CN1313611C - Novel tumor wilting matter 2 ligand gene, its expressed tumor wilting matter and its preparation method - Google Patents
Novel tumor wilting matter 2 ligand gene, its expressed tumor wilting matter and its preparation method Download PDFInfo
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- CN1313611C CN1313611C CNB021108803A CN02110880A CN1313611C CN 1313611 C CN1313611 C CN 1313611C CN B021108803 A CNB021108803 A CN B021108803A CN 02110880 A CN02110880 A CN 02110880A CN 1313611 C CN1313611 C CN 1313611C
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Abstract
The present invention relates to a novel tumor apoptosis 2 ligand complete gene obtained from Chinese's peripheral blood mononuclear cells, namely an Apo-2L I gene, which relates to the technical field of medical biological engineering. The novel tumor apoptosis 2 ligand complete gene is utilized to prepare human soluble tumor apoptosis protein, namely tumor apoptosis ligand or Apo-2L I 100. Compared with the existing tumor apoptosis 2 ligand complete gene(Apo-2L gene), besides the novel tumor apoptosis 2 ligand complete gene is lack of 42 nucleotides from number 271 to number 313 at the end 5' in the coding region of the existing Apo-2L gene, the rest part is the same as that of the existing Apo-2L gene, specifically, the encoded corresponding amino acids can not be changed even though several nucleotides are different. The encoded protein Apo-2L I is lack of 14 amino acids correspondingly but has the same apoptosis function for tumor cells. The Apo-2L I100 is completely same as the existing human soluble tumor apoptosis protein Apo-2L114. The Apo-2L I gene is used as a template and pBV220 is utilized to construct a prokaryotic expression vector pBV-Apo-2L I100, so that the prepared Apo-2L I100 can directly express biological activity. The preparation method has the advantages of convenience, high yield and high quality.
Description
Technical field
The present invention relates to the medical bioengineering technical field, be a kind of novel Tumor wilting extract 2 ligand genes that from Chinese's peripheral blood mononuclear cell, obtain, and utilize this gene to prepare the human soluble tumor apoptotic proteins---the method for Tumor wilting extract.
Background technology
Nineteen ninety-five, external report from the people express the sequence label library (
eXpressed
sEquence
tAg screens a kind of gene (GenBank No.:U57059 of the anti-tumor protein of encoding in EST); U37518; NM_003810; XM_045049), the protein of this genes encoding be called tumor necrosin relative death inducing ligand (
tUmor necrosis factor-
rElated
aPoptosis-
iNducing
lIgand, TRAIL), claim again Tumor wilting extract 2 parts (
ApoPtotin-2
lIgand. be called for short Apo-2L), be tumour necrosis factor (
tUmor
nEcrosis
fActor, TNF) one of family member, it is induced tumor and transformant apoptosis effectively, and normal cell is not had influence.The code sequence of people Apo-2L gene is shown 843 Nucleotide, and nucleotide sequence is seen GenBank No.:U57059; U37518; NM_003810; XM_045049, encoded protein matter Apo-2L is made up of 281 amino acid.Proved already that Apo-2L was typical II type transmembrane protein, its N-holds the extracellular region of the 114th beginning can form free solubility tumor apoptosis fibroin Apo-2L
114, also claim TRAIL
114, have the tumor death effect equally.At present both at home and abroad to Apo-2L
114Preparation all use the Apo-2L gene.Yet owing to carry out the recombinant expressed Apo-2L of protokaryon
114The time, that used is commercialization expression vector pET (Novagen company) or pEQ (Qiagen company) etc., and recombinant protein is inclusion body, and it must just might show biologic activity through renaturation, and renaturation process is a comparison difficulty and unsettled, and not every inclusion body recoverability all.
Summary of the invention
The present invention has obtained a kind of novel tumor apo 2 ligand gene from Chinese's peripheral blood mononuclear cell, be called Apo-2L I gene.The encoding sequence of Apo-2L I gene is made up of 801 Nucleotide, compare with existing Apo-2L I gene, except having lacked 42 Nucleotide that are positioned at the 271st～313 at latter coding region 5 ' end, rest part is basic identical, even indivedual Nucleotide differences, also do not change the corresponding amino acid of coding, the nucleotide sequence of Apo-2L I gene sees Table 1 SEQ ID No.1.Also lacked 14 amino acid by the albumen of Apo-2L I genes encoding is corresponding, be made up of 267 amino acid, aminoacid sequence sees Table 2 SEQ ID No.2.
The present invention prepares human soluble tumor apoptosis element with Apo-2L I gene.With Apo-2L I gene is template, pass through pcr amplification, obtain the human soluble tumor apoptin gene from encoding sequence 5 ' end 297-801 position Nucleotide, its amino acids coding is equivalent to Apo-2L I n-end of albumen the 100th～267 amino acids, so be called Apo-2L I
100, itself and Apo-2L I
114Gene is the same, is made up of 504 Nucleotide.Its encoded protein and Apo-2L I
114Identical, also form by 168 amino acid, be referred to as Apo-2L I
100Or Tumor wilting extract.
Preparation Apo-2L I
100The expression vector that is to use of key be improved pBV220.PBV220 is provided by Chinese preventive medicine, and this carrier is the prokaryotic expression carrier of temperature control type, and its expression amount is higher.The present invention replaces the sequence between former pBV220 carrier B gl II and the Xmn I, constructed expression vector called after pBV-Apo-2L I by synthetic oligonucleotide
100, this plasmid transformation escherichia coli, the engineering bacteria of gained can directly be expressed the Apo-2L I with biologic activity
100Make Apo-2L I
100Expression amount accounts for more than 40% of engineering bacteria total protein, accounts for more than 10% of broken back engineering bacteria supernatant total protein, has improved Tumor wilting extract Apo-2L I greatly
100Output and quality, and the preparation method is easy.
The clone of Apo-2L I gene of the present invention, Apo-2L I
100Preparation, specifically comprise the steps:
One, construction cDNA library, preparation Apo-2L I gene
1. prepare the total RNA of human peripheral blood single nucleus cell
(1) isolated lymphocytes from Chinese's peripheral blood according to a conventional method, and then obtain mononuclearcell with adherent method;
(2) activate the abundance of mononuclearcell with bacterial endotoxin, use RNA extraction agent box (Qiagen company) extracted total RNA again with irritation cell raising RNA.
2. screen Apo-2L I gene
(1) construction cDNA library
Being purified into total mRNA with Oligo-dT post (Qiagen company) from above-mentioned total RNA, is template with total mRNA, and (Clontech company) builds up the cDNA library with cDNA library construction test kit.
(2) filter out Apo-2L I gene
Prepare gene probe with random priming routinely, the above-mentioned cDNA of screening by hybridization library filters out the clone.These clones' insertion portion subclone is to the Not I site of pSPORT1.Through sequential analysis, its dna sequence dna of one of them cloned genes is made up of 1727bp, contains the 5 ' non-translational region of 87bp, the open reading frame of 801bp (267 amino acid of encoding, be total length Apo-2L I albumen), 3 ' non-translational region (contain polyadenylation signal or claim the polyA tail).This unnamed gene is an Apo-2L I gene, and its nucleotide sequence sees Table 1, and its amino acid sequence coded sees Table 2.This plasmid called after pSPORT1-Apo-2L I.The bacterial strain Escherichia coli K802 (genotype: supE hardgal metB) be preserved in Chinese typical culture collection center (preserving number: CCTCC No.:200037 on November 17th, 2000 that contains this plasmid; Wuhan University).
Two, make up Apo-2L I
100The prokaryotic expression engineering strain
1. prepare Apo-2L I
100Gene
Designing and synthesizing primer, is template with Apo-2L I gene, and pcr amplification goes out encoding sequence Apo-2L I
100Gene.
2. make up prokaryotic expression carrier pBV-Apo-2L I
100
Design and synthesize two oligonucleotide two strandss, the Bgl II of displacement pBV220 carrier and the sequence between the Xmn I, the metathetical sequence is: (1) is with the former promotor λ P of pBV220
LBe replaced into λ P
LP
RTandem promoter; (2) calling sequence is the SD sequence of 5 '-AGGAATTCACAATG-3 '.Then with Apo-2L I
100Gene links to each other with foreign gene downstream regulating and controlling sequence.Constructed like this pBV-Apo-2L I
100Carrier, the distance between its ribosome binding sequence and the protein translation initiator codon is 7 Nucleotide, is beneficial to Apo-2L I
100Expression in bacterium.
3. set up Apo-2L I
100Express engineering bacteria
With above-mentioned pBV-Apo-2L I
100Transformed into escherichia coli, the clone that screening obtains containing recombinant plasmid is Apo-2L I
100Engineering bacteria.
Three, preparation Apo-2L I
100
1. the fermentation culture engineering bacteria makes it express Apo-2L I
100
2. ultrasonication gained thalline, supernatant row metal affinity chromatography, gel-filtration are further purified, and promptly get Apo-2L I
100
Description of drawings
Fig. 1 is expression vector pBV-Apo-2L I of the present invention
100Make up schema
Embodiment
One, sets up the cDNA library, screening Apo-2L I gene
1. prepare the total RNA of Chinese's peripheral blood mononuclear cell
Separate Chinese's peripheral blood lymphocyte with lymphocyte separation medium routinely, with RPMI-1640 substratum (GIBCO company), adding 10% newborn calf serum (Hangzhou folium ilicis chinensis biotech firm) and penicillin and Streptomycin sulphate is cultured to adherent, get mononuclearcell, use the intracellular toxin (Biochemistry and Molecular Biology teaching and research room of basic courses department of Second Military Medical University, PLA) of the intestinal bacteria R595 of 10 μ g/L to stimulate then 2 hours, to activate mononuclearcell, collect 10
7Cell with total RNA extraction agent box (Qiagen company) extracted total RNA, gets the total RNA of mononuclearcell.
2. set up the cDNA library, screening Apo-2L I gene
Routinely with total RNA of above-mentioned gained, get total mRNA with Oligo-dT post (Qiagen company) purifying, with total mRNA is template, carry out the synthetic of cDNA first chain and second chain with cDNA synthetic agent box (Clontch company), be connected in the λ gt10 carrier after adding the EcoRI joint, and be packaged into the lambda particles phage library with lambda particles phage package kit (Clontech company), and build up λ gt10cDNA library, the titre in library is 6 * 10
6
Prepare gene probe with random priming, this λ gt10cDNA library of screening by hybridization (seeing " molecular cloning " the 396th～601 page for details).Obtain 12 positive plaques.Its DNA of extracting, the λ DNA of 12 purifying, with Not I digestion, 1% agarose gel electrophoresis, the Southern trace is confirmed and probe hybridization.Select the big clone of intensity for hybridization, its DNA of extracting is again the Not I site of the insertion portion subclone of this DNA to pSPORT1 (GIBCO company).Measure the dna sequence dna of insertion portion in the plasmid.One of them sequence is made up of 1727bp, comprises the 5 ' non-translational region of 87bp, 801bp open reading frame and 3 ' non-translational region (contain polyadenylation signal or claim the polyA tail), and this fragment is an Apo-2L I gene, nucleotide sequence such as table 1.The plasmid that contains this gene is pSPORT1-Apo-2L I, and the bacterial strain that contains this plasmid has been preserved in Chinese typical culture collection center (preserving number: CCTCC No.:200037, Chinese Wuhan University).According to the translation initiation site requirement of Kozak definition, 88-90 position Nucleotide (ATG, coding methionine(Met)) is translation initiation site, 267 aminoacid sequences such as tables 2 that the maximum open reading frame that obtains thus is coded.
Two, make up Apo-2L I
100Prokaryotic expression carrier
Based on the pBV220 plasmid, make up the plain Apo-2L I of solubility tumor apoptosis
100Prokaryotic expression carrier pBV-Apo-2L I
100Concrete steps are as follows:
1. design and synthesize primer P1, P2 and oligonucleotide double-stranded P3, P4
P1：5′-AC
G?AAT?TCA?CA
A?TGG?TGA?GAG?AAA?GAG?GTC-3′；
P2：5′-AC
G?GAT?CCT?TAG?CCA?ACT?AAA?AAG-3′
P1, P2 are amplification coding Apo-2L I
100The primer of gene.P1 introduces synthetic initiator codon ATG of albumen and EcoR I restriction enzyme site
GAATTCP2 introduces BamH I restriction enzyme site
GGATCC
Synthetic oligonucleotide double-stranded P3, P4:
P3：5′-AA
A?GAT?CTC?TCA?CCT?ACC?AAA?CAA?TGC?CCC?CCT?GCA?AAA?AAT?AAA?TTC?ATA?TAA?AAA?ACA
3′-TT
T?CTA?GAG?AGT?GGA?TCG?TTT?CTT?ACC?GGG?GGA?CCT?TTT?TTA?TTT?AAG?TAT?ATT?TTT?TGT
GCA?CAT?CAG?CAG?GAC?GCA?CTG?ACC?ACC?ATG?AAG?GTG?ACG?CTC?TTA?AAA?ATT?AAG?CCC?TGA?AGA?AGG?GCA?GCA
CGT?GTA?GTC?GTC?CTG?CGT?GAC?TGG?TGG?TAC?TTC?CAC?TGC?GAG?AAT?TTT?TAA?TTC?GGG?ACT?TCT?TCC?CGT?CGT
TTC?AAA?GCA?GAA?GGC?TTT?GGG?GTG?TGT?GAT?ACG?AAA?CGA?AGC?ATT?GGT?TAA?AAA?TTA?AGG?AG
G?AAT?TLA?C-3′
AAG?TTT?CGT?CTT?CCG?AAA?CCC?CAC?ACA?CTA?TGC?TTT?GCT?TCG?TAA?CCA ATT TTT?AAT?TCC?TC
C?TTA?AGT?G-5′
P4：5′-AA
G?GAT?CCG?TCG?ACC?TGC?AGC?CAA?GCT TGG CTG?TTT?TGG?CGG?ATG?AGA?
GAA?GAT?TTT?CAG-3′
3′-TT
C?CTA?GGC?AGC?TGG?ACG?TCG?GTT?CGA?ACC?GAC?AAA?ACC?GCC?TAC?TCT?
CTT?CTA?AAA?GTC-5′
P3, P4 are foreign gene upstream regulatory sequence and downstream sequence between original pBV220 carrier B gl II of displacement and the Xmn I.The italicized item of P3 is λ P
LP
RTandem promoter, dash area are cI arrestin binding site, and black matrix partly is a ribosome binding sequence, and 5 ' end is introduced Bgl II restriction enzyme site
AGATCT, 3 ' end is introduced EcoR I restriction enzyme site
GGATTC5 ' the end of P4 has been introduced BamH I restriction enzyme site
GGATCC, 3 ' end is introduced Xmn I restriction enzyme site
GAANNNNTTC
2. construction of expression vector pBV-Apo-2L I
100
Construction of expression vector pBV-Apo-2L I
100Flow process such as Fig. 1, concrete steps are as follows: with P1, P2 is primer, is template with plasmid pSPORT1-Apo-2L I, pcr amplification Apo-2L I
100Gene coded sequence, product is cut (NEB company) with EcoR I and BamH I enzyme.Cut P3 with Bgl II and EcoR I enzyme, BamH I and Xmn I enzyme are cut P4.With Bgl II and Xmn I double digestion pBv220 plasmid.Each endonuclease bamhi sepharose reclaims the fragment of corresponding size, connects each fragment with the T4 dna ligase, and resulting recombinant plasmid is Tumor wilting extract Apo-2L I
100Expression vector pBV-Apo-2L I
100
Three, transformed into escherichia coli is set up engineering bacteria
Routinely with pBV-Apo-2L I
100Transformed into escherichia coli BL21[genotype: hsdS gal (λ cIts857ind1 Sam7 nin5lacUV5-T7)], separation quality grain from ammonia benzyl resistance bacterium colony, enzyme is cut evaluation, and pBV-Apo-2L I is confirmed as in order-checking
100The bacterium of gained is engineering bacteria of the present invention.
Four, preparation Apo-2L I
100
Engineering bacteria bacterium colony of picking inserts in the test tube of the 5ml LB nutrient solution contain penbritin (100 μ g/ml) in 32 ℃ of overnight incubation.LB culture medium prescription (g/L) is a peptone: yeast powder: NaCl=10: 5: 5.
The bacterium of incubated overnight is contained in 2 * YT substratum of penbritin (100 μ g/ml) by access in 1: 100, cultivated 6 hours.2 * YT culture medium prescription (g/L) is a peptone: yeast powder: NaCl=16: 10: 5.
Again the bacterium of cultivating was inserted in the semisynthetic medium that contains penbritin (100 μ g/ml) by 1: 40 and fermented.Cultivated 5-7 hour for 32 ℃; Be warming up to 42 ℃ and cultivated 4-5 hour, add feed supplement with constant flow pump with the 0.2ml/min/L substratum, dissolved oxygen is controlled between 30～50%.Obtain the wet thallus of about 20-30 grams per liter fermented liquid.Prescription (g/L) is a peptone in the semi-synthetic fermention medium: yeast powder: KH
2PO
4: K
2HPO
4: Na
2HPO
412H
2O: (NH
4)
2SO
4: NH
4Cl=5: 5: 2: 4: 7: 1.2: 0.2; Feeding medium during fermentation prescription (g/L) is a glucose: yeast powder: peptone: MgSO
47H
2O=200: 70: 70: 5.7.
Ultrasonication fermentation thalline, supernatant is with the membrane filtration of 0.22 μ m; Use TALON Metal AffinityRsins (Clontech company) the post row metal affinity chromatography of 3 * 20cm again, 10mmol/L imidazoles, pH7.0, wash 5 bed volumes with the wash-out foreign protein, with the 100mmol/L imidazoles, pH7.0 washes 5 bed volumes with wash-out Apo-2L I again
100Siphacryl S-200 is further purified Apo-2L I
100, can obtain high purity Apo-2L I
100
Table 1 Apo-2L I gene nucleotide series table SEQ ID No.1
1?CCT?CAC?TGA?CTA?TAA?AAG?AAT?AGA?GAA?GGA?AGG?GCT?TCA?GTG?ACC?GGC?TGC?CTG?GCT?GAC 60
61?TTA?CAG?CAG?TCA?GAC?TCT?GAC?AGG?ATC?ATG?GCT?ATG?ATG?GAG?GTC?CAG?GGG?GGA?CCC?AGC 120
121?CTG?GGA?CAG?ACC?TGC?GTG?CTG?ATC?GTG?ATC?TTC?ACA?GTG?CTC?CTG?CAG?TCT?CTC?TGT?GTG 180
181?GCT?GTA?ACT?TAC?GTG?TAC?TTT?ACC?AAC?GAG?CTG?AAG?CAG?ATG?CAG?GAC?AAG?TAC?TCC?AAA 240
241?AGT?GGC?ATT?GCT?TGT?TTC?TTA?AAA?GAA?GAT?GAC?AGT?TAT?TGG?GAC?CCC?AAT?GAC?GAA?GAG 300
301?AGT?ATG?AAC?AGC?CCC?TGC?TGG?CAA?GTC?AAG?TGG?CAA?CTC?CGT?CAG?CTC?GTT?AGA?AAG?GAA 360
361?AAG?CAA?CAA?AAT?ATT?TCT?CCC?CTA?GTG?AGA?GAA?AGA?GGT?CCT?CAG?AGA?GTA?GCA?GCT?CAC 420
421?ATA?ACT?GGG?ACC?AGA?GGA?AGA?AGC?AAC?ACA?TTG?TCT?TCT?CCA?AAC?TCC?AAG?AAT?GAA?AAG 480
481?GCT?CTG?GGC?CGC?AAA?ATA?AAC?TCC?TGG?GAA?TCA?TCA?AGG?AGT?GGG?CAT?TCA?TTC?CTG?AGC 540
541?AAC?TTG?CAC?TTG?AGG?AAT?GGT?GAA?CTG?GTC?ATC?CAT?GAA?AAA?GGG?TTT?TAC?TAC?ATC?TAT 600
601?TCC?CAA?ACA?TAC?TTT?CGA?TTT?CAG?GAG?GAA?ATA?AAA?GAA?AAC?ACA?AAG?AAC?GAC?AAA?CAA 660
661?ATG?GTC?CAA?TAT?ATT?TAC?AAA?TAC?ACA?AGT?TAT?CCT?GAC?CCT?ATA?TTG?TTG?ATG?AAA?AGT 720
721?GCT?AGA?AAT?AGT?TGT?TGG?TCT?AAA?GAT?GCA?GAA?TAT?GGA?CTC?TAT?TCC?ATC?TAT?CAA?GGG 780
781?GGA?ATA?TTT?GAG?CTT?AAG?GAA?AAT?GAC?AGA?ATT?TTT?GTT?TCT?GTA?ACA?AAT?GAG?CAC?TTG 840
841?ATA?GAC?ATG?GAC?CAT?GAA?GCC?AGT?TTT?TTT?GGG?GCC?TTT?TTA?GTT?GGC?TAA?CTG?ACC?TGG 900
901?AAA?GAA?AAA?GCA?ATA?ACC?TCA?AAG?TGA?CTA?TTC?AGT?TTT?CAG?GAT?GAT?ACA?CTA?TGA?AGA 960
961?TGT?TTC?AAA?AAA?TCT?GAC?CAA?AAC?AAA?CAA?ACA?GAA?AAC?AGA?AAA?CAA?AAA?AAC?CTC?TAT?1020
1021?GCA?ATC?TGA?GTA?GAG?CAG?CCA?CAA?CCA?AAA?AAT?TCT?ACA?ACA?CAC?ACT?GTT?CTG?AAA?GTG?1080
1081?ACT?CAC?TTA?TCC?CAA?GAA?AAT?GAA?ATT?GCT?GAA?AGA?TCT?TTC?AGG?ACT?CTA?CCT?CAT?ATC?1140
1141?AGT?TTG?CTA?GCA?GAA?ATC?TAG?AAG?ACT?GTC?AGC?TTC?CAA?ACA?TTA?ATG?CAA?TGG?TTA?ACA?1200
1201?TCT?TCT?GTC?TTT?ATA?ATC?TAC?TCC?TTG?TAA?AGA?CTG?TAG?AAG?AAA?GCG?CAA?CAA?TCC?ATC?1260
1261?TCT?CAA?GTA?GTG?TAT?CAC?AGT?AGT?AGC?CTC?CAG?GTT?TCC?TTA?AGG?GAC?AAC?ATC?CTT?AAG?1320
1321?TCA?AAA?GAG?AGA?AGA?GGC?ACC?ACT?AAA?AGA?TCG?CAG?TTT?GCC?TGG?TGC?AGT?GGC?TCA?CAC?1380
1381?CTG?TAA?TCC?CAA?CAT?TTT?GGG?AAC?CCA?AGG?TGG?GTA?GAT?CAC?GAG?ATC?AAG?AGA?TCA?AGA?1440
1441?CCA?TAG?TGA?CCA?ACA?TAG?TGA?AAC?CCC?ATC?TCT?ACT?GAA?AGT?GCA?AAA?ATT?AGC?TGG?GTG?1500
1501?TGT?TGG?CAC?ATG?CCT?GTA?GTC?CCA?GCT?ACT?TGA?GAG?GCT?GAG?GCA?GGA?GAA?TCG?TTT?GAA?1560
1561?CCC?GGG?AGG?CAG?AGG?TTG?CAG?TGT?GGT?GAG?ATC?ATG?CCA?CTA?CAC?TCC?AGC?CTG?GCG?ACA?1620
1621?GAG?CGA?GAC?TTG?GTT?TCA?AAA?AAA?AAA?AAA?AAA?AAA?AAC?TTC?AGT?AAG?TAC?GTG?TTA?TTT?1680
1681?TTT?TCA?ATA?AAA?TTC?TAT?TAC?AGT?ATG?TCA?AAA?AAA?AAA?AAA?AAA?AA 1727
Table 2 Apo-2L I Argine Monohydrochloride sequence table SEQ ID No.2
1?MAMMEVQGGP?SLGQTCVLIV?IFTVLLQSLC?VAVTYVYFTN?ELKQMQDKYS?KSGIACFLKE?DDSYWDPNDE 70
71?ESMNSPCWQV?KWQLRQLVRK?EKQQNISPLV?RERGPQRVAA?HITGTRGRSN?TLSSPNSKNE?KALGRKINSW 140
141?ESSRSGHSFL?SNLHLRNGEL?VIHEKGFYYI?YSQTYFRFQE?EIKENTKNDK?QMVQYIYKYT?SYPDPILLMK 210
211?SARNSCWSKD?AEYGLYSIYQ?GGIFELKEND?RIFVSVTNEH?LIDMDHEASF?FGAFLVG 267
Claims (4)
1. the full gene of Tumor wilting extract 2 parts is an Apo-2L I gene, it is characterized in that it has the sequence of SEQ ID No.1.
2. the Tumor wilting extract 2 part Apo-2L I of the described Apo-2L I of claim 1 genes encoding is characterized in that it has the sequence of SEQ ID No.2.
3. Tumor wilting extract Apo-2L I
100The preparation method, it is characterized in that with the described Apo-2L I of claim 1 gene be that template amplification gets Apo-2L I
100Gene makes up pBV-Apo-2L I again
100Expression plasmid and engineering bacteria, expressed Apo-2L I
100Biologic activity is directly arranged.
4. the Tumor wilting extract Apo-2L I that obtains of described Tumor wilting extract 2 part Apo-2L I of claim 2 or the described method of claim 3
100Application in preparation killing tumor cell medicine or preparation.
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|---|---|---|---|
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|---|---|---|---|
| CNB021108803A CN1313611C (en) | 2002-02-22 | 2002-02-22 | Novel tumor wilting matter 2 ligand gene, its expressed tumor wilting matter and its preparation method |
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| CN100586961C (en) * | 2006-05-16 | 2010-02-03 | 中国人民解放军军事医学科学院野战输血研究所 | To tumour cell inhibited fusion rotein and encoding gene and application |
| GB201406705D0 (en) | 2014-04-15 | 2014-05-28 | Univ Liverpool | Fusion proteins,polynucleotides,expression vectors, and their uses |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999036535A1 (en) * | 1998-01-15 | 1999-07-22 | Genentech, Inc. | Apo-2 ligand |
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2002
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999036535A1 (en) * | 1998-01-15 | 1999-07-22 | Genentech, Inc. | Apo-2 ligand |
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