CN111647557A - Exosome with surface coupled with S protein, and preparation method and application thereof - Google Patents

Abstract

The invention provides an exosome with surface-coupled coronavirus S protein, a preparation method and application thereof, and belongs to the technical field of biopharmaceuticals. A stable cell line was obtained by transient transfection of human cells or recombinant lentivirus infection to achieve recombinant expression of the aptamer CP05-S protein, which was then incubated with human plasma. S-protein-linked exosomes. The invention uses exosomes to present antigen S protein, so that the prepared vaccine avoids the toxicity of immune adjuvant and the risk of virus vaccine infection, and is an ideal vaccine development strategy and an ideal strategy for closing the virus invasion route. At the same time, the delivery of S protein through exosomes can competitively inhibit the binding of the S protein of the new coronavirus to the receptor, play a blocking effect, and effectively block the invasion of the new coronavirus. Therefore, the exosomes coupled with the S protein can be used as an inhibitor of the new coronavirus infection. , may be used for the treatment of acute stage new coronary pneumonia.

Description

A kind of surface-coupled S protein exosome and preparation method and application thereof

technical field

The invention belongs to the technical field of biopharmaceuticals, and in particular relates to a surface-coupled S protein exosome and a preparation method and application thereof.

Background technique

At present, there is no confirmed and effective anti-disease treatment method for the clinical treatment of new coronary pneumonia. Interferon alpha, protease inhibitors or nucleotide ribavirin can be used, but the above treatment strategies have certain limitations, except for pregnant women. In addition to careful use with children and the elderly, patients with allergies to interferon and excipients, patients with autoimmune diseases, patients with heart, liver disease, renal insufficiency or bone marrow dysfunction, and patients with epilepsy and central nervous system function Not applicable to injured persons. Therefore, the task of developing new drugs to treat the new coronavirus is very urgent.

SUMMARY OF THE INVENTION

In view of this, the object of the present invention is to provide a surface-coupled S protein exosome and its preparation method and application, and the exosome has better antigen presentation effect and ideal immune effect.

The present invention provides a kind of preparation method of the exosome of surface coupling S protein, comprises the following steps:

1) insert the DNA fragment shown in SEQ ID No.1 into the pWPI vector after Pme1 digestion, to form the recombinant vector pWPI-S-RBD-SD-CP05;

2) The recombinant vector pWPI-S-RBD-SD-CP05 and psPAX2, pMD2.G were co-transfected into eukaryotic cells, and packaged into lentivirus;

3) after infecting the eukaryotic cells with the lentivirus, screening green fluorescent protein-positive cells to continue culturing to establish a stable transfection cell line;

4) culturing the stably transfected cell line, lysing the cells, and separating the protein lysate containing the CP05-S protein;

5) Mixing and incubating the protein lysate containing CP05-S protein and human plasma to extract exosomes to obtain surface-coupled S protein exosomes.

Preferably, the DNA fragment in step 1) comprises the nucleotide sequence encoding the CP05-S protein; the nucleotide sequence encoding the CP05-S protein is shown in SEQ ID No.2.

Preferably, the eukaryotic cells in step 2) include 293 cells.

Preferably, the volume ratio of the protein lysate containing CP05-S protein and human plasma in step 5) is 0.8-1.2:0.8-1.2.

Preferably, the incubation temperature in step 5) is 23-28°C; the incubation time is 50-70 min.

Preferably, the centrifugal force for extracting exosomes in step 5) is 80,000-120,000 g, and the centrifugation time for extracting exosomes is 4-5h.

The present invention provides the S protein-coupled exosomes prepared by the preparation method.

The present invention provides the application of the S protein-coupled exosome in the preparation of a vaccine against novel coronavirus.

The present invention provides a novel coronavirus infection inhibitor, including the exosome coupled to the S protein.

The preparation method of the surface-coupled S protein exosome provided by the present invention is to obtain a stably transfected cell line stably expressing the CP05-S protein through virus infection, recombinantly express the CP05-S protein, and combine the extracted CP05-S protein with human plasma. Mix and incubate to extract exosomes. Since there are CD63 membrane molecules on the surface of exosomes, they can bind with CP05-S protein with high affinity. During the incubation process, CP05-S protein binds to exosomes and settles together to obtain surface-coupled proteins. S-protein-linked exosomes. The method provided by the present invention eliminates complicated protein purification steps and simplifies the operation. Compared with the surface-coupled S protein exosomes prepared by direct protein injection, the protein attached to the exosomes has better antigen presentation effect; at the same time, the exosomes are derived from human plasma products, The clinical application is good, plasma-derived exosomes are safe as carriers, and the in vivo action rules of exosomes are similar to those of virus particles, which can better play a sealing role. Therefore, exosomes present antigens, avoiding the toxicity of immune adjuvants (traditional protein + adjuvant immunization) and the risk of virus (traditional attenuated or inactivated vaccines) infection.

The present invention provides the application of the S protein-coupled exosome in the preparation of a vaccine against novel coronavirus. This application immunizes mice with surface-coupled S protein exosomes. The test results show that compared with naked S protein and blank control, the immunized mice can produce ideal immune effects in vivo, and the generated antibodies can be used to detect S protein. protein. It can be seen that the engineering of S protein loaded on the surface of exosomes to prepare vaccines is an ideal vaccine development strategy and an ideal strategy for blocking the virus invasion pathway.

The present invention also provides a novel coronavirus infection inhibitor, including the exosome coupled to the S protein. The present invention delivers the S protein through exosomes, which can competitively inhibit the binding of the S protein of the new coronavirus to its receptor, play a role in blocking the virus, and may be used for the medicine of acute stage new coronary pneumonia.

Description of drawings

Figure 1 shows the results of Western blot detection of S protein coupled to the surface of exosomes;

Fig. 2 is a graph showing the results of titer detection of antisera obtained after immunizing mice with different antigens.

Detailed ways

The present invention provides a kind of preparation method of the exosome of surface coupling S protein, comprises the following steps:

1) insert the DNA fragment shown in SEQ ID No.1 into the pWPI vector after Pme1 digestion, to form the recombinant vector pWPI-S-RBD-SD-CP05;

2) The recombinant vector pWPI-S-RBD-SD-CP05 and psPAX2, pMD2.G were co-transfected into eukaryotic cells, and packaged into lentivirus;

3) after infecting the eukaryotic cells with the lentivirus, screening green fluorescent protein-positive cells to continue culturing to establish a stable transfection cell line;

4) culturing the stably transfected cell line, lysing the cells, and separating the protein lysate containing the CP05-S protein;

5) Mixing and incubating the protein lysate containing CP05-S protein and human plasma to extract exosomes to obtain surface-coupled S protein exosomes.

In the present invention, the DNA fragment shown in SEQ ID No. 1 is digested by Pme1 and inserted into the pWPI vector to form a recombinant vector pWPI-S-RBD-SD-CP05.

In the present invention, the DNA fragment preferably comprises a nucleotide sequence encoding CP05-S protein; the nucleotide sequence encoding CP05-S protein is preferably as shown in SEQ ID No. 2. The present invention does not specifically limit the method for inserting the digested DNA fragment into the pWPI vector, and a ligation method well known in the art can be used.

After obtaining the recombinant vector pWPI-S-RBD-SD-CP05, the present invention co-transfects the recombinant vector pWPI-S-RBD-SD-CP05 with psPAX2 and pMD2.G into eukaryotic cells, and packaged into lentivirus.

The present invention has no particular limitation on the co-transfection method, and a transfection method well known in the art can be used. In the present invention, there is no particular limitation on the type of eukaryotic cells to be transfected, and mature cell lines well known in the art can be used, such as 293 cells.

After obtaining the lentivirus, the present invention infects the eukaryotic cells with the lentivirus, and then selects green fluorescent protein-positive cells to continue culturing to establish a stably transfected cell line.

In the present invention, the method for screening green fluorescent protein-positive cells preferably adopts flow cytometry screening. The method for transfecting the coding gene of CP05-S protein into eukaryotic cells of the present invention can also adopt transient transfection to obtain a cell line with high expression of CP05-S protein.

After obtaining the stably transfected cell line, in the present invention, the stably transfected cell line is cultured, the cells are lysed, and the protein lysate containing the CP05-S protein is separated.

In the present invention, the culture is preferably cultured with serum replacement. The separation method is preferably centrifuged at a centrifugal force of 10000g for 5min to remove impurities such as cell debris to obtain a protein lysate containing CP05-S protein.

After obtaining the protein lysate containing the CP05-S protein, the present invention mixes and incubates the protein lysate containing the CP05-S protein and human plasma, extracts exosomes, and obtains the surface-coupled exosomes of the S protein.

In the present 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, more preferably 1:1. The incubation temperature is preferably 23-28°C, more preferably 25°C; the incubation time is preferably 50-70 min, more preferably 60 min. The purpose of the incubation is to make the CD63 membrane molecules on the surface of the exosomes bind with CP05 with high affinity, and the S protein and the exosomes are precipitated together by centrifugation. The centrifugal force for extracting exosomes is preferably 80,000-120,000 g, more preferably 100,000 g. The centrifugation time for extracting exosomes is preferably 4 to 5 hours, more preferably 4 hours.

The present invention provides the S protein-coupled exosomes prepared by the preparation method. The exosomes bind to the S protein through the CD63 membrane molecules on the surface of the exosomes. Experiments have shown that compared with the direct injection of S protein to present antigen, the S protein attached to exosomes has better antigen presentation effect, and the antibody titer produced by immunized animals is higher.

The present invention provides the application of the S protein-coupled exosome in the preparation of a vaccine against novel coronavirus. Exosomes are used to replace conventional virus particles. In view of the biosafety and antigen presentation function of exosomes, the S protein-coupled exosomes innovate a safe and efficient immune adjuvant, and its immune effect is better than Naked antigen is better.

The present invention provides a novel coronavirus infection inhibitor, including the exosome coupled to the S protein. The use of exosomes to deliver the S protein can competitively inhibit the binding of the new coronavirus S protein to the protein receptor and play a blocking role, which may be used for the acute phase of the new coronavirus. In the present invention, there is no special limitation on the preparation method of the inhibitor, and the preparation method of biological products well-known in the art can be used.

Below in conjunction with embodiment, a kind of surface-coupled S protein exosome provided by the present invention and its preparation method and application are described in detail, but they can not be construed as the limitation to the protection scope of the present invention.

Example 1

(1) Human cells express CP05-S protein:

The specific method is as follows: the following nucleotide sequence shown in SEQ ID No.1 is synthesized by Gene Synthesis Company, and then inserted into the pWPI vector using the Pme1 restriction site, and the constructed vector is pWPI-S-RBD-SD-CP05, wherein The sequence of CP05 is shown in SEQ ID No.2 (tgcaggcatagccagatgac ggtgacaagcaggct); then the recombinant vector pWPI-S-RBD-SD-CP05, psPAX2 and pMD2.G were co-transfected into 293 cells and packaged into lentivirus; the obtained lentivirus was infected 293 cells were screened by flow cytometry for green fluorescent protein-positive cells and continued to be cultured to establish a stably transfected cell line for use.

(2) Extract the protein containing CP05-S

The above stably transfected cell line was cultured with a medium containing 10% serum replacement, and then directly centrifuged at 10,000 g for 5 min to remove cell debris, and the supernatant rich in CP05-S protein was collected.

(3) Incubation of human plasma and supernatant containing CP05-S protein

Human plasma and CP05-S protein-containing supernatant were mixed in a volume ratio of 1:1 and shaken for 1 h at room temperature.

(4) Extracting exosomes

The incubation solution of the above-incubated exosomes and proteins was extracted by ultra-high-speed centrifugation for 4 h under the action of 100,000 g centrifugal force to obtain surface-coupled S protein exosomes. Using the commercialized S protein antibody Westernblot detection, it was found that the surface of the obtained exosomes was indeed coupled to the S protein (see Figure 1).

Example 2

The mice were immunized with PBS, naked S protein and the surface-coupled S protein exosomes prepared in Example 1, specifically intraperitoneal injection of 200 micrograms (protein concentration) of exosomes, once a week, twice in a row, Three weeks after immunization, blood was collected to prepare antiserum, and the antiserum was subjected to antibody titer detection. The specific method is as follows:

First, the purified S protein extracellular region (purchased from a commercialized antigen such as GenScript) was used as the antigen, and the antigen coating solution (50 mM carbonate coating buffer, pH 9.6) was used; the formula is: NaHCO ₃ 1.59g, NaHCO ₃ 2.93g and distilled water to 1000ml) were diluted to 5μg/mL, and then 100μL was added to each well of a 96-well plate, and coated overnight at 4°C, so that the extracellular region of S protein was attached to the plate. The next day, the coating solution was discarded, 200 μL of 5% BSA was added to block, and incubated at 37°C for 2 h. After washing with PBST, 100 μL of doubling diluted primary antibody solutions of different titers (see Figure 1) were added to each well (that is, mouse immunization). Serum, 3 replicates for each titer), incubate at 37°C for 120 min, wash with PBST, add 100 μL of secondary antibody solution (1:1500 dilution, commercial peroxidase-labeled rabbit anti-mouse IgG) to each well, 37°C Incubate for 60 min, wash thoroughly with PBST, add 50 μL of color developing solution, and develop color at 37°C for 10 min in the dark; add 50 μL of stop solution, and measure the OD value at 450 nm of the microplate reader.

The results are shown in Figure 2. As can be seen from Figure 2, the use of exosomes to present the S protein produced higher antibody titers compared with the naked antigen and blank control group. At the same time, according to the above-mentioned antiserum titer detection method, the antiserum produced by exosome immunization with surface-coupled S protein can be used to detect the new coronavirus.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

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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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