CN113559284B - Product based on non-tuberculous mycobacterium pure protein derivative and recombinant tubercle bacillus fusion protein, application and use method - Google Patents
Product based on non-tuberculous mycobacterium pure protein derivative and recombinant tubercle bacillus fusion protein, application and use method Download PDFInfo
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Abstract
The invention discloses a product based on a non-tubercular mycobacterium pure protein derivative and a recombinant tubercular bacillus fusion protein, an application and a use method, and belongs to the technical field of biological proteins. The non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein (EEC) are used in a combined way, so that tuberculosis and NTM diseases can be identified quickly and accurately, and when the delayed hypersensitivity reactions of the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein are all positive, the tuberculosis is formed; NTM disease is determined when delayed hypersensitivity of non-tuberculous mycobacterium pure protein derivative is positive and delayed hypersensitivity of recombinant tubercle bacillus fusion protein is negative. And a product based on the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein and application thereof are provided, so that tuberculosis and NTM diseases are rapidly and accurately identified, and reliable basis is provided for early diagnosis, treatment and the like of patients.
Description
Technical Field
The invention relates to a protein product and application thereof, in particular to a product based on a non-tuberculous mycobacterium pure protein derivative and recombinant tuberculous mycobacterium fusion protein, application and a use method thereof, belonging to the technical field of biological protein.
Background
Tuberculosis is a chronic infectious disease which seriously harms the health of human beings and animals, and 140 thousands of tuberculosis deaths are caused in 2019 all over the world. According to 2020 annual report of tuberculosis worldwide released by WTO: the number of the worldwide tuberculosis morbidity is estimated to be about 1000 thousands (890-1100 thousands) in 2019; among them, 264 million in India accounts for 26%; indonesia 84.6 ten thousand, 8.5%; 83.3 thousands in China, accounting for 8.4 percent. The number of Chinese tuberculosis patients is the third world rank, and is a tuberculosis high-burden country.
For nontuberculous mycobacteriosis (NTM), epidemiological data of China show that NTM infection is in an ascending trend; the tuberculosis epidemiological survey data in China all the times show that the NTM separation rate in 1990 is 4.9%, the NTM separation rate in 2000 is 11.1%, and the NTM separation rate in 2010 is increased to 22.9% in all the mycobacteria patient isolates. NTM infection is a major public health problem in China, NTM has great regional difference in strain distribution in China, and the overall distribution is characterized in that the coastal area is higher than the inland, the southern area is higher than the northern area, and the climate mild area is higher than the cold area.
Non-tuberculous mycobacteriosis (NTM disease) is close to the clinical symptoms of tuberculosis and can be easily treated as tuberculosis, but the treatment schemes of the non-tuberculous mycobacteriosis and the NTM disease are completely different, and the resistance of the NTM disease is very strong.
Wherein the treatment scheme of the NTM disease comprises: resistance to NTM disease is high for Isoniazid (INH), rifampicin (RFP), ethambutol (EMB) and pyrazinamide to treat tuberculosis. Quinolones are commonly used, such as: ciprofloxacin (Ciprofloxacin), ofloxacin (Ofloxacin), or Fleroxacin (Fleroxacin); macrolides, such as: azithromycin (agiithromycin AZ) or clarithromycin (Cear); cephalosporins, such as: cephalo tincture (Cefoxintin, cet) or Cefixime (Cefixime); aminoglycosides, such as: amikacin (amikacin, AM), capreomycin (Cap), gentamicin (Gentamicin, gm), rifabutin (Rifabutin, rb), cycloserine (Cy), or Ethionamide (Ethionamide, ethion). Further, as follows: imipenem (Imipenen), tetracycline (Tz), doxycycline (Dox), sodium aminosalicylate (PAS) and Sulfamethoxazole (Sulfamethoxazole) also have certain curative effects.
Treatment regimens for tuberculosis include: the common first-line antitubercular drugs mainly comprise Isoniazid (INH), rifampin (RFP), ethambutol (EMB) and pyrazinamide, the common first-line antitubercular treatment scheme is a 6-month treatment scheme, and the first 2 months are the four-drug combination of isoniazid, rifampin, ethambutol and pyrazinamide; the last 4 months is the combination of two drugs, mainly the combination of isoniazid and rifampicin.
Currently, the diagnostic methods for NTM disease are: high Performance Liquid Chromatography (HPLC), molecular biology methods (including acridinium ester labeled DNA probe assay, PCR or multiplex PCR method, PCR-restriction fragment length polymorphism analysis, DNA sequencing technology, reverse hybridization DNA amplification technology), and the like.
Based on the fact that clinical symptoms of NTM and tuberculosis are close to each other, but treatment schemes of the NTM and the tuberculosis are quite different, the conventional NTM diagnosis method generally takes longer time (more than 30 days) and can seriously affect the treatment of patients, and therefore, a simple and rapid detection reagent is urgently needed.
The prior art CN111905110A discloses a method, a system and an application for identifying mycobacterium tuberculosis infection and nontuberculous mycobacterium infection, wherein the method specifically comprises the following steps: providing an allergen consisting of recombinant mycobacterium tuberculosis Esat-6 protein and CFP-10 protein and an NTM mycoprotein consubstantiality double-arm skin test, and injecting the allergen consisting of recombinant mycobacterium tuberculosis Esat-6 protein and CFP-10 protein and the NTM mycoprotein into a to-be-detected organism for carrying out skin test reaction; and judging the infection conditions of the tubercle bacillus and the NTM of the organism to be detected according to the result of the skin test reaction. The system for detecting the mycobacterium tuberculosis infection and the nontuberculous mycobacterium infection provided by the invention can be used jointly for differential diagnosis, and can also be used for detecting the mycobacterium tuberculosis infection or NTM infection respectively and independently. CN102229999A discloses a kit for identifying mycobacterium tuberculosis and nontuberculous mycobacteria and a use method thereof, wherein, the fluorescence quantitative nucleic acid detection technology which can distinguish and identify the mycobacterium tuberculosis and the nontuberculous mycobacteria at one time is utilized by utilizing the advantage of double-channel fluorescence quantitative; provides a kit for identifying mycobacterium tuberculosis and nontuberculous mycobacteria, which comprises a primer for carrying out PCR amplification on a strain to be detected and a probe for carrying out fluorescence quantitative detection. The fluorescent probe is designed according to the difference of gene sequences of different mycobacteria, the mycobacterium tuberculosis and nontuberculous mycobacteria are distinguished, the gene sequences and molecular structure levels of bacteria are distinguished and identified, and the classification is more accurate and reliable. Solves the problem that the traditional identification method needs to take 4 to 6 weeks for distinguishing through the growth form of bacteria. CN101413031A discloses a method for identifying mycobacterium tuberculosis and nontuberculous mycobacteria and a special kit thereof, wherein the kit comprises primers and probes, the primers comprise 4 primers, and nucleotide sequences of the primers are respectively a sequence 1, a sequence 2, a sequence 4 and a sequence 5 in a sequence table; the probe comprises 2 probes, and the nucleotide sequences of the probes are respectively a sequence 3 and a sequence 6 in a sequence table; the 5 'end of the probe is provided with a fluorescent substance, and the 3' end of the probe is provided with a quenching substance; the 5' end of the 2 probes was different in fluorescent substance.
Disclosure of Invention
The inventor finds in long-term research that tuberculosis and NTM diseases can be rapidly identified (within 24 h) by combining the non-tuberculous mycobacterium pure protein derivatives and the recombinant tubercle bacillus fusion protein (EEC), and tuberculosis is identified when delayed hypersensitivity of the non-tuberculous mycobacterium pure protein derivatives and the recombinant tubercle bacillus fusion protein is positive (except pathogenic bacteria-Kansas mycobacterium); NTM disease when delayed hypersensitivity of non-tuberculous mycobacterium pure protein derivative is positive and delayed hypersensitivity of recombinant tubercle bacillus fusion protein (EEC) is negative;
however, it is found that the sensitivity of the pure protein derivative of nontuberculous mycobacteria to NTM diseases caused by pathogenic bacteria of nontuberculous mycobacteria group III (slow growth and no chromogenesis) is high, and the sensitivity to NTM diseases caused by pathogenic bacteria of nontuberculous mycobacteria group I (slow growth and no chromogenesis) is low, and the possibility of misjudgment is easy to occur, so that nontuberculous mycobacteria group I (slow growth and no chromogenesis) Kansasi mycobacteria (ATCC 12478), group II (slow growth and dark chromogenesis) Gordonia mycobacteria (ATCC 14770), group III (slow growth and no chromogenesis) intracellular mycobacteria (ATCC 13950) and group IV (fast growth) abscesses mycobacteria (ATCC 19977) are taken as strains to respectively produce proteins corresponding to four groups, and then the four types of proteins are mixed in a ratio of 1:1:1:1, the mixed protein is mixed into mixed protein, namely, a pure protein derivative of nontuberculous mycobacteria, thereby ensuring the sensitivity of identification;
based on the product, the application and the use method, the product, the application and the use method are provided based on the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein, so that tuberculosis and NTM (tuberculosis and NTM) diseases can be identified quickly and accurately, and reliable basis is provided for early diagnosis, treatment and the like of patients.
In order to achieve the technical purpose, the following technical scheme is proposed:
first, the present technical solution provides: a product based on a non-tuberculous mycobacterial pure protein derivative and a recombinant tuberculous bacillus fusion protein, the product comprises the non-tuberculous mycobacterial pure protein derivative and the recombinant tuberculous bacillus fusion protein, wherein the non-tuberculous mycobacterial pure protein derivative is as follows: the Mycobacterium kansasii, mycobacterium gordonii, mycobacterium intracellulare and Mycobacterium abscessus are taken as strains respectively, and the produced corresponding proteins are expressed in a ratio of 1:1:1:1 mixing the protein mixture.
Furthermore, the use concentration of the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein is 5 mug/mL.
Further, the preparation method of the non-tuberculous mycobacterium pure protein derivative comprises the following steps:
the method comprises the following steps of taking Mycobacterium kansasii ATCC12478, mycobacterium gordonii ATCC14770, mycobacterium intracellulare ATCC13950 and Mycobacterium abscessus ATCC19977 as strains, and obtaining corresponding bacterial liquid containing metabolic proteins after amplification and inactivation;
salting out and settling, acid denaturation and settling, desalting and ultra-filtering and sterilizing and filtering corresponding bacterial liquid to obtain corresponding protein stock solution;
the corresponding protein stock was tested, 1:1:1:1 mixing, diluting and packaging to obtain the finished product of the non-tuberculous mycobacterium pure protein derivative.
Wherein, in the amplification, amplification of the Roche egg culture medium is firstly carried out, and then amplification of the semi-solid culture medium is carried out. Preparing a large batch of thalli in amplification of a Roche egg culture medium, realizing large-scale production and preparing for subsequent batch finished products; in the amplification of the semi-solid culture medium, the metabolic protein of the thalli is convenient to collect, on one hand, the metabolic protein is connected with a large batch of thalli formed in the amplification of the Roche egg culture medium, and on the other hand, the method is suitable for the preparation of subsequent protein stock solution and finished products;
wherein the semi-solid culture medium is a potato Souton semi-solid culture medium, and comprises the following components in 1000 mL: 8.0g of sodium glutamate, 0.5g of dipotassium phosphate, 2.0g of citric acid, 0.5g of magnesium sulfate, 0.05g of ferric ammonium citrate, 60mL of glycerol and 940mL of water for injection. Subpackaging 500mL triangular bottles and 180mL bottles, and placing potato blocks in each bottle.
In the desalting ultrafiltration, an ultrafiltration device with the molecular weight cutoff of 50KD is adopted for desalting, ammonium sulfate and trichloroacetic acid residues introduced in the sedimentation are removed, and the protein purity in the finished product is improved.
Wherein, in the degerming filtration, degerming filtration equipment with the pore diameter of 0.2 mu m is adopted, and the degerming filtration equipment with the precision is further limited, so that the protein purity in a finished product is effectively improved.
Wherein in the dilution, 0.01mol/L PBS solution including 0.0005% polysorbate-80 at pH7.3 is used.
Wherein, the sub-packaging of the non-tuberculous mycobacterium pure protein derivative finished product adopts penicillin bottles and a pre-encapsulation mode.
Secondly, this technical scheme provides: the combination of the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein, and the application in the preparation of products for identifying tuberculosis and non-tuberculous mycosis.
Wherein, in the product application, when delayed hypersensitivity of the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein is positive (except pathogenic bacteria-Mycobacterium kansasii), the product is tuberculosis; nontuberculous mycobacteria disease when delayed hypersensitivity of the pure protein derivative of nontuberculous mycobacteria is positive and delayed hypersensitivity of the recombinant tuberculous bacillus fusion protein (EEC) is negative.
Finally, the technical solution provides: a method of using a product based on a non-tubercular mycobacterium pure protein derivative and a recombinant tubercular bacillus fusion protein, comprising:
A. injecting non-tubercular mycobacterium pure protein derivative and recombinant tubercular bacillus fusion protein into the organism to be tested to perform skin test reaction;
B. and judging whether the organism to be detected is tuberculosis or non-tuberculosis mycobacteriosis according to the result of the skin test reaction.
Wherein the judgment standard is that when delayed hypersensitivity of the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein are positive, the non-tuberculous mycobacterium pure protein derivative is tuberculosis;
when the delayed hypersensitivity of the non-tuberculous mycobacterium pure protein derivative is positive and the delayed hypersensitivity of the recombinant tubercle bacillus fusion protein is negative, the non-tuberculous mycobacterium disease is detected.
Further, the judgment standard of the skin test reaction is as follows:
measuring the transverse diameter and the longitudinal diameter of the induration of the injection part 24-48 hours after subcutaneous injection, taking the mean value of the transverse diameter and the longitudinal diameter as the skin test reaction diameter of the injection sample, and defining the skin test reaction diameter as a positive reaction if the skin test reaction diameter is more than or equal to 5 mm; otherwise, the reaction is negative.
By adopting the technical scheme, the beneficial technical effects brought are as follows:
the invention combines the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein, is applied to the preparation of products for identifying tuberculosis and non-tuberculous mycosis, has higher significance, high accuracy and short identification time (within 24h, the result appears); provides reliable theoretical basis and reference basis for accurate treatment scheme, clinical research and the like of patients.
Wherein, the non-tuberculous mycobacterium pure protein derivatives are: the Mycobacterium kansasii, mycobacterium gordonii, mycobacterium intracellulare and Mycobacterium abscessus are respectively taken as strains, and the produced corresponding proteins are mixed according to the proportion of 1:1:1:1, the mixed protein has larger hypersensitive reaction size than a single protein (such as a pure protein derivative of the mycobacterium intracellulare), so that high sensitivity can be effectively ensured.
Detailed Description
The present invention is further described in the following description of the specific embodiments, which is not intended to limit the invention, but various modifications and improvements can be made by those skilled in the art according to the basic idea of the invention, within the scope of the invention, as long as they do not depart from the basic idea of the invention.
For nontuberculous mycobacteriosis (NTM disease), NTM prevalence in south and east of China is higher than that in other regions, and partial literature data of major thoracic hospitals and colleges and universities throughout the country are shown in Table 1 below.
TABLE 1
In the following examples, M.kansasii (ATCC 12478), M.gordonii (ATCC 14770), M.intracellulare (ATCC 13950) and M.abscessus (ATCC 19977) were all from American type culture Collection collections;
mycobacterium tuberculosis attenuated strains (CMCC 93020), mycobacterium kansasii (CMCC 95013), mycobacterium marinum (CMCC 95014), mycobacterium simiae (CMCC 95015), mycobacterium gordonii (CMCC 95018), su Jia (CMCC 95019), mycobacterium scrofulae (CMCC 95017), mycobacterium intracellulare (CMCC 95002), mycobacterium avium (CMCC 95001), mycobacterium abscessus (CMCC 95021), mycobacterium cheloniae (CMCC 95020) and Mycobacterium fortuitum (CMCC 95022), which are all from the Chinese medical science collection and management center.
Example 1
This example discusses the use of non-tubercular mycobacterium pure protein derivatives in combination with recombinant tubercular bacillus fusion proteins to induce delayed hypersensitivity in guinea pigs sensitized with a tubercular mycobacterium attenuated strain (CMCC 93020) for further illustration of this protocol.
S1: guinea pig
Guinea pigs weighing 300-500g spf grade were kept in IVC cages (3 per cage, change litter 2 times per week, free access to water).
S2: skin test
Tuberculin pure protein derivative skin test negative guinea pigs.
S3: sensitization source
Sterilizing Mycobacterium tuberculosis attenuated strain (CMCC 93020) sensitization source (50 mg/mL) at 121 ℃ for 20min.
S4: sensitization
The inguinal region was injected with 0.2mL of allergen bacteria solution (50 mg/mL), and the skin test was performed 3 weeks after the first sensitization, and 2 weeks after the second sensitization.
S5: delayed hypersensitivity reaction
Removing hair on the back of a guinea pig, injecting 0.1mL of non-tuberculous mycobacterium pure protein derivative (5 mu g/mL) and 0.1mL of recombinant tubercle bacillus fusion protein (5 mu g/mL) into the skin, recording the transverse diameter and the longitudinal diameter of each part at 24h and 48h respectively, calculating the total average diameter of 24h and 48h, detecting the effect of the sensitization induction delayed hypersensitivity of the tubercle mycobacterium attenuated strain (CMCC 93020) of the guinea pig, and knowing that the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein are all positive to the sensitization induction delayed hypersensitivity of the tubercle mycobacterium attenuated strain (CMCC 93020) of the guinea pig. The results obtained are shown in table 2 below:
TABLE 2 results of experiments on non-tubercular mycobacterium pure protein derivatives and recombinant tubercular bacillus fusion proteins for sensitizing animals to mycobacterium tuberculosis attenuated strain (CMCC 93020)
Wherein, the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein are all positive for the induction delayed hypersensitivity of guinea pig sensitized by the tubercle mycobacterium attenuated strain, and meet the judgment standard of tuberculosis; the model is consistent with the judgment result.
Example 2
This example discusses the further demonstration of the present protocol that non-tuberculous mycobacterial pure protein derivatives in combination with recombinant tuberculous bacillus fusion proteins induce delayed hypersensitivity in guinea pigs sensitized to Mycobacterium kansasii (CMCC 95013), mycobacterium marinum (CMCC 95014) and Mycobacterium ape (CMCC 95015) major pathogenic bacteria of non-tuberculous mycobacterial group I (slow growth, photogenesis).
S1: guinea pig
Guinea pigs weighing 300-500g spf grade were kept in IVC cages (3 per cage, change litter 2 times per week, free access to water).
S2: skin test
Tuberculin pure protein derivative skin test negative guinea pigs.
S3: sensitization source
A sensitization source (50 mg/mL) of Mycobacterium kansasii (CMCC 95013), mycobacterium marinum (CMCC 95014) and Mycobacterium simian (CMCC 95015) was sterilized at 121 ℃ for 20min.
S4: sensitization
The inguinal region is injected with 0.2mL of allergen bacteria solution (50 mg/mL), and the skin test can be performed 3 weeks after the first sensitization and 2 weeks after the second sensitization.
S5: delayed hypersensitivity reaction
Removing hair from the back of a guinea pig, injecting 0.1mL of nontuberculous mycobacterium pure protein derivative (5 mu g/mL) and 0.1mL of recombinant tubercle bacillus fusion protein (5 mu g/mL) intradermally, recording transverse diameter and longitudinal diameter of each part at 24h and 48h respectively, calculating the total average diameter of 24h and 48h, detecting the effect of the guinea pig on the induction of delayed hypersensitivity reaction by the sensitization of Mycobacterium kansasii (CMCC 95013), mycobacterium marini (CMCC 95014) and Mycobacterium simiae (CMCC 95015), and obtaining that the nontuberculous mycobacterium pure protein derivative has positive delayed hypersensitivity reaction on the guinea pig of major pathogenic bacteria of nontuberculous mycobacterium I group (slow growth and photogenesis) (CMCC 95013), mycobacterium marini (CMCC 95014) and Mycobacterium simian (CMCC 95015); the recombinant tubercle bacillus fusion protein is negative to its delayed hypersensitivity (M.kansasii). The results obtained are shown in tables 3 to 5 below:
TABLE 3 results of experimental results of the non-tuberculous mycobacteria pure protein derivatives and recombinant tubercle bacillus fusion proteins on the sensitization of Mycobacterium kansasii (CMCC 95013)
TABLE 4 results of experiments on the sensitization of non-tuberculous mycobacteria pure protein derivatives and recombinant tubercle bacillus fusion proteins to Mycobacterium marinum (CMCC 95014)
TABLE 5 animal experiment results of sensitization of non-tuberculous mycobacteria pure protein derivatives and recombinant tubercle bacillus fusion protein to simian mycobacteria (CMCC 95015)
Wherein, the pure protein derivative of nontuberculous mycobacteria is all positive to the delayed hypersensitivity reaction induced by guinea pigs sensitized by Mycobacterium kansasii, mycobacterium marinum and Mycobacterium simian; the recombinant mycobacterium tuberculosis fusion protein is all positive to delayed hypersensitivity induced by guinea pigs sensitized by mycobacterium kansasii (the judgment standard is not met, the model and the judgment result are inconsistent, because the mycobacterium kansasii contains the same antigens ESAT-6 and CFP-10 as the mycobacterium tuberculosis, the delayed hypersensitivity of the recombinant mycobacterium tuberculosis melting protein (EEC) is positive); the recombinant mycobacterium tuberculosis fusion protein is negative to guinea pig induced delayed hypersensitivity sensitized by mycobacterium marinum and mycobacterium simiae, and meets the judgment standard of non-tuberculosis; the model is consistent with the judgment result.
Example 3
This example discusses the delayed hypersensitivity induced by the combination of pure protein derivatives of nontuberculous mycobacteria and recombinant tuberculous bacillus fusion protein in guinea pigs sensitized to Mycobacterium gordonii (CMCC 95018), su Jia (CMCC 95019) and Mycobacterium scrofulae (CMCC 95017) as the main pathogenic bacteria of nontuberculous mycobacteria group II (slow growth and dark color production), and further illustrates the present technical solution.
S1: guinea pig
Guinea pigs weighing 300-500g spf grade were kept in IVC cages (3 per cage, change litter 2 times per week, free access to water).
S2: skin test
Tuberculin pure protein derivative skin test negative guinea pigs.
S3: sensitization source
A sensitization source (50 mg/mL) of Mycobacterium kansasii (CMCC 95013), mycobacterium marinum (CMCC 95014) and Mycobacterium simian (CMCC 95015) was sterilized at 121 ℃ for 20min.
S4: sensitization
The inguinal region is injected with 0.2mL of allergen bacteria solution (50 mg/mL), and the skin test can be performed 3 weeks after the first sensitization and 2 weeks after the second sensitization.
S5: delayed hypersensitivity reaction
Removing hair from the back of a guinea pig, injecting 0.1mL of a pure protein derivative of nontuberculous mycobacteria (5 mu g/mL) and 0.1mL of a recombinant tuberculous bacillus fusion protein (EEC) (5 mu g/mL) into the skin, recording the transverse diameter and the longitudinal diameter of each part at 24h and 48h respectively, calculating the total average diameter of 24h and 48h, detecting the effect of the Gordon mycobacteria (CMCC 95018), su Jia mycobacteria (CMCC 95019) and Mycobacterium scrofulaceus (CMCC 95017) on inducing delayed hypersensitivity of the guinea pig, and knowing that the pure protein derivative of nontuberculous mycobacteria presents delayed hypersensitivity to the guinea pig induced positive hypersensitivity of the main pathogenic bacteria of nontuberculous mycobacteria I group (slow growth and photochromatogenesis) (CMCC 95018), su Jia mycobacteria (CMCC 95019) and Mycobacterium scrofulaceus (CC 95017); and the recombinant tubercle bacillus fusion protein (EEC) is negative to the delayed hypersensitivity (except Kansas). The results obtained are shown in tables 6 to 8 below:
TABLE 6 results of experiments on animals sensitized with Mycobacterium non-tuberculosis (CMCC 95018) recombinant Mycobacterium tuberculosis fusion protein and purified protein derivatives of Mycobacterium non-tuberculosis
TABLE 7 results of experiments on the sensitization of Su Jia Mycobacterium (CMCC 95019) to animals by non-tuberculous mycobacterial pure protein derivatives and recombinant tuberculous mycobacterial fusion proteins
TABLE 8 results of animal experiments on Mycobacterium scrofulae (CMCC 95017) sensitization by non-tubercle bacillus pure protein derivatives and recombinant Mycobacterium tuberculosis fusion protein
The non-tuberculous mycobacterium pure protein derivative is positive to induction delayed hypersensitivity of guinea pigs sensitized by Mycobacterium gordonae, su Jia and Mycobacterium scrofulae; the recombinant mycobacterium tuberculosis fusion protein is negative to late hypersensitivity induced by guinea pigs sensitized by mycobacterium gordoniae, su Jia and mycobacterium scrofulaceum, and meets the judgment standard of non-tuberculosis; the model is consistent with the judgment result.
Example 4
This example discusses the further demonstration of the present protocol by combining pure protein derivatives of non-tuberculous mycobacteria with recombinant tuberculous bacillus fusion proteins to induce delayed hypersensitivity in guinea pigs sensitized with M.intracellulare (CMCC 95002) and M.avium (CMCC 95001) which are the major pathogenic bacteria of the non-tuberculous mycobacterial group III (slow-growing, non-chromogenic).
S1: guinea pig
Guinea pigs weighing 300-500g spf grade were kept in IVC cages (3 per cage, change litter 2 times per week, free access to water).
S2: skin test
Tuberculin pure protein derivative skin test negative guinea pigs.
S3: sensitization source
A sensitization source (50 mg/mL) of Mycobacterium gordonae (CMCC 95018), mycobacterium Su Jia (CMCC 95019) and Mycobacterium scrofulaceum (CMCC 95017) was taken and sterilized at 121 ℃ for 20min.
S4: sensitization
The inguinal region is injected with 0.2mL of allergen bacteria solution (50 mg/mL), and the skin test can be performed 3 weeks after the first sensitization and 2 weeks after the second sensitization.
S5: delayed hypersensitivity reaction
Removing hair from the back of a guinea pig, injecting 0.1mL of a pure protein derivative (5 mu g/mL) of non-tuberculous mycobacteria and 0.1mL of a recombinant tubercle bacillus fusion protein (EEC) (5 mu g/mL) into the skin, recording the transverse diameter and the longitudinal diameter of each part at 24h and 48h respectively, calculating the total average diameter of 24h and 48h, detecting the effect of the delayed hypersensitivity induced by sensitization of the guinea pig of the intracellular mycobacteria (CMCC 95002) and the avian mycobacteria (CMCC 95001), and obtaining that the pure protein derivative of the non-tuberculous mycobacteria has positive induction delayed hypersensitivity to the guinea pig sensitized by the intracellular mycobacteria (CMCC 95002) and the avian mycobacteria (CMCC 95001) which are main pathogenic bacteria of the non-tuberculous mycobacteria II group (slow growth and dark color generation); the recombinant tubercle bacillus fusion protein (EEC) is negative to the delayed hypersensitivity reaction. The results obtained are shown in tables 9 to 10 below:
TABLE 9 results of animal experiments for sensitization of non-tuberculous mycobacteria pure protein derivatives and recombinant tuberculous bacillus fusion proteins to M.intracellulare (CMCC 95002)
TABLE 10 results of animal experiments on Mycobacterium avium (CMCC 95001) sensitization by non-tuberculous mycobacterial pure protein derivatives and recombinant tubercle bacillus fusion proteins
The non-tuberculous mycobacterium pure protein derivative is positive to delayed hypersensitivity induced by guinea pigs sensitized by intracellular mycobacterium and mycobacterium avium; the recombinant mycobacterium tuberculosis fusion protein is negative to guinea pig induced delayed hypersensitivity sensitized by intracellular mycobacteria and avian mycobacteria, and meets the judgment standard of non-tuberculosis; the model is consistent with the judgment result.
Example 5
This example discusses the use of pure protein derivatives of Mycobacterium nontuberculosis in combination with recombinant Mycobacterium tuberculosis fusion proteins to induce delayed hypersensitivity in guinea pigs sensitized with Mycobacterium nontuberculosis IV (fast growing) group (CMCC 95021), mycobacterium chelonii (CMCC 95020) and Mycobacterium fortuitum (CMCC 95022) for further elucidation of the present technical scheme.
S1: guinea pig
Guinea pigs weighing 300-500g spf grade were kept in IVC cages (3 per cage, change litter 2 times per week, free access to water).
S2: skin test
Tuberculin pure protein derivative skin test negative guinea pigs.
S3: sensitization source
Sterilizing Mycobacterium abscessus (CMCC 95021), mycobacterium chelonii (CMCC 95020) and Mycobacterium fortuitum (CMCC 95022) allergen (50 mg/mL) at 121 deg.C for 20min.
S4: sensitization
The inguinal region was injected with 0.2mL of allergen bacteria solution (50 mg/mL), and the skin test was performed 3 weeks after the first sensitization, and 2 weeks after the second sensitization.
S5: delayed hypersensitivity reaction
Removing hair from the back of a guinea pig, injecting 0.1mL of non-tuberculous mycobacterium pure protein derivative (5 mu g/mL) and 0.1mL of recombinant tubercle bacillus fusion protein (EEC) (5 mu g/mL) intradermally, recording the transverse diameter and the longitudinal diameter of each hard knot at 24h and 48h respectively, calculating the total average diameter of 24h and 48h, detecting the effect of sensitization induction of delayed hypersensitivity of guinea pigs by mycobacterium abscessus (CMCC 95021), mycobacterium cheloniae (CMCC 95020) and mycobacterium fortuitum (CMCC 95022), and knowing that the non-tuberculous mycobacterium pure protein derivative has positive delayed hypersensitivity of guinea pigs sensitized by non-tuberculous mycobacterium IV group (fast-growing) main mycobacterium abscessus (CMCC 95021), mycobacterium tryanus (CMCC 95020) and mycobacterium fortuitum (CMCC 95022); the recombinant tubercle bacillus fusion protein (EEC) is negative to the delayed hypersensitivity reaction. The results obtained are shown in tables 11 to 13 below:
TABLE 11 results of animal experiments on the sensitization of non-tuberculous mycobacteria pure protein derivatives and recombinant tubercle bacillus fusion proteins to mycobacterium abscessus (CMCC 95021)
TABLE 12 results of animal experiments on Mycobacterium cheloniae (CMCC 95020) sensitization by non-tubercle bacillus pure protein derivatives and recombinant tubercle bacillus fusion proteins
TABLE 13 results of animal experiments for sensitization of Mycobacterium fortuitum (CMCC 95022) by non-tuberculous mycobacterial pure protein derivatives and recombinant tuberculous bacillus fusion proteins
The non-tuberculous mycobacterium pure protein derivative is completely positive to delayed hypersensitivity induced by guinea pigs sensitized by mycobacterium abscessus, mycobacterium cheloni and mycobacterium fortuitum; the recombinant mycobacterium tuberculosis fusion protein is negative to delayed hypersensitivity induced by guinea pigs sensitized by mycobacterium abscessus, mycobacterium cheloni and mycobacterium fortuitum, and meets the judgment standard of non-tuberculosis; the model is consistent with the judgment result.
Claims (1)
1. The non-tuberculous mycobacterium protein derivative and the recombinant tubercle bacillus fusion protein are combined to be applied to the preparation of the identification products of tuberculosis and non-tuberculous mycosis, wherein the non-tuberculous mycosis is caused by simian mycobacterium CMCC 95015; the recombinant tubercle bacillus fusion protein EEC consists of recombinant tubercle bacillus Esat-6 protein and CFP-10 protein; the non-tuberculous mycobacterium pure protein derivative takes Mycobacterium kansasii ATCC12478, mycobacterium gordonii ATCC14770, mycobacterium intracellulare ATCC13950 and Mycobacterium abscessus ATCC19977 as strains to respectively produce corresponding proteins, and then the four types of proteins are expressed by a ratio of 1:1:1:1, mixing to obtain;
the preparation method of the non-tuberculous mycobacterium pure protein derivative comprises the following steps: the method comprises the following steps of taking Mycobacterium kansasii ATCC12478, mycobacterium gordonii ATCC14770, mycobacterium intracellulare ATCC13950 and Mycobacterium abscessus ATCC19977 as strains, and obtaining corresponding bacterial liquid containing metabolic proteins after amplification and inactivation;
salting out and settling, acid denaturation and settling, desalting and ultra-filtering, and sterilizing and filtering the corresponding bacterial liquid to obtain a corresponding protein stock solution;
the corresponding protein stock was tested, 1:1:1:1, mixing, diluting and subpackaging to obtain a finished product of the pure protein derivative of the nontuberculous mycobacterium;
the standard of the identification is that when the delayed hypersensitivity reaction of the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein is positive, the non-tuberculous mycobacterium pure protein derivative and the recombinant tubercle bacillus fusion protein are tuberculosis; when the delayed hypersensitivity of the non-tuberculous mycobacterium pure protein derivative is positive and the delayed hypersensitivity of the recombinant tubercle bacillus fusion protein is negative, the non-tuberculous mycobacterium disease is detected.
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