Gene for coding soluble VZV-gB-SpyTag protein and application thereof

Abstract

The invention discloses a gene for coding soluble VZV-gB-SpyTag protein and application of the gene, and belongs to the technical field of genetic engineering and immunology. The nucleotide sequence of the gene is as shown in SEQ ID NO. 8. The invention provides a gene for coding soluble VZV-gB-SpyTag as well as a recombinant plasmid and a recombinant microorganism thereof, soluble VZV-gB-SpyTag protein is prepared, Fe-gB nanoparticles are further constructed, the nanoparticles are uniform in size, good in dispersity and stable in structure, the cellular immune effect of the VZV-gB protein can be remarkably improved, and the VZV-gB-SpyTag protein can be used for preparing the soluble VZV-gB-SpyTag protein. The resistance of the body to varicella-zoster virus is enhanced. The invention provides technical support for prevention and treatment of varicella-zoster virus.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of genetic engineering and immunology, in particular to a gene encoding soluble VZV-gB-SpyTag protein and application thereof. BACKGROUND

[0002] Varicella-zoster virus (VZV), also known as human herpesvirus 3, belongs to the alpha herpesvirus and is a double-stranded DNA virus with a capsule. In a natural state, VZV only infects humans and has no other animal hosts. After infection, it can cause two diseases, varicella and herpes zoster (HZ). Primary VZV infection causes varicella, which mainly occurs in childhood. During varicella, water blisters appear on the skin and are accompanied by itching symptoms. Scratching can cause VZV to aerosolize and then spread to others. After varicella, the virus does not completely disappear, but a small amount of VZV is latent in the host trigeminal ganglion (TG) and dorsal root ganglion (DRG). When the host's immunity is low, the virus is reactivated, causing herpes zoster and a variety of complications, such as fever, local lymph pain, skin burning, and post-herpetic neuralgia (PHN). The herpes zoster rash gradually weakens, but the pain caused by PHN continues to occur, usually for more than 90 days or even longer, which has a serious negative impact on the patient's quality of life. Therefore, the development of varicella-zoster virus vaccine is of great significance for the prevention and control of varicella-zoster virus.

[0003] VZV encodes at least 7 glycoproteins, and the gB glycoprotein binds to cellular heparan sulfate proteoglycans, which is related to virus replication, virus invasion of cells, and virus transmission between cells. The varicella-zoster virus gB protein is a type I membrane protein encoded by ORF31, which contains about 868 amino acids and is highly conserved in all herpes virus families. The gB protein monomer contains an extracellular region with five subdomains (DI-DV), a hydrophobic transmembrane domain, and a cytoplasmic domain containing 125 amino acids. The cytoplasmic domain of gB protein is the longest domain among the membrane proteins encoded by VZV so far. However, the immune effect of the vaccine developed based on gB protein is not ideal.

[0004] Ferritin is a kind of biological self-assembled nanoparticles (BNP), which is safe and low toxicity. It can not only self-assemble into a spherical nanoparticle cage, display the fused antigen protein on the particle surface, but also promote the recognition and uptake of the antigen by antigen-presenting cells, and then induce a stronger immune response. Most importantly, compared with synthetic nanoparticles, ferritin has significant advantages. First, ferritin nanoparticles are uniform in size and good in dispersity. Second, it has good thermal stability, and it is reported that its spherical cage structure can withstand a temperature as high as 80 DEG C for a short time. Therefore, ferritin as an attractive protein nanocarrier has important significance for the development of new vaccines. At present, there is no report on the preparation of varicella-zoster virus vaccine by using ferritin. SUMMARY

[0005] The purpose of the present application is to provide a gene encoding soluble VZV-gB-SpyTag protein and its application, in order to solve the problems existing in the prior art. The present application provides a gene encoding soluble VZV-gB-SpyTag and its recombinant plasmid, recombinant microorganism, which is prepared to obtain soluble VZV-gB-SpyTag protein, and further constructs Fe-gB nanoparticles. The nanoparticles are uniform in size, good in dispersity and stable in structure, which can significantly improve the cellular immune effect of VZV-gB protein and enhance the resistance of the body to varicella-zoster virus. The present application provides technical support for the prevention and treatment of varicella-zoster virus.

[0006] To achieve the above purpose, the present application provides the following scheme:

[0007] The present application provides a gene encoding soluble VZV-gB-SpyTag protein, the nucleotide sequence of which is shown in SEQ ID NO. 8.

[0008] The present application also provides a recombinant vector, which comprises the above-mentioned gene.

[0009] The present application also provides a recombinant microorganism, which comprises the above-mentioned recombinant vector.

[0010] The present application also provides the application of the above-mentioned gene, the above-mentioned recombinant vector or the above-mentioned recombinant microorganism in the preparation of soluble VZV-gB-SpyTag protein.

[0011] The present application also provides a soluble VZV-gB-SpyTag protein, the amino acid sequence of which is shown in SEQ ID NO. 9.

[0012] The present application also provides a Fe-SpyCatcher protein, the amino acid sequence of which is shown in SEQ ID NO. 13.

[0013] The present invention also provides the use of the above-mentioned soluble VZV-gB-SpyTag protein and the above-mentioned Fe-SpyCatcher protein in the preparation of varicella-zoster virus immunomodulators.

[0014] The present invention also provides a varicella-zoster virus immunomodulator, wherein the varicella-zoster virus immunomodulator is prepared by mixing the above-mentioned Fe-SpyCatcher protein with the above-mentioned soluble VZV-gB-SpyTag protein at a molar ratio of 1:8, reacting for 12-16 hours, and then purifying.

[0015] The present invention also provides the use of the above-mentioned varicella-zoster virus immunizing agent in the preparation of varicella-zoster virus vaccine.

[0016] The present invention also provides a varicella-zoster virus vaccine comprising the above-mentioned varicella-zoster virus immunizing agent and vaccine adjuvant.

[0017] The present invention discloses the following technical effects:

[0018] This invention provides a soluble, eukaryotically expressed VZV-gB-SpyTag recombinant protein with good solubility and structural stability. Utilizing a SpyTag-SpyCatcher linkage system, the VZV-gB protein is linked to Helicobacter pylori ferritin to construct Fe-gB nanoparticles. These nanoparticles are uniform in size, well-dispersed, and structurally stable, allowing for large-scale production. The VZV-gB protein is displayed on the surface of Helicobacter pylori ferritin, significantly enhancing the immunogenicity of VZV-gB protein and strengthening the body's resistance to varicella-zoster virus. This invention provides technical support for the prevention and control of varicella-zoster virus. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The images show the PCR identification results of the recombinant plasmid pCGS3-gB-SpyTag. In image A, the PCR product identification results of the gB-SpyTag gene are shown, with lane M representing the marker, lane 1 representing the PCR product, and lane 2 representing the negative control (water). Image B shows the PCR product identification results of the bacterial culture, with lane M representing the marker, lanes 1-4 representing the PCR product, and lane 5 representing the negative control (water).

[0021] Figure 2 Figure 1 is a picture of the identification of VZV-gB-SpyTag protein expression; wherein lane M is Marker, lane 1 is positive control cell supernatant, and lane 2 is negative control cell supernatant;

[0022] Figure 3 Figure 2 is a picture of the gel filtration chromatography purification results and SDS-PAGE identification results of VZV-gB-SpyTag protein; wherein A is the gel filtration chromatography results, and peak 1 is the VZV-gB-SpyTag protein; B is the SDS-PAGE identification results, lane M is the protein standard molecular weight Marker, and lane 1 is the VZV-gB-SpyTag protein;

[0023] Figure 4 Figure 3 is a picture of the preliminary purification identification results of Fe-SpyCatcher protein; wherein lane M is the protein standard molecular weight Marker, lane 1 is the supernatant of the lysate before heat denaturation, lane 2 is the supernatant of the lysate after heat denaturation, lane 3 is the flow-through of anion exchange chromatography, lanes 4-7 are elution components with NaCl concentrations of 100 mM, 200 mM, 300 mM, and 400 mM, respectively, and lane 8 is the negative control (water);

[0024] Figure 5 Figure 4 is a picture of the gel filtration chromatography purification results and SDS-PAGE identification results of Fe-SpyCatcher protein; wherein A is the gel filtration chromatography results, and peak 1 is the Fe-SpyCatcher protein; B is the SDS-PAGE identification results, lane M is the protein standard molecular weight Marker, and lane 1 is the Fe-SpyCatcher protein;

[0025] Figure 6 Figure 5 is a picture of the gel filtration chromatography purification results and SDS-PAGE identification results of Fe-gB nanoparticles; wherein A is the gel filtration chromatography results, peak 1 is the Fe-gB nanoparticles, and peak 2 is the gB-Spytag protein; B is the SDS-PAGE identification results, lane M is the protein standard molecular weight Marker, lane 1 is the Fe-gB nanoparticle protein, and lane 2 is the gB-Spytag protein;

[0026] Figure 7 Figure 6 is a picture of the transmission electron microscopy of Fe-gB nanoparticles and ferritin nanoparticles; wherein A is the ferritin nanoparticles, and B is the Fe-gB nanoparticles; the scale bar is 200 nm;

[0027] Figure 8 Figure 7 is the cell immunization detection results of each group of mice; wherein A is the flow cytometry detection results, B is the number of CD4+T cells producing IFN-γ, and C is the number of CD4+T cells producing IL-2. DETAILED DESCRIPTION

[0028] The following detailed description of various example embodiments of the application will not be considered limiting of the application, but rather as a description of certain aspects, features and embodiments of the application.

[0029] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. Additionally, the use of "including", "comprising", "having" and "with" are not intended to be limiting of the application. It is to be understood that the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Under no circumstances can the patent be interpreted to be limited to the specific examples described herein. Under no circumstances can the patent be interpreted to be limited to the specific embodiments described herein.

[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described herein. In the case of conflict between the present specification and any document incorporated herein by reference, the present specification will control.

[0031] Various modifications and changes can be made to the specific embodiments of the application described herein without departing from the scope or spirit of the application. Other embodiments of the application will be apparent to those of ordinary skill in the art from the description and examples herein. The description and examples are illustrative of the application and are not intended to limit the scope of the application.

[0032] As used herein, the terms "comprise", "comprising", "include", "including", "have", "having" and the like are open-ended and do not exclude additional elements or steps.

[0033] The experimental methods in the following examples are all routine methods unless specifically indicated otherwise. The instruments and equipment used in the following examples are all routine laboratory instruments and equipment unless specifically indicated otherwise; the test materials used in the following examples are all purchased from routine biochemical reagent stores unless specifically indicated otherwise.

[0034] Example 1 Construction of recombinant expression plasmid

[0035] 1. Construction of pCGS3-gB-SpyTag recombinant vector

[0036] 1.1 Design and synthesis of gB-SpyTag gene primers

[0037] The VZV-gB gene sequence of the present application is a DNA sequence optimized by CHO cell preferred codons. All the amino acids thereof are all used with the most frequently used nucleotide codons in CHO; meanwhile, in order to avoid the GC proportion of translated mRNA being too high, the secondary structure of mRNA affecting the translation efficiency, and avoiding some commonly used enzyme cutting sites, the optimal codon frequency is corrected, and finally the VZV-gB gene sequence is designed. The nucleotide sequence is as follows:

[0038]

[0039] The amino acid sequence of which is as follows:

[0040] MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAGSGFNGPGVFITSVTGVWLCF

[0041] LCIFSMFVTAVVSVSPSSFYESLQVEPTQSEDITRSAHLGDGDEIREAIHKSQDAETKPTFYV

[0042] CPPPTGSTIVRLEPTRTCPDYHLGKNFTEGIAVVYKENIAAYKFKATVYYKDVIVSTAGAGSS

[0043] GTQITNRYADRVPIPVSEITDTIDKFGKCSSKATYVRNNHKVEAFNEDKNPQDMPLIASKYN

[0044] SVGSKAWHTTNDTYMVAGTPGTYRTGTSVNCIIEEVEARSIFPYDSFGLSTGDIIYMSPFFGL

[0045] RDGAYREHSNYAMDRFHQFEGYRQRDLDTRALLEPAARNFLVTPHLTVGWNWKPKRTEV

[0046] CSLVKWREVEDVVRDEYAHNFRFTMKTLSTTFISETNEFNLNQIHLSQCVKEEARAIINRIYT

[0047] TRYNSSHVRTGDIQTYLARGGFVVVFQPLLSNSLARLYLQELVRENTNHSPQKHPTRNTGS

[0048] GGSVPVELRANRTITTTSSVEFAMLQFTYDHIQEHVNEMLARISSSWCQLQNRERALWSGL

[0049] FPINPSALASTILDQRVKARILGDVISVSNCPELGSDTRIILQNSMRVSGSTTRCYSRPLISIVSL

[0050] NGSGTVEGQLGTDNELIMSRDLLEPCVANHKRYFLFGHHYVYYEDYRYVREIAVHDVGMI

[0051] STYVDLNLTLLKDREFMPLQVYTRDELRDTGLLDYSEIQRRNQMHSLRFYDIDKVVQ

[0052] (SEQ ID NO.2).

[0053] The nucleotide sequence of the SpyTag gene of the application is as follows:

[0054] GCTCATATTGTTATGGTTGATGCTTACAAGCCAACTAAG (SEQ ID NO. 3) ;

[0055] The amino acid sequence thereof is as follows: AHIVMVDAYKPTK (SEQ ID NO. 4).

[0056] The gB gene sequence is connected with the SpyTag gene through 2xG4S-Linker, and the synthesis of the pCGS3-gB-SpyTag recombinant vector is simulated by SnapGene. The primers containing the enzyme digestion sites Hind III and PacI are designed according to the gB-SpyTag target gene, as shown in Table 1.

[0057] Table 1 Primer sequence

[0058]

[0059] 1.2 gB-SpyTag target gene amplification

[0060] The gB-2xG4S-Linker gene is amplified by using the artificially synthesized gB gene sequence as a template, and using F-gB-SpyTag and R-gB-SpyTag 1 as the upper and lower primers. Then, the gB-SpyTag gene sequence is amplified by using the gB-2xG4S-Linker gene PCR product as a template, and using F-gB-SpyTag and R-gB-SpyTag 2 as the upper and lower primers. The amplification reaction system is shown in Table 2.

[0061] Table 2 PCR reaction system

[0062]

[0063]

[0064] After the reaction is completed, the gB-SpyTag gene PCR product is identified by agarose gel electrophoresis to determine whether the target gene is successfully amplified. The results are shown in Figure 1 Fig. 1A, and the results show that there is an obvious band at about 2100bp, which is consistent with the expected size.

[0065] The gB-SpyTag gene sequence is as follows:

[0066] ATGAGCCCTTGTGGATACTACTCTAAGTGGCGGAATCGGGACCGGCCTGAGTACCG

[0067] GCGGAACCTGCGGTTTCGCCGCTTCTTCTCTTCTATCCACCCTAACGCCGCTGCCGGCTC

[0068] CGGCTTTAATGGACCTGGTGTGTTCATCACCTCTGTGACCGGCGTGTGGCTGTGCTTCCT

[0069] GTGCATCTTCTCCATGTTCGTGACAGCCGTGGTGAGCGTGAGCCCTTCCAGCTTTTATGA

[0070] GTCCCTGCAGGTGGAGCCTACCCAGTCTGAAGATATCACACGCTCTGCCCATCTGGGGG

[0071] ATGGGGACGAGATCCGGGAGGCCATCCACAAGTCTCAGGACGCCGAGACCAAGCCAAC

[0072] CTTCTACGTGTGCCCTCCACCTACCGGCTCTACCATCGTCCGCCTGGAGCCTACCCGCAC

[0073] CTGCCCAGACTACCACCTGGGCAAGAACTTTACCGAGGGCATCGCCGTGGTGTATAAGG

[0074] AGAATATCGCTGCCTATAAGTTTAAGGCAACCGTGTACTATAAGGATGTGATCGTGAGCA

[0075] CAGCTGGGGCCGGGTCATCTGGTACACAGATTACAAATCGCTATGCCGACCGCGTGCCTA

[0076] TCCCAGTGAGCGAGATCACAGACACTATCGATAAGTTTGGCAAGTGTTCCTCCAAGGCC

[0077] ACCTACGTCCGTAACAACCACAAGGTGGAGGCCTTTAATGAGGATAAGAACCCTCAGGA

[0078] CATGCCACTGATCGCCTCTAAGTATAACAGCGTGGGAAGTAAGGCTTGGCATACCACTAA

[0079] CGATACCTATATGGTGGCTGGCACCCCAGGCACCTACCGGACCGGAACCTCTGTGAACT

[0080] GTATCATCGAAGAGGTGGAGGCTCGGTCAATCTTCCCATATGACAGCTTCGGCCTCTCTA

[0081] CCGGGGATATCATCTACATGTCTCCATTCTTCGGCCTGCGGGATGGCGCCTACCGCGAAC

[0082] ATTCTAACTACGCCATGGATCGCTTCCACCAGTTTGAGGGCTACCGCCAGCGCGATCTGG

[0083] ACACCCGCGCCCTGCTGGAGCCTGCCGCTCGGAACTTCCTGGTGACCCCTCACCTGACC

[0084] GTGGGCTGGAACTGGAAGCCTAAGCGCACCGAGGTGTGTTCCCTCGTGAAGTGGCGCG

[0085] AGGTGGAGGACGTGGTCCGGGACGAGTACGCTCATAACTTCCGGTTCACAATGAAAAC

[0086] CCTGAGCACCACATTCATTAGTGAGACCAATGAGTTTAACCTGAACCAGATCCATTTGAG

[0087] CCAGTGTGTGAAGGAAGAGGCCCGTGCTATCATCAACCGGATCTACACCACACGGTATA

[0088] ATAGCTCCCATGTGCGGACCGGAGACATCCAGACCTACCTGGCTCGTGGCGGCTTTGTG

[0089] GTGGTGTTTCAGCCTCTGCTGTCTAACTCTCTCGCTCGGCTGTATCTGCAGGAGCTCGTG

[0090] CGCGAGAACACTAACCACAGCCCACAGAAGCACCCTACACGTAACACAGGGTCAGGTG

[0091] GGTCCGTCCCTGTCGAGCTGCGCGCTAATCGCACAATCACAACTACCTCCAGCGTGGAG

[0092] TTTGCTATGCTGCAGTTCACATATGATCACATCCAGGAGCACGTGAACGAGATGCTGGCC

[0093] CGGATTAGCTCTAGCTGGTGCCAGCTGCAGAATCGGGAGCGCGCCCTGTGGAGCGGCCT

[0094] GTTTCCAATTAACCCTTCTGCTCTGGCCTCTACCATCCTGGATCAGCGTGTGAAGGCCCG

[0095] CATCCTCGGCGACGTGATCTCTGTGTCTAATTGTCCTGAGCTCGGCAGCGATACACGGAT

[0096] CATCCTGCAGAAcTCAATGCGCGTGAGCGGCTCCACTACTCGCTGCTATAGCCGGCCACT

[0097] GATCTCCATCGTGAGCCTGAACGGTAGCGGCACCGTGGAGGGCCAGCTGGGCACAGAC

[0098] AATGAACTGATCATGTCCCGTGATCTGCTCGAGCCATGCGTGGCTAACCATAAGCGGTAT

[0099] TTTCTGTTTGGACACCACTACGTGTACTACGAGGACTACCGTTACGTGCGGGAGATCGCT

[0100] GTGCATGACGTGGGCATGATCTCTACCTACGTGGACCTGAATCTGACCCTGCTGAAGGA

[0101] CCGGGAGTTTTATGCCACTGCAGGTGTACACCCGCGACGAGCTGCGGGACACCGGACTG

[0102] CTGGACTACTCCGAGATCCAGCGCCGGAATCAGATGCATTCCCTGCGGTTTTATGACATC

[0103] GACAAGGTGGTGCAG GGAGGAGGCGGCAGCGGAGGAGGCGGCAGC GCTCATATTGTT

[0104] ATGGTTGATGCTTACAAGCCAACTAAGtaaTTAATTAAGG (SEQ ID NO. 8).

[0105] Double digestion and recovery of the target fragment from the 1.3pCGS3 plasmid and gB-SpyTag.

[0106] The pCGS3 plasmid and the gB-SpyTag gene fragment were double-digested with Hind III and Pac I. The digestion system consisted of 1 μL each of Hind III and Pac I, 2 μL of gB-SpyTag gene and 7 μL of pCGS3 vector, 5 μL of 10×Cutsmart, and ddH2O to a final volume of 50 μL. After micro-incubation, the mixture was digested at 37°C for 4 h.

[0107] 2. Construction of pCGS3-gB-SpyTag recombinant expression bacteria

[0108] The double-digested product gB-SpyTag gene and pCGS3 vector were ligated overnight at 16°C using T4 ligase. The ligation system consisted of 2 μL of the target gene, 3 μL of the vector, 2 μL of 10×T4 DNA Ligase Buffer, 1 μL of T4 DNA Ligase, and ddH2O to a final volume of 20 μL.

[0109] The pCGS3-gB-SpyTag ligation product was transformed into DH5α competent cells, and positive clones were screened. After removing the plates, single colonies were selected and labeled, then sequentially inoculated into LB agar and incubated overnight at 37°C. PCR identification of the cultured single-clone cultures was performed using F-gB-SpyTag and R-gB-SpyTag 2 as primers. Results are shown below. Figure 1B, positive clones that can amplify the target gene band by PCR were selected from the monoclonal bacterial solution for sequence alignment, and the correct recombinant vector was named pCGS3-gB-SpyTag.

[0110] Example 2 Expression and identification of VZV-gB-SpyTag recombinant protein

[0111] SMM293-T11 medium was preheated in a 37°C water bath, and HEK 293F cells were taken out from the liquid nitrogen tank, thawed to the size of a soybean particle at 37°C, and resuscitated. The cells were continuously cultured for more than 3 generations, and the cell state was observed from time to time. After the cell state was stable, the cells were ready for transient transfection. 24h before transfection, the density was adjusted to 2x10 6 cells / mL, and placed in a constant temperature shaking incubator for culture. The cell density was adjusted to 3x10 6 cells / mL, and the total volume was 200mL of culture medium. 200μg of pCGS3-gB-SpyTag plasmid and 1mL of Sinofection transfection reagent were diluted with 150mM NaCl solution, respectively, so that the total volume of each was kept at 5mL, and they were left to stand at room temperature for about 8min; after standing, the diluted pCGS3-gB-SpyTag plasmid and transfection reagent were mixed, and the mixture was left to stand at room temperature for another 10min, and then the mixture was added dropwise to the cell suspension with adjusted density. The cell suspension was fed every 24h, and 7mL of SMS-293-SUPI was added each time. The cell viability was monitored in real time by microscope. When the cell viability was less than 60%, the cells were no longer cultured. The collected cell culture supernatant was centrifuged at 1250rpm for the first time for 5min to remove cell debris; then centrifuged at 4200rpm for another 30min to remove insoluble substances.

[0112] The reaction of gB-SpyTag protein in HEK 293F cell culture supernatant was identified by Western blot, and the HEK 293F cell culture supernatant transfected with pCGS3-MCS plasmid was used as positive control, and the HEK 293F cell culture supernatant without transfection of pCGS3-MCS plasmid was used as negative control. The results are shown in Figure 2 , it can be seen that there is an obvious protein band in the HEK293F cell culture supernatant transfected with pCGS3-MCS plasmid near 110kDa, that is, the gB-SpyTag protein expressed in the cell supernatant can specifically react with positive VZV serum, while the negative control cell supernatant has no reaction band. The results show that HEK 293F cells successfully expressed VZV-gB-SpyTag recombinant protein.

[0113] The amino acid sequence of the VZV-gB-SpyTag recombinant protein is as follows:

[0114] MSPCGYYSKWRNRDRPEYRRNLRFRRFFSSIHPNAAAGSGFNGPGVFITSVTGVWLCF

[0115] LCIFSMFVTAVVSVSPSSFYESLQVEPTQSEDITRSAHLGDGDEIREAIHKSQDAETKPTFYV

[0116] CPPPTGSTIVRLEPTRTCPDYHLGKNFTEGIAVVYKENIAAYKFKATVYYKDVIVSTAGAGSS

[0117] GTQITNRYADRVPIPVSEITDTIDKFGKCSSKATYVRNNHKVEAFNEDKNPQDMPLIASKYN

[0118] SVGSKAWHTTNDTYMVAGTPGTYRTGTSVNCIIEEVEARSIFPYDSFGLSTGDIIYMSPFFGL

[0119] RDGAYREHSNYAMDRFHQFEGYRQRDLDTRALLEPAARNFLVTPHLTVGWNWKPKRTEV

[0120] CSLVKWREVEDVVRDEYAHNFRFTMKTLSTTFISETNEFNLNQIHLSQCVKEEARAIINRIYT

[0121] TRYNSSHVRTGDIQTYLARGGFVVVFQPLLSNSLARLYLQELVRENTNHSPQKHPTRNTGS

[0122] GGSVPVELRANRTITTTSSVEFAMLQFTYDHIQEHVNEMLARISSSWCQLQNRERALWSGL

[0123] FPINPSALASTILDQRVKARILGDVISVSNCPELGSDTRIILQNSMRVSGSTTRCYSRPLISIVSL

[0124] NGSGTVEGQLGTDNELIMSRDLLEPCVANHKRYFLFGHHYVYYEDYRYVREIAVHDVGMI

[0125] STYVDLNLTLLKDREFMPLQVYTRDELRDTGLLDYSEIQRRNQMHSLRFYDIDKVVQ GGG

[0126] GSGGGGS AHIVMVDAYKPTK (SEQ ID NO. 9).

[0127] Example 3 Purification of VZV-gB-SpyTag recombinant protein

[0128] After the cell culture supernatant obtained in Example 2 was enriched by ammonium sulfate precipitation, gel filtration chromatography (filler is gel particles) was used for purification. The column was pre-equilibrated with buffer PBS, eluted with buffer PBS at a rate of 1 ml / min, and the elution peak size was 12.55 mL (A). The VZV-gB-SpyTag protein was identified by SDS-PAGE, and the results showed that the peak was composed of VZV-gB-SpyTag protein, which was consistent with the molecular weight of VZV-gB-SpyTag protein, and had high purity (B). Figure 3 Figure 3

[0129] Example 4 Expression and purification of Fe-SpyCatcher recombinant protein

[0130] The H. pylori ferritin gene sequence (SEQ ID NO. 10) was added with a SpyCatcher fragment (SEQ ID NO. 11) at its 5' end through a 4×G2S linker to obtain a Fe-SpyCatcher gene fragment (SEQ ID NO. 12). The gene fragment was inserted between the XbaI-XhoI enzyme cutting sites of the pET-28a vector, and the pET-28a-Fe-SpyCatcher recombinant vector map was simulated synthesized by SnapGene, and then the general biology (Anhui) Co., Ltd. synthesized the recombinant plasmid.

[0131] SEQ ID NO. 10:

[0132] ​​ATGCTGAGCAAAGATATTATTAAGCTGCTGAATGAACAGGTTAATAAGGAAATGAATAGCAGCAATCTGTATATGAGTATGAGCAGTTGGTGTTATACCCATAGCCTGGATGGTGCCGGCCTGTTTCTGTTTGATCATGCAGCAGAAGAATATGAACATGCCAAAAAACTGATTATCTTCCTGAATGAAAACAACGTGCCGGTGCAGCTGACCAGCATTAGTGCCCCGGAACATAAATTTGAAGGCCTGACCCAGATTTTTCAGAAAGCATATGAACATGAACAGCATATTAGCGAAAGTATTAATAACATCGTGGATCATGCCATTAAGAGCAAAGATCATGCAACCTTTAATTTTCTGCAGTGGTATGTGGCCGAACAGCATGAAGAAGAAGTGCTGTTTAAAGATATTCTGGATAAAATCGAGCTGATTGGTAATGAAAATCATGGCCTGTATCTGGCAGATCAGTATGTGAAAGGTATTGCCAAAAGCCGCAAAAGC.

[0133] SEQ ID NO. 11:

[0134] ATGGATAGTGCCACCCATATTAAGTTTAGTAAACGTGATGAAGACGGTAAAGAACTGGCAGGCGCAACCATGGAACTGCGTGATAGCAGCGGTAAAACCATTAGCACCTGGATTAGCGATGGTCAGGTTAAAGATTTTTATCTGTATCCGGGCAAATATACCTTTGTTGAAACCGCCGCACCGGATGGCTATGAAGTGGCAACCGCCATTACCTTTACCGTGAATGAACAGGGTCAGGTGACCGTGAATGGCAAAGCAACCAAAGGTGACGCACATATT.

[0135] SEQ ID NO. 12:

[0136] ATGGATAGTGCCACCCATATTAAGTTTAGTAAACGTGATGAAGACGGTAAAGAACTGGCAGGCGCAACCATGGAACTGCGTGATAGCAGCGGTAAAACCATTAGCACCTGGATTAGCGATGGTCAGGTTAAAGATTTTTATCTGTATCCGGGCAAATATACCTTTGTTGAAACCGCCGCACCGGATGGCTATGAAGTGGCAACCGCCATTACCTTTACCGTGAATGAACAGGGTCAGGTGACCGTGAATGGCAAAGCAACCAAAGGTGACGCACATATT GGCGGTAGCGGTGGTAG TGGTGGTAGTGGCGGTAGT ATGCTGAGCAAAGATATTATTAAGCTGCTGAATGAACAGGTTAATAAGGAAATGAATAGCAGCAATCTGTATATGAGTATGAGCAGTTGGTGTTATACCCATAGCCTGGATGGTGCCGGCCTGTTTCTGTTTGATCATGCAGCAGAAGAATATGAACATGCCAAAAAACTGATTATCTTCCTGAATGAAAACAACGTGCCGGTGCAGCTGACCAGCATTAGTGCCCCGGAACATAAATTTGAAGGCCTGACCCAGATTTTTCAGAAAGCATATGAACATGAACAGCATATTAGCGAAAGTATTAATAACATCGTGGATCATGCCATTAAGAGCAAAGATCATGCAACCTTTAATTTTCTGCAGTGGTATGTGGCCGAACAGCATGAAGAAGAAGTGCTGTTTAAAGATATTCTGGATAAAATCGAGCTGATTGGTAATGAAAATCATGGCCTGTATCTGGCAGATCAGTATGTGAAAGGTATTGCCAAAAGCCGCAAAAGC.

[0137] The pCGS3-Fe-SpyCatcher recombinant plasmid was transformed into BL21 competent cells. Single positive clones were marked on the plate, inoculated into LB medium containing kanamycin, and cultured overnight; the activated bacterial liquid was inoculated into LB medium containing kanamycin, and the culture was continued; a small amount of cultured bacterial liquid was taken to measure the absorbance at 600 nm, and IPTG was added to induce expression; the supernatant was discarded, the bacterial body was collected, and after resuspension, ultrasonic disruption was performed. At the end of ultrasonic disruption, the previously turbid bacterial liquid became clear and transparent. Since ferritin has thermal stability, the supernatant of the lysate was heated at 70°C for 15 minutes to remove part of the impure proteins. After cooling in ice water for 15 min, centrifugation was performed at 10,000 rpm for 30 min to remove the precipitated proteins. The supernatant after centrifugation was further purified by anion exchange chromatography, and it was found that the Fe-SpyCatcher protein was eluted at a NaCl concentration of 300 mM, and the band was at 30 kDa, which was consistent with the expected molecular weight. Figure 4 The eluted protein was concentrated and finally purified by gel filtration chromatography (SuperdexTM 6 Increase 10 / 300 GL, GE Health Sciences), and the column was pre-equilibrated with 20 mM Tris buffer (pH 8.5) and eluted with the same buffer at a rate of 0.5 ml / min, and the elution peak size was about 16 mL Figure 5 The identification by SDS-PAGE showed a band at about 30 kD, which was consistent with the expected molecular weight Figure 5 The protein at the elution peak was collected and concentrated to obtain the Fe-SpyCatcher recombinant protein.

[0138] The amino acid sequence of the Fe-SpyCatcher recombinant protein is as follows:

[0139] MDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVET

[0140] AAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI GGSGGSGGSGGS MLSKDIIKLLNEQV

[0141] NKEMNSSNLYMSMSSWCYTHSLDGAGLFLFDHAAEEYEHAKKLIIFLNENNVPVQLTSISA

[0142] PEHKFEGLTQIFQKAYEHEQHISESINNIVDHAIKSKDHATFNFLQWYVAEQHEEEVLFKDIL

[0143] DKIELIGNENHGLYLADQYVKGIAKSRKS (SEQ ID NO. 13).

[0144] Purification and identification of Fe-gB nanoparticles

[0145] The purified Fe-SpyCatcher recombinant protein was connected with the VZV-gB-Spytag recombinant protein at a molar ratio of 1:8 in PBS overnight, and the connection product was purified by gel filtration chromatography (filler was gel particles). The column was pre-equilibrated with buffer PBS, and eluted with buffer PBS at a rate of 1 ml / min. The elution volume of peak 1 was 9.5 mL, and the elution volume of peak 2 was about 16 mL, which was consistent with the expected size Figure 6 (A), and identified by SDS-PAGE, with bands appearing near 130 kD and 110 kD, respectively, consistent with the molecular weight sizes of Fe-gB protein and gB-SpyTag protein Figure 6 (B). The protein at peak 1 was collected to obtain Fe-gB nanoparticles.

[0146] Under high-pressure vacuum conditions, air discharge made the copper mesh carbon film hydrophilic, and Fe-gB nanoparticles and ferritin nanoparticles (recombinant vector constructed according to the sequence shown in SEQ ID NO. 10, expressed and purified according to the steps of Example 4) were adsorbed on it. After staining with phosphotungstic acid negative staining solution, transmission electron microscopy observation was performed. It was found that the sizes of ferritin nanoparticles and Fe-gB nanoparticles were about 14 nm and 27 nm, respectively, and the surface of Fe-gB nanoparticles had obvious "bifurcation" structure, which was the gB protein displayed on the surface of ferritin Figure 7 (A, B). Therefore, Fe-gB can form relatively uniform size, good dispersion and stable structure nanoparticles.

[0147] Example 6 Verification of immunization effect

[0148] Twenty 6-8 week old healthy BALB / c mice were selected and randomly divided into four groups (5 mice / group), namely Fe-gB group, gB group, adjuvant group and PBS group. To simulate natural VZV infection, the experimental mice were injected subcutaneously with varicella attenuated live vaccine on the back of the neck before the study. After 35 days, the immunogen Fe-gB nanoparticles and gB protein (recombinant vectors constructed according to the sequence shown in SEQ ID NO. 1, expressed and purified by transfecting HEK 293F cells according to the steps of Examples 2 and 3) were diluted with 20 mM Tris-120 mM NaCl, and then mixed with CpG and aluminum hydroxide adjuvant in equal volumes to prepare vaccines. After incubation at room temperature for 30 min, the mice were immunized by hind limb muscle injection. Each mouse in the experimental group was injected with 5 μg of immunogen, and PBS buffer and blank adjuvant were used as control groups, and the immunization dose was 100 μL. A total of two immunizations were performed with an interval of 14 days.

[0149] Two weeks after the second immunization, the mouse spleen cells were taken for cell immune detection, and the frequency of gB-specific CD4+T cells secreting IFN-γ or IL-2 was detected by intracellular factor staining and flow cytometry (ICS-FCM). The results are shown in Figure 8 Compared with the gB group, Fe-gB enhanced the CD4+T cell response, and induced a higher number of gB-specific CD4+T cells producing IL-2 and IFN-γ positive cytokines. Specifically, the number of CD4+T cells producing IL-2 was 2.15 times higher, and the number of CD4+T cells producing IFN-γ was 3.24 times higher, with significant differences. This indicates that the Fe-gB nanoparticles prepared by the present application have higher immune effect.

[0150] The above-described examples only describe the preferred modes of the present application and do not limit the scope of the present application. Without departing from the design spirit of the present application, various modifications and improvements to the technical solutions of the present application made by those of ordinary skill in the art shall fall within the scope of protection determined by the claims of the present application.

Claims

1. A gene encoding a soluble VZV-gB-SpyTag protein, characterized in that, Its nucleotide sequence is shown in SEQ ID NO.

8.

2. A recombinant vector, characterized in that, The recombinant vector contains the gene described in claim 1.

3. A recombinant microorganism, characterized in that, The recombinant vector comprises the recombinant vector of claim 2.

4. The use of the gene of claim 1, the recombinant vector of claim 2, or the recombinant microorganism of claim 3 in the preparation of soluble VZV-gB-SpyTag protein.

5. A soluble VZV-gB-SpyTag protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

9.

6. A Fe-SpyCatcher protein, characterized in that, Its amino acid sequence is shown in SEQ ID NO.

13.

7. The use of the soluble VZV-gB-SpyTag protein of claim 5 and the Fe-SpyCatcher protein of claim 6 in the preparation of varicella-zoster virus immunomodulators.

8. A varicella-zoster virus immunomodulator, characterized in that, The method for preparing the varicella-zoster virus immunomodulator is as follows: the Fe-SpyCatcher protein of claim 6 and the soluble VZV-gB-SpyTag protein of claim 5 are mixed at a molar ratio of 1:8, reacted for 12-16 hours, and then purified.

9. The use of the varicella-zoster virus immunizing agent according to claim 8 in the preparation of varicella-zoster virus vaccine.

10. A varicella-zoster virus vaccine, characterized in that, It includes the varicella-zoster virus immunizing agent and vaccine adjuvant as described in claim 8.

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