CN101497883B - Expression production and separation purification of recombinant placenta growth factor and chemical marker thereof - Google Patents
Expression production and separation purification of recombinant placenta growth factor and chemical marker thereof Download PDFInfo
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Abstract
The invention belongs to the field of biological technique, i.e. gene recombination engineering. Placental growth factors (PLGFs) play important roles in neovascularization and vascular proliferation; however, natural PLGF content in vivo is low and the natural PLGFs have short half-times, thus to extract sufficient entogenous PLGF proteins so as to satisfy the increasingly demands of experimental study and clinical practice is difficult. The invention discloses a method for efficiently expressing, producing and recombining a PLGF system in vitro by gene recombination engineering and cell culture technology and for extracting and purifying the PLGF proteins from expressed supernatant by chromatographic separating technology. The invention also discloses a nonradioactive chemical coupling labeling treatment method for the purified PLGF proteins in vitro. The PLGF proteins (including the unlabeled or the chemical coupling labeled) have wide application in the experimental study and the clinical practice.
Description
Technical field
The invention belongs to biotechnology-recombination engineering field, relate to genetically engineered and eukaryotic cell fermentation culture technology and come the bioactive recombinant placenta growth factor of Expression product tool (placental growth factor, PLGF).The invention still further relates to separation and purification, chemical labeling in vitro and its application of restructuring human placental growth factor.
Background technology:
Blood vessel hyperplasia or new life (angiogenesis) refer to that the blood vessel (such as capillary vessel and Venule) in the body produces the process of new blood vessel by the mode of sprouting or divide.Angiogenesis is grown at the many normal physiological process of keeping body such as Embryo tissue, the hysteria of trauma wounds is closed with repairing etc. is useful and essential.But excessive angiogenesis or hyperplasia are also closely bound up with many pathological changes (such as the hyperplasia diffusion of tumour, inflammatory reaction etc.).The key of constantly hyperplasia and growth of the blood vessel in the body is that the vascular endothelial cell of its liner has division and proliferation and directional migration is inserted the ability of existing vascular wall.Just (namely stimulate blood vessel hyperplasia) in the hyperplasia acceptor of vascular endothelial cell, the regulation and control of negative (namely suppressing blood vessel hyperplasia) two broad aspect factors.Wherein the of paramount importance factor of front regulating vascular endothelial cell hyperplasia is vascular endothelial growth factor (vascular endothelial growth factor, hereinafter to be referred as VEGF) and placenta growth factor (placentalgrowth factor is hereinafter to be referred as PLGF).
VEGF (comprising VEGF-A, B, C, D and E etc.), PLGF (comprising PLGF-1, PLGF-2 and PLGF-3) together, consist of the PDGF family member jointly with Thr6 PDGF BB (platelet-derived growth factor, PDGF).Similar aminoacid sequence and structure are arranged between the PDGF family protein, and its common trait is that its N-end contains the peptide sequence with 8 cysteine residues of high conservative.The PDGF family protein mainly is present in extracellular matrix or the body fluid with dimer soluble glycoprotein form, and comprises such as a series of biological activitys such as short vascular endothelial cell division and proliferation, migration and vascular permeability enhancings by mediating out with the special receptors bind with Tyrosylprotein kinase of expression on vascular endothelial cell.At present the special acceptor of known VEGF/PLGF mainly contains three kinds: i.e. VEGFR-1 (fms-liketyrosine kinase is called for short FLT-1); (Kinase-insert Domain Receptor is called for short KDR to VEGFR-2; In mouse, be called Flk-1) and VEGFR-3 (being called for short FLT-4).Wherein VEGF-A can with VEGFR-1 and VEGFR-2 receptors bind; And VEGF-B and PLGF only with the VEGFR-1 receptors bind.
Both homologies of VEGF and PLGF, and its structure is close with biological activity are VEGF but research in the past more pays close attention to.And for PLGF, (Maglione D et al.PNAS 1991,88:9267-9271), its meaning in blood vessel hyperplasia is to be in subordinate or unsettled condition for a long time always although its gene just cloned as far back as 1991.Along with going deep into of studying, increasing result proves that in fact PLGF participates in the blood vessel hyperplasia under many physiology or the pathological conditions in recent years.Support the evidence of this conclusion comprise at least following some: 1) PLGF also detects the expression of PLGF gene in other histoorgans in the body (such as lungs etc.) and the cell except high level expression in placenta; 2) its angiogenesis that after wound or transplantation tumor, brings out of the mouse of PLGF gene knockout than its in normal mouse, obviously reduce (Carmeliet P et al, Nature Medicine 2001,7:575-583); 3) the insane generation of the level of PLGF and solubility FLT-1 acceptor (being VEGFR-1) albumen thereof and the tendency among high risk pregnancy women is closely related with development in blood or the urine; 4) in the animal body experiment proved injection anti--antibody of PLGF after, the growth of tumour can be controlled (Fischer C et al.Cell 2007,131:463-475).Therefore, PLGF and acceptor thereof should be good drug target or systems, can be used for researching and developing the pharmaceutical preparation of various diseases relevant with blood vessel hyperplasia.
But take PLGF and acceptor thereof as the Field of Drug Discovery of target spot, also there are at present some technical barriers.Wherein a large technical barrier is because the interior natural PLGF content of body is low and the transformation period is short, therefore be difficult to extract with the human placental growth factor that is separated to q.s to satisfy the needs of growing experiment and clinical study from histocyte or other conventional biological samples (as blood plasma).The ideal scheme that addresses this problem is the human placental growth factor with genetic engineering technique Expression product restructuring in eukaryotic cell or protokaryon bacterium.The technical scheme of Expression product restructuring human placental growth factor only sees that indivedual reported in literature are arranged in the protokaryon bacterium.As at a publication number be CN1620465 (day for announcing: in Chinese invention patent document 2005.05.25) (patent documentation: patented invention title: the preparation method of recombinant placenta growth factor, the patent applicant: Italian Ghemona S.P.A.) reported a kind of in the protokaryon bacterium renaturation and the method for extracting purifying of abduction delivering restructuring PLGF and expressing protein.But the method Restruction human placental growth factor of this patent documentation report exists some latent defects.Its defective main manifestations is: the human placental growth factor of 1) expressing in the protokaryon bacterium exists mainly with the inclusion body form, and its renaturation process complexity is loaded down with trivial details, and generally is difficult to obtain the albumen such as natural PLGF sample activity; The human placental growth factor of 2) expressing in the protokaryon bacterium does not contain glycosyl, and natural PLGF is the albumen that contains glycosyl, and the existing glycosyl that studies show that plays an important role in the Stability Analysis of Structures of keeping PLGF and biological activity thereof.
Because above-mentioned factor, will be with ideal at eukaryotic expression as the Expression product of the restructuring human placental growth factor with potential pharmaceutical use.The common eukaryotic host cell that can be used for expressing recombinant protein has mammalian cell, insect cell and yeast cell etc.But with mammalian cell such as expressing cho cell Restruction albumen, its expression amount is general lower and be used for cultivating that the culture medium cost of such cell is high, price, therefore not too be suitable for large-scale industrialization production.And eucaryon unicellular yeast expression system is quick owing to the growth that has prokaryotic cell prokaryocyte concurrently, cultivation is convenient economical, with low cost, with eukaryotic cell system the two-fold advantage of modification (such as glycosylation) is reprocessed in the translation of expression product afterwards, then be the most desirable host that suitability for industrialized production is expressed the restructuring human placental growth factor.
Prepare the bioactive human placental growth factor of tool in order to obtain economy and to be convenient to scale operation, the present invention discloses the method that a kind of system with genetic engineering technique secreting, expressing restructuring human placental growth factor in the eucaryon yeast cell reaches fast separating and purifying restructuring human placental growth factor from the supernatant liquor of secreting, expressing at this.With this system and method express and prepare the Restruction human placental growth factor have efficient fast, the advantages such as economical and convenient, especially be suitable for large-scale industrialization production.The invention also discloses a kind of method of the human placental growth factor of separation and Extraction being carried out external nonradioactive chemical coupling labeling treatment.Human placental growth factor among the present invention (comprise chemical coupling mark or unlabelled) has widely purposes at experimental study and clinical diagnosis and treatment.
Summary of the invention
One of the object of the invention is to disclose a kind of system with genetic engineering technique bioactive restructuring human placental growth factor of efficient secretory expression tool in eukaryotic host cell (especially at yeast cell).The system is characterized in that it adopts the mode with eucaryon yeast cell secretion expression, the restructuring human placental growth factor that expression obtains maintains such as the natural structure of endogenous human placental growth factor and higher biological activity.In addition, because the recombinant protein of secreting, expressing is collected in the cell culture fluid, be more convenient for the restructuring human placental growth factor is reclaimed and the downstream process operation such as separation and purification.
Another large purpose of the present invention is the method that discloses easy sharp separation purification restructuring human placental growth factor from the yeast cell to express supernatant liquor.The method is characterized in that it has adopted ion exchange column.
The invention also discloses a kind of restructuring human placental growth factor to separating-purifying and carry out method and the preparation process thereof that external on-radiation chemicals mark is processed.The chemicals that wherein are used for the mark human placental growth factor are vitamin H (Biotin).Biotin labeled human placental growth factor is when merge using with avidin (avidin), and the biotin-avidin detection system of formation has rapid, sensitive, single-minded, stable, the multistage scale effect of detection and without characteristics such as radiocontaminations.
Human placental growth factor of the present invention (mark or unlabelled) is because maintaining the biological activity of being combined with its receptor-specific, therefore it has widely purposes at experimental study and clinic diagnosis.Its obvious purposes includes, but is not limited at least:
1) as pharmaceutical cpd, is used for intervening or treating various and the blood vessel hyperplasia diseases related;
2) be used in the body as detection reagent and the expression of external qualitative and detection by quantitative PLGF acceptor;
3) close sorbent material in the conduct for separating of the cell (such as vascular endothelial cell) of the PLGF expression of receptor positive and tissue etc.;
4) as developer, be used for cell and the organ-tissue of the location tracing display PLGF expression of receptor positive in the body;
5) as antigen for the preparation of and the screening anti-human placental growth factor antibody (comprising mono-clonal and polyclonal antibody etc.).
Particular content of the present invention is described below:
1. the structure and the secreting, expressing in eucaryon host (yeast cell) thereof that contain the expression vector of PLGF encoding gene
One of purpose of the present invention is the System and method for that is based upon efficient secretory expression restructuring PLGF in the eucaryon host (especially yeast cell).Guarantee that restructuring human placental growth factor large key factor of efficient secretory expression in eucaryon host (yeast cell) is to need a segment signal peptide (signal peptide) guiding the restructuring PLGF that newly synthesizes to penetrate endoplasmic reticulum, and finally be secreted into extracellular matrix.The gene of this signal peptide of encoding can come from PLGF gene itself, or other foreign genes such as IL-2, immunoglobulin (Ig) (Ig) etc.In implementation of the present invention because of the preferred yeast cell expression system, therefore its signal peptide has then been selected α secretion signal peptide sequence known in the yeast cell.A plurality of known expression vectors that contain yeast α secretion signal peptide sequence such as pPic9k (Invitrogen) etc. are available.Structure contains concrete technology and the working method of the recombinant gene expression vector of PLGF and yeast α secretion signal peptide sequence can be referring to books such as " molecular clonings ".Wherein relevant operation comprises the steps:
1) adopt first RT-PCR method clonal expansion from cell tissue such as human peripheral blood mononuclear cell (PBMCs) to obtain to contain the cDNA (comprising PLGF131, the forms such as 152) of people's human placental growth factor coding region (coding region).The upstream and downstream primer that is used for pcr amplification can be with reference to published PLGF cDNA sequence external synthetic.
2) the PLGF cDNA that obtains of PCR clone processes through digestion with restriction enzyme, again with the T4 ligase enzyme with its with warp accordingly the enzyme expression vector pPic9k (Invitrogen) that cuts processing link to each other, experience bacterium through being transformed into;
3) again through screening, amplification is extracted plasmid DNA and enzyme and is cut evaluation, namely obtains to contain the eukaryotic expression vector pPic9k-PLGF of restructuring PLGF gene.
Because the copy number positive correlation that restructuring PLGF the gene expression amount in the cell and PLGF gene after electricity transforms are integrated in host's (yeast) karyomit(e).And the yeast cell after expression plasmid carrier pPic9k-PLGF transforms has obtained the resistance to G418, the copy number positive correlation in yeast karyomit(e) of its medicine resistance ability and expression plasmid.Therefore can obtain by the clone that screening has a high drug-resistance to G418 the bacterial strain of high expression level goal gene.
After obtaining to contain the yeast cell of multiple copied PLGF gene, several clones of picking are used for abduction delivering PLGF.Abduction delivering carries out in shaking table is cultivated, with 1% methanol induction.After abduction delivering 2-3 days, collect the yeast cell culture supernatant, after centrifugal, again with SDS-polyacrylamide gel electrophoresis (polyacrylamide gel electrophoresis, PAGE) (SDS-PAGE) and Western-blot identification and analysis supernatant in contained restructuring human placental growth factor.
2.PLGF the separation and purification of albumen
The present invention's one large purpose is to set up the method for sharp separation extraction restructuring human placental growth factor from the yeast cell to express supernatant liquor.The method is characterized in that it has adopted ion exchange column.
Wherein relevant operation comprises the steps:
1) the PLGF cultivation and fermentation supernatant liquor of collecting, centrifugal and membrane filtration;
2) filter rear supernatant liquor and binding buffer liquid balance;
3) loading is to the DEAE-SEPHAROSE chromatography column;
4) chromatography column is removed foreign protein with elutriant;
5) albumen that is adsorbed with the recovery buffer solution elution is also collected the sample under the wash-out step by step; The sample of collecting namely separates the acquisition human placental growth factor through dialysis again; Separating the human placental growth factor that obtains can be again through analyses such as physics and chemistry and Biological Activity Identification and determination of protein concentration.Identification experiment comprises: SDS-polyacrylamide gel electrophoresis (polyacrylamide gel electrophoresis, PAGE (SDS-PAGE), coomassie brilliant blue staining; Immunoblotting/ELISA test; Be combined test and external short vascular endothelial cell proliferation experiment etc. with vegf receptor.
3.PLGF the chemical labeling in vitro of albumen is processed
Another large purpose of the present invention is to adopt on-radiation coupling marking method that the human placental growth factor of separation and purification is carried out chemical labeling in vitro to process.The chemical markers commonly used that can be used for mark includes, but is not limited to enzyme (horseradish peroxidase, alkaline phosphatase, glucose oxidase, microperoxisome); Fluorescein (fluorescein isothiocyanate (FITC); Vitamin H-antibiotin and Radioactive colloidal gold etc.; Wherein, the preferred marker of external chemical coupling mark human placental growth factor is vitamin H (Biotin) in the present invention.
Vitamin H is a kind of VITAMIN of small molecular weight, and it and avidin and avidin sample protein have very high binding ability.By suitable method, vitamin H can be coupled to numerous protein, does not change the original biologic activity of albumen.Biotin labeled albumen can with avidin height of formation affinity, single-minded stable combination, can be used for many tests such as ELISA, dot-blot or Western-blot.
In the invention process, the vitamin H chemistry reagent that is used for the mark human placental growth factor is the vitamin H of N-hydroxy-succinamide (NHS) ester acyl activation.In the damping fluid of pH 7-9, the vitamin H of NHS activation can be very effectively (NH2) reaction forms stable amido linkage with the primary amino of protein.The effect of biotinylated protein matter or efficiency then depend on the size of protein, the distribution of protein surface primary amino and the usage quantity of biotin labeling reagent.In the present invention, flag condition desirable or that optimize is as follows:
1) the whole solubility that is used for the reagent (vitamin H of NHS activation) of mark is 9mM;
2) mark vitamin H: the mol ratio of human placental growth factor matter to be marked is controlled at 10: 1 to 50: 1 for the reaction mol ratio;
3) pH of buffer of mark is 7-9;
4) temperature and time of mark: be reaction under room temperature 30-60 minute.
Under the flag condition of this optimization, but average 1-4 vitamin H in the general coupling of each human placental growth factor molecule obtains comparatively ideal mark effect.
Wherein, with biology usually the method for mark human placental growth factor comprise the steps:
1) dissolving human placental growth factor matter is in PBS;
3) add an amount of labelled reagent (NHS-vitamin H) and human placental growth factor sample mix, room temperature reaction 30-60 minute;
3) remove unreacted excessive vitamin H by dialysis or desalination, namely obtain biotin labeled human placental growth factor.
4. application mark or unlabelled human placental growth factor
Another large content of the present invention also relates to the application of human placental growth factor (comprise mark or unlabelled).
The restructuring human placental growth factor is because maintaining the biological activity (show to be combined with PLGF/VEGF acceptor high special and also can stimulate the vascular endothelial cell division and proliferation) such as endogenous PLGF, therefore it has widely purposes at experimental study and clinic diagnosis.Its obvious purposes includes, but is not limited to following several at least:
1) is used in the body as the detection kit composition and the expression of external qualitative and detection by quantitative PLGF/VEGF acceptor;
2) as sorbent material for separating of the cell (such as vascular endothelial cell) of the PLGF/VEGF expression of receptor positive or tissue etc.;
3) as the on-radiation developer, be used for location and cell and the organ-tissue of following the tracks of the PLGF/VEGF expression of receptor positive in the body;
4) as antigen for the preparation of and the screening anti-human placental growth factor antibody (comprising mono-clonal and polyclonal antibody etc.).
5) as pharmaceutical cpd, be used for intervening or treating various and the blood vessel hyperplasia diseases related;
Wherein several of relevant this human placental growth factor application specifically describe as follows:
4.1PLGF albumen is applied to measure the expression of PLGF acceptor as the detection kit composition
PLGF of the present invention (comprise mark or unlabelled) can be used as the expression that the detection kit composition is used for external qualitative or detection by quantitative PLGF acceptor.Detected PLGF acceptor can be to be expressed in the culturing cell cell or tissue with membranin, also can be that shla molecule that wander about as a refugee or degradation is present in various body fluid (such as serum) and the cell culture supernatant.
Being coated antibody such as the antibody with anti-PLGF-R, is detection reagent with biotin labeled human placental growth factor, comes the PLGF acceptor of wandering about as a refugee in the test sample with the ELISA method, and its step comprises:
1) with the coated elisa plate of the antibody (such as anti-human Flt1 antibody) of anti-PLGF-R, 4 ℃ are spent the night;
2) wrapper sheet sealed incubation 1 hour with the confining liquid that contains bovine serum albumin (BSA) in room temperature;
3) add the sample to be checked (such as serum, cell conditioned medium liquid etc.) that contains the PLGF acceptor of wandering about as a refugee, hatch 1-2h for 37 ℃;
4) after PBS-T washes plate, add and contain biotin labeled human placental growth factor reagent, incubation 1-2 hour;
5) after PBS-T washes plate, add again the avidin of horseradish peroxidase-labeled, hatch 1h for 37 ℃;
6) after PBS-T washes plate, then add nitrite ion (such as O-Phenylene Diamine ,-3%h202), room temperature leaves standstill to colour developing, adds stop buffer again, measures the extinction in each hole with microplate reader.Compare according to colour developing OD result and with object of reference, can measure the solubility of the PLGF acceptor of wandering about as a refugee (such as the Flt1 acceptor) in the sample to be checked.
And as take biotin labeled PLGF as reagent, detect the PLGF acceptor that is expressed in cell or tissue (such as organization chip or the routine pathology section) sample with methods such as immunohistochemical methodss, its detecting step comprises:
1) drips the confining liquid that contains BSA in section and sealed incubation 1 hour in room temperature;
2) fully wash with distilled water, contain biotin labeled human placental growth factor reagent, incubation 1-2 hour in the section dropping;
3) with the unnecessary sample of elutriant flush away, drip the avidin of enzyme labelling, incubation 30-60 minute;
4) clean with elutriant, drip substrate, lucifuge incubation 5-10 divides to colour developing;
5) in microscopically interpretation detected result.The detected result of organization chip, section compares with clinical data again, can obtain as detecting coincidence rate, relevant diagnosis or the detect parameters such as susceptibility, stability and specificity.
4.2.PLGF albumen is used for the inside and outside and separates absorption PLGF expression of receptor positive cell or tissue
Human placental growth factor also can be used for inside and outside absorption or separates cell and the tissue that contains the PLGF acceptor.Be example such as the cell or tissue that separates the PLGF expression of receptor positive with biotin labeled human placental growth factor vitro Adsorption, its step comprises:
1) first avidin is adsorbed on (such as Tissue Culture Plate, Tissue Culture Dish, nitrocellulose filter or nylon membrane etc.) on the suitable solid phase carrier;
2) drip the confining liquid incubation 1 hour contain bovine serum albumin (BSA) at solid phase carrier;
3) with the PBS washing, drip the human placental growth factor of vitamin H coupling mark at solid phase carrier, incubation 1-2 hour;
4) add again cell or other samples to be separated, incubation 1-2 hour;
5) with after the PBS washing, namely obtain the cell or tissue of the PLGF expression of receptor positive.
4.3.PLGF other purposes of albumen
Human placental growth factor among the present invention can be used as antigen for the preparation of the antibody that reaches the anti-human placental growth factor of screening.The antibody of preparation comprises polyclone, mono-clonal and genetic engineering modified antibody thereof (such as humanized antibody etc.).The preparation of anti-human placental growth factor antibody can be carried out with reference to the known method of routine.Wherein the animal for the preparation of polyclonal antibody comprises mouse, sheep, rabbit, chicken etc.; And comprise mouse for the preparation of the animal of monoclonal antibody, rabbit etc.The screening method of anti-human placental growth factor monoclonal antibody antibody comprises ELISA, immunohistochemical methods etc.
Human placental growth factor of the present invention, is introduced in the body for intervening or treating various and the blood vessel hyperplasia diseases related therefore can be used as pharmaceutical cpd (merging use with other suitable carriers or solution) because maintaining the biological activity of being combined with its receptor-specific; Or as developer, be used for cell and the organ-tissue of the location tracing display PLGF expression of receptor positive in the body.
In addition, biotin labeled human placental growth factor among the present invention can also have the little strain of magnetic grain of avidin to merge use with coupling, reaches the purpose of the cell or tissue of easy and sharp separation PLGF receptor protein or the PLGF expression of receptor positive by the effect of magnetic field absorption.And the cell (such as vascular endothelial cell) of the PLGF receptor protein that separation obtains or the PLGF expression of receptor positive or tissue can be used for further experimental study (as screening anti-angiogenic hyperplasia pharmaceutical cpd for setting up the extracorporeal blood vessel model of hyperplasia; For the preparation of antibody of anti-PLGF acceptor etc.).
Description of drawings
Fig. 1 is the schematic diagram of the expression plasmid of yeast pPic9-PLGF1 of structure.The cDNA fragment that wherein contains encoding human PLGF131 is inserted between the EcoR I and Not I restriction enzyme site of expression vector.
Fig. 2 is that SDS-PAGE detects the restructuring human placental growth factor of analyzing the positive yeast cell secreting, expressing of plasmid pPic9-PLGF1 transfection.Wherein band 1 (negative control yeast cell supernatant liquor) and 2 (the positive yeast cell supernatant liquor of pPic9-PLGF1 transfection) is not for adding unreduced two supernatant samples of DTT; Band 3 (negative control yeast cell supernatant liquor) and 4 (the positive yeast cell supernatant liquor of pPic9-PLGF131 transfection) is for adding two supernatant samples after DTT reduces; Band M is molecular weight of albumen standard (molecular weight is respectively 66,44,29,20 and 14KD).
Fig. 3 detects and the restructuring PLGF131 albumen of identifying in the yeast expression supernatant with the Salmonella method.Wherein the PLGF131 in the diagram represents the coated sample sets of yeast fermentation supernatant liquor of plasmid pPic9-PLGF1 transfection; And neg represents the coated sample sets of negative control yeast supernatant; Sample is all with double serial dilution.
Fig. 4 detects amount and the relative reactivity thereof that separates the restructuring PLGF131 albumen that obtains from the yeast expression supernatant with the ELISA standard measure.Wherein the representative of the VEGF (10ug/ml) in the diagram is with the coated sample of restructuring VEGF165 albumen; PLGF131 represents to separate the coated sample of restructuring PLGF131 albumen of acquisition; Yeast represents the coated sample of negative control yeast supernatant; Sample is all with double serial dilution.
The specific embodiment of the invention
Embodiment one: the structure of PLGF expression vector.
1.1PCR amplification people PLGF gene
The primer that is used for pcr amplification people PLGF gene is as follows:
Forward upstream primer (PLGF-F): ggattc
GAA TTCGccttgtctgctgggaacggc
Reverse downstream primer (PLGF-R): ggattc
GC GGCC GCGccgggtgcggggtctctctc
The pcr amplification system:
5×Primestar buffer 10ul
dNTPs(2.5mM each) 4ul
Primer F-EcoR I(10uM) 5ul
Primer R-Not I(10uM) 5ul
Template DNA 2ul
Primerstar polysaccharase 0.5ul
DD water 2ul
50ul
The pcr amplification condition: 94 ℃ 5 minutes/94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 40 seconds, totally 20 circulations.
Gene PCR amplification is complete, takes a morsel through the agarose gel electrophoresis analysis, in about 400bp place one amplified band is arranged, and clip size conforms to the PLGF131 gene fragment of expection.
1.2PLGF-pPic9K the structure of plasmid and evaluation
Above-mentioned pcr amplification segment and pPic9K plasmid are used respectively EcoRI and NotI double digestion, separate with agarose gel electrophoresis subsequently and recovery purpose fragment.The endonuclease bamhi of above-mentioned two kinds of recovery is respectively got approximately 10ng to be mixed rear 16 ℃ of connections and spends the night.Connect product and transform the DH5a competent cell.The random choose positive colony is some, extracts in a small amount plasmid DNA, the expression vector of structure with EcoR1 and Not1 double digestion after, through the agarose gel electrophoresis analysis, in about 400bp place an enzyme slitting band is arranged, big or smallly conform to expection.Selecting subsequently two cuts through enzyme and to identify that correct clone is sent to Shanghai ancient cooking vessel peace company and carries out determined dna sequence, sequencing primer is selected universal primer α-factor primer, sequencing result proof PLGF131 gene fragment has been inserted into the desired location of plasmid pPic9K, its reading frame conforms to α-factor secretion signal, and full length gene has no sudden change.
Embodiment two: expression vector transforms the abduction delivering of GS115 Host Strains and human placental growth factor
2.1 adopt preparation pPic9-PLGF1 plasmid in alkaline lysis-PEG purifying normal complex 100ml bacterium liquid.Concrete steps are carried out with reference to " molecular cloning three " related Sections.
2.2 get two the pipe each approximately 15ug pPic9-PLGF1 plasmid use respectively 30U Sal I and the thorough linearization for enzyme restriction of 30U Bgl II.After enzyme was cut end, plasmid was with the ethanol precipitation and weigh molten with the ultrapure aseptic deionized water of 5ul respectively.
2.3GS115 the preparation of competent cell
(1) inoculates mono-clonal in 3ml YPD substratum, 30 ℃ of overnight incubation
(2) press the above-mentioned fresh bacterium liquid of 300ul in 300ml YPD substratum, cultivated 18 hours for 30 ℃.This moment, the OD value was about 1.3.
(3) 1500g collected bacterium in centrifugal 5 minutes, used subsequently the ice-cold aseptic deionized water of 300ml resuspended.
(4) 1500g collected bacterium in centrifugal 5 minutes, used subsequently the ice-cold aseptic deionized water of 150ml resuspended.
(5) 1500g collected bacterium in centrifugal 5 minutes, used subsequently the ice-cold 1M sorbyl alcohol of 20ml resuspended.
(6) 1500g collected bacterium in centrifugal 5 minutes, used subsequently the ice-cold 1M sorbyl alcohol of 500ul resuspended, was positioned on ice.
2.4 electricity transforms
Adopt the Gene pusler of Bio-Rad company to carry out the electricity conversion.Electricity revolving cup gap 1mm,
Electricity turns condition 1000KV 25uF 200 Ω.Electric shock adds 1ml 1M sorbyl alcohol after finishing at once, and bacterium is transferred in the aseptic centrifuge tube of 50ml, and 30 ℃ left standstill 1 hour.Then add 1ml YPD, 30 ℃ of shaking tables were cultivated 4 hours.Bacterium is coated the RD-His flat board, cultivated 3 days.
2.5 screening is integrated with multiple copied PLGF131 gene cloning
Usually the expression amount of goal gene and the copy number positive correlation that goal gene is integrated in Host Strains karyomit(e).Yeast after transforming has simultaneously obtained the resistance to G418, and its medicine resistance ability also with the copy number positive correlation of expression plasmid in yeast karyomit(e).Therefore the clone who by screening G418 is had a high drug-resistance just may obtain the bacterial strain of high expression level goal gene.
The cloning process of screening high drug-resistance is as follows:
(1) preparation contain 0,0.25,0.5,0.75,1.0,1.5,2.0,3.0, the YPD of 4.0mg/ml G418 dull and stereotyped each two for subsequent use.
(2) draw 5ml YPD substratum and drip in transforming planar surface, with aseptic Tips used positive colony is all scraped, and be transferred to abundant mixing in the aseptic centrifuge tube of 15ml.
(3) adding aseptic glycerine to final concentration is 20%, and fully 1ml packing behind the mixing stays a pipe for subsequent use, and all the other are frozen in-20 ℃ immediately.
(4) get the above-mentioned bacterium liquid of 10ul, with coating respectively the YPD flat board behind the sterilized water gradient dilution, 30 ℃ of overnight incubation, second day calculates the titre (cfu/ml) of bacterium liquid by enumeration.
(5) get the frozen bacterium of a pipe and melt (the 3rd step gained), the titre fixed according to the 4th pacing.Get an amount of bacterium liquid and be diluted to 106cfu/ml with sterilized water, then get on the G418 flat board of gradient concentration that 100ul coats respectively the 1st step preparation.Cultivated 2-5 days for 30 ℃.
3, abduction delivering
Test for abduction delivering from lower concentration G418 flat board and dull and stereotyped several clones of respectively random picking of high density G418.Induce step as follows.
(1) the picking mono-clonal is inoculated in the 20ml BMGY substratum, is cultured to OD600=2-6;
(2) 1500g collected thalline in centrifugal 5 minutes, and resuspended with 2ml BMMY substratum;
(3) 1500g collected thalline in centrifugal 5 minutes, and resuspended with 2ml BMMY substratum;
(4) get the resuspended bacterium liquid of 1ml and be transferred in the 15ml round bottom centrifuge tube, put into shaking table and carry out abduction delivering.
(5) the resuspended bacterium liquid of 1ml is transferred in the aseptic triangular flask of 250ml in addition, adds the fresh BMMY substratum of 19ml, puts into shaking table and carries out abduction delivering.
(6) adding 100% methyl alcohol to final concentration in per 12 hours is 0.5%, and sampling in per 24 hours is an amount of; Sample 12000rpm is frozen in-20 ℃ respectively with upper cleer and peaceful thalline after centrifugal 2 minutes.
Embodiment three: SDS-PAGE detects the restructuring human placental growth factor of analyzing the yeast cell secreting, expressing
Adopt SDS-PAGE and ELISA method method to detect the restructuring human placental growth factor of secreting, expressing in the identification yeast cell supernatant liquor.Transform the GS115 yeast host after abduction delivering 48-96 hour, collect supernatant liquor and loading (add the DTT reduction, or do not add DTT do not reduce) to 12%SDS-PAGE gel electrophoresis and analyze.After electrophoresis finishes, PAGE glue dyes colour developing albumen with silver, its result is as shown in Figure 2: compare (swimming is with 1 and 3) with negative control yeast supernatant, the yeast supernatant samples that pPic9-PLGF1 transforms under the condition that adds the DTT reduction (swimming is with 4) at about visible specific proteins band about 20-25KD, this molecular weight with the restructuring human placental growth factor monomer of predicting conforms to; And not adding do not go back (swimming is with 2) in the raw sample of DTT, the specific proteins band then about 40-50KD, conforms to the dimeric molecular weight of restructuring human placental growth factor.This result has proved yeast secreted expression that pPic9-PLGF1 transforms restructuring human placental growth factor.
Embodiment four: the ELISA method detects the restructuring human placental growth factor of analyzing in the yeast cell supernatant liquor
Detect and the restructuring human placental growth factor of identifying in the yeast expression supernatant with the ELISA method, its step is as follows:
1) with the positive PLGF cultivation and fermentation supernatant liquor of plasmid pPic9-PLGF1 transfection and the coated elisa plate of negative control yeast supernatant liquor;
2) elisa plate is after the sealing of the rinsing of PBS liquid and 2%BSA (in PBS-0.1%tween20 liquid) room temperature, adds the reagent that contains recombinant soluble VEGF/PLGF receptor protein (be sflt1, or sVEGFR-1), hatches 2h for 37 ℃;
3) PBS-T wash-out, the antibody (being human VEGFR-3 resistant-1) of the anti-PLGF receptor protein of adding mouse is hatched 2h for 37 ℃;
4) PBS-T wash-out adds biotin labeled goat anti-mouse human placental growth factor, hatches 1h for 37 ℃;
5) PBS-T wash-out adds the Avidin of horseradish peroxidase-labeled again, hatches 1h for 37 ℃;
6) PBS-T wash-out adds nitrite ion (O-Phenylene Diamine)-3% hydrogen peroxide, and room temperature 10-20min is to colour developing;
7) add 1mol/L HCL termination reaction, measure the light absorption value in each hole, 492nm wavelength place with the enzyme linked immunological instrument.
The ELISA detected result as shown in Figure 3; The coated sample sets of the yeast fermentation supernatant liquor (PLGF131) of the plasmid pPic9-PLGF1 transfection positive is positive, and negative yeast controls supernatant (neg) sample is negative.This result prove conversion yeast secreted expression the restructuring human placental growth factor, and the restructuring human placental growth factor of expressing can acceptor special with it) (flt-1, i.e. VEGFR-1) combination.
Embodiment five: the separation and purification of human placental growth factor
The PLGF cultivation and fermentation supernatant liquor of collecting behind centrifugal and membrane filtration, adds 1.8M ammoniacal liquor and makes fermentation supernatant pH value reach 8.0, then adds equal-volume binding buffer liquid (binding buffer liquid: 50mM TrisHCl+50mM NaCl PH=7.4).After the balance, loading is (1L/10ml chromatography column) to the DEAE-SEPHAROSE chromatography column.Chromatography column is first removed foreign protein with elutriant, and the albumen that is adsorbed with damping fluid (50mM NaAc+500mM NaCl, PH=5.0) wash-out is more also collected sample (every 500ul one pipe substep is collected, altogether the 8-12 pipe) under the wash-out.Wash-out is collected liquid and namely separate acquisition PLGF131 albumen again after dialysis.The albumen that separates is measured protein concentration with PIERCE-BCA (bicinchoninic acid) method again, SDS-PAGE and Xylene Brilliant Cyanine G (R-250) dyeing Observe and measure purifying, the activity of vitro detection itself and acceptor (flt-1, or VEGFR-1) specific combination.
Wherein the vitro detection method of activity of separating restructuring human placental growth factor itself and special acceptor (flt-1, or the VEGFR-1) combination of acquisition with identification and detection adopts ELISA, and its step is as follows:
1) with containing restructuring human placental growth factor or VEGF positive control albumen, negative control yeast supernatant liquor is coated with elisa plate;
2) elisa plate is after the sealing of the rinsing of PBS liquid and 2%BSA (in PBS-0.1%tween20 liquid) room temperature, adds the reagent that contains recombinant soluble VEGF/PLGF receptor protein (be sflt1, or sVEGFR-1), hatches 2h for 37 ℃;
3) PBS-T wash-out, the antibody (being human VEGFR-3 resistant-1) of the anti-PLGF receptor protein of adding mouse is hatched 2h for 37 ℃;
4) PBS-T wash-out adds biotin labeled goat anti-mouse human placental growth factor, hatches 1h for 37 ℃;
5) PBS-T wash-out adds the Avidin of horseradish peroxidase-labeled again, hatches 1h for 37 ℃;
6) PBS-T wash-out adds nitrite ion (O-Phenylene Diamine)-3% hydrogen peroxide, and room temperature 10-20min is to colour developing;
7) add 1mol/L HCL termination reaction, measure the light absorption value in each hole, 492nm wavelength place with the enzyme linked immunological instrument.
The result is as shown in Figure 3: the same with the positive control protein sample that contains VEGF, and contain the coated sample of restructuring human placental growth factor sample and be positive; , and negative yeast controls supernatant (neg) sample is for being negative.This result proves and separates the activity that the human placental growth factor that obtains maintains acceptor special with it (flt-1, or VEGFR-1) combination.
The external biological element chemical labeling of embodiment six PLGF131 albumen
In example of the present invention, the preparation solubility of biotin labeling reagent is 9mM, and the reaction mol ratio of the labelled reagent of employing and vegf protein is 50: 1.Its mark may further comprise the steps:
1) dissolving 50-200ug PLGF131 protein is in 200-700ul PBS;
2) cut a pipe biotin labeling reagent, remaining reagent put into-20 degree frozen.Add 200ul reagent (water, DMF or DMSO) dissolving, and use the pipettor mixing;
3) by above-mentioned calculation result, add an amount of labelled reagent and mix with protein sample, and mixing;
4) room temperature reaction 30-60 minute;
5) remove unreacted excessive vitamin H by dialysis or desalination, namely obtain biotin labeled PLGF131 albumen.
Embodiment seven human placental growth factors are for detection of (or solubility) the PLGF acceptor of wandering about as a refugee and the level of detection by quantitative PLGF
Human placental growth factor among the present invention (comprise biotin labeling process human placental growth factor) also can merge to use with other reagent compositions and forms test kit, is used for the level of the material that the various biological samples of vitro detection (such as serum, cell pyrolysis liquid, cell culture supernatant etc.) can be combined with vegf protein (as wander about as a refugee or soluble VEGF-receptor) or detection by quantitative PLGF or VEGF.Wherein, merge the test kit composition that uses with human placental growth factor and comprise anti-PLGF antibody, solubility PLGF/VEGF acceptor etc.Such test kit can both can be used for detecting solubility PLGF/VEGF acceptor and level thereof with methods such as two-step approach/sandwich ELISA method or immunoblottings.
Be reagent such as human placental growth factor and human VEGFR-3 resistant 1 antibody processed with mark, adopt the sandwich ELISA method to come that soluble VEGFR 1 acceptor is example in the test sample, its detecting step comprises:
1) with anti-human VEGR1 antibody sandwich 96-well-ELISA plate (2 μ g/ml, 50 μ l/ holes), 4 ℃ are spent the night;
2) behind 1x PBS liquid rinsing and 2%BSA (in PBS-0.1%tween20 liquid) room temperature sealing 1～2h, add respectively sample to be checked, hatch 2h for 37 ℃;
3) behind the PBS-T wash-out, add biotin labeled human placental growth factor (1: 4), hatch 1h for 37 ℃;
4) behind the PBS-T wash-out, add the Avidin (1: 5000) of horseradish peroxidase-labeled, hatch 1h for 37 ℃;
5) behind the PBS-T wash-out, add nitrite ion (O-Phenylene Diamine)-3% hydrogen peroxide, room temperature 10-20min is to colour developing;
6) add 1mol/L HCL termination reaction, measure the light absorption value in each hole, 492nm wavelength place with the enzyme linked immunological instrument; Compare according to the OD value and with known normal object of reference (such as recombinant soluble VEGFR1 receptor protein), can calculate the level of contained that wander about as a refugee or soluble VEGF 1 receptor protein in the sample to be checked.
In like manner, biotin labeled human placental growth factor merges use such as the PLGF/VEGF albumen object of reference (such as restructuring PLGF or VEGFR albumen) with other reagent compositions (such as anti-human VEGF antibody) and known solubility, consist of the competitive ELISA test kit, also can be used for the level of VEGF in the various biological samples of vitro detection (such as serum, cell pyrolysis liquid, cell culture supernatant etc.).
As example, its detecting step comprises such as the level of coming PLGF in the test sample serum or VEGF take the competitive ELISA method:
1) with anti-human VEGF antibody sandwich 96-well-ELISA plate (2 μ g/ml, 50 μ l/ holes), 4 ℃ are spent the night;
2) after the rinsing of PBS liquid and the sealing of 2%BSA (in PBS-0.1%tween20 liquid) room temperature, add respectively sample to be checked, hatch 2h for 37 ℃;
3) behind the PBS-T wash-out, add the biotin labeled human placental growth factor (1: 4000) of known fixed solubility and sample (serum) to be checked, hatch 2h for 37 ℃; Other establishes PLGF or vegf protein contrast reference group, and this group adds the biotin labeled PLGF of known fixed solubility and unlabelled PLGF or the VEGF object of reference of different solubility, hatches 2h for 37 ℃;
4) behind the PBS-T wash-out, add the Avidin (1: 5000) of horseradish peroxidase-labeled, hatch 1h for 37 ℃;
5) behind the PBS-T wash-out, add nitrite ion (O-Phenylene Diamine)-3% hydrogen peroxide, room temperature 10-20min is to colour developing;
6) add the HCL termination reaction, measure the light absorption value in each hole, 492nm wavelength place with the enzyme linked immunological instrument;
7) compare according to the OD value and with the PLGF that contains known solubility or vegf protein, can calculate the level of vegf protein in the sample to be checked.Because of the competition of vegf protein and biotin labeled human placental growth factor in conjunction with anti-human VEGF antibody therefore, the amount of contained vegf protein and OD value are inverse relation in the sample, namely the amount of vegf protein is higher, the OD value is lower.
Claims (3)
1. express with recombination engineering, cell cultures and chromatographic technique and the systems approach of separation and Extraction recombinant placenta growth factor albumen for one kind, it is characterized in that it comprises the following steps:
1) from cell or tissue, clones the cDNA that obtains to contain the human placental growth factor of encoding with polymerase chain reaction;
2) cDNA that obtains of amplification is after restriction enzyme is processed, and with the T4 ligase enzyme itself and the expression vector processed through corresponding restriction enzyme linked to each other the acquisition recombinant expression vector again;
3) change above-mentioned recombinant expression vector over to host cell, behind screening, amplification and inducing culture, obtain the host cell of efficient stable secreting, expressing recombinant placenta growth factor albumen; Described inducing culture carries out in shaking table is cultivated, with 1% methanol induction; Described host cell is yeast cell;
4) collect the expression host cell culture supernatant, behind centrifugal and membrane filtration, add 1.8M ammoniacal liquor and make fermentation supernatant pH value reach 8.0, then add equal-volume binding buffer liquid, after the balance, with the filtrate loading to the DEAE-SEPHAROSE chromatography column; The 50mM TrisHCl that described binding buffer liquid is PH=7.4 and 50mM NaCl mixed solution;
5) chromatography column is first removed foreign protein with elutriant, and again with 50mM NaAc+500mM NaCl, the albumen that the buffer solution elution of PH=5.0 is adsorbed is also collected sample under the wash-out;
6) albumen of collecting wash-out namely obtains the recombinant placenta growth factor albumen of purifying again through regulating pH to neutrality and dialysis and physics and chemistry evaluation.
2. method according to claim 1, the cDNA of the human placental growth factor that it is characterized in that encoding comes from tissue or the cell of mammals biology.
3. method as claimed in claim 2, the cDNA of the human placental growth factor that it is characterized in that encoding comes from people's tissue or cell.
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| CN1325958A (en) * | 2000-05-26 | 2001-12-12 | 长春奇龙生物技术研究所 | Process for preparing genetically engineered recombinant human lysozyme |
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| CN1325958A (en) * | 2000-05-26 | 2001-12-12 | 长春奇龙生物技术研究所 | Process for preparing genetically engineered recombinant human lysozyme |
| CN1620465A (en) * | 2002-02-05 | 2005-05-25 | 盖莫纳股份公司 | Process for preparing recombined placental growth factor |
| CN1733929A (en) * | 2005-07-29 | 2006-02-15 | 华东理工大学 | Recombinant human composite alpha interferon preparation method |
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