阅读说明：本技术 结核杆菌蛋白质内含子剪接抑制剂筛选系统、构建方法及其应用 (Screening system, construction method and application of mycobacterium tuberculosis protein intron splicing inhibitor ) 是由 齐兴梅 熊思东 李弈阳 章佳兰 于 2021-07-01 设计创作，主要内容包括：本发明涉及一种结核杆菌蛋白质内含子剪接抑制剂筛选系统,以耻垢分枝杆菌为模式菌,将受蛋白质内含子调控的蛋白质的基因序列插入卡那抗性蛋白基因的65S处,以具有第一抗生素抗性的载体构建重组质粒,转入耻垢分枝杆菌后于含有第一抗生素和卡那霉素的培养基中培养,得到结核杆菌蛋白质内含子剪接抑制剂筛选系统,其中,第一抗生素为非卡那霉素的任一抗生素。通过抑制intein的剪接活性影响卡那抗性蛋白活性,从而影响重组耻垢分枝杆菌在含有卡那霉素的培养基中的生长,筛选出对intein具有抑制作用的药物,实现简单、高效、快速地筛选抗结核新药。(The invention relates to a tubercle bacillus protein intron splicing inhibitor screening system, which takes mycobacterium smegmatis as a model bacterium, inserts a gene sequence of a protein regulated and controlled by a protein intron into a 65S position of a kana resistance protein gene, constructs a recombinant plasmid by using a vector with first antibiotic resistance, and cultures the recombinant plasmid in a culture medium containing first antibiotic and kanamycin after transferring mycobacterium smegmatis to obtain the tubercle bacillus protein intron splicing inhibitor screening system, wherein the first antibiotic is any antibiotic other than kanamycin. The activity of the kanamycin-resistant protein is influenced by inhibiting the splicing activity of intein, so that the growth of recombinant mycobacterium smegmatis in a culture medium containing kanamycin is influenced, a medicament with an inhibiting effect on intein is screened, and a new antituberculosis medicament is simply, efficiently and quickly screened.)

1. A tubercle bacillus protein intron splicing inhibitor screening system is characterized in that: inserting a gene sequence of a protein regulated by a protein intron into a 65S position of a kana resistance protein gene by using mycobacterium smegmatis as a model bacterium, constructing a recombinant plasmid by using a vector with first antibiotic resistance, transferring the recombinant plasmid into mycobacterium smegmatis, and culturing the mycobacterium smegmatis in a culture medium containing first antibiotic and kanamycin to obtain a mycobacterium tuberculosis protein intron splicing inhibitor screening system;

the protein regulated by the protein intron is selected from full-length or micro-fragment of tubercle bacillus RecA intein, DnaB intein or SufB intein; the first antibiotic is any antibiotic other than kanamycin.

2. The screening system of claim 1, wherein: the carrier is PMV261 carrier.

3. A method of constructing a screening system according to claim 1, comprising the steps of:

(1) inserting a gene sequence of a protein regulated by a protein intron into a 65S position of a kana resistance protein gene to construct a fusion gene;

(2) constructing the fusion gene in a vector with first antibiotic resistance to obtain a recombinant plasmid;

(3) transferring the recombinant plasmid into mycobacterium smegmatis to obtain recombinant mycobacterium smegmatis;

(4) and inoculating the recombinant mycobacterium smegmatis into a culture medium containing a first antibiotic and kanamycin to obtain the mycobacterium tuberculosis protein intron splicing inhibitor screening system.

4. The construction method according to claim 3, wherein: in the step (2), the enzyme cutting sites of the fusion gene and the vector are both BamHI and HindIII.

5. The construction method according to claim 3, wherein: in step (4), the concentration of kanamycin in the medium is 30 to 50. mu.g/mL.

6. The construction method according to claim 3, wherein: in step (4), the concentration of the first antibiotic in the medium is 40-60. mu.g/mL.

7. Use of the screening system of claim 1 for screening for inhibitors of protein intron splicing in mycobacterium tuberculosis comprising the steps of: and adding a mycobacterium tuberculosis protein intron splicing inhibitor into the screening system, and carrying out high-throughput screening on the mycobacterium tuberculosis protein intron splicing inhibitor according to the growth condition of the recombinant mycobacterium smegmatis.

8. Use according to claim 7, characterized in that: and screening the mycobacterium tuberculosis protein intron splicing inhibitor according to the OD value of the recombinant mycobacterium smegmatis bacterial liquid.

9. Use according to claim 7, characterized in that: adding the mycobacterium tuberculosis protein intron splicing inhibitor and then culturing for 90-110 h.

10. Use of the screening system of claim 1 for screening anti-tubercular drugs.

Technical Field

The invention relates to the technical field of biology, in particular to a tubercle bacillus protein intron splicing inhibitor screening system, a construction method and application thereof.

Background

In recent years, the problems of co-infection of tuberculosis and AIDS and multi-drug resistant tuberculosis are increasingly serious, so that the clinical treatment of the tuberculosis faces huge challenges, and the development of novel anti-tuberculosis drugs becomes an urgent priority for preventing and treating the tuberculosis. Three important enzymes, namely RecA, DnaB and SufB, in a tubercle bacillus (MTB) body are respectively regulated and controlled by an intein (intein), and can play a role only by completing protein splicing after translation, and the three enzymes play an important role in the growth and the propagation of the MTB. Depending on the host gene inserted, the 3 protein introns contained in MTB are called Mtu RecA, Mtu DnaB and Mtu SufB, respectively. Since the amino acids in the active center of intein splicing are highly conserved, it is possible that the activity of any intein which it is capable of inhibiting may also act on the remaining inteins. Therefore, the protein splicing inhibitor aiming at intein can become a new action target of anti-tuberculosis drugs, and can minimize the occurrence of drug-resistant mutant strains. An intein (intein) is an insertion sequence present in a precursor protein that self-cleaves from the precursor protein during post-translational processing and maturation of the protein, joining the two protein exons to form a mature functional protein by natural peptide bonds, a process known as protein splicing. Protein splicing occurs only in bacteria, archaea and unicellular eukaryotic cells, but not in higher eukaryotes, and therefore inhibitors that specifically target intein are biologically safe for humans. The research for directly applying MTB to screen new antitubercular drugs against intein is greatly limited due to the high infectivity (BSL-3) and the long growth cycle (100 days) of MTB. Screening systems for intein inhibitors have been reported to include in vitro screening systems that rely on fluorescent proteins and in vivo screening systems that rely on bacterial thymidylate synthase, bacterial CcdB toxin, and bacterial DNA helicase subunit a, but in vitro screening systems require purification of the proteins for in vitro screening, which is complicated and time-consuming; and other screening systems all use escherichia coli as a host, the difference of screening results is large, and meanwhile, the reported screening systems all have low splicing activity, so that the screening sensitivity is low. Therefore, it is imperative to establish a simple and reliable screening system for protein intron inhibitors.

Disclosure of Invention

In order to solve the technical problems, the invention provides a mycobacterium tuberculosis protein intron splicing inhibitor screening system, which influences the activity of kanamycin-resistant protein by inhibiting the splicing activity of intein, thereby influencing the growth of mycobacterium smegmatis in a culture medium containing kanamycin. By observing the growth condition of the mycobacterium smegmatis, the medicine with inhibitory action on intein is screened out, and the simple, high-efficiency and rapid screening of a splicing inhibitor or a new anti-tuberculosis medicine is realized.

The invention relates to a tubercle bacillus protein intron splicing inhibitor screening system, which is characterized in that mycobacterium smegmatis is taken as a model bacterium, a gene sequence of a protein regulated by a protein intron is inserted into a 65S position of a kanamycin-resistant protein gene, a recombinant plasmid is constructed by a carrier with first antibiotic resistance, the recombinant plasmid is transferred into mycobacterium smegmatis and then cultured in a culture medium containing first antibiotic and kanamycin to obtain the tubercle bacillus protein intron splicing inhibitor screening system, wherein the protein regulated by the protein intron is selected from full-length or micro-fragment of tubercle bacillus RecA intein, DnaB intein or SufB intein, and the first antibiotic is any antibiotic other than kanamycin.

In the present application, the miniature fragment (mini type) refers to the removal of the homing endonuclease activity of the full-length intein, leaving only its splicing active region.

The mycobacterium smegmatis (Msm) adopted by the invention has high similarity with tubercle bacillus genes, and has the characteristics of high growth speed (3-5 days), low infectivity (BSL-l) and no intein sequence in a thallus genome, so that the mycobacterium smegmatis is taken as a model organism, a protein intron splicing inhibitor screening system depending on kanamycin resistance is externally introduced, and the tubercle bacillus is simulated to establish an in vivo screening system for rapidly screening a splicing inhibitor or an antituberculosis drug. Addition of intein inhibitors to the medium of the screening system resulted in no recovery of the activity of the kanamycin resistance protein due to inhibition of the splicing activity of intein, and thus M.smegmatis could not grow on kanamycin-containing medium.

Aiming at the problem of false positive or selection omission caused by weak sensitivity and specificity of a screening result in the existing screening system using escherichia coli as a model bacterium, the invention has the innovation points that: (1) the splicing activity of protein introns is greatly influenced by host bacteria, and sites that can be spliced in model bacteria such as E.coli are not necessarily suitable for M.smegmatis. The invention inserts the protein intron gene sequence into the kanamycin resistance protein, which can ensure that the activity of the kanamycin resistance protein is destroyed, ensure that intein has certain splicing activity at the site, and can generate protein splicing to rebuild the activity of the kanamycin resistance protein, so that the inventor finds that the 65S site has better effect and the highest shearing activity through different site attempts; (2) in a screening system using escherichia coli as a model bacterium, full-length type and micro type (mini type) of Rec A intein are respectively inserted, and the result shows that the splicing efficiency of the full-length type is about 30 percent, while the mini type has almost no splicing activity. In practical application, the activity of the homing endonuclease of the full-length intein is removed, and only the splicing active region is reserved to construct the mini-type intein, so that the sequence of the intein can be greatly shortened, and the construction and the expression of genes are facilitated; (3) in the screening system, the mini type of the Rec A intein is inserted into the 65S site, the splicing efficiency of the mini type is greatly improved to about 90 percent, the improvement of the splicing efficiency is beneficial to improving the sensitivity of the screening system, and the intein is more suitable for exerting the activity in the mycobacterium smegmatis. Therefore, the screening result of the screening system using the mycobacterium smegmatis as the host is more reliable, so that the new antituberculosis drugs with specific inhibition effect on intein are screened out.

Further, vectors useful in the present invention include, but are not limited to, PMV261 vector.

The construction method of the screening system comprises the following steps:

(1) inserting a gene sequence of a protein regulated by a protein intron into a 65S position of a kana resistance protein gene to construct a fusion gene;

(2) constructing the fusion gene in the step (1) in a vector with first antibiotic resistance to obtain a recombinant plasmid;

(3) transferring the recombinant plasmid in the step (2) into mycobacterium smegmatis to obtain recombinant mycobacterium smegmatis;

(4) and (4) inoculating the recombinant mycobacterium smegmatis in the step (3) into a culture medium containing a first antibiotic and kanamycin to obtain the mycobacterium tuberculosis protein intron splicing inhibitor screening system.

Further, in the step (2), the cleavage sites of the fusion gene and the vector are both BamHI and HindIII.

Further, in step (4), the concentration of kanamycin in the medium is 30 to 50. mu.g/mL, preferably 50. mu.g/mL. This concentration range has inhibitory activity on intein splicing, too low a concentration will result in screening insensitivity, and too high a concentration will affect growth of mycobacterium smegmatis.

Further, in step (4), the concentration of the first antibiotic in the medium is 40-60. mu.g/mL.

The invention claims the application of the screening system in screening the mycobacterium tuberculosis protein intron splicing inhibitor, which comprises the following steps: adding a mycobacterium tuberculosis protein intron splicing inhibitor into the screening system, and carrying out high-throughput screening on the mycobacterium tuberculosis protein intron splicing inhibitor according to the growth condition of the recombinant mycobacterium smegmatis.

Further, the concentration of the mycobacterium tuberculosis protein intron splicing inhibitor is 1-100. mu.M.

Further, adding a mycobacterium tuberculosis protein intron splicing inhibitor and then culturing for 90-110 h. The mycobacterium smegmatis is grown to a proper concentration, so that the OD value detection is convenient.

Further, an inhibitor having a splicing inhibition effect on intein is screened out by detecting the OD value of the bacterial liquid.

The invention also claims the application of the screening system in screening antituberculosis drugs.

By the scheme, the invention at least has the following advantages:

(1) the invention establishes a screening system depending on kanamycin resistance by taking mycobacterium tuberculosis as a model bacterium, has higher splicing activity on both full-length intein and mini-type intein by matching with a proper splicing site, breaks through the limitation that the existing screening system has no splicing activity or lower splicing activity on the mini-type intein, and improves the sensitivity and accuracy of the screening system.

(2) The invention provides a screening system of a protein intron inhibitor, and the strategy can be applied to screening of other substances or medicines.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 is the plasmid PMV261-Kana^RAnd plasmid PMV261-Kana^R-65S-RecA intein construction process schematic diagram;

FIG. 2 shows a plasmid containing PMV261-Kana^RBacterial liquid OD of-65S-RecA intein Mycobacterium smegmatis at different kanamycin concentrations₆₀₀And the effect of the addition of 20uM cisplatin on its growth;

FIG. 3 shows the plasmid PMV261-Kana^R65S-RecA intein and PMV261-Kana^RThe growth of Mycobacterium smegmatis was influenced by the addition of cisplatin at different concentrations to 50ug/ml kanamycin;

FIG. 4 shows a plasmid containing PMV261-Kana^RWestern-blot results of intein splicing efficiency at kanamycin concentrations 25ug/ml and 50ug/ml for 65S-RecA intein, respectively.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

Respectively using PKH-Kana^RAnd PKH-Kana^R-65 s-RecAmin-intein plasmid as template to amplify Kana resistance gene (Kana)^R) And Kana^RThe two sides of the primer are respectively added with HindIII and BamHI enzyme cutting sites, and the PCR product is subjected to double enzyme cutting of HindIII and BamHI and then is respectively connected with a vector PMV261 subjected to the same enzyme cutting to construct a plasmid PMV261-Kana^RAnd plasmid PMV261-Kana^R65S-RecA intein (schematic diagram in FIG. 1), and plasmids were separately electroporated into M.smegmatis and positive clones were selected by hygromycin.

Example 2

The plasmid PMV261-Kana was picked^RTransferring the positive clone of-65S-RecA intein into a hygromycin resistance-containing culture medium for amplification culture, placing the culture medium in a 37 ℃ special pubic dirt incubator for standing culture for 3-4 days, and taking the bacterial liquid to perform amplification culture according to the ratio of 1: 100 percent of the total amount of the kanamycin is transferred into 5ml of fresh liquid culture medium containing different kanamycin concentrations (10-100ug/ml), and the OD value is detected after 100 hours of culture, so as to obtain the influence of different kanamycin concentrations on the growth of the mycobacterium smegmatis (see figure 2, No Cisplatin). The effect of the addition of Cisplatin at a final concentration of 20uM at different kanamycin concentrations (10-100ug/ml) on the growth of M.smegmatis was also examined (see FIG. 2, 20. mu.M Cisplatin).

FIG. 2 shows that kanamycin concentration below 50ug/ml had a small effect on growth of M.smegmatis and greater effect than 50 ug/ml; after addition of 20uM cisplatin, the inhibition increased with increasing kanamycin concentration, so that a kanamycin concentration of 50ug/ml was preferred in the subsequent screening system.

Example 3

To examine that the inhibitory effect of cisplatin on the growth of Mycobacterium smegmatis was specific for intein, plasmid PMV261-Kana was included^RThe M.smegmatis was used as a control group and cisplatin at different concentrations at 50ug/ml kanamycin concentration was tested against itThe effect of growth. The expanded culture respectively contains PMV261-Kana^RAnd PMV261-Kana^R-65S-RecA intein, M.smegmatis in a ratio of 1: 100 to 5ml containing the double antibiotic Hyg⁺kana⁺(50ug/ml) culture medium, adding cisplatin (0-20uM) with different concentrations, standing in 37 deg.C incubator for 100 hr, and detecting bacterial liquid OD₆₀₀Values were used to verify the feasibility of this splicing system (FIG. 3).

FIG. 3 shows that the plasmid PMV261-Kana is contained with the increase of the concentration of cisplatin^RThe growth inhibition effect of the mycobacterium smegmatis of the-65S-RecA intein is gradually enhanced, the inhibition rate reaches 62% at 20uM, the mycobacterium smegmatis containing the control group plasmid is milder in inhibition effect, and the inhibition rate is only 14% at 20uM, which shows that the cis-platinum specifically inhibits the splicing of the intein, thereby influencing the growth of the mycobacterium smegmatis in a kanamycin-resistant culture medium, thereby showing that the screening system in the invention is feasible and can specifically screen out the drugs with the inhibition effect on the intein.

Example 4

To examine the splicing activity of the mini-form of RecA intein in M.smegmatis, M.smegmatis grown in 25ug/ml and 50ug/ml kanamycin-resistant media, respectively, was examined for its splicing activity by Western blot (FIG. 4).

The results in FIG. 4 show that the mini type of RecA intein has higher splicing efficiency, which can reach about 90 percent, at the site of kanamycin resistance 65S by taking Mycobacterium smegmatis as a host, so that the screening system has higher sensitivity.

Through the experimental results, the screening method for the tubercle bacillus intein inhibitor is shown that the built cisplatin inhibitor is added to show specific inhibition on intein under the concentration of 50ug/ml kanamycin, and the screening system can be used for intuitively, quickly and simply screening the substance with inhibitory activity on intein splicing.

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.