CN106397579A - Thrombin activated fibrinolysis inhibitor (TAFI) recombinant antigen efficiently expressed by escherichia coli as well as construction method and application of TAFI recombinant antigen - Google Patents
Thrombin activated fibrinolysis inhibitor (TAFI) recombinant antigen efficiently expressed by escherichia coli as well as construction method and application of TAFI recombinant antigen Download PDFInfo
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- C07K14/81—Protease inhibitors
- C07K14/8107—Endopeptidase (E.C. 3.4.21-99) inhibitors
- C07K14/811—Serine protease (E.C. 3.4.21) inhibitors
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Abstract
The invention belongs to the technical field of bioengineering and in particular relates to a thrombin activated fibrinolysis inhibitor (TAFI) recombinant antigen efficiently expressed by escherichia coli as well as a construction method and application of the TAFI recombinant antigen. An amino acid of the recombinant antigen is shown in a sequence table Seq ID No. 1. A recombinant antigen base is shown in a sequence table Seq ID No. 2. According to the TAFI recombinant antigen as well as the construction method and the application thereof disclosed by the invention, a TAFI amino acid sequence is reasonably changed by a genetic engineering method; after the sequence is optimized, the TAFI recombinant antigen is efficiently expressed by an escherichia coli expression system.
Description
Technical field
The invention belongs to technical field of bioengineering is and in particular to a kind of fibre of escherichia coli high-level expression thrombin activation
Molten mortifier (TAFI) recombinant antigen and its construction method and application.
Background technology
With the process of social continuous development and aging population, thrombotic diseases have become as the incidence of disease and death
A kind of all higher disease of rate, it has seriously threatened health and the life of patient, has been subject to the extensive concern of researcher, becomes
Research emphasis for domestic and international medical science.Thrombotic diseases occur main with body intravascular coagulation and fibrinolytic system unbalance relevant.
Thrombin activation inhibitors of fibrinolysis (thrombin activated fibrinolysis inhibitor,
TAFI) be internal fibrinolytic system important composition composition, it is to be synthesized by liver, and containing 423 amino acid residues, molecular weight is about
Single chain glycoprotein for 56kD.In blood, TAFI is not to have activated zymogen forms presence, after blood coagulation, TAFI quilt
Thrombomodulin, fibrinolysin and protease etc. activate into activated TAFIa.TAFIa is in blood clotting and fibrin
Play an important role in the balance of dissolving, it can excise fibrin c-terminus Arg or Lys, suppress fibrinolytic protein proenzyme
Activation, thus reducing the activation of fibrinolysin, have lower fibrinolytic system function.
The TAFI resource-constrained of natural origin, the merchandized handling for TAFI for the DNA recombinant technique provides a kind of economy to be had
The mode of effect, DNA recombinant technique is that foreign gene and carrier are carried out splicing restructuring in vitro, then proceeds to by internal, is allowed to
Wish according to people is stablized heredity and is given expression to new product or the molecule clone technology of new proterties.Many albumen have led at present
Cross gene recombination technology successful expression in multiple heterologous hosts.Escherichia expression system is the application of expressing heterologous recombinant protein
Most commonly used system, with other expression systems compared with yeast expression system etc., it has can Gao Shui in the short period of time
Level land expression multiple protein, fermentation condition are easily controlled and are expressed the low advantage of cost.
Natural TAFI sequence directly proceeds in escherichia expression system to be expressed, and can produce and inactive forgive the bodily form
Formula.The formation of inclusion body has favourable one side, also has unfavorable one side.Favourable one side be inclusion body formation eliminate several
Whole intracellular soluble proteins.Meanwhile, avoid the degraded to expression product for the proteolytic enzyme because inclusion body is formed
And greatly improve yield.Unfavorable one side is to need with denaturant and de-sludging during dissolving inclusion body carries out renaturation folding
Agent, and cause the irreversible modification of protein and property to change, these reagent are expensive, and the operating process of renaturation is bad
Control；Another aspect renaturation process is often accompanied by proteolysis and precipitation, and some also form isomers.Therefore, by gene work
It is very necessary that destination protein is directly expressed as activated soluble protein by escherichia expression system by Cheng Fangfa
's.
Content of the invention
It is an object of the invention to provide a kind of inhibitors of fibrinolysis (TAFI) of escherichia coli high-level expression thrombin activation
Recombinant antigen and its construction method and application.
For achieving the above object, the present invention using technical scheme is：
A kind of inhibitors of fibrinolysis (TAFI) recombinant antigen of escherichia coli high-level expression thrombin activation, recombinant antigen amino
Acid is as shown in sequence table Seq ID No.1.
Described recombinant antigen base is as shown in sequence table Seq ID No.2.
A kind of preparation method of inhibitors of fibrinolysis (TAFI) recombinant antigen of escherichia coli high-level expression thrombin activation：
Step (1) designs restriction enzyme site at the gene order two ends shown in Seq ID No.2 through codon optimization, respectively
BamH I and Xho I, by this gene be connected to pET-41a (+) on carrier, obtain recombinant vector (pET41a-p.MD-TAFI)；
The recombinant expression carrier pET41a-p.MD-TAFI that step (1) gained successfully constructs is transformed into large intestine by step (2)
Bacillus competent cell, builds and obtains recombination bacillus coli, by gained recombination bacillus coli through culture purified, obtain recombinant antigen.
Further,
(1) seed culture：The recombination bacillus coli of the inhibitors of fibrinolysis (TAFI) that thrombin activation is produced in described restructuring connects
Plant in seed culture medium, in 30～37 DEG C of shaken cultivation 8～12h, shaking speed 150～200r/min；
(2) Liquid Culture：The inoculation that gained activated seed liquid presses percent by volume 3～10% will be cultivated through step (1)
Amount is seeded to expression culture medium, in 30～37 DEG C of shaken cultivation 4～6h hours, shaking speed 150～200r/min；Then then at
20～30 DEG C of vibration Fiber differentiation 14～22h, shaking speed 150～200r/min expression finishes；
(3) slightly carry recombinant antigen：By the expression liquid centrifugation of step (2) gained；Collect supernatant, as extracellular slightly to propose restructuring anti-
Former；Collects thalline cell precipitation ultrasonication, gained ultrasonication liquid is centrifuged, and takes supernatant, and as intracellular slightly proposes restructuring and resists
Former.
Described expression medium component is by g/l being calculated as：Peptone 9～11g, dusty yeast 5～24g, MgCl20～
0.83g, KCl 0～0.186g, ZnCl20～0.186g, 50% glycerine 0～20%, 50% sucrose 0～20%, 50%
Glucose 0～20%, remaining composition be 0.25mol/L Tris-HCl.
Described seed culture based component is by g/l being calculated as：Peptone 9～11g, dusty yeast 4～6g, NaCl 9～11g, its
Remaining composition is water.
Described step (2) activated seed liquid, through expressing culture medium, after 30～37 DEG C of shaken cultivation, adds in culture medium
Derivant, carries out Fiber differentiation；Wherein, the addition of derivant is every liter of described expression culture medium 0～5g.
A kind of engineering bacteria of inhibitors of fibrinolysis (TAFI) recombinant antigen of high efficient expression thrombin activation, described restructuring is carried
(pET41a-p.MD-TAFI) it is transformed into competent escherichia coli cell, that is, obtain the fibrinolytic suppression of high efficient expression thrombin activation
The engineering bacteria of thing (TAFI) recombinant antigen.
Described engineering bacteria obtains inhibitors of fibrinolysis (TAFI) recombinant antigen of thrombin activation through high efficient expression.
Advantage for present invention：
Inhibitors of fibrinolysis (TAFI) amino acid sequence of thrombin activation is closed by the present invention by gene engineering method
Physics and chemistry changes, and by the fibrinolytic suppression of escherichia expression system high efficient expression thrombin activation after this sequence is optimized
Thing (TAFI) recombinant antigen.
Wherein, inhibitors of fibrinolysis (TAFI) recombinant antigen of the thrombin activation that the recombinant plasmid of employing is obtained shows
Higher activity, is that the inhibitors of fibrinolysis (TAFI) producing highly active thrombin activation in industrial level is laid a good foundation.
Brief description
Fig. 1 is the inhibitors of fibrinolysis (TAFI) of wild thrombin activation and the inhibitors of fibrinolysis optimizing thrombin activation
(TAFI) comparison chart of gene.
Fig. 2 be optimize after with optimize before protein tertiary structure figure, a be optimize after prognostic chart, b be optimize before prognostic chart.
Fig. 3 is culture dish positive colony.White circle note is satisfactory positive colony bacterial strain.
Fig. 4 be optimize after with optimize before expression product electrophoresis comparison diagram, wherein, M be protein standard, 1-2 be optimize amino
TAFI expression precipitation after acid and supernatant, 3-4 is the precipitation and supernatant before optimizing.
Fig. 5 is the purification result figure of the inhibitors of fibrinolysis (TAFI) of thrombin activation expressed by recombinant bacterium, and wherein, M is
Protein standard；1 is the inhibitors of fibrinolysis (TAFI) of the thrombin activation purifying；2 is the 12h supernatant of induction.
Specific embodiment
The description below is only the general introduction of technical solution of the present invention, in order to better understand the technological means of the present invention,
And can be practiced according to the content of specification, after being described in detail such as with presently preferred embodiments of the present invention below.
The present invention, according to the Preference of e. coli codon, optimizes inhibitors of fibrinolysis (TAFI) base of thrombin activation
Cause, is connected on expression vector, and achieves expression in Escherichia coli.Its step is：A, optimization amino acid：Rule of thumb,
Destination protein partial amino-acid series are optimized；B, codon optimization：According to the Preference of e. coli codon, optimize
Inhibitors of fibrinolysis (TAFI) gene of thrombin activation simultaneously synthesizes this sequence；C, inhibitors of fibrinolysis (TAFI) base of thrombin activation
The preparation of cause：Genes of interest is synthesized according to optimization by company.D, the structure of recombinant plasmid：Through BamH I and Xho I digestion
Obtain inhibitors of fibrinolysis (TAFI) fragment of the thrombin activation that size is 1203bp, be cloned into high copy number plasmid pET-41a
On (+), obtain recombinant plasmid pET41a-p.MD-TAFI；E, the structure of engineering bacteria：With method for transformation by plasmid pET41a-
P.MD-TAFI imports to E.coilBL21 (DE3), is expressed.
Embodiment 1
The optimization of inhibitors of fibrinolysis (TAFI) gene codon of thrombin activation
1) the Preference table according to e. coli codon and experience, inhibitors of fibrinolysis (TAFI) base to thrombin activation
Because of the optimization of codon, optimizing base sequence is the (inhibitors of fibrinolysis of the thrombin activation of optimization shown in Seq ID No.2
(TAFI) synthesized by Bioisystech Co., Ltd), result is as shown in Figure 2.
Seq ID No.1
(a) sequence signature：
* length：401aa
* type：Amino acid
* chain：Single-stranded
* topological structure：Straight-chain
(b) molecule type：Albumen
C () is assumed：No
(d) antisense：No
E () is initially originated：Engineer
Seq ID No.2
(a) sequence signature：
* length：1206bp
* type：Nucleotides
* chain：Single-stranded
* topological structure：Straight-chain
(b) molecule type：Gene
C () is assumed：No
(d) antisense：No
E () is initially originated：Engineer
2) design restriction enzyme site, respectively BamH I and Xho I at the two ends of this gene, this gene is connected to pET-
41a (+) on carrier.The recombinant expression carrier successfully constructing pET41a-p.MD-TAFI is transformed into E. coli competent BL21
(DE3) in, screening positive clone (referring to 3) on culture plate.
Correct for above-mentioned identification recombinant clone is inoculated in seed culture medium, 37 DEG C of shaken cultivation 12h hours.Will
Above-mentioned culture gained activated seed liquid is seeded to expression culture medium by the inoculum concentration of percent by volume 6%, in 30 DEG C of shaken cultivation 4
～6h hour, shaking speed 180r/min；Cultivate to OD600=0.6～0.8, then add the derivant IPTG of 1mM, shake in 20 DEG C
Swing Fiber differentiation 18h, the expression of shaking speed 160r/min finishes；8000r/min is centrifuged 5min collects thalline, adds wet after weighing
The PBS of bacterial classification (g) × 20 times, carries out ultrasonication；12000r/min, 4 DEG C of centrifugation 10min, collect supernatant then pure
Change (referring to Fig. 4).
Above-mentioned, seed culture based component is by g/l being calculated as：Peptone 10g, dusty yeast 5g, NaCl 10g, remaining composition is
Water.
Expression medium component is by g/l being calculated as：Peptone 20g, dusty yeast 5g, NaCl0.5g, MgCl20.39g, KCl
0.186g, 50% glycerine 2%, cysteine 80mM, remaining composition is 0.25mol/L Tris-HCl.
Embodiment 2 protein purification and concentration mensuration
Above-mentioned supernatant is purified, method is as follows：
(1) buffer, formula such as following table are configured：
(2) Ni-NTA Column prepares
1. the Ni-NTA Agarose in gentle bottle inverted is for several times；
2. the resin drawing 2mL adds in 15mL centrifuge tube, makes resin natural subsidence under gravity, soft sucking-off
Supernatant；
3. add the sterile distilled water of 5mL, gentle reverse pillar 3min, 500xg are centrifuged 5min, in soft sucking-off
Clearly.
4. bingding buffer is used to repeat above step.
5. add isopyknic bingding buffer in Ni post, make 50% homogenate.
(3) sample is combined with Ni post
According to the sample adding 4mL in the homogenate y of every 1mL 50%, under room temperature, it is incubated 1h.
(4) wash and elute
1. 500xg centrifugation 5min, soft sucking-off supernatant.
2. wash pillar with 1mL bingding buffer, gentle concussion 2min, then 500xg centrifugation on shaking table
5min, carefully suctions out supernatant.
3. use 0.5mL elution buffer to elute pillar, be repeated 4 times, be in charge of 4 eluents of collection, deposit 4 DEG C.
(5) adopting BSA method to measure protein concentration is 6mg/mL.
The inhibitors of fibrinolysis (TAFI) of embodiment 3 detection restructuring thrombin activation
Same concentrations are detected on automatic clinical chemistry analyzer respectively using TAFI biochemical reagents box (immunoturbidimetry)
The restructuring inhibitors of fibrinolysis (TAFI) of thrombin activation and the restructuring TAFI of certain company, contrast degree of reaction.
(1) two kinds of albumen of dilution are to 10,15,20 μ g/mL；
(2) detected with automatic clinical chemistry analyzer, checked degree of reaction (table 1)；
Table 1
Conclusion：As it can be seen from table 1 the degree of reaction of self-produced three concentration of recombinant protein is above certain company, illustrate in phase
With under the conditions of self-produced restructuring thrombin activation inhibitors of fibrinolysis (TAFI) compared with certain company, activity is high.
Claims (8)
1. a kind of escherichia coli high-level expression thrombin activation inhibitors of fibrinolysis (TAFI) recombinant antigen it is characterised in that：Weight
Group antigen amino acid is as shown in sequence table Seq ID No.1.
2. inhibitors of fibrinolysis (TAFI) recombinant antigen of the escherichia coli high-level expression thrombin activation as described in claim 1,
It is characterized in that：Described recombinant antigen base is as shown in sequence table Seq ID No.2.
3. inhibitors of fibrinolysis (TAFI) recombinant antigen of the escherichia coli high-level expression thrombin activation described in a kind of claim 1
Preparation method it is characterised in that：
Step (1) is in the design of gene order two ends shown in the Seq ID No.2 restriction enzyme site through codon optimization, respectively BamH
I and Xho I, by this gene be connected to pET-41a (+) on carrier, obtain recombinant vector (pET41a-p.MD-TAFI)；
The recombinant expression carrier pET41a-p.MD-TAFI that step (1) gained successfully constructs is transformed into Escherichia coli by step (2)
Competent cell, builds and obtains recombination bacillus coli, by gained recombination bacillus coli through culture purified, obtain recombinant antigen.
4. inhibitors of fibrinolysis (TAFI) recombinant antigen of the escherichia coli high-level expression thrombin activation as described in claim 3
Preparation method it is characterised in that：
(1) seed culture：The recombination bacillus coli of the inhibitors of fibrinolysis (TAFI) that thrombin activation is produced in described restructuring is inoculated in
In seed culture medium, in 30～37 DEG C of shaken cultivation 8～12h, shaking speed 150～200r/min；
(2) Liquid Culture：To connect by the inoculum concentration of percent by volume 3～10% through step (1) culture gained activated seed liquid
Plant to expressing culture medium, in 30～37 DEG C of shaken cultivation 4～6h hours, shaking speed 150～200r/min；Then then at 20～
30 DEG C of vibration Fiber differentiation 14～22h, shaking speed 150～200r/min expression finishes；
(3) slightly carry recombinant antigen：By the expression liquid centrifugation of step (2) gained；Collect supernatant, as extracellular slightly carry recombinant antigen；
Collects thalline cell precipitation ultrasonication, gained ultrasonication liquid are centrifuged, take supernatant, as intracellular slightly carries recombinant antigen.
5. inhibitors of fibrinolysis (TAFI) recombinant antigen of the escherichia coli high-level expression thrombin activation as described in claim 4
Preparation method it is characterised in that：Described expression medium component is by g/l being calculated as：Peptone 9～11g, dusty yeast 5～24g,
MgCl20～0.83g, KCl 0～0.186g, ZnCl20～0.186g, 50% glycerine 0～20%, 50% sucrose 0～
20%, 50% glucose 0～20%, remaining composition is 0.25mol/L Tris-HCl.
6. inhibitors of fibrinolysis (TAFI) recombinant antigen of the escherichia coli high-level expression thrombin activation as described in claim 4
Preparation method it is characterised in that：Described step (2) activated seed liquid through express culture medium, after 30～37 DEG C of shaken cultivation, to
Add derivant in culture medium, carry out Fiber differentiation；Wherein, the addition of derivant is every liter of described expression culture medium 0～5g.
7. a kind of inhibitors of fibrinolysis (TAFI) recombinant antigen of high efficient expression thrombin activation engineering bacteria it is characterised in that：Will
Described restructuring carries (pET41a-p.MD-TAFI) and is transformed into competent escherichia coli cell, that is, obtain high efficient expression thrombin activation
Inhibitors of fibrinolysis (TAFI) recombinant antigen engineering bacteria.
8. the engineering bacteria of inhibitors of fibrinolysis (TAFI) recombinant antigen of the high efficient expression thrombin activation as described in claim 7
Application it is characterised in that：Described engineering bacteria obtains inhibitors of fibrinolysis (TAFI) recombinant antigen of thrombin activation through high efficient expression.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5474901A (en) * | 1991-02-01 | 1995-12-12 | Genentech, Inc. | Antibodies to human carboxypeptidase B and methods of use thereof |
| CN101058805A (en) * | 2007-03-21 | 2007-10-24 | 北京贯虹科技有限公司 | Method of producing mutation procarboxypeptidase B and mutation carboxypeptidase B |
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- 2016-08-31 CN CN201610782811.0A patent/CN106397579A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5474901A (en) * | 1991-02-01 | 1995-12-12 | Genentech, Inc. | Antibodies to human carboxypeptidase B and methods of use thereof |
| CN101058805A (en) * | 2007-03-21 | 2007-10-24 | 北京贯虹科技有限公司 | Method of producing mutation procarboxypeptidase B and mutation carboxypeptidase B |
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Application publication date: 20170215 |