CN118108816B - Rv0767c protein, antibody and application thereof - Google Patents

Abstract

The present invention relates to the field of polypeptides, and specifically to a Rv0767c protein, an antibody thereof and an application thereof. The amino acid sequence of the transcription factor Rv0767c is shown in SEQ ID NO: 1. The present invention also provides an application of the transcription factor Rv0767c in directly regulating the rv3133c gene of Mycobacterium tuberculosis, and provides an Rv0767c antibody and an application of the Rv0767c antibody. The present invention clarifies for the first time that the transcription factor Rv0767c directly regulates the expression of rv3133c (dosR) , which can promote the growth of MTB, as well as colonization and organ damage in the host; the regulatory mechanism of Rv0767c provides a theoretical basis for the treatment of tuberculosis and the screening of drug molecular targets.

Description

A Rv0767c protein, its antibody and application

Technical Field

The present invention relates to the field of polypeptides, and in particular to an Rv0767c protein, an antibody thereof and applications thereof.

Background technique

Mycobacterium tuberculosis (MTB) is the pathogen that causes tuberculosis (TB). With the emergence of extensively drug-resistant and multidrug-resistant tuberculosis strains, MTB resistance continues to rise, making TB treatment more complicated and difficult. The fundamental reason why TB recurs and is difficult to cure is that MTB has a strong ability to adapt to the harsh environment of hypoxia and acidity in host cells. This adaptability depends largely on the unique transcriptional regulation system of MTB. This system helps MTB survive and maintain a latent state by using transcriptional regulatory proteins to regulate the rapid expression of target genes, thereby retaining the infection in the host for a long time.

The MTB genome encodes more than 4,000 proteins, including more than 200 transcriptional regulatory proteins, which play a regulatory role in bacterial replication, transcription, translation, transport and other aspects. The MTB genome encodes 13 σ factors, 12 two-component systems, 11 protein kinases, and 8 response regulatory elements. At present, among the more than 200 transcriptional regulatory factors encoded by MTB, only some transcriptional regulatory mechanisms have been analyzed. Mining, identifying and studying more transcriptional regulatory proteins and deeply clarifying the transcriptional regulatory mechanisms of pathogens will play an important role in the development of new prevention and control agents and effective treatment of tuberculosis. The MTB genome encodes 51 TetR family transcriptional regulatory factors. The TetR regulatory family is a common single-component signal transduction system and a large family of transcriptional repressors, which are mainly related to antibiotic resistance and the output of intracellular small molecules. TetR family proteins usually bind to DNA as transcriptional repressors, inhibit transcription in the absence of specific ligands, and bind to the C-terminus of TetR in the presence of ligands to cause conformational changes, resulting in the separation of proteins from target DNA, thereby causing regulatory changes.

The inventors explored the regulatory genes on the MTB genome through various methods such as chromatin immunoprecipitation, in vitro gel hysteresis test, and biomembrane interferometry technology, screened proteins that affect the promoter of the target gene, clarified its regulatory mechanism, and provided a theoretical basis for the screening of molecular drug targets for tuberculosis.

Summary of the invention

The inventors have found through research that the transcription factor Rv0767c (or HpoR protein, Rv0767c protein) is a broad-spectrum regulatory protein that can directly regulate the expression of the rv3133c ( dosR ) gene and plays an important role in the dormancy survival and pathogenicity of Mycobacterium tuberculosis (MTB).

The present invention provides the following technical solutions:

The present invention provides a transcription factor Rv0767c for directly regulating the rv3133c gene of Mycobacterium tuberculosis, and the amino acid sequence thereof is shown in SEQ ID NO: 1.

The present invention also provides the application of the transcription factor Rv0767c in directly regulating the rv3133c gene of Mycobacterium tuberculosis.

The present invention provides an Rv0767c antibody, which is prepared by the following method: injecting Rv0767c protein subcutaneously into a rabbit, collecting blood from the heart to separate serum, and then purifying to obtain the Rv0767c antibody; the amino acid sequence of the Rv0767c protein is shown in SEQ ID NO: 1. Preferably, the Rv0767c antibody is a polyclonal antibody. Obtaining the Rv0767c antibody is not limited to the method of the present invention, and artificially prepared hybridomas are also a common method. When prepared by hybridomas, monoclonal antibodies can also be obtained.

The present invention also provides the use of the Rv0767c antibody in inhibiting the in vitro growth of Mycobacterium tuberculosis, by inhibiting the binding of the transcription factor Rv0767c to the promoter of the rv3133c gene to inhibit the growth of mycobacteria. According to the experimental results of the present invention, the rv3133c gene can promote the growth of MTB, and the antibody binds to the transcription factor Rv0767c, inhibiting the binding of the transcription factor to the promoter of the rv3133c gene, inhibiting the expression of the rv3133c gene, thereby inhibiting the growth of MTB.

The present invention also provides the use of the Rv0767c antibody in preparing a drug for inhibiting the growth of Mycobacterium tuberculosis.

The present invention is based on the regulatory mechanism of transcription factor Rv0767c and also provides a method for inhibiting the growth of Mycobacterium tuberculosis, comprising: inhibiting the transcription factor Rv0767c from binding to the promoter of the rv3133c gene.

Furthermore, the binding of transcription factor Rv0767c to the promoter of rv3133c gene is inhibited by one or more of Lys (lysine), Arg (arginine), Tyr (tyrosine), Cu ²⁺ , Fe ³⁺ , Pb ²⁺ , and Rv0767c antibody; Fe ³⁺ and Rv0767c antibody are preferred.

Furthermore, the Lys (lysine) concentration is greater than 40 mM; the Arg (arginine) concentration is greater than 10 mM; the Tyr (tyrosine) concentration is greater than 40 mM; the Cu ²⁺ concentration is greater than 2.5 mM; the Fe ³⁺ concentration is greater than 0.05 mM; and the Pb ²⁺ concentration is greater than 5 mM.

Compared with the prior art, the beneficial effects of the present invention include at least:

1. The present invention has clarified for the first time that the transcription factor Rv0767c directly regulates the expression of rv3133c , which can promote the growth of MTB, as well as its colonization and organ damage in the host.

2. The present invention screens small molecules that affect the interaction between the transcription factor Rv0767c and the promoter of the target gene, and obtains a Rv0767c polyclonal antibody, thereby providing a new method for inhibiting the growth of MTB.

3. The present invention clarifies the regulatory mechanism of Rv0767c, providing a theoretical basis for the treatment of tuberculosis and the screening of drug molecular targets.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is the SDS-PAGE electrophoresis and Western-blot identification results of the purified Rv0767c protein;

FIG2 is the result of the specific competitive inhibition test between the cold probe and Cy-5 labeled rv3133c ;

Figure 3 is a diagram of the purification results of the Rv0767c polyclonal antibody and the ChIP-PCR results, A is the SDS-PAGE band of the Rv0767c polyclonal antibody, B is the Western Blot band of the Rv0767c polyclonal antibody, and C is the ChIP-PCR result;

Figure 4 is an EMSA verification of the effects of small molecules and metal ions on the binding of Rv0767c to the rv3133c promoter. In Figure 4, AF are the effects of Lys, Arg, Tyr, CuCl ₂ , FeCl ₃ and PbCl ₂ on the binding of Rv0767c to the rv3133c promoter, respectively;

FIG. 5 shows the growth curves of pMV262-Rv0767c-Ms and pMV262-Ms strains.

Detailed ways

In order to make the purpose, technical scheme and beneficial effect of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. The examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary illustrations of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention. Reagents and instruments used in the embodiments can be purchased from the market conventionally unless otherwise specified.

The endpoints of ranges and any values disclosed herein are not limited to the exact range or value, and these ranges or values should be understood to include approximations to these ranges.

Example 1 Expression, purification and identification of transcription factor Rv0767c

1.1 E. coli transformation

The coding sequence of Rv0767c was loaded on the pET22b plasmid to obtain the pET22b-Rv0767c plasmid, and the pET22b-Rv0767c plasmid was transformed into E. coli BL21 (DE3) competent cells. The sequence of the pET22b plasmid is shown in SEQ ID NO: 2, and the coding sequence of Rv0767c is shown in SEQ ID NO: 6.

The transformation steps are as follows: (1) Take out the E. coli BL21 (DE3) competent cells from the -80℃ refrigerator and place them on ice to thaw. (2) Take 50μL of competent cells, add 3-4μL of pET22b-Rv0767c plasmid, use a pipette to gently blow and mix, and place it on ice for 25 minutes. Turn on the water bath in advance and set the temperature to 42℃. (3) Place the competent cells in a 42℃ water bath for 90 seconds, quickly return them to ice and let them stand for 2 minutes. Be careful not to shake them violently during this process. (4) Add 700μL of non-resistant liquid LB, mix well, and then culture them in a shaking incubator at 37℃, 200rpm for 1h. (5) Centrifuge at 5000rpm for 1min to collect the bacteria. Keep about 100μL of culture medium to resuspend the bacteria and spread them on the Amp-resistant LB solid culture medium plate. (6) Invert the plate and incubate it in a 37°C incubator overnight. Pick a single colony within 12 hours.

1.2 Identification of monoclonal colonies

Extraction and identification of recombinant plasmid: Select the monoclonal colony obtained in the previous step from the plate in time, add it to 7mL Amp-resistant LB liquid culture medium, and culture it at 37℃, 150r/min shaking for 12h. Take 5mL of bacterial liquid and use Tiangen Plasmid Mini Extraction Kit (centrifugal column type) to extract plasmid, operate according to the instructions, and identify the extracted plasmid by agarose gel electrophoresis.

1.3 Rv0767c induction expression conditions

The identified monoclonal bacteria were activated in 10mL of Amp-resistant LB liquid medium, cultured at 37°C, 200r/min for 2h, and then 5mL was transferred to 500mL of Amp-resistant LB liquid medium for further culture. When the OD _600nm value of the bacterial solution was between 0.8 and 0.9, IPTG was added, with final concentrations of 0.7mM and 1mM, respectively, and then induced at 16°C, 150rpm overnight. The bacterial cells were collected by centrifugation at 4000rpm for 10min for subsequent protein purification.

1.4 Purification and desalting of Rv0767c protein

(1) Add 35 mL of lysis buffer to every 500 mL of bacterial liquid, resuspend and add 20 μL of lysozyme, and react on a rotating mixer at 360°, 20 rpm, at room temperature for 30 min. (2) Fix the centrifuge tube containing the preliminary lysed bacterial liquid in a beaker filled with ice and use an ultrasonic cell crusher for ultrasonic disruption. The amplitude was Φ6, the power was 50%, the ultrasound was turned on for 2 seconds, and the ultrasound was turned off for 3 seconds, and the total ultrasound was 15 minutes. Immediately after the ultrasound, use a refrigerated centrifuge to centrifuge at 4°C, 10,000g, for 30 minutes. From this step onwards, all operations were performed on ice. (3) Ni-agarose gel HP (Beijing Solebao Technology Co., Ltd.) was used to purify the Rv0767c protein according to the instructions. (4) Desalt the protein using a GE PD-10 desalting column according to the instructions to remove salt ions that affect protein activity. The protein buffer was replaced with PBS, and the protein buffer used for BLI experiments was replaced with PBST. (5) Add 80% glycerol to preserve the protein solution, adjust the final glycerol concentration to 30%, and then transfer the protein to a -80°C ultra-low temperature freezer for storage. No glycerol was added to the samples used for BLI experiments. (6) Use the Biotech BCA Protein Assay Kit to determine the protein concentration. Follow the instructions for the procedure.

1.5 Identification of Rv0767c protein

The purified Rv0767c protein was subjected to SDS-PAGE electrophoresis and Western-blot identification. The results are shown in Figure 1, where A in Figure 1 is the SDS-PAGE identification result, and B in Figure 1 is the Western-blot identification result. The results show that there is a specific band with a size of 28.4 kDa, which is consistent with the predicted protein size (the amino acid sequence of the Rv0767c protein is shown in SEQ ID NO: 1). This indicates that the Rv0767c protein purified by affinity chromatography has high purity and good specificity.

Example 2 Interaction between Rv0767c protein and promoter of target gene

The EMSA test method was used to preliminarily prove that Rv0767c binds to the promoter of potential regulatory genes in vitro. According to the NCBI database (https://www.ncbi.nlm.nih.gov), the primers of the promoter sequence of the target gene were designed using the genome sequence of Mycobacterium bovis BCG str.Tokyo 172 strain (GenBank: AP010918.1) and amplified by conventional PCR, and purified for later use.

According to the results of transcriptomics, 28 target genes were preliminarily selected, namely rv0166 , rv0217c , rv1086 , rv1814 , rv2526 , rv3133c , rv2031c , rv2007c , rv0575c , rv1085c , rv1403c , rv1404 , rv1405c , rv1682 , rv1963 , rv1964 , rv2415c , rv2515c , rv3197 , rv3425 , rv3520c , rv3746c , rv0765c , rv0902c , rv1706c , rv2212 , rv2638 and rv2902 c. The binding of these target genes to the Rv0767c regulatory protein was further verified by EMSA experiments, and the results showed that all of them were bound. Considering the important role of rv3133c in the life activities of MTB such as latent infection and growth, further research was carried out on this gene.

Example 3 Specific binding of Rv0767c to the rv3133c promoter region in vitro

In order to detect whether Rv0767c binds to rv3133c promoter specifically, EMSA test method was used for verification. The 5' end of the primer sequence of rv3133c gene promoter was labeled with Cy-5, PCR amplified, purified and collected. The primer sequence is: SN441-F: CTTTACCACCAGGGCACCAC (SEQ ID NO: 3); SN441-R: TGCTGCGGCACGCATTCGAG (SEQ ID NO: 4). Add ddH ₂ O to adjust the cold probe concentration to 8000nM. Add a large amount of cold probe (see Table 1) to the reaction system of Rv0767c and Cy5-labeled p/o rv3133c , and react in a water bath at 37℃ for 20min. After the reaction, add 4μL 6× Loading buffer to the sample, use a hand-held centrifuge to briefly centrifuge and then load the sample. Electrophoresis was performed at 200V for 3h under light-proof conditions. After the electrophoresis, the gel was taken out and scanned by Laser900 fully automatic multifunctional imaging system, and the results are shown in Figure 2. The results showed that the addition of cold probe made Cy-5 labeled rv3133c unable to bind to protein and was competed off.

Table 1 Reaction system for specific competitive inhibition assay

The affinity of Rv0767c and rv3133c promoter was determined by biofilm interferometry test. The 5' end of the upstream and downstream primers of rv3133c promoter was labeled with biotin. The DNA probe was PCR purified with an annealing temperature of 60°C. After purification, the concentration was determined using an ultra-micro spectrophotometer. The DNA probe with biotin label was adjusted to 60nM using 10×PBST and ddH ₂ O for later use, so that the final buffer of the DNA probe was 1×PBST.

The BLI experimental method was operated as follows: the protein concentration was measured by the BCA method, the protein concentration was adjusted to 10000nM, and the protein was diluted from high to low concentration. The BLI experiment used a sensor labeled with Streptavidin (SA) combined with a biotin-labeled nucleic acid probe. The reaction volume of all sample wells was 200μL, the reaction temperature was 30℃, and the concentration of the biotin-labeled p/o rv3133c DNA probe was 60nM. The data were processed using Data Analysis 12.0 software. The BLI results showed that the binding activity of the two was good, and the dissociation constant was 4.017× ^10-9 . The above results all indicate that Rv0767c specifically binds to the rv3133c gene promoter in MTB in vitro.

Example 4 ChIP-PCR verification of the binding of Rv0767c and rv3133c promoters in MTB

4.1 Animal immunization and titer testing

Before immunization, blood was collected from the ear vein of two adult New Zealand white rabbits and serum was separated. Then, Rv0767c protein was mixed with Freund's incomplete adjuvant in a ratio of 1:1 and emulsified. 400 μg protein was injected subcutaneously at multiple points on the back for immunization, once every 14 days. After immunization 3 times, blood was collected from the ear vein and serum was separated. The titer was detected by indirect ELISA method. The operation was carried out according to the instructions of the kit. According to the ELISA results, rabbits with antibody titers that met the requirements were selected for heart blood collection, and serum was separated for subsequent polyclonal antibody purification.

4.2 Polyclonal antibody purification

The polyclonal antibody was purified by protein A+G. The steps were as follows: the protein A+G column was washed with 2 column volumes of ddH ₂ O and then equilibrated with a phosphate buffer column. The serum was diluted 5 times with 20 mM phosphate buffer and filtered. The filtrate was slowly loaded onto the column and then equilibrated with 10 column volumes of phosphate buffer column. Finally, the Rv0767c polyclonal antibody was eluted with 5 column volumes of 0.1 M Gly-HCl at pH 2.7, and then Western-blot was performed for identification. The prepared Rv0767c rabbit polyclonal antibody was purified by protein A affinity and then detected by SDS-PAGE and Western Blot. The band size was correct (as shown in A in Figure 3) and specific (as shown in B in Figure 3).

4.3 Chromatin immunoprecipitation assay (ChIP)

BCG in the logarithmic growth phase was fixed with 1% formaldehyde for 10 minutes to crosslink proteins and nucleic acids. After adding 125mM/L Gly for 5 minutes, the cells were centrifuged at 8000rpm for 10 minutes at 4°C to collect the cells. The cell pellet was resuspended in IP buffer and then ultrasonically disrupted, with an amplitude of 20%, on for 3s, off for 5s, and 5min. Centrifuged at 12000rpm for 20 minutes, the supernatant was aspirated, and DNA fragments were detected by agarose gel electrophoresis. 20μL Rv0767c polyclonal antibody was coupled with M-280 goat anti-rabbit antibody to prepare coated magnetic beads. 100μL supernatant was taken as an experimental control, and the remaining supernatant after ultrasonication was incubated with coated magnetic beads at 4°C overnight. After washing the magnetic beads, the Rv0767c-DNA complex was collected and purified using the iPure DNA extraction kit. The collected DNA was used as a template for PCR amplification of primers SN441-F/SN441-R.

The results of ChIP-PCR are shown in Figure 3C, where input represents the supernatant of lysed cells (experimental group), P (preimmune) represents the serum before immunization (negative control group), and I (immune) represents the serum after immunization (positive control group). Figure 3C shows that the serum after immunization of the experimental group and the positive control group can detect a band of the same size as rv3133c , while the serum before immunization of the negative control group cannot detect it, indicating that Rv0767c binds to the rv3133c promoter under MTB in vivo conditions, thereby playing a regulatory role.

Example 5 Small molecules that affect the binding of Rv0767c to the rv3133c promoter

The EMSA test method was used to preliminarily screen small molecules that affect the binding of Rv0767c to the rv3133c promoter. Different small molecules were added to the EMSA reaction system and incubated at 37°C for 20 minutes to detect the effect of small molecules on binding. The specific operation steps are as follows: (1) Small molecule preparation: 20 amino acids were prepared with vitamins Vb ₁ , Vb ₃ , Vb ₅ , Vb ₆ and Vc and metal ions LiCl, CuCl ₂ , FeCl ₃ and PbCl ₂ into a mother solution with a concentration of 200mM, and the loading volume was adjusted according to different concentrations. The results are shown in Figure 4, where AF in Figure 4 are the effects of Lys, Arg, Tyr, CuCl ₂ , FeCl ₃ and PbCl ₂ on the binding of Rv0767c to the rv3133c promoter. The results showed that Lys, Arg, Tyr, CuCl ₂ , PbCl ₂ and FeCl ₃ could inhibit the binding of Rv0767c to rv3133c promoter. Lys and Tyr began to inhibit the binding of Rv0767c to rv3133c promoter at 40mM, Arg inhibited the binding of Rv0767c to rv3133c promoter at 10mM, CuCl ₂ showed an inhibitory effect at 2.5mM, PbCl ₂ showed an inhibitory effect at 5mM, and FeCl ₃ had the most obvious effect on the binding, showing an inhibitory effect at 0.05mM. Therefore, these small molecules can be used to inhibit the binding of Rv0767c to rv3133c promoter.

Example 6 Detection of the growth curve of the recombinant pMV262-Rv0767c-Ms strain

In order to explore the effect of Rv0767c on bacterial growth, pMV262-Rv0767c-Ms and pMV262-Ms strains were used to transform the constructed pMv262-Rv0767c recombinant plasmid into Mycolicibacterium smegmatis (Ms) competent cells to construct the pMV262-Rv0767c-Ms strain, and the shuttle plasmid pMv262 was transformed into Mycolicibacterium smegmatis (Ms) competent cells to construct the pMV262-Ms strain. The sequence of the pMv262 plasmid is shown in SEQ ID NO: 5. Spread on Kan-resistant LB solid medium and culture at 37°C for about 3 days until bacterial lawns appear. Scrape the bacterial lawn and inoculate it in Kan-resistant 7H9 liquid medium, and activate it in a shaker at 37°C and 120rpm.

The activated bacterial liquid was inoculated into Kan-resistant 7H9 liquid medium and cultured at 37°C, 120rpm until OD _600nm was 0.5. Three replicates were added to each group and heat-shocked for 2h at 45°C, 120rpm in a shaker. After the heat shock, the culture was continued at 37°C, 120rpm. Samples were taken every 12h for dilution and spotting (dilution gradient 10 ^-4 -10 ^-7 ), and Kan-resistant LB solid medium was used for the plate and cultured for 5 days. The plate was read on the third day after spotting, the number of colonies on the plate was counted, and the viability curve was drawn.

The results are shown in Figure 5. The growth curve results showed that in the first 48 hours, the growth trends of pMV262-Ms and pMV262-Rv0767c-Ms were consistent. Between 48 hours and 84 hours, the growth of pMV262-Rv0767c-Ms was better than that of pMV262-Ms, that is, overexpression of Rv0767c can promote the growth of Ms.

The sequence involved in the present invention is as follows:

Rv0767c amino acid sequence (SEQ ID NO: 1):

MSSDVLVTTPAQRQTEPHAEAVSRNRRQQATFRKVLAAAMATLREKSYADTVRLVAARAKVAPATAYTYFSSKNHLIAEVYLDLVRQVPCVTDVNVPMPIRVTTSSLRHLALVVADEPEIGAACTAALLDGGADPAVRAVRDRIGAEIHRRITSAIGPGADPGTVFALEMAFFGALVQAGSGTFTYHEIADRLGYVVGLILAGANEPSTGGSE

pET22b plasmid sequence (SEQ ID NO: 2):

ATCCGGATATAGTTCCTCCTTTCAGCAAAAAACCCCTCAAGACCCGTTTAGAGGCCCCAAGGGGTTATGCTAGTTATTGTCCAGCGGTGGCAGCAGCCAACTCAGCTTCCTTTCGGGCTTTGTTAGCAGCCGGATCTCAGTGGTGGTGGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATCCGAATTAATTCCGATATCCATGGCCATCGCCGGCTGGGCAGCGAGGAGCA GCAGACCAGCAGCAGCGGTCGGCAGCAGGTATTTCATATGTATATCTCCTTCTTAAAGTTAAACAAAATTATTTCTAGAGGGGAATTGTTA TCCGCTCACAATTCCCCTATAGTGAGTCGTATTAATTTCGCGGGATCGAGATCTCGATCCTCTACGCCGGACGCATCGGTGGCCGGCATCACCGGCGCCACAGGTGCGGTTGCTGGCGCCTATATCGCCGACATCACCGATGGGGAAGATCGGGCTCGCCACTTCGGGCTCATGAGCGCTTGTTTCGGCGTGGGTATGGTGGCAGGCCCCGTGGCCGGGGGACTGTTGGGCGCCATCTCCTTGCATGCACCATTCC TTGCGGCGGCGGTGCTCAACGGCCTCAACCTACTACTGGGCTGCTTCCTAATGCAGGAGTCGCATAAGGGAGAGCGTCGAGATCCCGG ACACCATCGAATGGCGCAAAACCTTTCGCGGTATGGCATGATAGCGCCCGGAAGAGAGTCAATTCAGGGTGGTGAATGTGAAACCAGTAACGTTATACGATGTCGCAGAGTATGCCGGTGTCTCTTATCAGACCGTTTCCCGCGTGGTGAACCAGGCCAGCCACGTTTCTGCGAAAACGCGGGAAAAAGTGGAAGCGGCGATGGCGGAGCTGAATTCATTCCCAACCGCGTGGCACAACAACAACTGGCGGGCAAACAGTCG TTGCTGATTGGCGTTGCCACCTCCAGTCTGGCCCTGCACGCGCCGTCGCAAATTGTCGCGGCGATTAAATCTCGCGCCGATCA ACTGGGTGCCAGCGTGGTGGTCGATGGTAGAACGAAGCGGCGTCGAAGCCTGTAAAGCGGCGGTGCACAATCTTCGCGCAACGCGTCAGTGGGCTGATCATTAACTATCCGCTGGATGACCAGGATGCCATTGCTGTGGAAGCTGCCTGCACTAATGTTCCGGCGTTATTTCTTGATGTCTCTGACCAGACACCCATCAACAGTATTATTTTCTCCCATGAAGACGGTACGCGACTGGGCGTGGAGCATCTGG TCGCATTGGGTCACCAGCAAATCGCGCTGTTAGCGGGCCCATTAAGTTCTGTCTCGGCGCGTCTGCGTCTGGCTGGCTGGCATAAAT ATCTCACTCGCAATCAAATTCAGCCGATAGCGGAACGGGAAGGCGACTGGAGTGCCATGTCCGGTTTTCAACAAACCATGCAAATGCTGAATGAGGGCATCGTTCCCACTGCGATGCTGGTTGCCAACGATCAGATGGCGCTGGGCGCAATGCGCGCCATTACCGAGTCCCGGGCTGCGTTGGTGCGGATATCTCGGTAGTGGGATACGACGATACCGAAGACAGCTCATGTTATATCCCGCCGTTAACCACCATCAAACA GGATTTTCGCCTGCTGGGGCAAACCACGGTGGACCGCTTGCTGCAACTCTCTCAGGGCCAGGCGGTGAAGGGCAATCAGCT GTTGCCCGTCTCACTGGTGAAAAGAAAAACCACCCTGGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGACAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTAAGTTAGCTCACTCATTAGGCACCGGGATCTCGACCGATGCCCTTGAGAGCCTTCAACCCAGTCAGCTCCTTCCGGTGGGCGCGGGGCATGACTATCGTCGCCGCACTTATG ACTGTCTTCTTTATCATGCAACTCGTAGGACAGGTGCCGGCAGCGCTCTGGGTCATTTTCGGCGAGGACCGCTTTCGCTGGAG CGCGACGATGATCGGCCTGTCGCTTGCGGTATTCGGAATCTTGCACGCCCTCGCTCAAGCCTTCGTCACTGGTCCCGCCACCAAACGTTTCGGCGAGAAGCAGGCCATTATCGCCGGCATGGCGGCCCCACGGGTGCGCATGATCGTGCTCCTGTCGTTGAGGACCCGGCTAGGCTGGCGGGGTTGCCTTACTGGTTAGCAGAATGAATCACCGATACGCGAGCGAACGTGAAGCGACTGCTGCTGCAAA ACGTCTGCGACCTGAGCAACAACATGAATGGTCTTCGGTTTCCGTGTTTCGTAAAGTCTGGAAACGCGGAAGTCAGCGCCCTGCACCATTATG TTCCGGATCTGCATCGCAGGATGCTGCTGGCTACCCTGTGGAACACCTACATCTGTATTAACGAAGCGCTGGCATTGACCCTGAGTGATTTTTCTCTGGTCCCGCCGCATCCATACCGCCAGTTGTTTACCCTCACAACGTTCCAGTAACCGGGCATGTTCATCATCAGTAACCCGTATCGTGAGCATCCTCTCTCGTTTCATCGGTATCATTACCCCCATGAACAGAAATCCCCTCTACACGGAGGCATCAGTGACCAAACAGG AAAAAACCGCCCTTAACATGGCCCGCTTTATCAGAAGCCAGACATTAACGCTTCTGGAGAAACTCAACGAGCTGGACGC GGATGAACAGGCAGACATCTGTGAATCGCTTCACGACCACGCTGATGAGCTTTACCGCAGCTGCCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCGCAGCCATGACCCAGTCACGTAGCGATAGCGGAGTGTAACTGGCTTAACT ATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAATACCGCATCAG GCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACA GGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTG TCCGCCTTTCTCCCTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGA AGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTA GCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTG ACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAG ATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGATGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTGCAGGCATCGTGGTGTCACGCTCG TCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCT CCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCT CAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACT TTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGAAATTGTAAACGTTAATATTTTGTTAAAATTCGCGTTAAATTTTTGTTAAATC AGCTCATTTTTTAACCAATAGGCCGAAATCGGCAAAATCCCTTATAAATCAAAAGAATAGACCGAGATAG GGTTGAGTGTTGTTCCAGTTTGGAACAAGAGTCCACTATTAAAGAACGTGGACTCCAACGTCAAAGGGCGAAAAACCGTCTATCAGGGCGATGGCCCACTACGTGAACCATCACCCTAATCAAGTTTTTTGGGGTCGAGGTGCCGTAAAGCACTAAATCGGAACCCTAAAGGGAGCCCCCGATTTAGAGCTTGACGGGGAAAGCCGGCGAACGTGGCGAGAAAGGAAGGGAAGAAAGCGAAAGGAGCGGGCGCTAGGGCGCT GGCAAGTGTAGCGGTCACGCTGCGCGTAACCACCACACCCGCCGCGCTTAATGCGCCGCTACAGGGCGCGTCCCATTCGCCA

SN441-F (SEQ ID NO: 3):

CTTTACCACCAGGGCACCAC

SN441-R (SEQ ID NO: 4):

TGCTGCGGCACGCATTCGAG

pMV262 plasmid (SEQ ID NO: 5):

AAAGCCACGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCGAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGCTTGTATGGGAAGCCCCATGCGCCAGAGTTGTTTC TGAAACATGGCAAAG GTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCTGACGGAATTTATGCCTCTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGGAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGT ATTTCGTCTCGCTCAGGCGCAATC ACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAATCTTTTGCCATTCTCACCGGATTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCT TCATTACAGAAACGGCTTTTCAA AAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCTAATCAGAATTGGTTAATTGGTTGTAACACTGGCAGAGCATTACGCTGACTTGACGGGACGGCGGCTTGTTGAATAAATCGAACTTTTGCTGAGTTGAAGGATCAGATCACGCATCTTCCCGACAACGCAGACCGTTCCGTGGCAAAGCAAAAGTTCAAAATCACCAACTGGTCCACCTACAACAAAGCTCTCAT CAACCGTGGCTCCCTCACTTTCTG GCTGGATGATGGGGCGATTCAGGCCTGGTATGAGTCAGCAACACCTTCTTCACGAGGCAGACCTCACTAGTTCCACTGAGCGTCAGACCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAAT ACTGTCCTTCTAGTGTAGCCGTAGTTAGG CCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCATTGAGAAAGCGCCACGCTTCCCGAAGGGAGAAA GGCGGACAGGTATCCGGTAAGCGGCAGGGTC GGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACC GCTCGCCGCAGCCGAACGACCGAGCGCA ACGCGTGAGCCCACCAGCTCCGTAAGTTCGGGTGCTGTGTGGCTCGTACCCGCGCATTCAGGCGGCAGGGGGTCTAACGGGTCTAAGGCGGCGTGTACGGCCGCCACAGCGGCTCTTAGCGGCCCGGAAACGTCCTCGAAACGACGCATGTGTTCCTCCTGGTTGGTACAGGTGGTTGGGGGTGCTCGGCTGTCGCTGGTGTTTCATCATCAGGGCTCGACGGGAGAGCGGGGGAGTGTGCAGTTGTGGGGTGGC CCCTCAGCGAAATATCTGACTTGGAGCTCGT GTCGGACCATACACCGGTGATTAATCTGGTTTATTATCAAGCGTGAGCCACGTCGCCGACGAATTTGAGCAGCTCTGGCTGCCGTACTGGTCCCTGGCAAGCGACGATCTGCTCGAGGGGATCTACCGCCAAAGCCGCGCGTCGGCCCTAGGCCGCCGGTACATCGAGGCGAACCCAACAGCGCTGGCAAACCTGCTGGTCGTGGACGTAGACCATCCAGACGCAGCGCTCCGAGCGCTCAGCGCCCGGGGGTCC CATCCGCTGCCCAACGCGATCGTGGGCAA TCGCGCCAACGGCCACGCACACGCAGTGTGGGCACTCAACGCCCCTGTTCCACGCACCGAATACGCGCGGCGTAAGCCGCTCGCATACATGGCGGCGTGCGCCGAAGGCCTTCGGCGCGCCGTCGATGGCGACCGCAGTTACTCAGGCCTCATGACCAAAAACCCCGGCCACATCGCCTGGGAAACGGAATGGCTCCACTCAGATCTCTACACACTCAGCCACATCGAGGCCGAGCTCGGCGCGAACATGCCACCGCCGCG CTGGCGTCAGCAGAACCACGTACAAA GCGGCTCCGACGCCGCTAGGGCGGAATTGCGCACTGTTCGATTCCGTCAGGTTGTGGGCCTATCTTCCCGCCCTCATGCGGATCTACCTGCCGACCCGGAACGTGGACGGACTCGGCCGCGCGATCTATGCCGAGTGCCACGCGCGAAACGCCGAATTTCCGTGCAACGACGTGTGTCCCGGACCGCTACCGGACAGCGAGGTCCGCGCCATCGCCAACAGCATTTGGCGTTGGATCACAACCAAGTCGCGCATTTGG GCGGACGGGATCGTGGTCTACGAGGCCA CACTCAGTGCGCGCCATGCGGCCATCTCGCGGAAGGGCGCAGCAGCGCGCACGGCGGCGAGCACAGTTGCGCGGCGCGCAAAGTCCGCGTCAGCCATGGAGGCATTGCTATGAGCGACGGCTACAGCGACGGCTACAGCGACGGCTACAACTGGCAGCCGACTGTCCGCAAAAAGCGGCGCGTGACCGCCGCCGAAGGCGCTCGAATCACCGGACTATCCGAACGCCACGTCGTCCGGCTCGTGGCGCAGGAACGC AGCGAGTGGTTCGCCGAGCAGGCTGCACGC CGCGAACGCATCCGCGCCTATCACGACGACGAGGGCCACTCTTGGCCGCAAACGGCCAAACATTTCGGGCTGCATCTGGACACCGTTAAGCGACTCGGCTATCGGGCGAGGAAAGAGCGTGCGGCAGAACAGGAAGCGGCTCAAAAGGCCCACAACGAAGCCGACAATCCACCGCTGTTCTAACGCAATTGGGGAGCGGGTGTCGCGGGGGTTCCGTGGGGGGTTCCGTTGCAACGGGTCGGACAGGTAAAAGTCCTG GTAGACGCTAGTTTTCTGGTTTGGGCCA TGCCTGTCTCGTTGCGTGTTTCGTTGCGTCCGTTTTGAATACCAGCCAGACGAGACGGGGTTCTACGAATCTTGGTCGATACCAAGCCATTTCCGCTGAATATCGTGGAGCTCACCGCCAGAATCGGTGGTTGTGGTGATGTACGTGGCGAACTCCGTTGTAGTGCTTGTGGTGGCATCCGTGGCGCGGCCGCGGTACCAGATCTTTAAATCTAGAGGGTGACCACAACGCCGCCCGCTTTGATCGGG GACGTCTGCGGCCGACCATTTACGGGTCTTGTTGTCGTT GGCGGTCATGGGCCGAACATACTCACCCGGATCGGAGGGCCGAGGACAAGGTCGAACGAGGGGCATGACCCGGTGCGGGGCTTCTTGCACTCGGCATAGGCGAGTGCTAAGAATAACGTTGGCACTCGCGACCGGTGAGTGCTAGGTCGGGACGGTGAGGCCAGGCCCGTCGTCGCAGCGAGTGGCAGCGAGGACAACTTGAGCCGTCCGTCGCGGGCACTGCGCCCGGCCAGCGTAAGTAGCGGGGTTGCCGTCACC CGGTGACCCCCGTTTCATCCCCGATCCG GAGGAATCACTTCGCAATGGCCAAGACAATTGCGGATCCGTGGTCGCACCCGCAGTTCGAGAAGGGCGGCGGCTCGCAGCTGCAGAATTCGAAGCTTCATATGATCGATGTCGACGTGGGCTCGGGCCACCACCACCACCACCACCACCACCACGGTTAACTAGCGTACGATCGACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATGCCATCATGGCCGCGGTGATCAGCTAGCC ACCTGACGTCGGGGGGGGGGG

Coding sequence of Rv0767c (SEQ ID NO: 6):

ATGAGCAGCGATGTGCTGGTGACCACCCCGGCGCAGCGCCAGACCGAACCGCATGCGGAAGCGGTGAGCCGCAACCGCCGCCAGCAGGCGACCTTTCGCAAAGTGCTGGCGGCGGCGATGGCGACCCTGCGCGAAAAAAGCTATGCGGATACCGTGCGCCTGGTGGCGGCGCGCGCGAAAGTGGCGCCGGCGACCGCGTATAACCTATTTTAGCAAAAACCATCTGATTGCGGAAGTGTATCTGGATCTGGTGCG CCAGGTGCCGTGCGTGACCGATGTGAACGTGCCGATGCCGATTCGCGTGACCAGCAGCCTG CGCCATCTGGCGCTGGTGGTGGCGGATGAACCGGAAATTGGCGCGGCGTGCACCGCGGCGCTGCTGGATGGCGGCGCGGATCCGGCGGTGCGCGCGGTGCGCGATCGCATTGGCGCGGAAATTCATCGCCGCATTACCAGCGCGATTGGCCCGGGCGCGGATCCGGGCACCGTGTTTGCGCTGGAAATGGCGTTTTTTGGCGCTGGTGCAGGCGGGCAGCGGCACCTTTTACCTATCATGAAATTGCGGATCG CCTGGGCTATGTGGTGGGCCTGATTCTGGCGGGCGCGAACGAACCGAGCACCGGCGGCAGCGAA

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims (1)

1. An application of a transcription factor Rv0767c in promoting the in vitro growth of Mycobacterium tuberculosis, characterized in that the amino acid sequence of the transcription factor Rv0767c is shown in SEQ ID NO: 1.