CN1500808A - Recombination of human soluble TRAIL protein, the preparing method and the application in preparing antineoplastic medicine - Google Patents
Recombination of human soluble TRAIL protein, the preparing method and the application in preparing antineoplastic medicine Download PDFInfo
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Abstract
The present invention discloses recombinant soluble human tumore necrosis factor related death inducing ligand and its preparation process and application in preparing antitumor medicine. The present invention adopts human tonsil tissue mRNA as template to amplify TRAIL coding sequence, constitute engineering colibacillus for expressing rhsTRAIL and engineering saccharomycete, establish the rhsTRAIL engineering colibacillus fermentation, occlusion body washing and destination protein purification process and the engineering saccharomycete and destination protein purification process separately, and obtain pure rhsTRAIL product, which has molecular weight of 19.6-24 KD and exists in both monomer and dimmer. The rhsTRAIL can induce death of several kinds of cancer cells outside body, inhibit amplification of liver cancer cell in tumor loading mouse obviously and kill tumor cell selectively, so that it may be used in preparing effective antitumor medicine.
Description
Invention field
The present invention relates to recombinant human soluble trail protein, its preparation method and the application in the preparation antitumor drug thereof, specifically, the present invention relates to relevant its gene clone of apoptosis part (to call trail protein in the following text) of tumour necrosis factor, preparation method and the application in the preparation antitumor drug thereof, described trail protein has the effect of external evoked apoptosis of tumor cells and anti-tumor in vivo, can be used for the treatment of kinds of tumors, in the treatment of clinical tumor, have the potential using value.
Background technology
The M ﹠ M of malignant tumour still rises year by year at present, becomes first place or second fatal disease.Tumor treatment mainly depends on radiotherapy, chemotherapy and excision three big therapies.Wherein excision has its limitation, and radiation and chemotherapy has following deficiency: 1. pair normal cell has tangible lethal effect; 2. many tumour cells have resistance to treatment.Therefore, develop a kind of high specificity, effectively, the antitumor drug of low toxicity is the main goal in research of oncotherapy.
The biotherapy of tumour more and more is subjected to expert and scholar's attention.Along with illustrating and the continuous discovery of the cell death inducing factor of apoptosis mechanism, the research of the apoptosis induction ligand that tumour necrosis factor is relevant extremely people is paid close attention to.It is the another apoptosis inducing factor of finding after TNF, FasL, extensively is present in the multiple healthy tissues of body.Because the structure of its acceptor on normal cell and tumour cell is different with distribution, thereby make it have the selective killing tumour cell and characteristic that normal cell is had no side effect.
WO9701633 discloses cDNA and the monoclonal antibody of TRAIL.
Chinese patent application 99111039 discloses derivative TRAILD and the antitumor action thereof of TRAIL, be to be template with Chinese's peripheral blood lymphocyte mRNA, adopt the cDNA of RT-PCR amplification TRAILD, obtain the recombinant prokaryotic expression vector of TRAILD, change intestinal bacteria over to after, obtain the engineering bacteria of stably express TRAILD recombination fusion protein, through the screening of going down to posterity, this albumen is expressed in the bacterium endochylema with soluble form, through fermentation, broken, chromatography with separate, obtain the TRAILD albumen of purifying.
Proved that now (recombinant human soluble tumor necrosisfactor-related apoptosis-inducing ligand rhsTRAIL) can induce the kinds of tumor cells apoptosis external to rhsTRAIL; The people's tumour that is used for the treatment of the bare mouse different species subcutaneous transplantation in vivo, can obviously suppress the propagation and the diffusion of tumour cell, without any detectable toxicity, more can not cause caused fatal vascular inflammation syndromes of TNF and the severe liver injury that FasL caused to healthy tissues, organ.Therefore, TRAIL has broad application prospects as antineoplastic biologic preparation of new generation.
Summary of the invention
The object of the present invention is to provide a kind of recombinant human soluble trail protein, described albumen can be induced the kinds of tumor cells apoptosis effectively, suppresses the propagation of transplanted tumor in the mouse body, has good anti-tumor biological.
Another object of the present invention is to provide a kind of production method of recombinant human soluble trail protein, adopt people's trail protein extracellular soluble outskirt encoding sequence gene in the described method, can pass through prokaryotic expression system---colibacillus, eukaryotic expression system---pichia spp is expressed its extracellular region protein respectively, prokaryotic expression system can reach and efficiently express, and Yeast engineering bacteria is a secretion type expression, and preparation technology is simple.
A further object of the present invention is to provide the application of trail protein in the preparation antitumor drug.
Recombinant human soluble trail protein of the present invention has the Seq1 sequence of colibacillus expression or yeast expressed Seq2 sequence.
The Seq1 sequence:
＂MVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGF
YYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELK
ENDRIFVSVTNEHLIDMDHEASFFGAFLVG＂
The Seq2 sequence:
EAEAEFVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIH
EKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGI
FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG
The production method of recombinant human soluble trail protein of the present invention is to adopt gene recombination technology, extracting mRNA from human tonsil's tissue is template, increase through RT-PCR, obtain people's trail protein encoding sequence gene and its extracellular soluble outskirt albumen coded sequence gene, pass through prokaryotic expression system---colibacillus or eukaryotic expression system---pichia spp is expressed its extracellular region protein respectively; The former is non-fusion rotein, mainly exists with the inclusion body form; The latter is through methanol induction, high-level secretory expression in nutrient solution.
Wherein the preparation method of Seq1 comprises the steps:
(1) human cloning soluble TRAIL protein gene;
Extracting mRNA from human tonsil's tissue is template, through the RT-PCR amplification, obtains people's trail protein encoding sequence gene and its extracellular soluble outskirt albumen coded sequence gene,
(2) at prokaryotic expression system---express and produce the human soluble trail protein in the colibacillus,
At first make up engineering bacteria and abduction delivering, high density fermentation engineering bacteria and abduction delivering then, inclusion body separate and washing after, with target protein matter renaturation, purifying.
Specifically, the preparation method of Seq1 of the present invention is:
(1) with upstream primer:
①——AG?GAATTC?TGAC?AGG?ATC?ATG?GCT?ATG?ATG——
②——AG?GAATTC?TATG?GTG?AGA?GAA?AGA?GGT?CCTC——
And downstream primer:
--AC GGATCC AGG TCAG TTA GCC AACT---is combined, through reverse transcriptase polymerase chain reaction increase respectively people's trail protein encoding sequence and the proteic encoding sequence cDNA of its extracellular soluble outskirt, the 114-281 amino acids of this cDNA coding trail protein, the process enzyme is cut, electrophoresis is connected with carrier with ligase enzyme after reclaiming dna fragmentation, product transforms the colibacillus competent cell, the picking positive colony has obtained trail protein encoding sequence gene (trail) and its extracellular soluble outskirt albumen coded sequence gene (strail);
(2) strail and expression vector plasmid are cut through enzyme, reclaim the corresponding DNA fragment, linked enzyme connects the back and transforms the colibacillus competent cell, the positive transformed clone of picking, the extracting plasmid carries out enzyme and cuts evaluation, obtain positive recombinant expression vector plasmid pBV220-hstrail, transform host bacterium DH5 α, obtain to express the colibacillus genetic engineering bacterium of non-fusion rhsTRAIL.
(3) the colibacillus genetic engineering bacterium seed liquor of the non-fusion of expression rhsTRAIL is inoculated in the substratum and cultivates, and fermented liquid pH is 6.9 ± 0.1, is cultured to OD
600=1.5 o'clock, intensification abduction delivering 4 hours was collected thalline;
(4) the adding lysis buffer is frozen, melts the back and adopts ultrasonic disruption under ice bath, centrifugal collection inclusion body.With the inclusion body that the washings washing is extracted, remove impurity, lipid and foreign protein, obtain refining inclusion body;
(5) will make with extra care solubilization of inclusion bodies in denaturing agent, centrifugal, dialysis is centrifugal again after the renaturation, supernatant is the rhsTRAIL sample of renaturation;
(6) with the rhsTRAIL of anion-exchange column purification renaturation, the target protein peak is further purified with molecular sieve column behind ultrafiltration and concentration, obtains the pure product of rhsTRAIL that colibacillus is expressed.
Further, the preparation method of Seq1 of the present invention also comprises MONOCLONAL ANTIBODIES SPECIFIC FOR, and its method is:
With the immune BALB/C female mice of the pure product of rhsTRAIL that colibacillus is expressed, extracting spleen cell and SP2/0 myelomatosis are carried out cytogamy, clone under the polyoxyethylene glycol condition, obtain the cell strain of monoclonal antibody of the anti-rhsTRAIL of 5 strains after testing.
The preparation method of Seq2 of the present invention comprises the steps:
(1) gene clone human soluble trail protein
Extracting mRNA from human tonsil's tissue is template, through the RT-PCR amplification, obtains people's trail protein encoding sequence gene and its extracellular soluble outskirt albumen coded sequence gene;
(2) abduction delivering and production human soluble trail protein in eukaryotic expression system-pichia spp
Comprise and make up rhsTRAIL yeast expression engineering bacteria, fermentation and abduction delivering and purifying target protein matter.
Specifically, the preparation method of Seq2 of the present invention is:
(1) gene clone human soluble trail protein
With upstream primer:
①——AG?GAATTC?TGAC?AGG?ATC?ATG?GCT?ATG?ATG——
②——AG?GAATTC?TATG?GTG?AGA?GAA?AGA?GGT?CCTC——
And downstream primer:
--AC GGATCC AGG TCAG TTA GCC AACT---is combined, through reverse transcriptase polymerase chain reaction increase respectively people's trail protein encoding sequence and the proteic encoding sequence cDNA of its extracellular soluble outskirt, the 114-281 amino acids of this cDNA coding trail protein, the process enzyme is cut, electrophoresis reclaims dna fragmentation and is connected with carrier, product transforms the colibacillus competent cell, the picking positive colony has obtained trail protein encoding sequence gene (trail) and its extracellular soluble outskirt albumen coded sequence gene (strail);
(2) structure of rhsTRAIL yeast expression engineering bacteria
Designing following primer modifies the strail end:
Upstream primer:---GTA GAATTC GTG AGA GAA AGA GGT C---
Downstream primer:---TAC GC GGCCGC CAG TTA GCC AAC TAA---
Use PCR method amplification stail gene, PCR product directed cloning after double enzymolysis, electrophoresis reclaim makes up the expression vector plasmid pPICZ α A-strail of sTRAIL to the alpha factor signal peptide encoding sequence downstream of expression vector plasmid pPICZ α A;
Get recombinant expression plasmid pPICZ α A-strail through linearizing, precipitation concentrates the back and transforms methyl alcohol nutritional type yeast GS115 bacterial strain (available from Invitrogen company), and screening positive clone obtains about 500 transformed bacterias on the flat board;
Positive colony is seeded on MDH and the MMH flat board correspondingly cultivates, Screening and Identification Mut phenotype, with the recombination yeast engineering strain of derivational expression method screening high level expression rhsTRAIL,
(3) fermentation of engineering bacteria and abduction delivering
In the BMGY substratum, jolting is cultured to OD with bacterial classification inoculation
600=5.0, collect thalline, be resuspended in the BMMY substratum, abduction delivering is collected the nutrient solution supernatant after 4 days;
Adopt the method for high-density, fed-batch fermentation.Get seed liquor and be inoculated in the batch fermentation substratum and cultivate, and carry out abduction delivering, results nutrient solution supernatant;
(4) purifying of target protein matter
After the substratum supernatant is removed foreign protein, can be with 40%-80% saturation ratio ammonium sulfate with 80% target protein precipitation, dissolve above-mentioned precipitation with the Tris-HCl damping fluid of pH8.8, with SephadexG-50 chromatographic column purifying, collection target protein peak.
Further, the preparation method of Seq2 of the present invention also comprises target protein separated with the reinforcing yin essence ion exchange column and obtains pure target protein.
The present invention is had aminoacid sequence consistent with natural trail protein and amino acid and is formed by the rhsTRAIL albumen of escherichia coli expression; Colibacillary efficiently expressing makes rhsTRAIL albumen have little time to be folded into natural correct structure in born of the same parents, and exists with the inclusion body form of non-activity, and target protein sex change, renaturation, purifying process through the later stage obtain highly purified rhsTRAIL albumen.
Yeast engineering bacteria is an eukaryotic expression system, because natural trail protein is a glycoprotein, (as glycosylation) can be processed, modify to the yeast eukaryotic expression system to target protein, therefore have natural structure and biological activity, adopt secreted expression carrier in addition, target protein is secreted in the substratum, helps separation and purification, therefore, we think that the structure of expressing rhsTRAIL protein yeast engineering bacteria is the ground zero of recombinant human trail protein research.In addition, for the non-fusion rotein of escherichia coli expression, Yeast engineering bacteria of the present invention is a secretion type expression, so target protein sequence of N end is connected with the yeast signal peptide sequence, the amino acid recognition sequence site that proteolytic enzyme is arranged between the two, after target protein was secreted into the extracellular, its N end also kept the amino acid of 2-6 signal peptide.
Experimental results show that in external, the body, recombinant human soluble trail protein of the present invention can more effectively be induced the kinds of tumor cells apoptosis with respect to the trail protein of other present application, suppress the propagation of transplanted tumor in the mouse body, have more superior anti-tumor biological.
Specific embodiment
Describe the present invention in detail below in conjunction with the drawings and specific embodiments, described embodiment is used to describe the present invention, rather than restriction the present invention.
Description of drawings
Accompanying drawing 1a, Fig. 1 b are respectively the positive and negative chain nucleotide sequence analysis of strail gene results;
The SDS-PAGE analytical results of accompanying drawing 2 escherichia coli expression rhsTRAIL
A, cellular lysate liquid; The rhsTRAIL of B, inclusion body C, purifying
The nucleotide sequence analysis result of accompanying drawing 3 recombinant yeast expression vector plasmid pPICZ α A-strail
Accompanying drawing 4 Yeast engineering bacterias are expressed the SDS-PAGE analytical results of rhsTRAIL
Accompanying drawing 5 Yeast engineering bacterias are expressed the Western blot detected result of rhsTRAIL.
Recombinant human soluble trail protein of the present invention is its extracellular region polypeptide portion (114-281 position Amino acid). E. coli expression product adds the methionine of initiation codon coding, amounts to 169 amino acid groups Become, molecular weight is 19.6KD. Yeast expressed product is because the point of contact difference of protease has N end heterogeneity The property, formed by 170-174 amino acid, but most of product is 174 amino acid, the about 24KD of molecular weight, Form dimer, isoelectric point 4.5-5.5 at about 48KD place.
The preparation method of recombinant human soluble trail protein of the present invention comprises Gene cloning, human soluble TRAIL---colibacillus or eukaryotic expression system---expression and production in the Pichia pastoris at prokaryotic expression system of human soluble TRAIL, and is specific as follows:
One, the gene clone of human soluble trail protein
Getting the human tonsil and organize 100mg, press mRNA separating kit operational manual (Boehringer Mannheim company) and extract mRNA, is template with it, respectively with
Upstream primer:
①——AG?GAATTC?TGAC?AGG?ATC?ATG?GCT?ATG?ATG——
②——AG?GAATTC?TATG?GTG?AGA?GAA?AGA?GGT?CCTC——
Downstream primer:
--AC GGATCC AGG TCAG TTA GCC AACT---is combined, through reverse transcriptase polymerase chain reaction (RT-PCR) increase respectively people's trail protein encoding sequence and the proteic encoding sequence cDNA of its extracellular soluble outskirt, the encode 114-281 amino acids of trail protein of the latter.Modify restriction enzyme site EcoRI/BamHI digestion PCR product and pBluscript KS (-) plasmid with the primer both sides, electrophoresis reclaims dna fragmentation after T
4Ligase enzyme connects, and product transforms uses CaCl
2The colibacillus JM109 of sensitization (available from Beijing ancient cooking vessel state biotech development center) competent cell, the picking positive colony, the extracting plasmid, cut through enzyme and to identify and the sequential analysis confirmation, obtained trail protein encoding sequence gene (trail) and its extracellular soluble outskirt albumen coded sequence gene (strail) is as follows:
The trail gene order:
tctga?caggatcatg?gctatgatgg?aggtccaggg?gggacccagc
ctgggacaga?cctgcgtgct?gatcgtgatc?ttcacagtgc?tcctgcagtc?tctctgtgtg
gctgtaactt?acgtgtactt?taccaacgag?ctgaagcaga?tgcaggacaa?gtactccaaa
agtggcattg?cttgtttctt?aaaagaagat?gacagttatt?gggaccccaa?tgacgaagag
agtatgaaca?gcccctgctg?gcaagtcaag?tggcaactcc?gtcagctcgt?tagaaagatg
attttgagaa?cctctgagga?aaccatttct?acagttcaag?aaaagcaaca?aaatatttct
cccctagtga?gagaaagagg?tcctcagaga?gtagcagctc?acataactgg?gaccagagga
agaagcaaca?cattgtcttc?tccaaactcc?aagaatgaaa?aggctctggg?ccgcaaaata
aactcctggg?aatcatcaag?gagtgggcat?tcattcctga?gcaacttgca?cttgaggaat
ggtgaactgg?tcatccatga?aaaagggttt?tactacatct?attcccaaac?atactttcga
tttcaggagg?aaataaaaga?aaacacaaag?aacgacaaac?aaatggtcca?atatatttac
aaatacacaa?gttatcctga?ccctatattg?ttgatgaaaa?gtgctagaaa?tagttgttgg
tctaaagatg?cagaatatgg?actctattcc?atctatcaag?ggggaatatt?tgagcttaag
gaaaatgaca?gaatttttgt?ttctgtaaca?aatgagcact?tgatagacat?ggaccatgaa
gccagttttt?tcggggcctt?tttagttggc?taactgacct?gga
The strail gene order:
gtga?gagaaagagg?tcctcagaga?gtagcagctc?acataactgg?gaccagagga
agaagcaaca?cattgtcttc?tccaaactcc?aagaatgaaa?aggctctggg?ccgcaaaata
aactcctggg?aatcatcaag?gagtgggcat?tcattcctga?gcaacttgca?cttgaggaat
ggtgaactgg?tcatccatga?aaaagggttt?tactacatct?attcccaaac?atactttcga
tttcaggagg?aaataaaaga?aaacacaaag?aacgacaaac?aaatggtcca?atatatttac
aaatacacaa?gttatcctga?ccctatattg?ttgatgaaaa?gtgctagaaa?tagttgttgg
tctaaagatg?cagaatatgg?actctattcc?atctatcaag?ggggaatatt?tgagcttaag
gaaaatgaca?gaatttttgt?ttctgtaaca?aatgagcact?tgatagacat?ggaccatgaa
gccagttttt?tcggggcctt?tttagttggc?taactgacct?gga
Referring to being the positive and negative chain nucleotide sequence analysis of strail gene result shown in accompanying drawing 1a, Fig. 1 b.
Two, soluble TRAIL protein is at prokaryotic expression system---expression and production in the colibacillus
1. the structure of engineering bacteria and abduction delivering
With pBluscript KS (-)-strail and expression vector plasmid pBV220 (viral journal, 1990,6 (2) 111-6) digest through EcoRI/BamHI, reclaim the corresponding DNA fragment, after connecting, the T4 ligase enzyme transforms colibacillus JM109 (available from Beijing ancient cooking vessel state biotech development center) competent cell of CaCl2 sensitization, the positive transformed clone of picking, the extracting plasmid carries out enzyme and cuts evaluation, obtain positive recombinant expression vector plasmid pBV220-strail, transform host bacterium DH5 α (available from Beijing ancient cooking vessel state biotech development center), positive monoclonal is inoculated in (every liter of tryptone 10g in the 5ml LB nutrient solution, yeast extract 5g, NaCl 10g), contain penbritin (Amp) 100 μ g/ml, at 30 ℃, 250rpm jolts and is cultured to OD
600=0.8-1.0 improves culture temperature to 42 and ℃ carried out abduction delivering 5 hours, collects thalline.SDS-PAGE detects, and compares with blank, and visible significantly 19.6KD target protein is expressed band (referring to accompanying drawing 2A), conforms to theoretical value, and the colibacillus genetic engineering bacterium of non-fusion rhsTRAIL is expressed in acquisition.
2. the high density fermentation of engineering bacteria and abduction delivering
The colibacillus genetic engineering bacterium seed liquor of expressing non-fusion rhsTRAIL is inoculated in the LB substratum (100 μ g/ml Amp) in 1: 10 ratio, at 30 ℃, cultivates under the 100rpm condition.Fall 10% by dissolved oxygen, rotating speed improves 100rpm and adjusts rotating speed, the highest 700rpm that is no more than.Regulating fermented liquid pH with 0.2mol/L NaOH is 6.9 ± 0.1, cultivates to begin to add glucose 0.25g/Lh after 3 hours, LB substratum 150ml/h.Be cultured to OD
600=1.5 o'clock, be warming up to 42 ℃, abduction delivering 4 hours is collected thalline, and the about 30g/L of thalline weight in wet base, target protein expression amount account for the 25%-30% (referring to Fig. 2 A) of bacterial protein.
3. the separation of inclusion body and washing
The wet bacterium of every gram adds lysis buffer 10ml (cracked solution consists of: 50mmol/L Tris-HCl, pH8.0,1mmol/L EDTA), and-30 ℃ frozen down.Melt the back and adopt ultrasonic disruption under ice bath, microscopy bacterial cell disruption rate is more than 90%, and centrifugal 20min under 12000rpm collects inclusion body.
To consist of 50mmol/L Tris-HCl, pH8.0, the inclusion body that the TE liquid washing of 1mmol/L EDTA is extracted, remove impurity, with 20mmol/L Tris-HCl, 10mmol/L EDTA, pH8.0,0.5%TritonX-100,2mmol/L beta-mercaptoethanol washing inclusion body 1 time is to remove lipid, again with the washing of TE liquid once; Use the 2M urea at last, 20mmol/L Tris-HCl, 2mmol/L EDTA, the pH8.0 washing is removed foreign protein 1 time, obtains refining inclusion body (referring to Fig. 2 B) at last.
The non-fusion rotein of escherichia coli expression of the present invention because it efficiently expresses, makes it have little time to be folded into natural correct structure in born of the same parents, and exists with the inclusion body form of non-activity.
4. the renaturation of target protein matter
Get refining inclusion body 50mmol/L NH
4AC (ammonium acetate), 7mol/L Guanidinium hydrochloride, 1% mercaptoethanol fully dissolves, centrifugal 10min under the 8000rpm, supernatant makes protein concentration be lower than 500 μ g/ml with the dilution of 2mol/L Guanidinium hydrochloride, uses 50mmol/L NH again
4Ac does 10 times of dilutions, to damping fluid 1mmol/L NH
4The Ac 24h that dialyses, centrifugal, supernatant is the rhsTRAIL sample of renaturation.
5. the column chromatography purification of recombinant protein matter
With 50mmol/L NH
4Ac balance DEAE-Sepharose Fast Flow anion-exchange column (Parmacia company product), the rhsTRAIL of renaturation goes up sample, uses 50mmol/L NaCl, 50mmol/L NH
4AC, pH8.0 wash-out target protein detects rhsTRAIL in main peak through SDS-PAGE.The target protein peak of receiving through ultrafiltration and concentration to 2.0mg/ml, last SuperdeX 75 molecular sieve columns with the physiological saline wash-out, are collected the target protein peak, purity is 97% (Fig. 2 C).
6. MONOCLONAL ANTIBODIES SPECIFIC FOR
With the immune BALB/C female mice of the pure product of rhsTRAIL that colibacillus is expressed, extracting spleen cell and SP2/0 myeloma cell carry out cytogamy, clone under the polyoxyethylene glycol condition, obtain the cell strain of monoclonal antibody of the anti-rhsTRAIL of 5 strains after testing.
Three, abduction delivering and the production of human soluble trail protein in eukaryotic expression system-pichia spp:
1.rhsTRAIL the structure of yeast expression engineering bacteria
Designing following primer modifies the strail end:
Upstream primer:---GTA GAATTC GTG AGA GAA AGA GGT C---
Downstream primer:---TAC GC GGCCGC CAG TTA GCC AAC TAA---
Use PCR method amplification stail gene, the PCR product is through the EcoRI/NotI double enzymolysis, electrophoresis reclaims the alpha factor signal peptide encoding sequence downstream of back directed cloning to expression vector plasmid pPICZ α A, makes up the expression vector plasmid pPICZ α A-strail of sTRAIL, and enzyme is cut evaluation.The sequencing results is:
--AAAAGAGAGG?CTGAAGCT?GAATTCgtga?gagaaagagg?tcctcagaga?gtagcagctc
acataactgg?gaccagagga?agaagcaaca?cattgtcttc?tccaaactcc?aagaatgaaa
aggctctggg?ccgcaaaata?aactcctggg?aatcatcaag?gagtgggcat?tcattcctga
gcaacttgca?cttgaggaat?ggtgaactgg?tcatccatga?aaaagggttt?tactacatct
attcccaaac?atactttcga?tttcaggagg?aaataaaaga?aaacacaaag?aacgacaaac
aaatggtcca?atatatttac?aaatacacaa?gttatcctga?ccctatattg?ttgatgaaaa
gtgctagaaa?tagttgttgg?tctaaagatg?cagaatatgg?actctattcc?atctatcaag
ggggaatatt?tgagcttaag?gaaaatgaca?gaatttttgt?ttctgtaaca?aatgagcact
tgatagacat?ggaccatgaa?gccagttttt?tcggggcctt?tttagttggc?taactg
GCGGCCGC?CAGCTTTCTA--
Be written as carrier pPICZ α A partial sequence greatly, small letter is the strail sequence.
Referring to Fig. 3, measurement result is consistent with design.
Get recombinant expression plasmid pPICZ α A-strail 10 μ g through the SacII linearizing, precipitation concentrates the back and transforms methyl alcohol nutritional type yeast GS115 bacterial strain (available from Invitrogen company) with the LiCl method, and working method is pressed the Easy Select of invitrogen
TMPichia Expression Kit specification sheets carries out.Contain that screening positive clone obtains about 500 transformed bacterias on YPDS (1% yeast extract, 2% tryptone, 2% glucose, the 1M sorbyl alcohol) flat board of Zeocin (invitrogen company product) 100mg/100ml.Detect the strail gene with PCR method again, obtain 20 positive colonies through evaluation.
With MDH[1.34%YNB (no ammonium persulphate does not have amino acid whose yeast nitrogen base), 4 * 10
-5The % vitamin H, 2% glucose] and MMH (1.34%YNB, 4 * 10
-5The % vitamin H, 0.5% methyl alcohol) the dull and stereotyped Mut phenotype of determining positive colony, positive colony is seeded on MDH and the MMH flat board correspondingly, cultivate after 2 days, determine that 20 strain positive colonies are Mut for 28-30 ℃
-sPhenotype, promptly methyl alcohol utilizes slow type.Screen the recombination yeast engineering strain of high level expression rhsTRAIL at last with derivational expression method, be about to above-mentioned positive colony and be inoculated in 10mlBMGY substratum (consisting of of BMGY substratum: 10g/L yeast extract respectively, the 20g/L peptone, 100mmol/L potassiumphosphate pH6.0,13.4g/LYNB, 4 * 10
-5G/L vitamin H and 1% glycerine), at 30 ℃, the 250rpm jolting is cultured to OD
600=5.0.Collect thalline, resuspended with 2ml BMMY (1% glycerine among the BMGY is replaced by 1% methyl alcohol) substratum.The same terms is cultivated down, and sampling in per 24 hours is used for the electrophoresis check, replenishes methyl alcohol simultaneously to final concentration 0.5%.Electrophoresis detection result shows: methanol induction began to express target protein in 1 day, induced to reach the peak on the 4th day.Expression amount is 50mg/L, target protein accounts for more than 80% of nutrient solution supernatant total protein, exist with monomer and two kinds of forms of dimer, molecular weight is respectively 20KD and 40KD (referring to Fig. 4), do western blot detection with the rhsTRAIL monoclonal antibody that Chinese People's Anti-Japanese Military and Political College enterobacteria is expressed, target protein has specific reaction, confirms that expression product is rhsTRAIL albumen (Fig. 5), and sequential structure is as follows:
ekr↓ea↑ea↑ef
VRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKG
FYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGI
FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG
Small letter is a carrier signal peptide moiety sequence, is written as the target protein sequence greatly.
↓: be Kex2 signal cleavage; ↑: be Ste13 signal cleavage
2. the fermentation of engineering bacteria and abduction delivering
1). shake-flask culture: bacterial classification is inoculated in the 500ml BMGY substratum by 2% concentration, and 30 ℃, the 300rpm jolting is cultured to OD
600=5.0.Collect thalline, be resuspended in the 100ml BMMY substratum, every 24h adds 1% volumes methanol, and abduction delivering is collected the nutrient solution supernatant after 4 days.SDS-PAGE electrophoresis detection result shows, the about 50mg/L of expression amount (figure slightly).
2). ferment tank: B.Braun5L fermentor tank, the method for employing high-density, fed-batch fermentation.Get seed liquor, be inoculated into 2.5L batch fermentation substratum (containing 4% glycerine and 4ml/L PTM1 trace element solution in the salt of FM21 basis) (referring to Molecular Cloning:A Laboratory Manual2 in 1: 10 ratio
Nd, 1989) in carry out batch culture, NH
4H
2O (ammoniacal liquor) transfers pH to about 5.5, and dissolved oxygen (DO) detects carbon source.When glycerine exhausted, flow feeding growth medium (50% glycerine, 12ml/L PTM1 solution) was kept dissolved oxygen greater than 10%, works as OD
600During=500 left and right sides, stop feed supplement.After glycerine exhausts, add inducing culture (100% methyl alcohol, 12ml/L PTM1 solution) and the pH value is controlled at 5.5-6.0 and carry out abduction delivering, regulate rotating speed, tank pressure, air flow quantity and stream and add methyl alcohol speed, make DO greater than 20%, methanol concentration is less than 1%.Abduction delivering 4 days, results nutrient solution supernatant, the target protein expression amount reaches 400mg/L.
3. the purifying of target protein matter
Ammonium sulfate precipitation: with 40% saturation ratio ammonium sulfate precipitation substratum supernatant, can remove most foreign proteins, 40%-80% saturation ratio ammonium sulfate can be with 80% target protein precipitation.Use 20mmol/L, the Tris-HCl damping fluid of pH8.8 dissolves above-mentioned precipitation 2mg/ml, with on 4% column volume through 20mmol/L, the SephadexG-50 chromatographic column of the Tris-HCl damping fluid dissolution equilibrium of pH8.8, use the same buffer wash-out, collect the target protein peak, purity can reach 90%.
Sample continues to separate through Q-Sepharose Fast Flow reinforcing yin essence ion exchange column, and the target protein peak is through non-reduced type SDS-PAG electrophoresis detection, and scanning result shows target protein purity＞97% (Fig. 4).
Four, the anti-tumor biological effect of group human soluble trail protein
The external evoked apoptosis of tumor cells activity of recombinant human soluble trail protein
Adopt the MTT detection method, respectively with SMMC7721, BEL7402 liver cancer cell, the G/C82 lung adenocarcinoma cell, the SW480 colon cancer cell is with 1 * 10
4/ ml is inoculated in 96 porocyte culture plates, the conventional 24h that cultivates abandons supernatant, dilutes recombinant human soluble trail protein of the present invention respectively to the concentration shown in the table 1 with 1640 (Sigma company) nutrient solution that contains 1% calf serum, add in the culture plate (n=8), continue to cultivate 48 hours.Control group is the RPMI-1640 that contains 1% calf serum.The MTT[3 that adds 10 μ l/ hole 1.5mg/ml, (4,5-methylthiozol-2-yl-2,5-diphenyltetrazolium brode assay)] cultivate after 5 hours, abandon supernatant, every hole adds 100 μ l DMSO (dimethyl sulfoxide (DMSO)), measure its A value with the ELX-800 microplate reader, measure wavelength 570nm, the absorption of reference wavelength 630nm the results are shown in Table 1.
Table 1 rhsTRAIL is to the effect (n=8) of culture of tumor cell
Grouping concentration (μ g/ml) SMMC7721 BEL7402 G/C82 SW480
Contrast-0.485 ± 0.031 0.375 ± 0.024 0.431 ± 0.011 0.532 ± 0.021
rhsTRAIL 0.1 0.491±0.035 0.365±0.018 0.440±0.043 0.533±0.031
1.0 0.452±0.021* 0.338±0.021* 0.401±0.032* 0.501±0.025*
5.0 0.317±0.017** 0.281±0.016** 0.347±0.024** 0.413±0.018**
10 0.301±0.020** 0.254±0.020** 0.315±0.014** 0.415±0.013**
Annotate: compare * P＜0.05, * * P＜0.01 with control group
By table 1 as seen, the pure product of 1 μ g/ml can be induced above-mentioned tumour cell generation apoptosis in various degree.Along with concentration improves, tumour cell is produced effect of apoptosis improve.
Same procedure detects the effect of rhsTRAIL to the normal rat primary hepatocyte, and the result shows there is not tangible apoptosis-induced effect.(seeing Table 2)
Table 2 rhsTRAIL is to the influence (n=8) of rat primary cultured hepatocyte
Grouping dosage (μ g/ml) OD
Contrast--0.345 ± 0.018
rhsTRAIL 0.1 0.351±0.022*
1.0 0.347±0.016*
5.0 0.344±0.015*
10 0.357±0.025*
Annotate: compare * P＞0.05 with control group
1. suppress the tumor cell proliferation activity in the recombinant human soluble trail protein body
Adopt 2X10
5The BEL-7402 liver cancer cell, 0.1ml it is subcutaneous that volume is inoculated in mouse (18-24g) the small of the back, make lotus BEL-7402 liver cancer cell mouse tumor animal model, intraperitoneal administration, the dosage of recombinant human soluble trail protein is respectively 0.1,0.5,1.0mg/kg/ days, does negative control with physiological saline.After the administration 10 days, measure certainly size of knurl, the result shows that rhsTRAIL can significantly suppress the BEL-7402 liver cancer cell in the intravital propagation of mouse.The results are shown in Table 3.
Table 3 rhsTRAIL is to the restraining effect (n=10) of mouse-borne tumor
Grouping dosage (mg/kg) lotus tumor weight (g) tumor control rate (%)
Contrast--0.832 ± 0.25
rhsTRAIL 0.1 0.854±0.27 -
0.5 0.5?35±0.25* 35.7
1.0 0.467±0.18** 43.9
Annotate: with control group * P＜O.05 relatively, * * P＜0.01
In a word, the present invention has made up proteic colibacillus engineering of expression rhsTRAIL and Yeast engineering bacteria, and has set up a whole set of production method.The rhsTRAIL that is characterized in escherichia coli expression is non-fusion rotein, has aminoacid sequence consistent with natural trail protein and amino acid and forms; Because natural trail protein is a glycoprotein, so we have adopted the yeast eukaryotic expression system, its advantage is: 1. the target protein of Biao Daing can be processed, modify glycosylation, have natural structure and biological activity, 2. adopt secreted expression carrier, target protein is secreted in the substratum, helps separation and purification.Therefore, we think that the structure of expressing rhsTRAIL protein yeast engineering bacteria is the ground zero of recombinant human trail protein research.
Claims (10)
1. recombinant human soluble trail protein, its sequence is:
Seq1：
＂MVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIHEKGF
YYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGIFELK
ENDRIFVSVTNEHLIDMDHEASFFGAFLVG＂，
Perhaps Seq2:
EAEAEFVRERGPQRVAAHITGTRGRSNTLSSPNSKNEKALGRKINSWESSRSGHSFLSNLHLRNGELVIH
EKGFYYIYSQTYFRFQEEIKENTKNDKQMVQYIYKYTSYPDPILLMKSARNSCWSKDAEYGLYSIYQGGI
FELKENDRIFVSVTNEHLIDMDHEASFFGAFLVG
2. a kind of recombinant human soluble trail protein according to claim 1 is characterized in that the 114-281 amino acids of described Seq1 sequence behaviour soluble TRAIL protein extracellular region polypeptide portion, and initiation codon is encoded to methionine(Met), and molecular weight is 19.6KD; Described Seq2 sequence is made up of 170-174 amino acid.
3. a kind of recombinant human soluble trail protein according to claim 3 is characterized in that described Seq2 sequence is made up of 174 amino acid, and the about 24KD of molecular weight forms dimer, iso-electric point 4.5-5.5 at about 48KD place.
4. the preparation method of the described recombinant human soluble trail protein of claim 1, the preparation that it is characterized in that having the trail protein of Seq1 sequence comprises the steps:
(1) gene clone human soluble trail protein encoding sequence;
Extracting mRNA from human tonsil's tissue is template, through the RT-PCR amplification, obtains people's trail protein encoding sequence gene and its extracellular soluble outskirt albumen coded sequence gene;
(2) at prokaryotic expression system---express and produce the human soluble trail protein in the colibacillus;
At first make up engineering bacteria and abduction delivering, high density fermentation engineering bacteria and abduction delivering then, inclusion body separate and washing after, with target protein matter renaturation, purifying.
5. the preparation method of recombinant human soluble trail protein according to claim 4 is characterized in that
(1) gene clone human soluble trail protein
With upstream primer:
①——AG?GAATTC?TGAC?AGG?ATC?ATG?GCT?ATG?ATG——
②——AG?GAATTC?TATG?GTG?AGA?GAA?AGA?GGT?CCTC——
And downstream primer:
--AC GGATCC AGG TCAG TTA GCC AACT---is combined, through reverse transcriptase polymerase chain reaction increase respectively people's trail protein encoding sequence and the proteic encoding sequence cDNA of its extracellular soluble outskirt, the 114-281 amino acids of this cDNA coding trail protein, the process enzyme is cut, electrophoresis reclaims dna fragmentation and is connected with ligase enzyme, product transforms the colibacillus competent cell, the picking positive colony has obtained trail protein encoding sequence gene (trail) and its extracellular soluble outskirt albumen coded sequence gene (strail);
(2) strail and expression vector plasmid are through digestion, reclaim the corresponding DNA fragment, linked enzyme connects the back and transforms the colibacillus competent cell, the positive transformed clone of picking, the extracting plasmid carries out enzyme and cuts evaluation, obtains positive recombinant expression vector plasmid pBV220-strail, transform host bacterium DH5 α, positive monoclonal is inoculated in cultivation and abduction delivering in the nutrient solution, collects thalline, obtain to express the colibacillus genetic engineering bacterium of the non-rhsTRAIL of fusion;
(3) the colibacillus genetic engineering bacterium seed liquor of the non-fusion of expression rhsTRAIL is inoculated in the substratum and cultivates, and fermented liquid pH is 6.9 ± 0.1, and when being cultured to OD600=1.5, intensification abduction delivering 4 hours is collected thalline;
(4) the adding lysis buffer is frozen, melts the back and adopts ultrasonic disruption under ice bath, centrifugal collection inclusion body.With the inclusion body that the washings washing is extracted, remove impurity, lipid and foreign protein, obtain refining inclusion body;
(5) will make with extra care solubilization of inclusion bodies in denaturing agent, centrifugal, dialysis is centrifugal again after the renaturation, supernatant is the rhsTRAIL sample of renaturation;
(6) with the rhsTRAIL of anion-exchange column purification renaturation, the target protein peak is further purified with molecular sieve column behind ultrafiltration and concentration, obtains the pure product of rhsTRAIL that colibacillus is expressed.
6. according to the preparation method of the described recombinant human soluble trail protein of claim 5, it is characterized in that comprising the steps:
(1) gets refining inclusion body 50mmol/L NH
4AC, 7mol/L Guanidinium hydrochloride, 1% mercaptoethanol fully dissolve, centrifugal 10min under the 8000rpm, and supernatant makes protein concentration be lower than 500 μ g/ml with the dilution of 2mol/L Guanidinium hydrochloride, uses 50mmol/L NH again
4Ac does 10 times of dilutions, to damping fluid 1mmol/L NH
4The Ac 24h that dialyses, centrifugal, supernatant is the rhsTRAIL sample of renaturation.
(2) column chromatography purification of recombinant protein matter
With 50mmol/L NH
4Ac balance DEAE-Sepharose Fast Flow anion-exchange column, the rhsTRAIL of renaturation goes up sample, uses 50mmol/L NaCl, 50mmol/L NH
4AC, pH8.0 wash-out target protein detects rhsTRAIL in main peak through SDS-PAGE.The target protein peak of receiving through ultrafiltration and concentration to 2.0mg/ml, last SuperdeX 75 molecular sieve columns with the physiological saline wash-out, are collected the target protein peak, purity is 97%.
7. the preparation method of the described recombinant human soluble trail protein of claim 1 is characterized in that the preparation of Seq2 sequence trail protein comprises the steps:
(1) gene clone human soluble trail protein encoding sequence
Extracting mRNA from human tonsil's tissue is template, through the RT-PCR amplification, obtains people's trail protein encoding sequence gene and its extracellular soluble outskirt albumen coded sequence gene;
(2) abduction delivering and production human soluble trail protein in eukaryotic expression system-pichia spp
Comprise and make up rhsTRAIL yeast expression engineering bacteria, fermentation, abduction delivering and purifying target protein matter.
8. the preparation method of recombinant human soluble trail protein according to claim 7 is characterized in that the preparation of Seq2 sequence trail protein comprises the steps:
(1) gene clone human soluble trail protein
With upstream primer:
①——AG?GAATTC?TGAC?AGG?ATC?ATG?GCT?ATG?ATG——
②——AG?GAATTC?TATG?GTG?AGA?GAA?AGA?GGT?CCTC——
And downstream primer:
--AC GGATCC AGG TCAG TTA GCC AACT---is combined, through reverse transcriptase polymerase chain reaction increase respectively people's trail protein encoding sequence and the proteic encoding sequence cDNA of its extracellular soluble outskirt, the 114-281 amino acids of this cDNA coding trail protein, the process enzyme is cut, electrophoresis reclaims dna fragmentation and is connected with carrier, product transforms the colibacillus competent cell, the picking positive colony has obtained trail protein encoding sequence gene and its extracellular soluble outskirt albumen coded sequence gene;
(2) structure of rhsTRAIL yeast expression engineering bacteria
Designing following primer modifies the strail end:
Upstream primer:---GTA GAATTC GTG AGA GAA AGA GGT C---
Downstream primer:---TAC GC GGCCGC CAG TTA GCC AAC TAA---
Use PCR method amplification stail gene, PCR product directed cloning after double enzymolysis, electrophoresis reclaim makes up the expression vector plasmid pPICZ α A-strail of sTRAIL to the alpha factor signal peptide encoding sequence downstream of expression vector plasmid pPICZ α A;
Get recombinant expression plasmid pPICZ α A-strail through linearizing, precipitation concentrates the back and transforms methyl alcohol nutritional type yeast GS115 bacterial strain, and screening positive clone obtains about 500 transformed bacterias on the flat board;
Positive colony is seeded on MDH and the MMH flat board correspondingly cultivates, its Mut phenotype of Screening and Identification is with the recombination yeast engineering strain of derivational expression method screening high level expression rhsTRAIL;
(3) fermentation of engineering bacteria and abduction delivering
In the BMGY substratum, jolting is cultured to OD with bacterial classification inoculation
600=5.0, to collect thalline, and be resuspended in the BMMY substratum, abduction delivering is collected the nutrient solution supernatant after 4 days;
Adopt the method for high-density, fed-batch fermentation.Get seed liquor and be inoculated in the batch fermentation substratum and cultivate, and carry out abduction delivering, results nutrient solution supernatant;
(4) purifying of target protein matter
After the substratum supernatant is removed foreign protein, can be with 40%-80% saturation ratio ammonium sulfate with 80% target protein precipitation, dissolve above-mentioned precipitation with the Tris-HCl damping fluid of pH8.8, with SephadexG-50 chromatographic column purifying, collection target protein peak.
9. the preparation method of a kind of recombinant human soluble trail protein according to claim 8 is characterized in that: target protein is separated obtaining pure target protein with the reinforcing yin essence ion exchange column, the reinforcing yin essence ion exchange column is Q-Sepharose Fast Flow.
10. as claim 1 to 9 recombinant human soluble trail protein application in preparation medicine for treating tumor thing as described in any one.
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