CN113308485B - Vaccine adjuvant and application thereof - Google Patents

Abstract

The invention relates to a vaccine adjuvant and application thereof. The vaccine adjuvant is a plasmid adjuvant, pVAX1 is used as a vector, a eukaryotic promoter is knocked out, and the vaccine adjuvant comprises: tandem of different species of CpG ODN, each CpG ODN being independently selected from any class of human or mouse CpG ODN; the plasmid contains 2-10 CpG ODN, and each CpG ODN is repeated 0-3 times. The invention selects different species specificity CpG ODN to be inserted into the pVAX1 vector in series to obtain a broad-spectrum plasmid adjuvant, which can effectively improve the vaccine titer when being used as a vaccine adjuvant and has important significance for the development of the vaccine adjuvant in the prior art.

Description

Vaccine adjuvant and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a vaccine adjuvant and application thereof.

Background

Vaccines are the most effective and economical way to control infectious diseases. The first generation of vaccines were attenuated live and inactivated vaccines, which contain a full spectrum of antigens that themselves induce immunoprotection against pathogens, but also contain components that produce side effects. The second generation vaccine is a subunit vaccine and a genetic engineering vaccine which are prepared from natural components of microorganisms or have the immunization function, the structure of the recombinant protein is confirmed, the purity is high, the recombinant protein is safe, the immunogenicity is weak, and an adjuvant is needed to enhance the antigen immune response. However, the types of clinically available adjuvants are few at present, aluminum hydroxide is a main adjuvant of subunit vaccines and plays a significant role in infectious disease control, but the aluminum hydroxide adjuvant still has certain limitations, and the infectious diseases mainly based on cellular immunity still lack effective control vaccines at present, such as tuberculosis, malaria and the like. The third generation vaccine is to recombine antigen gene to expression vector, and to introduce the recombinant antigen into body's cell by means of certain method to express antigen and induce body to produce specific humoral immunity and/or cellular immunity response, and is also called gene immunity, nucleic acid vaccine or DNA vaccine. DNA vaccines also require adjuvants to enhance the immune response, such as cloning cytokine adjuvants and antigen genes into the same vector for simultaneous expression, so-called intramolecular adjuvants [ Yang Y, et al.Antitumole effect of a DNA vaccine binding protein cancer-specific antigen with IL-12 as an intramolecular adjuvant, J.Mol Microbiol Biotechnol,2017,27(3): 168-; or an adjuvant and the DNA vaccine simultaneously immunize the organism, the adjuvant enhances the antigen specific immune response [ Danko JR, et al.safety and immunogenicity of a fourth viral DNA vaccine supplemented with a cationic lipid-based adjuvant in a phase 1clinical trial. am J Trop Med Hyg, 2018, 98(3): 849-. Therefore, in a certain sense, the adjuvant becomes the bottleneck of modern vaccine development, and the safe and effective adjuvant has important significance for vaccine development.

Vaccine adjuvants (adjuvants) refer to adjuvants used in combination with vaccine antigens, either alone or in combination with multiple adjuvants, to enhance or modulate (e.g., switch a Th1 immune response to a Th2 immune response) antigen-specific immune responses, ultimately with the aim of enhancing the clinical effectiveness of the vaccine. Adjuvants can be classified by their role as immunostimulants and vaccine antigen delivery systems, and immunostimulants generally include substances that activate the innate immune system to enhance a specific immune response to a vaccine antigen, such as Toll-like receptor (TLR) agonists, cytokines, and the like; vaccine antigen delivery systems can enhance antigen delivery to regional lymph nodes, such as liposomes, nanoparticles, and the like. Wherein, the TLR9 agonist is used alone or in combination with other adjuvants to be clinically researched, for example, CpG 7909 artificially synthesized with CpG oligonucleotide has been used as an adjuvant of infectious disease (hepatitis B, AIDS, influenza, etc.) vaccines and cancer vaccines to be clinically researched [ Belani CP, et al. phase 2 tertiary of erlotinib with or without PF-3512676(CPG 7909, a Toll-receptor 9 agonist) in Patients with advanced recovery EGFR-positive non-small cell lung cancer. cancer Biol Ther, 2013,14(7):557-63. ]; TLR4 agonist MPLA formulated with other adjuvants AS composite adjuvants AS01 and AS02 into clinical studies or marketed [ Tait DR, et al, final analysis of a trial of M72/AS01(E) vaccine to present tissue J Med,2019,381(25):2429 2439. ]; MF59, an oil-in-water nanoemulsion, is an adjuvant approved for human use following aluminum hydroxide adjuvant, and was first approved as an influenza vaccine for the elderly [ Squarrione S, et al, Complex of the reactogenicity and immunogenicity of a site and a study-added flu vaccine in elurly subjects, vaccine,2003, 21(11-12): 1268-. As a result of studies on the action mechanism of MF59, MF59 was introduced into clinical studies as another Vaccine adjuvant [ Nitayaphan S, et al. A phase I/II tertiary of HIV SF2 gp120/MF59 Vaccine in continuous Vaccine. AFRIMS-RIHES Vaccine Evaluation group. intensive Research Institute of Medical Sciences and the Research Institute for Health Sciences. Vaccine,2000, 18(15):1448 1455 ].

The CpG in the genome DNA of the vertebrate is mostly methylated, the bacterial DNA contains a large amount of unmethylated CpG dinucleotide sequences, researches show that the bacterial DNA containing unmethylated CpG and artificially synthesized oligonucleotide (CpG-ODN) containing unmethylated CpG are good immunostimulants, and the combined adjuvant which is used alone or combined with other adjuvants can enhance the cellular immune response of the cells mainly of Th1 type by being immunized together with various antigens, thereby playing an important role in resisting intracellular infection. The structural characteristics and the immune effects of different types of CpG ODN are different, and the CpG ODN is generally divided into A, B, C three types, wherein the A type CpG ODN takes a palindromic sequence containing CpG dinucleotide as a core, two ends of the palindromic sequence are poly G tails, a phosphodiester bond skeleton is partially sulfo-modified, a high-level structure is formed by the palindromic sequence and poly G, a large amount of I type interferon can be induced by activating plasmacytoid dendritic cells, and the activity of the plasma cell dendritic cells on B cells is weak. The B class CpG ODN is a linear CpG ODN which is modified by total sulfur, has strong immune stimulation activity to B cells, but can not activate plasmacytoid dendritic cells. The C-class CpG-ODN is a class of full-sulfo modified CpG ODN, can form a dimer through a palindromic sequence, has the activity of both A-type and B-type CpG-ODN, and can activate both plasmacytoid dendritic cells and B cells. CpG-ODN is unstable and easy to be enzymolyzed by 3' end exonuclease; in order to prevent the degradation of CpG-ODN, the phosphodiester skeleton is modified into a phosphorothioate skeleton, so that the synthesis cost is increased by 3-4 times; with the increase of the synthesis amount, the cost of synthesizing single base and modifying single phosphodiester skeleton into phosphorothioate skeleton is multiplied, so that the cost of artificially synthesized CpG-ODN is high, and the artificially synthesized CpG-ODN is not suitable for the preventive vaccines for the vast healthy population. Bacterial DNA containing CpG is also a TLR9 agonist and has good adjuvant function, such as BC02 adjuvant (consisting of BCG CpG-DNA, aluminum hydroxide adjuvant and phosphate buffer system) recombinant tuberculosis protein vaccine AEC/BC02 approved clinical research (WHO. Global tuberculosis report. 2020). The preparation cost of the BCG CpG-DNA is low, but the BCG CpG-DNA is composed of a series of DNA fragments with different base numbers, and the random breakage condition occurs in the extraction process of the bacterial genome DNA, so the problem of batch consistency of the BCG CpG-DNA is difficult to solve.

Plasmids are small circular DNA molecules that are found in many organisms such as bacteria and yeast and are capable of autonomous extrachromosomal replication. The cloning plasmid vector is artificially synthesized or modified plasmid DNA with a replicon, a selective marker and a cloning site, and the expression vector not only has the cloning vector elements, but also needs to be inserted with a prokaryotic expression promoter and a eukaryotic expression promoter to form a corresponding prokaryotic expression vector and a eukaryotic expression vector. The coding gene of the antigen is cloned to a eukaryotic expression vector and introduced into eukaryotic cells to express the antigen, and the expressed antigen stimulates immune cells to induce specific cell and humoral immune response, which is the theoretical basis of DNA vaccine design. Prokaryotic and eukaryotic expression vectors can be replicated in escherichia coli, and plasmid DNA with high purity, uniform structure and controllable batch quality is prepared in large quantity by adopting an escherichia coli fermentation technology.

Disclosure of Invention

In order to solve the problems in the prior art, the invention provides a vaccine adjuvant and application thereof. The invention connects a plurality of different species of CpG ODN in series to obtain a plasmid adjuvant which can be used alone or in combination with other adjuvants to effectively enhance the antigen immunogenicity.

In a first aspect, the present invention provides a plasmid comprising:

concatenating different classes of CpG ODN, each CpG ODN being independently selected from any class of human or mouse CpG ODN;

the plasmid contains 2-10 CpG ODN, and each CpG ODN is repeated 0-3 times.

Further, the eukaryotic promoter in the plasmid was knocked out.

Further, the plasmid comprises the nucleotide sequence of any one of SEQ ID NO 1-4.

Researches find that the partial CpG motif contained in the DNA vaccine vector can be used as an adjuvant for the antigen expression, and after different species of CpG-ODN sequences are connected in series, the CpG-ODN sequences are cloned to a plasmid vector pVAX1 approved by FDA to construct a recombinant plasmid containing the CpG-ODN; and in view of safety, the eukaryotic promoter is knocked out by a DNA recombination technology, so that plasmid DNA proliferation is guaranteed, but no gene is expressed, and the safety of the plasmid adjuvant is improved.

The invention further provides a vaccine adjuvant comprising the plasmid.

Further, the vaccine adjuvant also comprises an Al adjuvant or an SE adjuvant.

Further, the mass ratio of the plasmid to the SE adjuvant is 5-200 mug: 0.025ml to 0.1 ml.

The invention further provides application of the vaccine adjuvant in improving the titer of a vaccine.

Further, the vaccine is one or more of herpes virus vaccine, rabies virus vaccine or tuberculosis vaccine.

In a second aspect, the invention provides a vaccine comprising the vaccine adjuvant.

The invention has the following beneficial effects:

the construction and purification of the invention can obtain the plasmid DNA containing CpG ODN motif, which can effectively enhance antigen immunogenicity when used alone or combined with other adjuvant.

The broad-spectrum plasmid adjuvant which has high purity, uniform structure, stable batch quality, safety, effectiveness, low cost, easy production and suitability for mouse immune evaluation and clinic is obtained by the research of the invention, and the bottleneck problems of high cost of artificially synthesized CpG ODN and poor batch consistency of bacterial CpG-DNA are solved.

Drawings

FIG. 1 shows a plasmid extracted from a single colony culture solution selected by a PC plasmid-transformed DH5 alpha host strain provided by embodiment 1 of the present invention and a double digestion pattern (2% agarose electrophoresis) thereof; wherein, lane 1: pVAX1 plasmid, lane 2: PC04 plasmid extracted from colony 2 (PC04-2), lane 3: PC04 plasmid (PC04-1) extracted from colony No. 1, lane 4: PC03 plasmid (PC03-2) extracted from colony 2, lane 5: the PC03 plasmid extracted from colony No. 1 (PC03-1), the PC02 plasmid extracted from colony No. 6: 1 (PC02-1), and the DNA standard molecular weight (1kb plus DNA Ladder: 300, 500, 800, 1000, 1500, 2000, 3000, 4000, 5000, 6000, 8000, 10000bp) in lane 7; and (4) a swimming channel 8: the PC04-3 plasmid MluI and APaI were double digested, Lane 9: PC04-2 plasmid MluI and APaI double digested, lane 10: the PC04-1 plasmid MluI and APaI were double digested, Lane 11: the PC03-3 plasmid MluI and APaI were double digested, lane 12: the PC03-2 plasmid MluI and APaI were double digested, lane 13: PC03-1 MluI and APaI double digested, lane 14: PC02-3 plasmid NheI and APaI double digested, lane 16: the PC02-1 plasmid NheI and APaI were double digested, lane 17: the PC01-3 plasmid NheI and APaI are subjected to double digestion.

FIG. 2 is an electropherogram of the clarification and concentration effect of plasmids provided in example 2 of the present invention; wherein, lane 1: cytosolic lysate, lane 2: CaCl ₂ Pellet supernatant, lane 3: CaCl ₂ Precipitate, lane 4: 100KD membrane ultrafiltration permeate, lane 5: 100KD membrane ultrafiltration concentrate.

FIG. 3 is a 6% agarose gel chromatography sample electropherogram as provided in example 2 of the present invention; wherein, lane 1: sample loading, lane 2: first peak sample (peak 1), lanes 3 and 4: and then a peak sample (peak 2).

FIG. 4 shows the results of the single adjuvant gE vaccine immunization of mice on the log of serum IgG antibody titer.

FIG. 5 shows the results of comparing the serum IgG antibody titer of the gE vaccine immunized mice with different dosages of plasmid adjuvants and SE or Al as composite adjuvants provided in example 3 of the present invention.

FIG. 6 is a comparison of Th1 type immune responses of gE vaccine composed of SE or Al and different doses of plasmid adjuvant provided in example 3 of the present invention; wherein A: 1/2SE + gE, B: 1/3SE + gE, C: 10 μ g PC04+ gE, D: 20 μ g PC04+ gE, E: 40 μ G PC04+ gE, F: 1/2SE-10 μ G PC04+ gE, G: 1/2 SE-20. mu.g PC04+ gE, H: 1/2 SE-40. mu.g PC04+ gE, I: 1/3 SE-10. mu.g PC04+ gE, J: 1/3 SE-20. mu.g PC04+ gE, K: 1/3 SE-40. mu.g PC04+ gE, L: gE, M: and (6) comparison.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 CpG ODN tandem and Whole Gene Synthesis by inserting pVAX1 vector

The invention selects A, B specific to human and mouse and 6 CpG ODNs with different structures of C class to be connected in series into two DNA fragments (fragment 1 and fragment 2 for short) with different times and different positions; fragment 1 is a 304bp DNA fragment composed of 4 CpG ODN3 times of tandem connection and 2 CpG ODN 1 times of tandem connection, and fragment 2 is a 256 bp DNA fragment composed of 6 CpG ODN 2 times of tandem connection. NheI or MluI enzyme cutting sites are added to the 5 ' end of the fragment 1 and the 3 ' end of the fragment 2 respectively, ApaI enzyme cutting sites are added to the 3 ' end of the fragment, and the Shanghai biological whole gene is entrusted to synthesize 4 DNA fragments (the enzyme cutting site of the fragment 1+ is 316bp, and the enzyme cutting site of the fragment 2+ is 268 bp).

The fragment 1 recovered by double enzyme digestion of NheI and ApaI is connected with NheI and ApaI double enzyme digested pVAX1 (the nucleotide sequence of the pVAX1 vector is shown in SEQ ID NO: 5) and positive clones are screened, and a recombinant vector proved by enzyme digestion and sequencing is screened, namely PC01(SEQ ID NO:1) which contains 6 CpG ODNs, wherein 4 of the CpG ODNs are repeated for 3 times, and 2 of the CpG ODNs are repeated for 1 time. The fragment 1 is obtained by double enzyme digestion of MluI and ApaI, the recovered fragment is connected with double enzyme digestion of MluI and ApaI pVAX1, positive clones are screened, and a recombinant vector proved by enzyme digestion and sequencing is screened, wherein the recombinant vector is named as PC03(SEQ ID NO:3), and contains 6 CpG ODNs, 4 of the CpG ODNs are repeated for 3 times, and 2 of the CpG ODNs are repeated for 1 time.

The fragment 2 is obtained by double enzyme digestion of NheI and ApaI, the recovered fragment is connected with pVAX1 obtained by double enzyme digestion of NheI and ApaI, positive clones are screened, and a recombinant vector proved by enzyme digestion and sequencing is screened, wherein the recombinant vector is named as PC02(SEQ ID NO:2), and the recombinant vector contains 6 CpG ODNs, and 6 CpG ODNs are repeated for 2 times.

The fragment 2 is obtained by double enzyme digestion of MluI and ApaI, the recovered fragment is connected with pVAX1 obtained by double enzyme digestion of MluI and ApaI, positive clones are screened, and a recombinant vector proved by enzyme digestion and sequencing is screened, and is named as PC04(SEQ ID NO:4), and the recombinant vector contains 6 CpG ODNs, wherein 6 CpG ODNs are repeated for 2 times.

The fragments 1 and 2 in PC01 and PC02 were inserted at the multiple cloning site of pVAX1, without changing the various components of the original plasmid; fragment 1 and fragment 2 of PC03 and PC04 were inserted at the multiple cloning site and MluI site of pVAX1, replacing the CMV promoter sequence completely.

Further transforming DH5 alpha competent bacteria (purchased from Tiangen biotechnology limited company) by PC01, PC02, PC03 and PC04, selecting 3 colonies each on LB culture medium, shaking and culturing at 37 ℃ overnight, extracting plasmids and double digestion plasmids by a kit (purchased from Tiangen biotechnology limited company), displaying by 2% agarose electrophoresis (figure 1) because the inserted fragments are smaller, wherein the 316bp of the inserted fragments of double digestion of PC01 and PC03 is consistent with theory, and the plasmid fragment of double digestion of PC01 is larger than that of double digestion of PC03, which is consistent with design; the 268bp insert of PC02 and PC04 by double enzyme digestion is consistent with theory, and the plasmid fragment of PC02 by double enzyme digestion is larger than that of PC04 by double enzyme digestion, which is consistent with design.

EXAMPLE 2 Mass preparation of plasmid exemplified by PC04

(1) The bacteria preserved at the temperature of glycerol-80 ℃ are inoculated in an LB culture medium (containing 50 mu g/ml kanamycin) according to a ratio of 1:1000, shake-cultured overnight at the temperature of 37 ℃ and at the speed of 180-200rpm, inoculated with 2.5L of the LB culture medium (containing 50 mu g/ml kanamycin) according to a ratio of 4 percent for fermentation culture, and the fermentation conditions are as follows: stirring at 37 deg.C from 200rpm to no more than 700 rpm, automatically supplementing ammonia water to control pH 6.8-7.2, and feeding logarithmic growth phase (peptone 50g/L, yeast extract 50g/L, glycerol 120ml/L and MgSO 2) ₄ .7H ₂ O1 g/L), taking the fermentation liquor every hour, measuring the light absorption value at 600nm, and stopping fermentation when the light absorption value is not changed greatly. The fermentation liquor is centrifuged at 6000 rpm/min at 4 ℃ for 15min, the trace culture solution is dried, and then the thalli are transferred to 4-5 sealing membranes and weighed, and the thalli are preserved at-80 ℃.

(2) Storing thallus at-80 deg.C, adding solution I (50mM glucose, 25mM Tris-HCl (pH 8.0) and 10mM EDTA) at a ratio of 1:1-1:10(w: v), thawing thallus at room temperature, stirring with glass rod or magnetic stirrer to make thallus fully suspend aseptic block, adding 2 times volume of solution II (prepared fresh, 0.2M NaOH and 1% SDS) of the suspension, mixing by turning upside down for about 20 times, ice-water bathing for 3-5min, adding solution III (60ml of 5M potassium acetate, 11.5 ml glacial acetic acid, 28.5ml ultrapure water) of the same volume of the suspension, turning upside down for about 20 times, and mixing by ice-water bathing for 30 min. Adding 2M CaCl ₂ Mixing the solution to final concentration of 0.2M, precipitating at 4 deg.C for 1-3 hr, filtering the liquid with 40 mesh stainless steel screen or centrifuging at 12000 r and 4 deg.C for 15-30min, filtering to obtain filtrateOr clarifying the centrifugal supernatant by a 0.22 mu m membrane package; the clear solution was concentrated by ultrafiltration through a 100kD membrane.

(3) Separating DNA and RNA from the concentrated solution by 6% crosslinking degree agarose gel chromatography, collecting DNA peak, separating supercoiled and linear plasmid DNA by plasma select Xtra affinity chromatography, collecting supercoiled plasmid DNA, and purifying by SOURCE ^TM Collecting crossing peak by 30Q ion chromatography, ultrafiltering the sample, changing PBS buffer solution, concentrating, filtering with 0.22 μm membrane, sterilizing, packaging at-20 deg.C, and measuring DNA concentration, supercoiled structure DNA content, host bacterial protein and nucleic acid content, and endotoxin content.

Figure 2 results show that: 0.2M CaCl ₂ The precipitate was RNA, and when concentrated at 100kD, part of the RNA permeated the membrane into the permeate and the sample was concentrated.

Figure 3 the results show: 6% crosslinking degree agarose gel chromatography will completely separate plasmid DNA and RNA.

EXAMPLE 3 subunit vaccine immunogenicity characteristics of plasmids as adjuvant against herpesvirus glycoprotein gE 3.1 Effect of plasmid adjuvant alone or in combination with adjuvant on gE specific IgG antibodies

In this example, the plasmid (PC04) prepared in example 2 was used as adjuvant for herpesvirus glycoprotein gE, and the specific procedure was as follows:

SPF grade female Balb/c mice, 6-8 weeks old. The mice were divided into 16 groups of 5 mice each, each group was: the group comprises a gE antigen group, a gE + SE adjuvant group, a gE + low-dose PC04(PC04-L), a gE + medium-dose PC04(PC04-M), a gE + high-dose PC04(PC04-H), a gE + aluminum hydroxide adjuvant (Al for short), a gE + composite adjuvant SE-PC04-L, gE + composite adjuvant SE-PC04-M, gE + composite adjuvant SE-PC04-H, gE + composite adjuvant Al-PC04-L, gE + composite adjuvant Al-PC04-M, gE + composite adjuvant Al-PC04-H, gE + linear PC04-L, gE + composite adjuvant SE-linear PC04-L, gE + composite adjuvant Al-linear PC04-L, PBS control group.

Wherein the Al adjuvant is commercial aluminum hydroxide adjuvant, and the SE adjuvant is squalene nanoemulsion (10 mM citric acid buffer solution (pH6.5) containing 4.3-5% squalene, 0.5% Tween80, and 0.5% SPAN 85).

The mice were bled from orbital venous plexus for 3 days, then injected with 0.2ml of immunogen or PBS through the medial muscle of hind legs, immunized 1 time each for 0 and 21 days, bled from orbital venous plexus 3 days before the second immunization, and bled from eyeball 21 days after the last immunization. Centrifuging at 8000 rpm for 15min at room temperature for 3 hr, sucking serum at-20 deg.C, and storing.

ELISA detection of serum antibodies: in the preliminary experiment, the enzyme-labeled secondary antibody, the diluent and the antigen dosage are determined by adopting an orthogonal design. And (3) formal test: according to the determination of enzyme-labeled secondary antibody, diluent and antigen dosage, the serum is diluted according to the ratio of 1:100 and 1: 200 before test, and the serum collected for 1 time of immunization is selected from the ratio of 1: 200 is diluted by a multiple ratio, 2 times of immunization is carried out, and the serum is diluted by a multiple ratio from 1: 10000 and is diluted by 8 concentrations. The average value of the measured OD values of the control group sera is plus or minus 2SD according to the dilution ratio of 1:100 and is used as the critical value for judging positive; the reciprocal number of the maximum dilution multiple of the OD value measured by the experimental group which is larger than the critical value is taken as the antibody titer of the mouse serum; the mean value of the antibody titer of each group was calculated as a logarithmic value with the antibody titer of each mouse being base 10.

The results shown in table 1 were obtained and showed: the serum antibody level of the single antigen, the single adjuvant antigen vaccine and the compound adjuvant antigen vaccine after 3 weeks of second needle immunization is obviously higher than that of the serum antibody level of the single antigen, the single adjuvant antigen vaccine and the compound adjuvant antigen vaccine after 3 weeks of 1 needle immunization (all P is less than 0.01). The general trend of antibody changes of all immunogens immunized 1 time and 2 times is basically consistent, the antibody titer specific to the antigen is enhanced by the adjuvant, and the composite adjuvant is superior to a single adjuvant. Plasmid adjuvants, like the commercially available Al and SE adjuvants, can increase gE antigen-specific IgG antibody titers.

TABLE 1 Effect of plasmid adjuvants on the logarithm of antibody titers (lg antibody titers) specific for the gE antigen or in combination with other adjuvants

Note: and gE: mice were immunized with gE of 5 μ g per mouse, PC 04-L: each mouse immunized PC04 was 5 μ g, PC 04-M: the immune PC04 was 10 μ g per mouse, PC 04-H: each mouse was immunized with 20 μ g of PC04, Al: each mouse was immunized with 20 μ g of Al, SE: the immune SE per mouse was 0.1 ml.

Figure 4 shows that the SE adjuvant gE vaccine immunized mice both 1 and 2 times produced significantly higher serum IgG antibody titers than the other adjuvant vaccines. The high-dose plasmid adjuvant or Al adjuvant vaccine is immunized 1 time, and the serum IgG antibody titer is obviously higher than that of the low-dose linear plasmid adjuvant vaccine. The low-dose or high-dose plasmid adjuvant gE vaccine is immunized for 2 times, and the serum IgG antibody titer is obviously higher than that of the low-dose linear plasmid adjuvant gE vaccine. Increasing doses of plasmid adjuvant helped to increase gE antigen-specific antibody titers.

FIG. 5 shows that the effect of different doses of plasmid adjuvant in combination with SE or Al on the serum antibody titer of gE-immunized mice, the antibody level induced by the gE vaccine with the composite adjuvant of Al and PC04-L compared with that induced by the gE vaccine with the PC04-L adjuvant had no statistical significance (P >0.05) except for 2 immunizations, and the gE vaccine with the composite adjuvant of Al and PC04 and the gE vaccine with the composite adjuvant of SE and PC04 induced antibody levels significantly higher than those of the gE vaccine with the same dose of PC04-L adjuvant; the induction antibody level of the composite adjuvant gE vaccine of SE and PC04-H is obviously higher than that of the composite adjuvant gE vaccine of Al and PC 04-H.

The results of table 1, fig. 4-5 show that: the low, medium and high dose of plasmid adjuvant enhances the recombinant protein gE immune mice to generate antibody level, but the antibody level is lower than SE adjuvant gE vaccine; the humoral immunity effect induced by the mice immunized by the composite adjuvant prepared by the SE and the Al and the plasmid adjuvant is better than that of a single plasmid adjuvant, but is equivalent to SE adjuvant gE vaccine. The combined adjuvant prepared by the high-dose plasmid adjuvant and the SE enhances the antibody level generated by mice immunized by the recombinant protein gE to be better than the composite adjuvant prepared by the high-dose plasmid adjuvant and the Al.

3.2 Effect of plasmid adjuvant alone or its composite adjuvant on gE-specific Th1 type cellular immunity

SE induces mainly a Th2 type immune response. This subsection clarifies the characteristics of plasmid adjuvants and their complex adjuvants in enhancing the gE-specific Th1 cellular immune response.

SPF grade male Balb/c mice, 6-8 weeks old. The mice are divided into 13 groups, each group comprises 5 mice, and the groups respectively comprise: the gE antigen group, the gE +1/2SE adjuvant group, the gE +1/3SE adjuvant group, the gE +10 mu g PC04(LPC04), the gE +20 mu g PC04(MPC04), the gE +40 mu g PC04(HPC04), the gE + composite adjuvant 1/2SE-LPC04, the gE + composite adjuvant 1/2SE-MPC04, the gE + composite adjuvant 1/2SE-HPC04, the gE + composite adjuvant 1/3SE-LPC04, the gE + composite adjuvant 1/3SE-MPC04, the gE + composite adjuvant 1/3 and the PBS control group, wherein the gE is 5 mu g.

Intramuscular injection is carried out on the inner side of the hind leg, the immunization volume is 0.2ml, 1 time of immunization is carried out on 0 day and 21 days respectively, and the mice are dissected 21 days after the last immunization to carry out cellular immune index detection. ELISPOT measures the number of IFN-gamma secreting cell spots: mice were sacrificed by cervical dislocation and soaked in 75% alcohol for 3 min. Aseptically taking spleen, grinding spleen with syringe piston, separating spleen lymphocyte with mouse organ lymph separating medium, counting, adjusting cell concentration to 2 × 10 ⁶ And/ml. 3 wells were added to each mouse spleen lymphocytes and 100. mu.l of cell suspension was taken per well, 50. mu.l of medium (negative control well) in one well, 50. mu.l of pooled gE polypeptide (antigen-stimulated well), and 50. mu.l of 15g/ml CoA in one well (positive control well). 37 ℃ and 5% CO ₂ The color is developed after 24-48h of culture in the incubator. Color development was performed according to kit instructions. The developed spots are scanned by a spot scanner and counted, the number of the spots with antigen specificity is obtained by subtracting the spots of the negative control wells from the spots of the antigen stimulation wells, the number of the spots of the positive control wells is evaluated as a system, and the spots of the positive control wells are basically paved in the whole wells to prove that the activity of the cells is normal; otherwise the result needs to be discarded.

FIG. 6 (the abscissa of FIG. 6 corresponds to letters A: 1/2SE + gE, B: 1/3SE + gE, C: 10. mu.g PC04+ gE, D: 20. mu.g PC04+ gE, E: 40. mu.g PC04+ gE, F: 1/2 SE-10. mu.g PC04+ gE, G: 1/2 SE-20. mu.g PC04+ gE, H: 1/2 SE-40. mu.g PC04+ gE, I: 1/3 SE-10. mu.g PC04+ gE, J: 1/3 SE-20. mu.g PC04+ gE, K: 1/3 SE-40. mu.g PC04+ gE, L: gE, M: control. the bars show that the two groups of comparative differences are not statistically significant, and the bars show that the two groups of comparative differences are statistically not statistically significant: none of the mice immunized with the single SE and PC04 adjuvant gE vaccines were effective in enhancing antigen-specific Th 1-type immune responses, but the two were effective in enhancing antigen-specific Th 1-type immune responses in combination at appropriate doses, with the best combination of complex adjuvants 1/3SE +40 μ g PC04, followed by 1/2SE +40 μ g PC04, followed by 1/3SE +20 μ g PC 04. The result shows that the composite adjuvant consisting of 1/3-1/2 SE + 20-40 mu g of PC04 can effectively enhance the humoral and cellular immune response induced by the recombinant protein gE.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Beijing Enyuanhua Biotech Co., Ltd

<120> vaccine adjuvant and application thereof

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gatcgtcggg gggtcgtcgt ttttcgtgcg ttttttcgtc gttttcggcg cgcgccgggg 900

gtcaacgttg aggggggtcg tcgttttgtc gttttgtcgt tgggggacga tcgtcggggg 960

gtcgtcgttt ttcgtgcgtt ttttcgtcgt tttcggcgcg cgccggggcc cgtttaaacc 1020

cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc 1080

gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa 1140

attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac 1200

agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg 1260

gcttctactg ggcggtttta tggacagcaa gcgaaccgga attgccagct ggggcgccct 1320

ctggtaaggt tgggaagccc tgcaaagtaa actggatggc tttctcgccg ccaaggatct 1380

gatggcgcag gggatcaagc tctgatcaag agacaggatg aggatcgttt cgcatgattg 1440

aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg 1500

actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg 1560

ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg 1620

aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg 1680

ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc 1740

tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc 1800

tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc 1860

gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc 1920

aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg 1980

atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct 2040

tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt 2100

tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc 2160

tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt 2220

tcttctgaat tattaacgct tacaatttcc tgatgcggta ttttctcctt acgcatctgt 2280

gcggtatttc acaccgcata caggtggcac ttttcgggga aatgtgcgcg gaacccctat 2340

ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata 2400

aatgcttcaa taatagcacg tgctaaaact tcatttttaa tttaaaagga tctaggtgaa 2460

gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc 2520

gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat 2580

ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 2640

gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 2700

ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata 2760

cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac 2820

cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 2880

ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg 2940

tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag 3000

cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 3060

ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc 3120

aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctgggctt 3180

ttgctggcct tttgctcaca tgttctt 3207

<210> 2

<211> 3159

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag ctcgtcgttt tgtcgttttg 720

tcgtttccat gacgttcctg acgttggggg acgatcgtcg gggggggggt caacgttgag 780

gggggtcgtc gtttttcgtg cgttttttcg tcgttttcgg cgcgcgccgt cgtcgttttg 840

tcgttttgtc gtttccatga cgttcctgac gttgggggac gatcgtcggg gggggggtca 900

acgttgaggg gggtcgtcgt ttttcgtgcg ttttttcgtc gttttcggcg cgcgccgggg 960

cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt 1020

tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt cctttcctaa 1080

taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg 1140

gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggatgcg 1200

gtgggctcta tggcttctac tgggcggttt tatggacagc aagcgaaccg gaattgccag 1260

ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt aaactggatg gctttctcgc 1320

cgccaaggat ctgatggcgc aggggatcaa gctctgatca agagacagga tgaggatcgt 1380

ttcgcatgat tgaacaagat ggattgcacg caggttctcc ggccgcttgg gtggagaggc 1440

tattcggcta tgactgggca caacagacaa tcggctgctc tgatgccgcc gtgttccggc 1500

tgtcagcgca ggggcgcccg gttctttttg tcaagaccga cctgtccggt gccctgaatg 1560

aactgcaaga cgaggcagcg cggctatcgt ggctggccac gacgggcgtt ccttgcgcag 1620

ctgtgctcga cgttgtcact gaagcgggaa gggactggct gctattgggc gaagtgccgg 1680

ggcaggatct cctgtcatct caccttgctc ctgccgagaa agtatccatc atggctgatg 1740

caatgcggcg gctgcatacg cttgatccgg ctacctgccc attcgaccac caagcgaaac 1800

atcgcatcga gcgagcacgt actcggatgg aagccggtct tgtcgatcag gatgatctgg 1860

acgaagagca tcaggggctc gcgccagccg aactgttcgc caggctcaag gcgagcatgc 1920

ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg cttgccgaat atcatggtgg 1980

aaaatggccg cttttctgga ttcatcgact gtggccggct gggtgtggcg gaccgctatc 2040

aggacatagc gttggctacc cgtgatattg ctgaagagct tggcggcgaa tgggctgacc 2100

gcttcctcgt gctttacggt atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc 2160

ttcttgacga gttcttctga attattaacg cttacaattt cctgatgcgg tattttctcc 2220

ttacgcatct gtgcggtatt tcacaccgca tacaggtggc acttttcggg gaaatgtgcg 2280

cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc tcatgagaca 2340

ataaccctga taaatgcttc aataatagca cgtgctaaaa cttcattttt aatttaaaag 2400

gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc 2460

gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt 2520

tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt 2580

gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat 2640

accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga actctgtagc 2700

accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa 2760

gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg 2820

ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag 2880

atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag 2940

gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa 3000

cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt 3060

gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg 3120

gttcctgggc ttttgctggc cttttgctca catgttctt 3159

<210> 3

<211> 2540

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gactcttcgc gatgtacggg ccagatatac gcgttcgtcg ttttgtcgtt ttgtcgttgg 60

gggacgatcg tcggggggtc gtcgtttttc gtgcgttttt tcgtcgtttt cggcgcgcgc 120

cgtccatgac gttcctgacg tttcgtcgtt ttgtcgtttt gtcgttgggg gacgatcgtc 180

ggggggtcgt cgtttttcgt gcgttttttc gtcgttttcg gcgcgcgccg ggggtcaacg 240

ttgagggggg tcgtcgtttt gtcgttttgt cgttggggga cgatcgtcgg ggggtcgtcg 300

tttttcgtgc gttttttcgt cgttttcggc gcgcgccggg gcccgtttaa acccgctgat 360

cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt 420

ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat 480

cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg 540

gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttcta 600

ctgggcggtt ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa 660

ggttgggaag ccctgcaaag taaactggat ggctttctcg ccgccaagga tctgatggcg 720

caggggatca agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga 780

tggattgcac gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc 840

acaacagaca atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc 900

ggttcttttt gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc 960

gcggctatcg tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac 1020

tgaagcggga agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc 1080

tcaccttgct cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac 1140

gcttgatccg gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg 1200

tactcggatg gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct 1260

cgcgccagcc gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt 1320

cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg 1380

attcatcgac tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac 1440

ccgtgatatt gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg 1500

tatcgccgct cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg 1560

aattattaac gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat 1620

ttcacaccgc atacaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta 1680

tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt 1740

caataatagc acgtgctaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt 1800

tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac 1860

cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc 1920

ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca 1980

actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta 2040

gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct 2100

ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg 2160

gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc 2220

acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta 2280

tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg 2340

gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt 2400

cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg 2460

cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggg cttttgctgg 2520

ccttttgctc acatgttctt 2540

<210> 4

<211> 2492

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gactcttcgc gatgtacggg ccagatatac gcgttcgtcg ttttgtcgtt ttgtcgtttc 60

catgacgttc ctgacgttgg gggacgatcg tcgggggggg ggtcaacgtt gaggggggtc 120

gtcgtttttc gtgcgttttt tcgtcgtttt cggcgcgcgc cgtcgtcgtt ttgtcgtttt 180

gtcgtttcca tgacgttcct gacgttgggg gacgatcgtc gggggggggg tcaacgttga 240

ggggggtcgt cgtttttcgt gcgttttttc gtcgttttcg gcgcgcgccg gggcccgttt 300

aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct 360

cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg 420

aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc 480

aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct 540

ctatggcttc tactgggcgg ttttatggac agcaagcgaa ccggaattgc cagctggggc 600

gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct cgccgccaag 660

gatctgatgg cgcaggggat caagctctga tcaagagaca ggatgaggat cgtttcgcat 720

gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga ggctattcgg 780

ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc 840

gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga atgaactgca 900

agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg cagctgtgct 960

cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc cggggcagga 1020

tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg atgcaatgcg 1080

gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga aacatcgcat 1140

cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc tggacgaaga 1200

gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca tgcccgacgg 1260

cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg 1320

ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct atcaggacat 1380

agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg accgcttcct 1440

cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc gccttcttga 1500

cgagttcttc tgaattatta acgcttacaa tttcctgatg cggtattttc tccttacgca 1560

tctgtgcggt atttcacacc gcatacaggt ggcacttttc ggggaaatgt gcgcggaacc 1620

cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc 1680

tgataaatgc ttcaataata gcacgtgcta aaacttcatt tttaatttaa aaggatctag 1740

gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 1800

tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 1860

gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 1920

caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 1980

actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 2040

acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 2100

cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 2160

gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 2220

cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 2280

gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 2340

tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 2400

tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 2460

ggcttttgct ggccttttgc tcacatgttc tt 2492

<210> 5

<211> 2999

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

gactcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga ctagttatta 60

atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata 120

acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat 180

aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga 240

ctatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc 300

ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt 360

atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat 420

gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag 480

tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc 540

aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga 600

ggtctatata agcagagctc tctggctaac tagagaaccc actgcttact ggcttatcga 660

aattaatacg actcactata gggagaccca agctggctag cgtttaaact taagcttggt 720

accgagctcg gatccactag tccagtgtgg tggaattctg cagatatcca gcacagtggc 780

ggccgctcga gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta 840

gttgccagcc atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca 900

ctcccactgt cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc 960

attctattct ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata 1020

gcaggcatgc tggggatgcg gtgggctcta tggcttctac tgggcggttt tatggacagc 1080

aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt 1140

aaactggatg gctttctcgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca 1200

agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc 1260

ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc 1320

tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga 1380

cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac 1440

gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct 1500

gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa 1560

agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc 1620

attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct 1680

tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc 1740

caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg 1800

cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct 1860

gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct 1920

tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca 1980

gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attattaacg cttacaattt 2040

cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacaggtggc 2100

acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat 2160

atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatagca cgtgctaaaa 2220

cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa 2280

atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga 2340

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 2400

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 2460

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 2520

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 2580

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 2640

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 2700

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 2760

gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 2820

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 2880

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 2940

agcaacgcgg cctttttacg gttcctgggc ttttgctggc cttttgctca catgttctt 2999

Claims (7)

1. A plasmid is characterized in that the nucleotide sequence of the plasmid is shown as SEQ ID NO. 2 or 4.

2. A vaccine adjuvant, characterized in that it comprises the plasmid of claim 1.

3. The vaccine adjuvant according to claim 2, wherein the vaccine adjuvant further comprises an Al adjuvant or an SE adjuvant.

4. The vaccine adjuvant according to claim 3, wherein said vaccine adjuvant consists of said plasmid and said SE adjuvant;

the mass-to-volume ratio of the plasmid to the SE adjuvant is 5-200 [ mu ] g: 0.025ml to 0.1 ml.

5. Use of a vaccine adjuvant according to any one of claims 2 to 4 for the preparation of a vaccine, wherein the vaccine adjuvant is used to increase the vaccine potency.

6. The use according to claim 5, wherein the vaccine is one or more of a herpes virus vaccine, a rabies virus vaccine or a tuberculosis vaccine.

7. A vaccine comprising the vaccine adjuvant of any one of claims 2 to 4.

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