CN110590958B

CN110590958B - Tandem polypeptide and application thereof in immune protection against mycobacterium tuberculosis

Info

Publication number: CN110590958B
Application number: CN201910858314.8A
Authority: CN
Inventors: 龚文平; 吴雪琼; 梁艳; 王兰; 王杰; 薛勇; 米洁
Original assignee: 8th Medical Center of PLA General Hospital
Current assignee: 8th Medical Center of PLA General Hospital
Priority date: 2019-09-11
Filing date: 2019-09-11
Publication date: 2021-04-13
Anticipated expiration: 2039-09-11
Also published as: CN110590958A

Abstract

The invention discloses a tandem polypeptide and application thereof in immune protection against mycobacterium tuberculosis. The tandem polypeptide contains amino acid sequences shown as SEQ ID NO: 2, and the amino acid sequence of the polypeptide A is shown as SEQ ID NO: 3 and the amino acid sequence of the polypeptide B is shown as SEQ ID NO: 4. Experiments prove that the tandem polypeptide is used for strengthening immunity after the BCG priming to induce the remarkable reduction of the colony number of the mycobacterium tuberculosis in the liver and the lung of a humanized C57BL/6 mouse, and the immune protection efficiency of the mouse is superior to that of a BCG single immune group; there was no significant difference in the number of colonies of M.tuberculosis in the liver and lung of wild type C57BL/6 mice. Therefore, the tandem polypeptide can inhibit the proliferation of the mycobacterium tuberculosis in a humanized animal body; the Mycobacterium tuberculosis vaccine can be prepared according to the method. The invention has important application value.

Description

Tandem polypeptide and application thereof in immune protection against mycobacterium tuberculosis

Technical Field

The invention belongs to the field of immunology, and particularly relates to a tandem polypeptide and application thereof in immune protection against mycobacterium tuberculosis.

Background

Vaccination is the best means of eliminating Tuberculosis (TB) infection, but there is still a lack of effective vaccines. The earliest vaccines for preventing tuberculosis were BCG vaccines, but a number of studies have shown that BCG, although having a preventive effect on severe tuberculosis in children (such as miliar tuberculosis and tubercular meningitis), has a diminishing protective effect with age, and that BCG is essentially ineffective in people infected with tubercle bacillus. Compared with BCG vaccine, the subunit vaccine only uses certain protein antigens which can induce organisms to generate protective immune response, but not whole bacterial antigens, so that side effects after immunization are obviously reduced, and the preparation of the subunit vaccine is safer and more efficient compared with the traditional vaccine. However, as the research progresses, the subunit vaccine is found to have disadvantages, such as multiple vaccinations, adjuvant assistance, limited immune protection effect and the like.

After entering the body, the foreign substances are usually recognized by the body's immune system and mount an immune response to eliminate the foreign substances or pathogens. Traditional subunit vaccines vaccinate the whole pathogen protein or one or more protein antigens to induce the body to generate specific immune response against pathogen infection. However, the surface receptors of immune cells usually recognize only certain specific amino acids, i.e., epitopes (epitopes) or Antigenic determinants (Antigenic determinants), on Antigenic peptide molecules. Mycobacterium tuberculosis is an intracellular parasitic bacterium, and protective immunity against intracellular bacteria mainly depends on T cell-mediated specific cellular immune response. The T cell epitope vaccine can be recognized by more MHC molecules in the body and efficiently presented, and can solve the problems of immune failure and the like caused by the mutation of a certain dominant epitope of a pathogen. Because of these unique advantages, T cell epitope vaccines are gradually being focused on by more and more scholars, and become one of the hot spots in the field of vaccine research.

Therefore, the design and research of a novel and efficient T cell epitope vaccine of mycobacterium tuberculosis have important significance for preventing and treating tuberculosis.

Disclosure of Invention

The invention aims to provide a mycobacterium tuberculosis vaccine.

The invention firstly protects a tandem polypeptide which can be a1) or a 2):

a1) a tandem polypeptide comprising polypeptide A, polypeptide B and polypeptide C;

the amino acid sequence of the polypeptide A can be shown as SEQ ID NO: 2 is shown in the specification;

the amino acid sequence of the polypeptide B can be shown as SEQ ID NO: 3 is shown in the specification;

the amino acid sequence of polypeptide C can be shown as SEQ ID NO: 4 is shown in the specification;

a2) the N-terminal and/or C-terminal of a1) is linked to a tag to obtain a tagged tandem polypeptide.

In a2), the tag can be His tag (with the amino acid sequence of HHHHHHHHHH), Poly-Arg tag (with the amino acid sequence of RRRRR), FLAG tag (with the amino acid sequence of DYKDDDDK), c-myc tag (with the amino acid sequence of EQKLISEEDL) or Strep-tag II tag (with the amino acid sequence of WSHPQFEK).

Any of the tandem polypeptides described above may be any of b1) -b 6):

b1) the amino acid sequence is shown as SEQ ID NO: 1 ACP;

b2) the amino acid sequence is shown as SEQ ID NO: 5 tandem polypeptide APC;

b3) the amino acid sequence is shown as SEQ ID NO: 6, a tandem polypeptide CAP;

b4) the amino acid sequence is shown as SEQ ID NO: 7, a tandem polypeptide CPA;

b5) the amino acid sequence is shown as SEQ ID NO: 8 is a tandem polypeptide PAC;

b6) the amino acid sequence is shown as SEQ ID NO: 9, PCA.

The invention also provides a nucleic acid molecule encoding any of the tandem polypeptides described above.

The invention also protects the application of any one of the tandem polypeptides or the nucleic acid molecule for encoding any one of the tandem polypeptides, which can be c1) or c2) or c 3):

c1) preparing a mycobacterium tuberculosis vaccine;

c2) inhibiting proliferation of Mycobacterium tuberculosis;

c3) preparing a product for preventing and/or treating tuberculosis.

The invention also protects the application of any tandem polypeptide or nucleic acid molecule for coding any tandem polypeptide in combination with BCG, which can be c1), c2) or c 3):

c1) preparing a mycobacterium tuberculosis vaccine;

c2) inhibiting proliferation of Mycobacterium tuberculosis;

c3) preparing a product for preventing and/or treating tuberculosis.

The invention also protects the polypeptide A, the polypeptide B or the polypeptide C.

The invention also protects the application of any two or any one of the polypeptide A, the polypeptide B and the polypeptide C, which can be c1), c2) or c 3):

c1) preparing a mycobacterium tuberculosis vaccine;

c2) inhibiting proliferation of Mycobacterium tuberculosis;

c3) preparing a product for preventing and/or treating tuberculosis.

The invention also provides an anti-mycobacterium tuberculosis preparation which can contain any tandem polypeptide or a nucleic acid molecule for encoding any tandem polypeptide.

The anti-mycobacterium tuberculosis preparation can specifically consist of any tandem polypeptide or a nucleic acid molecule for encoding any tandem polypeptide.

The anti-mycobacterium tuberculosis preparation may further comprise bcg.

The anti-mycobacterium tuberculosis preparation can specifically consist of any tandem polypeptide or nucleic acid molecule for encoding any tandem polypeptide and BCG.

The invention also provides a product for preventing and/or treating tuberculosis, which can contain any tandem polypeptide or a nucleic acid molecule for coding any tandem polypeptide.

The product for preventing and/or treating tuberculosis can specifically consist of any tandem polypeptide or a nucleic acid molecule for encoding any tandem polypeptide.

The product for preventing and/or treating tuberculosis may further comprise BCG.

The product for preventing and/or treating tuberculosis can specifically consist of any tandem polypeptide or nucleic acid molecule for encoding any tandem polypeptide and BCG.

The product may specifically be a mycobacterium tuberculosis vaccine.

Any of the above tuberculosis may specifically be pulmonary tuberculosis.

Any of the tuberculosis may be hepatic tuberculosis.

Any one of the mycobacterium tuberculosis vaccines can be specifically a human mycobacterium tuberculosis vaccine.

Any one of the above methods for inhibiting proliferation of Mycobacterium tuberculosis may specifically be the method for inhibiting proliferation of Mycobacterium tuberculosis in a humanized animal or a human.

The humanized animal can be specifically a humanized mouse. The humanized mouse can be specifically an HLA-A11/DR1 transgenic C57BL/6 mouse (Zengyang. establishment of a human MHC transgenic mouse model and basic research of application thereof. military medical science college of people's liberation force, China.2016).

Any one of the above-mentioned M.tuberculosis specifically can be M.tuberculosis standard strain H37 Rv. Mycobacterium tuberculosis Standard strain H37Rv is described in the following documents: yan L, Xiaoyan Z, Li X, et al. immunological and Therapeutic Effects of pVAX1-rv1419 DNA from Mycobacterium tuberculosis [ J ]. Current Gene Therapy, 2016, 16 (4): 249-255.

In one embodiment of the present invention, humanized mice (i.e., HLA-A11/DR1 transgenic C57BL/6 mice) 6-7 weeks old and 16-18g in weight were randomly divided into an ACP group, a PBS group, a BCG group, and a BCG + ACP group, the ACP group was immunized with the tandem polypeptide ACP, the PBS group was immunized with PBS buffer, the BCG group was immunized with BCG vaccine, and the BCG + ACP group was primed with BCG and then boosted with the tandem polypeptide ACP; then, using the mycobacterium tuberculosis for counteracting toxic substances, and counting the number of the mycobacterium tuberculosis in the liver and the lung of the mouse. The result shows that in the liver of the humanized mouse, the immunoprotection of the serial polypeptide ACP accounts for 32.5 percent of the immunoprotection of BCG, and the combined immunoprotection of the BCG vaccine and the serial polypeptide ACP accounts for 94.5 percent of the immunoprotection of BCG; in the lung of the humanized mouse, the immunoprotection of the tandem polypeptide ACP accounts for 66.2% of the immunoprotection of BCG, and the immunoprotection of the combination of BCG vaccine and the tandem polypeptide ACP accounts for 103.4% of the immunoprotection of BCG. The lung is the most main target organ of mycobacterium tuberculosis, so that the immunoprotection of the polypeptide ACP in series can reach more than 66.2 percent of the BCG immunoprotection, and the combined immunoprotection of the BCG vaccine and the polypeptide ACP in series can reach more than 103.4 percent of the BCG immunoprotection. Therefore, the tandem polypeptide provided by the invention can induce the colony number of mycobacterium tuberculosis in the liver and lung of the humanized mouse to be remarkably reduced after the BCG is initially immunized, and the immune protection efficiency is superior to that of a BCG single immunization group.

The tandem polypeptide provided by the invention can inhibit the proliferation of mycobacterium tuberculosis in a humanized animal body; the Mycobacterium tuberculosis vaccine can be prepared according to the method. The invention has important application value.

Drawings

FIG. 1 is a schematic diagram of the screening process of positive epitope peptide.

FIG. 2 shows the results of four independent ELISOPT experiments screening positive epitope peptides.

FIG. 3 is a graph showing the immunoprotective effect of the tandem polypeptide ACP assessed by the number of Mycobacterium tuberculosis colonies in the liver and lung of immunized mice.

FIG. 4 is a graph of the prediction of tandem polypeptide ACP by the Protean module of DNASTAR software.

FIG. 5 is a graph of the prediction of tandem polypeptide APC by the Protean module of DNASTAR software.

FIG. 6 is a graph of the prediction of tandem polypeptide CAP by the Protean module of DNASTAR software.

Figure 7 is the results of prediction of tandem polypeptide CPA by the Protean module of DNASTAR software.

FIG. 8 is a graph of the results of prediction of tandem polypeptide PAC by the Protean module of DNASTAR software.

FIG. 9 shows the results of prediction of tandem polypeptide PCA by the Protean module of DNASTAR software.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

HLA-A11/DR1 transgenic C57BL/6 mice are described in the following documents: the basic research of establishing human MHC transgenic mouse model and its application, 2016. hereinafter, HLA-A11/DR1 transgenic C57BL/6 mouse is named humanized mouse.

The C57BL/6 mouse is a product of the Experimental animals technology of Beijing Wittingerhua. Hereinafter, the C57BL/6 mouse is referred to as a wild-type mouse.

CpG adjuvants are products of the company of Industrial bioengineering (Shanghai) and have a catalog number of ODN 2395. The Roche medium is a product of Behcet Biotechnology Co., Ltd, and the catalog number is BA 7005C-2. BCG is a product of Chengdu biological products, Inc.

In the following examples, all the M.tuberculosis are M.tuberculosis Standard strain H37Rv, which is publicly available from the applicant and can be used only for the experiments of the duplicated invention under the condition of meeting the biosafety operating specification. Mycobacterium tuberculosis Standard strain H37Rv is described in the following documents: yan L, Xiaoyan Z, Li X, et al. immunological and Therapeutic Effects of pVAX1-rv1419 DNA from Mycobacterium tuberculosis [ J ]. Current Gene Therapy, 2016, 16 (4): 249-255. in the literature, Mycobacterium tuberculosis Standard strain H37Rv is named Mycobacterium tuberculosis, H37Rv strain.

A schematic diagram of the screening process for positive epitope peptide is shown in FIG. 1.

Example 1 Artificial Synthesis of epitope peptide of Mycobacterium tuberculosis Th1

Method for predicting dominant epitope Th1 of mycobacterium tuberculosis by bioinformatics

1. The amino acid sequences of target proteins such as mycobacterium tuberculosis Ag85A, Ag85B, CFP21, PPE18 and the like are obtained from an NCBI database, and then an IEDB MHC-II database (the website is http:// tools. IEDB. org/mhcii /) is used for predicting HLA-DRB1 x 01:01 restriction epitopes.

2. The HLA-DRB 1:01 restriction epitope predicted in the step 1 is predicted by using IEDB recommended (website: http:// www.iedb.org /), Consensus method, Combinatorial library, NN-align (netMHCII-2.2), SMM-align (netMHCII-1.1), Sturniolo and NetMHCIIpan, respectively, and then the comprehensive ranking is scored, wherein the epitope with the score below 10 in the comprehensive ranking is the Th1 dominant epitope, and the lower the score is, the higher the affinity is.

Consensus methods are described in the following documents: wang P, Sidney J, Kim Y, set A, Lund O, Nielsen M, pets B.2010.peptide binding ligands for HLA DR, DP and DQ molecules. BMC bioinformatics.11:568.Wang P, Sidney J, Dow C, Moth é B, set A, pets B.2008.A systematic association of MHC class II peptide binding ligands and evaluation of a consensus approach. PLoS Comp. 4(4): e1000048.

Combinatorial library is described in the following documents: sidney J, Assarsson E, Moore C, Ngo S, Pinilla C, set A, pets B.2008.quantitative peptide binding kinetics for 19 man and mouse MHC class I molecules derived using positional peptide libraries Immunome Res 4:2.

NN-align (netMHCII-2.2) is described in the following documents: nielsen M, Lund O.2009.NN-alignment.an anatomical neural network-based alignment algorithm for MHC class II peptide binding prediction.BMC biologics.10: 296.

SMM-align (netMHCII-1.1) is described in the following documents: nielsen M, Lundigaard C, Lund O.2007.prediction of MHC class II binding affinity using SMM-alignment, a novel stabilization matrix alignment method BMC Bioinformatics.8:238.

Sturniolo is described in the following documents: sturniolo T, Bono E, Ding J, Raddrizani L, Tuereci O, Sahin U, Braxenhaler M, Gallazzi F, Protti MP, Sinigaglia F, Hammer J.1999 Generation of tissue-specific and pro-scientific HLA ligand using DNA microarrays and virtual HLA class II substrates Nat Biotechnol.17(6): 555-.

NetMHCIIpan is described in the following documents: andreatta M, Karosiene E, Rasmussen M, Stryhn A, Buus S, and Nielsen M.2015.accurate pan-specific prediction of peptide-MHC class II binding affinity. immunogenetics.67 (11-12): 641-50.

Specific information of the dominant epitope (i.e., candidate epitope) of Mycobacterium tuberculosis Th1 is shown in Table 1.

TABLE 1

II, artificially synthesizing the Mycobacterium tuberculosis Th1 epitope peptide

The Th1 epitope peptide of each mycobacterium tuberculosis is obtained by in vitro synthesis by a solid phase synthesis method and purification by a high pressure liquid chromatography.

The amino acid sequence of each Mycobacterium tuberculosis Th1 epitope peptide is specifically shown in the 5 Th column in Table 1.

Example 2 screening of Positive epitope peptides Using four independent ELISOPT experiments

First, first ELISPOT experiment (repeat 2 times)

A. Epitope peptide-stimulated group

1. Preparation of spleen cell suspension

(1) 100. mu.L of inactivated Mycobacterium tuberculosis bacterial solution (about 5X 10)⁶PFU) and 100. mu.L of complete Freund's adjuvant were mixed and then C57BL/6 mice were immunized.

A total of 10 were immunized.

(2) Mice were sacrificed at day 15 post immunization, then placed in 75% (v/v) aqueous ethanol, removed after 10min, and spleens dissected.

(3) 10mL 1640 cell culture medium was added to a sterile petri dish, a sterile 200 mesh copper mesh was placed in the petri dish, the spleen was placed in the copper mesh, and the spleen cells were spread by gentle squeezing with the syringe plunger tip (sterile).

(4) An ELISOT plate was prepared, washed 4 times (200. mu.L/well) with PBS buffer, and then incubated with 1640 medium containing 10% (v/v) FBS in an amount of 200. mu.L wells for at least 30min to obtain a spleen cell suspension.

2. Erythrocyte lysis

(1) Taking the spleen cell suspension prepared in the step 1, centrifuging at 4 ℃ for 5min at 500g, and discarding the supernatant.

(2) Adding erythrocyte lysate (20-30 mL/spleen), gently blowing, mixing, and lysing at room temperature for 4-5 min. During which the mixture was gently shaken every 1 min.

(3) Centrifuge at 500g for 5min at 4 ℃ and discard the supernatant.

If the red blood cells are found to be incompletely lysed, steps (2) and (3) may be repeated.

(4) Washing for 1-2 times. The steps of each washing are as follows: adding appropriate amount of serum-free culture solution, resuspending the precipitate, centrifuging at 4 deg.C and 500g for 2-3min, and collecting the precipitate.

The amount of serum-free medium used in the washing should generally be at least 5 times the volume of the precipitate.

3. Counting and paving plate

(1) Resuspending the pellet (i.e., cell pellet) collected in step 2 in serum-free 1640 cell culture medium, counting cells, and adjusting the cell concentration to 3X 10⁶one/mL.

(2) ELISPOT plates were plated at 100. mu.L/well, giving a 3X 10 cell count per well⁵And (4) respectively.

(3) Adding Th1 epitope peptide of Mycobacterium tuberculosis at a concentration of 10 μ L/well, placing ELISPOT plate in cell culture box, 37 deg.C, and 5% CO₂And (5) incubating for 12-48 h.

4. ELISpot detection mouse IFN-gamma reaction point

(1) The ELISPOT plate that completed step 3 was removed, and the cells in the ELISPOT plate were gently removed, followed by 5 washes with PBS buffer at 200. mu.L/well.

(2) The R4-6A 2-labeled monoclonal antibody was diluted to 1. mu.g/mL with 0.5% (v/v) FBS-containing PBS buffer, and then added to an ELISPOT plate in an amount of 100. mu.L/well, and incubated at room temperature for 2 h.

(3) PBS buffer wash 5 times, 200 u L/hole.

(4) streptavidin-ALP was diluted 1:1000 with PBS buffer containing 0.5% (v/v) FBS, and then added to the ELISPOT plate at 100. mu.L/well and incubated at room temperature for 1 h.

(5) PBS buffer wash 5 times, 200 u L/hole.

(6) The color developing solution was filtered through a 0.45 μm filter, and then an ELISPOT plate was added in an amount of 100. mu.L/well to observe the change of spots in the well. After the spots meet the requirements, quickly washing with a large amount of tap water, patting dry the water, and naturally drying the spots in dark. Reading the plate, scanning and counting the result.

B. Blank control group

And (3) in the steps A and 3 is replaced by the step (3A), and the other steps are not changed and are used as blank control groups. The step (3A) is as follows: the ELISPOT plate was placed in a cell culture chamber at 37 ℃ with 5% CO₂And (5) incubating for 12-48 h.

C. Statistical Stimulation Index (SI)

The Stimulation Index (SI) was calculated. Stimulation index is the number of spots in the epitope peptide-stimulated group/the number of spots in the blank control group.

When the SI of an epitope peptide is more than 2, the epitope peptide is considered as a positive epitope peptide.

Second, second ELISPOT experiment (repeat 2 times)

A. Epitope peptide-stimulated group

1. Preparation of spleen cell suspension

(1) Taking an inactivated mycobacterium tuberculosis solution with the concentration of 5mg/mL, carrying out 400W ultrasonic treatment for 20min, and then immunizing 10 humanized mice according to the dose of 200 mu L/mouse.

(2) The humanized mice were sacrificed at day 15 after immunization, then placed in 75% (v/v) ethanol aqueous solution, removed after 10min, and the spleen was dissected.

2. Erythrocyte lysis

The same as step 2 in the first step A.

3. Counting and paving plate

The same as step 3 in the first step A.

4. ELISpot detection mouse IFN-gamma reaction point

The same as 4 in the step A.

B. Blank control group

C. Statistical Stimulation Index (SI)

The same as step C.

Third, third ELISPOT experiment (repeat 3 times)

A. Epitope peptide-stimulated group

1. Preparation of spleen cell suspension

(1) Taking an inactivated mycobacterium tuberculosis solution with the concentration of 5mg/mL, performing ultrasonic treatment at 400W for 20min, and extracting total protein; then mixing the mixture with equivalent volume of Freund's complete adjuvant to obtain mixed solution; then, 10 humanized mice were immunized at a dose of 200. mu.L/mouse.

2. Erythrocyte lysis

The same as step 2 in the first step A.

3. Counting and paving plate

The same as step 3 in the first step A.

4. ELISpot detection mouse IFN-gamma reaction point

The same as 4 in the step A.

B. Blank control group

Replacing the step (3) in the steps A and 3 with the step (3A), keeping the other steps unchanged,as a blank control group. The step (3A) is as follows: the ELISPOT plate was placed in a cell culture chamber at 37 ℃ with 5% CO₂And (5) incubating for 12-48 h.

C. Statistical Stimulation Index (SI)

The same as step C.

Fourth and fourth ELISPOT experiment (repeat 3 times)

A. Epitope peptide-stimulated group

1. Preparation of spleen cell suspension

(1) Taking an inactivated mycobacterium tuberculosis solution with the concentration of 5mg/mL, performing ultrasonic treatment at 400W for 20min, and extracting total protein; then mixing the mixture with a non-Freund complete adjuvant with the same volume to obtain a mixed solution; then, 10 humanized mice were immunized at a dose of 200. mu.L/mouse.

2. Erythrocyte lysis

The same as step 2 in the first step A.

3. Counting and paving plate

The same as step 3 in the first step A.

4. ELISpot detection mouse IFN-gamma reaction point

The same as 4 in the step A.

B. Blank control group

C. Statistical Stimulation Index (SI)

The same as step C.

The results of 10 elispot experiments are shown in figure 2.

After four independent ELISOPT experiments for 10 times of repetition, 4 epitope peptides are finally determined to be positive epitope peptides. The 4 epitope peptides are respectively Ag85B_12-26、CFP21_14-28、PPE18_149-163And CFP21_12-26。CFP21_14-28And CFP21_12-26With slight difference, CFP21 was used subsequently_12-26Experiments were performed.

Ag85B_12-26、CFP21_12-26And PPE18_149-163The information of the stimulation index and the amino acid sequence is shown in Table 2.

TABLE 2

Protein	Initiation of	Terminate	Sequences and their positions in the sequence Listing	Irritation index (SI)
					Ag85B	12	26	GRRLMIGTAAAVVLP(SEQ ID NO：2)	21.94±2.55
CFP21	12	26	VVVATTLALVSAPAG(SEQ ID NO：3)	13.5±1.37
					PPE18	149	163	AAAMFGYAAATATAT(SEQ ID NO：4)	3.61±0.56

Fifthly, obtaining of tandem polypeptide ACP

Artificially synthesizing a polypeptide ACP with an amino acid sequence shown as SEQ ID NO: 1 is shown.

SEQ ID NO: 1 is: GRRLMIGTAAAVVLPVVVATTLALVSAPAGAAAMFGYAAATATAT

Example 3 protective assessment of tandem polypeptide ACP

First, mouse immunization

Female humanized mice or female wild-type mice with the body weight of 16-18g and the age of 6-7 weeks are taken and randomly divided into 4 groups of an experimental group (namely, ACP group), a negative control group (namely, PBS group), a positive control group (namely, BCG group) and a boosting group (namely, BCG + ACP group), and each group has 6 or 7 mice. Each group was treated as follows:

experimental groups: on the 28 th day of adaptive feeding, 30 mu g of the tandem polypeptide ACP is prepared in 200 mu of LCpG adjuvant and is immunized 1 time subcutaneously, and then 20 mu g of the tandem polypeptide ACP is prepared in 200 mu of LCpG adjuvant and is immunized 2 times in abdominal cavity, wherein the immunization interval is 14 days;

negative control group: on day 28 of adaptive feeding, subcutaneously immunizing with 200 μ L of mixed solution (prepared by mixing 30 μ g of lyophilized powder of CpG adjuvant and 200 μ L of PBS buffer solution) for 1 time, and then intraperitoneally immunizing with 200 μ L of mixed solution for 2 times, each time at an interval of 14 days;

positive control group: adaptive feeding on day 28, 200. mu.L BCG (about 1X 10)⁵CFU) (product of Chengdu biological products, Limited liability company) Immunizing subcutaneously for 1 time, and then immunizing intraperitoneally with 200 μ L BCG vaccine for 2 times, wherein each immunization interval is 14 days;

and (3) reinforcing group: adaptive feeding on day 28, 200. mu.L BCG (about 1X 10)⁵CFU) were immunized 1 time subcutaneously, followed by 2 intraperitoneal immunizations with 20 μ g of the tandem polypeptide ACP formulated in 200 μ LCpG adjuvant, each immunization separated by 14 days.

Second, counteracting toxic pathogen

On 14 days after the completion of the step one, a humanized mouse or a wild type mouse is taken, and the tail vein is injected with mycobacterium tuberculosis every day for counteracting the toxin, wherein the counteracting dose is 1.75 multiplied by 10⁵CFU/only.

Continuously counteracting toxic substance for 28 days.

Third, detection of the number of Mycobacterium tuberculosis colonies

1. After the second step, the mice were sacrificed by cervical-amputation, gross pathology of the lung and liver was observed, and then the lung and liver were aseptically harvested respectively.

2. Taking the lung or liver of a mouse, adding a proper amount of normal saline, and grinding by using a grinder to obtain the grinding fluid.

3. And (3) after the step 2 is finished, adding 4% (m/v) NaOH aqueous solution into the grinding liquid, digesting for 30min, and shaking up to obtain a digestion solution.

4. And 3, after the step 3 is finished, taking the digestive juice, and diluting the digestive juice by using normal saline in an equal ratio of 10, 100 and 1000 times to obtain a diluent. The dilutions were spread evenly in Roche medium (3 replicates per dilution) and incubated at 37 ℃ for 4 weeks.

5. After completing step 4, the number of colonies of Mycobacterium tuberculosis was counted and averaged by group.

The results are shown in Table 3, Table 4 and FIG. 3(A is humanized mouse liver, B is humanized mouse lung, C is wild type mouse liver, and D is wild type mouse lung).

TABLE 3 statistical results of the humanized mouse liver

	Mycobacterium tuberculosis colony Count (CFU)
		PBS group	28351.060
BCG group	2659.802
		ACP group	20022.060
BCG + ACP group	4071.732

TABLE 4 statistical results of humanized mouse lungs

	Mycobacterium tuberculosis colony Count (CFU)
		PBS group	87124
BCG group	3975
		ACP group	32040
BCG + ACP group	1094

The results are as follows:

in the humanized mouse liver, the colony numbers of mycobacterium tuberculosis in the BCG group, the ACP group and the BCG + ACP group are reduced to different degrees compared with the PBS group;

in the humanized mouse lung, compared with the PBS group, the bacterial colony numbers of the mycobacterium tuberculosis in the BCG group and the BCG + ACP group are obviously reduced, and the bacterial colony number of the mycobacterium tuberculosis in the CFP21 group is also reduced to a certain degree; the colony number of mycobacterium tuberculosis in the BCG + ACP group was also significantly reduced compared to the BCG group; therefore, the combined immunity of the BCG vaccine and the ACP is better than the single immunity of BCG;

the colonies of mycobacterium tuberculosis in the liver of the wild mouse have no significant difference among the PBS group, the BCG group, the ACP group and the BCG + ACP group (P is more than 0.05);

the number of mycobacterium tuberculosis colonies in the lungs of the wild-type mice was not significantly different among the PBS group, the BCG group, the ACP group and the BCG + ACP group (P > 0.05);

compared with the PBS group, the ACP group reduced the number of mycobacterium tuberculosis in the humanized mouse liver and lung by 29.4% and 63.2%, the BCG + ACP group reduced the number of mycobacterium tuberculosis in the humanized mouse liver and lung by 85.6% and 98.7%, and the BCG group reduced the number of mycobacterium tuberculosis in the humanized mouse liver and lung by 90.6% and 95.4%. Thus, in the liver of the humanized mouse, the immunoprotection of the tandem polypeptide ACP accounted for 32.5% (29.4%/90.6%) of the BCG immunoprotection, and the immunoprotection of the combination of BCG vaccine and the tandem polypeptide ACP accounted for 94.5% (85.6%/90.6%); in the lungs of the humanized mice, the immunoprotection of the tandem polypeptide ACP accounted for 66.2% (63.2%/95.4%) of BCG immunoprotection, and the immunoprotection of the combination of BCG vaccine and the tandem polypeptide ACP accounted for 103.4% (98.7%/95.4%). The lung is the most main target organ of mycobacterium tuberculosis, so that the immunoprotection of the polypeptide ACP in series can reach more than 66.2 percent of the BCG immunoprotection, and the combined immunoprotection of the BCG vaccine and the polypeptide ACP in series can reach more than 103.4 percent of the BCG immunoprotection.

Therefore, the tandem polypeptide ACP is used for strengthening immunity after the BCG priming to induce that the colony number of the mycobacterium tuberculosis in the liver and the lung of the humanized C57BL/6 mouse is obviously reduced, and the immune protection efficiency is superior to that of the BCG single immune group. However, the advantage is not shown in the liver and lung of the wild type C57BL/6 mouse, and further shows that the tandem polypeptide ACP has good specificity.

Example 4 Ag85B_12-26、CFP21_12-26And PPE18_149-163Effect of three epitope peptide arrangement sequences on tandem Polypeptides

To understand Ag85B_12-26、CFP21_12-26And PPE18_149-163The influence of the sequence of the arrangement of the three epitope peptides on the antigenicity, hydrophobicity, hydrophilicity, alpha helix, beta folding and the like of the tandem polypeptide is predicted by the inventor of the invention by adopting a Protean module of DNASTAR software to respectively predict the tandem polypeptide ACP, the tandem polypeptide APC, the tandem polypeptide CAP, the tandem polypeptide CPA, the tandem polypeptide PAC and the tandem polypeptide PCA.

The amino acid sequence of the tandem polypeptide APC is shown as SEQ ID NO: 5, respectively. SEQ ID NO: 5 is as follows: GRRLMIGTAAAVVLPAAAMFGYAAATATATVVVATTLALVSAPAG

The amino acid sequence of the tandem polypeptide CAP is shown as SEQ ID NO: and 6. SEQ ID NO: 6 is as follows: VVVATTLALVSAPAGGRRLMIGTAAAVVLPAAAMFGYAAATATAT

The amino acid sequence of the tandem polypeptide CPA is shown as SEQ ID NO: shown at 7. SEQ ID NO: 7 is as follows: VVVATTLALVSAPAGAAAMFGYAAATATATGRRLMIGTAAAVVLP

The amino acid sequence of the tandem polypeptide PAC is shown as SEQ ID NO: shown in fig. 8. SEQ ID NO: 8 is as follows: AAAMFGYAAATATATGRRLMIGTAAAVVLPVVVATTLALVSAPAG

The amino acid sequence of the tandem polypeptide PCA is shown as SEQ ID NO: shown at 9. SEQ ID NO: 9 is as follows: AAAMFGYAAATATATVVVATTLALVSAPAGGRRLMIGTAAAVVLP

The predicted results of the tandem polypeptide ACP are shown in FIG. 4.

The prediction of tandem polypeptide APCs is shown in FIG. 5.

The results of tandem polypeptide CAP prediction are shown in FIG. 6.

The results of the prediction of the tandem polypeptide CPA are shown in FIG. 7.

The predicted results for the tandem polypeptide PAC are shown in FIG. 8.

The results of the prediction of the tandem polypeptide PCA are shown in FIG. 9.

The results showed that Ag85B_12-26、CFP21_12-26And PPE18_149-163The sequence of the arrangement of the three epitope peptides has no obvious influence on the overall antigenic property, and the change is only reflected in the change of position. The above data suggest that Ag85B_12-26、CFP21_12-26And PPE18_149-163The tandem polypeptide consisting of the three epitope peptides has similar effect on inhibiting the proliferation of the mycobacterium tuberculosis.

<110> eighth medical center of general hospital of people liberation force of China

<120> tandem polypeptide and application thereof in immune protection against mycobacterium tuberculosis

<160> 9

<170> PatentIn version 3.5

<210>1

<211>45

<212>PRT

<213>Artificial sequence

<400>1

Gly Arg Arg Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro Val

1 5 10 15

Val Val Ala Thr Thr Leu Ala Leu Val Ser Ala Pro Ala Gly Ala Ala

20 25 30

Ala Met Phe Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr

35 40 45

<210>2

<211>15

<212>PRT

<213>Artificial sequence

<400>2

Gly Arg Arg Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro

1 5 10 15

<210>3

<211>15

<212>PRT

<213>Artificial sequence

<400>3

Val Val Val Ala Thr Thr Leu Ala Leu Val Ser Ala Pro Ala Gly

1 5 10 15

<210>4

<211>15

<212>PRT

<213>Artificial sequence

<400>4

Ala Ala Ala Met Phe Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr

1 5 10 15

<210>5

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<212>PRT

<213>Artificial sequence

<400>5

Gly Arg Arg Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro Ala

1 5 10 15

Ala Ala Met Phe Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr Val Val

20 25 30

Val Ala Thr Thr Leu Ala Leu Val Ser Ala Pro Ala Gly

35 40 45

<210>6

<211>45

<212>PRT

<213>Artificial sequence

<400>6

Val Val Val Ala Thr Thr Leu Ala Leu Val Ser Ala Pro Ala Gly Gly

1 5 10 15

Arg Arg Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro Ala Ala

20 25 30

Ala Met Phe Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr

35 40 45

<210>7

<211>45

<212>PRT

<213>Artificial sequence

<400>7

Val Val Val Ala Thr Thr Leu Ala Leu Val Ser Ala Pro Ala Gly Ala

1 5 10 15

Ala Ala Met Phe Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr Gly Arg

20 25 30

Arg Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro

35 40 45

<210>8

<211>45

<212>PRT

<213>Artificial sequence

<400>8

Ala Ala Ala Met Phe Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr Gly

1 5 10 15

Arg Arg Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro Val Val

20 25 30

Val Ala Thr Thr Leu Ala Leu Val Ser Ala Pro Ala Gly

35 40 45

<210>9

<211>45

<212>PRT

<213>Artificial sequence

<400>9

Ala Ala Ala Met Phe Gly Tyr Ala Ala Ala Thr Ala Thr Ala Thr Val

1 5 10 15

Val Val Ala Thr Thr Leu Ala Leu Val Ser Ala Pro Ala Gly Gly Arg

20 25 30

Arg Leu Met Ile Gly Thr Ala Ala Ala Val Val Leu Pro

35 40 45

Claims

1. A tandem polypeptide that is a1) or a 2):

a1) the amino acid sequence is shown as SEQ ID NO: 1 ACP;

2. A nucleic acid molecule encoding the tandem polypeptide of claim 1.

3. Use of the tandem polypeptide according to claim 1 or the nucleic acid molecule according to claim 2 as c1) or c 3):

c1) preparing a mycobacterium tuberculosis vaccine;

c3) preparing a product for preventing and/or treating tuberculosis.

4. Use of a tandem polypeptide according to claim 1 or a nucleic acid molecule according to claim 2 in combination with a bcg, c1) or c 3):

c1) preparing a mycobacterium tuberculosis vaccine;

c3) preparing a product for preventing and/or treating tuberculosis.

5. An anti-mycobacterium tuberculosis preparation comprising the tandem polypeptide of claim 1 or the nucleic acid molecule of claim 2.

6. The anti-mycobacterium tuberculosis formulation of claim 5, wherein: the anti-mycobacterium tuberculosis preparation also contains BCG.

7. A product containing a tandem polypeptide according to claim 1 or a nucleic acid molecule according to claim 2 for use in the prevention and/or treatment of tuberculosis.

8. The product of claim 7, wherein: the product also contains BCG.