CN1603415A - Fusion expression method of mycobacterium tuberculosis ESAT-6 protein in pichia - Google Patents
Fusion expression method of mycobacterium tuberculosis ESAT-6 protein in pichia Download PDFInfo
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- CN1603415A CN1603415A CN 200410067343 CN200410067343A CN1603415A CN 1603415 A CN1603415 A CN 1603415A CN 200410067343 CN200410067343 CN 200410067343 CN 200410067343 A CN200410067343 A CN 200410067343A CN 1603415 A CN1603415 A CN 1603415A
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Abstract
The invention belongs to the biological technology domain, is concrete is in one tuberculosis difference bacillus Esat-6 protein in Bi Chishi yeast fusion expression method. He (Arab League sends) the esat-6 gene and person the -2a disturbance factorConnects, two linker manner enterokinase recognition sequence, will reorganize the fusion gene the DNA fragment insertion to express carrier pPIC9K, the electric shock inducts Bi Chishi in yeast strain SMD1168, will realize the Esat-6 fusion protein highly effective secretion expression. Obtain pure Esat-6 after the sparse water chromatographic analysis and the ionic exchange.
Description
Technical field
The invention belongs to biological technical field, is the proteic fusion expression method of a kind of mycobacterium tuberculosis (Mycobacterium tuberculosis) ESAT-6.Specifically be by making up the pichia recombinant strain,, obtain the ESTA-6 fusion rotein through separation and purification through shake flask fermentation efficient secretory expression ESAT-6 fusion rotein.
Background technology
ESAT-6 albumen is a kind of secreted protein that is separated in mycobacterium tuberculosis Short-term Culture filtrate, is made up of 95 amino-acid residues, and molecular weight is 9.9KD.ESTA-6 albumen exists only in tubercule bacillus group and a few the pathogenic mycobacterium, and all bacterial strains of bacille Calmette-Guerin vaccine (BCG vaccine) lack this albumen; Non-virulent mycobacterium strain disappearance esat-6 gene more than 90%.Among four kinds of secreted protein MPB59, MPB64, MPB70 and the ESTA-6 of mycobacterium bovis, only ESAT-6 albumen can distinguish BCG immune animal and mycobacterium bovis institute infected animals; The proteic specificity IFN-r of ESAT-6 only replied in the caused lung desease patient of mycobacterium tuberculosis, exist, all do not observe this reaction among lung desease patient that mycobacterium avium group (MAC) causes and the normal healthy controls person.Therefore ESAT-6 albumen can be used as the specific diagnosis reagent of diagnosis animal and human's pathogenic m tuberculosis infection.
Against mycobacterium tuberculosis immune anamnestic reaction is mediated by immune effector cell, in outbreeding system guinea pig model and ox TB animal model, ESAT-6 albumen is one of main target antigen of this T effector cell, can be at tuberculosis infection once more early stage, induce it to breed rapidly and discharge high-level IFN-r, thus activating macrophage effectively.Make subunit vaccine and ESAT-6 albumen is aided with adjuvant-phosphoryl lipid A (MPL), can induce to produce reaction of intensive specific T-cells and the protective effect suitable with BCG.Therefore ESAT-6 albumen can be used as the component of subunit vaccine to reach, effectively prevention long-term to tuberculosis.
Using gene engineering method, make up reorganization ESAT-6 protein engineering bacterial strain, is the proteic optimal path of scale operation ESAT-6.The domestic people of having attempts utilizing escherichia expression system to express ESAT-6 albumen at present, though can obtain high-caliber expression, but expressed reorganization ESAT-6 albumen mainly exists with inclusion body (inclusion body) form, and does not see the report of this albumen biologically active so far.
Summary of the invention
The purpose of this invention is to provide a kind of method that efficiently expresses mycobacterium tuberculosis ESAT-6-6 fusion rotein.Specifically be to adopt the DNA recombinant technology to make up the pichia recombinant strain, identify, prove reorganization bacterium energy efficient secretory expression ESAT-6 fusion rotein through shake flask fermentation and SDS-PAGE; This fusion rotein is identified through biological activity analysis and western blot, shows that it has higher interferon activity, and the serum generation specificity combination of energy and tuberculosis patient.
The proteic fusion expression method of mycobacterium tuberculosis ESAT-6-6 that the present invention proposes, the esat-6 gene and the people α-2a interferon gene of mycobacterium tuberculosis are together in series, linker between the two is people's enteropeptidase recognition sequence, the dna fragmentation of reorganization fusion gene is inserted expression vector p PIC9K, electric shock imports among pichia (Pichiapastoris) the bacterial strain SMD1168, realizes the efficient secretory expression of ESAT-6 fusion rotein.Obtain pure ESAT-6 albumen through hydrophobic chromatography and ion-exchange.
Concrete operations step of the present invention is as follows:
1. structure recombinant expression plasmid
(contain people a-2a interferon gene with α F9 plasmid respectively, this gene order is published) and the E1f plasmid (contain mycobacterium tuberculosis esat-6 gene, this gene order is published) be template, obtain people α-2a interferon gene and mycobacterium tuberculosis esat-6 gene by pcr amplification.5 ' the end and the 3 ' end of α-2a interferon gene contain XhoI and NotI restriction enzyme site respectively.5 ' end of esat-6 gene contains NotI restriction enzyme site and people's enteropeptidase Linker, and 3 ' end contains the EcoRI restriction enzyme site.With α-2a interferon gene fragment XhoI and NotI double digestion, esat-6 gene fragment NotI and EcoRI double digestion, plasmid pPIC9 (INVITROGEN company product) connects with the T4DNA ligase enzyme then with XhoI and EcoRI double digestion, obtains recombinant plasmid pPIC9-α 2a-esat6.With this recombinant plasmid BamHI and SalI double digestion, resulting small segment inserts expression plasmid pPIC9K (INVITROGEN company product), obtains recombinant expression plasmid pPIC9K-α 2a-esat6.
2. screening recombinant strain
Behind the SalI single endonuclease digestion, electricity transforms pichia SMD1168 (INVITROGEN company product) with recombinant expression plasmid pPIC9K-α 2a-esat6, and conversion fluid coating RDB flat board is cultivated the back and obtained transformant.Transformant is through the G418 resistance screening, and (containing on the YPD flat board of 4mg/mlG418) obtains high resistant strain.High resistant strain detects through shake flask fermentation and SDS-PAGE, proves that energy efficient secretory expression molecular weight size is about the ESAT-6 fusion rotein of 30KD.
3. shake flask fermentation
High expression level bacterial strain BMGY substratum with above-mentioned gained carries out shake flask fermentation.Specifically can be in optimizing the BMGY substratum, about 30 ℃ of following shaking culture 24h, the centrifugal 6000r/min 6min of bacterium liquid collects thalline, suspends with optimizing BMMY substratum (shaking bottle), and shaking culture 96h under the similarity condition, every 24h add methyl alcohol one time.Supernatant liquor after testing, ESAT-6 expressing fusion protein level is about 60mg/L.
4. separation and purification
Fermented supernatant fluid obtains pure ESAT-6 fusion rotein through phenyl-Sepharose hydrophobic chromatography and DEAE-Sephadex ion-exchange.
The biological activity analysis and the method for detecting specificity of ESAT-6 fusion rotein are as follows:
Get the sample determination interferon biological activity behind the 2nd, 3,4 day fermented liquid supernatant and the preliminary purification respectively, prove ESAT-6
Fusion rotein has higher interferon activity.Anti-with tuberculosis patient serum and mouse-anti human alpha interferon monoclonal antibody respectively in addition as one, the engineering strain expressed products is carried out western blot identify, prove that it is the ESAT-6 fusion rotein.
Among the present invention, on recombinant expression plasmid pPIC9K-α 2a-esat6, α-2a interferon gene is positioned at 5 ' end of recombinant DNA, and the esat-6 gene is positioned at 3 ' end, and Linker between the two is people's enteropeptidase recognition sequence.The dna sequence dna of this enteropeptidase Linker can design as follows:
5’…
GCGGCCGCT
GATGACGATGACAAA…3’
NotI people's enteropeptidase Linker
The N end of therefore expressing the fusion rotein that obtains is recombinant alpha-2a Interferon, rabbit, and the C end is the enteropeptidase identification polypeptide for ESAT-6 albumen between the two.
Known person α-2a Interferon, rabbit has antiviral and effect enhancing body immunizing power, and the esat-6 gene is connected with people α-2a interferon gene, helps the expression of esat-6 gene in pichia.In the present invention, the N end of yeast expressed fusion protein is recombinant alpha-2a Interferon, rabbit, the C end is reorganization ESTA-6 albumen, if directly fusion rotein is imported in the body, utilize the separable α of the enteropeptidase-2a Interferon, rabbit and the ESTA-6 albumen of human body self, also can just realize separating by enzymolysis external.Discover that the ESAT-6 fusion rotein not only has higher interferon activity, and can with the serum generation specificity combination of tuberculosis patient, therefore in the development of quick diagnosis lungy and vaccine, have application prospect.
Description of drawings
The design of graphics of Fig. 1 recombinant expression plasmid pPIC9K-of the present invention α 2a-esat6.
Fig. 2 SDS-PAGE electrophoretogram of the present invention: 1, protein molecular quality standard; 2～4, the fermented liquid supernatant of recombinant strain SMD1168/pPIC9K-α 2a-esat6; 5, the fermented liquid supernatant of pichia SMD1168.
The proteic western blot of Fig. 3 ESAT-6 of the present invention detects.
The western blot of Fig. 4 α of the present invention-2a Interferon, rabbit detects.
Embodiment
The structure of embodiment 1, recombinant expression plasmid and evaluation
Plasmid pPIC9 is with XhoI and EcoRI double digestion, and the PCR product that contains α-2a interferon gene is with XhoI and NotI double digestion, and the PCR product that contains the esat-6 gene is with NotI and EcoRI double digestion.Connect with the T4DNA ligase enzyme then, transformed into escherichia coli Top10 competent cell extracts recombinant plasmid pPIC9-α 2a-esat6 enzyme and cuts evaluation.And then with this recombinant plasmid with BamHI and SalI double digestion and reclaim small segment, plasmid pPIC9K is also with BamHI and SalI double digestion and reclaim big fragment, connect with the T4DNA ligase enzyme, transformed into escherichia coli Top10, extract recombinant expression plasmid pPIC9K-α 2a-esat6, enzyme is cut and is identified and determined dna sequence, proves that the structure of recombinant expression plasmid is entirely true.
Inoculation pichia SMD1168 (pep4, his4) in the YPD substratum, 30 ℃ of shaking culture to OD600 be 1.3～1.5,4 ℃ of centrifugal 5min of 5000r/min, collect thalline, respectively wash once with precooling sterilized water and 1mol/L sorbyl alcohol, suspend with 1mol/L sorbyl alcohol 200 μ l at last, promptly obtain the SMD1168 competent cell.Get 80 μ l SMD1168 competent cells and mix with the recombinant expression plasmid of SalI single endonuclease digestion with 5 μ g, at 1300v, 25 μ F; electric shock transforms under the 200 Ω conditions, after suspending with 1mol/L sorbyl alcohol 1ml, coats the RDB flat board; cultivate 3～4d, a transformant surplus the result obtains 1000 for 30 ℃.
The screening of the high resistance yeast transformant of embodiment 3, G418
Will on the RDB flat board surplus in the of long 1000 a yeast conversion daughter colony dibbling G418 concentration is 1.5,3.0 to containing, on the YPD flat board of 4.0mg/ml, 30 ℃ of cultivations, step-sizing G418 resistant strain.The result is containing 28 bacterial strains of acquisition on the 4mg/ml G418 flat board.
The screening of embodiment 4, high expression level recombinant bacterial strain
The high resistance transformant of G418 is inoculated in the test tube that contains 3ml BMGY, and 30 ℃ of vibration 24h get 1.5ml bacterium liquid and change in the 50ml optimization BMGY substratum, and under 30 ℃ of conditions, about shaking culture 24h, the centrifugal 6000r/min6min of bacterium liquid collects thalline.Optimize the BMMY substratum with 15ml and suspend, similarity condition shaking culture 96h added one time methyl alcohol in per 24 hours.Get the 4th day fermented liquid supernatant of fermentation, detect with 15%SDS-PAGE.The result obtains 6 plant height expression strains, and wherein No. 32 bacterial strains are as experimental strain in choosing, and its ESAT-6 expressing fusion protein level is about the 60mg/L fermented liquid.
The western blot of embodiment 5, expression product identifies
About the proteic specific detection of ESAT-6: the SDS-PAGE gel behind the electrophoresis is peeled off, electrotransfer to the NC film, 200mA constant current transferase 12 h; The NC film is put into 5% skimmed milk confining liquid, and 37 ℃ of vibration 1h wash 3 times with the vibration of TBS room temperature; Tuberculosis patient serum 2ml is anti-as one, and incubated at room 1h washes 3 times with the TBS vibration; Anti-with the anti-human IgG antibody of the goat of horseradish peroxidase-labeled (10000 times of the milk dilutions with 5%) as two, hatch 1h, wash 3 times with the TBS vibration; Add 0.1mol/L TrisHCl (PH7.5) 5ml, 100 μ l DAB storage liquid (40mg/ml), 25 μ l NiCl
2Storage liquid (40mg/ml) and 3%H
2O
215 μ l behind the black out colour developing 5min, wash termination reaction with water.
Specific detection about α-2a Interferon, rabbit: method is the same substantially.Anti-with mouse-anti human alpha interferon monoclonal antibody (with 500 times of 5% skimmed milk dilutions) as one, rabbit anti-mouse igg antibody (with 10000 times of 5% skimmed milk dilutions) with horseradish peroxidase-labeled is anti-as two, after in the darkroom, adding DAB damping fluid 10ml, add Peroxide Buffer700 μ l and Ehancer Solution 700 μ l again, the fluorescent colour developing.
The mensuration of the interferon biological activity of embodiment 6, expression product
Get the 2nd, 3,4 day fermented liquid supernatant and the sample behind preliminary purification respectively and measure the biological activity of Interferon, rabbit, in contrast with the yeast SMD1168 fermented liquid supernatant of not inserting foreign gene by Wanxing Biological Pharmaceutical Co., Ltd., Shanghai.
Record interferon activity such as following table:
| Sample | The 2nd day sample | The 3rd day sample | The 4th day sample | The preliminary purification sample | Contrast |
| Active (U/ml) | ??3.15×10 5 | ??1.24×10 6 | ??2.10×10 6 | ??2.97×10 6 | ????0 |
As seen from the above table: from fermenting the 2nd day to the 4th day, the activity of α-2a Interferon, rabbit raises gradually, and wherein the active difference of the 3rd day and the 4th day is little, and contrast does not have activity.
The separation and purification of embodiment 7, expression product
Adding ammonium sulfate to final concentration in fermented supernatant fluid is 1M, puts 4 ℃ of 2h, gets supernatant after centrifugal, adds the Phenyl-Sepharase post that balance is good, and flow velocity is 0.5ml/min.With 5 times of volume 1M ammonium sulfate washings, use redistilled water wash-out and 30% Virahol wash-out more respectively, collect and flow out sample, make electrophoresis detection.With the protein sample dialysis tubing of packing into, place the 0.1M sodium phosphate buffer to dialyse.After protein sample after the dialysis is centrifugal, add in the DEAE-Sephadex post that balance is good, flow velocity is 0.5ml/min.With 10 times of volume 0.1M sodium phosphate buffers (PH7.6) washing, contain 0.1M sodium phosphate buffer (PH7.6) wash-out of NaCl again with 5 times of volumes earlier, collect elution samples and make electrophoresis detection.
Claims (3)
1, the proteic fusion expression method of a kind of mycobacterium tuberculosis ESAT-6-6, it is characterized in that the esat-6 gene and the people α-2a interferon gene of mycobacterium tuberculosis are together in series, Linker between the two is people's enteropeptidase recognition sequence, recombinant DNA inserts secreted expression carrier p PIC9K, electric shock imports among the pichia bacterial strain SMD1168 after the linearizing, realize the efficient secretory expression of ESAT-6 fusion rotein, obtain pure ESAT-6 fusion rotein through hydrophobic chromatography and ion-exchange.
2, amalgamation and expression mode according to claim 1 is characterized in that the concrete operations step is as follows:
(1) makes up recombinant expression plasmid
Be template with α F9 plasmid that contains people α-2a interferon gene and the E1f plasmid that contains mycobacterium tuberculosis esat-6 gene respectively, obtain people α-2a interferon gene and mycobacterium tuberculosis esat-6 gene by pcr amplification; 5 ' the end and the 3 ' end of α-2a interferon gene contain XhoI and NotI restriction enzyme site respectively, and 5 ' end of esat-6 gene contains NotI restriction enzyme site and people's enteropeptidase Linker, and 3 ' end contains the EcoRI restriction enzyme site; With α-2a interferon gene fragment XhoI and NotI double digestion, esat-6 gene fragment NotI and EcoRI double digestion, plasmid pPIC9 connects with the T4DNA ligase enzyme then with XhoI and EcoRI double digestion, obtains recombinant plasmid pPIC9-α 2a-esat6; With this recombinant plasmid BamHI and SalI double digestion, resulting small segment inserts expression plasmid pPIC9K, obtains recombinant expression plasmid pPIC9K-α 2a-esat6;
(2) screening recombinant strain
Behind the SalI single endonuclease digestion, electricity transforms pichia SMD1168 with recombinant expression plasmid pPIC9K-α 2a-esat6, and conversion fluid coating RDB flat board is cultivated the back and obtained transformant; Transformant obtains high resistant strain through the G418 resistance screening;
(3) shake flask fermentation
The high expression level bacterial strain of above-mentioned gained is inserted optimization BMGY substratum, carry out shake flask fermentation;
(4) separation and purification
Fermented supernatant fluid obtains pure ESAT-6 fusion rotein through phenyl-Sepharose hydrophobic chromatography and DEAE-Sephadex ion-exchange.
3, the proteic fusion expression method of ESAT-6 according to claim 2 is characterized in that the dna sequence dna of described enteropeptidase Linker is as follows:
5’......
GCGGCCGC?T
?GATGACGATGACAAA......3’
NotI enteropeptidase Linker
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100497592C (en) * | 2005-12-31 | 2009-06-10 | 重庆市肺科医院 | Mycobactrium tuberculosis protein for diagnosing rifampicin dependent mycobacterium tuberculosis |
| CN111303301A (en) * | 2020-03-18 | 2020-06-19 | 上海晶诺生物科技有限公司 | Preparation method of mycobacterium tuberculosis immunogen protein ESAT6 |
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2004
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100497592C (en) * | 2005-12-31 | 2009-06-10 | 重庆市肺科医院 | Mycobactrium tuberculosis protein for diagnosing rifampicin dependent mycobacterium tuberculosis |
| CN111303301A (en) * | 2020-03-18 | 2020-06-19 | 上海晶诺生物科技有限公司 | Preparation method of mycobacterium tuberculosis immunogen protein ESAT6 |
| CN111303301B (en) * | 2020-03-18 | 2022-04-05 | 上海晶诺生物科技有限公司 | Preparation method of mycobacterium tuberculosis immunogen protein ESAT6 |
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