CN101210045B - Mycobacterium tuberculosis Rv1009 protein used for accelerating mycobacterium growth and application thereof - Google Patents
Mycobacterium tuberculosis Rv1009 protein used for accelerating mycobacterium growth and application thereof Download PDFInfo
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Abstract
The invention relates to a mycobacterium tuberculosis Rv1009 protein applied to promoting mycobacterium growth and application thereof, pertaining to the diagnosis technology field of medical bacteriology. The prepared Rv1009 protein by using the gene engineering technique can be used for mycobacterium culturing testing, which can promote growth and metabolism, shorten culturing time and improve positive rate.
Description
Technical field
The invention belongs to Medical Bacteriology diagnostic techniques field, particularly a kind of mycobacterium tuberculosis Rv 1009 protein and application thereof that is used to promote mycobacterium growth.
Background technology
Still one of main transmissible disease of harm humans health in whole world tuberculosis, the immigrant of popular, the tuberculosis infection of acquired immune deficiency syndrome (AIDS) and the groups of people all living creatures reason such as poverty of living makes American-European developed country incidence of tuberculosis such as U.S. be rise trend since 1985, and especially the sickness rate of tubercule bacillus resistance problem and non-tuberculous mycobacteria disease rises year by year tuberculotherapy is made the matter worse especially.At present the whole world has tuberculosis patient about 2,000 ten thousand, annual newly-increased tuberculosis patient 800～1,000 ten thousand, annual death toll about 3,000,000.
Tuberculosis epidemic situation of China and resistance situation are all quite serious, and non-tuberculous mycobacteria (being called for short NTM) disease also can not be ignored.China is one of the high burden of 22 tuberculosis in whole world country, and the tuberculosis patient numerical digit occupies the second in the world, is only second to India.2000 the 4th time national tuberculosis epidemiological random sampling survey PRELIMINARY RESULTS shows, in state-owned 5.5 hundred million people infected tubercule bacillus; Existing pulmonary tuberculosis patient 4,510,000, wherein the infectivity pulmonary tuberculosis patient 1,960,000; Annual death toll about 130,000.Therefore, early diagnosis lungy, differential diagnosis all have extremely important meaning to early stage, effective chemotherapy lungy and the propagation of control tubercule bacillus.
It is the key of controlling tuberculosis that the antitubercular agent of early diagnosis, discovery patient, selection sensitivity is effectively treated.Traditional bacteriology checking is main path and means of finding contagium, be " gold standard " of diagnosis of tuberculosis, it is the important evidence of determining the tuberculosis chemotherapy regimen, it also is the safe criterion of examination curative effect, evaluation prevention effect, be the important component part of national tuberculosis control planning (NTP), in tuberculosis prevention and treatment work, play indispensable vital role.Wherein, though clinical samples smear, acid-fast stain, microscopy are easy, quick, the sensitivity that detects is low, needs 10
4～10
5Individual bacterium/ml phlegm just can detect, and positive rate has only 20-30%; Traditional mycobacterium Luo Shi cultivates positive rate low (having only about 30%), and needs the 4-8 time-of-week; Traditional drug sensitive test needs 1～2 month, can not satisfy clinical early stage, the effective needs of chemotherapy, make resistance especially many resistant tuberculosis people adopt the chemotherapy regimen of present standard to fail to respond to any medical treatment, resistant organism is sent out.Obviously traditional bacteriology checking method can not satisfy the needs of tuberculosis prophylaxis, control.
The new mycobacterium substratum of hitherto reported mainly contains two big classes: liquid nutrient medium is used more with Middlebrook7H9 and Su Tong liquid nutrient medium; Solid medium is that the substratum (as acid Russell medium, coulee egg medium) of main ingredient and nutrient agar (as Kuang Shi substratum, Middlebrook7H10,7H11 nutrient agar) are used more with ovum gallinaceum liquid.The positive rate of the culture medium culturing that these are new is apparently higher than modified Russell medium, and positive report time obviously shortens, but still needs the time about 2 weeks.The full-automatic culture systems of the quick mycobacterium of BacT/Alert 3D of the Bactec 960 full-automatic culture systems of quick mycobacterium of U.S. BectonDickinson company development and the development of Mei Liai company, the positive rate that mycobacterium is cultivated can be brought up to 50-60%, report time is 1-42 days; Can carry out the drug sensitive test of 4 kind of one line antitubercular agent; Easy, quick, "dead" pollution has become one of standard reference system of diagnosis of tuberculosis at home and abroad.But can only do the drug sensitive test and the preliminary strain identification of 4 kinds of medicines, instrument costs an arm and a leg, and needs special import patent liquid nutrient medium, and expense is higher, promotes difficulty in China low developed area.Therefore, need the further novel mycobacterium substratum of research,, improve positive rate to shorten incubation time.
Summary of the invention
One of purpose of the present invention is for overcoming the weak point of existing mycobacterium substratum, a kind of mycobacterium growth promotor is provided, can be used for the mycobacterium culture experiment, promote growth, the metabolism of mycobacterium, shorten incubation time, improving positive rate.
The objective of the invention is to reach by the following technical programs:
A kind of mycobacterium tuberculosis Rv 1009 protein that is used to promote mycobacterium growth.
A kind of optimal technical scheme is characterized in that: the aminoacid sequence of the described mycobacterium tuberculosis Rv 1009 protein that is used to promote mycobacterium growth is as follows:
MLRLVVGALLLVLAFAGGYAVAACKTVTLTVDGTAMRVTTMKSR
VIDIVEENGFSVDDRDDLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGMALPVVSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPIVEGMQIQVTRNRIKKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVIDGSIWDAIAGCEAGGNWAINTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREQIAVAEVTRLRQGWGAWPVCAARAG。
Another object of the present invention provides the above-mentioned preparation that is used to promote the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth, and its step is as follows:
(1) prepare Rv1009 albumen by genetic engineering technique:
1, design of primers and synthetic
Upstream primer 5 '-CATATGTTGCGCCTGGTAGTCGGTGC-3 '
Downstream primer 5 '-GAATTCCCCGCTCGTGCAGCACATACCG-3 '
Amplified fragments: 1088bp
2, pcr amplification Rv1009 gene
Using downstream primer, under the effect of Taqplus I archaeal dna polymerase, is template with mycobacterium tuberculosis H37Rv genomic dna, amplification Rv1009 gene.PCR response procedures: 95 ℃ of 5min; 94 ℃ of 1min, 68 ℃ of 1min, 72 ℃ of 2min circulate 30 times; Last 72 ℃ are extended 7min.Identify the amplification of DNA fragments of 1088bp in 1% agarose gel electrophoresis;
3, reclaim target gene fragment:
After agarose gel electrophoresis finishes, under the long wave ultraviolet irradiation, on glue, downcut the agar block that will reclaim DNA, put into aseptic centrifuge tube with clean knife blade.The specification sheets that reclaims in the test kit with reference to agarose DNA reclaims target gene fragment;
4, goal gene is connected with the pGEM-T carrier:
PCR product (50ng) behind the purifying is connected with cloning vector pGEM-T, and target gene fragment was mixed with the carrier segment in 3: 1 in molar ratio, and the 10ul reaction system is as follows:
2 * connection damping fluid, 5 μ l
PGEM-T carrier 1 μ l
PCR product 0.6 μ l
T
4Dna ligase 1 μ l
Sterilized water is mended to 10 μ l
Mixing is placed on 4 ℃ of refrigerator reaction 12h, and 75 ℃ of deactivation 10min directly transform behind the ice bath;
5. the preparation of bacillus coli DH 5 alpha and e. coli bl21 (DE3) competent cell:
The single colony inoculation of picking bacillus coli DH 5 alpha (or BL21) is put in 37 ℃ of shaking culture casees in the 200rpm overnight incubation in 5ml LB nutrient solution; , in 100ml LB nutrient solution in put in 37 ℃ shaking culture casees in 200rpm continue cultivate 2-3h by 1/100 transferred species inferior morning, treats bacteria concentration OD
600Be 0.6～0.8 o'clock, put into ice-cold big centrifuge tube, 4 ℃, 4000rpm, centrifugal 10min abandon supernatant liquor; The resuspended bacterium precipitation of the 0.1mol/LCaCl2 of 20ml ice precooling, ice bath 30min; In 4 ℃, 6000rpm, centrifugal 10min, abandon supernatant liquor; With the resuspended bacterium precipitation of 4ml 0.1mol/L CaCl2, put 4 ℃ of refrigerators placements and spend the night; Add the aseptic glycerine of 1ml inferior morning, piping and druming mixes, and per 100 μ l are sub-packed in the centrifuge tube of a 1.5ml, and it is standby to put-70 ℃ of preservations;
6. connect the conversion of product:
Target gene fragment is added in the centrifuge tube that contains 100 μ l bacillus coli DH 5 alpha competent cells ice bath 0.5h respectively with each the 5 μ l of product that are connected of PGEM-T; Put into 42 ℃ of water bath heat-shocked 90s, take out ice bath 2min rapidly; Add LB nutrient solution 400 μ l, 37 ℃ of constant temperature shaking tables are cultivated 1h; Add X-Gal 60 μ l, IPTG 4 μ l, mixing takes out 200-400 μ l and coats on the LB flat board that contains the 60ug/ml penbritin.Be inverted flat board, put 37 ℃ of constant incubators and cultivate 14h;
7. the extraction of plasmid:
According to blue hickie screening, the picking white colony is 6 at random, is inoculated in 5ml respectively and contains in the LB substratum of 60 μ g/ml penbritins, and 37 ℃ of shaking culture are spent the night; According to " molecular cloning " alkaline lysis method, extract plasmid in a small amount;
(1) plasmid extracts the preparation of reagent in a small amount
Solution I: 50mmol/L glucose
25mmol/L?Tris·CL(pH?8.0)
10mmol/L?EDTA(pH?8.0)
At 10lbf/in
2(6.895 * 10
4Pa) steam sterilizing 15 minutes under the high pressure; Be stored in 4 ℃ standby;
Solution II: 0.2mmol/L NaOH, 1%SDS face and use preceding preparation
Solution III: 5mol/L potassium acetate 60ml
Glacial acetic acid 11.5ml
Deionized water 28.5ml
Be stored in 4 ℃, standby;
(2) extract plasmid in a small amount
Get about 3.0ml bacterium liquid respectively and put in the centrifuge tube, in 12, the centrifugal 1min of 000rpm abandons supernatant liquor; Bacterial precipitation is resuspended in the 200 μ l solution I, ice bath 10min; The solution II that adds the new preparation of 400 μ l, ice bath 5min; Add 300 μ l solution III, ice bath 10min; In 4 ℃ 12, the centrifugal 10min of 000rpm; Supernatant liquor moves in the clean centrifuge tube, adds each extracting of equal-volume phenol, chloroform and chloroform once; The dehydrated alcohol thorough mixing that adds 2 times of volumes is placed 2h deposit D NA in-20 ℃; In 4 ℃ 12, the centrifugal 10min of 000rpm abandons supernatant liquor; With 1ml 70% washing with alcohol precipitation once, room temperature thorough drying; Precipitation is dissolved in the 20 μ l TE damping fluids, adds the Pancreatic RNase of no DNA enzyme, making its final concentration is 20 μ g/ml, in 37 ℃ of water-bath 30min, and digestion RNA; Get 2 μ l and carry out 1% agarose gel electrophoresis detection, quantitative, put-20 ℃ and store for future use;
8. evaluation recombinant plasmid:
(1) pcr amplification is identified: with the bacterium colony plasmid DNA of selecting is template, carries out pcr amplification with P1, P4 primer and identifies.Amplified production is electrophoresis in 1% sepharose, positive recombinant plasmid called after PGEM-Rv1009;
(2) enzyme is cut evaluation: get recombinant plasmid 5 μ l, use restriction enzyme Nde I, EcoRI double digestion 2h respectively; Electrophoresis in 1% sepharose.With dna molecular amount standard and amplified production is contrast, and enzyme is cut the back and produced two fragments, and one consistent with carrier segment size, and one consistent with this gene amplification segment;
(3) sequencing: directly select a clone and send order-checking, sequencing result is consistent with the genome sequence of report;
9. the structure of recombinant expression plasmid:
With restriction enzyme Nde I, EcoRI double digestion pGEM-Rv1009 recombinant plasmid dna and expression vector pET24b plasmid DNA, in 1% agarose gel electrophoresis, cut the Rv1009 gene fragment of 1088bp and the pET24b plasmid DNA fragment of 5265bp, reclaim the test kit purifying with agarose gel electrophoresis.Quantitatively, Rv1009 gene fragment and pET24b plasmid DNA fragment are pressed 2: 1 mixed in molar ratio, at T
4Under the dna ligase catalysis, spend the night, get connection product 5 μ l transformed into escherichia coli DH5 α competent cells next day, put 37 ℃ of thermostat containers and hatch 14h in 16 ℃ of connections; Select 5 and clone respectively that transferred species contains in the LB nutrient solution of 50ug/ml sulphuric acid kanamycin in 5ml, put 37 ℃ of constant temperature oscillator overnight incubation, use the alkaline lysis method of extracting plasmid;
10. the evaluation of recombinant expression plasmid
Transfer 6 clones at random with toothpick respectively, extract plasmid, adopt pcr amplification and double digestion authentication method to select recon; Identify correct positive colony called after pET24b-Rv1009;
11, the abduction delivering of pET24b-Rv1009 engineering bacteria and evaluation
With pET24b-Rv1009 plasmid transformation escherichia coli BL21 (DE3), choosing the mono-clonal transferred species contains in the LB nutrient solution of 50ug/ml sulphuric acid kanamycin in 5ml, put 37 ℃ of constant temperature oscillator overnight incubation, contain in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins to 10ml by 1% transferred species then, put 37 ℃ of constant temperature oscillators and be cultured to OD
600During for 0.6-0.8, add IPTG, induce 3-4hr;
With the precipitation sample of 1 * load sample damping fluid, 150 μ l adding from 1ml bacterium liquid, behind the suspendible, put 100 ℃ of boiling water bath 5min, in the centrifugal 10min of 12000rpm, get supernatant liquor 40 μ l and carry out SDS-PAGE, deposition condition is: spacer gel constant current 10mA, separation gel constant voltage 15mA, treat that the tetrabromophenol sulfonphthalein electrophoresis to the gel bottom, stops electrophoresis; With coomassie brilliant blue R250 staining fluid dyeing 6h; It is clear to band to decolour with destainer; Compare with contrast bacterium pET24b-BL21 (DE3), pET24b-Rv1009-BL21 (DE3) thalline has dense band of expression to occur near relative molecular mass 40kDa position, induces 3-4 hour expression amount maximum;
12.Rv1009 the purifying of recombinant protein
After will inducing the bacterium ultrasonication, the His.Bind protein purification test kit that adopts Novagen company to produce is pressed test kit specification sheets purifying Rv1009 albumen under the sex change condition, the Rv1009 albumen of the visible purifying of SDS-PAGE electrophoresis is a band, does not see other foreign proteins;
(2) preparation of Rv1009 protein formulation and storage
1. diluent (0.5M sodium-chlor-20mM Sodium phosphate dibasic-2% N.F,USP MANNITOL, pH 7.4) preparation: take by weighing 29.22g sodium-chlor, 7.1628g Sodium phosphate dibasic in 700ml distilled water, add 20g N.F,USP MANNITOL, on magnetic stirring apparatus, stir, add salt acid for adjusting pH value to 7.4, add bi-distilled water and be settled to 1000ml;
2. with the described mycobacterium tuberculosis reorganization of diluted Rv1009 albumen, making its concentration is 1mg/ml;
3. degerming filters behind the mixing, divides the container of packing into, and 1ml/ props up, and lyophilize is put 4 ℃ and kept in Dark Place standby.
Another object of the present invention provides the above-mentioned application that is used to promote the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth.
Above-mentioned purpose of the present invention reaches by the following technical programs:
One of scheme:
The aforesaid application that is used to promote the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth, may further comprise the steps: mycobacterium tuberculosis Rv 1009 protein is dissolved in the sterile diluent, add in the mycobacterium substratum of liquid, making its final concentration is 0.01mg/ml-0.1mg/ml, inoculation mycobacterium bacterium liquid or clinical samples to be cultivated put incubator and cultivated then.
Two of scheme:
The aforesaid application that is used to promote the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth, may further comprise the steps: mycobacterium tuberculosis Rv 1009 protein is dissolved in the sterile diluent, be mixed with the mycobacterium tuberculosis Rv 1009 protein solution of 0.01mg/ml-0.1mg/ml, be added on solid mycobacterium media surface, inoculation mycobacterium bacterium liquid or clinical samples to be cultivated put incubator and cultivated then.
Characteristics of the present invention and technique effect:
In micrococcus luteus (M.luteus), find to exist recovery to promote the factor (Resuscitation-promotingfactor, be called for short Rpf), can promote resurrection and the growth of the bacterium and the dormancy bacterium of normal growth, its mechanism may be to have participated in intercellular signal transduction.Find in the gram-positive microorganism of high G+C% content, extensively to exist similar Rpf gene at present, find also that in the mycobacterium tuberculosis genome 5 are similar to the gene that the micrococcus luteus recovery promotes the factor: rpfA (Rv0867c), rpfB (Rv1009), rpfC (Rv1884c), rpfD (Rv2389c) and rpfE (Rv2450c), the function of its proteins encoded it be unclear that, infer the effect that also has Rpf, can promote the growth of tubercule bacillus, especially recovery of dormancy bacterium and growth.
The applicant studies have shown that the Rv1009 albumen by the genetic engineering technique preparation is used for the mycobacterium culture experiment, can promote growth, the metabolism of tubercule bacillus, shortens incubation time, improves positive rate.
Of the present inventionly be used to promote that the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth also can be the natural Rv1009 albumen of mycobacterium tuberculosis or other mycobacterium or other mycobacterium reorganization Rv1009 albumen.
The present invention will be further described below by the drawings and specific embodiments, but and do not mean that limiting the scope of the invention.
Description of drawings
Fig. 1 is the SDS-PAGE result of the Rv1009 recombinant protein of purifying.
Embodiment
One, be used to promote the preparation of the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth:
(1) prepare Rv1009 albumen by genetic engineering technique:
1, design of primers and synthetic
Upstream primer 5 '-CATATGTTGCGCCTGGTAGTCGGTGC-3 '
Downstream primer 5 '-GAATTCCCCGCTCGTGCAGCACATACCG-3 '
Amplified fragments: 1088bp
2, pcr amplification Rv1009 gene
Using downstream primer, under the effect of Taq plus I archaeal dna polymerase, is template with mycobacterium tuberculosis H37Rv genomic dna, amplification Rv1009 gene.PCR response procedures: 95 ℃ of 5min; 94 ℃ of 1min, 68 ℃ of 1min, 72 ℃ of 2min circulate 30 times; Last 72 ℃ are extended 7min.Identify the amplification of DNA fragments of 1088bp in 1% agarose gel electrophoresis.
3, reclaim target gene fragment:
After agarose gel electrophoresis finishes, under the long wave ultraviolet irradiation, on glue, downcut the agar block that will reclaim DNA, put into aseptic centrifuge tube with clean knife blade.The specification sheets that reclaims in the test kit with reference to agarose DNA reclaims target gene fragment, and concrete grammar is as follows:
Xiang Guanzhong adds isopyknic sol solutions (about 0.4ml), melts fully until agarose; Xiang Guanzhong adds 0.6ml agarose DNA purifying resin, fully mixing; Syringe is inserted the microcentrifugation post tighten, extract syringe piston, resin compound is added injection tube, insert syringe piston, slowly, discharge all liquids and gases firmly to pressing down; Pull out syringe from the microcentrifugation post, extract syringe piston, syringe is inserted the microcentrifugation post tighten, 2ml I type pillar scavenging solution is added injection tube, slowly firmly to pressing down, discharge all liquids and gases with syringe piston; Take off the microcentrifugation post, the microcentrifugation post is inserted a new 1.5ml centrifuge tube tighten, add 150 μ l II type pillar scavenging solutions in centrifugal post, centrifugal 1200rpm 2-3min is to clean and dry resin; The microcentrifugation post is inserted a new 1.5ml centrifuge tube tighten, in centrifugal post, add 40 μ l TE damping fluids, left standstill one minute, 12, the centrifugal 20s of 000rpm; Reclaim the DNA elutriant, quantitatively, concentration is about 50ng/ μ l.Be stored in-20 ℃ standby.
4, goal gene is connected with the pGEM-T carrier:
With reference to the pGEM-T vector System-I of the Promega company description of product, PCR product (50ng) behind the purifying is connected with cloning vector pGEM-T, target gene fragment was mixed with the carrier segment in 3: 1 in molar ratio, and the 10ul reaction system is as follows:
2 * connection damping fluid, 5 μ l
PGEM-T carrier 1 μ l
PCR product 0.6 μ l
T
4Dna ligase 1 μ l
Sterilized water is mended to 10 μ l
Mixing is placed on 4 ℃ of refrigerator reaction 12h, and 75 ℃ of deactivation 10min directly transform behind the ice bath.
5. the preparation of bacillus coli DH 5 alpha and e. coli bl21 (DE3) competent cell:
The single colony inoculation of picking bacillus coli DH 5 alpha (or BL21) is put in 37 ℃ of shaking culture casees in the 200rpm overnight incubation in 5ml LB nutrient solution; , in 100ml LB nutrient solution in put in 37 ℃ shaking culture casees in 200rpm continue cultivate 2-3h by 1/100 transferred species inferior morning, treats bacteria concentration OD
600Be 0.6～0.8 o'clock, put into ice-cold big centrifuge tube, 4 ℃, 4000rpm, centrifugal 10min abandon supernatant liquor; The resuspended bacterium precipitation of the 0.1mol/LCaCl2 of 20ml ice precooling, ice bath 30min; In 4 ℃, 6000rpm, centrifugal 10min, abandon supernatant liquor; With the resuspended bacterium precipitation of 4ml 0.1mol/L CaCl2, put 4 ℃ of refrigerators placements and spend the night; Add the aseptic glycerine of 1ml inferior morning, piping and druming mixes, and per 100 μ l are sub-packed in the centrifuge tube of a 1.5ml, and it is standby to put-70 ℃ of preservations.
6. connect the conversion of product:
Target gene fragment is added in the centrifuge tube that contains 100 μ l bacillus coli DH 5 alpha competent cells ice bath 0.5h respectively with each the 5 μ l of product that are connected of PGEM-T; Put into 42 ℃ of water bath heat-shocked 90s, take out ice bath 2min rapidly; Add LB nutrient solution 400 μ l, 37 ℃ of constant temperature shaking tables are cultivated 1h; Add X-Gal 60 μ l, IPTG 4 μ l, mixing takes out 200-400 μ l and coats on the LB flat board that contains the 60ug/ml penbritin.Be inverted flat board, put 37 ℃ of constant incubators and cultivate 14h.
7. the extraction of plasmid:
According to blue hickie screening, the picking white colony is 6 at random, is inoculated in 5ml respectively and contains in the LB substratum of 60 μ g/ml penbritins, and 37 ℃ of shaking culture are spent the night; According to " molecular cloning " alkaline lysis method, extract plasmid in a small amount.
(1) plasmid extracts the preparation of reagent in a small amount
Solution I: 50mmol/L glucose
25mmol/L?Tris·CL(pH?8.0)
10mmol/L?EDTA(pH?8.0)
At 10lbf/in
2(6.895 * 10
4Pa) steam sterilizing 15 minutes under the high pressure; Be stored in 4 ℃ standby.
Solution II: 0.2mmol/L NaOH, 1%SDS face and use preceding preparation
Solution III: 5mol/L potassium acetate 60ml
Glacial acetic acid 11.5ml
Deionized water 28.5ml
Be stored in 4 ℃, standby.
(2) extract plasmid in a small amount
Get about 3.0ml bacterium liquid respectively and put in the centrifuge tube, in 12, the centrifugal 1min of 000rpm abandons supernatant liquor; Bacterial precipitation is resuspended in the 200 μ l solution I, ice bath 10min; The solution II that adds the new preparation of 400 μ l, ice bath 5min; Add 300 μ l solution III, ice bath 10min; In 4 ℃ 12, the centrifugal 10min of 000rpm; Supernatant liquor moves in the clean centrifuge tube, adds each extracting of equal-volume phenol, chloroform and chloroform once; The dehydrated alcohol thorough mixing that adds 2 times of volumes is placed 2h deposit D NA in-20 ℃; In 4 ℃ 12, the centrifugal 10min of 000rpm abandons supernatant liquor; With 1ml 70% washing with alcohol precipitation once, room temperature thorough drying; Precipitation is dissolved in the 20 μ l TE damping fluids, adds the Pancreatic RNase of no DNA enzyme, making its final concentration is 20 μ g/ml, in 37 ℃ of water-bath 30min, and digestion RNA; Get 2 μ l and carry out 1% agarose gel electrophoresis detection, quantitative, put-20 ℃ and store for future use.
8. evaluation recombinant plasmid:
(2) pcr amplification is identified: with the bacterium colony plasmid DNA of selecting is template, carries out pcr amplification with P1, P4 primer and identifies.Amplified production is electrophoresis in 1% sepharose, positive recombinant plasmid called after PGEM-Rv1009.
(2) enzyme is cut evaluation: get recombinant plasmid 5 μ l, use restriction enzyme Nde I, EcoRI double digestion 2h respectively; Electrophoresis in 1% sepharose.With dna molecular amount standard and amplified production is contrast, and enzyme is cut the back and produced two fragments, and one consistent with carrier segment size, and one consistent with this gene amplification segment.
(3) sequencing: directly select a clone and send order-checking.Sequencing result is consistent with the genome sequence of report.
9. the structure of recombinant expression plasmid:
With restriction enzyme Nde I, EcoRI double digestion pGEM-Rv1009 recombinant plasmid dna and expression vector pET24b plasmid DNA, in 1% agarose gel electrophoresis, cut the Rv1009 gene fragment of 1088bp and the pET24b plasmid DNA fragment of 5265bp, reclaim the test kit purifying with agarose gel electrophoresis.Quantitatively, Rv1009 gene fragment and pET24b plasmid DNA fragment are pressed 2: 1 mixed in molar ratio, at T
4Under the dna ligase catalysis, spend the night, get connection product 5 μ l transformed into escherichia coli DH5 α competent cells next day, put 37 ℃ of thermostat containers and hatch 14h in 16 ℃ of connections.Select 5 and clone respectively that transferred species contains in the LB nutrient solution of 50ug/ml sulphuric acid kanamycin in 5ml, put 37 ℃ of constant temperature oscillator overnight incubation, use the alkaline lysis method of extracting plasmid.
10. the evaluation of recombinant expression plasmid
Transfer 6 clones at random with toothpick respectively, extract plasmid, adopt pcr amplification and double digestion authentication method to select recon; Identify correct positive colony called after pET24b-Rv1009.
11, the abduction delivering of pET24b-Rv1009 engineering bacteria and evaluation
With pET24b-Rv1009 plasmid transformation escherichia coli BL21 (DE3), choosing the mono-clonal transferred species contains in the LB nutrient solution of 50ug/ml sulphuric acid kanamycin in 5ml, put 37 ℃ of constant temperature oscillator overnight incubation, contain in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins to 10ml by 1% transferred species then, put 37 ℃ of constant temperature oscillators and be cultured to OD
600During for 0.6-0.8, add IPTG, induce 3-4hr.
With the precipitation sample of 1 * load sample damping fluid, 150 μ l adding from 1ml bacterium liquid, behind the suspendible, put 100 ℃ of boiling water bath 5min, in the centrifugal 10min of 12000rpm, get supernatant liquor 40 μ l and carry out SDS-PAGE, deposition condition is: spacer gel constant current 10mA, separation gel constant voltage 15mA, treat that the tetrabromophenol sulfonphthalein electrophoresis to the gel bottom, stops electrophoresis.With coomassie brilliant blue R250 staining fluid dyeing 6h; It is clear to band to decolour with destainer.Compare with contrast bacterium pET24b-BL21 (DE3), pET24b-Rv1009-BL21 (DE3) thalline has dense band of expression to occur near relative molecular mass 40kDa position, induces 3-4 hour expression amount maximum.
12.Rv1009 the purifying of recombinant protein
After will inducing the bacterium ultrasonication, the His.Bind protein purification test kit that adopts Novagen company to produce is pressed test kit specification sheets purifying Rv1009 albumen under the sex change condition, the Rv1009 albumen of the visible purifying of SDS-PAGE electrophoresis is a band, does not see other foreign protein (see figure 1)s.
Wherein 1 contains the intestinal bacteria of pET24b-Rv1009 plasmid for inductive not; 2 contain the intestinal bacteria of pET24b-Rv1009 plasmid for the IPTG inductive; 3 is the Rv1009 recombinant protein of purifying.
It is as follows to test its aminoacid sequence by the testing method of routine:
MLRLVVGALLLVLAFAGGYAVAACKTVTLTVDGTAMR
VTTMKSRVIDIVEENGFSVDDRDDLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGMALPVVSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPIVEGMQIQVTRNRIKKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVIDGSIWDAIAGCEAGGNWAINTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREQIAVAEVTRLRQGWGAWPVCAARAG。
(2) preparation of Rv1009 protein formulation and storage
1. diluent (0.5M sodium-chlor-20mM Sodium phosphate dibasic-2% N.F,USP MANNITOL, pH 7.4) preparation: take by weighing 29.22g sodium-chlor, 7.1628g Sodium phosphate dibasic in 700ml distilled water, add 20g N.F,USP MANNITOL, on magnetic stirring apparatus, stir, add salt acid for adjusting pH value to 7.4, add bi-distilled water and be settled to 1000ml.
2. with the described mycobacterium tuberculosis reorganization of diluted Rv1009 albumen, making its concentration is 1mg/ml;
3. degerming filters behind the mixing, divides the container of packing into, and 1ml/ props up, and lyophilize is put 4 ℃ and kept in Dark Place standby.
Two, the application of Rv1009 albumen in mycobacterium is cultivated:
1. draw sterilized water 1ml, add in 1 above-mentioned mycobacterium Rv1009 protein formulation, dissolve freeze dried Rv1009 albumen after, obtain the Rv1009 protein solution of 1mg/ml, based on Middlebrook 7H9 liquid nutrient medium, press the substratum of table 1 preparation control group and experimental group:
The proteic content of Rv1009 in the table 1Middlebrook 7H9 liquid nutrient medium
(2) preparation of bcg bacteria liquid: (specification is per ampoule 5 person-portions with bacille Calmette-Guerin vaccine to get the intradermal injection that 3 Shanghai Vaccine and Serum Institute of Chinese biological technology group company produce, contain bacille Calmette-Guerin vaccine 0.2-0.3mg), be dissolved in the 3ml Middlebrook 7H9 liquid nutrient medium, every culture medium inoculated bcg bacteria liquid 0.1ml, if 1 simple liquid substratum contrast is put 37 ℃ of incubators and was cultivated for 1 week.
(3) result observes: the optical density value (OD that measures bacteria suspension 600nm place with visible spectrophotometer
600) after, in 4000 rev/mins of centrifugal 20min, abandon supernatant liquor, get precipitation smear, acid-fast stain after, examine under a microscope and have or not acid-fast bacilli or other assorted bacterium.The results are shown in Table 2.
Optical density value (the OD of bacterial growth in each culture tube of table 2 different times
600) and the smear staining result
(4) conclusion: when bacille Calmette-Guerin vaccine grew for 1 week in containing the proteic Middlebrook 7H9 of Rv1009 liquid nutrient medium, the bacterial optical density value that contains in the proteic substratum of 0.01-0.08mg/ml Rv1009 obviously increased, and illustrates that it impels the effect of bacterial growth obvious.
A kind of application that is used to promote the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth:
(1) draws sterilized water 1ml, add in the prepared Rv1009 protein formulation of 1 embodiment 1, dissolve freeze dried Rv1009 albumen after, obtain the Rv1009 protein solution of 1mg/ml, based on Middlebrook 7H9 liquid nutrient medium, press the substratum of table 3 preparation control group and experimental group:
The proteic content of Rv1009 in the table 3Middlebrook 7H9 liquid nutrient medium
(2) preparation of bcg bacteria liquid: (specification is per ampoule 5 person-portions with bacille Calmette-Guerin vaccine to get the intradermal injection that 2 Shanghai Vaccine and Serum Institute of Chinese biological technology group company produce, contain bacille Calmette-Guerin vaccine 0.2-0.3mg), be dissolved in the 6ml Middlebrook 7H9 liquid nutrient medium, every culture medium inoculated bcg bacteria liquid 0.1ml, if 1 simple liquid substratum contrast is put 37 ℃ of incubators and was cultivated 9 days.
(3) result observes: the optical density value (OD that measures bacteria suspension 600nm place with visible spectrophotometer
600) after, in 4000 rev/mins of centrifugal 20min, abandon supernatant liquor, get precipitation smear, acid-fast stain after, examine under a microscope and have or not acid-fast bacilli or other assorted bacterium.The results are shown in Table 4.
Optical density value (the OD of bacterial growth in each culture tube of table 4 different times
600) and the smear staining result
(4) conclusion: the bcg vaccination bacterium is measured more after a little while, growth is in the time of 9 days in containing the proteic Middlebrook 7H9 of Rv1009 liquid nutrient medium, in containing the proteic substratum of 0.01-0.08mg/ml Rv1009, prolongation along with the increasing of Rv1009 protein concentration, incubation time, the bacterial optical density value also obviously increases, and illustrates that Rv1009 albumen impels the effect of bacterial growth obvious.
A kind of application that is used to promote the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth:
(1) draws sterilized water 1ml, adds in the prepared Rv1009 protein formulation of 1 embodiment 1, make it to dissolve, obtain the Rv1009 protein solution of 1mg/ml;
(2) culture tube is divided into following 3 groups, each component sees Table 5:
A, Bactec 960 liquid nutrient medium doping groups: Bactec 960 liquid nutrient mediums and additive are the U.S. company BD product;
B, Bactec 960 liquid nutrient medium dopings add the Rv1009 protein groups;
C, Bactec 960 liquid nutrient mediums add the Rv1009 protein groups.
Each culture tube component of table 5
(3) from 4 ℃ of refrigerators, take out to store the mycobacterium tuberculosis type strain H37Rv on the modified Russell medium of being grown in of half a year, scrape and get tubercule bacillus, be mixed with 1mg/ml with physiological saline, then 10 times of serial dilutions to 10
-6, respectively get 0.1ml 10
-4To 10
-6Bacterial suspension inoculation in each the group substratum in, put in the full-automatic culture systems of 37 ℃ of Bactec, 960 quick mycobacteriums and cultivate.
(4) result observes: automatically cultivate Bactec 960 quick mycobacteriums, the cultivation male is reported bacterial growth unit automatically in the detection system, the results are shown in Table 6.
Table 6 is respectively organized the mycobacterium tuberculosis cultivation results
(5) conclusion: on Bactec 960 conventional cultivation bases, add Rv1009 albumen and can make bacteria containing amount be less than 10
-6The bacterium of mg/ml was cultivated positive in the time of 14 days, had shortened incubation time, had improved the cultivation positive rate.
Embodiment 4
A kind of application that is used to promote the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth:
(1) draw sterilized water 12.5ml, add in the prepared Rv1009 protein formulation of 1 embodiment 1, dissolve freeze dried Rv1009 albumen after, obtain the Rv1009 protein solution of 0.08mg/ml.
(2) from 4 ℃ of refrigerators, take out to store the strain of BCG vaccine on the modified Russell medium of being grown in of half a year, be mixed with the bacteria suspension of 1mg/ml with physiological saline.One group of bacteria suspension of getting 0.25ml is inoculated on the modified Russell medium, puts 37 ℃ of incubators and cultivates.Two groups of bacteria suspensions of getting 0.25ml mix with the Rv1009 protein solution of 0.25ml 0.08mg/ml, are inoculated in then on the modified Russell medium, put 37 ℃ of incubators and cultivate.
(3) result observes: saw bacterial growth not yet on the 12nd day cultivating for one group, and two groups the 7th day be visible colony growth, illustrate that Rv1009 albumen also can impel bacille Calmette-Guerin vaccine to grow significantly on modified Russell medium.
Claims (4)
1. mycobacterium tuberculosis Rv 1009 protein that is used to promote mycobacterium growth is characterized in that its aminoacid sequence is as follows:
MLRLVVGALLLVLAFAGGYAVAACKTVTLTVDGTAMRVTTMKSRVIDIVEENGFSVDDRDDLYPAAGVQVHDADTIVLRRSRPLQISLDGHDAKQVWTTASTVDEALAQLAMTDTAPAAASRASRVPLSGMALPVVSAKTVQLNDGGLVRTVHLPAPNVAGLLSAAGVPLLQSDHVVPAATAPIVEGMQIQVTRNRIKKVTERLPLPPNARRVEDPEMNMSREVVEDPGVPGTQDVTFAVAEVNGVETGRLPVANVVVTPAHEAVVRVGTKPGTEVPPVIDGSIWDAIAGCEAGGNWAINTGNGYYGGVQFDQGTWEANGGLRYAPRADLATREQIAVAEVTRLRQGWGAWPVCAARAG。
2. the preparation method who is used to promote the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth as claimed in claim 1, its step is as follows:
(1) prepare Rv1009 albumen by genetic engineering technique:
(a), design of primers and synthetic
Upstream primer 5 '-CATATGTTGCGCCTGGTAGTCGGTGC-3 '
Downstream primer 5 '-GAATTCCCCGCTCGTGCAGCACATACCG-3 '
Amplified fragments: 1088bp
(b), pcr amplification Rv1009 gene
Using downstream primer, under the effect of Taq plus I archaeal dna polymerase, is template with mycobacterium tuberculosis H37Rv genomic dna, amplification Rv1009 gene; PCR response procedures: 95 ℃ of 5min; 94 ℃ of 1min, 68 ℃ of 1min, 72 ℃ of 2min circulate 30 times; Last 72 ℃ are extended 7min; Identify the amplification of DNA fragments of 1088bp in 1% agarose gel electrophoresis;
(c), reclaim target gene fragment:
After agarose gel electrophoresis finishes, under the long wave ultraviolet irradiation, on glue, downcut the agar block that will reclaim DNA, put into aseptic centrifuge tube with clean knife blade; The specification sheets that reclaims in the test kit with reference to agarose DNA reclaims target gene fragment;
(d), goal gene is connected with the pGEM-T carrier:
PCR product 50ng behind the purifying is connected with cloning vector pGEM-T, and target gene fragment was mixed with the carrier segment in 3: 1 in molar ratio, and the 10ul reaction system is as follows:
2 * connection damping fluid, 5 μ l
PGEM-T carrier 1 μ l
PCR product 0.6 μ l
T
4Dna ligase 1 μ l
Sterilized water is mended to 10 μ l
Mixing is placed on 4 ℃ of refrigerator reaction 12h, and 75 ℃ of deactivation 10min directly transform behind the ice bath;
(e). the preparation of bacillus coli DH 5 alpha and e. coli bl21 (DE3) competent cell:
The single colony inoculation of picking bacillus coli DH 5 alpha or BL21 is put in 37 ℃ of shaking culture casees in the 200rpm overnight incubation in 5ml LB nutrient solution; , in 100ml LB nutrient solution in put in 37 ℃ shaking culture casees in 200rpm continue cultivate 2-3h by 1/100 transferred species inferior morning, treats that bacteria concentration OD600 is at 0.6～0.8 o'clock, puts into ice-cold big centrifuge tube, and 4 ℃, 4000rpm, centrifugal 10min abandon supernatant liquor; The resuspended bacterium precipitation of the 0.1mol/L CaCl2 of 20ml ice precooling, ice bath 30min; In 4 ℃, 6000rpm, centrifugal 10min, abandon supernatant liquor; With the resuspended bacterium precipitation of 4ml 0.1mol/L CaCl2, put 4 ℃ of refrigerators placements and spend the night; Add the aseptic glycerine of 1ml inferior morning, piping and druming mixes, and per 100 μ l are sub-packed in the centrifuge tube of a 1.5ml, and it is standby to put-70 ℃ of preservations;
(f). connect the conversion of product:
Target gene fragment is added in the centrifuge tube that contains 100 μ l bacillus coli DH 5 alpha competent cells ice bath 0.5h respectively with each the 5 μ l of product that are connected of PGEM-T; Put into 42 ℃ of water bath heat-shocked 90s, take out ice bath 2min rapidly; Add LB nutrient solution 400 μ l, 37 ℃ of constant temperature shaking tables are cultivated 1h; Add X-Gal 60 μ l, IPTG 4 μ l, mixing takes out 200-400 μ l and coats on the LB flat board that contains the 60ug/ml penbritin; Be inverted flat board, put 37 ℃ of constant incubators and cultivate 14h;
(g). the extraction of plasmid:
According to blue hickie screening, the picking white colony is 6 at random, is inoculated in 5ml respectively and contains in the LB substratum of 60 μ g/ml penbritins, and 37 ℃ of shaking culture are spent the night; According to " molecular cloning " alkaline lysis method, extract plasmid in a small amount;
1. plasmid extracts the preparation of reagent in a small amount
Solution I: 50mmol/L glucose
25mmol/L?Tris·CL，pH?8.0
10mmol/L?EDTA，pH?8.0
At 10lbf/in
2, promptly 6.895 * 10
4Steam sterilizing is 15 minutes under the Pa high pressure; Be stored in 4 ℃ standby;
Solution II: 0.2mmol/L NaOH, 1%SDS face and use preceding preparation
Solution III: 5mol/L potassium acetate 60ml
Glacial acetic acid 11.5ml
Deionized water 28.5ml
Be stored in 4 ℃, standby;
2. extract plasmid in a small amount
Get about 3.0ml bacterium liquid respectively and put in the centrifuge tube, in 12, the centrifugal 1min of 000rpm abandons supernatant liquor; Bacterial precipitation is resuspended in the 200 μ l solution I, ice bath 10min; The solution II that adds the new preparation of 400 μ l, ice bath 5min; Add 300 μ l solution III, ice bath 10min; In 4 ℃ 12, the centrifugal 10min of 000rpm; Supernatant liquor moves in the clean centrifuge tube, adds each extracting of equal-volume phenol, chloroform and chloroform once; The dehydrated alcohol thorough mixing that adds 2 times of volumes is placed 2h deposit D NA in-20 ℃; In 4 ℃ 12, the centrifugal 10min of 000rpm abandons supernatant liquor; With 1ml 70% washing with alcohol precipitation once, room temperature thorough drying; Precipitation is dissolved in the 20 μ l TE damping fluids, adds the Pancreatic RNase of no DNA enzyme, making its final concentration is 20 μ g/ml, in 37 ℃ of water-bath 30min, and digestion RNA; Get 2 μ l and carry out 1% agarose gel electrophoresis detection, quantitative, put-20 ℃ and store for future use;
(h). identify recombinant plasmid:
1. pcr amplification is identified: with the bacterium colony plasmid DNA of selecting is template, carries out pcr amplification with upstream and downstream 4 primers and identifies; Amplified production is electrophoresis in 1% sepharose, positive recombinant plasmid called after PGEM-Rv1009;
2. enzyme is cut evaluation: get recombinant plasmid 5 μ l, use restriction enzyme Nde I, EcoRI double digestion 2h respectively; Electrophoresis in 1% sepharose; With dna molecular amount standard and amplified production is contrast, and enzyme is cut the back and produced two fragments, and one consistent with carrier segment size, and one consistent with this gene amplification segment;
3. sequencing: directly select a clone and send order-checking;
(i). the structure of recombinant expression plasmid:
With restriction enzyme Nde I, EcoRI double digestion pGEM-Rv1009 recombinant plasmid dna and expression vector pET24b plasmid DNA, in 1% agarose gel electrophoresis, cut the Rv1009 gene fragment of 1088bp and the pET24b plasmid DNA fragment of 5265bp, reclaim the test kit purifying with agarose gel electrophoresis; Quantitatively, Rv1009 gene fragment and pET24b plasmid DNA fragment are pressed 2: 1 mixed in molar ratio, under the catalysis of T4DNA ligase enzyme, spend the night in 16 ℃ of connections, get connection product 5 μ l transformed into escherichia coli DH5 α competent cells next day, put 37 ℃ of thermostat containers and hatch 14h; Select 5 and clone respectively that transferred species contains in the LB nutrient solution of 50ug/ml sulphuric acid kanamycin in 5ml, put 37 ℃ of constant temperature oscillator overnight incubation, use the alkaline lysis method of extracting plasmid;
(j). the evaluation of recombinant expression plasmid
Transfer 6 clones at random with toothpick respectively, extract plasmid, adopt pcr amplification and double digestion authentication method to select recon; Identify correct positive colony called after pET24b-Rv1009;
(k), the abduction delivering of pET24b-Rv1009 engineering bacteria and evaluation
With pET24b-Rv1009 plasmid transformation escherichia coli BL21 (DE3), choosing the mono-clonal transferred species contains in the LB nutrient solution of 50ug/ml sulphuric acid kanamycin in 5ml, put 37 ℃ of constant temperature oscillator overnight incubation, contain in the LB nutrient solution of 50 μ g/ml sulphuric acid kanamycins to 10ml by 1% transferred species then, put 37 ℃ of constant temperature oscillators and be cultured to OD
600During for 0.6-0.8, add IPTG, induce 3-4hr;
With the precipitation sample of 1 * load sample damping fluid, 150 μ l adding from 1ml bacterium liquid, behind the suspendible, put 100 ℃ of boiling water bath 5min, in the centrifugal 10min of 12000rpm, get supernatant liquor 40 μ l and carry out SDS-PAGE, deposition condition is: spacer gel constant current 10mA, separation gel constant voltage 15mA, treat that the tetrabromophenol sulfonphthalein electrophoresis to the gel bottom, stops electrophoresis; With coomassie brilliant blue R250 staining fluid dyeing 6h; It is clear to band to decolour with destainer; Compare with contrast bacterium pET24b-BL21 (DE3), pET24b-Rv1009-BL21 (DE3) thalline has dense band of expression to occur near relative molecular mass 40kDa position, induces 3-4 hour expression amount maximum;
(l) purifying of .Rv1009 recombinant protein
After will inducing the bacterium ultrasonication, the His.Bind protein purification test kit that adopts Novagen company to produce is pressed test kit specification sheets purifying Rv1009 albumen under the sex change condition, the Rv1009 albumen of the visible purifying of SDS-PAGE electrophoresis is a band, does not see other foreign proteins;
(2) preparation of Rv1009 protein formulation and storage
1. diluent 0.5M sodium-chlor-20mM Sodium phosphate dibasic-2% N.F,USP MANNITOL, the preparation of pH 7.4: take by weighing 29.22g sodium-chlor, 7.1628g Sodium phosphate dibasic in 700ml distilled water, add 20g N.F,USP MANNITOL, on magnetic stirring apparatus, stir, add salt acid for adjusting pH value to 7.4, add bi-distilled water and be settled to 1000ml;
2. with the described mycobacterium tuberculosis reorganization of diluted Rv1009 albumen, making its concentration is 1mg/ml;
3. degerming filters behind the mixing, divides the container of packing into, and 1ml/ props up, and lyophilize is put 4 ℃ and kept in Dark Place standby.
3. the application that is used to promote the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth as claimed in claim 1, may further comprise the steps: mycobacterium tuberculosis Rv 1009 protein is dissolved in the sterile diluent, add in the mycobacterium substratum of liquid, making its final concentration is 0.01mg/ml-0.1mg/ml, inoculation mycobacterium bacterium liquid or clinical samples to be cultivated put incubator and cultivated then.
4. the application that is used to promote the mycobacterium tuberculosis Rv 1009 protein of mycobacterium growth as claimed in claim 1, may further comprise the steps: mycobacterium tuberculosis Rv 1009 protein is dissolved in the sterile diluent, be mixed with the mycobacterium tuberculosis Rv 1009 protein solution of 0.01mg/ml-0.1mg/ml, be added on solid mycobacterium media surface, inoculation mycobacterium bacterium liquid or clinical samples to be cultivated put incubator and cultivated then.
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| 苏明权等.结核分枝杆菌Rv1009的生物信息学分析及克隆、表达.生物技术通讯14 6.2003,14(6),第557页摘要. |
| 苏明权等.结核分枝杆菌Rv1009的生物信息学分析及克隆、表达.生物技术通讯14 6.2003,14(6),第557页摘要. * |
| 薛莹等.结核分枝杆菌Rv1009基因的克隆、表达及亲和层析纯化.科学技术与工程6 14.2006,6(14),第2031页摘要. |
| 薛莹等.结核分枝杆菌Rv1009基因的克隆、表达及亲和层析纯化.科学技术与工程6 14.2006,6(14),第2031页摘要. * |
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