CN107502616B

CN107502616B - Soluble recombinant protein CTA-CD154 and preparation method and application thereof

Info

Publication number: CN107502616B
Application number: CN201710969969.3A
Authority: CN
Inventors: 侯立婷; 郑其升; 张元鹏; 于晓明; 陈瑾; 乔绪稳; 侯继波
Original assignee: Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu Agricultural Science and Technology Transfer Center Co.,Ltd.
Priority date: 2017-10-18
Filing date: 2017-10-18
Publication date: 2020-08-04
Anticipated expiration: 2037-10-18
Also published as: CN107502616A

Abstract

The invention discloses a soluble recombinant protein CTA-CD154, and a nucleotide sequence and an amino acid sequence thereof. The invention also discloses a preparation method of the soluble recombinant protein CTA-CD 154. The invention also discloses application of the soluble recombinant protein CTA-CD 154. The soluble recombinant protein CTA-CD154 can be used as a novel immunopotentiator, can specifically target DC cells, and can remarkably enhance the immune response of an organism, and the effect of the fused immunopotentiation CTA-CD154 is remarkably higher than that of an individual component.

Description

Soluble recombinant protein CTA-CD154 and preparation method and application thereof

Technical Field

The invention belongs to the field of biological pharmacy, and particularly relates to a soluble recombinant protein CTA-CD154, and a preparation method and application thereof.

Background

Mucosal immunity is the first line of defense for resisting invasion of pathogenic microorganisms, and enhancing mucosal immunity efficacy is one of effective ways for improving body resistance. Therefore, more and more researchers are exploring how to improve the mucosal immunity of the body, and some researchers find that Cholera Toxin (CT) has better mucosal adjuvant activity and is a multi-subunit macromolecule consisting of 1 a subunit and 5 identical B subunits. A large number of researches prove that cholera toxin A subunit (CTA) can induce an organism to generate early, efficient and lasting immune response by a small amount of antigen; the immunopotentiator can effectively enhance the mucosal immunoreaction of organisms and can obviously improve the level of IgG antibodies generated by the stimulation of related antigens. However, CTA has no function of specifically targeting DC cells as an immunopotentiator, and thus its immunopotentiating effect is limited.

Dendritic Cells (DCs) are the professional Antigen Presenting Cells (APCs) that have attracted much attention in recent years and can take up, process and present antigens to initiate T cell-mediated immune responses T lymphocytes play an important role in the activation of the immune system, and the surface receptor CD40 interacts with CD154 to promote cell-mediated immunity through the activation of Dendritic Cells and the generation of natural killer Cells CD154 is also called CD 40L, is the ligand of CD40 and is also a type II transmembrane glycoprotein, and is expressed as a key stimulatory molecule in CD4⁺T cell surface, after activation, can also be expressed on the surface of CD8T cells, B cells, macrophages, mast cells, dendritic cells. CD154 can promote T, B cell differentiation and macrophage activation after being combined with its receptor as a ligand, has the function of specifically targeting DC cells, and plays an important role in humoral and cellular immune response. Therefore, a targeted DC immunopotentiator is designed, and a new idea is provided for subsequent experimental research.

Disclosure of Invention

The technical problem is as follows: the technical problem to be solved by the invention is to provide a DNA molecule for coding a soluble recombinant protein CTA-CD 154.

The technical problem to be solved by the invention is to provide a soluble recombinant protein CTA-CD 154.

The technical problem to be solved by the invention is to provide a recombinant expression vector, a transgenic cell line or a transgenic recombinant bacterium containing the DNA molecule of the soluble recombinant protein CTA-CD 154.

The technical problem to be solved by the invention is to provide an immunopotentiator.

The invention finally aims to solve the technical problem of providing the application of the immunopotentiator in the preparation of vaccines.

The technical scheme is as follows: in order to solve the technical problems, the invention discloses a DNA molecule for coding a soluble recombinant protein CTA-CD154, which has a nucleotide sequence shown as SEQ ID NO: 1 is shown.

The invention also discloses a soluble recombinant protein CTA-CD154, the amino acid sequence of which is shown in SEQ ID NO: 2, respectively.

The invention also comprises a recombinant expression vector, a transgenic cell line or a transgenic recombinant bacterium containing the DNA molecule of the soluble recombinant protein CTA-CD 154.

Wherein, the recombinant expression vector is a recombinant expression vector which is obtained by inserting the DNA molecule into an Escherichia coli expression vector to obtain the DNA molecule containing the soluble recombinant protein CTA-CD 154.

The invention also comprises a transgenic recombinant bacterium, wherein the recombinant bacterium is obtained by introducing the recombinant expression vector into escherichia coli and screening.

The invention also relates to a preparation method of the soluble recombinant protein CTA-CD154, which comprises the following steps:

1) and (2) optimizing and synthesizing a CTA-CD154 gene sequence according to the codon preference of escherichia coli, connecting the CTA-CD154 gene sequence with a vector, and constructing a recombinant plasmid, wherein the CTA-CD154 gene sequence is shown as SEQ ID NO: 1 is shown in the specification;

2) the recombinant plasmid is transformed, induced and purified to obtain the soluble recombinant expression protein CTA-CD 154.

Specifically, the preparation method of the soluble recombinant protein CTA-CD154 comprises the following steps:

(1) according to the codon preference optimization of Escherichia coli, a CTA-CD154 gene sequence is synthesized and connected with a vector pET32a to construct a recombinant plasmid.

(2) B L21 competent cells are transformed by the recombinant plasmid, a recombinant expression plasmid pET32a-CTA-CD154/B L21 is obtained after identification, recombinant expression protein is obtained after IPTG induction, after expression, thalli are subjected to ultrasonic disruption and SDS-PAGE analysis, the recombinant protein is mainly in a soluble form, the molecular weight of the protein is about 46kDa, and Western-blot shows that a specific band capable of being combined with the His monoclonal antibody appears at the position of 46 kDa.

The invention also comprises the application of the DNA molecule, the soluble recombinant protein CTA-CD154, the recombinant expression vector, the transgenic cell line or the transgenic recombinant bacterium in the aspect of preparing the immunopotentiator.

The present disclosure also includes an immunopotentiator comprising the soluble recombinant protein CTA-CD 154.

The immunopotentiator of the invention is an immunopotentiator targeting DC cells, can target DC cells, improve the activation efficiency of the DC cells, enhance the immune response reaction of organisms to antigens, and further improve the immune efficacy of vaccines.

The invention also comprises the application of the immunopotentiator in the preparation of vaccines.

The vaccine of the invention includes but is not limited to foot-and-mouth disease inactivated vaccine, circular ring vaccine or pseudorabies vaccine. The vaccine of the present invention may also comprise other inactivated vaccines, preferably for pigs.

Has the advantages that: compared with the prior art, the invention has the following advantages: according to the invention, CTA and CD154 gene are fused and then expressed by using an escherichia coli soluble expression system. The research result proves that the soluble recombinant protein CTA-CD154 can be used as a novel immunopotentiator, can specifically target DC cells, and obviously enhances the immune response of an organism, and the effect of the fused immunopotentiation CTA-CD154 is obviously higher than that of the immunopotentiation of a single component.

Drawings

FIG. 1 shows the restriction enzyme digestion identification of recombinant plasmid; 1-standard Marker with the molecular weight of 10000bp, 2-pET32a-CTA-CD154 restriction enzyme products, 3-pET32a-CTA restriction enzyme products and 4-pET32a-CD154 restriction enzyme products;

FIG. 2 is a SDS-PAGE analysis chart of soluble recombinant proteins, 1-protein standard Marker, 2-B L21 empty bacterium, 3-CTA-CD154 whole bacterium, inclusion body after 4-CTA-CD154 induced expression, supernatant after 5-CTA-CD154 induced expression, 6-purified CTA-CD154 protein, 7-purified CTA protein, 8-purified CD154 protein;

FIG. 3 is a Western-blot identification chart of soluble recombinant proteins; 1-protein standard Marker, 2-CTA-CD154 holobacteria, inclusion body after 3-CTA-CD154 induction expression, supernatant after 4-CTA-CD154 induction expression, 5-purified CTA-CD154 protein, 6-purified CTA protein, and 7-purified CD154 protein;

FIG. 4 shows the detection result of the liquid phase blocking E L ISA antibody level after the foot and mouth disease vaccine immunization;

FIG. 5 is a graph of the proportion of CD11C in DC cells after immunization with the foot and mouth disease vaccine;

FIG. 6 is a graph of the proportion of CD11C + CTA-CD154 in DC cells after immunization with a foot and mouth disease vaccine;

FIG. 7 shows the result of detecting E L ISA antibody after circular vaccine immunization;

FIG. 8, a graph of the proportion of CD11C in DC cells after immunization with the circular ring vaccine;

FIG. 9 is a graph of the proportion of CD11C + CTA-CD154 in DC cells after immunization with the circular vaccine;

FIG. 10, E L ISA antibody detection results after pseudorabies vaccine immunization;

FIG. 11, a graph of the proportion of CD11C in DC cells after pseudorabies vaccine immunization;

FIG. 12, a graph of the proportion of CD11C + CTA-CD154 in DC cells after pseudorabies vaccine immunization.

Detailed Description

To further illustrate the details of the present invention, several examples are set forth below, but the present invention should not be limited thereto.

The reagents used in the invention are DNA Marker, restriction enzymes Nde I and Xho I which are purchased from Dalibao biology, DH5 α and B L21 competent cells which are purchased from Beijing Quanjin biotechnology, Inc., protein molecular weight Marker and BCA kit are products of Thermo Fisher Scientific, PVDF membrane is a product of Millipore, DAB color developing solution and HIS monoclonal antibody are purchased from Nanjing Shengxing biotechnology, Inc.

EXAMPLE 1 preparation of immunopotentiator CTA-CD154

1. Cloning of CTA-CD154 target Gene

According to the gene sequences of CTA (accession number D30053.1) and CD154 (accession number HQ110108.1) registered by GenBank, designing a target gene sequence, respectively naming the target gene sequence as CTA, CD154 and CTA-CD154, and after optimizing according to the codon preference of escherichia coli, adding Nde I and Xho I enzyme cutting sites at two ends of the sequence, entrusting Nanjing Jinslei Biotech limited company to clone into a pUC57 vector to form recombinant cloning vectors pUC57-CTA, pUC57-CD154 and pUC57-CTA-CD 154.

2. Construction of recombinant plasmid

pUC57-CTA, pUC57-CD154, pUC57-CTA-CD154 and the recombinant prokaryotic expression vector pET32a were subjected to double digestion and ligation with Nde I and Xho I, respectively. The plasmid extracted from the transformed bacteria is subjected to enzyme digestion identification, and the band size of CTA is about 774bp, the band size of CD154 is about 474bp, and the band size of CTA-CD154 is about 1248bp through 1% agarose gel electrophoresis detection. The correctly identified recombinant expression plasmids were designated pET32a-CTA, pET32a-CD154, and pET32a-CTA-CD154, respectively. The cleavage identification map is shown in FIG. 1.

3. Expression of the protein of interest

Recombinant plasmids pET32a-CTA, pET32a-CD154 and pET32a-CTA-CD154 were transformed into E.coli B L21, respectively, ampicillin-containing L B plates were coated, single colonies were picked to obtain recombinant bacteria pET32a-CTA/B L21, pET32a-CD154/B L21 and pET32a-CTA-CD154/B L21, and empty plasmid pET32a was transformed into competent E.coli B L21 in the same manner to obtain control strains pET32a/B L21.

Recombinant bacteria pET32a-CTA/B L21, pET32a-CD154/B L21, pET32a-CTA-CD154/B L21 and pET32a/B L21 are selected as single colonies, the single colonies are respectively inoculated into L B liquid culture medium (containing ampicillin and chloramphenicol) of 5m L, the single colonies are subjected to shaking culture at 37 ℃ overnight, the mother liquor is transferred to new L B liquid culture medium (containing ampicillin and chloramphenicol) according to the ratio of 1:100 the next day, the mother liquor is subjected to shaking culture for 1.5 to 2h (OD600 reaches 0.5 to 1) at 37 ℃ at 220r/min, the mother liquor is added with IPTG with the final concentration of 0.4 mmol/L, the single colonies are induced for 24h at 15 ℃, the bacteria are harvested by centrifugation, the bacterial precipitates are collected and are respectively subjected to resuspension by 1m L buffer solution, ultrasonic lysis is carried out in an ice bath, the complete induced supernatant and the samples and the precipitates are subjected to SDS-electrophoresis at 28kDa, 18kDa and the sizes of PBS (see the expected target bands of the kDa).

And detecting the processed sample by using Western-blot, and transferring the protein on the separation gel to a PVDF membrane after carrying out SDS-PAGE on the sample. The transferred membrane was taken out and placed in TBST containing 5% skimmed milk powder in a blocking solution at 4 ℃ overnight. Adding His monoclonal antibody diluted by 1:2000, incubating at 37 deg.C for 1h, washing with TBST for 5 times and 5 min/time, adding HRP-labeled goat anti-mouse IgG diluted by 1:3000, incubating at 37 deg.C for 1h, washing with TBST for 5 times and 5 min/time, and performing DAB color development. Western-blot confirmed the presence of specific bands capable of binding to His mAb at 46kDa, 28kDa and 18kDa, respectively (see FIG. 3).

4. Protein purification

The recombinant proteins pET32a-CTA/B L, pET32a-CD154/B L and pET32a-CTA-CD154/B L are expressed in a large amount according to the method, thalli precipitates are collected by centrifugation, soluble proteins are obtained after repeated freezing and thawing and ultrasonic treatment, the proteins are purified by using a Ni-Charged Resin affinity chromatography column, the specific operation steps are carried out according to the instruction, the purified proteins are concentrated by an ultrafiltration concentration tube for 3 times, and then are subjected to salt removal, and are verified by SDS-PAGE and Western-blot, the concentrations of the purified proteins are determined by using a BCA kit, the concentration of the purified CTA-CD154 recombinant protein is determined to be 1.2mg/m L (see the 6 th lane in FIG. 2 and the 5 th lane in FIG. 3), the concentration of the purified CTA-CD154 protein is determined to be 1.35mg/m L (see the 7 th lane in FIG. 2 and the 6 th lane in FIG. 3), and the concentration of the purified CD154 protein is determined to be 1.25mg/m (see the 7 th lane in FIG. 3 and the lane in the immune enhancer in the lane 3).

Example 2 Effect of immunopotentiator CTA-CD154 on the immunopotency of inactivated vaccine for foot-and-mouth disease

1. Experimental Material

Inactivated O-type Foot and Mouth Disease Virus (FMDV) of pigs, the 146s content of which is 6 mu g/m L, is a premium from the inner Mongolia Jinyu group.

4-5 week old ICR mice were purchased from Yangzhou university center of comparative medicine and had fluid phase blocking E L ISA antibody titers of no greater than 1: 4.

The O-type foot-and-mouth disease liquid phase blocking E L ISA antibody detection kit is purchased from Lanzhou veterinary research institute.

GM-CSF, I L-4, was purchased from Gibco.

FITC-labeled anti-mouse CD11C mAb was purchased from Nanjing Automation Biotechnology, Inc.

FITC-CTA-CD154 was labeled by this laboratory preparation.

CD154 protein, CTA protein were provided by the laboratory (adjusted concentrations during vaccine production were consistent with the concentration of the immunopotentiator CTA-CD 154).

Adjuvant ISA206 was purchased from france, saibec.

2. Vaccine formulation

Mixing immunopotentiators CTA, CD154 and CTA-CD154 with inactivated FMDV according to the dose of 50 mu g/head part respectively to obtain an aqueous phase solution, mixing the aqueous phase solution with adjuvant ISA206 according to the volume ratio of 1:1, and emulsifying by using an emulsifying instrument to obtain the foot-and-mouth disease inactivated vaccine containing the immunopotentiators CTA, CD154 and CTA-CD 154.

3. Grouping, immunization and post-immunization correlation detection

3.1 grouping and immunization of test mice

50 ICR mice of 5 weeks old are purchased from the comparative medicine center of Yangzhou university and are randomly divided into 5 groups, each group is 10 and respectively marked as G1, G2, G3, G4 and G5, the G1 group is FMDV + CTA-CD154 group, the G2 group is FMDV + CTA group, the G3 group is FMDV + CD154 group, the G4 group is FMDV control group, the G5 group is PBS control group, the immunization dose of each mouse is 0.25m L/mouse, the boosting immunization is carried out once after 21d, and sera are respectively collected at 14d, 21d, 35d, 49d, 56d and 63d and used for detecting E L ISA antibodies, which is specifically shown in Table 1.

TABLE 1 mice grouping and immunization

3.2 post-immunization liquid phase blocking antibody detection

Blood was collected at 14d, 21d, 35d, 49d, 56d, and 63d after immunization, and serum was separated and subjected to liquid phase blocking antibody detection.

The detection results of the liquid blocking antibodies of each immunization group are shown in fig. 4, and the liquid blocking antibodies of each immunization group are in a rising trend at 35d after immunization, except for the PBS blank control group. Although the liquid phase blocking antibody of the FMDV control group increased, a significant decrease in the post-immunization 35d appeared. The antibody levels of the FMDV + CTA group and FMDV + CD154 group increased by one titer compared to the FMDV control group, and decreased slowly for the entire duration. The results show that the CTA and the CD154 alone can improve the immune response of the test mice to the antigen and have the immune enhancement effect. The antibody level of the FMDV + CTA-CD154 group was significantly higher than that of the FMDV control group, and the antibody titers of the two groups differed by 2 days after immunization³. The difference in antibody titer was 2 between the FMDV + CTA group and the FMDV + CD154 group². The result shows that the immunopotentiator CTA-CD154 can obviously improve the antibody level of a test mouse, has better immunopotentiation effect, and the immunopotentiation effect is obviously higher than that of FMDV + CTA and FMDV + CD154 immune groups.

3.3 detection of Targeted DC cells

Aseptically collecting mouse bone marrow cells, stimulating with GM-CSF and I L-4, culturing for 7 days to obtain enriched mouse bone marrow-derived dendritic cells, adjusting cell density to 1 × 10⁶The detection results are shown in fig. 5 and 6, the ratio of CD11C DC cells in an FMDV + CTA-CD154 immune group is 34.7%, the ratio of CD11C DC cells in an FMDV + CD154 immune group is 15.3%, but the ratio of CD11C + CTA-CD154 cells in an FMDV + CD154 immune group is 21.5%, while the ratio of CD11C + CD154 cells in an FMDV + CD154 immune group is 4%, compared with the two groups, the difference is very significant (P5639 + CTA-CD154 cells in the two groups are very significant (P-CTA is significant) (P-CTA-CD 154 immune group is 21.5%)<0.05). This result indicates that the immunopotentiator CTA-CD154 can specifically expressTargeting DC cells, while CD154 alone also has the function of targeting DC cells, but the targeting ability is weak.

In this embodiment, the volume of the immunopotentiator used may also be adjusted according to actual needs, which is not specifically listed here.

Example 3 Effect of the immunopotentiator CTA-CD154 on the immunopotency of a commercial porcine circovirus type 2 (PCV2) vaccine 1, Experimental materials

Porcine circovirus type 2 inactivated vaccine (DBN-SX07 strain) was purchased from Duty Bang.

ICR mice, 4-5 weeks old, were purchased from the university of Yangzhou comparative medicine center.

The porcine circovirus type 2E L ISA antibody detection kit is purchased from Wuhan Keshi Probiotics GmbH.

2. Vaccine formulation

Mixing immunopotentiators CTA, CD154 and CTA-CD154 with commercial porcine circovirus type 2 inactivated vaccine (DBN-SX07 strain) according to the dose of 50 mu g/head part respectively to obtain the circular vaccine containing the immunopotentiators CTA, CD154 and CTA-CD 154.

3. Grouping, immunization and post-immunization correlation detection

3.1 grouping and immunization of test mice

50 ICR mice of 5 weeks old are purchased from the comparative medicine center of Yangzhou university and are randomly divided into 5 groups, each group is 10 and respectively marked as Z1, Z2, Z3, Z4 and Z5, the Z1 group is PCV2+ CTA-CD154 group, the Z2 group is PCV2+ CTA group, the Z3 group is PCV2+ CD154 group, the Z4 group is PCV2 control group, the Z5 group is PBS control group, the immune dose of each mouse is 0.2m L/mouse, the mice are boosted once after 14d, and serum is respectively collected at 14d and 35d for detecting E L ISA antibodies, which is specifically shown in Table 2.

TABLE 2 grouping and immunization of mice

3.2 post-immunization detection of circular specific antibodies

Blood was collected at 14d and 35d after immunization, and serum was separated and subjected to PCV 2-specific antibody detection. The specific operation is carried out according to the kit instructions.

After the serum of each immune group is diluted by 400 times, the antibody detection result is shown in fig. 7, the serum collected by the prime immune group at 14d is used for detecting PCV2 specific antibody, the antibody level of the PCV2+ CTA-CD154 immune group is higher than that of the other immune groups, but the difference is not significant, after the boost immunity, the antibody level of the mouse PCV2 specific antibody is significantly increased, and the antibody level of the PCV2+ CTA-CD154 immune group is significantly higher than that of the PCV2+ CTA immune group, PCV2+ CD154 immune group and PCV2 commercial vaccine control group, and the difference is significant (p < 0.05). The detection of the PCV 2-specific antibody, which is a PBS negative control, was negative in the test. Experiments show that the immunopotentiator CTA + CD154 can obviously improve the immune efficacy of PCV2 inactivated vaccine and has good immune enhancement effect.

3.3 detection of Targeted DC cells

Aseptically collecting mouse bone marrow cells at 21d after immunization, stimulating with GM-CSF and I L-4, culturing for 7 days to obtain enriched mouse bone marrow-derived dendritic cells, and adjusting cell density to 1 × 10⁶And m/m L, adding an anti-mouse CD11C monoclonal antibody labeled by FITC with a final concentration of 5 mu g/m L FITC and FITC-CTA-CD154, incubating for three hours, washing with PBS for 3 times, adding 300 mu L1% paraformaldehyde, transferring to a flow-type loading tube, and performing FACS (fluorescence detection) to detect the positive proportion of DC cells in each tube, wherein the detection result is shown in figures 8 and 9, the proportion of CD11C DC cells in a PCV2+ CTA-CD154 immune group is 18.5%, and the difference is very obvious compared with other immune groups (P is P) (the P is P<0.05). The proportion of CD11C + CTA-CD154DC cells in PCV2+ CTA-CD154 immune group is 10.98%, and the difference is very significant compared with other immune groups (P<0.05). The results indicate that the immunopotentiator CTA-CD154, after immunization with the circular ring vaccine, is able to specifically target DC cells.

Example 4 Effect of the immunopotentiator CTA-CD154 on the immunopotency of porcine Pseudorabies (PRV) vaccine

1. Experimental Material

Pseudorabies vaccine (pseudorabies gene deletion vaccine) was purchased from northern university group.

The PRV gB E L ISA kit is purchased from Biochek, the Netherlands.

2. Vaccine formulation

Mixing immunopotentiators CTA, CD154 and CTA-CD154 with commercial pseudorabies vaccine according to the dose of 50 mu g/head part respectively to obtain the pseudorabies vaccine containing the immunopotentiators CTA, CD154 and CTA-CD 154.

3. Grouping, immunization and post-immunization correlation detection

3.1 grouping and immunization of test mice

50 ICR mice of 5 weeks old are purchased from the comparative medicine center of Yangzhou university and are randomly divided into 5 groups, each group is 10 and respectively marked as P1, P2, P3, P4 and P5, the P1 group is a PRV + CTA-CD154 group, the P2 group is a PRV + CTA group, the P3 group is a PRV + CD154 group, the P4 group is a PRV control group, the P5 group is a PBS control group, the immune dose of each mouse is 0.2m L/mouse, and serum is collected at 21d for detecting E L ISA antibodies, which is concretely shown in Table 3.

TABLE 3 mice grouping and immunization

3.2 post-immunization Pseudorabies-specific antibody detection

Blood was collected at 21d after immunization, and serum was separated and subjected to PRV-specific antibody detection. The specific operation is carried out according to the kit instructions.

Each immunization group serum was administered as 1: the detection is carried out after 10-fold dilution, the detection result of the antibody is shown in figure 10, the serum is collected 3 weeks after immunization for PRV specific antibody detection, the antibody level of a PRV + CTA immune group and a PRV + CD154 immune group is higher than that of a PRV commercial vaccine control group, and the difference is not obvious. Compared with the other three immune groups, the PRV + CTA-CD154 immune group has obviously higher antibody level, obvious difference and better antibody detection uniformity. The test result of the PBS control group in the test is negative. Experiments show that the immunopotentiator CTA-CD154 can obviously improve the immune efficacy of PRV attenuated vaccines and has good immune enhancement effect.

3.3 detection of Targeted DC cells

Sterile collection of postexempt 21d is littleStimulating mouse bone marrow cells with GM-CSF and I L-4, culturing for 7 days to obtain enriched mouse bone marrow-derived dendritic cells, and adjusting cell density to 1 × 10⁶The detection result is shown in figure 11 and figure 12, the proportion of CD11C DC cells of a PRV + CTA-CD154 immune group is 13.54%, the proportion of CD11C DC cells of a PRV + CD154 immune group is 4.05%, but the proportion of CD11C + CTA-CD154 cells of a PRV + CTA-CD154 immune group is 8.54%, and the proportion of CD C + CTA-CD154 cells of a PRV + CD154 immune group is 2.05%, compared with other immune groups, the difference is remarkable, and the result shows that the DC cells can be specifically targeted by the special enhancer CTA-CD154 after the immune enhancer CTA-CD154 vaccine is added in the pseudorabies.

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325 330 335

Ala Asn Thr His Ser Ser Ser Lys Pro Cys Gly Gln Gln Ser Ile His

340 345 350

Leu Gly Gly Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn

355 360 365

Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe

370 375 380

Gly Leu Leu Lys Leu His His His His His His

385 390 395

Claims

1. A DNA molecule for encoding the soluble recombinant protein CTA-CD154, having the nucleotide sequence set forth in SEQ ID NO: 1 is shown.

2. A soluble recombinant protein CTA-CD154, the amino acid sequence of which is shown in SEQ ID NO: 2, respectively.

3. A recombinant expression vector or a transgenic recombinant bacterium comprising the DNA molecule encoding the soluble recombinant protein CTA-CD154 according to claim 1.

4. The recombinant expression vector of claim 3, wherein the DNA molecule of claim 1 is inserted into an E.coli expression vector to obtain a recombinant expression vector comprising a DNA molecule encoding the soluble recombinant protein CTA-CD 154.

5. A transgenic recombinant bacterium obtained by introducing the recombinant expression vector of claim 3 or 4 into Escherichia coli and screening the recombinant bacterium.

6. The method of producing the soluble recombinant protein CTA-CD154 as claimed in claim 2, comprising the steps of:

1) a CTA-CD154 gene sequence is synthesized according to the codon preference optimization of Escherichia coli, and is connected with a vector to construct a recombinant plasmid, wherein the CTA-CD154 gene sequence is shown in SEQ ID NO: 1 is shown in the specification;

7. Use of the DNA molecule of claim 1, the soluble recombinant protein CTA-CD154 of claim 2, the recombinant expression vector of claim 3, or the transgenic recombinant bacterium for the preparation of an immunopotentiator.

8. An immunopotentiator comprising the soluble recombinant protein CTA-CD154 according to claim 2.

9. Use of an immunopotentiator according to claim 8 for the preparation of a vaccine.

10. The use according to claim 9, wherein the vaccine is an inactivated foot and mouth disease vaccine, a circovirus vaccine or a pseudorabies vaccine.