CN103255160A - Preparation method of recombinant human soluble TNF (Tumor Necrosis Factor)-related apoptosis-including ligand - Google Patents

Abstract

The invention discloses a preparation method of a recombinant human soluble TNF (Tumor Necrosis Factor)-related apoptosis-including ligand (TRAIL). The preparation method is used for preparing recombinant human soluble TRAIL with higher bioactivity by constructing prokaryotic expression plasmid of the extracellular functional area of the recombinant human soluble TRAIL and optimizing related conditions of inducing protein expression.

Description

The preparation method of the apoptosis induction ligand that recombination human soluble tumor necrosis factor is relevant

Technical field

The invention belongs to biological field, be specifically related to a kind of recombination human soluble tumor necrosis factor relevant apoptosis induction ligand (TNF-related apoptosis-inducing ligand, preparation method TRAIL).

Background technology

TRAIL is the tumor necrosis factor superfamily newcomer, is expressed in human multiple tissue and the cell, is found first and clones out by people such as Wiley [1] in nineteen ninety-five.People's trail dna is positioned at karyomit(e) 3q26, and the cDNA total length is 1769bp, and molecular weight is 32.5KD, is made up of 281 amino acid, and functional part is 114～281 amino acids residue iso-electric points 7.6, belongs to II type membrane-spanning protein.The reactive site of TRAIL outside after birth C-terminal and have stronger conservative property, wherein the 230th halfcystine (C230) has vital role for keeping its structure and biological activity.TRAIL can be become free monomer by the protease hydrolysis in the body, thereby 3 TRAIL monomer molecules can form active stronger tripolymer by zine ion of C230 chelating.Then can not form tripolymer when C230 suddenlys change, this moment, the ability of TRAIL and receptors bind reduced, and the ability of inducing apoptosis of tumour cell also reduces.Receptors bind on TRAIL energy specificity and the tumour cell causes apoptosis of tumor cells, and normal cell is not almost had influence, is considered to a kind of very promising anti-tumor agent.The preparation method of conventional trail protein goes into behind the intestinal bacteria 37 ℃ with the recombinant plasmid transformed for preparing to be cultured to OD and 0.6 to add and continue under 37 ℃ condition inducible protein behind the IPTG and express.Also can induce target protein to express with this understanding, but mostly be the inclusion body of lifeless matter activity, even there is soluble proteins to express but the also very easily sex change of instability of very low its character of output.

Summary of the invention

The purpose of this invention is to provide a kind of preparation method that can obtain to have the trail protein of inducing apoptosis of tumour cell.For achieving the above object, technical scheme of the present invention is as follows:

One aspect of the present invention relates to the preparation method of the relevant apoptosis induction ligand of a kind of recombination human soluble tumor necrosis factor, it comprises the steps: to use upstream primer (5 '-CG GGA TCC GTG AGA GAA AGA GGT C-3 ') and downstream primer (5 '-CCC CCA GGT CAG TTA GC-3 '), be that template is carried out pcr amplification with the TRAIL full length cDNA sequence, the PCR product is designated as TRAIL _114-281

With TRAIL _114-281Make up recombinant expression plasmid pET-28a (+)-TRAIL with being connected after carrier pET-28a (+) use restriction enzyme BamH I and the recovery of Hind III double digestion _114-281, in its transformed into escherichia coli E.coli DH5 α, cut evaluation and screening by enzyme and go out positive transformant, with recombinant plasmid pET-28a (+)-TRAIL of sequence verification _114-281Be transformed in the e. coli bl21 (DE3), cultivate the single bacterium colony of back picking, be inoculated into overnight incubation in the LB substratum that contains kantlex (Kan); The bacterium liquid that will spend the night next day is inoculated among the LB, the kantlex of adding, and 36-38 ℃ of cultivation when cell density OD value is 0.5-0.7, adds inductor IPTG and zinc sulfate, cultivates 3-5h for 24-26 ℃, takes out bacterium liquid, the centrifugal results bacterium that wets;

Wet bacterium adds and comprises 8-12% glycerine, 0.3-0.6mol/L NaCl, 10-30mmol/L tris-Hcl, 4-6mmol/L DTT, 0.5-1.5mmol/L the buffered soln of PMSF, the ultrasonic degradation bacterium, centrifugal, the results supernatant removes by filter impurity, and it is 15-25mmol/L that the adding imidazoles makes its final concentration, adopt affinity chromatography ni-sepharose purification trail protein, with 400-600mmol/L imidazoles eluant solution, to collect elutriant and place 0-4 ℃ of dialysed overnight of PBS damping fluid, collection next day is the trail protein behind the purifying.

In a preferred embodiment of the present invention, described trail protein has the molecular weight about 24-26kDa.

In another preferred embodiment of the present invention, the induced concentration of described inductor IPTG and zinc sulfate is 0.8-1.2mmol/L.

Than prior art, method of the present invention has following advantage at least: the temperature that inducible protein is expressed changes 25 ℃ into by traditional 37 ℃, than the albumen of 37 ℃ of abduction deliverings, thereby at also the slow down trail protein of the biologically active that is folded to form solubility that is beneficial to albumen space structure picture of the speed that 25 ℃ of cold condition following times slow down bacterial growth speed target protein is expressed; In addition, add 1mM zinc sulfate at the induction period of albumen and can make TRAIL molecule chelating go into zine ion to make its activity more epistasis matter is more stable, thereby and influence the expression of albumen in the growth that the cell growth phase just adds zinc sulfate and can influence bacterium; Secondly, the damping fluid of resuspended wet bacterium has added DTT and makes the TRAIL activity stronger when purifying, and adds proteinase inhibitor PMSF and can prevent the TRAIL degraded.

Description of drawings

Fig. 1: recombinant plasmid vector gel electrophoresis analysis: 1, albumen Marker; 2, control group BL21-pET-28a (+); 3, BL21-pET-28a (+)-TRAIL _114-281

Fig. 2: purifying protein gel electrophoresis analysis: 1, albumen Marker; 2, the TRAIL behind the purifying;

Fig. 3: the apoptosis rate of H460 cell (right lower quadrant is early apoptosis, and right upper quadrant is the middle and advanced stage apoptosis).

Embodiment

The present invention is described in detail below in conjunction with specific examples.

Embodiment 1:

According to people's trail dna sequence number (NCBI Reference Sequence:NM_003810.2) according to its extracellular fragment coding region 114-281 amino acids sequences Design upstream primer (5 '-CG GGA TCC GTG AGA GAA AGA GGT C-3 ') and downstream primer (5 '-CCC CCA GGT CAG TTA GC-3 ').Be template with TRAIL full length cDNA sequence (Shanghai Invitrogen company synthetic), use, downstream primer carries out pcr amplification, reaction system is: cDNA template (1.5ng/ml) 1.6 μ l, upstream primer (10 μ M) 1 μ l, downstream primer (10 μ M) 1 μ l, Taq enzyme 2.5 μ l, water 21.4 μ l; 94 ℃ of pre-sex change 4min, 94 ℃ of sex change 45s, 60 ℃ of annealing 40s, 72 ℃ are extended 70s, 35 circulations, 72 ℃ are extended 5min again, and the PCR product is designated as TRAIL _114-281With TRAIL _114-281Be connected after carrier pET-28a (+) (purchasing the company in Novagen) uses restriction enzyme BamH I and Hind III double digestion to reclaim.Recombinant expression plasmid pET-28a (+)-TRAIL that builds _114-281Among the transformed into escherichia coli E.coli DH5 α, cut evaluation and screening by enzyme and go out positive transformant, with recombinant plasmid pET-28a (+)-TRAIL of sequence verification _114-281Be transformed in the e. coli bl21 (DE3) and (arrange one simultaneously and do not connect TRAIL _114-281The control group of gene), cultivates the single bacterium colony of back picking, be inoculated into overnight incubation in the LB substratum that contains kantlex (Kan).The 10ml bacterium liquid that will spend the night next day is inoculated in the Luria-Bertani substratum (LB) of 1000ml, adds the Kan of 1ml, and 37 ℃ of shaking tables are cultivated.When cell density OD value is about 0.6, add inductor sec.-propyl-β-D-sulfo-galactopyranoside (Isopropyl β-D-1-Thiogalactopyranoside, IPTG), to make its final concentration be 1mmol/L to zinc sulfate, 25 ℃, 200rpm.Take out bacterium liquid behind the 4h, the centrifugal results bacterium that wets.Wet bacterium adds 50ml Buffer (10% glycerine, 0.5mol/L NaCl, 20mmol/L tris-Hcl, 5mmol/L DTT (DL-Dithiothreitol, dithiothreitol (DTT)), 1mmol/L PMSF (phenylmethylsulfonyl fluoride, phenylmethylsulfonyl fluoride)) ultrasonic degradation bacterium, centrifugal, the results supernatant, the SDS-polyacrylamide gel electrophoresis is analyzed, and the electrophoretic analysis result compares the expression that control group visible one tangible protein band proof has target protein as shown in Figure 1 at swimming lane 3 about 25KDa places.Remove by filter impurity, it is 20mmol/L that the adding imidazoles makes its final concentration.The affinity chromatography ni-sepharose purification has six histidine-tagged trail proteins to be undertaken by GE Healthcare company specification sheets, roughly process is to add the 20mM imidazoles earlier in sample, with 20mM imidazoles solution equilibria pillar, last sample, collection penetrates liquid, add 20,40,60,80 more successively, collect after the rinsing of 100mM imidazoles solution and penetrate liquid, use 500mM imidazoles eluant solution at last, collect and penetrate liquid.SDS-polyacrylamide gel electrophoresis analytical results has only single band proof purification effect good as can be seen from Figure 2.The elutriant of 7ml is placed 4 ℃ of dialysed overnight of 1000ml PBS damping fluid, and collection next day is the trail protein behind the purifying, and-70 ℃ of preservations are standby.The trail protein that separation is obtained is with 0,50,100,200,400,800ng/ml acts on the cellularity lung carcinoma cell H460 of National People's Congress cell 24h, Annexin V-FITC/PI is two to be dyed apoptosis rate of each group of Flow cytometry (experimental result right lower quadrant is as shown in Figure 3 represented early apoptosis, right upper quadrant is represented the middle and advanced stage apoptosis), the apoptosis rate that does not add the control group of TRAIL as can be seen from Figure only is 0.9%, and the apoptosis rate of medicine group obviously is respectively 34.65% than control group height, 56.8%, 58.58%, 62.2%, 63.1%, along with the also rising gradually of increase apoptosis rate of TRAIL concentration, this trail protein that has confirmed preparation has good biological activity really.

The above only is the specific embodiment of the present invention, but protection scope of the present invention is not limited thereto, and any variation or replacement of expecting without creative work all should be encompassed within protection scope of the present invention.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claims were limited.

Claims (3)

1. the preparation method of the apoptosis induction ligand (TRAIL) that a recombination human soluble tumor necrosis factor is relevant, it comprises the steps: to use upstream primer (5 '-CG GGA TCC GTG AGA GAA AGA GGT C-3 ') and downstream primer (5 '-CCC CCA GGT CAG TTA GC-3 '), be that template is carried out pcr amplification with the TRAIL full length cDNA sequence, the PCR product is designated as TRAIL _114-281

With TRAIL _114-281Make up recombinant expression plasmid pET-28a (+)-TRAIL with being connected after carrier pET-28a (+) use restriction enzyme BamH I and the recovery of Hind III double digestion _114-281, in its transformed into escherichia coli E.coli DH5 α, cut evaluation and screening by enzyme and go out positive transformant, with recombinant plasmid pET-28a (+)-TRAIL of sequence verification _114-281Be transformed in the e. coli bl21 (DE3), cultivate the single bacterium colony of back picking, be inoculated into overnight incubation in the LB substratum that contains kantlex (Kan); The bacterium liquid that will spend the night next day is inoculated among the LB, adds kantlex, and 36-38 ℃ of cultivation when cell density OD value be 0.5-0.7, adds inductor IPTG and zinc sulfate, 24-26 ℃ of cultivation 3-5h, taking-up bacterium liquid, the centrifugal results bacterium that wets;

Wet bacterium adds and comprises 8-12% glycerine, 0.3-0.6mol/L NaCl, 10-30mmol/L tris-Hcl, 4-6mmol/L DTT, 0.5-1.5mmol/L the buffered soln of PMSF, the ultrasonic degradation bacterium, centrifugal, the results supernatant removes by filter impurity, and it is 15-25mmol/L that the adding imidazoles makes its final concentration, adopt affinity chromatography ni-sepharose purification trail protein, with 400-600mmol/L imidazoles eluant solution, to collect elutriant and place 0-4 ℃ of dialysed overnight of PBS damping fluid, collection next day is the trail protein behind the purifying.

2. preparation method according to claim 1, described trail protein has the molecular weight about 24-26kDa.

3. preparation method according to claim 1, the induced concentration of described inductor IPTG and zinc sulfate is 0.8-1.2mmol/L.

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