A gene recombinant VZV fusion protein and its preparation method and application

Abstract

The present invention provides a genetically recombinant varicella-zoster virus fusion protein and a corresponding preparation method and application. The fusion protein is a fusion protein that utilizes genetic recombination technology to recombinant the amino acid 31-546 (i.e., gE1) of the VZVgE protein in series with a non-toxic toxin carrier protein or its functionally active fragment, and an artificial signal peptide is added to construct a target fusion gene. The target fusion gene is expressed by constructing a recombinant expression vector, transforming, and transfecting CHO cells, and then purifying to obtain a genetically recombinant VZV fusion protein. Compared with simple VZVgE protein, a genetically recombinant VZV fusion protein, especially a genetically recombinant VZV vaccine prepared by combining a genetically recombinant VZV fusion protein with an AS01B adjuvant, can cause higher antibody titers, have better immune activity, and induce stronger cellular immunity, thereby significantly improving the immune-promoting effect of the vaccine, and there is no risk of causing potential infection.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, especially the field of genetic engineering vaccines, and particularly relates to a fusion protein constructed by tandem recombination of varicella-zoster virus antigen and toxin carrier protein, and a preparation method and application thereof. Background Art

[0002] Varicella-zoster virus (VZV) belongs to the α subfamily of herpesviruses. It is a double-stranded DNA virus with a length of about 125,000 bp and encodes about 70 open reading frames. The virus nucleocapsid is about 80 to 120 nm in diameter and is icosahedral, surrounded by an inner membrane and an envelope that is partially derived from the cell membrane. VZV primary infection causes chickenpox. After the systemic symptoms and rash of the child subside, a small amount of residual virus will remain dormant in the host's spinal dorsal root ganglia, cranial ganglia, and enteric ganglia nerve cells for life. In the future, when the body's immunity is reduced, the virus lurking in the nerve cells will be reactivated, leading to a painful skin disease - herpes zoster (HZ).

[0003] For varicella and herpes zoster, the main treatment is symptomatic. There is still no specific treatment. Antiviral drugs, such as acyclovir, valacyclovir, and famciclovir, can help patients with varicella and herpes zoster recover quickly, but they cannot stop the virus from being excreted and prevent latent infection. Giving an overdose of immunoglobulin after exposure to VZV is also limited in stopping or reducing the burden of the disease. Therefore, vaccines play an important role in controlling varicella infection. Vaccination is the most effective and reliable means of preventing and controlling varicella.

[0004] The varicella vaccine was first developed by the Takahashi M research group in Japan in 1974 and is called the Oka vaccine (vOka). The live attenuated varicella vaccine is currently the only vaccine approved for the prevention of varicella, but it still has a certain risk of causing latent infection. Compared with the live attenuated vaccine, the inactivated vaccine is relatively safe, but the vaccination dose is larger, and generally 2-3 doses are needed to produce a better protective effect.

[0005] VZV glycoprotein E (gE) subunit vaccine is the mainstream research direction of varicella vaccine at present. gE is encoded by the virus ORF68 gene. This gene consisting of 1872 bases is located in the short fragment region of the VZV genome. gE exists on the surface of virus particles and the surface and cytoplasm of infected cells. It is an essential glycoprotein for the generation of infectious virus particles and the most abundant envelope glycoprotein on the viral envelope and host cell membrane. In the serum of patients with varicella and herpes zoster in the recovery period, VZV antibodies are mainly directed against gE, gB and gH. In particular, cellular immunity and humoral immunity induced by gE as the main target can protect animals from virus attack. Therefore, gE is also an important target of cellular and humoral immune responses. There have been quite a few studies on VZV gE protein: the wild-type or full-length gE protein is generally 623 amino acids. It consists of the main part of the gE protein containing the signal peptide, the hydrophobic anchor region (residues 546-558) and the C-terminal tail. Different studies have slightly different distinctions on the protein molecular structure of gE protein. Some researchers divide the protein molecule into a hydrophilic extracellular region (including a signal peptide), a hydrophobic transmembrane region (residues 545-561) and an intracellular tail (for example, see Grose C. Glycoproteins encoded by varicella-zoster virus: biosynthesis, phosphorylation and intracellular trafficking. Annu Rev Microbil. 1990, 44: 59-80), but the above different distinctions do not produce substantial differences in the application and preparation of gE protein. The protein in a typical pharmaceutical composition will be different from the full-length protein, but a truncated protein. For example, when using modern biomolecular technology to prepare recombinant VZV gE protein, the gE protein is generally truncated to lack the carboxyl-terminal hydrophobic anchor region. At present, there are many literatures and patents reporting the modification and expression of VZV glycoprotein E, such as truncated gE aa1-511 (Vafai et al. 1993, 1994, 1995); truncated gE aa1-546 (Haumont et al. 1996, Jacquet et al. 2002; GlaxoSmithKline EP0405867B1 1990, WO0043527A12000); truncated gE aa1-539 (CN102517302 2011); expression of fusion protein containing gE aa37-161 (CN105906721 2016), etc.

[0006] Tetanus toxin (TT) is a strong neurotoxin produced by Clostridium tetani. It consists of 1315 amino acids and has a molecular weight of 150kD. It can be cleaved by papain into a light chain and a heavy chain connected by two disulfide bonds. According to the role of the toxin in the body, it is divided into three parts: A, B, and C, each with a molecular weight of 50kD. Tetanus Toxin Fragment C (TTC) is the C-terminal gene of the heavy chain that binds to target cells. The natural C fragment is non-toxic in animals and retains many properties such as the binding of the complete toxin to gangliosides. The immune titer is equivalent to that of the toxin, and the allergic reaction is lower than that of the complete toxin. It can be used as a future vaccine development direction. In addition, with the development of polysaccharide protein conjugate vaccines, carrier proteins are also constantly developing. At present, diphtheria toxoids and tetanus toxoids are commonly used as carrier proteins, and these carrier proteins are all immunogenic.

[0007] Some literature has reported the use of gene recombination technology to express gE subunit vaccines, but most of them are still under development, and their effectiveness and safety need to be further determined. Some scholars have also been committed to studying the expression of gE protein by fusion protein, but no substantial progress has been made. Therefore, it is necessary to design and study a fusion protein that can be stably expressed with high quality and can enhance both humoral and cellular immune responses, and apply it to the development of VZV vaccines or therapeutic drugs. Summary of the invention

[0008] The purpose of the present invention is to provide a gene recombinant VZV fusion protein and its preparation method and application based on the fusion gene of varicella-zoster virus antigen and toxin carrier protein.

[0009] The present invention is achieved through the following technical solutions:

[0010] The first object of the present invention is to provide a genetically recombinant varicella-zoster virus fusion protein, which includes a varicella-zoster virus antigen and a toxin carrier protein, and the fusion protein is constructed by tandem recombination of the varicella-zoster virus antigen and the toxin carrier protein, and the resulting fusion protein is not a naturally occurring protein or fragment.

[0011] The fusion protein of the present invention, when the varicella-zoster virus antigen is at the N-terminus of the fusion protein, the toxin carrier protein is at the C-terminus of the fusion protein; when the varicella-zoster virus antigen is at the C-terminus of the fusion protein, the toxin carrier protein is at the N-terminus of the fusion protein.

[0012] In certain embodiments, the varicella-zoster virus antigen is at the N-terminus of the fusion protein, and the toxin carrier protein is at the C-terminus of the fusion protein.

[0013] The varicella-zoster virus antigen of the present invention is a truncated fragment of natural VZV gE protein.

[0014] The specific amino acid sequence of the natural VZV gE protein of the present invention is the amino acid sequence of glycoprotein E of the varicella-zoster virus Oka strain with GenBank accession number AAK19946.1 (SEQ ID NO: 23):

[0015] .

[0016] The truncated fragment of the natural VZV gE protein of the present invention is the fragment gE1 obtained by removing the signal peptide (aa1-30) and the carboxyl terminal hydrophobic anchor region (aa547-623) of the gE protein, and its amino acid sequence is shown in SEQ ID NO:1.

[0017] The gE1 described in the present invention includes but is not limited to the content disclosed in the prior art EP0405867B (the suitable VZVgE antigen is the VZV glycoprotein gE truncated to remove the carboxyl terminal anchor region (starting from amino acid position 547)), and the protein sequence generally obtained by expressing the protein in eukaryotic cells will lack the leading sequence (also called signal peptide, amino acids 1-30).

[0018] The toxin carrier protein of the present invention is selected from one of the polypeptides P2, P30, TTC or TT218 of tetanus toxoid, or the non-toxic mutant CRM197 of diphtheria toxoid DT.

[0019] The tetanus toxoid TT described in the present invention is produced by Clostridium tetani and has a total of 1315 amino acids, and its amino acid sequence is:

[0020]

[0021] The polypeptide P2 of the tetanus toxoid of the present invention is the 830-844 amino acids of the tetanus toxin, and the sequence (as shown in SEQ ID NO: 3) is "QYIKANSKFIGITEL". P30 is the 947-967 amino acids of the tetanus toxin, and the sequence (as shown in SEQ ID NO: 4) is "FNNFTVSFWLRVPKVSASHLE".

[0022] The tetanus toxoid C fragment TTC of the present invention is the 865-1315 amino acids of tetanus toxin, and the sequence (as shown in SEQ ID NO: 5) is:

[0023] KNLDCWVDNEEDIDVILKKSTILNLDINNDIISDISGFNSSVITYPDAQLVPGINGKAIHLVNNESSEVIVHKAMDIEYNDMFNNFTVSFWLRVPKVSASHLEQYGTNEYSII SSMKKHSLSIGSGWSVSLKGNNLIWTLKDSAGEVRQITFRDLPDKFNAYLANKWVFITITNDRLSSANLYINGVLMGSAEITGLGAIREDNNITLKLDRCNNNNQYVSIDKFR IFCKALNPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYN NNEHIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND.

[0024] The tetanus toxoid polypeptide TT218 of the present invention is amino acids 1097-1315 of tetanus toxin, and the sequence (as shown in SEQ ID NO: 6) is:

[0025] NPKEIEKLYTSYLSITFLRDFWGNPLRYDTEYYLIPVASSSKDVQLKNITDYMYLTNAPSYTNGKLNIYYRRLYNGLKFIIKRYTPNNEIDSFVKSGDFIKLYVSYNNNE HIVGYPKDGNAFNNLDRILRVGYNAPGIPLYKKMEAVKLRDLKTYSVQLKLYDDKNASLGLVGTHNGQIGNDPNRDILIASNWYFNHLKDKILGCDWYFVPTDEGWTND.

[0026] The amino acid sequence of the non-toxic mutant CRM197 of diphtheria toxoid DT of the present invention (as shown in SEQ ID NO: 7) is:

[0027] .

[0028] In certain embodiments, the recombinant VZV fusion protein is produced by:

[0029] (1) gE1, and (2) tetanus toxoid polypeptide P2 are constructed by tandem recombination. The amino acid sequence is shown in SEQ ID NO: 13.

[0030] In certain embodiments, the recombinant VZV fusion protein is produced by:

[0031] (1) gE1, and (2) tetanus toxoid polypeptide P30 are constructed by tandem recombination. The amino acid sequence is shown in SEQ ID NO:14.

[0032] In certain embodiments, the recombinant VZV fusion protein is produced by:

[0033] (1) gE1, and (2) tetanus toxin C fragment TTC are constructed by tandem recombination. The amino acid sequence is shown in SEQ ID NO: 15.

[0034] In certain embodiments, the recombinant VZV fusion protein is produced by:

[0035] (1) gE1, and (2) tetanus toxoid polypeptide TT218 are constructed by tandem recombination. The amino acid sequence is shown in SEQ ID NO: 16.

[0036] In certain embodiments, the recombinant VZV fusion protein is produced by:

[0037] (1) gE1, and (2) the non-toxic mutant CRM197 of diphtheria toxoid DT were constructed by tandem recombination. The amino acid sequence is shown in SEQ ID NO: 17.

[0038] The second object of the present invention is to provide a method for preparing a genetically recombinant VZV fusion protein, which comprises the following steps:

[0039] S1. Artificially synthesize the gene sequence of the above fusion protein, and connect the gene of the fusion protein into an expression vector to construct a recombinant expression plasmid.

[0040] S2. The constructed recombinant expression plasmid is transformed into the host bacteria, and the correct recombinant plasmid is selected and identified through double enzyme digestion and DNA sequencing.

[0041] S3. The recombinant VZV fusion protein was expressed using Chinese hamster ovary cells (CHO) and purified.

[0042] The fusion protein gene sequence was commissioned to be synthesized by WuXi Biologics.

[0043] The construction of the recombinant expression plasmid is as follows:

[0044] Take gE1-TTC as an example. Add a signal peptide (amino acid sequence as shown in SEQ ID NO: 25) and an 8×His tag. Select pcDNA3.1(+) as the vector and perform codon optimization according to the host CHO cells. Recombine the gene of the artificially synthesized fusion protein into the target vector pcDNA3.1(+) to obtain a pcDNA3.1(+)-gE1-TTC-8×His recombinant expression plasmid.

[0045] Preferably, S2 is specifically:

[0046] The recombinant expression plasmid obtained in S1 was transformed into E. coli DH5α competent cells, coated with LB plates containing 100 μg / mL ampicillin, and placed at 37°C overnight. The next day, the transformed colonies were randomly picked, and the fusion protein gE1-TTC gene fragment was amplified by PCR. The positive single colony plasmid was extracted, double enzyme digestion was performed for identification, and the plasmid containing the exogenous gene was subjected to DNA sequence analysis.

[0047] Preferably, S3 is specifically:

[0048] One day before transfection, CHO cells (EXPICHO from Thermo) were seeded with serum-free cell expression medium OptiPRO at an appropriate concentration. TM SFM, suspension culture, 37°C, 8% CO2. TM The transfection reagent and pcDNA3.1(+)-gE1-TTC-8×His recombinant expression plasmid complex were prepared in SFM medium, and the recombinant plasmid was transfected, wherein the transfection reagent was ExpiFectamine CHO reagent. The cell supernatant was collected 8 days after transfection, and the protein expression level was detected for subsequent purification. The fusion protein was purified by a combination of immobilized metal ion affinity chromatography (IMAC) and size exclusion chromatography (SEC)-HPLC to obtain the purified gene recombinant VZV fusion protein gE1-TTC.

[0049] Gene recombinant VZV fusion proteins gE1-P2, gE1-P30, gE1-TT218 and gE1-CRM197 can all be obtained by the above-mentioned preparation method. The specific operations of the above experimental steps can be carried out with reference to the existing technology and will not be described in detail here.

[0050] The third object of the present invention is to provide an immunogenic composition, which comprises the above-mentioned fusion protein.

[0051] The immunogenic composition of the present invention may comprise the fusion protein gE1-P2 and one or more selected from the group consisting of gE1-P30, gE1-TTC, gE1-TT218 and gE1-CRM197.

[0052] The immunogenic composition of the present invention may comprise the fusion protein gE1-P30 and one or more selected from the group consisting of gE1-P2, gE1-TTC, gE1-TT218 and gE1-CRM197.

[0053] The immunogenic composition of the present invention may comprise the fusion protein gE1-TTC and one or more selected from gE1-P2, gE1-P30, gE1-TT218 and gE1-CRM197.

[0054] The immunogenic composition of the present invention may comprise the fusion protein gE1-TT218 and one or more selected from gE1-P2, gE1-P30, gE1-TTC and gE1-CRM197.

[0055] The immunogenic composition of the present invention may also comprise the fusion protein gE1-CRM197 and one or more selected from gE1-P2, gE1-P30, gE1-TTC and gE1-TT218.

[0056] The fourth object of the present invention is to provide a genetically recombinant VZV vaccine prepared from the above fusion protein or immunogenic composition.

[0057] The gene recombinant VZV vaccine of the present invention comprises the above-mentioned fusion protein, or immunogenic composition, and optionally a pharmaceutically acceptable adjuvant.

[0058] The pharmaceutically acceptable adjuvant described in the present invention is at least one of aluminum salt adjuvants, Freund's complete adjuvant, propolis adjuvant, oil-water emulsion, cytokine, CpG, DNA, genetically engineered toxin-reducing agent, immunostimulatory complex, and liposome.

[0059] In certain embodiments, the liposomal adjuvant is AS01B.

[0060] The formula of AS01B adjuvant is as follows: each dose (0.5 mL) contains MPL 50 μg, QS-21 50 μg, NaCl 4.385 mg, DOPC 1 mg, cholesterol 0.25 mg, KH2PO4 0.54 mg, Na2HPO4 (anhydrous) 0.15 mg, and water for injection.

[0061] The beneficial effects of the present invention are:

[0062] The present invention utilizes gene recombination technology to remove the signal peptide and the C-terminal hydrophobic anchor region, and the amino acids 31-546 (i.e., gE1) of the VZV gE protein obtained by tandem recombination with TTC or TT218, and adds an artificial signal peptide and an 8×His tag to construct a target fusion gene, insert it into a pcDNA3.1(+) expression vector, transform Escherichia coli DH5α, and verify the recombinant expression plasmid. Then, the correctly identified recombinant expression plasmid is transfected into CHO cells to express the target fusion gene, and then purified to obtain the gene recombinant VZV fusion proteins gE1-TTC and gE1-TT218. The toxin carrier protein is non-toxic and can stimulate a strong immune response as a carrier protein, so that the fusion protein has both the immunogenicity and adjuvant activity of the gE protein. Vaccination of the recombinant VZV vaccine of the present invention can form a strong Th1 type cellular immune memory, and there is no risk of inducing potential infection. It can significantly improve the immune promotion effect of the vaccine, and lay a good immunological foundation for the preparation of a new varicella protein vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 The SDS-PAGE detection results of the purified fusion proteins gE1-TTC and gE1-TT218. DETAILED DESCRIPTION OF THE INVENTION

[0065] In order to better understand the present invention, definitions and explanations of relevant terms are provided below.

[0066] The term "toxin carrier protein" as used herein refers to an inactivated toxin, such as tetanus toxoid or a polypeptide fragment of tetanus toxoid, diphtheria toxoid and diphtheria toxin CRM197 (natural mutant) or a functionally active fragment thereof.

[0067] The term "tetanus toxin or TT" as used herein is produced by Clostridium tetani. It can bind to peripheral nerve synapses and inhibit the release of neurotransmitters. The total length of tetanus toxin is 1315 amino acids. Because TT is toxic, it needs to be inactivated by formaldehyde when used as a vaccine vector. The term "P2" refers to the 830-844 amino acids of tetanus toxin. The term "P30" refers to the 947-967 amino acids of tetanus toxin. The term "TTC" is the approximately 47kD TT toxin C fragment produced by enzymatic cleavage of TT toxin with papain protease, and TTC is the 865-1315 amino acids of tetanus toxin. The term "TT218" refers to the 1097-1315 amino acids of tetanus toxin, a total of 218 amino acids. The term "diphtheria toxin or DT" is produced by a bacteriophage infected with Corynebacterium diphtheriae, and is a protease with a total of 567 amino acids, and 1-32 is a signal peptide sequence. The term "atoxic mutant of diphtheria toxin CRM197" is the G52E mutation of DT.

[0068] The term "fusion protein" used herein generally refers to a protein molecule with biological functional activity obtained by genetic engineering technology. In this article, the fusion protein is a polypeptide fragment gE1 obtained by removing the signal peptide (aa1-30) and the carboxyl terminal hydrophobic anchor region (aa547-623) of the gE protein of the varicella-zoster virus Oka strain, and a fusion protein gE1-P2, gE1-P30, gE1-TTC, gE1-TT218 or gE1-CRM197 selected from the polypeptides P2, P30, TTC or TT218 of tetanus toxoid, or the non-toxic mutant CRM197 of diphtheria toxoid DT.

[0069] The term "include" as used herein generally means including, encompassing, containing or encompassing. In some cases, it also means "for", "consisting of...". DETAILED DESCRIPTION

[0070] In order to better understand the present invention, the present invention is further described below in conjunction with specific examples and drawings. The following examples are only explanations of the present invention and cannot be understood as limiting the present invention. In addition, whether or not there is a His tag or an N-terminal signal peptide in the examples does not affect the protection scope. Those skilled in the art can make non-essential improvements and adjustments to the examples based on the content of the present invention, which still fall within the protection scope of the present invention.

[0071] The experimental methods in the following examples, unless otherwise specified, are all conventional experimental methods understandable to those skilled in the art. The experimental materials used in the following examples, unless otherwise specified, are all purchased from the market.

[0072] Example 1 Synthesis of fusion gene gE1-TTC

[0073] WuXi Biologics was commissioned to artificially synthesize the gene sequence of the fusion protein gE1-TTC according to the sequence of SEQ ID NO: 20 described in the present invention.

[0074] Example 2 Construction of pcDNA3.1(+)-gE1-TTC-8×His recombinant expression plasmid

[0075] A signal peptide (amino acid sequence as shown in SEQ ID NO: 25) was added, and an 8×His tag was added. The vector was selected as pcDNA3.1(+), and codon optimization was performed according to the host CHO cells. The gene sequence of the synthesized fusion protein was recombined into the target vector pcDNA3.1(+) to obtain a pcDNA3.1(+)-gE1-TTC-8×His recombinant expression plasmid. The reaction system is shown in Table 1.

[0076] Table 1. The reaction system of the treated target fragment and the vector ligation

[0077] name volume Vector after enzyme digestion 5μL Purified PCR products 5μL Seamless assembly MIX 10μL Total volume 20μL

[0078] The above ligation solution was connected at a constant temperature of 52°C for 30 minutes to obtain the recombinant expression plasmid pcDNA3.1(+)-gE1-TTC-8×His.

[0079] Example 3 Screening and identification of recombinant plasmid pcDNA3.1(+)-gE1-TTC-8×His

[0080] Transformation of recombinant expression plasmid:

[0081] (1) Pipette 1-3 μL of the recombinant expression plasmid at a concentration of 100 ng / μL into 100 μL of E. coli DH5α competent cells, gently shake and rotate to mix, and place on ice for 3 minutes. (2) Place in a 42℃ water bath for 90 seconds without shaking. (3) Place in an ice bath for about 3 minutes. (4) Add 500-800 μL of 37℃ pre-warmed LB medium to each tube and culture gently at 37℃ on a shaker at 200 rpm for about 40 minutes.

[0082] Verification of recombinant expression plasmid:

[0083] (1) Prepare LB plates containing ampicillin. (2) Take 100 μL of bacterial solution and spread it on an LB plate containing 100 μg / mL ampicillin. Use a sterile glass spreader to gently spread the bacteria on the surface of the plate and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours until colonies appear. (4) Pick the bacteria from the plate and shake at 37°C at 250 rpm for 14 hours. Use the bacterial solution for PCR identification and send the positive clone bacterial solution for sequencing.

[0084] Method for identification of cloned plasmids:

[0085] PCR amplification of gE1-TTC fragment, identification primer sequence was synthesized by the company's internal primer department, PCR reaction used 20μL system: primer 0.5μL, template bacterial solution 2μL, polymerase buffer 0.5μL, buffer 3μL, ddH2O 14μL. Cycle parameters: 96℃ pre-denaturation 3min; 95℃15s, 58℃15s, 72℃20s, 23 cycles, 72℃ final extension 1min. Positive clones were screened by bacterial solution PCR method, and the obtained positive bacterial solution was shaken at 37℃ for plasmid sequencing. The correct plasmid was sequenced and double-digested with BamHI-XhoI, and the two fragments obtained were consistent with the expected fragment size.

[0086] Recombinant expression plasmid extraction:

[0087] Inoculate 1% E. coli DH5α competent cells containing pcDNA3.1(+)-gE1-TTC-8×His recombinant expression plasmid in 2 mL LB medium and shake culture at 37°C overnight. After treating the cells, extract 100 μg of plasmid using a plasmid extraction kit. Sequencing was performed to verify the target gene, and the sequencing results were consistent with the designed genome sequence.

[0088] Example 4 Cell transfection and gE1-TTC protein purification

[0089] Cell transfection:

[0090] CHO cells were cultured and expanded in serum-free cell expression medium in a conical flask at 37°C and 8% CO2. The viable cell density and survival rate were determined before transfection. The cell density should reach 7×10 6 ~10×10 6 / mL, survival rate 95-99% according to ExpiFectamine TM Transfection was performed using the CHO transfection kit. Fresh cell expression medium was preheated to 37°C and the cells were diluted to a final density of 6 × 10 6 / mL, 50mm amplitude incubator, 90rpm shaking culture, 37℃, 8% CO2. TM Prepare transfection reagent and pcDNA3.1(+)-gE1-TTC-8×His recombinant expression plasmid complex in SFM medium (4°C), i.e., prepare 40 μL OptiPRO for 1 mL CHO cells TM Add 0.8 μg of recombinant expression plasmid to SFM, mix well and let stand for 5 minutes. Prepare 40 μL OptiPRO TM SFM plus 3 μL ExpiFectamine TM CHO reagent, mix well and place for 5 minutes, then place at room temperature for another 1 to 5 minutes, then slowly transfer the solution to a shake flask and shake it, gently shaking the shake flask during the addition process. Place the cells in a 50mm amplitude incubator at 90rpm for shaking culture, 37°C, 8% CO2. 18-22 hours after transfection, add Enhancer and ExpiCHO Feed according to the kit instructions. Example: Add 6μL Enhancer and 0.24mL ExpiCHO Feed to 1mL of cells, and place the cells in a 50mm amplitude incubator at 90rpm for culture, 37°C, 8% CO2. Collect 8 days after transfection for subsequent purification of the recombinant fusion protein.

[0091] Protein purification:

[0092] A Hi Trap 5ml Chelating HP column loaded with NiSO4 (GE, USA) was used. After pretreatment of the chelating chromatography medium, the column was loaded and washed with 50mmol / L EDTA, 0.2mol / L NaOH, and ultrapure water, respectively. After balancing with more than 5 times the lysis buffer, the cell lysis supernatant was directly loaded onto the column and eluted with solution A (20mmol / L PB, pH7.4, 0.15mol / L NaCl) and solution B (20mmol / L PB, pH7.4, 0.15mol / L NaCl, 0.25mol / L imidazole) as the mobile phase. The target components were collected and SEC separation was continued. A HiLoad 26 / 60 Superdex-200 pre-grade gel column (GE, USA) was used. One column volume (about 320mL) was eluted with solution C (20mmol / L PB, pH7.4, 0.15mol / L NaCl) as the mobile phase, and the elution rate was 2.5mL / min. The target protein fractions were collected and the purity was tested by SDS-PAGE. The purified protein of gE1-TTC was obtained and analyzed by SDS-PAGE. Figure 1 As shown, the purity of the purified target protein can reach 85.4%.

[0093] Example 5 Synthesis of fusion gene gE1-TT218

[0094] WuXi Biologics was commissioned to artificially synthesize the gene sequence of the fusion protein gE1-TT218 according to the sequence of SEQ ID NO: 21 described in the present invention.

[0095] Example 6 Construction of pcDNA3.1(+)-gE1-TT218-8×His recombinant expression plasmid

[0096] Add a signal peptide (amino acid sequence as shown in SEQ ID NO: 25) and add an 8×His tag. Select pcDNA3.1(+) as the vector and optimize the codons according to the host CHO cells. Recombine the synthesized fusion protein gene sequence into the target vector pcDNA3.1(+) to obtain the pcDNA3.1(+)-gE1-TT218-8×His recombinant expression plasmid. The reaction system is as shown in Table 1. Connect the connection liquid at a constant temperature of 52°C for 30 minutes to obtain the recombinant expression plasmid pcDNA3.1(+)-gE1-TT218-8×His.

[0097] Example 7 Screening and identification of recombinant plasmid pcDNA3.1(+)-gE1-TT218-8×His

[0098] Transformation of recombinant expression plasmid:

[0099] (1) Pipette 1-3 μL of the recombinant expression plasmid at a concentration of 100 ng / μL into 100 μL of E. coli DH5α competent cells, gently shake and rotate to mix, and place on ice for 3 minutes. (2) Place in a 42℃ water bath for 90 seconds without shaking. (3) Place in an ice bath for about 3 minutes. (4) Add 500-800 μL of 37℃ pre-warmed LB medium to each tube and culture gently at 37℃ on a shaker at 200 rpm for about 40 minutes.

[0100] Verification of recombinant expression plasmid:

[0101] (1) Prepare LB plates containing ampicillin. (2) Take 100 μL of bacterial solution and spread it on an LB plate containing 100 μg / mL ampicillin. Use a sterile glass spreader to gently spread the bacteria on the surface of the plate and incubate the plate at 37°C for 15 minutes. (3) Invert the plate and incubate at 37°C for 12-16 hours until colonies appear. (4) Pick the bacteria from the plate and shake at 37°C at 250 rpm for 14 hours. Use the bacterial solution for PCR identification and send the positive clone bacterial solution for sequencing.

[0102] Method for identification of cloned plasmids:

[0103] PCR amplification of gE1-TT218-8×His fragment, identification primer sequence was synthesized by the company's internal primer department, PCR reaction used 20μL system: primer 0.5μL, template bacterial solution 2μL, polymerase buffer 0.5μL, buffer 3μL, ddH2O 14μL. Cycle parameters: 96℃ pre-denaturation 3min; 95℃15s, 58℃15s, 72℃20s, 23 cycles, 72℃ final extension 1min. Positive clones were screened by bacterial solution PCR method, and the obtained positive bacterial solution was shaken at 37℃ for plasmid sequencing. The correct plasmid was sequenced and double-digested with BamHI-XhoI, and the two fragments obtained were consistent with the expected fragment size.

[0104] Recombinant expression plasmid extraction:

[0105] Inoculate 1% E. coli DH5α competent cells containing pcDNA3.1(+)-gE1-TT218-8×His recombinant expression plasmid in 2 mL LB medium and shake culture at 37°C overnight. After treating the cells, extract 100 μg of plasmid using a plasmid extraction kit. Sequencing was performed to verify the target gene, and the sequencing results were consistent with the designed genome sequence.

[0106] Example 8 Cell transfection and gE1-TT218 protein purification

[0107] Cell transfection:

[0108] CHO cells were cultured and expanded in serum-free cell expression medium in a conical flask at 37°C and 8% CO2. The viable cell density and survival rate were determined before transfection. The cell density should reach 7×10 6 ~10×10 6 / mL, survival rate 95-99% according to ExpiFectamine TM Transfection was performed using the CHO transfection kit. Fresh cell expression medium was preheated to 37°C and the cells were diluted to a final density of 6 × 10 6 / mL, 50mm amplitude incubator, 90rpm shaking culture, 37℃, 8% CO2. TM Prepare transfection reagent and pcDNA3.1(+)-gE1-TT218-8×His recombinant expression plasmid complex in SFM medium (4°C), i.e., prepare 40 μL OptiPRO for 1 mL CHO cells TM Add 0.8 μg of recombinant expression plasmid to SFM, mix well and let stand for 5 minutes. Prepare 40 μL OptiPRO TM SFM plus 3 μL ExpiFectamine TM CHO reagent, mix well and place for 5 minutes, then place at room temperature for another 1 to 5 minutes, then slowly transfer the solution to a shake flask and shake it, gently shaking the shake flask during the addition process. Place the cells in a 50mm amplitude incubator at 90rpm for shaking culture, 37°C, 8% CO2. 18-22 hours after transfection, add Enhancer and ExpiCHO Feed according to the kit instructions. Example: Add 6μL Enhancer and 0.24mL ExpiCHO Feed to 1mL of cells, and place the cells in a 50mm amplitude incubator at 90rpm for culture, 37°C, 8% CO2. Collect 8 days after transfection for subsequent purification of the recombinant fusion protein.

[0109] Protein purification:

[0110] A Hi Trap 5ml Chelating HP column loaded with NiSO4 (GE, USA) was used. After pretreatment of the chelating chromatography medium, the column was loaded and washed with 50mmol / L EDTA, 0.2mol / L NaOH, and ultrapure water, respectively. After balancing with more than 5 times the lysis buffer, the cell lysis supernatant was directly loaded onto the column and eluted with solution A (20mmol / L PB, pH7.4, 0.15mol / L NaCl) and solution B (20mmol / L PB, pH7.4, 0.15mol / L NaCl, 0.25mol / L imidazole) as the mobile phase. The target components were collected and SEC separation was continued. A HiLoad 26 / 60 Superdex-200 pre-grade gel column (GE, USA) was used. One column volume (about 320mL) was eluted with solution C (20mmol / L PB, pH7.4, 0.15mol / L NaCl) as the mobile phase, and the elution rate was 2.5mL / min. The target protein fraction was collected and the purity was tested by SDS-PAGE. The purified protein of gE1-TT218 was obtained and analyzed by SDS-PAGE. Figure 1 As shown, the purity of the purified target protein can reach 87.5%.

[0111] Example 9 Construction of vector and protein expression

[0112] With reference to the above examples, the following target fusion gene to be expressed was constructed, and the purified protein was expressed, as shown in Table 2.

[0113] Table 2 Different target fusion genes

[0114]

[0115]

[0116] Example 10 Preparation of immunogenic components

[0117] 50 μg of recombinant VZV fusion protein gE1-P2, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were dispensed into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0118] Example 11 Preparation of immunogenic components

[0119] 50 μg of recombinant VZV fusion protein gE1-P30, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were dispensed into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0120] Example 12 Preparation of immunogenic components

[0121] 50 μg of recombinant VZV fusion protein gE1-TTC, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were dispensed into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0122] Example 13 Preparation of immunogenic components

[0123] 50 μg of recombinant VZV fusion protein gE1-TT218, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were dispensed into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0124] Example 14 Preparation of immunogenic components

[0125] 50 μg of recombinant VZV fusion protein gE1-CRM197, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0126] Example 15 Preparation of immunogenic components

[0127] 50 μg of VZV gE protein, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were dispensed into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0128] Example 16 Preparation of immunogenic composition

[0129] 25 μg of recombinant VZV fusion protein gE1-TTC, 25 μg of recombinant VZV fusion protein gE1-TT218, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0130] Example 17 Preparation of immunogenic composition

[0131] 25 μg of recombinant VZV fusion protein gE1-P2, 25 μg of recombinant VZV fusion protein gE1-TT218, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0132] Example 18 Preparation of immunogenic composition

[0133] 25 μg of recombinant VZV fusion protein gE1-P2, 25 μg of recombinant VZV fusion protein gE1-TTC, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0134] Example 19 Preparation of immunogenic composition

[0135] 25 μg of recombinant VZV fusion protein gE1-P2, 25 μg of recombinant VZV fusion protein gE1-P30, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0136] Example 20 Preparation of immunogenic composition

[0137] 25 μg of recombinant VZV fusion protein gE1-TTC, 25 μg of recombinant VZV fusion protein gE1-CRM197, 20 mg of sucrose, 0.160 mg of sodium dihydrogen phosphate dihydrate, 0.116 mg of dipotassium phosphate, and 0.08 mg of polysorbate 80 were divided into 2 mL vials, 0.5 mL per vial, and freeze-dried in a freeze dryer.

[0138] Example 21 Preparation of vaccine

[0139] The immunogenic components described in any one of Examples 10-15 and the immunogenic compositions described in any one of Examples 16-20 further comprise an AS01B adjuvant. The adjuvant bottle is 0.5 mL, containing 50 μg of MPL, 50 μg of QS21, 4.385 mg of sodium chloride, 1 mg of DOPC, 0.54 mg of potassium dihydrogen phosphate, 0.25 mg of cholesterol, 0.15 mg of anhydrous disodium phosphate, and water for injection to 0.5 mL. Refrigerate at 2-8 ° C and keep away from light. Redissolve the antigen with the adjuvant solution before vaccination. After reconstitution, each human dose is 0.5 ml, containing 50 μg of antigen protein.

[0140] Example 22 Detection of mouse serum specific IgG antibodies

[0141] The mice were immunized with the vaccine prepared with the immunogenic components gE (1-546aa) described in Example 15, the immunogenic components gE1-TTC and gE1-TT218 described in Example 12 and Example 13, and the AS01B adjuvant, and the specific IgG antibody of the mouse serum was detected. In this study, C57BL / 6 mice were divided into different antigen groups, with 6 mice in each group, and immunized with a 1 / 10 human dose. First, 2000pfu of inactivated varicella vaccine (purchased from Shanghai Rongsheng Bio-Pharmaceutical Co., Ltd.) was inoculated, and then the recombinant herpes zoster vaccine was immunized on the 35th and 49th days of inoculation, and the serum was separated 14 days (63d) after the second immunization with the recombinant herpes zoster vaccine, and the gE protein-specific IgG antibody was detected by ELISA.

[0142] The results are shown in Table 3. Mice immunized with recombinant gE1-TTC and gE1-TT218 vaccines can produce higher titers of specific IgG antibodies, and the recombinant gE1-TT218 vaccine has more advantages than the recombinant gE-TTC vaccine.

[0143] Table 3 Specific IgG antibody detection results

[0144] Antigen name S1 S2 S3 S4 S5 S6 TT218 1000 250 500 250 250 2000 TTC 250 1000 4000 500 250 1000 E 2560000 1280000 1280000 1280000 2560000 1280000 gE1-TTC 320000 640000 640000 320000 320000 640000 gE1-TT218 2560000 2560000 2560000 1280000 1280000 1280000

[0145] Example 23 Cellular Immunoassay

[0146] The mice were immunized with the vaccine prepared with the immunogenic components gE (1-546aa) described in Example 15, the immunogenic components gE1-TTC and gE1-TT218 described in Example 12 and Example 13, and the AS01B adjuvant, and the cellular immunity was detected. In this study, C57BL / 6 mice were divided into different antigen groups, immunized with a 1 / 10 human dose, and a saline group was set as a negative control, with 6 mice in each group. First, 2000pfu of inactivated varicella vaccine (purchased from Shanghai Rongsheng Biopharmaceutical Co., Ltd.) was inoculated, and then the recombinant herpes zoster vaccine was immunized on the 35th and 49th days of inoculation, and samples were taken on the 63rd day to evaluate the cellular immunity effects of different adjuvants. 14 days (63d) after the second immunization of the recombinant herpes zoster vaccine, the mouse spleen was taken to separate the mouse spleen lymphocytes, and the VZV gE (1-546aa) peptide library was used as a stimulus, and the levels of intracellular cytokines IFN-γ and IL-2 were detected by flow cytometry. Since there was no significant difference in the results of CD8+T cells, the final results presented were the percentages of cells secreting different cytokines in CD4+T cells.

[0147] Table 4 Results of mouse cellular immunity detection

[0148] antigen IL-2+ IL-2+IFN-γ+ IFN-γ+ total Normal saline 0.062 — 0.05706 0.12 TTC / 5μg 0.045 — 0.1059 0.15 TT218 / 5μg 0.052 — 0.09531 0.15 gE / 5μg 0.93 0.8 0.452 2.18 gE1-TTC / 5μg 0.54 0.93 0.366 1.84 gE1-TT218 / 5μg 0.48 1.6 0.7807 2.86

[0149] The results showed that the recombinant gE1-TTC and gE1-TT218 vaccines can induce stronger cellular immunity than the simple VZV gE protein vaccine. These results show that vaccination with the recombinant VZV gE1-TTC and gE-TT218 vaccines of the present invention may form a stronger Th1 type cellular immune memory, and the recombinant gE1-TT218 vaccine has more advantages than the genetic recombinant gE1-TTC vaccine.

[0150] The above embodiments are merely descriptions of preferred implementations of the present invention, and are not intended to limit the protection scope of the present invention. Without departing from the design spirit of the present invention, any non-substantial modifications made by those skilled in the art on the basis of the present invention shall fall within the protection scope of the present invention.

Claims

1. A genetically recombinant VZV fusion protein, characterized in that: The fusion protein is constructed by tandem recombination of a varicella-zoster virus antigen and a polypeptide TT218 of a tetanus toxoid, wherein the amino acid sequence of the varicella-zoster virus antigen is shown in SEQ ID NO: 1, and the amino acid sequence of TT218 is shown in SEQ ID NO: 6, the varicella-zoster virus antigen is at the N-terminus of the fusion protein, and the polypeptide TT218 is at the C-terminus of the fusion protein.

2. A genetically recombinant VZV fusion protein, characterized in that: The amino acid sequence of the fusion protein is shown in SEQ ID NO:

16.

3. A method for preparing the genetically recombinant VZV fusion protein according to claim 1 or 2, characterized in that: The following steps are involved: S1. Artificially synthesizing the gene sequence of the fusion protein according to claim 1 or 2, and connecting the gene of the fusion protein into an expression vector to construct a recombinant expression plasmid; S2, transforming the constructed recombinant expression plasmid into the host bacteria, and selecting and identifying the correct recombinant plasmid through double enzyme digestion and DNA sequencing; S3. Using Chinese hamster ovary cells to express the recombinant VZV fusion protein, and purifying it.

4. The method for preparing the genetically recombinant VZV fusion protein according to claim 3, characterized in that: The expression vector described in S1 is pcDNA3.1(+).

5. The method for preparing the genetically recombinant VZV fusion protein according to claim 3, characterized in that: The host bacteria described in S2 is Escherichia coli DH5α.

6. An immunogenic composition, characterized in that The immunogenic composition comprises the fusion protein of claim 1 or 2.

7. A genetically recombinant VZV vaccine, characterized in that: The vaccine comprises the fusion protein of claim 1 or 2 or the immunogenic composition of claim 6, and optionally a pharmaceutically acceptable adjuvant.

8. The genetically recombinant VZV vaccine according to claim 7, characterized in that: The adjuvant is at least one of aluminum salt adjuvant, Freund's complete adjuvant, propolis adjuvant, oil-water emulsion, cytokine, CpG-DNA, genetically engineered attenuated toxin, immunostimulatory complex or liposome.

9. The genetically recombinant VZV vaccine according to claim 8, characterized in that: The liposome adjuvant is AS01B.

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