WO2018121499A1

WO2018121499A1 - Drug resistant to mycobacterium tuberculosis and to infection with mycobacterium tuberculosis, and application of drug

Info

Publication number: WO2018121499A1
Application number: PCT/CN2017/118497
Authority: WO
Inventors: 丘小庆
Original assignee: 畿晋庆三联（北京）生物技术有限公司
Priority date: 2016-12-26
Filing date: 2017-12-26
Publication date: 2018-07-05
Also published as: CN108341869A

Abstract

A drug resistant to Mycobacterium tuberculosis and to infection with Mycobacterium tuberculosis, and an application of the drug, relating to the field of pharmaceuticals. All or some of the active ingredients of the drug are a recombinant polypeptide consisting of bacteriocins, such as colicins E1, Ia, Ib, A, B, and N, or an antibody mimetic polypeptide having its aqueous pore domain and carboxyl-terminus of the peptide chain linked. The drug has strong bactericidal effect against multidrug-resistant Mycobacterium tuberculosis, and can cure a macaque fatally infected with a drug-resistant Mycobacterium tuberculosis strain.

Description

Mycobacterium tuberculosis and its infection and its application

Technical field

The invention relates to the field of biomedicine, in particular to a medicament for preventing Mycobacterium tuberculosis and infection thereof and application thereof

Background technique

Since the introduction of antibiotics such as penicillin, the lives of countless people have been saved, but with the widespread use of antibiotics, more and more pathogenic bacteria have developed resistance, and existing antibiotics have gradually failed to cure these resistant pathogens. According to reports published by the Centers for Disease Control and Prevention (CDC) over the years, these antibiotics will probably fail completely after 10 to 20 years, resulting in no drug availability.

Tuberculosis is a chronic infectious disease caused by the Mycobacterium tuberculosis complex (Mycobacterium tuberculosis complex, M. tuberculosis or tuberculosis), which can affect the whole body multi-organ system. The most common diseased part is the lung, which accounts for tuberculosis in various organs. 80-90% of the total. It can also involve organs such as liver, kidney, brain, and lymph nodes. The main routes of transmission are the respiratory tract, the digestive tract, the skin and the uterus, but mainly through the respiratory tract. After the sputum of the sterilized tuberculosis patient is dried, the bacteria fly with the dust and are inhaled by others to cause infection. Whether the human body inhales the droplets containing M. tuberculosis is mainly caused by the amount of inhaled tuberculosis, the virulence, and the body's resistance. And so on a variety of factors.

The existing drugs for treating tuberculosis include isoniazid, rifampicin, pyrazinamide, ethambutol, and streptomycin, rifapentine, etc., which have side effects on liver and kidney damage, for the lungs. The treatment of external tuberculosis such as bone tuberculosis and tuberculous meningitis is usually treated with chemotherapy or surgery, and the condition is repeated and even amputation treatment is required to bring physical and mental pain to the patient. In recent years, a large number of drug-resistant/drug-resistant tuberculosis strains have been produced. The existing drugs have no effect or little effect on them, which leads to the need to increase the dose of drugs in the clinic, which in turn induces more stubborn drug-resistant strains, due to new drugs. The development cycle is long, and the speed of research and development of new drugs is far behind the speed of drug-resistant strains. This has made tuberculosis have now become a problem for the world's medicine.

Inventor Qiu Xiaoqing's patent No. ZL200910092128.4, entitled "A New Antibiotic Containing Antibody Mimics", published in 2009, discloses a class of peptide drugs with anti-meningococcal and anti-resistant Phytophthora Enterococcus faecalis, anti-methicillin-resistant Staphylococcus aureus or anti-multi-drug resistant Pseudomonas aeruginosa activity. The recombinant polypeptide drug has the characteristics of strong targeting, safety and high efficiency, and is not easy to produce drug resistance, but the polypeptide drug has not been found to have any effect on the antituberculosis bacillus and its infection.

Summary of the invention

The inventors found in an accidental experiment that the antibody mimetic short peptide disclosed in ZL200910092128.4 has recognition and binding effect on multiple drug-resistant Mycobacterium tuberculosis, and the antibody mimetic and the colicin linked recombinant polypeptide are many The drug-resistant Mycobacterium tuberculosis has strong killing activity and no damage to the animal body of the infected bacteria. Based on this and subsequent extensive experimental research, the following technical solutions are provided:

A method for identifying Mycobacterium tuberculosis (Mycobacterium tuberculosis) antibody mimetic, characterized in that an immunoglobulin V _{_H} CDR1, V _H FR2 and V _L CDR3 region NV _{_H} CDR1-V _H FR2- _L CDR3-C V connected in a manner that the amino acid sequence as shown in Seq ID No.2.

The use of the above antibody mimic in the preparation of a reagent targeting M. tuberculosis, by coupling the antibody mimetic to an action molecule to form a coupled molecule, the antibody mimetic in the coupled molecule retaining immunity The recognition, binding and/or affinity activity of the globulin on the target cell for directing the action molecule to or near the cell membrane of M. tuberculosis;

The acting molecule is a chemical molecule or a polypeptide molecule;

The chemical molecule comprises a label, a bacterial cytotoxic agent, a growth inhibitor; the polypeptide molecule comprises an enzymatic active toxin, a bacteriocin;

The bacteriocin is selected from the group consisting of coenzyme E1, Ia, Ib, A, B, N, an aqueous channel domain polypeptide thereof, and pyocyanin.

In some embodiments, the acting molecule is a polypeptide molecule and the ligation is by a protein coupling agent or recombinant expression.

In another aspect of the present disclosure, a recombinant polypeptide of Mycobacterium tuberculosis is provided, wherein the recombinant polypeptide is linked to a peptide chain of the above antibody mimetic by a carboxy terminus of a peptide chain of a polypeptide-acting molecule. The amino terminal linkage is composed; the polypeptide-acting molecule is selected from the group consisting of colicin E1, Ia, Ib, A, B, N or an aqueous channel domain polypeptide thereof, and pyocyanin.

In some embodiments, the colicin is Ia.

In some preferred embodiments, the recombinant polypeptide has an amino acid sequence such as Seq ID No. 6.

In still another aspect of the present disclosure, a method for preparing a drug against Mycobacterium tuberculosis or an infection thereof, characterized in that

In the production process of the anti-tuberculosis drug, the recombinant polypeptide of any of the above or the agent comprising the recombinant polypeptide is used as the entire pharmaceutically active ingredient or a part of the pharmaceutically active ingredient of the anti-tuberculosis drug.

In some embodiments, the reagent comprising the recombinant polypeptide refers to a protein recombinant expression product that has been purified initially or multiple times, wherein the recombinant polypeptide has a purity of 30-99.5%.

Preferably, the preparation method further comprises a recombinant expression step and a purification step of the recombinant polypeptide, wherein the recombinant expression vector for expressing any of the recombinant polypeptides is transformed into an expression cell to induce expression of the recombinant polypeptide. And purification.

In a further aspect of the present invention, the use of any of the above recombinant polypeptides as anti-tuberculosis drugs, characterized in that the recombinant polypeptide or an agent comprising the recombinant polypeptide is used as the total drug activity of the anti-tuberculosis drug Ingredients or parts of pharmaceutically active ingredients.

According to still another aspect of the present invention, an anti-tuberculosis bacterium or an agent thereof is provided, wherein all or a pharmaceutically active ingredient thereof is a recombinant polypeptide of any of the above or an agent comprising the recombinant polypeptide.

In some implementations, the medicament further comprises a pharmaceutically acceptable ingredient, which is formulated to:

Enteral administration agents, such as powders, tablets, granules, capsules, solutions, emulsions, suspensions;

Injectable agents, such as intravenous, intramuscular, subcutaneous, intradermal, and intraluminal; or

Respiratory agents, such as sprays, aerosols, powders.

In one aspect, the present disclosure provides a recombinant expression vector for the preparation of a drug against Mycobacterium tuberculosis or an infection thereof, which is loaded with an open reading frame expressing any of the above recombinant polypeptides.

Preferably, the coding gene sequence in the open reading frame is as shown in Seq ID No. 5.

In one aspect, the invention provides a method of treating a Mycobacterium tuberculosis infection, characterized in that any one of the above agents is provided to an infected individual.

Antibody mimics of the present disclosure for identifying Mycobacterium tuberculosis, which was originally based on specific V _H CDR1 antibody anti Meningococcal porin A, V _H FR2 and V _L CDR3 designed to retain the original The recognition, binding or affinity activity of an antibody. In the prior patent, a recombinant polypeptide linked to a bacteriocin, such as colicin, in addition to recognizing the killing of meningococcus, also against vancomycin-resistant intestinal Cocci, anti-methicillin-resistant Staphylococcus aureus or anti-multi-drug resistant Pseudomonas aeruginosa have recognition and sterilizing activity. Based on the existing known studies on colicin, bacteriocins such as colicin have only the ability to recognize E. coli, and they do not have the ability to specifically recognize other bacteria. Therefore, it is clear that the recombinant polypeptide is resistant to Van Gogh. In the bactericidal process of Enterococcus faecalis, methicillin-resistant Staphylococcus aureus or anti-multi-drug resistant Pseudomonas aeruginosa, the antibody mimetic recognizes the colicin and directs it to the cell membrane of the identified target. Attack.

In the present disclosure, the inventors have found that the recombinant polypeptide obtained by linking the antibody mimetic to bacteriocin has superior killing activity against hundreds of strains of currently resistant strains of Mycobacterium tuberculosis. However, antibody simulation or colicin was co-cultured with these strains alone, and no antibacterial activity was found.

Therefore, the above antibody mimetic can be used as a targeting molecule targeting a cell of Mycobacterium tuberculosis, and some of the acting molecules are linked at one end thereof to form a coupling molecule, which can be guided to the cell membrane of Mycobacterium tuberculosis cells. Specific effects on Mycobacterium tuberculosis, such as labeling, killing, inhibiting growth and proliferation, and the like. The acting molecule includes, but is not limited to, the aforementioned bacteriocin such as colicin or a well-known fragment of the aqueous channel region thereof. It may also be a cytotoxic chemical reagent such as a radioisotope (for example, a typical radioactive element comprising At211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 and cesium radioisotopes), growth inhibition Agent, toxin (eg, a protein toxin, an enzymatically active toxin of a bacterial, fungal, plant or animal source or a fragment thereof). The method of coupling these functional molecules with a polypeptide is a conventional technique in the art. When the acting molecule is a polypeptide molecule, the coupling can be accomplished by polypeptide synthesis techniques or recombinant expression. There are no technical obstacles to those skilled in the art.

Methods of making recombinant polypeptides of the present disclosure are also optional. It is not limited to the description in the examples.

A specific aspect of the present disclosure is to claim the use of the above antibody mimetic in the preparation of a drug against Mycobacterium tuberculosis or an infection, the use of which mainly comprises the imitation of the antibody and the bacteriocin, selected from the large intestine a group consisting of an amino acid domain polypeptide, a pyocyanin, a recombinant polypeptide, and a recombinant polypeptide obtained as a whole or a part of a pharmaceutical active ingredient to prepare and produce antituberculosis Bacillus or its infected drugs.

The experiments in the present disclosure demonstrate the in vitro antibacterial activity, in vivo protective effects, and animal therapeutic effects of the above agents or recombinant polypeptides.

The disclosure of the Chinese patent application ZL2910092128.4 is incorporated herein by reference.

DRAWINGS

Figure 1. Structure of recombinant plasmid pBHC-PorA1 containing antibody mimics and colicin Ia

Wherein the peptide chain of the antibody mimetic is linked to the carboxy terminus of the peptide chain of Ia, and the amino acid sequence of the antibody mimetic

As shown in Seq ID No. 2.

Figure 2. Structure of recombinant plasmid pBHC-PorA2 containing antibody mimetic peptide chain and colicin Ia

Wherein the antibody mimetic peptide chain is ligated at the carboxy terminus of Ia, and the antibody mimetic peptide chain is: the carboxy terminus of the first complementarity determining region of the heavy chain variable region is linked to the amino terminus of the second backbone region of the heavy chain, and the light chain variable region The peptide-terminal carboxy-terminal amino acid of the third complementarity determining region is linked to the carboxy-terminal amino acid of the second backbone region of the heavy chain, as shown by Seq ID No. 4.

Figure 3. Structure of the novel antibiotic of the present invention

Wherein T and R are two signal recognition domains at the amino terminus of colicin Ia; channel-forming is the formation of an ion channel domain at the carboxy terminus of coenzyme Ia; AM is an antibody mimetic.

Figure 4. Colony counts in the lungs of a mouse tuberculosis infection model:

From left to right: early control group, late control group, isoniazid group, low-dose pheromone group, high-dose pheromone group.

Figure 5. Statistical results of therapeutic survival of the genomicin for the cynomolgus tuberculosis infection model.

Figure 6. Statistical results of effective control rates for tuberculosis infection in macaques that survived 150 days after treatment with genomicin.

Figure 7. Results of blood biochemical tests in a model of cynomolgus tuberculosis infection.

Figure 8. Results of histopathological examination of cynomolgus tuberculosis infection model:

The picture on the left is 40x magnification, and the image on the right is 200x magnification.

The untreated group of monkeys (A) showed typical miliary tuberculosis lesions in the lungs;

Isoniazid/rifampicin treatment group monkeys (B) showed typical tuberculous granulomatous lesions in the lungs;

In the pheromone-treated group, only the interstitial pneumonia lesions appeared in the lungs of the monkeys (C), and no such tuberculosis lesions were observed.

detailed description

The present disclosure is specifically described by the description of the preferred embodiments of the present invention, but not by way of limitation. Experimental methods performed in the present application, such as animal experimental methods, unless otherwise specified, indicate routine manipulation in the art. The reagents used in the experiments of the present invention, unless otherwise specified, indicate the use of conventional reagents in the art.

Example 1. The active molecule was selected from colistin Ia to prepare a recombinant polypeptide.

Original plasmid pSELECT ^TM -1 loaded colicin plasmids and immunity protein gene (8.3kb). A double-stranded oligonucleotide point mutation technique (QuickChange ^TM Kit, Strategene) will encode a gene fragment encoding an antibody mimetic: 5-tcttattggctgcattggat taaacagaga cctggtcagg gactgtggat cggatctcagtccacgcatg tgccgagaacc-3 (Seq ID No. 1) or 5-tcttattggc tgcattggat Taaacagaga cctggtcagg gactgtggat cggaaccaga ccggtgcata cgtcccagtct-3 (Seq ID No. 3) was inserted into the 626 site of the colicin polypeptide gene to obtain the mutant plasmids pBHC-PorA1 and pBHC-PorA2 for the preparation of novel antibiotics (Figs. 1 and 2). Show). The mutant plasmid was transfected into E. coli BL-21 engineered bacteria to prepare a new antibiotic.

The mutation program was performed according to the Strategene QuickChange SiteDirected Mutagenesis Kit (catalog #200518) kit manual, ie:

1. Prepare the point mutation reactant:

5ul 10X buffer

2ul (10ng) of the original plasmid loaded with the colicin allosteric polypeptide and the immunogenic protein gene

1.25 ul (125 ng) of designed 5'-3' oligonucleotide primers (see primer sequences listed)

1.25 ul (125 ng) of designed 3'-5' oligonucleotide primers (see primer sequences listed)

1ul dNTP

Double steamed water 50ul

1ul pfu

(Reagents prepared in the kit except for plasmids, primers and double distilled water)

2. Perform PCR amplification, amplification conditions: denaturation 95 ° C, 35 seconds, annealing 53 ° C, 70 seconds, extension 68 ° C, 17 points, a total of 20 cycles;

3. After adding Dpn 1 endonuclease 1 ul to digest the maternal DNA strand (37 ° C, 1 hour), 1 ul of the reaction was incubated with XL1-Blue competent cells for 50 minutes, heat shocked for 42 ° C, 45 seconds, and then placed. 2 minutes in the ice;

4. Add NZY Pepsi 0.5 ml, 220 rpm, shake at 37 ° C for 1 hour, take 50-100 ul of the reaction plate (LB PBS plus 1% agar, add 50 ug / ml ampicillin, overnight at 37 ° C);

5. After 18 hours, the bacteria were picked, and the plasmid was extracted and sequenced to confirm the mutation was successful;

6. Incubate the mutated plasmid 100 ng with the prepared BL-21 engineering bacteria competent cells for 40 minutes, heat shock at 42 ° C for 30 seconds, then place in ice for 2 minutes, add SOC broth 160 ul, 220 rpm, shake at 37 ° C After 1 hour, the bacteria were plated (LB-based plus 1% agar, plus 50 ug/ml ampicillin, overnight at 37 ° C), and the monoclonal colonies were picked up to increase the bacteria;

7. A large number of bacteria, 8-10 liters of FB medium, 250 rpm, 30 ° C, 3-4 hours; warmed to 42 ° C, 250 rpm growth for 0.5 hours; cooled to 37 ° C, 250 rpm growth for 1.5 hours; 4 ° C, 6000 g, 20 Precipitating the cells by centrifugation;

8. Take 80-100 ml of suspended cells in 50 mM boric acid buffer (pH 9.0, 2 mM EDTA) at 4 ° C. After adding 50 ug of PMSF, the cells were sonicated (4 ° C, 400 W, 1 minute, repeated 4-5 times, intermittent 2- 3 minutes to ensure the temperature of the bacterial liquid), high-speed centrifugation to precipitate the broken cells (4 ° C, 75000g, 90 minutes), take the supernatant to add 5 million units of streptomycin sulfate precipitation DNA (4 ° C stirring for 1 hour), 10,000g, 4 After centrifugation at 10 °C for 10 minutes, the supernatant was placed in a dialysis bag with a molecular weight of 15,000 at 4 ° C, and dialyzed against 10 liters of 50 mM boric acid buffer overnight. After centrifugation again at 10,000 g, 4 ° C, 10 minutes, the supernatant was applied to CM. The ion exchange column was fully washed and eluted with 0.3 M NaCl + 50 mM boric acid buffer to obtain the prepared informationin.

Corresponding to the above two kinds of plasmids, PMC-AM1 and PMC-AM2 can be obtained, respectively, and the amino acid sequences thereof are as shown in Seq ID No. 6, Seq ID No. 8.

Wherein PMC-AM1 is the first complementarity determining region of the heavy chain variable region, the second heavy chain region of the heavy chain, and the third complementarity determining region of the light chain variable region, and the three regions are sequentially linked to the amino terminus of the next region by the carboxy terminus. , the amino acid sequence is shown as Seq ID No. 2; PMC-AM2 is the carboxy-terminal linked heavy chain second framework region of the first complementarity determining region of the heavy chain variable region, and the third complementarity determining region of the light chain variable region The carboxy terminal amino acid of the peptide chain is linked to the carboxy terminal amino acid of the second backbone region of the heavy chain, and the amino acid sequence is shown as Seq ID No. 4.

PMC-AM2 was used as a control for PMC-AM1 to verify the function of antibiotics produced in the different linkages between the amino acid stretches of the antibody mimics designed in the present invention.

The oligonucleotide primer sequences designed in the above preparation plasmids are as follows:

5'-3' (SEQ ID NO. 9)

3'-5' (SEQ ID NO. 10)

5'-3' (SEQ ID NO. 11)

3'-5' (SEQ ID NO. 12)

5'-3' (SEQ ID NO. 13)

3'-5' (SEQ ID NO. 14)

pBHC-PorA 2

5'-3' (SEQ ID NO. 15)

3'-5' (SEQ ID NO. 16)

5'-3' (SEQ ID NO. 17)

3'-5' (SEQ ID NO. 18)

5'-3' (SEQ ID NO. 19)

3'-5' (SEQ ID NO. 20)

Experimental Example 1 Comparison of Minimum Inhibitory Concentrations of Lethal Drug-Resistant Strains

Experimental strain:

Sensitive strain: Mtb Erdman is sensitive to current anti-tuberculosis drugs, SUNY Upstate Medical University, State University of New York

Drug-resistant strain: PUMC-94789 is a Beijing genotype strain, resistant to isoniazid and rifampicin, with a classic isoniazid resistance to katG315 gene mutation and rifampicin rpoB531, 526 gene mutation, PUMC-94789 The virulence is strong, and the survival time of LD50 mice is 7 days (the survival rate of tuberculosis standard strain H37Rv is 14 days). Peking Union Medical College, Peking Union Medical College.

506 multidrug-resistant tuberculosis (MDR-TB) was provided by the National Laboratory of Tuberculosis Reference, China CDC, China National Center for Disease Control and Prevention.

The above strains are well-known strains, and the applicant's laboratory also has a preservation. The appropriate amount can be provided to the public within 20 years from the date of application for verification of this application.

Experimental reagents:

The genomicin PMC-AM1 was prepared in Example 1;

Isoniazid and other commonly used anti-tuberculosis drugs are commercially available.

7H9-S medium: containing 0.47% 7H9 (purchased from BD Company, USA, Cat. No. 271310), 0.2% glycerol, 0.05% Tween-80, 0.085% sodium chloride, 0.5% calf serum component V (Roche, Cat. No. 10735094001), 0.2% glucose, 0.003% catalase (Sigma-Aldrich, Cat. No. C9322-1G).

Gradient dilution before each drug test;

Take 100 μl of each concentration gradient for each drug, place it in the calibrated microwells in a 96-well plate, place 100 μl of 7H9-S broth in the control wells, and then add 100 μl to each well. Inoculum (10 ⁷ CFU containing tuberculosis per ml of 7H9-S medium).

The final concentration gradient of each drug was: 80, 40, 20, 10, 5, 2.5, 1.25, 0.62, 0.31, 0.16, 0.08 μg/ml, and other anti-tuberculosis drugs were 256, 128, 64, 32, 16, 8 , 4, 2, 1, 0.5 μg/ml

The final concentration of tuberculosis in each well was adjusted to 5x10 ⁵ CFU/ml.

The microplate was cultured for 10 days (35 to 37 ° C). The minimum concentration that inhibits the growth of tuberculosis visible to the naked eye is the minimum inhibitory concentration of the drug. The results are as follows:

Table 1

Minimum inhibitory concentration ( nM ( ug /ml)) of 506 strains of multidrug-resistant tubercle bacilli (10% of which are super-resistant XDR)

Note The test data between pheromone and other agents were compared by T test, and the P value <0.05 was considered to be statistically significant.

As can be seen from Table 1 above, the MIC _{50 of} pheromone to 506 multidrug-resistant tuberculosis (MDR-TB) including PUMC-94789 averaged around 1 nM, and the MIC ₉₀ was around 143 nM, while 11 of the same period were tested. The MIC ₅₀ and MIC _{90 of} anti-tuberculosis drugs (isoniazid, rifampicin, ethambutol, capreomycin, kanamycin, levofloxacin, etc.) are in the range of hundreds to hundreds of thousands of nM. That is, the bactericidal effect of the pheromone on the hundreds of MDR-TB tested is hundreds to hundreds of thousands times greater than the bactericidal efficacy of the 11 existing anti-tuberculosis drugs.

Experimental Example 2 Mouse lung tuberculosis infection model colony count in the lung

Female BLAB/c mice were vaccinated with 6.8x10 ² CFU Mtb Erdman tuberculosis;

Each group of 6 was divided into five groups: early control group, late control group, isoniazid group, low dose pheromone group, high dose pheromone group;

After 21 days of infection, each group of rats was treated as follows:

The early control group and the late control group were intraperitoneally injected with physiological saline;

Isoniazid group oral isoniazid 183μmol/kg/d (25mg/kg/d);

The low-dose pheromone group and the high-dose pheromone group were intraperitoneally injected with 0.286 or 0.572 μmol/kg/d (

ie

20 or 40 mg/kg/d) pheromone.

Four weeks later, the right lung was removed, and the tuberculosis colonies were counted after milling and homogenization. The statistical results are shown in Figure 4. The lung counts of the mice treated with low-dose and high-dose pheromone-NM were compared with those in the control group. Colony counts are two to three pairs of orders of magnitude lower. The counts of colons in the lungs of mice treated with isoniazid were also two to three orders of magnitude lower than those in the control group. However, the dose used for isoniazid is 320 to 640 times larger than that used for genomicin-NM. Therefore, the effect of genomicin-NM on inhibiting tuberculosis in the lung of mice is 320-640 times greater than that of isoniazid.

Experimental Example 3 Therapeutic rate of informationin on the model of rhesus pulmonary tuberculosis infection

Male rock crab monkeys (Macacca fasicularis) were randomly divided into three groups after healthy screening:

Group 1. Untreated group (n=4),

Group 2. Isoniazid/rifampicin treatment control group (n=4),

Group 3. The pheromone treatment group (n=8).

300CFU multidrug-resistant tuberculosis PUMC-94789 was inoculated into the right lower lung by bronchoscopy.

Twenty-one days after the inoculation, the pulmonary tuberculosis test and lung CT examination confirmed that the lung infection was successful, treated with each drug for more than 150 days, and after stopping the drug for more than 180 days, the test totaled 52 weeks (about one year).

The therapeutic doses were as follows: Group 1. No treatment; Group 2. Isoniazid/rifampicin 21.8/3.65 μmol/kg/d (3/3 mg/kg/d), intraperitoneal injection; Group 3. Informationin 43nM/kg/d (3mg/kg/d), intraperitoneal injection.

In the experiment, the animal's body weight, body temperature, appetite, metabolism, excretion and other physiological indicators were observed. CT scan was performed every 30 days to calculate the volume of tuberculosis lung lesions, and the progress of tuberculosis and the progress of informational bacteriocin treatment were quantitatively observed.

After the end of the experiment, pathological anatomy and histopathological examination were performed. The lung tissue and homogenate were collected and cultured to calculate the CFU (bacterial burden); blood biochemical specimens were collected to observe the presence or absence of drug toxicity.

As shown in Figure 5, all untreated monkeys died within 17 weeks of tuberculosis infection. All isoniazid/rifampicin treated control monkeys also died within 17 weeks of tuberculosis infection. This indicates that this experiment did cause a lethal multi-drug resistant infection in monkeys, and the most powerful current anti-tuberculosis drug, isoniazid / rifampicin treatment is ineffective. At the same time, genomicin-NM showed a powerful therapeutic effect on lethal multidrug-resistant infections: only 2 of 8 monkeys died, and 6 survived at the end of 150 days of treatment, and the treatment was successful. The rate is 75%.

As shown in Figure 6, after the end of the 150-day treatment, 5 pheromone-NM-treated monkeys (6 were originally, but one was sacrificed after the end of treatment to facilitate treatment with the untreated group and isoniazid/rifampicin). The treatment control group was compared. After more than 30 days of stopping the drug, the CT was reexamined. It was found that the two monkeys relapsed. The sputum was again given the pheromone-NM 43nM/kg/d (3mg/kg/d) treatment, and the condition was relieved once. More than 120 days later died. The remaining 3 monkeys did not find any recurrence of the disease during the 180-month follow-up observation of the drug withdrawal; and the appetite and behavior were good during the observation period of withdrawal, and the body weight gradually increased. Therefore, after 150 days of informationin treatment, 60% (3/5) of the tuberculosis infection in the monkey was effectively controlled, and there was no recurrence in the 180-day withdrawal observation period, which should be clinically cured.

Blood biochemical test results

Figure 7 shows the blood biochemical test indicators of the untreated group (n=4) and the pheromone treated group (n=8), compared with the untreated monkeys, after 150 days of pheromone treatment:

In the treatment of monkey blood, urea nitrogen BUM, creatinine CRE, total bilirubin TBIL, direct bilirubin DBIL, total bile acid TBA level did not increase, but also lower than the untreated group;

Serum enzymes, such as alanine aminotransferase ALT, serum pancreatic amylase AMY, alkaline phosphatase ALP and other indicators and serum, globular protein levels such as total protein TP, serum albumin ALB, globulin Glob no significant difference;

The serum glucose Glu and triglyceride TG of untreated monkeys were significantly lower than that of the informationin treatment group, which should be due to the aggravation of tuberculosis, poor eating and malnutrition in the untreated group;

In summary, the 150-day pheromone treatment did not cause toxicity to the liver, kidney, and metabolic functions of the experimental monkeys.

Histopathological examination results

As shown in Figure 8, the untreated group of monkeys (A) showed typical miliary tuberculosis lesions in the lungs, isoniazid/rifampicin treatment group, and monkeys (B) showed typical tuberculous granulomatous lesions in the lungs. In the pheromone treatment group, only the interstitial pneumonia was present in the lungs of the monkey (C), and no TB lesions were observed. (40x magnification on the left and 200x magnification on the right)

Claims

A method for identifying Mycobacterium tuberculosis (Mycobacterium tuberculosis) antibody mimetic, characterized in that an immunoglobulin V H CDR1, V H FR2 and V L CDR3 region NV H CDR1-V H FR2- L CDR3-C V connected in a manner that the amino acid sequence as shown in Seq ID No.2.
The use of the antibody mimetic of claim 1 for the preparation of a reagent targeting M. tuberculosis, which comprises forming a coupling molecule by linking the antibody mimetic to an action molecule, the antibody in the coupled molecule The mimetic retains the recognition, binding and/or affinity activity of the immunoglobulin on the target cell for directing the action molecule to or near the cell membrane of M. tuberculosis;

The acting molecule is a chemical molecule or a polypeptide molecule;

The chemical molecule comprises a label, a bacterial cytotoxic agent, a growth inhibitor; the polypeptide molecule comprises an enzymatic active toxin, a bacteriocin;

The bacteriocin is selected from the group consisting of coenzyme E1, Ia, Ib, A, B, N, an aqueous channel domain polypeptide thereof, and pyocyanin.
The use according to claim 2, characterized in that the acting molecule is a polypeptide molecule and the ligation is carried out by a protein coupling agent or recombinant expression.
A recombinant polypeptide against Mycobacterium tuberculosis, characterized in that the recombinant polypeptide is linked by a peptide chain carboxy terminus of a polypeptide-acting molecule to the amino acid terminal of the peptide chain of the antibody mimetic of claim 1. The polypeptide-acting molecule is selected from the group consisting of colicin E1, Ia, Ib, A, B, N or an aqueous channel domain polypeptide thereof, pyocyanin.
The recombinant polypeptide according to claim 1, wherein the colicin is Ia.
The recombinant polypeptide according to claim 4 or 5, which has an amino acid sequence such as Seq ID No. 6.
A method for preparing a drug against Mycobacterium tuberculosis or an infection thereof, characterized in that

In the process of producing an anti-tuberculosis drug, the recombinant polypeptide of any one of claims 4 to 6 or an agent comprising the recombinant polypeptide is used as the entire pharmaceutically active ingredient or a part of the pharmaceutically active ingredient of the anti-tuberculosis drug.
The preparation method according to claim 7, wherein the reagent comprising the recombinant polypeptide refers to a recombinant expression product of a preliminary purification or a plurality of purifications, wherein the purity of the recombinant polypeptide is 30-99.5%. .
The production method according to claim 7, comprising a recombinant expression step and a purification step comprising the recombinant polypeptide, characterized in that the recombinant expression for expressing the recombinant polypeptide of any one of claims 4-6 is used The vector is transformed into an expression cell, and the recombinant polypeptide is induced to be expressed and purified.
The use of the recombinant polypeptide according to any one of claims 4 to 6 as a drug against Mycobacterium tuberculosis, characterized in that the recombinant polypeptide or an agent comprising the recombinant polypeptide is used as a whole pharmaceutically active ingredient of the anti-tuberculosis drug Or part of the active ingredient of the drug.
The use of the recombinant polypeptide according to any one of claims 4 to 6 as a drug against Mycobacterium tuberculosis, characterized in that the recombinant polypeptide or an agent comprising the recombinant polypeptide is used as a whole pharmaceutically active ingredient of the anti-tuberculosis drug Or part of the active ingredient of the drug.
An agent against Mycobacterium tuberculosis or an infection thereof, characterized in that all or a pharmaceutically active ingredient thereof is a recombinant polypeptide according to any one of claims 4 to 6 or an agent comprising the recombinant polypeptide.
The agent according to claim 12, wherein the agent comprising the recombinant polypeptide refers to a recombinant expression product of a protein which is initially purified or repeatedly purified, wherein the recombinant polypeptide has a purity of 30 to 99.5%.
The medicament according to claim 12 or 13, further comprising a pharmaceutically acceptable ingredient, which is prepared:

Enteral administration agents, such as powders, tablets, granules, capsules, solutions, emulsions, suspensions;

Injectable agents, such as intravenous, intramuscular, subcutaneous, intradermal, and intraluminal; or

Respiratory agents, such as sprays, aerosols, powders.
A recombinant expression vector for the preparation of a drug against Mycobacterium tuberculosis or an infection thereof, which is loaded with an open reading frame which expresses the recombinant polypeptide of any of claims 4-6.
The nucleotide molecule according to claim 15, wherein the nucleotide sequence of the coding gene in the open reading frame is as shown in Seq ID No. 5.
A method of treating a Mycobacterium tuberculosis infection, characterized in that an effective amount of the agent according to any one of claims 12-14 is provided to an infected individual.