CN113416746A

CN113416746A - Integrated plasmid for aTc induced expression of mycobacterium tuberculosis gene

Info

Publication number: CN113416746A
Application number: CN202110322375.XA
Authority: CN
Inventors: 郭铦; 周亚凤; 申兆兴
Original assignee: Shanghai Gene Optimal Biotechnology Co ltd
Current assignee: Shanghai Gene Optimal Biotechnology Co ltd
Priority date: 2021-03-25
Filing date: 2021-03-25
Publication date: 2021-09-21

Abstract

The invention discloses an integrated plasmid for expressing mycobacterium tuberculosis genes by aTc induction, establishes an integrated mycobacterium expression system based on micromolecule aTc induction, and has wide regulation and control dynamic range and wide application range. The regulated gene is detected by qRT-PCR: the expression level of the Rv3875 gene is improved by 51.68 times, and the expression level of the MSMEG0129 gene is reduced by 6.53 times. The expression system provides a stable platform for proteins which are difficult to express by mycobacteria, such as membrane proteins and the like, and provides a method for knocking down essential genes which cannot be knocked out by the mycobacteria, thereby providing a thought for gene function research.

Description

Integrated plasmid for aTc induced expression of mycobacterium tuberculosis gene

Technical Field

The invention relates to the technical field of mycobacterium tuberculosis, in particular to an integrated plasmid for expressing mycobacterium tuberculosis genes by induction of aTc.

Background

Tuberculosis is an infectious disease caused by infection of mycobacterium tuberculosis, and estimated by world health organization '2020 Global tuberculosis report', about 1000 million new tuberculosis patients in 2019 worldwide, wherein about 120 million tuberculosis dead patients and 20.8 ten thousand HIV positive tuberculosis patients die. In 2019 years in China, about 83.3 ten thousand new tuberculosis patients exist, and about 3.1 ten thousand dead tuberculosis patients exist.

Tuberculosis has historically been a major infectious disease control problem in all countries around the world. With the improvement and development of the economic and technical development level of China, under the condition of normative and general tuberculosis prevention and control management, the current situation and situation of tuberculosis in China are more specifically and objectively known. The research and the need of new drugs, new vaccines and effective public health management measures which are necessary for the clinical and prevention and control of tuberculosis are different from the research interests of scientists in other developed countries, and the research and the need are all determined by the important social requirement of tuberculosis prevention and control reality faced by China.

The genome of the mycobacterium tuberculosis H37Rv consists of 4411532 basic groups, the content of G + C is 65.6 percent, and 4043 genes encode 3993 proteins and 50 RNAs. Because the commonly used prokaryotic expression system is difficult to express mycobacterial proteins, particularly difficult-to-express proteins such as membrane proteins, and a lot of tuberculosis proteins are difficult to obtain expression and purification (Seniya S P, Yadav P, Jain V.restriction of E.coli-Mycobacterium proteins vectors with a variety of expression systems and polypeptide tags for gene expression in Mycobacterium [ J ]. PLoS ONE,2020,15(3): e0230282.), the lack of an excellent expression system aiming at the mycobacterial proteins leads to insufficient cognition on the expressed protein functions, consequently, the cognition deficiency on the pathogenesis of the mycobacterial proteins, and also hinders the development and development of novel vaccines. The tetracycline induction expression system is a regulation system for inducing gene expression, which is established on the basis of a specific Tet resistance operon in a Tn10 transposon of escherichia coli. Through the development of recent years, two sets of Tet-on and Tet-off regulation and control systems are derived; with the improvement of the Tet induction regulation expression system, the stringency, induction efficiency, safety and other aspects of gene expression regulation are gradually improved, and the Tet induction regulation expression system is widely applied to basic research and in-vivo and in-vitro experimental treatment research.

Disclosure of Invention

The present invention aims at overcoming the demerits of available technology and providing one kind of integrated plasmid for aTc inducing expression of tubercle mycobacillus gene. The problem of obtaining proteins which are difficult to express such as membrane proteins in mycobacteria is solved by applying an inducible expression method of a Tet-on system; the Tet-off system is used to solve the problem that the essential gene can not be knocked out, the expression quantity of the essential gene is regulated and controlled to reduce the expression quantity, the gene knocking-down effect is achieved, and the research on the function of the essential gene is solved.

The first purpose of the invention is to provide an integrated plasmid for aTc induced expression of mycobacterium tuberculosis genes.

The second purpose of the invention is to provide the application of any one of the integrative plasmids in establishing a gene expression system of Mycobacterium tuberculosis.

The third purpose of the invention is to provide an expression system for induction expression of Mycobacterium tuberculosis genes by aTc.

In order to achieve the purpose, the invention is realized by the following scheme:

the invention optimizes two sets of Tet-on and Tet-off regulation and control systems, and the principle is that the small molecular compound aTc changes the conformation of the regulation and control protein, so that the expression of the target protein is controlled, the regulation and control of the gene expression quantity of the mycobacterium is suitable, and a new thought is provided for the gene function research, the protein expression and the like of the mycobacterium tuberculosis.

Specifically, the invention takes pMV361 plasmid (mycobacterial integrative plasmid) as a vector framework, inserts a transcription regulatory factor TetR or rTetR after codon optimization at a polyclonal site, and then reversely inserts a mashup promoter hsp60-tetO at the upstream of an attp site₄And a multiple cloning site sequence, thereby obtaining an inducible expression plasmid (hereinafter referred to as a small molecule inducible plasmid) based on the small molecule aTc (anhydrotetracycline): the nucleotide sequence is shown as SEQ ID NO.1 (codon-optimized inserted transcription regulatory factor TetR), and the plasmid map is shown as figure 1; or the nucleotide sequence is shown in SEQ ID NO.2 (codon-optimized inserted transcription regulatory factor rTetR), and the plasmid map is shown in figure 2. The working scheme of the mycobacterial expression system is shown in FIG. 3.

The small molecule inducing plasmid is used as a carrier, and a Mycobacterium tuberculosis H37Rv gene Rv3875 or Mycobacterium smegmatis MC is inserted into a reverse multiple cloning site²155 Gene MSMEG0129, and the constructed gene expression plasmids were introduced into the corresponding genesThe expression level of the Rv3875 gene of the host is improved by 51.68 times and the expression level of the MSMEG0129 gene is reduced by 6.53 times through qRT-PCR detection. The induction expression system lays a foundation for protein function analysis of mycobacteria and novel vaccine development.

Specifically, the mashup promoter hsp60-tetO₄And the design principle of the sequence of the multiple cloning sites, including the prediction of the functional region of the promoter hsp60, the linkage of the hybrid tag of 4 × tetO and the downstream multiple sites.

The mashup promoter hsp60-tetO₄And the nucleotide sequence of the multiple cloning site sequence is shown as SEQ ID NO. 3.

The mashup promoter hsp60-tetO₄And de novo synthesis to pUC57 vector after the sequence design of the multiple cloning site.

The mashup promoter hsp60-tetO₄And the design principle of the reverse multiple cloning site sequence is that the core functional region of the promoter hsp60 is detected and annotated by promoter prediction software, 4 tetO sequences are continuously inserted into the transcription initiation site, and random sequences are inserted among the tetO sequences to ensure that the number of the base separation number of 2 tetO sequences is multiple of 10. Mashup promoter hsp60-tetO₄The downstream 18bp inserts NcoI, KpnI and Acc65I enzyme cutting sites to form a multiple cloning site.

The insertion design of the transcription regulatory factor TetR and rTetR sequences: the transcription regulation factor TetR and rTetR nucleic acid sequences are respectively subjected to codon optimization and then synthesized to a pUC57 vector from the beginning, a target fragment is amplified through polymerase chain reaction and is seamlessly cloned to a pMV361 plasmid vector which is subjected to EcoRI and HindIII double digestion and purification after purification, and the sequences of the TetR and rTetR nucleic acid sequences which are subjected to codon optimization are respectively connected between EcoRI sites and HindIII sites of the pMV361 plasmid vector to respectively obtain pMV361-Phsp60-tetR plasmids and pMV361-Phsp60-rtetR plasmids. Wherein, the upstream primer sequence amplified by the transcription regulating factor TetR sequence polymerase chain reaction after codon optimization is SEQ ID NO.4, and the downstream primer sequence is SEQ ID NO. 5; the upstream primer sequence amplified by the codon-optimized transcription regulatory factor rTetR sequence polymerase chain reaction is SEQ ID NO.4, and the downstream primer sequence is SEQ ID NO. 6.

The above-described mashup promoter hsp60-tetO₄And the vector connection design of the reverse multiple cloning site sequence, amplifying a target fragment by a polymerase chain reaction, purifying, and then respectively cloning seamlessly to the pMV361-Phsp60-tetR/rtetR plasmid vector which is subjected to double enzyme digestion and purification by KpnI and NotI to obtain pMV361-Phsp60-tetO₄-MCS-Phsp60-tetR/rtetR plasmid. Wherein, the mashup promoter hsp60-tetO₄And the upstream primer sequence amplified by the reverse multiple cloning site sequence polymerase chain reaction is SEQ ID NO.7, and the downstream primer sequence is SEQ ID NO. 8.

The insertion connection design of the Rv3875 gene and the MSMEG0129 gene is that target fragments are amplified by polymerase chain reaction respectively and are cloned seamlessly to pMV361-Phsp60-tetO which is subjected to KpnI and NcoI double enzyme digestion and purification respectively after purification₄-MCS-Phsp60-tetR plasmid and pMV361-Phsp60-tetO₄-MCS-Phsp60-rtetR plasmid to obtain pMV361-Phsp60-tetO₄the-Rv 3875-Phsp60-tetR plasmid and pMV361-Phsp60-tetO₄MSMEG0129-Phsp60-rtetR plasmid.

The small molecule aTc is added to induce and express each gene design, and after the small molecule aTc with concentration gradient is added to the corresponding host of the inducible expression system, the qRT-PCR is used for detecting the relation between the expression quantity change of the corresponding gene and the concentration gradient of the small molecule aTc.

Therefore, the invention is to protect a plasmid for expressing mycobacterium tuberculosis genes by induction of aTc, a nucleotide sequence of a transcription regulatory factor TetR or rTetR which is optimized or not optimized by a codon is inserted at a multi-cloning site by taking a mycobacterium integrated plasmid as a carrier framework, a sequence containing a mashup promoter hsp60-tetO4 and a multi-cloning site is reversely inserted at the upstream of an attp site, 2-4 tetO sequences are continuously inserted at the transcription starting site of the mashup promoter hsp60 by the mashup promoter hsp60-tetO4, random sequences are inserted among the tetO sequences, and the number of the interval bases of each 2 tetO sequences is integral multiple of 10.

Specifically, the plasmid contains a TetR protein which is a Tet-on system, and a reporter gene is expressed after an inducer is added;

the plasmid contains TetR protein and is a Tet-off system, and the expression of a reporter gene is inhibited after an inducer is added.

Preferably, the mashup promoter hsp60-tetO4 is a sequence of 4 tetO sequences inserted consecutively at the transcription start site of the promoter hsp 60. 4 tetO sequences are inserted continuously, and the binding strength of the tetR protein and the tetO sequences is better than that of 2 or 3 tetO sequences inserted continuously; however, the inhibition effect is too strong due to the excessive insertion of the tetO sequence, and the inhibition effect cannot be completely released even when an inducer is added.

Preferably, the sequence comprising the mashup promoter hsp60-tetO4 and the multiple cloning site is located 18bp downstream of the mashup promoter hsp60-tetO 4.

More preferably, the sequence of the multiple cloning site consists of three enzyme cutting sites of NcoI, KpnI and Acc 65I.

Preferably, a nucleotide sequence of a codon optimized transcription regulatory factor TetR or rTetR is inserted at a multi-cloning site by taking a mycobacterium integration type plasmid as a carrier framework.

More preferably, the nucleotide sequence of the transcription regulatory factor TetR after codon optimization is shown as SEQ ID NO. 13.

More preferably, the nucleotide sequence of the transcription regulatory factor rTetR after codon optimization is shown as SEQ ID NO. 14.

Preferably, the mycobacterial integrative plasmid is the pMV361 plasmid.

More preferably, the nucleotide sequence of the sequence containing the mashup promoter hsp60-tetO4 and the multiple cloning site is shown in SEQ ID NO. 3.

Most preferably, the nucleotide sequence of the plasmid is shown as SEQ ID No.1 or 2.

The application of any one of the integrated plasmids in establishing a gene expression system of Mycobacterium tuberculosis also belongs to the protection scope of the invention.

Preferably, a target gene is inserted into the multiple cloning site sequence of any one of the integrative plasmids, Mycobacterium tuberculosis is transferred to obtain a recombinant strain, and the aTc induces the expression of the target gene.

The invention also claims an expression system for expressing the gene of mycobacterium tuberculosis by the induction of the aTc, and the expression of the gene of the mycobacterium tuberculosis by utilizing the integrated plasmid and the induction of the target gene by the aTc.

Compared with the prior art, the invention has the following beneficial effects:

the integrated small molecule aTc induction-based mycobacterium expression system provided by the invention has the advantages of wide regulation and control dynamic range and wide application range. The regulated gene is detected by qRT-PCR: the expression level of the Rv3875 gene is improved by 51.68 times, and the expression level of the MSMEG0129 gene is reduced by 6.53 times. The expression system provides a stable platform for proteins which are difficult to express by mycobacteria, such as membrane proteins and the like, and provides a method for knocking down essential genes which cannot be knocked out by the mycobacteria, thereby providing a thought for gene function research.

Drawings

FIG. 1 shows pMV361-Phsp60-tetO₄-MCS-Phsp60-tetR plasmid map.

FIG. 2 shows pMV361-Phsp60-tetO₄-MCS-Phsp60-rtetR plasmid map.

FIG. 3 is a schematic diagram of the operation of the integrated aTc-induced expression system for Mycobacteria.

FIG. 4 shows Mycobacterium smegmatis MC²155 in mashup promoter hsp60-tetO₄And (5) qualitatively characterizing the expression intensity.

Detailed Description

The present invention will be described in further detail with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the present invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.

EXAMPLE 1 construction of Small molecule aTc inducible expression plasmids

First, experiment method

1. Mashup promoter hsp60-tetO4 and reverse multiple cloning site sequence design, synthesis and amplification

Detecting and annotating the promoter hsp60 sequence by promoter prediction software, markingThe core functional region of the sequence and the transcription initiation site. 4 tetO sequences are designed and inserted in sequence at the +1 position of the transcription starting site, random sequences are designed and inserted between the connected tetO sequences, but the number of the interval bases between the 2 tetO sequences is required to be multiple of 10. In the mashup promoter hsp60-tetO₄The downstream 18bp is inserted with three enzyme cutting sites NcoI \ KpnI \ Acc65I to form a multiple cloning site. The designed sequence was submitted to a Gene Synthesis company, DNA was synthesized de novo, and the DNA was cloned into a pUC57 vector to obtain pUC57-Phsp60-tetO₄-an MCS plasmid. Using this plasmid as a template, Phsp60-tetO was amplified by PCR using primers having the nucleotide sequences shown in SEQ ID Nos. 7 and 8₄MCS sequence (nucleotide sequence shown in SEQ ID NO. 3), separating the target DNA fragment from the PCR product by nucleic acid gel electrophoresis, and recovering the target DNA fragment for later use by using Omega gel electrophoresis kit.

2. Construction of pMV361-Phsp60-tetO4-eGFP plasmid

The Escherichia coli DH5 alpha strain containing pMV361 plasmid was cultured in LB, the plasmid was extracted using Omega plasmid extraction kit, the pMV361 plasmid obtained was digested with NotI and KpnI restriction enzymes, nucleic acid gel electrophoresis was performed, and the linearized plasmid was recovered using Omega gel recovery kit.

Recovered Phsp60-tetO₄The MCS fragment (nucleotide sequence shown in SEQ ID NO. 3) and linearized plasmid pMV361 were seamlessly cloned at 50 ℃ for 1h, the ligation product was transformed into E.coli DH 5. alpha. competent cells and spread on LB solid plate (containing 100. mu.g/ml kanamycin sulfate), and the plate was incubated overnight in a 37 ℃ incubator. And (3) selecting a monoclonal colony growing on the plate, inoculating the colony to an LB liquid culture medium, shaking the colony at a shaker of 200rpm at 37 ℃ overnight for culture, and verifying that the sequence of the bacterial liquid has no mutation through sequencing of the department company. Then, the plasmid was extracted using the Omega plasmid extraction kit to obtain the pMV361-Phsp60-tetO4-MCS plasmid.

This plasmid was digested with restriction enzymes NcoI and KpnI, subjected to nucleic acid gel electrophoresis, and the linearized plasmid pMV361-Phsp60-tetO was recovered using the Omega gel recovery kit₄-MCS backup.

eGFP gene was produced by Gene Synthesis Inc. by codon optimization and de novo DNA synthesisCloned into pUC57 vector. This plasmid was digested with restriction enzymes NcoI and KpnI, subjected to nucleic acid gel electrophoresis, and the recovered eGFP fragment and linearized plasmid pMV361-Phsp60-tetO were recovered using the Omega gel recovery kit₄MCS, reacted with T4 ligase overnight at 4 ℃, the ligation product transformed E.coli DH 5. alpha. competent cells and plated on LB solid plate (containing 100. mu.g/ml kanamycin sulfate), which was incubated overnight in a 37 ℃ incubator.

And (3) selecting a monoclonal colony growing on the plate, inoculating the colony to an LB liquid culture medium, shaking the colony at a shaker of 200rpm at 37 ℃ overnight for culture, and verifying that the sequence of the bacterial liquid has no mutation through sequencing of the department company. Then, the plasmid was extracted using the Omega plasmid extraction kit to obtain pMV361-Phsp60-tetO₄-eGFP plasmid.

3. promoter Strength detection of pMV361-Phsp60-tetO4-eGFP plasmid

Taking a Mycobacterium smegmatis MC²155 competent cells were incubated with 5. mu. LpMV361-Phsp60-tetO₄-eGFP plasmid mixing, ice-bath for 10min, electroporation using 2mm electroporation cuvette Bio-rad electrotransfer, electroporation parameters: voltage 2.5kV, resistance 1000 Ω, capacitance 25 μ F. After electric shock, 7H9 medium was added and incubated overnight in an incubator at 37 ℃. Centrifuging at 5000rpm for 10min, removing supernatant after leaving 100 μ L, gently blowing the thallus, mixing well, and coating with 7H10 resistant plate (containing 100 μ g/ml kanamycin sulfate). After the plate was incubated in a 37 ℃ incubator for 2 to 3 days, the monoclonal antibody was picked up in 7H9 medium (containing 100. mu.g/ml kanamycin sulfate), and shake-incubated at 37 ℃ for 2 to 3 days with 200rpm shaking. After the sequencing of the Optimalaceae company verifies that the sequence has no mutation, the bacteria liquid is centrifuged at 5000rpm for 10min, the supernatant is removed, the bacteria are placed in a blue light plate for detection and photographing, as shown in figure 4, and the expression intensity effect of the mixed promoter is superior to that of the hsp60 promoter. Since an excellent induction system generally requires a promoter with high expression strength, a mashup promoter is more suitable for the system.

4. Construction of pMV361-Phsp60-tetR and pMV361-Phsp60-rtetR plasmids

After the nucleotide sequences of the transcription regulating factors TetR and rTetR are respectively subjected to codon optimization, the nucleotide sequence of the transcription regulating factor TetR after the codon optimization is shown as SEQ ID NO.13, and the nucleotide sequence of the transcription regulating factor rTetR after the codon optimization is shown as SEQ ID NO.14, are synthesized to a pUC57 vector from the beginning, and thus pUC57-tetR and pUC57-rtetR plasmids are obtained. The plasmid is used as a template, primers with nucleotide sequences shown as SEQ ID No. 4-5 are respectively used for carrying out PCR amplification tetR sequence, and primers with nucleotide sequences shown as SEQ ID No.4 and 6 are respectively used for carrying out PCR amplification rtetR sequence. The PCR product was subjected to nucleic acid gel electrophoresis to separate the target DNA fragment, and the target DNA fragment was recovered by using an Omega gel recovery kit.

The Escherichia coli DH5 alpha strain containing pMV361 plasmid was cultured in LB, the plasmid was extracted using Omega plasmid extraction kit, the obtained pMV361 plasmid was digested with EcoRI and HindIII restriction enzymes, nucleic acid gel electrophoresis was performed, and linearized plasmid was recovered using Omega gel recovery kit.

The tetR and rtetR fragments recovered, respectively, and the linearized plasmid pMV361 were reacted for 1h at 50 ℃ by seamless cloning, the ligation product transformed E.coli DH 5. alpha. competent cells and spread on LB solid plates (containing 100. mu.g/ml kanamycin sulfate), which were incubated overnight in a 37 ℃ incubator. And (3) selecting a monoclonal colony growing on the plate, inoculating the colony to an LB liquid culture medium, shaking the colony at a shaker of 200rpm at 37 ℃ overnight for culture, and verifying that the sequence of the bacterial liquid has no mutation through sequencing of the department company. Then, plasmids were extracted using an Omega plasmid extraction kit to obtain pMV361-Phsp60-tetR and pMV361-Phsp60-rtetR plasmids.

5、pMV361-Phsp60-tetO₄MCS-Phsp60-tetR and pMV361-Phsp60-tetO₄Construction of the-MCS-Phsp 60-rtetR plasmid

Respectively culturing Escherichia coli DH5 alpha strain containing pMV361-Phsp60-tetR and pMV361-Phsp60-rtetR plasmids by LB, extracting the plasmids by using an Omega plasmid extraction kit, performing nucleic acid gel electrophoresis after the obtained pMV361-Phsp60-tetR and pMV361-Phsp60-rtetR plasmids are cut by using NotI and KpnI restriction enzymes, and recovering linearized pMV361-Phsp60-tetR and pMV361-Phsp60-rtetR plasmids by using an Omega gel recovery kit.

Thus Phsp60-tetO₄-PCR amplification of Phsp60-tetO using the MCS plasmid as template and primers having the nucleotide sequences shown in SEQ ID No.7 and 8₄-MCAnd an S sequence (the nucleotide sequence is shown as SEQ ID NO. 3).

Recovered Phsp60-tetO₄The MCS fragment and linearized plasmids pMV361-Phsp60-tetR and pMV361-Phsp60-rtetR were reacted for 1h at 50 ℃ by seamless cloning, the ligation products transformed E.coli DH 5. alpha. competent cells and plated on LB solid plates (containing 100. mu.g/ml kanamycin sulfate), which were incubated overnight in a 37 ℃ incubator. And (3) selecting a monoclonal colony growing on the plate, inoculating the colony to an LB liquid culture medium, shaking the colony at a shaker of 200rpm at 37 ℃ overnight for culture, and verifying that the sequence of the bacterial liquid has no mutation through sequencing of the department company. Then, the plasmid was extracted using the Omega plasmid extraction kit to obtain pMV361-Phsp60-tetO₄MCS-Phsp60-tetR and pMV361-Phsp60-tetO₄-MCS-Phsp60-rtetR plasmid.

Wherein pMV361-Phsp60-tetO₄The nucleotide sequence of the-MCS-Phsp 60-tetR plasmid is shown in SEQ ID NO.1, and the plasmid map is shown in figure 1.

To obtain pMV361-Phsp60-tetO₄The nucleotide sequence of the-MCS-Phsp 60-rtetR plasmid is shown in SEQ ID NO.2, and the plasmid map is shown in figure 2.

Example 2 construction of the pMV361-Phsp60-tetO4-Rv3875-Phsp60-tetR plasmid

The genome of Mycobacterium tuberculosis H37Rv is used as a template, primers with the nucleotide sequences shown in SEQ ID NO. 9-10 are used for PCR amplification of an Rv3875 sequence, a PCR product is subjected to nucleic acid gel electrophoresis to separate a target DNA fragment, and a gel back kit of Omega company is used for recovering the target DNA fragment for later use. LB culture containing pMV361-Phsp60-tetO₄Escherichia coli DH 5. alpha. strain of-MCS-Phsp 60-tetR plasmid, plasmid was extracted using Omega plasmid extraction kit, and pMV361-Phsp60-tetO was obtained₄the-MCS-Phsp 60-tetR plasmid was digested with restriction enzymes NcoI and KpnI, subjected to nucleic acid gel electrophoresis and the linearized plasmid was recovered using the Omega gel recovery kit.

Recovered Rv3875 fragment and linearized plasmid pMV361-Phsp60-tetO₄MCS-Phsp60-tetR, seamless cloning at 50 ℃ for 1h, transformation of E.coli DH 5. alpha. competent cells with the ligation product and plating of LB solid plates (containing 100. mu.g/ml kanamycin sulfate) which were incubated overnight in a 37 ℃ incubator. Picking plateAnd inoculating the grown monoclonal colony to an LB liquid culture medium, shaking by a shaking table at the speed of 200rpm at the temperature of 37 ℃ overnight for culture, and verifying that the sequence of the bacterial liquid has no mutation by sequencing of the department company. Then, the plasmid was extracted using the Omega plasmid extraction kit to obtain pMV361-Phsp60-tetO₄-Rv3875-Phsp60-tetR plasmid.

Example 3 pMV361-Phsp60-tetO₄Detection of the Rv3875-Phsp60-tetR inducible expression System

First, experiment method

1、pMV361-Phsp60-tetO₄-Rv3875-Phsp60-tetR plasmid transformation of Mycobacterium tuberculosis H37 Rv. delta. Rv3875

One Mycobacterium tuberculosis H37 Rv. delta. Rv3875 competent cell was taken and 5. mu.L of pMV361-Phsp60-tetO prepared in example 2 was added₄Rv3875-Phsp60-tetR plasmid mixture, ice bath for 10min, transformation by electric shock using a 2mm electric rotor Bio-rad electrotransfer, parameters of electric shock: voltage 2.5kV, resistance 1000 Ω, capacitance 25 μ F. After electric shock, 7H9 medium was added and incubated overnight in an incubator at 37 ℃. Centrifuging at 5000rpm for 10min, removing supernatant after retaining 100. mu.L, gently blowing the thallus, mixing well, and coating with 7H10 resistant plate (containing 10% OADC + 25. mu.g/ml kanamycin sulfate + 75. mu.g/ml hygromycin). After the plate was incubated in a 37 ℃ incubator for 3 to4 weeks, the monoclonal antibody was picked up in a 7H9 medium (containing 10% OADC + 25. mu.g/ml kanamycin sulfate + 75. mu.g/ml hygromycin), and incubated in a 37 ℃ incubator for 2 to 3 weeks.

2. Small molecule aTc induces Rv3875

Culturing until OD of the bacterial liquid is about 0.6, adding small molecular aTc respectively to the bacterial liquid to obtain final concentration gradients of 0,1 and 2 mu g/ml, and enabling 2 tubes of each concentration gradient to be parallel.

3. qRT-PCR detection of Rv3875 gene expression quantity

After reverse transcription, cDNA samples are diluted by 10 times and used as templates for qRT-PCR detection.

Wherein, the housekeeping gene primer:

16S rRNA-F：5'-GGATAAGCCTGGGAAACTGG-3'，

16S rRNA-R：5'-GCCGTATCTCAGTCCCAGTGT-3'；

rv3875 gene primer:

Rv3875-F：5'-TTGCCTTCGGTCGAAGCCAT-3'，

Rv3875-R：5'-AGGGAAATGTCACGTCCATT-3'。

the qRT-PCR reaction system is as follows:

putting the 96-well plate added with the sample into an ABI Stepone plus type fluorescent quantitative PCR instrument for reaction, wherein a qRT-PCR reaction system is as follows:

second, experimental results

The results are shown in Table 1. The qRT-PCR detection shows that the expression level of the Rv3875 gene of the strain added with the 2 mug/mL micromolecular aTc inducer is improved by 51.68 times compared with the strain not added with the aTc inducer.

Table 1:

note: "Rv 3875-0" is an inducer to which 0 μ g/mL of small molecule aTc is added; "Rv 3875-1" is an inducer with the addition of 1 μ g/mL small molecules aTc; "Rv 3875-2" is an inducer with 2 μ g/mL small molecules aTc added; "-" is not determined.

Example 4 construction of pMV361-Phsp60-tetO4-MSMEG0129-Phsp60-rtetR plasmid

With Mycobacterium smegmatis MC²155 genome as a template, using primers with nucleotide sequences shown in SEQ ID NO. 11-12 to perform PCR amplification on the MSMEG0129 sequence, separating a target DNA fragment from a PCR product through nucleic acid gel electrophoresis, and recovering the target DNA fragment for later use by using an Omega colloidal nucleic acid kit. LB culture containing pMV361-Phsp60-tetO₄Escherichia coli DH 5. alpha. strain of-MCS-Phsp 60-rtetR plasmid, plasmid was extracted using Omega plasmid extraction kit to obtain pMV361-Phsp60-tetO₄-MCS-Phsp60-rtetR plasmidAfter restriction with NcoI/KpnI restriction enzymes, nucleic acid gel electrophoresis was performed and the linearized plasmid was recovered using the Omega gel recovery kit.

Recovered MSMEG0129 fragment and linearized plasmid pMV361-Phsp60-tetO₄MCS-Phsp60-rtetR, seamless cloning at 50 ℃ for 1h, transforming E.coli DH 5. alpha. competent cells with the ligation product and plating LB solid plate (containing 100. mu.g/ml kanamycin sulfate), placing the plate in a 37 ℃ incubator for overnight culture. And (3) selecting a monoclonal colony growing on the plate, inoculating the colony to an LB liquid culture medium, shaking the colony at a shaker of 200rpm at 37 ℃ overnight for culture, and verifying that the sequence of the bacterial liquid has no mutation through sequencing of the department company. Then, the plasmid was extracted using the Omega plasmid extraction kit to obtain pMV361-Phsp60-tetO₄MSMEG0129-Phsp60-rtetR plasmid.

Example 5 pMV361-Phsp60-tetO₄Detection of MSMEG0129-Phsp60-rtetR inducible expression System

First, experiment method

1、pMV361-Phsp60-tetO₄-MSMEG0129-Phsp60-rtetR plasmid transformation of Mycobacterium smegmatis MC²155

Taking a Mycobacterium smegmatis MC²155 competent cells and 5. mu.L of pMV361-Phsp60-tetO prepared in example 4₄MSMEG0129-Phsp60-rtetR plasmid, ice bath for 10min, electroporation using 2mm electric rotor Bio-rad electrotransfer, electroporation parameters: voltage 2.5kV, resistance 1000 Ω, capacitance 25 μ F. After electric shock, 7H9 medium was added and incubated overnight in an incubator at 37 ℃. Centrifuging at 5000rpm for 10min, removing supernatant after retaining 100. mu.L, gently blowing the thallus, mixing well, and coating with 7H10 resistant plate (containing 10% OADC + 25. mu.g/ml kanamycin sulfate). After the plate was incubated in a 37 ℃ incubator for 1 to 2 days, the monoclonal antibody was picked up in a 7H9 medium (containing 10% OADC + 25. mu.g/ml kanamycin sulfate), and incubated in a 37 ℃ incubator for 1 to 2 days.

2. Small molecule aTc-induced MSMEG0129

Culturing until OD of bacteria liquid is about 0.6, adding small molecule aTc respectively to obtain final concentration gradient of 0, 10, 20, 50 μ g/ml, each concentration gradient is 3 tubes in parallel.

3. qRT-PCR detection of MSMEG0129 gene expression quantity

Wherein, the housekeeping gene primer:

16S rRNA-F：5'-GGATAAGCCTGGGAAACTGG-3'，

16S rRNA-R：5'-GCCGTATCTCAGTCCCAGTGT-3'；

MSMEG0129 gene primer:

MSMEG0129-F：5'-TGCTGCAGCATCGTGAAGAT-3'，

MSMEG0129-R：5'-ATCACGTCGATCGTCTCGGA-3'。

the qRT-PCR reaction system is as follows:

second, experimental results

The results are shown in Table 2. The qRT-PCR detection shows that the strain added with 10 mug/mL of small-molecule aTc inducer reduces the expression level of MSMEG-0129 gene by 6.53 times compared with the wild-type strain WT.

Table 2:

note: "MSMEG 0129-0" is added with 0 μ g/mL small molecule aTc inducer; "MSMEG 0129-10" is small molecule aTc inducer added with 10 mug/mL; "MSMEG 0129-20" is small molecule aTc inducer added with 2 mug/mL; "MSMEG 0129-50" is small molecule aTc inducer added with 50 μ g/mL; "WT" is wild control; "-" is not determined.

It should be finally noted that the above examples are only intended to illustrate the technical solutions of the present invention, and not to limit the scope of the present invention, and that other variations and modifications based on the above description and thought may be made by those skilled in the art, and that all embodiments need not be exhaustive. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the claims of the present invention.

Sequence listing

<120> an integrated plasmid for aTc induced expression of mycobacterium tuberculosis gene

<160> 14

<170> SIPOSequenceListing 1.0

<210> 1

<211> 5354

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc 60

ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga 120

tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg 180

ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt 240

ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg 300

ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag 360

aaatgcataa tcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac 420

ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg 480

gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc 540

cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat 600

tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac 660

actggcagag cattacgctg acttgacggg acggcggctt tgttgaataa atcgaacttt 720

tgctgagttg aaggatcaga tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa 780

gcaaaagttc aaaatcacca actggtccac ctacaacaaa gctctcacca accgtggctc 840

cctcactttc tggctggatg atggggcgat tcaggcctgg tatgagtcag caacaccttc 900

ttcacgaggc agacctcact agttccatga gcgtcagacc ccgtagaaaa gatcaaagga 960

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 1020

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 1080

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 1140

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 1200

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 1260

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 1320

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 1380

gaggggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 1440

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 1500

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 1560

agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 1620

cctgcgttat cccctgattc tgtggataac cgtattaccg cctggtacct ttttgccatg 1680

gcaattgtct tggccattgc gaagtgattc ctccggactc tatcactgat agggagactc 1740

tatcactgat agggagactc tatcactgat agggagactc tatcactgat agggatcggg 1800

gatgaaacgg gggtcaccgg gtgacggcaa ccccgctact tacgctggcc gggcgcagtg 1860

cccgcgacgg acggctcaag ttgtcctcgc tgccactcgc tgcgacgacg ggcctggcct 1920

caccgtcccg acctacgact caccggtcgc gagtgccaac gttattctta gcactcgcct 1980

atgccgagtg caagaagccc cgcaccgggt catgcccctc gttcgacctt gtcctcggcc 2040

ctccgatccg ggtgagtatg ttcggcccat gaccgccaac gacaacaaga cccgtaaatg 2100

gtcggccgca gacgtccccg atcaaagcgg gcgcgtcgtt gtggtcacca actcccggtg 2160

caaccttgtc ccggtctatt ctcttcactg caccagctcc aatctggtgt gaatgcccct 2220

cgtctgttcg cgcaggcggg gggctctatt cgtttgtcag catcgaaagt agccagatca 2280

gggatgcgtt gcaaccgcgt atgcccaggt cagaagagtc gcacaagagt tgcagacccc 2340

tggaaagaaa aatggccaga gggcgaaaac accctctgac cagcggagcg ggcgacggga 2400

atcgaacccg cgtagctagt ttggaagaat gggtgtctgc cgaccacata tgggccggtc 2460

aagataggtt tttaccccct ctcggctgca tcctctaagt ggaaagaaat tgcaggtcgt 2520

agaagcgcgt tgaagcctga gagttgcaca ggagttgcaa cccggtagcc ttgttcacga 2580

cgagaggaga cctagttggc acgtcgcgga tggggatcgc tgaagactca gcgcagcggg 2640

aggatccaag cctcatacgt caacccgcag gacggtgtga ggtactacgc gctgcagacc 2700

tacgacaaca agatggacgc cgaagcctgg ctcgcgggcg agaagcggct catcgagatg 2760

gagacctgga cccctccaca ggaccgggcg aagaaggcag ccgccagcgc catcacgctg 2820

gaggagtaca cccggaagtg gctcgtggag cgcgacctcg cagacggcac cagggatctg 2880

tacagcgggc acgcggagcg ccgcatctac ccggtgctag gtgaagtggc ggtcacagag 2940

atgacgccag ctctggtgcg tgcgtggtgg gccgggatgg gtaggaagca cccgactgcc 3000

cgccggcatg cctacaacgt cctccgggcg gtgatgaaca cagcggtcga ggacaagctg 3060

atcgcagaga acccgtgccg gatcgagcag aaggcagccg atgagcgcga cgtagaggcg 3120

ctgacgcctg aggagctgga catcgtcgcc gctgagatct tcgagcacta ccggatcgcg 3180

gcatacatcc tggcgtggac gagcctccgg ttcggagagc tgatcgagct tcgccgcaag 3240

gacatcgtgg acgacggcat gacgatgaag ctccgggtgc gccgtggcgc ttcccgcgtg 3300

gggaacaaga tcgtcgttgg caacgccaag accgtccggt cgaagcgtcc tgtgacggtt 3360

ccgcctcacg tcgcggagat gatccgagcg cacatgaagg accgtacgaa gatgaacaag 3420

ggccccgagg cattcctggt gaccacgacg cagggcaacc ggctgtcgaa gtccgcgttc 3480

accaagtcgc tgaagcgtgg ctacgccaag atcggtcggc cggaactccg catccacgac 3540

ctccgcgctg tcggcgctac gttcgccgct caggcaggtg cgacgaccaa ggagctgatg 3600

gcccgtctcg gtcacacgac tcctaggatg gcgatgaagt accagatggc gtctgaggcc 3660

cgcgacgagg ctatcgctga ggcgatgtcc aagctggcca agacctcctg aaacgcaaaa 3720

agcccccctc ccaaggacac tgagtcctaa agaggggggt ttcttgtcag tacgcgaaga 3780

accacgcctg gccgcgagcg ccagcaccgc cgctctgtgc ggagacctgg gcaccagccc 3840

cgccgccgcc aggagcattg ccgttcccgc cagaaatcta gacggtgacc acaacgcgcc 3900

cgctttgatc ggggacgtct gcggccgacc atttacgggt cttgttgtcg ttggcggtca 3960

tgggccgaac atactcaccc ggatcggagg gccgaggaca aggtcgaacg aggggcatga 4020

cccggtgcgg ggcttcttgc actcggcata ggcgagtgct aagaataacg ttggcactcg 4080

cgaccggtga gtgctaggtc gggacggtga ggccaggccc gtcgtcgcag cgagtggcag 4140

cgaggacaac ttgagccgtc cgtcgcgggc actgcgcccg gccagcgtaa gtagcggggt 4200

tgccgtcacc cggtgacccc cgtttcatcc ccgatccgga ggaatcactt cgcaatggcc 4260

aagacaattg cggatccagc tgcagaattc atgtcccgcc tggacaagtc caaggtcatc 4320

aactccgccc tggagctgct gaacgaggtc ggcatcgagg gcctgaccac ccgcaagctg 4380

gcccagaagc tgggcgtcga gcagccgacc ctgtactggc acgtcaagaa caagcgggcc 4440

ctgctcgacg ccctggccat cgagatgttg gaccgccacc acacccactt ctgcccgctg 4500

gagggcgaga gctggcagga cttcctgcgc aacaacgcca agagcttccg ctgcgccttg 4560

ctgagccacc gcgacggcgc caaggtgcac ctgggcaccc ggccgaccga gaagcagtac 4620

gagaccctgg agaaccagtt ggccttcctg tgccagcagg gcttctcgct ggagaacgcg 4680

ttgtacgccc tgtcggccgt gggccacttc accctgggct gcgtgttgga ggaccaggag 4740

caccaggtcg ccaaggagga gcgcgagacc ccgaccaccg acagcatgcc gccgttgctg 4800

cgccaggcca tcgagctgtt cgaccaccag ggcgccgagc cggccttctt gttcggcctg 4860

gagctgatca tctgcggctt ggagaagcag ctgaagtgcg agagcggctc ctaaaagctt 4920

atcgatgtcg acgtagttaa ctagcgtacg atcgactgcc aggcatcaaa taaaacgaaa 4980

ggctcagtcg aaagactggg cctttcgttt tatgccatca tggccgcggt gatcagctag 5040

ccacctgacg tcgggggggg gggaaagcca cgttgtgtct caaaatctct gatgttacat 5100

tgcacaagat aaaaatatat catcatgaac aataaaactg tctgcttaca taaacagtaa 5160

tacaaggggt gttatgagcc atattcaacg ggaaacgtct tgctcgaggc cgcgattaaa 5220

ttccaacatg gatgctgatt tatatgggta taaatgggct cgcgataatg tcgggcaatc 5280

aggtgcgaca atctatcgct tgtatgggaa gccccatgcg ccagagttgt ttctgaaaca 5340

tggcaaaggt agcg 5354

<210> 2

<211> 5357

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ttgccaatga tgttacagat gagatggtca gactaaactg gctgacggaa tttatgcctc 60

ttccgaccat caagcatttt atccgtactc ctgatgatgc atggttactc accactgcga 120

tccccgggaa aacagcattc caggtattag aagaatatcc tgattcaggt gaaaatattg 180

ttgatgcgct ggcagtgttc ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt 240

ttaacagcga tcgcgtattt cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg 300

ttgatgcgag tgattttgat gacgagcgta atggctggcc tgttgaacaa gtctggaaag 360

aaatgcataa tcttttgcca ttctcaccgg attcagtcgt cactcatggt gatttctcac 420

ttgataacct tatttttgac gaggggaaat taataggttg tattgatgtt ggacgagtcg 480

gaatcgcaga ccgataccag gatcttgcca tcctatggaa ctgcctcggt gagttttctc 540

cttcattaca gaaacggctt tttcaaaaat atggtattga taatcctgat atgaataaat 600

tgcagtttca tttgatgctc gatgagtttt tctaatcaga attggttaat tggttgtaac 660

actggcagag cattacgctg acttgacggg acggcggctt tgttgaataa atcgaacttt 720

tgctgagttg aaggatcaga tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa 780

gcaaaagttc aaaatcacca actggtccac ctacaacaaa gctctcacca accgtggctc 840

cctcactttc tggctggatg atggggcgat tcaggcctgg tatgagtcag caacaccttc 900

ttcacgaggc agacctcact agttccatga gcgtcagacc ccgtagaaaa gatcaaagga 960

tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg 1020

ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact 1080

ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac 1140

cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg 1200

gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg 1260

gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga 1320

acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc 1380

gaggggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg 1440

agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc 1500

tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc 1560

agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt 1620

cctgcgttat cccctgattc tgtggataac cgtattaccg cctggtacct ttttgccatg 1680

gcaattgtct tggccattgc gaagtgattc ctccggactc tatcactgat agggagactc 1740

tatcactgat agggagactc tatcactgat agggagactc tatcactgat agggatcggg 1800

gatgaaacgg gggtcaccgg gtgacggcaa ccccgctact tacgctggcc gggcgcagtg 1860

cccgcgacgg acggctcaag ttgtcctcgc tgccactcgc tgcgacgacg ggcctggcct 1920

caccgtcccg acctacgact caccggtcgc gagtgccaac gttattctta gcactcgcct 1980

atgccgagtg caagaagccc cgcaccgggt catgcccctc gttcgacctt gtcctcggcc 2040

ctccgatccg ggtgagtatg ttcggcccat gaccgccaac gacaacaaga cccgtaaatg 2100

gtcggccgca gacgtccccg atcaaagcgg gcgcgtcgtt gtggtcacca actcccggtg 2160

caaccttgtc ccggtctatt ctcttcactg caccagctcc aatctggtgt gaatgcccct 2220

cgtctgttcg cgcaggcggg gggctctatt cgtttgtcag catcgaaagt agccagatca 2280

gggatgcgtt gcaaccgcgt atgcccaggt cagaagagtc gcacaagagt tgcagacccc 2340

tggaaagaaa aatggccaga gggcgaaaac accctctgac cagcggagcg ggcgacggga 2400

atcgaacccg cgtagctagt ttggaagaat gggtgtctgc cgaccacata tgggccggtc 2460

aagataggtt tttaccccct ctcggctgca tcctctaagt ggaaagaaat tgcaggtcgt 2520

agaagcgcgt tgaagcctga gagttgcaca ggagttgcaa cccggtagcc ttgttcacga 2580

cgagaggaga cctagttggc acgtcgcgga tggggatcgc tgaagactca gcgcagcggg 2640

aggatccaag cctcatacgt caacccgcag gacggtgtga ggtactacgc gctgcagacc 2700

tacgacaaca agatggacgc cgaagcctgg ctcgcgggcg agaagcggct catcgagatg 2760

gagacctgga cccctccaca ggaccgggcg aagaaggcag ccgccagcgc catcacgctg 2820

gaggagtaca cccggaagtg gctcgtggag cgcgacctcg cagacggcac cagggatctg 2880

tacagcgggc acgcggagcg ccgcatctac ccggtgctag gtgaagtggc ggtcacagag 2940

atgacgccag ctctggtgcg tgcgtggtgg gccgggatgg gtaggaagca cccgactgcc 3000

cgccggcatg cctacaacgt cctccgggcg gtgatgaaca cagcggtcga ggacaagctg 3060

atcgcagaga acccgtgccg gatcgagcag aaggcagccg atgagcgcga cgtagaggcg 3120

ctgacgcctg aggagctgga catcgtcgcc gctgagatct tcgagcacta ccggatcgcg 3180

gcatacatcc tggcgtggac gagcctccgg ttcggagagc tgatcgagct tcgccgcaag 3240

gacatcgtgg acgacggcat gacgatgaag ctccgggtgc gccgtggcgc ttcccgcgtg 3300

gggaacaaga tcgtcgttgg caacgccaag accgtccggt cgaagcgtcc tgtgacggtt 3360

ccgcctcacg tcgcggagat gatccgagcg cacatgaagg accgtacgaa gatgaacaag 3420

ggccccgagg cattcctggt gaccacgacg cagggcaacc ggctgtcgaa gtccgcgttc 3480

accaagtcgc tgaagcgtgg ctacgccaag atcggtcggc cggaactccg catccacgac 3540

ctccgcgctg tcggcgctac gttcgccgct caggcaggtg cgacgaccaa ggagctgatg 3600

gcccgtctcg gtcacacgac tcctaggatg gcgatgaagt accagatggc gtctgaggcc 3660

cgcgacgagg ctatcgctga ggcgatgtcc aagctggcca agacctcctg aaacgcaaaa 3720

agcccccctc ccaaggacac tgagtcctaa agaggggggt ttcttgtcag tacgcgaaga 3780

accacgcctg gccgcgagcg ccagcaccgc cgctctgtgc ggagacctgg gcaccagccc 3840

cgccgccgcc aggagcattg ccgttcccgc cagaaatcta gacggtgacc acaacgcgcc 3900

cgctttgatc ggggacgtct gcggccgacc atttacgggt cttgttgtcg ttggcggtca 3960

tgggccgaac atactcaccc ggatcggagg gccgaggaca aggtcgaacg aggggcatga 4020

cccggtgcgg ggcttcttgc actcggcata ggcgagtgct aagaataacg ttggcactcg 4080

cgaccggtga gtgctaggtc gggacggtga ggccaggccc gtcgtcgcag cgagtggcag 4140

cgaggacaac ttgagccgtc cgtcgcgggc actgcgcccg gccagcgtaa gtagcggggt 4200

tgccgtcacc cggtgacccc cgtttcatcc ccgatccgga ggaatcactt cgcaatggcc 4260

aagacaattg cggatccagc tgcagaattc atgtcccgcc tggacaagtc caaggtcatc 4320

aactccgccc tggccctggg caacgaggtc ggcatcgagg gcgtcaccac ccgcaagctg 4380

gcccagaagc tgggcgtcga gcagccgacc ctgtactggc acgtcaagaa caagcgggcc 4440

ctgctggacg ccctggcggt ggagatcctg gcgcgccacc acgactactc gctgccggcg 4500

gccggcgagt cgtggcagtc gttcctgcgc aacaacgcca tgtcgttccg ccgcgcgctg 4560

ctgcgctacc gcgacggcgc caaggtgcac ctgggcaccc gcccggacga gaagcagtac 4620

gacaccgtgg agacccagct gcgcttcatg accgagaacg gcttctcgct gcgcgacggc 4680

ttgtacgcga tctcggcggt cagccacttc accttgggcg ccgtgctgga gcagcaggag 4740

cacaccgccg ccctgaccga ccgcccggcc gcgccggacg agaacctgcc gccgctgttg 4800

cgggaggcgc tgcagatcat ggactcggac gacggcgagc aggccttcct gcacggcctg 4860

gagagcctga tccggggctt cgaggtgcag ctgaccgccc tgttgcagat cgtgtaaaag 4920

cttatcgatg tcgacgtagt taactagcgt acgatcgact gccaggcatc aaataaaacg 4980

aaaggctcag tcgaaagact gggcctttcg ttttatgcca tcatggccgc ggtgatcagc 5040

tagccacctg acgtcggggg ggggggaaag ccacgttgtg tctcaaaatc tctgatgtta 5100

cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt acataaacag 5160

taatacaagg ggtgttatga gccatattca acgggaaacg tcttgctcga ggccgcgatt 5220

aaattccaac atggatgctg atttatatgg gtataaatgg gctcgcgata atgtcgggca 5280

atcaggtgcg acaatctatc gcttgtatgg gaagccccat gcgccagagt tgtttctgaa 5340

acatggcaaa ggtagcg 5357

<210> 3

<211> 485

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gtgaccacaa cgacgcgccc gctttgatcg gggacgtctg cggccgacca tttacgggtc 60

ttgttgtcgt tggcggtcat gggccgaaca tactcacccg gatcggaggg ccgaggacaa 120

ggtcgaacga ggggcatgac ccggtgcggg gcttcttgca ctcggcatag gcgagtgcta 180

agaataacgt tggcactcgc gaccggtgag tcgtaggtcg ggacggtgag gccaggcccg 240

tcgtcgcagc gagtggcagc gaggacaact tgagccgtcc gtcgcgggca ctgcgcccgg 300

ccagcgtaag tagcggggtt gccgtcaccc ggtgaccccc gtttcatccc cgatccctat 360

cagtgataga gtctccctat cagtgataga gtctccctat cagtgataga gtctccctat 420

cagtgataga gtccggagga atcacttcgc aatggccaag acaattgcca tggcaaaaag 480

gtacc 485

<210> 4

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

tccagctgca gaattcatgt cccgcctgga caagtccaa 39

<210> 5

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cgacatcgat aagcttttac acgatctgca acagggcgg 39

<210> 6

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cgacatcgat aagcttttag gagccgctct cgcacttca 39

<210> 7

<211> 57

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

taaccgtatt accgcctggt acctttttgc catggcaatt gtcttggcca ttgcgaa 57

<210> 8

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

gttgcaccgg gagttggtga ccacaacgac gcgcccg 37

<210> 9

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

aaccgtatta ccgcctccta cgtcgactta gtgatggtg 39

<210> 10

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gccaagacaa ttgccatgac agagcagcag tggaatttc 39

<210> 11

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tattaccgcc tggtactcag ctctgggtga gctgctcga 39

<210> 12

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

ggccaagaca attgccatga gcaagactgt cgaggtcgc 39

<210> 13

<211> 624

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

atgtcccgcc tggacaagtc caaggtcatc aactccgccc tggagctgct gaacgaggtc 60

ggcatcgagg gcctgaccac ccgcaagctg gcccagaagc tgggcgtcga gcagccgacc 120

ctgtactggc acgtcaagaa caagcgggcc ctgctcgacg ccctggccat cgagatgttg 180

gaccgccacc acacccactt ctgcccgctg gagggcgaga gctggcagga cttcctgcgc 240

aacaacgcca agagcttccg ctgcgccttg ctgagccacc gcgacggcgc caaggtgcac 300

ctgggcaccc ggccgaccga gaagcagtac gagaccctgg agaaccagtt ggccttcctg 360

tgccagcagg gcttctcgct ggagaacgcg ttgtacgccc tgtcggccgt gggccacttc 420

accctgggct gcgtgttgga ggaccaggag caccaggtcg ccaaggagga gcgcgagacc 480

ccgaccaccg acagcatgcc gccgttgctg cgccaggcca tcgagctgtt cgaccaccag 540

ggcgccgagc cggccttctt gttcggcctg gagctgatca tctgcggctt ggagaagcag 600

ctgaagtgcg agagcggctc ctaa 624

<210> 14

<211> 627

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgtcccgcc tggacaagtc caaggtcatc aactccgccc tggccctggg caacgaggtc 60

ggcatcgagg gcgtcaccac ccgcaagctg gcccagaagc tgggcgtcga gcagccgacc 120

ctgtactggc acgtcaagaa caagcgggcc ctgctggacg ccctggcggt ggagatcctg 180

gcgcgccacc acgactactc gctgccggcg gccggcgagt cgtggcagtc gttcctgcgc 240

aacaacgcca tgtcgttccg ccgcgcgctg ctgcgctacc gcgacggcgc caaggtgcac 300

ctgggcaccc gcccggacga gaagcagtac gacaccgtgg agacccagct gcgcttcatg 360

accgagaacg gcttctcgct gcgcgacggc ttgtacgcga tctcggcggt cagccacttc 420

accttgggcg ccgtgctgga gcagcaggag cacaccgccg ccctgaccga ccgcccggcc 480

gcgccggacg agaacctgcc gccgctgttg cgggaggcgc tgcagatcat ggactcggac 540

gacggcgagc aggccttcct gcacggcctg gagagcctga tccggggctt cgaggtgcag 600

ctgaccgccc tgttgcagat cgtgtaa 627

Claims

1. An integrated plasmid for inducible expression of mycobacterium tuberculosis genes by using aTc is characterized in that codon optimized or non-optimized transcription regulatory factor TetR or rTetR nucleotide sequences are inserted at a plurality of cloning sites by using the mycobacterium integrated plasmid as a carrier framework, sequences containing mashup promoter hsp60-tetO4 and a plurality of cloning sites are reversely inserted at the upstream of attp sites, 2-4 tetO sequences are continuously inserted at the transcription starting sites of promoter hsp60 by using the mashup promoter hsp60-tetO4, random sequences are inserted among the tetO sequences, and the number of spaced bases of each 2 tetO sequences is integral multiple of 10.

2. The integrative plasmid of claim 1, wherein the sequence comprising the mashup promoter hsp60-tetO4 and the multiple cloning site is located 18bp downstream of the mashup promoter hsp60-tetO 4.

3. The integrative plasmid of claim 1, wherein a codon optimized transcriptional regulator TetR or rTetR nucleotide sequence is inserted at a multiple cloning site by using the Mycobacterium integrative plasmid as a vector backbone.

4. The integrative plasmid of claim 1, wherein the nucleotide sequence of the transcription regulatory factor TetR after codon optimization is shown as SEQ ID NO. 13; the nucleotide sequence of the transcription regulatory factor rTetR after codon optimization is shown in SEQ ID NO. 14.

5. The integrative plasmid of claim 1, wherein the Mycobacterium integrative plasmid is the pMV361 plasmid.

6. The integrative plasmid of claim 2, wherein the nucleotide sequence of the sequence comprising the mashup promoter hsp60-tetO4 and the multiple cloning site is shown in SEQ ID No. 3.

7. The integrative plasmid of claim 1, wherein the nucleotide sequence of the plasmid is shown as SEQ ID No.1 or 2.

8. Use of the integrative plasmid of any one of claims 1 to 7 for the establishment of a gene expression system for Mycobacterium tuberculosis.

9. The use according to claim 8, wherein the desired gene is inserted into the multiple cloning site sequence of the integrative plasmid of any one of claims 1 to 7, and transformed into Mycobacterium tuberculosis to obtain a recombinant strain, and the aTc induces the expression of the desired gene.

10. An expression system for aTc-induced expression of Mycobacterium tuberculosis genes, characterized in that any one of the integrative plasmids of claims 1 to 7 is linked with a target gene, and the aTc is utilized to induce the expression of the target gene in Mycobacterium tuberculosis.