CN106188311A - The preparation method of a kind of recombinant tumor necrosis factor related apoptosis-inducing ligand albumen and application - Google Patents
The preparation method of a kind of recombinant tumor necrosis factor related apoptosis-inducing ligand albumen and application Download PDFInfo
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Abstract
The invention discloses a kind of recombined human HM trail protein, its aminoacid sequence is as shown in SEQ ID NO.1.First the present invention constructs one and comprises His6The people of Myc label recombinates HM trail protein expression vector, and utilize it to construct a kind of people of expression to recombinate the recombinant bacteria of HM trail protein, recombinant expressed recombined human HM trail protein, the most extracted, purification is induced, it is thus achieved that highly purified HM trail protein by IPTG.The apoptosis of this recombined human HM trail protein alternative inducing mammary cancerous cell in vitro, and do not cause the Normocellular apoptosis of mammary gland.The feature that recombined human HM trail protein prepared by the present invention has good stability, dissolubility is high, can the apoptosis of obvious inducing tumor cell, application prospect is good.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of recombinant tumor necrosis factor related apoptosis-inducing and join
The preparation method of body protein and application.
Background technology
Tumor has become one of healthy serious threat of human life, although achieving very based on the Comprehensive Treatment of operation
Good curative effect, but postoperative recurrence transfer is still the principal element affecting survival rate and mortality rate.In recent years, biotechnology is to tumor
Treatment open new approach.
The apoptosis-inducing ligand (TRAIL) that tumor necrosis factor (TNF) is relevant is a member of TNF superfamily,
Can be apoptosis-induced by activating exogenous apoptosis pathway.TRAIL can be with two receptor (TRAILR1/ comprising death domain
DR4 and TRAILR2/DR5) combine, thus trigger cell is dead, TRAIL also can be with two Decoy receptor (TRAILR3/ in addition
DcR1 and TRAILR4/DcR2) combine, play a protective role.Recently, wither due to TRAIL significant selective induction tumor cell
The characteristic (not killing normal cell) died and be considered as a kind of potential anti-tumor medicine.TRAIL can effectively induce
The apoptosis of kinds of tumor cells, currently carries out showing that TRAIL is a kind of safety with completed I phase and II clinical trial phase
Effective medicine.
But the TRAIL of monomer structure can not inducing cell apoptosis, for realizing the purpose of TRAIL inducing cell apoptosis, existing
After technology forms dimer or polymer by rsTRAIL, combine with death receptor and play apoptotic effect.
But poor, the highest, the easy precipitation of renaturing inclusion bodies rate of the equal existence and stability of preparation process etc. of the rsTRAIL albumen reported at present
Problem.
Summary of the invention
For above-mentioned prior art, it is an object of the invention to provide a kind of recombinant tumor necrosis factor related apoptosis and lure
Lead ligandin and preparation method thereof.The present invention will be with His6The aminoacid sequence of-Myc label is inserted into and is positioned at human TNF related apoptosis-inducing ligand
After 95-281 aminoacid sequence of albumen, the recombiant protein obtained is referred to as His6-Myc-TRAIL, is called for short HM-TRAIL.System
The standby recombined human HM-TRAIL albumen obtained has the effect of significant inducing cell apoptosis；And preparation process good stability,
Renaturing inclusion bodies rate is high, is difficult to precipitation.
It is a further object to provide above-mentioned recombined human HM-TRAIL albumen answering in preparing antitumor drug
With.
For achieving the above object, the present invention uses following technical proposals:
A first aspect of the present invention, it is provided that a kind of recombined human HM-TRAIL albumen, its aminoacid sequence such as SEQ ID NO.1
Shown in.
A second aspect of the present invention, it is provided that a kind of sequence encoding above-mentioned recombined human HM-TRAIL albumen, its nucleotides sequence
Row are as shown in SEQ ID NO.2.
A third aspect of the present invention, it is provided that a kind of recombinant expression carrier, it is to be inserted through double digestion by above-mentioned nucleotide sequence
Enter and be prepared from the multiple clone site of pQE16 expression vector.
A fourth aspect of the present invention, it is provided that a kind of recombinant bacteria, it is to be converted to E.coli by above-mentioned recombinant expression carrier
TOP10 competence antibacterial is prepared from.
A fifth aspect of the present invention, it is provided that the preparation method of a kind of recombined human HM-TRAIL albumen, comprises the steps:
(1) above-mentioned recombinant bacteria is inoculated in LB culture medium, carries out incubated overnight；
(2) strain of the incubated overnight LB culture medium containing ammonia benzyl mycin and kanamycin is diluted, continues to cultivate
OD value to bacterium solution is 0.6-0.8 at 600nm；
(3) add IPTG as derivant so that it is final concentration of 40 μ g/ml, inducing culture, make recombinant bacteria express restructuring
People's HM-TRAIL albumen.
In said method:
In step (1), the condition of incubated overnight is: 37 DEG C of shaking tables are cultivated, and shaking speed is 225-250rpm.
In step (2), described dilution operation is particularly as follows: by the strain of incubated overnight with containing 1:1000 ammonia benzyl mycin and Ka Na
The LB culture medium of mycin carries out 200 times of dilutions.
In step (3), the condition of described inducing culture is: 20-22 DEG C, 225-250rpm, cultivates 16-20h.If running into molten
The problems such as solution, can cultivate in 18 DEG C of environment, to improve the expression of albumen.
Said method also includes the separation of expression product and the step of purification；
Described separating step is: the antibacterial 6000rpm after inducing culture is centrifuged 15min, collects bacterial precipitation；To antibacterial
Precipitation adds bacterial lysate, mixing, obtains cell suspension；The bacterial suspension obtained is carried out ultrasonic degradation, room temperature 14,
000rpm is centrifuged 15min, separates the supernatant containing soluble protein, by being diluted with buffer, or by cutoff value is
The micropore centrifuge tube of 10KD swaps, and the supernatant filter of 0.22 μm filters.
The step of described purification is: use GE HiTrap to chromatograph chelate column, by isolated upper containing soluble protein
Clear loading, with the elution buffer eluting target protein of 2-5 times of chelate column volume；
Consisting of of described elution buffer: 1MTris-HCl (pH=7.4) 10ml, sodium chloride 8.19g, beta-mercaptoethanol
0.39065g, imidazoles 34.0385g, ddH2O to 1L.
In above-mentioned purification step, before with elution buffer eluting target protein, also include: with 5 times of chelating cylinders
Long-pending distilled water rinse pillar and the step with the combination buffer balance pillar of at least 5 times of chelate column volumes；
Consisting of of described combination buffer: 1MTris-HCl (pH=7.4) 10ml, sodium chloride 29.25g, β-sulfydryl second
Alcohol 0.39065g, ddH2O to 1L.
In above-mentioned purification step, after with elution buffer eluting target protein, also include: with 5 times of chelating cylinders
Long-pending regeneration buffer rebalancing pillar, then by the step of the distilled water rinse pillar of 5-10 times of chelate column volume；
Consisting of of described regeneration buffer: 1MTris-HCl (pH=7.4) 10ml, sodium chloride 29.25g,
EDTA14.612g, ddH2O to 100ml.
The application in preparing antitumor drug of the above-mentioned recombined human HM-TRAIL albumen is also protection scope of the present invention；Excellent
Choosing, the application in the medicine of preparation treatment breast carcinoma of the above-mentioned recombined human HM-TRAIL albumen.
A sixth aspect of the present invention, it is provided that a kind of antitumor drug, it contains above-mentioned recombined human HM-TRAIL albumen.
Beneficial effects of the present invention:
(1) feature that the recombined human HM-TRAIL albumen that prepared by the present invention has good stability, dissolubility is high, can substantially lure
Leading the apoptosis of tumor cell, application prospect is good.
(2) His that the application is inserted when preparing recombined human HM-TRAIL albumen6-Myc label is positioned at people HM-TRAIL
After 95-281 aminoacid sequence of albumen, stability and the recoverability of recombined human HM-TRAIL albumen can be improved, again can
Thering is provided two kinds of protein tag for follow-up experiment, it is simple to purification and detection, application prospect is good simultaneously.
(3) escherichia expression system is the most conventional exogenous protein expression system, but colibacillary difference
At identical conditions, the characteristic shown is far the most different for bacterial strain, therefore, for making exogenous gene obtain optimal expression, needs
Host Strains in escherichia expression system and expression vector are screened and optimizes.The present invention is with recombined human HM-TRAIL
The expression of albumen is inspection target, is screened the multiple expression vector in escherichia expression system and Host Strains,
It was found that the expression vector pQE16 selected by the present invention, its molecular weight is relatively small, when intracellular amplification to carefully
The load that born of the same parents cause can be smaller, and then the copy number of the exogenous gene connected on carrier is the most, therefore, and recombiant protein
Expression also can relatively increase；It addition, the expression of recombined human HM-TRAIL albumen also significant difference between bacterial strain and bacterial strain,
Compared with other Host Strains in escherichia expression system, Host Strains E.coli TOP10 competence of the present invention is thin
Bacterium, the expression of its recombined human HM-TRAIL albumen substantially increases.
(4) expression of recombined human HM-TRAIL albumen is also had an impact by induction mode, the addition of IPTG, inducing culture
Temperature and time is the key process parameter affecting recombined human HM-TRAIL protein expression.Wherein, IPTG is in finite concentration scope
The interior expression that can promote gene, but, IPTG has potential toxicity, and price is higher, and the addition of IPTG also should not mistake
Many；The addition of IPTG is optimized by the present invention, it was found that during the final concentration of 40 μ g/ml of IPTG, recombined human HM-
The expression of trail protein is the highest, and process costs and the potential toxicity of IPTG have also been obtained preferably control.
The temperature of inducing culture is also a key parameter, and the regulating and controlling effect of gene expression can be occurred replicating, turning by it
Record, translation molecular level and cellular level.The induction carrying out target protein under conditions of temperature is relatively low is beneficial to solubility egg
White production, but the aggregate velocity of albumen is slow；Temperature is higher, accelerates the aggregate velocity of albumen, if but protein folding speed
Do not catch up with albumen aggregate velocity, then will cause the formation of a large amount of inclusion body protein.The temperature of inducing culture is carried out by the present invention
Investigating, aggregate balancing carves aggregate velocity and the content of soluble protein of albumen, and the temperature of the inducing culture finally determined is 20-
22℃。
On the one hand the length of inducing culture time affect the yield of foreign protein, on the other hand also can affect fermentation period；
The present invention has investigated different induction times to expressing the Changing Pattern of recombined human HM-TRAIL albumen, so that it is determined that optimal
The inducing culture time is 16-20h.
(5) present invention is by expression vector, Host Strains and the optimization of protein induced expression condition, first improve total
The expression of recombined human HM-TRAIL albumen (including the recombined human HM-TRAIL albumen of inclusion body and soluble form), at this base
Improve the expression of solubility HM-TRAIL albumen on plinth again, thus reach to improve recombinant soluble HM-TRAIL in unit cell
The purpose of protein yield.The method using the present invention, the expression of total recombined human HM-TRAIL albumen reaches as high as 32.6%,
Up to 21.4%, (expression in the present invention refers to recombined human HM-TRAIL expressed to the expression of solubility HM-TRAIL albumen
Albumen accounts for the mass percent of bacterial protein).
(6) difficult point place prepared by the purification of recombiant protein always recombiant protein, the present invention uses GE HiTrap to chromatograph
Chelate column has carried out purification to the recombined human HM-TRAIL albumen of preparation, by purification step, and elution buffer, combination
Buffer and the optimization of regeneration buffer, improve the purity of the recombiant protein of preparation.
Accompanying drawing explanation
Fig. 1: the SDS-PAGE of recombined human HM-TRAIL albumen and the result of coomassie brilliant blue staining；Wherein, WCL: the thinnest
Cellular lysate product；FL: uncombined outflow product；W:10mM imidazole wash liquid；E1: imidazole elution first pass；E2: imidazoles eluting
Liquid second time；
Fig. 2: mtt assay detection recombined human HM-TRAIL induces the effect of various cell line of mammary gland apoptosis.
Detailed description of the invention
The present invention is further illustrated in conjunction with the embodiments, it should explanation, and the description below is merely to explain this
Invention, is not defined its content.
Reagent used in the embodiment of the present invention, enzyme, expression vector etc. are conventional products of the prior art.
Embodiment 1: the preparation of recombination human source HM-TRAIL albumen
The structure of 1.pQE16-HM-TRAIL expression vector
Will be with His6The aminoacid sequence (MRGSHHHHHHGSEQKLISEEDLNLQ) of-Myc label is inserted into and is positioned at people
(sequence such as SEQ ID NO.1), above-mentioned recombined human HM-of PCR amplification coding after 95-281 aminoacid sequence of trail protein
The nucleotide sequence (sequence such as SEQ ID NO.2) of trail protein, and double digestion connects, and to be inserted into pQE16 expression vector many
In cloning site, and sequence verification sequence.
The conversion (structure of recombinant bacteria) of 2.pQE16-HM-TRAIL expression vector
(1) E.coli TOP10 competence antibacterial (100ul) taking-up being stored in-80 DEG C is placed in thawed on ice.
(2) product (about 20ul) will be connected to add in competence antibacterial, flick bottom EP pipe, ice bath 30min after mixing.
(3) EP pipe is put in heat shock 90s in 42 DEG C of water-baths, inserts quickly cooling 2-3min on ice.
(4) often adding the 900ul LB culture medium without antibiotic in pipe, 37 DEG C, 220rpm vibrates 1h.
(5), after 1h, room temperature 3,000rpm is centrifuged 5min, after discarding 950ul supernatant, resuspended by remaining 50ulLB culture medium
Antibacterial is applied on the flat board containing antibiotic.
(7) LB agar plate is just being placed in 37 DEG C of incubators, is being absorbed to surface liquid, about 0.5-1h.
(8) flat-plate inverted is placed in 37 DEG C of incubators, incubated overnight, observes the growing state of bacterium colony on flat board next day, it is possible to
On flat board, the bacterial clone of growth is transformant, i.e. converts successful antibacterial.
3. bacterium solution amplification and preservation
To the LB containing antibiotic (the ammonia benzyl mycin of 1/1000 and kanamycin) shaking often pipe addition about 5～8ml in tube
Culture medium, with the monoclonal on the picking LB flat board of 20ul sterilizing rifle head tip in test tube, 37 DEG C, 220rpm vibration 12～16h,
Until bacterium solution OD value is 0.4-0.6.The bacterium solution obtained can subpackage to-80 DEG C long-term preservations in 1.5ml EP pipe.
4. the recovery of strain
For recovery strain, scrape the upper surface of the frozen bacterial strain somewhat melted with the inoculating loop of sterilizing gently, then stand
Flat board, at the flat lining out of the LB containing antibiotic, is placed on 37 DEG C of incubated overnight by the antibacterial that will dip on inoculating loop.
5. antibacterial culturing and protein induced
(1) from the Bacterial Plate that the 4th step is cultivated, take a monoclonal with sterilizing rifle choicest, squeeze into 5ml and contain LB cultivation
The test tube of base is cultivated, culture medium adds suitable antibiotic, this experiment adds ammonia benzyl mycin and the card of 1/1000
That mycin, incubated overnight is in 37 DEG C of shaking tables (225-250rpm).
(2) strain of the incubated overnight LB culture medium containing 1:1000 ammonia benzyl mycin and kanamycin carries out 200 times of dilutions,
Using the sterilizing flask of 1L as shaking bacterium container, 37 DEG C are shaken 3h, treat that at 600nm, OD value is 0.6-0.8.
(3) express with IPTG induced protein, in the bacterium solution shaken, add the IPTG of 100mg/ml so that it is final concentration of 40
μg/ml.Induction needs 20-22 DEG C to cultivate 18h (225-250rpm).If running into the problems such as dissolving, can carry out at 18 DEG C of environment, can carry
The expression of high dissolubility albumen.
6. target protein extracts
(1) the antibacterial 6000rpm induced is centrifuged 15min.This step bacterial precipitation can directly carry out lower step or freeze
It is stored in-80 DEG C.
(2) bacterial lysate is prepared: every 1mlBacterial lysate (Thermo Fisher) adds 2 μ lDNA enzyme I+
2 μ l lysozyme, need in lysate to add the appropriate protease inhibitor without EDTA.
(3) weigh bacterial precipitation, add the ratio cracking bacterial precipitation of 4ml bacterial lysate, whirlpool in every 1mg bacterial precipitation
Rotation is shaken or blendes together homogeneous bacterial suspension with pipettor, continues softly to shake 10min after mixing.
(4) bacterial suspension that step (3) obtains is carried out ultrasonic degradation, to be sufficiently destroyed bacteria cell wall, carry further
The concentration of high dissolubility albumen.The detailed process of ultrasonic degradation is: be placed in by bacterial suspension on ice, ultrasonic probe is inserted suspension
Below liquid level, carry out ultrasonic with intermediate frequency, the most persistently 20s, intermittently 10s, continuous 10 circulations.
(5) room temperature 14,000rpm is centrifuged 15min, and (insoluble proteins is centrifugal to separate the supernatant containing soluble protein
In precipitation), supernatant is collected in new centrifuge tube.(note: under normal circumstances, the soluble protein of general collection general 90%
Matter.Again extract and perhaps can increase yield, but typically no necessity.)
(6) in order to reduce the Miscellaneous ingredients in solution, sample should adjust the composition of buffer.Can be by entering with buffer
Row dilution, or swapped by the micropore centrifuge tube that cutoff value is 10KD.(note:Reagent uses specific gentleness
PH value is the non-ionic detergent of the 20mM TrisHCl of 7.5, does not has the composition of enzyme.According to the most protein-based
Type, can add such as salt, lysozyme, protease inhibitor, the composition such as reducing agent and chelating agen.)
(7) the supernatant filter of 0.22 μm filters, and waits to be purified.
7. target protein purification (GE HiTrap chromatographs chelate column)
(1) in 10ml syringe, fill distilled water, remove the stopping valve of chelate column, connect syringe and chelate column,
Avoid during connection producing bubble.With the distilled water rinse pillar of 10 times of chelate column volumes.
(2) by 0.1M NiSO4Solution 2.5ml injects in the 5ml chromatographic column of distilled water rinse.
(3) with the distilled water rinse pillar of 5 times of chelate column volumes.
(4) with the combination buffer balance pillar of at least 5 times of chelate column volumes.
(5) the albumen upper prop of purification will be needed, it is recommended that flow be 5ml/min.
(6) with lavation buffer solution-1 rinse pillar of at least 2 times of chelate column volumes, then with at least 2 times of chelate columns
Rinse buffer-2 rinse pillar of volume, leaves and takes often step effluent and runs PAGE glue.
(7) with the elution buffer eluting target protein of 2-5 times of chelate column volume, from each step, leave and take 40 μ l run
PAGE glue.
(8) with the regeneration buffer rebalancing pillar of 5 times of chelate column volumes, then with 5-10 times of chelate column volume
Distilled water rinse pillar.The ethanol that chelate column can add 20% is stored in+4～+30 DEG C.
(9) protein requirement of purification is dialysed in the PBS containing 10mM beta-mercaptoethanol, and the bag filter cutoff value of selection is
15kD；Albumen is stored in-80 DEG C, it is to avoid multigelation.
8. solution needed for protein purification
(1) 1MTris-HCl solution
(2) 0.1M nickel sulfate solution
(3) lavation buffer solution-1
(4) lavation buffer solution-2
(5) elution buffer
(6) buffer is combined
(7) regeneration buffer
The SDS-PAGE of recombined human HM-TRAIL albumen and the result of coomassie brilliant blue staining are as shown in Figure 1.
Comparative example: the preparation of recombination human source trail protein
Using pQE9 as expression vector, M15pRep4 competence antibacterial is as host bacteria, His6-Myc label is with implementing
Example 1, builds recombinant bacteria in conventional manner.Carry out cultivating and protein induced to the recombinant bacteria built, and to target protein
It is purified.
Use SDS-PAGE and Shimadzu thin-layer chromatogram scanner that the expression efficiency of embodiment 1 and the recombiant protein of comparative example is carried out
Detection, result is: the method using embodiment 1, the expression of total recombined human trail protein is 32.6%, soluble TRAIL
The expression of albumen is 21.4%.And the method using comparative example, the expression of total recombined human trail protein is 27.1%,
The expression of soluble TRAIL protein is 16.5%.Result shows, uses the preparation method of the recombiant protein of the present invention, hence it is evident that
Improve the expression of destination protein and soluble protein.
Embodiment 2: experiment in vitro
In order to study the inducing action to apoptosis of tumor cells in vitro of recombined human HM-TRAIL albumen, we are numerous
Breast cancer cell line and normal breast epithelial MCF-10A utilize mtt assay have detected various cell line to HM-TRAIL
Sensitivity.
1. cell is cultivated and condition of culture
MCF-7 cell strainHJ2mm, MDA-MB-231 and MDA-MB-468 cell strain be incubated at containing 10% hyclone,
In DMEM (Dulbecco's the improves MEM culture medium) high glucose medium of 100U/ml penicillin and 100 μ g/ml streptomycins.Human milk
Adenocarcinoma cell strain SK-BR-3 and ZR-75-1 is incubated in RPMI 1640 culture medium containing 10% hyclone and antibiotic.
T47D breast carcinoma cell strain is incubated in RPMI 1640 culture medium containing 10 μ g/ml bovine insulins and 10% hyclone.All
Cell all at 37 DEG C and 5%CO2Under the conditions of cellar culture.
The detection HM-TRAIL impact on apoptosis of tumor cells of 2.MTT method
(1) in the 96 every holes of orifice plate, 100ul is added containing 1 × 103～3 × 103The cell suspension of individual target cell, if 3-5 multiple
Hole, at 37 DEG C, 5%CO2Incubator in overnight incubation.
(2), after cultivating 1-5 days, every hole adds 20ul MTT dye liquor (5mg/ml is dissolved in PBS), at 37 DEG C, 5%CO2Training
Support and case is cultivated 4～6h.
(3) slowly sucking-off supernatant culture medium, tries not to pick up purple precipitation, and after exhaustion supernatant, every hole adds 100ul's
DMSO dissolves purple precipitation, after abundant dissolving to be precipitated, with the absorbance in every hole under microplate reader detection 490nm and 550nm wavelength
Value.
(4) with absorbance, the time (natural law) is mapped, draw cell proliferation curve.
Recombined human HM-TRAIL processes the survival rate detecting various breast cancer cells after 48h with mtt assay, result such as Fig. 2 institute
Show.Show that recombined human HM-TRAIL that the present invention synthesizes can significant inducing apoptosis of tumour cell.
Although the detailed description of the invention of the present invention is described by the above-mentioned accompanying drawing that combines, but not the present invention is protected model
The restriction enclosed, one of ordinary skill in the art should be understood that on the basis of technical scheme, and those skilled in the art are not
Need to pay various amendments or deformation that creative work can make still within protection scope of the present invention.
Claims (10)
1. a recombined human HM-TRAIL albumen, its aminoacid sequence is as shown in SEQ ID NO.1.
2. encode a gene for recombined human HM-TRAIL albumen described in claim 1, its nucleotide sequence such as SEQ ID
Shown in NO.2.
3. a recombinant expression carrier, it is to be inserted into pQE16 by the nucleotide sequence described in claim 2 through double digestion to express
The multiple clone site of carrier is prepared from.
4. a recombinant bacteria, it is to be converted to E.coli TOP10 competence by the recombinant expression carrier described in claim 3
Antibacterial is prepared from.
5. a preparation method for the recombined human HM-TRAIL albumen described in claim 1, comprises the steps:
(1) above-mentioned recombinant bacteria is inoculated in LB culture medium, carries out incubated overnight；
(2) strain of the incubated overnight LB culture medium containing ammonia benzyl mycin and kanamycin is diluted, continues to cultivate to bacterium
The OD value of liquid is 0.6-0.8 at 600nm；
(3) add IPTG as derivant so that it is final concentration of 40 μ g/ml, inducing culture, make recombinant bacteria express recombined human
HM-TRAIL albumen.
6. preparation method as claimed in claim 5, it is characterised in that in step (3), the condition of described inducing culture is: 20-
22 DEG C, 225-250rpm, cultivate 16-20h.
7. preparation method as claimed in claim 5, it is characterised in that also include the step of the separation of expression product；
Described separating step is: the antibacterial 6000rpm after inducing culture is centrifuged 15min, collects bacterial precipitation；To bacterial precipitation
Middle additionBacterial lysate, mixing, obtain cell suspension；The bacterial suspension obtained is carried out ultrasonic degradation, room temperature 14,
000rpm is centrifuged 15min, separates the supernatant containing soluble protein, by being diluted with buffer, or by cutoff value is
The micropore centrifuge tube of 10K swaps, and supernatant filters.
8. preparation method as claimed in claim 5, it is characterised in that also include the step of the purification of expression product；
The step of described purification is: use GE HiTrap to chromatograph chelate column, by the isolated supernatant containing soluble protein
Sample, with the elution buffer eluting target protein of 2-5 times of chelate column volume；
Consisting of of described elution buffer: 1MTris-HCl (pH=7.4) 10ml, sodium chloride 8.19g, beta-mercaptoethanol
0.39065g, imidazoles 34.0385g, ddH2O to 1L.
9. the application in preparing antitumor drug of the recombined human HM-TRAIL albumen described in claim 1.
10. the medicine treating tumor, it is characterised in that contain the weight described in claim 1 of effective dose in described medicine
Group people's HM-TRAIL albumen.
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| CN109400711A (en) * | 2017-05-31 | 2019-03-01 | 四川大学华西医院 | PDGFR β targeting tumor necrosis factor related apoptosis inducing ligand variant and preparation method and application thereof |
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Cited By (2)
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| CN109400711A (en) * | 2017-05-31 | 2019-03-01 | 四川大学华西医院 | PDGFR β targeting tumor necrosis factor related apoptosis inducing ligand variant and preparation method and application thereof |
| CN109400711B (en) * | 2017-05-31 | 2022-02-08 | 四川大学华西医院 | PDGFR beta targeting tumor necrosis factor related apoptosis inducing ligand variant and preparation method and application thereof |
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