CN111647557A - Exosome with surface coupled with S protein, and preparation method and application thereof - Google Patents
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Abstract
The invention provides an exosome with a surface coupled with coronavirus S protein, a preparation method and application thereof, belonging to the technical field of biological pharmacy. The recombinant expression of aptamer CP05-S protein is realized by obtaining a stable cell strain through transient transfection of human cells or recombinant lentivirus infection, the recombinant expression is mixed with human plasma for incubation, and the exosome is combined with the aptamer CP05 through surface CD63 membrane molecules to form the exosome of surface coupling S protein. The invention adopts exosome to present antigen S protein, so that the prepared vaccine avoids the toxicity of immunologic adjuvant and the risk of virus vaccine infection, is an ideal vaccine development strategy and is also an ideal strategy for closing virus invasion paths. Meanwhile, S protein is delivered through exosome, so that the combination of the S protein of the new coronavirus and a receptor can be competitively inhibited, a sealing effect is exerted, and the invasion of the new coronavirus is effectively blocked, so that the exosome coupled with the S protein is used as a novel coronavirus infection inhibitor and is possibly used for treating acute stage new coronary pneumonia.
Description
Technical Field
The invention belongs to the technical field of biological pharmacy, and particularly relates to an exosome with a surface coupled with S protein, and a preparation method and application thereof.
Background
At present, alpha-interferon, protease inhibitor or ribavirin as an effective disease-resistant treatment method which is not confirmed clinically for the treatment of the new coronary pneumonia can be used, but the treatment strategy has certain limitations, and is not suitable for patients allergic to interferon and auxiliary materials, patients suffering from autoimmune diseases, patients with heart and liver diseases, renal insufficiency or abnormal bone marrow function, patients suffering from epilepsy and central nervous system function injury, besides being used with caution to pregnant women, children and old people. The task of developing new drugs for the treatment of new coronaviruses is therefore very urgent.
Disclosure of Invention
In view of this, the present invention aims to provide an exosome with a surface coupled with S protein, and a preparation method and an application thereof, wherein the exosome has a good antigen presentation effect and an ideal immune effect.
The invention provides a preparation method of an exosome with a surface coupled with S protein, which comprises the following steps:
1) the DNA fragment shown in SEQ ID No.1 is cut by enzyme Pme1 and then inserted into a pWPI vector to form a recombinant vector pWPI-S-RBD-SD-CP 05;
2) co-transfecting the recombinant vector pWPI-S-RBD-SD-CP05, psPAX2 and pMD2.G into eukaryotic cells, and packaging into lentiviruses;
3) after the lentivirus infects eukaryotic cells, screening green fluorescent protein positive cells for continuous culture, and establishing a stable transfer cell strain;
4) culturing the stable cell strain, cracking the cell, and separating protein hydrolysate containing CP05-S protein;
5) and mixing and incubating the protein lysate containing the CP05-S protein and human plasma, and extracting an exosome to obtain the exosome with the surface coupled with the S protein.
Preferably, the DNA fragment in step 1) comprises a nucleotide sequence encoding CP05-S protein; the nucleotide sequence of the coding CP05-S protein is shown in SEQ ID No. 2.
Preferably, the eukaryotic cells in step 2) comprise 293 cells.
Preferably, the volume ratio of the protein lysate containing CP05-S protein to human plasma in the step 5) is 0.8-1.2: 0.8-1.2.
Preferably, the incubation temperature in the step 5) is 23-28 ℃; the incubation time is 50-70 min.
Preferably, the centrifugal force for extracting the exosomes in the step 5) is 80000-120000 g, and the centrifugal time for extracting the exosomes is 4-5 h.
The invention provides an exosome of a coupled S protein prepared by the preparation method.
The invention provides application of the S protein-coupled exosome in preparation of a vaccine for resisting a novel coronavirus.
The invention provides a novel inhibitor of coronavirus infection, comprising exosomes coupled with the S protein.
The preparation method of the exosome of surface coupled S protein provided by the invention comprises the steps of obtaining a stable transgenic cell strain for stably expressing the CP05-S protein through virus infection, recombining and expressing the CP05-S protein, mixing and incubating the extracted CP05-S protein and human plasma, and extracting the exosome, wherein the surface of the exosome is provided with a CD63 membrane molecule which can be combined with the CP05-S protein in a high affinity manner, and the CP05-S protein and the exosome are combined and settled together in the incubation process, so that the exosome of the surface coupled S protein is obtained. The method provided by the invention omits a complicated protein purification step and simplifies the operation. Compared with the method for extracting the antigen by direct protein injection, the prepared exosome with the surface coupled with the S protein has better antigen extraction effect by the protein attached to the exosome; meanwhile, the exosome is derived from a human plasma product, the clinical application is good, the safety of the exosome derived from the plasma as a carrier is high, the in-vivo action rule of the exosome is similar to that of the virus particles, and the better sealing effect is achieved. Thus, exosomes present antigens, avoiding the toxicity of immune adjuvants (traditional protein + adjuvant immunization) and the risk of viral (traditional attenuated or inactivated vaccines) infection.
The invention provides application of the S protein-coupled exosome in preparation of a vaccine for resisting a novel coronavirus. The application immunizes a mouse with the exosome of which the surface is coupled with the S protein, and the test result shows that compared with the naked S protein and a blank control, the mouse can generate ideal immune effect in vivo, and the generated antibody can be used for detecting the S protein. Therefore, the S protein is loaded on the surface of the exosome in an engineering way to prepare the vaccine, so that the S protein is an ideal vaccine development strategy and an ideal strategy for closing a virus invasion path.
The invention also provides a novel inhibitor of coronavirus infection, comprising the exosome coupled with the S protein. The invention delivers the S protein through the exosome, can competitively inhibit the combination of the S protein of the new coronavirus to the receptor thereof, plays a role in virus blocking, and can be possibly used for the medication of the acute stage new coronary pneumonia.
Drawings
FIG. 1 is a diagram showing the results of Westernblot detection of exosome surface-coupled S-protein;
FIG. 2 is a graph showing the results of the measurement of the titers of antisera obtained by immunizing mice with different antigens.
Detailed Description
The invention provides a preparation method of an exosome with a surface coupled with S protein, which comprises the following steps:
1) the DNA fragment shown in SEQ ID No.1 is cut by enzyme Pme1 and then inserted into a pWPI vector to form a recombinant vector pWPI-S-RBD-SD-CP 05;
2) co-transfecting the recombinant vector pWPI-S-RBD-SD-CP05, psPAX2 and pMD2.G into eukaryotic cells, and packaging into lentiviruses;
3) after the lentivirus infects eukaryotic cells, screening green fluorescent protein positive cells for continuous culture, and establishing a stable transfer cell strain;
4) culturing the stable cell strain, cracking the cell, and separating protein hydrolysate containing CP05-S protein;
5) and mixing and incubating the protein lysate containing the CP05-S protein and human plasma, and extracting an exosome to obtain the exosome with the surface coupled with the S protein.
The DNA fragment shown in SEQ ID No.1 is cut by enzyme by Pme1 and then inserted into a pWPI vector to form a recombinant vector pWPI-S-RBD-SD-CP 05.
In the present invention, the DNA fragment preferably comprises a nucleotide sequence encoding CP05-S protein; the nucleotide sequence for coding the CP05-S protein is preferably shown as SEQ ID No. 2. The method of inserting the enzymatically cleaved DNA fragment into the pWPI vector of the present invention is not particularly limited, and a ligation method well known in the art may be used.
After obtaining the recombinant vector pWPI-S-RBD-SD-CP05, the invention transfects eukaryotic cells with the recombinant vector pWPI-S-RBD-SD-CP05, psPAX2 and pMD2.G together and packages the eukaryotic cells into lentiviruses.
The co-transfection method is not particularly limited in the present invention, and a transfection method well known in the art may be used. The type of eukaryotic cell transfected in the present invention is not particularly limited, and a mature cell line known in the art, for example, 293 cell, may be used.
After the lentivirus is obtained, the invention screens green fluorescent protein positive cells for continuous culture after the lentivirus infects eukaryotic cells, and establishes stable transfer cell strains.
In the present invention, the method for screening green fluorescent protein positive cells preferably employs flow cytometry screening. The method for transfecting the coding gene of the CP05-S protein into the eukaryotic cell can also adopt transient transfection to obtain a cell strain with high expression of the CP05-S protein.
After obtaining the stable cell strain, the invention cultures the stable cell strain, cracks the cell, and separates the protein lysate containing CP05-S protein.
In the present invention, the culture is preferably a serum replacement culture. The separation method preferably comprises centrifuging at 10000g centrifugal force for 5min to remove impurities such as cell debris, to obtain protein lysate containing CP05-S protein.
After the protein lysate containing the CP05-S protein is obtained, the protein lysate containing the CP05-S protein and human plasma are mixed and incubated, and an exosome is extracted to obtain the exosome of which the surface is coupled with the S protein.
In the invention, the volume ratio of the protein lysate containing CP05-S protein to human plasma is preferably 0.8-1.2: 0.8-1.2, and more preferably 1: 1. The incubation temperature is preferably 23-28 ℃, and more preferably 25 ℃; the incubation time is preferably 50-70 min, and more preferably 60 min. The incubation aims to enable the CD63 membrane molecule on the surface of the exosome to be combined with CP05 with high affinity, and the S protein and the exosome are sedimented together through centrifugation. The centrifugal force for extracting the exosome is preferably 80000-120000 g, and more preferably 100000 g. The centrifugation time for extracting the exosome is preferably 4-5 h, and more preferably 4 h.
The invention provides an exosome of a coupled S protein prepared by the preparation method. The exosome is combined with the S protein through a CD63 membrane molecule on the surface of the exosome. Experiments prove that compared with the method of directly injecting the S protein to present the antigen, the S protein attached to the exosome has better antigen presenting effect, and the titer of the antibody generated by the immune animal is higher.
The invention provides application of the S protein-coupled exosome in preparation of a vaccine for resisting a novel coronavirus. The exosome is adopted to replace the conventional virus particle, and in view of the biosafety and the antigen presenting function of the exosome, the exosome coupled with the S protein creates a safe and efficient immune adjuvant, and the immune effect of the exosome is better than that of a naked antigen.
The invention provides a novel inhibitor of coronavirus infection, comprising exosomes coupled with the S protein. The S protein is delivered by adopting an exosome, so that the combination of the new coronavirus S protein to a protein receptor can be competitively inhibited, a sealing effect is achieved, and the novel coronavirus S protein can be possibly used for acute-phase medication of the new coronavirus. The method for preparing the inhibitor is not particularly limited, and the preparation method of the biological product well known in the art can be adopted.
The following examples are provided to illustrate the surface-coupled S protein exosomes of the present invention and their preparation method and application, but they should not be construed as limiting the scope of the present invention.
Example 1
(1) Human cells express the CP05-S protein:
the specific method comprises the following steps: synthesizing a nucleotide sequence shown in SEQ ID No.1 by a gene synthesis company, and then inserting a Pme1 enzyme cutting site into a pWPI vector to construct a vector pWPI-S-RBD-SD-CP05, wherein the CP05 sequence is shown in SEQ ID No.2 (tgcaggcatagccagatgac ggtgacaagcaggct); then the recombinant vector pWPI-S-RBD-SD-CP05, psPAX2 and pMD2.G are transfected into 293 cells together to be packaged into lentivirus; infecting 293 cells with the obtained lentivirus, screening green fluorescent protein positive cells by adopting a flow cytometer, continuously culturing, and establishing a stable cell strain for later use.
(2) Extraction of protein containing CP05-S
Culturing the above-mentioned stably transformed cell strain in a culture medium containing 10% serum substitute, then directly centrifuging at 10000g for 5min to remove cell debris, and collecting the supernatant rich in CP05-S protein.
(3) Incubation of human plasma with supernatant containing CP05-S protein
Mixing human plasma and supernatant containing CP05-S protein at a volume ratio of 1:1, and shaking at room temperature for 1 h.
(4) Extraction of exosomes
And (3) carrying out ultrahigh-speed centrifugal extraction on the incubated exosome and protein incubation liquid for 4 hours under the action of 100000g of centrifugal force to obtain the exosome with the surface coupled with the S protein. The obtained exosome surface is exactly coupled with the S protein (shown in figure 1) by using a commercial S protein antibody Westernblot detection.
Example 2
Specifically, an immune mouse with exosome of which the surface is coupled with S protein, which is prepared by respectively adopting PBS, naked S protein and exosome immune mice of which the surface is coupled with S protein prepared in example 1, is injected with 200 micrograms (protein concentration) of exosome in the abdominal cavity, is continuously collected for 2 times in 1 week, and is immunized for three weeks to prepare antiserum, and the antiserum is subjected to antibody titer detection, wherein the specific method comprises the following steps:
the purified S protein extracellular region (purchased from Kinsley and other commercial antigens) is used as an antigen, and an antigen coating solution (50 mM carbonate coating buffer solution, pH 9.6) is used; the formula is NaHCO31.59g，NaHCO32.93g distilled water to 1000mL) to 5. mu.g/mL, and then 100. mu.L per well in a 96-well plate, coated overnight at 4 ℃ to attach the S protein extracellular region to the plate. Discarding the coating solution the next day, adding 200 μ L of 5% BSA for blocking, incubating at 37 ℃ for 2h, washing PBST, adding 100 μ L of primary antibody solution (shown in figure 1) diluted in multiple times with different titers (each titer is selected to be 3 times), incubating at 37 ℃ for 120min, washing PBST, adding 100 μ L of secondary antibody solution (diluted 1:1500, commercial peroxidase-labeled rabbit anti-mouse IgG) to each well, incubating at 37 ℃ for 60min, sufficiently washing PBST, adding 50 μ L of developing solution, and developing at 37 ℃ for 10min in a dark place; add stop solution 50. mu.L, OD 450nm of microplate reader.
The results are shown in FIG. 2. As can be seen from FIG. 2, the use of exosomes to present the S protein resulted in higher titers of antibody compared to the naked antigen and blank control. Meanwhile, according to the method for detecting the titer of the antiserum, the antiserum generated by exosome immunization of the surface coupled S protein can be used for detecting the novel coronavirus.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Sequence listing
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<120> exosome of surface coupled S protein, preparation method and application thereof
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attaccctga caaagttttc agatcctcag ttttacattc aactcaggac ttgttcttac 180
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agaggtttga taaccctgtc ctaccattta atgatggtgt ttattttgct tccactgaga 300
agtctaacat aataagaggc tggatttttg gtactacttt agattcgaag acccagtccc 360
tacttattgt taataacgct actaatgttg ttattaaagt ctgtgaattt caattttgta 420
atgatccatt tttgggtgtt tattaccaca aaaacaacaa aagttggatg gaaagtgagt 480
tcagagttta ttctagtgcg aataattgca cttttgaata tgtctctcag ccttttctta 540
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ttgatggtta ttttaaaata tattctaagc acacgcctat taatttagtg cgtgatctcc 660
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gttggacagc tggtgctgca gcttattatg tgggttatct tcaacctagg acttttctat 840
taaaatataa tgaaaatgga accattacag atgctgtaga ctgtgcactt gaccctctct 900
cagaaacaaa gtgtacgttg aaatccttca ctgtagaaaa aggaatctat caaacttcta 960
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cagattcatt tgtaattaga ggtgatgaag tcagacaaat cgctccaggg caaactggaa 1260
agattgctga ttataattat aaattaccag atgattttac aggctgcgtt atagcttgga 1320
attctaacaa tcttgattct aaggttggtg gtaattataa ttacctgtat agattgttta 1380
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aatgtgtcaa tttcaacttc aatggtttaa caggcacagg tgttcttact gagtctaaca 1680
aaaagtttct gcctttccaa caatttggca gagacattgc tgacactact gatgctgtcc 1740
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Claims (9)
1. A preparation method of an exosome with a surface coupled with an S protein is characterized by comprising the following steps:
1) the DNA fragment shown in SEQ ID No.1 is cut by enzyme Pme1 and then inserted into a pWPI vector to form a recombinant vector pWPI-S-RBD-SD-CP 05;
2) transfecting eukaryotic cells with the recombinant vector pWPI-S-RBD-SD-CP05, psPAX2 and pMD2.G together, and packaging into lentiviruses;
3) after the lentivirus infects eukaryotic cells, screening green fluorescent protein positive cells for continuous culture, and establishing a stable transfer cell strain;
4) culturing the stable cell strain, cracking the cell, and separating a protein lysate containing CP05-S protein;
5) and mixing and incubating the protein lysate containing the CP05-S protein and human plasma, and extracting an exosome to obtain the exosome with the surface coupled with the S protein.
2. The method according to claim 1, wherein the DNA fragment in step 1) comprises a nucleotide sequence encoding CP05-S protein; the nucleotide sequence of the code CP05-S protein is shown in SEQ ID No. 2.
3. The method according to claim 1, wherein the eukaryotic cells in step 2) comprise 293 cells.
4. The preparation method according to claim 1, wherein the volume ratio of the protein lysate containing CP05-S protein in step 5) to human plasma is 0.8-1.2: 0.8-1.2.
5. The preparation method according to claim 1, wherein the incubation temperature in the step 5) is 23-28 ℃; the incubation time is 50-70 min.
6. The preparation method according to any one of claims 1 to 5, wherein the centrifugal force of the extracted exosomes in the step 5) is 80000 to 120000g, and the centrifugal time of the extracted exosomes is 4 to 5 h.
7. An exosome coupled to an S protein prepared by the preparation method of any one of claims 1 to 5.
8. Use of an exosome coupled to the S protein of claim 7 in the preparation of a vaccine against a novel coronavirus.
9. A novel inhibitor of coronavirus infection comprising an exosome coupled with the S protein of claim 7.
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| CN202010418002.8A CN111647557A (en) | 2020-05-18 | 2020-05-18 | Exosome with surface coupled with S protein, and preparation method and application thereof |
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| CN202010418002.8A CN111647557A (en) | 2020-05-18 | 2020-05-18 | Exosome with surface coupled with S protein, and preparation method and application thereof |
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| CN112646781A (en) * | 2020-12-25 | 2021-04-13 | 广东省人民医院 | Exosome containing human ACE2 protein and application thereof |
| CN112933220A (en) * | 2020-11-25 | 2021-06-11 | 深圳市第二人民医院(深圳市转化医学研究院) | Nano vaccine of recombinant exosome |
| WO2022077968A1 (en) * | 2020-10-14 | 2022-04-21 | 苏州大学 | Targeted exosome based on rbd region of sars-cov-2 s protein and preparation method therefor |
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| WO2022077968A1 (en) * | 2020-10-14 | 2022-04-21 | 苏州大学 | Targeted exosome based on rbd region of sars-cov-2 s protein and preparation method therefor |
| CN112439058A (en) * | 2020-11-25 | 2021-03-05 | 深圳市第二人民医院(深圳市转化医学研究院) | Recombinant novel coronavirus nano vaccine method based on exosome as vector |
| CN112933220A (en) * | 2020-11-25 | 2021-06-11 | 深圳市第二人民医院(深圳市转化医学研究院) | Nano vaccine of recombinant exosome |
| CN112646781A (en) * | 2020-12-25 | 2021-04-13 | 广东省人民医院 | Exosome containing human ACE2 protein and application thereof |
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| CN112626030A (en) * | 2020-12-31 | 2021-04-09 | 深圳市第二人民医院 | Production method for neutralizing new coronavirus by using nano antibody of spinous process protein Spike displayed on surface of exosome |
| WO2022231116A1 (en) * | 2021-04-27 | 2022-11-03 | 주식회사 씨케이엑소젠 | Exosome platform-based antiviral vaccine |
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