Respiratory syncytial virus membrane protein and preparation method thereof

Abstract

The invention discloses a preparation method of a respiratory syncytial virus membrane protein, which at least comprises the following steps: taking cells containing RSV (Respiratory Syncytial Virus), splitting, centrifuging, and taking precipitate; adding the precipitate into a first buffer solution containing a detergent, incubating, centrifuging, and collecting supernate; wherein the mass concentration of the detergent in the first buffer solution is 0.5-1.0%, and the mass ratio of the precipitate to the first buffer solution is 1: (10-50); putting the supernatant into a dialysis bag, dialyzing in a second buffer solution, taking out dialysate after dialysis, centrifuging, and collecting the supernatant; carrying out cation chromatography on the dialyzed supernatant, and collecting the eluent; carrying out ultrafiltration concentration on the eluent to obtain a concentrated solution; and carrying out molecular sieve chromatography on the concentrated solution to obtain the respiratory syncytial virus membrane protein. The preparation method disclosed by the invention can be used for successfully extracting the RSV membrane protein with relatively high purity, has important significance and value for RSV structure confirmation and verification, and can also be popularized to extraction and purification of other macromolecular membrane proteins.

Description

Technical Field

[0001] The present invention belongs to the technical field of membrane protein extraction and purification, and particularly relates to a respiratory syncytial virus membrane protein and a preparation method thereof. Background Art

[0002] Respiratory syncytial virus (RSV) is one of the main causes of hospitalization for infants, the elderly, patients in long-term care facilities, and other vulnerable patients (such as immunocompromised patients, transplant patients, patients with chronic lung and / or heart diseases, actively treated cancer patients, and asthma / chronic obstructive pulmonary disease patients).

[0003] Currently, nirsevimab (Beyfortus) has been approved for the prevention of respiratory syncytial virus (RSV) in all infants during their first season of life, and the monoclonal antibodies palivizumab (Synagis) and nirsevimab (Beyfortus) are approved for the prevention of RSV in high-risk infants during the second season. They have shown 40% to 80% effectiveness in preventing hospitalization, depending on the underlying risk profile. Currently, two RSV vaccines are approved for protecting the elderly and young children through maternal immunization in the third trimester of pregnancy, and one vaccine has recently been approved for the elderly in the United States. However, only ribavirin, whether administered by nebulization or orally, despite safety concerns and complex efficacy, is still approved for treatment in some countries. Therefore, there is an obvious unmet medical need for the treatment of RSV.

[0004] In recent decades, the production technology of mRNA has gradually matured, and lipid nanoparticles (LNPs) have become one of the effective carriers for in vivo delivery of mRNA. Compared with traditional polypeptide and protein vaccines, mRNA vaccines have a simple production process, a short R & D cycle, low cost, are convenient for standardized production, and are suitable for vaccine development and production during large-scale epidemics and infectious disease outbreaks. They are vaccines with broad application prospects.

[0005] After the RSV vaccine produced using mRNA technology is injected into the human body, the protein structure and function produced cannot be accurately and clearly understood. Therefore, after the mRNA vaccine is transferred into cells, it is necessary to extract high-purity RSV protein for structure verification to facilitate a more accurate determination of what may occur after the vaccine enters the human body. Currently, a method for extracting and purifying high-purity RSV membrane protein is urgently needed to be established.

[0006] The information disclosed in this background art section is only intended to increase the understanding of the overall background of the present invention and should not be regarded as an admission or any form of implication that this information constitutes prior art already known to those of ordinary skill in the art. Summary of the Invention

[0007] The object of the present invention is to provide a respiratory syncytial virus membrane protein and a preparation method thereof, and the preparation method can extract RSV membrane protein with high purity.

[0008] In order to achieve the above object, the technical solution provided by a specific embodiment of the present invention is as follows:

[0009] A preparation method of respiratory syncytial virus membrane protein, at least including:

[0010] Take cells containing RSV, lyse them and centrifuge, and take the precipitate;

[0011] Add the precipitate to the first buffer containing a detergent and incubate, centrifuge and collect the supernatant; wherein, the mass concentration of the detergent in the first buffer is 0.5-1.0%, and the mass ratio of the precipitate to the first buffer is 1:(10-50);

[0012] Load the supernatant into a dialysis bag, perform dialysis in the second buffer, take out the dialysis solution after dialysis, centrifuge and collect the supernatant;

[0013] Perform cation exchange chromatography on the dialyzed supernatant and collect the eluate;

[0014] Ultrafiltrate and concentrate the eluate to obtain a concentrated solution;

[0015] Perform size exclusion chromatography on the concentrated solution to obtain respiratory syncytial virus membrane protein.

[0016] In one or more embodiments of the present invention, the detergent is at least one of LMNG / CHS, TritionX-100, and DDM.

[0017] In one or more embodiments of the present invention, after the precipitate is mixed with the first buffer, it is incubated on ice for 0.8-1.2 h, and after incubation, it is centrifuged at a speed of 14000-16000 g for 30-60 min.

[0018] In one or more embodiments of the present invention, the cut-off molecular weight of the dialysis bag used in the dialysis operation is less than 100KDa.

[0019] In one or more embodiments of the present invention, the chromatography packing material used for cation exchange chromatography is Capto SPFF, Capto S ImpAct or Capto SPImpRes.

[0020] In one or more embodiments of the present invention, the multiple of ultrafiltration concentration is 3-5 times.

[0021] In one or more embodiments of the present invention, the chromatography column used for size exclusion chromatography is Superdex200pg or Superdex 200Increase.

[0022] In one or more embodiments of the present invention, the lysis operation is as follows: mix the cells with a third buffer solution, add sucrose to make the final concentration of sucrose in the solution 3-5% (v / v), and then perform lysis by repeated freezing and thawing.

[0023] In one or more embodiments of the present invention, after the cell lysis, centrifuge at a speed of 14000-16000g for 30-60 min.

[0024] A respiratory syncytial virus membrane protein is prepared by the preparation method of the above-mentioned respiratory syncytial virus membrane protein.

[0025] Compared with the prior art, the preparation method of the respiratory syncytial virus membrane protein of the present invention is simple in operation, and the extracted protein has a relatively high purity. The purified protein can be used to detect the structure, which has important significance and value for the confirmation and verification of the RSV structure, and this method can also be extended to the extraction and purification of other macromolecular membrane proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only some embodiments recorded in the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0027] Figure 1 Results of RSV membrane protein extraction in Example 1, Example 2 and Comparative Example 1 of the present invention;

[0028] Figure 2 Results of the first SDS-Page detection of the sample obtained after cation exchange chromatography in Example 1 of the present invention;

[0029] Figure 3 Results of the second SDS-Page detection of the sample obtained after cation exchange chromatography in Example 1 of the present invention;

[0030] Figure 4 Results of the third SDS-Page detection of the sample obtained after cation exchange chromatography in Example 1 of the present invention;

[0031] Figure 5 Results of the first WB detection of the sample obtained after cation exchange chromatography in Example 1 of the present invention;

[0032] Figure 6 Results of the second WB detection of the sample obtained after cation exchange chromatography in Example 1 of the present invention;

[0033] Figure 7 This is the result of the third WB test on the sample obtained after cation exchange chromatography in Example 1 of the present invention;

[0034] Figure 8 This is the result of the first SDS-Page test on the sample obtained after size exclusion chromatography in Example 1 of the present invention;

[0035] Figure 9 This is the result of the second SDS-Page test on the sample obtained after size exclusion chromatography in Example 1 of the present invention;

[0036] Figure 10 This is the result of the third SDS-Page test on the sample obtained after size exclusion chromatography in Example 1 of the present invention;

[0037] Figure 11 This is the result of the first WB test on the sample obtained after size exclusion chromatography in Example 1 of the present invention;

[0038] Figure 12 This is the result of the second WB test on the sample obtained after size exclusion chromatography in Example 1 of the present invention;

[0039] Figure 13 This is the result of the third WB test on the sample obtained after size exclusion chromatography in Example 1 of the present invention. Detailed implementation mode

[0040] In order to enable those skilled in the art to better understand the technical solutions in the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0041] A specific implementation mode of the present invention provides a method for preparing respiratory syncytial virus membrane protein, including steps 1-8.

[0042] Step 1, obtain cells containing RSV.

[0043] Specifically, the cells containing RSV can be obtained by transfection with a preparation. The obtained cells are mixed with a third buffer solution and stored in a -80°C refrigerator. The third buffer solution is specifically a 25 mM acetic acid-sodium acetate solution with a pH of 5.5.

[0044] Step 2, take the cells containing RSV, lyse them and centrifuge, and take the precipitate.

[0045] Specifically, the cells are taken out of the refrigerator and thawed at room temperature. After complete thawing, they are frozen again in an -80°C refrigerator. After repeating this process several times, they are thawed. At this time, the cells are broken, and the intracellular substances are dissolved in the third buffer. Then, centrifugation is carried out at a speed of 14,000 - 16,000 g for 30 - 60 min. The supernatant is discarded, and the precipitate is retained. The precipitate contains the broken cell membranes.

[0046] Furthermore, to prevent degradation during the freeze-thaw process, an appropriate amount of sucrose needs to be added to the cell solution to protect the proteins. Specifically, a protective solution of 25 mM acetic acid-sodium acetate and 50% (w / w) sucrose is prepared. The pH of the protective solution is 5.5. According to the final concentration of sucrose in the cell solution being 3 - 5% (v / v), the protective solution is added to the cell solution. Here, 50% (w / w) sucrose means that every 100 g of the solution contains 50 g of sucrose, and 3 - 5% (v / v) means that every 100 mL contains 3 - 5 mL of sucrose.

[0047] Step 3: Add the precipitate to the first buffer containing a detergent and incubate. After centrifugation, collect the supernatant; among them, the mass concentration of the detergent in the first buffer is 0.5 - 1.0%, and the mass ratio of the precipitate to the first buffer is 1:(10 - 50).

[0048] Specifically, the detergent is at least one of LMNG / CHS, TritionX-100, and DDM. By selecting the above types of detergents and limiting the mass concentration and dosage of the detergent, the RSV membrane protein can be effectively extracted and the RSV membrane protein can be fully dissolved in the solution.

[0049] Furthermore, after mixing the precipitate with the first buffer, incubate on ice for 0.8 - 1.2 h. After incubation, centrifuge at a speed of 14,000 - 16,000 g for 30 - 60 min. Centrifuging at an appropriate speed can effectively remove insoluble substances such as cell membrane tissues, which is beneficial to obtaining a higher-purity RSV membrane protein subsequently.

[0050] Step 4: Load the supernatant into a dialysis bag and place it in the second buffer for dialysis. After dialysis, centrifuge the dialysis solution and collect the supernatant.

[0051] Specifically, the second buffer is a 25 mM acetic acid-sodium acetate, 0.1% TritonX-100 solution, with a pH of 5.5. TritonX-100 can also be replaced by LMNG / CHS or DDM. The supernatant and the second buffer are dialyzed at a mass ratio of 1:(20 - 100) and dialyzed overnight in a 2 - 8°C refrigerator. The cut-off molecular weight of the dialysis bag used for dialysis is less than 100 KDa, and the centrifugation conditions are 10,000 - 15,000 g for 10 min. Dialyzing the supernatant first can simply and quickly remove impurities preliminarily and at the same time reduce the possibility of protein denaturation or inactivation.

[0052] Step 5: Subject the supernatant after dialysis to cation exchange chromatography and collect the eluate.

[0053] Specifically, the chromatography packing material used for cation exchange chromatography is Capto SP FF, Capto S ImpAct or Capto SPImpRes. Cation exchange chromatography is used to further remove impurities to improve the purity of the RSV membrane protein. In the steps of chromatography that contact the sample, such as equilibration, washing, and elution, the solutions all contain detergents. If conventional solutions without detergents are used, there is a risk of product precipitation, resulting in column clogging and experimental failure. In this step, the added content of the detergent is equal to or slightly less than that in Step 3. If the added content of the detergent is further reduced, a stability test needs to be conducted in advance to ensure that the RSV membrane protein will not precipitate.

[0054] Furthermore, during chromatography, a solution containing 0.1% Triton X-100 is selected for chromatography. Triton X-100 can also be replaced by LMNG / CHS or DDM. Selecting a specific solution for chromatography can ensure that the eluate contains a relatively high amount of RSV membrane protein.

[0055] Step 6: Ultrafilter and concentrate the eluate to obtain a concentrated solution.

[0056] Specifically, the eluate is concentrated using an ultrafiltration tube with a cut-off molecular weight below 100 KDa, and the concentration factor is 3 to 5 times to concentrate the RSV membrane protein and remove small molecule impurities. Before concentration, the ultrafiltration tube is wetted with the cation exchange chromatography equilibration solution to prevent precipitation of the RSV membrane protein.

[0057] Step 7: Subject the concentrated solution to size exclusion chromatography to obtain the respiratory syncytial virus membrane protein.

[0058] Specifically, the chromatography column used for size exclusion chromatography is Superdex 200 pg or Superdex 200 Increase. As the last step for purifying the membrane protein, by selecting the above types of chromatography columns, the membrane protein can be efficiently separated, further improving the purity of the membrane protein.

[0059] Another specific embodiment of the present invention provides a respiratory syncytial virus membrane protein prepared by the above-described method for preparing a respiratory syncytial virus membrane protein.

[0060] The following further elaborates on the present invention with specific examples.

[0061] Example 1

[0062] The method for preparing a respiratory syncytial virus membrane protein includes:

[0063] (1) Formulation transfection

[0064] Culture HEK293 cells. When the confluence reaches 90%, seed the cells in a T225 cell flask and incubate in a 37°C incubator for 20 h. Prepare a dilution of the transfection reagent Lipo. Mix RSV in the form of mRNA with the Lipo dilution to obtain the transfection working solution, and then evenly add it to the seeded cells. Gently mix and place in a 37°C, CO 2 incubator for culture. After 24 h of transfection, take out the transfected cells, discard the culture medium supernatant, and wash the cells.

[0065] (2) Centrifugation and washing

[0066] Centrifuge the collected cells, wash the cells to remove impurities in the culture medium and change the solution system. The centrifugation conditions are 500 g for 10 min. Pour off the upper layer solution, add 25 mM acetic acid - sodium acetate, pH 5.5 solution to the precipitate. Add 2 mL of the solution to each T225 cell flask. Centrifuge again under the same conditions and wash the cells twice repeatedly. Then add the solution to the centrifuge tube. Additionally, prepare 25 mM acetic acid - sodium acetate, 50% (w / w) sucrose, pH 5.5 solution. Add it to the cell solution according to 5% (v / v) sucrose in the final sample, mix well, and freeze the cells in an -80°C refrigerator.

[0067] (3) Freeze - thaw lysis

[0068] Take the washed cells out of the -80°C refrigerator, thaw them at room temperature. After complete thawing, freeze them again in the -80°C refrigerator. Repeat this three times, then thaw. The cells are lysed and the intracellular substances are dissolved in the solution. Centrifuge at 15000 g for 30 min, discard the supernatant, and retain the precipitate, which contains the broken cell membrane.

[0069] (4) Extract membrane proteins with detergent

[0070] Weigh the mass of the centrifuged precipitate and add the prepared 25 mM acetic acid - sodium acetate, 1% Triton X - 100, 300 mM sodium chloride, pH 5.5 solution according to a mass ratio of 1:20. Mix well and place in an ice box. Put the ice box on a shaker and incubate for 1 h. After incubation, centrifuge at 15000 g for 30 min. Take the supernatant after centrifugation and discard the precipitate.

[0071] (5) Dialysis

[0072] Prepare a 25 mM acetic acid - sodium acetate, 0.1% Triton X - 100, pH 5.5 solution. According to a mass ratio of 1:50, pour the centrifuged supernatant into a dialysis bag with a molecular weight cut - off of 10 KDa. Place the dialysis bag in the weighed solution and dialyze overnight in a 5°C refrigerator. After dialysis, measure the conductivity of the sample. If the conductivity is within the range of the conductivity of the cation exchange chromatography equilibration buffer, it can be centrifuged for the next step; if not, replace the dialysis solution and continue dialysis until the conductivity is within the range. After dialysis, if the sample is turbid, centrifuge it under the conditions of 10000 g for 10 min, take the supernatant, and perform the next chromatography step.

[0073] (6) Cation exchange chromatography

[0074] Perform cation - binding mode chromatography on the supernatant sample after dialysis and centrifugation. The chromatography packing material is Capto SPFF, and the chromatography parameters are shown in Table 1. To collect a sample with higher purity, maintain isocratic elution when the B pump is at 20%. When the UV value decreases, that is, after the UV in the chromatogram drops and levels off, continue to increase the gradient.

[0075] Table 1 Cation exchange chromatography parameters

[0076]

[0077]

[0078] (7) Ultrafiltration concentration

[0079] After combining the cation exchange chromatography elution samples containing the target protein, concentrate them using a 30 KDa ultrafiltration tube. Before concentration, moisten the ultrafiltration tube with the cation exchange chromatography equilibration buffer to prevent membrane protein precipitation, and the concentration factor is 3 times.

[0080] (8) Size - exclusion chromatography

[0081] After concentration, filter the sample and load it. Select a Superdex 200 Increase chromatography column, and the chromatography parameters are shown in Table 2.

[0082] Table 2 Size - exclusion chromatography parameters

[0083]

[0084] Example 2

[0085] The difference between this example and Example 1 is only that in step (4) for extracting membrane proteins with detergents, the solution used is a 25 mM acetic acid - sodium acetate, 0.5% Triton X - 100, 300 mM sodium chloride, pH 5.5 solution.

[0086] Example 3

[0087] The difference between this example and Example 1 is only that in step (8) of molecular sieve chromatography, a Superdex 200pg chromatography column is used.

[0088] Comparative Example 1

[0089] The difference between this comparative example and Example 1 is only that in step (4) of detergent extraction of membrane proteins, Triton X-100 is not added when preparing the solution.

[0090] The sources of the main materials and reagents used in the present invention are shown in Table 3, and the specific preparation formulas of the solutions used are shown in Table 4, where the only function of acetic acid is to adjust the pH of the solution.

[0091] Table 3 Sources of Materials and Reagents

[0092]

[0093]

[0094] Table 4 Solution Preparation Formulas

[0095]

[0096] The results of detecting the preparation methods of respiratory syncytial virus membrane proteins in each example and comparative example are as follows:

[0097] (1) RSV Membrane Protein Extraction Results

[0098] Take the whole cell lysate, supernatant and precipitate after centrifugation of the cell mixture after freeze-thaw disruption in step (3) of Example 1, the whole cell lysate, supernatant and precipitate after incubation in step (4) of Example 1, the whole cell lysate, supernatant and precipitate after incubation in step (4) of Example 2, and the whole cell lysate, supernatant and precipitate after incubation in step (4) of Comparative Example 1, and perform WB experiments (Western blotting). The results are as Figure 1 shown, Figure 1 where M refers to PageRuler Prestained Protein Marker.

[0099] It can be seen from Figure 1 that after freeze-thaw disruption, RSV membrane proteins are present in the whole cell lysate, supernatant and precipitate. Since there are some incompletely glycosylated RSV membrane proteins in the cells, and the molecular weights of these incompletely glycosylated membrane proteins are relatively close to those of the glycosylated membrane proteins, corresponding bands are also present in the whole cell lysate and supernatant. After extraction with 0.5% Triton X-100 and 1% Triton X-100, the corresponding bands in the whole cell lysate and supernatant are clearer, indicating that the RSV membrane proteins have been successfully extracted.

[0100] (2) Results of cation exchange chromatography purification

[0101] In Examples 1 - 3, after dialysis, the samples were turbid. After centrifugation, the precipitate and supernatant were respectively detected, and it was found that there was basically no precipitate of RSV membrane protein. The supernatant was used for cation exchange chromatography. During elution, the samples were collected in separate tubes, and each tube of sample was subjected to SDS-Page and WB detection, with each detection repeated 3 times. It was determined from the detection results of Examples 1 - 3 that the RSV membrane protein was successfully eluted. The detection results of Example 1 are as shown in Figures 2-7 . According to the detection results, it was confirmed that RSV membrane protein with relatively high purity could be collected when the elution gradient conductivity was between 6.8 - 23.0 mS / cm (the proportion of pump B was between 10 - 23%).

[0102] (3) Results of size exclusion chromatography purification

[0103] In Examples 1 and 3, during size exclusion chromatography, the samples were collected in separate tubes, and each tube of sample was subjected to SDS-Page and WB detection, with each detection repeated 3 times. According to the WB detection results, when using a Superdex 200 Increase chromatography column, the sample collection time was 10.80 - 12.60 min; when using a Superdex 200 pg chromatography column, the sample collection time was 47.00 - 57.50 min. At the same time, combined with the SDS-Page results, the samples collected in the above stages had relatively high purity, clear bands, and no impurity bands, meeting the requirements for structural verification.

[0104] In summary, after extraction with detergent, dialysis, cation exchange chromatography, and size exclusion chromatography in the present invention, the RSV membrane protein obtained was detected by SDS-Page, showing no obvious impurity bands and clear target protein bands, proving its relatively high purity and being suitable for structural verification. Moreover, the method in the present invention is simple to operate, and the materials used are relatively common, applicable to the extraction and purification of various macromolecular membrane proteins. Therefore, the method disclosed in the present invention is of great significance and value for the extraction of RSV membrane protein and other macromolecular membrane proteins.

[0105] For those skilled in the art, it is obvious that the present invention is not limited to the details of the above exemplary embodiments, and without departing from the spirit or basic characteristics of the present invention, the present invention can be implemented in other specific forms. Therefore, in any regard, the embodiments should be regarded as exemplary and non-limiting. The scope of the present invention is defined by the appended claims rather than the above description. Therefore, all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be encompassed within the present invention. Any reference signs in the claims should not be regarded as limiting the claimed rights.

[0106] In addition, it should be understood that although this specification is described in terms of embodiments, not every embodiment contains only one independent technical solution. This narrative style of the specification is only for clarity. Those skilled in the art should regard the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing respiratory syncytial virus membrane protein, characterized in that: At least: Take the cells containing RSV, lyse them, centrifuge them, and take the precipitate; The precipitate is added to a first buffer solution containing a detergent for incubation, and the supernatant is collected after centrifugation; wherein the mass concentration of the detergent in the first buffer solution is 0.5-1.0%, and the mass ratio of the precipitate to the first buffer solution is 1:(10-50); The supernatant is placed in a dialysis bag and dialyzed in a second buffer solution. After dialysis, the dialyzate is taken out and the supernatant is collected after centrifugation. The dialyzed supernatant was subjected to cation chromatography, and the eluate was collected; The eluate is concentrated by ultrafiltration to obtain a concentrate; The concentrated solution was subjected to molecular sieve chromatography to obtain respiratory syncytial virus membrane protein.

2. The method for preparing respiratory syncytial virus membrane protein according to claim 1, characterized in that: The detergent is at least one of LMNG / CHS, TritionX-100 and DDM.

3. The method for preparing respiratory syncytial virus membrane protein according to claim 1, characterized in that: The precipitate is mixed with the first buffer solution and then incubated on ice for 0.8 to 1.2 hours. After incubation, the precipitate is centrifuged at a speed of 14,000 to 16,000 g for 30 to 60 minutes.

4. The method for preparing respiratory syncytial virus membrane protein according to claim 1, characterized in that: The molecular weight cut-off of the dialysis bag used in the dialysis operation is less than 100 KDa.

5. The method for preparing respiratory syncytial virus membrane protein according to claim 1, characterized in that: The chromatographic filler used in the cationic chromatography is Capto SP FF, Capto S ImpAct or Capto SP ImpRes.

6. The method for preparing respiratory syncytial virus membrane protein according to claim 1, characterized in that: The ultrafiltration concentration is performed at a multiple of 3 to 5 times.

7. The method for preparing respiratory syncytial virus membrane protein according to claim 1, characterized in that: The chromatographic column used for the molecular sieve chromatography is Superdex 200pg or Superdex 200Increase.

8. The method for preparing respiratory syncytial virus membrane protein according to claim 1, characterized in that: The lysis operation is as follows: the cells are mixed with the third buffer, and sucrose is added to make the final concentration of sucrose in the solution 3-5% (v / v), and then the cells are repeatedly frozen and thawed for lysis.

9. The method for preparing respiratory syncytial virus membrane protein according to claim 1, characterized in that: After the cells are lysed, they are centrifuged at a speed of 14,000 to 16,000 g for 30 to 60 minutes.

10. A respiratory syncytial virus membrane protein, characterized in that: The respiratory syncytial virus membrane protein is prepared by the method for preparing the respiratory syncytial virus membrane protein according to any one of claims 1 to 9.

Images