CN103665116A - Mycobacterium tuberculosis rTPA38 protein inclusion body primary purification method applicable to mass production - Google Patents
Mycobacterium tuberculosis rTPA38 protein inclusion body primary purification method applicable to mass production Download PDFInfo
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Abstract
The invention belongs to the field of bioengineering and relates to a mycobacterium tuberculosis rTPA38 protein inclusion body primary purification method. The specific technical scheme is that the primarily purified inclusion bodies are obtained through obtaining and crushing of thalli as well as four times of washing. The method is suitable for the industrial production and large in batch, 100-5000 g of rTPA38 thalli can be treated once, the crushing effect is good, the crushing rate can reach above 99.9%, the yield is high, the yield of the inclusion bodies reaches above 90%, the purity of the rTPA38 protein in the inclusion bodies is high, the purity (electrophoresis) of target protein of the rTPA38 inclusion bodies reaches above 50%, the difficulty is reduced for follow-up purification work, and the activity of the rTPA38 protein is not affected.
Description
Technical field
The present invention relates to just pure method of mycobacterium tuberculosis rTPA38 albumen inclusion body.
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.
Escherichia expression system becomes medicinal restructuring TPA38KDa(recombinant TPA38KD, rTPA38KDa because genetic background is clear, genetic manipulation is simple, expression level is high, fermentation costs is low and be easy to the advantages such as technical scale amplification) desirable host system.The nearly source on evolving because of MTB and intestinal bacteria, so the TPA38KDa encoding sequence natural or the simple transformation of warp in MTB source is easy to obtain high efficient expression under the control of strong promoter (as T7 promotor), expression level generally can reach more than 30%.The same with other target protein of expressing at intestinal bacteria system high efficiency, the efficient expression product of TPA38KDa is also inclusion body protein a kind of non-activity, water-insoluble, must take suitable renaturation and purification process could obtain the product of pharmaceutical grade.But yield is low in the preliminary purification of inclusion body in prior art, and cost is high, be difficult to be applied to scale operation.
Therefore,, for deficiency of the prior art, needing badly provides a kind of and can industrialization amplifies, is convenient to that Quality Control, efficiency are high, the purification process of the mycobacterium-tuberculosis recombinant inclusion-body protein of the pharmaceutical grade that saves production cost.
Summary of the invention
The object of the present invention is to provide: a kind of mycobacterium tuberculosis rTPA38 albumen inclusion body is pure method just, thereby collect a large amount of inclusion bodys that amplification obtains, obtain the target protein after a large amount of purifying, promoted r T P A38 albumen for extensive use.
Technical scheme of the present invention: a kind of mycobacterium tuberculosis rTPA38 albumen inclusion body is pure method just, comprises the steps:
One. thalline pre-treatment:
(1) thalline suspends: the initial engineering bacteria thalline of the expression rTPA38 albumen of fermentation culture is thawed, after thawing, with the dilution of rTPA38 thalline buffer suspension liquid, then in stainless steel thalline treatment tank, be stirred to and suspend completely evenly, agitator motor frequency 45Hz.In inclusion body thalline treatment tank chuck, pass into-5 ℃ of-0 ℃ of refrigerated waters thallus suspension liquid is down to temperature lower than below 15 ℃;
(2) bacterial cell disruption: stainless steel thalline treatment tank discharge port is connected with high-pressure homogeneous machine inlet capable with silicone tube, thallus suspension liquid is passed into high pressure homogenizer with the flow velocity of 1.35L per minute, with the pressure breaking of 800bar, process, the about 80MPa of cracking pressure, in bacterium liquid access stainless steel cask after processing, return again in stainless steel thalline treatment tank to stir and be cooled to lower than again broken after 15 ℃, repeat continuous three times;
(3) centrifugal: the bacterial cell disruption liquid after high-pressure homogeneous broken three times is centrifugal, discarded supernatant liquor, collecting precipitation obtains thick inclusion body;
Two. inclusion body washing:
(1) inclusion body washing for the first time: 1. stirring suspension: thick inclusion body is pressed 1:20 inclusion body weight (g)/damping fluid volume (mL) (2%Triton-X100,100mM NaCl, 1mM EDTA, pH8.020mM Tris-HCl, 0.5% mercaptoethanol) ratio is by the thick inclusion body dilution of rTPA38, centrifugal collection inclusion body precipitation, discarded supernatant liquor;
(2) inclusion body washing for the second time: 1. stirring suspension: will wash for the first time rear inclusion body precipitation by 1:20 inclusion body weight (g)/damping fluid volume (mL) (1%Triton-X100,100mM NaCl, 1mM EDTA, pH8.020mM Tris-HCl, 0.1% mercaptoethanol) ratio is by the thick inclusion body dilution of rTPA38, centrifugal collection inclusion body precipitation, discarded supernatant liquor;
(3) inclusion body washing for the third time: 1. stirring suspension: will wash for the second time rear inclusion body precipitation by 1:20 inclusion body weight (g)/damping fluid volume (mL) (1mM EDTA2Na, pH8.020mM Tris-HCl) ratio is diluted centrifugal collection inclusion body precipitation, discarded supernatant liquor by the thick inclusion body of rTPA38;
(4) the 4th inclusion body washings: a. stirring suspension: will wash for the third time rear inclusion body precipitation by 1:20 inclusion body weight (g)/damping fluid volume (mL) (1mM EDTA, pH8.020mM Tris-HCl) ratio is by the thick inclusion body dilution of rTPA38, the centrifugal inclusion body obtaining after just pure, and-30 ℃ of preservations.
Beneficial effect of the present invention: method of the present invention, be applicable to industrialized production, large in batches, disposable 100g to the 5000g rTPA38 thalline of processing, crushing effect is high, percentage of damage can reach more than 99.9%, and yield is high, and inclusion body yield reaches more than 90%, in inclusion body, rTPA38 purity of protein is high, rTPA38 inclusion body target protein purity (electrophoresis) reaches more than 50%, and work has reduced difficulty to subsequent purification, and does not affect the activity of rTPA38 albumen.
Accompanying drawing explanation
Opticmicroscope figure after gramstaining before Fig. 1 rTPA38 bacterial cell disruption
Opticmicroscope figure after gramstaining after Fig. 2 rTPA38 bacterial cell disruption
Fig. 3 rTPA38 inclusion body electrophoretogram (1 swimming lane: the thick inclusion body obtaining after bacterial cell disruption; 2 swimming lanes: the inclusion body obtaining after washing for the first time; 3 swimming lanes: the inclusion body obtaining after washing for the third time; 4 swimming lanes: the inclusion body obtaining after the 4th washing)
Fig. 4 rTPA38 inclusion body is pure method process flow sheet just
Embodiment
Embodiment 1: thalline pre-treatment:
(1) mycobacterium tuberculosis rTPA38 albumen is reached to carrier with pet sheet and be connected, after abduction delivering, obtain the recombination bacillus coli thalline of expressing in the mode of inclusion body, freezing preservation.
Thalline suspends: the initial engineering bacteria thalline of the expression rTPA38 albumen of fermentation culture is put in 37 ℃ of thermostat water baths and thawed, after thawing, in 1:20(thalline weight (g)/damping fluid volume (mL)) rTPA38 thalline buffer suspension liquid (100mM NaCl for ratio, 1mM EDTA, 50mM Tris-HCl, pH8.0) dilution is then stirred to and suspends completely evenly, agitator motor frequency 45Hz in stainless steel thalline treatment tank.In inclusion body thalline treatment tank chuck, pass into-5 ℃ of-0 ℃ of refrigerated waters thallus suspension liquid is down to temperature lower than below 15 ℃;
(2) bacterial cell disruption: stainless steel thalline treatment tank discharge port is connected with high-pressure homogeneous machine inlet capable with silicone tube, thallus suspension liquid is passed into high pressure homogenizer with the flow velocity of 1.35L per minute, with the pressure breaking of 800bar, process, the about 80MPa of cracking pressure, in bacterium liquid after processing access stainless steel cask, then return in stainless steel thalline treatment tank, stir cold, but to lower than again broken after 15 ℃, repeat continuous three times;
(3) centrifugal: by the bacterial cell disruption liquid after high-pressure homogeneous broken three times centrifugal (4 ℃, 8000rpm, 12min), discarded supernatant liquor, collecting precipitation obtains thick inclusion body.As can be seen from Figure 2, bacterial cell disruption is very complete, and existing does not hardly have broken thalline, illustrates that broken method is extremely successful, Fig. 1 be after thawing before broken, observe thalline image.
Embodiment 2: inclusion body washing
(1) inclusion body washing for the first time: 1. stirring suspension: thick inclusion body is pressed 1:20 inclusion body weight (g)/damping fluid volume (mL) (2%Triton-X100,100mM NaCl, 1mM EDTA, pH8.020mM Tris-HCl, 0.5% mercaptoethanol) ratio is by the thick inclusion body dilution of rTPA38, then pour in stainless steel cask with lower magnetic force agitator stirring suspension 15-20 minute to completely evenly approximately 30 ℃ ± 5 ℃ of temperature into.2. thick inclusion body suspensions is centrifugal: centrifugal rotational speed is 8000rpm, and centrifuging temperature is 4 ℃, and centrifugation time is 15min to 20min, collects inclusion body precipitation, discarded supernatant liquor;
(2) inclusion body washing for the second time: 1. stirring suspension: will wash for the first time rear inclusion body precipitation by 1:20 inclusion body weight (g)/damping fluid volume (mL) (1%Triton-X100,100mM NaCl, 1mM EDTA, pH8.020mM Tris-HCl, 0.1% mercaptoethanol) ratio is by the thick inclusion body dilution of rTPA38, then pour in stainless steel cask with lower magnetic force agitator stirring suspension 15-20 minute to completely evenly approximately 30 ℃ ± 5 ℃ of temperature into.2. thick inclusion body suspensions is centrifugal: centrifugal rotational speed is 8000rpm, and centrifuging temperature is 4 ℃, and centrifugation time is 15min to 20min, collects inclusion body precipitation, discarded supernatant liquor;
(3) inclusion body washing for the third time: 1. stirring suspension: will wash for the second time rear inclusion body precipitation by 1:20 inclusion body weight (g)/damping fluid volume (mL) (1mM EDTA, pH8.020mM Tris-HCl) ratio is by the thick inclusion body dilution of rTPA38, then pour in stainless steel cask with lower magnetic force agitator stirring suspension 15-20 minute to completely evenly approximately 30 ℃ ± 5 ℃ of temperature into.2. thick inclusion body suspensions is centrifugal: centrifugal rotational speed is 8000rpm, and centrifuging temperature is 4 ℃, and centrifugation time is 15min to 20min, collects inclusion body precipitation, discarded supernatant liquor;
(4) the 4th inclusion body washings: a. stirring suspension: will wash for the third time rear inclusion body precipitation by 1:20 inclusion body weight (g)/damping fluid volume (mL) (1mM EDTA, pH8.020mM Tris-HCl) ratio is by the thick inclusion body dilution of rTPA38, then pour in stainless steel cask with lower magnetic force agitator stirring suspension 15-20 minute to completely evenly approximately 30 ℃ ± 5 ℃ of temperature into; B. thick inclusion body suspensions is centrifugal: centrifugal rotational speed is 8000rpm, and centrifuging temperature is 4 ℃, and centrifugation time is 15min to 20min, and c. collects: collect inclusion body precipitation, discarded supernatant liquor, obtains the inclusion body after just pure, and-30 ℃ of preservations.By Fig. 3 electrophoresis detection, can find out, after the 4th washing, inclusion body is very pure, almost there is no the interference of foreign protein.
The inclusion body that the method for embodiment 3 routines obtains
Thalline construction and expression is with embodiment 1.Take out the thalline after freezing, with 30ml/mg, be resuspended in broken damping fluid (the 50mmol/LTrisHCl pH8.0 of cell supersonic wave; 1mmol/L EDTA; 50mmol/L NaCl; 5% glycerine), under condition of ice bath, carry out ultrasonic disruption, 12000rpm, 4 ℃ of centrifugal 15min, collecting precipitation; With inclusion body lavation buffer solution (50mmol/L TrisHCl pH8.0; 2mmol/L urea; 0.5%Triton-X-100) washing is 3 times, and the precipitation of acquisition is the inclusion body of purifying.
As can be seen from the above results, the inclusion body that uses present method to obtain can guarantee the activity of rTPA38 albumen to greatest extent, compared with prior art, has significant progress.
Claims (2)
1. the first pure method of mycobacterium tuberculosis rTPA38 albumen inclusion body that is applicable to scale operation, comprises the following steps:
One, thalline pre-treatment: (1) thalline suspends: the initial engineering bacteria thalline of the expression rTPA38 albumen of fermentation culture is put in 37 ℃ of thermostat water baths and thawed, after thawing, in rTPA38 thalline buffer suspension liquid dilution for the ratio of 1:20 thalline weight (g)/damping fluid volume (mL), then in stainless steel thalline treatment tank, be stirred to and suspend completely evenly, agitator motor frequency 45Hz.In inclusion body thalline treatment tank chuck, pass into-5 ℃ of-0 ℃ of refrigerated waters thallus suspension liquid is down to temperature lower than below 15 ℃; RTPA38 thalline buffer suspension liquid: 100mM NaCl, 1mM EDTA, 50mM Tris-HCl, pH8.0;
(2) bacterial cell disruption: stainless steel thalline treatment tank discharge port is connected with high-pressure homogeneous machine inlet capable with silicone tube, thallus suspension liquid is passed into high pressure homogenizer with the flow velocity of 1.35L per minute, with the pressure breaking of 800bar, process, the about 80MPa of cracking pressure, in bacterium liquid after processing access stainless steel cask, then return in stainless steel thalline treatment tank, stir cold, but to lower than again broken after 15 ℃, repeat continuous three times;
(3) centrifugal: by centrifugal 4 ℃ of the bacterial cell disruption liquid after high-pressure homogeneous broken three times, 8000rpm, 12min, discarded supernatant liquor, collecting precipitation obtains thick inclusion body.
Two, inclusion body washing
(1) inclusion body washing for the first time: a. stirring suspension: thick inclusion body dilutes the thick inclusion body of rTPA38 in the ratio of 1:20 inclusion body weight (g)/damping fluid volume (mL), then pour in stainless steel cask with lower magnetic force agitator stirring suspension 15-20 minute to completely evenly approximately 30 ℃ ± 5 ℃ of temperature into.B. thick inclusion body suspensions is centrifugal: centrifugal rotational speed is 8000rpm, and centrifuging temperature is 4 ℃, and centrifugation time is 15min to 20min, collects inclusion body precipitation, discarded supernatant liquor; Buffer formulation is: 2%Triton-X100,100mM NaCl, 1mM EDTA, pH8.020mMTris-HCl, 0.5% mercaptoethanol;
(2) inclusion body washing for the second time: a. stirring suspension: will wash for the first time rear inclusion body precipitation and in the ratio of 1:20 inclusion body weight (g)/damping fluid volume (mL), the thick inclusion body of rTPA38 be diluted, then pour in stainless steel cask with lower magnetic force agitator stirring suspension 15-20 minute to completely evenly approximately 30 ℃ ± 5 ℃ of temperature into; B. thick inclusion body suspensions is centrifugal: centrifugal rotational speed is 8000rpm, and centrifuging temperature is 4 ℃, and centrifugation time is 15min to 20min, collects inclusion body precipitation, discarded supernatant liquor; Buffer formulation is: 1%Triton-X100,100mM NaCl, 1mM EDTA, pH8.020mM Tris-HCl, 0.1% mercaptoethanol.
(3) inclusion body washing for the third time: a. stirring suspension: will wash for the second time rear inclusion body precipitation and in the ratio of 1:20 inclusion body weight (g)/damping fluid volume (mL), the thick inclusion body of rTPA38 be diluted, then pour in stainless steel cask with lower magnetic force agitator stirring suspension 15-20 minute to completely evenly approximately 30 ℃ ± 5 ℃ of temperature into.B. thick inclusion body suspensions is centrifugal: centrifugal rotational speed is 8000rpm, and centrifuging temperature is 4 ℃, and centrifugation time is 15min to 20min, collects inclusion body precipitation, discarded supernatant liquor; Buffer formulation is: 1mM EDTA, pH8.020mM Tris-HCl; (4) the 4th inclusion body washings: a. stirring suspension: will wash for the third time rear inclusion body precipitation and in the ratio of 1:20 inclusion body weight (g)/damping fluid volume (mL), the thick inclusion body of rTPA38 be diluted, then pour in stainless steel cask with lower magnetic force agitator stirring suspension 15-20 minute to completely evenly approximately 30 ℃ ± 5 ℃ of temperature into; Buffer formulation is: 1mM EDTA, pH8.020mM Tris-HCl; B. thick inclusion body suspensions is centrifugal: centrifugal rotational speed is 8000rpm, and centrifuging temperature is 4 ℃, and centrifugation time is 15min to 20min, and c. collects: collect inclusion body precipitation, discarded supernatant liquor, obtains the inclusion body after just pure, and-30 ℃ of preservations.
2. according to the method for claim 1, the amount that it is characterized in that the processing of thalline can be disposable processing 100g to 5000g rTPA38 thalline.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1407085A (en) * | 2001-09-10 | 2003-04-02 | 北京东康龙病毒生物技术工程研究中心 | Modular automatic production line for biopharmaceutics |
| CN1793373A (en) * | 2005-11-04 | 2006-06-28 | 中国人民解放军第三军医大学 | Process for preparing human leucocyte interleukin 24 by genetic engineering and it expressing carrier and engineering bacterium |
| CN101139388A (en) * | 2007-09-26 | 2008-03-12 | 华东理工大学 | Purification renaturation method for antineoplastic medicine TAT(m)-Survivin(T34A) |
| CN101220081A (en) * | 2008-01-28 | 2008-07-16 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Extraction and purification process for recombinant protein |
| CN101220082A (en) * | 2008-01-28 | 2008-07-16 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Extraction and purification process for recombinant protein |
| CN101843899A (en) * | 2010-05-24 | 2010-09-29 | 中国人民解放军第三军医大学 | Methicillin-resistant staphylococcus aureus (MRSA) recombinant multivalent subunit genetic engineering vaccine and method for preparing same |
| 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 |
-
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Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1407085A (en) * | 2001-09-10 | 2003-04-02 | 北京东康龙病毒生物技术工程研究中心 | Modular automatic production line for biopharmaceutics |
| CN1793373A (en) * | 2005-11-04 | 2006-06-28 | 中国人民解放军第三军医大学 | Process for preparing human leucocyte interleukin 24 by genetic engineering and it expressing carrier and engineering bacterium |
| CN101139388A (en) * | 2007-09-26 | 2008-03-12 | 华东理工大学 | Purification renaturation method for antineoplastic medicine TAT(m)-Survivin(T34A) |
| CN101220081A (en) * | 2008-01-28 | 2008-07-16 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Extraction and purification process for recombinant protein |
| CN101220082A (en) * | 2008-01-28 | 2008-07-16 | 江苏吴中医药集团有限公司苏州中凯生物制药厂 | Extraction and purification process for recombinant protein |
| CN101843899A (en) * | 2010-05-24 | 2010-09-29 | 中国人民解放军第三军医大学 | Methicillin-resistant staphylococcus aureus (MRSA) recombinant multivalent subunit genetic engineering vaccine and method for preparing same |
| 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 |
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