CN107970444B

CN107970444B - Composite adjuvant and vaccine containing same

Info

Publication number: CN107970444B
Application number: CN201610949182.6A
Authority: CN
Inventors: 何秀云; 黄香玉; 庄玉辉; 张春青; 宋庆德; 朱传智; 邵进士; 李军丽
Original assignee: 309th Hospital of PLA
Current assignee: 309th Hospital of PLA
Priority date: 2016-10-25
Filing date: 2016-10-25
Publication date: 2022-03-01
Anticipated expiration: 2036-10-25
Also published as: CN107970444A

Abstract

The invention discloses an immune composite adjuvant, a preparation method thereof and application of the immune composite adjuvant in enhancing vaccine immunity. The invention also relates to a vaccine composition containing the immune composite adjuvant, and application of the vaccine composition in treating related diseases.

Description

Composite adjuvant and vaccine containing same

Technical Field

The invention relates to a compound adjuvant and a preparation method thereof. The composite adjuvant induces a shift of Th1 type immune response relative to a single adjuvant, and the composite adjuvant enhances immune response against intracellular bacteria such as Mycobacterium tuberculosis and the like and viruses.

Background

The first generation vaccines were typically live attenuated or whole killed vaccines, which were immunogenic but induced immunopathological responses in addition to protective immune responses. The new vaccines studied to date are mainly protective antigens or epitopes, which are relatively poorly immunogenic compared to the first generation vaccines and require adjuvants to enhance their immunogenicity. Adjuvants as part of vaccines enhance the immune response induced by antigens in vivo.

Protein vaccines are approved for their safety because of their clear components, but because of the inherent poor immunogenicity of antigens, adjuvants (adjuvants) are generally required for their assistance. Therefore, protein vaccines generally consist of an adjuvant and an antigen, and the adjuvant is injected together with the antigen or an adjuvant is injected into the body in advance to enhance the immune response of the body to the antigen or to change the type of the immune response.

The adjuvants are various, and can be divided into inorganic adjuvants (such as aluminum hydroxide and alum), organic adjuvants (such as microorganisms and their components, including BCG, Corynebacterium parvum, muramyl dipeptide, BCG polysaccharide nucleic acid, etc.) and synthetic adjuvants (such as antibacterial peptide and synthetic TLR agonist, etc.) according to their components. However, the number of adjuvants approved for clinical studies is very limited, MF59, AS03, AS04 and liposomes are adjuvants approved for use in humans following aluminum adjuvants [ Mlow ML et al, Curr Opin Immunol,2010,22: 411-.

An adjuvant used for a preventive vaccine described in the third pharmacopoeia of the people's republic of china published in 2010 is basically aluminum hydroxide (compiled by the national committee of pharmacopoeia, pharmacopoeia of the people's republic of china, P21-132, chemical industry press, 2010). Aluminum hydroxide is one of the aluminum adjuvants, which primarily enhances antigen-induced humoral immune responses, while for intracellular bacteria, such as tubercle bacillus, the body is cleared primarily through cellular immune responses. The aluminum adjuvant is used as an adjuvant of intracellular mycoprotein vaccines such as tuberculosis protein vaccines, and the effect of the aluminum adjuvant is difficult to meet the requirements of people.

The water-oil microspheres enhance natural immune response in a unique way. MF59 is an oil-in-water nano-scale emulsion, the vaccine composed of adjuvant and antigen mainly adopts intramuscular injection immunization, MF59 adjuvant regulates the temporary expression of muscle cells to genes related to immune activation and the induced transient release of endogenous ATP to promote cell recruitment, neutrophils, monocytes, macrophages and CD11b + cells are rapidly recruited to an injection site to form an immune enhancement microenvironment, and meanwhile, the recruited antigen presenting cells take up antigen and are conveyed to a drainage lymph node to present to T cells to start acquired immune response [ Seubert A, J Immunol,2008,180: 5402-type 5412; the cellular immunity induced by the vaccine with Vono M et al, Proc Natl Acad Sci USA,2013,110: 21095-. Whereas intraperitoneal injection of MF59 adjuvant human papillomavirus type 16 high dose antigen E2 produced Th 1-type immune response drift and cytotoxic activity [ Heinemann L et al, Viral Immunol,2008,21: 225-. Although experiments have shown that the TLR4 agonist E6020 and TLR9 agonist CpG ODN respectively form a composite adjuvant with MF59, which can enhance Th1 type immune response drift [ Baudner BC, et al, Pharm Res,2009,26: 1477-; singh M, et al, Hum vaccine immunolther, 2012,8: 486-; yang M, et al, Int Immunopharmacol,2012,13: 408-. GLA-SE has similar composition and adjuvanticity to MF59-E6020, SE mainly induces Th2 type immune response as adjuvant of Mycobacterium tuberculosis recombinant protein ID93, the same protein induces Th1 type immune response and protective immune response with GLA-SE or GLA-SE/CpG as adjuvant, but both initiate natural immunity through different signal pathways [ Bertholet S, et al, Sci Transl Med,2010,2:53ra 74; orr MT, et al, PLoS ONE,2014,9: e 83884; desbien AL, et AL, Eur J Immunol,2015,45: 407-; orr MT, et al, Eur J Immunol,2013,43: 2398-.

Disclosure of Invention

The present invention relates to the following:

1. an adjuvant cocktail comprising sorbitan trioleate, squalene, a surfactant and heat inactivated bacillus calmette-guerin (hbg) or high pressure homogeneous break BCG.

2. The composite adjuvant of item 1, wherein the amount of the sorbitol trioleate is 0.1% to 1% (w/v).

3. The composite adjuvant of

item

1 or 2, wherein the squalene is 1-10% (v/v).

4. The composite adjuvant of any one of items 1 to 3, wherein the surfactant is a nonionic surfactant; preferably tween; more preferably tween 80.

5. The composite adjuvant of any one of items 1 to 4, further comprising a buffer; preferably, the buffer solution is a sodium citrate buffer solution, a phosphate buffer solution and a histidine buffer solution; more preferably, the buffer has a concentration of 5-10mM and a pH of 6-7.

6. The adjuvant combination of any one of items 1-5, wherein the amount of sorbitol trioleate is 0.3% to 0.7%; preferably 0.4-0.6%, more preferably 0.5%.

7. The composite adjuvant of any one of claims 1 to 6, wherein the squalene is 3-7%; preferably 4 to 6%; more preferably 5%.

8. The composite adjuvant of any one of items 1 to 7, wherein the concentration of the surfactant is 0.3 to 0.7% (w/v); preferably 0.4-0.6%%; more preferably 0.5%.

9. The composite adjuvant of any one of items 1 to 8, wherein the amount of heat inactivated BCG (hBCG) or high pressure homogeneous crushed BCG is 5 to 500 μ g, more preferably 25 to 500 μ g; more preferably 50-500. mu.g; more preferably 50-450 μ g; more preferably 50-400 μ g; more preferably 50-400 μ g; more preferably 50-300. mu.g; most preferably 50-250. mu.g.

10. A method for preparing the composite adjuvant of any one of items 1 to 9, comprising

1) Adding a surfactant into the buffer solution, and uniformly stirring to obtain a water phase;

2) weighing sorbitan trioleate, adding squalene, and fully and uniformly mixing to obtain an oil phase;

3) adding the oil phase into the water phase, and fully and uniformly mixing;

4) fully emulsifying to form water-oil microspheres with the particle size of 160-220nm, preferably 170-210nm, more preferably 180-200nm, and adding heat-inactivated BCG (hBCG) or high-pressure homogeneous BCG after sterilization.

11. The method of item 10, wherein the amount of sorbitol trioleate is 0.1% to 1% (w/v).

12. The method of item 10 or 11, wherein the squalene is 1-10% (v/v).

13. The method of any of items 10-12, wherein the surfactant is a nonionic surfactant; preferably tween; more preferably tween 80.

14. The method of any one of items 10-13, further comprising a buffer; preferably, the buffer solution is a sodium citrate buffer solution, a phosphate buffer solution and a histidine buffer solution; more preferably, the buffer has a concentration of 5-10mM and a pH of 6-7.

15. The method of any of items 10-14, wherein the amount of sorbitol trioleate is 0.3% to 0.7%; preferably 0.4-0.6%, more preferably 0.5%.

16. The process of any one of claims 10 to 15, wherein the squalene is 3-7%; preferably 4 to 6%; more preferably 5%.

17. The method of any of items 10 to 16, wherein the concentration of the surfactant is 0.3 to 0.7% (w/v); preferably 0.4-0.6%%; more preferably 0.5%.

18. The method of any one of items 10-17, wherein the amount of heat inactivated bacillus calmette-guerin (hbg) or high pressure homogeneous disrupted BCG is 5-500 μ g, more preferably 25-500 μ g; more preferably 50-500. mu.g; more preferably 50-450 μ g; more preferably 50-400 μ g; more preferably 50-400 μ g; more preferably 50-300. mu.g; most preferably 50-250. mu.g.

19. The present invention also relates to a mycobacterium tuberculosis antigen or a fusion protein having mycobacterium tuberculosis immunoreactivity selected from the group consisting of:

(a) comprises or consists of an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 4 and yet is immunogenic;

(b) 4, one or more amino acids are substituted, deleted, inserted and/or added in the amino acid sequence shown in SEQ ID NO. 4, and still have immunogenicity;

(c) Is encoded by a nucleotide sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 3;

(d) is encoded by a nucleotide sequence which substitutes, deletes, inserts and/or adds one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 3; and

(e) encoded by a nucleotide sequence that hybridizes under at least medium stringency conditions, at least medium-high stringency conditions, at least high stringency conditions, or at least very high stringency conditions with the polynucleotide set forth in SEQ ID NO. 3.

20. The invention also relates to a nucleic acid comprising or consisting of:

(a) a polynucleotide having a nucleotide sequence that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID No. 3;

(b) a polynucleotide, the nucleotide sequence of which is substituted, deleted, inserted and/or added with one or more nucleotides in the nucleotide sequence shown in SEQ ID NO. 3;

(c) a polynucleotide whose nucleotide sequence hybridizes under at least medium stringency conditions, at least medium-high stringency conditions, at least high stringency conditions, or at least very high stringency conditions with the polynucleotide set forth in SEQ ID No. 3;

(d) A polynucleotide encoding a fusion protein having an amino acid sequence with at least 90%, 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID No. 4;

(e) a polynucleotide encoding a fusion protein in which one or more amino acids are substituted, deleted, inserted and/or added in the amino acid sequence shown in SEQ ID NO. 4; or

(f) A polynucleotide encoding SEQ ID NO. 4 but differing from SEQ ID NO. 3 due to codon degeneracy.

21. The invention also relates to a method for producing a mycobacterium tuberculosis antigen of item 19 or a fusion protein with mycobacterium tuberculosis immunoreactivity, comprising expressing the nucleic acid of item 20 in a suitable host cell.

The invention further relates to a nucleic acid construct or expression vector comprising the above polynucleotide, preferably pET28a-PstS 1-LEP. The present invention further relates to a host cell comprising the above-described construct or expression vector, preferably a cell of the family Enterobacteriaceae, more preferably an E.coli cell, still more preferably an E.coli BL21(DE3) cell.

In another aspect, the present invention also provides a method for preparing the above fusion protein having immunoreactivity with Mycobacterium tuberculosis or the above nucleic acid construct or expression vector (preferably PstS1-LEP), which comprises using the polynucleotide, nucleic acid construct, expression vector or host cell of the present invention. Specifically, the method comprises (a) culturing a host cell of the invention under conditions conducive for production of the fusion protein; and (b) recovering the fusion protein.

More preferably, the above preparation method comprises the steps of:

1) obtaining a linear vector-PstS 1 recombinant plasmid, preferably a linear PET28a-Pst S1 recombinant plasmid;

2) obtaining an LEP amino acid coding nucleic acid sequence shown as SEQ ID NO. 2, synthesizing, connecting the synthesized LEP gene to a vector, preferably a PUC19 vector, and forming a vector-LEP recombinant plasmid;

3) transforming a suitable host cell, preferably escherichia coli, by using the obtained vector-LEP recombinant plasmid, screening, selecting a monoclonal colony for culturing, extracting the plasmid for enzyme digestion to obtain an LEP gene segment, preferably performing double enzyme digestion, and then performing electrophoresis recovery to obtain the LEP gene segment;

4) connecting the linear vector PstS1 recombinant plasmid and the LEP gene segment, preferably by T4DNA ligase; introducing the vector PstS1-LEP recombinant plasmid obtained by the connection into a suitable host cell, preferably an Escherichia coli host cell, more preferably an Escherichia coli cell BL21(DE 3);

5) culturing said host cell under conditions conducive to expression of the sequence of said vector-PstS 1-LEP; and

6) recovering, purifying, renaturing and/or identifying the expressed fusion protein.

22. The invention also relates to a pharmaceutical composition comprising an immunocomplex adjuvant according to any of items 1 to 9 and an antigen, preferably the antigen is an intracellular bacterial or viral antigen, more preferably a mycobacterium tuberculosis antigen or a cytomegalovirus antigen, more preferably an antigen or fusion protein according to item 19 or a Cytomegalovirus (CMV) gB antigen (CMV gB).

23. The pharmaceutical composition of item 22, which is a vaccine, further, which is a prophylactic and/or therapeutic vaccine for intracellular bacterial infection or viral infection, more preferably a prophylactic and/or therapeutic vaccine for mycobacterium tuberculosis infection or cytomegalovirus infection.

24. Use of the immune complex adjuvant of any one of items 1-9 to increase the immune reactivity of the body, preferably the immune reactivity is against intracellular bacteria or viruses, preferably the immune reactivity is against mycobacterium tuberculosis or cytomegalovirus.

25. Use of the adjuvant multiplex immunoassay of any one of items 1 to 9 for the preparation of a pharmaceutical composition or vaccine, preferably a prophylactic and/or therapeutic pharmaceutical composition or vaccine against intracellular bacteria or viruses, preferably against mycobacterium tuberculosis or cytomegalovirus.

26. Use of the immune complex adjuvant of any one of items 1-9 for the prevention and/or treatment of intracellular bacterial or viral infections, preferably the prevention and/or treatment of infections with mycobacterium tuberculosis or cytomegalovirus.

27. A method of preventing and/or treating an intracellular bacterial or viral infection comprising administering to a patient a prophylactically or therapeutically effective amount of the pharmaceutical composition or vaccine of item 22 or 23.

28. In the uses and methods of the above, the pharmaceutical composition or vaccine is administered subcutaneously or intramuscularly.

29. The fusion protein is a multi-epitope antigen, has good immunoreaction and can be used for diagnosing tuberculosis. Therefore, the invention also relates to the use of the fusion protein in diagnosing tuberculosis. In one embodiment, the fusion protein is used to detect serum IgG for diagnosis of tuberculosis; in another embodiment, the fusion protein is used to detect serum IgM for diagnosis of extrapulmonary tuberculosis. Both assays have high sensitivity and specificity. Furthermore, in one embodiment, the fusion protein diagnoses tuberculosis and extrapulmonary tuberculosis by detecting IgG and IgM in the same well.

30. The present invention relates to a kit for diagnosing tuberculosis comprising the antigen or fusion protein described in the above item 19.

31. The invention also relates to the use of the antigen or fusion protein described in item 19 above in the preparation of a tuberculosis diagnostic kit.

The composite adjuvant and the composition containing the composite adjuvant and the antigen overcome the defects of common immunogenicity deficiency of the tubercle bacillus and common and weak immune response enhancement effect of the existing adjuvant on intracellular bacteria or viruses. By utilizing the compound adjuvant, the immunoreactivity and the immune effect of intracellular bacteria or viruses, particularly mycobacterium tuberculosis or cytomegalovirus are effectively improved, and the effective prevention and/or treatment of tuberculosis are promoted.

The intracellular bacteria are pathogenic bacteria which stay in host cells and propagate after invading human body for most of time. Such as tubercle bacillus, leprosy bacillus, brucella, etc. The role of humoral immunity in such bacterial infections is limited because antibodies cannot enter cells, and the defense function against intracellular infections is dominated by cellular immunity. For example, the body is infected with tubercle bacillus for the first time, because cellular immunity is not established, although phagocytes can phagocytize the tubercle bacillus, the tubercle bacillus cannot be effectively digested and killed, so pathogenic bacteria are easy to spread and spread along with the phagocytes in the body, and the whole body infection is caused. However, in the infection process, the body gradually forms cellular immunity under the stimulation of pathogenic bacteria, and various lymphokines released by sensitized lymphocytes activate phagocytes, so that the phagocytic digestive ability of the body can be greatly enhanced, the survival of the pathogenic bacteria in the phagocytes is inhibited, and the immunity for defending against the reinfection of the same pathogenic bacteria is obtained.

The term "Mycobacterium tuberculosis" as used herein may also be referred to simply as "Mycobacterium tuberculosis".

The virus of the invention is a microorganism without cell structure and with vital characteristics of heredity, replication and the like. It is smaller than bacteria, has no cell structure, consists of protein and nucleic acid, and can only proliferate in living cells, and this characteristic is similar to that of intracellular bacteria.

Bacillus Calmette-Guerin (BCG for short, Chinese name comes from inventor's Calmette-Meerz) is a vaccine for preventing tuberculosis, live Mycobacterium bovis (Mycobacterium bovis) is used for in vitro 230 generations of culture on a special artificial culture medium for attenuation, loses the pathogenic capability to human, but still maintains enough immunogenicity, so the BCG is an attenuated live vaccine. The inoculation of BCG vaccine has obvious effect in preventing tuberculosis, especially serious tuberculosis possibly endangering children life, such as tuberculous meningitis, and tuberculosis of millet. "hBCG" (heat-aggregated bacillus Calmette-Guerin) is the abbreviation of heat inactivated BCG vaccine, so "hBCG", "heat inactivated BCG vaccine" and "heat inactivated BCG vaccine hBCG" can be used interchangeably.

"high pressure homogeneous disruption BCG" is a cell-free product resulting from a high pressure homogeneous disruption operation following heat inactivation of BCG. For example, the method of embodiment 1 of the present invention can be adopted to thermally inactivate BCG collected by culture at 80 ℃ for 1h, crush the BCG with a low-temperature ultrahigh-pressure continuous flow cell crusher (JN3000Plus), and cycle for five times under the condition of power of 1500-1700 Bar; fully crushing the thallus, centrifuging for 10min at 5000 rpm to remove the BCG thallus which is not crushed, and irradiating and sterilizing by cobalt 60 to obtain the product, namely the high-pressure homogeneous crushed BCG.

"Heat inactivated BCG vaccine (hBCG)" is heat inactivated BCG, structurally intact non-toxic Mycobacterium bovis, and "high pressure homogeneous disrupted BCG" is a disrupted product of inactivated non-toxic Mycobacterium bovis.

The term "fusion protein" as used herein refers to a region of one protein fused to the N-or C-terminus of a region of another protein. Fusion proteins are typically produced by fusing a polynucleotide encoding one protein to a polynucleotide encoding another protein. Techniques for producing fusion proteins are known in the art and include ligating the coding sequences encoding the polypeptides so that they are in frame (in frame) and that expression of the fusion protein is under the control of the same promoter and terminator. Fusion proteins can also be constructed using the intein (intein) technique, where the fusion protein is produced post-translationally (Cooper et al, 1993, EMBO J.12: 2575-. The fusion protein may also comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved, releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, Martin et al, 2003, J.Ind.Microbiol.Biotechnol.3: 568-576; svetina et al, 2000, J.Biotechnol.76: 245-; Rasmussen-Wilson et al 1997, appl.environ.Microbiol.63: 3488-; ward et al, 1995, Biotechnology 13:498- > 503; and Contreras et al, 1991, Biotechnology 9: 378-; eaton et al, 1986, biochem.25: 505-; Collins-Racie et al, 1995, Biotechnology 13: 982-; carter et al, 1989, Proteins: Structure, Function, and Genetics 6: 240-; and the sites disclosed in Stevens,2003, Drug Discovery World 4: 35-48.

The term "codon degeneracy" as used herein refers to the phenomenon that an amino acid is encoded by more than one triplet code.

The term "immunoreactivity" as used herein refers to the ability of an antigenic molecule to specifically bind to a corresponding immune response product (antibody or sensitized lymphocytes) in vitro or in vivo, also referred to as antigenicity.

The term "complex adjuvant" as used herein refers to a novel adjuvant consisting of two or more adjuvants having different mechanisms of action.

The term "epitope" as used herein, also called antigenic determinant, refers to a chemical group in an antigenic molecule that determines the specificity of an antigen, which is the basic unit capable of specifically binding to the T cell antigen receptor TCR or B cell antigen receptor BCR.

The term "immunogenicity" as used herein refers to the ability of the body to be stimulated to form specific antibodies or to sensitize lymphocytes. It also refers to the property of antigen to stimulate specific immune cells, activate, proliferate, differentiate, and finally produce immune effector antibodies and sensitized lymphocytes.

The term "host cell" as used herein includes any cell type that is susceptible to transformation, transfection, transduction, and the like using a nucleic acid construct or expression vector comprising a polynucleotide of the present invention.

The term "expression vector" as used herein is defined as a linear or circular DNA molecule comprising a polynucleotide encoding a polypeptide of the present invention linked to other nucleotides for expression thereof. The term "construct" or "nucleic acid construct" refers to a nucleic acid molecule, either single-or double-stranded, that is isolated from a naturally occurring gene or that is modified to contain a non-naturally occurring gene, the construct may contain the regulatory sequences required for expression of the coding sequence of the invention.

The term "sequence identity" or "identity" as used herein is used to describe the relatedness of two nucleotide or amino acid sequences, expressed as a percentage, using conventional algorithms and penalties rules for sequence alignment as known in the art.

The term "substitution" as used herein means that a nucleotide/amino acid occupying a certain position is replaced with a different nucleotide/amino acid; "deletion" means the removal of the nucleotide/amino acid occupying a position; "insertion" means the addition of nucleotides/amino acids in the middle of a sequence, next to and immediately after the nucleotide/amino acid occupying a position; "addition" means the addition of nucleotides/amino acids at both ends of the sequence.

The terms "very high stringency conditions", "medium-high stringency conditions", "medium stringency conditions" as used herein mean prehybridization and hybridization at 42 ℃ in 5X SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured salmon sperm DNA, and 50%, 35%, and 35% formamide, respectively, according to standard Southern blotting procedures for 12 to 24 hours for probes of at least 100 nucleotides in length, respectively. The carrier material is finally washed three times each for 15 minutes using 2 XSSC, 0.2% SDS at 70 ℃, 65 ℃, 60 ℃ and 55 ℃ respectively.

The term "kit" used in the present invention refers to a kit prepared by using the protein of the present invention to complete the diagnosis of tuberculosis. The kit is used for diagnosing tuberculosis including pulmonary tuberculosis, extrapulmonary tuberculosis and the like. The kit comprises a container containing the fusion protein of the present invention. The fusion proteins of the invention may be packaged in any convenient and suitable packaging means. The kit of the present invention may further comprise other containers, which may contain standards, antibodies or labeled antibodies, enzymes, substrates or buffers, etc., for detection, respectively. In the kit, a label and a package insert are included to provide instructions for use of the kit. Other materials may also be included to suit the needs of the user, such as microtiter plates and the like.

The term "adjuvant" of the present invention refers to a non-specific immunopotentiator, which is clinically used for enhancing the immune response of animal bodies to antigen substances or changing the immune response type thereof by single inoculation or simultaneous inoculation after mixing with a vaccine before vaccine immunization, and particularly after secondary immunization (secondary immunization), the immune enhancement effect is more obvious. The term "co-adjuvant" refers to the combination of two or more adjuvants or adjuvants with other agents to induce a stronger immune response.

The term "tuberculosis" in the present invention refers to a chronic infectious disease caused by infection with mycobacterium tuberculosis. The term "tuberculosis" refers to an infectious disease caused by infection of the lung by Mycobacterium tuberculosis. The term "extrapulmonary tuberculosis" refers to tuberculosis occurring in various parts outside the lung due to pulmonary lesions spreading to various organs of the human body through the blood or lymphatic system.

The term "vaccine" of the present invention refers to a preventive biological preparation for preventive vaccination of a subject for the purpose of preventing, controlling the occurrence and/or prevalence of infectious diseases, and specifically includes preparations for prevention, diagnosis and treatment prepared from microorganisms or toxins thereof, enzymes, human or animal sera, cells, and the like.

Brief Description of Drawings

FIG. 1 shows the cleavage map of the recombinant plasmid. In the figure, lane 1 is the lambda DNA/HindIII marker; lane 2, pET-28a-PstS1-LEP digested with NcoI; lane 3 shows pET-28a-PstS1-LEP digested with BamHI; lane 4, pET-28a-PstS1-LEP digested with NcoI + BamHI; lane 5, pET-28a-PstS1-LEP digested with NcoI + HindIII; lane 6 shows pET-28a-PstS1-LEP digested with BamHI + HindIII; lane 7 is a 100bp DNA marker (where bands indicate 200, 300, 400, 500, 600, 700, 800, 900, 1000 and 1500bp, respectively). As shown in FIG. 1, the desired gene of the fragment of about 1.1kb and the linear plasmid DNA of about 5.9kb were obtained by double digestion with the recombinant plasmids NcoI and BamHI, the desired gene of about 1.7kb and the linear plasmid DNA of 5.3kb were obtained by double digestion with NcoI and HindIII, the desired gene of about 0.6kb and the linear plasmid DNA of 6.4kb were obtained by double digestion with BamHI and HindIII, and the linear plasmid DNA of about 7.0kb was obtained by single digestion with NcoI and BamHI, respectively. The NcoI and BamHI double digestion target gene fragment is PstS1 coding gene, the BamHI and HindIII double digestion target gene fragment is multi-epitope protein LEP coding gene, therefore, the DNA sequence of the coding protein is successfully connected with the carrier at the accurate digestion site, the recombinant plasmid contains the fusion gene of the pre-expression protein, and the size of the inserted exogenous gene is consistent with the theory.

FIG. 2 shows the SDS-polyacrylamide gel electrophoresis pattern of the recombinant engineered bacteria and the purified protein and the immunoblotting pattern of the purified renaturated protein. In fig. 2 a: m1 is protein standard molecular weight; 1 is pET-28a-PstS1-LEP/BL21(DE3) host strain which is not induced by IPTG; 2 is IPTG induced pET-28a-PstS1-LEP/BL21(DE3) host strain; 3 is IPTG induced pET-28a-PstS1-LEP/BL21(DE3) host bacteria ultrasonic supernatant; 4 IPTG induced pET-28a-PstS1-LEP/BL21(DE3) host bacteria ultrasonic precipitation; 5 purifying protein with anion chromatographic column; 6 is renaturation protein. In fig. 2 b: m2 is the molecular weight of prestained protein standard; 1, reaction of PstS1-LEP protein with anti-PstS 1 monoclonal antibody; 2, reaction of PstS1-LEP protein and serum IgG of a tuberculosis patient; 3 reaction of PstS1-LEP protein with serum Ig (G + M) of tuberculosis patients; and 4, reaction of PstS1-LEP protein with serum IgM of tuberculosis patients.

The FIG. 2a shows that the engineering bacteria express the recombinant protein under IPTG induction and express in the form of inclusion body, and the engineering bacteria hardly express the recombinant protein without IPTG induction. Purifying and renaturing the chromatographic column to obtain the purer recombinant protein. FIG. 2b shows that the renatured recombinant protein positively reacted with anti-PstS 1 monoclonal antibody, IgG and IgM in the serum of tuberculosis patients and Ig (G + M).

FIG. 3 shows the particle size detection results of two batches of water-oil microspheres prepared at different times. FIG. 3A shows the particle size measurement of the water-oil microsphere prepared in 10 months of 2014, in which the average particle size is 1846.4 nm; fig. 3B shows the particle size measurement of the water-oil microsphere prepared in 2015 in 4 months, wherein the average particle size is 193 nm.

FIG. 4 shows that the water-oil microsphere, hBCG alone or in combination externally stimulate the RAW264.7 cells to secrete TNF-alpha, MCP-1 and IL-1 beta, and neither the water-oil microsphere, hBCG alone nor in combination can stimulate the RAW264.7 cells to secrete IL-1 beta; except for the low dose of hBCG, other cells can stimulate RAW264.7 cells to secrete TNF-alpha in vitro; only the composite adjuvant water-oil microspheres/hBCG can stimulate the RAW264.7 cells to secrete MCP-1. Different letters on the bars in the graph indicate that different combinations of in vitro stimulated cells secrete the same cell/chemokine comparison shows statistical differences, where a indicates that the difference from the unstimulated wells is less significant; b shows that the difference is significant compared with 25 mug/ml hBCG; c shows significant difference compared with 50 mug/ml hBCG; d represents a reaction with 1: the comparison of the 250 water-oil microspheres is remarkably different.

FIG. 5A shows that mice immunized with aqueous-oil microspheres/50. mu.g/ml hBCG subcutaneously or intramuscularly for 24 hours, the percentage of DC cells, macrophages, neutrophils and monocytes were recruited to the muscle tissue at the injection site. FIG. 5B shows that mice immunized with aqueous-oil microspheres/250. mu.g/ml hBCG subcutaneously or intramuscularly for 24 hours, the percentage of DC cells, macrophages, neutrophils and monocytes were recruited to the muscle tissue at the injection site. Both intramuscular and subcutaneous immunization promote recruitment of immune cells to the muscle tissue at the site of injection.

FIG. 6 shows that when different adjuvants are used to immunize mice with the protein PstS1-LEP of the invention, compared with the PstS1-LEP immunization group, only the spleen cells of the mice immunized with the water-oil microsphere/hBCG and the protein PstS1-LEP of the invention secrete significantly higher numbers of IFN-gamma and IL-2 with specificity to PstS1-LEP antigen, while Al (OH)₃The number of cells secreting IL-4 from splenocytes of mice immunized with the protein PstS1-LEP of the invention is obviously increased. FIG. 6 shows different letters on the column indicate that different adjuvants are combined with PstS1-LEP to immunize mice, and the comparison of the level of the same cytokine secreted by the spleen cells of the mice shows statistical significance, wherein a indicates the difference compared with the water-oil microsphere immunization groupIs remarkable; b shows significant difference compared with the hBCG immune group; c indicates significant difference compared with the PstS1-LEP immune group.

FIG. 7, the different letters in the bars indicate that the mice immunized with different adjuvants in combination with PstS1-LEP exhibit statistical significance in comparison to the level of the same cytokine in the culture supernatant stimulated in vitro by the splenocytes, wherein a indicates a significant difference from the water-oil microsphere immunization group; b shows significant difference compared with the hBCG immune group; c indicates significant difference compared with the PstS1-LEP immune group. This figure shows that mice immunized with different adjuvants in combination with the protein of the invention PstS1-LEP, compared to the PstS1-LEP immunization group: the water-oil microspheres and the water-oil microspheres/hBCG are respectively compatible with the protein PstS1-LEP to immunize a mouse, so that the spleen cells of the mouse secrete PstS1-LEP antigen specific IFN-gamma; the water-oil microsphere/hBCG is matched with the protein PstS1-LEP to immunize a mouse, so that the secretion of PstS1-LEP antigen specific IL-2 by the spleen cell of the mouse is obviously enhanced.

FIG. 8 shows different letters on the bars representing different adjuvants in combination with PstS1-LEP to immunize mice, and the mice exhibited statistical significance in comparison of the levels of the same cytokine in vitro stimulation of macrophage cells in abdominal cavity of the mice in culture supernatants, wherein a represents significant difference compared with the water-oil microsphere immunization group; b shows significant difference compared with the hBCG immune group; c indicates significant difference compared with the PstS1-LEP immune group. This figure shows that mice immunized with different adjuvants in combination with the protein of the invention PstS1-LEP, compared to the PstS1-LEP immunization group: the water-oil microspheres and the protein PstS1-LEP are matched to immunize a mouse, so that the secretion of PstS1-LEP antigen specific IL-12 by abdominal macrophages of the mouse is obviously enhanced; hBCG, water oil microsphere, Al (OH)₃The hBCG is respectively matched with the protein PstS1-LEP to immunize a mouse, so that the secretion of PstS1-LEP antigen specific IL-1 beta by abdominal cavity macrophages of the mouse is obviously enhanced.

FIG. 9 shows that different doses of hBCG alone or in combination with water and oil microsphere immunized mice, mice splenocyte secretion BCG-PPD specific IFN-gamma and IL-2 all showed rising trend with hBCG dose increase, same dose of hBCG, composite adjuvant water and oil microsphere/hBCG were superior to single hBCG. In the case of IL-4, there is no obvious rule.

FIG. 10 shows that different doses of hBCG alone or in combination with water and oil microspheres immunized mice, mouse peritoneal macrophages secreted BCG-PPD specific IL-12 and IL-1 beta, low and medium doses of hBCG and water and oil microspheres composed of composite adjuvant immunized mice enhanced macrophage secretion of IL-12 and IL-1 beta, and high doses of hBCG and water and oil microspheres composed of composite adjuvant immunized mice inhibited macrophage secretion of IL-12.

FIGS. 11A-C show water-oil microspheres and/or hBCG, as K6 antigen-adjuvanted mice, and the results of IgG subtype detection of serum antibodies show that: hBCG, Water-emulsion microsphere/hBCG, Al (OH) compared with K6 antigen immunization group₃Respectively, the hBCG and K6 antigen are compatible to immunize mice to obviously induce K6 specific IgG1 antibody, but only Al (OH)₃The mice immunized by the combination of the hBCG and the K6 antigen remarkably induce the K6 specific IgG2a antibody. The different letters on the column in the figure indicate that the comparison between different immune groups shows statistical significance, and a indicates that the difference is significant compared with the water-oil microsphere immune group (P)<0.05); b shows significant difference compared with the hBCG immune group (P)<0.05); c shows significant difference compared with K6 immune group (P)<0.05)。

FIGS. 12A-C show the induction of IFN-. gamma.IL-2 and IL-4 secretion by splenocytes from mice immunized with K6 antigen in combination with various adjuvants.

FIGS. 13A-B show hBCG, Water-in-oil microspheres/hBCG, Al (OH)₃The hBCG is used as a K6 antigen adjuvant to immunize mice, and has the function of secreting K6 specific IL-12 and IL-1 beta to abdominal cavity macrophages. The results showed that water emulsion microspheres, water oil microspheres/hBCG, Al (OH) compared to the K6 immunization group₃The hBCG and K6 are respectively combined to immunize mice to enhance the secretion of IL-12 by macrophages. The different letters on the bars in the graph indicate that the comparison between different immune groups shows statistical significance, and a indicates that the difference is significant compared with the water-oil microsphere group (P) <0.05); b shows significant difference compared with the hBCG group (P)<0.05); c shows significant difference compared with K6 (P)<0.05)。

FIG. 14 shows the effect of water-oil microspheres and/or hBCG in promoting CMV gB induction of anti-CMV gB IgG antibody production. Wherein, A: mice immunized with water-oil microspheres, B: water oil microsphere/hbbcg immunized mice, C: CMV gB antigen immunized mice, D: water-oil microsphere + CMV gB immunized mice, E: water oil microsphere/hbbcg + CMV gB immunized mice, F: water and oil microsphere/hbbcg + CMV gB immunized mice. Groups A-D and F were immunized intramuscularly and group E was immunized subcutaneously. In the figure,: p <0.01

FIGS. 15a-b show the effect of water-oil microspheres and/or hBCG on CMV gB-specific IFN-. gamma.and IL-4 secretion. Wherein, A: mice immunized with water-oil microspheres, B: water oil microsphere/hbbcg immunized mice, C: CMV gB antigen immunized mice, D: water-oil microsphere + CMV gB immunized mice, E: water oil microsphere/hbbcg + CMV gB immunized mice, F: water and oil microsphere/hbbcg + CMV gB immunized mice. Groups A-D and F were immunized intramuscularly and group E was immunized subcutaneously. In the figure: p <0.05, x: p < 0.01.

FIGS. 16A-C show the humoral immune response induced by the composite adjuvant prepared by different preparation processes, adjuvanted with the protein PstS1-LEP of the present invention. IgG and IgG1 are absorbance values (A and B) measured at a serum 1:50000 dilution; IgG2a is the absorbance (C) measured at a serum 1:2500 dilution.

FIGS. 17A-C show the cellular immune response induced by the composite adjuvant prepared by different preparation processes and assisted by the protein PstS1-LEP of the invention. The figure shows the number of spot-forming cells secreting IFN-. gamma.IL-4, IL-17 specific for the protein PstS1-LEP of the invention.

Examples

Example 1: preparation of water-oil microsphere composite adjuvant

(1) Preparation of water-oil microspheres

Taking 456ml 10mmol/L citric acid buffer solution with pH6.5, adding Tween 80 (0.5%), and stirring uniformly (water phase); weighing 2.5g sorbitol trioleate, adding 21.5ml-25ml, preferably 21.5ml of squalene, and mixing well (oil phase); magnetically stirring the aqueous phase, slowly adding the oil phase, and continuing stirring for 1 h. Performing ultrasonic treatment for three times and 30min each time, then crushing by a low-temperature ultrahigh-pressure continuous flow cell crusher (JN3000Plus), and circulating for five times under the condition of power of 1500-1700 Bar; is milk white, does not delaminate, has no obvious oil drops and is even to hang on the wall. 0.22 μm filter sterilized, packaged, and stored at 4 deg.C (O' Hagan DT, Ott GS, Nest GV, etc. The history of microspheres in water and oil

adjuvant:a phoenix that arose from the ashes.Expert Rev Vaccines,2013,12(1): 13-30). The effective period is 6-12 months. The average particle size is 180-200nm measured by particle size test, and the results of particle size test of two batches of water-oil microspheres prepared in different periods are shown in FIG. 3A and FIG. 3B.

(2) Preparation of hBCG

The strain is the same as BCG vaccine for intradermal injection (Chengdu biological products research institute, LLC), namely BCG D2PB 302. Dissolving 1 lyophilized BCG vaccine for intradermal injection in 0.2ml sterile water for injection, inoculating on 1 improved slope of Roche medium, and adsorbing for 30 min; culturing at 37 deg.C for 4-5 weeks until BCG colony grows over the slant. And (3) subculturing and expanding the recovered BCG vaccine in an improved Roche medium at 37 ℃ for about 3 weeks, then subculturing and expanding the recovered BCG vaccine in the improved Roche medium at 37 ℃ for about 3 weeks. Collecting thallus on slant, adding Sutong culture medium containing 10% glycerol, suspending, and subpackaging in freezing tube at-70 deg.C for use as seed bacteria. Transferring the rest thallus to SUTONG potato culture medium, culturing at 37 deg.C for 2-3 weeks, collecting BCG, grinding, washing, adjusting bacteria concentration to 100mg/ml, heat inactivating at 80 deg.C for 1 hr, and storing at 4 deg.C. The BCG is heat inactivated, namely the hBCG used in the composite adjuvant of the invention.

(3) Compound adjuvant and preparation for compatibility with antigen

When the composition is used, the water-oil microspheres and the hBCG are mixed uniformly, the water-oil microspheres account for 50 percent (V/V), the final concentration of the hBCG is 25-50 mu g/ml (in vitro stimulation) or 50-250 mu g/0.2ml (immunized mice), and PBS with corresponding volume is added; mixing tip head for more than 30 times.

When the composition is used, the water-oil microspheres and the hBCG are mixed uniformly, and simultaneously, the antigen is added; the vaccine contains 50% (V/V) of water-oil microspheres, the final concentration of hBCG is 5-250 mug/0.2 ml, the final concentration of antigen is 10 mug/0.2 ml, and PBS with corresponding volume is added; mixing tip head for more than 30 times.

Example 2: the compound adjuvant stimulates macrophages to secrete monocyte chemotactic protein-1 (MCP-1) in vitro

DMEM high-sugar medium adjusted to 1 x 10⁶The/ml passage cells RAW264.7 (purchased from the cell resource center of the institute of basic medicine of Chinese academy of medical sciences) are distributed into 12-well culture plates, and each well is divided intoLoading 1ml of cell suspension at 37 deg.C with 5% CO₂And culturing for 5 h. Washing non-adherent cells, adding 1ml DMEM high-sugar complete medium (containing 10% high-quality fetal calf serum and diabodies (i.e. penicillin and streptomycin diabodies)) to each well, adding 50 μ l of stimulus, repeating 2 wells for each stimulus, adding 50 μ l of DMEM high-sugar complete medium to control wells, 37 deg.C, and 5% CO₂Culturing for 24h, collecting cell culture supernatant, and storing at-80 deg.C. The stimulating agent is prepared from DMEM high-sugar complete culture medium, and comprises 1:5 and 1:12.5 diluted water-oil microspheres, 0.5mg/ml hBCG, 1:5 water-oil microspheres and 0.5mg/ml hBCG, 1:5 water-oil microspheres and 1mg/ml hBCG, 1:12.5 water-oil microspheres and 0.5mg/ml hBCG, and 1:12.5 water-oil microspheres and 1mg/ml hBCG. Detecting the content of cell culture supernatant cytokines by a sandwich ELISA method: TNF-. alpha.IL-1. beta. and MCP-1ELISA kit instructions (BD Bioscience, USA). The water-oil microspheres and hBCG alone or in combination stimulated TNF-alpha, IL-1 beta and MCP-1 secretion from RAW264.7 as shown in FIG. 4.

50 mu g/ml of hBCG, 1:100 water-oil microspheres, 1:250 water-oil microspheres and any dosage of composite adjuvant water-oil microspheres/hBCG can effectively stimulate RAW264.7 cells to secrete TNF-alpha, while only any dosage of composite adjuvant water-oil microspheres/hBCG can effectively stimulate RAW264.7 cells to secrete MCP-1, and any adjuvant and combination thereof cannot stimulate RAW264.7 cells to secrete IL-1 beta.

Example 3: the compound adjuvant of the invention is injected subcutaneously and intramuscularly to enhance the recruitment of natural immune cells of muscle tissues at the injection site

(1) Intramuscular injection of single or compound adjuvant to recruit natural immune cells in muscle tissue at different time points

BALB/c mice were divided into 8 groups of 9 mice each. PBS, water and oil microspheres, 50 mu g of hBCG, PstS1-LEP, 250 mu g of hBCG, water and oil microspheres/50 mu g of hBCG/PstS1-LEP and water and oil microspheres/250 mu g of hBCG are respectively injected at the abundant part of the right hind leg muscle. After injection, 3 mice are killed in each group at 24 hours, 48 hours and 72 hours respectively, and muscle tissues at the injection part of the right leg of the mice are taken and cut into 2-4 mm; dissolving collagenase according to the specification of collagenase A, D, P (product of Tian and whirly company in Germany and America), sequentially adding 9 mul of collagenase A, 50 mul of collagenase D, 12 mul of collagenase P and 1.4ml of cell culture solution DEME into a C tube of a Gentle mild tissue processor (Gentle MACS), mixing uniformly, putting the muscle tissue cut by one mouse into the enzymolysis mixed solution of the C tube of the Gentle MACS, carrying out water bath at 37 ℃ for 60min, carrying out slight oscillation continuously, and treating the water bath for 1 time by adopting the program muscle-01 of the Gentle MACS after the water bath is finished; the 37 ℃ water bath was continued for another 30min with Gentle shaking and the programme of Gentle MACS, muscle-01, was reprocessed 1 time. And adding 3ml of DEME culture solution into the muscle homogenate suspension, uniformly mixing, filtering the homogenate solution to a 15ml centrifuge tube through a 200-mesh nylon membrane, and adding DMEM to 10 ml. The cells were collected by centrifugation, washed 1 time with 10ml DMEM, pelleted with 5ml DMEM suspension cells, filtered 1 time through a 200 mesh nylon membrane, centrifuged to suspend the cells, pelleted with PBS 1 time, and suspended in 700. mu.l PBS. 300 μ l of cell suspension was labeled with the following fluorescent antibodies: CD11b-FITC, F4/80-APC, Ly6G-PE and CD14-Percp-CY5.5, and another 300. mu.l cell suspension was labeled with the following fluorescent antibodies: CD14-Percp-CY5.5, CD11c-APC, CD45R/B220-FITC, CD 83-PE. After washing, the labeled cells were loaded on a flow cytometer for analysis, and the results were analyzed using the software on the instrument (tables 1 and 2).

Table 1: recruitment of DC cells at the site of injection

Note: CD11c + refers to the percentage of CD11c + positive cells to the first round gate of CD14 positive cells, and CD11c + CD83+ refers to the percentage of CD11c + CD83+ positive cells to the first round gate of CD14 positive cells.

Table 2: macrophage, neutrophil and monocyte recruited at injection site

Note: the neutrophil is the percentage of the number of Ly6G and CD11b double positive cells in the first round of gate, the monocyte is the percentage of the number of CD14 single positive cells in the first round of gate, and the macrophage is the percentage of the number of F4/80 and CD11b double positive cells in the first round of gate CD14 single positive cells.

Table 1 shows: CD11c + cells and CD11c + CD83+ cells represent two different types of Dendritic Cells (DC) from different sources, and DC cells at the injection site show a trend of decreasing with the prolonging of the immunization time. The content of CD11c + cells recruited by 50 mu g of hBCG immunized mice for 24 hours is the highest, while the content of CD11c + CD83+ cells immunized mice for 24 hours is the highest.

Table 2 shows: the neutrophil and monocyte show a growing trend along with the prolonging of the immunization time, the percentage content of the neutrophil recruited at the injection site of the mice in the water-oil microsphere/50 mug hBCG/antigen immunization group is higher than that of the neutrophil recruited at the injection site of the mice in the PBS, 50 mug hBCG, antigen, 250 mug hBCG and water-oil microsphere/50 mug hBCG immunization group (P is less than 0.05), and the percentage content of the monocyte recruited at the injection site of the mice in the antigen and the water-oil microsphere/50 mug hBCG immunization group is obviously higher than that of the water-oil microsphere immunization group (P is less than 0.05) and slightly higher than that of the PBS group (P is 0.075 and P is 0.061), besides the statistical difference of the comparison between the neutrophil content and the monocyte content of each group, the comparison difference between the immunization 24 hours of other groups and the comparison of each group at other times has no statistical significance. Macrophages show relatively stable trend at three detection time points, and the macrophage recruitment effect is the best in 24 hours by using water and oil microspheres/50 mu g of hBCG/antigen immune group; the macrophage recruitment effect of the water-oil microsphere/50 mu g of hBCG immune group is best at 48 hours; macrophage recruitment was best in the 72 hour water oil microsphere/250 μ g hcbcg immunized group.

Thus, tables 1 and 2 show that: immunization for 24 hours, water oil microspheres/50 μ g of hBCG enhanced DC cell and monocyte recruitment, while water oil microspheres/50 μ g of hBCG/antigen enhanced neutrophil and macrophage recruitment. Immunization for 48 hours, water and oil microspheres/50 μ g of hBCG enhanced DC cell, macrophage, monocyte recruitment.

(2) Water-oil microsphere/hBCG composite adjuvant muscle and subcutaneous two injection ways for immunizing muscle tissue at 24-hour injection site to recruit natural immune cells

BALB/c mice were divided into 4 groups of 9 mice each. Respectively injecting immune water-oil microspheres/50 mug hBCG and water-oil microspheres/250 mug hBCG subcutaneously at the abundant part of the right hind leg muscle, or respectively injecting water-oil microspheres/50 mug hBCG and water-oil microspheres/250 mug hBCG intramuscularly at the same position of subcutaneous injection. Muscle tissue at the injection site is taken after 24 hours of injection, single cells are separated by the enzymolysis-mechanical action of the muscle tissue, and the operation of the flow cytometry analysis test is the same as the above.

Fig. 5A shows: mice immunized with hBCG subcutaneously or intramuscularly at 50. mu.g/ml had an increased tendency to recruit DC cells and monocytes and decreased tendency to macrophages and neutrophils to the muscle tissue at the subcutaneous site compared to the intramuscular injection, but the differences were not statistically significant (P > 0.05). Fig. 5B shows: mice immunized with the hBCG injection or the intramuscular injection of the water-oil microspheres/250 mu g/ml have an increasing trend of DC cells and monocytes recruited at the subcutaneous injection site and a decreasing trend of neutrophils compared with the intramuscular injection, but the difference is not statistically significant (P > 0.05).

In conclusion, intramuscular injection and subcutaneous injection can cause immune cells to be recruited to the muscle tissue at the injection site, and the adjuvant provided by the invention is prompted to recruit cells related to natural immunity to the muscle tissue at the injection site, so that antigen presentation is facilitated and an acquired immune response is induced.

Example 4: the compound adjuvant of the invention has the function of nonspecific resistance to mycobacterium tuberculosis infection

(1) Guinea pig challenge and immunotherapy: SPF-grade Hartley guinea pigs (350 + -50 g), available from the Chinese food and drug testing institute; production license number of experimental animal: SCXK (Jing) 2014-; quality certification of experimental animals: 11400500008759. skin test of guinea pig (0.1 ml TB-PPD/mouse) and skin test results were observed at 24 hours and 48 hours, and 36 guinea pigs negative to skin test were infected with Mycobacterium tuberculosis 10²-10³CFU/guinea pigs were divided into 3 groups of 12 animals each. 3 days, 10 days, 24 days and 38 days after the infection of the mycobacterium tuberculosis, the group A is injected with 0.5ml PBS/mouse subcutaneously, the group B is injected with 0.5ml water-oil microspheres/50 mu g hBCG/mouse subcutaneously, and the group C is injected with 0.5ml water-oil microspheres/250 mu g hBCG/mouse subcutaneously; 1 week after the last immunization, 2 pre-dissects each group, and observes lesions; the formal dissection time is determined from the lesion.

(2) Guinea pig dissection, organ lesions, and colony counts: the guinea pig is killed, and the guinea pig is dissected to observe spleen, liver and pulmonary tuberculosis lesions; half of the spleens were ground and the spleen suspension was diluted 10-fold with 0.05% Tween80 in saline, 2 for each dilution and 0.1ml for each slant of Roche medium. After 4 weeks at 37 ℃ the number of Colonies (CFU) was counted. The liver, lungs and remaining spleen were soaked in neutral formaldehyde solution.

(3) Pathological changes of liver, lung and spleen organs: taking viscera for dehydration, embedding in paraffin, slicing, HE staining and microscopic examination.

As a result, the combination of the water-oil microspheres and different doses of hBCG has a certain anti-mycobacterium tuberculosis infection effect, wherein the water-oil microspheres/250 mu g of hBCG obviously reduce the visceral lesion of guinea pigs infected with mycobacterium tuberculosis and reduce the bacterial carrying amount of spleen of the guinea pigs (Table 3).

Table 3: liver, spleen and lung comprehensive lesion scoring and splenic bacteria separation number logarithm value of guinea pig

To summarize: the water-oil microsphere/hBCG composite adjuvant has the function of nonspecific anti-mycobacterium tuberculosis infection, and the tuberculosis new-generation protein vaccine with better effect and clinical application value is developed by searching the mycobacterium tuberculosis antigen with strong immunogenicity which is properly compatible with the water-oil microsphere/hBCG composite adjuvant.

Example 5: preparation of PstS1-LEP fusion protein

PstS1(38kDa or Rv0934) was expressed efficiently in E.coli and was very soluble in 8M urea buffer (He xiyun et al, J. China tuberculosis and Res. 1999,22(3): 161-163). The PET28a-PstS1-ESAT6 vector was preserved by this chamber (Heluoxicloud et al, Mycobacterium tuberculosis fusion protein and its application, ZL200610000710. X). The PET28a-PstS1-ESAT6 plasmid was subjected to double digestion with BamHI and HindIII, the digested product was electrophoresed, an agarose block containing the linear plasmid PET28a-PstS1 fragment was cut, and the linear recombinant plasmid PET28a-PstS1 was recovered using a SanPrep column DNA gel recovery kit (Biotechnology engineering, Shanghai, Ltd.).

20 mycobacterium tuberculosis proteins are screened through comparative genomics, the Th linear epitope (http:// www.syfpeithi.de) of the 20 proteins is analyzed by an online database SYFPEITHI, 12 Th epitope peptides which have high scores and aim at different HLA sites and have 15 amino acids are selected, and the 12 Th epitope peptides are connected in series to form a polypeptide which is called a linear multi-epitope protein (LEP). The LEP amino acid sequence was translated into a base sequence by a translation tool (primer5) and modified into a codon preferred for E.coli according to the degeneracy of the codon, BamHI cleavage site and HindIII cleavage site were introduced into the N-terminus and C-terminus of the sequence, respectively, and a termination code UAA was added before the HindIII cleavage site. The designed base sequence of the coding base of the LEP protein is synthesized by the whole gene of Shanghai biological engineering company Limited, and the synthesized LEP gene is cloned into a PUC19 vector and provided by a PUC19-LEP recombinant plasmid. PUC19-LEP is transformed into competent Escherichia coli DH5 alpha (purchased from Tiangen biotechnology limited), blue and white spot screening is carried out, white colony culture is selected, PUC19-LEP plasmid is extracted by an alkaline lysis method and is subjected to double enzyme digestion by BamHI and HindIII, and the enzyme digestion product electrophoresis has two DNA bands, and the size of the DNA bands is consistent with that of linear PUC19 and LEP genes. The agarose block of the LEP gene fragment was cut, and the LEP gene fragment was recovered using SanPrep column DNA gel recovery kit (bio-engineering (shanghai) gmbh).

BamHI and HindIII double digested linear recombinant plasmid PET28a-PstS1 and LEP gene at T₄The recombinant plasmid pET28a-PstS1-LEP is formed by connecting the recombinant plasmid pET28a-PstS1-LEP under the action of DNA ligase, the recombinant plasmid pET28a-PstS1-LEP is transformed into competent Escherichia coli DH5 alpha, the recombinant plasmid pET28a-PstS1-LEP is identified by selecting a recombinant Escherichia coli colony, extracting the plasmid by an alkaline lysis method and carrying out plasmid digestion, and the single digestion map and the double digestion map of the recombinant plasmid pET28a-PstS1-LEP are required to be in accordance with the figure 1. The target gene inserted into the recombinant plasmid pET28a-PstS1-LEP is proved to be completely consistent with the designed sequence through DNA full-automatic sequencing. The recombinant plasmid pET28a-PstS1-LEP with the correct sequence of the inserted LEP gene was transformed into competent Escherichia coli BL21(DE3) (purchased from Tiangen Biotechnology Co., Ltd.), plated on a kanamycin-resistant LB agar plate, and cultured overnight at 37 ℃; selecting single colony in LB culture medium with kanamycin resistance, culturing overnight at 37 ℃, and performing culture according to the formula of 1% inoculated in LB medium resistant to kanamycin, cultured at 37 ℃ to OD_600nmAdding IPTG (0.6-0.8 mmol/L) for induction culture for 4 hr, centrifuging to collect thallus, suspending thallus in lysate, ultrasonic breaking thallus, centrifuging at 12000rpm/min for 15min, and culturing under induction condition to express target protein as inclusion body and to obtain molecular weight of about 50 KD.

Example 6: purification, renaturation and identification of the fusion protein PstS1-LEP according to the invention

IPTG-induced BL21(DE3) thallus containing recombinant plasmid pET28a-PstS1-LEP is added with lysis solution (1g of wet bacteria and 3ml of lysis buffer solution) to fully suspend the thallus, lysozyme and PMSF are sequentially added, water bath at 37 ℃ is carried out for 60min, the thallus is subjected to low-temperature ultrasonic crushing for 40min (ultrasonic conditions: power 540W, working time 6 sec, and gap 8 sec), centrifugation is carried out at 12000rpm/min for 15min at 4 ℃, and supernatant is discarded. The pellet was washed 2 times with a cell lysate containing 2% Triton X-100 (Sigma) and 1 time with 20mmol/L Tris.Cl buffer (pH8.5) containing 2M urea and 2 times with 20mmol/L Tris.Cl buffer (pH8.5), respectively. After each time of thorough washing, the mixture is centrifuged at 12000rpm/min for 20min at 4 ℃ and the supernatant is discarded. The precipitate is the inclusion body which is primarily purified. The inclusion body is fully dissolved by 20mmol/L Tris.Cl buffer solution (pH8.5) containing 8M urea, centrifuged at 12000rpm/min for 20min at normal temperature, and the supernatant is filtered by a 0.45 mu M filter to obtain a chromatography sample.

Packing the Q-FF anion exchange column with a packing, and balancing the distilled water by 5 column volumes; equilibrating with 20mmol/L Tris.Cl buffer (pH8.5) containing 8M urea for 5-6 column volumes, loading and collecting the cross-over peak, and after loading is complete, continuing to equilibrate the column with 20mmol/L Tris.Cl buffer (pH8.5) containing 8M urea until the UV absorbance falls to baseline. Gradient elution is carried out by 8M urea 20mmol/L Tris.Cl buffer solution (pH8.5) containing 0-1mol/L NaCl, elution peaks are collected step by step, and SDS-PAGE detects the purity of proteins passing through the peaks and collected tubes. The target protein mainly passes through a peak, the protein passing through the peak passes through a Source-30Q anion exchange filler chromatographic column, and the chromatographic method is basically the same as that of Q-FF chromatography. Collecting protein, detecting by SDS-PAGE, finding that the target protein with higher purity mainly exists in an elution collecting pipe near 200mmol/L NaCl, combining the collecting pipes containing the target protein and with high purity, filling a protein solution into a dialysis bag, renaturing by a dialysis method, and sequentially putting the dialysis bag into the following renaturation buffer solutions, wherein each buffer solution is dialyzed for 24 hours, and the renaturation buffer solutions are sequentially: buffer I: 6mol/L urea, 50mmol/L Tris-HCl (pH8.5), 50mmol/L NaCl, 5mmol/L EDTA, 5mmol/L reductive glutathione, 1mmol/L oxidative glutathione; and (2) buffer solution II: 4mol/L urea, 50mmol/L Tris-HCl (pH8.5), 50mmol/L NaCl, 5mmol/L EDTA, 5mmol/L reductive glutathione, 1mmol/L oxidative glutathione; buffer III: 2mol/L urea, 50mmol/L Tris-HCl (pH8.5), 50mmol/L NaCl, 5mmol/L EDTA, 5mmol/L reductive glutathione, 1mmol/L oxidative glutathione; and (3) buffer solution IV: 50mmol/L Tris-HCl (pH8.5), 50mmol/L NaCl, 5mmol/L EDTA, 5mmol/L reduced glutathione, 1mmol/L oxidized glutathione; and (3) buffer solution V: 50mmol/L Tris-HCl (pH 8.5); buffer VI: 50mmol/L Tris-HCl (pH 8.5). The renatured protein was filtered through a 0.22 μm filter and dispensed, and the protein concentration was measured by Lowry method (the national Committee of pharmacopoeia, pharmacopoeia of the people's republic of China, chemical industry Press, 2010) and the protein was stored at-30 ℃.

SDS-PAGE of the purified protein revealed that the protein was nearly 95% pure by scanning the optical density of the gel (FIG. 2 a). The purified protein was electrophoresed through SDS-PAGE and electrically transferred to a PVDF membrane. 1 XBS buffer containing 5% skim milk (25mmol/L Tris, 0.1% Tween-20 and 150mmol/L NaCl) blocks the PVDF membrane for 2 hours at room temperature, and the 1 XBS buffer washes the membrane thoroughly, which is mixed with 1 XBS buffer containing 5% skim milk 1: Anti-PstS1 monoclonal antibody (Anti-PstS1 rat monoclonal antibody (Anti-PstS1 rat monoclonal antibody), Jackson Immuno Research Laboratories Inc. USA) was diluted 10000, or mixed with 1 × TBS buffer 1 containing 5% skim milk: 100 diluting the serum of tuberculosis patients, and incubating for 2 hours at room temperature; washing the membrane, and incubating with the corresponding horseradish peroxidase-labeled secondary antibody for 1 hour at room temperature (1 × TBS buffer containing 5% skim milk 1: 10000 dilution enzyme-labeled secondary antibody, Jackson Immuno Research Laboratories Inc. USA); washing the membrane, adding a chemiluminescent Substrate (Pierce ECL Western Blotting Substrate; Thermo Fisher Scientific Inc., USA) to the PVDF membrane under dark conditions, developing the color, and exposing the luminescent signal to X-ray film. The PstS1-LEP protein positively reacts with anti-PstS1 monoclonal antibody, tuberculosis patient serum IgG and IgM (figure 2b), and the fusion protein PstS1-LEP has immunoreactivity and simultaneously retains PstS1 immunoreactivity.

PstS1-LEP reacts with serum antibodies from tuberculosis patients: 1) the coating solution diluted PstS1-LEP to 5. mu.g/ml, 100. mu.l per well, and coated overnight at 4 ℃.

2) Plates were washed 5 min/time three times in total (wash solution 0.05% Tween-20 in PBS, PBST). Mu.l of blocking solution (PBST containing 1% Bovine Serum Albumin (BSA), PBST-B) was added to each well and blocked at 37 ℃ for 1 h.

3) Wash plate, as above. The serum to be detected is diluted by a confining liquid with the dilution ratio of 1: 100, 100. mu.l of diluted serum was added to each well, two wells were set for each sample, and incubated at 37 ℃ for 2 h.

4) Wash plate, as above. Mu.l of HRP-labeled rabbit anti-human IgG secondary antibody diluent, IgM secondary antibody diluent or IgG and IgM secondary antibody diluent mixture (diluted according to the secondary antibody specification, the diluent is a confining solution) is added into each hole, and the incubation is carried out for 1h at 37 ℃.

5) Wash plate, as above. Adding 100 μ l of developing solution into each well, standing in dark at room temperature for 30min, adding 50 μ l of stop solution (2N H)₂SO₄) The color development was stopped and the absorbance of OD450nm was measured.

The results of the detection of serum anti-PstS 1-LEP IgG, IgM and IgG and IgM in the same well from different sources are shown in Table 4.

TABLE 4 human serum anti-PstS 1-LEP antibody assay

Table 4 the results show: the positive rate of the tuberculosis patient serum against PstS1-LEP IgG, Ig (G + M) and IgG + IgM is higher than that of the extrapulmonary tuberculosis patient serum corresponding antibody (x 2 ═ 38.6, x 2 ═ 9.9 and x 2 ═ 11.6; all P < 0.001). And the positive rate of the serum of tuberculosis patients to the PstS1-LEP IgM is lower than that of the corresponding antibody of the serum of extrapulmonary tuberculosis patients (x 2 is 14.1, and P is less than 0.001). The positive rate of serum anti-PstS 1-LEP Ig (G + M) and IgG + IgM of the extrapulmonary tuberculosis patients is higher than that of IgG (x 2 is 18.8 and x 2 is 7.4, all P is less than 0.001), and the positive rate of serum anti-PstS 1-LEP IgG, Ig (G + M) and IgG + IgM of the extrapulmonary tuberculosis patients is compared, and the difference is not statistically significant (P is more than 0.05). Comparison of anti-PstS 1-LEP Ig (G + M) and IgG + IgM detection: the former increases the detection positive rate but does not decrease the specificity, and the latter increases the detection positive rate but decreases the specificity.

Example 7: immunogenicity of the recombinant protein of the invention in combination with different adjuvants

The composite adjuvant water-oil microsphere/hBCG enhances the recombinant protein PstS1-LEP to induce Th1 type immune response, and is represented by the fact that the serum IgG2a antibody level is increased, the number of cells for stimulating spleen cells to secrete IL-2 and IFN-gamma in vitro by PstS1-LEP is increased, and the level of secreted IL-2 and IFN-gamma is increased.

BALB/c mice were divided into 7 groups of 8 mice each. Respectively, subcutaneous immune water-oil microspheres, hBCG, PstS1-LEP, hBCG + PstS1-LEP, water-oil microspheres + PstS1-LEP, water-oil microspheres/hBCG + PstS1-LEP, Al (OH)₃The ratio of the concentration of the hBCG to the concentration of the PstS1-LEP is/hBCG + PstS 1-LEP. Each mouse was injected subcutaneously with 0.2ml, 1 booster at 3 and 6 weeks, respectively, at a dose of PstS1-LEP 10. mu.g/mouse and hBCG 50. mu.g/mouse. The eye was removed 3 weeks after the last immunization, bled, sacrificed and dissected.

(1) And (3) serum antibody detection: the indirect ELISA method detects the IgG, IgG1 and IgG2a levels of serum anti-PstS 1-LEP of the immunized mice.

1) The coating solution diluted PstS1-LEP to 5. mu.g/ml, 100. mu.l per well, and coated overnight at 4 ℃.

3) Wash plate, as above. The serum to be detected is diluted by a confining liquid with the dilution ratio of 1: 5000, 100. mu.l of diluted serum was added to each well, and two wells were set for each sample, and incubated at 37 ℃ for 2 hours.

4) Wash plate, as above. Mu.l of HRP-labeled secondary antibody dilution (dilution according to secondary antibody specification, dilution as blocking solution) was added to each well and incubated at 37 ℃ for 1 h.

5) Wash plate, as above. Adding 100 μ l of developing solution into each well, standing in dark at room temperature for 30min, adding 50 μ l of stop solution (2N H)₂SO₄) TerminateColor development was performed and OD450nm absorbance was measured.

Different adjuvants are combined with the antigen PstS1-LEP of the invention to immunize mice, and the detection results of IgG, IgG1 and IgG2a of the mouse serum anti-PstS 1-LEP are shown in Table 5.

Table 5: the detection of the serum anti-PstS 1-LEP antibody of the mouse immunized by the recombinant protein PstS1-LEP and different adjuvants

^*Shows that the difference is obvious compared with the water-oil microsphere immunity group,

^#shows that the difference is obvious compared with the hBCG immune group,

^&shows significant difference compared with PstS1-LEP immune group

Immunization of mice with PstS1-LEP alone or in combination with adjuvant resulted in IgG and IgG1 antibodies against PstS1-LEP, but adjuvant/PstS 1-LEP immunized mice resulted in significantly higher levels of IgG and IgG1 antibodies against PstS1-LEP than PstS1-LEP immunized alone (P1-LEP alone)<0.05). Mice immunized with hydro-oleo microspheres, hbg alone or both as PstS1-LEP adjuvant induced a significantly higher water mean for IgG2a than for the PstS1-LEP group (P0.001 and P0.0002) against PstS1-LEP, for the hbg + PstS1-LEP group and for the hydro-oleo microspheres/hbg + PstS1-LEP group; al (OH) ₃The + hBCG + PstS1-LEP group IgG2a level is significantly higher than the hBCG group (P ═ 0.036), while the difference compared with the PstS1-LEP group is not statistically significant (P ═ 0.036)>0.05)。

(2) Detecting immune indexes of spleen cells: aseptically taking spleen, placing 200 mesh nylon net on disposable culture dish, placing spleen on nylon net, cutting with scissors, grinding spleen with 5ml syringe piston, collecting cell suspension filtered by nylon net, centrifuging at 1500rpm for 10min, discarding supernatant, suspending cells in 1ml RPMI-1640 culture medium, slowly adding into 2ml liquid surface of mouse organ lymphocyte tissue separating mediumAnd centrifuging at 1700rpm for 20 min. After centrifugation, the second layer of opalescent lymphocytes was pipetted into a 15ml centrifuge tube and RPMI-1640 medium was added to 10 ml. Centrifuging at 1500rpm for 10min, discarding the supernatant, and washing the cells once in RPMI-1640 medium. Suspending lymphocytes in RPMI-1640 complete culture medium (containing 10% high-quality fetal bovine serum and double antibody), counting, and adjusting cell concentration to 2 × 10⁶/ml。

Enzyme Linked Immunospot (ELISPOT) assay: the number of spot-forming cells secreting IFN-. gamma.IL-2, IL-4 from splenic lymphocytes was determined according to the instructions of Mouse IFN-. gamma.IL-2, IL-4ELISpot PLUS kit (ALP) kit (Mabtech, Sweden), and the detailed procedures were as follows: (1) coating ELISPOT plate: non-precoated ELISPOT plates were coated with ELISPOT plates overnight in dissected mice (precoated ELISPOT does not require this operation): each well of the 96-well ELISPOT plate was first activated for 1min by adding 15. mu.l of 35% ethanol. Wash 5 times with 200 μ l deionized water, add 100 μ l of coating antibody per well (dilute according to instructions) and coat overnight at 4 ℃. (2) Stimulation of splenocytes in vitro culture: splenocytes were prepared as above, the plate was removed, the liquid was discarded, 200. mu.l PBS (0.22 μm filtration) was added to each well to wash the plate 5 times, 200. mu.l complete medium was added to each well, and incubation was carried out at room temperature for 30 min. The medium was discarded and 100. mu.l of lymphocyte suspension and 5. mu.l of PstS1-LEP antigen (final concentration 5. mu.g/ml) were added in 5% CO ₂And incubating at 37 ℃ for 24-48 h. (3) Color development: the plate was removed, the liquid was discarded, 200. mu.l PBS (0.22 μm filtration) was added to each well to wash the plate 5 times, 100. mu.l detection antibody was added to each well in sequence, and incubation was carried out at room temperature for 2 h. The well solution was discarded, and the plate was washed 5 times, and 100. mu.l of Streptavidin-ALP was added to each well in sequence, and incubated at room temperature for 1 h. Discarding the solution in the wells, washing the plate for 5 times, adding 100 μ l BCIP/NBT substrate solution (0.45 μm filtration), standing at room temperature for 10-30min, washing each well with distilled water after spots appear, and drying the microplate in the ventilated place away from light. (4) Detection of number of spot-forming cells: the plate was read by an enzyme-linked immunosorbent assay (CTL, USA) and the number of Spot Forming Cells (SFC) of IFN-. gamma., IL-2, and IL-4 was automatically analyzed. The results are shown in FIG. 6.

FIG. 6 shows: water and oil microsphere/hBCG + PstS1-LEP immunized mice enhanced their spleen cells to secrete PstS1-LEP specific IFN-. gamma.and IL-2 (FIGS. 6A and B), while Al (OH)₃Small/hBCG + PstS1-LEP immuneThe mice enhanced secretion of PstS 1-LEP-specific IL-4 by their spleen cells (FIG. 6C).

Detecting the contents of the cytokines IFN-gamma, IL-2 and IL-4 by sandwich ELISA: (1) stimulation of splenocytes in vitro culture: adding 1ml of 2X 10 cells per well of 24-well cell culture plate⁶Spleen lymphocyte suspension/ml, four wells were added to the same mouse spleen cells, two of which were not stimulated with antigen, and two wells were stimulated with 10. mu.l of PstS1-LEP antigen (final concentration 5. mu.g/ml), 37 ℃ with 5% CO ₂And culturing for 72 h. The lymphocyte culture supernatant was collected by centrifugation. (2) ELISA: the cytokine content in the culture supernatant was measured according to the kit instructions (BD Bioscience, usa), and the specific procedures were as follows: 1) coating: the coating antibody was diluted to the desired concentration and 100. mu.l was added to each well of a 96-well microplate and coated overnight at 4 ℃. 2) And (3) sealing: discard the coating solution and wash the plate 3 times (PBST wash solution). Mu.l of blocking solution (PBST containing 10% high-quality fetal calf serum) was added to each well, and blocked at 37 ℃ for 1 hour. 3) Primary antibody incubation: wash plate, as above. The culture supernatant to be tested was diluted with PBST (dilution ratio 1: 5), 100. mu.l was added to each well, and two wells were repeated and incubated at 37 ℃ for 2 hours. 4) And (3) secondary antibody incubation: and washing the plate for 5 times. Add 100. mu.l of detection antibody to each well and incubate for 1h at 37 ℃. 5) Color development: and washing the plate for 7 times. Adding 100 μ l of color developing solution into each well, standing at room temperature in dark place for 30min, adding 50 μ l of stop solution 2N H₂SO₄The color development was stopped and the absorbance of OD450nm was measured. The results are shown in FIG. 7.

The water-oil microspheres and the hBCG are used as PstS1-LEP antigen adjuvant alone or in combination for immunizing mice to enhance the spleen cells to secrete PstS1-LEP antigen specific IFN-gamma, and the composite adjuvant Al (OH)₃The hBCG can not effectively enhance spleen cells of mice immunized with the PstS1-LEP antigen to secrete PstS1-LEP antigen specific IFN-gamma; comparison between groups with statistical differences showed: the hbg + PstS1-LEP group was significantly higher than the hbg group (P0.013), the water and oil microspheres + PstS1-LEP group and the water and oil microspheres/hbg + PstS1-LEP group were significantly higher than the water and oil microspheres group (P0.013 and P0.003), the hbg group (P0.001 and P0.0002) and the PstS1-LEP group (P0.037 and P0.008); al (OH) ₃The/hbg + PstS1-LEP group was significantly lower than the water oil microspheres/hbg + PstS1-LEP group (P ═ 0.016) (fig. 7A).

Water-oil microspheres, water-oil microspheres/hBCG and Al (OH)₃/hBCG as PstS1-LEP antigen adjuvantThe spleen cells of the immunized mice with the PstS1-LEP antigen can not be effectively enhanced by the hBCG adjuvant to secrete the PstS1-LEP antigen specific IL-2; comparison between groups with statistical differences showed: water and oil microspheres + PstS1-LEP group and Al (OH)₃The/hcg + PstS1-LEP group is significantly higher than the water and oil microsphere group (P0.007 and P0.004), the hcg group (P0.004 and P0.002); the water oil microsphere/hcg + PstS1-LEP group was significantly higher than the water oil microsphere group (P ═ 0.0001), the hcg group (P ═ 0.0003), the PstS1-LEP group (P ═ 0.007), and the hcg + PstS1-LEP group (P ═ 0.013) (fig. 7B).

Comparison of the level of IL-4 secretion stimulated by PstS1-LEP antigen in vitro by splenocytes from mice immunized with different immunogens: the hBCG + PstS1-LEP group is significantly higher than the water-oil microsphere group (P is 0.020) and the hBCG group (P is 0.010); the other groups compared the difference to be statistically insignificant (P >0.05) (fig. 7C).

(3) Mouse peritoneal macrophages secrete IL-1 β and IL-12: mice were intraperitoneally injected with 1ml of 6% soluble starch broth 3 days before dissection. After 3 days, the mouse picks up eyeballs and takes blood, and the neck is cut off and then the mouse is soaked in 75% alcohol for 1 min. Under aseptic conditions, the skin was cut open, the peritoneum was exposed, 8ml of ice-cold peritoneal lavage fluid was taken with a 10ml syringe, injected into the abdominal cavity of the mouse, the abdomen of the mouse was gently, the tip of the needle was adjusted to face down obliquely, the peritoneum was gently lifted up, and the peritoneal lavage fluid (containing macrophages) was slowly aspirated back into a 15ml centrifuge tube. Centrifuging at 1500rpm for 10min, discarding supernatant, washing macrophage twice with RPMI-1640 medium, suspending macrophage in DMEM medium, counting, and adjusting macrophage concentration to 5x10 ⁵And/ml. Adding 1ml macrophage suspension into each well of 24-well cell culture plate, and culturing at 37 deg.C with 5% CO₂Adherent for 2-4h, washing nonadherent cells with DMEM medium, adding 1ml DMEM complete medium (containing 10% high quality fetal bovine serum and double antibody) and 10 μ l PstS1-LEP antigen (final concentration of 5 μ g/ml), and culturing at 37 deg.C and 5% CO₂And culturing for 72 h. Macrophage culture supernatant was collected. The results of the sandwich ELISA method for detecting IL-1 beta and IL-12 levels in the macrophage culture supernatant are shown in FIG. 8, and the detection method is the same as that for detecting the content of the splenocyte culture supernatant cytokines in the embodiment.

hBCG, water oil microsphere, Al (OH)₃/hBCG respectively used as PstS1-LEP adjuvant to immunize mice and enhance miceMouse peritoneal macrophages secrete PstS1-LEP antigen specific IL-12, and the specific difference comparison: hBCG + PstS1-LEP group and Al (OH)₃The/hcbcg + PstS1-LEP group is significantly higher than the water and oil microspheres group (P ═ 0.050 and P ═ 0.033), the hcbcg group (P ═ 0.011 and P ═ 0.006); the water-oil microsphere + PstS1-LEP group is significantly higher than the water-oil microsphere group (P is 0.002), the hBCG group (P is 0.0006) and the PstS1-LEP group (P is 0.017); the water-oil microsphere/hBCG + PstS1-LEP group is significantly lower than the hBCG + PstS1-LEP group (P is 0.008), the water-oil microsphere + PstS1-LEP group (P is 0.0003) and Al (OH)₃The/hcbcg + PstS1-LEP group (P ═ 0.004) (fig. 8A).

hBCG, water-oil microsphere/hBCG, Al (OH)₃The hBCG is respectively used as PstS1-LEP adjuvant to immunize mice, and the macrophages in the abdominal cavity of the mice are enhanced to secrete PstS1-LEP antigen specific IL-1 beta, and the specific difference is compared: hBCG + PstS1-LEP group, water and oil microsphere + PstS1-LEP group and Al (OH)₃The hcbcg + PstS1-LEP group is significantly higher than the water-oil microsphere group (P ═ 0.0002, P ═ 0.0005 and P ═ 0.0004), the hcbcg group (P ═ 0.0003, P ═ 0.0001 and P ═ 0.0007) and the PstS1-LEP group (P ═ 0.001, P ═ 0.005 and P ═ 0.006); the water oil microsphere/hbg + PstS1-LEP group was significantly higher than the water oil microsphere group (P0.011) and the hbg group (P0.003) (fig. 8B).

Example 8: effect of hBCG dose on Effect of composite adjuvant water-oil microsphere/hBCG adjuvant

BALB/c mice were randomly grouped into 7 groups of 8 mice each. Respectively as follows: water-oil microspheres, low-dose hBCG (5 mug/hBCG (L)), medium-dose hBCG (50 mug/hBCG (M)), high-dose hBCG (500 mug/hBCG (H)), water-oil microspheres/hBCG (L), water-oil microspheres/hBCG (M), and water-oil microspheres/hBCG (H). Each mouse in each experimental group was injected subcutaneously with 0.2ml, boosted 1 time each at 2 and 4 weeks, and 2 weeks after the last immunization, the eye was removed, bled, sacrificed and dissected.

The indirect ELISA method for measuring the serum antibody level and the sandwich ELISA method for detecting the cell factor content of the mouse spleen cell in vitro stimulated culture supernatant and the cell factor content of the macrophage cell in vitro stimulated culture supernatant are the same as those in example 7, except that the antigen for stimulating the cell culture in vitro is a pure protein derivative (BCG-PPD) prepared from the BCG culture supernatant, and the preparation method is disclosed in the literature (the national Committee of pharmacopoeia, the pharmacopoeia of the people's republic of China, P21-132, the chemical industry Press, 2010). Briefly describing the preparation method: inoculating BCG into a sutong culture medium, and culturing at 37 ℃ for 6 weeks; sterilizing at 121 deg.C for 30min, centrifuging at 12000 rpm/min at 4 deg.C for 10min, and filtering the supernatant with 0.45 μm membrane; adding 40% trichloroacetic acid into the filtrate to a final concentration of 2-4%, mixing, precipitating at 4 deg.C for 6 h; centrifuging at 4 deg.C at 12000 rpm for 10min, and washing the precipitate with 1% trichloroacetic acid for 3 times; dissolving the precipitate with phosphate buffer (pH8.2), adding saturated ammonium sulfate, and precipitating at 4 deg.C overnight; centrifuging at 4 deg.C at 12000 rpm for 10min, dissolving the precipitate with phosphate buffer (pH8.2), dialyzing the solution with PBS; after thorough dialysis, sterile filtration, measurement of protein concentration by Lowry, split charging and preservation at-20 ℃.

The hBCG alone with different doses or the compound adjuvant combined with the water-oil microspheres is used for immunizing mice, and the OD value of the mouse serum is extremely low and is close to that of a blank control hole when the mouse serum is detected by IgG, IgG1 and IgG2a antibodies of the BCG-PPD.

The results of the secretion of IFN-gamma, IL-2 and IL-4 by mouse splenocytes are shown in FIG. 9: spleen cells of low, medium and high dose hBCG immune mice secrete BCG-PPD specific IFN-gamma level which shows a rising trend along with the increase of hBCG dose, but three groups of comparison differences have no statistical significance; the spleen cells of the water-oil microsphere/hBCG immunized mice also exhibited an increasing trend in the secretion of BCG-PPD-specific IFN- γ with increasing hcg dose, and the level of IFN- γ production was significantly higher in the water-oil microsphere/hbg (h) group than in the water-oil microsphere/hbg (L, M) two groups (P ═ 0.0005 and P ═ 0.001) (fig. 9A). The comparison difference of the levels of IFN-gamma specific to BCG-PPD secreted by spleen cells of mice immunized by the water-oil microspheres, the hBCG (L), the water-oil microspheres/the hBCG (L) has no statistical significance; the comparison of the level differences of BCG-PPD specific IFN-gamma secreted by spleen cells of mice immunized by the water-oil microspheres, the hBCG (M), the water-oil microspheres/the hBCG (M) has no statistical significance; the spleen cells of the water-oil microsphere/hbcg (h) immunized mice secreted BCG-PPD-specific IFN- γ levels significantly higher than the water-oil microsphere and hbcg (h) immunized mice groups (P ═ 0.0002 and P ═ 0.001) (fig. 9A).

Spleen cells of low, medium and high dose hbg-immunized mice secrete BCG-PPD-specific IL-2 levels that tend to increase with increasing hbg dose, and the hbg (h) immunized group has significantly higher IL-2 levels than the hbg (l) group (P ═ 0.044); spleen cells of mice immunized with the water-oil microsphere/hBCG composite adjuvant secreted BCG-PPD-specific IL-2 level also showed an increasing trend with increasing hBCG dose, but three groups of comparative differences were not statistically significant (FIG. 9B). Spleen cells of the water-oil microsphere/hBCG (L) immune mice secrete BCG-PPD specific IL-2 level which is obviously higher than that of the water-oil microsphere or hBCG (L) immune mice (P ═ 0.002 and P ═ 0.001); spleen cells of the water-oil microsphere/hBCG (M) immune mice secrete BCG-PPD specific IL-2 levels which are significantly higher than those of the water-oil microsphere or hBCG (M) immune mice (P ═ 0.002 and P ═ 0.001); spleen cells of water-oil microsphere/hbbcg (h) immunized mice secreted BCG-PPD-specific IL-2 levels significantly higher than those of the water-oil microsphere or hbbcg (h) immunized mouse group (P ═ 0.001 and P ═ 0.005) (fig. 9B).

Comparison of the differences in the levels of BCG-PPD-specific IL-4 secretion by splenocytes from each group of mice was not statistically significant (FIG. 9C).

The results of the mouse peritoneal macrophages secreting IL-12 and IL-1 beta are shown in FIG. 10. The level of IL-12 production by peritoneal macrophages of mice immunized with the water emulsion microsphere/hBCG (L) or water emulsion microsphere/hBCG (M) through stimulation of BCG-PPD antigen is higher than that of mice immunized with hBCG (L) or hBCG (M), but the difference of IL-12 level in each group has no statistical significance (figure 10A). The IL-1 β levels in the water-oil microsphere/hcg (l) group were all significantly higher than those in the water-oil microsphere (P ═ 0.05), hcg (l) group (P ═ 0.022) and hcg (m) group (P ═ 0.01), and the IL-1 β levels in the water-oil microsphere group (P ═ 0.016), hcg (l) group (P ═ 0.006), hcg (m) group (P ═ 0.003) and hcg (h) group (P ═ 0.048) (fig. 10B).

Combined splenocyte and macrophage immune responses: the dosage range of the hBCG is 5 mu g/dose-500 mu g/dose, and the optimal range is 50 mu g/dose-250 mu g/dose.

Example 9: water-oil microsphere/hBCG adjuvant for enhancing immunogenicity of mycobacterium tuberculosis fusion protein

The Mycobacterium tuberculosis fusion protein is PstS1-EAST6 and is named as K6(ZL200610000710. X). Female BALB/c mice (SPF grade) (purchased from Experimental animals center of military medical science institute of people's liberation force of China) 6-8 weeks old were divided into 7 groups of 8 mice each. Mice of different groups are respectively immunized with water-oil microspheres, hBCG, K6, hBCG + K6, water-oil microspheres + K6, water-oil microspheres/hBCG + K6, Al (OH)₃/hBCG + K6. Each mouse injected subcutaneously0.2ml injection, 1 booster at 2 and 4 weeks, 610. mu.g/dose of K and 50. mu.g/dose of hBCG. 2 weeks after the last immunization, mice were bled from the eye, sacrificed and dissected. ELISA for serum antibody detection, sandwich ELISA for detection of cytokine content in supernatants collected from in vitro K6 stimulated culture of splenocytes and peritoneal macrophages, example 7 was repeated except that the stimulating antigen used in vitro culture of splenocytes and peritoneal macrophages was K6 (final concentration: 5 (g/ml)).

(1) Humoral immunity

The K6 antigen immunized mice induce certain level of anti-K6 IgG, IgG1 and IgG2a antibodies, but the level of IgG and IgG1 is obviously lower than that of water-oil microspheres + K6, water-oil microspheres/hBCG + K6 or Al (OH) ₃The group of/hBCG + K6 immunized mice (FIG. 11). Compared with the K6 immune group, the hBCG, the water emulsion microsphere and the water emulsion microsphere/hBCG are used as K6 antigen adjuvants to mainly induce K6 specific IgG1 antibody, but not K6 specific IgG2a antibody; and Al (OH)₃The/hcbcg induced IgG1 and IgG2a antibodies as a K6 antigen adjuvant (fig. 11B and C).

(2) Cytokines secreted by splenocytes

The K6 antigen combined with different adjuvants can not effectively induce spleen cells to secrete IFN-gamma, IL-2 and IL-4, Al (OH)₃the/hBCG compound adjuvant K6 vaccine enhances IL-4 secretion of mouse splenocytes (figure 12).

(3) Cell factor secreted by macrophage in abdominal cavity of mouse

Water-oil microsphere, water-oil microsphere/hBCG, Al (OH)₃The mice immunized with/hBCG as K6 antigen adjuvant enhanced secretion of K6 specific IL-12, but not IL-1 β, by peritoneal macrophages (FIG. 13).

Example 10 Water-oil microsphere/hBCG adjuvant enhanced Cytomegalovirus (CMV) Virus gB antigen (CMV gB) immunogenicity

CMV virus gB antigen (1mg/ml) was purchased from daceae biotechnology limited with a purity of greater than 95%. BALB/c mice were divided into 6 groups of 6 mice each. Group A-D and group F respectively comprise muscle immunity water-oil microspheres, water-oil microspheres/hBCG, CMV gB, water-oil microspheres + CMV gB, and water-oil microspheres/hBCG + CMV gB; group E subcutaneous immune water oil microspheres/hBCG + CMV gB. Each experimental group was injected intramuscularly or subcutaneously with 0.2ml, 1 booster each at

week

2 and 4, with an immunization dose of CMV gB 5. mu.g/dose and hBCG 50. mu.g/dose. The eye was removed 2 weeks after the last immunization, bled, sacrificed and dissected.

(1) Serum antibody detection

The indirect ELISA method detects serum IgG levels against CMV gB in immunized mice:

1) CMV gB was diluted to 5. mu.g/ml in coating solution and 100. mu.l was added to each well and coated overnight at 4 ℃.

2) The plate was washed 5 min/time for three times (PBST wash). Mu.l of blocking solution (PBST containing 1% Bovine Serum Albumin (BSA)) was added to each well and blocked at 37 ℃ for 1 h.

3) Wash plate, as above. The serum to be detected is diluted by a sealing liquid according to the dilution ratio: the serum of mice in the water-oil microsphere/hBCG immune group is diluted by 1:100 and 1:500 respectively, the serum of CMV gB immune mice is diluted by 1:500, 1:2500 and 1:5000 respectively, the serum of water-oil microsphere + CMV gB and water-oil microsphere/hBCG + CMV gBC (including intramuscular injection and subcutaneous injection) immune mice is diluted by 1:5000, 1:25000 and 1:30000 respectively, 100 mu l of serum diluent is added into each hole, each sample is provided with two multiple holes per dilution degree, and the samples are incubated for 2 hours at 37 ℃.

4) Wash plate, as above. Mu.l of HRP-labeled secondary antibody dilution was added to each well and incubated at 37 ℃ for 1 h.

The maximum dilution which is larger than the OD average value measured by the dilution of the water-oil microsphere immune mouse serum 1:100 +/-2 SD is taken as the serum antibody titer. The CMV gB immunized mice induced anti-CMV gB IgG levels were lower, both water and oil microspheres and composite adjuvant water and oil microspheres/hcbcg were able to significantly enhance CMV gB induced anti-CMV gB IgG antibody production (fig. 14). The two adjuvants enhance the CMV gB induced anti-CMV gB IgG antibody titer comparison, and the difference has no statistical significance; the anti-CMV gB IgG antibody is induced to be generated by immunizing a mouse with the composite adjuvant water-oil microspheres/hBCG + CMV gB through subcutaneous injection and intramuscular injection, and the comparison difference of the antibody titer has no statistical significance (figure 14).

(2) ELISPOT (enzyme-linked immunosorbent assay) for detecting the number of spot-forming cells secreting IFN-gamma and IL-4 from splenic lymphocytes

The experimental procedure was the same as in example 7, and the results showed that: the number of spot-forming cells of the mice immunized by intramuscular injection of the water-oil microspheres/hBCG + CMV gB for enhancing the secretion of CMV gB specific IFN-gamma by spleen cells is obviously higher than that of other groups (P < 0.01); the number of spot-forming cells of the mice immunized by the water-oil microsphere/hBCG + CMV gB subcutaneous injection, which can enhance the spleen cells to secrete CMV gB specific IFN-gamma, is obviously higher than that of the adjuvant group, and the difference is not statistically significant compared with the mice immunized by the CMV gB, water-oil microsphere and CMV gB (figure 15 a). The number of spot-forming cells secreting CMV gB-specific IFN- γ by splenocytes of mice in the water and oil microsphere + CMV gB immunized group was significantly higher than in the water and oil microsphere immunized mice group (P <0.05), slightly higher than in the water and oil microsphere/hBCG immunized mice group (P ═ 0.052) (fig. 15 a).

The number of spot-forming cells secreting CMV gB-specific IL-4 from splenocytes of CMV gB immunized mice was significantly higher than water-oil microspheres, water-oil microspheres/hcbg, water-oil microspheres + CMV gB or water-oil microspheres/hcbg + CMV gB intramuscular injected (group F) immunized mice (fig. 15 b); the number of spot-forming cells secreting CMV gB-specific IL-4 from splenocytes of water-oil microsphere/hbbcg + CMV gB subcutaneous immunized mice (group E) was significantly higher than that of water-oil microsphere/hbbcg immunized mice (fig. 15 b).

And (4) conclusion: the water-oil microsphere/hBCG and the water-oil microsphere adjuvant can enhance the CMV gB immune mice to generate antibodies, and the water-oil microsphere/hBCG adjuvant and CMV gB intramuscular injection immune scheme can effectively enhance and induce Th1 type immune response, and particularly IFN-gamma secretion is beneficial to the body to eliminate viruses. Therefore, the antiviral effect of the CMV gB enhanced by the water-oil microspheres/hBCG is better than that of the water-oil microspheres.

EXAMPLE 11 comparative adjuvanticity of Water-oil microspheres/hBCG composite adjuvants for two different preparation methods

The preparation process of the composite adjuvant water-oil microsphere/hBCG-1 (abbreviated as adjuvant 1) is shown in the relevant part of example 1; the formula of the composite adjuvant water-oil microsphere/hBCG-2 (called adjuvant 2 for short) is completely consistent with that of the water-oil microsphere/hBCG-1, and the preparation process is different from that of the composite adjuvant water-oil microsphere/hBCG-1: the preparation process of the water-oil microsphere/hBCG-1 comprises homogenizing the water-oil microsphere under high pressure to form a nano emulsion, filtering by 0.22 μm, wherein the hBCG is a complete inactivated thallus, and mixing the two at a certain ratio to form the composite adjuvant water-oil microsphere/hBCG-1; the water-oil microsphere/hBCG-2 is prepared by mixing water-oil microsphere and hBCG, homogenizing under high pressure, wherein hBCG is broken thallus.

BALB/c mice were divided into 2 groups of 6 mice each; 0.2ml of water-oil microsphere/hBCG-1 + PstS1-LEP and water-oil microsphere/hBCG-2 + PstS1-LEP are injected subcutaneously respectively, the booster immunization is carried out for 1 time in the 2 nd week and the 4 th week, the immunization dose is PstS1-LEP for 5 mu g/time, and the immunization dose is hBCG for 50 mu g/time (the water-oil microsphere/hBCG-1 is 50 mu g of inactivated BCG thallus, and the water-oil microsphere/hBCG-2 is 50 mu g of inactivated BCG thallus fragmentations). The eye was removed 2 weeks after the last immunization, bled, sacrificed and dissected.

Serum IgG, IgG1, and IgG2a were detected as in "serum antibody detection" in example 7. As shown in fig. 16: the tuberculosis protein vaccines of the adjuvant 1 and the adjuvant 2 which are respectively compatible with the PstS1-LEP are used for immunizing mice to generate IgG, IgG1 and IgG2a which resist the PstS1-LEP, and the difference has no statistical significance (P is more than 0.05).

The method for detecting the spot-forming cells secreting IFN-gamma, IL-4 or IL-17 from splenocytes by ELISPOT is the same as that of the "detection of immune index of splenocytes" in example 7.

As shown in fig. 17: tuberculosis protein vaccine (adjuvant 2+ PstS1-LEP) is used for immunizing mice, and IFN-gamma and IL-17 are induced to be secreted by splenocytes, which is superior to tuberculosis protein vaccine (adjuvant 1+ PstS 1-LEP).

As can be seen from the above examples, the composite adjuvant of the present invention can enhance the induction of humoral and cellular immunity by Mycobacterium tuberculosis protein, and can also enhance the induction of humoral and cellular immunity by viral protein (CMV virus). The single water-oil microspheres mainly enhance Th2 type immune response, and the composite adjuvant combined with the water-oil microspheres and the heat inactivated BCG (hBCG) induces the immune response to drift to Th1 type cellular immune response; enhances the capability of organisms to eliminate tubercle bacillus, and is an important candidate adjuvant for tuberculosis prevention and therapeutic vaccines.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, these descriptions and examples should not be construed as limiting the scope of the invention. Accordingly, all suitable modifications or equivalents may be resorted to, falling within the scope of the invention as defined by the claims. The disclosures of all patent and scientific documents cited herein are expressly incorporated herein by reference in their entirety.

Sequence listing

<110> three good and nine hospitals of liberation military of Chinese people

<120> composite adjuvant and vaccine containing the same

<130> PBK00886

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 1050

<212> DNA

<213> Artificial Sequence

<220>

<223> coding sequence of amino acid 25 to amino acid 374 of PstS1 antigen

<400> 1

ggctcgaaac caccgagcgg ttcgcctgaa acgggcgccg gcgccggtac tgtcgcgact 60

acccccgcgt cgtcgccggt gacgttggcg gagaccggta gcacgctgct ctacccgctg 120

ttcaacctgt ggggtccggc ctttcacgag aggtatccga acgtcacgat caccgctcag 180

ggcaccggtt ctggtgccgg gatcgcgcag gccgccgccg ggacggtcaa cattggggcc 240

tccgacgcct atctgtcgga aggtgatatg gccgcgcaca aggggctgat gaacatcgcg 300

ctagccatct ccgctcagca ggtcaactac aacctgcccg gagtgagcga gcacctcaag 360

ctgaacggaa aagtcctggc ggccatgtac cagggcacca tcaaaacctg ggacgacccg 420

cagatcgctg cgctcaaccc cggcgtgaac ctgcccggca ccgcggtagt tccgctgcac 480

cgctccgacg ggtccggtga caccttcttg ttcacccagt acctgtccaa gcaagatccc 540

gagggctggg gcaagtcgcc cggcttcggc accaccgtcg acttcccggc ggtgccgggt 600

gcgctgggtg agaacggcaa cggcggcatg gtgaccggtt gcgccgagac accgggctgc 660

gtggcctata tcggcatcag cttcctcgac caggccagtc aacggggact cggcgaggcc 720

caactaggca atagctctgg caatttcttg ttgcccgacg cgcaaagcat tcaggccgcg 780

gcggctggct tcgcatcgaa aaccccggcg aaccaggcga tttcgatgat cgacgggccc 840

gccccggacg gctacccgat catcaactac gagtacgcca tcgtcaacaa ccggcaaaag 900

gacgccgcca ccgcgcagac cttgcaggca tttctgcact gggcgatcac cgacggcaac 960

aaggcctcgt tcctcgacca ggttcatttc cagccgctgc cgcccgcggt ggtgaagttg 1020

tctgacgcgt tgatcgcgac gatttccagc 1050

<210> 2

<211> 540

<212> DNA

<213> Artificial Sequence

<220>

<223> coding sequence of multi-epitope antigen LEP amino acid

<400> 2

gtgccgctct atggacagct gggagccaaa ggggtagtcg gtaccggcgc cgaattgctc 60

gacgacatta gggcattctt gcggcggttc cggacccagt tcgaggatgc gtggtcccgg 120

tatctctctg ccgacaccac ccgggtgacg atcctgaccg gcagacggat gaccgatttg 180

gtggtcatct tcgtgacact gggcgccgcg gccatcccgg cgtggccgat cgcgctggct 240

tacttcgtgg cgttgtcccg acagcggtgg cgagagtttt tgacaaagct cactggcgca 300

ggcgcagcgg cattccccgc cacgttcacc gaagacgtcc gccaggcgtt gtacgcctcc 360

aagggccgct acttccggct gttgaccggg tgggtcgggg gcggaacctg gccgtacccc 420

gaaatcgcta ccgcagccat gatttcgcca ttcaaggact actttggcct ggcgcacgac 480

ctgccgaagt gggcgtggta cgcggtcgac gtcttttcca ctcttttggt agtccctggg 540

<210> 3

<211> 1596

<212> DNA

<213> Artificial Sequence

<220>

<223> coding sequence of amino acids of PstS1-LEP fusion protein (without PstS 1N-terminal 24 amino acids)

<400> 3

ggctcgaaac caccgagcgg ttcgcctgaa acgggcgccg gcgccggtac tgtcgcgact 60

acccccgcgt cgtcgccggt gacgttggcg gagaccggta gcacgctgct ctacccgctg 120

ttcaacctgt ggggtccggc ctttcacgag aggtatccga acgtcacgat caccgctcag 180

ggcaccggtt ctggtgccgg gatcgcgcag gccgccgccg ggacggtcaa cattggggcc 240

tccgacgcct atctgtcgga aggtgatatg gccgcgcaca aggggctgat gaacatcgcg 300

ctagccatct ccgctcagca ggtcaactac aacctgcccg gagtgagcga gcacctcaag 360

ctgaacggaa aagtcctggc ggccatgtac cagggcacca tcaaaacctg ggacgacccg 420

cagatcgctg cgctcaaccc cggcgtgaac ctgcccggca ccgcggtagt tccgctgcac 480

cgctccgacg ggtccggtga caccttcttg ttcacccagt acctgtccaa gcaagatccc 540

gagggctggg gcaagtcgcc cggcttcggc accaccgtcg acttcccggc ggtgccgggt 600

gcgctgggtg agaacggcaa cggcggcatg gtgaccggtt gcgccgagac accgggctgc 660

gtggcctata tcggcatcag cttcctcgac caggccagtc aacggggact cggcgaggcc 720

caactaggca atagctctgg caatttcttg ttgcccgacg cgcaaagcat tcaggccgcg 780

gcggctggct tcgcatcgaa aaccccggcg aaccaggcga tttcgatgat cgacgggccc 840

gccccggacg gctacccgat catcaactac gagtacgcca tcgtcaacaa ccggcaaaag 900

gacgccgcca ccgcgcagac cttgcaggca tttctgcact gggcgatcac cgacggcaac 960

aaggcctcgt tcctcgacca ggttcatttc cagccgctgc cgcccgcggt ggtgaagttg 1020

tctgacgcgt tgatcgcgac gatttccagc ggatccgtgc cgctctatgg acagctggga 1080

gccaaagggg tagtcggtac cggcgccgaa ttgctcgacg acattagggc attcttgcgg 1140

cggttccgga cccagttcga ggatgcgtgg tcccggtatc tctctgccga caccacccgg 1200

gtgacgatcc tgaccggcag acggatgacc gatttggtgg tcatcttcgt gacactgggc 1260

gccgcggcca tcccggcgtg gccgatcgcg ctggcttact tcgtggcgtt gtcccgacag 1320

cggtggcgag agtttttgac aaagctcact ggcgcaggcg cagcggcatt ccccgccacg 1380

ttcaccgaag acgtccgcca ggcgttgtac gcctccaagg gccgctactt ccggctgttg 1440

accgggtggg tcgggggcgg aacctggccg taccccgaaa tcgctaccgc agccatgatt 1500

tcgccattca aggactactt tggcctggcg cacgacctgc cgaagtgggc gtggtacgcg 1560

gtcgacgtct tttccactct tttggtagtc cctggg 1596

<210> 4

<211> 532

<212> PRT

<213> Artificial Sequence

<220>

<223> amino acid sequence of PstS1-LEP fusion protein (without PstS 1N-terminal 24 amino acids)

<400> 4

Gly Ser Lys Pro Pro Ser Gly Ser Pro Glu Thr Gly Ala Gly Ala Gly

1 5 10 15

Thr Val Ala Thr Thr Pro Ala Ser Ser Pro Val Thr Leu Ala Glu Thr

20 25 30

Gly Ser Thr Leu Leu Tyr Pro Leu Phe Asn Leu Trp Gly Pro Ala Phe

35 40 45

His Glu Arg Tyr Pro Asn Val Thr Ile Thr Ala Gln Gly Thr Gly Ser

50 55 60

Gly Ala Gly Ile Ala Gln Ala Ala Ala Gly Thr Val Asn Ile Gly Ala

65 70 75 80

Ser Asp Ala Tyr Leu Ser Glu Gly Asp Met Ala Ala His Lys Gly Leu

85 90 95

Met Asn Ile Ala Leu Ala Ile Ser Ala Gln Gln Val Asn Tyr Asn Leu

100 105 110

Pro Gly Val Ser Glu His Leu Lys Leu Asn Gly Lys Val Leu Ala Ala

115 120 125

Met Tyr Gln Gly Thr Ile Lys Thr Trp Asp Asp Pro Gln Ile Ala Ala

130 135 140

Leu Asn Pro Gly Val Asn Leu Pro Gly Thr Ala Val Val Pro Leu His

145 150 155 160

Arg Ser Asp Gly Ser Gly Asp Thr Phe Leu Phe Thr Gln Tyr Leu Ser

165 170 175

Lys Gln Asp Pro Glu Gly Trp Gly Lys Ser Pro Gly Phe Gly Thr Thr

180 185 190

Val Asp Phe Pro Ala Val Pro Gly Ala Leu Gly Glu Asn Gly Asn Gly

195 200 205

Gly Met Val Thr Gly Cys Ala Glu Thr Pro Gly Cys Val Ala Tyr Ile

210 215 220

Gly Ile Ser Phe Leu Asp Gln Ala Ser Gln Arg Gly Leu Gly Glu Ala

225 230 235 240

Gln Leu Gly Asn Ser Ser Gly Asn Phe Leu Leu Pro Asp Ala Gln Ser

245 250 255

Ile Gln Ala Ala Ala Ala Gly Phe Ala Ser Lys Thr Pro Ala Asn Gln

260 265 270

Ala Ile Ser Met Ile Asp Gly Pro Ala Pro Asp Gly Tyr Pro Ile Ile

275 280 285

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

290 295 300

Ala Gln Thr Leu Gln Ala Phe Leu His Trp Ala Ile Thr Asp Gly Asn

305 310 315 320

Lys Ala Ser Phe Leu Asp Gln Val His Phe Gln Pro Leu Pro Pro Ala

325 330 335

Val Val Lys Leu Ser Asp Ala Leu Ile Ala Thr Ile Ser Ser Gly Ser

340 345 350

Val Pro Leu Tyr Gly Gln Leu Gly Ala Lys Gly Val Val Gly Thr Gly

355 360 365

Ala Glu Leu Leu Asp Asp Ile Arg Ala Phe Leu Arg Arg Phe Arg Thr

370 375 380

Gln Phe Glu Asp Ala Trp Ser Arg Tyr Leu Ser Ala Asp Thr Thr Arg

385 390 395 400

Val Thr Ile Leu Thr Gly Arg Arg Met Thr Asp Leu Val Val Ile Phe

405 410 415

Val Thr Leu Gly Ala Ala Ala Ile Pro Ala Trp Pro Ile Ala Leu Ala

420 425 430

Tyr Phe Val Ala Leu Ser Arg Gln Arg Trp Arg Glu Phe Leu Thr Lys

435 440 445

Leu Thr Gly Ala Gly Ala Ala Ala Phe Pro Ala Thr Phe Thr Glu Asp

450 455 460

Val Arg Gln Ala Leu Tyr Ala Ser Lys Gly Arg Tyr Phe Arg Leu Leu

465 470 475 480

Thr Gly Trp Val Gly Gly Gly Thr Trp Pro Tyr Pro Glu Ile Ala Thr

485 490 495

Ala Ala Met Ile Ser Pro Phe Lys Asp Tyr Phe Gly Leu Ala His Asp

500 505 510

Leu Pro Lys Trp Ala Trp Tyr Ala Val Asp Val Phe Ser Thr Leu Leu

515 520 525

Val Val Pro Gly

530

Claims

1. A mycobacterium tuberculosis antigen or fusion protein with mycobacterium tuberculosis immunoreactivity has a sequence shown in SEQ ID NO. 4.

2. A nucleic acid encoding a mycobacterium tuberculosis antigen or a fusion protein with mycobacterium tuberculosis immunoreactivity, having the polynucleotide sequence: (a) a polynucleotide sequence as shown in SEQ ID NO. 3; or (b) a polynucleotide sequence encoding the amino acid sequence shown in SEQ ID NO. 4 which differs from SEQ ID NO. 3 due to codon degeneracy.

3. A method of preparing a mycobacterium tuberculosis antigen or fusion protein with mycobacterium tuberculosis immunoreactivity of claim 1, comprising expressing the nucleic acid of claim 2 in a suitable host cell.

4. A vector expressing the polynucleotide of claim 2.

5. A host cell comprising the construct or expression vector of claim 4.

6. A method of making a fusion protein of claim 1 that is immunoreactive with mycobacterium tuberculosis comprising (a) culturing a host cell of claim 5 of the invention under conditions conducive to production of the fusion protein; and (b) recovering the fusion protein.

7. A pharmaceutical composition comprising an immune complex adjuvant and an antigen, wherein the immune complex adjuvant comprises: sorbitan trioleate, squalene, a surfactant and heat inactivated bacillus calmette-guerin (hBCG) or high pressure homogeneous disruption BCG, wherein the antigen is a mycobacterium tuberculosis antigen or a fusion protein with mycobacterium tuberculosis immunoreactivity, and the fusion protein is an amino acid sequence shown as SEQ ID NO: 4.

8. The pharmaceutical composition of claim 7, which is a vaccine.

9. The pharmaceutical composition according to claim 7 or 8, wherein the amount of sorbitol trioleate is 0.1% to 1% (w/v).

10. The pharmaceutical composition of any one of claims 7-8, wherein the squalene is 1-10% (v/v).

11. The pharmaceutical composition of any one of claims 7-8, wherein the surfactant is a non-ionic surfactant.

12. The pharmaceutical composition of claim 11, wherein said surfactant is tween.

13. The pharmaceutical composition of claim 12, wherein said tween is tween 80.

14. The pharmaceutical composition of any one of claims 7-8, 12-13, further comprising a buffer.

15. The pharmaceutical composition of claim 14, wherein the buffer is a sodium citrate buffer.

16. The pharmaceutical composition of claim 15, wherein said sodium citrate buffer has a concentration of 5-10mM and a pH of 6-7.

17. The pharmaceutical composition of any one of claims 7-8, 12-13, 15-16, wherein the amount of sorbitol trioleate is 0.3% to 0.7%.

18. The pharmaceutical composition of claim 17, wherein said sorbitol trioleate is in an amount of 0.4-0.6%.

19. The pharmaceutical composition of claim 18, wherein said sorbitol trioleate is in an amount of 0.5%.

20. The pharmaceutical composition of any one of claims 7-8, 12-13, 15-16, 18-19, wherein the squalene is 3-7%.

21. The pharmaceutical composition of claim 20, wherein the squalene is 4-6%.

22. The pharmaceutical composition of claim 21, wherein the squalene is 5%.

23. The pharmaceutical composition of any one of claims 7-8, 12-13, 15-16, 18-19, 21-22, wherein the concentration of the surfactant is 0.3-0.7% (w/v).

24. The pharmaceutical composition of claim 23, wherein the surfactant is present at a concentration of 0.4-0.6%.

25. The pharmaceutical composition of claim 23, wherein the surfactant is present at a concentration of 0.5%.

26. The pharmaceutical composition of any one of claims 7-8, 12-13, 15-16, 18-19, 21-22, 24-25, wherein the amount of heat inactivated bacillus calmette-guerin (hbg) or high pressure homogeneous disrupted BCG is 5-500 μ g.

27. The pharmaceutical composition of claim 26, wherein the amount of heat inactivated BCG (hBCG) or high pressure homogeneous disrupted BCG is 25-500 μ g.

28. The pharmaceutical composition of claim 26, wherein the amount of heat inactivated BCG (hBCG) or high pressure homogeneous disrupted BCG is 50-500 μ g.

29. The pharmaceutical composition of claim 26, wherein the amount of heat inactivated BCG (hBCG) or high pressure homogeneous disrupted BCG is 50-400 μ g.

30. The pharmaceutical composition of claim 26, wherein the amount of heat inactivated BCG (hBCG) or high pressure homogeneous disrupted BCG is 50-300 μ g.

31. The pharmaceutical composition of claim 26, wherein the amount of heat inactivated BCG (hBCG) or high pressure homogeneous disrupted BCG is 50-250 μ g.

32. The pharmaceutical composition of any one of claims 7-8, 12-13, 15-16, 18-19, 21-22, 24-25, 27-31, wherein the method of making the composite adjuvant comprises:

3) adding the oil phase into the water phase, and fully and uniformly mixing;

4) adding heat inactivated BCG vaccine (hBCG) or high pressure homogeneous crushed BCG after fully emulsifying and sterilizing.

33. Use of the antigen or fusion protein of claim 1 in the preparation of a tuberculosis diagnostic kit.