CN110092841B

CN110092841B - Recombinant virus-like particle expressed based on inclusion body form and preparation method and application thereof

Info

Publication number: CN110092841B
Application number: CN201910406257.XA
Authority: CN
Inventors: 刘永东; 苏志国; 张耀
Original assignee: Institute of Process Engineering of CAS
Current assignee: Institute of Process Engineering of CAS
Priority date: 2019-05-15
Filing date: 2019-05-15
Publication date: 2021-06-08
Anticipated expiration: 2039-05-15
Also published as: CN110092841A

Abstract

The invention relates to a recombinant virus-like particle expressed based on an inclusion body form, a preparation method and application thereof, wherein the preparation method comprises the following steps: expressing recombinant plasmids containing recombinant virus capsid protein genes or recombinant virus capsid protein genes fused with other antigen genes in an exogenous expression system to obtain recombinant virus capsid proteins existing in the form of inclusion bodies or recombinant virus capsid proteins fused with other antigens; and dissolving the inclusion body by using a surfactant, diluting, adding an amphiphilic cosolvent, desalting, realizing the assembly of the virus-like particles, and purifying to obtain the recombinant virus-like particles. The method is suitable for all recombinant virus-like particles expressed in the form of inclusion bodies, is simple and easy to operate, and the prepared recombinant virus-like particles have a form similar to that of natural viruses, thereby providing a strategy for the development of preventive vaccines and therapeutic vaccines.

Description

Recombinant virus-like particle expressed based on inclusion body form and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to recombinant virus-like particles expressed based on inclusion body forms, and a preparation method and application thereof.

Background

Virus-like particles (VLPs) refer to hollow particles composed of one or more capsid proteins of a virus, free of viral nucleic acids, and having diameters between tens to hundreds of nanometers. The VLP has a form similar to that of a virus and a way of entering an organism, can greatly improve immunogenicity, stimulate strong humoral immunity and cellular immunity of the organism, and has wide application prospect in the aspects of vaccine carriers and drug delivery. Wherein, the hepatitis B virus core antigen (HBc) is composed of single-chain capsid protein, can be expressed in the form of assembly in eukaryotic cells and prokaryotic cells, and is the VLP which is most widely applied at present. Although HBc allows insertion and replacement of other epitopes, when exogenous systems, particularly prokaryotic expression systems represented by Escherichia coli, express the epitopes, structural errors are easily generated, and a fermentation product-inclusion body without biological activity is generated, so that the application of the inclusion body is severely limited.

Although some methods can be used to promote the soluble expression of recombinant proteins in expression systems such as E.coli, such as fusion expression of recombinant proteins with chaperones or folding enzymes, low temperature induction, reduction of inducer concentration, optimization of host bacteria, culture medium types, and the like. However, these methods are not effective for all recombinant proteins, or even if soluble expression is achieved, the problem of low expression level still remains. Therefore, it is necessary to start with how to recover the activity of the inclusion bodies. The process of recovering the activity of the inclusion bodies is called renaturation or in vitro refolding of the inclusion bodies, namely, after the inclusion bodies are collected, the inclusion bodies are dissolved by using a proper denaturant, and then proper solution environmental conditions are provided to ensure that the peptide chain in an extended state is folded into a correct structure, the natural conformation of the protein is recovered, and then the VLP is formed by assembly.

To achieve renaturation of the inclusion bodies, the inclusion bodies first need to be solubilized. The reagents most commonly used to solubilize inclusion bodies include chaotropes and surfactants. The most important chaotropic agents for dissolving the inclusion body protein are guanidine hydrochloride and urea, but at present, there is no precedent for successful assembly after the guanidine hydrochloride is used for dissolving the virus capsid protein, which is probably because the protein denatured by guanidine hydrochloride is easy to aggregate in the renaturation process and the HBc is very hydrophobic for the protein with strong hydrophobicity. Therefore, other reagents for solubilizing inclusion bodies have to be sought. The surfactant is another and most economical agent for solubilizing the inclusion body protein, and the solubilized protein has the possibility of retaining part or all of the biological activity, indicating that the lower structure and even the tertiary structure of the protein are retained under such conditions, and furthermore, the protein aggregation can be effectively inhibited. However, the problem is that the surfactant is easy to combine with protein and not easy to remove, and further assembly of the assembly unit to form the virus-like particles is seriously influenced. Therefore, complete removal of the surfactant from the recombinant protein is required to achieve assembly of the virus-like particles.

CN103898070A discloses an H3N8 subtype equine influenza recombinant virus-like particle, a preparation method and an application thereof, and the recombinant virus-like particle is prepared by the following method: cloning the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 or the nucleotide sequences with homology of more than 95 percent with the nucleotide sequences shown in SEQ ID NO.1 and SEQ ID NO.2 or the nucleotide sequences which encode the same protein and have degeneracy of genetic codes with the same protein into a baculovirus transfer vector, transforming DH10Bac competent cells by the obtained recombinant baculovirus transfer vector to obtain recombinant bacmid, transfecting Sf9 insect cells by the recombinant bacmid and enabling the recombinant bacmid to express H3N8 subtype equine influenza recombinant virus-like particle protein, and carrying out gradient density centrifugation and purification on sucrose to obtain the recombinant bacmid. The vaccine prepared by the method can induce mice to generate good protective immune response after the mice are immunized, and has no any risk of live viruses.

CN102839183A discloses a preparation method and application of recombinant enterovirus 71-type virus-like particles, the DNA molecule composition for preparing the recombinant virus-like particles provided by the invention comprises DNA molecules I, DNA, DNA molecules II, DNA molecules III and DNA molecules IV, the DNA molecule composition is introduced into host bacteria to obtain recombinant bacteria, and the recombinant bacteria are subjected to sucrose gradient density centrifugal separation and purification to obtain the recombinant virus-like particles. The recombinant enterovirus 71 type virus-like particle is similar to real virus in morphology and does not contain potential pathogenic virus genomes, so that an ideal alternative is provided for the development of antiviral vaccines.

The preparation methods of the recombinant virus-like particles disclosed in the prior art are all based on the soluble form of the assembly, and the recombinant virus-like particles are directly purified by methods such as gradient density centrifugation, so that the application range is narrow. In the process of preparing the vaccine with the virus-like particles as the carriers, when the antigen and the virus capsid protein are expressed by an exogenous expression system, particularly an escherichia coli expression system after being fused, inclusion bodies with wrong structures and no biological activity are easily generated, and the recombinant virus-like particles cannot be obtained by a direct purification mode, so that the development of the preparation method of the recombinant virus-like particles based on the inclusion body form expression, which has the advantages of simple operation and wide application range, is very significant.

Disclosure of Invention

Aiming at the defects and practical needs of the prior art, the invention aims to provide a recombinant virus-like particle expressed based on an inclusion body form, and a preparation method and application thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

in one aspect, the present invention provides a method for preparing a recombinant virus-like particle expressed based on inclusion body form, comprising the following steps: expressing recombinant plasmids containing recombinant virus capsid protein genes or recombinant virus capsid protein genes fused with other antigen genes in an exogenous expression system to obtain recombinant virus capsid proteins existing in the form of inclusion bodies or recombinant virus capsid proteins fused with other antigens; and dissolving the inclusion body by using a surfactant, diluting, adding an amphiphilic cosolvent, desalting, realizing the assembly of the virus-like particles, and purifying to obtain the recombinant virus-like particles.

The method of the present invention recovers the natural spatial conformation of the inclusion body on the basis of obtaining the inclusion body, and finally obtains the recombinant virus-like particle with biological activity. The preparation method is simple and easy to operate, and the prepared virus-like particles have a form similar to that of natural viruses, so that a strategy is provided for developing preventive or therapeutic vaccines.

In the invention, the preparation method comprises the following steps:

(1) obtaining recombinant plasmids containing recombinant virus capsid protein genes or recombinant virus capsid protein genes fused with other antigen genes by a gene recombination technology, and expressing the recombinant virus capsid protein or the recombinant virus capsid protein fused with other antigens in an exogenous expression system to obtain fermentation products;

(2) centrifugally collecting the fermentation product obtained in the step (1), crushing cells, centrifugally collecting inclusion bodies, and washing;

(3) dissolving the inclusion bodies washed in the step (2) by using SDS solution;

(4) diluting the inclusion body dissolved in the step (3), mixing the inclusion body with an amphiphilic cosolvent for reaction, desalting, and assembling virus-like particles;

(5) and (4) carrying out ultrafiltration concentration and purification on the product obtained in the step (4) to obtain the recombinant virus-like particles.

The preparation method related by the invention expresses the recombinant virus capsid protein or the recombinant virus capsid protein fused with other antigens by a gene recombination technology, the capsid protein in a fermentation product exists in the form of an inclusion body, the spatial structure of the inclusion body is wrong, the inclusion body does not have biological activity, and the subsequent application is seriously influenced. The invention adopts the anionic surfactant SDS to dissolve and depolymerize the inclusion body, the SDS can efficiently dissolve the inclusion body and promote the formation of protein alpha helix, but SDS molecules are easy to combine with protein and are difficult to remove by methods such as ion exchange chromatography, ultrafiltration or dialysis, and the like, thereby influencing further assembly of capsid protein to form VLP. Therefore, the invention utilizes the function of the amphiphilic cosolvent to separate the protein from the SDS and restore the natural space conformation of the protein, thereby completing the assembly of the virus-like particles and finally obtaining the recombinant virus-like particles with biological activity.

The reason for selecting SDS solution as the reagent for dissolving inclusion bodies is that SDS can ensure the complete dissolution of inclusion bodies on one hand, and can promote the recombinant capsid protein to generate alpha helical structure, which is beneficial to the generation of subsequent assembly, but guanidine hydrochloride and urea can not generate the above effect on the other hand.

In the invention, the recombinant virus capsid protein in the step (1) comprises a recombinant hepatitis B virus core antigen HBc, a recombinant parvovirus capsid protein B19, a recombinant cowpea chlorotic speck virus capsid protein CCMW, a recombinant cytomegalovirus capsid protein CMV or a recombinant rabbit hemorrhagic virus capsid protein RHDV.

Preferably, the sequence of the recombinant hepatitis B virus core antigen includes a truncated sequence or a full-length sequence.

The full-length sequence of the recombinant hepatitis B virus core antigen is SEQ ID NO. 1: mdidpykefgafvellsflpsdfffpsiirdlldtasalallelespeeethephshlalcrqalalcwgellmwvgslnwglsnledvisnsvnmglkirqllfwfhisclfgrylvlesfvwirtppyrapyrppnapitlpirlpirlpirttvvrrrgprrrrsqprrrrrrrsqsresqc.

The truncated sequence of the recombinant hepatitis B virus core antigen is at least 140 amino acids from the N end of the recombinant hepatitis B virus core antigen, preferably 144-149 amino acids.

The truncated sequence of the recombinant hepatitis B virus core antigen comprises an amino acid sequence shown in SEQ ID NO. 2: mdidpykefgafvellsflpsdfffpsiirdlldtasalallelepipes phlalrgqailcweglmnwslvntwlvgsnlgsnlepsepsepsepsepsilonsvlnsvmnfrwshwhitchregretvllevllevllevllevlesfgirlpipnapil.

Preferably, the other antigens comprise any one or a combination of at least two of melanoma related gene antigen MAGE, influenza virus antigen M2E, hand-foot-and-mouth disease virus antigen VP1, hepatitis B virus antigen PreS1, hepatitis B virus antigen PreS2, hepatitis B virus antigen HBsAg or human papilloma virus antigen E7.

The recombinant virus capsid protein gene can be fused with a plurality of antigens of the same type at the same time, and has synergistic effect, such as a plurality of different genes in a melanoma antigen MAGE family, hepatitis B virus antigen PreS1 and PreS2, and the like.

The other antigens are fused to the recombinant viral capsid proteins by means of insertions or substitutions at the N-terminus, the C-terminus or the MIR (major immunodominant region).

Preferably, the exogenous expression system comprises a eukaryotic expression system or a prokaryotic expression system.

Preferably, the eukaryotic expression system comprises a chinese hamster oocyte, insect cell or pichia pastoris.

Preferably, the prokaryotic expression system comprises E.coli.

In the present invention, the means for disrupting the cells in step (2) comprises ultrasonication and/or high-pressure homogenization.

Preferably, the wash uses a solution comprising 15-25mM phosphate, 1-3M urea, 4-6mM EDTA and 0.4% -0.6% Triton X-100.

The phosphate may be present in a molarity of 15mM, 17mM, 18mM, 20mM, 22mM or 25mM, etc.

The molar concentration of the urea may be 1M, 1.2M, 1.5M, 2M, 2.5M, 2.8M, 3M, or the like.

The molar concentration of EDTA may be 4mM, 4.2mM, 4.5mM, 5mM, 5.2mM, 5.5mM, or 6mM, etc.

The Triton X-100 accounts for 0.4%, 0.42%, 0.45%, 0.5%, 0.52%, 0.55%, 0.6% or the like in percentage by mass.

Preferably, the pH of the solution used for washing is 6.5-7.5, such as 6.5, 6.7, 6.8, 7.0, 7.1, 7.2, 7.4, 7.5, or the like.

Since the lipid material and partially disrupted cell membranes and membrane proteins adhere to the inclusion bodies, the inclusion bodies are washed prior to solubilization of the inclusion bodies.

In the present invention, the final concentration of the protein after the inclusion body is solubilized is 15 to 25mg/mL (e.g., 15mg/mL, 18mg/mL, 20mg/mL, 22mg/mL, or 25mg/mL, etc.) when the concentration of SDS in the SDS solution of step (3) is 0.5 to 5% (e.g., 0.5%, 1%, 2%, 3%, 4%, or 5%, etc.).

Preferably, the concentration of SDS is 1%, and the final concentration of protein after the inclusion body is dissolved is 20 mg/mL.

As described above, the SDS solution is used to dissolve the excess inclusion bodies, thereby reducing the ratio of SDS to protein, facilitating effective and complete removal of SDS by adding the amphiphilic cosolvent, and achieving assembly of capsid protein.

The reason why the concentration range of SDS was selected relative to the concentration of inclusion bodies was: if the concentration exceeds the above range, SDS cannot be completely removed even if MPD is added, and the assembly of virus-like particles is affected; if the concentration is less than the above range, the SDS has a poor solubilizing effect.

The forces that maintain the protein structure in inclusion bodies are intramolecular non-covalent forces that also maintain the stable structure of the native protein. Disulfide bonds, whether correct or incorrect, do not play a direct role in maintaining the compact structure of the internal proteins. Thus, SDS solution is used here to solubilize these inclusion body proteins, which, when solubilized, then proceed to the in vitro folding process of the protein.

Preferably, the SDS solution further comprises 0.4% to 6% beta-mercaptoethanol (e.g., at a concentration of 0.4%, 0.42%, 0.45%, 0.5%, 0.52%, 0.55%, or 0.6% by mass, etc.).

Beta-mercaptoethanol was added to the SDS solution to ensure a reducing environment to ensure no aggregation due to disulfide mismatch.

In the present invention, the solution used for the dilution in step (4) comprises 15-25mM Tris-HCl (e.g., 15mM, 18mM, 20mM, 21mM, 22mM, 24mM, or 25mM, etc.) and 0.4% -6% beta-mercaptoethanol (at a concentration of, for example, 0.4%, 0.42%, 0.45%, 0.5%, 0.52%, 0.55%, or 0.6% by mass, etc.).

Studies have shown that disulfide bonds do not affect the assembly of HBc VLPs, so addition of β -mercaptoethanol to dilute solutions ensures a reducing environment to ensure that aggregation or extracellular assembly is not affected due to disulfide bond mismatches.

The molar concentration of Tris-HCl may be 15mM, 17mM, 18mM, 20mM, 22mM, 23mM, 25mM, or the like.

Preferably, the pH of the solution used for dilution is 7.5-8.5, such as 7.5, 7.7, 7.8, 8.0, 8.1, 8.2, or 8.5, etc.

Preferably, the temperature of the dilution is 2-8 ℃, such as 2 ℃,4 ℃,5 ℃, 6 ℃ or 8 ℃ and the like.

Preferably, the dilution is 80-120 times, such as 80 times, 90 times, 100 times, 110 times, 120 times, etc., preferably 100 times.

In order to achieve the assembly of recombinant virus-like particles, the interaction between the protein and the SDS needs to be reduced as much as possible, and therefore, the ratio of the SDS to the amphiphilic cosolvent is reduced as much as possible, i.e., the SDS is reduced or the amphiphilic cosolvent is increased. Since proteins do not dissolve at low SDS concentrations, the inclusion bodies are first denatured by SDS dissolution, then diluted and finally reacted with an amphiphilic cosolvent.

The dilution factor is specifically selected in the range of 80-120 fold, where addition of MPD leads to the final formation of a large number of assemblies of protein, below which the protein will exhibit different degrees of aggregation, and above which the cost of use will increase.

Preferably, the amphiphilic cosolvent in the step (4) comprises n-butanol, isobutanol, sec-butanol, tert-butanol, 1, 2-pentanediol, 1, 5-pentanediol, 2, 4-pentanediol, glycerol, 2-methyl-2, 4-pentanediol or 2, 4-dimethyl-2, 4-pentanediol, preferably 2-methyl-2, 4-pentanediol.

Preferably, the final concentration of 2-methyl-2, 4 pentanediol is 0.5M to 2M, such as 0.5M, 0.7M, 1M, 1.2M, 1.5M, 1.8M, 2M, or the like, preferably 1M.

Although SDS is effective in solubilizing inclusion bodies, SDS at high concentrations presents micelles, and even when diluted below micelle concentrations, SDS will still bind to proteins, affecting assembly of VLPs, and thus removal of SDS is necessary for assembly of VLPs. The invention utilizes 2-methyl-2, 4-pentanediol (MPD) to shield the negative charge of the head of SDS in aqueous solution and protect the hydrophobic tail, thereby separating SDS from each other and causing micelle to disappear. Finally, the inclusion bodies can be dissolved and the natural conformation of the protein can be restored in the system, and the protein can be further assembled into VLPs along with the separation of SDS and the protein.

The reason why the final concentration of 2-methyl-2, 4-pentanediol was selected to be in the range of 0.5M to 2M was: if the concentration exceeds the range, the protein can be precipitated; if the concentration is less than the above range, the protein will not be separated from SDS.

Preferably, the temperature of the reaction in step (4) is 4-30 ℃, e.g., 4 ℃,5 ℃, 10 ℃, 12 ℃, 15 ℃, 20 ℃ or 30 ℃, etc., preferably 20 ℃.

Preferably, the reaction of step (4) is carried out for a period of 1-3d, such as 1d, 2d or 3d, etc., preferably 2 d.

Preferably, the desalting column used in the desalting of step (4) is a G25 desalting column.

Preferably, the buffer used for desalting in step (4) is a 20mM Tris-HCl solution with pH 7.4.

Preferably, the temperature of the desalting in step (4) is maintained at 2-6 deg.C, such as 2 deg.C, 3 deg.C, 4 deg.C, 5 deg.C or 6 deg.C.

Preferably, the time for standing after desalting in step (4) is 2-4d, such as 2d, 3d or 4 d.

In the invention, the ultrafiltration tube used in the ultrafiltration concentration in the step (5) is a 50mL ultrafiltration tube with the molecular weight cut-off of 10 KD.

Preferably, the method of purification in step (5) comprises gel filtration or combined chromatography, preferably combined chromatography.

Preferably, the medium used in the combined chromatography is Capto core 700.

The concentration of the capsid protein after desalting is very low (less than 0.1mg/mL), so that the complete virus-like particles are obtained by purification after ultrafiltration concentration, and the concentration mode used in the invention does not damage the assembled particle structure.

After the recombinant virus-like particles are obtained by purification, the recombinant virus-like particles need to be characterized, wherein the characterization means comprises any one or a combination of at least two of colorimetric method determination of SDS content, SDS-PAGE, circular dichroism spectrum, fluorescence spectrum, dynamic light scattering, transmission electron microscopy, gel filtration chromatography, agarose gel electrophoresis or matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Wherein the gel filtration chromatography medium comprises Superdex 20010/300 GL and/or TSK G4000 SWxl. Wherein the concentration of SDS-PAGE separating gel is 8-15%.

As a preferred technical scheme of the invention, the preparation method comprises the following steps:

(2) centrifuging the fermentation product obtained in step (1), disrupting the cells by ultrasonication and/or high pressure homogenization, centrifuging to collect inclusion bodies, and washing with a solution having a pH of 6.5-7.5 comprising 15-25mM phosphate, 2M urea, 3mM EDTA and 0.5% Triton X-100;

(3) dissolving the inclusion bodies washed in the step (2) by using an SDS solution containing 0.5% of beta-mercaptoethanol, wherein the concentration of SDS in the SDS solution is measured by 0.5% -5%, and the final concentration of the protein after the inclusion bodies are dissolved is 15-25 mg/mL;

(4) diluting the inclusion body dissolved in the step (3) by 80-120 times by using a solution with the pH value of 7.5-8.5 and comprising 20mM Tris-HCl and 0.5% beta-mercaptoethanol at the temperature of 2-8 ℃, mixing with an amphiphilic cosolvent, reacting at the temperature of 4-30 ℃ for 1-3d, desalting to 20mM Tris-HCl with the pH value of 7.4 by using a G25 desalting column, standing at the temperature of 2-6 ℃ for 2-4 days, and assembling virus-like particles;

(5) and (3) carrying out ultrafiltration concentration on the product obtained in the step (4) by using a 50mL ultrafiltration tube with the molecular weight cutoff of 10KD, and purifying by using a gel filtration method or a combined chromatography method to obtain the recombinant virus-like particles.

In another aspect, the present invention provides a recombinant virus-like particle expressed based on inclusion body form, which is prepared by the preparation method as described above.

In a further aspect, the present invention provides a use of the recombinant virus-like particle as described above for the preparation of a prophylactic vaccine or a therapeutic vaccine.

The recombinant virus-like particle prepared by the invention belongs to a nano-scale particle, can be used as a vaccine carrier, improves the immunogenicity of other antigens by inserting other antigen epitopes into a main immunodominant region of the recombinant virus-like particle, has good biocompatibility, can stimulate an organism to generate immune response without an adjuvant, and can be used for preparing a preventive or therapeutic vaccine.

Compared with the prior art, the invention has the following beneficial effects:

the preparation method provided by the invention firstly plays a role in preliminarily purifying the inclusion body by washing the inclusion body; an anionic surfactant SDS is adopted to dissolve and depolymerize the inclusion body, and the inclusion body can be efficiently dissolved by the SDS, so that the formation of protein alpha helix is promoted; separating protein from SDS by using the function of amphiphilic cosolvent to restore natural space conformation; and removing redundant SDS and the amphiphilic cosolvent through desalting, and further finishing the assembly of the virus-like particles. Finally, pure virus-like particles with natural virus forms are obtained through ultrafiltration concentration and purification. And the whole process is carried out under the reducing condition, so that aggregation caused by mismatching of disulfide bonds can be avoided, and the assembly of capsid protein is not influenced.

The method of the present invention recovers the spatial protein conformation of inclusion body to obtain bioactive recombinant virus-like particle, and is suitable for all recombinant virus-like particles expressed in the form of inclusion body. The preparation method is simple and easy to operate, and the prepared recombinant virus-like particles have a form similar to that of natural viruses, so that a strategy is provided for development of preventive vaccines and therapeutic vaccines.

Drawings

FIG. 1 is a graph showing the growth of bacterial cells in a 20L fermenter according to example 1;

FIG. 2 is a graph showing the results of SDS-PAGE in example 1 (wherein M is marker, lane 1 is whole pre-induction bacteria, lane 2 is whole post-induction bacteria, lane 3 is disrupted supernatant, lane 4 is disrupted precipitate,

lanes

5 and 6 are first-wash supernatant and precipitate, respectively,

lanes

7 and 8 are second-wash supernatant and precipitate, respectively, and the arrow indicates the theoretical molecular weight of HBc-MAGE3 II);

FIG. 3 is a graph showing the dissolution efficiency of various denaturants in example 2;

FIG. 4 is a circular dichroism spectrum of the recombinant hepatitis B virus core antigen in example 2 (wherein: the sample is wild-type HBc virus-like particles; the sample is renatured after the inclusion body is dissolved by guanidine hydrochloride; the sample is obtained after the inclusion body is dissolved by SDS; the sample is 100-fold diluted after the inclusion body is dissolved by SDS; and the sample is obtained after the inclusion body is dissolved by SDS and is subjected to MPD treatment after dilution);

FIG. 5 is a TEM representation of the effect of different dilution times on protein assembly in example 3 (10-, 20-, 50-and 100-fold for a, b, c, d, respectively, scale representing 100 nm);

FIG. 6 is a TEM representation of the effect of different SDS contents on protein assembly in example 3 (FIG. A, B, C shows initial SDS contents of 0.01%, 0.03%, 0.05%, respectively, and D shows a statistical plot of the number of moles of SDS bound per mole of protein molecules, with the scale representing 100 nm);

FIG. 7 is a graph of the results of DLS characterization of the sample after purification in example 4;

FIG. 8 is a TEM representation of the sample after purification in example 4 (scale represents 100 nm).

Detailed Description

The technical solution of the present invention is further explained by the following embodiments. It should be understood by those skilled in the art that the examples are only for the understanding of the present invention and should not be construed as the specific limitations of the present invention.

Example 1

Expression and identification of recombinant hepatitis B virus core antigen fused with other antigens

The specific operation method comprises the following steps:

(1) a major histocompatibility complex MHC II antigen (amino acid sequence is SEQ ID NO. 3: AELVHFLLLKYRAR) in a human melanoma related gene MAGE A3 gene is inserted into a hepatitis B virus core antigen HBc MIR region (inserted between HBc 78aa and HB79 aa) by using a genetic engineering means, and is connected into a plasmid pET28a which is named as pET28a-HBc-MAGE3 II. The recombinant plasmid is subcloned into a competent Escherichia coli cell BL21(DE3), and the amino acid sequence of HBc-MAGE3II is shown in SEQ ID NO. 4. Inoculating into 20L fermentation tank, fermenting until OD₆₀₀When the strain reaches 5-8 hours, 0.1mM isopropyl thiogalactoside (IPTG) is added to induce for 4 hours to finish fermentation, and a strain growth curve is drawn, as shown in figure 1.

SEQ ID NO.4：

Mdidpykefgasvellsflpsdffpsirdlldtasalyrealespehcsphhtalrqailcwgelmnlatwvgsnledaelvhflllkyrarpasrelvvsyvnvnmglkirqllwfhiscltfgretvleylvsfgvwirtppayrppnapilstlpettvvrrrgrsprrrtpsprrrrsqsprrrrsqsresqc。

(2) Collecting 185g of thallus by centrifugation, adding a disruption solution (20mM Tris-HCl, 3mM EDTA, 0.1% Triton X-100, 1mM PMSF, pH 8.0) according to a ratio of 1:10, circularly disrupting three times by a high-pressure homogenization method under a pressure of 800MPa, centrifuging at 10000rpm for 40min to collect precipitates (inclusion bodies), detecting the expression of whole bacteria before and after induction and supernatant after disruption and HBc-MAGE 353 3II in the precipitates by SDS-PAGE (the theoretical molecular weight of HBc-MAGE3II is about 28kDa), and obtaining the result shown in FIG. 2 (wherein M is marker, lane 1 is whole bacteria before induction, lane 2 is whole bacteria after induction, lane 3 is supernatant after disruption, and lane 4 is disrupted precipitate).

(3) The inclusion body obtained above was washed twice with a washing solution, and then washed once with deionized water to remove the remaining washing solution, the washing solution being a solution containing 20mM phosphate, 2M urea, 3mM EDTA and 0.5% Triton X-100 at pH 7.0. The unwashed inclusion bodies, supernatant after the first washing, pellet after the first washing, supernatant after the second washing, and pellet after the second washing were identified by SDS-PAGE, and the results are shown in FIG. 2 (wherein

lanes

5 and 6 are the first-washing supernatant and pellet, respectively, and

lanes

7 and 8 are the second-washing supernatant and pellet, respectively).

The experimental results are as follows:

(1) from the results of FIG. 1, it can be seen that: when the density of the cells OD₆₀₀When the strain reaches 4.95, the strain enters the logarithmic phase, IPTG is added for induction for 4h, and the escherichia coli grows normally in the whole fermentation process.

(2) From the results of FIG. 2, it can be seen that: the obvious increase of the protein amount of HBc-MAGE3II can be seen after the induction of the whole bacteria; after the bacteria are broken, the target protein (indicated by a black arrow) is expressed in both the supernatant and the precipitate, but is mainly expressed in an inclusion body form (precipitate); after the inclusion body is washed, the impurities are greatly reduced, the target protein is not lost, an obvious purification effect is obtained, and the inclusion body which has the purity of more than 85 percent and is expressed with HBc-MAGE3II is prepared.

Example 2

Solubilization of Inclusion bodies

The specific operation method comprises the following steps:

(1) 0.1g of the inclusion bodies obtained in example 1 was weighed, 1mL of a denaturant containing 8M urea, 6M guanidine hydrochloride or 1% SDS was added thereto, the mixture was shaken overnight at room temperature, and the supernatant was centrifuged to measure the protein concentration and calculate the dissolution rate. The results are shown in FIG. 3.

(2) The inclusion bodies washed in example 1 were added to a pH 8.0 lysis buffer containing 20mM Tris-HCl, 1% SDS, 0.5% beta-mercaptoethanol at a ratio of 1g:1mL, and the inclusion bodies were in excess, saturated in solubility, and the protein concentration was about 20mg/mL as measured by the Bradford method.

The embodiment also explores the influence of different types of dissolving solvents and subsequent treatment on the secondary structure of the protein, and the specific method comprises the following steps: the buffer solutions of the respective protein solutions are used as references, and the circular dichroism method is adopted to characterize the protein secondary structures of different types of dissolving solvents and samples subjected to subsequent processing, and the results are shown in fig. 4.

The experimental results are as follows:

(1) from the results of FIG. 3, it can be seen that: even when the pH was raised to 9.0, 8M urea was still unable to effectively solubilize inclusion bodies; guanidine hydrochloride and SDS can completely solubilize the inclusion bodies.

(2) From the results of FIG. 4, it can be seen that: the samples (not assembled) renatured after dissolution of guanidine hydrochloride are mainly irregular coils, while the samples dissolved by SDS are mainly in alpha-helical structures (consistent with natural HBc virus-like particles), so that the subsequent assembly is facilitated, and the alpha-helical structures of the samples dissolved by SDS cannot be influenced by the post-treatment.

Example 3

Extracellular Assembly of Inclusion bodies

The specific operation method comprises the following steps:

(1) the inclusion bodies obtained in example 2 after SDS solubilization were diluted with 15-25mM Tris-HCl and 0.4% -6% beta-mercaptoethanol at pH 8.0 at 4 ℃ over 100X overnight, reacted with 2-methyl-2, 4 pentanediol at a final concentration of 1M at 20 ℃ for 2d, desalted and then placed at 4 ℃ for 3d, the desalting column used for desalting was a G25 desalting column, and the buffer used for desalting was a 20mM Tris-HCl solution at pH 7.4.

The present example also explores the effect of different dilution times on protein, and the specific method is as follows: the recombinant core antigen of hepatitis B virus fused with other antigens in the form of inclusion bodies was prepared according to the method of example 1-3, and the inclusion bodies were washed, dissolved in 1% SDS solution, diluted 10-fold, 20-fold, 50-fold and 100-fold respectively, and then overnight at 4 deg.C, after which 2-methyl-2, 4-pentanediol was added to a final concentration of 1M, and reacted at 20 deg.C for 2d, followed by desalting. The four samples were characterized by transmission electron microscopy, respectively, and the results are shown in fig. 5.

The present example also explores the influence of different residual amounts of SDS on protein assembly, and the specific method is as follows: the recombinant core antigen of hepatitis B virus fused with other antigens in the form of inclusion bodies was prepared according to the method of example 1-3, and the inclusion bodies were washed, solubilized with 1% SDS solution, diluted 100-fold with dilutions having different SDS concentrations to a final protein concentration of 0.2mg/mL, SDS concentrations of 0.01%, 0.03%, and 0.05%, and then overnight at 4 ℃, followed by addition of 2-methyl-2, 4-pentanediol to a final concentration of 1M, reacted at 20 ℃ for 2d, and desalted. The three samples were characterized by transmission electron microscopy, respectively, with the results shown in FIG. 6, and by colorimetric SDS trace determination, with the results shown in Table 1.

The experimental results are as follows:

(1) from the results of FIG. 5, it can be seen that: the samples were diluted 10 and 20 times and the proteins were mainly present in a random aggregation; the sample diluted 50 times presents hollow circular structures less than 10nm inside the aggregates while a large amount of random aggregates are present, which may be pentamers of HBc; whereas hollow round structures of about 30nm, i.e. recombinant virus-like particles, appear in the 100-fold diluted sample.

(2) From the results of FIG. 6, it can be seen that: 0.01% SDS group, a large number of intact virus-like particles (hollow round structures with a size of about 30 nm) were present in the sample; 0.03% SDS, samples were predominantly in aggregated form, but a large number of hollow round structures less than 10nm were present, possibly as HBc pentamers; whereas in the 0.05% SDS group, aggregation of SDS and protein was predominant.

(3) From the results of table 1 in combination with the results of fig. 6, it can be seen that: the lower the amount of residual SDS, the less SDS bound per protein subunit, the greater the chance of VLP assembly and conversely the greater the degree of aggregation. VLPs are produced in large quantities when each protein subunit binds 0.14 SDS molecules.

TABLE 1

Example 4

Ultrafiltration concentration and purification of inclusion bodies

The specific operation method comprises the following steps:

(1) the desalted product obtained in example 3 was concentrated by ultrafiltration using a 50mL ultrafiltration tube with a molecular weight cut-off of 10 KD.

(2) And (3) utilizing the Capto core to finely purify the concentrated sample, enabling the assembly to flow through, and combining the unassembled or wrongly assembled structure with a medium to achieve the purpose of fine purification. The breakthrough peak was collected and the purified product was characterized by DLS, the results are shown in figure 7. The purified product was characterized by TEM and the results are shown in figure 8.

The experimental results are as follows:

(1) from the results of FIG. 7, it can be seen that: the particle size of 99.2% of the purified product measured by a Malvern particle sizer is 27.6nm, the three measurements have no obvious difference, the particle size is uniform, and the particle size is consistent with the theory.

(2) From the results of FIG. 8, it can be seen that: the purified product is basically completely of a nano spherical structure of about 30nm, and the shape is similar to the shape of the natural virus, which indicates that the prepared recombinant virus-like particle has a complete structure.

The applicant states that the present invention is illustrated by the above examples, but the present invention is not limited to the above examples, i.e. it does not mean that the present invention must be implemented by the above examples. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.

It should be noted that the various technical features described in the above embodiments can be combined in any suitable manner without contradiction, and the invention is not described in any way for the possible combinations in order to avoid unnecessary repetition.

SEQUENCE LISTING

<110> institute of Process engineering of Chinese academy of sciences

<120> recombinant virus-like particle expressed based on inclusion body form, preparation method and application thereof

<130> 2019

<160> 4

<170> PatentIn version 3.3

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Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu

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Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

35 40 45

Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

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Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala

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Ser Arg Glu Leu Val Val Ser Tyr Val Asn Val Asn Met Gly Leu Lys

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Ile Arg Gln Leu Leu Trp Phe His Ile Ser Cys Leu Thr Phe Gly Arg

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Glu Thr Val Leu Glu Tyr Leu Val Ser Phe Gly Val Trp Ile Arg Thr

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Pro Pro Ala Tyr Arg Pro Pro Asn Ala Pro Ile Leu Ser Thr Leu Pro

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Glu Thr Thr Val Val Arg Arg Arg Gly Arg Ser Pro Arg Arg Arg Thr

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Pro Ser Pro Arg Arg Arg Arg Ser Gln Ser Pro Arg Arg Arg Arg Ser

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Gln Ser Arg Glu Ser Gln Cys

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Met Asp Ile Asp Pro Tyr Lys Glu Phe Gly Ala Ser Val Glu Leu Leu

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Ser Phe Leu Pro Ser Asp Phe Phe Pro Ser Ile Arg Asp Leu Leu Asp

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Thr Ala Ser Ala Leu Tyr Arg Glu Ala Leu Glu Ser Pro Glu His Cys

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Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

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Leu Met Asn Leu Ala Thr Trp Val Gly Ser Asn Leu Glu Asp Pro Ala

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Ser Pro His His Thr Ala Leu Arg Gln Ala Ile Leu Cys Trp Gly Glu

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Arg Glu Ser Gln Cys

195

Claims

1. A preparation method of recombinant virus-like particles expressed based on inclusion body form is characterized by comprising the following steps:

(1) obtaining recombinant plasmid containing recombinant hepatitis B virus core antigen HBc gene or recombinant hepatitis B virus core antigen HBc gene fused with other antigen genes by gene recombination technology, and expressing the recombinant hepatitis B virus core antigen HBc or the recombinant hepatitis B virus core antigen HBc fused with other antigens in a prokaryotic expression system to obtain a fermentation product;

the other antigens comprise any one or the combination of at least two of melanoma related gene antigen MAGE, influenza virus antigen M2E, hand-foot-and-mouth disease virus antigen VP1, hepatitis B virus antigen PreS1, hepatitis B virus antigen PreS2, hepatitis B virus antigen HBsAg or human papilloma virus antigen E7;

(3) dissolving the inclusion bodies washed in the step (2) by using SDS solution; the concentration of SDS in the SDS solution is measured by 0.5% -1%, and the final concentration of the protein after the inclusion body is dissolved is 15-25 mg/mL; the SDS solution further comprises 0.4% -0.6% beta-mercaptoethanol;

(4) diluting the inclusion body dissolved in the step (3), wherein the dilution multiple is 80-120 times; mixing the virus-like particles with an amphiphilic cosolvent for reaction, desalting, and assembling the virus-like particles; the amphiphilic cosolvent is 2-methyl-2, 4 pentanediol;

2. The method of claim 1, wherein the prokaryotic expression system comprises E.coli.

3. The method of claim 1, wherein the means for disrupting the cells of step (2) comprises ultrasonication and/or high pressure homogenization.

4. The method of claim 1, wherein the washing solution comprises 15-25mM phosphate, 1-3M urea, 2-4mM EDTA, and 0.4% -0.6% Triton X-100.

5. The method of claim 1, wherein the pH of the solution used for washing is 6.5 to 7.5.

6. The method according to claim 1, wherein the final concentration of the protein after the inclusion body is solubilized is 20mg/mL, when the concentration of SDS is 1%.

7. The method of claim 1, wherein the diluted solution used in step (4) comprises 15-25mM Tris-HCl and 0.4% -0.6% β -mercaptoethanol.

8. The method of claim 1, wherein the pH of the solution used for dilution is 7.5 to 8.5.

9. The method of claim 1, wherein the dilution temperature is 2-8 ℃.

10. The method of claim 1, wherein the dilution factor is 100-fold.

11. The method according to claim 1, wherein the final concentration of 2-methyl-2, 4-pentanediol is 0.5M to 2M.

12. The method according to claim 1, wherein the final concentration of 2-methyl-2, 4-pentanediol is 1M.

13. The method according to claim 1, wherein the temperature of the reaction in the step (4) is 4 to 30 ℃.

14. The method of claim 1, wherein the temperature of the reaction in step (4) is 20 ℃.

15. The method according to claim 1, wherein the reaction time in the step (4) is 1 to 3 days.

16. The method according to claim 1, wherein the reaction time in the step (4) is 2 days.

17. The method according to claim 1, wherein the desalting column used in the desalting in the step (4) is a G25 desalting column.

18. The method according to claim 1, wherein the buffer used for desalting in step (4) is a 20mM Tris-HCl solution having a pH of 7.4.

19. The method according to claim 1, wherein the sample is allowed to stand at a temperature of 2 to 6 ℃ after desalting in the step (4).

20. The method according to claim 1, wherein the sample is allowed to stand for 2 to 4 days after desalting in the step (4).

21. The method of claim 1, wherein the ultrafiltration tube used in the ultrafiltration concentration in step (5) is a 50mL ultrafiltration tube with a molecular weight cut-off of 10 kD.

22. The method according to claim 1, wherein the purification method of step (5) comprises gel filtration or combined chromatography.

23. The method according to claim 1, wherein the purification method in step (5) is a combined chromatography.

24. The method of claim 23, wherein the combined chromatography medium is Capto core 700.