CN108410899A - A kind of expression and purification method of recombination human muscle hemoglobin - Google Patents
A kind of expression and purification method of recombination human muscle hemoglobin Download PDFInfo
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Abstract
The present invention relates to clinical medicine detect and diagnose field, in particular to a kind of expression and purification method of recombination human muscle hemoglobin, the method includes：Recombination human myoglobin gene's sequence is connected into coli expression carrier, human muscle hemoglobin expression plasmid is obtained, the plasmid is transformed into Escherichia coli and obtains transformant；To the transformant expand culture induced expression after by clasmatosis, supernatant is collected after centrifugation；The recombination human muscle hemoglobin N-terminal given expression to contains Trx (His)6Fusion tag；The supernatant is used into Ni column affinity chromatographys after membrane filtration, up to recombination human muscle hemoglobin after elution.Recombination human muscle hemoglobin prepared by the method for the invention shows the feature of high yield, high activity and high stability, is suitable for the requirement of in-vitro diagnosis raw material.
Description
Technical field
The present invention relates to clinical medicine detect and diagnose fields, in particular to a kind of table of recombination human muscle hemoglobin
Up to purification process.
Background technology
Myoglobins (Myoglobin, Mb) is a kind of oxygen combination hemoglobin, by a peptide chain and a prosthetic heme group
Composition, molecular weight 16.7kDa are widely present in the cardiac muscle and skeletal muscle of people and other mammals.Prosthetic heme group is
Ferriporphyrin compound is connected to form a big ring, Fe by 4 pyrroles by 4 methenyls2+Positioned at pyrrole ring center.Flesh red eggs
White molecules align is close, and a molecule protein combines a molecular oxygen, this structure to make myoglobins in the mistake for storing and conveying oxygen
Journey faster plays a role.
In acute myocardial injury, since myoglobins is plasmosin, molecular weight is smaller, can be released in blood at first
In, the Mb after 2-3h in blood exceeds normal upper limit, and 6-9h peaks, therefore detection Mb levels can quickly be made a definite diagnosis and control in time
It treats, to reduce case fatality rate.Detection method in relation to Mb has very much, such as ELISA method, RIA methods, fluorescence immunoassay, is immunized
Turbidimetry etc., domestic detection kit is mostly that import or imported raw material assemble at present, expensive, is largely limited
The promotion and application of Mb detections.Therefore what is be currently badly in need of is independent development detection kit, breaks away from the present situation for relying on import.
In recent years, the relevant report of some domestic extraction myoglobins shows that natural method for extraction and purification is asked there are following
Topic：Source is difficult, extraction step is cumbersome, quality and activity are unstable etc.；Although recombination myoglobins solves native purified
Subproblem, but there is also low yield, the problems such as stability is bad, activity is low.
In view of this, special propose the present invention.
Invention content
It is an object of the invention to solve human muscle hemoglobin traditional native purified method cardiac muscular tissue source difficulty, yield
Low, quality and activity are difficult to stable defect, provide genetic engineering bacterium and the purifying of a kind of efficiently expressing recombinant human myoglobins
Scheme can obtain the recombination human muscle hemoglobin of high activity and high stability, provide and examine for clinical detection myocardial damage and prognosis
Disconnected reagent raw material, the preparation for quick diagnosis reagent kits of heart diseases such as acute myocardial infarction AMIs are laid a good foundation.
In order to realize that the above-mentioned purpose of the present invention, spy use following technical scheme：
The present invention relates to a kind of expression and purification methods of recombination human muscle hemoglobin, including：
Recombination human myoglobin gene's sequence is connected into coli expression carrier, obtains human muscle hemoglobin expression plasmid,
The plasmid is transformed into Escherichia coli and obtains transformant；
To the transformant expand culture induced expression after by clasmatosis, supernatant is collected after centrifugation；The recombination given expression to
Human muscle hemoglobin N-terminal contains Trx- (His)6Fusion tag；The supernatant is used into Ni- column affinity chromatographys after membrane filtration, is washed
Up to recombination human muscle hemoglobin after de-.
Recombination human muscle hemoglobin prepared by the method for the invention shows the spy of high yield, high activity and high stability
Sign is suitable for the requirement of in-vitro diagnosis raw material.
Compared with prior art, beneficial effects of the present invention are：
(1) present invention uses expression of recombinant e. coli Mb, N-terminal to have (His)6Label, one-step method obtain high-purity, high yield
Rate destination protein, yield reach 40mg/L；
(2) present invention designs Trx labels in destination protein N-terminal, and optimizes albumen and preserve system, improves the preservation of Mb
Stability, in 37 DEG C of accelerated tests, stability is more than 1 week.
Description of the drawings
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in being described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, other drawings may also be obtained based on these drawings.
Fig. 1 is metal chelating chromatography purifying protein electrophoretogram in one embodiment of the invention；
Fig. 2 is that Mb accelerates one week electrophoretogram in 37 DEG C in one embodiment of the invention；
Mb accelerates one week concentration mensuration figure in 37 DEG C in Fig. 3 one embodiment of the invention；
Mb accelerates one week activity stability in 37 DEG C in Fig. 4 one embodiment of the invention.
Specific implementation mode
The present invention relates to a kind of expression and purification methods of recombination human muscle hemoglobin, including：
Recombination human myoglobin gene's sequence is connected into coli expression carrier, recombination human muscle hemoglobin is obtained and expresses matter
Grain, the expression plasmid is converted to Escherichia coli and obtains transformant；
To the transformant expand culture induced expression after by clasmatosis, supernatant is collected after centrifugation；The recombination given expression to
Human muscle hemoglobin N-terminal contains Trx- (His)6Fusion tag；The supernatant is used into Ni- column affinity chromatographys after membrane filtration, is washed
Up to recombination human muscle hemoglobin after de-.
Fusion tag technology is to merge the encoding gene of certain label at the ends 3' or 5' of target gene, is a kind of recombination
DNA technique, common fusion tag have：MBP-tag, GST-tag, SBP-tag, SUMO-tag, Trx-tag etc..The present invention adopts
With fusion tag Trx, the solubility and stability of destination protein are finally improved.
The present invention overcomes the traditional native purified method cardiac muscular tissue source difficulty of human muscle hemoglobin, low yield, quality and
Activity is difficult to stable defect, provides a kind of genetic engineering bacterium and purification schemes of efficiently expressing recombinant human myoglobins, can
The recombination human muscle hemoglobin for obtaining high activity and high stability provides diagnostic reagent original for clinical detection myocardial damage and prognosis
Material, the preparation for quick diagnosis reagent kits of heart diseases such as acute myocardial infarction AMIs are laid a good foundation.
Preferably, method as described above, the Escherichia coli are BL21 (DE3).
Preferably, method as described above, the coli expression carrier are pET-32a (+).
PET-32a (+) expression vector N-terminal has Trx labels and His labels, and stabilization and the separation for being conducive to expression product are pure
The facility of change.
Preferably, the condition of method as described above, the transfection includes：
It will be added in the competent Escherichia coli under the human muscle hemoglobin expression plasmid condition of ice bath, ice
15min~25min, heat shock 80s~100s are placed in bath, 36 DEG C~38 DEG C, 200rpm~240rpm shake cultures 40min~
Coated plate screens transformant after 60min；
It is furthermore preferred that the condition of the transfection includes：
It will be added in the competent Escherichia coli under the human muscle hemoglobin expression plasmid condition of ice bath, ice
20min, heat shock 90s are placed in bath, 37 DEG C, coated plate screens transformant after 220rpm shake cultures 50min.
Preferably, method as described above, it is described induction for 0.05mM~0.80mM isopropylthiogalactosides into
Row induced expression；
It is furthermore preferred that the induction is to carry out induced expression with 0.10mM isopropylthiogalactosides.
Preferably, method as described above, when the isopropylthiogalactoside is added, bacterium solution culture to OD600=
0.6~0.8, more preferably 0.7.
Preferably, method as described above further includes after the isopropylthiogalactoside is added：16 DEG C~38
DEG C, 200rpm~240rpm shake cultures 3h~20h；
It is furthermore preferred that after the isopropylthiogalactoside is added, further include：37 DEG C, 200rpm~240rpm shakes
Swing culture 3h~5h.
Preferably, method as described above, the clasmatosis carry out in buffer solution, the buffer solution include 15mM~
25mM imidazoles；
More preferably 20mM imidazoles；
It is furthermore preferred that the ingredient of the buffer solution includes：
40mM~60mM Tris-HCl, 0.4M~0.6M NaCl, 15mM~25mM imidazoles of pH 8.2~8.8；
It is furthermore preferred that the ingredient of the buffer solution includes：
50mM Tris-HCl, 0.5M NaCl, the 20mM imidazoles of pH 8.5.
Preferably, method as described above, the parameter of the centrifugation be 8000rpm~12000rpm centrifuge 10min~
30min；
It is furthermore preferred that the parameter of the centrifugation, which is 10000rpm, centrifuges 20min.
Preferably, the aperture of method as described above, the filter membrane is 0.2 μm~0.24 μm；
More preferably 0.22 μm.
Preferably, method as described above, the eluent used in the elution includes 90mM~500mM imidazoles, preferably
90mM~110mM imidazoles；More preferably 100mM；
It is furthermore preferred that the ingredient of the eluent includes：
40mM~60mM Tris-HCl, 0.4M~0.6M NaCl, 90mM~500mM imidazoles of pH 8.2~8.8；
It is furthermore preferred that the ingredient of the eluent includes：
50mM Tris-HCl, 0.5M NaCl, the 100mM imidazoles of pH 8.5.
Unless otherwise defined, all technical and scientific terms that the present invention uses have with belonging to disclosed embodiment
The normally understood identical meaning of those of ordinary skill in field.Although similar or equivalent with method of the present invention and material
Method and material can be used in the practice or test of present embodiment, but hereafter still describe suitable method and material.
All publications, patent application, patent and other bibliography that the present invention refers to are incorporated into this by quoting full content
Wen Zhong.In the case of a conflict, this specification (including definition) will play dominating role.In addition, material, method and embodiment are only
It is merely illustrative, and is not intended to be limited to.Other feature and advantage of embodiment will be from following detailed description of book and right
Become in it is required that apparent.
In order to promote to understand implementations described herein this purpose, certain embodiments will be referred to, and will use
Language-specific describes these embodiments.Term as used herein is only used for description specific implementation mode purpose, without purport
It is limiting the scope of the present disclosure.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment
1) it is reference with the gene of the NCBI Autopsy Cases myoglobins provided, in conjunction with the experimental design requirement of the present invention, really
Determine SEQ ID NO:Gene shown in 1 simultaneously entrust Shanghai life work synthesized, carrier be pET-32a (+), N-terminal have Trx and
(His)6Label, is conducive to the stabilization of expression product, and facility is provided for fast separating and purifying.
2) recombinant plasmid imports host e. coli
Take 1 μ L expression plasmids, under condition of ice bath, be added in E. coli competent BL21 (DE3), ice bath is placed
20min, heat shock 90s are added 700 μ L LB culture mediums, 37 DEG C, 220rpm, shake culture 50min, 100 μ L bacterium solutions are taken to be applied to
In ammonia benzyl resistant panel.
3) destination gene expression process
The monoclonal in step 2) is chosen, sterile working is inoculated in LB culture mediums of the 5ml containing 100 μ g/ml, 37 DEG C,
220rpm, shake culture to OD600 is between 0.6~0.8, addition 0.1mM IPTG progress induced expressions, 37 DEG C, 220rpm,
Continue shake culture 4h.Sampling carries out SDS-PAGE identifications, is control with the bacterium solution not induced.
4) expression product purifies
Shaking flask culture 5L bacterium solutions, 8000rpm centrifuge 5min, collect thalline, prepare buffer solution 250ml (50mM Tris-
HCl, 0.5M NaCl, 20mM imidazoles, pH 8.50), thalline is resuspended, ultrasonication, 10000rpm, 20min is centrifuged, in collection
Clearly, Ni- column affinity chromatographys are crossed after 0.22 μm of membrane filtration, with 50mM Tris-HCl, 0.5M NaCl, 100mM imidazoles, pH
The albumen of 8.50 elutions is destination protein.
The electrophoretogram of albumen is as shown in Figure 1 after elution.
5) protein stability is tested
The destination protein of acquisition is sub-packed in respectively in 2mL EP pipes, 1mL/ branch is sealed with sealed membrane.Every batch of 5,
In 1 be placed in 4 DEG C as a contrast, remaining 4 be placed in 37 DEG C do acceleration one week test, respectively at 0,1,3,5,7 day sampling progress
Identification carries out electroresis appraisal and concentration mensuration.
Mb accelerates one week electrophoretogram as shown in Figure 2 in 37 DEG C.
Mb accelerates one week concentration mensuration figure as shown in Figure 3 in 37 DEG C.
6) chemoluminescence method identified activity
It is detected using double antibody sandwich method：1. the myoglobins monoclonal antibody of sample, biotin labeling, a word used for translation heavy stone used as an anchor ester
The other one plant of Mb monoclonal antibody and the coated magnetic particle of streptavidin of label are reacted under incubation conditions, antibody capture
Antigen in sample forms antigen-antibody sandwich complex.Sample-adding system：+ 100 μ L L4+10 μ L L1 of 20 μ L samples, 37 DEG C of temperature
Educate 10min；100 μ L L3,37 DEG C incubate 15min, and magnetic bead washing lotion is washed 3 times, measure luminous value.2. detection reading：Incubation terminates
Afterwards, add magnetic field to precipitate, remove supernatant, clean sediment composite with cleaning solution, and blot waste liquid, remove not with magnetic particle knot
The substance of conjunction, then reaction cup is sent into measuring chamber.Instrument is pumped into two kinds of exciting liquids automatically, and compound is made to generate chemiluminescence letter
Number, luminous intensity is measured by photoelectric multiplier.
Using chemiluminescence determination Mb active concentrations, as a result active concentration is the 40% of albumen concentration, far above diagnosis
Kit calibration object height limits.
7) chemoluminescence method identifies stability
Experimental program：It dispenses the several antigens of 100ul Mb and places 37 DEG C of insulating boxs, take out within the 0/4/7th day 10ul detections,
It is operated according to antigen mother liquor effective concentration validation criteria operating instruction, investigates raw material stability
Experimental result is as shown in Figure 4, the results showed that, the Mb containing 50% glycerine is accelerating a peripheral stability good.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but it will be understood by those of ordinary skill in the art that：Its
It still can be with technical scheme described in the above embodiments is modified, either to which part or all technical features
Carry out equivalent replacement；And these modifications or replacements, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
SEQUENCE LISTING
<110>The biotech inc Ji Dan
<120>A kind of expression and purification method of recombination human muscle hemoglobin
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 477
<212> DNA
<213>Artificial sequence
<400> 1
ggatccatgg gtctgagcga cggtgaatgg cagctggtgc tgaacgtctg gggtaaggtg 60
gaggctgaca tcccaggcca tggtcaggaa gtcctgatca ggctgtttaa gggtcaccca 120
gagactctgg agaagtttga caagttcaag cacctgaagt ctgaggacga gatgaaggcg 180
tctgaggacc tgaagaagca tggtgccacc gtgctgaccg ccctgggtgg catcctgaag 240
aagaagggtc atcatgaggc agagattaag ccgctggcac agtctcatgc caccaagcac 300
aagatcccgg tgaagtacct ggagttcatc tctgaatgca tcatccaggt tctgcagagc 360
aagcatccgg gtgactttgg tgctgatgcc cagggtgcca tgaacaaagc tctggagctg 420
ttccgtaagg acatggcctc caactacaag gagctgggct tccagggcta aaagctt 477
Claims (10)
1. a kind of expression and purification method of recombination human muscle hemoglobin, which is characterized in that including：
Recombination human myoglobin gene's sequence is connected into coli expression carrier, human muscle hemoglobin expression plasmid is obtained, by institute
It states plasmid and is transformed into Escherichia coli and obtain transformant；
To the transformant expand culture induced expression after by clasmatosis, supernatant is collected after centrifugation；The recombined human flesh given expression to
Lactoferrin N-terminal contains Trx- (His)6Fusion tag；The supernatant is used into Ni- column affinity chromatographys after membrane filtration, after elution
Up to recombination human muscle hemoglobin.
2. according to the method described in claim 1, it is characterized in that, the nucleotides sequence of recombination human myoglobin gene's sequence
Row such as SEQ ID NO:Shown in 1.
3. according to the method described in claim 1, it is characterized in that, the coli expression carrier is pET-32a (+).
4. according to the method described in claim 1, it is characterized in that, the Escherichia coli are BL21 (DE3).
5. according to the method described in claim 1, it is characterized in that, the conversion condition includes：
It will be added in the competent Escherichia coli under the human muscle hemoglobin expression plasmid condition of ice bath, ice bath is put
Set 15min~25min, heat shock 80s~100s, 36 DEG C~38 DEG C, after 200rpm~240rpm shake cultures 40min~60min
Coated plate screens transformant.
6. according to the method described in claim 1, it is characterized in that, the induction is with final concentration 0.05mM~0.8mM isopropyls
Base thiogalactoside carries out induced expression；
Preferably, the induction is to carry out induced expression with final concentration 0.10mM isopropylthiogalactosides.
7. according to the method described in claim 6, it is characterized in that, when the isopropylthiogalactoside is added, bacterium solution is trained
It supports to OD600=0.6~0.8.
8. according to the method described in claim 6, it is characterized in that, after the isopropylthiogalactoside is added, also wrap
It includes：16 DEG C~38 DEG C, 200rpm~240rpm shake cultures 3h~20h；
Preferably, after the isopropylthiogalactoside is added, further include：37 DEG C, 200rpm~240rpm shake cultures
3h~5h.
9. according to the method described in claim 1, it is characterized in that, the clasmatosis carries out in buffer solution, the buffering
Liquid includes 15mM~25mM imidazoles；
Preferably, the ingredient of the buffer solution includes：
40mM~60mM Tris-HCl, 0.4M~0.6M NaCl, 15mM~25mM imidazoles of pH 8.2~8.8.
10. according to the method described in claim 1, it is characterized in that, the eluent used in the elution includes 90mM~500mM
Imidazoles；
Preferably, the ingredient of the eluent includes：
40mM~60mM Tris-HCl, 0.4M~0.6M NaCl, 90mM~500mM imidazoles of pH 8.2~8.8.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109254153A (en) * | 2018-08-27 | 2019-01-22 | 广东菲鹏生物有限公司 | The antioxidant of myoglobins and its application |
| CN110240646A (en) * | 2019-06-26 | 2019-09-17 | 广东菲鹏生物有限公司 | The purification process of myoglobins and application |
| CN114703213A (en) * | 2022-03-07 | 2022-07-05 | 桂林英美特生物技术有限公司 | Preparation method of recombinant human cardiac troponin I and monoclonal antibody thereof |
| CN115417918A (en) * | 2022-03-24 | 2022-12-02 | 桂林英美特生物技术有限公司 | Preparation method of recombinant human beta 2 microglobulin and monoclonal antibody thereof |
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| JP3355049B2 (en) * | 1994-10-07 | 2002-12-09 | オリエンタル酵母工業株式会社 | Method for producing recombinant human myoglobin |
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| LIAPIS H 等: "myoglobin [Homo sapiens]，Accession Number：NP_001349775.1", 《NCBI GENBANK》 * |
| 刘闻: "人肌红蛋白基因合成、原核表达及单克隆抗体制备", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109254153A (en) * | 2018-08-27 | 2019-01-22 | 广东菲鹏生物有限公司 | The antioxidant of myoglobins and its application |
| CN110240646A (en) * | 2019-06-26 | 2019-09-17 | 广东菲鹏生物有限公司 | The purification process of myoglobins and application |
| CN110240646B (en) * | 2019-06-26 | 2021-04-09 | 广东菲鹏生物有限公司 | Purification method and application of myoglobin |
| CN114703213A (en) * | 2022-03-07 | 2022-07-05 | 桂林英美特生物技术有限公司 | Preparation method of recombinant human cardiac troponin I and monoclonal antibody thereof |
| CN115417918A (en) * | 2022-03-24 | 2022-12-02 | 桂林英美特生物技术有限公司 | Preparation method of recombinant human beta 2 microglobulin and monoclonal antibody thereof |
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Application publication date: 20180817 |
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