttagg gaatttgtgt ttaagaatat tgatggttat 600
tttaaaatat attctaagca cacgcctatt aatttagtgc gtgatctccc tcagggtttt 660
tcggctttag aaccattggt agatttgcca ataggtatta acatcactag gtttcaaact 720
ttacttgctt tacatagaag ttatttgact cctggtgatt cttcttcagg ttggacagct 780
ggtgctgcag cttattatgt gggttatctt caacctagga cttttctatt aaaatataat 840
gaaaatggaa ccattacaga tgctgtagac tgtgcacttg accctctctc agaaacaaag 900
tgtacgttga aatccttcac tgtagaaaaa ggaatctatc aaacttctaa ctttagagtc 960
caaccaacag aatctattgt tagatttcct aatattacaa acttgtgccc ttttggtgaa 1020
gtttttaacg ccaccagatt tgcatctgtt tatgcttgga acaggaagag aatcagcaac 1080
tgtgttgctg attattctgt cctatataat tccgcatcat tttccacttt taagtgttat 1140
ggagtgtctc ctactaaatt aaatgatctc tgctttacta atgtctatgc agattcattt 1200
gtaattagag gtgatgaagt cagacaaatc gctccagggc aaactggaaa tattgctgat 1260
tataattata aattaccaga tgattttaca ggctgcgtta tagcttggaa ttctaacaat 1320
cttgattcta aggttggtgg taattataat taccggtata gattgtttag gaagtctaat 1380
ctcaaacctt ttgagagaga tatttcaact gaaatctatc aggccggtag cacaccttgt 1440
aatggtgttc aaggttttaa ttgttacttt cctttacaat catatggttt ccaacccact 1500
tatggtgttg gttaccaacc atacagagta gtagtacttt cttttgaact tctacatgca 1560
ccagcaactg tttgtggacc taaaaagtct actaatttgg ttaaaaacaa atgtgtcaat 1620
ttcaacttca atggtttaac aggcacaggt gttcttactg agtctaacaa aaagtttctg 1680
cctttccaac aatttggcag agacattgct gacactactg atgctgtccg tgatccacag 1740
acacttgaga ttcttgacat tacaccatgt tcttttggtg gtgtcagtgt tataacacca 1800
ggaacaaata cttctaacca ggttgctgtt ctttatcagg gtgttaactg cacagaagtc 1860
cctgttgcta ttcatgcaga tcaacttact cctacttggc gtgtttattc tacaggttct 1920
aatgtttttc aaacacgtgc aggctgttta ataggggctg aacatgtcaa caactcatat 1980
gagtgtgaca tacccattgg tgcaggtata tgcgctagtt atcagactca gactaattct 2040
cgtcggcgg 2049
<210> 2
<211> 683
<212> PRT
<213> recombinant CHO expression ()
<400> 2
Met Phe Val Phe Leu Val Leu Leu Pro Leu Val Ser Ser Gln Cys Val
1 5 10 15
Asn Leu Thr Thr Arg Thr Gln Leu Pro Pro Ala Tyr Thr Asn Ser Phe
20 25 30
Thr Arg Gly Val Tyr Tyr Pro Asp Lys Val Phe Arg Ser Ser Val Leu
35 40 45
His Ser Thr Gln Asp Leu Phe Leu Pro Phe Phe Ser Asn Val Thr Trp
50 55 60
Phe His Ala Ile His Val Ser Gly Thr Lys Gly Thr Lys Arg Phe Asp
65 70 75 80
Asn Pro Val Leu Pro Phe Asn Asp Gly Val Tyr Phe Ala Ser Thr Glu
85 90 95
Lys Ser Asn Ile Ile Arg Gly Trp Ile Phe Gly Thr Thr Leu Asp Ser
100 105 110
Lys Thr Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile
115 120 125
Lys Val Cys Glu Phe Gln Phe Cys Asn Asp Pro Phe Leu Gly Val Tyr
130 135 140
Tyr His Lys Asn Asn Lys Ser Trp Met Glu Ser Glu Phe Arg Val Tyr
145 150 155 160
Ser Ser Ala Asn Asn Cys Thr Phe Glu Tyr Val Ser Gln Pro Phe Leu
165 170 175
Met Asp Leu Glu Gly Lys Gln Gly Asn Phe Lys Asn Leu Arg Glu Phe
180 185 190
Val Phe Lys Asn Ile Asp Gly Tyr Phe Lys Ile Tyr Ser Lys His Thr
195 200 205
Pro Ile Asn Leu Val Arg Asp Leu Pro Gln Gly Phe Ser Ala Leu Glu
210 215 220
Pro Leu Val Asp Leu Pro Ile Gly Ile Asn Ile Thr Arg Phe Gln Thr
225 230 235 240
Leu Leu Ala Leu His Arg Ser Tyr Leu Thr Pro Gly Asp Ser Ser Ser
245 250 255
Gly Trp Thr Ala Gly Ala Ala Ala Tyr Tyr Val Gly Tyr Leu Gln Pro
260 265 270
Arg Thr Phe Leu Leu Lys Tyr Asn Glu Asn Gly Thr Ile Thr Asp Ala
275 280 285
Val Asp Cys Ala Leu Asp Pro Leu Ser Glu Thr Lys Cys Thr Leu Lys
290 295 300
Ser Phe Thr Val Glu Lys Gly Ile Tyr Gln Thr Ser Asn Phe Arg Val
305 310 315 320
Gln Pro Thr Glu Ser Ile Val Arg Phe Pro Asn Ile Thr Asn Leu Cys
325 330 335
Pro Phe Gly Glu Val Phe Asn Ala Thr Arg Phe Ala Ser Val Tyr Ala
340 345 350
Trp Asn Arg Lys Arg Ile Ser Asn Cys Val Ala Asp Tyr Ser Val Leu
355 360 365
Tyr Asn Ser Ala Ser Phe Ser Thr Phe Lys Cys Tyr Gly Val Ser Pro
370 375 380
Thr Lys Leu Asn Asp Leu Cys Phe Thr Asn Val Tyr Ala Asp Ser Phe
385 390 395 400
Val Ile Arg Gly Asp Glu Val Arg Gln Ile Ala Pro Gly Gln Thr Gly
405 410 415
Asn Ile Ala Asp Tyr Asn Tyr Lys Leu Pro Asp Asp Phe Thr Gly Cys
420 425 430
Val Ile Ala Trp Asn Ser Asn Asn Leu Asp Ser Lys Val Gly Gly Asn
435 440 445
Tyr Asn Tyr Arg Tyr Arg Leu Phe Arg Lys Ser Asn Leu Lys Pro Phe
450 455 460
Glu Arg Asp Ile Ser Thr Glu Ile Tyr Gln Ala Gly Ser Thr Pro Cys
465 470 475 480
Asn Gly Val Gln Gly Phe Asn Cys Tyr Phe Pro Leu Gln Ser Tyr Gly
485 490 495
Phe Gln Pro Thr Tyr Gly Val Gly Tyr Gln Pro Tyr Arg Val Val Val
500 505 510
Leu Ser Phe Glu Leu Leu His Ala Pro Ala Thr Val Cys Gly Pro Lys
515 520 525
Lys Ser Thr Asn Leu Val Lys Asn Lys Cys Val Asn Phe Asn Phe Asn
530 535 540
Gly Leu Thr Gly Thr Gly Val Leu Thr Glu Ser Asn Lys Lys Phe Leu
545 550 555 560
Pro Phe Gln Gln Phe Gly Arg Asp Ile Ala Asp Thr Thr Asp Ala Val
565 570 575
Arg Asp Pro Gln Thr Leu Glu Ile Leu Asp Ile Thr Pro Cys Ser Phe
580 585 590
Gly Gly Val Ser Val Ile Thr Pro Gly Thr Asn Thr Ser Asn Gln Val
595 600 605
Ala Val Leu Tyr Gln Gly Val Asn Cys Thr Glu Val Pro Val Ala Ile
610 615 620
His Ala Asp Gln Leu Thr Pro Thr Trp Arg Val Tyr Ser Thr Gly Ser
625 630 635 640
Asn Val Phe Gln Thr Arg Ala Gly Cys Leu Ile Gly Ala Glu His Val
645 650 655
Asn Asn Ser Tyr Glu Cys Asp Ile Pro Ile Gly Ala Gly Ile Cys Ala
660 665 670
Ser Tyr Gln Thr Gln Thr Asn Ser Arg Arg Arg
675 680
Claims (3)
1. A recombinant protein K-S, characterized in that: the protein sequence is as shown in SEQ ID NO:2.
2. the method for preparing the recombinant protein K-S according to claim 1, wherein the method comprises the following steps: the method comprises the following steps:
(1) Selecting a nucleotide sequence of the S1 protein according to the published gene sequence MT 226610.1;
(2) On the nucleotide sequence of the S1 protein, 6 key sites are mutated to construct a new sequence, and the mutation sites and bases are respectively: G1251T, T1355G, G1450C, A1501T, A1841G and C2042G, the corresponding sites after encoding the proteins are: K417N, L452R, E484Q, N501Y, D614G and P681R;
(3) Two ends of the new sequence are added with enzyme cutting sites NheI, namely GCTAGC and KpnI, namely GGTACC, and are connected to a eukaryotic expression vector pcDNA3.1 for expression by utilizing CHO-S cells;
(4) Obtaining the supernatant after expression, and purifying the protein to obtain recombinant protein K-S; the recombinant protein K-S nucleotide sequence is as shown in SEQ ID NO: 1.
3. Use of recombinant protein K-S according to claim 1 for the preparation of a novel coronavirus SARS-CoV-2 vaccine.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112076315A (en) * | 2020-08-25 | 2020-12-15 | 中国农业科学院生物技术研究所 | Nano antigen particle fused with new coronavirus S protein and ferritin subunit, new coronavirus vaccine, and preparation method and application thereof |
| WO2021156267A1 (en) * | 2020-02-04 | 2021-08-12 | Curevac Ag | Coronavirus vaccine |
| CN113321739A (en) * | 2021-02-04 | 2021-08-31 | 广东克冠达生物医药科技有限公司 | COVID-19 subunit vaccine and preparation method and application thereof |
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2021
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021156267A1 (en) * | 2020-02-04 | 2021-08-12 | Curevac Ag | Coronavirus vaccine |
| CN112076315A (en) * | 2020-08-25 | 2020-12-15 | 中国农业科学院生物技术研究所 | Nano antigen particle fused with new coronavirus S protein and ferritin subunit, new coronavirus vaccine, and preparation method and application thereof |
| CN113321739A (en) * | 2021-02-04 | 2021-08-31 | 广东克冠达生物医药科技有限公司 | COVID-19 subunit vaccine and preparation method and application thereof |
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| Preclinical immunological evaluation of an intradermal heterologous vaccine against SARS-CoV-2 variants;Shengtao Fan等;《Emerging Microbes & Infections》;第11卷;第212-226页 * |
| Present variants of concern and variants of interest of severe acute respiratory syndrome coronavirus 2: Their significant mutations in S‐glycoprotein, infectivity, re‐infectivity, immune escape and vaccines activity;Chiranjib Chakraborty等;《Rev Med Virol. 2022》;第1-14页 * |
| surface glycoprotein [Severe acute respiratory syndrome coronavirus 2];GenBank;《GenBank》;GenBank: QUQ44927.1 * |
| 中和抗体和细胞免疫在病毒疫苗有效性评价中的相互关系;张志晓等;《中国生物制品学杂志 》;第28卷(第1期);第91-94页 * |
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