CN104288753B - Lampetra japonica (Martens). CRBGP application in preparing anti-angiogenic drugs - Google Patents
Lampetra japonica (Martens). CRBGP application in preparing anti-angiogenic drugs Download PDFInfo
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- CN104288753B CN104288753B CN201410528552.XA CN201410528552A CN104288753B CN 104288753 B CN104288753 B CN 104288753B CN 201410528552 A CN201410528552 A CN 201410528552A CN 104288753 B CN104288753 B CN 104288753B
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- martens
- crbgp
- lampetra japonica
- aac
- gtg
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Abstract
The present invention discloses a kind of Lampetra japonica (Martens). CRBGP application in preparing anti-angiogenic drugs.Present invention finds that Lampetra japonica (Martens). is natural and recombinant C RBGP can suppress the propagation of the human umbilical vein endothelial cell (Human Umbilical Vein Endothelial Cells, HUVEC) induced by bFGF, and in dose dependent, its IC50It is 4.8 μm ol/L；Find six age in days chick chorioallantoic membranes (the chicken chorioallantoic membrane that Lampetra japonica (Martens). is natural and recombinant C RBGP is to being induced by bFGF simultaneously, CAM) angiogenesis is inhibited, can be applicable to prepare low toxicity, efficient anti-angiogenic drugs.
Description
Technical field
The present invention relates to the purposes of a kind of Lampetra japonica (Martens). CRBGP, especially a kind of Lampetra japonica (Martens). CRBGP is preparing anti-angiogenic rebirth
Application in medicine.
Background technology
Angiogenesis refers to develop the blood capillary made new advances from already present blood vessel, (all in some normal physiological process
As fetal development, wound healing, corpus luteum are formed) play an important role.But, unexpected angiogenesis also results in multiple disease
Sick occurring, the retinopathy that causes such as chronic inflammatory disease, diabetes, choroidal neovascularization, psoriasis, myeloproliferative disorder are comprehensive
Disease, rheumatoid arthritis and tumor etc., the life and health of the serious harm mankind and quality of life.Therefore, exploitation low toxicity, height
The anti-angiogenic drugs of effect is the focus for the treatment of at present and inflammation relevant blood vessel regenerative anomaly disease.
It is that a class exists rich in cysteine secretory protein (Cysteine rich secretory proteins, CRISP)
The protein of high conservative in evolution, containing the cysteine residues of 16 high conservatives, can form 8 to disulfide bond.Space is tied
Structure is mainly by the PR-1 domain (pathogenesis-related group 1 domain) of N end, the CRD domain of C end
The hinge area three part composition of (cysteine-rich domain) and connection the two domain.Recent study is sent out
Existing, each member of CRISP family in mammal is distributed mainly in reproductive tract, at maturation, the sperm-egg fusion of sperm and exempt from
Epidemic disease system plays very important effect；CRISP family member in nonmammalian is then distributed mainly on body of gland
In juice, it is possible to block K+、Ca2+And cyclic nucleotide gate passage etc., and closely related with inflammatory reaction.
Lampetra japonica (Martens). CRBGP be from jamprey-ell (Lampetra japonica) oral glands isolated and purified go out one
Novel rich in cysteine secretory protein, because there is high homology with CRISP family member, there are 16 high conservatives equally
Cysteine residues, therefore by its named oral glands secretory protein (cysteine-rich rich in cysteine
Buccal gland protein, CRBGP).
The cDNA sequence (open reading frame) of Lampetra japonica (Martens). CRBGP contains 774bp, and its protein has 257 aminoacid groups
Becoming, mass spectroscopy molecular weight is 25.6 kDa, and its cDNA sequence and the protein amino acid sequence derived by it are as follows:
Atg ttc act aac ctc gtg acc ccg gcg gcg ttg gcg ttg gtg ttc 45
M F T N L V T P A A L A L V F
1 5 10 15
Atg gcg agc gtc gtg gcg gcg aca tcc gtc aac gac tgg aag ctc 90
M A S V V A A T S V N D W K L
20 25 30
Ctg gac acg aag ctg tcg gcg aac cgg aag gtc atc gtg gac gtt 135
L D T K L S A N R K V I V D V
35 40 45
Cac aac gag ctg cgg cgc ggc gtg gtg ccc acc gcc agc aac atg 180
H N E L R R G V V P T A S N M
50 55 60
Ctc aag atg gcg tac aac gaa cag gca gca gag acc agc cgc ttg 225
L K M A Y N E Q A A E T S R L
65 70 75
Tgg gcc gcc gcc tgc agc ttc tcg cac agc ccc agc aac acg cgc 270
W A A A C S F S H S P S N T R
80 85 90
Acc tgg aag acg ccg caa gca gag tgg gac tgc gga gag aac ctc 315
T W K T P Q A E W D C G E N L
95 100 105
Ttc atg tcc agc aac cca cgg tcg tgg gac gag gca gtg cgc agc 360
F M S S N P R S W D E A V R S
110 115 120
Tgg tac gac gag gtc acc tcc ccc ggc ttc cag tac ggc acg ggg 405
W Y D E V T S P G F Q Y G T G
125 130 135
Gct gtg ggg ccc ggg gcc gtg gga cac tac act cag gtg gtg tgg 450
A V G P G A V G H Y T Q V V W
140 145 150
Tac aag tcc cac cag gtg ggc tgc gcc gtc aac tac tgc ccc aac 495
Y K S H Q V G C A V N Y C P N
155 160 165
Cac ccc ggc gcc ctc aag ttc ctc tac gtg tgc cac tac tgc ccc 540
H P G A L K F L Y V C H Y C P
170 175 180
Gca ggg aac ctg gtc acc agg atc aac aaa ccc tac gac ctg ggg 585
A G N L V T R I N K P Y D L G
185 190 195
Act ccg tgc cag gcc tgc ccc cat agc tgc gac aac aac ctg tgc 630
T P C Q A C P H S C D N N L C
200 205 210
Acc aac ccg tgc ccc tac gtg gac cag ttc agc aac tgc ccg cag 675
T N P C P Y V D Q F S N C P Q
215 220 225
Ctg ttc agc gcc cac ggc tgc gcg aac gac ggc gcg ggg gga acc 720
L F S A H G C A N D G A G G T
230 235 240
Ttc gtg cag aca aac tgc cct gcc acg tgc agc tgc acg acg gat 765
F V Q T N C P A T C S C T T D
245 250 255
Gtg cag tga 774
V Q *
257
At present, it has been found that the natural CRBGP of Lampetra japonica (Martens). is Na+Channel blocker, it is possible to block hippocampal neuron and Dorsal root
The Na of neuron+Electric current, reduces hippocampal neuron and the action potential of Dorsal ganglion unit thus the irritability of inhibitory neuron；This
The outer also discovery Lampetra japonica (Martens). CRBGP K to hippocampal neuron+Passage also has blocking effect.Up to now, there is not yet relevant rich in
The report of cysteine secretory protein angiogenesis inhibiting function.
Summary of the invention
Present invention finds the anti-angiogenic rebirth function of Lampetra japonica (Martens). CRBGP, thus it is anti-in preparation to have invented Lampetra japonica (Martens). CRBGP
Application in angiogenesis medicament.
The technical solution of the present invention is a kind of Lampetra japonica (Martens). CRBGP application in preparing anti-angiogenic drugs.
Present invention finds that Lampetra japonica (Martens). is natural and recombinant C RBGP can suppress the human umbilical vein endothelial that induced by bFGF
The propagation of cell (Human Umbilical Vein Endothelial Cells, HUVEC), and in dose dependent, its IC50
It is 4.8 μm ol/L；Find the six age in days chick chorioallantoic membranes that Lampetra japonica (Martens). is natural and recombinant C RBGP is to being induced by bFGF simultaneously
(chicken chorioallantoic membrane, CAM) angiogenesis is inhibited, can be applicable to prepare low toxicity,
Efficient anti-angiogenic drugs.
Accompanying drawing explanation
The inhibitory action design sketch of Fig. 1 Lampetra japonica (Martens). CRBGP HUVEC cell proliferation to being induced by bFGF.
The inhibitory action effect of Fig. 2 Lampetra japonica (Martens). CRBGP six age in days chick chorioallantoic membrane angiogenesiss to being induced by bFGF
Fruit figure.
Detailed description of the invention
The present invention is by sieve chromatography technology (Sephadex G75) isolated electricity from Japanese lamprey oral gland
The natural CRBGP of Lampetra japonica (Martens). of swimming purity.
Utilize gene cloning approach Lampetra japonica (Martens). CRBGP gene is connected to pEGX-4T-1 expression vector (or other forms carry
Body) in E.coli Rosetta, carry out abduction delivering, and use GST affinity chromatograph method purification of Recombinant Lampetra japonica (Martens). CRBGP,
12% polyacrylamide gel electrophoresis and Mass Spectrometer Method destination protein matter, its purity and accuracy are consistent with target protein.
Experiment:
1.MTT method measures the Lampetra japonica (Martens). CRBGP inhibitory action to HUVEC cell proliferation.
(1) HUVEC cell in good condition is inoculated in 96 orifice plates, and containing the final concentration of 3ng/mL of bFGF()
RPMI RPMI-1640 is cultivated 24 hours；
(2) experimental group is sequentially added into the natural CRBGP of Lampetra japonica (Martens). of variable concentrations gradient (5 μ L, 10 μ L, 15 μ L, 20 μ L), right
Add isopyknic PBS according to group, continue to cultivate 24 hours；
(3) the MTT solution 10 μ L of final concentration of 0.5 mg/mL, in 37 DEG C, CO are added2Incubator continues cultivate 4 little
Time；
(4) suck culture fluid gently, and in 96 orifice plates, add DMSO(100 μ L), shaken cultivation in the shaking table of 37 DEG C
10 minutes；
(5) microplate reader is utilized to measure matched group and experimental group cell absorption value at 492nm；
(6) often group experiment is 3 experiments and averages；
(7) the killing rate of cell=(meansigma methods of the meansigma methods of matched group absorbance-experimental group absorbance)/matched group is inhaled
Meansigma methods × 100% of luminosity.
Experimental result is as shown in Figure 1: show that Lampetra japonica (Martens). CRBGP can suppress the human umbilical vein endothelial induced by bFGF
The propagation of cell (Human Umbilical Vein Endothelial Cells, HUVEC), and in dose dependent, its IC50
It is 4.8 μm ol/L.
2. utilize chicken allantocherion system to carry out internal anti-angiogenic rebirth functional examination.
Select chicken allantocherion (chicken chorioallantoic membrane, CAM) model views Lampetra japonica (Martens).
The CRBGP inhibitory action to Embryo Gallus domesticus angiogenesis.CAM model is current screening study anti-angiogenic drugs and mechanism of action
One of best model, has the characteristics such as experimental period is short, result is fast, is approved by numerous scholars.Under normal circumstances, normal Embryo Gallus domesticus
Blood vessel started just quickly form and expand from the 6th day, and now vascular development is vigorous, mainly generates with blastogenesis, intussusception mode.First
Use bFGF(200 ng/embryo) induction Embryo Gallus domesticus angiogenesis, use after 24 hours variable concentrations Lampetra japonica (Martens). CRBGP or
The PBS of equivalent acts on Embryo Gallus domesticus.Specifically comprise the following steps that
(1) cotton ball soaked in alcohol of employing 75% is by clean for the instar chicken embryo surface wipes on the six just bought；
(2) file is used to frustrate out 1cm at the 1/3 of distance embryo head2Fenestella；
(3) have at blood vessel in aseptic glass fiber filter paper sheet being placed in CAM, and on filter paper, add bFGF(200
Or equivalent PBS ng)；
(4) with adhesive tape, fenestella is sealed, in 37 DEG C of calorstats, hatch 24 hours；
(5) on filter paper, variable concentrations gradient (0.5 μ g, 1 μ g, 5 μ g, 10 μ g, the Lampetra japonica (Martens). sky of 20 μ g are added
So CRBGP or the PBS of equivalent, and seal with adhesive tape；
(6) continue to put in 37 DEG C of calorstats and hatch 24 hours；
After (7) 24 hours, filter paper is carefully removed, takes pictures immediately.
Result is as shown in Figure 2: compared with PBS control group, and the CAM blood vessel processed through bFGF becomes apparent from, in vein shape and
Branch is various, and number and the diameter of blood vessel the most significantly increase.When adding the Lampetra japonica (Martens). CRBGP of 0.5 μ g on 7th, Embryo Gallus domesticus
The number of blood vessel reduces, sparse and tiny without radial thick blood vessels network, blood vessel around drug treating.Along with Lampetra japonica (Martens).
The increase (1 μ g, 5 μ g, 10 μ g) of CRBGP concentration, rupturing significantly occurs in the major branch of Embryo Gallus domesticus blood vessel, and the number of blood vessel is the brightest
Reducing, blood vessel color is light and in pale yellow aobviously.When adding the Lampetra japonica (Martens). CRBGP of 20 μ g on 7th, the blood vessel of Embryo Gallus domesticus quilt completely
Destroy, occur in that territory, obvious avascular area, and blood vessel wall is completely dissolved.This shows that Lampetra japonica (Martens). CRBGP is to being induced by bFGF
Embryo Gallus domesticus angiogenesis has obvious inhibitory action, and in dose dependent.
Sequence table
<110>Liaoning Normal University
<120>Lampetra japonica (Martens). CRBGP application in preparing anti-angiogenic drugs
<130> Q. Gao, Y. Pang, Y. Wu, F. Ma, Q.W. Li, [Expressed sequence tags
(ESTs) analysis of the oral gland of Lampetra japonica], Yi
Chuan Xue Bao 32 (2005) 1045-1052.
<160> 1
<170> PatentIn version 3.2
<210> 1
<211> 207
<212> DNA
<213> Lampetra japonica
<220>
<221> exon
<222> (1)..(207)
<400> 1
Atg ttc act aac ctc gtg acc ccg gcg gcg ttg gcg ttg gtg ttc 45
M F T N L V T P A A L A L V F
1 5 10 15
Atg gcg agc gtc gtg gcg gcg aca tcc gtc aac gac tgg aag ctc 90
M A S V V A A T S V N D W K L
20 25 30
Ctg gac acg aag ctg tcg gcg aac cgg aag gtc atc gtg gac gtt135
L D T K L S A N R K V I V D V
35 40 45
Cac aac gag ctg cgg cgc ggc gtg gtg ccc acc gcc agc aac atg180
H N E L R R G V V P T A S N M
50 55 60
Ctc aag atg gcg tac aac gaa cag gca gca gag acc agc cgc ttg225
L K M A Y N E Q A A E T S R L
65 70 75
Tgg gcc gcc gcc tgc agc ttc tcg cac agc ccc agc aac acg cgc270
W A A A C S F S H S P S N T R
80 85 90
Acc tgg aag acg ccg caa gca gag tgg gac tgc gga gag aac ctc315
T W K T P Q A E W D C G E N L
95 100 105
Ttc atg tcc agc aac cca cgg tcg tgg gac gag gca gtg cgc agc360
F M S S N P R S W D E A V R S
110 115 120
Tgg tac gac gag gtc acc tcc ccc ggc ttc cag tac ggc acg ggg405
W Y D E V T S P G F Q Y G T G
125 130 135
Gct gtg ggg ccc ggg gcc gtg gga cac tac act cag gtg gtg tgg450
A V G P G A V G H Y T Q V V W
140 145 150
Tac aag tcc cac cag gtg ggc tgc gcc gtc aac tac tgc ccc aac495
Y K S H Q V G C A V N Y C P N
155 160 165
Cac ccc ggc gcc ctc aag ttc ctc tac gtg tgc cac tac tgc ccc540
H P G A L K F L Y V C H Y C P
170 175 180
Gca ggg aac ctg gtc acc agg atc aac aaa ccc tac gac ctg ggg585
A G N L V T R I N K P Y D L G
185 190 195
Act ccg tgc cag gcc tgc ccc cat agc tgc gac aac aac ctg tgc630
T P C Q A C P H S C D N N L C
200 205 210
Acc aac ccg tgc ccc tac gtg gac cag ttc agc aac tgc ccg cag675
T N P C P Y V D Q F S N C P Q
215 220 225
Ctg ttc agc gcc cac ggc tgc gcg aac gac ggc gcg ggg gga acc720
L F S A H G C A N D G A G G T
230 235 240
Ttc gtg cag aca aac tgc cct gcc acg tgc agc tgc acg acg gat765
F V Q T N C P A T C S C T T D
245 250 255
Gtg cag tga 774
V Q *
257
Claims (1)
1. the Lampetra japonica (Martens). CRBGP application in preparing anti-angiogenic drugs.
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| CN106589100B (en) * | 2016-12-14 | 2020-04-21 | 辽宁师范大学 | Anti-angiogenesis lamprey recombinant PR-1 protein and preparation method thereof |
| CN107137697B (en) * | 2017-04-28 | 2019-12-03 | 辽宁师范大学 | Lamprey CRBGP is preparing the application in anti-glioblastoma tumor medicine |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 日本七鳃鳗(Lampetra japonica)口腔腺分泌蛋白BGSP-1和CRBGP功能的研究;肖蓉 等;《中l闷细胞生物学学会全体会员代表大会暨第十二次学术人会论文摘要》;20110716;第190页 * |
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