CN104419710B - A kind of resistance new gene of mycobacterium tuberculosis and its application - Google Patents
A kind of resistance new gene of mycobacterium tuberculosis and its application Download PDFInfo
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- CN104419710B CN104419710B CN201310398783.9A CN201310398783A CN104419710B CN 104419710 B CN104419710 B CN 104419710B CN 201310398783 A CN201310398783 A CN 201310398783A CN 104419710 B CN104419710 B CN 104419710B
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Abstract
The invention provides a kind of drug resistance of Mycobacterium tuberculosis new gene and its application, following any one albumen of the gene code:i)The protein sequence includes the SEQ ID No.2 sequences in amino acid and/or nucleotides sequence list;ii)Sequence i)Through substitution, lack or be superimposed one or several amino acid derived protein sequences and with sequence i)Identical function.Susceptibility detection finds that the gene enables to bacterium to produce drug resistance to various antituberculotics.
Description
Technical field
The present invention relates to a kind of genetic fragment, more particularly to a kind of mycobacterium tuberculosis resistance new gene.
Background technology
Tuberculosis is a kind of chronic infectious disease of serious harm people's health, wherein, mycobacterium tuberculosis
(M.tuberculosis, is commonly called as mycobacterium tuberculosis), it is to cause pathogen lungy.Mycobacterium tuberculosis can invade whole body
Each organ, but with pulmonary tuberculosis as most common, China is one of serious country of 22, whole world TB endemic.Although, with life
Running water is flat to be improved and hygienic state improvement, has particularly carried out mass prevention and mass treatment, the universal bcg vaccination of children so that lungy
Morbidity and mortality are greatly lowered.But the appearance of resistance and substance of medicines-resistant branched tubercle bacillus causes situation lungy increasingly
Sternness, the how resistance to bacterial strain of mycobacterium tuberculosis gradually increases all over the world in recent years, or even causes outbreak of epidemic, China the 4th time
Tuberculosis epidemiological random sampling survey shows, the total resistant rate of China's tuberculosis is with close to 30%.
The resistance of mycobacterium tuberculosis can be produced by spontaneous mutation(Primary drug resistance)Or by the mutated selection of inappropriate medication
Produce(Secondary resistance), but how resistance to generation may be mainly due to the latter.With going deep into for resistance Journal of Sex Research, it has been found that
The generation of Drug Resistance of Mycobacterium Tuberculosis is mostly the resistance base having now been found that caused by specific gene site is undergone mutation
Because include KatG, inhA, kasA, ndh, oxyR-ahpC intergenic region, rpoB, embB, embC, embA, pncA, rrs,
The generation of the resistance to isoniazid of gyrA and gyrB genes etc., such as mycobacterium tuberculosis encodes base with Catalase-peroxidase
Because the missing of KatG genes is relevant with mutation, and also there is correlation with the mutation of inhA genes;The generation of resistance to ethambutol and EMB
The mutation of gene is relevant;The generation of resistance to streptomycin is then due to its ribosomal protein S1 2 encoding gene rpsL and 16SrRNA coding
Rrs missing and mutation caused by;The generation of resistance to rifampin is relevant with the rpoB gene mutations of its nucleus.Therefore, to tuberculosis
The research of mycobacteria drug resistant gene and its encoding proteins helps further to strengthen the treatment and prevention to mycobacterium tuberculosis.
Although having announcement for the drug resistant gene of single antituberculotic, known drug resistant gene is only capable of explanation portion more
Divide the resistance situation of clinical strains, whole resistances can not be explained, especially the multi-drug resistant situation of clinical strains.Current resistance
The propagation of mycobacterium tuberculosis, especially substance of medicines-resistant branched tubercle bacillus and the problem for being still Tubercufosis control is spread,
Therefore, the resistance new gene occurred in bacterium is found in time, and especially those are related to the multi-drug resistant generation of bacterium
Gene, exploitation and preventing and treating lungy to new antituberculotic have great importance.
The content of the invention
Based on background above, the invention provides a kind of resistance new gene of new mycobacterium tuberculosis, and by described
The albumen of gene code, and their application.
One side of the invention is to provide a kind of resistance new gene of mycobacterium tuberculosis, and the gene code is appointed as follows
Anticipate a kind of protein sequence:
i)The protein sequence includes the SEQ ID No.2 sequences in amino acid and/or nucleotides sequence list;
ii)Sequence i)By replacing, lacking or being superimposed one or several amino acid derived protein sequences and can make
Mycobacterium tuberculosis produces the protein sequence of resistance.
In the resistance new gene described in present invention one side, the protein sequence is preferably sequence i).
In the resistance new gene described in present invention one side, the coding region nucleotide sequence of the gene includes ammonia
SEQ ID No.1 sequences in base acid and/or nucleotides sequence list.
Second aspect of the invention is to provide a kind of protein sequence, and the protein sequence is selected from following any one albumen sequence
Row:
i)The protein sequence includes the SEQ ID No.2 sequences in amino acid and/or nucleotides sequence list;
ii)Sequence i)Through substitution, lack or be superimposed one or several amino acid derived protein sequences and with
Sequence i)Identical function.
In the resistance new gene described in second aspect of the invention, the protein sequence is preferably sequence i).
Third aspect of the present invention is to provide a kind of recombinant vector, and the carrier contains described in present invention one side
Any one resistance new gene.
4th aspect of the invention is to provide a kind of transformant, and the transformant contains described in present invention one side
Any one resistance new gene, or the recombinant vector described in third aspect of the present invention.
Preferably, the transformant is antibody-resistant bacterium.
5th aspect of the invention is to provide a kind of method for preparing above-mentioned transformant, and step includes:By the present invention first
Resistance new gene described in individual aspect is imported into host.
The 6th aspect of the present invention is to provide a kind of engineered antibody prepared for above-mentioned arbitrary protein sequence, wherein, institute
Stating engineered antibody being capable of mycobacterium tuberculosis described in immune detection, and/or the caused sense of mycobacterium tuberculosis described in immunization therapy
Dye disease.
The 7th aspect of the present invention is to provide a kind of above-mentioned any one transformant and is preparing prevention mycobacterium tuberculosis
Application in immune formulation.
It will be apparent to a skilled person that immune formulation of the present invention can be the vaccine being made up of bacterium,
And be made up of virus, Richettsia, conveyor screw is vaccine etc..
The present invention isolates 3 from the tuberculosis section patient culture positive, the sputum specimen of smear-positive of Shanghai Pulmonary Hospital
Strain is to 4 kind of one line antituberculotic such as rifampin, ethambutol, isoniazid, streptomysin while the mycobacterium tuberculosis of resistance, leads to
Cross the coding region nucleotide sequence that PCR amplification sequencings obtain a kind of drug resistant gene(SEQ ID NO.1)And its response egg of coding
Bai Xulie(SEQ ID NO.2), by the genetic transformation to the wild type mycobacterium smegmatis without the gene(ATCC19420)
In, susceptibility detection finds, the gene enables to mycobacterium smegmatis to produce drug resistance to various antituberculotics.
Resistance new gene encoding proteins of the present invention, can realize to rifampin, ethambutol, isoniazid, strepto-
The resistance to the action of a drug of the one line antituberculotics such as element, therefore, for the albumen preparation engineering antibody, can play tied described in immune detection
The purpose of infectious disease caused by mycobacterium tuberculosis described in core mycobacteria, and/or immunization therapy.Additionally, preparing resistive connection
During nuclear pharmaceuticals, can be with the treating tuberculosis performance of the medicine prepared by more convenient checking by the encoding proteins.
Brief description of the drawings
Fig. 1 is the functional sequence schematic diagram of plasmid transduction experiment and prokaryotic expression experimental verification new gene.
Specific embodiment
3 plants are isolated to 4 one from tuberculosis section of Shanghai Pulmonary Hospital patient culture positive, the sputum specimen of smear-positive
Line antituberculotic rifampin, ethambutol, isoniazid, streptomysin are while the mycobacterium tuberculosis of resistance.
The bacterial strain of above-mentioned acquisition is cultivated 2 weeks in Michaelis 7H9 fluid nutrient mediums, collects thalline, obtain genomic DNA and make
It is template, design primer enters performing PCR amplification, amplified production is sequenced, and obtains the coding region nucleotide sequence of the drug resistant gene
(SEQ ID NO.1)And the corresponding protein sequence of coding(SEQ ID NO.2).Compared by sequence homology, finding should
Sequence be due in mycobacterium tuberculosis gene group DNA RD105 areas missing produce fusion MFG71/74, the gene by
The nucleotide sequence in the 79486-795567 areas of mycobacterium tuberculosis reference culture H37Rv and the nucleotides in 83034-83983 areas
Sequence is constituted;Repetitive sequence containing 9bp in 79486-795567 areas(GGTGGACCC), in H37Rv bacterial strains, the 9bp weights
The repeat number of complex sequences is 5, and its repeat number is 9 in new fusion(Corresponding amino acid is 9 VDP of repetition), by this
Unnamed gene is MFG71/74-9.
It is of the invention by MFG71/74-9 gene weights in order to confirm that the resistance that recipient bacterium obtains derives from MFG71/74-9 genes
Group is transformed into the mycobacterium smegmatis of wild type in PVV16 carriers(ATCC19420, the bacterium does not contain MFG71/74
The homologous gene of gene)In, by the Michaelis 7H10 solid plate screening positive clones containing hygromycin(Contain PVV16-
The bacterium of MFG71/74-9 recombinant plasmids), positive colony is done into drug sensitive test, as a result show that MFG71/74-9 genes can make shame
Dirty mycobacteria has to following Drug-resistant:Ethambutol, streptomysin, rifampicin isoniazid, amikacin, capreomycin,
Linezolid, Rifabutin, cefoxitin sodium, TOB.
Embodiment 1
First, material and method
1.Axgen plasmids are small to take out kit, the PMD18-T support agents box of TakaRa companies, the DNA of Beijing Tiangeng company
Gel reclaims kit, the high-fidelity enzyme of TakaRa companies,
2nd, method
The separation of 2.1 mycobacterium tuberculosis containing MFG71/74-9 genes and the susceptibility detection for the treatment of tuberculosis first-line drug
By the sputum specimen from tuberculosis section of Shanghai Pulmonary Hospital patient, the NaOH solution of isometric 4wt%, whirlpool are accessed
After rotation vibration 15-20min, the PBS of 20-40mL0.067mol/L is added to mix.3000g is centrifuged 30min, removes supernatant, sediment
0.5mL PBS are added to mix;Take the treatment sample of 0.1ml, sterile working inoculation acid Russell medium, 37 DEG C of cultures to bacterium colony
Grow, then write according to basis Professional Committee of Chinese anti-tuberculosis association《Diagnosis of tuberculosis laboratory inspection code》Carry out bacterium
Plant identification and drug sensitive experiment.
2.2PCR expands resistance new gene
(1)Extracting genome DNA:The bacterial strain of above-mentioned acquisition is cultivated 2 weeks in Michaelis 7H9 fluid nutrient mediums, 80 DEG C go out
1h living, 10000r/min centrifugation 10min, collects thalline abandon nutrient solution.Bacterial sediment is resuspended with physiological saline, 10000r/
Min centrifuge washing 5min, abandon supernatant, repeated washing 1 time.Addition DNA extraction lysates, 50 DEG C of water-bath 1h, 100 DEG C are boiled
10min, 12000r/min are centrifuged 10min, supernatant is moved into new centrifuge tube and obtains genomic DNA as template;
(2)Amplimer is designed according to MFG71/74-9 gene orders.Primer sequence is as follows:
F:5’-CGGGATCCATGAGTTCGATCACGGTGTC-3’
R:5’-CGAAGCTTTTATGACGGGGTGTTGTAGCC-3’
It is template with the genomic DNA for extracting, PCR reaction systems are as follows:5×PrimerSTAR buffer (Mg2+
plus)10μL;dNTP Mixture(Each 2.5mM)4 μ L, primer(10μM)Each 1 μ L;DNA1μL;PrimerSTAR HS DNA
Polymerase(2.5U/μL)0.5μL;ddH2O complements to 50 μ L.
PCR amplification conditions are:98℃、10sec;68℃、1min;72 DEG C, 1min, totally 30 circulation;Last 72 DEG C of extensions
10min;PCR primer is detected with 1% agarose gel electrophoresis, and reclaims DNA with the DNA gel QIAquick Gel Extraction Kit of Tiangeng.
(3)Cloning and sequencing
After the PCR primer of recovery is added into A using TakaRa companies plus A kits, connection18-T simple
Vector, and be cloned into E.coli DH5 α, it is applied on the Amp LB culture mediums containing 50 μ g/mL, 37 DEG C of incubated overnights.Picking
Positive colony(Bacterium containing MFG71/74-9 recombinant plasmids)Serve Hai Shenggong, using universal primer PrimerRv-M and
Primer M13-47 are sequenced.
3rd, result
1)The identification of separated antibody-resistant bacterium and the susceptibility testing result for the treatment of tuberculosis first-line drug
Separation strains biochemical characteristic:The elongated Gram-positive bacillus for slightly bending, acid-fast stain is positive, and colonial morphology is dish
It is flower-shaped, paranitrobenzoic acid(PNB)Growth is negative, thiophene-2-carboxylic acid hydrazine(TCH)Growth is positive, not chromogenic, meets tuberculosis branch
The biochemical characteristic of bacillus, through 16SrRNA sequencing identifications, is mycobacterium tuberculosis, be respectively designated as FK9m-1, FK9m-2,
FK9m-3。
Separation strains treating tuberculosis first-line drug drug sensitive experiment, from minimal inhibitory concentration(MIC)Value(Table 1)Display:3 plants of clinics point
From strain to 4 kinds of equal resistances of first-line drug, wherein most strong to the drug resistance of rifampin, streptomysin.
The one line antituberculotic drug sensitivity tests of 1,3 clinical separation strains of table
2)The determination of MFG71/74-9 new genes:
Cloning and sequencing machine sequence alignment shows that FK9m-1, FK9m-2, FK9m-3 have identical MFG71/74 nucleotides sequences
Row(Shown in SEQ ID NO.1), the protein sequence of its coding is SEQ ID NO.2.Compared by sequence homology, finding should
Sequence is due to the new fusion MFG71/74 that the missing in RD105 areas in mycobacterium tuberculosis gene group DNA is produced, the gene
By the nucleotide sequence and the nucleotides in 83034-83983 areas in mycobacterium tuberculosis reference culture H37Rv79486-795567 areas
Sequence is constituted;Repetitive sequence containing 9bp in 79486-795567 areas(GGTGGACCC), in H37Rv bacterial strains, 9bp repeats sequence
The repeat number of row is 5, and 5 VDP of correspondence repeat amino acid sequence, in the new of FK9m-1, FK9m-2, FK9m-3 Clinical isolation
Its repeat number is 9 in fusion, and 9 VDP of correspondence repeat amino acid, therefore are MFG71/74-9 by the unnamed gene.
Embodiment 2, bacterial plasmid transduction experiment and Resistance detection
1. material
E.coli DH5α;Mycobacterium smegmatis(ATCC19420);Axgen plasmids are small to take out kit;TakaRa companies
PMD18-T support agent boxes;E.coli- mycobacterial shuttle plasmids PVV16;The DNA gel reclaim reagent of Beijing Tiangeng company
Box, the T4DNA ligases and high-fidelity enzyme of the endonuclease BamHI and HindIII TakaRa companies of TakaRa companies.
2. method
The 2.1 PMD18-T-MFG71/74-9 recombinant plasmids that will be obtained in embodiment 1, are extracted using plasmid extraction kit
Afterwards, BamHI and HindIII double digestions are carried out simultaneously with PVV16, digestion products are reclaimed with DNA gel QIAquick Gel Extraction Kit, Ran Houjing
The connection of T4DNA ligases obtains PVV16-MFG71/74-9 recombinant plasmids, and recombinant plasmid transformed E.coli DH5 α are containing
There are 37 DEG C of culture screening positive clones on the flat board of Kan resistances.Acquisition PVV16- is extracted again using plasmid extraction kit
MFG71/74-9 recombinant plasmids, mycobacterium smegmatis is turned for electricity.
Structure and the susceptibility detection of the 2.2 mycobacterium smegmatis engineering bacterias containing PVV16-MFG71/74-9 recombinant plasmids
1)The preparation of mycobacterium smegmatis competent cell:The fresh single bacterium colony of picking is inoculated in 5mL Michaelis 7H9 culture mediums
In, 37 DEG C of shaken cultivations to OD600 values 0.6 or so are seeded in the Michaelis 7H9 culture mediums of 200mL, and 37 DEG C of incubated overnights are extremely
OD600 values 0.4 or so, add acetamide(0.2%)Continue to cultivate 2-3h.Culture is placed into 0.5-1h on ice, 4 DEG C,
5000rpm is centrifuged 10min collects thallines, and thalline is resuspended with the sterile glycerol of 100mL precoolings 10%, 4 DEG C, 5000rpm centrifugations
10min collects thallines, repeated washing thalline 3 times, used 10% glycerine, after blowing even thalline, with 0.2mL/ pipes packing be stored in-
80℃。
2)Shock parameters:Voltage 2.5kV, the Ω of resistance 1000, the μ F of electric capacity 25.After electric shock, the Michaelis of 1mL is added immediately
7H9 culture mediums, 37 DEG C of shaken cultivation 24h are applied to the Michaelis 7H10 resistance solid plates containing 50 μ g/mL Kan, 37 DEG C of culture 3-
5d.Picking positive colony(Bacterium containing PVV16-MFG71/74-9 recombinant plasmids).
3)The drug sensitive test of positive colony:
A. the U-shaped plate in hole of micro MIC medicines gradient 96 is prepared:Medicine dissolving is configured to 10mg/mL, 0.22 μm of sterile filters
Filtration sterilization, with Michaelis 7H9 fluid nutrient medium gradient dilutions after, according to the μ L of every hole 100 add the U-shaped plate in 96 holes.
B. by wild type mycobacterium smegmatis, the recombinant bacterial strain containing PVV16 empty carriers and PVV16-MFG71/ is contained
The positive colony bacterial strain of 74-9 recombinant plasmids in after Russell medium culture 2 weeks, with Michaelis 7H9 fluid nutrient mediums it is resuspended than it is turbid extremely
1mg/mL, is then diluted to 10 with fluid nutrient medium-3.In the U-shaped plate in 96 holes containing medicine gradient, 100 μ L are added to contain per hole
There is the fluid nutrient medium of bacterium solution, while setting growth control hole, negative control hole.After 37 DEG C of culture 4d, the U-shaped plate in 96 holes is placed in
It is inverted on magnifying glass and observes result.What naked eyes visible white bacterial sediment occurred in bottom hole is the positive, is visible by naked eyes bacterial sediment
Be feminine gender.And by positive colony drug sensitivity tests and wild type mycobacterium smegmatis and recombinant bacterial strain containing PVV16 empty carriers
Drug sensitivity tests compare, obtain positive colony bacterium resistance result.
Testing result is shown in Table 2.
Table 2, the drug sensitivity tests of the recombinant Mycobacterium smegmatis containing PVV16-FP71/74-9 plasmids
In table:
71/74-5 and 71/74-7 are by the repetitive sequence of the 9bp in FP71/74-9 genes of the present invention(GGTGGACCC)Weight
Plural number is reduced to gained gene order after 5 and 7, and its sequence and coding protein sequence are respectively as shown in SEQ ID No.5-8;Adopt
Recombinant Mycobacterium smegmatis are carried out with the methods described of embodiment 2 and carry out Resistance detection.
4)Result show recombinant bacterial strain to streptomysin, isoniazid, rifampin, ethambutol, amikacin, capreomycin,
Linezolid, cefoxitin sodium, Rifabutin resistance, TOB(It is shown in Table 2), this illustrates that MFG71/74-9 genes can make carefully
Bacterium has to various antituberculotic resistances.
The repetitive sequence of the 9bp of MFG71/74-9 genes(GGTGGACCC)After repeat number is reduced, its encoding proteins(VDP phases
Should reduce)Remain able to play the resistance to the action of a drug, but effect is not so good as MFG71/74-9 gene coded proteins.
Specific embodiment of the invention has been described in detail above, but it is intended only as example, and the present invention is not limited
It is formed on particular embodiments described above.To those skilled in the art, any equivalent modifications carried out to the present invention and
Replacement is also all among scope of the invention.Therefore, the impartial conversion made without departing from the spirit and scope of the invention and
Modification, all should be contained within the scope of the invention.
Claims (2)
1. a kind of MFG71/74-9 genes of mycobacterium tuberculosis as drug resistant gene application, it is characterised in that it is described
MFG71/74-9 gene codes sequence as shown in the SEQ ID No.2 in amino acid and/or nucleotides sequence list;Wherein, it is described
Resistance refers to following Drug-resistant:Ethambutol, streptomysin, isoniazid, rifampin, amikacin, capreomycin, profit how azoles
Amine, Rifabutin, cefoxitin sodium, TOB.
2. application according to claim 1, it is characterised in that the coding region nucleotide sequence of the MFG71/74-9 genes
The sequence shown in the SEQ ID No.1 in amino acid and/or nucleotides sequence list.
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CN105219860B (en) * | 2015-10-20 | 2019-04-16 | 中山大学 | A kind of mycobacterium tuberculosis KatG resistant mutational site detection kit |
CN111471632B (en) * | 2020-02-28 | 2022-03-08 | 中国科学院广州生物医药与健康研究院 | Construction method and application of recombinant drug-resistant BCG strain |
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AU2002337287A1 (en) * | 2001-10-12 | 2003-04-28 | Health Protection Agency | Mycobacterial antigens expressed under high oxygen tension |
CN101144099A (en) * | 2006-09-11 | 2008-03-19 | 中山大学达安基因股份有限公司 | Method and kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation thereof |
WO2010132054A1 (en) * | 2009-05-13 | 2010-11-18 | Immport Therapeutics, Inc. | Compositions and methods for immunodominant antigens of mycobacterium tuberculosis |
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AU2002337287A1 (en) * | 2001-10-12 | 2003-04-28 | Health Protection Agency | Mycobacterial antigens expressed under high oxygen tension |
CN101144099A (en) * | 2006-09-11 | 2008-03-19 | 中山大学达安基因股份有限公司 | Method and kit for detecting mycobacterium tuberculosis and drug-resistant gene mutation thereof |
WO2010132054A1 (en) * | 2009-05-13 | 2010-11-18 | Immport Therapeutics, Inc. | Compositions and methods for immunodominant antigens of mycobacterium tuberculosis |
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Accession NO:WP_016810372,hypothetical protein[Mycobacterium tuberculosis];None;《GenBank》;20130627;features,origin * |
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