CN117003896A - gE fusion protein containing P2 and PADRE epitope, and preparation method and application thereof - Google Patents
gE fusion protein containing P2 and PADRE epitope, and preparation method and application thereof Download PDFInfo
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- CN117003896A CN117003896A CN202310999201.6A CN202310999201A CN117003896A CN 117003896 A CN117003896 A CN 117003896A CN 202310999201 A CN202310999201 A CN 202310999201A CN 117003896 A CN117003896 A CN 117003896A
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- fusion protein
- padre
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Classifications
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Abstract
The invention discloses gE fusion protein containing P2 and PADRE epitopes, a preparation method and application thereof, and solves the technical problem of weak immunogenicity of genetic engineering recombinant subunit vaccine antigens in the prior art. Comprising a fusion protein encoding a polypeptide comprising the varicella zoster virus glycoprotein E outer membrane amino acid sequence (gE), tetanus toxin universal T cell epitope peptide P2 gene sequence (P2), universal DR Th epitope sequence (PADRE), said gE fusion protein gene being capable of expressing gE fusion protein in CHO cells. The gE fusion protein PADRE-gE-P2 has excellent immunogenicity, and can enhance humoral immune response and cellular immune response while maintaining safety.
Description
Technical Field
The invention belongs to the technical field of biological vaccines, and particularly relates to gE fusion protein containing P2 and PADRE epitopes, and a preparation method and application thereof.
Background
Varicella-zoster virus (varicella zoster virus, VZV), also known as human herpesvirus type 3, is a human alpha herpesvirus. Primary infection of varicella-zoster virus in normal populations can lead to varicella during childhood. After VZV infection, the virus is latent in the ganglion of the human body, and when immunity is reduced due to various reasons, the virus is activated again and replicated in large quantities, which leads to shingles (HZ) and is often accompanied by postherpetic neuralgia (Post-herpetic neuralgia, PHN). VZV infection may be accompanied by severe complications such as pulpitis, encephalomyelitis in immunocompromised populations. The genomic size of VZV is about 125kb, encoding about 69 proteins, 8 of which include gB, gC, gE, gH, gI, gK, gL, gM; among them, glycoprotein E (gE) is the main glycoprotein recognized by the host immune system, and is the most antigenic and abundant glycoprotein on the viral envelope and infectious cell membrane, and 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.
Varicella-zoster virus is extremely contagious, transmitted mainly by air droplets and direct contact, and it is reported that about 90% of all adults over 50 years of age worldwide are positive for serum VZV detection. Children infected with VZV may develop fever and develop global red maculopapules, herpes and scabs, with self-limitation. The incidence of herpes zoster gradually increases with the aging, the life pressure increases with the current increase of the life rhythm, and the incidence of the herpes zoster tends to be younger. The onset of varicella-zoster severely affects the quality of life of people, especially the presence of PHN, which is the most common sequelae of shingles, with a incidence of 10% -30% in shingles, and pain lasting from months to years, up to 10 years.
There are two types of vaccines currently approved for the prevention of VZV infection, one is VZV attenuated live vaccine (Oka strain), low dose for the prevention of varicella in children and high dose for the prevention of shingles; one is a genetically engineered recombinant subunit vaccine for use with an adjuvant system. The high dose (about 20000 PFU) attenuated live vaccine product Zostavax of the Mongolian company is approved in the year 2005 for preventing herpes zoster in adults over 50 years old, and adverse effects of the vaccine are mainly caused by pain, erythema and swelling of inoculated parts, and large-scale clinical experiments prove that the protection rate of the vaccine is 69.8% in the people of the age of 50-59 years old, 51% in the people of more than 60 years old, gradually decreases with the increase of the age, and only 18% in the people of more than 80 years old. In the aspect of preventing PHN, the vaccine protection rate is 39% for people over 60 years old. There is a literature report that the efficacy of the vaccine is continuously reduced after 5-8 years of Zostavax vaccination, and is not statistically significant after more than 8 years, and domestic live attenuated herpes zoster vaccine with vinca and hundred grams is currently marketed in batch in month 02 of 2023.
The adjuvant shingles subunit vaccine shintrix from the company glazin smith was approved by the FDA in 2017 for use in preventing shingles in adults over 50 years of age. The vaccine consists of two parts, namely truncated VZV glycoprotein E expressed by CHO cells and AS01B adjuvant system. The side effects of the adjuvant subunit vaccine shintrix were more severe than those of the live attenuated vaccine Zostavax, with at least 1 occurrence of symptomatic symptoms of 84.5% and 33.7% and at least 1 occurrence of grade 3 AE of 16.0% and 2.5% in the test and placebo group subjects, respectively, within 7 days after vaccination. Clinical experiments prove that the vaccine has the overall protection rate of 97.16 percent on HZ in subjects more than or equal to 50 years old. According to layering analysis of 50-59 years old, 60-69 years old and more than or equal to 70 years old, the protection rate of the product to HZ is equivalent in all ages, wherein the 50-59 years old is 96.57%, the 60-69 years old is 97.36%, and the more than or equal to 70 years old is 97.93%. In the case of preventing PHN, in normal population of 50 years old or more, the occurrence rate of PHN can be reduced by 91.2% by Sringrix. In normal people aged more than or equal to 70 years, the feed additive is reduced by 88.8 percent. Continuous studies on vaccine efficacy have shown that vaccine effectiveness can last for more than 10 years. AS01 B The adjuvant system comprises 3D-MPL, QS-21 and phosphorusThe components of fatty acyl choline, cholesterol, etc. can obviously improve the effectiveness of the vaccine, but have the great disadvantage of high side reaction degree and incidence rate.
Clinical experiments prove that the attenuated live vaccine has smaller side reaction, but lower protection efficiency and protection durability, complex vaccine production process and limited amplified production; the recombinant subunit vaccine matched with the adjuvant system has good protection efficiency and high durability, but has strong side reaction and strong vaccine side reaction, 3D-MPL in the adjuvant system is derived from lipopolysaccharide detoxification products of salmonella, the yield is low, and the vaccine cost is high. A large number of researches prove that the cell immunity function plays an extremely important role in VZV infection, and other genetic engineering recombinant subunit vaccines reported in literature have weaker antigen immunogenicity due to antigen design reasons or weaker cell immunity function because the used adjuvant system is a traditional aluminum adjuvant, so that the vaccine has weaker effect in preventing varicella zoster.
Patent publication No. CN110343722 discloses a method for recombinant expression of truncated glycoprotein E of varicella-zoster virus v-Oka strain, which comprises introducing truncated gE protein gene into baculovirus and infecting insect cells with the recombinant baculovirus to express soluble gE protein. The method is easy to screen, stable in batches, but the protein expressed by the insect cells has larger difference compared with the glycosylation of the mammalian cells, and the protein alone cannot effectively activate the humoral and cellular immune functions of the human body.
Publication No. CN112870344 discloses a method for preparing recombinant varicella zoster vaccine, which expresses fusion protein gE-Fc of truncated gE and IgG antibody Fc segment in CHO cells. The recombinant protein is purified by a series of chromatography and virus inactivated to obtain highly purified fusion protein. The fusion protein can be matched with aluminum adjuvant to immunize animals so as to generate high-titer serum neutralizing antibodies. A great deal of researches prove that the effect of the herpes zoster vaccine is mainly determined by the cellular immunity function, and although Fc in the fusion protein can improve the cellular immunity function at a certain level, the invention uses an aluminum adjuvant as the adjuvant, and the single fusion protein is matched with the aluminum adjuvant, so that the cellular immunity function of a human body can not be activated efficiently.
Disclosure of Invention
The invention aims to provide gE fusion protein containing P2 and PADRE epitopes, and a preparation method and application thereof, so as to solve the technical problems that in the prior art, the immunogenicity of a genetic engineering recombinant subunit vaccine antigen is weak, an used adjuvant system is a traditional aluminum adjuvant, the cellular immunity function is weak, and a composite adjuvant system is used, and the side reaction is high.
In order to achieve the above purpose, the present invention provides the following technical solutions:
the gE fusion protein containing P2 and PADRE epitopes provided by the invention comprises a coding gene containing varicella-zoster virus glycoprotein E outer membrane amino acid sequence (gE), tetanus toxin universal T cell epitope peptide P2 amino acid sequence (P2) and universal DR Th epitope amino acid (PADRE), wherein the gE fusion protein gene can express the gE fusion protein in CHO cells.
Furthermore, the combination mode of the amino acid sequences of the fusion proteins of the outer segment of the gE membrane, P2 and PADRE is P2-PADRE-gE, P2-gE-PADRE, PADRE-gE-P2, PADRE-P2-gE, gE-P2-PADRE or gE-PADRE-P2, the preferred combination is PADRE-gE-P2, and the preferred combination of the amino acid sequences of the PADRE-gE-P2 is shown as SEQ ID NO:1, the nucleotide sequence of the gene coding is shown as SEQ ID NO: 2.
Further, the gE extra-membrane segment sequence, the P2 sequence and the PADRE sequence are connected through a connecting peptide, wherein the connecting peptide is GGS and/or GGGS and/or GGGGS and/or GSGSG connecting peptide.
Furthermore, the gE fusion protein containing P2 and PADRE epitopes is applied to the preparation of varicella-zoster vaccine.
The varicella-zoster vaccine provided by the invention is formed by combining gE fusion protein containing P2 and PADRE epitopes and an adjuvant.
Further, the adjuvant is any one or a combination of more than one of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, neutral liposome adjuvant containing saponin, cationic liposome adjuvant containing saponin and anionic liposome adjuvant containing saponin, cpG adjuvant, nanoemulsion and adjuvant containing 3D-MPL.
Further, the varicella-zoster virus vaccine contains 5 to 200 μg of the fusion protein per dosage unit, preferably 10 to 100 μg of the fusion protein per dosage unit, and more preferably 20 to 80 μg of the fusion protein per dosage unit.
The invention provides a preparation method of fusion protein, which comprises the following steps:
s1, carrying out total gene synthesis on a fusion protein gene sequence subjected to codon optimization to obtain a fusion protein gene;
s2, connecting the fusion protein gene into a mammalian expression vector pXNM3.0 to obtain a recombinant expression plasmid;
s3, transfecting the recombinant expression plasmid into CHO cells, and obtaining cell strains for stably expressing recombinant proteins through screening of mini cell groups and monoclonal screening;
s4, culturing the cell strain for stably expressing the recombinant protein, and collecting supernatant of the fermentation product for purification treatment to obtain the fusion protein.
Further, in step S2, the expression vector is a plasmid expression vector carrying the GS screening system and/or carrying the bleomycin resistance gene.
Further, in step S3, the CHO cells are preferably CHO-K1 cells.
Based on the technical scheme, the embodiment of the invention at least has the following technical effects:
(1) The gE fusion protein containing P2 and PADRE epitope provided by the invention has excellent immunogenicity, and the antibody titer generated after mice are immunized is far higher than the immune effect of pure gE protein, and intracellular cytokine staining experiments prove that the fusion protein can induce stronger T cell immune response than that of pure gE antigen.
(2) The varicella-zoster vaccine provided by the invention consists of fusion protein and an adjuvant system. The adjuvant system matched with the vaccine is correspondingly innovated, and the vaccine is applied to GSK AS01 B On the basis of an adjuvant system, 3D-MPL components with complex production process, low yield and high side reaction are removed. AdjuvantUnder the condition that the content of QS-21 (Quillaja saponaria saponin QS-21) and the content of liposome (DOPC and cholesterol) in the system are consistent with that of GSK company, the antibody titer generated after the mice are immunized is higher than that of the miceIs a natural immune system. Intracellular cytokine staining experiments prove that the innovative vaccine induces the ratio +.>Stronger T cell immune response.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to the structures shown in these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a block diagram of an expression vector PXNM3.0 according to an embodiment of the present invention;
FIG. 2 is a block diagram of a fusion protein (PADRE-gE-P2) expression vector according to an embodiment of the present invention;
FIG. 3 shows the GMT titers of serum specific for two immunization gE proteins according to an embodiment of the invention;
FIG. 4 is a CD4+ T cell response specific for example gE of the invention.
Detailed Description
The following description of the technical solutions in the embodiments of the present invention will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. In addition, the technical solutions of the embodiments may be combined with each other, but it is necessary to base that the technical solutions can be realized by those skilled in the art, and when the technical solutions are contradictory or cannot be realized, the technical solutions should be considered that the combination does not exist and is not within the scope of protection claimed by the present invention.
The aim of the invention is realized by the following technical scheme:
embodiment one: preparation of fusion proteins comprising the steps of
S1, fusion protein codon optimization and total gene synthesis
Codon optimization is carried out on the gene of the fusion protein, and the optimization principle is as follows: 1. avoiding the common enzyme cutting sites; 2. according to the preference of codons in CHO cells, replacing synonymous codons with low frequency with codons with high frequency, and controlling rare codons; 3. the GC content in the control sequence is 40% -60% so as to improve the transcription efficiency of mRNA, and meanwhile, the influence of high GC content on the secondary structure of mRNA is avoided, and further the translation efficiency is influenced. And adding a signal peptide sequence in front of the optimized sequence, introducing HindIII restriction enzyme sites at the upstream of the sequence, adding a stop codon and BamHI restriction enzyme sites at the downstream of the sequence, and performing complete genome synthesis of the nucleotide sequence.
The sequences involved are as follows:
the PADRE-gE-P2 fusion protein has an amino acid sequence shown in a sequence table SEQ ID NO:1, the nucleotide sequence of the gene coding of the PADRE-gE-P2 fusion protein is shown in a sequence table SEQ ID NO:2 is shown in the figure; the amino acid sequence (gE) of the outer membrane region of the varicella-zoster virus glycoprotein E is shown in a sequence table SEQ ID NO:3, as shown in the sequence table SEQ ID NO:4 is shown in the figure; the amino acid sequence (P2) of the tetanus toxin universal T cell epitope peptide P2 is shown in a sequence table SEQ ID NO:5, the nucleotide sequence of P2 is shown as a sequence table SEQ ID NO:6 is shown in the figure; the PADRE amino acid sequence is shown in a sequence table SEQ ID NO:7, the nucleotide sequence of the PADRE is shown as a sequence table SEQ ID NO: shown at 8.
S2, construction of fusion protein expression plasmid
The cloning vector containing the total gene synthesis sequence is used for transforming DH5 alpha competent bacteria, then a large amount of bacteria are amplified, the cloning vector is digested with restriction enzymes HindIII and BamHI, meanwhile, the cloning vector is digested with restriction enzymes HindIII and BamHI to form an expression vector pXNM3.0, the structure diagram of the expression vector PXNM3.0 is shown in figure 1, and the structure diagram of the gE fusion protein (PADRE-gE-P2-Fc) expression vector is shown in figure 2. The double enzyme-cut cloning vector cuts the gel to recover the fusion protein gene part, and the expression vector cuts the gel to recover the skeleton part. The two are connected by T4 enzyme, DH5 alpha competent bacteria are transformed, plates containing ampicillin resistance are coated for screening, positive colonies are picked up to amplify plasmids, hindIII and BamHI are used for double enzyme digestion identification, and correct recombinant expression vectors are sequenced and verified.
S3, construction of stable transgenic cell strain
Identifying correct recombinant expression vectors, carrying out enrichment, carrying out single enzyme digestion on the recombinant expression vectors by using Pvu I, recovering the enzyme tangential vector by gel digestion, and filtering and sterilizing for later use. CHO-K1 cells are serially passaged more than twice after resuscitation and used for electrotransformation at cell viability greater than 95%. In a clean bench, 0.6ml of cell suspension (about 1X 107 cells), 200ul of linearized recombinant expression vector (about 50. Mu.g) was added to a 4mm electrocuvette, and the electrotransport conditions were set to 300V, capacitance 900. Mu.F. After the electrotransformation, the cells were transferred to a cell shake flask containing 30ml of CD CHO medium and incubated at 37℃for 24 hours at a carbon dioxide concentration of 5% and at 125 rpm. After centrifugation and electrotransformation for 10 minutes, 100g of cell suspension was used, the supernatant was discarded, cells were resuspended in CD CHO medium containing 25. Mu.M MSX and 200. Mu.g/ml bleomycin, then inoculated into 24 empty plates, the amount of expression was detected by ELISA after 3 weeks of culture, 3 cells with high amount were mixed and then inoculated into 96-well plates by limiting dilution for monoclonal screening, and photographs were taken with single cell imaging equipment on days 0, 1, 2, 3, 7, 15. After 15 days, the expression level is detected by ELISA method, and 3 cells with high expression level are frozen. After stability studies, cell lines for vaccine preparation were determined and then two-stage cell banks were established.
S4, expression of target product
Resuscitates a frozen working seed with OPM-CHO CDP9 culture medium, gradually amplifies in shake flask, and finally transfers into 5L bioreactor for culturing with inoculation density of 0.8X106 cells/ml, and culture parameters are set to 37 deg.C, pH7.0, rotation speed of 150r/min, and dissolved oxygen concentration of 40%. The cell viability, density, lactic acid content, glucose content were measured by daily sampling, and when the viable cell density reached 3X 106cells/ml by 3 days of culture, feed media CDF18 and CDF26 were added, and 250ml and 25ml were inoculated, respectively. The same volume of feed medium was then added every 1 day. The glucose content in the culture solution is maintained to be more than 2g/L, and when the concentration is lower than the glucose content, the glucose concentration is supplemented to be 4g/L. Culturing for about 15 days, and stopping culturing when the cell viability is as low as 70%. The depth filter filters out cells and cell debris and collects cell culture supernatants.
S5, purifying target products (stock solution preparation)
Cell culture supernatant was adjusted to pH7.5, anion exchange column Capto Q was equilibrated to UV absorption baseline level with pH7.5 mM PB buffer, pH was stabilized, then cell supernatant was passed through the column, equilibrated to UV absorption baseline level with the same equilibration buffer, and then subjected to linear elution with pH7.5 mM PB elution buffer containing 1M sodium chloride, and the objective was collected. The anion purified product is inactivated at low pH (pH 3.0-4.0, standing at 18-25 ℃ for 60 min), 1M ammonium sulfate is added to the inactivated product, the pH is adjusted to 7.5, a pH7.5 mM PB buffer and a 1M ammonium sulfate buffer are used for balancing a hydrophobic chromatography column Capto Phenyl ImpRes to the ultraviolet absorption baseline level, the pH is stable, then the inactivated solution after the pH adjustment is passed through the column, the same buffer is used for balancing the chromatography column, and finally the target object is eluted linearly by the pH7.5 mM PB buffer. The hydrophobic purification product is purified by Sephacryl S-300High Resolution molecular sieve chromatography and is changed to obtain the purified protein. The purified protein is subjected to nanofiltration through a 15nm filter and sterilization filtration through a 0.22 mu m filter membrane, and then vaccine stock solution is obtained.
Embodiment two: formulation of vaccines
(1) Preparation of liposomes (ethanol infusion; 100ml volume, DOPC and cholesterol concentrations of 4mg/ml and 1mg/ml, respectively): respectively weighing 400mgDOPC and 100mg cholesterol; completely dissolving DOPC and cholesterol in 10ml of absolute ethyl alcohol, and uniformly mixing to obtain an organic phase; 10ml of the organic phase was poured into 90ml of 10mM PBS buffer salt solution (pH 7.0) to prepare liposome colostrum; then using a high-pressure micro-jet homogenizer to carry out granulation until the particle size of the liposome is about 100 nm; then, ethanol is removed by a dialysis method; finally, filtering and sterilizing by using a 0.22 mu m sterilizing filter to obtain the finished liposome.
(2) Preparation of gE fusion protein adjuvant vaccine (10 ml volume; gE fusion protein concentration 100. Mu.g/ml; immunopotentiator saponin QS-21 100. Mu.g/ml; liposomal composition: DOPC and cholesterol 2mg/ml and 0.5mg/ml, respectively): taking 5ml of liposome, adding 1ml of saponin QS-21 solution (the concentration is 1 mg/ml), and uniformly stirring to obtain the adjuvant; then adding the gE fusion protein solution into the adjuvant, and then using 10mM PBS buffer salt solution (pH 7.0) to complement the total volume to 10ml, and uniformly stirring to obtain the gE fusion protein adjuvant vaccine; the volume (ml) of the added gE fusion protein solution is calculated as: the mass of the gE fusion protein (μg)/the concentration of the gE fusion protein (μg/ml) =the theoretical molecular weight of the gE fusion protein (μg/ml) =the concentration of the gE in the vaccine (μg/ml)/(the theoretical molecular weight of the gE fusion protein (μg/ml) ]=the theoretical molecular weight of the gE fusion protein (μg/ml) ×100×10/(the theoretical molecular weight of the gE fusion protein).
Embodiment III: vaccine immunized animals
The experimental animal is C57BL/6 female mice with the age of 6-8 weeks; animal immunization and feeding were performed in the absence of specific pathogens. To mimic infection with varicella-zoster virus in natural environment, mice were presoaked subcutaneously with varicella-zoster virus (containing not less than 3.3lg PFU live virus, 0.5 ml) at the neck 35 days before immunization with the candidate vaccine. On days 0 and 28, candidate vaccine was injected intramuscularly in the leg at a dose of 50ul per mouse. Blood was collected by eye drop method 28 days after the second-day, and mice were sacrificed after blood collection. The collected whole blood is kept stand at 2-8 ℃ overnight, and centrifuged at 3000rpm for 30min in the next day, and the top serum is sucked. Mouse serum titers were measured by indirect ELISA and GMT values were calculated. Spleen cells were isolated from mice, the numbers of gE-specific INF-gamma and IL-2 secreting CD4+ T cells were measured by intracellular cytokine staining, cellular immune levels were assessed, and information about vaccine animal immunogenicity studies is shown in Table 1.
TABLE 1 information related to vaccine animal immunogenicity studies
Embodiment four: vaccine immunogenicity analysis
(1) Detection of gE protein binding antibodies in serum
Total gE-specific antibodies in serum samples 28 days after total individual mice were double-immunized were detected by means of an indirect ELISA. The procedure is that gE protein is coated into a 96-well plate by carbonate buffer solution, the coating amount is 500 ng/well, the coating is carried out overnight at 4 ℃, the plate is closed by a TPBS solution containing BSA, the TPBS solution is washed 3 times, serum of all individual mice is diluted according to different dilutions, the diluted serum is added into the well plate, the diluted serum is incubated for 1 hour at 37 ℃, the TPBS solution is washed 3 times, the plate is incubated for 1 hour at 37 ℃ by an HRP-marked goat anti-mouse secondary antibody, the plate is washed 3 times by the TPBS solution, the color development is carried out for 10 minutes by TMB color development liquid, the reaction is stopped by adding 0.2M sulfuric acid, and the value at OD450 is read by an enzyme-labeling instrument. The post-immunization serum titers were determined using 3-fold of the reading of a pre-immune serum mix as Cut-Off, and the two immunization gE protein-specific serum GMT titers are shown in FIG. 3.
(2) Intracellular cytokine flow cytometer detection
The spleen of the test mouse is taken 28 days after the second immunity, spleen single cell suspension is prepared, after the cell concentration is regulated, red blood cells in the single cell suspension are lysed by using a lysis solution, spleen cells are stimulated by using a specific peptide pool to secrete cytokines, secretion is blocked after Containing Brefeldin A secretion blocking agents are added, then the spleen cells are subjected to steps of cell living dying, surface receptor FcR blocking, CD3, CD45 and CD4 surface dying, membrane rupture fixation, intracellular dying of IL-2 and IFN-gamma and the like, and the quantity of CD4+ T cells secreting IL-2 and IFN-gamma is detected by using a flow cytometry, wherein the detection quantity is shown in a figure 4.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (10)
1. A gE fusion protein comprising P2 and PADRE epitopes, characterized in that: comprising a sequence encoding E-membrane outer region (gE) containing varicella zoster virus glycoprotein, a tetanus toxin universal T cell epitope peptide P2 sequence (P2), a universal DR Th epitope peptide sequence (PADRE), wherein the gE fusion protein gene can express gE fusion protein in CHO cells.
2. The gE fusion protein comprising P2 and PADRE epitopes of claim 1, wherein: the combination mode of the fusion protein amino acid sequences of the gE extra-membrane segment sequence, the P2 sequence and the PADRE sequence is P2-PADRE-gE, P2-gE-PADRE, PADRE-gE-P2, PADRE-P2-gE, gE-P2-PADRE or gE-PADRE-P2, the preferable combination is PADRE-gE-P2, and the preferable combination of the PADRE-gE-P2 has the amino acid sequence shown as SEQ ID NO:1, the nucleotide sequence of the gene coding is shown as SEQ ID NO: 2.
3. The gE fusion protein comprising P2 and PADRE epitopes of claim 2, characterized in that: the gE extra-membrane segment sequence, the P2 sequence and the PADRE sequence are connected through a connecting peptide, wherein the connecting peptide is GGS and/or GGGS and/or GGGGS and/or GSGSGSG connecting peptide.
4. Use of a gE fusion protein comprising P2 and PADRE epitopes according to any one of claims 1 to 3 for the preparation of varicella-zoster vaccine.
5. A varicella zoster vaccine characterized by: the vaccine is formed by combining the gE fusion protein containing the P2 and PADRE epitopes and an adjuvant according to any one of claims 1-3.
6. The varicella zoster vaccine according to claim 5, characterised in that: the adjuvant is any one or a combination of any more of aluminum hydroxide adjuvant, aluminum phosphate adjuvant, neutral liposome adjuvant containing saponin, cationic liposome adjuvant containing saponin and anionic liposome adjuvant containing saponin, cpG adjuvant, nanoemulsion and adjuvant containing 3D-MPL.
7. The varicella zoster vaccine according to claim 5, characterised in that: the varicella-zoster vaccine contains 5 to 200 μg of fusion protein per dosage unit, preferably 10 to 100 μg of fusion protein per dosage unit, more preferably 20 to 80 μg of fusion protein per dosage unit.
8. A method for preparing a fusion protein for preparing a gE fusion protein comprising P2 and PADRE epitopes according to any one of claims 1 to 3, comprising the steps of:
s1, carrying out total gene synthesis on a fusion protein gene sequence subjected to codon optimization to obtain a fusion protein gene;
s2, connecting the fusion protein gene into a mammalian expression vector pXNM3.0 to obtain a recombinant expression plasmid;
s3, transfecting the recombinant expression plasmid into CHO cells, and obtaining cell strains for stably expressing recombinant proteins through screening of mini cell groups and monoclonal screening;
s4, culturing the cell strain for stably expressing the recombinant protein, and collecting supernatant of the fermentation product for purification treatment to obtain the fusion protein.
9. The method for producing a fusion protein according to claim 8, wherein: in the step S2, the expression vector is a plasmid expression vector carrying a GS screening system and/or carrying a bleomycin resistance gene.
10. The method for producing a fusion protein according to claim 8, wherein: in step S3, the CHO cells are preferably CHO-K1 cells.
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