CN106188311B - Preparation method and application of recombinant tumor necrosis factor-related apoptosis-inducing ligand protein - Google Patents
Preparation method and application of recombinant tumor necrosis factor-related apoptosis-inducing ligand protein Download PDFInfo
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Abstract
The invention discloses a recombinant human HM-TRAI L protein, the amino acid sequence of which is shown in SEQ ID NO.16-Myc-labeled human recombinant HM-TRAI L protein expression vector, constructing recombinant bacteria for expressing human recombinant HM-TRAI L protein by using the vector, inducing and expressing recombinant human HM-TRAI L protein through IPTG, extracting and purifying to obtain high-purity HM-TRAI L protein, and selectively inducing apoptosis of breast cancer cells in vitro by the recombinant human HM-TRAI L protein without causing apoptosis of normal breast cellsThe recombinant human HM-TRAI L protein has the characteristics of good stability and high solubility, can obviously induce the apoptosis of tumor cells, and has good application prospect.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and application of recombinant tumor necrosis factor-related apoptosis-inducing ligand protein.
Background
Although comprehensive treatment mainly based on operation has good curative effect, relapse and metastasis after operation still remain main factors influencing survival rate and death rate. In recent years, biotechnology has opened up new avenues for the treatment of tumors.
TRAI L binds to two receptors containing death domains (TRAI L R1/DR4 and TRAI L R2/DR5) to trigger cell death, and also TRAI L binds to two decoy receptors (TRAI L R3/DcR1 and TRAI L R4/DcR2) to play a protective role.
However, TRAI L with a monomer structure cannot induce apoptosis, and in order to achieve the purpose of inducing apoptosis by TRAI L, in the prior art, after dimer or polymer is formed by recombinant TRAI L, the recombinant TRAI L is combined with death receptor to play a role in apoptosis.
Disclosure of Invention
Aiming at the prior art, the invention aims to provide a recombinant tumor necrosis factor-related apoptosis-inducing ligand protein and a preparation method thereof. The invention will have His6The insertion of the amino acid sequence of the Myc tag into the 95-281 amino acid sequence of the human TRAI L protein, resulting in a recombinant protein called His6The recombinant human HM-TRAI L protein prepared from Myc-TRAI L, HM-TRAI L for short, has significant effect of inducing apoptosis, and has good stability in preparation process and inclusion body recoveryHigh sexual rate and difficult precipitation.
The invention also aims to provide the application of the recombinant human HM-TRAI L protein in preparing anti-tumor drugs.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided recombinant human HM-TRAI L protein, the amino acid sequence of which is shown in SEQ ID NO. 1.
In a second aspect of the present invention, there is provided a sequence encoding the above recombinant human HM-TRAI L protein, the nucleotide sequence of which is shown in SEQ ID NO. 2.
In a third aspect of the present invention, a recombinant expression vector is provided, which is prepared by inserting the above nucleotide sequence into the multiple cloning site of the pQE16 expression vector by double digestion.
In a fourth aspect of the present invention, a recombinant bacterium is provided, which is prepared by transforming the recombinant expression vector into an e.
In a fifth aspect of the present invention, there is provided a process for preparing recombinant human HM-TRAI L protein, comprising the steps of:
(1) inoculating the recombinant bacteria to L B culture medium, and culturing overnight;
(2) diluting the overnight cultured strain with L B culture medium containing ampicillin and kanamycin, and continuously culturing until OD value of bacterial liquid is 0.6-0.8 at 600 nm;
(3) IPTG was added as an inducer to a final concentration of 40. mu.g/ml, and induction culture was carried out to express recombinant human HM-TRAI L protein in the recombinant bacteria.
The method comprises the following steps:
in the step (1), the overnight culture conditions are as follows: shaking culture at 37 ℃ and shaking rotation speed of 225 and 250 rpm.
In the step (2), the dilution is specifically performed by diluting the overnight-cultured strain 200-fold with L B medium containing 1: 1000 ampicillin and kanamycin.
In the step (3), the conditions of the induction culture are as follows: culturing at 20-22 deg.C and 225-250rpm for 16-20 h. If the protein is dissolved, the protein can be cultured in an environment of 18 ℃ to improve the expression level of the protein.
The method also comprises the steps of separating and purifying the expression product;
the separation steps are as follows: centrifuging the bacteria subjected to induction culture at 6000rpm for 15min, and collecting bacterial precipitates; adding a bacterial lysate into the bacterial precipitate, and uniformly mixing to obtain a cell suspension; the resulting bacterial suspension was subjected to ultrasonic lysis, centrifuged at 14,000rpm for 15min at room temperature, and the supernatant containing soluble proteins was separated, either by dilution with buffer or by exchange through a millipore centrifuge tube with a cut-off of 10kD, and the supernatant was filtered through a 0.22 μm filter.
The purification steps are as follows: loading the separated supernatant containing soluble protein by adopting a GE HiTrap chromatography chelating column, and eluting the target protein by using elution buffer solution with the volume 2-5 times that of the chelating column;
the elution buffer comprises 10ml of 1M Tris-HCl (pH 7.4), 8.19g of sodium chloride, 0.39065g of β -mercaptoethanol, 34.0385g of imidazole and ddH2O to 1L.
In the above purification step, before eluting the target protein with the elution buffer, the method further comprises: a step of rinsing the column with distilled water in a volume of 5 times that of the chelate column and equilibrating the column with a binding buffer in a volume of at least 5 times that of the chelate column;
the binding buffer consisted of 1M tris-HCl (pH 7.4)10ml, sodium chloride 29.25g, β -mercaptoethanol 0.39065g, ddH2O to 1L.
In the above purification step, after eluting the target protein with the elution buffer, the method further comprises: a step of re-equilibrating the column with a regeneration buffer solution of 5 times the volume of the chelate column and then rinsing the column with distilled water of 5 to 10 times the volume of the chelate column;
the regeneration buffer solution comprises the following components: 10ml of 1M Tris-HCl (pH 7.4), 29.25g of sodium chloride, EDTA14.612g of sodium chloride, ddH2O to 100 ml.
The application of the recombinant human HM-TRAI L protein in preparing antitumor drugs is also within the protection range of the invention, and preferably, the recombinant human HM-TRAI L protein is applied in preparing drugs for treating breast cancer.
In a sixth aspect, the present invention provides an anti-tumor drug comprising the above recombinant human HM-TRAI L protein.
The invention has the beneficial effects that:
(1) the recombinant human HM-TRAI L protein prepared by the invention has the characteristics of good stability and high solubility, can obviously induce the apoptosis of tumor cells, and has good application prospect.
(2) His inserted in the preparation of recombinant human HM-TRAI L protein6The Myc label is positioned behind 95-281 amino acid sequences of human HM-TRAI L protein, so that the stability and recoverability of the recombinant human HM-TRAI L protein can be improved, two protein labels can be provided for subsequent experiments at the same time, purification and detection are facilitated, and the application prospect is good.
(3) The expression vector pQE16 selected by the invention has relatively small molecular weight, causes less load to cells when being amplified in cells, further has relatively more copy number of the foreign genes connected on the vector, therefore, the expression quantity of the recombinant human HM-TRAI L protein between strains is also relatively increased, and compared with other host bacteria in the escherichia coli expression system, the expression quantity of the recombinant human HM-TRAI L protein between strains is also obviously different, and the expression quantity of the recombinant human HM-TRAI protein is obviously increased by E.coli TOP10 state sensitive bacteria adopted by the invention.
(4) The invention optimizes the addition of IPTG, and the result shows that when the final concentration of IPTG is 40 mu g/ml, the expression of the recombinant human HM-TRAI L protein is the highest, and the process cost and the potential toxicity of IPTG are better controlled.
The temperature of the induction culture is also a critical parameter, and its regulation of gene expression can occur at the replication, transcription, translation molecular level and cellular level. The induction of the target protein under the condition of lower temperature is beneficial to the production of soluble protein, but the synthesis speed of the protein is slower; higher temperatures increase protein synthesis, but if the protein folding rate does not follow the protein synthesis rate, a large amount of inclusion body protein will be formed. The invention inspects the temperature of the induction culture, comprehensively balances the synthesis speed of the carved protein and the content of the soluble protein, and finally determines the temperature of the induction culture to be 20-22 ℃.
The invention considers the change rule of different induction time to the expression recombinant human HM-TRAI L protein, thereby determining the best induction culture time to be 16-20 h.
(5) The invention improves the expression quantity of total recombinant human HM-TRAI L protein (including inclusion body and soluble recombinant human HM-TRAI L protein) by optimizing the expression vector, host bacteria and protein induction expression conditions, and improves the expression quantity of soluble HM-TRAI L protein on the basis, thereby achieving the purpose of improving the yield of recombinant soluble HM-TRAI L protein in unit cells.
(6) The purification of the recombinant protein is always the difficult point of the preparation of the recombinant protein, the invention adopts a GE HiTrap chromatography chelating column to purify the prepared recombinant human HM-TRAI L protein, and improves the purity of the prepared recombinant protein by optimizing the purification step, the elution buffer solution, the combination buffer solution and the regeneration buffer solution.
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FIG. 1 shows the results of SDS-PAGE and Coomassie blue staining of recombinant human HM-TRAI L protein, in which WC L is whole cell lysate, F L is unbound effluent, W is 10mM imidazole wash, E1 is first imidazole eluate, and E2 is second imidazole eluate;
FIG. 2 MTT method for detecting the effect of recombinant human HM-TRAI L in inducing apoptosis of various mammary gland cell lines.
Detailed Description
The present invention will be further described with reference to examples, but the following description is only for the purpose of explaining the present invention and does not limit the contents thereof.
Reagents, enzymes, expression vectors and the like used in the embodiments of the present invention are conventional products in the prior art.
Example 1 preparation of recombinant human HM-TRAI L protein
Construction of pQE16-HM-TRAI L expression vector
Will carry His6After the amino acid sequence of the Myc tag (MRGSHHHHHHGSEQK L ISEED L N L Q) is inserted into 95-281 amino acid sequences of human TRAI L protein (the sequence is shown as SEQ ID NO.1), the nucleotide sequence (the sequence is shown as SEQ ID NO.2) for coding the recombinant human HM-TRAI L protein is amplified by PCR, and is inserted into the polyclonal site of a pQE16 expression vector by double-enzyme digestion connection, and the sequence is sequenced and verified.
Transformation of pQE16-HM-TRAI L expression vector (construction of recombinant bacteria)
(1) Coli TOP10 competent bacteria (100ul) stored at-80 ℃ were removed and thawed on ice.
(2) The ligation product (approx. 20ul) was added to competent bacteria, flicked off the bottom of the EP tube, mixed well and ice-cooled for 30 min.
(3) The EP tube was heat shocked in a 42 ℃ water bath for 90s and inserted onto ice for rapid cooling for 2-3 min.
(4) 900ul of L B medium without antibiotics was added to each tube and shaken at 220rpm for 1h at 37 ℃.
(5) After 1h, centrifuge at 3,000rpm for 5min at room temperature, discard 950ul of supernatant, resuspend the bacteria with the remaining 50ul of L B medium and smear on antibiotic-containing plates.
(7) The L B agar plates were placed in an incubator at 37 ℃ until surface fluid was absorbed for about 0.5-1 h.
(8) The plates were placed upside down in an incubator at 37 ℃ for overnight culture, and the growth of colonies on the plates was observed the next day, and bacteria that could grow on the plates were cloned as transformants, i.e., bacteria that were successfully transformed.
3. Amplification and preservation of bacterial solution
Adding about 5-8 ml of L B culture medium containing antibiotics (1/1000 ampicillin and kanamycin) into each tube of a bacteria shaking tube, picking a single clone on a L B plate by using a tip of a 20ul sterilizing gun head, placing the single clone in a test tube, oscillating at 37 ℃ and 220rpm for 12-16 h until the OD value of bacterial liquid is 0.4-0.6, and subpackaging the obtained bacterial liquid into 1.5ml of EP tubes at-80 ℃ for long-term storage.
4. Recovery of bacterial species
To recover the strain, the upper surface of the slightly thawed cryopreserved strain was gently scraped using a sterilized inoculating loop, and then the bacteria dipped in the inoculating loop were immediately streaked on L B plates containing antibiotics, which were incubated overnight at 37 ℃.
5. Bacterial culture and protein induction
(1) A single clone was picked up from the bacterial plate cultured in step 4 using a sterilized tip and cultured in 5ml of a test tube containing L B medium to which appropriate antibiotics were added, 1/1000 of ampicillin and kanamycin were added in this experiment, and cultured overnight in a shaker at 37 ℃ (225-.
(2) The overnight cultured strain was diluted 200-fold with L B medium containing 1: 1000 ampicillin and kanamycin, and the strain was shaken at 37 ℃ for 3 hours in a 1L sterilized flask to an OD of 0.6 to 0.8 at 600 nm.
(3) Protein expression was induced by IPTG, and 100mg/ml of IPTG was added to the shaken solution to a final concentration of 40. mu.g/ml. Induction took place at 20-22 ℃ for 18h (225-250 rpm). If the protein is dissolved, the protein can be carried out in the environment of 18 ℃, and the expression quantity of the soluble protein can be improved.
6. Extraction of target protein
(1) The induced bacteria were centrifuged at 6000rpm for 15 min. This bacterial pellet can be directly used next step or frozen at-80 ℃.
(2) Preparing a bacterial lysate: each 1ml
Mu.l DNase I + 2. mu.l lysozyme was added to the bacterial lysate (Thermo Fisher) and the lysate required the addition of the appropriate amount of EDTA-free protease inhibitor.
(3) Weighing bacterial precipitates, adding 4ml of bacterial lysate into each 1mg of bacterial precipitates to crack the bacterial precipitates, performing vortex oscillation or mixing by using a pipettor to obtain uniform bacterial suspension, and continuing to perform gentle oscillation for 10min after uniformly mixing.
(4) And (4) carrying out ultrasonic lysis on the bacterial suspension obtained in the step (3) so as to fully destroy the bacterial cell wall and further improve the concentration of soluble protein. The specific process of ultrasonic cracking is as follows: the bacterial suspension was placed on ice, an ultrasound probe was inserted below the level of the suspension, and ultrasound was performed at moderate frequency, each for 20s, with 10s intervals, for 10 consecutive cycles.
(5) The supernatant containing the soluble proteins (non-soluble proteins in the pellet) was separated by centrifugation at 14,000rpm for 15min at room temperature and collected in a fresh centrifuge tube. (Note: usually, approximately 90% of the soluble protein is generally collected. Re-extraction may increase yield, but is generally not necessary.)
(6) To reduce confounding components in the solution, the sample should be adjusted for the composition of the buffer. The exchange can be performed by dilution with buffer or by micro-porous centrifuge tubes with a cut-off of 10 kD. (Note:
the reagent was a 20mM TrisHCl non-ionic detergent with a specific mild pH of 7.5, no enzyme components were present. Depending on the specific protein type, ingredients such as salts, lysozymes, protease inhibitors, reducing agents and chelating agents may be added. )
(7) The supernatant was filtered through a 0.22 μm filter and was awaited for purification.
7. Purification of target protein (GE HiTrap chromatography chelating column)
(1) A10 ml syringe was filled with distilled water, the stop valve of the chelating column was removed, and the syringe and the chelating column were connected without generation of air bubbles. The column was rinsed with 10 times the volume of the chelating column of distilled water.
(2) Mixing 0.1M NiSO42.5ml of the solution was poured into a 5ml column rinsed with distilled water.
(3) The column was rinsed with 5 times the volume of the chelating column of distilled water.
(4) The column is equilibrated with at least 5 times the volume of the chelating column of binding buffer.
(5) The protein to be purified is applied to the column at a recommended flow rate of 5 ml/min.
(6) The column was rinsed with at least 2 times the volume of the chelating column of washing buffer-1, followed by rinsing the column with at least 2 times the volume of the chelating column of rinsing buffer-2, leaving the effluent from each step running on PAGE gels.
(7) The target protein was eluted with 2-5 times the volume of the chelating column of elution buffer, leaving 40. mu.l of running PAGE gel from each step.
(8) The column was re-equilibrated with 5 times the volume of the regeneration buffer solution of the chelate column, and then rinsed with 5 to 10 times the volume of distilled water of the chelate column. The chelating column can be added with 20% ethanol and stored at +4 to +30 ℃.
(9) The purified protein was dialyzed against PBS containing 10mM β -mercaptoethanol, with a cut-off of 15kD using dialysis bags, and the protein was stored at-80 ℃ without repeated freezing and thawing.
8. Solution required for protein purification
(1)1M Tris-HCl solution
(2)0.1M Nickel sulfate solution
(3) Washing buffer-1
(4) Washing buffer-2
(5) Elution buffer
(6) Binding buffer
(7) Regeneration buffer
The results of SDS-PAGE and Coomassie blue staining of recombinant human HM-TRAI L protein are shown in FIG. 1.
Comparative example preparation of recombinant human TRAI L protein
pQE9 was used as expression vector, M15pRep4 competent bacterium was used as host bacterium, His6The Myc tag recombinant bacteria were constructed in the same manner as in example 1. Culturing and inducing the constructed recombinant bacteria, and purifying the target protein.
The expression efficiency of the recombinant proteins of example 1 and comparative example was examined by SDS-PAGE and Shimadzu thin-layer scanner, and the results were 32.6% for total recombinant human TRAI L protein and 21.4% for soluble TRAI L protein by the method of example 1, 27.1% for total recombinant human TRAI L protein and 16.5% for soluble TRAI L protein.
Example 2: in vitro experiments
In order to study the induction effect of recombinant human HM-TRAI L protein on tumor cell apoptosis in vitro, we tested the sensitivity of various cell lines to HM-TRAI L by using MTT method in a plurality of breast cancer cell lines and normal breast epithelial cell line MCF-10A.
1. Cell culture and culture conditions
Human breast cancer cell lines MCF-7, MDA-MB-231 and MDA-MB-468 were cultured in DMEM (Dulbecco's modified MEM medium) high-glucose medium containing 10% fetal bovine serum, 100U/ml penicillin and 100. mu.g/ml streptomycin. Human breast cancer cell lines SK-BR-3 and ZR-75-1 are cultured in RPMI 1640 medium containing 10% fetal calf serum and antibiotics. T47D breast cancer cell line was cultured in RPMI 1640 medium containing 10. mu.g/ml bovine insulin and 10% fetal bovine serum. All cells were at 37 ℃ and 5% CO2Culturing under conventional conditions.
MTT method for detecting effect of HM-TRAI L on tumor cell apoptosis
(1) Adding 100ul of 1 × 10 in each well of 96-well plate3～3×103Cell suspension of target cells, 3-5 multiple wells, 5% CO at 37 deg.C2Was cultured overnight in an incubator.
(2) After 1-5 days of incubation, 20ul of MTT stain (5mg/ml in PBS) was added to each well at 37 deg.C with 5% CO2The culture is carried out for 4-6 h in the incubator.
(3) Slowly sucking out the culture medium of the supernatant, avoiding sucking up the purple precipitate as much as possible, adding 100ul of DMSO into each hole after sucking up the supernatant to dissolve the purple precipitate, and detecting the absorbance value of each hole at the wavelengths of 490nm and 550nm by using an enzyme-labeling instrument after the precipitate is fully dissolved.
(4) Cell proliferation curves were plotted as absorbance versus time (days).
The survival rate of various breast cancer cells is detected by an MTT method after the recombinant human HM-TRAI L is treated for 48 hours, and the result is shown in figure 2.
Although the embodiments of the present invention have been described with reference to the accompanying drawings, it is not intended to limit the scope of the present invention, and it should be understood by those skilled in the art that various modifications and variations can be made without inventive efforts by those skilled in the art based on the technical solution of the present invention.
Claims (1)
1. A method for preparing recombinant human HM-TRAI L protein is characterized in that the amino acid sequence of the recombinant human HM-TRAI L protein is shown as SEQ ID NO.1, and the preparation method comprises the following steps:
(1) inoculating the recombinant bacteria on L B culture medium for overnight culture;
(2) diluting the overnight cultured strain with L B culture medium containing ampicillin and kanamycin, and continuously culturing until OD value of bacterial liquid is 0.6-0.8 at 600 nm;
(3) adding IPTG as inducer to make the final concentration 40 mug/ml, inducing and culturing to make the recombinant bacteria express recombinant human HM-TRAI L protein, wherein the inducing and culturing conditions are 20-22 ℃, 225 and 250rpm, culturing for 16-20 h;
wherein the recombinant bacterium is transformed from a recombinant expression vector toE.coliTOP10 competent bacteria; the recombinant expression vector is prepared by inserting a nucleotide sequence of a code SEQ ID NO.1 into a multiple cloning site of a pQE16 expression vector through double enzyme digestion;
further comprising the steps of isolation and purification of the expression product;
the separation steps are as follows: centrifuging the bacteria subjected to induction culture at 6000rpm for 15min, and collecting bacterial precipitates; adding B-PER bacteria lysate into the bacteria sediment, and uniformly mixing to obtain cell suspension; carrying out ultrasonic lysis on the obtained bacterial suspension, centrifuging at room temperature of 14,000rpm for 15min, separating supernatant containing soluble protein, diluting with buffer solution, or exchanging through a microporous centrifuge tube with a cut-off value of 10KD, and filtering the supernatant;
the purification step comprises adopting a GE HiTrap chromatography chelating column, loading the separated supernatant containing soluble protein, and eluting the target protein with an elution buffer solution with the volume 2-5 times that of the chelating column, wherein the elution buffer solution comprises 1M Tris-HCl10 ml with the pH =7.4, 8.19g of sodium chloride, β -mercaptoethanol 0.39065g, 34.0385g of imidazole and ddH2O to 1L.
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| Trail resistance induces epithelial-mesenchymal transition and enhances invasiveness by suppressing PTEN via miR-221 in breast cancer;Wang H 等;《PLoS One》;20140606;第9卷(第6期);摘要，第2页左栏第3段 * |
| tumor necrosis factor ligand superfamily member 10 isoform 1 [Homo sapiens]，NP_003801.1;GenBank;《GenBank》;20160114;NP_003801.1 * |
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