A gE fusion protein containing P2, Fc and PADRE, and its preparation method and application

Abstract

The present invention discloses a gE fusion protein containing P2, Fc and PADRE, and its preparation method and application. The present invention also discloses a preparation method of an immunogenic gE fusion protein: the fusion protein is expressed in CHO K1 cells by using the mammalian cell expression vector pXNM3.0, and after purification by affinity chromatography Protein A, hydrophobic chromatography Capto Phenyl ImpRes, and size exclusion chromatography Sephacryl S-300 High Resolution, the fusion protein is obtained. Compared with the simple gE protein, the gE fusion protein of the present invention has excellent immunogenicity, can enhance both the humoral immune response and the cellular immune response, and has good clinical application potential.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedicine, and in particular to a gE fusion protein containing P2, Fc and PADRE, and a preparation method and application thereof. Background Art

[0002] Varicella zoster virus (VZV), also known as human herpesvirus type 3, is a human alpha herpesvirus. Primary infection with varicella zoster virus in normal people will cause chickenpox in childhood. After infection with VZV, the virus will lurk in the human ganglia. When immunity is reduced due to various reasons, the virus will be reactivated and replicate in large quantities, causing herpes zoster (HZ), which is often accompanied by post-herpetic neuralgia (PHN). In people with impaired immune function, VZV infection may be accompanied by serious complications such as myelitis and encephalomyelitis. The genome size of VZV is about 125kb, encoding about 69 proteins, including 8 glycoproteins, including gB, gC, gE, gH, gI, gK, gL, and gM. Among them, glycoprotein E (gE) is the main glycoprotein recognized by the host immune system. It is also the glycoprotein with the strongest antigenicity and the most abundant content on the viral envelope and infected cell membrane. It is also widely present on the surface of VZV particles and in the cell membrane and cytoplasm of host cells, and can induce cellular immunity and humoral immunity.

[0003] The varicella-zoster virus is highly contagious and is mainly transmitted through air droplets and direct contact. It is reported that about 90% of adults over 50 years old worldwide test positive for serum VZV. Children infected with VZV will develop fever and a systemic red maculopapular rash, herpes and scabs, which are self-limiting. With age, the body's immunity decreases, and the incidence of herpes zoster gradually increases. With the accelerated pace of life and increased life pressure, the incidence of herpes zoster is showing a trend of younger people. The onset of varicella-zoster seriously affects people's quality of life, especially the presence of PHN. PHN is the most common sequelae of herpes zoster, with an incidence of 10% to 30% in herpes zoster. The pain can last for months to years, up to 10 years.

[0004] The immunogenicity of gE molecules expressed alone is poor. To achieve a better immune effect, they must be used together with adjuvants / immunoenhancers with strong immunostimulatory ability, such as GSK's It contains 50 μg of gE and AS01 adjuvant [containing two immune enhancers: 50 μg of saponin QS-21 and 50 μg of monophosphoryl lipid A (MPLA)]. Fused expression of antigenic epitopes such as P2 and PADRE can enhance the immunogenicity of the fusion protein. When used in combination with an adjuvant with extremely strong immune stimulation ability, the immune effect is better. When the fusion protein contains Fc, it can increase the protein expression yield, make the fusion protein easy to purify, significantly improve the half-life of the fusion protein, strengthen the protein stability. Because of the Fc effect, the antigen forms a dimer, and the immunogenicity is enhanced. The Fc receptor on the surface of antigen-presenting cells binds to the Fc region of immunoglobulin, which can promote the processing and presentation of antigens by antigen-presenting cells. When used in combination with an adjuvant with extremely strong immune stimulation ability, a better immune effect can be achieved.

[0005] The patent with publication number CN11462356 discloses a varicella-zoster subunit vaccine using IL18 as a molecular adjuvant. The fusion expression of IL-18, gE and Fc can increase the yield of gE fusion protein (IL18-gE-Fc), make the gE fusion protein easy to purify, and has a good immune effect. The disadvantage of IL18 is that the safety risk for human use is relatively high: IL18 may mediate virus-induced superinflammation by amplifying CD8+ T cell responses and promoting overexpression of IFN-γ, and may also increase the production of amyloid related to Alzheimer's disease in human neuronal cells. Currently, no IL18 drugs are on the market for sale.

[0006] The patent with publication number CN110343722 discloses a method for recombinantly expressing truncated glycoprotein E of varicella-zoster virus v-Oka strain. This method introduces the truncated gE protein gene into baculovirus and uses the reconstructed baculovirus to infect insect cells to express soluble gE protein. This method is easy to screen and batch-to-batch stable, but the protein expressed by insect cells has significant differences in glycosylation compared with mammalian cells, and the single protein cannot effectively activate the humoral and cellular immune functions of the human body. Summary of the Invention

[0007] The purpose of the present invention is to provide a gE fusion protein containing P2, Fc and PADRE, its preparation method and application. The nucleotide sequences of truncated gE (removing the transmembrane region and intracellular sequence of gE), P2, Fc and PADRE are linked with a linker and inserted into an expression vector, and then transfected into mammalian cells CHOK1 to express the gE fusion protein PADRE-gE-P2-Fc. The gE fusion protein PADRE-gE-P2-Fc has excellent immunogenicity and can induce high levels of humoral and cellular immune responses.

[0008] To achieve the above purpose, the present invention provides the following technical solutions:

[0009] A gE fusion protein containing P2, Fc and PADRE, wherein the gE fusion protein contains the extracellular region of varicella-zoster virus glycoprotein E (gE), the nucleotide sequence of which is shown in SEQ ID NO:1, and the amino acid sequence is shown in SEQ ID NO:2; the tetanus toxin universal T cell epitope peptide P2 (P2), the nucleotide sequence of which is shown in SEQ ID NO:3, and the amino acid sequence is shown in SEQ ID NO:4; the universal DR Th epitope peptide (PADRE), the nucleotide sequence of which is shown in SEQ ID NO:5, and the amino acid sequence is shown in SEQ ID NO:6; the Fc segment of human IgG1 antibody (Fc), the nucleotide sequence of which is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8.

[0010] Furthermore, the amino acid sequence combination mode of the fusion protein is any order combination of P2, Fc, PADRE and gE. The preferred combination mode is PADRE-gE-P2-Fc. The amino acid sequence of the preferred combination PADRE-gE-P2-Fc is shown in SEQ ID NO:10, and the nucleotide sequence is shown in SEQ ID NO:9.

[0011] Furthermore, the P2 sequence, gE sequence, PADRE sequence and Fc sequence are connected by a linker peptide, and the linker peptide is a GGS and / or GGGS and / or GGGGS and / or GSGSG linker peptide.

[0012] A gE fusion protein gene containing P2, Fc and PADRE, wherein the gE fusion protein gene encodes a fusion protein containing gE, P2, PADRE and Fc, and the gE fusion protein gene can express the gE fusion protein in CHO cells.

[0013] Preferably, the CHO cell is a CHOK1 cell.

[0014] A preparation method of a gE fusion protein containing P2, Fc and PADRE, comprising the following steps:

[0015] S1. After codon optimization of the gene of the fusion protein, total gene synthesis of the nucleotide sequence is carried out;

[0016] S2. Clone the synthesized total gene synthesis sequence into an expression vector;

[0017] S3. Transfect the expression vector obtained in step S2 into CHO cells, and through the screening of mini cell populations and monoclonal screening, obtain a cell line stably expressing the gE fusion protein.

[0018] S4. Culture the cell line described in step S3, collect the supernatant of the cell culture for purification, and obtain the purified gE fusion protein.

[0019] Furthermore, in step S2, the expression vector is a plasmid expression vector carrying a GS screening system and / or a bleomycin resistance gene.

[0020] Use of a gE fusion protein containing P2, Fc, and PADRE in the preparation of a varicella-zoster vaccine.

[0021] A varicella-zoster vaccine, the vaccine comprising a gE fusion protein and an adjuvant.

[0022] Furthermore, the adjuvant is any one or any combination of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, neutral liposome adjuvant containing saponin, cationic liposome adjuvant containing saponin, anionic liposome adjuvant containing saponin, CpG adjuvant, nanoemulsion, adjuvant containing 3D-MPL.

[0023] Furthermore, each dose unit of the varicella-zoster virus vaccine contains 5 - 200 μg of the fusion protein. Preferably, each dose unit contains 10 - 100 μg of the fusion protein. More preferably, each dose unit contains 20 - 80 μg of the fusion protein.

[0024] Based on the above technical solutions, the present invention can at least produce the following technical effects:

[0025] The present invention fuses and expresses the fusion protein PADRE-gE-P2-Fc of the truncated varicella-zoster virus (VZV) envelope glycoprotein E (removing the transmembrane region and the intracellular sequence of gE), the tetanus toxin universal T cell epitope peptide P2, the Fc terminus of human immunoglobulin γ (IgG1) of type I, and the pan-DR Th epitope in CHO cells. The addition of the two epitope peptides in this fusion protein can enhance the immunogenicity of the protein. The immunoglobulin Fc can increase the half-life of the antigen, enhance the protein stability, and at the same time, Fc can promote antigen presentation. This fusion protein greatly improves the problem of insufficient immunogenicity of the simple gE protein. The fusion protein is combined with a single-component immunopotentiator adjuvant. In mouse experiments, its humoral immunity and cellular immunity levels are higher than those of the simple gE protein and also higher than those of the Shin An Li Shi vaccine containing two immunopotentiator adjuvants. Description of the Drawings

[0026] Figure 1 Schematic diagram of the composition of the expression vector PXNM3.0;

[0027] Figure 2 Schematic diagram of the composition of the expression vector of the gE fusion protein PADRE-gE-P2-Fc;

[0028] Figure 3 Schematic diagram for comparing the GMT titers of sera specific for the gE protein after two immunizations;

[0029] Figure 4 Schematic diagram for comparing the CD4+ T cell responses specific for gE. Specific implementation manners

[0030] To make the objectives, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention will be described in detail below. Obviously, the described embodiments are only a part rather than all of the embodiments of the present invention.

[0031] Example 1: Preparation of fusion protein

[0032] Codon optimization and gene synthesis of the fusion protein: The gene of the fusion protein was codon-optimized. The optimization principles were as follows: 1. Avoid common restriction enzyme sites; 2. According to the codon preference in CHO cells, replace synonymous codons with low frequencies with codons with high frequencies to control rare codons; 3. Control the GC content in the sequence at 40% - 60% to improve the transcription efficiency of mRNA, and at the same time avoid the high GC content affecting the secondary structure of mRNA and further affecting the translation efficiency. A signal peptide sequence was added in front of the optimized sequence, a HindⅢ restriction enzyme site was introduced upstream of the sequence, a stop codon and a BamHⅠ restriction enzyme site were added downstream of the sequence, and the whole gene combination synthesis of the nucleotide sequence was carried out.

[0033] Construction of the fusion protein expression plasmid: First, the synthesized whole gene synthesis sequence was ligated to the cloning expression vector PXNM3.0. The composition schematic diagram of the expression vector PXNM3.0 is as Figure 1 shown, and the composition schematic diagram of the gE fusion protein expression vector P2-PADRE-gE-Fc-PXNM3.0 is as Figure 2 shown; then the cloning vector containing the whole gene synthesis sequence was transformed into DH5α competent bacteria, and then amplified in large quantities; after plasmid extraction, the cloning vector was double digested with the restriction enzymes HindⅢ and BamHⅠ, and at the same time the expression vector pXNM3.0 was double digested with the restriction enzymes HindⅢ and BamHⅠ. The gel of the double-digested cloning vector was recovered to obtain the fusion protein gene part, and the gel of the expression vector was recovered to obtain the backbone part. The two were ligated with T4 enzyme and then transformed into DH5α competent bacteria, and spread on a plate containing ampicillin resistance for screening. Positive colonies were picked for amplification and plasmid extraction, and then identified by double digestion with HindⅢ and BamHⅠ, and the correct recombinant expression vector was verified by sequencing.

[0034] Construction of stable cell line: Identify the correct recombinant expression vector, amplify the bacteria and extract a large amount of plasmid. Digest the recombinant expression vector with Pvu I alone, recover the digested and linearized vector by gel cutting, filter and sterilize it for later use. After resuscitation, passage the CHO-K1 cells continuously for more than two times. When the cell viability is greater than 95%, use them for electroporation. In a clean bench, add 0.6 ml of cell suspension (about 1×10 7 cells) and 200 μl of linearized recombinant expression vector (about 50 μg) into a 4 mm electroporation cuvette. Set the electroporation conditions as voltage 300 V and capacitance 900 μF. After electroporation, transfer the cells to a cell shake flask containing 30 ml of CD CHO medium and culture them at 37 °C, 5% carbon dioxide concentration and 125 rpm for 24 hours. Centrifuge the electroporated cell suspension at 100 g for 10 minutes, discard the supernatant, resuspend the cells with CD CHO medium containing 25 μM MSX and 200 μg / ml bleomycin, then inoculate them into a 24-well plate. After 3 weeks of culture, detect the expression level by ELISA method. Mix the 3 cell wells with high expression levels and inoculate them into a 96-well plate by limiting dilution method for monoclonal screening. Take pictures on days 0, 1, 2, 3, 7 and 15 with a single-cell imaging device. After 15 days, detect the expression level by ELISA method. Freeze the 3 cell strains with high expression levels. After stability study, determine the cell line for vaccine preparation, and then establish a two-level cell bank.

[0035] Expression of target product: Resuscitate a cryopreserved working seed with OPM-CHO CDP9 medium, amplify it step by step in a shake flask, and finally transfer it into a 5 L bioreactor for culture. The inoculation density is 0.8×10 6 cells / ml. Set the culture parameters as temperature 37 °C, pH 7.0, rotation speed 150 r / min and dissolved oxygen concentration 40%. Take samples daily to detect cell viability, density, lactate content and glucose content. On the 3rd day of culture, when the viable cell density reaches 3×10 6 cells / ml, add feeding media CDF18 and CDF26, 250 ml and 25 ml respectively. Then add the same volume of feeding medium every 1 day. Maintain the glucose content in the culture medium above 2 g / L. When the concentration is lower than this, supplement the glucose concentration to 4 g / L. After about 15 days of culture, when the cell viability drops to 70%, terminate the culture. Filter the cells and cell debris with a depth filter, and collect the supernatant of the cell culture.

[0036] Purification of the target product (preparation of the stock solution): Adjust the pH of the cell culture supernatant to 7.5. Equilibrate Protein A with 40 mM PB buffer containing 150 mM sodium chloride at pH 7.5 to the baseline level of ultraviolet absorption with stable pH. Then pass the cell supernatant through the column. After equilibration to the baseline level of ultraviolet absorption with the same equilibration buffer, elute the target product with acetic acid-sodium acetate buffer at pH 3.0 - 4.0. Inactivate the purified product at low pH (pH 3.0 - 4.0, place at 18 - 25 °C for 60 min). After inactivation, add 1 M ammonium sulfate to the product and adjust the pH to 7.5. Equilibrate the hydrophobic chromatography column Capto Phenyl ImpRes with 50 mM PB buffer + 1 M ammonium sulfate buffer at pH 7.5 to the baseline level of ultraviolet absorption with stable pH. Then pass the inactivated solution after pH adjustment through the column and equilibrate the chromatography column with the same buffer. Finally, linearly elute the target product with 50 mM PB buffer at pH 7.5. Purify the hydrophobic purified product by Sephacryl S-300 High Resolution molecular sieve chromatography and exchange the solution to obtain the purified protein. The purified protein is nanofiltered through a 15 nm filter and sterilized by filtration through a 0.22 μm filter membrane to obtain the stock solution of the vaccine.

[0037] Example 2: Vaccine formulation

[0038] (1) Preparation of liposomes (ethanol injection method; 100 ml volume, DOPC and cholesterol concentrations are 4 mg / ml and 1 mg / ml respectively): Weigh 400 mg of DOPC and 100 mg of cholesterol respectively; then completely dissolve DOPC and cholesterol in 10 ml of absolute ethanol and mix evenly to obtain the organic phase; inject 10 ml of the organic phase into 90 ml of 10 mM PBS buffer solution (pH 7.0) to prepare the primary liposome emulsion; then use a high-pressure microfluidic homogenizer to size the liposomes to about 100 nm in diameter; then remove ethanol by dialysis method; finally, filter and sterilize through a 0.22 μm sterilizing filter to obtain the finished liposomes.

[0039] (2) Preparation of the gE adjuvant vaccine (10 ml volume; gE concentration is 100 μg / ml; immunopotentiator saponin QS-21 is 100 μg / ml; liposome components: DOPC and cholesterol are 2 mg / ml and 0.5 mg / ml respectively): Take 5 ml of liposomes, add 1 ml of saponin QS-21 solution (concentration 1 mg / ml), and stir evenly to obtain the adjuvant; then add the gE fusion protein solution to the adjuvant, and use 10 mM PBS buffer solution (pH 7.0) to make up the total volume to 10 ml and stir evenly to obtain the gE fusion protein adjuvant vaccine.

[0040] The calculation formula for the volume (ml) of the gE fusion protein solution added is: the mass (μg) of the gE fusion protein added / the concentration (μg / ml) of the gE fusion protein = the theoretical molecular weight of the gE fusion protein * the gE concentration (μg / ml) in the vaccine * the volume (ml) of the vaccine / [the theoretical molecular weight of gE * the concentration (μg / ml) of the gE fusion protein] = the theoretical molecular weight of the gE fusion protein * 100 * 10 / (the theoretical molecular weight of gE * the concentration of the gE fusion protein).

[0041] Experimental example: Immunization of animals with the vaccine

[0042] The experimental animals were 6 - 8 - week - old female C57BL / 6 mice; animal immunization and feeding were carried out under specific pathogen - free conditions. To simulate the infection of varicella - zoster virus in the natural environment, 35 days before the immunization with the candidate vaccine, the mice were pre - sensitized by subcutaneous injection of varicella - zoster virus (containing no less than 3.3 lg PFU of live virus, 0.5 ml) into the neck. On day 0 and day 28, the candidate vaccine was injected intramuscularly into the leg at a dose of 50 μl per mouse. Blood was collected by orbital bleeding 28 days after the second immunization, and the mice were sacrificed after blood collection. The collected whole blood was allowed to stand overnight at 2 - 8 °C, and centrifuged at 3000 rpm for 30 min the next day, and the top serum was aspirated. The serum titer of the mice was measured by indirect ELISA, and the GMT value was calculated. Spleen cells were isolated from the spleens of the mice, and the number of CD4+ T cells secreting INF - γ and P2 specifically for gE was detected by intracellular cytokine staining to evaluate the cellular immune level.

[0043] Table 1 Information related to the immunogenicity study of the vaccine in animals

[0044]

[0045] Analysis of vaccine immunogenicity

[0046] (1) Detection of gE protein - binding antibodies in serum

[0047] The total gE - specific antibodies in the serum samples of all individual mice 28 days after the second immunization were detected by indirect ELISA. The procedure was to coat the gE protein onto a 96 - well plate with carbonate buffer at a coating amount of 500 ng / well, and coat overnight at 4 °C. Then it was blocked with TPBS solution containing BSA, and the plate was washed 3 times with TPBS solution. Then, after diluting the sera of all individual mice at different dilution ratios, they were added to the wells, incubated at 37 °C for 1 hour, and the plate was washed 3 times with TPBS solution. Then, it was incubated with HRP - labeled goat anti - mouse secondary antibody at 37 °C for 1 hour, washed 3 times with TPBS solution, developed color with TMB chromogenic solution for 10 min, and the reaction was terminated by adding 0.2 M sulfuric acid. The absorbance value at OD450 was read with an enzyme - linked immunosorbent assay reader. Three times the reading of the pre - immunization serum mixed sample was used as the Cut - Off value to determine the serum titer after immunization. The experimental results are shown in Figure 3.

[0048] (2) Detection of intracellular cytokines by flow cytometry

[0049] On the 28th day after the second immunization, spleens were collected from the experimental mice to prepare single-cell suspensions of the spleen. After adjusting the cell concentration, the red blood cells in the single-cell suspension were lysed with lysing solution. The spleen cells were stimulated with specific peptide pools to secrete cytokines. After adding the secretion blocker Containing Brefeldin A to block secretion, and then through steps such as cell viability and dead staining, surface receptor FcR blocking, surface staining of CD3, CD45, and CD4, fixation and permeabilization, and intracellular staining of P2 and IFN-γ, the CD4 + T and CD8 + T cell numbers were detected using a flow cytometer, and the proportion of positive cells was calculated. The experimental results are shown in Figure 4.

[0050] As described above, the above are only specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention can easily think of changes or substitutions, which should all be covered within the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A gE fusion protein containing P2, Fc and PADRE, characterized in that: The gE fusion protein contains the extracellular region of varicella-zoster virus glycoprotein E, the universal T cell epitope peptide P2 of tetanus toxin, the universal DR Th epitope peptide, and the Fc segment of human IgG1 antibody. The gE fusion protein is PADRE-gE-P2-Fc, and its amino acid sequence is shown in SEQ ID NO:10, and its nucleotide sequence is shown in SEQ ID NO:

9.

2. A gE fusion protein gene containing P2, Fc and PADRE, wherein the gE fusion protein gene encodes a fusion protein containing gE, P2, padre and Fc, and the gE fusion protein gene can express the gE fusion protein in CHO cells. The gE fusion protein is PADRE-gE-P2-Fc, and its amino acid sequence is shown in SEQ ID NO:

10.

3. The gE fusion protein gene containing P2, Fc and PADRE according to claim 2, characterized in that: The CHO cells are CHOK1 cells.

4. A method for preparing a gE fusion protein containing P2, Fc and PADRE, characterized in that: The following steps are involved: S1. Codon optimization of the fusion protein gene was performed followed by whole genome synthesis of the nucleotide sequence; S2, cloning the synthesized full gene sequence into an expression vector; S3, transfecting the expression vector obtained in step S2 into CHO cells, and obtaining a cell line stably expressing the gE fusion protein through mini cell population screening and monoclonal screening; S4. Cultivate the cell line described in step S3, collect the cell culture supernatant for purification, and obtain the purified gE fusion protein. The gE fusion protein is PADRE-gE-P2-Fc, and its amino acid sequence is shown in SEQ ID NO:

10.

5. An application of a gE fusion protein containing P2, Fc and PADRE, characterized in that: Use of the gE fusion protein as claimed in claim 1 in the preparation of varicella-zoster vaccine.

6. A varicella-zoster vaccine, characterized in that: The vaccine comprises the gE fusion protein as claimed in claim 1 and an adjuvant.

7. A varicella-zoster vaccine according to claim 6, characterized in that: The adjuvant is any one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, neutral liposome adjuvant containing saponin, cationic liposome adjuvant containing saponin, anionic liposome adjuvant containing saponin, CpG adjuvant, nanoemulsion, and adjuvant containing 3D-MPL, or any combination of multiple thereof.

8. A varicella-zoster vaccine according to claim 7, characterized in that: Each dosage unit of the varicella-zoster virus vaccine contains 5 to 200 μg of fusion protein.

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