Mycobacterium protein with anti-inflammatory effect as well as preparation and application thereof

Abstract

The invention discloses a mycobacterium protein with an anti-inflammatory effect as well as preparation and application of the mycobacterium protein. The invention discloses a mycobacterium protein Rv2723 with an amino acid sequence as shown in SEQ ID NO.1. The protein of the sequence is reported for the first time. Activity studies show that the Rv2723 recombinant protein can significantly inhibit the expression of LPS-induced macrophage inflammatory factors, the Rv2723 overexpression can effectively inhibit the expression of Mycobacterium smegmatis-induced macrophage inflammatory factors, and the Rv2723 overexpression can down-regulate the expression of Mycobacterium smegmatis-infected mouse serum inflammatory factors IL-6. In addition, the Rv2723 does not generate excessive immunological rejection. Therefore, the mycobacterium protein Rv2723 with the amino acid sequence as shown in SEQ ID NO.1, the coding gene of the mycobacterium protein Rv2723, and a recombinant vector, recombinant bacteria or recombinant cells containing the coding gene have the prospect of being developed into anti-inflammatory drugs.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a mycobacterial protein with anti-inflammatory effect and preparation and application thereof. BACKGROUND

[0002] Mycobacterium tuberculosis (M.tb) is mainly transmitted through air, which enters the lungs from the respiratory tract and infects macrophages. These macrophages are the main initial effector cells against M.tb infection. Infected macrophages secrete inflammatory cytokines, which play a key role in the inflammatory response and the outcome of mycobacterial infection, and factors inhibiting the expression of these inflammatory cytokines are ideal targets for controlling M.tb infection.

[0003] The initiation of host immunity is based on the recognition of pathogen-associated molecular patterns (PAMPs), which trigger signaling through Toll-like receptors (TLRs). TLRs clear pathogens by inducing the production of inflammatory factors and interferons, maintaining the stability of the internal environment of the body. Inflammatory cytokines are signal molecules secreted by macrophages, T cells and other cells that promote inflammation and immunity, including IL-1, IL-12, TNF-α, IFN-γ, etc. They up-regulate the host immune response by activating macrophages, inducing apoptosis and recruiting other immune cells, etc. As the "sentinel" of innate immunity, the appropriate activation of TLRs is critical for host defense, but excessive activation can lead to an inflammatory factor storm and the destruction of immune tolerance, which is an important pathogenesis of inflammatory diseases and autoimmune diseases. Therefore, the activation of the TLR signaling pathway must be strictly regulated to avoid excessive activation, especially to prevent the excessive production of inflammatory factors, in order to avoid serious consequences such as shock and organ function damage caused by bacterial infection.

[0004] In summary, there is an urgent need in the art to develop an immunologically active substance that can effectively inhibit the inflammatory factor storm caused by mycobacterial infection. Based on this, the present application is proposed. SUMMARY

[0005] The first object of the present application is to provide a mycobacterial protein with an amino acid sequence as shown in SEQ ID NO. 1, the second object is to provide the application of the mycobacterial protein in the preparation of anti-inflammatory drugs, the third object is to provide a coding gene of the mycobacterial protein as shown in SEQ ID NO. 1, the fourth object is to provide the application of the coding gene in the preparation of anti-inflammatory drugs, the fifth object is to provide a recombinant vector, a recombinant bacteria or a recombinant cell containing the coding gene of the mycobacterial protein, and the sixth object is to provide the application of the recombinant vector, the recombinant bacteria or the recombinant cell in the preparation of anti-inflammatory drugs.

[0006] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0007] A mycobacterium protein, the amino acid sequence of which is shown in SEQ ID NO.1.

[0008] Application of the mycobacterial protein in the preparation of anti-inflammatory drugs.

[0009] Furthermore, the anti-inflammatory drug uses the above-mentioned mycobacterial protein as an active ingredient and is prepared into a pharmaceutically acceptable dosage form through pharmaceutically acceptable carriers or excipients.

[0010] A gene encoding the above-mentioned mycobacterium protein, whose nucleotide sequence is shown in SEQ ID NO.2.

[0011] The application of the above coding gene in the preparation of anti-inflammatory drugs.

[0012] Furthermore, the anti-inflammatory drug uses the above-mentioned coding gene as an active ingredient and is prepared into a pharmaceutically acceptable dosage form through pharmaceutically acceptable carriers or excipients.

[0013] A recombinant vector, recombinant bacteria or recombinant cell containing the gene encoding the mycobacterium protein.

[0014] The use of the above-mentioned recombinant vector, recombinant bacteria or recombinant cells in the preparation of anti-inflammatory drugs.

[0015] Furthermore, the anti-inflammatory drug uses the above-mentioned recombinant vector, recombinant bacteria or recombinant cells as an active ingredient and is prepared into a pharmaceutically acceptable dosage form through pharmaceutically acceptable carriers or excipients.

[0016] Beneficial effects:

[0017] The present invention discloses a mycobacterial protein Rv2723 with an amino acid sequence as shown in SEQ ID NO.1, and the protein of this sequence is reported for the first time. Activity studies have shown that the recombinant protein Rv2723 can significantly inhibit the expression of LPS-induced macrophage inflammatory factors, and overexpression of Rv2723 can effectively inhibit the expression of Mycobacterium smegmatis-induced macrophage inflammatory factors. Overexpression of Rv2723 can downregulate the expression of the inflammatory factor IL-6 in the serum of mice infected with Mycobacterium smegmatis. In addition, Rv2723 does not produce excessive immune rejection reactions. Therefore, the mycobacterial protein Rv2723 with an amino acid sequence as shown in SEQ ID NO.1, its encoding gene, and the recombinant vector, recombinant bacteria or recombinant cell containing the encoding gene have the prospect of being developed into anti-inflammatory drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The Rv2723 recombinant protein inhibits the transcription of the inflammatory factor IL-1β in macrophages induced by LPS;

[0019] Figure 2 The Rv2723 recombinant protein inhibits the transcription of the inflammatory factor IL-6 in macrophages induced by LPS;

[0020] Figure 3 The Rv2723 recombinant protein inhibits the transcription of TNF-α, an inflammatory factor in macrophages induced by LPS;

[0021] Figure 4 Overexpression of Rv2723 inhibits the expression of IL-1β, an inflammatory factor in macrophages induced by Mycobacterium smegmatis;

[0022] Figure 5 Overexpression of Rv2723 inhibits the expression of IL-6, an inflammatory factor in macrophages induced by Mycobacterium smegmatis;

[0023] Figure 6 Overexpression of Rv2723 inhibits the expression of TNF-α, an inflammatory factor in macrophages induced by Mycobacterium smegmatis;

[0024] Figure 7 Rv2723 overexpression downregulates the expression of serum inflammatory factor IL-6 in mice infected with Mycobacterium smegmatis. DETAILED DESCRIPTION

[0025] The following examples describe the essential contents of the present invention in detail, but are not intended to limit the scope of protection of the present invention. In the following examples, unless otherwise specified, the disclosed experimental methods, detection methods, and preparation methods 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 for example 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; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE 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. 119, Chromatin Protocols (PB Becker, ed.) Humana Press, Totowa, 1999, etc.

[0026] The amino acid sequence of the mycobacterial protein (named Rv2723) is shown in SEQ ID NO. 1:

[0027] MGASGLVWTLTIVLIAGLMLVDYVLHVRKTHVPTLRQAVIQSATFVGIAILFGIAVVVFGGSELAVEYFACYLTDEALSVDNLFVFLVIISSFGVPRLAQQKVLLFGIAFALVTRTGFIFVGAALIENFNSAFYLFGLVLLVMAGNLARPTGLESRDAETLKRSVIIRLADRFLRTSQDYNGDRLFTVSNNKRMMTPLLL VMIAVGGTDILFAFDSIPALFGLTQNVYLVFAATAFSLLGLRQLYFLIDGLLDRLVYLSYGLAVILGFIGVKLMLEALHDNKIPFINGGKPVPTVEVSTTQSLTVIIIVLLITTAASFWSARGRAQNAMARARRYATAYLDLHYETESAERDKIFTALLAAERQINTLPTKYRMQPGQDDDLMTLLCRAHAARDAHM(SEQ ID NO.1).

[0028] The nucleotide sequence of the gene encoding the mycobacterium protein whose amino acid sequence is shown in SEQ ID NO: 1 is shown in SEQ ID NO. 2:

[0029]

[0030] Example 1: Rv2723 recombinant protein inhibits LPS-induced expression of inflammatory cytokines

[0031] 1. Experimental Materials

[0032] The Mycobacterium tuberculosis H37Rv genome was purchased from Shanghai Jingnuo Biotechnology Co., Ltd.; the pCold plasmid was purchased from Novagen; DH5α Escherichia coli competent cells were purchased from TAKAR; mouse macrophage RAW264.7 cells were purchased from ATCC; the Ni-Agarose His tag protein purification kit and isopropyl-β-D-thiogalactopyranoside (IPTG) were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.; BamHI and Sal I were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; 24-well cell plates were purchased from Jiete Biofiltration Co., Ltd.; LPS was purchased from Beyotime Biotechnology Co., Ltd.; ampicillin was purchased from Solebao Technology Co., Ltd.; and an ultrasonic lyser was purchased from SONICS & MATERIALS.

[0033] 2. Experimental Methods

[0034] 1. Prokaryotic expression and identification of Rv2723 recombinant protein

[0035] First, primers with BamHI and SalI double restriction sites were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.:

[0036] Forward primer: 5′-CTCGGTACCCTCGAGGGATCCATGGGTGCTTCCGGCCTG-3′ (SEQ ID NO. 3);

[0037] Reverse primer: 5'-CTATCTAGACTGCAGGTCGACTCACATGTGCGCGTCGCG-3' (SEQ ID NO. 4).

[0038] Then, the Rv2723 gene (sequence shown in SEQ ID NO. 2) was amplified by PCR using the Mycobacterium tuberculosis H37Rv genome as a template and the sequences of SEQ ID NO. 3 and SEQ ID NO. 4 as amplification primers, and then subjected to agarose gel. Table 1 shows the amplification system and Table 2 shows the amplification procedure.

[0039] Table 1 PCR amplification system

[0040]

[0041] Table 2 PCR reaction procedure

[0042]

[0043] The Rv2723 product was connected to the pCold plasmid in one step by electrophoresis recovery (the enzyme digestion system of the plasmid is shown in Table 3, and the enzyme digestion was performed at 37°C for 3-5h), followed by transformation of DH5a E. coli competent cells and coating on 50 μg / mL ampicillin-resistant LB solid medium for screening. Positive clones were picked for identification, and after sequencing (Shanghai Gener Biotech Co., Ltd.) was correct, the prokaryotic expression plasmid pCold-Rv2723 was obtained. The results showed that the Rv2723 gene expression sequence in the prokaryotic expression plasmid pCold-Rv2723 was as shown in SEQ ID NO. 2.

[0044] Table 3 Enzyme digestion system of pCold vector

[0045]

[0046] The positive single colony was picked and placed in 4 mL of LB liquid medium containing 50 μg / mL ampicillin, and incubated at 37°C in a constant temperature shaker overnight. The next day, the recombinant bacteria were inoculated into LB liquid medium containing 50 μg / mL ampicillin at a ratio of 1:100, and incubated at 37°C in a constant temperature shaker until the OD value was between 0.4-0.6. IPTG was added to a final concentration of 0.5 mmol / L, and induced at 37°C for 6h in a constant temperature shaker at 150 rpm. The bacterial cells were collected by centrifugation and washed with sterile PBS three times. The bacterial cells were lysed in an ice-water mixture using an ultrasonic lysis instrument (30W, lysis for 3s, interval of 5s). Then the supernatant was collected by centrifugation at 10000 rpm for 10 min. The supernatant after centrifugation was taken out 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 then 7.5 mL of sterile ultrapure water, 12.5 mL of Charge buffer and 7.5 mL of Binding buffer were added to the His-tagged purification column in turn, followed by sample loading and finally 25 mL of Binding buffer, 15 mL of Washing buffer i and 15 mL of Elute buffer. The cap was closed and the mixture was incubated at room temperature for 20 min. The purified protein was collected in a sterile 1.5 mL pipette and subjected to SDS-PAGE and Western Blot analysis, respectively. In addition, sequencing was performed to confirm that the expressed protein amino acid sequence (as shown in SEQ ID NO. 1) was correct.

[0047] 2. Rv2723 recombinant protein inhibits transcription of LPS-induced inflammatory factors in macrophages

[0048] The mouse macrophage RAW264.7 was inoculated at 5×10 5Cells were plated into 24-well plates. Control groups included a blank culture medium group (Medium), groups stimulated with recombinant Rv2723 protein alone (10 μg / mL and 20 μg / mL Rv2723), and a group stimulated with LPS alone (1 μg / mL LPS). Experimental groups included protein and LPS stimulation. The experimental groups were stimulated with 10 μg / mL and 20 μg / mL recombinant Rv2723 protein (endotoxin-free) for 5 hours, followed by the addition of LPS (1 μg / mL) for another hour. Cells were then harvested for RNA extraction, and transcript levels of inflammatory cytokines such as IL-1β, IL-6, and TNF-α were measured.

[0049] 3. Experimental Results

[0050] The results are as follows Figures 1 to 3 As shown, compared with the LPS-only stimulation group, the transcriptional levels of inflammatory cytokines in the experimental group were significantly downregulated, indicating that the Rv2723 recombinant protein can significantly inhibit the upregulation of inflammatory cytokine transcription levels caused by LPS stimulation, and the inhibitory effect is dose-dependent. In addition, no changes in cell morphology were observed after stimulation of cells with different concentrations of Rv2723, indicating that Rv2723 does not cause excessive immune rejection.

[0051] Example 2: Overexpression of Rv2723 inhibits the expression of host inflammatory factors induced by recombinant Mycobacterium smegmatis

[0052] 1. Experimental Materials

[0053] EcoR I was purchased from Bio-Technology (Beijing) Co., Ltd.; pMV261 linear vector and Mycobacterium smegmatis mc 2 155 competent cells are kept in the laboratory.

[0054] 2. Experimental Methods

[0055] 1. Construction and identification of recombinant rMS::pMV261-Rv2723

[0056] First, design primers with BamHI and EcoRI double restriction sites:

[0057] Forward primer: 5′-GGCCAAGACAATTGCGGATCCATGGGTGCTTCCGGCCTG-3′ (SEQ ID NO. 5);

[0058] Reverse primer: 3'-ACATCGATAAGCTTCGAATTCTCACATGTGCGCGTCGCG-5' (SEQ ID NO. 6).

[0059] Then, using the H37Rv genome of Mycobacterium tuberculosis as a template and the sequences of SEQ ID NO.5 and SEQ ID NO.6 as amplification primers, PCR amplify the Rv2723 gene (sequence as shown in SEQ ID NO.2) (amplification system and procedure are the same as Table 1-2), and then recover it by agarose gel electrophoresis. The purified and recovered Rv2723 amplification product is cloned into the pMV261 linear vector (Table 5 is the plasmid enzyme digestion system, 37°C enzyme digestion for 3-5h), and transformed into DH5α competent bacteria. A single colony is picked, and the target fragment is amplified using the inner and outer primers for identification (outer primers are shown in Table 6, and the amplification system and procedure are shown in Table 1-2). The positive clone is sent to Shanghai Shenggong Biological Co., Ltd. for sequencing. The DH5α (pMV261-Rv2723) that has been sequenced correctly is expanded and cultured, and a single colony is picked to obtain the recombinant plasmid pMV261-Rv2723. The recombinant plasmid is then electroporated into Mycobacterium smegmatis mc 2 155 competent cells. Single colonies grown after electroporation transformation were picked for PCR identification (amplification system and procedure are shown in Table 1-2). The successfully identified strain was named rMS::pMV261-Rv2723.

[0060] Table 5 Enzyme digestion system of pMV261 vector

[0061]

[0062] Table 6 Target gene outer primers

[0063]

[0064] 2. Overexpression of Rv2723 inhibits the expression of host inflammatory factors induced by recombinant Mycobacterium smegmatis

[0065] Mouse macrophage RAW264.7 cells were cultured at 5 × 10 5 Cells / well were plated into 24-well cell plates, and blank group (Medium), control group (rMS::pMV261) and infection group (rMS::pMV261-Rv2723) were set up. The control group and infection group were infected with rMS::pMV261 empty vector bacteria and rMS::pMV261-Rv2723 recombinant bacteria at MOI=10, respectively. The cell supernatants were collected at 6h, 24h and 48h, respectively, and the protein expression levels of inflammatory cytokines IL-1β, IL-6 and TNF-α were determined by ELISA.

[0066] 3. Experimental Results

[0067] The results are as follows Figures 4-6As shown, compared with rMS::pMV261 empty vector bacteria, the secretion level of IL-1β, IL-6 and TNF-α of the recombinant bacteria rMS::pMV261-Rv2723 group was significantly down-regulated, which indicated that overexpression of Rv2723 could effectively inhibit the expression of host inflammatory factors induced by recombinant Mycobacterium smegmatis.

[0068] Example 3: Rv2723 overexpression inhibits the expression of serum inflammatory factors of mice infected with recombinant Mycobacterium smegmatis

[0069] I. Experimental materials

[0070] C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0071] II. Experimental methods

[0072] Six to eight-week-old female C57BL / 6 mice were randomly divided into three groups, and PBS group, control group (rMS::pMV261) and experimental group (rMS::pMV261-Rv2723) were set up. The infection steps were as follows: rMS::pMV261 empty vector bacteria and rMS::pMV261-Rv2723 recombinant bacteria were injected into mice at a dose of 5x10 7 CFU / 200 μL, and PBS (200 μL) was injected intraperitoneally as a blank control group, and 3-day and 6-day infection times were set up. The secretion level of inflammatory cytokines in mouse serum was collected, and the body weight of mice in each group was measured at 0, 3 and 6 days, and the activity and state of mice in each group were observed.

[0073] III. Experimental results

[0074] As shown in the results, Figure 7 compared with rMS::pMV261 empty vector bacteria, the secretion level of IL-6 in the serum of mice infected with rMS::pMV261-Rv2723 recombinant bacteria for 3 days and 6 days was significantly down-regulated. In addition, the body weight of mice infected with recombinant bacteria did not decrease, and there was no difference in activity and state compared with the blank group, indicating that Rv2723 did not produce excessive immune rejection.

[0075] In summary, the present invention discloses a mycobacterial protein Rv2723 with an amino acid sequence as shown in SEQ ID NO.1, and the protein of this sequence is reported for the first time. Activity studies have shown that the Rv2723 recombinant protein can significantly inhibit the expression of LPS-induced macrophage inflammatory factors, and the overexpression of Rv2723 can effectively inhibit the expression of Mycobacterium smegmatis-induced macrophage inflammatory factors. The overexpression of Rv2723 can downregulate the expression of the inflammatory factor IL-6 in the serum of mice infected with Mycobacterium smegmatis. In addition, Rv2723 does not produce excessive immune rejection reactions. Therefore, the mycobacterial protein Rv2723 with an amino acid sequence as shown in SEQ ID NO.1, its encoding gene, and the recombinant vector, recombinant bacteria or recombinant cell containing the encoding gene have the prospect of being developed into an anti-inflammatory drug.

[0076] The purpose of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to this specific embodiment.

Claims

1. A protein, the amino acid sequence of which is shown in SEQ ID NO.

1.

2. Use of the protein according to claim 1 in the preparation of anti-inflammatory drugs.

3. The use according to claim 2, wherein the anti-inflammatory drug uses the protein according to claim 1 as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier or excipient.

4. A gene encoding the protein according to claim 1, the nucleotide sequence of which is shown in SEQ ID NO.

2.

5. Use of the coding gene according to claim 4 in the preparation of anti-inflammatory drugs.

6. The use according to claim 5, wherein the anti-inflammatory drug uses the coding gene according to claim 4 as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier or excipient.

7. A recombinant vector, recombinant bacterium or recombinant cell containing a gene encoding the protein of claim 1.

8. Use of the recombinant vector, recombinant bacteria or recombinant cell according to claim 7 in the preparation of anti-inflammatory drugs.

9. The use according to claim 8, wherein the anti-inflammatory drug uses the recombinant vector, recombinant bacteria or recombinant cells according to claim 7 as an active ingredient and is prepared into a pharmaceutically acceptable dosage form with a pharmaceutically acceptable carrier or excipient.

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