阅读说明：本技术 结核分枝杆菌的重组蛋白、表达载体、重组工程菌及其应用 (Recombinant protein, expression vector, recombinant engineering bacterium and application of mycobacterium tuberculosis ) 是由 宋宁宁 李兆利 宋金妙 林红 王书贤 王慧 于 2021-07-06 设计创作，主要内容包括：本发明提供一种结核分枝杆菌的重组蛋白,其氨基酸序列为SEQ ID NO:2。本发明还提供了相应的表达载体、重组工程菌及其应用。本发明证明了结核分枝杆菌Rv0324重组蛋白能够直接调控rv3597c的表达,参与调控MTB的氧耗和代谢水平和毒力侵袭,对MTB能长期持续感染宿主有着重要的作用,有助于完整解析MTB调控机制,可以用于临床治疗的药物筛选。(The invention provides a recombinant protein of mycobacterium tuberculosis, the amino acid sequence of which is SEQ ID NO 2. The invention also provides a corresponding expression vector, recombinant engineering bacteria and application thereof. The invention proves that the mycobacterium tuberculosis Rv0324 recombinant protein can directly regulate and control the expression of Rv3597c, participates in regulating and controlling oxygen consumption, metabolic level and virulence invasion of MTB, has an important effect on long-term continuous infection of a host by MTB, is beneficial to completely analyzing an MTB regulation mechanism, and can be used for drug screening of clinical treatment.)

1. The mycobacterium tuberculosis recombinant protein is characterized in that the amino acid sequence is SEQ ID NO 2.

2. A recombinant gene for expressing the recombinant protein of Mycobacterium tuberculosis of claim 1, wherein the recombinant gene is obtained by PCR amplification using the genome of Mycobacterium bovis BCG Tokyo-172 strain as a template, and the primer pair used in the PCR amplification is SEQ ID NO. 3 and SEQ ID NO. 4.

3. The recombinant gene of claim 2 wherein the nucleotide sequence is SEQ ID NO 1.

4. An expression vector for expressing a recombinant protein of Mycobacterium tuberculosis comprising the recombinant gene of claim 2 or 3.

5. The expression vector of claim 4, further comprising a backbone plasmid, wherein the backbone plasmid is a pET-22b vector.

6. The expression vector of claim 4, prepared by a method comprising:

the recombinant gene and pET-22b vector as described in claim 2 or 3 are double digested with endonuclease Nde I and Xho I, and the digested rv0324 gene fragment is connected to double digested pET-22b vector to constitute recombinant plasmid, i.e. expression vector.

7. A recombinant engineered bacterium for expressing recombinant proteins of Mycobacterium tuberculosis, comprising the recombinant gene of claim 2 or 3, or the expression vector of any one of claims 4 to 6.

8. The recombinant engineered bacterium of claim 7, wherein the host bacterium is Escherichia coli, preferably E.coli BL21 or E.coli DH5 α.

9. Use of the recombinant protein according to claim 1, the recombinant gene according to claim 2 or 3, the expression vector according to any one of claims 4-6, or the recombinant engineered bacterium according to claim 7 or 8 for screening drugs for treating tuberculosis.

Technical Field

The invention relates to the technical field of biological medicines, in particular to a recombinant protein, an expression vector, a recombinant engineering bacterium and application of mycobacterium tuberculosis Rv 0324.

Background

Tuberculosis (TB) is a chronic wasting disease that is mainly caused by Mycobacterium Tuberculosis (MTB). After MTB infects a host, the human host resists infection and transcriptional control caused by the immune response of MTB to the host, so that MTB evolves to be a pathogenic bacterium which can continuously infect people for a long time. Therefore, the research on the regulation mechanism of MTB transcription regulation protein can provide a certain theoretical basis for the elucidation of the pathogenic mechanism of MTB and the treatment of tuberculosis.

It has been shown that there are more than about 200 regulatory genes in the MTB genome, and among them, Rv0324, a protein encoded by Rv0324, belongs to the ArsR family of proteins with highly conserved DNA recognition region helix-turn-helix (HTH). Rv0324 is a key node in the MTB transcriptional regulatory network and is the central regulator of the MTB hypoxia response. The Rv3597c gene encodes histone-like protein (H-NS) Rv3597c (Lsr2), which is a histone-like protein regulated by iron ions and is sensitive to changes in external environment, especially oxygen concentration. However, the regulatory relationship between Rv3597c and Rv0324 is not clear as it is a gene encoding a broad transcriptional regulatory protein that also varies in the oxygen concentration levels in response to MTB.

Disclosure of Invention

The mycobacterium tuberculosis Rv0324 recombinant protein provided by the invention can regulate and control the response oxygen concentration change of Rv3597C, a Rhodanese Homology Domain (RHOD) at the C end is a key region for recognizing and sensing environmental change and playing a protein function, and two cysteines (Cys177 and Cys182) in Rv0324 are both located at the RHOD, so that the RHOD at least comprises one cysteine site with catalytic activity.

The invention firstly provides a mycobacterium tuberculosis recombinant protein, the amino acid sequence of which is SEQ ID NO. 2.

The invention also provides a recombinant gene for coding the Mycobacterium tuberculosis recombinant protein, wherein the recombinant gene is obtained by taking the genome of the Mycobacterium bovis BCG Tokyo-172 strain as a template and performing PCR amplification, and primer pairs used in the PCR amplification are SEQ ID NO. 3 and SEQ ID NO. 4.

In one embodiment according to the invention, the nucleotide sequence is SEQ ID NO 1.

The invention further provides an expression vector for expressing the recombinant protein of the mycobacterium tuberculosis Rv0324, which comprises the recombinant gene.

In one embodiment according to the present invention, the expression vector further comprises a backbone plasmid, which is pET-22 b.

In one embodiment according to the present invention, the above expression vector is prepared by a method comprising the steps of:

carrying out double enzyme digestion on the recombinant gene and the pET-22b vector by using ligase Nde I and Xho I respectively, and then connecting the enzyme digested rv0324 gene fragment to the pET-22b vector to construct a recombinant plasmid, namely an expression vector.

The invention also provides a recombinant engineering bacterium for expressing the recombinant protein of the mycobacterium tuberculosis Rv0324, which is characterized in that the recombinant gene or the expression vector is adopted.

In one embodiment according to the invention, the host bacterium is e.coli, preferably e.coli BL21 or e.coli DH5 a.

The invention also provides application of the recombinant protein, the recombinant gene expression vector or the recombinant engineering bacterium in screening drugs for treating tuberculosis.

The technical scheme of the invention has the following beneficial effects:

the invention proves that the mycobacterium tuberculosis Rv0324 recombinant protein can directly regulate and control the expression of Rv3597c, participates in regulating and controlling oxygen consumption, metabolic level and virulence invasion of MTB, has an important effect on long-term continuous infection of a host by MTB, is beneficial to completely analyzing an MTB regulation mechanism, and can be used for drug screening of clinical treatment.

Drawings

FIG. 1 shows the Western blot identification result of recombinant protein Rv 0324;

FIG. 2A shows the EMSA detection of the binding ability of Rv0324 and p/o Rv3597c in vitro, wherein B is the binding hysteresis band of Rv0324 and p/o Rv3597c, and F is the free p/o Rv3597c band;

FIG. 2B is a graph showing the results of competitive inhibition experiments, wherein B is the band where Rv0324 and p/o Rv3597c bind to the hysteresis band, and F is the free p/o Rv3597c band;

FIG. 3 is an identification map of Rv0324 polyclonal antibody, wherein A is the result of SDS-PAGE; b is a Western Blot identification result;

FIG. 4 is a ChIP assay for the in vitro binding capacity of Rv0324 and p/o Rv3597 c;

FIG. 5 is a major groove interaction detection profile of Rv0324 and p/o Rv3597 c;

wherein, 1-4: methyl green and Cy5 p/o Rv3597c act together with Rv 0324; 7-10: actinomycin D and Cy5 p/o Rv3597c act together with Rv 0324; wherein B is Rv0324 and p/o Rv3597c binding hysteresis band, and F is free p/o Rv3597c band.

Detailed Description

In order to make the technical problems, technical solutions and advantages of the present invention more apparent, the following detailed description is given with reference to the accompanying drawings and specific embodiments.

Materials and methods:

strains, cell lines and vectors

The Mycobacterium bovis BCG (Tokyo-172) and pET-22b vectors were purchased from the center of ATCC strain,

coli DH5 α and e coli BL21(DE3) competent cells were purchased from tiangen biochemistry science co.

The main reagents are as follows:

both 7H9 and LB medium were purchased from BD biosciences,

nde I and Xho I restriction endonucleases, T4 DNA ligase and associated buffers, EMSA kit, M-280 goat anti-rabbit antibody magnetic beads were purchased from Thermo Fisher Scientific,

the agarose Gel DNA Gel recovery Kit Gel Extraction Kit and the small upgraded particle Kit Plasmid Mini Kit I were purchased from OMEGA,

IPTG, ampicillin and related amino acids were purchased from Amresco,

freund's incomplete adjuvant and methyl green were purchased from Sigma-ALDRICH,

IDDye goat anti-rabbit IgG and goat anti-mouse IgG were purchased from LI-COR,

mouse monoclonal anti-His antibodies were purchased from tiangen Biochemical technologies,

actinomycin D was purchased from Alphabio, SYBR Green mix from BIO-RAD, and ChIP IPURE kit from Diagenode.

Example 1 primer design and Synthesis

Specific primers SN221f and SN221r were designed with reference to the rv0324(Gene ID: 886548) Gene sequence SEQ ID NO:1 in GenBank database, and the primer information is shown in Table 1.

TABLE 1 primer information Table

Example 2 expression of recombinant protein Rv0324

1. BCG genome extraction

Extraction was performed using a bacterial genome extraction kit (Tiangen).

(1) Centrifuging the BCG bacterial liquid at 10000 rpm for 1min, discarding the supernatant, and collecting bacterial liquid precipitate.

(2) About 1mL of lysozyme (20mg/mL) was added to 500mL of the bacterial suspension, and the mixture was divided into two tubes, 500. mu.L/tube. And (3) crushing thalli: ultrasonic: 35% amplitude, on 3s, off 5s, 2min, followed by bar milling for 2 min. Water bath at 37 ℃ for 1 h.

(3) Add 4. mu.L RNase per tube, at room temperature, for 5 min.

(4) Add 20. mu.L proteinase K treatment.

(5) Adding 440 μ L GB, shaking for 15s, standing at 70 deg.C for 10min, and centrifuging briefly.

(6) Adding 440 μ L of absolute ethyl alcohol, shaking for 15s, and centrifuging at 10000 rpm for 5 min.

(7) Adding the supernatant into adsorption column CB3, centrifuging at 10000 rpm for 1min, pouring the effluent into the adsorption column, centrifuging at 10000 rpm for 1min, and discarding the effluent.

(8) Add 500. mu.L GD to the adsorption column, centrifuge at 12000 rpm for 1min, and discard the effluent.

(9) Adding 750 μ L of rinsing solution PW into the adsorption column, centrifuging at 12000 rpm for 1min, and discarding the effluent.

(10) Repeating the step (9) once.

(11) And (3) performing air separation at 12000 rpm for 2 min.

(12) Replacement of EP tubes, standing at room temperature for 2min, and incubation with ddH₂O (autoclaved) 50. mu.L/column and centrifuged at 10000 rpm for 1 min.

(13) Agarose gel electrophoresis was performed for validation and concentration was measured.

2. Expression vector construction and expression

The genome of Mycobacterium bovis BCG (Tokyo-172) is used as a template to amplify rv0324 gene, Nde I and Xho I double enzyme digestion rv0324 gene fragment and pET-22b vector are connected to construct recombinant plasmid pET-22b-rv0324, the recombinant plasmid pET-22b-rv0324 is converted into E.coli DH5 alpha, and double enzyme digestion and sequencing are carried out to determine the gene sequence of recombinant protein. The recombinant plasmid pET22b-rv0324 with correct sequencing is transformed into E.coli BL21(DE3), cultured in a shaking table at 37 ℃ and 180r/min until the OD600nm of the solution is 0.8, and the cells are collected after the induction expression of IPTG with the final concentration of 1mmol/L at 30 ℃ and 180r/min for 6 h. The supernatant was sonicated and collected and the protein Rv0324 was purified by affinity chromatography. After concentrating Rv0324, a mouse monoclonal antibody His antibody (1:5000) is used as a primary antibody, goat anti-mouse IDDye fluorescein IgG (1:10000) is used as a secondary antibody, an infrared fluorescence scanner is used for scanning, and the Rv0324 is separated and identified by a Western Blot method. The protein was stored at-80 ℃ for subsequent experiments.

3. Protein purification

1) The cells collected after induction expression were removed and resuspended in 80mL of lysate. And (3) carrying out ultrasonic disruption by using a cell ultrasonic disruptor, wherein the amplitude is 37 percent, the time is 3 seconds, the time is 5 seconds, and the ultrasonic disruption is carried out for 1 hour until the bacterial liquid is clear.

2) And transferring the bacteria liquid after ultrasonic treatment into a high-speed centrifugal tube, and centrifuging the bacteria liquid at 4 ℃ for 30min at 10000. The supernatant was retained and the precipitate was discarded.

3) The supernatant was filtered through a 0.22 μ M filter.

4) 2mL of Ni-NTA affinity resin is put into a chromatographic column, and a column lysate is added to naturally flow down to balance the resin.

5) Adding the supernatant into the chromatographic column, naturally flowing down, and repeating the above steps for 5 times to allow the protein to be fully bound with the resin.

6) Adding two columns of ddH₂And O, flowing down naturally.

7) The impurities were washed with 100mL of each of the impurity-washing solutions 1, 2, 3, 4 and 5.

Washing impurity liquid 1: 20mM Tris-HCl; 500mM NaCl; 20mM imidazole; 3% of glycerol; PH 8.0.

Washing impurity liquid 2: 20mM Tris-HCl; 500mM NaCl; 40mM imidazole; 3% of glycerol; PH 8.0.

Washing impurity liquid 3: 20mM Tris-HCl; 500mM NaCl; 50mM imidazole; 3% of glycerol; PH 8.0.

Washing impurity liquid 4: 20mM Tris-HCl; 500mM NaCl; 60mM imidazole; 3% of glycerol; PH 8.0.

Washing impurity liquid 5: 20mM Tris-HCl; 1M NaCl; PH 8.0.

8) Adding two columns of ddH₂And O, making the mixture naturally flow down.

9) Then 3mL of eluent is added into the chromatographic column, the chromatographic column is placed on ice and kept stand for 10min, the eluent flows down, and the step is repeated for 2 times to obtain the pure protein.

Eluent: 20mM Tris-HCl; 150mM NaCl; 500mM imidazole; 5% of glycerol; the pH value is 8.0, and the pH value is lower than the standard value,

after the protein is purified, the protein is identified by a Western Blot experiment, and the result shows that a band with the size of about 25ku (figure 1) is consistent with the size of the target protein Rv0324, which indicates that the protein is successfully expressed and Rv0324 is obtained.

Example 3 in vitro binding experiments for Rv0324 and p/o Rv3597c

PCR was performed using Mycobacterium bovis BCG (Tokyo-172) genome as template and SN395f/r as primer to obtain p/o rv3597c, and gel migration Experiment (EMSA) was performed. Preparing an EMSA reaction system with 30 mu L: 1 uL 40 ng/. mu. L p/o rv3597c, 6. mu.L of 5 XBinding Buffer, different final concentrations (0.16. mu. mol/L, 0.33. mu. mol/L, 0.67. mu. mol/L, 0.83. mu. mol/L, 0.99. mu. mol/L) of Rv0324 protein, and use ddH₂And (4) complementing O. After incubation at 37 ℃ for 30min, the complexes were separated by electrophoresis on 8% polyacrylamide gel at 200V for 3h, and after staining the complexes were imaged by uv transmission using a gel imaging system to verify that Rv0324 and p/o Rv3597c bind in vitro. 5' Cy5 labeled primer SN395f/r, the BCG (Tokyo-172) genome is amplified by PCR to obtain Cy5 labeled p/o Rv3597c, and the primer competes with unlabeled p/o Rv3597c for binding to Rv0324 for EMSA. EMSA reaction system 30. mu.L: 0.5. mu.L 0.1mmol/L Rv0324, 1. mu.L 20 ng/. mu.L Cy5 labeled p/o Rv3597c, 3. mu.L 10 × binding buffer, 3. mu.L 25mmol/L DTT, 1.5. mu.L 1mg/mL poly (dI-dC), 1.5. mu.L 50% glycerol, 1.5. mu.L 100mmol/L MgCl₂And higher than Cy 5-labeled p/o rv3597 c-fold and 120-fold unlabeled p/o rv3597c with ddH₂And (4) complementing O. Incubating at 37 ℃ in the dark for 30min, carrying out electrophoresis on 8% polyacrylamide gel at 200V for 3h, separating the compound, and analyzing by using a two-color infrared laser imaging system and scanning imaging to verify the in vitro specific binding of Rv0324 and p/o Rv3597 c.

EMSA was performed after incubation of 0. mu. mol/L, 0.16. mu. mol/L, 0.33. mu. mol/L and 0.67. mu. mol/L of Rv0324 with p/o Rv3597c, respectively, and the results showed that as the concentration of Rv0324 increased, the free p/o Rv3597c decreased, and at a final concentration of Rv0324 of 0.67. mu. mol/L, there was a significant hysteresis band, at which time p/o Rv3597c bound completely to Rv0324 (FIG. 2A), indicating that Rv0324 could bind to p/o Rv3597c in vitro.

EMSA was performed by competing 60-fold and 120-fold higher unlabeled p/orv3597c than Cy 5-labeled p/o Rv3597c with Cy 5-labeled p/o Rv3597c, respectively, and the results showed that after 60-fold addition of unlabeled p/o Rv3597c, the bound complex of Rv0324 and Cy 5-labeled p/o Rv3597c decreased and free Cy 5-labeled p/o Rv3597c appeared; after addition of 120-fold more unlabeled p/o Rv3597c, free Cy 5-labeled p/o Rv3597c increased (FIG. 2B), indicating that unlabeled p/o Rv3597c competitively inhibited the binding of Cy 5-labeled p/o Rv3597c to Rv0324, indicating that Rv0324 and p/o Rv3597c bind specifically in vitro.

Example 4 preparation of Rabbit anti-Rv 0324 protein polyclonal antibody

Two new zealand white rabbits were used as a blank control group, and the other two new zealand white rabbits were used as an immunization group. After mixing Rv0324 and Freund's incomplete adjuvant in equal volume ratio, 400 μ g of each of the two rabbits in the immunized group was injected subcutaneously three times at intervals of 14 d. The blank control group was not treated with injections. After the blank group and the immune group were subjected to heart blood collection, serum was prepared and stored at-80 ℃. Purifying immune group serum and blank control group serum by an octanoic acid-ammonium sulfate method and a protein G affinity chromatographic column to respectively obtain an Rv0324 polyclonal antibody and a negative control serum antibody. The recombinant protein Rv0324 is used as an antigen, Rv0324 polyclonal antibody (1:500) is used as a primary antibody, goat anti-rabbit IDDye fluorescein IgG (1:10000) is used as a secondary antibody, an infrared fluorescence scanner is used for scanning, and the Rv0324 polyclonal antibody is identified by a Western Blot method and is used for a subsequent chromatin co-immunoprecipitation experiment (ChIP).

The recombinant protein Rv0324 is emulsified and then used for immunizing a rabbit, and after the serum of the rabbit is purified, the Rv0324 protein polyclonal antibody is successfully obtained through SDS-PAGE gel electrophoresis and western blot identification (figure 3). ChIP is carried out by using Rv0324 polyclonal antibody, qRT-PCR experiment is carried out on DNA collected in the ChIP, and Ct difference comparison method is adopted for analysis, and the result shows that the enrichment degree of p/o Rv3597c in the negative control group is 0.53; the enrichment degree of p/o Rv3597c in the multiple antibody group is 1.57, which is 2.96 times that of the control group (figure 4), and the results show that Rv0324 can be combined with p/o Rv3597c in thalli, and Rv0324 directly regulates the transcription of Rv3597 c.

Example 5 in vivo binding experiments for Rv0324 and p/o Rv3597c

Performing chromatin co-immunoprecipitation (ChIP) experiment, culturing BCG to OD solution_600nmAt 0.8, cross-linking with 1% formaldehyde for 10min, reacting with 125mmol/L glycine for 5min to terminate the reaction, centrifuging at 3500r/min for 20min, and collecting thallus. 3mL of IP Buffer was added to resuspend the pellet, and after ultrasonication, the supernatant was transferred to a new EP tube. 100. mu.L of the supernatant was subjected to reverse crosslinking, and DNA in the cells was extracted by phenol chloroform method, followed by agarose gel electrophoresis to analyze the size of the DNA fragment. 20 μ L of the prepared Rv0324 polyclonal antibody and 20 μ L of the negative control serum antibody in 1.6 are respectively coupled with M-280 goat anti-rabbit antibodyAnd (3) incubating the coated magnetic beads at 4 ℃ to serve as a multi-antibody group and a negative control group, and incubating 1.2mL of the ultrasonically-crushed supernatant with the coated magnetic beads at 4 ℃ for 16 h. After washing the magnetic beads, the complexes on the magnetic beads were collected using an IPURE kit, and DNAs in the complexes were purified and separated, and then obtained by ethanol precipitation.

Diluting the collected DNA by 1000 times to be used as a template, taking sigA amplified by a primer SN9f/r as a housekeeping gene, carrying out fluorescent dye method qRT-PCR on a sample by primers SN398f and SN398r, repeating each group for 3 times, analyzing by using LightCycler 480 software, adopting a Ct difference comparison method, calculating the enrichment degree of p/o Rv3597c in a multi-antibody group and a negative control group by using a 2^ (-delta Ct) formula, and comparing the difference between the enrichment degrees so as to verify the combination of Rv0324 and p/o Rv3597c in vivo.

Example 6 Large-and small-groove specific binding experiments for Rv0324 and p/o Rv3597c

According to the specificity of methyl green and DNA major groove combination and actinomycin D and DNA minor groove combination, after mixing Cy5 labeled p/o Rv3597c and Rv0324, and mixing with methyl green or actinomycin D with different concentrations respectively, EMSA is carried out.

EMSA reaction system (30. mu.L):

mu.L of 0.1mmol/L Rv0324, 1. mu.L of 40 ng/. mu.L Cy5 labeled p/o Rv3597c, 3. mu.L of 10 × binding buffer, 3. mu.L of 25mmol/L DTT, 1.5. mu.L of 1mg/mL poly (dI-dC), 1.5. mu.L of 50% glycerol, 1.5. mu.L of 100mmol/L MgCl₂And methyl green or actinomycin D with final concentration of 0.01mmol/L, 0.05mmol/L, 0.25mmol/L, 1.25mmol/L respectively, and ddH₂And (4) complementing O.

Incubating at 37 ℃ in the dark for 30min, carrying out electrophoresis on 8% polyacrylamide gel at 200V for 3h, separating the compound, carrying out scanning imaging by using a two-color infrared laser imaging system, and analyzing the main region of Rv0324 combined on p/o Rv3597 c.

By utilizing the specificity of methyl green and actinomycin D for respectively combining with major and minor grooves of DNA, different concentrations of methyl green or actinomycin D are incubated with Rv0324 and p/o Rv3597c marked by Cy5 for EMSA, and the result shows that after 0.01mmol/L of methyl green acts, a free p/o Rv3597c band appears, and the formation of Rv0324 and p/o Rv3597c complexes is inhibited; when actinomycin D was added, the concentration of actinomycin was increased from 0.01mmol/L to 1.25mmol/L, and no change was observed in the free p/o rv3597c fragment (FIG. 5). This indicates that methyl green inhibits the interaction between p/o Rv3597c and Rv0324, with Rv0324 interacting with the major groove region of p/o Rv3597 c.

While the foregoing is directed to the preferred embodiment of the present invention, it will be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Applicant's information

<120> recombinant protein, expression vector, recombinant engineering bacterium of mycobacterium tuberculosis and application thereof

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