CN101671675A - Method for cloning, identifying and expressing fusion and non-fusion genes of angiostatin Kringle 5 - Google Patents
Method for cloning, identifying and expressing fusion and non-fusion genes of angiostatin Kringle 5 Download PDFInfo
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Abstract
The invention relates to a method for cloning, identifying and expressing fusion and non-fusion genes of angiostatin Kringle 5. The method comprises the following steps: 1, preparation of ThioA/L15/7and E. coli PET32 (vector) plasmid DNA; 2, PCR of the target gene Kringle5; 3, extraction and purification of a PCR product; 4, double digestion of the PCR product and the vector plasmid DNA; 5, reclamation of DNA gel; 6, preparation of the recombinant vector pET32/kringle5; 7, preparation of a competent cell; 8, conversion of the competent cell; 9, miniature fermentation and induction expressionof the recombinant protein; and 10, culture and plasmid extraction, and identification of agarose gel electrophoresis after double digestion. The method successfully constructs the non-fusion expression vector of the pET28a (+)-K5 gene, and establishes foundation for non-fusion expression of the K5 gene in colibacillus.
Description
Technical field
The present invention relates to clone, evaluation and the expression method of a kind of angiostatin Kringle 5 fusions and non-fusion gene.
Background technology
The whole world has 5,000,000 people to die from tumour every year approximately.
As far back as the seventies in 20th century, folkman just proposes, and tumor growth is that blood vessel is dependent.Vessel growth has two visibly different stages, promptly from avascular slow growth phase to the quick growth phase that blood vessel is arranged.The generation of blood vessel makes tumour can obtain enough nutritive substances, is the key link of facilitating above-mentioned transformation.The generation of blood vessel mainly relies on the adjusting of vasculogenesis stimulating factor and supressor.
Suppressing prevailing in the oncotherapy field is the strategy of direct killing tumour cell, promptly carries out tumor resection earlier or by radiotherapy primary tumor is disappeared, and carries out chemicotherapy again, to eliminate residual cancer cells in the body.The shortcoming of this strategy is that specificity is not high, side effect to patient is too big, even treatment means can be quickened patient's death sometimes, and cancer cells is exposed to repeatedly under the chemotherapy and can develops immunity to drugs, have a strong impact on result of treatment, so a lot of tumour experts' attention is transferred to overall tumor tissues, especially angiogenesis from tumour cell itself.
With the blood vessel of tumor tissues is target and directly compared two significant advantages at tumour cell itself: at first, because many solid knurls (perhaps also having some leukemia) are that vasculogenesis relies on, thereby this method needn't be formulated specific treatment plan for certain specific tumour.Secondly, anti-angiogenic medicaments just stops the growth of new vessel, does not attack healthy blood vessel, and is harmless and do not develop immunity to drugs to normal blood vessels in theory.The generation of new vessel is in the state of relative tranquillization in the normal adult human body, have only the endotheliocyte about 0.01% to be in division stage, will exceed 2-3 the order of magnitude and tumor vessel is in the endotheliocyte ratio of mitotic cycle, therefore, inhibition of endothelial cell proliferation is very little to normal tissue influence.
The fibrolan is former to be a kind of Angiostatin, is made of molecular weight 92 * 10 790 amino-acid residues
3It is a kind of 5 Kringle structural domains and secreting glycoprotein that serine protease goes of containing, and each ring is made up of 76-80 amino acid, and the K5 ring is one of them ring, and its molecular weight size is approximately 92kD.
Someone is separated to angiostatin from the mouse urine of lotus knurl and serum, there is the homology of height in it and the former Kringle 1-4 of fibrolan district.The propagation that experimental results show that the specific inhibition vascular endothelial cell of angiostatin energy is arranged subsequently, the vasculogenesis people such as Cao in 1996 that also can suppress in primary tumor and the metastatic tumor tissue have compared the anti-angiogenic proliferative activity in each Kringle district in the angiostatin, find each independently ringle fragment inhibition of endothelial cell proliferation to some extent, and the independent anti-angiogenic activity of k5 fragment is stronger, and it is inhibition of endothelial cell proliferation and the most effective proplasmin fragment of migration known at present.K1, K2, K3 have and suppress activity and k4 has low inhibition activity, and the inhibition activity of K1 and k3 is better than k2 again.Ironically, when research K2-K3 fragment was active, its activity was stronger and be weaker than K3. it suppresses active obviously rising when K2, K3 are acted on endotheliocyte simultaneously than K2.
Proplasmin can not spontaneously play a role, and needs the activation of plasminogen activator (PA).So-called fibrinolytic system of they common compositions.PA mainly is that tissue-type PA (tissue-typePA) abbreviation t-PA. also has urokinase type PA (being called for short u-PA) in addition in the body, and double-stranded u-PA is urokinase (urokinase).They all are glycoprotein also, belong to the serine stretch protein enzyme.People t-PA, relative molecular weight are 70 * 10
3, containing 527 residues, molecule is by 5 structural domains: the finger-type district, the somatomedin district, two ring cake districts and a serine protease district form.Proplasmin, breaks and goes the part peptide chain to be transformed into Tryptase as the t-PA effect through PA.The Tryptase peptide chain in the bar-shaped joining region of fibrin that ruptures specially, this enzyme can enter fibrin grumeleuse inside by the water-based passage, with near wherein bar-shaped joining region.The gene of coding t-PA has been cloned and has been expressed in the mammalian cell of cultivating.The long-living t-PA of recombinant DNA method is applied clinically.Adopt the coronary artery thrombosis that forms in the intravenous injection t-PA method 1h, can be opened, improve the survival rate of infraction significantly by molten.
Complete Kringle structure has great effect to the anti-angiogenic hyperplasia ability of Fibrinogen hydrolysis fragment.There is the disulfide linkage that studies confirm that between the destruction angiostatin Kringle ring and encircle inside can significantly reduce the activity of its anti-endothelial cell proliferation.But the Kringle structural research to K5 then has different views.The K5 that handles with reduction and alkylation has stronger anti-endotheli ocytosis ability than K5 itself.Its reason may be the useful effect that the Kringle structure " has been covered " some functional groups and endotheliocyte among the K5.In addition, K5 can also suppress the motion of endotheliocyte, and it can make the endothelial cell division cycle stop and promoting natural death of cerebral cells.
K5 plays a role by the regulatory factor of regulating vasculogenesis.K5 can make cells of vascular wall and and retina in vascular endothelial growth factor reduce, Angiostatin pigment Urogastron is increased, and have dose-dependence.The rise of the following mediation Angiostatin of endogenic vasculogenesis stimulating factor causes the vasculogenesis dysequilibrium, may be that k5 suppresses vasoactive mechanism.Because K5 has the vigor of strong inhibition endotheli ocytosis, and its aminoacid sequence is shorter, being considered to has bigger application potential in the treatment tumor disease.Use modern biotechnology, according to the preferences of amino acid residue sequence and the intestinal bacteria genetic code of the K5 that has reported, but the full gene of synthetic proplasmin K5.Utilization genetically engineered relative theory, the expression of K5 gene in intestinal bacteria might realize.But the production in enormous quantities that realizes K5 can run into some difficult problems, such as how to realize efficiently expressing of it, how to realize the optimization of the separating and purifying technology of its Optimizing Conditions of Fermentation and K5.Activity mechanism research and the exploitation of novel anti angiogenesis drug that K5 gene efficiently expressing in intestinal bacteria can be K5 lay the foundation.
Summary of the invention
The invention provides clone, evaluation and the expression method of a kind of angiostatin Kringle 5 fusions and non-fusion gene,
Carried out the aim sequence amplification, successfully be connected in carrier and transfered cell, double digestion is identified and is confirmed.The small-sized fermentation inducible protein is expressed, and SDS-PAGE identifies affirmation, and Kringle5 albumen exists with the inclusion body form.Obtain clone's product, expression product and the engineering bacteria thereof of tumor vascular growth supressor K (5) gene.
Success makes up the non-fusion expression carrier of pET28a (+)-K5 gene.For the research of the non-fusion expression of K5 gene in intestinal bacteria is laid a good foundation.
The technology of the present invention solution is as follows:
A kind of clone, evaluation and expression method of angiostatin Kringle 5 fusion genes, its special character is: comprise following steps:
1], the preparation of ThioA/L15/7 and E.coli PET32 (carrier) plasmid DNA:
(1) plasmid extracts reagent
Solution I: every bottle about 100 milliliters, contain 50mmol/L glucose, 25mmol/L Tris-HCL (PH8.0),
10mmol/L EDTA (PH8.0) is at 6.895*10
4The Pa 15min that sterilizes is stored in 4 ℃;
Solution II: 0.2mol/L NaOH (facing) 1%SDS with preceding stock solution matching while using;
Solution III: be made into 100ml and contain 5mol/Lkac 60ml Glacial acetic acid, 11.5ml, water 28.5ml;
Solution I, solution III were sterilized 20 minutes in 150kg/cm2;
Above reagent is homemade analytical pure;
(2) operation
In Bechtop 2ml being contained the antibiotic LB substratum of the 100ug/ml ammonia Bian capacity of joining is in the 15ml test tube, insert the single bacterium colony of ThioA/L15/7, overnight incubation under 37 ℃ of violent joltings, draw the 1.5ml culture in Eppendorf tube, centrifugal 30 seconds of 12000g, nutrient solution is removed in suction, makes bacterial precipitation dry as far as possible, and residue is stored in 4 ℃.Contain the antibiotic LB substratum of 100ug/ml ammonia Bian and add the ice-cold solution I of 100ul in above-mentioned precipitation, thermal agitation disperses precipitation fully; In the solution II that adds the new preparation of 200ul, cover the tight mouth of pipe, put upside down centrifuge tube fast 5 times, with the mixing content, centrifuge tube is placed on ice; Adding the ice-cold solution III of 150ul, cover the tight mouth of pipe, gentle vibration 10 seconds, solution III is uniformly dispersed in the heavy-gravity bacterial lysate, place 5min on ice afterwards, add RNAase 3ul, in 60 ℃ of water-bath 20min, the centrifugal 5min of 12000g, supernatant is changed in another centrifuge tube that adds 200ul phenol and 24: 1 chloroform of 200ul/primary isoamyl alcohol solution, and vibration mixes makes the double-stranded DNA precipitation, places 2min under the room temperature, be the centrifugal 3min of 12000g, draw supernatant liquor in another centrifuge tube,, shake up with 800ul straight alcohol washing double-stranded DNA, left standstill two hours in the refrigerator freeze space, the centrifugal 5min of 12000g removes supernatant, adherent slow adding 70% ice precooled ethanol, the centrifugal 5min of 12000g, remove supernatant as far as possible and be positioned over 37 ℃ of oven for drying, dissolve nucleic acid again with the 25ul sterilized water, vibration shakes up, get 5ul and do the detection of 1% agarose gel electrophoresis, residue is stored in-20 ℃;
Carrier pET32a plasmid DNA prepares the same;
2], the PCR of goal gene Kringle5:
(1) reagent
Primer sequence:
Upstream primer: 5 ' G
CCATGGTGCTGCTCCCGGATGTAG3 '
Nco??I??25bp
Downstream primer: 3 ' G
GAATTCTTACGGGGCCGCACACTGAGG3 '
EcoR?I??28bp
Taq-DNApolyase is the archaeal dna polymerase of a kind of heat-stable DNA of depending on, has 5 ' of polymerization of depending on → 3 ' exonuclease activity, the best use of temperature is 75 ℃--80 ℃, starting polymerization reaction under the condition of optimum temperuture need be lower than, in order to avoid primer dissociates from template, Taq-DNApolyase needs Mg as the time spent
2+. because primer: unwind and the annealing temperature of template crossbred is subjected to the influence of divalent cation, needs to consider the best Mg of amplified reaction
2+Concentration.Phosphate buffered saline buffer suppresses the Taq-DNApolyase activity, and amplified reaction carries out in the Tris damping fluid usually, and this damping fluid at room temperature pH is 8.3;
(2) operation
Add 10 * Buffer 5ul in the 200ul centrifuge tube, dNTP 4ul, primer P1 2ul, P2 2ul, template plasmid 2ul, Taq-DNApolyase 0.5ul, sterilized water 34.5ul, making the reaction system cumulative volume is 50ul, with the sealing of small amount of liquid paraffin, reacts on the PCR instrument at last;
The PCR parameter is
Get 5ul PCR reaction product and 1ul marker and carry out the detection of 2% agarose gel electrophoresis, identify the product size.
3], the extracting and purifying of PCR product
(1) reagent
TE(Ph＝8.0)：10mmol/L?Tris-Hcl(PH＝8.0)+1mmol/L?EDTA(PH＝8.0)
(2) operation
The PCR product is transferred to the 1.5ml centrifuge tube, add TE (PH8.0) and supply 140ul, add chloroform/each 70ul of primary isoamyl alcohol solution of phenol and 24: 1, jolting is in the centrifugal 10min of 15000g, get supernatant and to the chloroform/primary isoamyl alcohol solution 140ul that wherein adds 24: 1, jolting in the centrifugal 10min of 15000g, is got supernatant and to wherein adding 14ul 3M NaAc and 420ul (3 times of volumes) dehydrated alcohol, in-70 ℃ of refrigerators, left standstill 2 hours, take out the back in the centrifugal 15min of 15000g.Add 1ml 70% washing with alcohol,, put into oven drying, get 5ul and carry out the detection of 2% agarose gel electrophoresis in the centrifugal 15min of 15000g.
4], double digestion PCR product and vector plasmid DNA:
(1) reagent
Nco I restriction enzyme site: 5 ' ..
A T G G..3 '
3 ' ..G G T A C
▲65 ℃ of C..5 ' heat inactivations, 20min
EcoR I restriction enzyme site: 5 ' ..
A T T C..3 '
3 ' ..C T T A A
▲65 ℃ of G..3 ' heat inactivations, 20min
(2) operation
In centrifuge tube, add 10 * Buffer 2ul, 0.1%BSA 2ul, PCR product 14ul, Nco I1.2ul, EcoR I 0.8ul, total reaction volume is 20ul,
In centrifuge tube, add 10 * Buffer 2ul, 0.1%BSA 2ul, vector plasmid DNA 12ul, NcoI 1.2ul, EcoR I 0.8ul, sterilized water 2ul, total reaction volume is 20ul,
Above-mentioned two centrifuge tubes are spent the night in 36 ℃ ± water-bath, enzyme is cut product all carry out 2% agarose gel electrophoresis;
5], dna gel reclaims:
(1) reagent
Dna gel reclaims test kit and comprises DE-A: gel melting agent (containing the DNA protective material)
DE-B: high from liquid sequence solution (making dna fragmentation greater than 100bp optionally be attached to DNA prepares on the film)
W1: washings W2: washings
Elutriant: 2.5mM Tris-Hcl, Ph=8.5
(2) operation
At first ultraviolet lamp downcuts the gel that contains target DNA down, blot and shred with paper handkerchief, the heavy 0.08g of tumor vascular growth supressor K (5) dna gel, the heavy 0.11g of carrier DNA gel, respectively as a gel volume. respectively to the DE-A solution that wherein adds 5 gel volumes, mixed evenly back is in 75 ℃ of heating, be interrupted and mix, melt fully until gel. add the DE-B solution of 0.5 DE-A volume, mixing, because tumor vascular growth supressor K (5) dna molecular amount is 300bp, is 20%. to draw said mixture respectively to wherein adding Virahol to final concentration again, transferring to DNA prepares in the pipe, be positioned in the 2ml centrifuge tube, in the centrifugal 1min of 3600rpm, if any residual, the centrifugal again 1min that raises speed abandons filtrate.To prepare pipe and put back centrifuge tube, and add 0.5ml W1 solution, the centrifugal 30s of 3600rpm abandons filtrate.To prepare pipe and put back centrifuge tube, and add 0.7mlW2 solution, the centrifugal 30s of 3600rpm abandons filtrate.Repeat once to prepare pipe and put back centrifuge tube, add 0.7mlW2 solution, the centrifugal 30s of 3600rpm abandons filtrate.To prepare pipe and put back centrifuge tube, high speed centrifugation 1min.The preparation pipe is placed clean 1.5ml centrifuge tube, prepare the film centre at DNA and add the 25ul elutriant, room temperature leaves standstill 1min.High speed centrifugation 1min eluted dna,
Obtain the double digestion fragment of goal gene Kringle5 and the double digestion fragment of carrier pET32a respectively;
6], preparation recombinant vectors pET32/kringle5
(1) reagent
But form phosphodiester bond between 5 '-phosphoric acid and the 3 '-hydroxyl among T4 ligase catalysis double-stranded DNA or the RNA, can connect two flat ends, can repair dsDNA, dsRNA, RNA/DNA incises.65 ℃ of heat inactivations, 10min,
(2) operation
10 * Buffer 1ul, the double digestion fragment 4ul of Kringle5, the double digestion fragment 2ul of carrier pET32a, T4 ligase 1ul, H
2O 2ul. total reaction volume is that 10ul.16 ℃ of constant temperature spends the night enzyme even;
7], the preparation of competent cell
(1) reagent
0.2mol/L Cacl
2Solution
0.1mol/L CaCl
2-glycerine solution: 25% glycerine+0.1M CaCl
2
(2) operation
From 37 ℃ of single bacterium colonies of cultivating a diameter 2-3mm of picking on 16-20 hour the fresh flat board of BL21, transfer in 1 liter of flask that contains the 50ml substratum, 37 ℃ of 300 commentaries on classics/min vibrated 3 hours, in super clean bench, bacterium is transferred in the ice-cold 50ml polypropylene tube, continued to place 10 minutes on ice.Reclaim cell in 4 ℃ of centrifugal 10min of 4000rpm, remove nutrient solution, will manage inversion and residual nutrient solution be flow to end in 1 minute, with the ice-cold 0.1mol/L Cacl of 10ml
2Resuspended precipitation is positioned on ice, in 4 ℃ of centrifugal 10min of 4000rpm, pours out nutrient solution, is inverted 1min, and residual nutrient solution is flow to end.Every 50ml initial incubation thing ice-cold 0.1mol/L CaCl of 2ml
2The resuspended every part of cell precipitation of-glycerine is divided into the every pipe of 200ul/ with this solution,
If do conversion, directly use, if preserve, add 7ul DMSO (dimethyl sulfoxide (DMSO)) in then every pipe, place 15min on ice, add 7ul DMSO, directly be positioned in-70 ℃ of refrigerators, or directly add water in-70 ℃ of preservations;
8], the conversion of competent cell
From-70 ℃ of refrigerators, take out the competent cell BL21 of a pipe 200ul, holding is placed on ice in the hand, add 2ml recombinant vectors pET32/kringle5 in every pipe, rotation gently, place 30mim on ice, pipe is put in 42 ℃ of water-baths slowly jolting 90s, go to fast and cool off 2min in the ice bath, other gets the 5ml small test tube, every pipe adds the not LB substratum of added with antibiotic of 500ul, heat to 37 ℃ with water-bath, the top competent cell BL21 that transformed is added wherein, be positioned on 37 ℃ of shaking tables the following temperature of 220rpm and bathed 45 minutes, make the BL21 recovery and the express recombinant carrier pET32/kringle5 ammonia Bian resistant maker gene that have transformed, centrifugal 5 minutes of the nutrient solution 5000rpm that above-mentioned 1ml temperature is bathed, top 500ul is removed in suction, and following 200ul is used for bed board, on Bechtop, the competent cell that this 200ul has been transformed is transferred to and is put in advance on the LB nutrient agar that contains 100ug/ml ammonia Bian in 37 ℃ of baking ovens, and flat board is inverted in 37 ℃ and cultivates down and bacterium colony can occur in about 14 hours;
9], the abduction delivering of small-sized fermentation and recombinant protein
(1) reagent
Inductor IPTG, 0.1M Tris-HCl (PH8.0), 1mg/ml N,O-Diacetylmuramidase
The preparation of SDS-PAGE
Separation gel now is made into wherein H of 5ml
2O 1.434ml, 30% acrylamide 2.166ml, 1.5mol/LTris (Ph8.8) 1.3ml, 10%SDS 0.05ml, 10% (NH
4)
2S
2O
80.05ml, TEMED (peptizer) 0.002ml.
Concentrated glue now is made into wherein H of 2ml
2O 1.434ml, 30% acrylamide 0.33ml, 1.0mol/LTris (Ph6.5) 0.25ml, 10%SDS 0.02ml, 10% (NH
4)
2S
2O
80.02ml, TEMED (peptizer) 0.002ml.
(2) operation
Single bacterium colony of turning out after the above-mentioned conversion of picking is to the test tube that contains the 7ml nutrient solution, 37 ℃ of 300rpm shaken overnight, drawing the 200ul culture was inoculated in the LB substratum that contains 100ug/ml ammonia Bian with 1: 30,37 ℃ of 300rpm vibrated 3 hours, add inductor IPTG, making its final concentration is 1mmol/ml, again in 37 ℃ of 300rpm vibrations 3 hours
Get 1.5ml and induce bacterium, centrifugal collection thalline, with resuspended thalline of 200ul 0.1M Tris-HCl (PH8.0) and washing, in 5000rpm centrifugal 5 minutes, abandoning supernatant added the resuspended thalline of 100ul 0.1M Tris-HCl (PH8.0), add 50ul 1mg/ml N,O-Diacetylmuramidase again, place half an hour in-70 ℃ of refrigerators, 37 ℃ of water-baths 5 minutes were put into-70 ℃ of refrigerators 5 minutes again, behind the multigelation five times, in 12000rpm centrifugal 10 minutes, collect supernatant (being the tenuigenin part), be precipitated as the inclusion body part, supernatant and precipitation and do not add inductive contrast thalline and be SDS-PAGE respectively, dyeing, decolouring, imaging;
10], cultivate and extract plasmid, the laggard row agarose gel electrophoresis evaluation of double digestion
The single bacterium colony of BL21 after picking transforms contains in the antibiotic LB substratum of 100ug/ml ammonia Bian in 2ml, extracts plasmid according to 1.2.1, carry out double digestion according to 1.2.4 after, 2% agarose gel electrophoresis is identified.
A kind of clone, evaluation and expression method of angiostatin Kringle 5 non-fusion genes, its special character is: comprise following steps:
1], design the upstream and downstream primer of its coding region according to K5 gene complete sequence, design restriction enzyme site and protection base in the primer,
Upstream primer is 5 ' CAT G
CC ATG GCA GCT CCC GGA TGT AG 3 ' underscore partly is a Nco I restriction enzyme site,
Downstream primer is 5 ' CCC
AAG CTTTTA CGG GGC CGC ACA CTG AGG 3 ' underscore partly is the HindIII restriction enzyme site,
Primer is that Shanghai biotechnology company limited is synthetic.
2], the TD-PCR of K5 gene amplification
The TD-PCR reaction system
The TD-PCR program:
3], the agarose gel electrophoresis of PCR product
(1) glue
Take by weighing the agarose of 0.40g, put special-purpose triangular flask, add the distilled water of 50ml, be heated to boiling; After cooling, add a little ethidium bromide stock solution, pour into behind the mixing in the electrophoresis chamber of inserting good comb in advance, treat to add an amount of electrophoretic buffer after its cooled and solidified, carefully take out comb.(end of well is near negative electrode.) careful operation, must not allow ethidium bromide contact own or contaminate environment.
(2) go up sample
(3) electrophoresis
The power adjustment voltage of connecting tiselius apparatus is to 100v, treats that sample enters fully that regulating voltage is that 80v carries out electrophoresis in the gel.According to the needs of electrophoresis detection, treat that tetrabromophenol sulfonphthalein indicator swimming turns down to the appropriate location and cut off the electricity supply.
(4) analyze: under the gel imaging instrument, observe.
4], a small amount of of pET28a+ plasmid DNA preparation
(1) reagent
Solution I: every bottle (100ml) contains the glucose of 50mmol/L, the Tris-HCL of 25mmol/L (PH8.0) 10mmol/L EDTA (pH8.0), 6.895 * 10
4The Pa sterilization was stored in 4 ℃ after 15 minutes,
Solution II: 0.2mmol/L NaOH, 1%SDS. (matching while using),
Solution III: be made into 100ml and contain 5mol/L kac 60ml, Glacial acetic acid 11.5ml, water 28.5ml, 6.895 * 10
4The Pa sterilization was stored in 4 ℃ after 20 minutes.
Above reagent is homemade analytical pure
(2) operation
The LB substratum capacity of joining that 3ml is contained 100 μ g/ml Amp in the aseptic technique platform is in the test tube of 15ml, inserts pET28a+ bacterium liquid 30 μ l, spends the night in 37 ℃ of fierce shaking culture.Draw the 1.5ml culture in Eppendorf tube, centrifugal 30 seconds of 12000g inhales and removes nutrient solution, make bacterial precipitation dry as far as possible, the residue culture is stored in 4 ℃, adds the solution I (precooling) of 100 μ l in containing sedimentary test tube, and thermal agitation disperses precipitation fully; Add the solution II of the new preparation of 200 μ l again, cover the tight mouth of pipe, put upside down fast 5 times,, centrifuge tube is placed on ice with the mixing content; The solution III that adds 150 μ l precoolings again, cover the tight mouth of pipe, gentle vibration made solution III be uniformly dispersed in the heavy-gravity bacterial lysate in 10 seconds and places afterwards and added Rnase 3 μ l on ice in 5 minutes in 60 ℃ of water-baths 20 minutes, centrifugal 5 minutes of 12000g, supernatant is changed in another centrifuge tube that is added with 200 μ l phenol and 24: 1 chloroform of 200 μ l/primary isoamyl alcohol solution, it is the double-stranded DNA precipitation that vibration mixes, and places 2 minutes at 12000g centrifugal 3 minutes under the room temperature.The careful supernatant liquor of drawing is in another centrifuge tube, with 800 μ l straight alcohols washing double-stranded DNA, 70% ice precooled ethanol, the centrifugal 5min of 12000g, remove supernatant as far as possible and be positioned over 37 ℃ of oven for drying, dissolve nucleic acid again with 25 μ l sterilized waters, vibration shakes up, get 5 μ l and do the detection of 1% agarose gel electrophoresis, residue is stored in-20 ℃;
5], double digestion PCR product and vector plasmid DNA
Reaction system:
Enzyme Qie Wendu is 37 ℃, and the time is 3.5h.
6], the sepharose of dna segment detects and the glue recovery.
(1) reagent: (dna gel recovery test kit)
Buffer DE-A: gel melting agent (containing the DNA protective material)
Buffer DE-B:DNA binding soln
Buffer W1: washings
Buffer W2: demineralised liquid (pressing designated volume adding dehydrated alcohol on the reagent bottle before using)
Eluent:2.5mM Tris-HCl, pH8.5, DNA elute soln
(2) operation:
(2.1) at first under ultraviolet lamp, downcut the sepharose that contains target DNA, use Sillim's gel surface liquid so far.Reduce gel volume, the gel piece of large volume need be cut into small pieces as far as possible, to shorten the time that gel melts in the step 4.
(2.2) weighing gel weight is with 1mg=1 μ l conversion gel volume.According to gel strength, the parameter that is provided by table adds Buffer DE-A:
| Gel strength | Buffer DE-A volume |
| ??≤1.0％ | 3 gel volumes |
What this experiment needed recovery is double digestion pET28a+ and K5 fragment, and the glue of use all is 0.8%, so add the Buffer DE-A of 3 gel volumes of people.
(2.3) in 75 ℃ of heating, mixed once after suspending evenly, melt (about 6-8 minute) fully until gel piece every 2-3 minute.
(2.4) adding Buffer DE-B by 50% of Buffer DE-A volume mixes.When the dna fragmentation that reclaims during less than 500bp, add Virahol, the final concentration that makes Virahol is 20%.
(2.5) DNA-prep Tube is placed 2ml Microfuge Tube, the mixed solution in the step 5 is moved among the DNA-prep Tube centrifugal 1 minute of 5500rpm.
(2.6) abandon filtrate, DNA-prep Tube is put get back among the 2ml Microfuge Tube, add the Buffer W1 that 500ul has added dehydrated alcohol, centrifugal 1 minute of 5500rpm
(2.7) abandon filtrate, DNA-prep Tube is put get back among the 2ml Microfuge Tube, add the Buffer W2 that 700ul has added dehydrated alcohol, centrifugal 1 minute of 5500rpm, with same method more once with 700ulBuffer W2 washing.
(2.8) DNA-prep Tube is placed the 1.5ml centrifuge tube, centrifugal 1 minute of 12000 * g.
(2.9) DNA-prep Tube is placed another clean 1.5ml centrifuge tube, central authorities add 25-30ul Eluent or deionized water at the silica film, and room temperature left standstill 1 minute.Centrifugal 1 minute eluted dna of 12000 * g.
7], the preparation of recombinant vectors pET28a+/K5
Reaction system is gone into following table
16 ℃ of incubated overnight ,-20 ℃ of preservations are standby,
8], the preparation of competent cell
(1) reagent:
(1.1) CaCl
2Solution (ice-cold)
0.1mol/L CaCl
2Solution, after the filtration sterilization of use disposable filter ,-4 ℃ of refrigerator storages are standby,
(1.2) 0.1mol/L CaCl
2Glycerine solution
25% glycerine+0.1M CaCL
2After using the disposable filter filtration sterilization ,-4 ℃ of refrigerators are preserved standby
(2) operation:
(2.1) inoculation e. coli strain bl21 list bacterium colony is gone in the 5ml LB liquid nutrient medium 37 ℃ of overnight incubation under the aseptic condition.Second day, pour in the LB liquid nutrient medium of 250ml, 37 ℃ of vibration 3.5h-4.0h (600=0.4 ~ 0.6) stop to cultivate,
(2.2) the nutrient solution branch is installed in the aseptic polypropylene tube of 8 25ml precoolings, in putting 5-10 minute on ice, 4 ℃ then, the centrifugal 8min of 8000rmp,
(2.3) cell precipitation is resuspended with the ice-cold CaCl2 solution of 10ml, centrifugal 5 minutes of 4 ℃ of 6000rmp,
(2.4)) abandon supernatant after, add the ice-cold CaCl2 solution re-suspended cell of 10ml, ice bath is after half an hour, 4 ℃, centrifugal 5 minutes of 6000rmp,
(2.5) abandon supernatant after, add the ice-cold CaCl2 solution re-suspended cell of 2ml, be sub-packed in the 0.5ml centrifuge tube of precooling by every pipe 200 μ l amount, frozen immediately standby in-75 ℃ of refrigerator-freezers,
9], the conversion of recombinant plasmid and preservation
(1) 10 μ L are connected product and be added in the 200 μ L competent cell suspensions, mixing, ice bath 30-40 minute, 42 ℃ of water bath heat preservations 90 seconds were put 1-2 minute on ice immediately, and transfer to then and carry out contrast culture on the substratum that is added with microbiotic ammonia benzyl,
(2) will transform successful intestinal bacteria, picking list bacterium colony carries out the purifying amplification cultivation in liquid nutrient medium, and 37 ℃ of violent joltings are spent the night.The absorption culture 500ul that shaking table cultivates that spends the night adds in the centrifuge tube of 1.5ml, adds 40% glycerine 500ul again and mixes the back in-20 or-70 ℃ of preservations, regularly activates heavily preservation, in case spawn degeneration or plasmid loss.
10], the extraction of recombinant plasmid
Method is the same,
11], the PCR of recombinant plasmid detects
Used consistent in program thereby and the reaction system and 3.2, carry out the detection of agarose gel electrophoresis then and observe.
Advantage of the present invention is:
Carried out the aim sequence amplification, successfully be connected in carrier and transfered cell, double digestion is identified and is confirmed.The small-sized fermentation inducible protein is expressed, and SDS-PAGE identifies affirmation, and Kringle5 albumen exists with the inclusion body form.Obtain clone's product, expression product and the engineering bacteria thereof of tumor vascular growth supressor K (5) gene.
Success makes up the non-fusion expression carrier of pET28a (+)-K5 gene.For the research of the non-fusion expression of K5 gene in intestinal bacteria is laid a good foundation.
Description of drawings
Fig. 1 is the proplasmin structural representation.
Fig. 2 is a kind of clone, evaluation and expression method schema of angiostatin Kringle 5 fusion genes.
Fig. 3 is a kind of clone, evaluation and expression method schema of angiostatin Kringle 5 non-fusion genes.
Fig. 4 is ThioA/L15/7 and E.coli PET32a carrier.
Fig. 5 is albumen Kringle5 band.
Fig. 6 is a segment pcr amplification electrophoretogram, CR product wherein, and 2 is DNA Marker, 3 is the PCR product.
Fig. 7 is that the pulsating PCR of the purpose in the plasmid identifies electrophoretogram, and swimming lane 1 and swimming lane 3 are PCR products, and swimming lane 2 is DNA Marker,
Be followed successively by 2000bp from top to bottom, 1000bp, 750bp, 500bp, 250bp, 100bp.
Embodiment
A kind of clone, evaluation and expression method of angiostatin Kringle 5 fusion genes:
(1.) purpose: make up angiostatinK5 cDNA expression vector, and it is imported in the intestinal bacteria, extract the back enzyme and cut evaluation.Small-sized fermentation is induced competent cell expressing K 5 genes, and SDS-PAGE identifies expression product K5.(2.) method: angiostatinK5 cDNA is in the plasmid of ThioA/L15/7 bacterial strain, plasmid carries out the aim sequence pcr amplification after extracting, double digestion after the purification of products, then goal gene is connected in carrier pET32a, recombinant vectors pET32a/kringle5 imports competent cell E.coli BL21, cultivate and extract plasmid, the laggard row agarose gel electrophoresis of final double digestion is identified.Small-sized fermentation is induced competent cell BL21 expressing K 5 genes that transformed with IPTG, and SDS-PAGE identifies expression product K5 albumen behind the broken thalline of N,O-Diacetylmuramidase.(3.) result: carried out the aim sequence amplification, successfully be connected in carrier and transfered cell, double digestion is identified and is confirmed.The small-sized fermentation inducible protein is expressed, and SDS-PAGE identifies affirmation, and Kringle5 albumen exists with the inclusion body form.(4.) conclusion: the clone's product, expression product and the engineering bacteria thereof that obtain tumor vascular growth supressor K (5) gene.
1.1 material:
1.1.1 bacterial strain and plasmid
Bacterial strain: E.coli BL21 carrier: pET32a plasmid
Goal gene: Kringle-5 of Angiostatin
By this experimental center preservation, provide.
Kringle 5 protein sequences
CMFGNGKGYRGKRATTVTGTPCQDWAAQEPHRHSIFTPENPRAGLEKNYCRNPDGDVGGPWCYTT
NPRKLYDYCDVPQC
1.1.2 substratum
The LB substratum
Tryptones: 10 yeast extracts: 5 gram sodium-chlor: 10 grams
Add water to 1000 milliliters, 150kg/cm2 sterilized 20 minutes
Nutrient agar
Tryptones: 10 gram yeast extracts: 5 gram sodium-chlor: 10 grams
Agar powder: 15 grams
Add water to 1000 milliliters, 150kg/cm2 sterilized 20 minutes
1.1.3DNA gel reclaims test kit: the clean biochemical technology of DNA gel extraction kit Hangzhou Wei Te company limited
1.1.4 cutting with enzyme, enzyme is connected reagent: NcoI﹠amp; The precious biotechnology (Dalian) of EcoRI T4 ligase company limited
1.1.5 instrument
Whizzer: development Jiangsu Province Xinghua City analytical instrument factory of GTL-16AL high speed tabletop centrifuge Nanjing University produces
Whizzer: Avanti J-25 high speed freezing centrifuge Beckman company produces
The pcr amplification instrument: T-Gradient Thermoblock Germany Biometra company produces
Gel imaging system
1.2 method:
1.2.1 the preparation of ThioA/L15/7 and E.coli PET32 (carrier) plasmid DNA:
(1) plasmid extracts reagent
Solution I: every bottle about 100 milliliters, contain 50mmol/L glucose, 25mmol/L Tris-HCL (PH8.0), 10mmol/L EDTA (PH8.0) is at 6.895*10
4The Pa 15min that sterilizes is stored in 4 ℃
Solution II: 0.2mol/L NaOH (facing) 1%SDS with preceding stock solution matching while using
Solution III: be made into 100ml and contain 5mol/Lkac 60ml Glacial acetic acid, 11.5ml, water 28.5ml
Solution I, solution III were sterilized 20 minutes in 150kg/cm2
(above reagent is homemade analytical pure)
(2) operation
In Bechtop 2ml being contained the antibiotic LB substratum of the 100ug/ml ammonia Bian capacity of joining is in the 15ml test tube, inserts the single bacterium colony of ThioA/L15/7, overnight incubation under 37 ℃ of violent joltings.Draw the 1.5ml culture in Eppendorf tube, centrifugal 30 seconds of 12000g inhales and removes nutrient solution, makes bacterial precipitation dry as far as possible, and residue is stored in 4 ℃.Contain the antibiotic LB substratum of 100ug/ml ammonia Bian
Add the ice-cold solution I of 100ul in above-mentioned precipitation, thermal agitation disperses precipitation fully; In the solution II that adds the new preparation of 200ul, cover the tight mouth of pipe, put upside down centrifuge tube fast 5 times, with the mixing content, centrifuge tube is placed on ice; Adding the ice-cold solution III of 150ul, cover the tight mouth of pipe, gentle vibration 10 seconds is uniformly dispersed solution III in the heavy-gravity bacterial lysate, place 5min on ice afterwards, add RNAase 3ul, in 60 ℃ of water-bath 20min, the centrifugal 5min of 12000g, supernatant is changed in another centrifuge tube that adds 200ul phenol and 24: 1 chloroform of 200ul/primary isoamyl alcohol solution, vibration mixes makes the double-stranded DNA precipitation, places 2min under the room temperature, is the centrifugal 3min of 12000g.The careful supernatant liquor of drawing is in another centrifuge tube.With 800ul straight alcohol washing double-stranded DNA, shake up, left standstill two hours, the centrifugal 5min of 12000g in the refrigerator freeze space, remove supernatant, adherent slow adding 70% ice precooled ethanol, the centrifugal 5min of 12000g removes supernatant as far as possible and is positioned over 37 ℃ of oven for drying, again dissolve nucleic acid with the 25ul sterilized water, vibration shakes up, and gets 5ul and does the detection of 1% agarose gel electrophoresis, and residue is stored in-20 ℃.
Carrier pET32a plasmid DNA prepares the same.
1.2.2 the PCR of goal gene Kringle5:
(1) reagent
Primer sequence:
Upstream primer: 5 ' G
CCATGGTGCTGCTCCCGGATGTAG3 '
Nco??I??25bp
Downstream primer: 3 ' G
GAATTCTTACGGGGCCGCACACTGAGG3 '
EcoR?I??28bp
Taq-DNApolyase is the archaeal dna polymerase of a kind of heat-stable DNA of depending on, has 5 ' of polymerization of depending on → 3 ' exonuclease activity.The best use of temperature is 75 ℃--80 ℃, and need be being lower than starting polymerization reaction under the condition of optimum temperuture, in order to avoid primer dissociates from template.Taq-DNApolyase needs Mg as the time spent
2+. because primer: unwind and the annealing temperature of template crossbred is subjected to the influence of divalent cation, needs to consider the best Mg of amplified reaction
2+Concentration.Phosphate buffered saline buffer suppresses the Taq-DNApolyase activity, and amplified reaction carries out in the Tris damping fluid usually, and this damping fluid at room temperature pH is 8.3.
(2) operation
Add 10 * Buffer 5ul in the 200ul centrifuge tube, dNTP 4ul, primer P1 2ul, P2 2ul, template plasmid 2ul, Taq-DNApolyase 0.5ul, sterilized water 34.5ul, making the reaction system cumulative volume is 50ul, with the sealing of small amount of liquid paraffin, reacts on the PCR instrument at last.
The PCR parameter is:
Get 5ul PCR reaction product and 1ul marker and carry out the detection of 2% agarose gel electrophoresis, identify the product size.
1.2.3PCR the extracting and purifying of product
(1) reagent
TE(Ph＝8.0)：10mmol/L?Tris-Hcl(PH＝8.0)+1mmol/L?EDTA(PH＝8.0)
(2) operation
The PCR product is transferred to the 1.5ml centrifuge tube, adds TE (PH8.0) and supply 140ul.Chloroform/each 70ul of primary isoamyl alcohol solution that adds phenol and 24: 1, jolting.In the centrifugal 10min of 15000g.Get supernatant and to the chloroform/primary isoamyl alcohol solution 140ul that wherein adds 24: 1, jolting.In the centrifugal 10min of 15000g.Get supernatant and to wherein adding 14ul 3M NaAc and 420ul (3 times of volumes) dehydrated alcohol.In-70 ℃ of refrigerators, left standstill 2 hours.Take out the back in the centrifugal 15min of 15000g.Add 1ml 70% washing with alcohol, in the centrifugal 15min of 15000g.Put into oven drying.Get 5ul and carry out the detection of 2% agarose gel electrophoresis.
1.2.4 double digestion PCR product and vector plasmid DNA:
(1) reagent
Nco I restriction enzyme site: 5 ' ..
A T G G..3 '
3 ' ..G G T A C
▲65 ℃ of C..5 ' heat inactivations, 20min
EcoR I restriction enzyme site: 5 ' ..
A T T C..3 '
3 ' ..C T T A A
▲65 ℃ of G..3 ' heat inactivations, 20min
(2) operation
In centrifuge tube, add 10 * Buffer 2ul, 0.1%BSA 2ul, PCR product 14ul, Nco I 1.2ul, EcoR I 0.8ul,
Total reaction volume is 20ul.
In centrifuge tube, add 10 * Buffer 2ul, 0.1%BSA 2ul, vector plasmid DNA 12ul, Nco I1.2ul, EcoR I 0.8ul, sterilized water 2ul, total reaction volume is 20ul.
Above-mentioned two centrifuge tubes are spent the night in 36 ℃ ± water-bath, enzyme is cut product all carry out 2% agarose gel electrophoresis.
1.2.5DNA gel reclaims:
(1) reagent
Dna gel reclaims test kit and comprises DE-A: gel melting agent (containing the DNA protective material)
DE-B: high from liquid sequence solution (making dna fragmentation greater than 100bp optionally be attached to DNA prepares on the film)
W1: washings W2: washings
Elutriant: 2.5mM Tris-Hcl, Ph=8.5
(2) operation
At first ultraviolet lamp downcuts the gel that contains target DNA down, blot and shred with paper handkerchief, the heavy 0.08g of tumor vascular growth supressor K (5) dna gel, the heavy 0.11g of carrier DNA gel, respectively as a gel volume. respectively to the DE-A solution that wherein adds 5 gel volumes, mixed evenly back is in 75 ℃ of heating, be interrupted and mix, melt fully until gel. add the DE-B solution of 0.5 DE-A volume, mixing, because tumor vascular growth supressor K (5) dna molecular amount is 300bp, is 20%. to draw said mixture respectively to wherein adding Virahol to final concentration again, transferring to DNA prepares in the pipe, be positioned in the 2ml centrifuge tube, in the centrifugal 1min of 3600rpm, if any residual, the centrifugal again 1min that raises speed abandons filtrate.To prepare pipe and put back centrifuge tube, and add 0.5mlW1 solution, the centrifugal 30s of 3600rpm abandons filtrate.To prepare pipe and put back centrifuge tube, and add 0.7ml W2 solution, the centrifugal 30s of 3600rpm abandons filtrate.Repeat once to prepare pipe and put back centrifuge tube, add 0.7ml W2 solution, the centrifugal 30s of 3600rpm abandons filtrate.To prepare pipe and put back centrifuge tube, high speed centrifugation 1min.The preparation pipe is placed clean 1.5ml centrifuge tube, prepare the film centre at DNA and add the 25ul elutriant, room temperature leaves standstill 1min.High speed centrifugation 1min eluted dna.
Obtain the double digestion fragment of goal gene Kringle5 and the double digestion fragment of carrier pET32a respectively.
1.2.6 preparation recombinant vectors pET32/kringle5
(1) reagent
But form phosphodiester bond between 5 '-phosphoric acid and the 3 '-hydroxyl among T4 ligase catalysis double-stranded DNA or the RNA, can connect two flat ends, can repair dsDNA, dsRNA, RNA/DNA incises.65 ℃ of heat inactivations, 10min.
(2) operation
10 * Buffer 1ul, the double digestion fragment 4ul of Kringle5, the double digestion fragment 2ul of carrier pET32a, T4 ligase 1ul, H
2O 2ul. total reaction volume is that 10ul.16 ℃ of constant temperature spends the night enzyme even.
1.2.7 the preparation of competent cell
(1) reagent
0.3mol/L Cacl
2Solution
0.1mol/L CaCl
2-glycerine solution: 25% glycerine+0.1M CaCl
2
(2) operation
From 37 ℃ of single bacterium colonies of cultivating a diameter 2-3mm of picking on 16-20 hour the fresh flat board of BL21, transfer in 1 liter of flask that contains the 50ml substratum, 37 ℃ of 300 commentariess on classics/min vibrated 3 hours.In super clean bench, bacterium is transferred in the ice-cold 50ml polypropylene tube, continue to place 10 minutes on ice.Reclaim cell in 4 ℃ of centrifugal 10min of 4000rpm, remove nutrient solution, will manage inversion and residual nutrient solution be flow to end in 1 minute.With the ice-cold 0.1mol/L Cacl of 10ml
2Resuspended precipitation is positioned on ice, in 4 ℃ of centrifugal 10min of 4000rpm, pours out nutrient solution, is inverted 1min, and residual nutrient solution is flow to end.Every 50ml initial incubation thing ice-cold 0.1mol/L CaCl of 2ml
2The resuspended every part of cell precipitation of-glycerine is divided into the every pipe of 200ul/ with this solution.
If do conversion, directly use, if preserve, add 7ul DMSO (dimethyl sulfoxide (DMSO)) in then every pipe, place 15min on ice, add 7ul DMSO, directly be positioned in-70 ℃ of refrigerators, or directly add water in-70 ℃ of preservations.
1.2.8 the conversion of competent cell
Take out the competent cell BL21 of a pipe 200ul from-70 ℃ of refrigerators, holding is placed on ice in the hand.Add 2ml recombinant vectors pET32/kringle5 in every pipe, 30mim is placed in rotation on ice gently.Pipe is put in 42 ℃ of water-baths slowly jolting 90s, goes to fast and cool off 2min in the ice bath.Other gets the 5ml small test tube, and every pipe adds the not LB substratum of added with antibiotic of 500ul, heats to 37 ℃ with water-bath.The top competent cell BL21 that transformed is added wherein, be positioned on 37 ℃ of shaking tables the following temperature of 220rpm and bathed 45 minutes, make the BL21 recovery and the express recombinant carrier pET32/kringle5 ammonia Bian resistant maker gene that have transformed.Centrifugal 5 minutes of the nutrient solution 5000rpm that above-mentioned 1ml temperature is bathed, top 500ul is removed in suction, following 200ul is used for bed board, on Bechtop, the competent cell that this 200ul has been transformed is transferred to and is put in advance on the LB nutrient agar that contains 100ug/ml ammonia Bian in 37 ℃ of baking ovens, and flat board is inverted in 37 ℃ and cultivates down and bacterium colony can occur in about 14 hours.
1.2.9 the abduction delivering of small-sized fermentation and recombinant protein
(1) reagent
Inductor IPTG, 0.1M Tris-HCl (PH8.0), 1mg/ml N,O-Diacetylmuramidase
The preparation of SDS-PAGE
Separation gel now is made into wherein H of 5ml
2O 1.434ml, 30% acrylamide 2.166ml, 1.5mol/LTris (Ph8.8) 1.3ml, 10%SDS 0.05ml, 10% (NH
4)
2S
2O
80.05ml, TEMED (peptizer) 0.002ml.
Concentrated glue now is made into wherein H of 2ml
2O 1.434ml, 30% acrylamide 0.33ml, 1.0mol/LTris (Ph6.5) 0.25ml, 10%SDS 0.02ml, 10% (NH
4)
2S
2O
80.02ml, TEMED (peptizer) 0.002ml.
(2) operation
Single bacterium colony of turning out after the above-mentioned conversion of picking to the test tube that contains the 7ml nutrient solution, 37 ℃ of 300rpm shaken overnight.Draw the 200ul culture and be inoculated in the LB substratum that contains 100ug/ml ammonia Bian with 1: 30,37 ℃ of 300rpm vibrated 3 hours, added inductor IPTG, and making its final concentration is 1mmol/ml, again in 37 ℃ of 300rpm vibrations 3 hours.
Get 1.5ml and induce bacterium, centrifugal collection thalline, with 200ul 0.1M Tris-HCl (PH8.0) resuspended thalline and washing, in 5000rpm centrifugal 5 minutes, abandoning supernatant.Add the resuspended thalline of 100ul 0.1M Tris-HCl (PH8.0), add 50ul 1mg/ml N,O-Diacetylmuramidase again, place half an hour in-70 ℃ of refrigerators, 37 ℃ of water-baths 5 minutes, put into-70 ℃ of refrigerators again 5 minutes, behind the multigelation five times, in 12000rpm centrifugal 10 minutes, collect supernatant (being the tenuigenin part), be precipitated as the inclusion body part, supernatant and precipitation and do not add inductive contrast thalline and be SDS-PAGE respectively, dyeing, decolouring, imaging.
1.2.10 cultivate and extract plasmid, the laggard row agarose gel electrophoresis of double digestion is identified
The single bacterium colony of BL21 after picking transforms contains in the antibiotic LB substratum of 100ug/ml ammonia Bian in 2ml, extracts plasmid according to 1.2.1, carry out double digestion according to 1.2.4 after, 2% agarose gel electrophoresis is identified.
The result:
Obtain ThioA/L15/7 and E.coli PET32a (carrier) plasmid DNA, 1% agarose gel electrophoresis detects, and observes three bright bands under ultraviolet lamp respectively.Fig. 2 is a carrier
Behind the PCR of goal gene Kringle5, get 5ul PCR reaction product and 1ul marker and carry out the detection of 2% agarose gel electrophoresis, the bright band of observing the PCR reaction product under ultraviolet lamp is concordant with the 4th band of marker, is about 300bp, conforms to data.
Behind the extracting and purifying of PCR product, get 5ul and carry out the detection of 2% agarose gel electrophoresis.Under ultraviolet lamp, observe a bright band, concordant with the 4th band of marker, be the purpose purified product.See Fig. 2
Behind the double digestion enzyme is cut product and all carry out 2% agarose gel electrophoresis.Observing under ultraviolet lamp, is a very bright band that is about 5000bp behind the carrier double digestion; It behind the goal gene double digestion bright band that is about 300bp.Fig. 2
Dna gel reclaims, and obtains the double digestion fragment of goal gene Kringle5 and the double digestion fragment of carrier pET32a respectively. and enzyme obtains recombinant vectors pET32/kringle5 after connecting.
Transform 37 ℃ of following overnight incubation behind the preparation competent cell.Be coated with four flat boards altogether, on average grow bacterium colony 6-8,8 single bacterium colonies of picking are cultivated.Choose 3 pipe cultures and carry out small-sized fermentation.Behind the abduction delivering, collect broken thalline, supernatant and precipitation and do not add inductive contrast thalline and be SDS-PAGE respectively, dyeing, decolouring, imaging.Can clearly find out has a protein band in the precipitation, concordant with the 4th band of marker, conforms to Kringle5 albumen data in the data, confirms as Kringle5 albumen.Do not add in the inductive contrast then not this band in supernatant neutralization, thereby affirmation Kringle5 albumen exists with the inclusion body form.Fig. 3
Angiostatin and each Kringle thereof express in multiple systems successfully, and Tao Yimin etc. link to each other with carrier pPIC9K after with Kringle5 gene amplification, transform the engineering bacteria that pichia spp GS115 obtains stably express.People such as Cao utilize the PRC/CMV plasmid to be carrier, become in the fibrosarcoma cell to have expressed the recombined small-mouse angiostatin at mouse T241.Zhang Ju etc. insert Bac to Bac baculovirus expression system with the K1-4 gene, and transfection insect cell sf9 expresses angiostatin.Wu Jingwen etc. make up the carrier for expression of eukaryon pcDNA-SAK (1-3) of angiostatin K1-3, it is transfected into the SHG44 human glioma cell expresses.And utilize the example of escherichia coli expression a lot, and characteristics such as it is fast that reason is that intestinal bacteria have a breeding, and nutritional requirement is low, and is simple to operate, for not needing glycosylation, modifications such as phosphorylation promptly present bioactive albumen, are first-selected expression systems.Eukaryotic gene and prokaryotic gene merge, and make transcribe and translate initial by normal intestinal bacteria Nucleotide control, help the expression of eukaryotic gene and stablizing of product.Carrier is selected to necessarily require carrier can have the independently duplicated ability of DNA in recipient cell; Molecular weight is as far as possible little, is easy to purifying; Can recombinate with foreign DNA in the point of contact in the single point of contact that includes multiple restriction enzyme; Having in the carrier does not influence it and duplicates the inessential zone of growth; Has the multiple choices mark.Natural carrier need just can reach requirement as cloning vector through a series of transformation.PET32a is a protokaryon pattern of fusion efficient expression vector, and 5900bp is arranged, and expression efficiency is very high.The DNA that uses double digestion can avoid single endonuclease digestion to produce when enzyme is cut reverses simultaneously, is that translation can not normally be carried out, thereby has guaranteed the accuracy of expressing.Recombinant products forms inclusion body, is easy to purifying.Next step will carry out renaturation to expression product, and purifying and angiogenesis inhibiting activity are measured.
Test in the selected method at this, following experiment main points directly influence result of experiment
RNA digestion is fully very important when plasmid extracts, and it directly has influence on the effect of double digestion;
Time of repose should be long as far as possible after adding straight alcohol, and plasmid is precipitated fully.Should adherent slow adding when adding 70% ethanol, otherwise can make plasmid outwell with ethanol and run off.Dna gel should not be exposed to it under ultraviolet lamp when reclaiming for a long time, can be heated to 60 ℃ for raising the efficiency elutriant.Different owing to NcoI when double digestion with the required condition of EcoR I, and Nco I more depends on reaction conditions, so select the required condition of Nco I.The preparation key of competent cell is cold operation, to guarantee the biologic activity of competent cell.The catalytic activity of various enzymes all has optimum temperuture simultaneously, strict controlled temperature.When culturing cell, should gather in the crops in logarithmic phase, to obtain high reactivity and cell concentration.
The target cell of Angiostatin effect is an endotheliocyte, and it does not have the sort of height unstable of cell, therefore is difficult for Angiostatin is developed immunity to drugs.The endotheliocyte life-span is very long, and the tumor vascular endothelial cell rate of propagation more normally organizes endothelial cell proliferation speed high 50 times.Therefore for tumour patient, use Angiostatin within a certain period of time, can't cause obvious influence normal in-house blood vessel.Angiostatin has brought hope for defeating tumour, though still need further to confirm check by clinical experiment, Angiostatin and existing embolic chemotherapy combined utilization can be predicted, can aspect oncotherapy, obtain important breakthrough.
A kind of clone, evaluation and expression method of angiostatin Kringle 5 non-fusion genes,
Kringle5 is the inhibition vascular endothelial cell proliferation found at present and the strongest Profibrinolysin fragment of activity of tumor growth, specificity height and toxic side effect is little.Tool potential immense value and wide application prospect aspect tumor treatment.
The sequence map design PCR primer of the K5 gene that this experimental basis has been delivered, amplify the former K5 portion gene of fibrolan by TD-PCR from existing cloning vector, at Nco I and HindIII two restriction enzyme sites respectively to K5 and pET28a (+) gene order double digestion, again the two is linked to each other, successfully make up the non-fusion expression carrier of pET28a (+)-K5 gene.For the research of the non-fusion expression of K5 gene in intestinal bacteria is laid a good foundation.
Reagent and manufacturer are as follows:
All available from the precious biotechnology in Dalian Engineering Co., Ltd, the Taq enzyme is available from MBI for restriction enzyme, T4 dna ligase, high-fidelity Taq enzyme;
Dna gel reclaims test kit available from the clean biochemical technology of Hangzhou Wei Te company limited;
Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Test used bacterial strain and plasmid
(1) e. coli bl21 (DE3) is preserved by this laboratory
(2) plasmid pET28a (+) is preserved by this laboratory
Key instrument and manufacturer are as follows:
J-25 type high speed freezing centrifuge Backman company (U.S.)
New campus, GTL-16A desk centrifuge Nanjing technical institute (China)
PB2002-E type electronic balance Metler company (Germany)
TY-90S type decolorization swinging table Nanjing University (China)
PTC-150 type pcr amplification instrument MJ company (U.S.)
Slab-electrophoresis groove and electrophoresis apparatus Liuyi Instruments Plant, Beijing
Substratum:
2.2.1LB substratum: Trypsin freezes 10g, yeast extract 5g, sodium-chlor 10g, adds water to 1000ml.121.3 ℃ sterilization 20 minutes.
Nutrient agar: Trypsin freezes 10g, yeast extract 5g, sodium-chlor 10g, agar powder 15g, adds water to 1000ml.121.3 ℃ sterilization 20 minutes.
1, K5 gene upstream and downstream primer design is with synthetic
Design the upstream and downstream primer of its coding region according to the K5 gene complete sequence of having reported, design restriction enzyme site and protection base in the primer.
Upstream primer is 5 ' CAT G
CC ATG GCA GCT CCC GGA TGT AG 3 ' underscore partly is the NcoI restriction enzyme site
Downstream primer is 5 ' CCC
AAG CTTTTA CGG GGC CGC ACA CTG AGG 3 ' underscore partly is the HindIII restriction enzyme site
Primer is that Shanghai biotechnology company limited is synthetic.
2, the TD-PCR of K5 gene amplification
The TD-PCR reaction system
The TD-PCR program:
3, the agarose gel electrophoresis of PCR product
3.1 glue
Take by weighing the agarose of 0.40g, put special-purpose triangular flask, add the distilled water of 50ml, be heated to boiling; After cooling, add a little ethidium bromide stock solution, pour into behind the mixing in the electrophoresis chamber of inserting good comb in advance, treat to add an amount of electrophoretic buffer after its cooled and solidified, carefully take out comb.(end of well is near negative electrode.) careful operation, must not allow ethidium bromide contact own or contaminate environment.
3.2 last sample
3.3 electrophoresis
The power adjustment voltage of connecting tiselius apparatus is to 100v, treats that sample enters fully that regulating voltage is that 80v carries out electrophoresis in the gel.According to the needs of electrophoresis detection, treat that tetrabromophenol sulfonphthalein indicator swimming turns down to the appropriate location and cut off the electricity supply.
3.4 analyze: under the gel imaging instrument, observe.
4, a small amount of of pET28a+ plasmid DNA preparation
4.1 reagent
Solution I: every bottle (100ml) contains the glucose of 50mmol/L, the Tris-HCL of 25mmol/L (PH8.0) 10mmol/L EDTA (pH8.0), 6.895 * 10
4The Pa sterilization was stored in 4 ℃ after 15 minutes.
Solution II: 0.2mmol/L NaOH, 1%SDS. (matching while using).
Solution III: be made into 100ml and contain 5mol/L kac 60ml, Glacial acetic acid 11.5ml, water 28.5ml, 6.895 * 10
4The Pa sterilization was stored in 4 ℃ after 20 minutes.
(above reagent is homemade analytical pure)
4.2 operation
The LB substratum capacity of joining that 3ml is contained 100 μ g/ml Amp in the aseptic technique platform is in the test tube of 15ml, inserts pET28a+ bacterium liquid 30 μ l, spends the night in 37 ℃ of fierce shaking culture.Draw the 1.5ml culture in Eppendorf tube, centrifugal 30 seconds of 12000g inhales and removes nutrient solution, makes bacterial precipitation dry as far as possible, the residue culture
Be stored in 4 ℃.Add the solution I (precooling) of 100 μ l in containing sedimentary test tube, thermal agitation disperses precipitation fully; Add the solution II of the new preparation of 200 μ l again, cover the tight mouth of pipe, put upside down fast 5 times,, centrifuge tube is placed on ice with the mixing content; The solution III that adds 150 μ l precoolings again, cover the tight mouth of pipe, gentle vibration made solution III be uniformly dispersed in the heavy-gravity bacterial lysate in 10 seconds and places afterwards and added Rnase 3 μ l on ice in 5 minutes in 60 ℃ of water-baths 20 minutes, centrifugal 5 minutes of 12000g, supernatant is changed in another centrifuge tube that is added with 200 μ l phenol and 24: 1 chloroform of 200 μ l/primary isoamyl alcohol solution, it is the double-stranded DNA precipitation that vibration mixes, and places 2 minutes at 12000g centrifugal 3 minutes under the room temperature.The careful supernatant liquor of drawing is in another centrifuge tube.With 800 μ l straight alcohols washing double-stranded DNA, 70% ice precooled ethanol, the centrifugal 5min of 12000g, remove supernatant as far as possible and be positioned over 37 ℃ of oven for drying, dissolve nucleic acid again with 25 μ l sterilized waters, vibration shakes up, get 5 μ l and do the detection of 1% agarose gel electrophoresis, residue is stored in-20 ℃.
5, double digestion PCR product and vector plasmid DNA
Reaction system:
Enzyme Qie Wendu is 37 ℃, and the time is 3.5h.
6, the sepharose of dna segment detects and the glue recovery.
6.1 reagent: (dna gel recovery test kit)
Buffer DE-A: gel melting agent (containing the DNA protective material)
Buffer DE-B:DNA binding soln
Buffer W1: washings
Buffer W2: demineralised liquid (pressing designated volume adding dehydrated alcohol on the reagent bottle before using)
Eluent:2.5mM Tris-HCl, pH8.5, DNA elute soln
6.2 operation:
(1) at first under ultraviolet lamp, downcuts the sepharose that contains target DNA, use Sillim's gel surface liquid so far.Reduce gel volume, the gel piece of large volume need be cut into small pieces as far as possible, to shorten the time that gel melts in the step 4.
(2) weighing gel weight is with 1mg=1 μ l conversion gel volume.According to gel strength, the parameter that is provided by table adds Buffer DE-A:
| Gel strength | Buffer DE-A volume |
| ??≤1.0％ | 3 gel volumes |
What this experiment needed recovery is double digestion pET28a+ and K5 fragment, and the glue of use all is 0.8%, so add the Buffer DE-A of 3 gel volumes of people.
(3) in 75 ℃ of heating, mixed once after suspending evenly, melt (about 6-8 minute) fully until gel piece every 2-3 minute.
(4) adding Buffer DE-B by 50% of Buffer DE-A volume mixes.When the dna fragmentation that reclaims during less than 500bp, add Virahol, the final concentration that makes Virahol is 20%.
(5) DNA-prep Tube is placed 2ml Microfuge Tube, the mixed solution in the step 5 is moved among the DNA-prep Tube centrifugal 1 minute of 5500rpm.
(6) abandon filtrate, DNA-prep Tube is put get back among the 2ml Microfuge Tube, add the Buffer W1 that 500ul has added dehydrated alcohol, centrifugal 1 minute of 5500rpm
(7) abandon filtrate, DNA-prep Tube is put get back among the 2ml Microfuge Tube, add the Buffer W2 that 700ul has added dehydrated alcohol, centrifugal 1 minute of 5500rpm, with same method more once with 700ul Buffer W2 washing.
(8) DNA-prep Tube is placed the 1.5ml centrifuge tube, centrifugal 1 minute of 12000 * g.
(9) DNA-prep Tube is placed another clean 1.5ml centrifuge tube, central authorities add 25-30ul Eluent or deionized water at the silica film, and room temperature left standstill 1 minute.Centrifugal 1 minute eluted dna of 12000 * g.
7, the preparation of recombinant vectors pET28a+/K5
Reaction system is gone into following table
16 ℃ of incubated overnight ,-20 ℃ of preservations are standby.
8, the preparation of competent cell
8.1 reagent:
(1) CaCl
2Solution (ice-cold)
0.1mol/L CaCl
2Solution, after the filtration sterilization of use disposable filter ,-4 ℃ of refrigerators storages are standby.
(2) 0.1mol/L CaCl
2 -Glycerine solution
25% glycerine+0.1M CaCL
2After using the disposable filter filtration sterilization ,-4 ℃ of refrigerators are preserved standby 8.2 operations:
(1) inoculation e. coli strain bl21 list bacterium colony is gone in the 5ml LB liquid nutrient medium 37 ℃ of overnight incubation under the aseptic condition.Second day, pour in the LB liquid nutrient medium of 250ml, 37 ℃ of vibration 3.5h-4.0h (600=0.4 ~ 0.6) stop to cultivate.
(2) the nutrient solution branch is installed in the aseptic polypropylene tube of 8 25ml precoolings, in putting 5-10 minute on ice, 4 ℃ then, the centrifugal 8min of 8000rmp.
(3) cell precipitation is resuspended with the ice-cold CaCl2 solution of 10ml, centrifugal 5 minutes of 4 ℃ of 6000rmp.
(4)) abandon supernatant after, add the ice-cold CaCl2 solution re-suspended cell of 10ml, ice bath is after half an hour, 4 ℃, centrifugal 5 minutes of 6000rmp.
(5) abandon supernatant after, add the ice-cold CaCl2 solution re-suspended cell of 2ml, be sub-packed in the 0.5ml centrifuge tube of precooling by every pipe 200 μ l amount, frozen immediately standby in-75 ℃ of refrigerator-freezers.
9, the conversion of recombinant plasmid and preservation
(1) 10 μ L are connected product and be added in the 200 μ L competent cell suspensions, mixing, ice bath 30-40 minute, 42 ℃ of water bath heat preservations 90 seconds were put 1-2 minute on ice immediately, transferred to then and carried out contrast culture on the substratum that is added with microbiotic ammonia benzyl.
(2) will transform successful intestinal bacteria, picking list bacterium colony carries out the purifying amplification cultivation in liquid nutrient medium, and 37 ℃ of violent joltings are spent the night.The absorption culture 500ul that shaking table cultivates that spends the night adds in the centrifuge tube of 1.5ml, adds 40% glycerine 500ul again and mixes the back in-20 or-70 ℃ of preservations, regularly activates heavily preservation, in case spawn degeneration or plasmid loss.
10, the extraction of recombinant plasmid
Method is the same in 3.4 method.
11, the PCR of recombinant plasmid detects
Used consistent in program thereby and the reaction system and 3.2, carry out the detection of agarose gel electrophoresis then and observe.
Interpretation of result
The segmental TD-PCR amplification of 1 purpose
We amplify the segmental full gene of purpose by TD-PCR from existing cloning vector, include protection base and restriction enzyme site.Fig. 6 is that the PCR product passes through 0.8% the photo of agarose gel electrophoresis under ultraviolet gel imaging instrument.
Can learn that the sample of No. 1 swimming lane contains more purpose segment DNA from Fig. 7, be more satisfactory, can be used as the substrate that enzyme is cut, and No. 3 swimming lane purpose segment is fewer, is not suitable as enzyme and cuts substrate.The result of 2 recombinant plasmid transformed
In the substratum that is added with microbiotic ammonia benzyl, cultivate behind the recombinant plasmid transformed competence colibacillus cell, and do contrast with unconverted competent cell and blank.The result is presented at the growth that does not have bacterium colony in the contrast, and can grow bacterium colony in doing the substratum that transforms.
The selectable marker gene that contains anti-ammonia benzyl among the expression vector pET28a+, and primary BL21 bacterial classification does not contain this selectable marker gene, can grow bacterium colony and show that it is successful transforming in the substratum that is added with microbiotic ammonia benzyl.
3 extracting recombinant plasmids carry out the pulsating PCR of purpose and detect
Carry out the purifying amplification cultivation with transforming successful intestinal bacteria, the laggard performing PCR amplification of extracting plasmid is identified.Agarose gel electrophoretogram such as Fig. 6
As seen purpose segment expanding effect is relatively good from Fig. 6, and band is single and bright.This has proved that tentatively the structure of recombinant vectors is successful.The result need carry out the further evaluation of protein electrophorese and dna sequencing.
The utilization of TD-PCR in this test is successful.TD-PCR
[8]Represented a kind of diverse PCR optimization method.It is not with a lot of reaction tubess and every effective different reagent concentration and loop parameter, but with a reaction tubes or mustn't go to being suitable for increasing the purpose product in order to little set of reaction tubes under the cycling condition of artifacts or primer dimer and react.Design multicycle program so that the round-robin annealing temperature that links to each other is more and more lower.This strategy helps first primer-template hybridisation events and occurs between the complementary reactant, and promptly those produce between the reactant of purpose amplified productions, are the methods that a kind of potential single stage method finds best amplification condition.
Used PCR is TD-PCR in this test, and effect is reasonable, and this test finished very important effect smoothly.
Claims (2)
1, a kind of clone, evaluation and expression method of angiostatin Kringle 5 fusion genes is characterized in that: comprise following steps:
1], the preparation of ThioA/L15/7 and E.coli PET32 (carrier) plasmid DNA:
(1) plasmid extracts reagent
Solution I: every bottle about 100 milliliters, contain 50mmol/L glucose, 25mmol/L Tris-HCL (PH8.0), 10mmol/L EDTA (PH8.0) is at 6.895*10
4The Pa 15min that sterilizes is stored in 4 ℃;
Solution II: 0.2mol/L NaOH (facing) i%SDS with preceding stock solution matching while using;
Solution III: be made into 100ml and contain 5mol/Lkac 60ml Glacial acetic acid, 11.5ml, water 28.5ml;
Solution I, solution III were sterilized 20 minutes in 150kg/cm2;
Above reagent is homemade analytical pure;
(2) operation
In Bechtop 2ml being contained the antibiotic LB substratum of the 100ug/ml ammonia Bian capacity of joining is in the 15ml test tube, insert the single bacterium colony of ThioA/L15/7, overnight incubation under 37 ℃ of violent joltings, draw the 1.5ml culture in Eppendorf tube, centrifugal 30 seconds of 12000g, nutrient solution is removed in suction, makes bacterial precipitation dry as far as possible, and residue is stored in 4 ℃.Contain the antibiotic LB substratum of 100ug/ml ammonia Bian and add the ice-cold solution I of 100ul in above-mentioned precipitation, thermal agitation disperses precipitation fully; In the solution II that adds the new preparation of 200ul, cover the tight mouth of pipe, put upside down centrifuge tube fast 5 times, with the mixing content, centrifuge tube is placed on ice; Adding the ice-cold solution III of 150ul, cover the tight mouth of pipe, gentle vibration 10 seconds, solution III is uniformly dispersed in the heavy-gravity bacterial lysate, place 5min on ice afterwards, add RNAase 3ul, in 60 ℃ of water-bath 20min, the centrifugal 5min of 12000g, supernatant is changed in another centrifuge tube that adds 200ul phenol and 24: 1 chloroform of 200ul/primary isoamyl alcohol solution, and vibration mixes makes the double-stranded DNA precipitation, places 2min under the room temperature, be the centrifugal 3min of 12000g, draw supernatant liquor in another centrifuge tube,, shake up with 800ul straight alcohol washing double-stranded DNA, left standstill two hours in the refrigerator freeze space, the centrifugal 5min of 12000g removes supernatant, adherent slow adding 70% ice precooled ethanol, the centrifugal 5min of 12000g, remove supernatant as far as possible and be positioned over 37 ℃ of oven for drying, dissolve nucleic acid again with the 25ul sterilized water, vibration shakes up, get 5ul and do the detection of 1% agarose gel electrophoresis, residue is stored in-20 ℃; Carrier pET32a plasmid DNA prepares the same;
2], the PCR of goal gene Kringle5:
(1) reagent
Primer sequence:
Upstream primer: 5 ' G
CCATGGTGCTGCTCCCGGATGTAG3 '
Nco?I??25bp
Downstream primer: 3 ' G
GAATTCTTACGGGGCCGCACACTGAGG3 '
EcoR?I??28bp
Taq-DNApolyase is the archaeal dna polymerase of a kind of heat-stable DNA of depending on, has 5 ' of polymerization of depending on → 3 ' exonuclease activity, the best use of temperature is 75 ℃--80 ℃, starting polymerization reaction under the condition of optimum temperuture need be lower than, in order to avoid primer dissociates from template, Taq-DNApolyase needs Mg as the time spent
2+. because primer: unwind and the annealing temperature of template crossbred is subjected to the influence of divalent cation, needs to consider the best Mg of amplified reaction
2+Concentration.Phosphate buffered saline buffer suppresses the Taq-DNApolyase activity, and amplified reaction carries out in the Tris damping fluid usually, and this damping fluid at room temperature pH is 8.3;
(2) operation
Add 10 * Buffer 5ul in the 200ul centrifuge tube, dNTP 4ul, primer P12ul, P22ul, template plasmid 2ul, Taq-DNApolyase 0.5ul, sterilized water 34.5ul, making the reaction system cumulative volume is 50ul, with the sealing of small amount of liquid paraffin, reacts on the PCR instrument at last;
Get 5ul PCR reaction product and 1ul marker and carry out the detection of 2% agarose gel electrophoresis, identify the product size.
3], the extracting and purifying of PCR product
(1) reagent
TE(Ph＝8.0)：10mmol/L?Tris-Hcl(PH＝8.0)+1mmol/L?EDTA(PH＝8.0)
(2) operation
The PCR product is transferred to the 1.5ml centrifuge tube, add TE (PH8.0) and supply 140ul, add chloroform/each 70ul of primary isoamyl alcohol solution of phenol and 24: 1, jolting is in the centrifugal 10min of 15000g, get supernatant and to the chloroform/primary isoamyl alcohol solution 140ul that wherein adds 24: 1, jolting in the centrifugal 10min of 15000g, is got supernatant and to wherein adding 14ul 3M NaAc and 420ul (3 times of volumes) dehydrated alcohol, in-70 ℃ of refrigerators, left standstill 2 hours, take out the back in the centrifugal 15min of 15000g.Add 1ml 70% washing with alcohol,, put into oven drying, get 5ul and carry out the detection of 2% agarose gel electrophoresis in the centrifugal 15min of 15000g.
4], double digestion PCR product and vector plasmid DNA:
(1) reagent
Nco I restriction enzyme site: 5 ' ..
A T G G..3 '
3 ' ..G G T A C
▲65 ℃ of C..5 ' heat inactivations, 20min
EcoR I restriction enzyme site: 5 ' ..
A T T C..3 '
3 ' ..C T T A A
▲65 ℃ of G..3 ' heat inactivations, 20min
(2) operation
In centrifuge tube, add 10 * Buffer 2ul, 0.1%BSA 2ul, PCR product 14ul, Nco I1.2ul, EcoR I 0.8ul, total reaction volume is 20ul,
In centrifuge tube, add 10 * Buffer 2ul, 0.1%BSA 2ul, vector plasmid DNA 12ul, NcoI 1.2ul, EcoR I 0.8ul, sterilized water 2ul, total reaction volume is 20ul,
Above-mentioned two centrifuge tubes are spent the night in 36 ℃ ± water-bath, enzyme is cut product all carry out 2% agarose gel electrophoresis;
5], dna gel reclaims:
(1) reagent
Dna gel reclaims test kit and comprises DE-A: gel melting agent (containing the DNA protective material)
DE-B: high from liquid sequence solution (making dna fragmentation greater than 100bp optionally be attached to DNA prepares on the film)
W1: washings W2: washings
Elutriant: 2.5mM Tris-Hcl, Ph=8.5
(2) operation
At first ultraviolet lamp downcuts the gel that contains target DNA down, blot and shred with paper handkerchief, the heavy 0.08g of tumor vascular growth supressor K (5) dna gel, the heavy 0.11g of carrier DNA gel, respectively as a gel volume. respectively to the DE-A solution that wherein adds 5 gel volumes, mixed evenly back is in 75 ℃ of heating, be interrupted and mix, melt fully until gel. add the DE-B solution of 0.5 DE-A volume, mixing, because tumor vascular growth supressor K (5) dna molecular amount is 300bp, is 20%. to draw said mixture respectively to wherein adding Virahol to final concentration again, transferring to DNA prepares in the pipe, be positioned in the 2ml centrifuge tube, in the centrifugal 1min of 3600rpm, if any residual, the centrifugal again 1min that raises speed abandons filtrate.To prepare pipe and put back centrifuge tube, and add 0.5ml W1 solution, the centrifugal 30s of 3600rpm abandons filtrate.To prepare pipe and put back centrifuge tube, and add 0.7mlW2 solution, the centrifugal 30s of 3600rpm abandons filtrate.Repeat once to prepare pipe and put back centrifuge tube, add 0.7mlW2 solution, the centrifugal 30s of 3600rpm abandons filtrate.To prepare pipe and put back centrifuge tube, high speed centrifugation 1min.The preparation pipe is placed clean 1.5ml centrifuge tube, prepare the film centre at DNA and add the 25ul elutriant, room temperature leaves standstill 1min.High speed centrifugation 1min eluted dna,
Obtain the double digestion fragment of goal gene Kringle5 and the double digestion fragment of carrier pET32a respectively;
6], preparation recombinant vectors pET32/kringle5
(1) reagent
But form phosphodiester bond between 5 '-phosphoric acid and the 3 '-hydroxyl among T4 ligase catalysis double-stranded DNA or the RNA, can connect two flat ends, can repair dsDNA, dsRNA, RNA/DNA incises.65 ℃ of heat inactivations, 10min,
(2) operation
10 * Buffer 1ul, the double digestion fragment 4ul of Kringle5, the double digestion fragment 2ul of carrier pET32a, T4ligase 1ul, H
2O 2ul. total reaction volume is that 10ul.16 ℃ of constant temperature spends the night enzyme even;
7], the preparation of competent cell
(1) reagent
0.1mol/L Cacl
2Solution
0.1mol/L CaCl
2-glycerine solution: 25% glycerine+0.1M CaCl
2
(2) operation
From 37 ℃ of single bacterium colonies of cultivating a diameter 2-3mm of picking on 16-20 hour the fresh flat board of BL21, transfer in 1 liter of flask that contains the 50ml substratum, 37 ℃ of 300 commentaries on classics/min vibrated 3 hours, in super clean bench, bacterium is transferred in the ice-cold 50ml polypropylene tube, continued to place 10 minutes on ice.Reclaim cell in 4 ℃ of centrifugal 10min of 4000rpm, remove nutrient solution, will manage inversion and residual nutrient solution be flow to end in 1 minute, with the ice-cold 0.1mol/L Cacl of 10ml
2Resuspended precipitation is positioned on ice, in 4 ℃ of centrifugal 10min of 4000rpm, pours out nutrient solution, is inverted 1min, and residual nutrient solution is flow to end.Every 50ml initial incubation thing ice-cold 0.1mol/L CaCl of 2ml
2The resuspended every part of cell precipitation of-glycerine is divided into the every pipe of 200ul/ with this solution,
If do conversion, directly use, if preserve, add 7ul DMSO (dimethyl sulfoxide (DMSO)) in then every pipe, place 15min on ice, add 7ul DMSO, directly be positioned in-70 ℃ of refrigerators, or directly add water in-70 ℃ of preservations;
8], the conversion of competent cell
From-70 ℃ of refrigerators, take out the competent cell BL21 of a pipe 200ul, holding is placed on ice in the hand, add 2ml recombinant vectors pET32/kringle5 in every pipe, rotation gently, place 30mim on ice, pipe is put in 42 ℃ of water-baths slowly jolting 90s, go to fast and cool off 2min in the ice bath, other gets the 5ml small test tube, every pipe adds the not LB substratum of added with antibiotic of 500ul, heat to 37 ℃ with water-bath, the top competent cell BL21 that transformed is added wherein, be positioned on 37 ℃ of shaking tables the following temperature of 220rpm and bathed 45 minutes, make the BL21 recovery and the express recombinant carrier pET32/kringle5 ammonia Bian resistant maker gene that have transformed, centrifugal 5 minutes of the nutrient solution 5000rpm that above-mentioned 1ml temperature is bathed, top 500ul is removed in suction, and following 200ul is used for bed board, on Bechtop, the competent cell that this 200ul has been transformed is transferred to and is put in advance on the LB nutrient agar that contains 100ug/ml ammonia Bian in 37 ℃ of baking ovens, and flat board is inverted in 37 ℃ and cultivates down and bacterium colony can occur in about 14 hours;
9], the abduction delivering of small-sized fermentation and recombinant protein
(1) reagent
Inductor IPTG, 0.1M Tris-HCl (PH8.0), 1mg/ml N,O-Diacetylmuramidase
The preparation of SDS-PAGE
Separation gel now is made into wherein H of 5ml
2O 1.434ml, 30% acrylamide 2.166ml, 1.5mol/LTris (Ph8.8) 1.3ml, 10%SDS 0.05ml, 10% (NH
4)
2S
2O
80.05ml, TEMED (peptizer) 0.002ml.
Concentrated glue now is made into wherein H of 2ml
2O 1.434ml, 30% acrylamide 0.33ml, 1.0mol/LTris (Ph6.5) 0.25ml, 10%SDS 0.02ml, 10% (NH
4)
2S
2O
80.02ml, TEMED (peptizer) 0.002ml.
(2) operation
Single bacterium colony of turning out after the above-mentioned conversion of picking is to the test tube that contains the 7ml nutrient solution, 37 ℃ of 300rpm shaken overnight, drawing the 200ul culture was inoculated in the LB substratum that contains 100ug/ml ammonia Bian with 1: 30,37 ℃ of 300rpm vibrated 3 hours, add inductor IPTG, making its final concentration is 1mmol/ml, again in 37 ℃ of 300rpm vibrations 3 hours
Get 1.5ml and induce bacterium, centrifugal collection thalline, with resuspended thalline of 200ul 0.1M Tris-HCl (PH8.0) and washing, in 5000rpm centrifugal 5 minutes, abandoning supernatant added the resuspended thalline of 100ul 0.1M Tris-HCl (PH8.0), add 50ul 1mg/ml N,O-Diacetylmuramidase again, place half an hour in-70 ℃ of refrigerators, 37 ℃ of water-baths 5 minutes were put into-70 ℃ of refrigerators 5 minutes again, behind the multigelation five times, in 12000rpm centrifugal 10 minutes, collect supernatant (being the tenuigenin part), be precipitated as the inclusion body part, supernatant and precipitation and do not add inductive contrast thalline and be SDS-PAGE respectively, dyeing, decolouring, imaging;
10], cultivate and extract plasmid, the laggard row agarose gel electrophoresis evaluation of double digestion
The single bacterium colony of BL21 after picking transforms contains in the antibiotic LB substratum of 100ug/ml ammonia Bian in 2ml, extracts plasmid according to 1.2.1, carry out double digestion according to 1.2.4 after, 2% agarose gel electrophoresis is identified.
2, a kind of clone, evaluation and expression method of angiostatin Kringle 5 non-fusion genes is characterized in that: comprise following steps:
1], design the upstream and downstream primer of its coding region according to K5 gene complete sequence, design restriction enzyme site and protection base in the primer,
Upstream primer is 5 ' CAT G
CC ATG GCA GCT CCC GGA TGT AG 3 ' underscore partly is the NcoI restriction enzyme site,
Downstream primer is 5 ' CCC
AAG CTTTTA CGG GGC CGC ACA CTG AGG 3 ' underscore partly is the HindIII restriction enzyme site,
Primer is that Shanghai biotechnology company limited is synthetic.
2], the TD-PCR of K5 gene amplification
The TD-PCR reaction system
The TD-PCR program:
3], the agarose gel electrophoresis of PCR product
(1) glue
Take by weighing the agarose of 0.40g, put special-purpose triangular flask, add the distilled water of 50ml, be heated to boiling; After cooling, add a little ethidium bromide stock solution, pour into behind the mixing in the electrophoresis chamber of inserting good comb in advance, treat to add an amount of electrophoretic buffer after its cooled and solidified, carefully take out comb.(end of well is near negative electrode.) careful operation, must not allow ethidium bromide contact own or contaminate environment.
(2) go up sample
Loading buffer 3 μ l with micropipette rifle absorption 10x place the label paper back side, draw PCR product 6 μ l again, join in the well after the mixing.Add DNA Marker in the well aside and do contrast.Attention rifle head is not poked gel.
(3) electrophoresis
The power adjustment voltage of connecting tiselius apparatus is to 100v, treats that sample enters fully that regulating voltage is that 80v carries out electrophoresis in the gel.According to the needs of electrophoresis detection, treat that tetrabromophenol sulfonphthalein indicator swimming turns down to the appropriate location and cut off the electricity supply.
(4) analyze: under the gel imaging instrument, observe.
4], a small amount of of pET28a+ plasmid DNA preparation
(1) reagent
Solution I: every bottle (100ml) contains the glucose of 50mmol/L, the Tris-HCL of 25mmol/L (PH8.0) 10mmol/L EDTA (pH8.0), 6.895 * 10
4The Pa sterilization was stored in 4 ℃ after 15 minutes,
Solution II: 0.2mmol/L NaOH, 1%SDS. (matching while using),
Solution III: be made into 100ml and contain 5mol/L kac 60ml, Glacial acetic acid 11.5ml, water 28.5ml, 6.895 * 10
4The Pa sterilization was stored in 4 ℃ after 20 minutes.
Above reagent is homemade analytical pure
(2) operation
The LB substratum capacity of joining that 3ml is contained 100 μ g/ml Amp in the aseptic technique platform is in the test tube of 15ml, inserts pET28a+ bacterium liquid 30 μ l, spends the night in 37 ℃ of fierce shaking culture.Draw the 1.5ml culture in Eppendorf tube, centrifugal 30 seconds of 12000g inhales and removes nutrient solution, make bacterial precipitation dry as far as possible, the residue culture is stored in 4 ℃, adds the solution I (precooling) of 100 μ l in containing sedimentary test tube, and thermal agitation disperses precipitation fully; Add the solution II of the new preparation of 200 μ l again, cover the tight mouth of pipe, put upside down fast 5 times,, centrifuge tube is placed on ice with the mixing content; The solution III that adds 150 μ l precoolings again, cover the tight mouth of pipe, gentle vibration made solution III be uniformly dispersed in the heavy-gravity bacterial lysate in 10 seconds and places afterwards and added Rnase 3 μ l on ice in 5 minutes in 60 ℃ of water-baths 20 minutes, centrifugal 5 minutes of 12000g, supernatant is changed in another centrifuge tube that is added with 200 μ l phenol and 24: 1 chloroform of 200 μ l/primary isoamyl alcohol solution, it is the double-stranded DNA precipitation that vibration mixes, and places 2 minutes at 12000g centrifugal 3 minutes under the room temperature.The careful supernatant liquor of drawing is in another centrifuge tube, with 800 μ l straight alcohols washing double-stranded DNA, 70% ice precooled ethanol, the centrifugal 5min of 12000g, remove supernatant as far as possible and be positioned over 37 ℃ of oven for drying, dissolve nucleic acid again with 25 μ l sterilized waters, vibration shakes up, get 5 μ l and do the detection of 1% agarose gel electrophoresis, residue is stored in-20 ℃;
5], double digestion PCR product and vector plasmid DNA
Reaction system:
Enzyme Qie Wendu is 37 ℃, and the time is 3.5h.
6], the sepharose of dna segment detects and the glue recovery.
(1) reagent: (dna gel recovery test kit)
Buffer DE-A: gel melting agent (containing the DNA protective material)
Buffer DE-B:DNA binding soln
Buffer W1: washings
Buffer W2: demineralised liquid (pressing designated volume adding dehydrated alcohol on the reagent bottle before using)
Eluent:2.5mM Tris-HCl, pH8.5, DNA elute soln
(2) operation:
(2.1) at first under ultraviolet lamp, downcut the sepharose that contains target DNA, use Sillim's gel surface liquid so far.Reduce gel volume, the gel piece of large volume need be cut into small pieces as far as possible, to shorten the time that gel melts in the step 4.
(2.2) weighing gel weight is with 1mg=1 μ l conversion gel volume.According to gel strength, carry by table
The parameter of confession adds Buffer DE-A:
What this experiment needed recovery is double digestion pET28a+ and K5 fragment, and the glue of use all is 0.8%, so add the Buffer DE-A of 3 gel volumes of people.
(2.3) in 75 ℃ of heating, mixed once after suspending evenly, melt (about 6-8 minute) fully until gel piece every 2-3 minute.
(2.4) adding Buffer DE-B by 50% of Buffer DE-A volume mixes.When the dna fragmentation that reclaims during less than 500bp, add Virahol, the final concentration that makes Virahol is 20%.
(2.5) DNA-prep Tube is placed 2ml Microfuge Tube, the mixed solution in the step 5 is moved among the DNA-prep Tube centrifugal 1 minute of 5500rpm.
(2.6) abandon filtrate, DNA-prep Tube is put get back among the 2ml Microfuge Tube, add the Buffer W1 that 500ul has added dehydrated alcohol, centrifugal 1 minute of 5500rpm
(2.7) abandon filtrate, DNA-prep Tube is put get back among the 2ml Microfuge Tube, add the Buffer W2 that 700ul has added dehydrated alcohol, centrifugal 1 minute of 5500rpm, with same method more once with 700ulBuffer W2 washing.
(2.8) DNA-prep Tube is placed the 1.5ml centrifuge tube, centrifugal 1 minute of 12000 * g.
(2.9) DNA-prep Tube is placed another clean 1.5ml centrifuge tube, central authorities add 25-30ul Eluent or deionized water at the silica film, and room temperature left standstill 1 minute.Centrifugal 1 minute eluted dna of 12000 * g.
7], the preparation of recombinant vectors pET28a+/K5
Reaction system is gone into following table
16 ℃ of incubated overnight ,-20 ℃ of preservations are standby,
8], the preparation of competent cell
(1) reagent:
(1.1) CaCl
2Solution (ice-cold)
0.1mol/L CaCl
2Solution, after the filtration sterilization of use disposable filter ,-4 ℃ of refrigerator storages are standby,
(1.2) 0.1mol/LCaCl
2Glycerine solution
25% glycerine+0.1M CaCL
2After using the disposable filter filtration sterilization ,-4 ℃ of refrigerators are preserved standby
(2) operation:
(2.1) inoculation e. coli strain bl21 list bacterium colony is gone in the 5ml LB liquid nutrient medium 37 ℃ of overnight incubation under the aseptic condition.Second day, pour in the LB liquid nutrient medium of 250ml, 37 ℃ of vibration 3.5h-4.0h (600=0.4 ~ 0.6) stop to cultivate,
(2.2) the nutrient solution branch is installed in the aseptic polypropylene tube of 8 25ml precoolings, in putting 5-10 minute on ice, 4 ℃ then, the centrifugal 8min of 8000rmp,
(2.3) cell precipitation is resuspended with the ice-cold CaCl2 solution of 10ml, centrifugal 5 minutes of 4 ℃ of 6000rmp,
(2.4)) abandon supernatant after, add the ice-cold CaCl2 solution re-suspended cell of 10ml, ice bath is after half an hour, 4 ℃, centrifugal 5 minutes of 6000rmp,
(2.5) abandon supernatant after, add the ice-cold CaCl2 solution re-suspended cell of 2ml, be sub-packed in the 0.5ml centrifuge tube of precooling by every pipe 200 μ l amount, frozen immediately standby in-75 ℃ of refrigerator-freezers,
9], the conversion of recombinant plasmid and preservation
(1) 10 μ L are connected product and be added in the 200 μ L competent cell suspensions, mixing, ice bath 30-40 minute, 42 ℃ of water bath heat preservations 90 seconds were put 1-2 minute on ice immediately, and transfer to then and carry out contrast culture on the substratum that is added with microbiotic ammonia benzyl,
(2) will transform successful intestinal bacteria, picking list bacterium colony carries out the purifying amplification cultivation in liquid nutrient medium, and 37 ℃ of violent joltings are spent the night.The absorption culture 500ul that shaking table cultivates that spends the night adds in the centrifuge tube of 1.5ml, adds 40% glycerine 500ul again and mixes the back in-20 or-70 ℃ of preservations, regularly activates heavily preservation, in case spawn degeneration or plasmid loss.
10], the extraction of recombinant plasmid
Method is the same,
11], the PCR of recombinant plasmid detects
Used consistent in program thereby and the reaction system and 3.2, carry out the detection of agarose gel electrophoresis then and observe.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810150873A CN101671675A (en) | 2008-09-09 | 2008-09-09 | Method for cloning, identifying and expressing fusion and non-fusion genes of angiostatin Kringle 5 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810150873A CN101671675A (en) | 2008-09-09 | 2008-09-09 | Method for cloning, identifying and expressing fusion and non-fusion genes of angiostatin Kringle 5 |
Publications (1)
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2628748A1 (en) | 2012-02-14 | 2013-08-21 | Szilak Laboratories Bioinformatics & Molecule-Design Ltd. | Angiostatin chimeras and uses thereof |
| CN111621497A (en) * | 2020-05-29 | 2020-09-04 | 广西大学 | Rapid extraction method and application of chicken blood DNA |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2628748A1 (en) | 2012-02-14 | 2013-08-21 | Szilak Laboratories Bioinformatics & Molecule-Design Ltd. | Angiostatin chimeras and uses thereof |
| WO2013120959A1 (en) | 2012-02-14 | 2013-08-22 | Szilak Laboratories Bioinformatics & Molecule-Design Ltd. | Angiostatin chimeras and uses thereof |
| CN111621497A (en) * | 2020-05-29 | 2020-09-04 | 广西大学 | Rapid extraction method and application of chicken blood DNA |
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