CN101503697B - Preparation of recombinant protein of human vascular endothelial cell growth inhibition factor rhVEGI-192A - Google Patents

Preparation of recombinant protein of human vascular endothelial cell growth inhibition factor rhVEGI-192A Download PDF

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CN101503697B
CN101503697B CN 200910037702 CN200910037702A CN101503697B CN 101503697 B CN101503697 B CN 101503697B CN 200910037702 CN200910037702 CN 200910037702 CN 200910037702 A CN200910037702 A CN 200910037702A CN 101503697 B CN101503697 B CN 101503697B
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recombinant protein
rhvegi
protein
expression vector
endothelial cell
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CN101503697A (en
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黎孟枫
朱勋
李鲁远
袁洁
吴珏珩
何振健
古明晖
夏蕾
贺海朋
马剑达
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Sun Yat Sen University
National Sun Yat Sen University
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Abstract

The invention provides a method for preparing recombinant protein of a human vascular endothelial cell-growth inhibiting factor rhVEGI-192A. The method comprises the steps of using a nucleotide sequence of VEGI-192A gene coding to construct an expression vector and inducing the expression vector to express in a host cell. Particularly, total RNA of human umbilical vein vascular endothelial cell line HUVEC is amplified to obtain a VEGI-192A gene fragment; a target gene is obtained through digestion by use of restriction endonuclease double enzyme; the target gene is connected to the expression vector pET-30a to construct a recombinant protein expression vector; and the recombinant protein expression vector is transformed into escherichia coli host bacteria to induce expression so as to obtain purified protein. The method has the advantages of building a target-protein prokaryotic expression system with high efficiency, high yield, high activity and high purity on the basis of maintaining the natural spatial conformation of rhVEGI-192 recombinant protein, optimizing expression conditions and building a method for separating, purifying and renaturing target products.

Description

A kind of preparation method of recombinant protein of human vascular endothelial cell growth inhibition factor rhVEGI-192 A
Technical field
The present invention relates to biological technical field, be specifically related to the preparation method of the recombinant protein of a kind of human vascular endothelial growth inhibitor rhVEGI-192.
Background technology
Vascular endothelial cell growth supressor (VEGI) is a kind of new vessel supressor of recent findings, is mainly produced by vascular endothelial cell.Tan in 1997 etc. take the lead in having found people VEGI by screening Human umbilical vein endothelial cells cDNA library, at that time called after TLI (TNF-like ligand l).Bioactivity research subsequently finds that VEGI has the effect of obvious inhibition of endothelial cell proliferation, and gains the name therefrom.The VEGI full-length gene is 17Kb, is made of I, II, four exons of III, IV and three introns.Produce three kinds of mRNA, VEGI-251, VEGI-192 and the VEGI-174 isomer of encoding respectively and being formed by 251,192,174 amino-acid residues by different connecting method splicings.The three is all contained the IVb exon, and wherein the C-terminal of three kinds of splicing isomer proteins has 151 amino acid identical.
Because the poorly soluble of the VEGI of total length causes its anti-tumour effect not good.And VEGI-192A is a kind of soluble relatively endogenous factors.Experimental results show that VEGI-192A has identical inhibition endotheli ocytosis and the anti-tumor activity with solubility VEGI (being VEGI 29-147 amino acid).Experiment in vitro shows, VEGI-192A can significantly suppress the propagation of bovine aortic endothelial cell and Human umbilical vein endothelial cells, it suppresses active in the highest in three kinds of splicing isomer, and to the cell unrestraint proliferation function of other types such as human coronary artery's smooth muscle cell and mouse lewis lung carcinoma cell LLC, and kidney, liver shown nontoxicity.As the supressor of vascular endothelial cell autocrine, VEGI-192A has the important pathological physiological significance.In addition, and VEGI-192A and angiogenic growth statin (Angiostatin, AS), (Endostatin ES) waits and to compare endostatin, can the single-minded endotheliocyte that acts on.In view of effect high efficiency and the specificity of VEGI-192A, very likely become a kind of anti-angiogenic medicaments with development prospect.
Automatization and microminiaturized two kinds of key factors that become back era gene high-throughput research engineering.At present VEGI-192A is developed to the tumour medicine aspect, also lacks the mass production techniques of a kind of high-level efficiency, low cost, stable and controllable.
Summary of the invention
In order to overcome the deficiencies in the prior art, the invention provides a kind of preparation method of recombinant protein of human vascular endothelial cell growth inhibition factor rhVEGI-192 A, this preparation method can efficiently express foreign protein, the automatic inducible system of height optimization can be realized again, output, purity and the activity of the recombinant protein of human vascular endothelial cell growth inhibition factor rhVEGI-192 A can be increased.
Concrete technical scheme of the present invention is as follows:
The preparation method of the recombinant protein of a kind of human vascular endothelial cell growth inhibition factor rhVEGI-192 A of the present invention, comprise the nucleotide sequence construction of expression vector with the VEGI-192A genes encoding, and with the step of described expression vector abduction delivering in host cell.
Further, this preparation method is that the total RNA amplification of HUVEC obtains the VEGI-192A target gene fragment by RT-PCR from human umbilical vein endothelial cell, use restriction enzymes double zyme cutting to get goal gene, be connected to and make up recombinant protein rhVEGI-192A expression vector among the expression vector pET-30a, be converted into abduction delivering in the e. coli host bacteria again, final purification albumen.
Preferably, upstream primer and the downstream primer that uses among the above-mentioned RT-PCR is respectively:
Upstream primer is: 5 '-TTCCATATGCAA CTCACAAAGGGCCGTCT-3 ';
Downstream primer is: 5 '-CGCGGATCCCTATAGTAAGAAGGCTCCAAAGAAGGTT-3 ';
5 ' end of described upstream primer is Nde I restriction enzyme site, and 5 ' end of downstream primer is BamH I restriction enzyme site.
Preferably, above-mentioned e. coli host bacteria is OrigamiB (DE3).
Preferably, the calcium chloride transform mode is adopted in above-mentioned conversion.
Preferably, above-mentioned inducing adopted pET t7 rna polymerase composite automatic induction.
Preferably, above-mentioned purifying adopts the nickel ion metal chelate affinity chromatography
The present invention has following beneficial effect:
Preparation method of the present invention is on the basis that keeps the natural space conformation of rhVEGI-192 recombinant protein, set up efficient, high yield, high reactivity, high purity and expressed the prokaryotic expression system of target protein, optimize expression condition, set up the method for the separation of purpose product, purifying and renaturation.
The automatic inducible protein expression system of pET t7 rna polymerase that the present invention adopts is to be based upon on the basis of pET system, mainly is by changing the composition of substratum, thereby makes and do not need additionally to add inductor and can express target protein.Simultaneously, this cover system is specially adapted to high flux screening, because this cover system has solved the problem that the bacterium that contains different plasmids seldom shakes synchronously, thereby makes that carrying out high-energy expresses test and become possibility, and can improve the output of target protein.Specifically, the automatic inducible system of rhVEGI-192 recombinant protein of the present invention mainly has the outstanding advantage in two aspects: reduce production costs, improve productivity effect.The present invention uses lactose to replace traditional, expensive inductor IPTG, has saved culture medium cost greatly on the one hand, and Financial cost is the former about a thirtieth; Need not to monitor cell growth state on the other hand and manually add inductor IPTG, greatly made things convenient for the production operation of albumen; More crucial is the use of having replaced non-natural toxic substance IPTG, is more suitable for the application in field of medicaments.
The present invention uses OrigamiB (DE3) as the host bacterium of expressing, and not only can enlarge the advantage of self-induction pET system, improves the output of target protein, and the albumen of expressing can form the disulfide linkage that correctly folds, for follow-up purifying and renaturation bring convenience.Origami B bacterial strain integrates the advantage of BL21 and Origami host bacterium.Origami B host bacterium can accurately be regulated expression product according to the concentration of IPTG, makes the expression product amount present the IPTG concentration dependent.And OrigamiB host bacterium can form the correct folding albumen that contains disulfide linkage, for follow-up purifying and renaturation bring convenience.
The present invention adopts Ni 2+The method of-NTA post metal chelate affinity chromatography purifying protein is carried out purifying to the recombinant protein occlusion body lysate of collecting; And adopt column chromatography and two kinds of refolding methods of gradient dialysis to obtain highly active albumen.Use Ni 2+The metal-chelate that-NTA resin carries out is closed the fusion rotein that chromatographic separation purifying N-end has 6 continuous Histidines, compare the advantage with uniqueness with other protein purification extraction system: (1) fusion tag Histidine belongs to rare base in general protein, the fusion rotein of introducing 6 continuous Histidines has very high specificity, the foreign protein of weak affinity is less combined, in washing process, easily be eliminated, thereby the target protein purity of wash-out is higher relatively; (2) Ni 2+The reusable several of-NTA resin is conducive to the control of protein Preparation cost.This purification process is quick, easy, efficient simultaneously.
The invention will be further described below in conjunction with drawings and Examples.
Description of drawings
Fig. 1 is the figure as a result of a preferred embodiment of the SDS-PAGE of 16 clone's bacterium colony lysates obtaining of the screening of high-expression clone in the IPTG abduction delivering of the present invention system;
Fig. 2 is the figure as a result of a preferred embodiment of SDS-PAGE of the condition optimizing of IPTG abduction delivering of the present invention system;
Fig. 3 is the figure as a result of a preferred embodiment of the SDS-PAGE of 8 clone's bacterium colony lysates obtaining of the screening of high-expression clone in the automatic abduction delivering of the present invention system;
Fig. 4 is the figure as a result of a preferred embodiment of the SDS-PAGE of 2 clone's bacterium colony lysates obtaining of the screening of high-expression clone in the automatic abduction delivering of the present invention system;
Fig. 5 is under the different inducing temperatures of the automatic abduction delivering of the present invention system, seven figure as a result with the preferred embodiment of the SDS-PAGE of the target protein under the elutriant wash-out;
Fig. 6 is the figure as a result of the preferred embodiment of the SDS-PAGE under the different induction times of the automatic abduction delivering of the present invention system;
Fig. 7 is the optimal dissolution scheme that adopts the denaturing agent dissolving occlusion body detection of 8 kinds of different qualities among the present invention;
Fig. 8 adopts Ni-NTA post metal chelate affinity chromatography technology purifying to extract the figure as a result of a preferred embodiment of recombinant protein under the sex change condition among the present invention;
Fig. 9 is the figure as a result of the preferred embodiment of the Western Blotting of evaluation target protein among the present invention;
Figure 10 is the figure as a result of a preferred embodiment of the MTT colorimetry of the present invention biologic activity that detects the external dose-dependent inhibition bovine aortic endothelial cells propagation of recombinant protein rhVEGI-192A;
Figure 11 is the figure as a result of a preferred embodiment of the MTT colorimetry of the present invention biologic activity that detects the external dose-dependent inhibition Human umbilical vein endothelial cells propagation of recombinant protein rhVEGI-192A;
Figure 12 is the figure as a result of a preferred embodiment of the Annexin V-FITC/PI of the present invention dyeing biologic activity that detects rhVEGI-192A recombinant protein inducing endothelial cell apoptosis;
Figure 13 is that the present invention adopts chick chorioallantoic membrane angiogenesis inhibition test to measure the figure as a result of a preferred embodiment of angiogenesis inhibiting activity in the rhVEGI-192A recombinant protein body.
Embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention.
Embodiment 1: the structure of recombinant protein rhVEGI-192A expression vector
Obtain the rhVEGI-192A goal gene by PCR method from the total RNA amplification of human umbilical vein endothelial cell.The PCR upstream primer is 5 '-TTCCATATGCAACTCACAAAGGGCCGTCT-3 ', and downstream primer is: 5 '-CGCGGATCCCTATAGTAAGAAGGCTCCAAAGAA GGTT-3 '.5 ' end of described upstream primer is Nde I restriction enzyme site, and 5 ' end of downstream primer is BamH I restriction enzyme site.Re-use above two kinds of restriction enzymes double zyme cuttings and get goal gene, be connected at last and make up recombinant protein rhVEGI-192A expression vector among the expression vector pET-30a.Insert the nucleotide sequence that site 3 ' end contains 6 Histidines of encoding.The correct single open reading frame of the accuracy of rhVEGI-192A recombinant chou is all confirmed through sequencing.
Embodiment 2:rhVEGI-192A in intestinal bacteria the IPTG abduction delivering and the screening of high-expression clone.
1.IPTG induce: adopt expression vector pET-30a-rhVEGI-192A transformed competence colibacillus cell BL21 (DE3) pLysS, 16 clones of picking bacterium colony adds kantlex to final concentration 50 μ g/ml, 37 ℃ of recovery overnight incubation in 3mL LB substratum at random.Measure the liquid-tight degree of every pipe bacterium next day, between 0.6 to 1, just can add IPTG to certain final concentration as OD600, inducing culture 4 hours, negative control is induced for not adding IPTG.Analyze recombinant expressed situation by SDS-PAGE.Obtain 183 clone's bacterium colonies.
2.LB substratum: 1% peptone, 0.5% yeast extract, 1% sodium-chlor, pH7.0; Pyocianil: 50mg/ml; IPTG solution: 100mmol/L;
3. screen rhVEGI-192A albumen high-expression clone: use the method for high-throughput SDS-PAGE, from 183 clone's bacterium colonies, filter out 16 the highest single bacterium colonies of expression efficiency.Fig. 1 is the SDS-PAGE that carries out of 16 clone's bacterium colony lysates that the screening of high-expression clone in the IPTG abduction delivering system obtains figure as a result, wherein 1 to 16 is high-expression clone bacterium colony group, clone's bacterium colony group that C induces for no IPTG, M is protein molecular weight standard, and the host bacterium is BL21 (DE3) pLysS.
Low temperature is protected kind of the clone's bacterium colony that has filtered out for one 80 ℃ when carrying out follow-up expression, purifying procedure.Use gel scanning software analysis purposes expressing quantity behind the SDS-PAGE and account for 44% of total tropina.
The condition optimizing of embodiment 3:IPTG abduction delivering system
Different culture condition influences the target protein expression amount, comprises temperature, IPTG concentration, density (OD600 value), host bacterium, induction time and the inducible system of engineering bacteria when inducing, and gropes the optimum expression condition by preliminary experiment.Select clone's bacterium colony #3 number, carry out protein induced expression condition optimization experiment.Fig. 2 is the condition optimizing of IPTG abduction delivering of the present invention system, comprises before the bacteria-induction incubation time and adds the IPTG amount, and the host bacterium is BL21 (DE3) pLysS.A parameter is changed in each group screening, and when screening best bacterium colony, the host bacterium of using is BL21 (DE3) pLysS, and inductive condition is IPTG:1mM, 16h, temperature: 25 ℃; When screening best IPTG concentration, the host bacterium of using is E.coli BL21 (DE3) pLysS, and inductive condition is 16h, temperature: 25 ℃; When screening best induction time, the host bacterium of using is BL21 (DE3) pLysS, and inductive condition is IPTG:1mM, temperature: 25 ℃; When screening best inducing temperature, the host bacterium of using is BL21 (DE3) pLysS, and inductive condition is IPTG:1mM, 16h; When screening best host bacterium, inducible system is the self-induction system, and temperature is 25 ℃, and the time is 16h.
We reach a conclusion as a result according to above: rhVEGI-192A albumen is IPTG addition: 1mM in the top condition of BL21 (DE3) pLysS host bacterium abduction delivering, selected incubation time: 16h.
Embodiment 4:rhVEGI-192A in intestinal bacteria automatic abduction delivering and the screening of high-expression clone.
1. induce automatically: adopt expression vector pET-30a-rhVEGI-192A transformed competence colibacillus cell BL21 (DE3) pLysS, picking some amount clone bacterium colony is in 3mL LB substratum at random, add kantlex to final concentration 50 μ g/ml, 37 ℃ of recovery overnight incubation.Next day, centrifugal collection bacterial sediment was analyzed recombinant expressed situation by SDS-PAGE.
2.1000 * trace metal solution: 0.05M FeCl 30.12M HCl; 0.02M CaCl 20.01M MnCl 2-4H 2O; 0.01M ZnSO 4-7H 2O; 0.002M CoCl 2-6H 2O; 0.002MCuCl 2-2H 2O; 0.002M NiCl 2-6H 2O; 0.002M Na 2MoO 4-5H 2O; 0.002MNa 2SeO 3-5H 2O; 0.002M H 3BO 3. add distilled water and quantitatively arrive 100ml.
3. self-induction is expressed substratum: 1% Tryptones (tryptone), 0.5% yeast extract (yeastextract), 25mM Na 2HPO 4, 25mM KH 2PO 4, 50mM NH 4Cl, 5mM Na 2SO 4, 2mM MgSO 4, 0.2 * metals (10 μ M Fe+9), 0.5% glycerine (glycerol) (54mM), 0.05%glucose (glucose) (2.8mM), 0.2%a-lactose (a-lactose) is (5.6mM).
4. use the method for high-throughput SDS-PAGE, from 136 clone's bacterium colonies, filter out 8 the highest single bacterium colonies of expression efficiency.Fig. 3 is the SDS-PAGE that carries out of 8 clone's bacterium colony lysates that the screening of high-expression clone in the automatic abduction delivering of the present invention system obtains figure as a result, wherein 1 to 8 is high-expression clone bacterium colony group, C is the clone's bacterium colony group that has transformed the empty plasmid carrier, M is protein molecular weight standard, and the host bacterium is BL21 (DE3) pLysS.
Low temperature-80 ℃ guarantor kind of the clone's bacterium colony that has filtered out when carrying out follow-up expression, purifying work.Use gel scanning software analysis purposes expressing quantity behind the SDS-PAGE and account for 50% of total tropina.
Embodiment 5: the condition optimizing of automatic abduction delivering system
1. the host bacterium is optimized: simultaneously for improving the solubility of rhVEGI-192, we change the host bacterium is OrigamiB (DE3), Fig. 4 is the SDS-PAGE that carries out of 2 clone's bacterium colony lysates that the screening of high-expression clone in the automatic abduction delivering system obtains figure as a result, wherein 1 to 2 is high-expression clone bacterium colony group, C is the clone's bacterium colony group that has transformed the empty plasmid carrier, M is protein molecular weight standard, and the host bacterium is OrigamiB (DE3).
Its trxB and gor gene have sudden change, thereby for recombinant protein provides balanced oxidation-reduction potential in the host bacterium, the correct space that is beneficial to albumen is folding.According to above-mentioned result of experiment, in two kinds of host bacterium, induce the output of rh-VEGI-192A recombinant protein to be more or less the same automatically, but in host bacterium OrigamiB (DE3), induce in the system background foreign protein littler automatically, so we finally select the host bacterium is the #1 high-expression clone bacterium colony of OrigamiB (DE3), incubation time is 16h, culture temperature is 25 ℃, adopts self-induction culture systems high-density culture to prepare target protein.
2. inducing temperature optimization: under 10 ℃, 25 ℃, 37 ℃ three kinds of temperature, induce down great expression rhVEGI-192A recombinant protein automatically, and reclaim rhVEGI-192A with the occlusion body form.Inducing temperature is more low as can be known by the result, and highly active rhVEGI-192A recombinant protein (existing with dimer or polymer form) is more many.Comprehensive output and activity are considered, and we select 25 ℃ of inductive conditions.Fig. 5 is the inducing temperature condition optimizing of automatic abduction delivering system, and the temperature that is divided into three gradients is induced automatically: 10 ℃, 25 ℃, 37 ℃.E1 to E7 represents the SDS-PAGE electrophoretic band that carries out with the target protein under the elutriant wash-out respectively seven times, and BSA application of sample amount is 2 μ g, and M is protein molecular weight standard, and the host bacterium is OrigamiB (DE3).
3. induction time optimization: at following six time point: 2h, 4h, 8h, 12h, 16h, 24h collects bacterium liquid respectively and carries out SDS-PAGE electrophoresis detection target protein with lysis buffer cracking bacterium and induce output.Fig. 6 is the induction time condition optimizing of automatic abduction delivering system, is divided into following six time point: 2h, 4h, 8h, 12h, 16h, 24h.BSA application of sample amount is 2 μ g, and M is protein molecular weight standard, and the host bacterium is OrigamiB (DE3).
Show that by figure best induction time is 16 hours.
Embodiment 6: the purifying of recombinant protein and renaturation
1. at first with the denaturing agent of 8 kinds of different qualities dissolving occlusion body.Fig. 7 adopts the denaturing agent of 8 kinds of different qualities to dissolve the comparison diagram of the optimal dissolution scheme of occlusion body detection.1 is expressed as respectively to 9: 1:1%Triton X-100; 2:0.1%Triton X-100; 3:CHAPS (3-[(3-courage amido propyl group) dimethylamino] propanesulfonic acid salt); 4:PBS; 5:Non-detergent sulfobetaines-195 (drone salt-195 in 1-(3-sulfopropyl) pyridine); 6:8M urea (Urea); 7:4M urea (Urea); 8:0.3%N-lauroyl sarkosine (N-Lauroylsarcosine); 9:BSA (2 μ g), M is protein molecular weight standard, the host bacterium is OrigamiB (DE3).Figure draws the optimal dissolution scheme: utilize 8mM urea dissolution of bacteria and occlusion body thus.
2. adopt Ni-NTA post metal chelate affinity chromatography technology purifying to extract recombinant protein:
1) gets the bacterium liquid of inducing culture, 15000r/min, the centrifugal collection bacterial sediment of 1min; The resuspended bacterium of bacterial lysate that adds 1/100 times of inoculum volume, 15000r/min, 1min is centrifugal, collects supernatant liquor;
2) the 1st) add the 50%Ni-NTA HisBind chromatography agent of 1/4 times of inoculum volume in the step in the supernatant liquor collected, then mixed solution is put on the shaking table, mix 60min under the room temperature.(in order to reach better in conjunction with effect, can mix the longer time, if but chronic, should avoid the degraded of albumen in operation on ice).Mixture is adorned post, treat that the sedimentation of post material fully after, collect residue 1mL supernatant, be used for SDS-PAGE and analyze.
3) with 4ml washings washing chromatography column, the about 28-30 of flow velocity drips/min, washs altogether 8 times, collects the liquid that each washings flows out from chromatography column, is used for SDS-PAGE and analyzes.
4) with 0.5ml elutriant wash-out target protein, the about 28-30 of flow velocity drips/min, and co-elute 8 times is collected the liquid that each washings flows out from chromatography column, is used for SDS-PAGE and analyzes.Utilize 8mM urea dissolution of bacteria and occlusion body, adopt Ni-NTA post affinity chromatography technology that the recombinant protein occlusion body lysate of collecting is carried out purifying.SDS-PAGE result shows that the binding ability of Ni2+-NTA affinity column and target protein is very strong, so almost do not have target protein in the effluent liquid behind the mistake post.In view of our protein yield very high, so wash eight times.Wash-out dry straight, SDS-PAGE result is shown as electrophoresis list band, does not have other foreign protein band, sees Fig. 8, and Fig. 8 is that the present invention adopts Ni-NTA post metal chelate affinity chromatography technology purifying to extract recombinant protein under the sex change condition.W1 to W8 is eight washings point samples, and E1 to E8 is eight elutriant point samples, and FT was effluent liquid behind the post, and BSA application of sample amount is 2 μ g, and M is protein molecular weight standard, and the host bacterium is OrigamiB (DE3).
3. the dialysis renaturation of recombinant protein
Dialysis renaturation buffer (50 * Dialysis buffer): available from Novagen company, be diluted to 1 * Dialysis buffer during use; 1mol/L DTT: available from Novagen company; 30% sarcosyl solution (N-lauroylsarcosine): available from Novagen company; Dialyzate A:1 * Dialysis buffer adds DTT to final concentration 0.1mmol/L; Dialyzate B:1 * Dialysis buffer; Dialyzate C:1 * Dialysis buffer adds 1mmol/L reduced glutathion and 0.2mmol/L Sleep-promoting factor B.
This experiment utilizes 8mol/L urea strong denaturant to keep the solvability of albumen, makes metaprotein natural renaturation gradually in dialysis procedure.To contain the elutriant of target protein in proportion with the deionized water dilution, add the 4mol/L urea soln, in the pretreated dialysis tubing of the above-mentioned process of packing into.Get 50 times to the dialyzate A of albumen dilution volume, dialysed 3 hours for 4 ℃, change dialyzate A and continue dialysis 3 hours.With dialyzate B dialysis 3 hours, change dialyzate B and continue dialysis 3 hours then.Get 25 times to the dialyzate C of albumen dilution volume, 4 ℃ of dialysed overnight.Collect dialysis back albumen, centrifugal collection supernatant liquor.Analyze the dialysis result by SDS-PAGE.This purifying protein is used Novagen Refolding test kit carry out renaturation in conjunction with the method for anionite-exchange resin, successfully set up the stable refolding method at rhVEGI-192A, obtain soluble recombining albumen, the renaturation yield is about 60%.
Embodiment 7:Western Blotting identifies target protein
SDS-PAGE can only get on to infer that what obtain is target protein from molecular weight, and what further determine to obtain is target protein, need carry out Western Blotting.Because VEGI is at bovine aortic endothelial cells (ABAE) high expression level, so using total protein in the cell of ABAE as positive control the rhVEGI1-192A that expresses in our experiment to be carried out Western Blotting, we analyze.Fig. 9 is the Western Blotting result who identifies target protein, wherein, the recombinant protein that VEGI-192 obtains for purifying of the present invention, ABAE is for collecting the expression level of VEGI-192 in the bovine aortic endothelial cells total protein.It is monomer that the result shows parallel with the ABAE band, and the top be speculated as polymer, insufficient about the higher structure denaturing treatment of this albumen, this has report at document still polymer form albumen.This is VEGI-192A with regard to proving the recombinant protein that our purifying extracts.
Western blotting experimental technique: protein example is carried out SDS-PAGE, treat to stop electrophoresis after tetrabromophenol sulfonphthalein is run out of glue; Be ready to two 3M filter paper and a pvdf membrane, immersed the methyl alcohol deionized water 5 minutes, be soaked in 1 * commentaries on classics film damping fluid with special filter paper and fiber mat then; Peel gel, remove concentrated glue part, and filter paper and pvdf membrane are cut into the gel size; Change film according to the sandwich sandwich assay, settle order as follows: negative pole-fiber mat-1 special filter paper-gel-pvdf membrane-1 special filter paper-fiber mat positive plate, putting it into changes in the film groove; Ice bath, 300mA changeed film 1 hour; Take out pvdf membrane, confining liquid sealing 2 hours; Primary antibodie was hatched 2 hours; Reclaim primary antibodie, TBST washes film, and each 15min repeats 3 times; The two anti-45-60min of hatching; Reclaim two and resist, TBST washes film, and each 15min repeats 3 times; Develop the color with ECL colour developing liquid at dark place.
Embodiment 8: the detection of concentration and purity
1. purity detecting: the SDS-PAGE method detects, 96% monomer, and 4% is polymer.95% is dimer in the polymer, and 3% is tripolymer and polymer.Use the dialysis method renaturation, and adopt liquid chromatography to remove intracellular toxin.Detect every batch of albumen endotoxin concns all less than 5EU/mg with limulus reagent test.
2. concentration detects: adopt the BCA albuminimetry quantitative to the rhVEGI-192A recombinant protein that extracts purifying, obtain quantitative criterion graphic representation and concentration formula Y=0.046X+0.0382, coefficient R 2 reaches 0.999, and result precision is very high.Calculate thus and obtain rhVEGI-192A recombinant protein maximum production and reach 50mg/L (optimum yield bacterial strain #1 number clone).
The measuring method of expressing quantity (adopting the BCA Protein Assay Kit of Novagen company):
1) the drawing standard curve sees Table 1.Table 1 is the data sheet of drawing standard curve application of sample amount.
Table 1
2) testing protein is got 2 μ L, added 23 μ L tri-distilled waters.Add respectively in each hole of 96 orifice plates.
3) configuration effort reaction solution: A liquid: B liquid is 50: 1, and every hole adds 200 μ L working fluids then, shakes 30 seconds with mixing, hatches 30min for 37 ℃.(providing in A liquid and the B liquid BCA Protein Assay Kit test kit for Novagen company)
4) use microplate reader and measure absorbancy, calculate the content of albumen then according to typical curve and absorbancy.
Embodiment 9: the biologic activity that detects rhVEGI-192A recombinant protein body external dose dependency inhibition of endothelial cell proliferation
1. cell cultures: bovine aortic endothelial cells (ABAE) is incubated in the DMEM perfect medium that contains 5% foetal calf serum, 2mML-glutamine, 0.1mM non-essential amino acid, 10mM Hepes (hydroxyethyl piperazine Qin Yi thiosulfonic acid solution), 100IU/ml penicillin and 100 μ g/ml Streptomycin sulphates, places 37 ℃, 5%CO 2Hatch cultivation in the incubator; With 0.05% trysinization that contains 0.02%EDTA, stop digestion with the DMEM perfect medium when going down to posterity.Human umbilical vein endothelial cells HUVEC is incubated at and contains in the 20% foetal calf serum HESFM substratum, places 37 ℃, 5%CO 2Hatch cultivation in the incubator; Use above-mentioned trysinization when going down to posterity, stop digestion with pancreatin inhibitor.
2.MTT colorimetric experiment: will count behind HUVEC and the BAEC cell dissociation, be seeded to respectively in the 96 porocyte culture plates by 1.0 * 104 cells in every hole, place 37 ℃, 5%CO2 incubator to hatch cultivation.Be replaced by the substratum of the rhVEGI-192A that contains different concns behind the 24h, each concentration is all established 3 parallel multiple holes, continues to cultivate 48h.Every hole adds 5mg/mL MTT solution 20 μ L subsequently, and 37 ℃ of effect 4h abandon supernatant liquor after centrifugal, add DMSO 150 μ L, vibrator vibration 10min is fully dissolving crystallized, and putting and measuring wavelength on the microplate reader is absorbance A value under the 570nm, calculates inhibiting rate as follows.Inhibitory rate of cell growth (%)=(control group average A value-experimental group average A value)/control group average A value * 100%.Control group is that rhVEGI-192A concentration is 0 culture hole.Use half-inhibition concentration (IC50) software for calculation Bliss's software to calculate IC50, use SPSS software and carry out data statistic analysis.
3. result: use MTT colorimetric test method and measure the rhVEGI-192A recombinant protein inhibition activity of endotheliocyte growing multiplication be the results are shown in Figure 10 and Figure 11.Figure 10 is the figure as a result of a preferred embodiment of the MTT colorimetry of the present invention biologic activity that detects the external dose-dependent inhibition bovine aortic endothelial cells propagation of recombinant protein rhVEGI-192A; Figure 11 is the figure as a result of a preferred embodiment of the MTT colorimetry of the present invention biologic activity that detects the external dose-dependent inhibition Human umbilical vein endothelial cells propagation of recombinant protein rhVEGI-192A.It is very strong that the rhVEGI-192 recombinant protein suppresses the endothelial cell growth activity, is 0.566185 μ g/mL to HUVEC clone IC50, is 0.100979 μ g/mL to the IC50 of bovine aortic endothelial cells system (ABAE).
Embodiment 10:Annexin V-FITC/PI dyeing detects rhVEGI-192A recombinant protein inducing endothelial cell apoptosis result
Use Annexin V cell dyeing analyzing and testing early apoptosis of cells index.In normal cell, phosphatidylserine only is distributed in the inboard of cytolemma lipid bilayer, and early stage at apoptosis, and the phosphatidylserine in the cytolemma by rollover in the adipose membrane laterally.Annexin V is a kind of Ca2+ dependency phospholipids incorporate albumen, with phosphatidylserine high affinity is arranged, so can be combined with the after birth of the early stage cell of apoptosis by the phosphatidylserine that the cell outside exposes.Therefore Annexin V is used as one of sensitive index that detects early apoptosis of cells.Annexin V is carried out fluorescein FITC mark, with mark Annexin V as fluorescent probe, utilize fluorescent microscope can detect apoptotic generation.(Propidium Iodide PI) is a kind of nucleic acid dye to propidium iodide, and it can not see through complete cytolemma, but to cell and the dead cell of apoptosis middle and advanced stage, and PI can permeate through cell membranes and nucleus is incarnadined.Therefore with Annexin V and the use of PI coupling, just the cell differentiation that is in different apoptosis period can be come.
Annexin V-FITC/PI dyeing process: after the HUVEC cell handled with 0.5 μ g/mLrhVEGI-192A albumen respectively, scrape to scrape from culture dish with cell and get cell, 1 * PBS washed cell secondary (the centrifugal 5min of 2000 * rpm) is collected 5 * 105 cells.After adding the BindingBuffer suspension cell of 500 μ L, add 5 μ L Annexin V-FITC mixings, add 5 μ L iodate third ingots (PI) again, mixing, room temperature lucifuge reaction 5~15min.In 1h, under fluorescent microscope, to observe and take pictures, it is green that Annexin V-FITC fluorescent signal is, and the PI fluorescent signal takes on a red color.
The HUVEC cell after 8 hours, be the results are shown in Figure 12 with rhVEGI-192A recombinant protein 0.5 μ g/mL processing, and as seen from the figure, the rhVEGI-192 recombinant protein can be induced the apoptosis of Human umbilical vein endothelial cells HUVEC.
The newborn biologic activity of the inhibition chicken embryo fine hair allantoic vessel of embodiment 11:rhVEGI-192A chimeric protein detects
Hatching of breeding eggs the 6th day disinfects the egg embryo in alcohol back and stabs an osculum with dental burr or emery wheel on egg embryo top on super clean bench, eggshell and shell membrane around removing carefully then make opening be about 1.5cm * 1.5cm size.Can see that air chamber bottom is the CAM film across a cameral mantle this moment, and can be clear that the size of vasoganglion on the CAM film and distributing position and the chicken blastophore of beating are dirty.Determine to needle cameral mantle with injection needles from air chamber and yolk separated place carefully behind the application of sample position, annotate people 1-2 and drip sterilized water, cameral mantle and CAM film are separated, remove the cameral mantle on upper strata then with tweezers gently, expose the CAM film of lower floor.Be ready to sample in advance, and be divided into three groups: the PBS control group, I group (adding rhVEGI-192A recombinant protein 10mg) II group (adding rhVEGI-192A recombinant protein 20mg), with sample with 10 μ L volumes mix air-dry on the gelfoam carrier that is added to 5mm * 5mm size after, gently load sample filter paper is placed CAM and the less position of blood vessel, yolk cyst membrane place with tweezers, seal with sterile transparent glue then, continue to hatch 48h or longer time.Add methyl alcohol by viewing window: the stationary liquid of acetone=1: 1 pre-fixes 15min, removes the chicken embryo, takes off CAM and separates with the egg skin, fixes finishing after, CAM is tiled on flat board or the filter paper, but prolonged preservation.
Identical magnification counting blood vessel is divided into large, medium and small three kinds of blood vessels according to diameter under the anatomical lens.To be the one-level blood vessel in experiment edge, the position 1mm scope, 5mm place, edge, position is the secondary blood vessel with experiment, and I and II pipe blood vessel is observation analysis respectively.Centered by checking matter, the radial growth conditions towards checking matter of surrounding blood vessel is called the blood vessel spoke and beats.Major blood vessel is to the crooked of carrier plate with near the attraction that is referred to as blood vessel.Under dissecting microscope was observed, according to blood vessel diameter blood vessel being divided into 3 classes: d>0.1mm was great vessels; 0.1mm>d>0.05mm is medium vessels; D<0.05mm be little blood vessel (Wang Lei, Zhang Shucheng, Wu Zhikui, etc., Chinese pharmacology and clinical, 2000,16 (6): 46-47).It is converge like the spokes of a wheel at the hub that positive criteria shows tangible blood vessel, and centered by the experiment position, the chorioallantoic membrane blood vessel is concentrated to the carrier growth in a large number, is arranged in the rung shape, wherein has 1 above medium vessels or little blood vessel to become the rung shape, but have medium above blood vessel to attract simultaneously.Negative standard peripheral vascularization is normal, or vascular reaction is lighter, and few it is messy to distribute and thin to the blood vessel of carrier growth, even generation vision visible avascular area territory.Occupy and also be decided to be feminine gender between the two.Vascular counts can be finished in conjunction with technology such as automated image analysis system, shootings.We observe around the gelfoam carrier in the 1cm scope and vessel density away from (greater than 1cm) position, calculate the chicken embryo number that angiogenesis suppresses to occur, and data are carried out variance analysis.Table 2 is that the rhVEGI-192A recombinant protein is to chick chorioallantoic membrane one-level blood vessel, the restraining effect analytical data of secondary blood vessel, table 3 be the rhVEGI-192A recombinant protein to the restraining effect analytical data of the large, medium and small blood vessel of chick chorioallantoic membrane, Figure 13 adopts the newborn inhibition test of chicken embryo fine hair allantoic vessel to measure angiogenesis inhibiting activity in the rhVEGI-192A recombinant protein body.Compare group with PBS, the I group: add rhVEGI-192A recombinant protein 10mg, II group: add rhVEGI-192A recombinant protein 20mg.
By we find that rhVEGI-192A recombinant protein group is compared to control group as a result, the great vessels number of variations is not remarkable, vessel branch obviously reduces, medium and small number of blood vessel obviously reduces, and along with increasing inhibition, concentration strengthens, illustrate that the equal concentration dependent of two histones significantly suppresses new vessel and generates, and the main new life who suppresses middle or small blood vessel.
Table 2
Figure DEST_PATH_G2009100377026D00151
*P<0.05
Table 3
Figure DEST_PATH_G2009100377026D00152
*P<0.05
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although with reference to preferred embodiment the present invention has been done detailed description; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from essence and the scope of technical solution of the present invention.

Claims (3)

1. the preparation method of the recombinant protein of a human vascular endothelial cell growth inhibition factor rhVEGI-192 A, it is characterized in that, be that the total RNA amplification of HUVEC obtains the VEGI-192A target gene fragment by RT-PCR from human umbilical vein endothelial cell, use restriction enzymes double zyme cutting to get goal gene, be connected to and make up recombinant protein rhVEGI-192A expression vector among the expression vector pET-30a, be converted into abduction delivering in the e. coli host bacteria again, final purification albumen; Described e. coli host bacteria is OrigamiB (DE3); Described inducing adopted pET t7 rna polymerase composite automatic induction, and self-induction is expressed substratum and is: 1% Tryptones (tryptone), 0.5% yeast extract (yeast extract), 25mM Na 2HPO 4, 25mM KH 2PO 4, 50mM NH 4Cl, 5mM Na 2SO 4, 2mM MgSO 4, 0.2 * trace metal solution, 0.5% glycerine (glycerol), and 0.05% glucose (glucose), 0.2% alpha-lactose (α-lactose); Wherein,
0.2 the prescription of * trace metal solution obtains by the prescription dilution of following solution: 1000 * trace metal solution: 0.05M FeCl 30.12M HCl; 0.02M CaCl 20.01M MnCl 2-4H 2O; 0.01M ZnSO 4-7H 2O; 0.002M CoCl 2-6H 2O; 0.002M CuCl 2-2H 2O; 0.002MNiCl 2-6H 2O; 0.002M Na 2MoO 4-5H 2O; 0.002M Na 2SeO 3-5H 2O; 0.002M H 3BO 3
2. preparation method according to claim 1 is characterized in that, the upstream primer and the downstream primer that use among the described RT-PCR are respectively:
Upstream primer is: 5 '-TTCCATATGCAA CTCACAAAGGGCCGTCT-3 ';
Downstream primer is: 5 '-CGCGGATCCCTATAGTAAGAAGGCTCCAAAGAAGGTT-3 ';
5 ' end of described upstream primer is Nde I restriction enzyme site, and 5 ' end of downstream primer is BamH I restriction enzyme site.
3. preparation method according to claim 1 is characterized in that, the calcium chloride transform mode is adopted in described conversion.
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