CN103695348A - Method for fermenting mycobacterium tuberculosis bacteria - Google Patents
Method for fermenting mycobacterium tuberculosis bacteria Download PDFInfo
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- CN103695348A CN103695348A CN201310692668.2A CN201310692668A CN103695348A CN 103695348 A CN103695348 A CN 103695348A CN 201310692668 A CN201310692668 A CN 201310692668A CN 103695348 A CN103695348 A CN 103695348A
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Abstract
The invention relates to a fermentation method of mycobacterium tuberculosis, belongs to the field of biological fermentation, and provides a bran-new method for fermenting mycobacterium tuberculosis. The results of ultra-high bacterial concentration of the mycobacterium tuberculosis and abundant expression of target protein are finally achieved by primary amplification, secondary amplification, primary fermentation and secondary fermentation of a bacterial strain. The bran-new fermentation method of the mycobacterium tuberculosis is obtained. The fermentation yield is not lower than 12g/L, the total rTPA (recombinant tissue plasminogen activator) 38 target protein in the bacteria can be up to over 15%, and the activity is high, which cannot be achieved by the prior art. The bacteria fermentation is large in batch quantity, and 10-200L of bacteria can be fermented one time. The method is fast in bacteria collection and high in production efficiency.
Description
Technical field
The present invention relates to the fermentation process of mycobacterium tuberculosis, belong to biological fermentation field.
Background technology
Tuberculosis is to infect by mycobacterium tuberculosis complex the multi organ infection disease causing, is mainly pulmonary tuberculosis.Wherein the annual new patient of China is up to 1,500,000.The EPDML latent infection that key character is mycobacterium tuberculosis of tubercule bacillus.Mycobacterium tuberculosis latent infection is a kind of sub-clinical state, and without actinoscopy sign, bacterium is dormant state, approximately has 10% mycobacterium tuberculosis latent infection crowd can develop into tuberculosis patient in life.
Tubercule bacillus latent infection crowd there is no diagnosis gold standard at present, the test of PPD skin test is diagnosing tubercle bacillus latent infection crowd's common method, but this method has certain defective, because test PPD antigen used, be that mycobacterium tuberculosis, non-virulent mycobacterium and bacille Calmette-Guerin vaccine (being BCG) are common, have cross reaction, specificity is poor.China is the country of general bcg vaccination, and because BCG (Bacille Calmette-Guerin) vaccination person and tubercule bacillus natural infection person are difficult to distinguish to the reaction of PPD test in performance, even if adopt above-mentioned PPD skin test test strong positive as judging criterion, still can some BCG (Bacille Calmette-Guerin) vaccination person and the infected of non-virulent mycobacterium be included in tubercule bacillus latent infection crowd, therefore cause the practical function of PPD test in tubercule bacillus latent infection crowd diagnosis very limited.Needing to find the preparation that a kind of new specificity is high, side effect is little replaces.Restructuring Mycobacterium tuberculosis albumen 38K D a (r T P A38) is a kind of lipoprotein and the major antigen of Mycobacterium tuberculosis, and its effect of bringing out cavy delayed type hypersensitivity D T H is better than other single antigen.The susceptibility and the specificity that for tuberculosis serological, detect are higher.Therefore, r T P A38 albumen is the albumen at present with better application prospect.
But, in the prior art, because mycobacterium tuberculosis is in the culturing process of cell, and expression target protein that can be a large amount of unlike intestinal bacteria, and there is with intestinal bacteria prediction scheme and expression r T P A38 albumen the defect that this folding activities is low, therefore, the defect of the inventor based on prior art, has designed the present invention.The present invention is by progressively optimizing mycobacterium tuberculosis cultural method of the prior art, a kind of brand-new mycobacterium tuberculosis fermentation process is provided, by one-level amplification, secondary amplification, one grade fermemtation, the second order fermentation of bacterial strain, has finally realized the result of the dense superelevation of mycobacterium tuberculosis bacterium, target protein great expression.
Summary of the invention
The object of the present invention is to provide: the method for mycobacterium tuberculosis thalline fermentation, thus collect a large amount of thalline that amplification obtains, obtain a large amount of target proteins, promoted the use of r T P A38 albumen for extensive vaccine.
Technical scheme of the present invention: the method for mycobacterium tuberculosis thalline fermentation, comprises the steps: one, the preparation of one-level amplification liquid: get inclined-plane list bacterium colony, be placed in the test tube of one-level amplification culture medium, overnight incubation in full temperature shaking culture case; In Bechtop, in every bottle of one-level amplification culture medium, add penbritin, then inoculate the bacterial classification in test tube, constant-temperature shaking culture in full temperature shaking culture case, it is about 1-2 that every bottle of sampling detects OD600 value;
Two, secondary amplification liquid: get primary seed solution and be inoculated in secondary amplification culture medium, constant-temperature shaking culture in full temperature shaking culture case, it is 1-2 that every bottle of sampling detects OD600 value;
Three, one grade fermemtation is cultivated: before inoculation, set ventilation for 2500L/h, tank pressure is 0.04Mp, and stirring velocity is 560rpm, and dissolved oxygen when temperature is 37 ℃ is 100%.With the inoculum size inoculation secondary amplification liquid of 5%-7%, in the 10L fermention medium in seeding tank, fermentation culture, cultivates 2-4h to OD
6002～3, in culturing process, every half an hour, measure the OD600 value of fermented liquid;
Four, second order fermentation is cultivated: the satisfactory one grade fermemtation bacterium of OD600 in seeding tank liquid is inoculated in fermentor tank in 120L fermention medium with the inoculum size of 5-10%; Aerated culture was received bacterium in OD600 >=8 after approximately 4 to 5 hours continuously, measured the OD600 value of fermented liquid in culturing process every about half an hour;
Five, receive bacterium: adopt tubular-bowl centrifuge to carry out centrifugal bacterium liquid and abandon supernatant liquor, microorganism collection is deposited in clean container.
Six, quality index: smear gramstaining, at optical microphotograph Microscopic observation, should be typical gram negative bacillus, in bacterial protein, (target protein rTPA38) is not less than 15%, fermentation yield: be not less than 12g/L, calculation formula: fermentation yield (g/L)=wet bacterium weight (g)/fermentation basic medium amount (L).
Beneficial effect of the present invention: the inventor is by having improved the fermentation process of mycobacterium tuberculosis, optimized the concrete conditional parameter of each step, obtain the fermentation process of brand-new mycobacterium tuberculosis, fermentation yield is not less than 12g/L, and in thalline, total rTPA38 target protein can reach more than 15%, activity is also higher, and these are all that prior art is beyond one's reach.The batch of thalline fermentation is large, and the disposable 10L to 200L that ferments receives bacterium speed fast, and production efficiency is high.
Accompanying drawing explanation
Fig. 1 zymocyte liquid gramstaining opticmicroscope (typical gram negative bacillus)
Fig. 2 thalline fermentation process process flow sheet
Embodiment
Embodiment 1:
Substratum preparation
(1) mycobacterium tuberculosis amplification culture medium is Roche egg slant medium, buys from Roche Holding Ag.
(2) fermention medium (1000mL): sodium selenate 10mg, potassium primary phosphate 2.4g, b) magnesium sulfate 0.24g, c) Citric Acid 0.6g, d) asparagine 3.6g, e) glycerine 20mL, f) yam starch 30g, g) fresh ovum gallinaceum liquid 940mL, h) Sodium Glutamate 0.15g surplus is water.
(3) preparation of other feed supplement liquid:
Dilute hydrochloric acid solution packing (1000mL): get each packing of dilute hydrochloric acid HCl1000mL 500mL and enter 2 500mL in sterilizing feed supplement bottle, liquor ammoniae fortis packing (1000mL): get each packing of liquor ammoniae fortis 1000mL 500mL and enter 2 500mL in sterilizing feed supplement bottle.
80% carbon source preparation (8L): take glycerine 6400g be dissolved in purified water to cumulative volume be 8L, be distributed in 5L feed supplement bottle every bottle of 4L, 121 ℃ of sterilizing 20min, airtight the depositing of cooling rear normal temperature.
50mg/mL penbritin (10mL): take 1g penbritin and dissolve by 20mL purified water.In Bechtop, with the filtration sterilization of aseptic syringe needle strainer, be distributed in sterilising vessel, packing specification is 1mL/ pipe (1.5ml EP pipe), totally 10 manages, and preserves stand-by in 2-8 ℃ of refrigerator.
Embodiment 2: thalline fermentation process
One, one-level amplification liquid preparation: get the mycobacterium tuberculosis (bacterial strain that this area is conventional, for example: ATCC93009) inclined-plane list bacterium colony, be placed in the test tube that contains 3mL amplification culture medium, in full temperature shaking culture case, 37 ± 1 ℃ of constant temperature, rotating speed 170r/min shaking culture are spent the night; In Bechtop, in every bottle of amplification culture medium (8mL), add 50mg/mL penbritin 8 μ l, then inoculate the bacterial classification in 80 μ l test tubes, in full temperature shaking culture case after 37 ± 1 ℃ of constant temperature, rotating speed 170r/min shaking culture 10-12h, every bottle of sampling 1-2mL, purified water detects and calculates OD600 value with visible spectrophotometer after suitably diluting and is about 1;
Two, secondary amplification liquid: before inoculation, in Bechtop, in 2 bottles of amplification culture mediums (500mL), add 50mg/mL penbritin 500 μ l, then getting primary seed solution 5mL is inoculated in secondary amplification culture medium, in full temperature shaking culture case after 37 ± 1 ℃ of constant temperature, rotating speed 170r/min shaking culture 12-14h, every bottle of sampling 1-2mL, purified water detects and calculates OD600 value with visible spectrophotometer after suitably diluting and is about 1.5;
Three, one grade fermemtation is cultivated: (1) electrode is proofreaied and correct: pH electrode is proofreaied and correct, and before seed culture medium sterilizing, at room temperature with the reference liquid of pH6.86 and pH9.18, proofreaies and correct.The setting of DO electrode: before substratum preparation, DO electrode being put into saturated sodium bisulfite solution, to set DO value be 0; Before inoculation, set ventilation for 2500L/h, tank pressure is 0.04Mp, and stirring velocity is 560rpm, and dissolved oxygen when temperature is 37 ℃ is 100%.(2) work before inoculation: connect 500mL liquor ammoniae fortis (concentration approximately 28%) solution feed supplement bottle and 500mL dilute hydrochloric acid (concentration approximately 10%) solution feed supplement bottle, regulate the pH to 6.8-7.0 of substratum; (3) inoculation: by the satisfactory secondary of OD600 amplification liquid under flame protection with the inoculum size inoculation secondary amplification liquid of 5%-7% in the 10L fermention medium in seeding tank; (4) fermentation culture: 37 ± 1 ℃ of culture temperature, initial stirring velocity is 100rpm, and air flow is 300L/h, tank pressure 0.02-0.04Mpa, pH6.8-7.0, in culturing process, automatic makeup adds liquor ammoniae fortis and dilute hydrochloric acid adjusting pH, and dissolved oxygen sets 35%, is controlled between 30%-40%, dissolved oxygen and rotating speed coupling, if dissolved oxygen, lower than 30%, strengthens air flow, cultivate 2-4h to OD
6002～3, in culturing process, every half an hour, measure the OD600 value of fermented liquid;
Four, second order fermentation is cultivated: (1) electrode is proofreaied and correct: pH electrode is proofreaied and correct: before medium sterilization, with the reference liquid of pH6.86 and pH9.18, proofread and correct pH electrode.The setting of DO electrode: before substratum preparation, DO electrode being put into saturated sodium bisulfite solution, to set DO value be 0; Before inoculation, set ventilation for 16m
3/ h, tank pressure is 0.04Mp, and stirring velocity is 400rpm, and dissolved oxygen when temperature is 37 ± 1 ℃ is 100%; (2) work before inoculation: connect 500mL liquor ammoniae fortis feed supplement bottle, the rare HCl solution of 500mL feed supplement bottle, 4L80% carbon source feed supplement bottle; (3) inoculation: the satisfactory one grade fermemtation liquid of OD600 in seeding tank is inoculated in fermentor tank in 120L fermention medium with the inoculum size of 5-10%; (4) fermentation culture: initial stirring velocity is 100rpm, and air flow is 1.8m
3/ h, pH6.8-7.0,37 ± 1 ℃ of cultivations, after dissolved oxygen drops to 35%, setting DO is 35% controls associated with rotating speed, is controlled at 30%-40%, if associated control dissolved oxygen still lower than 30%, cancellation association, adjustable rotating speed and tune up gradually air flow, maximum can be adjusted to 16m
3/ h, in culturing process, automatic makeup adds liquor ammoniae fortis and dilute hydrochloric acid solution to regulate pH, dissolved oxygen set(ting)value is 15%, when OD600>=3,, adjusting rotary speed is controlled dissolved oxygen at 10%-20%, and air flow still keeps the air flow before induction, when DO significantly rises, with carbon source restriction schema stream, add 80% carbon source of sterilizing.37 ± 1 ℃ of continuous aerated culture of continuation were received bacterium in OD600 >=8 after approximately 4 to 5 hours, measured the OD600 value of fermented liquid in culturing process every about half an hour;
Five, receive bacterium: adopt tubular-bowl centrifuge to carry out centrifugal bacterium liquid with 45Hz frequency (about 14000rpm) and abandon supernatant liquor, flow velocity is 1.4-1.8L/min, and microorganism collection is deposited in clean container.
Six, quality index: smear gramstaining, the thalline of observing under opticmicroscope after fermentation is typical gram negative bacillus, by SDS-PAGE, rTPA38 albumen in bacterial protein being detected is 19%, fermentation yield: be 12g/L, calculation formula: fermentation yield (g/L)=wet bacterium weight (g)/fermentation basic medium amount (L).
The cultural method that embodiment 3 is common is cultivated mycobacterium tuberculosis
Get inclined-plane list bacterium colony, be placed in the test tube that contains 3mL Roche egg slant medium, in full temperature shaking culture case, 37 ± 1 ℃ of constant temperature, rotating speed 170r/min shaking culture are spent the night; In Bechtop, in every bottle of amplification culture medium (8mL), add 50mg/mL penbritin 8 μ l, then inoculate the bacterial classification in 80 μ l test tubes, in full temperature shaking culture case after 37 ± 1 ℃ of constant temperature, rotating speed 170r/min shaking culture 10-12h, every bottle of sampling 1-2mL, purified water detects and calculates OD600 value with visible spectrophotometer after suitably diluting and is about 1; Inoculum size with 5%-7% is inoculated in the 10L Roche egg slant medium substratum in seeding tank, adopt conventional cultural method, in culturing process, automatic makeup adds liquor ammoniae fortis and dilute hydrochloric acid adjusting pH, through about 12h, cultivate, fermentation ends, fermentation yield: be 3g/L, in bacterial protein, target protein rTPA38 is 15%.
Claims (1)
1. the method that mycobacterium tuberculosis thalline ferments, comprises the following steps:
One, one-level amplification liquid preparation: get mycobacterium tuberculosis inclined-plane list bacterium colony, be placed in the test tube that contains 3mL amplification culture medium, 37 ± 1 ℃ of constant temperature, rotating speed 170r/min shaking culture are spent the night in full temperature shaking culture case; In Bechtop, in the triangular flask that contains 8mL amplification culture medium, add 50mg/mL penbritin 8 μ l, then inoculate the bacterial classification in 80 μ l test tubes, in full temperature shaking culture case after 37 ± 1 ℃ of constant temperature, rotating speed 170r/min shaking culture 10-12h, sampling, is about 1 to OD600 value and can stops cultivating;
Two, secondary amplification liquid: before inoculation, in Bechtop, in 2 bottles of triangular flasks that contain 500mL amplification culture medium, add 50mg/mL penbritin 500 μ l, then getting one-level amplification liquid 5mL is inoculated in secondary amplification culture medium, in full temperature shaking culture case after 37 ± 1 ℃ of constant temperature, rotating speed 170r/min shaking culture 12-14h, sampling, is about 1.5 to OD600 value;
Three, one grade fermemtation is cultivated: before inoculation, set ventilation for 2500L/h, tank pressure is 0.04Mp, and stirring velocity is 560rpm, and dissolved oxygen when temperature is 37 ℃ is 100%; (2) work before inoculation: connecting 500mL concentration is 28% liquor ammoniae fortis feed supplement bottle and 500mL concentration 10% dilute hydrochloric acid solution feed supplement bottle, regulates the pH to 6.8-7.0 of substratum; (3) inoculation: the inoculum size with 5%-7% is inoculated in the 10L fermention medium in seeding tank; (4) fermentation culture: 37 ± 1 ℃ of culture temperature, initial stirring velocity is 100rpm, and air flow is 300L/h, tank pressure 0.02-0.04Mpa, pH6.8-7.0, in culturing process, automatic makeup adds liquor ammoniae fortis and dilute hydrochloric acid adjusting pH, and dissolved oxygen sets 35%, is controlled between 30%-40%, dissolved oxygen and rotating speed coupling, if dissolved oxygen, lower than 30%, strengthens air flow, cultivate 2-4h to OD
6002～3, in culturing process, every half an hour, measure the OD600 value of fermented liquid;
Four, second order fermentation is cultivated: before (1) inoculation, set ventilation for 16m
3/ h, tank pressure is 0.04Mp, and stirring velocity is 400rpm, and dissolved oxygen when temperature is 37 ± 1 ℃ is 100%; (2) work before inoculation: connecting 500mL concentration is 28% liquor ammoniae fortis feed supplement bottle, the rare HCl solution of 500mL concentration 10% feed supplement bottle, 4L80% carbon source feed supplement bottle; (3) inoculation: the satisfactory seed liquor of the OD600 after one grade fermemtation is inoculated in fermentor tank in 120L fermention medium with the inoculum size of 5-10%; (4) fermentation culture: initial stirring velocity is 100rpm, and air flow is 1.8m
3/ h, pH6.8-7.0,37 ± 1 ℃ of cultivations, after dissolved oxygen drops to 35%, setting DO is 35% controls associated with rotating speed, is controlled at 30%-40%, if associated control dissolved oxygen still lower than 30%, cancellation association, adjustable rotating speed and tune up gradually air flow, maximum can be adjusted to 16m
3/ h, in culturing process, automatic makeup adds liquor ammoniae fortis and dilute hydrochloric acid solution to regulate pH, dissolved oxygen set(ting)value is 15%, when OD600>=3,, adjusting rotary speed is controlled dissolved oxygen at 10%-20%, and air flow still keeps the air flow before induction, when DO significantly rises, with carbon source restriction schema stream, add 80% carbon source of sterilizing.37 ± 1 ℃ of continuous aerated culture of continuation were received bacterium in OD600 >=8 after 4 to 5 hours, measured the OD600 value of fermented liquid in culturing process every about half an hour;
Receive bacterium: adopt tubular-bowl centrifuge to carry out centrifugal bacterium liquid with 45Hz frequency, 14000rpm and abandon supernatant liquor, flow velocity is 1.4-1.8L/min, and microorganism collection is deposited in clean container, remembers the object thalline fermenting;
Five, substratum is formulated as follows:
(1) mycobacterium tuberculosis amplification culture medium is Roche egg slant medium, buys from Roche Holding Ag.
(2) fermention medium (1000mL): sodium selenate 10mg, potassium primary phosphate 2.4g, b) magnesium sulfate 0.24g, c) Citric Acid 0.6g, d) asparagine 3.6g, e) glycerine 20mL, f) yam starch 40g, g) fresh ovum gallinaceum liquid 930mL, h) Sodium Glutamate 0.15g surplus is water.
(3) preparation of other feed supplement liquid:
Dilute hydrochloric acid solution packing (1000mL): get each packing of dilute hydrochloric acid HCl1000mL 500mL and enter 2 500mL in sterilizing feed supplement bottle, liquor ammoniae fortis packing (1000mL): get each packing of liquor ammoniae fortis 1000mL 500mL and enter 2 500mL in sterilizing feed supplement bottle;
80% carbon source preparation (8L): take glycerine 6400g be dissolved in purified water to cumulative volume be 8L, be distributed in 5L feed supplement bottle every bottle of 4L, 121 ℃ of sterilizing 20min, airtight the depositing of cooling rear normal temperature.
50mg/mL penbritin (10mL): take 1g penbritin and dissolve by 20mL purified water.In Bechtop, with the filtration sterilization of aseptic syringe needle strainer, be distributed in sterilising vessel, packing specification is 1mL/1.5ml EP pipe, totally 10 pipes, preserve stand-by in 2-8 ℃ of refrigerator.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105154356A (en) * | 2015-07-29 | 2015-12-16 | 程永娟 | Culture medium of mycobacterium tuberculosis |
Citations (2)
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| CN101423807A (en) * | 2007-11-02 | 2009-05-06 | 中国科学院沈阳应用生态研究所 | Method for preparing polycyclic aromatic hydrocarbon degrading bacteria |
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Patent Citations (2)
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| CN101423807A (en) * | 2007-11-02 | 2009-05-06 | 中国科学院沈阳应用生态研究所 | Method for preparing polycyclic aromatic hydrocarbon degrading bacteria |
| CN102295687A (en) * | 2011-08-12 | 2011-12-28 | 广东瀚森生物药业有限公司 | Renaturing and purifying method of mycobacterium-tuberculosis recombinant inclusion-body protein |
Non-Patent Citations (3)
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105154356A (en) * | 2015-07-29 | 2015-12-16 | 程永娟 | Culture medium of mycobacterium tuberculosis |
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