CN113388012A - Application of Uu-DnaJ protein and vaccine for resisting Uu infection - Google Patents

Abstract

The invention relates to the field of biotechnology, and discloses the application of Uu-DnaJ protein and a vaccine against Uu infection. The present invention provides a new application of Uu-DnaJ protein and its T/B cell dominant epitope amino acid fragment in the preparation of Uu immunogen or vaccine against Uu infection. Relevant experimental results show that Uu-DnaJ protein can be infected by Uu-infected small cells. Recognized by mouse serum, it can produce specific antibody response after immunization, and the antibody titer is as high as 1:640000; at the same time, after immunizing mice with Uu‑DnaJ, it stimulates the proliferation of spleen lymphocytes to increase, and induces transition from IgG1 to protective IgG2a subtype antibody response and Th1-type cellular immune response, thereby reducing the amount of Uu colonization, inflammatory response and lesions in the cervix of mice, fully demonstrated immune protection. Various results prove that Uu‑DnaJ protein has the potential to develop vaccines against Uu infection.

Description

Application of Uu-DnaJ protein and vaccine for resisting Uu infection

Technical Field

The invention relates to the technical field of biology, in particular to application of Uu-DnaJ protein and a vaccine for resisting Uu infection.

Background

Ureaplasma urealyticum (Uu) is a pathogenic bacterium which is first isolated from the urethra of a patient with nongonococcal urethritis by Shepard in 1954, and the formed colony tiny is also called as 'T strain' mycoplasma. Uu is closely related to human urogenital infections and is the smallest of the prokaryotic microorganisms found to date that have the ability to self-replicate in inanimate media. Although it evolved from a gram-positive ancestor, it did not have a cell wall and belongs to the order of mycoplasma of the mollicutes. When growing in urea-containing liquid mycoplasma culture medium, Uu can decompose urea to produce ammonia, change the environmental pH value and further discolor the indicator. Studies have shown that Uu is a low virulence symbiont that colonizes the adult urogenital tract and can be transmitted by sexual contact, often in the lower urogenital tract of sexually active women, with a worldwide incidence of between 40% and 80%. Uu first attaches to the surface of epithelial cells of the respiratory or genitourinary tract and colonizes them, and produces many toxic substances, such as urease, IgA protease and phospholipase, which undoubtedly increase the risk of some diseases of the genital tract, such as vaginosis, urethritis, cervicitis, pelvic inflammation, etc.

Although the pathogenic role of Uu in humans remains controversial as a common asymptomatic mycoplasma in humans, with the continuous and intensive knowledge of the biological group and serotype of Uu, researchers speculate that the pathogenicity of Uu may be related to its diverse biological groups and serotypes. Ureaplasma consists of two biological types and 14 serotypes, wherein the biological 1 group (or Parvo) is micro ureaplasma (U.parvum, UP) and comprises genotype 1 (serotype 1), genotype 3 (serotype 3 and 14) and genotype 6 (serotype 6); meanwhile, organism group 2 (or T960) is ureaplasma urealyticum (Uu, which is expressed as Uu in the present invention), and includes genotype 4 ( sera 4, 7, 10, 11, 12 and 13) and genotype 8 ( sera 2, 5, 8 and 9), and most of them are serological type mixed infections. Uu is mainly typed according to surface antigen, namely multi-band antigen (MBA), and the lengths of MBAs of all serotypes are not consistent, so that MBA is also one of the factors causing different diseases due to different biotypes and serotype infection of Uu. Studies have reported that the Uu organism 1 population is the normal carrier flora of the lower reproductive tract, whereas the Uu organism 2 population is associated with pathogenic infections of the lower reproductive tract. Although most Uu infected individuals do not have significant clinical symptoms, these infections increase the risk of adverse pregnancy outcomes such as premature rupture of membranes, spontaneous preterm labor, miscarriage and stillbirth. Female genital tracts are short and directly susceptible to Uu infection, and early symptoms of infection are not obvious and can be overlooked, resulting in chronic, persistent and cross-infection. During pregnancy, the whole immunity level of the pregnant woman body of the pregnant woman is reduced, Uu ascending infection is easily caused if immune balance is disturbed, intrauterine infection is caused by placenta, and adverse pregnancy fatalities such as premature rupture of fetal membranes, spontaneous premature birth, abortion and the like are caused. If pregnant women have no obvious infection symptoms, intervention measures are not advocated clinically. Studies have indicated that Uu is found to be the culprit for invasive infections in immunocompromised adults, and that Uu may further contribute to pneumonia, sepsis and meningitis in premature and term infants. Wherein the lower the gestational age, the higher the risk of disease. Neonatal meningitis and neuritis may cause severe brain damage and long term adverse effects on patient health. Uu colonization has also been associated with brain abscesses, prostatitis, rheumatoid arthritis, artificial joint infections, and hyperammonemia after lung transplantation. Although Uu infected individuals can be treated with antibiotics, the emergence and spread of multidrug resistant strains present a significant challenge to the treatment of first and second line antibacterials, and clinically, asymptomatic, chronic, persistent, and recurrent infections in Uu infected individuals. Aiming at the conditions that the genital tract infection caused by the traditional Chinese medicine composition is difficult to radically cure, the serotype is more, the pathogenic substance is difficult to define and the like, the prevention of the related diseases is more effective and economical than the simple medication. However, no ureaplasma associated vaccine has been approved for clinical treatment at home and abroad. Therefore, there is a need to develop a safe, efficient and economical broad-spectrum vaccine for preventing ureaplasma infection.

In the prior art, although a multiband antigen (MBA) protein is a main outer membrane protein of ureaplasma urealyticum (Uu), the antigenicity of the MBA protein between different serogroups of Uu is very different, and the MBA protein cannot generate a wide immune protection effect against clinical infection of different serotypes of Uu; meanwhile, only B cell epitopes of mycoplasma urealyticum serotype 3 and serotype 14 MBA proteins are predicted to be known in the prior art, but whether the epitopes have relevant immune protection effect against Uu infection is not evaluated, so that the MBA proteins are not ideal as a ureaplasma urealyticum broad-spectrum vaccine.

Disclosure of Invention

In view of the above, the present invention aims to provide the use of Uu-DnaJ protein in the preparation of Uu immunogens and vaccines against Uu infection, which can produce a high-efficiency and broad-spectrum immunoprotection during the application process;

it is another object of the present invention to provide a vaccine against Uu infection that provides high-potency broad-spectrum immune protection using Uu-DnaJ protein as an immunogen.

In order to achieve the above purpose, the invention provides the following technical scheme:

the use of Uu-DnaJ protein for the preparation of Uu immunogens and vaccines against Uu infection.

In this context, the Uu-DnaJ protein may be non-natural, e.g.synthetic or expressed from an artificial vector (often referred to in the art as recombinant Uu-DnaJ protein). The term "non-natural" means that the target substance is not naturally occurring in nature, which does not preclude the non-natural substance from having the same structure and/or composition as the naturally occurring substance. The recombinant Uu-DnaJ protein is obtained through prokaryotic expression, has completely the same amino acid sequence as the Uu-DnaJ protein, and is shown as SEQ ID NO. 1, and the Uu-DnaJ protein coding sequence used by the invention is shown as SEQ ID NO. 2.

The chaperone DnaJ is an accessory chaperone protein necessary for the activation of the ATPase and protein folding activities of DnaK, has a conserved J domain of 70 amino acids, and is necessary for interacting with the nucleotide binding domain of DanK to stimulate ATP hydrolysis; in addition, its C-terminus contains two zinc binding sites and an important domain for binding to a substrate. Although the role of DnaK in the host's immune response to bacterial infection has been extensively studied, the cofactor DnaJ has received relatively little attention and has not been reported at all in Uu infections, and it is unclear whether DnaJ can provide protection against Uu genital tract infections, nor can any conclusions be predicted.

The invention analyzes the conservation of the Uu-DnaJ protein in different serotypes of Uu through the immune informatics, constructs the Uu recombinant protein DnaJ by utilizing the molecular biology technology, injects BALB/C female mice into thigh muscles, and evaluates the immune response type of the Uu recombinant protein DnaJ and the immune protection mechanism for resisting Uu infection. The result shows that the Uu-DnaJ protein can be combined with the serum of a Uu infected mouse and generate a specific antibody reaction in the mouse, and the antibody titer is 1: 640000; after a Uu-DnaJ immunized mouse stimulates the proliferation of splenic lymphocytes to increase, the antibody reaction of transition from IgG1 to protective IgG2a subtype is induced, the Th1 type cellular immune reaction is induced, the cervical Uu colonization amount, the inflammatory reaction and the pathological change of the mouse are reduced, and the immune protection effect is fully shown. Furthermore, the conservation of Uu-DnaJ of serotype 10 is further analyzed by BLASTp software, and the protein of Uu-DnaJ is highly conserved in all serotypes of Uu, and the consistency is more than 99%, which shows that the protein can realize broad-spectrum immune protection against Uu, is suitable for Uu genotype 4 ( serotype 4, 7, 10, 11, 12 and 13) and Uu genotype 8 ( serotype 2, 5, 8 and 9), and also shows that Uu-DnaJ of other serotypes can be used as immunogen and for preparing related vaccines.

On the basis of the above, the invention uses a plurality of algorithms in a DNAStartv7.1 software Protern module to predict the antigen index, the surface accessibility, the hydrophilicity and the flexibility of polypeptide chains in each region of Uu-DnaJ to obtain the region of each polypeptide chain which is most likely to form B cell epitope. The combination of the protein Uu-DnaJ in AA_16-42，AA_55-65，AA_99-123，AA_130-160，AA_211-247，AA_257-260，AA_300-309，AA_319-334And AA_342-362Amino acid residue positions are all likely to form a B cell dominant epitope. Subsequently, it was also found that Th cells of the Uu-DnaJ protein, CTL and their common epitope capable of binding specifically to murine MHC molecules were located in approximately the same region as the dominant epitope of T cells using the AMPHI, Rothbard-Taylor and set MHC Motifs algorithms, namely: AA_9-18，AA_49-58，AA_107-120，AA_155-160，AA_270-298，AA_335-342，AA_352-363. Therefore, the invention also provides the application of the polypeptide formed by one or more than two of the amino acid fragments in the preparation of Uu immunogen or vaccine for resisting Uu infection, and the application of the polypeptide formed by partial amino acid sequence selected from one or more than one amino acid fragments in the preparation of Uu immunogen or vaccine for resisting Uu infection.

According to the application, the invention also provides a vaccine for resisting Uu infection, which takes the polypeptide formed by one or more than two of the Uu-DnaJ protein and/or the T/B cell dominant epitope amino acid segment thereof as immunogen, or takes the polypeptide formed by partial amino acid sequence selected from the Uu-DnaJ protein T/B cell dominant epitope amino acid segment as immunogen.

Preferably, the vaccine further comprises an immunological adjuvant. In a specific embodiment of the invention, the immunological adjuvant is Freund's incomplete adjuvant, Freund's complete adjuvant, saponin, CpG oligonucleotide adjuvant, aluminum adjuvant or cytokine adjuvant. In a specific embodiment of the invention, the immunogen is thawed with injection adjuvant, the volume of the thawing solution is 100 μ L, and the concentration of the immunogen protein is 50 μ g/mouse.

According to the technical scheme, the invention provides new application of the Uu-DnaJ protein and the T/B cell dominant epitope amino acid fragment thereof in preparation of Uu immunogen or vaccine for resisting Uu infection, and related experimental results show that the Uu-DnaJ protein can be recognized by serum of mice infected with Uu, a specific antibody reaction can be generated after immunization, and the antibody titer is as high as 1: 640000; meanwhile, after a Uu-DnaJ immunized mouse stimulates the proliferation of splenic lymphocytes to increase, the transition from IgG1 to protective IgG2a subtype antibody reaction and Th1 type cellular immune reaction are induced, so that the cervical Uu colonization amount, inflammatory reaction and pathological changes of the mouse are reduced, the immune protection effect is fully shown, and various results prove that the Uu-DnaJ protein has the potential of developing a vaccine for resisting Uu infection.

Drawings

FIG. 1 shows the B cell dominant epitope prediction of Uu-DnaJ by DNAStartv7.1 software;

FIG. 2 shows the T cell dominant epitope prediction of Uu-DnaJ by DNAStartv7.1 software;

FIG. 3 shows the recombinant protein DnaJ Immunoblotting identification; 1: anti-His; 2, Uu infected mouse serum; 3, normal mouse serum;

FIG. 4 shows the results of antibody titer detection;

FIG. 5 shows the antibody levels and subtype measurements for each group of mice; each group of columns sequentially represents PBS group, FA group and DnaJ group columns from left to right; p <0.05,; p <0.01,; p <0.001, >;

FIG. 6 shows the result of splenic lymphocyte proliferation reaction in each group of mice; p <0.001, >;

FIG. 7 shows ELISA detection of splenic lymphocyte supernatant cytokine levels; p <0.05,; p <0.01,; p <0.001, >; p <0.00001, >;

FIG. 8 shows T cell specific responses of immunization with recombinant protein DnaJ; p <0.05,; p <0.01,; p <0.001, >; p <0.00001, >;

FIG. 9 shows that the recombinant protein DnaJ immuno-decreases the colonization of the cervix by Uu; p <0.05,;

FIG. 10 shows the results of immunization with the recombinant protein DnaJ to reduce the inflammatory response of the cervix; each group of columns sequentially represents PBS group, FA group and DnaJ group columns from left to right; p <0.05,; p <0.01,; p <0.001, >;

FIG. 11 shows the cervical H & E lesion results of mice immunized with the recombinant protein DnaJ;

FIG. 12 shows cervical immunohistochemical lesion results of mice immunized with recombinant protein DnaJ;

FIG. 13 shows an ELISA analysis of the immunoreactivity of candidate dominant epitopes to DnaJ group and control group sera;

FIG. 14 shows experiments on lymphocyte proliferation stimulated by candidate dominant epitope polypeptides of DnaJ protein

FIG. 15 shows the detection of antibody titers in D1 and D4 immunized mice; each set of columns represents PBS, FA, KLH, D4, D1, and DnaJ columns from left to right in sequence;

FIG. 16 shows the detection of serum Ig antibodies and IgG subclass antibodies in immunized BALB/c mice; each set of columns represents PBS, FA, KLH, D4, and D1 columns from left to right; p <0.05,; p <0.01,;

FIG. 17 shows measurement of cytokine levels in mouse spleen lymphocyte supernatants by indirect ELISA; p <0.01,; p < 0.001.

Detailed Description

The invention discloses application of Uu-DnaJ protein and a vaccine for resisting Uu infection, and a person skilled in the art can realize the application by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The applications and vaccines of the present invention have been described by way of example, and it will be apparent to those skilled in the art that the techniques of the present invention may be implemented and applied by modifying or appropriately modifying or combining the applications and vaccines described herein without departing from the spirit, scope and spirit of the invention.

The invention analyzes restriction endonuclease sites by extracting Uu serotype 8 standard strain DNA and using DNMAN software. According to a map of an enzyme cutting site of a prokaryotic expression vector pET28a (the C end contains a 6 XHis tag ggatcc, the N end contains a 6 XHis tag ctcgag), a DnaJ specific primer is designed by utilizing PGTool-Win software and synthesized by Shanghai's company, DNA is amplified by PCR, an amplification product is introduced into an expression vector pET28a in an enzyme cutting connection mode, prokaryotic expression is carried out by using Escherichia coli E.coli BL21(DE3), and recombinant Uu-DnaJ is obtained by IPTG induction and purification.

The molecular formula of the Uu-DanJ protein is C₁₈₅₇H₂₉₁₂N₅₀₄O₅₇₅S₉Consisting of 5857 atoms in total and having a relative molecular mass of 41.79X 10³And the isoelectric point is 8.04. It consists of 375 amino acids, of which Lys (11.2%), Gly (8.5%), Glu (7.7%) and Ile (7.7%) were higher, and the ratio of the total number of positively and negatively charged residues (Arg + Lys)/(Asp + Glu) was 50/54. The instability index of the protein is 37.72, and the protein is stable (when it is<40 is defined as stable protein). The half-life of the protein is predicted to be 30h in vitro mammalian reticulocytes and to be in Escherichia coli>10h in the yeast body>And (5)20 h. The DanJ protein fat index is 71.73. The average hydrophilicity index of the protein is-0.0594, and the protein belongs to hydrophilic protein.

The antigenic index, surface accessibility, hydrophilicity and flexibility of the polypeptide chains of each region of Uu-DnaJ were predicted using the algorithms in the Protern module of DNAStartv7.1 software to obtain its respective most likely B cell epitope-forming region (FIG. 1). Binding to the protein in AA_16-42，AA_55-65，AA_99-123，AA_130-160，AA_211-247，AA_257-260，AA_300-309，AA_319-334And AA_342-362Amino acid residue positions are all likely to form a B cell dominant epitope. Subsequently, it was also found that Th cells of the Uu-DnaJ protein, CTL and their common epitope capable of binding specifically to murine MHC molecules were located in approximately the same region as the dominant epitope of T cells using the AMPHI, Rothbard-Taylor and set MHC Motifs algorithms, namely: AA_9-18，AA_49-58，AA_107-120，AA_155-160，AA_270-298，AA_335-342，AA_352-363(FIG. 2). Meanwhile, 6 DnaJ protein B cell structural epitopes are obtained by IEDB software prediction. According to the prediction result, 7 candidate T/B cell shared dominant epitopes of the DnaJ protein are screened out through multiple sequence alignment (Table 1);

TABLE 1

The seven polypeptide sequences D1-D7 (shown as SEQ ID NO:3-9 in sequence) simultaneously comprise partial amino acid sequences selected from the following two groups of amino acid fragments:

(1)AA_16-42，AA_55-65，AA_99-123，AA_130-160，AA_211-247，AA_257-260，AA_300-309，AA_319-334，AA_342-362(B cell dominant epitope);

(2)AA_9-18，AA_49-58，AA_107-120，AA_155-160，AA_270-298，AA_335-342，AA_352-363(T cell dominant epitope).

D1, D3, D4, D6 and D7 can stimulate the proliferation of lymphocytes of a DnaJ immune mouse, wherein two polypeptide sequences of D1 and D4 are shared dominant epitopes of DnaJ protein T/B cells, can be combined with DnaJ immune serum antibodies, and induce body-specific humoral immunity and cellular immunity.

Therefore, the invention also provides the application of a polypeptide formed by a partial amino acid sequence selected from one or more of the following amino acid fragments of the Uu-DnaJ protein in preparing a Uu immunogen or vaccine for resisting Uu infection:

AA_16-42，AA_55-65，AA_99-123，AA_130-160，AA_211-247，AA_257-260，AA_300-309，AA_319-334，AA_342-362，AA_9-18，AA_49-58，AA_107-120，AA_155-160，AA_270-298，AA_335-342，AA_352-363。

preferably, the formed polypeptide comprises a partial amino acid sequence selected from the following two groups of amino acid fragments:

(1)AA_16-42，AA_55-65，AA_99-123，AA_130-160，AA_211-247，AA_257-260，AA_300-309，AA_319-334，AA_342-362；

(2)AA_9-18，AA_49-58，AA_107-120，AA_155-160，AA_270-298，AA_335-342，AA_352-363。

in a specific embodiment of the invention, the sequence of the polypeptide formed is as shown in any one of SEQ ID NO 3-9.

Using the DnaJ protein sequence from Uu serotype 10, further analysis of DnaJ conservation by BLASTp software revealed that DnaJ protein is highly conserved among all serotypes of Uu (complete identity of DnaJ amino acid sequences of each serotype is 99% or more), and DnaJ is considered as a target protein of a broad-spectrum vaccine according to the experimental conclusion of the invention.

TABLE 2 evaluation of the conserved distribution of DnaJ protein sequences among common serotypes of Uu

The immunogenicity research of the recombinant protein DnaJ thigh muscle immunity adopts the following experimental method:

1. immunogenicity study of recombinant protein DnaJ

1.1, immunizing BALB/c mice by recombinant protein DnaJ:

(1) breeding 45 female BALB/c mice with week age of 6-8 weeks;

(2) randomly divided into 3 groups of 15, PBS and FA (adjuvant) groups injected with 100. mu.L of PBS and adjuvant, respectively, and DnaJ group injected with 100. mu.L of adjuvant melt (protein concentration 50. mu.g/body);

(3) the BALB/c mice of the control group and the experimental group are immunized 1 time and 3 times at intervals of 14 days;

(4) collecting the serum of tail vein at 0, 1, 2, 3, 4, 5 and 6 weeks after the BALB/c mouse is immunized for the first time;

1.2, detecting the titer of the recombinant protein DnaJ antibody:

(1)1 × protein coating solution DnaJ recombinant protein was diluted to 10. mu.g/mL, 100. mu.L/well, added to an ELISA test plate and incubated overnight at 4 ℃;

(2) discarding liquid in the hole, PBST washing the plate for 3 times, 3min each time, removing residual liquid in the hole by using absorbent paper, and drying the absorbent paper;

(3) PBST 200 muL/hole added with 5% skimmed milk is added into an ELISA plate, and the incubator is closed for 2h at 37 ℃;

(4) discarding liquid in the hole, PBST washing the plate for 3 times, 3min each time, and removing residual liquid in the hole by absorbent paper;

(5) BALB/c mouse serum collected at different times (1:100) was diluted with PBST fold ratio supplemented with 5% skim milk at 100. mu.L/well and incubated at 37 ℃ for 2 h;

(6) discarding liquid in the hole, PBST washing the plate for 5 times, 3min each time, and removing residual liquid in the hole by absorbent paper;

(7) diluting HRP-labeled goat anti-mouse IgG (1:20000), incubating at 37 ℃ for 2h at 100. mu.L/well;

(8) discarding liquid in the holes, washing the plate for 6 times by PBST (Poly-p-phenylene-terephthamide) for 3min each time, and removing residual liquid in the holes by using absorbent paper;

(9) adding TMB color development solution, reacting at room temperature for 35min (keeping out of the sun), and adding stop solution;

(10) and (3) placing the sample in a microplate reader to read the OD value (450nm) of each hole, wherein the positive result is obtained when the ratio of the experimental group to the negative control group is more than or equal to 2.1.

1.3 detection of recombinant protein DnaJ antibody level

(1)1 Xprotein coating solution DnaJ recombinant protein is diluted to 10 mug/mL, 100 mug/well is added into an ELISA detection plate, and the mixture is incubated overnight at 4 ℃;

(2) discarding liquid in the hole, PBST washing the plate for 3 times, 3min each time, removing residual liquid in the hole by using absorbent paper, and drying the absorbent paper;

(3) adding PBST (200 mu L/hole) of 5% skimmed milk, and sealing in an incubator at 37 ℃ for 2 h;

(4) discarding liquid in the hole, PBST washing the plate for 3 times, 3min each time, removing residual liquid in the hole by using absorbent paper, and drying the absorbent paper;

(5) the BALB/c mouse serum collected from the last immunization (1:100) was diluted with PBST (containing 5% skim milk), 100. mu.L/well, incubated at 37 ℃ for 2 h;

(6) discarding liquid in the hole, PBST washing the plate for 5 times, 3min each time, and removing residual liquid in the hole by absorbent paper;

(7) diluting 100 μ L/well of HRP-labeled goat anti-mouse IgG, IgG1, IgG2a, IgG3, IgA, and IgM (all 1:10000), and incubating at 37 deg.C for 2 h;

(8) discarding liquid in the holes, washing the plate for 6 times by PBST (Poly-p-phenylene-terephthamide) for 3min each time, and removing residual liquid in the holes by using absorbent paper;

(9) adding TMB color development solution, reacting at room temperature for 35min (keeping out of the sun), and adding stop solution; (10) placing the sample in a microplate reader to read the OD (450nm) value of each well, and comparing the experimental group with the negative control group

A positive result is obtained when the ratio is more than or equal to 2.1.

1.4 recombinant protein DnaJ immune mouse spleen lymphocyte proliferation experiment

(1) After 2 weeks of the last immunization of the mice, the spleens were aseptically isolated by anesthetizing the mice and loaded into sterile test tubes (containing 200 mesh nylon membranes);

(2) adding 5mL of incomplete RPMI1640 culture medium into nylon membrane, grinding spleen, centrifuging at 1000g for 8 min;

(3) discarding the supernatant in aseptic operation, sucking 3mL of erythrocyte lysate by a micropipette, adding into a centrifuge tube, mixing uniformly, and centrifuging for 8min at 1000 g;

(4) discarding supernatant in aseptic operation, sucking 5mL of incomplete RPMI1640 culture medium by a micropipette, adding into a centrifuge tube, mixing uniformly, and centrifuging for 8min at 1000 g;

(5) aseptically discarding the supernatant, sucking 5mL of Hank's solution (containing double antibiotics, i.e. streptomycin) by a micropipettor, adding into a centrifuge tube, uniformly mixing, and centrifuging for 8min at 1000 g;

(6) discarding the supernatant in aseptic operation, sucking 4mL of incomplete 1640 culture medium by a micropipette, adding the incomplete 1640 culture medium into a centrifuge tube, uniformly mixing, and centrifuging for 8min at 1000 g;

(7) discarding the supernatant by aseptic operation, sucking 4mL of complete 1640 culture medium by a micropipette, adding the medium into a centrifuge tube, uniformly mixing, sucking 20 mu L of medium and counting under a microscope;

(8) diluting the cell number to 6 × 106/well, and adding the diluted cell number into a 24-well plate and a 96-well plate respectively;

(9) DnaJ protein (10. mu.g/ml) was added and a control was set at 37 ℃ with 5% CO₂Adding 20 mu L of CCK-8 solution into each hole after culturing for 44 hours in a cell culture box, continuously culturing for 48 hours, collecting cell culture supernatant, and reading the A450 value by an enzyme-labeling instrument;

1.5 cytokine detection of spleen lymphocyte supernatant of mice immunized by recombinant protein DnaJ

Spleen lymphocytes were collected according to step 1.4 at 6X 10⁶The cells were transferred to a 24-well plate at a cell density of 10. mu.g DnaJ protein per well, mixed and placed at 37℃，5％CO₂After culturing for 48h in a cell culture box, centrifuging at 6000rpm for 15min, collecting cell culture supernatant, freezing at-20 ℃, and using for detecting IFN-gamma, TNF-alpha, IL-4 and IL-10 cytokine levels by a subsequent ELISA method.

(1) The cytokine capture antibody was diluted with 1 × Coating Buffer, added to a 96-well ELISA plate at 100 μ L/well, and placed in a refrigerator at 4 ℃ overnight;

(2) discarding liquid by using a micropipette, washing the plate for 4 times and 3 min/time by using 200 mu L of buffer solution, discarding liquid, and patting dry;

(3) adding 1 XELISA/ELISPOT solution (200. mu.L/well), and incubating at 37 ℃ for 1 h;

(4) washing the plate, and carrying out the synchronous step (2);

(5) establishing a standard curve, namely diluting the standard substance by using Diluent 1 multiplied by ELISA/ELISPOT multiple ratio, and 100 mu.L/hole; simultaneously, 100 mu L of the treated splenocyte supernatant is added into each hole, and the mixture is incubated for 2h in a constant temperature box at 37 ℃;

(6) repeating the step (2);

(7) add 100. mu.L of detection antibody (1 × ELISA/ELISPOT dilution) to each well, incubate 1h at 37 ℃ incubator;

(8) repeating the step (2);

(9) add 100. mu.L of HRP-labeled Avidin (1 × assay dilution well) to each well, incubate in a 37 ℃ incubator for 30min in the dark;

(10) repeating the step (2) once;

(11) adding 100 mu L/hole of TMB working solution, and incubating at room temperature for 15 min;

(12) stop buffer was added at 50. mu.L/well and the ELISA plate was read at 450nm after 5 min.

1.6 flow cytometry

(1) Preparing a flow tube, a cell culture plate and a centrifuge tube;

(2) the concrete operation for preparing the mouse spleen cell suspension is shown in 1.4. Transfer of prepared spleen cell suspension (1X 10)⁶/mL) into 24-well plates, protein-stimulated and unstimulated wells (DnaJ protein-stimulated), 37 ℃, 5% CO₂Incubating the cells in a cell incubator for 8 hours;

(3) preparing a 1.5mL centrifuge tube, culturing for 5h, sucking the cell suspension into the 1.5mL centrifuge tube, centrifuging at 3000rpm for 5min to collect cells, discarding the supernatant, adding 500. mu.L of complete 1640 culture medium to wash the cell culture plate, collecting the cell culture plate and the centrifuge tube together, centrifuging at 3000rpm for 5min, and discarding the supernatant;

(4) respectively adding 1mL of sterilized PBS, centrifuging at 3000rpm for 5min, discarding the supernatant, resuspending with 1mL of sterilized PBS, centrifuging at 3000rpm for 5min, discarding the supernatant, and then adding 100 mu L of PBS to resuspend the cells;

(5) blocking surface staining, namely adding 1 mu L of corresponding surface marker antibodies (CD4 and CD8) respectively, and incubating for 22min at room temperature in a dark place;

(6) adding 500 μ L PBS into each tube, and centrifuging at room temperature 2276rpm for 5 min;

(7) fixing and membrane rupture, namely removing the supernatant, adding 300 mu L of Fixation/cytofix, incubating at 4 ℃ in a dark place for 23min, washing cells for 2 times by using 1mL of 1 xBD perm Wash Buffer, and centrifuging at 3000rpm for 5 min; (8) intracellular staining, resuspending fixed membrane-broken cells with 100. mu.L of 1 XBD perm Wash Buffer. Correspondingly adding corresponding PE Rat Anti-Mouse IL-4 and APC Rat Anti-Mouse according to the management setting

IFN-gamma is subjected to intracellular staining, and incubated for 25min at room temperature in a dark place;

(9) washing the cells with 500 μ L PBS for 2 times, centrifuging at 3000rpm for 5min, and discarding the supernatant;

(10) after being resuspended in sterilized PBS, the cells are stored in a refrigerator at 4 ℃ and are detected on a flow cytometer.

2. Immunoprotective study of recombinant protein DnaJ

2.1 cultivation and quantification of Uu8 type Standard Strain to concentration

2.1.1 cultivation of the type Uu8 Standard Strain

Culturing a large amount of recovered and purified Uu8 type standard strain liquid, establishing a negative control (without Uu bacterial liquid), culturing at 37 ℃ for 1-2 days, and controlling the color change range of the phenol red indicator to be 6.8-8.4. Observing the color change of the bacterial liquid, and carrying out subsequent experiments when the color of the culture medium is changed from light yellow to red and the bacterial liquid is transparent and not turbid.

2.1.2 quantification of the Uu8 type Standard Strain

The positive culture medium was inoculated onto Uu solid medium at the following dilution ratio by sucking 100. mu.L of each of the positive culture medium.

Test tube 1 Uu positive bacteria liquid

Test tube 2 Uu positive bacteria liquid PBS 1:10

Test tube 3 Uu positive bacteria liquid PBS 1:100

Spreading diluted bacterial solution 100 μ L on Uu solid culture medium respectively, and adding 5% CO₂And culturing in a constant-temperature incubator at 37 ℃ for 1-3 days, and setting a solid culture medium without adding a bacterial liquid as a negative control. The solid culture is taken out and observed under an ordinary inverted microscope, the number of 10 visual field Uu colonies is counted under a low-power microscope, and the mycoplasma load (CFU/mL) is calculated by adopting the following formula for subsequent quantitative experiments.

2.1.3 concentration of the type Uu8 Standard Strain

Collecting the cultured Uu bacteria liquid, centrifuging at 10000rpm for 30min, discarding supernatant, and collecting Uu

And (4) precipitating. And sucking the sterilized PBS by a micropipette, washing for 2-3 times, centrifuging at 10000rpm for 10min, and leaving a precipitate. And (4) resuspending the precipitate with sterilized PBS, storing at-80 ℃ and performing subsequent experimental study on Uu genital tract infected mice.

2.2 Uu infected BALB/c mice

Two weeks after the last immunization PBS, FA and DnaJ groups, each mouse was vaginally injected with 1X 10⁷The CFU/mL Uu8 type bacterial liquid is 50 mu L, after injection, the mice are kept supine for 1min, and the normal control group is not injected. 7 days prior to infection, each mouse was injected subcutaneously in the neck once a week for three weeks with 0.5mg estradiol benzoate to synchronize the estrous cycle and increase the sensitivity of the mice to Uu infection. To identify whether mice were infected with Uu, on days 0, 7, 14 and 21 post infection, sterile swabs were inserted into the lower genital tract and uterus to take vaginal and cervical secretions, rotated and left for 1 minute. Next, the swab samples were washed into Uu broth and divided into two parts, one for Uu body culture and determination of Uu concentration and the other for PCR detection. Mice were monitored daily for signs of hair loss, increased secretions, edema, congestion and fluid accumulation at the site of infection, and weight changesAnd (5) forming and recording.

2.3 Uu Loading and cytokine detection in the cervix of mice after infection

2.3.1 Uu DNA extraction in cervical fractionation

The method is characterized in that the DNA extraction kit of MP company is used for extracting Uu DNA in a cervical secretion specimen, and the specific operation is as follows:

(1) adding 978 mu L of SPB into the lysing Matrix E tube, adding 122 mu L of MT solution, and fully stirring the swab in a tube to ensure that the secretion falls off;

(2) vortex, shaking and mixing for 1 min;

(3) centrifuging at 14000g for 5 min;

(4) sucking the supernatant into a new 2.0mL centrifuge tube, adding 250 μ L PPS solution, and reversing and mixing uniformly for 10 times;

(5)14000g, centrifuging for 5min, and sucking the supernatant into a new 2.0mL centrifuge tube;

(6) adding 1mL BMS solution, reversing and uniformly mixing for 2min, and standing for 3min at room temperature;

(7) discarding 500. mu.L of supernatant;

(8) taking 600 mu L of mixed solution for re-suspension, adding the mixed solution into an adsorption column, centrifuging for 2min at 14000g, repeating the steps, and discarding the waste liquid;

(9) adding 500 μ L SEWS-M solution to adsorption column, centrifuging at 14000g for 1min, discarding the waste liquid, repeating, and standing at room temperature for 5 min;

(10) adding 40 mul DES solution to dissolve, centrifuging for 2min at 14000g, repeating, discarding waste liquid, and storing the extracted DNA solution at-20 deg.C.

2.3.2 detection of cytokines in the cervix

The cytokine levels of IL-6, TNF-alpha, IL-1 beta, IL-10, IL-1 alpha, IL-17a, MCP-1 and IFN-gamma in the cervical secretion samples were determined using the Bio-Legend multi-cytokine detection kit, as follows:

(1) dilution of samples 25. mu.L of sample and 25. mu.L of Assay Buffer were added to each tube

(2) Diluting the standard substance, namely adding 25 mu L of standard substance and 75 mu L of Assay Buffer into a first hole, and then diluting 6 holes in a multiplying ratio in sequence for preparing a standard curve;

(3) vortex, shake, mix evenly, put on shaking table and shake for 2h in dark;

(4) centrifuging at 3000rpm for 5min, and discarding the supernatant;

(5)200 μ L Washing Buffer resuspension;

(6) centrifuging at 3000rpm for 5min, and discarding the supernatant;

(7) dripping 25 μ L of detection antibody, shaking in the dark for 1h, adding 25 μ L of SA-PE solution, and shaking in the dark for 30 min; (8) centrifuging at 3000rpm for 5min, discarding the supernatant, adding 200 μ L Washing Buffer for resuspension, centrifuging at 3000rpm for 5min, and discarding the supernatant;

(9) add 200. mu.L Washing Buffer for resuspension and transfer to flow tube for detection.

2.4 extraction of Uu genomic DNA from tissues after infection of mice

Extracting DNA in tissues by using an animal tissue genome DNA miniprep kit of Tiangen company, and specifically operating as follows:

(1) animal tissue treatment, weighing <10mg animal tissue, grinding into cell suspension, centrifuging at 12000g for 5min, collecting precipitate, adding 250 μ L GA buffer solution, and oscillating for suspension;

(2) adding 20 μ L protease K solution, mixing, dissolving in 56 deg.C water bath overnight, centrifuging for the next day to remove water drop on the inner wall of the tube cover;

(3) adding 200 μ L of GB buffer solution, fully reversing and mixing uniformly, placing in a 70 ℃ water bath box for 10min, clarifying the solution by strain, performing instantaneous centrifugation, and transferring the supernatant into a clean 1.5mL centrifuge tube;

(4) adding 200 μ L of anhydrous ethanol, shaking thoroughly, mixing for 15s, and centrifuging at 12000g for 2 min;

(5) adding the solution and flocculent precipitate obtained in the previous step into an adsorption column CB3, separating 12000g for 30s, and discarding the waste liquid;

(6) adding 500 mu L of buffer GD into an adsorption column CB3, centrifuging for 30s at 12000g, and discarding waste liquid;

(7) adding 600 mu L of rinsing liquid PW into an adsorption column CB3, centrifuging for 30s at 12000g, and discarding the waste liquid;

(8) repeating the step (7) once;

(9) placing adsorption column CB3 back into the collecting tube, centrifuging at 12000g for 2min, pouring off waste liquid, and standing the adsorption column at room temperature for 5 min;

(10) placing the adsorption column CB3 into a clean collecting tube, sucking 100 mu LTE buffer solution by a micropipette, suspending, adding into the adsorption, standing for 4min, centrifuging at 12000g for 2min, and collecting the liquid for storage at-20 ℃.

2.5 real-time fluorescent quantitative PCR (RT-PCR)

Shanghai bio corporation synthesizes a primer specific to the Uu8 type urease gene, and RT-PCR detects the Uu loading capacity in each tissue of the mouse. Calculation of the Uu load in tissues of BALB/c mice A standard curve was established by 10-fold dilution (10) of the Uu linearized plasmid DNA⁷To 10¹Linearization) to obtain a standard curve of Uu8 type urease gene; BALB/c mouse DNA concentration (100ng to 0.0488ng) was serially diluted in multiple times to obtain a standard curve of the internal reference (. beta. -actin) gene:

2.6 histopathological analysis of the cervix of the infected reproductive tract

2.6.1H & E analysis of cervical tissue of infected reproductive tract

(1) After 21 days of infection, separating mouse genital tract tissues, adding 10% formaldehyde fixing solution, dewaxing paraffin sections after 48 hours until water is obtained, sequentially placing the tissue sections into dimethylbenzene I, dimethylbenzene II, 100% I, 100% II and 75% ethanol for gradual dehydration for 7-9min respectively, and washing with tap water.

(2) Dyeing the dehydrated and dewaxed slices with hematoxylin for 2-6min, washing with water for a while, then placing in hydrochloric acid aqueous solution for differentiation, returning ammonia aqueous solution to blue, and washing with water;

(3) and (3) eosin staining, namely sequentially dehydrating the tissue slices with 85% and 95% gradient ethanol, and adding the dehydrated tissue slices into eosin staining solution for 6 min.

(4) Dehydrating and sealing, namely putting the sliced tissue into 100 percent I, 100 percent II, 100 percent III, xylene I and xylene II for 7-9min respectively to be transparent, drying or electrically drying the slices, sealing the slices with neutral gum, and sticking a label.

(5) And (5) microscopic examination and analysis of results.

(6) The result of the analysis was that the nucleus was blue and the cytoplasm was reddish.

2.6.2 immunohistochemical analysis of cervical tissue of infected reproductive tract

(1) After 21 days of infection, separating mouse genital tract tissues, adding 10% formaldehyde fixing solution, and dewaxing paraffin sections to water after 48 hours, namely sequentially putting the tissue sections into dimethylbenzene I, dimethylbenzene II, dimethylbenzene III, 100% I, 100% II, 85% and 75% ethanol for gradual dehydration for 7-10min respectively, and washing with tap water.

(2) Antigen retrieval, namely, antigen retrieval in a microwave oven, wherein the medium fire is 6min till the antigen is boiled, the fire is stopped for 6min, and the medium and low fire is turned for 6 min. After natural cooling, the cells were washed 4 times with shaking 7 min/time in PBS buffer (pH 7.4).

(3) Endogenous peroxidase blocking sections were placed in 3% H2O2 solution (H2O2: H2O ═ 1:9), protected from light for 20min, and slides were placed in PBS buffer at pH 7.4. Shaking and washing on decolorizing shaker for 3 times (7 min/time).

(4) And (5) serum blocking, namely dripping 3% BSA (bovine serum albumin) into a histochemical ring to uniformly cover the section, and blocking for 35min at room temperature.

(5) Add primary antibody-gently remove blocking solution, add primary antibody (non-immune mouse anti-Uu serum) to the sections, and place the sections flat in a wet box overnight at 4 ℃.

(6) Add secondary antibody in PBS buffer (pH7.4), shake wash 4 times, 7 min/time. After the section is slightly dried, a secondary goat anti-mouse IgG antibody marked by HRP is dripped into the circle. The sections were covered and left at room temperature for 55 min.

(7) DAB visualization, placing the slide in PBS buffer solution with pH7.4, and washing 5 times and 7 min/time on a decolorizing shaking table. After the DAB is slightly dried and sliced, a DAB coloration solution which is prepared freshly is dripped into the ring, the coloration is stopped when the positive color is brownish yellow under the observation of a microscope.

(8) Counterstaining cell nucleus, hematoxylin Harris counterstaining for 5min, water washing, differentiation, water washing, bluing and water washing.

(9) And (3) dehydrating and sealing 75%, 85%, 100% I and 100% II in dimethylbenzene I for 7-10min, taking out the slices from the dimethylbenzene, slightly drying the slices, and sealing the slices in neutral gum.

(10) Microscopic examination and result analysis.

(11) The paraffin section is analyzed for immunohistochemical results, wherein the nucleus is blue, and the positive expression is brown yellow granules.

3. Statistical processing method

The experimental data were statistically analyzed using GraphPad Prism 7.0 software, cytokine content and Uu loading in tissues were expressed as mean ± standard deviation (± S), comparisons between groups were analyzed using one-way variance, and pairwise comparisons were tested using Post Hoc. P <0.05, indicating that the difference is statistically significant, and NS, i.e., P >0.05, indicating that the difference is not statistically significant.

The use of the Uu-DnaJ protein and vaccines against Uu infection provided by the present invention are further described below.

Example 1: immunoblotting identification of purified recombinant protein DnaJ

The results of Immunoblotting analysis and identification of recombinant protein DnaJ by using a mouse anti-His monoclonal antibody, Uu infected mouse serum and normal mouse serum as primary antibodies and sheep anti-mouse IgG (H + L) as secondary antibodies show that the infected mouse serum and the anti-His antibody as primary antibodies show correspondingly obvious specific reaction bands which are consistent with the expected relative molecular weight and no specific band appears in a control group (normal mouse serum) (figure 3).

Example 2: specific antibody titer detection of recombinant protein DnaJ immunity

Researching whether the recombinant protein DnaJ can induce the antibody reaction of mice, respectively collecting tail vein blood at 0, 1, 2, 3, 4, 5 and 6 weeks after the mice of each group are immunized, and detecting the titer level of specific IgG antibody in serum by an ELISA method. The results showed that at 7 days, the level of IgG antibodies in DnaJ-immunized mice increased, with a gradual increase in the immunization time (FIG. 4), with an antibody titer of 1: 640000. The results show that intramuscular injection of the recombinant protein DnaJ into BALB/c mice results in a specific antibody response.

Example 3: recombinant protein DnaJ antibody level and IgG subtype detection

As shown in FIG. 5, the IgG and IgM antibody levels of the DnaJ group were significantly higher than those of the control group (PBS group and FA group). Further evaluation of the specific type of antibody response induced by DnaJ, the levels of IgG subclass antibodies (IgG1, IgG2a and IgG3) in serum were measured and the levels of DnaJ group IgG2a were found to be higher than IgG1, indicating that DnaJ immunized mice induced a transition from IgG1 to protective IgG2a subtype antibody response.

Example 4: proliferation of spleen lymphocytes of BALB/c mice immunized by recombinant protein DnaJ

After BALB/c mice are immunized for 2 weeks for the last time, splenocytes of all groups of mice are separated in a sterile mode, and the splenic lymphocyte proliferation level of all groups of BALB/c mice is detected by a CCK-8 method after the mice are stimulated by corresponding DnaJ recombinant protein in vitro. The results showed that splenic lymphocyte proliferation was increased in the DnaJ group mice, and that splenic lymphocyte stimulation index values (SI) were significantly different from those of the control group, as shown in fig. 6.

Example 5: stimulation of T cell phenotype in splenic lymphocytes of BALB/c mice by recombinant protein DnaJ

1. Indirect ELISA method for detecting level of splenic lymphocyte supernatant cytokine

Researching induced cell reaction types after the recombinant protein DnaJ immunizes mice, collecting splenocyte supernatants of various groups of mice after the protein stimulates the mice to culture, and detecting the level of the cell factors by an indirect ELISA method. Splenocytes from DnaJ-immunized mice showed higher levels of the Th 1-associated cytokines TNF- α and IFN- γ, with significant differences compared to the control group, see FIG. 7. The Th2 related cytokines IL-4 and IL-10 were not statistically different from the control group. Taken together, these results indicate that the immunogenicity of DnaJ is based on its ability to induce a protective T cell response, in particular a Th1 biased cellular immune response.

2. Flow cytometry analysis of T cell phenotype in splenic lymphocytes of BALB/c mice

Although CD4⁺And CD8⁺T cells are all essential for the prevention of Uu infection, but host immune responses can be identified and monitored by detecting the levels of IFN- γ, the most important Th 1-associated cytokine associated with early pathogen immune responses. Thus, IFN- γ production was studied by flow cytometry prior to Uu challenge. Both DnaJ-immunized groups had significantly increased IFN- γ production compared to the control group. IFN-gamma producing T cells (i.e., IFN-gamma producing CD4+ and CD8) in splenocytes from the immunized group⁺T cell) level, DnaJ immune group compared to control group CD4⁺T cells produced IFN-gamma significantly increased (FIG. 8a), and the DnaJ immune group compared with the control group CD8⁺The production of IFN- γ by T cells was significantly increased (fig. 8 c). On the contraryThere was no significant difference in IL-4 production between the immunized and control groups (FIG. 8 b). In addition, the results show that compared with the control group, the content of IFN-gamma in the DnaJ immune group is obviously increased, but the IL-4 level has no statistical significance, and further prove that the DnaJ immune BALB/c mice can induce Th1 type cell immune response.

Example 6: immunoprotection of the recombinant protein DnaJ

1. Recombinant protein DnaJ immunity reduces mouse cervix planting amount

To evaluate the protective effect of DnaJ-immunized mice, BALB/c mice were inoculated with Uu8 type strain. At the initial stage of infection, vaginal secretion is taken every three days, cervical secretion is taken every other week for microbial culture analysis and PCR identification, and the result shows that the culture is positive, which indicates that Uu infects mice successfully. In addition, one week after Uu infection, all mice showed vaginal hair loss, increased secretions, redness of vaginal opening and loss of appetite, but the mice in the DnaJ immunized group showed relatively mild symptoms (fig. 9a), and to confirm whether DnaJ immunized mice could attenuate Uu colonization at the site of colonization (cervix) in mice, Uu load at the site of colonization was assessed by quantitative PCR method 21 days after Uu inoculation in mice. The results showed that the Uu load level at the site of Uu colonization was significantly lower in DnaJ immunized mice than in the control group (FIG. 9b)

2. Immunization of recombinant protein DnaJ for reducing inflammatory response of mouse cervix

The levels of IL-6, TNF- α, IL-1 β, IL-10, IL-1 α, IL-17a, MCP-1 and IFN- γ in the cervical homogenate supernatants of the DnaJ immunized and control groups after vaginal infection of BALB/c mice with Uu were assessed using a multi-cytokine flow assay kit. As shown in FIG. 10a, there was no significant difference in the level of IL-17a, IL-6, IL-10 and IL-1 α between the DnaJ-immunized group mice and the control group, whereas the level of IFN-. gamma. (P <0.05), TNF-. alpha. (P <0.01), MCP-1 (. P <0.05) and IL-1. beta. (P <0.001) was significantly lower in the cervix of the DnaJ-immunized group mice than in the control group. Fig. 10b is a corresponding heatmap analysis.

3. The recombinant protein DnaJ immunity can obviously reduce the cervical lesion of mice

Uu infection caused severe pathological damage to cervical tissue in mice in the PBS (FIG. 11a, E, i) and FA (FIG. 11b, f, j) groups, and H & E results showed severe edema, hemorrhage and increased secretion in cervical tissue in mice in the PBS and FA groups. Inflammatory responses (massive inflammatory cell infiltration and exudation of inflammatory fluid within the glandular cavity) were shown in PBS and FA group mice after Uu infection (figure 11). While in the cervical tissue of the DnaJ group (fig. 11c, g, k) mice the glandular expansion, glandular secretion increase, pathological features such as edema, hemorrhage and inflammatory cell infiltration decrease, and the structures of the glandular and cervical tissues can be clearly identified and their integrity maintained compared to the normal control group mice (fig. 11d, h, l).

S-P immunohistochemical evaluation of cervical tissue sections of mice 21 days after Uu infection showed that pathological lesions were less in DnaJ protein immunized mice and Uu load (FIG. 12c, g) (brown; red arrows) was significantly lower in DnaJ immunized mice than in PBS and FA groups (FIG. 12a, b, e, f). The results also show and compared to normal control mice (fig. 12d, h) that Uu is mainly present in cervical glands, mesenchymal cells and cytoplasm.

Example 7: immunization of candidate dominant epitope polypeptides of DnaJ protein

7 candidate DnaJ protein dominant epitopes predicted in the table 1 are synthesized into corresponding peptide fragments and purified. Through HPLC analysis, the purity of each polypeptide reaches over 90 percent. The mass spectrometry identification shows that the measured value of the relative molecular weight of each polypeptide conforms to the theoretical value, which indicates that the synthesized polypeptide conforms to the requirements and can be used for subsequent experiments.

1. Identification of immunodominant epitopes of DnaJ proteins

The immunoreactivity of 7 DnaJ candidate dominant epitope polypeptides (D1-D7) synthesized above to DnaJ immune serum and control group serum was tested by ELISA method, and the results showed that D1 and D4 polypeptides could bind to the corresponding immune serum antibodies and are B cell dominant epitopes (FIG. 13). And D1, D3, D4, D6 and D7 in the candidate dominant epitope polypeptides can stimulate the proliferation of corresponding lymphocytes (CCK-8 method), and are T cell dominant epitopes (FIG. 14). Taken together, D1 and D4 are shared dominant epitopes of DnaJ protein T/B cells, while D3, D4, D6 and D7 are dominant epitopes of DnaJ protein T cells rather than B cells.

2. DnaJ protein immunodominant epitope polypeptide can induce body specific humoral immunity

The two naked peptides D1 and D4 synthesized above are coupled with KLH to increase the stability in the body, mixed with Freund's adjuvant and emulsified, and then injected into BALB/c mice through muscle, serum of 0, 14, 28 and 42 days after each group of mice is immunized is collected respectively as primary antibody, and the specific IgG antibody titer level is detected by indirect ELISA method by using corresponding polypeptide (naked peptide without KLH coupling) or DnaJ protein as coating antigen. The results showed that IgG antibody levels increased in D1 and D4 immunized mice at day 14, thereafter, with a gradual increase in immunization time, and both peaked at day 42 (fig. 15).

The levels of IgA, IgM, IgG and their subtypes of antibodies in the immune serum were further examined, and the results are shown in fig. 16, where IgG and IgM antibody levels in D1 and D4 immunized mice were significantly higher than those in PBS group, FA group and KLH group. Serum IgG antibody subtype levels (IgG1, IgG2a, IgG2b, IgG3) were also significantly higher in D1 and D4 immunized mice than in the negative control group, and IgG2a was also found to be higher than IgG 1.

3. DnaJ protein immunodominant epitope polypeptide can induce specific cellular immunity of organism

Splenic lymphocytes of mice in each immunization group were prepared aseptically, and splenic cell supernatants were collected after 48h stimulation with intact DnaJ protein, D1, D4, PBS, FA, KLH, respectively, and cytokine levels were measured by indirect ELISA. As shown in FIG. 17, splenocytes from D1 and D4 immunized mice exhibited higher levels of TNF- α and IFN- γ (Th1 cell-associated cytokines).

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of southern China

Application of <120> Uu-DnaJ protein and vaccine for resisting Uu infection

<130> MP21012603

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

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<212> PRT

<213> Ureaplasma urealyticum serovar 10 str. ATCC 33699

<400> 1

Met Ala Lys Arg Asp Tyr Tyr Glu Val Leu Gly Val Ser Lys Ser Ala

1 5 10 15

Ser Pro Glu Glu Ile Lys Thr Ala Phe Arg Lys Leu Ala Lys Glu His

20 25 30

His Pro Asp Arg Asn Lys Ser Ala Asp Asp Thr Val Phe Lys Glu Ile

35 40 45

Asn Glu Ala Tyr Glu Val Leu Ser Asp Pro Lys Lys Arg Ala Gln Tyr

50 55 60

Asp Gln Phe Gly His Asp Gly Pro Gln Gly Phe Ala Gly Ala Gly Gly

65 70 75 80

Phe Ser Gly Phe Ser Asp Gly Phe Gly Gly Val Asp Phe Asp Ile Asn

85 90 95

Asp Ile Phe Gly Ser Phe Phe Lys Asn Gly Ala Ser Ser Arg Ser Ser

100 105 110

Ser Ser Gln Tyr Glu Thr Tyr Asp Ile His Leu Arg Leu His Leu Glu

115 120 125

Phe Ile Glu Ala Ile Lys Gly Val Ser Lys Asn Ile Ser Tyr Asp Arg

130 135 140

Lys Ile Thr Cys Asn Lys Cys Gln Gly Thr Gly Ala Lys Asp Pro Lys

145 150 155 160

Asp Val Lys Thr Cys Thr Lys Cys His Gly Arg Gly Thr Thr Ile Glu

165 170 175

Asn Val His Ser Leu Phe Gly Thr Ile Gln Gln Glu Val Glu Cys His

180 185 190

Glu Cys Glu Gly Thr Gly Lys Val Ala Asn Ser Lys Cys Glu Gln Cys

195 200 205

Tyr Gly Lys Lys Val Ile Asn Glu Arg Val Asn Leu Thr Val Glu Ile

210 215 220

Pro Ala Gly Thr Gln Asp Asn Glu Lys Leu Val Val Ser Lys Lys Gly

225 230 235 240

Asn Ile Ile Asn Asn Gln Glu Phe Asp Leu Tyr Leu His Ile Ser Val

245 250 255

Lys Pro Ser Lys Tyr Phe Ala Phe Asp Gly Leu Asp Ile Tyr Ser Glu

260 265 270

Thr Tyr Val Asp Pro Ile Lys Ala Ile Val Gly Gly Val Ile Glu Val

275 280 285

Val Thr Thr Ser Gly Ile Lys Thr Ile Glu Ile Pro Pro Asn Thr Pro

290 295 300

Glu Gly Lys Lys Phe Arg Ile Ser Gly Ala Gly Ile Val Asn Lys Lys

305 310 315 320

Pro Asn Ile Phe Ser Lys Lys Asn Gly Asp Phe Tyr Thr Thr Ile Arg

325 330 335

Tyr Ala Lys Pro Leu Glu Leu Thr Lys Glu Glu Ile Ala Tyr Leu Lys

340 345 350

Asn Ile Ser Ala Arg Thr Asn Gln Ser Val Glu Tyr Tyr Lys Asn Lys

355 360 365

Leu Leu Lys Glu Val Asn Lys

370 375

<210> 2

<211> 1128

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggcaaaac gtgattacta tgaagtttta ggtgttagta aatcagcaag ccctgaagaa 60

attaaaactg cgtttcgtaa attagcaaaa gagcaccatc cagatcgaaa caaatctgct 120

gatgatactg tttttaaaga aattaatgaa gcttatgagg tgttatcaga tcctaaaaaa 180

cgtgctcaat acgaccaatt tggtcatgat ggaccgcaag ggtttgctgg agctggtggc 240

ttttctgggt ttagtgatgg atttggcggt gttgattttg acatcaacga catttttgga 300

agttttttta aaaatggtgc tagctcacgt agttcatcaa gccaatatga aacgtatgac 360

attcatttac gtttacattt agaatttata gaagctatta agggtgtttc taaaaatatt 420

agttatgatc gtaaaatcac atgtaacaag tgtcaaggaa caggagcaaa agatcctaaa 480

gatgttaaaa cttgtacaaa atgtcatggt cgtggaacta caattgaaaa cgttcattca 540

ttatttggaa caattcaaca agaagttgaa tgtcatgaat gtgagggaac tggaaaagtt 600

gctaatagca aatgcgaaca atgttatggc aaaaaagtta ttaatgaacg cgttaattta 660

acagttgaaa ttcctgctgg tacacaagat aatgaaaaat tagtagtaag taaaaaaggt 720

aatatcataa ataaccaaga atttgatctt tatttacata ttagcgttaa accatcaaaa 780

tattttgcat tcgatggttt agatatttac tcagaaactt atgttgatcc tattaaagca 840

attgttggtg gtgtaattga agtagtaaca acaagtggaa ttaaaactat tgaaattcca 900

ccaaatactc cagaaggtaa aaaatttaga attagtggcg ctggaattgt taataaaaaa 960

ccaaatattt ttagcaaaaa aaatggagat ttttacacaa caattcgtta tgcaaaacca 1020

cttgaattaa ctaaagaaga aattgcttat ttaaaaaata ttagtgctcg taccaaccaa 1080

agtgttgagt attataaaaa taaattatta aaagaggtta ataaataa 1128

<210> 3

<211> 25

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Thr Ile Glu Ile Pro Pro Asn Thr Pro Glu Gly Lys Lys Phe Arg Ile

1 5 10 15

Ser Gly Ala Gly Ile Val Asn Lys Lys

20 25

<210> 4

<211> 23

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Ala Ser Pro Glu Glu Ile Lys Thr Ala Phe Arg Lys Leu Ala Lys Glu

1 5 10 15

His His Pro Asp Arg Asn Lys

20

<210> 5

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 5

Glu Leu Thr Lys Glu Glu Ile Ala Tyr Leu Lys Asn Ile Ser Ala Arg

1 5 10 15

Thr Asn Gln Ser

20

<210> 6

<211> 26

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 6

Asn Ile Ser Tyr Asp Arg Lys Ile Thr Cys Asn Lys Cys Gln Gly Thr

1 5 10 15

Gly Ala Lys Asp Pro Lys Asp Val Lys Thr

20 25

<210> 7

<211> 18

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Phe Gly Ser Phe Phe Lys Asn Gly Ala Ser Ser Arg Ser Ser Ser Ser

1 5 10 15

Gln Tyr

<210> 8

<211> 19

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Asn Glu Ala Tyr Glu Val Leu Ser Asp Pro Lys Lys Arg Ala Gln Tyr

1 5 10 15

Asp Gln Phe

<210> 9

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 9

Ile Tyr Ser Glu Thr Tyr Val Asp Pro Ile Lys Ala Ile Val Gly Gly

1 5 10 15

Val Ile Glu Val

20

Claims (8)

1. The application of Uu-DnaJ protein or the polypeptide formed by one or more of the following amino acid fragments in the preparation of Uu immunogen or anti-Uu infection vaccine: AA _16-42 , AA _55-65 , AA _99-123 , AA _130-160 , AA _211-247 , AA _257-260 , AA _300-309 , AA _319-334 , AA _342-362 , AA _9-18 , AA _49-58 , AA _107-120 , AA _155-160 , AA _270-298 , AA _335-342 , AA _352-363 . 2 . The application according to claim 1 , wherein the amino acid sequence of the Uu-DnaJ protein is shown in SEQ ID NO: 1, or an amino acid sequence whose identity is more than 99%. 3 . 3. The application of the polypeptide formed by the partial amino acid sequence selected from the following one or more amino acid fragments of Uu-DnaJ protein in the preparation of Uu immunogen or anti-Uu infection vaccine: AA _16-42 , AA _55-65 , AA _99-123 , AA _130-160 , AA _211-247 , AA _257-260 , AA _300-309 , AA _319-334 , AA _342-362 , AA _9-18 , AA _49-58 , AA _107-120 , AA _155-160 , AA _270-298 , AA _335-342 , AA _352-363 . 4. application according to claim 3, is characterized in that, described formed polypeptide simultaneously comprises the partial amino acid sequence that is selected from following two groups of amino acid fragments: (1)AA _16-42 , AA _55-65 , AA _99-123 , AA _130-160 , AA _211-247 , AA _257-260 , AA _300-309 , AA _319-334 , AA _342-362 ; (2) AA _9-18 , AA _49-58 , AA _107-120 , AA _155-160 , AA _270-298 , AA _335-342 , AA _352-363 . 5. The application according to claim 3 or 4, wherein the sequence of the formed polypeptide is shown in any one of SEQ ID NOs: 3-9. 6. a vaccine against Uu infection, is characterized in that, with Uu-DnaJ protein in claim 1 and/or the polypeptide formed by one or more of its amino acid fragments as immunogen, or with claim 3-5 The resulting polypeptide of any one of the claims is an immunogen. 7. The vaccine according to claim 6, further comprising an immune adjuvant. 8. The vaccine according to claim 7, wherein the immune adjuvant is incomplete Freund's adjuvant, complete Freund's adjuvant, saponin, CpG oligonucleotide adjuvant, aluminum adjuvant or cytokine adjuvant.

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