CN1314807C - Gene clone and expression of RGD die body protein of oral gland in Japan lamprey possessing function for anti tumour - Google Patents
Gene clone and expression of RGD die body protein of oral gland in Japan lamprey possessing function for anti tumour Download PDFInfo
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- CN1314807C CN1314807C CNB2005100834377A CN200510083437A CN1314807C CN 1314807 C CN1314807 C CN 1314807C CN B2005100834377 A CNB2005100834377 A CN B2005100834377A CN 200510083437 A CN200510083437 A CN 200510083437A CN 1314807 C CN1314807 C CN 1314807C
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Abstract
The present invention relates to a gene clone and expression of a Japanese lamprey oral gland RGD model protein with an antitumor effect, which belongs to the technical field of biology. The present invention relates to a cDNA sequence and a clone with three RGD model proteins in the oral gland of Japanese lamprey, a protein sequence, the expression of the protein sequence in engineering bacteria, and the function of the three RGD proteins for closing a signal transduction pathway of integrin dielectric by resisting the functions of high-expression integrin on the surface of neogenesis endothelial cells of blood vessels and tumour cells. Consequently, the present invention inhibits the neogenesis of blood vessels, and the proliferation, the adhesion, the infiltration and the transference of tumor cells. Moreover, the present invention causes the apoptosis of the endothelial cells of blood vessels and tumor cells so as to achieve the function of resisting tumors.
Description
Technical field
The proteic gene cloning and expression of Japanese lamprey oral gland RGD die body belongs to biological technical field, relate in the Japanese lamprey oral gland three proteic cDNA sequences of tool RGD die body and clone, Dui Ying protein sequence and the expression in intestinal bacteria or pichia spp thereof with it, and these three kinds of recombinant proteins are by some integration element of antagonism new vessel endotheliocyte and tumor cell surface high expression level and combining of extracellular matrix, the signal transduction pathway of sealing mediated by integrin, thereby propagation with angiogenesis inhibiting and inhibition tumour cell, adhere to, soak into and transfer, and cause the apoptosis of vascular endothelial cell and tumour cell, and reach antineoplastic effect, can in preparation angiogenesis inhibitor series antineoplastic medicament, use.
Background technology
Jamprey-ell (Lampetra japonica) belongs to Cyclostomata (Cyclostomata), Peteromyzoni-form, Petromyzonidae, Lampetra, is the most ancient no jaw vertebrates of finding so far.It is the oceanodromous migration fish, and adult is lived in the ocean, is attached on other fish body through sucker commonly used and sucks its blood and meat, and The blood streamed down to make the fish that is adsorbed, and this is indicating in its oral gland composition should contain RGD die body toxin.
Malignant tumour is one of principal disease of current harm humans health, is a kind of common disease, frequently-occurring disease.According to World Health Organization's annual report 1998, the de novo malignant tumor patient in the whole world in 1997 is about 1,000 ten thousand, dies from the about 623.5 ten thousand of malignant tumour, accounts for 12% of whole death tolls.Conventional methods of treatment has operation, radiotherapy, chemotherapy, and certain limitation and side effect are all arranged, and has influenced the raising of curative effect.In the continuous progressive of science and technology today, it is the new way of conquering cancer 21 century that the theory and practice of controlling cancer has all had new breakthrough----" tumor therapy hungry to death ".Broad research is carried out over against numerous angiogenesis inhibitors in countries in the world, present many angiogenesis inhibitors are carrying out I～III clinical trial phase research, kinds more than 20 such as angiostatin, Endostatin, fumidil analogue and metalloprotein enzyme inhibition factor and urokinase, il-1 2, platelet factor, Bufotanine for example, and curative effect more definitely surely belongs to Bufotanine, partly the patient is with can be effective in cure in 7 days, and do not have the side effect of appearance.This therapy can make apoptosis of tumor cells speed up, downright bad and degenerate to initial dormant state, and then suppress tumour cell to shift and the treatment cancer.
RGD die body albumen has become the main force of antineoplastic vascular formation inhibitor class medicine, this be because: the RGD sequence can specificity antagonism new vessel endotheliocyte and some of tumor cell surface high expression level integrate combining of plain and extracellular matrix, the signal transduction pathway of sealing mediated by integrin, thereby have angiogenesis inhibiting and suppress propagation, adhesion, infiltration and the transfer of tumour cell, and cause the apoptosis of vascular endothelial cell and tumour cell and reach antineoplastic effect.Found to have the RGD die body toxin family in three different plant species sources up to now, these three extended familys derive from snake venom, bloodsucker leech and lice class salivary gland secretion (R.Manjunatha kini et al, 2001) respectively.The RGD die body toxin that derives from snake venom also calls to integrate plain, is that to be studied the most also be maximum gang, and kind can reach tens of kinds according to snake kind source difference; The RGD die body toxin that derives from leech sialisterium is also referred to as r-hirudin, only finds two class decorsin (Krezel AM et al, 2000) and ornatin (PMazur et al, 1991) so far; The RGD die body toxin that contains that derives from the lice class is called variabilin (Wang X et al., 1996). by the disintegrin Primary Structure Analysis as can be seen, come from homology higher (more than 60%) between the disintegrin of these species of snake venom, and the RGD die body toxin between species does not have homology on primary structure except that having RGD die body and many general character to halfcystine.The present invention has found to be present in the new gene of RGD die body albumen in the Japanese lamprey oral gland, and this gene does not still have research and report at present at home and abroad, the invention belongs to first and finds.And the Japanese lamprey oral gland RGD die body albumen of this project has typical angiogenesis inhibiting, causes the effect of apoptosis of tumor cells, can use in preparation angiogenesis inhibitor series antineoplastic medicament.
Summary of the invention
The present invention has found three open reading frame that can translate RGD (Arg-Gly-Asp) die body in Japanese lamprey oral gland cDNA library, through angling of its mRNA total length got, order-checking and cDNA clone, its recombinant protein has been carried out efficient abduction delivering in intestinal bacteria.The biologic activity of these three recombinant proteins relates to by some integration element of antagonism new vessel endotheliocyte and tumor cell surface high expression level and combining of extracellular matrix, the signal transduction pathway of sealing mediated by integrin, thereby have angiogenesis inhibiting and suppress propagation, adhesion, infiltration and the transfer of tumour cell, and cause the apoptosis of vascular endothelial cell and tumour cell and reach antineoplastic effect.
The present invention relates in the Japanese lamprey oral gland three proteic cDNA sequences of tool RGD die body and clone, corresponding protein sequence and the expression in intestinal bacteria or pichia spp thereof with it, and propagation, adhesion, infiltration and the transfer of angiogenesis inhibiting and inhibition tumour cell, and cause the biologic activity of the apoptosis of vascular endothelial cell and tumour cell.
The present invention is separation and Extraction mRNA from Japanese lamprey oral gland, biological information that obtains the cDNA library that utilization made up from early stage, the EST library and mRNA total length angle the sequencing result of getting to choose open reading frame design primer, adopt the RT-PCR method to obtain three proteic cDNA of RGD and sequence thereof, the present invention also provides the albumen and the sequence thereof of three successful expression in e. coli bl21, these three antineoplastic biologic activity of RGD die body recombinant protein tool can be used in preparation angiogenesis inhibitor series antineoplastic medicament.
Article three, RGD die body albumen is by difference called after Lampetrin1, Lampetrin2 and Lampetrin3.Its cDNA sequence and as follows by the protein amino acid sequence of its derivation:
1.Lampetrin1 cDNA sequence 417bp long, its protein is made up of 139 amino acid, contains a RGD die body, molecular weight of albumen is 14,178Da.The protein amino acid sequence that its cDNA sequence reaches by its derivation is:
1?atgagcagcaacgagtgtttccgatgcggaggctctggccactgg
M S S N E C F R C G G S G H W
46?gcccgcgagtgccccaatggagccggtggtggccgggggcccggc
A R E C P N G A G G G R G P G
91?ggccctgtcggacgtggaggccgcggccgaggagatggatgctat
G P V G R G G R G
R G D G C Y
136?cgctgcggcgaaggcggccatatagcgcgcgaatgcccgctgccc
R C G E G G H I A R E C P L P
181?caagacagtgtctcttcaaacaccgcagcatgctacaactgtggc
Q D S V S S N T A A C Y N C G
226?aaggggggccacatcgcgcgcgagtgccccgagggccggcaggac
K G G H I A R E C P E G R Q D
271?cgcggcggcggacccagctgctacacgtgtggcaagcagggccat
R G G G P S C Y T C G K Q G H
316?ctggcccgtgagtgcagcagtggcggcggtggacccggggacaac
L A R E C S S G G G G P G D N
361?aagtgctacgggtgcggccagcgcggccacatgcagcgcgactgc
K C Y G C G Q R G H M Q R D C
406?accaaggccagcagatag?423
T K A S R *
2.Lampetrin2 cDNA sequence 204bp long, its protein is made up of 68 amino acid, contains two RGD die bodys, molecular weight of albumen is 7,548Da.The protein amino acid sequence that its cDNA sequence reaches by its derivation is:
1?atgccatttgtcataagcagaattatcccatgggtacggagacac
M P F V I S R I I P W V R R H
46?agggagacacacgtggagacacacggacacacacggagacacaag
R E T H V E T H G H T R R H K
91?ctgaggcacggacacacgcagagacgcacggagacacacgtggag
L R H G H T Q R R T E T H V E
136?acacacggagacacacgtggagacacacgcggagacacacggaca
T H G D T
R G D T
R G D T R T
181?cacacggacacacacggagacacaagctga?210
H T D T H G D T S *
3.Lampetrin3 cDNA sequence 351bp long, its protein is made up of 117 amino acid, contains three RGD die bodys, molecular weight of albumen is 11, the protein amino acid sequence that its cDNA sequence of 934Da. reaches by its derivation is:
1?atgtcaacgttcatcaacggaacccaggaagtggatgccatttgt
M S T F I N G T Q E V D A I C
46?cataagcagaattatcccatgggtacggagacacagggagacaca
H K Q N Y P M G T E T Q G D T
91?cgtggagacacacggacacacacggagacacaagctgaggcacgg
R G D T R T H T E T Q A E A R
136?acacacgcagagacgcacggagacacacgtggagacacacggaga
T H A E T H G D T
R G D T R R
181?cacacgtggagacacacgcggagacacacggacacacacggacac
H T W R H T R R H T D T H G H
226?acacggagacacaagctgaggcacgaacacacgcagagacacacg
T R R H K L R H E H T Q R H T
271?ggggcccgcggagacgcacggagacacggacacaacaaacattta
G A
R G D A R R H G H N K H L
316?cacagaatgagtgcagcggtgagtgaatgtgttggggagtga?357
H R M S A A V S E C V G E *
Article three, the biologic activity of the angiogenesis inhibiting of RGD die body albumen Lampetrin is as follows:
The vasculogenesis experiment adopts chicken chorioallantoic membrane (CAM) model to carry out in the body.Behind the newborn 24h of bFGF (200ng/embryo) induction of vascular, act on the chicken embryo with the Lampetrin of the gradient concentration of 20 μ l or the PBS of equivalent.The result shows that the major branch blood vessel of the CAM that handled with PBS after bFGF induces is clear and flourishing, and capillary network forms gradually; And Lampetrin1, obviously fracture takes place in 2, the 3 CAM major branch blood vessels of handling, and the blood vessel number obviously reduces.Along with the increasing of Lampetrin dosage, even vessel wall also occurs and dissolve the phenomenon of only remaining tube wall groove fully.This explanation Lampetrin has the effect of angiogenesis inhibiting.
Description of drawings
Fig. 1 is experiment flow figure of the present invention;
Fig. 2 is the restraining effect (digital camera take, 5 times amplifications) of Lampetrin to chicken chorioallantoic membrane (CAM) angiogenesis. (A) bFGF contrast; (B) Lampetrin1 of 1 μ mol/L is to the restraining effect of CAM angiogenesis; (C) Lampetrin2 of 1 μ mol/L is to the restraining effect of CAM angiogenesis; (D) Lampetrin3 of 1 μ mol/L is to the restraining effect of CAM angiogenesis.
Embodiment
1. the extraction of total RNA: the Trizol reagent that adopts GIBCOBRL.Specific as follows:
(1) getting Japanese lamprey oral gland places liquid nitrogen to preserve rapidly;
(2) take by weighing 0.2g oral gland tissue, add the homogenate of 1ml TRIzol reagent preparation poison gland, hatch 5min for 4 ℃;
(3) add the 0.2ml chloroform, the tight lid of lid back is jolting 15sec firmly, places 5min on ice then;
(4) 4 ℃, 12000xg, centrifugal 15min;
(5) upper water is moved into another centrifuge tube mutually, add the 0.5ml Virahol, and hatch 10min on ice;
(6) 4 ℃, 12000xg, centrifugal 15min;
(7) abandon supernatant, in precipitation (containing RNA), add 1ml 75% washing with alcohol, the vortex mixing;
(8) 4 ℃, the centrifugal 5min of 10000xg obtains the RNA precipitation;
(9) after the dry air, with an amount of TE or not have the Rnase water dissolution standby.
2. the primer with design voluntarily carries out the RT-PCR amplification, to obtain the proteic cDNA of Japanese lamprey oral gland RGD die body;
Carry out reverse transcription reaction by following condition: 42 ℃ of 20min, 99 ℃ of 5min, 5 ℃ of 5min
Primer sequence is as follows:
P1-1：5’-XXcatatgagcagcaacgagtgtttccga-3’
P1-2：5’-XXaagctttctgctggccttggtgcagtc-3’
P2-1：5’-XXcatatgccatttgtcataagcagaatt-3’
P2-2：5’-XXaagctt?gcttgtgtctccgtgtgtgtcc-3’
P3-1：5’-XXcatatgtcaacgttcatcaacggaacc-3’
P3-2：5’-XXaagcttctccccaacacattcactcac-3’
3. the purpose cDNA that obtains is connected with pET23b (Novagen company product) carrier, is transformed into the Screening and Identification of carrying out positive transformant behind the clone bacterium DH5a;
For generation has histidine-tagged fusion rotein, select pET23b as gene cloning carrier, clone's concrete operations are as follows:
(1) recovery of goal gene and order-checking: the recovery of goal gene adopts TaKaRa PCR Fragment Recovery Kit to carry out; Check order to reclaiming DNA, this work is finished by the precious biotechnology in Dalian company limited.
(2) extraction of plasmid: adopt precious biological plasmid extraction kit to carry out.
(3) being connected of goal gene dna fragmentation and carrier pET23b: because designed primer has Nde I and Hind III restriction enzyme site respectively, and these two multiple clone site that restriction enzyme site also is pET23b, this makes the possibility that is connected to become of goal gene dna fragmentation and carrier pET23b.
(4) will connect product C aCl2 method is converted among the clone bacterium E.coli BL21.
(5) Screening and Identification of positive transformant: utilize T7 universal primer method and double digestion method to carry out the Screening and Identification of positive transformant.
4. positive recombinant is carried out the IPTG abduction delivering that final concentration is 1mmol/L.The abduction delivering condition is 30 ℃ and spends the night and induce.
5. the recombinant protein of expressing is carried out the Histidine affinitive layer purification.
1) the centrifugal 10min results of 10000g thalline is abandoned supernatant, and raffinate is flowed out as far as possible.The ratio re-suspended cell that adds the ice-cold 1X Binding buffer of 4ml with the original fluid of every 100ml.
2) above-mentioned sample is placed ultrasonic degradation cell on ice, until solution thickness no longer.
3) the centrifugal 20min of 14000g is to remove cell debris.
4) supernatant is with the membrane filtration of 0.45 μ m.
5) the storage liquid of chamber on the His.Bind Column is removed in suction, and opens the following mouth of pipe;
6) the 1x Binding Buffer with 10ml carries out balance to pillar;
7) will filter sample on the good supernatant liquor;
8) the 1x Binding Buffer with 10ml washes post;
9) the 1x Wash Buffer with 10ml washes post;
10) the 1x Elute Buffer wash-out target protein of usefulness 5ml.
6. utilize chicken chorioallantoic membrane CAM system and TUBE FORMATION experiment carrying out anti-angiogenic rebirth functional examination.
Concrete experimental procedure is as follows:
(1) gets instar chicken embryo on the six, bromogeramine sterilization with 0.1%;
(2), and saw out the fenestella of 1cm2 in the chorioallantoic membrane body surface projection location of windowing apart from embryo head 1cm place;
(3) aseptic glass fiber filter paper places on the CAM with the bFGF (200ng/disk) of 20 μ l or equivalent PBS is full closes, and scotch tape covers;
(4) in the water isolation type constant temperature incubator of 37 ℃ of 60% humidity, hatch 24h after, the PBS that adds the Lampetrin of gradient concentration or equivalent is on the filter paper that overlays on the CAM, scotch tape covers;
(5) continue to hatch behind the 24h to observe and take pictures with digital camera.
Claims (8)
1. RGD die body albumen Lampetrin1 who derives from Japanese lamprey oral gland, it is characterized in that: be made up of 139 amino acid, contain a RGD die body, molecular weight of albumen is 14,178Da, its proteinic aminoacid sequence be,
M S S N E C F R C G G S G H W
A R E C P N G A G G G R G P G
G P V G R G G R G?
R G D?G C Y
R C G E G G H I A R E C P L P
Q D S V S S N T A A C Y N C G
K G G H I A R E C P E G R Q D
R G G G P S C Y T C G K Q G H
L A R E C S S G G G G P G D N
K C Y G C G Q R G H M Q R D C
T K A S R
2. the proteic gene of Japanese lamprey oral gland RGD die body according to claim 1 of encoding, its gene order be,
1?atgagcagcaacgagtgtttccgatgcggaggctctggccactgg
46?gcccgcgagtgccccaatggagccggtggtggccgggggcccggc
91?ggccctgtcggacgtggaggccgcggccgaggagatggatgctat
136?cgctgcggcgaaggcggccatatagcgcgcgaatgcccgctgccc
181?caagacagtgtctcttcaaacaccgcagcatgctacaactgtggc
226?aaggggggccacatcgcgcgcgagtgccccgagggccggcaggac
271?cgcggcggcggacccagctgctacacgtgtggcaagcagggccat
316?ctggcccgtgagtgcagcagtggcggcggtggacccggggacaac
361?aagtgctacgggtgcggccagcgcggccacatgcagcgcgactgc
406?accaaggccagcagatag?423
3. recombinant plasmid is characterized in that it contains the gene of claim 2.
4. recombinant plasmid according to claim 3 is characterized in that described gene is connected on the pET23b carrier.
5. a genetic engineering bacterium is characterized in that its quilt is according to claim 3 or 4 described recombinant plasmid transformed.
6. genetic engineering bacterium according to claim 5 is characterized in that its host cell is E.coli BL21.
7. Japanese lamprey oral gland RGD die body albumen according to claim 1 is used for suppressing the application of the medicine that neovascularity generates in preparation.
8. application according to claim 7, wherein said to be used to suppress the medicine that neovascularity generates be anti-tumor drug.
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Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1995345B (en) * | 2006-08-31 | 2010-12-29 | 辽宁师范大学 | Recombinant L-251 protein with anti-inflammatory effect secreted by Japanese lamprey oral gland |
| CN101195656B (en) * | 2006-12-08 | 2012-05-09 | 辽宁师范大学 | Recombined Japanese lamprey glandulae oris excreted L-250 protein anticoagulant efficacy |
| CN102732524B (en) * | 2011-04-11 | 2014-02-26 | 辽宁师范大学 | Use of histidine-rich glycoprotein (HRG)-like lampetra japonica Lj-RGD3 all RGD deletion mutant Lj-112 in antitumor drug |
| CN103173453A (en) * | 2011-12-23 | 2013-06-26 | 辽宁师范大学 | Application of lamprey Lj-RGD2 in preparation of anti-angiogenesis antitumour drug |
| CN103520704B (en) * | 2013-09-10 | 2015-06-17 | 辽宁师范大学 | Application of lampetra japonica genetic recombinant protein rLj-RGD3 in preparation of cerebral ischemia reperfusion protective medicament |
| CN103536902B (en) * | 2013-10-17 | 2015-04-22 | 辽宁师范大学 | Application of recombinant protein of Lj-RGD3 full-RGD motif-deleted mutant Lj-112 in preparing antifungal drug |
| CN103694330B (en) * | 2013-11-25 | 2016-01-06 | 辽宁师范大学 | Go the preparation method of the recombinant lamprey Lj-RGD3 albumen of affinitive layer purification label |
| CN107320713A (en) * | 2016-04-28 | 2017-11-07 | 辽宁师范大学 | Applications of the recombinant protein rLj-RGD4 in antithrombotic reagent is prepared |
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2005
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Non-Patent Citations (1)
| Title |
|---|
| 日本七鳃鳗(Lampetrs japonica)唾液腺cDNA文库的构建 李旭霞等，辽宁师范大学学报(自然科学版)，第27卷第1期 2004 * |
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