Application of mycobacterium protein in preparation of antigen presentation inhibitor

Abstract

The invention discloses application of mycobacterium protein in preparation of an antigen presentation inhibitor. The amino acid sequence of the mycobacterium protein is as shown in SEQ ID NO: 1, and the protein does not generate excessive immunological rejection; secondly, the protein can inhibit the expression of surface molecules of antigen presenting cells and inhibit the occurrence of acquired immune response; the protein inhibits host antigen presentation through multiple factors and multiple ways, and inhibits acquired immunity.

Description

Technical Field

[0001] The invention relates to use of mycobacterium protein in preparing antigen presentation inhibitors, belonging to the field of biotechnology. Background Art

[0002] Mycobacterium tuberculosis (M.tb) enters the lungs through the respiratory tract and is phagocytosed by dendritic cells (DCs) and macrophages (MΦ), which control M.tb infection through similar pathways, such as phagocytosis and killing of invading pathogens; recognition of mycobacteria and their components through surface receptors, thereby causing a series of intracellular reactions to clear mycobacteria; mediating inflammatory responses and corresponding immune responses by secreting cytokines and other factors; taking up and processing antigens, transferring them to the membrane together with the class II major histocompatibility complex (MHC II), and presenting the antigens to CD4 + T lymphocytes, activate the acquired immune response.

[0003] After mycobacterial infection, antigen presenting cells (APCs) will change their expression levels of a series of molecules related to antigen presentation and T cell activation, such as CD40, CD80, CD86, and MHC II. Stimulation of inflammatory signals or the uptake of pathogen antigens will cause immature DCs to migrate to secondary lymphoid organs. Immature DCs will undergo a maturation process, including the secretion of cytokines and upregulation of the expression of surface MHC class II molecules and co-stimulatory molecules CD80, CD86, and CD40. The expression of MHC class II molecules on the cell surface is a necessary condition for antigen presentation, and its expression is regulated by the transcriptional regulator CIITA. T cell activation depends on these surface molecules and co-stimulatory signal molecules of APCs. When the T cell receptor (TCR) recognizes the specific antigen peptide presented by the MHC class II molecules expressed by APCs, CD4 + T cells will be activated.

[0004] Antigen presentation is a key step that connects innate and acquired immunity and involves unique mechanisms. M.tb's evasion of antigen presentation may be one of the key issues in the development of a truly effective tuberculosis vaccine. BCG is widely used, which is derived from Mycobacterium bovis, which is closely related to M.tb. This vaccine may retain many of the immune escape properties of M.tb, preventing it from inducing highly protective and long-lasting acquired immunity. Therefore, a deeper understanding of the mechanisms by which M.tb interferes with antigen presentation and subverts or escapes acquired immunity is crucial for the development of new host-directed therapies, disease biomarkers, and effective tuberculosis vaccines.

[0005] In summary, there is an urgent need in the art to develop an immunologically active substance that can effectively inhibit antigen presentation after mycobacterium infection. Summary of the invention

[0006] Purpose of the invention: The technical problem to be solved by the present invention is to provide the use of mycobacterial protein in the preparation of antigen presentation inhibitors, which can inhibit the expression of surface molecules of antigen presenting cells and have the function of inhibiting the occurrence of acquired immune response.

[0007] Technical solution: To solve the above technical problems, the present invention provides a method for preparing an antigen presentation inhibitor by using a mycobacterium protein; the amino acid sequence of the mycobacterium protein is shown in SEQ ID NO: 1: VPTYSYECTQCANRFDVVQAFTDDALTTCERCSGRLRKLFNAVGVVFKGTGFYRTDS RESGKKSKSQTNGSSTSESTKSSGSSGSSGSSESKASGSTEKSTSSTTAAAAV.

[0008] In the examples of the present invention, the protein of the present invention is named Rv0991c.

[0009] The present invention also provides use of the gene encoding the mycobacterium protein in preparing an antigen presentation inhibitor.

[0010] The polynucleotide encoding the protein may be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded.

[0011] Further, the polynucleotide sequence is such as SEQ ID As shown in NO:2, specifically: GTGCCAACCTACAGCTACGAGTGCACCCAGTGCGCCAACCGCTTCGATGTTGTGCAGGCCTTCACCGACGATGCGCTGACCACGTGCGAGCGGTGTTCTGGCCGGCTGCGCAAGCTGTTCAATGCGGTCGGCGTGGTGTTCAAAGGCACCGGCTTCTACCG CACCGACAGCCGCGAGTCGGGCAAGAAGTCCAAGAGCCAGACCAACGGATCCTCGACCAGCGAGTCGACGAAGAGTTCGGGGAGTTCGGGGAGTTCTGGCTCGAGTGAAAGCAAGGCTTCGGGGTCAACCGAGAAGTCAACCAGCTCCACAACCGCCGCCGCGGCCGTCTGA.

[0012] The present invention also provides the use of a recombinant vector, a recombinant bacterium or a recombinant cell containing the gene in preparing an antigen presentation inhibitor.

[0013] Among them, inhibition of antigen presentation includes downregulating the transcription levels of CD40, CD80, and MHC II.

[0014] Wherein, the expression vector contains the aforementioned polynucleotide.

[0015] Methods well known to those skilled in the art can be used to construct the expression vector. These methods include recombinant DNA technology, DNA synthesis technology, etc. The DNA encoding the protein can be effectively connected to an appropriate promoter in the expression vector to guide mRNA synthesis and then express the protein.

[0016] Preferably, the expression vector is a prokaryotic expression vector and a mycobacterial expression vector. Further, the prokaryotic expression vector can be stably expressed in Escherichia coli, such as the prokaryotic vector pET22, etc. The mycobacterial expression vector can be stably expressed in Mycobacterium smegmatis, such as the mycobacterial vector pALACE.

[0017] Wherein, the host cell contains or is integrated with the above expression vector.

[0018] The host cell may be a prokaryotic cell, such as a bacterial cell, representative examples of which include Escherichia coli and Mycobacterium smegmatis.

[0019] The present invention also provides the use of a recombinant vector, a recombinant bacterium or a recombinant cell containing a gene encoding a mycobacterial protein having an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of a drug for treating mycobacterial infection.

[0020] In the embodiments of the present invention, the host cells used are Escherichia coli and Mycobacterium smegmatis.

[0021] The fifth aspect of the present invention provides a method for preparing the above protein, which is selected from any one of the following:

[0022] a) synthesizing the protein using chemical synthesis methods; or

[0023] b) culturing the host cell under conditions suitable for expressing the protein, and isolating the protein from the culture.

[0024] The method for isolating proteins that inhibit antigen presentation from culture can be extracted using nickel column purification.

[0025] After stimulating cells with small molecule proteins (Rv0991c) of different concentrations, the cell morphology was found to be unchanged. At the same time, when mice were intraperitoneally injected with recombinant bacteria, the mice were observed on days 0, 3, and 6, and the weight did not decrease, and the mobility and state were no different from the blank group, indicating that no excessive immune rejection reaction would occur.

[0026] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: the small molecule protein (Rv0991c) of the present invention does not produce excessive immune rejection reactions; secondly, Rv0991c can inhibit the expression of surface molecules of antigen presenting cells and inhibit the occurrence of acquired immune responses. Rv0991c is different from the antigen presentation inhibitors used clinically in that they do not target a single antigen presentation process, but multiple factors and multiple pathways, and strongly inhibit the occurrence of acquired immunity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 It was shown that the Rv0991c protein inhibited the secretion of the T cell activation cytokine IL-2;

[0028] Figure 2 It was shown that Rv0991c overexpression inhibited the secretion of IL-2, a T cell activation cytokine induced by Mycobacterium smegmatis;

[0029] Figure 3a It was shown that overexpression of Rv0991c inhibited the transcription of CD40, a surface molecule of dendritic cells induced by Mycobacterium smegmatis; Figure 3b It was shown that overexpression of Rv0991c inhibited the transcription of CD80, a surface molecule of dendritic cells induced by Mycobacterium smegmatis; Figure 3c It was shown that Rv0991c overexpression inhibited the transcription of MHC II, a surface molecule of dendritic cells, induced by Mycobacterium smegmatis;

[0030] Figure 4a It was shown that Rv0991c overexpression inhibited the transcription of CD40, a macrophage surface molecule induced by Mycobacterium smegmatis; Figure 4b It was shown that Rv0991c overexpression inhibited the transcription of CD80, a macrophage surface molecule induced by Mycobacterium smegmatis; Figure 4c It was shown that overexpression of Rv0991c inhibited the transcription of MHC II, a surface molecule of macrophages, induced by Mycobacterium smegmatis;

[0031] Figure 5a It was shown that Rv0991c overexpression downregulated the secretion of MHC II on the surface molecule of dendritic cells induced by Mycobacterium smegmatis; Figure 5b It was shown that Rv0991c overexpression downregulated the secretion of MHC II on the macrophage surface molecule induced by Mycobacterium smegmatis. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0033] The following describes the embodiments of the present invention through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0034] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are for describing the specific embodiments rather than for limiting the scope of protection of the present invention; in the present specification and claims, unless otherwise expressly stated herein, the singular forms "a", "an" and "the" include plural forms.

[0035] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0036] Unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt conventional techniques in the field of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology, and related fields. These techniques are well described in the literature, see Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; these series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATINSTRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 304, Chromatin (PM Wassarman and AP Wolffe, eds.), Academic Press, San Diego, 1999. BIOLOGY, Vol. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0037] Example 1 Rv0991c protein inhibits the secretion level of T cell activation cytokine IL-2

[0038] (1) Prokaryotic expression and identification of Rv0991c protein

[0039] First, primers with Nde I and Xho I double restriction sites were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.:

[0040] Forward primer: 5′-TAAGAAGGAGATATACATATGCATATGGTGCCTACCTATAGTTATG AATG-3′ (SEQ ID NO: 3);

[0041] Reverse primer: 5'-GTGGTGGTGGTGGTGCTCGAGCTCGAGCACAGCTGCGGC-3' (SEQ ID NO: 4).

[0042] Then, the Mycobacterium tuberculosis H37Rv genome (purchased from Shanghai Jingnuo Biotechnology Co., Ltd.) was used as a template, and the sequences of SEQ ID NO: 3 and SEQ ID NO: 4 were used as amplification primers to PCR amplify the Rv0991c gene (sequence as shown in SEQ ID NO: 2) and then subjected to agarose gel. Table 1 is the amplification system, and Table 2 is the amplification program.

[0043] Table 1 PCR amplification system

[0044]

[0045] Table 2 PCR reaction program

[0046]

[0047] After electrophoresis recovery, the Rv0991c product was connected to the pET22a plasmid (purchased from Novagen) in one step (Table 3 is the plasmid restriction system, 37°C restriction for 3-5h), and then transformed into DH5a Escherichia coli competent cells (purchased from TAKAR), and coated on 50μg / mL ampicillin-resistant LB solid medium for screening. Positive clones were picked for identification, and the prokaryotic expression plasmid pET22a-Rv0991c was obtained after sequencing (Shanghai Shenggong Biotechnology Co., Ltd.). The confirmation results showed that in the prokaryotic expression plasmid pET22a-Rv0991c, the Rv0991c gene expression sequence was shown in SEQ ID NO: 2.

[0048] Table 3 Enzyme digestion system of pET22a vector

[0049]

[0050] Pick a positive single colony, place it in 4mL of LB liquid medium containing 50μg / mL ampicillin resistance, and culture it in a constant temperature shaker at 37℃ overnight. The next day, the recombinant bacteria were inoculated into LB liquid medium containing 50μg / mL ampicillin resistance at a ratio of 1:100, cultured in a constant temperature shaker at 37℃ with an OD value between 0.4-0.6, and IPTG was added to a final concentration of 0.5mmo1 / L, and induced in a constant temperature shaker at 37℃150rpm for 6h. The bacteria were collected by centrifugation and washed three times with sterile PBS. The bacteria were lysed using an ultrasonic lysator in an ice-water mixture (30W, lysis 3s, interval 5s). The supernatant was then collected by centrifugation at 10000rpm for 10min. The supernatant after centrifugation was removed and the precipitate was resuspended with an equal volume of sterile PBS. The supernatant collected after centrifugation was purified using a Ni-Agarose His tag protein purification kit. First, the column was pretreated, and 7.5 mL of sterile ultrapure water, 12.5 mL of Charge buffer, and 7.5 mL of Binding buffer were added to rinse the purification column with the His tag, and then the sample was added to the column, and finally 25 mL of Binding buffer, 15 mL of Washing bufferi, and 15 mL of Elute buffer were added, and the stopper was covered and allowed to stand at room temperature for 20 min. The purified protein was collected in a sterile 1.5 mL finger tube, and SDS-PAGE and Western Blot analysis were performed respectively. In addition, sequencing was performed to confirm that the amino acid sequence of the expressed protein was correct, as shown in SEQ ID NO: 1.

[0051] (2) Rv0991c protein inhibits the secretion of IL-2, a cytokine activated by T cells

[0052] C57BL / 6 mouse bone marrow-derived dendritic cells (BMDCs) were plated in 24-well cell plates and stimulated with 10 μg / mL Rv0991c protein (endotoxin-free). An unstimulated control group was also set up. After 2 h of culture, 100 μL of wild Mycobacterium smegmatis was added to each well. 2 155 (MOI = 10, Shanghai Jingnuo Biotechnology Co., Ltd., catalog number GOMY0001) was stimulated and cultured for 4 h, and then Ag85A stored in the laboratory was added 241-260Specific T cell hybridoma DE10 (Z Xu, C Meng, B Qiang, et al. Differential effects of Mycobacterium bovis BCG on macrophages and dendritic cells from murine spleen [J]. International Journal of Molecular Sciences, 2015, 16 (10): 24127-24138.) 250 μL was co-incubated for 24 hours, and the culture supernatant was collected and the secretion level of IL-2 was detected by ELISA. The results showed that Rv0991c could significantly inhibit the secretion level of IL-2 ( Figure 1 ). Among them, Medium+OVA represents the ovalbumin positive control group, Rv0991c+OVA represents the Rv0991c treatment group, Rv0991c+Trypsin+OVA represents the Rv0991c treatment group after trypsin digestion, BSA represents the BSA isotype control group, and Medium represents the negative control group.

[0053] Example 2 Overexpression of Rv0991c inhibits the secretion of IL-2, a T cell activation cytokine induced by Mycobacterium smegmatis

[0054] (1) Construction and identification of recombinant rMS smegmatis::pALACE-Rv0991c

[0055] First, design primers with BamH I and Hind III double restriction sites:

[0056] Forward primer: 5'-ACCACCCGATAAGAGAAAGGGAGTC-3' (SEQ ID NO: 5)

[0057] Reverse primer: 3'-CCAACTTCGATAACGTTCTCGGCTC-5' (SEQ ID NO: 6)

[0058] Then, the H37Rv genome of Mycobacterium tuberculosis was used as a template, and the sequences of SEQ ID NO: 5 and SEQ ID NO: 6 were used as amplification primers to PCR amplify the Rv0991c gene (the sequence is shown in SEQ ID NO: 2) (the amplification system and procedure are the same as Tables 1-2), and then the amplified product of Rv0991c was purified and recovered by agarose gel electrophoresis. The amplified product was cloned into the pALACE linear vector donated by Southwest University (Table 4 is the plasmid restriction system, 37°C restriction for 3-5h), and transformed into DH5a competent bacteria, and a single colony was picked. The target fragment was amplified using the inner and outer primers for identification (the inner and outer primers are shown in Table 5, and the amplification system and procedure are shown in Tables 1-2). The positive clone was sent to Shanghai Shenggong Biological Co., Ltd. for sequencing. The DH5a (pALACE-Rv0991c) that was correctly sequenced was expanded and cultured, and a single colony was picked to obtain the recombinant plasmid pALACE-Rv0991c. The recombinant plasmid was then electroporated into Mycobacterium smegmatis mc 2 155 competent state. Single colonies grown after electroporation transformation were picked for PCR identification (amplification system and procedure see Tables 1-2), and the successfully identified strain was named rMS::pALACE-Rv0991c.

[0059] Table 4 Enzyme digestion system of pALACE vector

[0060]

[0061] Table 5 Target gene primers

[0062]

[0063]

[0064] (2) Overexpression of Rv0991c recombinant Mycobacterium smegmatis inhibits the secretion of T cell activation cytokine IL-2

[0065] C57BL / 6 mouse BMDCs were plated in 24-well cell plates, and recombinant bacteria rMS::pALACE-Rv0991c were added to infect cells at MOI=10. The rMS::pALACE empty vector bacteria were used as the control group, and 2.5μg / mL Ag85A polypeptide (amino acid sequence: SEQ ID NO.9: QDAYNAGGGHNGVFDFPDSG) was used as the positive control (Ag85Apeptide). The cell supernatant was collected and the protein expression level of IL-2 was measured. The results showed that compared with the rMS::pALACE empty vector bacteria, the secretion level of IL-2 in the recombinant bacteria rMS::pALACE-Rv0991c group was significantly downregulated ( Figure 2 ). Among them, Medium represents the negative control.

[0066] Example 3 Overexpression of Rv0991c inhibits the transcription of surface molecules induced by Mycobacterium smegmatis antigen presenting cells

[0067] C57BL / 6 mouse BMDCs were plated in 24-well cell plates, and the recombinant bacteria rMS::pALACE-Rv0991c was added to infect the cells at MOI=10. The rMS::pALACE empty vector bacteria was used as the control group. The cells were collected at 6h, 24h, and 48h after infection to extract RNA, and the transcription levels of cell surface molecules such as CD40, CD80, and MHC II were measured. The results showed that in BMDCs, compared with the rMS::pALACE empty vector bacteria, the transcription levels of CD40, CD80, and MHC II in the recombinant bacteria rMS:pALACE-Rv0991c group were significantly downregulated ( Figure 3a-3c ).

[0068] At the same time, the transcription levels of CD40, CD80, and MHC II on the surface of peritoneal macrophages of C57BL / 6 mice were detected. The results showed that in mouse peritoneal macrophages, compared with rMS::pALACE empty vector bacteria, the transcription levels of CD40, CD80, and MHC II in the recombinant bacteria rMS::pALACE-Rv0991c group were significantly downregulated ( Figure 4a-4c ).

[0069] Example 4 Overexpression of Rv0991c inhibits the expression of MHC II on the surface of antigen presenting cells induced by Mycobacterium smegmatis

[0070] C57BL / 6 mouse BMDCs were cultured, and the recombinant bacteria rMS::pALACE-Rv0991c was added to infect the cells at MOI=10. The rMS::pALACE empty vector bacteria were used as the control group. Cell lysates were collected at 0h, 3h, and 6h after infection, and the protein expression level of MHCII was measured. The results showed that in BMDCs, the expression level of MHC II in the recombinant bacteria rMS::pALACE-Rv0991c group was significantly downregulated compared with the rMS::pALACE empty vector bacteria ( Figure 5a ). Among them, Medium represents the culture medium control group.

[0071] At the same time, the expression level of MHC II on the surface of peritoneal macrophages of C57BL / 6 mice was detected. The results showed that in mouse peritoneal macrophages, the expression level of MHCII in the recombinant bacteria rMS::pALACE-Rv0991c group was significantly downregulated compared with the rMS::pALACE empty vector bacteria ( Figure 5b ). Among them, Medium represents the culture medium control group.

Claims

1. Use of a mycobacterial protein having an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of an antigen presentation inhibitor.

2. Use of a gene encoding a mycobacterial protein having an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of an antigen presentation inhibitor.

3. The use according to claim 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

4. Use of a recombinant vector, recombinant bacteria or recombinant cell containing a gene encoding a mycobacterial protein having an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of an antigen presentation inhibitor.

5. The use according to claim 4, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

2.

6. The use according to any one of claims 1 to 5, characterized in that: Inhibition of antigen presentation includes downregulation of CD40, CD80, and MHC II transcription levels.

7. Use of a recombinant vector, recombinant bacteria or recombinant cell containing a gene encoding a mycobacterial protein having an amino acid sequence as shown in SEQ ID NO: 1 in the preparation of a drug for treating mycobacterial infection.

8. The use according to claim 7, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:2.

Images