CN114437237A

CN114437237A - Staphylococcus aureus TRAP targeted recombinant protein antigen and application thereof

Info

Publication number: CN114437237A
Application number: CN202210030392.0A
Authority: CN
Inventors: 佟春玉; 宋博翠; 韩先乐; 崔玉东; 梁益民
Original assignee: Heilongjiang Bayi Agricultural University
Current assignee: Heilongjiang Bayi Agricultural University
Priority date: 2022-01-12
Filing date: 2022-01-12
Publication date: 2022-05-06
Anticipated expiration: 2042-01-12
Also published as: CN114437237B

Abstract

The invention relates to a staphylococcus aureus TRAP recombinant protein antigen of a targeted dendritic cell DEC205 receptor, and the nucleotide sequence of the antigen is shown as SEQ ID No. 1. The invention also relates to a Staphylococcus aureus TRAP recombinant protein antigen of a targeted presenting cell FC receptor, and the nucleotide sequence of the antigen is shown as SEQ ID No. 2. According to the invention, TRAP protein and receptor peptide segments of targeted antigen presenting cells are recombined together, so that the humoral immune response level of a mouse organism is enhanced, the cellular immune response level of the mouse is obviously enhanced, and the problem of intracellular infection and parasitism of staphylococcus aureus is solved; the method not only obviously reduces the immune measurement of the vaccine and improves the utilization efficiency of the vaccine, but also has the immune protection rate which is increased to 90 percent and is obviously higher than that of the non-targeted TRAP protein, thereby being beneficial to reducing the immune cost and accelerating the popularization of the vaccine for preventing the cow mastitis.

Description

Staphylococcus aureus TRAP targeted recombinant protein antigen and application thereof

Technical Field

The invention belongs to the field of biomedicine, relates to a recombinant protein, and particularly relates to a targeted recombinant protein capable of enhancing cellular immune response and anti-infection effect.

Background

Studies have shown that s.aureus can survive extracellularly and also infect cells, survive within macrophages, and require both a good humoral immune response and a strong cellular immune response to prevent. Researches prove that the antigen connected by utilizing the targeting property of the ligand can not only enhance the humoral immune response level in vivo, but also obviously enhance the cellular immune response level.

The TRAP protein is expressed in all S.aureus, no homologous protein of TRAP is found except S.aureus at present, and the TRAP protein has good immunogenicity, so the TRAP can be used as a universal anti-staphylococcus vaccine epitope. Dendritic cells are the only antigen presenting cells that can induce long-lasting T cell responses through MHC molecules and can activate Th 2-induced thymus-dependent memory B cell immune responses. DEC205 is a C-type lectin receptor on the surface of dendritic cells, and has high targeting property. F c receptors mediate antigen processing and cytokine secretion on the surface of antigen presenting cells. Therefore, it is considered that the targeting peptide of the receptor is recombined with the TRAP protein to improve the targeting of the TRAP protein, thereby improving the immune level of the TRAP protein.

Disclosure of Invention

The invention aims to provide a staphylococcus aureus TRAP recombinant protein antigen targeting a dendritic cell DEC205 receptor, and solves the problems of insufficient targeting performance and low immunity level of the TRAP protein at present.

The second purpose of the invention is to provide the application of the recombinant protein antigen.

The third purpose of the invention is to provide a staphylococcus aureus TRAP recombinant protein antigen of a targeted presenting cell FC receptor, which overcomes the problems of insufficient targeting performance and low immunity level of the prior TRAP protein.

The fourth purpose of the invention is to provide the application of the recombinant protein antigen.

The invention is realized by the following technical scheme:

the staphylococcus aureus TRAP recombinant protein antigen of a targeted dendritic cell DEC205 receptor has a nucleotide sequence shown in SEQ ID No. 1.

Furthermore, the recombinant protein antigen is prepared by passing TRAP protein and targeting scFvDEC205 gene fragment through a section of flexible protein (G)₄S)₃Are connected together to prepare the product.

And secondly, the recombinant protein antigen is applied to preparation of a vaccine for resisting staphylococcus aureus infection.

A staphylococcus aureus TRAP recombinant protein antigen of a target presenting cell FC receptor, the nucleotide sequence of which is shown as SEQ ID No.2

Furthermore, the recombinant protein antigen is prepared by passing a TRAP protein and a targeting Fc gene fragment through a section of flexible protein (G)₄S)₃Are connected together to prepare the product.

And fourthly, the recombinant protein antigen is applied to preparation of a vaccine for resisting staphylococcus aureus infection.

Adopt above-mentioned technical scheme's positive effect: according to the invention, the TRAP protein and the peptide segment of the target antigen presenting cell receptor are recombined together, so that the humoral immune response level of a mouse body is enhanced, the cellular immune response level of the mouse is obviously enhanced, and the problem of intracellular infection and parasitism of staphylococcus aureus is solved; the method not only obviously reduces the immune dose of the vaccine and improves the utilization efficiency of the vaccine, but also has the immune protection rate which is increased to 90 percent and is obviously higher than that of the non-targeted TRAP protein, thereby being beneficial to reducing the immune cost and accelerating the popularization of the vaccine for preventing the cow mastitis.

Drawings

FIG. 1 is a schematic representation of a recombinant protein expression vector plasmid;

FIG. 2 is a primary alignment of murine and bovine derived DEC205 receptors;

FIG. 3 is a secondary alignment of murine and bovine derived DEC205 receptors;

FIG. 4 is a tertiary alignment of murine and bovine derived DEC205 receptors;

FIG. 5 shows RMSD scores for murine versus bovine DEC205 receptors;

FIG. 6 is a primary alignment of murine and bovine Fc receptors;

FIG. 7 is a secondary alignment of murine and bovine derived Fc receptors;

FIG. 8 shows the restriction enzyme identification of four protein plasmids; in the figure, M lanes: marker; 1: an empty plasmid; 2: carrying out double digestion on empty plasmids; 3: performing single enzyme digestion on the empty plasmid; 4: a TRAP-containing plasmid; 5: double digestion of TRAP-containing plasmid; 6: single enzyme digestion of TRAP-containing plasmid; 7: T-DEC 205-containing plasmid; 8: carrying out double digestion on plasmids containing T-DEC 205; 9: carrying out single enzyme digestion on plasmid containing T-DEC 205; 10: a T-FcR-containing plasmid; 11: carrying out double digestion on plasmid containing T-FcR; 12: carrying out single enzyme digestion on plasmid containing T-FcR;

FIG. 9 is a soluble assay of four proteins; in the figure, 1: carrying out ultrasonic treatment on the label bacteria; 2: centrifuging the labeled bacteria and then obtaining supernatant; 3: centrifuging the labeled bacteria and precipitating; 4: carrying out ultrasonic treatment on TRAP bacteria; 5: supernatant after centrifugation of TRAP bacteria; 6: centrifuging TRAP bacteria and precipitating; 7: centrifuging T-DEC205 strain; 8: centrifuging T-DEC205 strain to obtain supernatant; 9: centrifuging T-DEC205 strain and precipitating; 10: centrifuging T-FcR bacteria; 11: supernatant after centrifugation of T-FcR bacteria; 12: centrifuging T-FcR bacteria and precipitating;

FIG. 10 is a graph showing the results of purification of four proteins; in the figure, 1: before ultrasonic purification of pET-32 a; 2: pET-32a protein holothallus; 3: after pET-32a protein purification; 4: before ultrasonic purification of TRAP protein; 5: TRAP protein whole thallus; 6: after purifying TRAP protein; 7: T-DEC205 protein holothallus; 8: before ultrasonic purification of T-DEC205 protein; 9: after the T-DEC205 protein is purified; 10: T-FcR protein holothallus; 11: before T-FcR protein ultrasonic purification; 12: after purification of the T-FcR protein;

FIG. 11 is the WesternBlot results; in the figure, left: m: marker; 1: pET-32a protein; 2: a Trap protein; and (3) right: m: marker; 1: T-DEC205 protein; 2: a T-FcR protein;

FIG. 12 is detection of mRNA in spleen;

FIG. 13 is the transcription level of individual cytokine mRNA in spleen tissue;

FIG. 14 is the antigen-induced expression levels of specific cytokines;

FIG. 15 is the level of specific antibody induced by the antigen;

FIG. 16 is a graph of antigen-induced antibody subclass results;

FIG. 17 shows the results of antigen-induced proliferation of splenic lymphocytes;

FIG. 18 is a graph showing antigen-induced mouse cytokine expression levels;

FIG. 19 is the antigen-induced levels of specific IgA and IgG antibody subclasses;

FIG. 20 is bacterial load in the organ of immunized mice;

FIG. 21 is a pathological tissue section of each organ of a mouse after challenge;

figure 22 is survival of mice after challenge.

Detailed Description

The source of the biomaterial of the invention;

1. the designed primer and the recombination sequence are both synthesized in Shanghai.

The technical solution of the present invention is further described below with reference to specific examples, but the present invention should not be construed as being limited thereto:

example 1

This example is the construction and prokaryotic expression of TRAP antigen vector targeting DC

1. Alignment of murine and bovine DEC205 receptor protein structures

The murine DEC205 receptor structure and the Fc receptor structure, the bovine DEC205 receptor structure and the Fc receptor structure are found by consulting the literature and NCBI, and the primary structures of the proteins are compared on line through the Blast function (https:// Blast. NCBI. nlm. nih. gov/Blast. cgi) of NCBI, as shown in FIG. 2, the homology is 85%; using an online comparison website PROMALS3D (http/PROMata. swmed. edu/PROMALS3d/PROMALS3d. php), the secondary structure of the protein was compared, as shown in FIG. 3, and the folding pattern of the protein was highly similar; the tertiary structure of the proteins was compared using the RCSBPDB database (http:// www.rcsb.org /) and PyMOLWin software, as shown in FIG. 4, and the degree of overlap between the two proteins was very high. As can be seen in fig. 5, the overall RMSD score is only 1.41. In summary, two proteins are highly similar from the respective structures of the two proteins, and therefore, a single chain antibody targeting the murine DEC205 receptor may also target the bovine DEC205 receptor. The primary comparison result of the Fc receptor proteins is shown in fig. 6, and the primary comparison result is shown in fig. 7, which indicates that the primary structure of the murine Fc receptor protein has a structural similarity of up to about 75% with the structure of the bovine Fc receptor protein, and the secondary structure comparison shows that the murine Fc receptor protein and the bovine Fc receptor protein have highly similar protein folding structures. Thus, single chain antibodies targeting murine Fc receptors may also target bovine Fc receptors.

Construction of T-DEC205 and T-FcR plasmids

The full length of TRAP gene (GenBank ID: GU475118.1), scFvDEC205 gene (GenBank ID: ACB88023.1) and Fc gene (GenBank ID: X62916) fragments are obtained through reference of literature and Genebank respectively, and all the gene fragments are subjected to codon optimization by using the codon bias of Escherichia coli BL21(DE3) host bacteria so as to increase the yield of expressed protein. TRAP gene fragment, scFvDEC205 and Fc gene fragment are respectively connected by using flexible protein linker (GGGGS) and named as T-DEC205 and T-FcR, BamHI and Xhol cleavage sites are respectively added at two ends, and the TRAP gene fragment, the scFvDEC205 and the Fc gene fragment are cloned on pET-32a plasmid as shown in a figure 1 and are synthesized by Shanghai worker.

3. Restriction enzyme identification of recombinant plasmid

Activating a Top10 bacterial solution containing vectors T-DEC205 and T-FcR plasmid by adopting a scribing mode, carrying out liquid culture, extracting the plasmid according to the specification of a plasmid extraction kit, quantifying the plasmid, putting 1 mu g of plasmid DNA into a centrifuge tube, adding 1 mu LBamHI enzyme, 1 mu LXHol enzyme, 2 mu LCutsmartBuffer and supplementing ddH 2O-20 mu L; carrying out warm bath at 37 ℃ for 60min, and carrying out electrophoresis detection. The results are shown in FIG. 8, bands are generated at 1248bp and 1176bp respectively by T-DEC205 and T-FcR, and the sizes of the bands are the same as those of the empty vector band after double digestion, which shows that the T-DEC205 and T-FcR prokaryotic expression vector are successfully constructed, and then the extracted plasmid is transformed into BL21 bacteria for later use.

4. Solubility detection of targeted recombinant proteins

Activated recombinant bacteria of BL21(DE3) of pET-32a, TRAP, T-DEC205 and T-FcR were inoculated into LB liquid medium containing Amp + resistance at a ratio of 1: 100, and shake-cultured at 37 ℃ and 200rpm until the OD600 of the bacterial liquid was about 0.7. The recombinant protein was expressed by IPTG-induced bacterial solution, and SDS-PAGE electrophoresis was performed after cell disruption by ultrasonication, the results are shown in FIG. 9: the bands of the pET-32a label protein and the TRAP protein in the supernatant at the positions of 22kDa and 35kDa are obviously larger than those in the precipitate, which indicates that the pET-32a protein and the TRAP protein are both soluble proteins; the T-DEC205 protein is in contrast to the T-FcR protein, and the bands of protein in the pellet at 67kDa and 68kDa, respectively, are significantly larger than in the supernatant, indicating that both proteins are insoluble proteins.

5. Expression purification of targeted recombinant proteins

The strains capable of correctly expressing the four proteins of pET-32a, TRAP, T-DEC205 and T-FcR are reactivated, amplified, subjected to induced expression ultrasound, centrifuged, and purified by using a His tag purification column, and the results are shown in FIG. 10, wherein the four proteins have clear bands and no other miscellaneous bands at the positions with expected sizes.

6. Verification of immunogenicity of recombinant proteins

And performing SDS-PAGE electrophoresis on 5 mu L of the protein to verify whether a hybrid band is generated on the protein, performing WesternBlot experiment on another 5 mu L of protein solution after SDS-PAGE electrophoresis to verify the immunogenicity of the protein, and finally, generating obviously visible single bands at 35kDa, 67kDa and 68kDa of the protein respectively, wherein the results are consistent with the expectation in figure 11 and show that the recombinant protein is successfully expressed and has good immunogenicity.

In conclusion, the structural comparison of the DEC205 receptor and Fc gene fragment of murine and bovine origin is carried out in the experiment, and the result shows that the two groups of proteins have high similarity and the key binding proteins are the same, so that the experimental result on the mouse model is also applicable to cattle. The TRAP gene is respectively connected with the scFvDEC205 gene fragment and the Fc gene fragment through a linker to construct two recombinant plasmids, and two strains of recombinant proteins are expressed after induction. Purifying the protein by using a His tag protein purification column to obtain pure tag proteins pET-32a, TRAP protein, T-DEC205 protein and T-FcR protein.

Example 2

This example illustrates the optimization of T-DEC205 and T-FcR immunization conditions

1. Preparation of antigens

Respectively adjusting the concentrations of the purified pET-32a, TRAP, T-DEC205 and T-FcR proteins to 1mg/mL, 0.5mg/mL, 0.1mg/mL, 0.01mg/mL and 0.001mg/mL, uniformly mixing the purified pET-32a, TRAP, T-DEC205 and T-FcR proteins with Freund's complete adjuvant at a ratio of 1: 1, fully emulsifying by using an emulsifying instrument, and then keeping the mixture at 4 ℃ for later use.

2. Immunization of mice

Female BALB/c mice, 8 weeks old, were randomly divided into 20 groups of 4 mice each. The prepared pET-32a, TRAP, T-DEC205 and T-FcR protein antigens are used for primary immunization of mice by means of intramuscular injection of the rear legs, and the inoculation dose is 100 mu L per mouse.

3. Isolation of splenic lymphocytes from mice

Separating spleen of 3 mice in each group, preparing 10mL spleen cell suspension by using 1640 culture medium, inoculating 1mL erythrocyte lysate to resuspend cell sediment after centrifugation, centrifuging, discarding supernatant, washing three times, discarding supernatant for the last time, suspending cells by using complete culture medium, diluting the cells by multiple ratios, counting, and adding complete culture medium to dilute lymphocytes into 1 x10⁶The volume is/mL for standby.

4. Detection of cytokine mRNA transcript levels in Whole splenocytes

4.1 design of primers:

reference to Genbank and related literature; the nucleotide sequences of the existing mouse INF-. gamma.IL-4, IL-6, IL-10, IL-17a and mouse reference gene (. beta. -actin) were searched, and primers were designed using Primer5, with the results shown in Table 1.

4.2 extraction of Total RNA:

in order to preliminarily know the immune effect of the protein antigen, mice are immunized by intramuscular injection with the same dose of the protein antigens with different concentrations, 3 mice are randomly selected from each group after immunization for 6h, splenocytes are taken, the total RNA of tissues is extracted by using a Trizol method, the quality of RNA extraction is determined by agarose gel electrophoresis of the randomly extracted RNA, and as shown in figure 12, three bands are clear and have no other miscellaneous bands, which indicates that the RNA is successfully extracted and has better quality, and the method can be used for the next experiment.

4.3 Reverse Transcription (RT)

Removing a genome: putting 1 μ g of the total RNA extracted above into an enzyme-free centrifuge tube, placing the centrifuge tube on ice, sequentially adding 5 × Buffer1 and 1 μ L of genome to remove enzyme, adding enzyme-free double distilled water until the total volume is 10 μ L, transferring the centrifuge tube into a water bath kettle at 42 ℃ for 2min, and transferring the centrifuge tube to ice.

Reverse transcription reaction: mu.L of enzyme-free double distilled water and 4. mu.L of 5 XBuffer 2 were added to the above mixture in this order, 4. mu.L of LMix1 and 1. mu.L of reverse transcriptase were added to the above mixture in this order, the centrifuge tube was transferred to a PCR instrument, reacted at 37 ℃ for 15min, then heated to 85 ℃ for reaction for 5s, and the centrifuge tube was transferred to ice (or stored at-20 ℃).

4.4qRT-PCR detection

mu.L of the above reverse transcription product cDNA was taken into a new PCR tube, and added sequentially: 2 XMix 10. mu.L, pre-primer for each cytokine (10. mu. mol. mu.L)^-1) With the rear primer (10. mu. mol. L)^-1) Adding 0.5 μ L each, and adding enzyme-free double distilled water to make up 20 μ L. Transfer the centrifuge tube to a PCR instrument, amplification program: 30s at 94 ℃; 94 ℃ for 5 s; 61 ℃, 35 s; ct values for each cytokine were recorded for 40 cycles. By using 2^-ΔΔCtThe method calculates the relative transcription amount of the target gene in each detection sample. SI is the relative expression of the cytokine in the antigen group/the relative expression of the cytokine in the tag protein. As a result, as shown in FIG. 13, when the amount of protein injected was 50. mu.g and 10. mu.g, each cytokine in the T-DEC205 proteome was significantly higher than that in the pET-32a and TRAP groups, but the 10. mu.g proteome was significantly higher than that in the 50. mu.g proteome as seen in the cytokine IFN-. gamma.. The T-FcR protein has the advantages that the three cytokines of IL-4, IL-6 and IL-10 are increased at 50 mu g, but the increase range is small, even the cytokine IFN-gamma is lower than that of the TRAP group, and when the protein concentration is 10 mu g, the change amount of each cytokine except IL-17a is obviously increased. When the protein concentration is lower than 1 mu g, the expression of the T-DEC205 protein and the T-FcR protein is relatively increased, but the increase is smaller. The above data indicate that T-DEC205 and T-FcR proteins can more directly and rapidly induce immune response in the body than the tag protein pET-32a and TRAP proteins, and the dosage is most significant at 10 μ g.

5. Indirect ELISA for detecting cytokine expression level

In order to identify the level and type of the antigen-induced cellular immune response, specific cytokines induced by splenic lymphocytes of mice in pET-32a, TRAP, T-DEC205 and T-FcR immune groups were tested. Levels of cytokines (IFN-. gamma., IL-17a, IL-10 and IL-4) secreted by splenic lymphocytes following in vitro stimulation with TRAP proteins were detected by indirect ELISA methods. As shown in FIG. 14, the levels of each cytokine secreted from the T-DEC205 and T-FcR immune groups were significantly higher than those of the negative control group, wherein the level of each cytokine secreted from spleen lymphocytes of mice in the T-DEC205 and T-FcR immune groups was higher than that of the TRAP immune group. In conclusion, the results of cytokine detection of splenic lymphocytes of mice show that each antigen can induce specific cellular immune response, and the capacity of T-DEC205 and T-FcR to induce cellular immune response is superior to that of TRAP antigen. Meanwhile, the amount of the cytokine induced and expressed by the protein at 10 mu g is obviously higher than that of other groups.

ELISA for serum IgG and fecal IgA detection

To analyze the level of specific IgG produced by antigen-induced mice, two weeks after immunization, mouse sera were taken and assayed for IgG antibody levels using TRAP protein as the coating antigen. The specific IgG antibody level of the mouse serum of each immune group is shown in FIG. 15, the IgG antibody level of TRAP, T-DEC205 and T-FcR groups is obviously higher than that of pET-32a control group, the IgG level of the mouse serum of the T-DEC205 and T-FcR immune group is higher than that of the TRAP antigen group, and interestingly, the IgG level generated by the T-FcR immune group is higher than that of the T-DEC205 immune group and is 2 times higher than that of the TRAP immune group. At a protein dose of 10 μ g, IgG antibodies were elicited much higher than in the other groups. This shows that both the T-DEC205 protein antigen and the T-FcR protein antigen can cause good humoral immunity, and the degree is higher than the TRAP protein antigen immunity degree.

7. ELISA for detecting IgA and IgG subclasses of feces

In order to analyze the expression level of IgA and IgG subclasses generated by an antigen-induced mouse, fresh mouse excrement is taken, dissolved by PBS and centrifuged to take supernatant, the method is the same as the IgG subclass detection, the anti-subclass level of mouse serum is simultaneously researched, the mouse excrement supernatant and the blood serum are used as primary antibodies, and the IgA and IgG subclass antibodies are diluted as secondary antibodies according to the instructions. The results are shown in FIG. 16, and compared with the tag protein pET-32a protein immune group, the secretory IgA of IgA is increased in each protein immune group at a protein dose of 10 μ g, wherein the T-DEC205 protein immune group is lower than the T-FcR protein immune group, and the two groups of proteins are higher than the TRAP protein immune group, which indicates that the T-DEC205 protein group and the T-FcR protein group can better cause the body to produce IgA compared with the TRAP protein group. In the detection of the antibody subclasses, when the protein dose is 10 mu g, compared with pET-32a protein immunization, the antibody subclasses caused by the respective histone antigens are not obviously increased except the IgG2a subclass protein, and other respective antibody subclasses are all increased to different degrees, wherein the IgG1 antibody subclass of the T-DEC205 protein and T-FcR protein immunization group is obviously higher than that of the TRAP protein immunization group. When the protein concentration is lower than 1 mu g, although the immune group is increased relative to the tag protein pET-32a, the increase amplitude is not obvious.

8. Statistical analysis

Differences within the groups were expressed using mean and standard deviation, differences between different groups were analyzed for significant differences using the T test, experimental data was analyzed using originpro8.0 software, "+" indicates P < 0.05, "+" indicates P < 0.01, and "+" indicates P < 0.001.

Example 3

This example demonstrates the immune effect of targeted recombinant proteins.

1. Immunization of laboratory animals

100 female BALB/c mice, 8 weeks old, were randomly divided into four groups. After the diluted protein and complete Freund adjuvant are mixed uniformly according to the volume ratio of 1: 1, the mice are subjected to primary immunization by means of intramuscular injection of hind legs, and the inoculation dose is 100 mu L per mouse. 3 weeks after the primary immunization, the same protein and Freund's incomplete adjuvant are mixed uniformly in a volume ratio of 1: 1, and the mice are immunized secondarily in the same immunization mode. The tag protein expressed by pET-32a plasmid and Freund's adjuvant are mixed uniformly in equal volume, and the emulsified and immunized mouse is used as control.

2. Counteracting toxic pathogen of mice

At one week post booster immunization, different doses of Newman bacterial suspension were used for the same batchAnd (3) carrying out pre-challenge by intraperitoneal injection on the non-immunized mice, and calculating the maximum tolerance dose of the mice to Newman bacteria according to the death condition of the mice. Two weeks after booster immunization, mice of each group were injected with the highest tolerated dose of the s. aureus newman strain by intraperitoneal injection of the mice, with a challenge dose of 8 × 10⁸And (5) detecting the bacterial colonization condition of each tissue organ of the mouse 48 hours after the challenge. Two groups of mice were injected with the smallest lethal dose of 4 × 10 s⁹Survival of mice was recorded after challenge for CFU/mouse.

3. CCK-8 method for detecting spleen lymphocyte proliferation

After stimulating splenic lymphocytes with 1mg/ml of TRAP protein, proliferation of the cells was examined by the CCK-8 method. Compared with a tag protein pET-32a antigen group, the cell proliferation indexes of mouse lymphocytes of other three groups are obviously improved, wherein the SI values of the T-DEC205 antigen group and the T-FcR antigen group are both more than 3 and are obviously higher than the SI value of the TRAP group by 2.3, and meanwhile, the T-DEC205 antigen group is also higher than the T-FcR antigen group. This indicates that the T-DEC205, T-FcR antigen group has a higher ability to stimulate splenic lymphocyte proliferation than the TRAP protein group. The results are shown in FIG. 17. SI ═ (immunization OD value-blank OD value)/negative control OD value.

4. Indirect ELISA for detecting cytokine expression level

The level of the cell factor secreted by splenic lymphocytes of each group of immune mice after being stimulated by TRAP protein in vitro is respectively detected by using a cell factor detection kit and adopting an indirect ELISA method. The capacities of the T-DEC205 immune group and the T-FcR immune group for secreting IFN-gamma are equivalent to about 300pg, which are higher than those of the TRAP immune group and the tag protein pET-32a immune group, so that the T-DEC205 protein and the T-FcR protein induce cellular immune response to be remarkably higher than that of the TRAP protein, the secretion level of IL-10 cytokines is slightly higher than that of the TRAP immune group but is remarkably higher than that of the tag protein pET-32a immune group, and the protein immune groups induce inflammatory response of organisms. The secretion level of IL-17a and IL-4 is obviously higher than that of TRAP protein immune group, which shows that T-DEC205 protein and T-FcR protein induce humoral immune response obviously higher than that of TRAP protein. The results are shown in FIG. 18.

5. ELISA method for detecting serum IgG expression level

And (3) taking each immune group of mice 14 days after the second immunization, taking eyeballs, taking blood, collecting serum, and detecting the serum IgG antibody level of each immune group by using TRAP protein as a coating antigen. The serum IgG expression level of each group of mice is shown in Table 1, and the results show that the specific IgG production level of each group of protein immunity groups is obviously increased compared with that of the tag protein immunity group (1: 1000), wherein the specific IgG production level of the T-DEC205 protein immunity group and the specific IgG production level of the T-FcR protein immunity group are the same, and both the specific IgG production level and the specific IgG production level can reach 1: 128000 which are higher than those of the TRAP protein immunity group (1: 64000). Therefore, mice immunized by each histone can cause good humoral immunity, and the T-DEC205 protein immunization group and the T-FcR protein immunization group are higher than the TRAP protein immunization group. The results are shown in Table 2.

TABLE 2 mouse serum specific IgG antibody titers

6.ELISA method for detecting IgA and IgG subclasses of excrement

Fresh mouse feces are collected, dissolved by PBS and centrifuged to obtain supernatant, the anti-subclass level of the mouse serum after the second immunization is simultaneously researched, the mouse feces supernatant and the blood serum are used as primary antibodies, and IgA and IgG subclass antibodies are diluted as secondary antibodies according to the instructions. The results are shown in FIG. 17, and compared with the tag protein pET-32a protein immune group, IgA secreted by each protein immune group is increased, wherein the T-DEC205 protein immune group is higher than the T-FcR protein immune group, and the two groups of proteins are simultaneously higher than the TRAP protein immune group, which indicates that the T-DEC205 protein group and the T-FcR protein group can better cause the body to produce IgA compared with the TRAP protein group. In the detection of antibody subclasses, compared with pET-32a protein immunization, the antibody subclasses caused by various histone antigens are increased to different degrees except that IgG3 subclass protein is not obviously increased, wherein the IgG1 antibody of T-DEC205 and T-FcR immunization groups is obviously higher than that of TRAP immunization groups, which shows that T-DEC205 protein and T-FcR protein can induce good humoral immune response. The IgG2a antibody in the T-DEC205 and T-FcR immune group is obviously higher than that in the TRAP immune group, which indicates that the T-DEC205 protein and the T-FcR protein can induce good cellular immune response. The results are shown in FIG. 19.

7. Detection of bacterial colonization amount in organ of immunized mouse

Two weeks after the booster immunization of the mice, the mice were subjected to peritoneal challenge with the s. The results are shown in FIG. 18. The results show that S.aureus colonizes tissues and organs of mice in both the immune group and the labeled control group, but compared with the labeled protein pET-32a immune group, the bacterial colonizing amount of organs of the immune group of other proteins is obviously reduced, wherein the bacterial colonizing amount of the T-DEC205 protein and T-FcR protein immune group is lower than that of TRAP protein. Except that the bacterial colonization dose of the T-FcR protein immune group in the liver is lower than that of the T-DEC205 protein immune group, the bacterial colonization dose of the T-DEC205 protein immune group in other organs is higher than that of the T-FcR protein immune group. In general, better immune protection is obtained after various groups of antigen immunization compared with the tag protein pET-32a, wherein the immune protection effect on the body after T-DEC205 protein and T-FcR protein immunization is stronger than that of TRAP protein. The results are shown in FIG. 20.

8. Pathological tissue analysis of immunized mice

After one week of toxicity attack, the liver, spleen, lung and kidney of the mouse are taken to prepare pathological tissue sections, pathological tissues of all tissues of the mouse are analyzed by an HE staining method, the result is shown in figure 19, the blank group is all organs of the mouse without toxicity attack, the pathological tissues have complete tissue morphology, the hepatic portal vein morphology of the liver of the pET-32a immune group is very incomplete, the glomerulus of the kidney of the mouse is also seriously damaged, local adhesion of alveoli in the lung occurs, the tissue morphology is changed and damaged, and the spleen also has tissue lesion. And each group of immune groups has complete tissue structure and good form, which shows that each histone plays a good protection role on organisms. The results are shown in FIG. 21.

9. Antigen-induced immune protection in mice

Mice were challenged intraperitoneally with an absolute lethal dose of s. The results are shown in FIG. 22, the mortality rate of the mice in the tag protein pET-32a protein immunization group reaches 50% within one day after challenge, and the mortality rate reaches 90% within one week after challenge. The TRAP protein immunised group had a mortality rate of 50% within one week after challenge. The mortality rate of the T-DEC205 protein and the T-FcR protein is 20% within four days after challenge, and no mice die after 4 days after challenge. The experimental result shows that compared with a tag protein pET-32a immune group, the TRAP protein, the T-DEC205 protein and the T-FcR protein immune group can obviously enhance the immune response capability of an organism to resist S.aureus after antigen immunization, wherein the T-DEC205 protein immune group and the T-FcR protein immune group can more effectively improve the specific immune response capability of the organism compared with the TRAP protein immune group.

10. Statistical analysis

Differences within the groups were expressed as means and standard deviations, differences between different groups were analyzed for significant differences using the T test, experimental data were analyzed using originpro8.0 software, "' indicates P < 0.05," ' indicates P < 0.01, and ' indicates P < 0.001.

In conclusion, the TRAP protein and the receptor of the target antigen presenting cell are recombined together, so that the humoral immune response level of a mouse body is enhanced, the cellular immune response level of the mouse is obviously enhanced, and the problem of intracellular infection and parasitism of staphylococcus aureus is solved; the method not only obviously reduces the immune measurement of the vaccine and improves the utilization efficiency of the vaccine, but also has the immune protection rate which is increased to 90 percent and is obviously higher than that of the non-targeted TRAP protein, thereby being beneficial to reducing the immune cost and accelerating the popularization of the vaccine for preventing the cow mastitis.

Sequence listing

<110> university of eight agricultural reclamation of Heilongjiang

<120> staphylococcus aureus TRAP targeted recombinant protein antigen and application thereof

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<400> 1

ggatccgaag ttaaactgca gcagtctggc accgaagttg tgaaaccggg tgcgagcgtt 60

aaactgagct gtaaagcgag cggttacatc ttcacctcct acgatatcga ttgggttcgt 120

cagaccccgg aacagggtct ggaatggatc ggttggatct tcccaggtga aggctctacc 180

gaatacaacg aaaaattcaa aggccgtgcg accctgagcg ttgataaatc tagctctacc 240

gcgtacatgg aactgacccg tctgacctct gaagattctg cggtttactt ctgcgcgcgt 300

ggtgattact accgtcgtta cttcgatctg tggggccagg gtaccaccgt taccgtttct 360

tctggtggcg gcggttctgg cggcggtggc agcggcggtg gcggttctga catccagatg 420

acccagtctc cgagcttcct gagcaccagc ctgggtaaca gcatcaccat cacctgccac 480

gcgtcccaga acattaaagg ttggctggcg tggtaccagc agaaaagcgg taacgcgccg 540

cagctgctga tttacaaagc gtctagcctg cagtctggcg ttccgagccg tttctctggc 600

tctggctccg gtaccgatta catcttcacc atcagcaacc tgcagccgga agatatcgcg 660

acctactact gccagcacta ccagtctttc ccgtggacct tcggtggtgg taccaaactg 720

gaaattaaac gtgcggcggg tggcggtggt tctatgaaaa aactgtacac cagctacggc 780

acctacggtt tcctgcacca gatcaaaatc aacaacccga cccaccagct gttccagttc 840

tctgcttctg ataccagcgt tatcttcgaa gaaaccgatg gtgaaaccgt tctgaaatcc 900

ccgagcatct atgaagtgat caaagaaatc ggcgaattct ccgaacacca cttctactgc 960

gcgatcttca tcccgtccac cgaagatcac gcttaccagc tggaaaagaa actgatcagc 1020

gttgacgata acttccgtaa cttcggtggc ttcaaaagct accgtctgct gcgtccggct 1080

aaaggtacca cctacaaaat ctatttcggt ttcgctgatc gtcacgcgta cgaagatttc 1140

aaacagtccg atgcgttcaa cgatcacttc agcaaagatg cgctgagcca ctacttcggt 1200

agcagcggcc agcactcttc ttacttcgaa cgttacctgt acccgatcaa agaactcgag 1260

<210> 2

<211> 1188

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ggatccatga aaaaactgta cacctcttac ggtacctacg gtttcctgca ccagatcaaa 60

atcaacaacc cgacccacca gctgttccag ttcagcgcaa gcgataccag cgtgatcttc 120

gaagaaaccg atggtgaaac cgttctgaaa agcccgagca tctacgaagt gatcaaagaa 180

atcggcgaat tcagcgaaca ccacttctac tgcgcgatct tcatcccgag caccgaagat 240

cacgcatacc agctggaaaa gaaactgatt agcgttgacg ataacttccg taacttcggc 300

ggtttcaaaa gctatcgtct gctgcgtccg gcgaaaggca ccacctataa aatctacttc 360

ggcttcgctg atcgtcacgc gtacgaagat ttcaaacaga gcgatgcttt caacgaccat 420

ttcagcaaag atgcgctgag ccactacttc ggcagctccg gccagcactc cagctacttc 480

gaacgttacc tgtacccgat caaagaaggc ggcggcggta gcccgccgcc ggaactgccg 540

ggtggcccga gcgttttcat cttcccgccg aaaccgaaag acaccctgac catcagcggc 600

accccggaag ttacctgcgt ggttgtggat gtcggccacg atgatccgga agttaaattc 660

tcctggttcg ttgatgatgt tgaagttaac accgcgacca ccaaaccgcg cgaagaacag 720

ttcaacagca cctaccgtgt ggtttctgcg ctgcgtatcc agcaccagga ttggaccggt 780

ggtaaagaat tcaaatgcaa agttcacaac gaaggcctgc cggcgccgat cgttcgtacc 840

atttcccgta ccaaaggccc ggctcgtgaa ccgcaggttt acgttctggc tccgccgcag 900

gaagaactga gcaaaagcac cgtgagcctg acctgcatgg ttaccagctt ctacccggat 960

tacatcgcgg tggaatggca gcgtaacggt cagccggaaa gcgaagataa atacggcacc 1020

accccgccgc agctggatgc ggatagcagc tacttcctgt actctaaact gcgtgttgat 1080

cgtaacagct ggcaggaagg cgatacctac acctgcgttg tgatgcacga agcactgcac 1140

aaccactaca cccagaaatc tacctctaaa tctgcgggta aactcgag 1188

<210> 3

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

agttgtcatc ctgctcttct ttctc 25

<210> 4

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctcactctct gtggtgttct tcgt 24

<210> 5

<211> 12

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tagtccattt cc 12

<210> 6

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

ttggtcctta gccactcctt c 21

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gttcccctac tgtcatcccc 20

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aggcagacaa acaatacacc a 21

<210> 9

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

tttaactccc ttggcgcaaa a 21

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctttccctcc gcattgacac 20

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctgagacaat gaacgctaca cact 24

<210> 12

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cttttcttcc acatctatgc cactt 25

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgagagggaa atcgtgcgtg ac 22

<210> 14

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gctcgttgcc aatagtgatg acc 23

Claims

1. A staphylococcus aureus TRAP recombinant protein antigen of a targeted dendritic cell DEC205 receptor has a nucleotide sequence shown in SEQ ID No. 1.

2. The recombinant protein antigen of claim 1, which is prepared by passing TRAP protein and targeting scFvDEC205 gene fragment through a section of flexible protein (G)₄S)₃Are connected together to prepare the product.

3. Use of the recombinant protein antigen of claim 1 in the preparation of a vaccine against s.

4. A Staphylococcus aureus TRAP recombinant protein antigen of a target presenting cell FC receptor has a nucleotide sequence shown in SEQ ID No. 2.

5. The recombinant protein antigen as claimed in claim 4, which is prepared by passing TRAP protein and targeted Fc gene fragment through a flexible protein (G)₄S)₃Are connected together to prepare the product.

6. Use of the recombinant protein antigen of claim 4 in the preparation of a vaccine against S.