Method for producing varicella-zoster virus glycoprotein E
By recombinant expression in host cells and dissolving and purification with a combination of specific surfactants, the production problem of shingles glycoprotein E full-length protein is solved, and a high purity and high immunogenicity of full-length protein preparation is achieved, suitable for shingles vaccines.
Patent Information
- Application Number
- CN202111423496.X
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-11-26
- Publication Date
- 2025-08-05
- Estimated Expiration
- 2041-11-26
AI Technical Summary
The prior art cannot effectively produce the full-length intact protein of shingles glycoprotein E, resulting in low immunogenicity and incorrect antibodies.
The recombinant expression vector was used to express shingles glycoprotein E in the host cell and purified with specific surfactants such as DDM, LMNG and FC12 lysed proteins, combined with affinity chromatography and ion exchange chromatography to obtain a full-length intact protein.
A protein purity of more than 95% and a protein yield of more than 20 mg/L was achieved. The obtained full-length protein has significantly higher immunogenicity than the extracellular region and is suitable as a shingles vaccine antigen.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a method for recombinant production of herpes zoster glycoprotein E. Background Art
[0002] Varicella-zoster virus (VZV) is a highly contagious herpes virus. VZV typically causes primary infection in childhood, causing chickenpox. The virus can then remain dormant in the brain and dorsal root ganglia. With aging or other factors that weaken the immune system or impair immune function, VZV can reactivate and cause herpes zoster and other complications.
[0003] Shingles (shingles) has a significant negative impact on the quality of life of patients, particularly the elderly. However, for the prevention and treatment of shingles, only three vaccines are currently approved for marketing worldwide: the live attenuated shingles vaccines Zostavax and Skyzoster, and the recombinant shingles vaccine Shingrix. The limited availability of vaccines, their high cost, and the low protective efficacy and immune durability of live attenuated vaccines have resulted in these vaccines falling far short of meeting actual needs.
[0004] Glycoprotein E (gE) is the primary surface structural protein of VZV and its most important neutralizing antigen, and is highly conserved. Specifically, VZV-gE, encoded by ORF68, consists of 623 amino acids and is a type I transmembrane glycoprotein comprising four regions: a signal peptide, an extracellular domain, a transmembrane domain, and an intracellular domain. The gE protein is most abundantly expressed on the surface of virus-infected cells and possesses both T and B cell epitopes, making VZV-gE the most extensively studied.
[0005] Previous studies and literature have reported the use of truncated glycoprotein E, such as the extracellular domain, as vaccine antigens. However, it is well known that recombinant full-length, intact membrane proteins better preserve the structure of the original membrane protein when used as membrane protein antigens. Compared to recombinant truncated extracellular domains, neutralizing antibodies generated by full-length, intact membrane proteins as vaccine antigens are more accurately able to recognize the original viral antigens during infection. This suggests that the full-length, intact herpes zoster glycoprotein E protein possesses superior immunogenicity, and vaccines produced from it offer more sustained and effective protection.
[0006] Furthermore, the structure of the complete glycoprotein E has yet to be clearly characterized. Current bioinformatics methods for predicting and analyzing sites within the extracellular and transmembrane regions of glycoprotein E cannot guarantee accurate prediction of the true protein structure. In fact, different bioinformatics software often predicts different cutoff points. This hinders a full understanding of the structure of glycoprotein E, particularly the distribution of antigenic epitopes.
[0007] Therefore, there is still a need in the art to provide a full-length intact protein of herpes zoster glycoprotein E and use this full-length intact protein as a vaccine antigen. Summary of the Invention
[0008] The technical problem to be solved by the present invention is to provide a novel production method of herpes zoster glycoprotein E, by which the full-length intact protein of herpes zoster glycoprotein E can be obtained, rather than its truncated regions such as the extracellular region or other functional regions. The obtained full-length intact protein can thus be used as an antigen in the production of herpes zoster recombinant vaccines, avoiding the situation where epitope loss occurs due to the selection of a truncated extracellular region as an antigen, or avoiding the low immunogenicity caused by the structural inconsistency between the truncated protein and the original protein, or the incorrect antibodies produced and the inability to neutralize different strains.
[0009] In response to the above technical problems, the purpose of the present invention is to provide a method for producing herpes zoster glycoprotein E, which adopts the method of recombinantly expressing the encoding nucleic acid of full-length herpes zoster glycoprotein E in host cells, and then recovering the full-length intact protein of herpes zoster glycoprotein E from the host cells through effective membrane protein solubilization and separation means.
[0010] The technical solutions for achieving the above objectives are as follows:
[0011] A method for producing full-length herpes zoster glycoprotein E, comprising the following steps:
[0012] 1) constructing a recombinant expression vector comprising a nucleotide sequence encoding full-length herpes zoster glycoprotein E, transforming the obtained recombinant expression vector into host cells, and culturing the transformed host cells to express full-length herpes zoster glycoprotein E;
[0013] 2) centrifuging the cell culture obtained in step 1) to obtain host cells, adding a buffer to resuspend the cells, then disrupting the cells and centrifuging to obtain a supernatant;
[0014] 3) adding a surfactant to the supernatant obtained in step 2) and mixing uniformly, wherein the surfactant is selected from one or more of N-dodecyl-β-D-maltoside (DDM), lauryl maltose neopentyl glycol (LMNG), 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and n-dodeclyphosphocholine (Fos-choline 12, FC12).
[0015] In the production method provided by the present invention, the amino acid sequence of the full-length herpes zoster glycoprotein E in step 1) is shown in SEQ ID NO: 1.
[0016] In step 1), the recombinant expression vector can be constructed using an expression vector suitable for the host cell and suitable for expressing full-length herpes zoster glycoprotein E. For example, the host cell can be a bacterial cell such as Escherichia coli, a fungal cell such as Pichia pastoris, an insect cell, or a mammalian cell, and the expression vector can be a pET vector system, a pYE vector system, an insect baculovirus expression vector system, or the like.
[0017] Preferably, the nucleotide sequence in step 1) is codon-optimized to a nucleotide sequence suitable for expression in mammalian cells, and the mammalian cells are transformed to express the full-length herpes zoster glycoprotein E; further preferably, the carboxyl (C) terminus or amino (N) terminus of the expressed full-length herpes zoster glycoprotein E carries a tag for protein purification, such as a 6xHis tag. More preferably, the carboxyl (C) terminus of the expressed full-length herpes zoster glycoprotein E carries a tag for protein purification. According to a specific embodiment of the present invention, the nucleotide sequence encoding the full-length herpes zoster glycoprotein E after codon optimization is shown in SEQ ID NO: 2. For example, a mammalian cell expression vector such as a pcDNA vector system is used, and mammalian cells such as CHO cells are transformed. According to a specific embodiment of the present invention, the nucleotide sequence is constructed into the mammalian cell expression vector pcDNA3.4 to obtain a recombinant expression plasmid, which is then transformed into CHO cells.
[0018] According to a specific embodiment of the present invention, step 2) is ultrasonically broken. Preferably, the ratio of the buffer added in step 2) to the cells is 2-100mL:2E8 cells, preferably 5-30mL:2E8 cells, and then the cells are ultrasonically broken. Preferably, the cells are resuspended with a buffer containing 0-2mol / L sodium chloride, preferably with a buffer containing 0.5mol / L sodium chloride, and the cells are resuspended. The buffer may be Tris, PBS or HEPES buffer, such as 20mmol / L Tris, pH 8.0, 1xPBS, 50mmol / L HEPES. According to a specific embodiment of the present invention, the ratio of the buffer added in step 2) to the cells is 10mL:2E8 cells. Further, after ultrasonication, the cell lysate obtained is centrifuged at 3,000-20,000g for 5-60 minutes, preferably at 5,000-15,000g for 20-40 minutes. According to a specific embodiment of the present invention, after ultrasonic disruption, the obtained cell lysate is centrifuged at 10,000 g for 30 minutes.
[0019] In the production method provided herein, the surfactant in step 3) is two or three selected from the group consisting of DDM, LMNG, and FC12. For example, the surfactant comprises DDM and LMNG; preferably, the surfactant is a mixture of DDM and LMNG. Alternatively, the surfactant comprises DDM, LMNG, and FC12; preferably, the surfactant is a mixture of DDM, LMNG, and FC12. Alternatively, the surfactant comprises LMNG and FC12; preferably, the surfactant is a mixture of LMNG and FC12.
[0020] In the production method provided by the present invention, the ratio of the number of host cells disrupted in step 2) to the surfactant added in step 3) is 2E8 cells: 0.1-6g surfactant, preferably 0.1-1g surfactant. According to a specific embodiment of the present invention, the ratio of the number of host cells disrupted in step 2) to the surfactant added in step 3) is 2E8 cells: 0.2-0.3g surfactant. Preferably, the two or more surfactants are added to the supernatant at a weight ratio of 1:1.
[0021] Preferably, the surfactants are added in step 3) such that the concentration of each surfactant in the supernatant is 0.5-2% (w / v). According to a specific embodiment of the present invention, the surfactants are added in step 3) such that the concentration of each surfactant in the supernatant is 1% (w / v).
[0022] Preferably, after adding the surfactant in step 3), the mixture is placed at 4-30°C for 2-72 hours, preferably at 4-25°C for 12-50 hours, to fully dissolve the full-length herpes zoster glycoprotein E. According to a specific embodiment of the present invention, the mixture is placed at 4°C for 16-48 hours.
[0023] Optionally, the production method further comprises the following steps:
[0024] 4) subjecting the mixed solution obtained in step 3) to affinity chromatography to obtain the purified full-length herpes zoster glycoprotein E, wherein the affinity chromatography uses an eluent containing a surfactant for washing impurities and eluting the protein, and the surfactant is DDM.
[0025] In the production method provided by the present invention, during the affinity chromatography in step 4), an eluent containing 0.01-0.1% (w / v) DDM, preferably 0.05% DDM, is used for impurity washing and protein elution, respectively. According to a specific embodiment of the present invention, the eluent used for impurity washing further contains 0-50 mmol / L imidazole, and the eluent used for protein elution further contains 100-500 mmol / L imidazole. For example, when affinity chromatography is performed, impurities are washed with a buffer containing 0.01-0.1% (w / v), preferably 0.05% DDM and 0-50 mmol / L, preferably 20 mmol / L imidazole as an eluent, and proteins are eluted with a buffer containing 0.01-0.1% (w / v), preferably 0.05% DDM and 100-500 mmol / L, preferably 200 mmol / L imidazole as an eluent, wherein the buffer can be Tris, PBS or HEPES buffer, for example, 20 mmol / L Tris, pH 8.0, 1xPBS, 50 mmol / L HEPES.
[0026] Optionally, the production method further comprises the following steps:
[0027] 5) subjecting the obtained purified protein to ion exchange chromatography to obtain further purified full-length herpes zoster glycoprotein E, wherein the ion exchange chromatography uses an eluent containing a surfactant for washing impurities and eluting the protein, and the surfactant is DDM.
[0028] In the production method provided by the present invention, during the ion exchange chromatography in step 5), an eluent containing 0.01-0.1% (w / v), preferably 0.05% DDM, is used for impurity washing and protein elution, respectively. According to a specific embodiment of the present invention, the eluent used for impurity washing further contains 0-100 mmol / L sodium chloride, and the eluent used for protein elution further contains 300-1000 mmol / L sodium chloride. For example, when performing ion exchange chromatography, a buffer containing 0.01-0.1% (w / v), preferably 0.05% DDM and 0-100 mmol / L, preferably 50 mmol / L sodium chloride is used as an eluent for washing impurities, and a buffer containing 0.01-0.1% (w / v), preferably 0.05% DDM and 300-1000 mmol / L, preferably 500 mmol / L sodium chloride is used as an eluent for protein elution, wherein the buffer can be Tris, PBS or HEPES, for example 20 mmol / L Tris, pH 8.0, 1xPBS, 50 mmol / L HEPES.
[0029] Preferably, in the production method provided by the present invention, after affinity chromatography is performed in step 4) to obtain the eluted protein, dialysis is first performed to remove imidazole, and then ion exchange chromatography is performed in step 5).
[0030] Depending on actual needs, the obtained protein can be subjected to other purification methods, such as gel chromatography.
[0031] Compared with the prior art, the present invention provides a method for producing herpes zoster glycoprotein E. In this method, a nucleotide sequence encoding the full-length herpes zoster glycoprotein E is recombinantly expressed in a host cell. Then, after the host cells are fully disrupted, a combination of two or three specific surfactants is added to the glycoprotein E expressed on the cell membrane of the host cell, thereby effectively dissolving the protein from the small cell membrane fragments. The dissolved protein can then be separated and purified by affinity chromatography, ion exchange chromatography, etc.
[0032] Experiments have demonstrated that the production method of the present invention can effectively solubilize and isolate full-length herpes zoster glycoprotein E, achieving a protein purity exceeding 95% and a protein yield exceeding 20 mg / L. Furthermore, the resulting protein is intact and exhibits significantly higher immunogenicity than the extracellular region of herpes zoster glycoprotein E, suggesting its potential as a vaccine antigen for herpes zoster. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings, in which:
[0034] Figure 1The figure shows the SDS-gel electrophoresis analysis results of the full-length herpes zoster glycoprotein E and its extracellular region protein obtained by the production method of the present invention.
[0035] Figure 2 The antibody titers in the sera of mice immunized with full-length herpes zoster glycoprotein E and its extracellular domain protein are shown. P<0.05 indicates a significant difference between the two. DETAILED DESCRIPTION
[0036] The present invention is described below with reference to specific examples. Those skilled in the art will appreciate that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.
[0037] The experimental methods in the following examples are conventional methods unless otherwise specified. The raw materials, reagents, etc. used in the following examples are commercially available products unless otherwise specified.
[0038] Amino acid sequence of glycoprotein E (NCBI Reference Sequence: NP_040190.1; SEQ ID NO: 1):
[0039] MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHTDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDKSPYNQSMYYAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVYIDKTR
[0040] Optimized coding nucleic acid sequence SEQ ID NO:2:
[0041]
[0042] Example 1 Screening of surfactants
[0043] The following procedures were performed to screen the surfactants:
[0044] Step 1. The nucleotide sequence of the full-length herpes zoster glycoprotein E having the amino acid sequence shown in SEQ ID NO: 1 was obtained. Codon optimization was performed to obtain the mammalian codon sequence shown in SEQ ID NO: 2. A (GS)1-(His)6 tag sequence was added to the carboxyl (C) terminus. The resulting construct was then incorporated into the mammalian cell expression vector pcDNA3.4. The pcDNA3.4-full GE expression plasmid was transformed into CHO cells to express the full-length herpes zoster glycoprotein E.
[0045] Step 2. After expression is complete, remove approximately 100 mL of the cell culture containing 2E8 host cells and centrifuge at 3,000 g for 5 minutes to harvest the cells. Add 10 mL of buffer (20 mmol / L Tris, pH 8.0; 0.5 mol / L NaCl) to the cells, disrupt the cells by ultrasonication, and centrifuge at 10,000 g for 30 minutes. Collect the supernatant.
[0046] Step 3. The supernatant obtained by the same method was divided into different test groups, and different surfactants were added. The types of surfactants added and their concentrations in the supernatant are shown in Table 1 below. After mixing evenly, the mixture was placed at 4°C for 16 hours.
[0047] Step 4. The mixed solution was then subjected to affinity chromatography (HIS60) for a one-step protein purification. This one-step purification was performed sequentially using eluents containing different types and concentrations of surfactants for washing and elution. The protein content in the eluents was monitored to collect the protein-containing eluents, and the amount of glycoprotein E recovered was estimated. The types of surfactants contained in the eluents used for washing and elution and their concentrations are also shown in Table 1. The eluent used for washing was 20 mmol / L Tris, pH 8.0, and 20 mmol / L imidazole, and the eluent used for elution was 20 mmol / L Tris, pH 8.0, and 200 mmol / L imidazole.
[0048] The amount of protein recovered is shown in Table 1.
[0049] Table 1. Solubilization and purification of glycoprotein E using different surfactants
[0050]
[0051] During the screening process, the inventors first comprehensively studied all known membrane protein production methods and found that the top three most widely used surfactants were: N-dodecyl-β-D-maltoside (DDM); decylmaltoside (DM); and octyl glucoside (OG). However, experimental results showed that when these three surfactants were used for glycoprotein E solubilization and one-step affinity chromatography purification, the protein recovery yields were relatively low, at 0.5, 0.3, and 0.2 mg / 2E8 cells, respectively.
[0052] The above three surfactants are all non-ionic surfactants. In order to improve the recovery of glycoprotein E, the inventors expanded the screening of surfactants to other non-ionic, amphoteric and ionic surfactants. In addition to the three aforementioned surfactants, nonionic surfactants also included lauryl maltose neopentyl glycol (LMNG), dodecyl octaethylene glycol ether (C12E8), and Triton X-100. Amphoteric surfactants included N,N-dimethyldodecylamine N-oxide (LADO), 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), and n-dodeclyphosphocholine (Fos-choline 12, FC12). Cholesteryl hemisuccinate tris salt (CHS) was used as the ionic surfactant.
[0053] In addition to the type of surfactant, each surfactant has a different critical micelle concentration (CMC), and the solubilization of membrane proteins requires a concentration much higher than the CMC. Therefore, different solubilization and purification concentrations of different surfactants were set during the above screening process.
[0054] By studying the extraction of glycoprotein E using the above 10 different surfactants, the inventors were surprised to find that the extraction efficiency of glycoprotein E with different surfactants could differ by 15 times: LMNG had the highest efficiency, with approximately 1.5 mg of glycoprotein E obtained per 2E8 cells, while CHS had the lowest efficiency, with only approximately 0.1 mg of glycoprotein E obtained per 2E8 cells. This proves that the choice of surfactant has a very important impact on membrane protein production.
[0055] To further increase the recovery of glycoprotein E, the inventors combined different surfactants. The combination was based on the following principles: several surfactants with high efficiency when used alone were selected, the surfactants were mixed according to the combination method in Table 2, and steps 1 to 4 were repeated. In step 4, the surfactant used for protein purification was 0.05% DDM.
[0056] Table 2. Solubilization and purification of glycoprotein E using mixtures of different surfactants
[0057]
[0058]
[0059] In analyzing the efficacy of the above 8 combinations of surfactants, the inventors were pleasantly surprised to find that in the comparison of the combination of two surfactants, DDM+LMNG showed the best recovery efficiency, and the mixture of the two surfactants was more efficient than the sum of the efficiency of the two surfactants used alone, suggesting that there is a certain degree of synergistic strengthening effect between the two surfactants.
[0060] Even more surprising is that when the three surfactants (DDM+LMNG+FC12) are combined, the recovery of glycoprotein E can reach 3.8 mg / 2E8 cells (100 mL of cell culture), equivalent to 38 mg / L cells, which is a very high level for the yield of membrane proteins expressed in small-scale experiments in mammalian cells. This is also the first time that the inventors are aware of that three surfactants have been used to solubilize and purify membrane proteins. At the same time, the inventors found that when other combinations of three surfactants, such as DDM+LMNG+CHAPS, were selected, there was no difference compared to the combination of two surfactants, DDM+LMNG, and another combination of three surfactants, DDM+CHAPS+FC12, only showed limited improvement compared to two of the surfactants, DDM+FC12.
[0061] The inventors further investigated the three-surfactant combination of DDM, LMNG, and FC12. They reduced the concentration of the combination from 1% + 1% + 1% to 0.5% + 0.5% + 0.5%, repeating steps 1 through 4 above. The recovery of glycoprotein E decreased to 2.2 mg / 2E8 cells. Furthermore, the concentration was increased to 2% + 2% + 2%, but the recovery of glycoprotein E did not increase, indicating that a 1% concentration maximized glycoprotein E solubilization.
[0062] Example 2 Production of full-length herpes zoster glycoprotein E
[0063] Produce full-length herpes zoster glycoprotein E as follows:
[0064] Step 1. Same as Example 1.
[0065] Step 2. Same as Example 1.
[0066] Step 3. Add a mixture of DDM, LMNG and FC12 surfactants to the supernatant. The final concentration of the added surfactants in the supernatant is 1%. Mix well and place at 4°C for 16 hours.
[0067] Step 4. The mixture was subjected to affinity chromatography (HIS60), washed with 20 mmol / L Tris, pH 8.0; 0.05% DDM; 20 mmol / L imidazole, and eluted with 20 mmol / L Tris, pH 8.0; 0.05% DDM; 200 mmol / L imidazole.
[0068] Step 5. The protein-containing eluate was collected and dialyzed to remove imidazole. The protein was then further purified by ion exchange chromatography. For ion exchange chromatography, Q resin (Q Sepharose Fast Flow, Cytiva, 17-0510) was used, and a sodium chloride gradient was used for washing (20 mmol / L Tris, pH 8.0; 0.05% DDM; 50 mmol / L sodium chloride) and elution (20 mmol / L Tris, pH 8.0; 0.05% DDM; 500 mmol / L sodium chloride).
[0069] The protein purity was >95% as determined by SDS-PAGE gel electrophoresis, and the yield was 2 mg / 2E8 cells (100 ml cell culture medium).
[0070] The extracellular region of herpes virus glycoprotein E (amino acid sequence 1-539 of SEQ ID NO: 1) was expressed in CHO cells, and the resulting protein was subjected to SDS-gel electrophoresis with the proteins obtained in steps 1 to 5 of this example. The results are shown in FIG. Figure 1 .
[0071] Example 3 Production of full-length herpes zoster glycoprotein E
[0072] The same procedures were followed as in Example 2, except that in step 1, the recombinant expression plasmid constructed using pcDNA3.4 was transformed into HEK293 cells, another mammalian cell type commonly used for recombinant protein production, to express full-length herpes zoster glycoprotein E. The surfactant combination and concentration used for protein solubilization were the same as those used in Example 2, and the purification steps were also the same as in Example 2.
[0073] Surprisingly, the full-length membrane protein expressed by HEK293 cells has a higher protein content. After the same purification steps, the purity of the protein can reach 95% as determined by SDS-PAGE gel electrophoresis, and the yield can reach 2.5 mg / 2E8 cells.
[0074] Example 4 Production of full-length herpes zoster glycoprotein E
[0075] The same procedure as in Example 2 was followed, except that DDM and LMNG surfactants were added in step 3, with the final concentration of the added surfactants in the supernatant being 1%. The mixture was mixed well and then allowed to stand at 4°C for 48 hours. The purification steps were the same as in Example 2.
[0076] The protein purity was determined to be 95% by SDS-PAGE gel electrophoresis, and the yield was 1.6 mg / 2E8 cells (100 ml cell culture medium).
[0077] Example 5 Validation of the immunogenicity of full-length herpes zoster glycoprotein E
[0078] This example aims to verify the application of the product protein obtained by the production method of the present invention.
[0079] BALB / C mice aged 6-8 weeks were randomly divided into groups of 5 mice each. PBS, full-length herpes simplex virus glycoprotein E (5 μg / mouse), or the extracellular domain of glycoprotein E (see Example 2) were administered intramuscularly. Antigen was injected into the inner hind leg at 0 and 3 weeks of immunization. Blood was collected 14 days after the last immunization for antibody titer ELISA. The extracellular domain protein was diluted to 5 μg / ml as the coating antigen, and 100 μL was added to each well. Serum to be tested was diluted in a gradient.
[0080] Test results such as Figure 2The results showed that mice immunized with full-length herpesvirus glycoprotein E produced significantly higher IgG (GMT) levels than mice immunized with truncated extracellular domain proteins. This demonstrates that full-length herpesvirus glycoprotein E, as an antigen, has better immunogenicity than truncated extracellular domain proteins and is more suitable as a vaccine antigen for herpes zoster.
[0081] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art can make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should fall within the scope of the appended claims of the present invention. Sequence Listing <110> Anbo Zhilian (Beijing) Biotechnology Co., Ltd. <120> Production method of herpes zoster glycoprotein E <130> LC21110101 <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 623 <212> PRT <213> Human alphaherpesvirus 3 <400> 1 Met Gly Thr Val Asn Lys Pro Val Val Gly Val Leu Met Gly Phe Gly 1 5 10 15 Ile Ile Thr Gly Thr Leu Arg Ile Thr Asn Pro Val Arg Ala Ser Val 20 25 30 Leu Arg Tyr Asp Asp Phe His Thr Asp Glu Asp Lys Leu Asp Thr Asn 35 40 45 Ser Val Tyr Glu Pro Tyr Tyr His Ser Asp His Ala Glu Ser Ser Trp 50 55 60 Val Asn Arg Gly Glu Ser Ser Arg Lys Ala Tyr Asp His Asn Ser Pro 65 70 75 80 Tyr Ile Trp Pro Arg Asn Asp Tyr Asp Gly Phe Leu Glu Asn Ala His 85 90 95 Glu His His Gly Val Tyr Asn Gln Gly Arg Gly Ile Asp Ser Gly Glu 100 105 110 Arg Leu Met Gln Pro Thr Gln Met Ser Ala Gln Glu Asp Leu Gly Asp 115 120 125 Asp Thr Gly Ile His Val Ile Pro Thr Leu Asn Gly Asp Asp Arg His 130 135 140 Lys Ile Val Asn Val Asp Gln Arg Gln Tyr Gly Asp Val Phe Lys Gly 145 150 155 160 Asp 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Pro Phe Asp Leu Leu Leu Glu Trp Leu Tyr Val Pro Ile Asp 370 375 380 Pro Thr Cys Gln Pro Met Arg Leu Tyr Ser Thr Cys Leu Tyr His Pro 385 390 395 400 Asn Ala Pro Gln Cys Leu Ser His Met Asn Ser Gly Cys Thr Phe Thr 405 410 415 Ser Pro His Leu Ala Gln Arg Val Ala Ser Thr Val Tyr Gln Asn Cys 420 425 430 Glu His Ala Asp Asn Tyr Thr Ala Tyr Cys Leu Gly Ile Ser His Met 435 440 445 Glu Pro Ser Phe Gly Leu Ile Leu His Asp Gly Gly Thr Thr Leu Lys 450 455 460 Phe Val Asp Thr Pro Glu Ser Leu Ser Gly Leu Tyr Val Phe Val Val 465 470 475 480 Tyr Phe Asn Gly His Val Glu Ala Val Ala Tyr Thr Val Val Ser Thr 485 490 495 Val Asp His Phe Val Asn Ala Ile Glu Glu Arg Gly Phe Pro Pro Thr 500 505 510 Ala Gly Gln Pro Pro Ala Thr Thr Lys Pro Lys Glu Ile Thr Pro Val 515 520 525 Asn Pro Gly Thr Ser Pro Leu Leu Arg Tyr Ala Ala Trp Thr Gly Gly 530 535 540 Click Download to save Leu Ala Ala Val Val Leu Cys Leu Val Ile Phe Leu Ile Cys Thr 545 550 555 560 Ala Light Arg With Arg Val Light Ala Tyr Arg Val Asp Light Ser Pro Tyr 565 570 575 Asn Gln Ser Meth Thyr Tyr Ala Glyleu Proval Asp Asp Phe Glu Asp 580 585 590 Ser Glu Ser Thr Asp Thr Glu Glu Glu Phe Gly Asn Ala Ile Gly Gly 595 600 605 Ser His Gly Gly Ser Ser Tyr Thr Val Tyr Ile Asp Lys Thr Arg 610 615 620 <210> 2 <211> 1869 <212> DNA <213> singular sequence (artificial sequence) <220> <223> Built-in fertilizer-based E-buy <400> 2 atgggaaccg tgaacaagcc cgtcgtgggc gtgctgatgg gcttcggaat catcaccgga acgctgagaa tcaccaaccc cgtgcgggcc agcgtgctga gatacgacga ctttcacaca gatgaggaca agctggatac gactctgtg actaccactc tgaccacgct gagtcctctt gggtgaatag aggagaatct tctcggaagg cctacgacca caactcccct tacatctggc ctagaaacga ctacgacggc ttcctggaga atgctcacga gcatcatggc 300 gtgtacaacc agggcagagg catcgactcc ggcgagagac tgatgcagcc cacccagatg 360 tccgcccaag aggacctggg agacgatacc ggcatccacg tgatccctac cctgaatggc 420 480 gacctgaacc ccaagcctca aggccagaga ttgatcgaag tgtccgtgga agaaaaccac 540 cccttcacc tgcgggctcc catccagcgg atctacggcg ttcggtacac cgagacatgg 600 tccttcctgc cttccctgac ctgtaccggc gatgccgccc ctgctatcca gcacatctgt 660 ctgaagcaca ccacatgctt ccaggacgtg gttgtggacg tggactgcgc cgagaacacc 720 aaagaatc agctggccga aatctcttac agattccagg ccaagaagga ggctgaccag 780 ccttggatcg tggtcaacac ctccaccctg ttcgacgaac tggaactgga ccctccagag 840 attgagcccg gcgtgctgaa ggtgctgaga accgagaagc agtacctggg cgtctacatc 900 tggaacatgc ggggctccga cggaaaagc acctacgcca catttctggt gacctggaag 960 ggcgacgaga aaaccagaaa ccctacacct gccgtgaccc ctcagcctag aggcgccgag 1020 ttccacatgt ggaactacca ctcccatgtg ttcagcgttg gcgacacctt ctccctcgcc 1080 atgcacctgc agtacaaaat ccatgaggcc ccatttgatc tgctcttgga gtggctgtac 1140 gtgcctatcg atccaacctg ccagcctatg cggctgtact ccacctgcct gtaccaccct 1200 aacgctcctc agtgcctgtc ccacatgaac tctggctgca ccttcacatc ccctcacctg 1260 gcccagagag tggcctccac cgtgtaccag aactgcgagc acgccgacaa ctacaccgct 1320 tactgtctgg gcatctccca catggaacct tctttcggcc tgatcctgca cgacggcgga 1380 accaccttga agttcgtgga cacccctgag tctctgtctg gcctgtatgt gtttgtggtg 1440 tacttcaacg gccacgtgga agctgtggcc tatacagtgg tgtctaccgt ggatcacttc 1500 gtgaacgcca tcgaggaaag gggcttccct cctactgctg gccaaccccc agccaccacc 1560 aaaccaaaag agatcacccc tgtcaatcct ggcacatctc ctctgctgcg ctacgctgct 1620 tggaccggcg gcctggctgc tgtggtgctg ctgtgcctgg tcatcttcct gatctgcacc 1680 gccaagcgga tgagagtgaa ggcctaccgg gtggacaagt ccccctacaa ccagtctatg tactacgccg gcctgcctgt agcgacttc gaggactccg agtccacaga caccgaaga gagttcggca acgccatcgg cggcagccac ggcggctcct cctacaccgt gtatatcgac aagaccaga 1869
Claims
1. A method for producing full-length herpes zoster glycoprotein E, comprising the following steps: 1) constructing a recombinant expression vector comprising a nucleotide sequence encoding full-length herpes zoster glycoprotein E, transforming the resulting recombinant expression vector into a mammalian host cell, and culturing the transformed host cell to express full-length herpes zoster glycoprotein E; wherein the nucleotide sequence is shown in SEQ ID NO: 2; 2) Centrifuging the cell culture obtained in step 1) to obtain host cells, and resuspending the cells in buffer at a ratio of 5-30 mL of buffer to 2E8 cells; then disrupting the cells by sonication, and centrifuging the resulting cell lysate at 5,000-15,000 g for 20-40 minutes to obtain a supernatant; 3) adding a surfactant to the supernatant obtained in step 2) and mixing uniformly, wherein the ratio of the disrupted host cells in step 2) to the added surfactant is 2E8 cells: 0.1-1 g surfactant, and the surfactant is a mixture of DDM, LMNG, and FC12 in a weight ratio of 1:1:1, and the concentrations of DDM, LMNG, and FC12 in the supernatant are 1-2% (w / v), respectively; and then placing the mixture at 4-25° C. for 12-50 hours.
2. The production method according to claim 1, characterized in that The amino acid sequence of the full-length herpes zoster glycoprotein E in step 1) is shown in SEQ ID NO:
1.
3. The production method according to claim 1, characterized in that The expressed full-length herpes zoster glycoprotein E has a tag at its carboxyl (C) or amino (N) terminus for protein purification.
4. The production method according to claim 1, characterized in that In step 2), the cells are resuspended in a buffer containing 0-2 mol / L sodium chloride.
5. The production method according to claim 4, characterized in that Resuspend the cells in a buffer containing 0.5 mol / L sodium chloride.
6. The production method according to claim 4, characterized in that The buffer is Tris, PBS or HEPES buffer.
7. The production method according to claim 5, characterized in that The buffer is Tris, PBS or HEPES buffer.
8. The production method according to any one of claims 1 to 7, characterized in that The ratio of buffer added in step 2) to cells was 10 mL: 2E8 cells.
9. The production method according to claim 8, characterized in that The resulting cell lysate was centrifuged at 10,000 g for 30 minutes.
10. The production method according to claim 9, characterized in that The ratio between the disrupted host cells in step 2) and the surfactant added in step 3) is 2E8 cells: 0.2-0.3 g surfactant.
11. The production method according to claim 8, characterized in that After adding the surfactant in step 3), the mixture is placed at 4° C. for 16-48 hours.
12. The production method according to any one of claims 1 to 7, characterized in that The production method further comprises the following steps: 4) subjecting the mixed solution obtained in step 3) to affinity chromatography to obtain the purified full-length herpes zoster glycoprotein E, wherein the affinity chromatography is performed using an eluent containing 0.01-0.1% (w / v) surfactant for impurity washing and protein elution, respectively, and the surfactant is DDM; and the eluent for impurity washing further contains 0-50 mmol / L imidazole, and the eluent for protein elution further contains 100-500 mmol / L imidazole.
13. The production method according to claim 12, characterized in that During the affinity chromatography in step 4), the eluent for washing and protein elution contained 0.05% DDM.
14. The production method according to claim 13, characterized in that The eluent used for washing impurities further contained 20 mmol / L imidazole, and the eluent used for eluting proteins further contained 200 mmol / L imidazole.
15. The production method according to claim 12, characterized in that The eluent is Tris, PBS or HEPES buffer.
16. The production method according to claim 12, characterized in that The production method further comprises the following steps: 5) subjecting the obtained purified protein to ion exchange chromatography to obtain further purified full-length herpes zoster glycoprotein E, wherein the ion exchange chromatography uses an eluent containing 0.01-0.1% (w / v) surfactant for impurity washing and protein elution, respectively, and the surfactant is DDM; and the eluent for impurity washing further contains 0-100 mmol / L sodium chloride, and the eluent for protein elution further contains 300-1000 mmol / L sodium chloride.
17. The production method according to claim 16, characterized in that During the ion exchange chromatography in step 5), the eluent used for washing impurities and eluting proteins contained 0.05% DDM.
18. The production method according to claim 17, characterized in that The eluent used for washing impurities further contained 50 mmol / L sodium chloride, and the eluent used for eluting proteins further contained 500 mmol / L sodium chloride.
19. The production method according to any one of claims 16 to 18, characterized in that The eluent is Tris, PBS or HEPES buffer.
20. The production method according to claim 12, characterized in that In step 4), after affinity chromatography is performed to obtain the eluted protein, dialysis is first performed to remove imidazole, and then ion exchange chromatography is performed in step 5).