CN1428347A - Clone, expression and biological activity of stingray tumor suppressor gene - Google Patents
Clone, expression and biological activity of stingray tumor suppressor gene Download PDFInfo
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Abstract
The present invention utilizes the construction of dasytis akajei caudal spine cDNA library and DNA sequecing and designs primer according to the obtained IPL tumor inhibiting gene EST3' end and carrier nucleotide sequence, and uses PCR method to screen the dasytis akajei caudal spine cDNA library and clone the IPL total length gene. The length of new gene is 849 bp, it can code mature peptide of 136 amino acids (sequence nimber 1). The isoelectric point of the expressed protein is 9.68 and molecular weight is about 15,690 Dalton. The dasytis akajei caudal spine IPL gene is cloned in uxA gene 3' tail end of thioredoxin gene fusion expression vector pTRX and the fusion gene meeting reading frame is contructed, said fusion protein is existed in colibacillus, its expression amount can be up to 40-60 mg/L.
Description
Technical field
The present invention relates to clone, the Expression and biological activity of stingray tumor suppressor gene.More specifically say, the present invention relates to from the total plasmid of stingray caudal spine cDNA expression library, utilize the PCR method screening and cloning to go out the IPL full-length gene, utilize engineered method, in efficient escherichia expression system, express and be purified into the stingray caudal spine IPL albumen of biologically active and the medicine that is used to prevent and treat human malignancies that constitutes by this albumen.
Background technology
China marine site is vast, and marine organisms are numerous in variety, and this huge resource is undoubtedly our huge treasure-house.According to record, the mankind just recognized before several thousand at least and exist active substance in the marine organisms that can utilize it is that the mankind cure the disease.China is one of country that the earliest marine organisms is used as medicine, apart from the clinical application history of modern existing two thousand years.365 kinds of medicine monograph Shennong's Herbal medicine carryings the earliest, wherein marine drug is about 10 kinds, successive dynasties are continued development again, but until the marine organisms that Ming Dynasty LI Shi-Zhen work Compendium of Material Medica has been put down in writing hyoscine reach 90 surplus kind, to nearly 10 kinds of the Qing Dynasty's newly-increased ocean of Zhao Xuemin work supplementary Amplifications of the Compendium of Materia Medica Chinese medicine, sum reaches kind more than 100.About the pharmaceutical use of ray class anal spine, in Compendium of Material Medica, the time treasure say: extra large ray, eagle ray fish sound is angry, catch Barb fish sound shop compares pictograph.Few positive lotus, and the words and deeds look is also.Give birth to the South Sea, its anal spine people, very the person is till death.Meat: sweet, salty flat, nontoxic [curing mainly] not beneficial people.Gonorrhoea cream drenches, the puckery pain of beautiful stem.Tooth: nontoxic [curing mainly] malignant malaria, burn black grinding into powder, wine is obeyed two money seven.Tail: poisonous [curing mainly] dentalgia.The Chinese medicine of ray class anal spine is used except that above-mentioned Compendium of Material Medica book, and folk remedy is then more of long standing and well established.Ray class anal spine is used as medicine and is used to treat multiple disease, and malaria, infantile malnutrition, mazoitis, toothache, swelling and pain in the throat, lung cancer, esophagus cancer, cancer of the stomach and acanthotoxic fishes are stabbed all certain curative effect.Ray class anal spine tool poison gland tissue, its main active component comprises amino acid, polypeptide proteinoid, 5 '-hydroxy-tryptamine, 5 '-phosphonuclease and phosphodiesterase etc., animal experiment confirms to have certain central nervous system heart effect of unifying.We are cloned into an IPL homologous gene from stingray caudal spine cDNA library, for study stingray caudal spine IPL gene from molecular level, we have carried out corresponding bioinformatic analysis and function prediction.And it has been cloned in the efficient expression vector, grope its expression time, expression temperature, IPTG induced concentration etc. and efficiently expressed condition.The red ray IPL of the reorganization albumen that has obtained purifying carries out the function corresponding activity research.The be used as medicine The Molecular Biology Mechanism of treatment mammary cancer, cancer of the stomach, esophagus cancer, cancer of the stomach and lung cancer of stingray caudal spine may be partly explained in the discovery of red ray IPL gene.
The existing IPL of studies show that gene is a tumor suppressor gene and/or an imprinted genes with PH functional domain.Imprinted genes is meant and shows the genoid that monoallelic is expressed that in normal development promptly express male parent allelotrope person and then do not express maternal allelotrope, vice versa.Express maternal allelic imprinted genes and be tending towards suppressing cell proliferation; Express male parent allelotrope person and then be tending towards stimulating growth.IPL genetic expression be maternal allelotrope, consistent with its potential growth inhibitory effect.Mouse apoptosis-related genes TDAG51 is the analogue of IPL/TSSC3, and TDAG51 crosses to express and causes mouse T-quadroma FAS mediated Apoptosis.In the Fas of activation inducing cell death expressed, the expression that IPL/TSSC3 raises Fas came mediated apoptosis, and TDAG51 and TCR combined signal work.The IPL expression of gene has growth inhibitory effect, and its low expression may cause cell that apoptotic signal is produced resistance.Structure prediction shows that IPL/TSSC3/BWR1C has one and is similar to pleckstrin homology (pleckstrin-homology, PH) Qu center motif (motif).As some other imprinted genes, the IPL gene is less and its intron is also little.PH domain be one with the relevant functional domain of the interactional signal transduction path of protein-phosphatide.Red ray IPL gene is a first found tumor suppressor gene in chondrichthyes, the species scope that has expanded the quantity of known imprinted genes and included.
Bioinformatic analysis shows that the nucleotide sequence similarity of red ray CH123 gene and people and mouse homologue IPL gene reaches 78% and 72%; The protein sequence homology reaches 50% and 48%, and its functional domain PH district is conservative substantially.
Summary of the invention
The objective of the invention is: (1) utilizes the PCR method screening and cloning to go out the IPL full-length gene from the total plasmid of stingray caudal spine cDNA expression library; (2) utilize engineered method, in efficient escherichia expression system, express and be purified into the stingray caudal spine IPL albumen of biologically active; (3) by of the research of the red ray IPL albumen of reorganization in functions such as the activity of vitro inhibition human malignancies cell line growth propagation and the interactional signal conduction of mediating protein-phosphatide, prove conclusively its activity, study its characteristics and mechanism of action, for further drug development lays the foundation to the tumour cell effect.
The invention provides a kind of shown in serial number in the sequence table 1 DNA of nucleotide sequence.
The invention provides a kind of shown in serial number in the sequence table 1 protein of aminoacid sequence.
The invention provides a kind of recombinant vectors, this recombinant vectors comprises above-mentioned a kind of nucleotide sequence.
The invention provides a kind of protokaryon bacterium that contains above-mentioned recombinant vectors.
In one embodiment, above-mentioned protokaryon bacterium is intestinal bacteria.
In one embodiment, it is to serve as to merge the companion body with sulphur oxygen cyclase protein that above-mentioned carrier makes, the middle affinity labelling site of 6 His and the efficient expression vector pETTRX-IPL of protease 3 C recognition site of inserting.
In one embodiment, above-mentioned carrier make described DNA in intestinal bacteria with soluble formal representation in the born of the same parents, its expression amount is 40-60mg/L.
In one embodiment, the expression product Supersonic lysate of above-mentioned recombinant vectors is through the Ni-Chelating affinity column chromatography, and protease 3 C cutting and gel-filtration can obtain the maturation protein of purity in the stable in properties more than 95%, and yield is 5-8mg/L.
The present invention also provides a kind of dna fragmentation as primer, and this dna fragmentation is made up of a part of sequence of above-mentioned nucleotide sequence.
The present invention also provides a kind of pharmaceutical preparation that contains above-mentioned proteic prevention or treatment tumor disease.
Above-mentioned albumen of the present invention can be used to prepare the medicine of prevention and treatment tumor disease.
The selected ray fish of the present invention belongs to red ray (Dasytis akajei), and buying is from fishery products market, Zhanjiang, Guangdong Province.The structure in stingray caudal spine cDNA library: at first anatomical isolation is extracted total RNA to stingray caudal spine; It is synthetic to carry out first chain, LD PCR cDNA amplification, and enzyme cuts digestion and post reclaims cDNA construction expression library; The order-checking of picking a small amount of library clone obtains the EST of red ray IPL tumor suppressor gene at random.The method of using PCR then from the total plasmid of stingray caudal spine expression library screening and cloning to the IPL full-length gene, according to the tumor suppressor gene IPL3 ' end and the carrier core nucleotide sequence that are obtained, the design primer, pcr amplification obtains the purpose band, the purpose band is connected to the T-easy carrier, and Transformed E .coli selects recombinant clone.
The present invention is by carrying out sequencing to above recombinant clone, and the clone obtains red ray IPL tumor suppressor gene full-length gene, and gives expression to recombinant protein by gene engineering method.New 136 amino acid whose mature peptides of genes encoding, iso-electric point is about 9.68, and molecular weight is about 15,690 dalton, has the feature of typical IPL gene primary structure.PH structural domain primary structure distinctive single and almost constant C-terminal tryptophane (Trp, W) residue have appearred.Intramolecularly contains 3 halfcystines.
The present invention is by a pair of primer design, and the red ray IPL gene of will encoding comes out from the amplification of T-easy carrier with PCR method, is cloned on the prokaryotic fusion expression vector pTRX, is built into expression plasmid and with its transformed into escherichia coli BL21 (DE3) (see figure 3).This expression vector (pTRX-IPL3C) is promotor with T7, adopt molecular chaperones TRX for merging the companion body, can help recombinant protein correctly folding, with soluble formal representation, the C end of TRX has gentle sequence and 6 * His structure, is convenient to utilize immobilization metal part affinity chromatography to carry out purifying.Designed upstream primer contains protease 3 C site, so that the monomeric acquisition of foreign protein.By to incubation time, induction time, the groping and optimize of conditions such as temperature, IPL3C Expression of Fusion Protein amount can reach 60mg/L, is in the solvable state of part in Ecoli..
The present invention also gropes and has optimized the proteic purification condition of reorganization IPL, the ultrasonic degradation liquid of expression product is through Ni2+ Chelating Sepharose affinity chromatography, obtain the higher fusion rotein of purity, fusion rotein can obtain purity at the reorganization of the maturation more than 95% stingray caudal spine tumor suppressor gene IPL albumen through cutting of 3C proteolytic enzyme and further G50 gel filtration chromatography.
The recombinant tumor suppressor gene IPL albumen that the present invention obtains be have bioactive.
The reorganization stingray caudal spine tumor suppressor gene IPL albumen that the present invention obtains has the obvious growth restraining effect to some malignant cell of the mankind of vitro culture.The activity experiment that reorganization stingray caudal spine tumor suppressor gene IPL albumen is carried out, utilize mtt assay to detect of the effect of red ray IPL recombinant protein to culture of tumor cell, find that the red ray IPL albumen of reorganization has lethal effect to MGC803 (cancer of the stomach), RD (rhabdosarcoma), HL60 and Jurkat (leukemia) etc., does not then have effect to two lung carcinoma cells.(seeing FIG) the present invention utilizes molecular biological method, has found 1 new red ray IPL tumor suppressor gene, and adopts the amalgamation and expression system to produce the red ray IPL of the reorganization albumen with vitro inhibition malignant cell growth and breeding.The expression plasmid pETTRX-IPL3C (building process is seen Fig. 3) that contains stingray caudal spine tumor suppressor gene IPL albumen mature polypeptide coding sequence of the present invention has been preserved in Chinese typical culture collection center (CCTCC, China, Wuhan, Luo Jiashan, Wuhan University in the school), preserving number is: M201038, preservation day: October 26 calendar year 2001.
Through Kpn I/Not I double digestion, can obtain the fragment of 448bp by this expression plasmid carrier, be stingray caudal spine tumor suppressor gene IPL albumen mature polypeptide coding sequence (wherein comprising protease 3 C recognition site) (enzyme is cut and be the results are shown in Figure 4)
The clone method of expression plasmid carrier of the present invention:, press CaCl with reference to Sambrook (Sambrook, et al.1989, Molecular cloing.Cold Spring Harbor Labroratory Press.USA) method
2Method transforms plasmid in E.Coli.DH5 α or BL21 (DE3) bacterial strain, with the LB substratum transform bacteria that contains penbritin (100 μ g/mL), alkaline process extracts plasmid.
Description of drawings
Fig. 1 is the total RNA electrophoresis result of stingray caudal spine, wherein: the M.RNA mark; 1.RNA.
Fig. 2 is the PCR electrophoresis result of stingray caudal spine IPL tumor suppressor gene, wherein: 1.PCR purpose band; 2.200bp DNA indicates (Promega).
Fig. 3 is the recombinant plasmid pPETTRX-IPL3C expression plasmid design of graphics that contains red ray IPL gene.
Fig. 4 is the synoptic diagram of expression PH structural domain.
Fig. 5 contains among the BSK-IPL3C of stingray caudal spine IPL tumor suppressor gene interstitial granules and pPETTRX-IPL3C expression plasmid enzyme to cut evaluation figure.Wherein, M:1Kb dna ladder formula; Hurdle 1:pPETTRX-IPL3C vector plasmid KpnI, NotI double digestion product; Hurdle 2:pPETTRX-IPL3C vector plasmid; The KpnI of hurdle 3:BSK-IPL3C vector plasmid, BamHI double digestion product; Hurdle 4:BSK-IPL3C vector plasmid
Fig. 6 is for the protein induced expression of the red ray IPL3C of reorganization, affinity chromatography, enzyme is cut and the sieve chromatography electrophorogram.
Fig. 6 A represents the identical receipts bacterium time, the protein expression of different IP TG induced concentration and solubility, wherein, C: do not add the total thalline of IPTG inductive; M: protein hangs down molecular marker; 0,0.01,0.1,0.25,0.5 and the ultrasonic supernatant of 1mM:IPTG concentration gradient abduction delivering thalline.
Fig. 6 B represents the SDS-PAGE electrophoresis of the red ray rIPL of purifying, wherein, and C: the total thalline of inductive not; M: protein hangs down molecular marker; 1: express total thalline; 2: ultrasonic supernatant; 3:150mM imidazoles peak; 4: enzyme is cut; 5: purifying merges rIPL; 6: the rIPL of purifying.
Fig. 7 red ray rIPL albumen affinity chromatography collection of illustrative plates of representing to recombinate.Wherein, B:B liquid wash-out; C:C liquid wash-out; D:D liquid wash-out; E:E liquid wash-out.
Fig. 8 red ray rIPL protein molecular sieve chromatography collection of illustrative plates of representing to recombinate.A: the 1st peak; B: the 2nd peak; C: the 3rd peak.
Fig. 9 represents to measure the growth-inhibiting effect of the red ray rIPL of reorganization to cultured tumor cells in vitro with mtt assay.
Extraction, library construction and the PCR colony screening of the total RNA of embodiment embodiment 1 stingray caudal spine
The extraction of total RNA and cDNA are synthetic: dissect two stingray caudal spines, adopt guanidine isothiocyanate method to extract the total RNA of tentacle, protein is removed in phenol/chloroform extracting.Obtain the total RNA of 65 μ g anal spines, its A
260/ A
280=1.98, detect as seen 28s and 18s two bands clearly through 1% denaturing formaldehyde gel electrophoresis, ratio＞2, and mRNA smear (see figure 1) show that total RNA integrity is good.It is synthetic to carry out first chain, LD PCR cDNA amplification, and enzyme cuts digestion and post reclaims cDNA, construction cDNA library.
Design of primers and pcr amplification: clone the EST 3 ' end and the carrier core nucleotide sequence of the IPL tumor suppressor gene that the random sequencing result obtained on a small quantity according to the library, design primer, T7 primer: 5 '-TAA TAC GAC TCACTA TA-3 '; IPL primer: 5 '-TCA CTT CAG CCA GGT GTC A-3 '.Per 50 μ l PCR reaction volumes add the total plasmid in 1 μ l library as template, the PGR amplification, and electrophoresis detection is found near the specific amplification band (see figure 2) that appearance is expected 850bp, reclaims this band.The mensuration and the analysis of embodiment 2 reorganization stingray caudal spine IPL tumor suppressor gene sequences
The electrophoresis product that reclaims is connected to the T-easy carrier, transforms DH5 α intestinal bacteria, select the recombinant clone order-checking.Measured 16 clones altogether, Blast homology analysis revealed wherein has 10 to be IPL tumor suppressor gene sequence, and these 10 IPL tumor suppressor gene sequence lengths all are 849bp, and 1 length of encoding is 136 amino acid whose IPL albumen, is numbered Ch123.The nucleotide sequence similarity of it and people and mouse homologue IPL gene reaches 78% and 72%; The protein sequence homology reaches 50% and 48% but incomplete same, and its functional domain PH district is conservative substantially.Being reported first in red ray, is 1 new IPL tumor suppressor gene.
Utilize tool software DNAtools that its base sequence is analyzed, and obtain its maximum reading frame, initiator codon ATG is appearring near 5 ' end place, and before ATG-3 be A, can confirm that the ATG immediately following appearance thereafter is the initiator codon of red ray IPL full length gene sequence; Sequential analysis is also found, the polyA sequence occurred after 3 ' end terminator codon, can also confirm that thus this sequence terminates.So can judge that according to above multianalysis the red ray IPL gene order that this experiment is adopted is a full length sequence.
Can get following comparative result by the Clustalw analysis software in addition: Ch123_--------MRKTADSAQVMKEGVLEKRGDNLLQLWKKKYCVLTQDCLQLY PDSQKRSRSKTIH1---------MTAAATATVLKEGVLEKRSGGLLQLWKRKRCVL TERGLQLFEAKGTGGRPKHom:-----------MKSPDEVLREGELEKRSDSLFQL WKKKRGVLTSDRLSLFPAS-PRARPKMus_ MASKIVMSSKTVKTSDEILCEGELEKRSDSLFQVWKKKRCVLTADRLRLFSG-KTS PAK
. ::?**?****...*:*:**:* *** *?*: .?.?*Ch123_ DLSLQEIRTVDCVERTGKYIYFTVVTTDNKEMDFRCLAEG-SWNAAITMAVIEFKNKKAITIH1 ELSFARIKAVECVESTGRHIYFTLVTEGGGEIDFRCPLEDPGWNAQITLGLVKFKNQQAIHom： ELRFHSILKVDCVERTGKYVYFTIVTTDHKEIDFRCAGES-CWNAAIALALIDFQNRRALMus_ ELFFHSILKVDCVEHTSKYVYFTIVTNYYKEIDFRCTVES-CWNAAITMALIDFQNRRAL
:*?: * *:***?*.:::***:** *:**** *. ***?*::.::.*:*::*:Ch123_ QSFRSRQDAEMLSVGQQEKLLGRAP--------------------TIH1 QTVRARQSLGTGTLVS-----------------------------Hom： QDFRSRQERTAPAAPAEDAVAAAAAAPSEPSEPSRPSPQPKPRTPMus_ QDFPRYRYQRSESE-----------MPSEPGEQSALGP-------
*?. : :
" * " expression is identical; ": " expression difference is less; ". " expression obvious difference; Complete difference is represented in the space
By last analytical results as can be seen, the red ray IPL gene that this experiment is adopted and the homologous gene of other species have higher homology, have the feature of typical IPL gene primary structure.Its functional domain PH district is conservative substantially, PH structural domain primary structure distinctive single and almost constant C-terminal tryptophane (Trp, W) residue have occurred.Through the functional domain prediction, the from the 10th to 105 amino acid of the PH structural domain of red ray IPL gene, Fig. 4 is seen in the position.
In pcr amplification reaction, the mispairing of about 10-4 can appear with common Taq enzyme, because the purpose fragment is little, the cycle number of PCR is no more than 30, reduced the wrong probability that participates in, from experimental result, this sequence repeats in a plurality of cloning and sequencings, but debug participates in, and illustrates that the result of this experiment PCR has higher confidence level.The structure of embodiment 3 reorganization stingray caudal spine IPL tumor suppressor gene expression plasmids
According to the synthetic a pair of primer of two terminal sequences of red ray IPL gene, in upstream primer, introduce Kpn I restriction enzyme site (GGTACC), ATG initiator codon and a Prescission Protease cleavage site, introduce BamH I restriction enzyme site (GGATCC) and terminator codon TTA, CTA in the downstream primer.
Upstream primer (P1): 5 '-GG
GGTACC CTGGAAGTTCTGTTCCAAGGTCCA ATG
Kpn?I Precision Protease site
AGGAAGACAGCGGATAGT-3’
Downstream primer (P2): 5 '-CG
GGATCC TTA CTAGGGAGCCCTTCCCAACA-3 '
BamH?I
With the pGEM-T Easy plasmid that contains red ray IPL gene is template, and P1, P2 are the primer PCR amplification, obtains the single band of specific amplified, and the product size is about 450bp.Pcr amplification product earlier is built into BSK-IPL to be connected on the BSK carrier behind the Kpn I/BamHI double digestion, again with the enzyme cutting clone of Kpn I/Not I to prokaryotic fusion expression vector pPETTRX.Cloning vector and expression vector carry out double digestion with Kpn I/BamH I and Kpn I/Not I respectively and identify (enzyme is cut and be the results are shown in Figure 5).Foreign gene in cloning vector and the expression vector is identified correct through order-checking.Contain Pre-scission proteolytic enzyme cutting site in the designed upstream primer, so that excision fusion molecule companion is TRX.The expression of embodiment 4 reorganization stingray caudal spine tumor suppressor gene IPL fusion proteins
With pETTRX-IPL transformed into escherichia coli BL21 (DE3).Genetic engineering bacterium ultrasonic degradation supernatant liquor shows that through the SDS-PAGE electrophoretic analysis thalline has tangible specifically expressing product band after inducing, and molecular weight conforms to the theoretical value 30.1KD that predicts with software PROTEIN ANALYSIS.
Process is to incubation time, induced concentration, groping (see Fig. 6: abduction delivering compares behind the thalline enlarged culturing different time) of conditions such as temperature, the culture condition of genetic engineering bacterium is: the order bacterium colony is in 50ml ammonia Bian resistance LB substratum, 37 ℃, the 250rpm overnight incubation, get in the ammonia Bian resistance LB substratum that overnight culture 20ml is inoculated in 2L, 37 ℃, 250rpm is cultured to OD600=0.6 (about 2 h of enlarged culturing), add 100mM IPTG and 20% glucose to final concentration and be respectively 1mM and 0.2%, 25 ℃, centrifugal results thalline behind the 250rpm inducing culture 10h.Through SDS-PAGE electrophoretic analysis analysis revealed, stingray caudal spine Trx-IPL Expression of Fusion Protein amount accounts for more than 20% of bacterial protein with this understanding, is in the solvable state (see figure 6) of part.The purifying of embodiment 5 reorganization stingray caudal spine tumor suppressor gene IPL fusion roteins and the acquisition of maturation protein
Total thalline of results is washed with TE (pH8.0), and (50mM pH7.0) suspends, and after the supersound process, the cracking supernatant liquor is through Ni to use Tis again
2+Chelating Sepharose affinity chromatography single step purification is analyzed and the purity of thin layer scanning analysis revealed fusion rotein reaches 80% (Fig. 6 B, Fig. 7, Fig. 8) through SDS-PAGE.With 1L genetic engineering bacterium culture is material, the final fusion rotein that obtains about 60mg purifying.
For yield and the concentration that improves recombinant protein, simplify experimental procedure, shorten treatment time to recombinant protein, with Ni2+ affinity chromatography purification of recombinant proteins the time, we adopt single stage method to carry out purifying.According to the expressed foreign protein of vector plasmid pETTRX and the stronger characteristics of binding ability of Ni2+ affinity column, selected the elution requirement of simplifying for use: 50mMTris, 500mMNaCl, pH7.0 (A liquid); 50mMTris, 500mMNaCl, pH6.0 (B liquid); The 50mM imidazoles, 50mMTris, 500mMNaCl, pH6.0 (C liquid); The 100mM imidazoles, 50mMTris, 500mMNaCl, pH6.0 (D liquid); The 150mM imidazoles, 50mMTris, 500mMNaCl, pH6.0 (E liquid).Reorganization IPL albumen is eluted (Fig. 7) at D liquid and E liquid.Judge the separating effect The cream of the crop from SDS-PAGE result.
Utilize Folin-phenol method to measure reorganization PLA
2Protein concentration, the results elutriant of concentration more than 0.7mg/ml adds Precision Protease and cuts, and detects enzyme with SDS-PAGE and cuts the result, can obviously see the bands of a spectrum (seeing Fig. 6 B) of the ripe rIPL of the representative 16kD near.With reaction solution Ni2+ ChelatingSepharose affinity chromatography column purification (condition is the same), results contain the percolation liquid of ripe rIPL, and be further purified (elutriant is PBS) with Sephadex G-50, just purity in the stingray caudal spine rIPL albumen (see figure 8) of recombinating of the maturation 95% or more.Test 1 mtt assay and measure of the growth-inhibiting effect of the red ray rIPL albumen of reorganization cultured tumor cells in vitro
MTT can combine with the plastosome of viable cell to form and replace by the bluish voilet first moon, so the crystallization content of MTT is directly proportional with viable count.At the RPMI1640 of 10%FBS nutrient solution, 37 ℃, 5%CO
2Culture condition under, conventional MGC803 (cancer of the stomach), RD (rhabdosarcoma), HL60 and the Jurkat cells such as (leukemia) cultivated.The cell in vegetative period of taking the logarithm, the digestion numeration, inoculation equal amts cell is in 96 orifice plates.Added the red ray rIPL of the reorganization protein sample by the concentration gradient preparation in second day, blank is made with PBS in each concentration gradient three multiple hole.After the function cells 48 hours, every hole adds 20ul MTT (5mg/ml), and 37 ℃ of incubations 4 hours are inhaled and abandoned supernatant, and every hole adds the DMSO of 100ul.After shaking up, measure absorbance value at the 570nm place, calculate the IC50 value with ORIGIN5 with enzyme connection instrument, and curve plotting figure.It the results are shown in Table 1 and Fig. 9.
The red ray rIPL albumen of table 1 reorganization is to the variation (mtt assay) of cultured tumor cells in vitro effect OD57 value
Sequence table 1. general informations
(i) applicant: Beijing Boao Huanyu Bioisystech Co., Ltd
(ii) denomination of invention: the clone of stingray tumor suppressor gene, Expression and biological activity
(iii) sequence number: 2 sequence numbers: 1
Sequence length: 849
Sequence type: base pair
Chain number: two strands
Topology: straight chain shape
Sequence kind: DNA
Origin is biological: red ray (Dasytis akajei) strain name: e. coli bl21 (DE3) sequence signature:
The mark of representation feature: correct reading frame is arranged
Decision position: initial, terminator codon
The method of decision feature: experiment
The location: initiator codon is present in the 76-78 position, terminator codon 494-496 position GAGTCAGAGA GCTGCCGCTG TGGGAGAAGT CGGAGAGCCG GTTCTAGCTT CTTTACGGCA 60AGGCTTCAAC TTAAAAGTTC TT
AG
ATGAGG AAG ACA GCG GAT AGT GCC CAG GTG ATG 118
Met?Arg?Lys?Thr?Ala?Asp?Ser?Ala?Gln?Val?MetAAG?GAA?GGC?GTG?CTG?GAG?AAG?AGG?GGC?GAT?AAC?CTG?CTC?CAG?CTC?TGG?AAG?169Lys?Glu?Gly?Val?Leu?Glu?Lys?Arg?Gly?Asp?Asn?Leu?Leu?Gln?Leu?Trp?LysAAG?AAG?TAC?TGC?GTG?CTG?ACC?CAA?GAC?TGC?CTC?CAG?CTC?TAC?CCG?GAC?TCC?220Lys?Lys?Tyr?Cys?Val?Leu?Thr?Gln?Asp?Cys?Leu?Gln?Leu?Tyr?Pro?Asp?SerCAG?AAG?CGG?TCC?CGG?AGC?AAG?GAC?CTG?AGT?CTG?CAG?GAG?ATC?CGG?ACG?GTG?271Gln?Lys?Arg?Ser?Arg?Ser?Lys?Asp?Leu?Ser?Leu?Gln?Glu?Ile?Arg?Thr?ValGAC?TGC?GTG?GAA?AGG?ACG?GGC?AAA?TAC?ATC?TAC?TTC?ACC?GTG?GTC?ACC?ACC?322Asp?Cys?Val?Glu?Arg?Thr?Gly?Lys?Tyr?Ile?Tyr?Phe?Thr?Val?Val?Thr?ThrGAC?AAC?AAA?GAG?ATG?GAC?TTC?AGG?TGC?CTG?GCG?GAA?GGC?AGC?TGG?AAC?GCG?373Asp?Asn?Lys?Glu?Met?Asp?Phe?Arg?Cys?Leu?Ala?Glu?Gly?Ser?Trp?Asn?AlaGCC?ATC?ACC?ATG?GCT?GTC?ATT?GAG?TTC?AAG?AAC?AAG?AAA?GCC?ATC?CAG?AGC?424Ala?Ile?Thr?Met?Ala?Val?Ile?Glu?Phe?Lys?Asn?Lys?Lys?Ala?Ile?Gln?SerTTC?CGA?TCG?CGA?CAG?GAC?GCA?GAG?ATG?CTC?AGT?GTG?GGC?CAG?CAA?GAG?AAG?475Phe?Arg?Ser?Arg?Gln?Asp?Ala?Glu?Met?Leu?Ser?Val?Gly?Gln?Gln?Glu?LysCTG?TTG?GGA?AGG?GCT?CCC
TAGGCGT?ACTCCACAGG?TTGGTCATGT?TGATCGTGGA 530Leu?Leu?Gly?Arg?Ala?Pro
*AGAAACATCA GTAAGTGAAC AGTGGATGAA CACACCCAGG AGATTGAGAG CCATTTGAAG 590TGACTTCAGT TTCGTCCTCT GCTGCTGCTG TCAGAAGGAC CCAAGCTGCT GTAATCAGCC 650CCAGACATTC AGGACCTGAC ACCTGGCTGA AGTGAGCCTG TGGGGTTGAC TGGAAATCTG 710CTTTATTTAT TTACCTGTTG CAGAATCCAA GAACATTTAT NTNGNGTGGT CATCTTATAA 770ATTATTTNGT AATCTAAATT TNGCATATTT ATTAAAAGTT TTTAATATAA AAATGACCCC 830C
AAAAAAAAA AAAAAAAAA
Claims (11)
1. the DNA of a nucleotide sequence shown in serial number in the sequence table 1.
2. the protein of an aminoacid sequence shown in serial number in the sequence table 1.
3. recombinant vectors, this recombinant vectors comprises nucleotide sequence as claimed in claim 1.
4. protokaryon bacterium that contains recombinant vectors as claimed in claim 3.
5. protokaryon bacterium as claimed in claim 4 is characterized in that described protokaryon bacterium is intestinal bacteria.
6. recombinant vectors as claimed in claim 3 is characterized in that described carrier is serves as to merge the companion body with sulphur oxygen cyclase protein, the middle affinity labelling site of 6 His and the efficient expression vector pETTRX-IPL of protease 3 C recognition site of inserting.
7. as carrier as described in the claim 3, it is characterized in that this carrier make described DNA in intestinal bacteria with soluble formal representation in the born of the same parents, its expression amount is 40-60mg/L.
8. as recombinant vectors as described in the claim 3, it is characterized in that expression product Supersonic lysate process Ni-Chelating affinity column chromatography by this recombinant vectors, protease 3 C cutting and gel-filtration can obtain the maturation protein of purity in the stable in properties more than 95%, and yield is 5-8mg/L.
9. dna fragmentation as primer, its a part of sequence by the described nucleotide sequence of claim 1 is formed.
One kind contain just like the described proteic prevention of claim 2 or the treatment tumor disease pharmaceutical preparation.
11. the application of albumen as claimed in claim 2 in the medicine of preparation prevention and treatment tumor disease.
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| CNB011369396A CN100509836C (en) | 2001-12-25 | 2001-12-25 | Clone, expression and biological activity of stingray tumor suppressor gene |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103204904A (en) * | 2013-02-01 | 2013-07-17 | 浙江海洋学院 | Dasyatis akajei chondroprotein polypeptide capable of resisting prostate cancer, and preparation method and application thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103204904A (en) * | 2013-02-01 | 2013-07-17 | 浙江海洋学院 | Dasyatis akajei chondroprotein polypeptide capable of resisting prostate cancer, and preparation method and application thereof |
| CN103204904B (en) * | 2013-02-01 | 2015-07-22 | 浙江海洋学院 | Dasyatis akajei chondroprotein polypeptide capable of resisting prostate cancer, and preparation method and application thereof |
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