CN1420126A - Method for preparing recombinant human plasminogen kringle 5 mutant protein rhpk-5 - Google Patents

Method for preparing recombinant human plasminogen kringle 5 mutant protein rhpk-5 Download PDF

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CN1420126A
CN1420126A CN 02123925 CN02123925A CN1420126A CN 1420126 A CN1420126 A CN 1420126A CN 02123925 CN02123925 CN 02123925 CN 02123925 A CN02123925 A CN 02123925A CN 1420126 A CN1420126 A CN 1420126A
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rhpk
mutant
kringle
exist
preparation
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牛勃
陈显久
杨椅
杨涛
常冰梅
张东昌
李乐工
韩锐
阎占清
郭勇
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SHANGHAI KELUN BIO-TECH Co Ltd
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牛勃
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Abstract

A process for preparing recombinant human plasminogen kringle 5 (rhPK-5) and its mutant includes designing the gene of rhPK-5 by genetic engineering and protein engineering, constructing prokaryotic expression vector pBV220/rhPK-5, and respectively transforming 4 kinds of colibacillus to obtain their expressions. The resultant rhPK-5 has the bioactivity to suppress the neogenesis of blood vessel.

Description

The preparation method of a kind of recombinant human plasminogen Kringle 5 mutant protein rhPK-5
Technical field:
The present invention relates to the production technique of a kind of recombinant human plasminogen Kringle 5 (rhPK-5) mutant.This technology be utilize genetic engineering technique make up the rhPK-5 mutant prokaryotic expression carrier and cloning vector, utilize that microbial fermentation technology obtains the rhPK-5 mutant efficiently express, utilize that protein engineering obtains the pure product albumen of rhPK-5 mutant, it has wild activity to utilize the chick chorioallantoic membrane experiment confirm, thereby realized obtaining a kind of novel method of eucaryon mutator gene product with prokaryotic expression system, use this technology and can prepare the rhPK-5 mutant protein in a large number, verified this mutant protein has the wild activity that angiogenesis suppresses.
Background of invention:
Human plasminogen (human Plasminogen, hPgn) constituting essentially identical five Kringle loop domains by amino acid forms, each structural domain is formed by about 80 amino acid, and the hPK-5 factor is the 5th structural domain of Profibrinolysin.1994, O ' Reilly (O ' Reilly M.S. and Holmgren L., Cell, 1994S1,79 (2): 315-328) from mice with tumor serum and urine, extract Angiostatin (hPK1-4), can suppress a kind of endogenous albumen of vascular endothelial proliferation specifically.Studies show that the hPK-5 factor has the biologic activity that suppresses endotheli ocytosis, and its inhibition activity is better than Angiostatin[Cao Y. and Ji R.W., J.Biol.Chem., 1996,271:29461-29467, Cao Y. and Chen A., J.Biol.Chem., 1997,272:22924-22928, and O ' Reilly M.S. and Holmgren L., Cell, 1994 S1,79 (2): 315-328]; The hPK-5 factor also has biologic activity (Ji W-R. and the Barrientos L.G. that suppresses endothelial cell migration, Biochem.Biophys.Rrs.Comm., 1998,247:414-419 and Lu H. and Dhanabal M., Biochem.Biophys.Rrs.Comm., 1999,285:668-673).Have characteristics such as high reactivity, cellular type selectivity, short amino acid sequence just because of the hPK-5 factor, this factor is regarded as a kind of medicine [Cao Y. and Chen A. of potential neovascular diseases always, J.Biol.Chem., 1997,272:22924-22928, and Ji W-R., with Barrientos L.G., Biochem.Biophys.Rrs.Comm., 1998,247:414-419], particularly along with the successful exploitation of the U.S., more make the research of this factor apparent more important to neovascularization inhibitor-Endostatin.
Vascularization promptly forms the process of neovascularity, and it is to comprise that the normal body activity of reproduction, growth and wound repair is necessary.Although do not understand this process fully, it may comprise the molecule complex interactions of regulating endotheliocyte (primary cell of capillary vessel) growth.As if under normal operation, these molecules remain on microvasculature stationary state (promptly without any the capillary vessel growth), its hold-time can last up to several weeks, reaches the several years in some cases.(as during wound repair) in case of necessity, these molecules can experience fast breeding and conversion (Folkman in 5 days, J. and Shing, Y.The Journal of Biological Chemistry, 267 (16): 10931-10934, and Folkman, J. and Klagsbrum, M., Science, 1987 (135): 442-447).
Although vasculogenesis is the process for highly being regulated under normal circumstances, persistent non-adjusting vasculogenesis can cause numerous disease (is principal character with the vasculogenesis).Other has report, and the vasculogenesis of non-adjusting can cause specific disease, can make also that existing sb.'s illness took a turn for the worse.The clinical study result confirms, the common cause of patient's blinds such as diabetes is generations of eye new vessel, and this cause of disease is being controlled the generation of about 20 kinds of illness in eye.In arthritic development, new capillary vessel is invaded the joint and is destroyed cartilage and will make that sb.'s illness took a turn for the worse.
At present, developing several angiogenesis inhibitors (Gasparini, G. and Harris, the A.L. that is used for the treatment of the vasculogenesis disease, Clin, Oncol., 1995,13 (3): 765-782), but these compounds have some relevant shortcomings, and for example, Suramine is a kind of effective inhibitors.But under the required dosage of anti-tumor activity, cause the systemic toxicity that the people is serious; It is safe that the compound of Retinoid, Interferon, rabbit and estrogen antagonist and so on uses for the mankind, but has weak blood vessel formation against function; It is higher that other compound may be difficult to the cost for preparing or prepare.
From the angiogenesis inhibitor present Research, the angiogenesis inhibitor of exploitation high reactivity, hypotoxicity (high security) is the class medicine that present clinical treatment blood vessel hyperplasia class disease is badly in need of.
Recombinant human plasminogen Kringle 5 mutant proteins of the present invention (rhPK-5) are by optionally suppressing the propagation of new vessel endotheliocyte; simultaneously normal body cell is not had or hypotoxicity; pathological state is shown selective toxicity; and a large amount of easily preparations of this compound, for its clinical application is laid a good foundation.
Summary of the invention:
In facility scheme of the present invention, gene order, the aminoacid sequence of rhPK-5 are provided, make up the method for its expression vector, the optimization that obtains to express the engineering strain preparation method of rhPK-5 and efficiently express condition, the separation method of rhPK-5, purification process, refolding method and the experimental technique of measuring its angiogenesis inhibiting.
Kringle 5 described herein is meant that plasminogen in the mammalian plasma (is called for short Profibrinolysin, Plasminogen) the 5th loop domain, its space structure resembles a kind of cake Kringle of Denmark, so this albumen is called as Kringle 5 (Fig. 3).The present invention utilizes this protein factor of genetically engineered recombinant technology preparation, keep Kringle space conformation be to be positioned at Cys 460-Cys 510, Cys 482-Cys 523, Cys 511-Cys 535Three disulfide linkage (Fig. 4).
RhPK-5 of the present invention is that the gene order with Profibrinolysin in the human plasma is a template, and through after the genetic modification of multiple mode, the albumen based on sequence shown in Figure 1 of expressing in prokaryotic cell prokaryocyte (intestinal bacteria etc.), preparing is as described in embodiment 1.
RhPK-5 expression vector of the present invention is meant the rhPK-5 gene or passes through the recombinant plasmid expression vector that improved gene clone obtains behind expression vector such as pBV220.
Engineering cell strain of the present invention is meant that methods such as utilizing calcium chloride transformation changes the rhPK-5 expression vector over to prokaryotic cell prokaryocyte strain that the body that obtains in prokaryotic cell prokaryocyte (as the intestinal bacteria) body contains the rhPK-5 expression vector, and this prokaryotic cell prokaryocyte strain can give expression to rhPK-5 albumen or mutain under given conditions.
Expression condition of the present invention is meant the optimization external conditions of engineering cell strain expression rhPK-5 albumen or mutain, comprises engineering cell culture condition (pH, dissolved oxygen, incubation time etc.), engineering cell inductive condition (inducing temperature, inducing sustained time etc.).
Mutant preparation technology of the present invention is meant with rhPK-5 albumen or mutain the preparation technology of separation from obtain the engineering cell strain after rhPK-5 albumen or mutain are expressed, purifying, specifically comprises the separation and the purifying of the proteic removal of fragmentation, impurity, rhPK-5 albumen or the mutain of engineering cell strain.
The Determination of biological activity of angiogenesis inhibiting of the present invention is meant that with the rhPK-5 albumen of prepared of the present invention or mutain be experiment material, measures the biologic activity of the angiogenesis inhibiting of the rhPK-5 albumen of prepared of the present invention or mutain with chick chorioallantoic membrane experiment etc.
The preparation of embodiment 1:pBV220/hPK-5 recombinant plasmid expression vector
PCR design of primers: according to the gene order of the hPK-5 factor shown in Figure 2, design following primer: primer 1 is: 5 '-GCGAATTCCATGGACTGTATGTTT GGG-3 ', primer 2 is: 5 '-GCGGATCCCTATTAGTGGTGGTGGTGGTGGTGGGCACACTGAGGGAC-3 '.Wherein, GAATTC and GGAT CC are the restriction enzyme site of restriction enzyme EcoRI and BamHI; ATG is an initiator codon; TAATAG is a terminator codon; GTGGTGGTGGT GGTGGTG4 is 6 Histidine sequences; 5 ' GC is the protection sequence; GACTGTAT GTTTGGG and GGC ACACTGAGGGAC are exogenous gene sequence.
Pcr amplification hPK-5 factor gene: reaction system is: hPK-5 dna profiling 5ul, 4 * dNTPs 2ul, primer 1 1ul, primer 2 1ul, 10 * Buffer 2ul, Taq enzyme 1ul, dH 2O 8ul; Reaction conditions is: 95 ℃ of 10min, 94 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 1min, 30 circulations back 72 ℃, 1min.The PCR product carries out 2% agarose gel electrophoresis analysis (Fig. 5).
HPK-5 gene EcoRI/BamHI double digestion: reaction system: enzyme is cut substrate 5ul, 10 * Buffer 2ul, BSA 0.5ul, dH 2O 11.5ul, EcoRI 1ul.Mixing, slightly centrifugal, put 37 ℃ of water-bath 2h, dehydrated alcohol precipitation 1h, 1, the centrifugal 5min of 2000r/min abandons supernatant, and evaporation residue ethanol adds 10 * Buffer 2ul again, BSA 0.5ul, dH 2O 11.5ul, BamHI 1ul, mixing, slightly centrifugal, put 37 ℃ of water-bath 2h, the endonuclease reaction product all carries out 2% sepharose (LMP) electrophoresis.
The piece that digs of endonuclease bamhi reclaims and purifying: plasmid pBV220 forms open loop pBV220, flexible glue recovery gene fragment with the EcoRI/BamHI double digestion, and enzyme is cut product and analyzed with agarose gel electrophoresis, (Fig. 6).
HPK-5 gene endonuclease bamhi is connected with open loop pBV220's: reaction system is: 10 * T 4Connect Buffer 5ul, pBV220 open loop 2ul, the hPK-5 enzyme is cut gene fragment 1ul, T 4Dna ligase 1ul, dH 2O 6ul, slightly centrifugal behind the mixing, 4 ℃ are spent the night.
Flexible glue reclaims the recombinant plasmid gene fragment: the recombinant plasmid gene fragment called after pBV220/hPK-5 (Fig. 7) that recovery is obtained.
Embodiment 2: preparing the competent cell of multiple prokaryotic cell prokaryocyte strain, is example with colibacillary a kind of bacterial strain JM109.
The JM109 bacterial strain is rule on the nutrient agar flat board, and 37 ℃, 230r/min spend the night; The single bacterium colony that takes out 1 2-3mm size from flat board moves in the test tube that contains 50ml LB nutrient solution 37 ℃, 230rpm, 3h to OD600=0.2-0.4 (logarithmic phase); Put 20min on ice, change in the centrifuge tube, the centrifugal 10min of 4000r/min; Abandon supernatant, be inverted centrifuge tube 1min and flow to end remaining liq, add the ice-cold 0.1MCaCl of 10ml then 2Sensitization liquid suspension cell, 15min on ice; The centrifugal 10min of 4000r/min reclaims cell, abandons supernatant, is inverted 1min; The 0.1M CaCl of 2ml precooling on ice 2Suspension cell; According to the 200ul/Eppendorf packing; Every pipe adds 80% glycerine 29ul to final concentration 10%, mark, put-70 ℃ frozen; Can reserve 2 pipes as if conversion pBV220/hPK-5 in 24h places 4 ℃ of refrigerators to preserve.
Embodiment 3: expression plasmid carrier pBV220/hPK-5 is transformed the competent cell strain of above-mentioned preparation
Get each two pipe of JM109 competence bacteria, wherein a pipe adds reorganization plasmid (1 μ g/ μ L) 1 μ L, and other pipe adds empty plasmid pBV220 (2 μ g/ μ L) 1 μ L and compares, and thrum makes it mixing gently, places 30min on ice bath; 90s is left standstill in 42 ℃ of water-baths; Every pipe adds LB substratum 800 μ L, mixing, and 1h is placed in 37 ℃ of water-baths; Each pipe is got 200 μ L respectively, be tiled in and contain penbritin (60 μ g/mL) LB agar plate surface, behind the placing flat 20min, be inverted and cultivate more than the 12h, treat that bacterium colony forms the back and takes out 4 ℃ of preservations.
Embodiment 4: cultivate, induce engineering cell strain, express recombinant hPK-5 albumen or reorganization hPK-5 mutant protein.
With transfering loop picking list bacterium colony from the LB culture plate of the engineering bacteria JM109 that contains hPK-5 factor expression carrier, be inoculated in the test tube of the 5mL LB substratum that contains Amp (100mg/L), on 37 ℃ of shaking tables, with the 230r/min shaken overnight; Above-mentioned bacterium is changed over to respectively in the 250mL triangular flask that contains 50mL LB substratum with 1: 50 ratio, cultivated 4 hours for 30 ℃, rising culture temperature to 42 ℃ continues to cultivate 4 hours; Wash with STE after getting medium centrifugal, 1mL TE is resuspended, gets 20 μ L, add equal-volume 2 * sample-loading buffer (100mmol/L Tris-Cl, 200mmol/L DTT, 4% SDS, 0.2% bromjophenol blue, 20% glycerine, pH6.8) mixing, boil 3min in the boiling water bath, last sample 20 μ L carry out 15% SDS-PAGE electrophoresis (Fig. 8) (electrophoretic buffer: 25mmol/LTris-Cl, 250mmol/L glycine, 0.1% SDS, pH8.3).Deposition condition: 110V, about 6h adds the cold water circulation.
Embodiment 5: the Immunological Identification of expression product
Press document (J. Sa nurse Brooker, EF. Ritchie not, T. Manny A Disi chief editor, gold winter wild goose Li Meng maple is translated, molecular cloning experiment guide [M], Beijing: Science Press, 1999, second edition: membrane-transferring device (transfering buffering liquid: 25mmol/LTris is installed 681-683), the 192mmol/L glycine, 20% methyl alcohol), change the film condition: 9mA, 1-2h; Deionization washing film 2 times, each 10min; Nitrocellulose filter is placed in the plate, adds confining liquid (10mmol/L Tris-Cl, 100mmol/L NaCl, pH8.0,5% skim-milk) and soak film, room temperature is swayed 2h; Combine with first antibody: polyclone first antibody (His-Tag Monoclonal Antibody purchases the Novagen company in the U.S.) is pressed 1: 1000 dilution proportion, adds the 0.1mL ratio in every square centimeter and adds an anti-liquid, and room temperature is swayed 2h; Washing: wash 3 times each 10min with TBST (10mmol/L Tris-Cl, 100mmol/L NaCl, pH8.0,0.1% Tween-20); Combine with second antibody: change film over to second antibody (goat anti-mouse Ig-G-Ap, purchase in precious biotechnology (Dalian) company limited) in the solution (the doubling dilution second antibody was in confining liquid in 1: 200), 4 ℃ are swayed 5h, after the taking-up, wash 3 times each 10min with TBST; Alkaline phosphatase colour developing: get 0.5g NBT (nitroblue tetrazolium(NBT) nitroblue terazolium) and be dissolved in the dimethyl formamide of 10ml 70%, getting 0.5g BCIP (5 '-bromo-4-chloro-3-indoxyl phosphate) is dissolved in the dimethyl formamide of 10ml 70%, in 10ml alkaline phosphatase damping fluid (100mM NaCl, 5mM MgCl2,100mM TrisCl), add BCIP 4ul, NBT 4ul, when treating that band reaches desired depth, wash film at once with water, (Fig. 9).
Embodiment 6: adopt ultrasonic method to carry out cytoclasis
Cell culture fluid is at 4 ℃, and 12, the centrifugal supernatant of abandoning of 000r/min * 20min; Bacterial sediment is resuspended in the lysis buffer (50mM Tris-Cl pH8.0,100mM NaCl, 1mM EDTA) with 1: 10 ratio, and ice-bath ultrasonic is broken bacterium, 250W, 3 seconds/time, 3 seconds at interval, ultrasonic 200 times.
Embodiment 7: separate rhPK-5 albumen or its mutant protein from total protein
Cell culture fluid is at 4 ℃, and 12, the centrifugal supernatant of abandoning of 000r/min * 20min; Bacterial sediment is resuspended in the lysis buffer (50mM Tris-Cl pH8.0,100mM NaCl, 1mM EDTA) with 1: 10 ratio, and ice-bath ultrasonic is broken bacterium, 250W, 3 seconds/time, 3 seconds at interval, ultrasonic 200 times.
With Eppendorf centrifuge in 4 ℃, 12,000r/min * 20min is centrifugal; Inclusion body precipitation is resuspended in the lysis buffer that 9 times of volumes contain 0.5%Triton X-100 and 10mM EDTA (pH8.0) the static 5min of room temperature; 10000r/min * 20min is centrifugal with Eppendorf centrifuge; Repeat secondary, preparation inclusion body precipitation is used for following experiment.Deposit sample is got each 1ml of urea soln that adds 5M successively, suspension, static 10min; 4 ℃, 10,000r/min * 15min is centrifugal, repeats 1-3 time.
Last step inclusion body deposit sample adds each 1ml of Septochol sodium solution of 1-3%, suspension, static 10min; 4 ℃, 10,000r/min * 15min, centrifuging and taking precipitation, supernatant prepare the SDS-PAGE sample, (Figure 10).
Embodiment 8: with the Histidine affinity chromatography target protein is carried out purifying
Use the rhPK-5 albumen of this technology of preparing acquisition or the amino acid chain that mutant protein is made up of 3-10 Histidine at the carboxyl terminal of its aminoacid sequence.Can adopt the Histidine affinity chromatography that target protein is carried out purifying according to this feature.His-tag colum (280nm monitoring, flow rate of liquid is 0.5ml/min) uses Buffer A (1M urea, 100mM NaH 2PO 4, 10mM Tris-Cl, pH8.0) balance to baseline steadily after, the solubilization of inclusion bodies supernatant is directly gone up sample, the about 1mg/ml of protein concentration, last sample volume is 1ml/ time; With Buffer B (1M urea, 100mM NaH 2PO 4, 10mM Tris-Cl, pH6.3) the non-specific combination albumen of wash-out, steady once more until baseline; With Buffer C (1M urea, 100mM NaH 2PO 4, 10mM Tris-Cl, pH5.9) the rhPK-5 factor of elution of bound on Ni2+, the fraction collection elution peak; Continue wash-out, fraction collection elution peak with Buffer D (Buffer C, 250mM imidazoles); With Buffer E (1M urea, 100mM NaH 2PO 4, 10mM Tris-Cl, pH4.5) continue wash-out, steady until no baseline.
The Determination of biological activity of embodiment 9:rhPK-5 albumen or mutain angiogenesis inhibiting
Levy (Jiang Zhigang with reference to Jiang Zhigang, Hubei Province, E Zheng, the anatomy magazine, 1989, method 12:142-143) is with 0.5% methylcellulose gum membrane prepare, experiment component is crude samples group, the pure product of the rhPK-5 factor not renaturation group, the pure product renaturation of rhPK-5 factor group, establishes PBS group and solubilization of inclusion bodies, the negative control group of diluent group.The chicken embryo is hatched: after the benzalkonium bromide with 0.1% cleans new freshly-slaughtered poultry embryo, egg is put into 38-40 ℃, the incubator routine of 60% relative humidity hatches, air chamber up, egg major axis and egg holder are 80 and spend angles.Every day, egg-turning was 2-3 time.To the 7th day, the chicken embryo that selection survives had a distance to determine the position of windowing at two main intersecting blood vessels points of chorioallantoic membrane and from idiosome.Window: open an aperture decompression earlier on the air chamber top, after the position sterilization of determining, open the fenestella of about a 0.5 * 0.5cm then, carefully start shell membrane, expose the chorioallantoic membrane and the blood vessel thereof of its below.Apply tester: the methylcellulose gum film that will prepare places the test zone, seals fenestella with scotch tape.Continue to hatch to 10 days.Each dosage group guarantees to survive 8 chicken embryos at least.Get film and take pictures: add behind the test membrane the 3rd day, remove the above shell membrane in CAM plane, adds 1: 1 methanol/acetone stationary liquid 1.5ml/, room temperature is fixedly behind the 15min, treats that blood coagulation cuts chorioallantoic membrane, puts into water and launches.Chorioallantoic membrane is layered on the sheet glass, flattens with another sheet glass more above, under dissecting microscope, observe and take pictures.The result observes: with total, the large, medium and small number of blood vessel of dissecting microscope observation, counting and carrier plate edge contact, according to caliber blood vessel is divided three classes (Wang Lei, Zhang Shucheng, Wu Zhikui, etc., Chinese pharmacology and clinical, 2000,16 (6): 46-47): 1.＞0.1mm is a great vessels; 0.1mm＞1.＞0.05mm is a medium vessels; 1.＜0.05mm is little blood vessel.The blood vessel that goes out from the trunk branch is called the one-level blood vessel, from one-level again branch be the secondary blood vessel, and the like be three grades, level Four ..., record blood vessel classification situation.With the checking matter film is the center, and it is converge like the spokes of a wheel at the hub that the radial growth conditions towards checking matter of surrounding blood vessel is called blood vessel; Major blood vessel is to the crooked of loading film with near the attraction that is referred to as blood vessel.Observe around the loading film in the 1cm scope and vessel density away from (greater than 1cm) position, the chicken embryo number of blood vessel hyperplasia or inhibition appears in counting, and data are carried out variance analysis (table 1), (table 2), (Figure 11).
Table 1 rhPK-5 is to the one-level blood vessel, and the inhibitory action group N one-level blood vessel of secondary blood vessel number is counted the secondary blood vessel and counted PBS 8 2.46 ± 0.23 11.78 ± 3.56 solubilization of inclusion bodies, dilution group 8 2.51 ± 0.68 10.69 ± 4.27rhPK-5 crude product 25 2.09 ± 0.47 12.76 ± 4.58rhPK-5 sterling, 30 1.15 ± 0.24*, 7.21 ± 2.53**P＜0.05
With the pharmaceutical carrier film is the center, one-level, the secondary blood vessel number of counting and its side edge, compare and find relatively there was no significant difference of rhPK-5 crude product group and control group (PBS group, solubilization of inclusion bodies diluent group), pure product group of rhPK-5 and control group relatively have significant difference (P＜0.05).The effect that rhPK-5 factor angiogenesis inhibiting after the renaturation is described is remarkable.
Table 2 rhPK-5 is to greatly, in, little blood vessel is counted the little blood vessel PBS 8 5.80 of restraining effect group N great vessels medium vessels ± 2.20 5.50 ± 2.18 81.00 ± 11.56 solubilization of inclusion bodies, diluent group 8 5.63 ± 2.31 5.38 ± 2.54 79.26 ± 10.89hPK-5 crude products 25 4.95 ± pure product 30 5.61 of 3.24 4.14 ± 3.26 68.81 ± 12.53hPK-5 ± 1.10 2.02 ± 1.35*, 38.68 ± 10.52**P＜0.05
The middle or small blood vessel number of control group is obviously more, with experimental group (the pure product group of rhPK-5) significant difference is arranged relatively, and great vessels is counted there was no significant difference, illustrates that the rhPK-5 factor mainly suppresses the generation of middle or small blood vessel, and rhPK-5 crude product group and control group be not statistically significant relatively.
The preparation of embodiment 10:rhPK-5 mutant protein
With Kringle shown in Figure 15 for substantially series, press the experimental technique of embodiment 1, the A that arbitrary amino acid series can be arranged in its aminoterminal design, or the B of arbitrary amino acid series arranged in carboxyl terminal design, with aminoacid sequence shown in Figure 12 (1), base sequence shown in Figure 13 is example (2), makes up the rhPK-5 mutant protein.
(1)A-Kringle?5-B-H 1
(2)C-Kringle?5-D-H 2
Kringle 5 is meant aminoacid sequence shown in Figure 1 in the formula (1); A and B are the aminoacid sequence of 0-20 arbitrary sequence; H 1Amino acid chain for 3-10 Histidine composition.A can not exist, and B can not exist, or A and B all do not exist, or A and B all exist.
Kringle 5 is meant base sequence shown in Figure 1 in the formula (2); C and D are the base sequence of 0-60 encode respectively A and B; H 2Alkali basic sequence for continuous 3-10 the Histidine chain of encoding.C can not exist, and D can not exist, or C and D all do not exist, or A and B all exist.A is identical with the B sequence in the present embodiment, and C is identical with the D sequence, in the design of the carboxyl terminal of mutant protein 6 Histidine chains is arranged, and scheme is as follows:
PCR design of primers: according to the gene order of the hPK-5 factor shown in Figure 12, design following primer: primer 1 is: 5 '-GCGAATTCCATGACCGGTCATGC TAGCG-3 ', primer 2 is: 5 '-GCGGATCCCTATTAGTGGTGGTGGTGGTGGTGTGCTTGAGCTAGCGAAGC-3 '.Wherein, GAATTC and GGATCC are the restriction enzyme site of restriction enzyme EcoRI and BamHI; ATG is an initiator codon; TAATAG is a terminator codon; GTGGTGGTGGT GGTGGTG4 is 6 Histidine sequences; 5 ' GC is the protection sequence; GACTGTAT GTTTGGG and GGC ACACTGAGGGAC are exogenous gene sequence.
Pcr amplification hPK-5 factor mutant gene: reaction system is: Profibrinolysin dna profiling (or contain other gene order of Kringle 5 dna sequence dnas) 5ul, 4 * dNTPs2ul, primer 1 1ul, primer 2 1ul, 10 * Buffer 2ul, Taq enzyme 1ul, dH2O 8ul; Reaction conditions is: 95 ℃ of 10min, 94 ℃ of 1min, 58 ℃ of 1min, 72 ℃ of 1min, 30 circulations back 72 ℃, 1min.The PCR product carries out 2% agarose gel electrophoresis analysis.
HPK-5 gene EcoRI/BamHI double digestion: reaction system: enzyme is cut substrate 5ul, 10 * Buffer 2ul, BSA 0.5ul, dH 2O 11.5ul, EcoRI 1ul.Mixing, slightly centrifugal, put 37 ℃ of water-bath 2h, dehydrated alcohol precipitation 1h, 1, the centrifugal 5min of 2000r/min abandons supernatant, and evaporation residue ethanol adds 10 * Buffer 2ul again, BSA 0.5ul, dH 2O 11.5ul, BamHI 1ul, mixing, slightly centrifugal, put 37 ℃ of water-bath 2h, the endonuclease reaction product all carries out 2% sepharose (LMP) electrophoresis.
The piece that digs of endonuclease bamhi reclaims and purifying: plasmid pBV220 forms open loop pBV220, flexible glue recovery gene fragment with the EcoRI/BamHI double digestion, and enzyme is cut product and analyzed with agarose gel electrophoresis.
HPK-5 gene endonuclease bamhi is connected with open loop pBV220's: reaction system is: 10 * T 4Connect Buffer 5ul, pBV220 open loop 2ul, the hPK-5 enzyme is cut gene fragment 1ul, T 4Dna ligase 1ul, dH 2O 6ul, slightly centrifugal behind the mixing, 4 ℃ are spent the night.
Flexible glue reclaims the recombinant plasmid gene fragment: the recombinant plasmid gene fragment called after pBV220/hPK-5 mutant expression plasmid that recovery is obtained.
The engineering cell strain of expressing the rhPK-5 mutant protein is as described in the embodiment 2, and the method that obtains the rhPK-5 mutant protein is as described in the embodiment 3,4,5,6,7,8, and the part experimental technique can improve to some extent according to the feature of rhPK-5 mutant protein.
The invention effect:
The invention has the advantages that having established a kind of easier preparing in a large number has biology Active recombinant human plasminogen Kringle 5 (rhPK-5) and the preparation of mutant thereof Technology, it utilizes technique for gene engineering keeping Kringle 5 natural space conformations On the basis, made up at prokaryotic expression system, optimized expression condition, set up purpose The method of product separation, purifying and renaturation, and confirm that the rhPK-5 of the method preparation has The activity of angiogenesis inhibiting.
Description of drawings:
The primary amino acid sequence of Fig. 1 Kringle 5;
The base sequence of the primary amino acid sequence of Fig. 2 encoded K ringle 5;
The 5th loop domain of Fig. 3 Kringle 5 plasminogen in mammalian plasma;
Fig. 4 is positioned at Cys in Profibrinolysin aminoacid sequence and Kringle aminoacid sequence 460-Cys 510, Cys 482-Cys 523, Cys 511-Cys 535Three disulfide linkage;
Behind Fig. 5 pcr amplification hPK-5 factor gene, the PCR product carries out 2% agarose gel electrophoresis analysis, and wherein: 1,2 is pcr amplification product, and M is the DL-2000 molecular weight standard;
Fig. 6 plasmid pBV220 is segmental behind double digestion to dig that piece reclaims and purifying, and enzyme is cut product and analyzed with agarose gel electrophoresis, wherein, 1 be pBV220 through EcoRI, BamHI double digestion product, M is the DL-2000 molecular weight standard;
Fig. 7 the technological line of construction expression recombinant plasmid pBV220/hPK-5;
The polyacrylamide gel electrophoresis result that Fig. 8 recombinant expression pBV220/rhPK-5 expresses at JM109, wherein, M is a protein molecular weight standard, 1,6 is 5 hours bacterial protein of abduction delivering, 2,5 is 4 hours bacterial protein of abduction delivering, and 3,4 is the bacterial protein before the abduction delivering;
Fig. 9 shows the Immunological Identification result that recombinant plasmid pBV220/rhPK-5 expresses at JM109, and wherein, 1,3 be the result of preceding bacterial protein of abduction delivering and His-Tag antibody response, and 2 is the result of bacterial protein and His-Tag antibody response behind the abduction delivering;
The polyacrylamide gel electrophoresis result of Figure 10 rhPK-5 albumen or its mutant protein purge process, wherein, 1,4,7 is the first step " ultrasonic, lysis buffer washed twice; Second step: 5M urea washed twice; The 3rd step: 3% Septochol is received washing once " method washing inclusion body result; 2,5,8 for adopting " ultrasonic, lysis buffer washed twice; Second step: 5M urea washing three times; The 3rd step: 3% Septochol is received washing once " method washing inclusion body result; 3,6,9 is that " the first step: ultrasonic+lysis buffer washs once in employing; Second step: 3% Septochol is received washing once; The 3rd step: 5M urea washing three times " method washing inclusion body result;
Figure 11 adopts the biologic activity of chick chorioallantoic membrane measuring rhPK-5 albumen or its mutant protein angiogenesis inhibiting;
Wherein: A is the PBS group; B is solubilization of inclusion bodies, diluent group; C is a rhPK-5 crude samples group; D is the pure product group of rhPK-5;
Figure 12 rhPK mutant arbitrary amino acid sequence;
The base sequence of Figure 13 rhPK mutant arbitrary amino acid sequence correspondence.

Claims (6)

1, the preparation method of a kind of recombinant human plasminogen Kringle-5 (rhPK-5) mutant protein, it is characterized in that with Kringle-5 being its mutant of primary amino acid sequence construct, by setting up recombinant plasmid expression vector, transformed into escherichia coli obtains the prokaryotic expression cell strain, efficiently express the rhPK-5 mutant, and carry out the isolation and purification of rhPK-5 mutant.
2, preparation method as claimed in claim 1 is characterized in that said mutant has the aminoacid sequence of formula (1) feature or the base sequence with formula (2) feature:
A-Kringle-5-B-H 1 (1)
C-Kringle-5-D-H 2 (2)
Kringle-5 is meant and contains 82 amino acid whose primary amino acid sequences in the formula (1);
A, B are the aminoacid sequence of 0-20 arbitrary sequence;
H 1Amino acid chain for 3-10 Histidine composition;
A can not exist, and B can not exist, or A and B all do not exist, or A and B all exist;
Kringle-5 is meant the basic base sequence that contains 246 bases in the formula (2);
C and D are the base sequence of 0-60 encode respectively A and B;
H 2Base sequence for continuous 3-10 the Histidine chain of encoding;
C can not exist, and D can not exist, or C and D all do not exist, or C and D all exist;
By the PCR reaction, obtain hPK-5 factor mutant gene.
3, preparation method as claimed in claim 1 is characterized in that the hPK-5 mutant gene is connected with protokaryon plasmid expression vector such as pBV220 with BamHI by proper restriction site such as EcoRI, obtains the rhPK-5 recombinant plasmid expression vector.
4, preparation method as claimed in claim 1 is characterized in that the acquisition of rhPK-5 mutant prokaryotic expression cell strain, is the rhPK-5 recombinant plasmid expression vector is transformed prokaryotic cell prokaryocyte such as intestinal bacteria.
5, preparation method as claimed in claim 1 is characterized in that rhPK-5 mutant prokaryotic expression cell strain being cultivated and being induced through temperature control, obtains efficiently expressing of rhPK-5 mutant protein.
6, preparation method as claimed in claim 1 is characterized in that adopting carrying out ultrasonic bacteria breaking, cracking, and urea and Sodium desoxycholate wash inclusion body, carry out the purifying of rhPK-5 mutant protein again with the Histidine affinity chromatography.
CN 02123925 2002-07-10 2002-07-10 Method for preparing recombinant human plasminogen kringle 5 mutant protein rhpk-5 Pending CN1420126A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101880312A (en) * 2010-06-13 2010-11-10 山西医科大学 Method for obtaining crude protein of genic recombinant inclusion body
CN101649312B (en) * 2009-09-04 2011-11-16 厦门大学 Human plasminogen K5 modified by RGD and preparation method and application thereof
US8318661B2 (en) 2006-08-28 2012-11-27 Omnio Healer Ab Candidates against infection
US10086052B2 (en) 2006-08-28 2018-10-02 Omnio Healer Ab Drug target for preventing and treating periodontal disease, improving healing of periodontal wounds and promoting oral health

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8318661B2 (en) 2006-08-28 2012-11-27 Omnio Healer Ab Candidates against infection
CN101563100B (en) * 2006-08-28 2013-08-07 李季男 Novel drug target of preventing and treating periodontal disease, improving healing of periodontal wounds and promoting oral health
US10086052B2 (en) 2006-08-28 2018-10-02 Omnio Healer Ab Drug target for preventing and treating periodontal disease, improving healing of periodontal wounds and promoting oral health
US10729750B2 (en) 2006-08-28 2020-08-04 Omnio Healer Ab Candidates against infection
CN101649312B (en) * 2009-09-04 2011-11-16 厦门大学 Human plasminogen K5 modified by RGD and preparation method and application thereof
CN101880312A (en) * 2010-06-13 2010-11-10 山西医科大学 Method for obtaining crude protein of genic recombinant inclusion body

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