CN1401785A - Human recombinant secretor type endostatin protein, preparing process and use thereof - Google Patents
Human recombinant secretor type endostatin protein, preparing process and use thereof Download PDFInfo
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Abstract
A human recombinant secretory endostatin protein is prepared by RT-PCR process, which includes cloning endostatin gene from Chinese fetus liver tissue, constructing expression plasmid pGAPZoA-endostatin, agrose electrophoresis to recover plasmid DNA, mixing with yeast GS115 liquid, electric conversion, screening recombinant positive expression yeast by SDS-polyacrylamide gel electrophoresis and affinity chromatography for separating and purifying the product. It can be used to treat the disease caused by nascent blood vessel formation in cornea.
Description
One, technical field:
The present invention relates to a kind of endostatin protein and its production and application, relate in particular to a kind of human recombinant secretor type endostatin protein and its production and application.
Two, background technology:
The normal cornea transparency of organization does not have blood vessel.But clinically, because of wearing of contact lens, corneal wound, operation, infection, soda acid burn etc., cause invading cornea gradually, cause cornea rebirth blood vessel (corneal neovascularization, appearance CNV) from the new capillary vessel that the corneal limbus vasoganglion forms.Although under given conditions, CNV has certain active effect to disease.But the excessive formation of CNV often causes corneal transparence to descend, and influences patient's vision to some extent, even causes the patient blind, is the one of the main reasons of blinding clinically.CNV has also changed the anatomical features of cornea " premunition absolution " simultaneously, is the first cause of corneal transplantation failure.Therefore, the prevention of CNV and treatment all have great importance to the success ratio of keeping corneal transparency and increase corneal transplantation.In addition, new vessel also can appear at iris, choroid, retina and with hemorrhage, ooze out, a series of pathological changes such as hyperplasia, the 26S Proteasome Structure and Function of havoc eye causes the patient blind, also is the major cause of present blinding.
The method that is usually used in preventing and treat CNV and other eye neovascular diseases at present mainly comprises:
(1) medicine: glucocorticosteroid (as dexamethasone), non-hormone antiphlogiston (as INDOMETHACIN), anti-proliferative drugs (as ametycin), vascular endothelial growth factor (VEGF) monoclonal antibody, interleukin 1 receptor (IL-1R) antagonist etc.There is toxic action (can cause keratohelcosis) but above medicine has as ametycin, what have causes more complication (can cause corticosteroid glaucoma, cataract and infection as glucocorticosteroid), what have is restricted (as the VEGF monoclonal antibody is mouse source property, can cause allergy or cause curative effect to reduce because of producing anti-antibody in the human body application) in clinical application.
(2) laser: laser light is coagulated the treatment that therapy can be used for CNV.As utilize argon laser can solidify directly that the list that is positioned at the corneal limbus place props up or the less new vessel that coincide, but coincide to intensive that then curative effect is relatively poor for new vessel into the net, and cause damage easily to normal adjacent tissue.
(3) isotropic substance:
90The strontium cornea of applying ointment or plaster, the β ray of its release can suppress new vessel, but relatively poor to deep-level blood vessel and thick blood vessel effect, and might cause radiational injury to its hetero-organization of eye.
(4) operation: as the limbal stem cell transplantation of limbal transplantation, limbal transplantation associating flaggy or penetrating keratoplasty, cultivation, amnion transplantation etc., more than the formation that can stop CNV of eye table reconstruction operations, but the inconvenience of drawing materials, and curative effect instability sometimes.
Therefore, how searching out a kind of evident in efficacy, specificity good, side effect the is little treatment CNV and the method for other eye neovascular diseases is focuses that domestic and international investigator pays close attention to.
Endostatin (endostatin) be a kind of endogenous vascular endothelial cell proliferation of finding first by the auspicious thunder of Australia (O ' Relly) in 1997 potent inhibitor [O ' Reilly MS, BoehmT, Shing Y, et al.Endostatin:Anendogenous inhibitor of angiognesis and tumor growth.Cell, 1997,88 (2): 277].Discover that Endostatin energy specificity suppresses to be in the new vessel endotheliocyte of vegetative state, and with apoptosis-induced effect [Dhanabal M, Ramchaneran R, Waterman MJ, et al.Endostatin induces endothelialcell apoptosis.J Biol Chem, 1999,274 (17): 11721].In recent years, Chinese scholars mainly concentrates on it to the tumor treatment effect to the research of Endostatin.Result of study shows that it can specificity suppress the growth of new vessel endotheliocyte, thereby blocks the blood supply and the nutrition of tumour cell, makes tumor regression.Now entered II phase clinical experiment.The outstanding feature of Endostatin effect is: 1, evident in efficacy, and high specificity.2, no obvious toxic-side effects.Immobilized endotheliocyte or other cell under people's normal mature state there is not influence.3, medication repeatedly, do not produce resistance [Boehm T, Folkman J, Browder T, et al.Antiangiogenic therapy of experomantalcancer does not induce acquired drug resistance.Nature.1997,390 (6658): 404].
Endostatin content in liver is maximum, but too high from liver extraction purification process complexity and expense.At present, in the success of Chinese people Endostatin clonal expression, but be mostly, as expression in escherichia coli [Fan Yanrong, Xu Genxing, Liu Xinjuan etc. at prokaryotic cell prokaryocyte.The inside and outside antitumor action research of recombinant human Endostatin/MBP fusion rotein.China's tumor biotherapy magazine, 2000,7 (2): 150].This Endostatin of expressing with prokaryotic expression system exists with non-folding form, poorly water-soluble, and the activity of its inhibition of endothelial cell proliferation can be influenced.Express even have in eukaryotic cell such as yeast, also mostly be the nonsecreting type fusion rotein, be unfavorable for purifying.[Sun Luguo, Jiang Ying, Yu Yongli.Human endostatin in yeast cell expression and facilitate the fibrocyte mitogen activation.China's Journal of Immunology, 2000,16 (5): 231] or the Yeast expression carrier that uses be that pPIC9 needs methanol induction [Feng Yi, Cui Libin, Liu Chuanxuan etc.Expression, purifying and the biological function research of human endostatin in Pichia yeast.The biotechnology journal, 2001,17 (3): 278].
The formation of eye new vessel and the propagation of vascular endothelial cell are closely related, belong to local application in ophthalmic applications, and dosage is few, and can be prepared into the local eye droppings of collyrium, have so both made things convenient for the patient, can alleviate patient's economical load again.Thereby the eye neovascular diseases will be to use the good indication that Endostatin is treated.Yet, up to now, yet there are no the report that people's recombinant secretor type endostatin protein is used for the treatment of the eye neovascular diseases.
Three, summary of the invention:
At the deficiency of above-mentioned acquisition reorganization Endostatin method and application, the problem that the present invention mainly solves provides a kind of human recombinant secretor type endostatin protein and preparation method thereof and its application in the medicine of preparation treatment eye neovascular diseases.
Human recombinant secretor type endostatin protein of the present invention can make by following method:
(1) extracted total RNA from the fresh fetal liver cells tissue;
(2) utilize Endostatin complementary DNA (cDNA) sequence, design upstream and downstream primer P1, P2 wherein add EcoR I restriction enzyme site in the upstream, and the downstream adds Not I restriction enzyme site;
(3) be template with above-mentioned extractive total RNA, carry out reverse transcription polymerase chain reaction,PCR (RT-PCR), obtain Endostatin cDNA;
(4) by gel electrophoresis, plasmid extraction, reclaiming by centrifuge Endostatin cDNA;
(5) with the cDNA of above-mentioned recovery, (pGEM-T) is connected with cloned plasmids, transformed into escherichia coli JM109.
(6) be that primer carries out pcr amplification with P1, P2, screening amplification male contains the clone of endostatin gene, the extracting plasmid DNA, and order-checking is identified;
(7) utilize restriction enzyme EcoR I, Not I double digestion to contain the cloned plasmids (pGEM-endostatin) of Endostatin (endostatin) gene with ordinary method, realize and pGAPZ
αThe connection of A expression plasmid of yeast;
(8) get the connection product transformed into escherichia coli JM109 of step 7, screening contains the positive recombinant expression vector engineering bacteria of Endostatin (endostatin) gene, the extracting plasmid DNA, and order-checking is identified;
(9) contain the expression of recombinant yeast plasmid pGAPZ of endostatin gene with restriction enzyme A vr II single endonuclease digestion
αA-endostatin, 0.8% agarose electrophoresis reclaims complete linearizing plasmid DNA, mixes with the competence bacteria liquid of yeast GS115 and carries out the electricity conversion;
(10) utilize SDS-polyacrylamide gel electrophoresis screening reorganization positive expression yeast;
(11) get above-mentioned reorganization positive expression yeast and cross with the heparin affinity chromatography post through Sepharose G25 that post separates, purifying, make Endostatin (endostatin) expressing protein, be human recombinant secretor type endostatin protein, molecular weight is about: 32KD comprises at least 183 amino acid.Above-mentioned reverse transcription polymerase chain reaction,PCR (RT-PCR), amplification Endostatin cDNA the primer P1, P2 are:
P1：5’T?GGT?
GAA?TTC?CAC?AGC?CAC?CGC?GAC?TT?3’
EcoR?I
P2：5’TAA?
TGC?GGC?CGC?CTT?GGA?GGC?AGT?CAT?G?3’
Not?I
The reaction conditions of above-mentioned reverse transcription polymerase chain reaction,PCR (RT-PCR) is: 95 ℃ of pre-sex change 5 minutes; 95 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute; After carrying out 35 circulations, 72 ℃ were extended 5 minutes.
Above-mentioned yeast expressed carrier is pGAPZ
αThe A plasmid DNA.
The preparation method of above-mentioned human recombinant secretor type endostatin protein comprises the step of following order:
(1) clone of endostatin gene:
1. design of primers:
According to Endostatin complementary DNA (cDNA) sequence (http://www.ncbi.nlm.nih.gov/
Genebank/), design upstream and downstream primer P1, P2 (Primer1, Primer2) and restriction endonuclease sites,
Wherein add EcoR I restriction enzyme site in the upstream, the downstream adds Not I restriction enzyme site
P1：5’T?GGT?
GAA?TTC?CAC?AGC?CAC?
CGC?GAC?TT?3’
EcoR?I
P2：5’TAA?
TGC?GGC?CGC?CTT?GGA?GGC?AGT?CAT?G?3’
Not?I
2. the extracting of the total RNA of tire hepatic tissue:
Get the fresh fetal liver cells 0.75g of liquid nitrogen cryopreservation, add Trizol reagent 7.5ml rapidly, be ground to the liquid clarification
After, it is centrifugal to add equal-volume chloroform/primary isoamyl alcohol 12000rpm 10 minutes, draws supernatant, precipitates with dehydrated alcohol
After, being dissolved in the water of 50 μ l DEPC processing ,-20 ℃ of preservations are standby;
3. the preparation of Endostatin (endostatin) cDNA:
Adopt reverse transcription polymerase chain reaction,PCR (RT-PCR) amplification endostatin gene, be with extractive total RNA
Template is carried out reverse transcription reaction, amplification cDNA first chain, and reaction system is:
RNA template 18 μ l
5 * damping fluid, 10 μ l
Few deoxythymidine Oligo (dT) 2.5 μ l (150ng)
Dithiothreitol (DTT) (DTT) 5 μ l (0.01M)
RNA enzyme inhibitors (Rnasin) 1.5 μ l (60U)
Reversed transcriptive enzyme (M-MuLV) 3 μ l (600U)
Deoxidation ATP, TTP, GTP, CTP (dNTP) 10 μ l (0.5mM)
Behind the mixing, 37 ℃, 100 ℃ of terminations in 5 minutes are put in amplification in 1 hour again, get reverse transcription reaction product 25 μ l, are primer with P1, P2, pcr amplification Endostatin (endostatin) cDNA, and reaction system is:
Reverse transcription (RT) reaction product 25 μ l
10 * damping fluid, 10 μ l
P1 2.5μl(0.5μM)
P2 2.5μl(0.5μM)
Deoxidation ATP, TTP, GTP, CTP (dNTP) 8 μ l (0.2 μ M)
Heat-resisting high-fidelity DNA polymerase (pfu Taq enzyme) 1 μ l (5u)
Supply distilled water to 100 μ l
Reaction conditions is: 95 ℃ of pre-sex change 5 minutes; 95 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, was extended 1 minute by 72 ℃; After carrying out 35 circulations, 72 ℃ were extended 5 minutes;
Do not add the negative contrast of reverse transcription (RT) reaction product;
4. the recovery of Endostatin (endostatin) cDNA:
The RT-PCR amplified production is through 2% agarose gel electrophoresis, downcut the target DNA band of 550bp, behind 25 ℃ ,-20 ℃ multigelations, place plasmid extraction box particulate (Pu Luomaige, Promega company product), behind centrifugal 5 minutes of the 10000rpm, collect filtered solution, add 2 times of volume dehydrated alcohols, place-20 ℃, after 30 minutes, under the 12000rpm4 ℃ of condition centrifugal 10 minutes, precipitation was the target DNA of purifying;
5. the T-A of Endostatin (endostatin) gene clones:
Add dATP and Taq enzyme and each 1 μ l of 10 * damping fluid among the cDNA that 1-2 μ l is reclaimed, supply distilled water to 10 μ l, under 72 ℃ of conditions, extend and added the A tail in 15 minutes, and purifying and recycling cDNA; Get each 1 μ l of this kind goal gene DNA and pGEM-T carrier (Pu Luomaige, Promega company product), add T4 ligase enzyme 1 μ l, 10 * damping fluid, 1 μ l and distilled water 6 μ l under 4 ℃ of conditions, 16-18 hour, finish the T-A pairing;
6. the screening of recombinant clone, evaluation:
Conventional preparation e. coli jm109 competence is got the connection product that 5. 2 μ l steps prepare, with CaCl
2Method transformed into escherichia coli JM109 competence, converted product is coated in the LA plate that contains 200mg/ml x-gal (5-bromo-4-chloro-3-indoles-β-D-semi-lactosi) 40 μ l and 20mg/ml IPTG (isopropyl-) 4 μ l, 37 ℃ of activation of random choose 7-12 white single bacterium colony are after 12 hours, the extracting plasmid DNA, with P1, P2 is that primer carries out pcr amplification, select amplification male clone, extracting plasmid DNA, order-checking;
(2) Endostatin (endostatin) expression of gene
1. pGAPZ
αThe amplification of A expression plasmid of yeast, extraction:
E. coli jm109 activates back preparation competence routinely, gets pGAPZ
αA plasmid DNA (Invitrogen company product) 1 μ l, conventional CaCl
2Method transforms JM109, and the JM109 bacterium liquid after transforming is coated in the less salt LB flat board that contains 25 μ g/ml Zeocin (microbiotic, Invitrogen company product), get single bacterium colony after 18-22 hour, amplification, and with plasmid extraction test kit (Pu Luomaige, Promega company product) extracting plasmid;
The prescription of above-mentioned less salt LB flat board is: yeast extract 0.5%, and peptone 1%, NaCl 0.5%, agar 1.5%, pH7.5;
2. the subclone of Endostatin (endostatin) gene:
Restriction enzyme EcoR I, Not I (Shanghai bio-engineering corporation product) double digestion respectively contain the cloned plasmids (pGEM-endostatin) and the empty expression vector pGAPZ of endostatin gene
αA, through 0.8% agarose electrophoresis, the expression vector dna gel piece of the target DNA of the about 550bp of recovery and 3kb is in the 1.5ml pipe respectively, behind-20 ℃, 25 ℃ multigelations three times, place plasmid extraction box particulate, 10000rpm is after centrifugal 5 minutes, collect filtered solution, add 2 times of volume dehydrated alcohols, place-20 ℃ after 30 minutes, under 4 ℃ of conditions of 12000rpm, centrifugal 10 minutes, deposit D NA added 1ml 75% ethanol rinsing, under 4 ℃ of conditions of 12000rpm, centrifugal 10 minutes, abandon supernatant; The target DNA and the expression vector dna that reclaim are dissolved in the 8 μ l distilled waters together, add 1 μ l T4 ligase enzyme and 2 μ l, 5 * damping fluid, after room temperature connects 16-18 hour, get and connect product 10 μ l conversion JM109, and coat in the less salt LB flat board that contains 25 μ g/mlZeocin, simultaneously to transform the pGAPZ that does not contain the endostatin gene
αThe A plasmid is as blank;
3. screening, the evaluation of Endostatin (endostatin) subclone:
Select mono-clonal from Zeocin less salt LB flat board, be inoculated in the less salt LB substratum that contains Zeocin, 37 ℃ of lucifuges are cultivated, and the extracting plasmid DNA is respectively with the positive reorganization bacterium that enzyme is cut, the PCR method is screened the above-mentioned endostatin of containing gene, extracting plasmid DNA, order-checking;
The prescription of above-mentioned less salt LB substratum is: yeast extract 0.5%, and peptone 1%, NaCl 0.5%, pH7.5;
4. the competence of yeast GS115 (Invitrogen company product) preparation:
Get Pichia yeast (GS115) bacterial classification 10 μ l, be inoculated in the 2ml YPD substratum, the vibration of 30 ℃ of shaking tables was got GS115 bacterium liquid 100 μ l after 12-16 hour, be inoculated among the 100mlYPD, 30 ℃ shaking culture 12-16 hour; When bacterium liquid OD600 is 1.3-1.5, take out, centrifugal 7 minutes of 4 ℃ of 2500rpm precipitate resuspendedly with the 1M D-sorbyl alcohol of 200 μ l, put in the ice bath;
Above-mentioned YPD culture medium prescription is: yeast extract 1%, Tryptones 2%, D-glucose 2%;
5. electric transformation experiment:
The expression of recombinant yeast plasmid pGAPZ that contains the endostatin gene with restriction enzyme A vrII (TaKaRa company product) single endonuclease digestion
αA-endostatin with preceding method, reclaims complete linearizing plasmid DNA with 0.8% agarose electrophoresis, is dissolved in the 10 μ l distilled waters; Getting GS115 competence bacteria liquid 80 μ l mixes with 10 μ l linearization plasmid DNA, behind the ice bath 5 minutes, the electric revolving cup that adds the 0.2cm specification, with electroporation (Bole company product), 1500V voltage electric shock, the 1M Sorbitol Solution USP that adds 1ml immediately, be transferred in the sterile tube, 30 ℃ leave standstill 1-2 hour after, get 50,100,200,400 μ l respectively and coat on the YPDS flat board that contains 100 μ g/ml Zeocin, 30 ℃ of lucifuges were cultivated after three days, simultaneously not contain the empty plasmid pGAPZ of endostatin
αThe A electricity transforms GS115 and compares, and the picking mono-clonal is to 1ml YPD, and 30 ℃ of shaking culture are to logarithmic phase, and-20 ℃ frozen;
The prescription of above-mentioned YPDS flat board is: yeast extract 1%, Tryptones 2%, D-glucose 2%, sorbyl alcohol 1M, agar 2%;
6. the SDS-polyacrylamide gel electrophoresis screens the positive expression bacterium:
After the frozen bacterium 10 μ l of mono-clonal after electricity changes are activated, be inoculated in the 100ml YPD nutrient solution with 1: 100,30 ℃ of shaking culture were got bacterium liquid 5ml every 24 hours, remove bacterial sediment behind the high speed centrifugation, and with the supernatant packing ,-20 ℃ of preservations are to be measured; Before the electrophoresis, get 500 μ l culture supernatant, add 2 times of volume dehydrated alcohols ,-20 ℃ 30 minutes, centrifugal 5 minutes of 12000rpm, the precipitation protein concentrate, albumen is dissolved in 20 μ l, 6 * load sample damping fluid after concentrating, after 100 ℃ of sex change in 3 minutes, be splined on 12% polyacrylamide gel, the 50mA electrophoresis;
7. the purifying of Endostatin (endostatin) expressing protein:
Getting the yeast that positive yeast (name and be yeast B1-3) of reorganization that 6. the 2L step screen and 2L transform empty carrier pGAPZ α A (names and is yeast SC3-1, the 4th day culture supernatant in contrast), respectively through 8000rpm after centrifugal 15 minutes, remove precipitation, supernatant concentrates (name respectively and be B1-3 concentrated solution and SC3-1 concentrated solution) for 20 times through ultrafiltration, Sepharose G25 post is earlier after 50mM NaCl 10mM Tris.Cl balance, with sample on the concentrated solution, after crossing post, SepharoseG25 desalts and lower-molecular substance, collect protein peak, heparin (Heparin) affinity column (Fa Moxiya, Phamacia company product) by after the explanation installation, earlier through 50mM NaCl 10mM Tris.Cl balance, with sample on each crest behind the G25 wash-out, with 50mM-2M NaCl 10mM Tris.Cl gradient buffering liquid wash-out, collect two protein peaks, wherein the protein peak of 1M NaCl 10mM Tris.Cl wash-out (naming the eluted protein for 1M) is human recombinant secretor type endostatin protein, molecular weight is about: 32KD comprises at least 183 amino acid; 0.5M the protein peak of NaCl 10mM Tris.Cl wash-out (naming the eluted protein into 0.5M) is non-target protein.
The application of above-mentioned human recombinant secretor type endostatin protein in the medicine of preparation treatment eye neovascular diseases.
The application of above-mentioned human recombinant secretor type endostatin protein in the medicine of preparation treatment cornea rebirth blood vessel disease.
Above-mentioned human recombinant secretor type endostatin protein is in the medicine of preparation treatment cornea rebirth blood vessel disease, and employed effective concentration is 40 μ g/ml-60 μ g/ml.
Above-mentioned human recombinant secretor type endostatin protein is in the medicine of preparation treatment cornea rebirth blood vessel disease, and the scope of particularly suitable is a mammals.
Adopt technique scheme, the present invention to utilize Endostatin (endostatin) gene of cloning in the Chinese fetus liver therefrom, construct a kind of novel endostatin secretor type Yeast expression carrier pGAPZ
αA-endostatin.This expression vector has α-signal peptide that pilot protein goes out born of the same parents, can make product secrete born of the same parents, helps the purifying and the production of product; PGAPZ
αA utilizes the promotor of encoding glycerol aldehyde-3-phosphate dehydrogenase (GAPDH) gene, but constructive expression's foreign gene and do not need methanol induction, and expression amount is than induction type carrier height; And the sequence with poly Histidine tail and part myc helps expressing the detection and the purifying of after product.By expressing in the electric method for transformation importing Pichia yeast, from saccharomycetic culture supernatant, obtain activated endostatin albumen, can be used for after purified treating that retina hyperplasia that cornea rebirth blood vessel formation, neovascular glaucoma, age-related macular degeneration, diabetes causes etc. is a series of forms relevant eye disease with new vessel, have more significant effect.Concrete implementation result is as follows: (one) experiment in vitro-human recombinant secretor type endostatin protein suppresses experiment to vascular endothelial cell proliferation
The Human umbilical vein endothelial cells in vegetative period of taking the logarithm (ECV-304 cell) 5 * 10
4/ ml is inoculated in 96 orifice plates, 100 μ l/ holes, 37 ℃, 5%CO
2Cultivate in the incubator to inhale after 24 hours and abandon supernatant, add the different dilution 0.5M eluted proteins of 100 μ l, 1M eluted protein, B1-3 concentrated solution and SC3-1 concentrated solution respectively, continue to cultivate after 2 days, measure cell proliferation inhibition rate with mtt assay (tetramethyl-azo azoles salt).Inhibiting rate=1-(experimental group OD/ control group OD).Result: MTT tests demonstration, the propagation that all can effectively suppress vascular endothelial cell through the B1-3 of 2,3,4,5 times of dilutions concentrated solution, the highest inhibiting rate can reach 74% (seeing Table 1), morphologic observation shows the apoptosis of the restraining effect of B1-3 with cell, act on after 20 minutes, cell promptly all becomes circle, and concentrating appears in nuclear, blank bacterium SC3-1 after the 4th day culture supernatant then do not have this effect (see figure 1).Can obviously suppress ECV-304 propagation equally through the 1M of 2,3,4,5 times of dilutions eluted protein, the highest inhibiting rate can reach 86% (seeing Table 1).
The B1-3 concentrated solution of 10 times of dilutions and 1M eluted protein are to the propagation unrestraint effect of ECV-304.
Under the similarity condition, the MTT experiment confirm of liver cancer cell (HepG2.2.15 clone), the culture supernatant of B1-3 and SC3-1 all can not suppress the propagation (see figure 2) of liver cancer cell.
Show by above-mentioned experimental result, all can effectively suppress the propagation of vascular endothelial cell before and after the human recombinant secretor type endostatin protein purifying, and other cell in non-endotheliocyte source is not had influence.(2) the interior experiment-human recombinant secretor type endostatin protein of body is to the restraining effect of rabbit corneal new vessel
Utilize cornea suture legal system to be equipped with rabbit cornea new vessel animal model, be divided into 2 groups at random, be respectively:
(1) human recombinant secretor type endostatin protein treatment group: postoperative same day, at sub-conjunctival injection 40-60 μ g/ml reorganization Endostatin 0.4ml, the next day once, 8-12 time continuously.
(2) physiological saline control group: postoperative same day, at sub-conjunctival injection physiological saline 0.4ml, the next day once, 8-12 time continuously.
Establish the blank group in addition: be left intact.The result: blank group corneal transparency does not have blood vessel, and the treatment group is than physiological saline control group new vessel poor growth and more sparse (see Table 2 and Fig. 3).Treatment group bulbar conjunctiva is not seen hyperemia, oedema, and cornea is not seen muddiness, oedema yet.Pathological examination shows that treatment group corneal epithelium is normal, and new vessel is few around the hypothallus suture, and minority plasmocyte and inoblast are arranged.Physiological saline control group corneal epithelium is normal, and the hypothallus new vessel is many, and tube chamber is big, assembles a large amount of plasmocyte, and inoblast is few.
Pathological examination shows, other internal organs: the heart, liver,kidney,spleen, lung, testis, treatment group and physiological saline control group do not have obvious difference.Human recombinant secretor type endostatin protein can effectively suppress the growth of rabbit cornea new vessel, and eyeball and other important organ are not had obvious toxic-side effects.
Four, description of drawings: Fig. 1 human recombinant secretor type endostatin protein is to the restraining effect of umbilical vein vascular endothelial cell (ECV-304)
A wherein: the normal blood vessels endotheliocyte B:B1-3 concentrated solution effect before the effect 20 minutes,
1 day D:B1-3 concentrated solution of C:B1-3 concentrated solution effect effect 2 days
2 days Fig. 2 human recombinant secretor type endostatin proteins of E:SC3-1 concentrated solution effect are to the effect of liver cancer cell (HepG2.2.15 clone)
Wherein the effect of A:B1-3 concentrated solution is 2 days
B: normal hepatocytes cancer cells contrast Fig. 3 human recombinant secretor type endostatin protein is to the restraining effect of rabbit corneal new vessel
Fig. 3 (A) human recombinant secretor type endostatin protein treatment group rabbit corneal new vessel growing state wherein
Fig. 3 (B) physiological saline control group rabbit corneal new vessel growing state
Fig. 3 (C) blank group rabbit corneal Fig. 4 RT-PCR amplification endostatin gene result
A:RT-PCR product B wherein: Marker C: negative control Fig. 5 expression vector pGAPZ
αThe restriction enzyme digestion and electrophoresis qualification result of A-endostatin
1:pGAPZ wherein
αA-endostatin EcoR I single endonuclease digestion
2：Marker
3:pGAPZ
αA-endostatin EcoR I, Not I double digestion Fig. 6 expression vector pGAPZ
αThe sequencing result of A-endostatin
Fig. 6 (A) wherein: expression vector pGAPZ
αThe sequence chart of A-endostatin
Fig. 6 (B): expression vector pGAPZ
αSDS-polyacrylamide gel electrophoresis figure before and after sequence chart Fig. 7 B1-3 of A concentrates
Wherein 1: standard protein 2:B1-3 concentrates preceding 3: low molecular weight protein (LMWP) Marker 4:B1-3 concentrates the SDS-polyacrylamide gel electrophoresis figure behind back Fig. 8 B1-3 concentrated solution purifying
Wherein 1:1M eluted protein 2:0.5M eluted protein 3: low molecular weight protein (LMWP) Marker
Five, embodiment: embodiment 1: human recombinant secretor type endostatin protein is to vitro inhibition effect (1) design of primers of vascular endothelial cell: according to Endostatin complementary DNA (cDNA) sequence (http://www.ncbi.nlm.nih.gov/Genebank/), design upstream and downstream primer P1, P2 (Primer1, Primer2) and restriction endonuclease sites, wherein add EcoR I restriction enzyme site in the upstream, the downstream adds Not I restriction enzyme site
P1：5’T?GGT?
GAA?TTC?CAC?AGC?CAC?CGC?GAC?TT?3’
EcoR?I
P2：5’TAA?
TGC?GGC?CGC?CTT?GGA?GGC?AGT?CAT?G?3’
The extracting of the total RNA of Not I (2) tire hepatic tissue: the fresh fetal liver cells 0.75g that gets liquid nitrogen cryopreservation, add Trizol reagent 7.5ml rapidly, after being ground to the liquid clarification, it is centrifugal to add equal-volume chloroform/primary isoamyl alcohol 12000rpm 10 minutes, draw supernatant, with the dehydrated alcohol post precipitation, be dissolved in the water of 50 μ l DEPC processing ,-20 ℃ of preservations are standby; (3) preparation of Endostatin (endostatin) cDNA: adopting reverse transcription polymerase chain reaction,PCR (RT-PCR) amplification endostatin gene, is template with extractive total RNA, carries out reverse transcription reaction, amplification cDNA first chain, and reaction system is:
RNA template 18 μ l
5 * damping fluid, 10 μ l
Few deoxythymidine Oligo (dT) 2.5 μ l (150ng)
Dithiothreitol (DTT) (DTT) 5 μ l (0.01M)
RNA enzyme inhibitors (Rnasin) 1.5 μ l (60U)
Reversed transcriptive enzyme (M-MuLV) 3 μ l (600U)
Deoxidation ATP, TTP, GTP, CTP (dNTP) 10 μ l (0.5mM)
Behind the mixing, 37 ℃, 100 ℃ of terminations in 5 minutes are put in amplification in 1 hour again, get reverse transcription reaction product 25 μ l, are primer with P1, P2, pcr amplification Endostatin (endostatin) cDNA, and reaction system is:
Reverse transcription (RT) reaction product 25 μ l
10 * damping fluid, 10 μ l
P1 2.5μl(0.5μM)
P2 2.5μl(0.5μM)
Deoxidation ATP, TTP, GTP, CTP (dNTP) 8 μ l (0.2 μ M)
Heat-resisting high-fidelity DNA polymerase (pfu Taq enzyme) 1 μ l (5u)
Supply distilled water to 100 μ l
Reaction conditions is: 95 ℃ of pre-sex change 5 minutes; 95 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, was extended 1 minute by 72 ℃; After carrying out 35 circulations, 72 ℃ were extended 5 minutes;
Do not add the negative contrast of reverse transcription (RT) reaction product; The recovery of (the results are shown in Figure 4) (4) Endostatins (endostatin) cDNA: the RT-PCR amplified production is through 2% agarose gel electrophoresis, downcut the target DNA band of 550bp, behind 25 ℃ ,-20 ℃ multigelations, place plasmid extraction box particulate (Pu Luomaige, Promega company product), behind centrifugal 5 minutes of the 10000rpm, collect filtered solution, add 2 times of volume dehydrated alcohols, place-20 ℃, after 30 minutes, under 4 ℃ of conditions of 12000rpm centrifugal 10 minutes, precipitation was the target DNA of purifying; (5) T-A of Endostatin (endostatin) gene clone: add dATP and Taq enzyme and each 1 μ l of 10 * damping fluid among the cDNA that 1 μ l is reclaimed, supply distilled water to 10 μ l, under 72 ℃ of conditions, extend and added the A tail in 15 minutes, and purifying and recycling cDNA; Get each 1 μ l of this kind goal gene DNA and pGEM-T carrier (Pu Luomaige, Promega company product), add T4 ligase enzyme 1 μ l, 10 * damping fluid, 1 μ l and distilled water 6 μ l under 4 ℃ of conditions, 17 hours, finish the T-A pairing; (6) screening of recombinant clone, evaluation: conventional preparation e. coli jm109 competence, get the connection product that 5. 2 μ l steps prepare, with CaCl
2Method transformed into escherichia coli JM109 competence, converted product is coated in the LA plate that contains 200mg/ml x-gal (5-bromo-4-chloro-3-indoles-β-D-semi-lactosi) 40 μ l and 20mg/ml IPTG (isopropyl-) 4 μ l, 8 single bacterium colonies of white of random choose.37 ℃ of activation are after 12 hours, and the extracting plasmid DNA is that primer carries out pcr amplification with P1, P2, select amplification male clone, the extracting plasmid DNA, and order-checking is identified; (7) pGAPZ
αThe amplification of A expression plasmid of yeast, extraction: e. coli jm109 activates back preparation competence routinely, gets pGAPZ
αA plasmid DNA (Invitrogen company product) 1 μ l, conventional CaCl
2Method transforms JM109, and the JM109 bacterium liquid after transforming is coated in the less salt LB flat board that contains 25 μ g/ml Zeocin (microbiotic, Invitrogen company product), get single bacterium colony after 20 hours, amplification, and with plasmid extraction test kit (Pu Luomaige, Promega company product) extracting plasmid;
The prescription of above-mentioned less salt LB flat board is: yeast extract 0.5%, and peptone 1%, NaCl 0.5%, agar 1.5%, pH7.5; (8) subclone of Endostatin (endostatin) gene: restriction enzyme EcoR I, Not I (Shanghai bio-engineering corporation product) double digestion respectively contain the cloned plasmids (pGEM-endostatin) and the empty expression vector pGAPZ of endostatin gene
αA, through 0.8% agarose electrophoresis, the expression vector dna gel piece of the target DNA of the about 550bp of recovery and 3kb is in the 1.5ml pipe respectively, behind-20 ℃, 25 ℃ multigelations three times, place plasmid extraction box particulate, 10000rpm is after centrifugal 5 minutes, collect filtered solution, add 2 times of volume dehydrated alcohols, place-20 ℃ after 30 minutes, under 4 ℃ of conditions of 12000rpm, centrifugal 10 minutes, deposit D NA added the rinsing of 1ml75% ethanol, under 4 ℃ of conditions of 12000rpm, centrifugal 10 minutes, abandon supernatant; The target DNA and the expression vector dna that reclaim are dissolved in the 8 μ l distilled waters together, add 1 μ l T4 ligase enzyme and 2 μ l, 5 * damping fluid, after room temperature connects 17 hours, get and connect product 10 μ l conversion JM109, and coat in the less salt LB flat board that contains 25 μ g/ml Zeocin, simultaneously to transform the pGAPZ that does not contain the endostatin gene
αThe A plasmid is as blank; (9) screening, the evaluation of Endostatin (endostatin) subclone: from Zeocin less salt LB flat board, select mono-clonal, be inoculated in the less salt LB substratum that contains Zeocin, 37 ℃ of lucifuges are cultivated, the extracting plasmid DNA, respectively with enzyme cut, the positive reorganization bacterium of the above-mentioned endostatin of the containing gene of PCR method screening, the extracting plasmid DNA, order-checking is identified; (the results are shown in Figure 5, Fig. 6)
The prescription of above-mentioned less salt LB substratum is: yeast extract 0.5%, peptone 1%, NaCl0.5%, pH7.5; (10) competence of yeast GS115 (Invitrogen company product) preparation: get Pichia yeast GS115 bacterial classification 10 μ l, be inoculated in the 2ml YPD substratum, 30 ℃ of shaking table vibrations were got GS115 bacterium liquid 100 μ l after 14 hours, be inoculated among the 100mlYPD 30 ℃ of shaking culture 14 hours; As bacterium liquid OD
600Be about at 1.4 o'clock, take out, centrifugal 7 minutes of 4 ℃ of 2500rpm precipitate resuspendedly with the 1M D-sorbyl alcohol of 200 μ l, put in the ice bath;
Above-mentioned YPD culture medium prescription is: yeast extract 1%, Tryptones 2%, D-glucose 2%; (11) electric transformation experiment: the expression of recombinant yeast plasmid pGAPZ that contains the endostatin gene with restriction enzyme A vr II (TaKaRa company product) single endonuclease digestion
αA-endostatin with preceding method, reclaims complete linearizing plasmid DNA with 0.8% agarose electrophoresis, is dissolved in the 10 μ l distilled waters; Getting GS115 competence bacteria liquid 80 μ l mixes with 10 μ l linearization plasmid DNA, behind the ice bath 5 minutes, the electric revolving cup that adds the 0.2cm specification, with electroporation (Bole company product), 1500V voltage electric shock, the 1M Sorbitol Solution USP that adds 1ml immediately, be transferred in the sterile tube, 30 ℃ leave standstill 1 hour 30 minutes kinds after, get 50,100,200,400 μ l respectively and coat on the YPDS plate that contains 100 μ g/ml Zeocin, 30 ℃ of lucifuges were cultivated after 3 days, simultaneously not contain the empty plasmid pGAPZ of endostatin
αThe A electricity transforms GS115 and compares, and the picking mono-clonal is to 1ml YPD, and 30 ℃ of shaking culture are to logarithmic phase, and-20 ℃ frozen;
The prescription of above-mentioned YPDS flat board is: yeast extract 1%, Tryptones 2%, D-glucose 2%, sorbyl alcohol 1M, agar 2%; (12) SDS-polyacrylamide gel electrophoresis screening positive expression bacterium: after the frozen bacterium 10 μ l of mono-clonal after electricity changes are activated, be inoculated in the 100ml YPD nutrient solution with 1: 100,30 ℃ of shaking culture, got bacterium liquid 5ml every 24 hours, remove bacterial sediment behind the high speed centrifugation, with the supernatant packing ,-20 ℃ of preservations are to be measured; Before the electrophoresis, get 500 μ l culture supernatant, add 2 times of volume dehydrated alcohols ,-20 ℃ 30 minutes, centrifugal 5 minutes of 12000rpm, the precipitation protein concentrate, albumen is dissolved in 20 μ l, 6 * load sample damping fluid after concentrating, after 100 ℃ of sex change in 3 minutes, be splined on 12% polyacrylamide gel, the 50mA electrophoresis; (13) purifying of Endostatin (endostatin) expressing protein: get positive yeast (name and be yeast B1-3) of reorganization and 2L conversion empty carrier pGAPZ that 6. the 2L step screens
αThe yeast of A (is named and is yeast SC3-1, the 4th day culture supernatant in contrast), respectively through 8000rpm after centrifugal 15 minutes, remove precipitation, supernatant concentrates (name respectively and be B1-3 concentrated solution and SC3-1 concentrated solution) for 20 times through ultrafiltration, Sepharose G25 post is earlier after 50mM NaCl 10mMTris.Cl balance, with sample on the concentrated solution, after crossing post, Sepharose G25 desalts and lower-molecular substance, collect protein peak, heparin (Heparin) affinity column (Fa Moxiya, Phamacia company product) by after the explanation installation, earlier through 50mM NaCl 10mM Tris.Cl balance, with sample on each crest behind the G25 wash-out, with 50mM～2M NaCl 10mMTris.Cl gradient buffering liquid wash-out, collect two protein peaks, wherein the protein peak of 1M NaCl 10mM Tris.Cl wash-out (naming the eluted protein for 1M) is human recombinant secretor type endostatin protein, and molecular weight is about: 32KD comprises at least 183 amino acid; 0.5M the protein peak of NaCl 10mM Tris.Cl wash-out (naming the eluted protein into 0.5M) is non-target protein.(seeing Fig. 7, Fig. 8) (14) experiment in vitro-human recombinant secretor type endostatin protein suppresses experiment to vascular endothelial cell proliferation: selects Human umbilical vein endothelial cells ECV-304, goes down to posterity in the DMEM substratum that contains 10% foetal calf serum, and 37 ℃, 5% CO
2Cultivate in the incubator.The cell 5 * 10 in vegetative period of taking the logarithm
4/ ml is inoculated in 96 orifice plates, and 100 μ l/ holes place 37 ℃, 5%CO
2Cultivate in the incubator to inhale after 24 hours and abandon supernatant, add 100 μ l respectively through the 0.5M of 2,3,4,5,10 times of dilutions eluted protein, 1M eluted protein, B1-3 concentrated solution and SC3-1 concentrated solution, continue to cultivate after 2 days, each hole adds the MTT (tetramethyl-azo azoles salt) of 20 μ l5mg/ml, after cultivating 6 hours again, each hole is sucking-off 100 μ l supernatants gently, add 100 μ l DMSO (dimethyl sulfoxide (DMSO)), after room temperature was vibrated 10 minutes, OD
570Measure cell proliferation inhibition rate.Inhibiting rate=1-(experimental group OD/ control group OD)
During experiment, in kind above-mentioned each sample is acted on the HepG2.2.15 hepatoma cell line, as the contrast of non-vascular endothelial cell.Result: MTT tests demonstration, the propagation that all can effectively suppress vascular endothelial cell through the B1-3 of 2,3,4,5 times of dilutions concentrated solution, the highest inhibiting rate can reach 74% (seeing Table 1), morphologic observation shows the apoptosis of the restraining effect of B1-3 with cell, act on after 20 minutes, cell promptly all becomes circle, and concentrating appears in nuclear, blank bacterium SC3-1 after the 4th day culture supernatant then do not have this effect (see figure 1).Can obviously suppress ECV-304 propagation equally through the 1M of 2,3,4,5 times of dilutions eluted protein, the highest inhibiting rate can reach 86% (seeing Table 1).
The B1-3 concentrated solution of 10 times of dilutions and 1M eluted protein are to the propagation unrestraint effect of ECV-304.
The table 1 people restraining effect of endostatin protein of recombinating to ECV-304 cell proliferation
The diluted sample multiple
2 3 4 5 10
The B1-3 concentrated solution
*74% 39% 33% 22% 0
The 1M eluted protein
*86% 72% 70% 64% 0
0.5M eluted protein 00000
SC3-1 concentrated solution 00000
*P＜0.05
Under the similarity condition, the MTT experiment confirm of liver cancer cell (HepG2.2.15 clone), the culture supernatant of B1-3 and SC3-1 all can not suppress the propagation (see figure 2) of liver cancer cell.
Show by above-mentioned experimental result, all can effectively suppress the propagation of vascular endothelial cell before and after the human recombinant secretor type endostatin protein purifying, and other cell in non-endotheliocyte source is not had influence.Embodiment 2: human recombinant secretor type endostatin protein is to restraining effect (1) design of primers of rabbit cornea new vessel: according to Endostatin complementary DNA (cDNA) sequence (http://www.ncbi.nlm.nih.gov/Genebank/), design upstream and downstream primer P1, P2 (Primer1, Primer2) and restriction endonuclease sites, wherein add EcoR I restriction enzyme site in the upstream, the downstream adds Not I restriction enzyme site
P1：5’T?GGT?
GAA?TTC?CAC?AGC?CAC?CGC?GAC?TT?3’
EcoR?I
P2：5’TAA?
TGC?GGC?CGC?CTT?GGA?GGC?AGT?CAT?G?3’
The extracting of the total RNA of Not I (2) tire hepatic tissue: the fresh fetal liver cells 0.75g that gets liquid nitrogen cryopreservation, add Trizol reagent 7.5ml rapidly, after being ground to the liquid clarification, it is centrifugal to add equal-volume chloroform/primary isoamyl alcohol 12000rpm 10 minutes, draw supernatant, with the dehydrated alcohol post precipitation, be dissolved in the water of 50 μ l DEPC processing ,-20 ℃ of preservations are standby; (3) preparation of Endostatin (endostatin) cDNA: adopting reverse transcription polymerase chain reaction,PCR (RT-PCR) amplification endostatin gene, is template with extractive total RNA, carries out reverse transcription reaction, amplification cDNA first chain, and reaction system is:
RNA template 18 μ l
5 * damping fluid, 10 μ l
Few deoxythymidine Oligo (dT) 2.5 μ l (150ng)
Dithiothreitol (DTT) (DTT) 5 μ l (0.01M)
RNA enzyme inhibitors (Rnasin) 1.5 μ l (60U)
Reversed transcriptive enzyme (M-MuLV) 3 μ l (600U)
Deoxidation ATP, TTP, GTP, CTP (dNTP) 10 μ l (0.5mM)
Behind the mixing, 37 ℃, 100 ℃ of terminations in 5 minutes are put in amplification in 1 hour again, get reverse transcription reaction product 25 μ l, are primer with P1, P2, pcr amplification Endostatin (endostatin) cDNA, and reaction system is:
Reverse transcription (RT) reaction product 25 μ l
10 * damping fluid, 10 μ l
P1 2.5μl(0.5μM)
P2 2.5μl(0.5μM)
Deoxidation ATP, TTP, GTP, CTP (dNTP) 8 μ l (0.2 μ M)
Heat-resisting high-fidelity DNA polymerase (pfu Taq enzyme) 1 μ l (5u)
Supply distilled water to 100 μ l
Reaction conditions is: 95 ℃ of pre-sex change 5 minutes; 95 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, was extended 1 minute by 72 ℃; After carrying out 35 circulations, 72 ℃ were extended 5 minutes;
Do not add the negative contrast of reverse transcription (RT) reaction product; The recovery of (the results are shown in Figure 4) (4) Endostatins (endostatin) cDNA: the RT-PCR amplified production is through 2% agarose gel electrophoresis, downcut the target DNA band of 550bp, behind 25 ℃ ,-20 ℃ multigelations, place plasmid extraction box particulate (Pu Luomaige, Promega company product), behind centrifugal 5 minutes of the 10000rpm, collect filtered solution, add 2 times of volume dehydrated alcohols, place-20 ℃, after 30 minutes, under 4 ℃ of conditions of 12000rpm centrifugal 10 minutes, precipitation was the target DNA of purifying; (5) T-A of Endostatin (endostatin) gene clone: add dATP and Taq enzyme and each 1 μ l of 10 * damping fluid among the cDNA that 1 μ l is reclaimed, supply distilled water to 10 μ l, under 72 ℃ of conditions, extend and added the A tail in 15 minutes, and purifying and recycling cDNA; Get each 1 μ l of this kind goal gene DNA and pGEM-T carrier (Pu Luomaige, Promega company product), add T4 ligase enzyme 1 μ l, 10 * damping fluid, 1 μ l and distilled water 6 μ l under 4 ℃ of conditions, 17 hours, finish the T-A pairing; (6) screening of recombinant clone, evaluation: conventional preparation e. coli jm109 competence, get the connection product that 5. 2 μ l steps prepare, with CaCl
2Method transformed into escherichia coli JM109 competence, converted product is coated in the LA plate that contains 200mg/ml x-gal (5-bromo-4-chloro-3-indoles-β-D-semi-lactosi) 40 μ l and 20mg/ml IPTG (isopropyl-) 4 μ l, 8 single bacterium colonies of white of random choose.37 ℃ of activation are after 12 hours, and the extracting plasmid DNA is that primer carries out pcr amplification with P1, P2, select amplification male clone, the extracting plasmid DNA, and order-checking is identified; (7) pGAPZ
αThe amplification of A expression plasmid of yeast, extraction: e. coli jm109 activates back preparation competence routinely, gets pGAPZ
αA plasmid DNA (Invitrogen company product) 1 μ l, conventional CaCl
2Method transforms JM109, and the JM109 bacterium liquid after transforming is coated in the less salt LB flat board that contains 25 μ g/ml Zeocin (microbiotic, Invitrogen company product), get single bacterium colony after 20 hours, amplification, and with plasmid extraction test kit (Pu Luomaige, Promega company product) extracting plasmid;
The prescription of above-mentioned less salt LB flat board is: yeast extract 0.5%, and peptone 1%, NaCl 0.5%, agar 1.5%, pH7.5; (8) subclone of Endostatin (endostatin) gene: restriction enzyme EcoR I, Not I (Shanghai bio-engineering corporation product) double digestion respectively contain the cloned plasmids (pGEM-endostatin) and the empty expression vector pGAPZ of endostatin gene
αA, through 0.8% agarose electrophoresis, the expression vector dna gel piece of the target DNA of the about 550bp of recovery and 3kb is in the 1.5ml pipe respectively, behind-20 ℃, 25 ℃ multigelations three times, place plasmid extraction box particulate, 10000rpm is after centrifugal 5 minutes, collect filtered solution, add 2 times of volume dehydrated alcohols, place-20 ℃ after 30 minutes, under 4 ℃ of conditions of 12000rpm, centrifugal 10 minutes, deposit D NA added the rinsing of 1ml75% ethanol, under 4 ℃ of conditions of 12000rpm, centrifugal 10 minutes, abandon supernatant; The target DNA and the expression vector dna that reclaim are dissolved in the 8 μ l distilled waters together, add 1 μ l T4 ligase enzyme and 2 μ l, 5 * damping fluid, after room temperature connects 17 hours, get and connect product 10 μ l conversion JM109, and coat in the less salt LB flat board that contains 25 μ g/ml Zeocin, simultaneously to transform the pGAPZ that does not contain the endostatin gene
αThe A plasmid is as blank; (9) screening, the evaluation of Endostatin (endostatin) subclone: from Zeocin less salt LB flat board, select mono-clonal, be inoculated in the less salt LB substratum that contains Zeocin, 37 ℃ of lucifuges are cultivated, the extracting plasmid DNA, respectively with enzyme cut, the positive reorganization bacterium of the above-mentioned endostatin of the containing gene of PCR method screening, the extracting plasmid DNA, order-checking is identified; (the results are shown in Figure 5, Fig. 6)
The prescription of above-mentioned less salt LB substratum is: yeast extract 0.5%, and peptone 1%, NaCl 0.5%, pH7.5; (10) competence of yeast GS115 (Invitrogen company product) preparation: get Pichia yeast GS115 bacterial classification 10 μ l, be inoculated in the 2ml YPD substratum, 30 ℃ of shaking table vibrations were got GS115 bacterium liquid 100 μ l after 14 hours, be inoculated among the 100mlYPD 30 ℃ of shaking culture 14 hours; As bacterium liquid OD
600Be about at 1.4 o'clock, take out, centrifugal 7 minutes of 4 ℃ of 2500rpm precipitate resuspendedly with the 1M D-sorbyl alcohol of 200 μ l, put in the ice bath;
Above-mentioned YPD culture medium prescription is: yeast extract 1%, Tryptones 2%, D-glucose 2%; (11) electric transformation experiment: the expression of recombinant yeast plasmid pGAPZ that contains the endostatin gene with restriction enzyme A vr II (TaKaRa company product) single endonuclease digestion
αA-endostatin with preceding method, reclaims complete linearizing plasmid DNA with 0.8% agarose electrophoresis, is dissolved in the 10 μ l distilled waters; Getting GS115 competence bacteria liquid 80 μ l mixes with 10 μ l linearization plasmid DNA, behind the ice bath 5 minutes, the electric revolving cup that adds the 0.2cm specification, with electroporation (Bole company product), 1500V voltage electric shock, the 1M Sorbitol Solution USP that adds 1ml immediately, be transferred in the sterile tube, 30 ℃ leave standstill 1 hour 30 minutes kinds after, get 50,100,200,400 μ l respectively and coat on the YPDS plate that contains 100 μ g/ml Zeocin, 30 ℃ of lucifuges were cultivated after 3 days, simultaneously not contain the empty plasmid pGAPZ of endostatin
αThe A electricity transforms GS115 and compares, and the picking mono-clonal is to 1ml YPD, and 30 ℃ of shaking culture are to logarithmic phase, and-20 ℃ frozen;
The prescription of above-mentioned YPDS flat board is: yeast extract 1%, Tryptones 2%, D-glucose 2%, sorbyl alcohol 1M, agar 2%; (12) SDS-polyacrylamide gel electrophoresis screening positive expression bacterium: after the frozen bacterium 10 μ l of mono-clonal after electricity changes are activated, be inoculated in the 100ml YPD nutrient solution with 1: 100,30 ℃ of shaking culture, got bacterium liquid 5ml every 24 hours, remove bacterial sediment behind the high speed centrifugation, with the supernatant packing ,-20 ℃ of preservations are to be measured; Before the electrophoresis, get 500 μ l culture supernatant, add 2 times of volume dehydrated alcohols ,-20 ℃ 30 minutes, centrifugal 5 minutes of 12000rpm, the precipitation protein concentrate, albumen is dissolved in 20 μ l, 6 * load sample damping fluid after concentrating, after 100 ℃ of sex change in 3 minutes, be splined on 12% polyacrylamide gel, the 50mA electrophoresis; (13) purifying of Endostatin (endostatin) expressing protein: get positive yeast (name and be yeast B1-3) of reorganization and 2L conversion empty carrier pGAPZ that 6. the 2L step screens
αThe yeast of A (is named and is yeast SC3-1, the 4th day culture supernatant in contrast), respectively through 8000rpm after centrifugal 15 minutes, remove precipitation, supernatant concentrates (name respectively and be B1-3 concentrated solution and SC3-1 concentrated solution) for 20 times through ultrafiltration, Sepharose G25 post is earlier after 50mM NaCl 10mMTris.Cl balance, with sample on the concentrated solution, after crossing post, Sepharose G25 desalts and lower-molecular substance, collect protein peak, heparin (Heparin) affinity column (Fa Moxiya, Phamacia company product) by after the explanation installation, earlier through 50mM NaCl 10mM Tris.Cl balance, with sample on each crest behind the G25 wash-out, with 50mM～2M NaCl 10mMTris.Cl gradient buffering liquid wash-out, collect two protein peaks, wherein the protein peak of 1M NaCl 10mM Tris.Cl wash-out (naming the eluted protein for 1M) is human recombinant secretor type endostatin protein, and molecular weight is about: 32KD comprises at least 183 amino acid; 0.5M the protein peak of NaCl 10mM Tris.Cl wash-out (naming the eluted protein into 0.5M) is non-target protein.The preparation of (seeing Fig. 7, Fig. 8) (14) cornea rebirth blood vessel animal model: healthy new zealand white rabbit is got 12 rabbits, and is male, body weight 2-2.5kg, and eye is no abnormal.Eyes are tested simultaneously, through Patients Under Ketamine Anesthesia, and eye droppings before the tetracaine art, aseptic condition and microscopically, 2mm place seam is put 2 on the black silk thread of 5-0 in the corneal limbus of top, and spacing is 2mm, and suture is embedded in long 3mm in the corneal stroma.(15) suppress experiment in the body: above-mentioned 12 rabbits, divide 2 groups at random, 6 every group, 12 eyes.
1. human recombinant secretor type endostatin protein treatment group: postoperative same day, at sub-conjunctival injection 50 μ g/ml human recombinant secretor type endostatin protein 0.4ml, the next day once, continuous 8 times.
2. physiological saline control group: postoperative same day, at sub-conjunctival injection physiological saline 0.4ml, the next day once, continuous 8 times.
Establish the blank group in addition: 3 rabbits, totally 6 eyes are left intact.(16) observation index:
I. begin under slit-lamp microscope, to observe every day cornea rebirth blood vessel growing state, continuous 16 days behind the cornea suture.And under operating microscope, took the cornea rebirth blood vessel upgrowth situation in the 3rd day after surgery, the 4th day, the 5th day, the 7th day, the 10th day, the 13rd day, the 16th day with digital camera location, and directly import computer, new vessel is changed carry out image analysis.
II. postoperative was put to death rabbit on the 16th day, carried out eyeball excise, drew materials; Other gets the important organ heart, liver,kidney,spleen, lung, testis and send pathology, carries out light microscopy checking.
III. observe simultaneously after the medication that bulbar conjunctiva is whether congested, oedema, cornea has or not muddy oedema and aqueous humor situation.The result: blank group corneal transparency does not have blood vessel, and the treatment group is than physiological saline control group new vessel poor growth and more sparse (see Table 2 and Fig. 3).Treatment group bulbar conjunctiva is not seen hyperemia, oedema, and cornea is not seen muddiness, oedema yet.Pathological examination shows that treatment group corneal epithelium is normal, and new vessel is few around the hypothallus suture, and minority plasmocyte and inoblast are arranged.Physiological saline control group corneal epithelium is normal, and the hypothallus new vessel is many, and tube chamber is big, assembles a large amount of plasmocyte, and inoblast is few.
Pathological examination shows, other internal organs: the heart, liver,kidney,spleen, lung, testis, treatment group and physiological saline control group do not have obvious difference.Human recombinant secretor type endostatin protein can effectively suppress the growth of rabbit cornea new vessel, and eyeball and other important organ are not had obvious toxic-side effects.
Table 2 cornea rebirth blood vessel maximum growth area and the comparison (n=12) of long-living length
Group new vessel maximum growth area (mm
2) the most long-living length of new vessel (mm) Endostatin treatment group 10.88 ± 3.78 4.52 ± 1.08 physiological saline injection groups 24.83 ± 4.31 6.78 ± 2.25*P＜0.05
Claims (9)
1. a human recombinant secretor type endostatin protein is characterized in that, can make by following method:
(1) organizes extracted total RNA from fresh fetal liver cells;
(2) utilize Endostatin complementary DNA (cDNA) sequence, design upstream and downstream primer P1, P2 wherein add EcoR I restriction enzyme site in the upstream, and the downstream adds Not I restriction enzyme site;
(3) be template with above-mentioned extractive total RNA, carry out reverse transcription polymerase chain reaction,PCR (RT-PCR), obtain Endostatin cDNA;
(4) by gel electrophoresis, plasmid extraction, reclaiming by centrifuge Endostatin cDNA;
(5) with the cDNA of above-mentioned recovery, (pGEM-T) is connected with cloned plasmids, transformed into escherichia coli JM109.
(6) be that primer carries out pcr amplification with P1, P2, screening amplification male contains the clone of endostatin gene, the extracting plasmid DNA, and order-checking is identified;
(7) utilize restriction enzyme EcoR I, Not I double digestion to contain the cloned plasmids (pGEM-endostatin) of Endostatin (endostatin) gene with ordinary method, realize and pGAPZ
αThe connection of A expression plasmid of yeast;
(8) get the connection product transformed into escherichia coli JM109 of step 7, screening contains the positive recombinant expression vector engineering bacteria of Endostatin (endostatin) gene, the extracting plasmid DNA, and order-checking is identified;
(9) contain the expression of recombinant yeast plasmid pGAPZ of endostatin gene with restriction enzyme A vr II single endonuclease digestion
αA-endostatin, 0.8% agarose electrophoresis reclaims complete linearizing plasmid DNA, mixes with the competence bacteria liquid of yeast GS115 and carries out the electricity conversion;
(10) utilize SDS-polyacrylamide gel electrophoresis screening reorganization positive expression yeast;
(11) get above-mentioned reorganization positive expression yeast and cross with the heparin affinity chromatography post through Sepharose G25 that post separates, purifying, make Endostatin (endostatin) expressing protein, be human recombinant secretor type endostatin protein, molecular weight is about: 32KD comprises at least 183 amino acid.
2. human recombinant secretor type endostatin protein according to claim 1 is characterized in that, described reverse transcription polymerase chain reaction,PCR (RT-PCR), and amplification Endostatin cDNA the primer P1, P2 are:
P1：5’T?GGT?
GAA?TTC?CAC?AGC?CAC?CGC?GAC?TT?3’
EcoR?I
P2：5’TAA?
TGC?GGC?CGC?CTT?GGA?GGC?AGT?CAT?G?3’
Not?I
3. human recombinant secretor type endostatin protein according to claim 1 is characterized in that, the reaction conditions of described reverse transcription polymerase chain reaction,PCR (RT-PCR) is: 95 ℃ of pre-sex change 5 minutes; 95 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, 72 ℃ were extended 1 minute; After carrying out 35 circulations, 72 ℃ were extended 5 minutes.
4. human recombinant secretor type endostatin protein according to claim 1 is characterized in that, described yeast expressed carrier is pGAPZ
αThe A plasmid DNA.
5. the preparation method of a human recombinant secretor type endostatin protein as claimed in claim 1 comprises the step of following order:
(1) clone of endostatin gene:
1. design of primers:
According to Endostatin complementary DNA (cDNA) sequence, design upstream and downstream primer P1, P2 and restriction endonuclease sites wherein add EcoR I restriction enzyme site in the upstream, and the downstream adds Not I restriction enzyme site
P1：5’T?GGT?
GAA?TTC CAC?AGC?CAC?
CGC?GAC?TT?3’
EcoR?I
P2：5’TAA?
TGC?GGC?CGC?CTT?GGA?GGC?AGT?CAT?G?3’
Not?I
2. the extracting of the total RNA of tire hepatic tissue:
Get the fresh fetal liver cells 0.75g of liquid nitrogen cryopreservation, add Trizol reagent 7.5ml rapidly, be ground to liquid clarification after, it is centrifugal to add equal-volume chloroform/primary isoamyl alcohol 12000rpm 10 minutes, draws supernatant, with the dehydrated alcohol post precipitation, be dissolved in the water of 50 μ l DEPC processing ,-20 ℃ of preservations are standby;
3. the preparation of Endostatin (endostatin) cDNA:
Adopting reverse transcription polymerase chain reaction,PCR (RT-PCR) amplification endostatin gene, is template with extractive total RNA earlier, carries out reverse transcription reaction, amplification cDNA first chain, and reaction system is:
RNA template 18 μ l
5 * damping fluid, 10 μ l
Few deoxythymidine Oligo (dT) 2.5 μ l (150ng)
Dithiothreitol (DTT) (DTT) 5 μ l (0.01M)
RNA enzyme inhibitors (Rnasin) 1.5 μ l (60U)
Reversed transcriptive enzyme (M-MuLV) 3 μ l (600U)
Deoxidation ATP, TTP, GTP, CTP (dNTP) 10 μ l (0.5mM)
Behind the mixing, 37 ℃, 100 ℃ of terminations in 5 minutes are put in amplification in 1 hour again, get reverse transcription reaction product 25 μ l, are primer with P1, P2, pcr amplification Endostatin (endostatin) cDNA, and reaction system is:
Reverse transcription (RT) reaction product 25 μ l
10 * damping fluid, 10 μ l
P1 2.5μl(0.5μM)
P2 2.5μl(0.5μM)
Deoxidation ATP, TTP, GTP, CTP (dNTP) 8 μ l (0.2 μ M)
Heat-resisting high-fidelity DNA polymerase (pfu Taq enzyme) 1 μ l (5u)
Supply distilled water to 100 μ l
Reaction conditions is: 95 ℃ of pre-sex change 5 minutes; 95 ℃ of sex change 1 minute, 65 ℃ of annealing 1 minute, was extended 1 minute by 72 ℃; After carrying out 35 circulations, 72 ℃ were extended 5 minutes;
Do not add the negative contrast of reverse transcription (RT) reaction product;
4. the recovery of Endostatin (endostatin) cDNA:
The RT-PCR amplified production is through 2% agarose gel electrophoresis, downcut the target DNA band of 550bp, behind 25 ℃ ,-20 ℃ multigelations, place plasmid extraction box particulate, 10000rpm is after centrifugal 5 minutes, collect filtered solution, add 2 times of volume dehydrated alcohols, place-20 ℃, after 30 minutes, under 4 ℃ of conditions of 12000rpm centrifugal 10 minutes, precipitation was the target DNA of purifying;
5. the T-A of Endostatin (endostatin) gene clones:
Add dATP and Taq enzyme and each 1 μ l of 10 * damping fluid among the cDNA that 1-2 μ l is reclaimed, supply distilled water to 10 μ l, under 72 ℃ of conditions, extend and added the A tail in 15 minutes, and purifying and recycling cDNA; Get each 1 μ l of this kind goal gene DNA and pGEM-T carrier, add T4 ligase enzyme 1 μ l, 10 * damping fluid, 1 μ l and distilled water 6 μ l under 4 ℃ of conditions, 16-18 hour, finish the T-A pairing;
6. the screening of recombinant clone, evaluation:
Conventional preparation e. coli jm109 competence is got the connection product that 5. 2 μ l steps prepare, with CaCl
2Method transformed into escherichia coli JM109 competence, converted product is coated in the LA plate that contains 200mg/ml x-gal (5-bromo-4-chloro-3-indoles-β-D-semi-lactosi) 40 μ l and 20mg/ml IPTG (isopropyl-) 4 μ l, 8 single bacterium colonies of white of random choose.37 ℃ of activation are after 12 hours, and the extracting plasmid DNA is that primer carries out pcr amplification with P1, P2, select amplification male clone, the extracting plasmid DNA, and order-checking is identified;
(2) Endostatin (endostatin) expression of gene
1. pGAPZ
αThe amplification of A expression plasmid of yeast, extraction:
E. coli jm109 activates back preparation competence routinely, gets pGAPZ
αA plasmid DNA 1 μ l, conventional CaCl
2Method transforms JM109, and the JM109 bacterium liquid after transforming is coated in the less salt LB flat board that contains 25 μ g/ml Zeocin, gets single bacterium colony after 18-22 hour, amplification, and with plasmid extraction test kit extracting plasmid;
The prescription of above-mentioned less salt LB flat board is: yeast extract 0.5%, and peptone 1%, NaCl 0.5%, agar 1.5%, pH7.5;
2. the subclone of Endostatin (endostatin) gene:
Restriction enzyme Not I, EcoR I double digestion respectively contain the cloned plasmids (pGEM-endostatin) and the empty expression vector pGAPZ of endostatin gene
αA, through 0.8% agarose electrophoresis, the expression vector dna gel piece of the target DNA of the about 550bp of recovery and 3kb is in the 1.5ml pipe respectively, behind-20 ℃, 25 ℃ multigelations three times, place plasmid extraction box particulate, 10000rpm is after centrifugal 5 minutes, collect filtered solution, add 2 times of volume dehydrated alcohols, place-20 ℃ after 30 minutes, under 4 ℃ of conditions of 12000rpm, centrifugal 10 minutes, deposit D NA added the rinsing of 1ml75% ethanol, under 4 ℃ of conditions of 12000rpm, centrifugal 10 minutes, abandon supernatant; The target DNA and the expression vector dna that reclaim are dissolved in the 8 μ l distilled waters together, add 1 μ l T4 ligase enzyme and 2 μ l, 5 * damping fluid, after room temperature connects 16-18 hour, get and connect product 10 μ l conversion JM109, and coat in the less salt LB flat board that contains 25 μ g/ml Zeocin, simultaneously to transform the pGAPZ that does not contain the endostatin gene
αThe A plasmid is as blank;
3. screening, the evaluation of Endostatin (endostatin) subclone:
Select mono-clonal from Zeocin less salt LB flat board, be inoculated in the less salt LB substratum that contains Zeocin, 37 ℃ of lucifuges are cultivated, the extracting plasmid DNA, respectively with the positive reorganization bacterium that enzyme is cut, the PCR method is screened the above-mentioned endostatin of containing gene, extracting plasmid DNA, order-checking evaluation;
The prescription of above-mentioned less salt LB substratum is: yeast extract 0.5%, peptone 1%, NaCl0.5%, pH7.5;
4. the competence of yeast GS115 preparation:
Get Pichia yeast GS115 bacterial classification 10 μ l, be inoculated in the 2ml YPD substratum, the vibration of 30 ℃ of shaking tables was got GS115 bacterium liquid 100 μ l after 12-16 hour, be inoculated among the 100mlYPD, 30 ℃ shaking culture 12-16 hour; When bacterium liquid OD600 is 1.3～1.5, take out, centrifugal 7 minutes of 4 ℃ of 2500rpm precipitate resuspendedly with the 1M D-sorbyl alcohol of 200 μ l, put in the ice bath;
Above-mentioned YPD culture medium prescription is: yeast extract 1%, Tryptones 2%, D-glucose 2%;
5. electric transformation experiment:
The expression of recombinant yeast plasmid pGAPZ that contains the endostatin gene with restriction enzyme A vr II single endonuclease digestion
αA-endostatin with preceding method, reclaims complete linearizing plasmid DNA with 0.8% agarose electrophoresis, is dissolved in the 10 μ l distilled waters; Getting GS115 competence bacteria liquid 80 μ l mixes with 10 μ l linearization plasmid DNA, behind the ice bath 5 minutes, the electric revolving cup that adds the 0.2cm specification, with electroporation, 1500V voltage electric shock, the 1M Sorbitol Solution USP that adds 1ml immediately, be transferred in the sterile tube, 30 ℃ leave standstill 1-2 hour after, get 50,100,200,400 μ l respectively and coat on the YPDS plate that contains 100 μ g/ml Zeocin, 30 ℃ of lucifuges were cultivated after three days, simultaneously not contain the empty plasmid pGAPZ of endostatin
αThe A electricity transforms GS115 and compares, and the picking mono-clonal is to 1ml YPD, and 30 ℃ of shaking culture are to logarithmic phase, and-20 ℃ frozen;
The prescription of above-mentioned YPDS flat board is: yeast extract 1%, Tryptones 2%, D-glucose 2%, sorbyl alcohol 1M, agar 2%;
6. the SDS-polyacrylamide gel electrophoresis screens the positive expression bacterium:
After the frozen bacterium 10 μ l of mono-clonal after electricity changes are activated, be inoculated in the 100ml YPD nutrient solution with 1: 100,30 ℃ of shaking culture were got bacterium liquid 5ml every 24 hours, remove bacterial sediment behind the high speed centrifugation, and with the supernatant packing ,-20 ℃ of preservations are to be measured; Before the electrophoresis, get 500 μ l culture supernatant, add 2 times of volume dehydrated alcohols ,-20 ℃ 30 minutes, centrifugal 5 minutes of 12000rpm, the precipitation protein concentrate, albumen is dissolved in 20 μ l, 6 * load sample damping fluid after concentrating, after 100 ℃ of sex change in 3 minutes, be splined on 12% polyacrylamide gel, the 50mA electrophoresis;
7. the purifying of endostatin expressing protein:
Get positive yeast of reorganization and 2L conversion empty carrier pGAPZ that 6. the 2L step screens
αThe 4th day culture supernatant of the yeast of A, respectively through 8000rpm after centrifugal 15 minutes, remove precipitation, supernatant concentrates for 20 times through ultrafiltration, Sepharose G25 post is earlier after 50mM NaCl 10mM Tris.Cl balance, with sample on the concentrated solution, after crossing post, Sepharose G25 desalts and lower-molecular substance, collect protein peak, after the heparin affinity chromatography post is installed by explanation, earlier through 50mM NaCl 10mMTris.Cl balance, with sample on each crest behind the G25 wash-out, with 50mM-2M NaCl 10mM Tris.Cl gradient buffering liquid wash-out, collect two protein peaks, wherein the protein peak of 1M NaCl 10mM Tris.Cl wash-out is human recombinant secretor type endostatin protein, and molecular weight is about: 32KD comprises at least 183 amino acid; 0.5M the protein peak of NaCl 10mM Tris.Cl wash-out is non-target protein.
6. the application of any one described human recombinant secretor type endostatin protein in the medicine of preparation treatment eye neovascular diseases among the claim 1-5.
7. the application of human recombinant secretor type endostatin protein according to claim 6 in the medicine of preparation treatment cornea rebirth blood vessel disease.
8. human recombinant secretor type endostatin protein according to claim 7 is in the medicine of preparation treatment cornea rebirth blood vessel disease, and employed effective concentration is 40 μ g/ml-60 μ g/ml.
9. human recombinant secretor type endostatin protein according to claim 8 is in the medicine of preparation treatment cornea rebirth blood vessel disease, and the scope of particularly suitable is a mammals.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100494376C (en) * | 2005-09-23 | 2009-06-03 | 四川大学 | Recombinant human esoderma colyone adenovirus, and its preparing method and use |
| CN100593420C (en) * | 2006-04-26 | 2010-03-10 | 山东大学 | Endostatin conjugate and its preparation method |
| CN105440138A (en) * | 2014-09-30 | 2016-03-30 | 复旦大学 | Preparation method for long-acting endostatin fusion protein and use |
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2002
- 2002-09-26 CN CN 02135574 patent/CN1401785A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100494376C (en) * | 2005-09-23 | 2009-06-03 | 四川大学 | Recombinant human esoderma colyone adenovirus, and its preparing method and use |
| CN100593420C (en) * | 2006-04-26 | 2010-03-10 | 山东大学 | Endostatin conjugate and its preparation method |
| CN105440138A (en) * | 2014-09-30 | 2016-03-30 | 复旦大学 | Preparation method for long-acting endostatin fusion protein and use |
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