CN1884527A - Method for preparing protein using endogenetic angiogenesis inhibitive factor Arresten gene - Google Patents
Method for preparing protein using endogenetic angiogenesis inhibitive factor Arresten gene Download PDFInfo
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Abstract
The invention relates to a gene recombination and expression technology, and especially to a method for preparation of protein by using autogenous vessel to produce inhibitor Arresten gene, which has solves the problem existed in current technology namely that the process for preparation of protein which can prevent the tumour vessel from producing by using human Arresten gene is inefficiency. The steps include: extracting total RNA from human body, adding upstream, downstream primer and Arresten gene DNA obtained by RT-PCR reverse transcription augmentation, then connecting plasmid carrier pBV220 by enzyme digestion and RT-PCR augmentation product Arresten DNA by enzyme digestion, obtaining coupled reaction product pBV220-Arr, finally switching the products into host bacillus coli to express, and obtaining Arresten protein. Compared with the current technology, the invention adopts a plasmid pBV220 not only as union carrier but also as expression carrier, which is simple and economic and saves costs, and the expression effect is good and the composite efficiency is improved, which provides a new approach for needed purpose protein synthesis.
Description
Technical field
The present invention relates to a kind of gene recombination, expression technology, be specially a kind of endogenetic angiogenesis inhibitive factor Arresten gene that utilizes and prepare proteic method.
Background technology
Tumour is a kind of typical blood vessel dependence disease, and early 1970s, Folkman proposes " generation that growth of tumor and transfer all depend on new vessel ".New vessel not only provides competent oxygen and nutritive substance for tumor growth, and in time transport the objectionable impurities that metabolism produces, but also stimulate growth of tumor with the form of paracrine, promote that tumour cell enters blood circulation, thereby to other histoorgan infiltration metastasis.Therefore Folkman proposes solid tumor is adopted the methods of treatment that suppresses vasculogenesis, people's angiogenesis inhibitor treatment research starts from 1988, the angiogenesis inhibitor Arresten that derives from people's basilar membrane collagen of latest find, Tumstatin, Canstatin, Endostatin and Vastatin, Restin etc. have the proliferate effect that specificity suppresses tumor vascular endothelial cell, itself and the angiogenic growth factor of tumor cell secretion antagonism mutually, thereby reach the prevention tumor-blood-vessel growth, make tumour capillary vessel stunted growth, cut off the purpose of tumors of nutrients supply.Arresten is a kind of strong effectively Angiostatin of being discovered in 2000 by Colorado etc., and expression amount height in placenta also has expression in the tire liver.It is the C-terminal NCl structural domain polypeptide fragment of IV Collagen Type VI α 1 chain, total length 690bp, and 229 amino acid of encoding, iso-electric point is 6.4, molecular weight is about 26KD.So utilizing Arresten gene preparation in the human body to have the protein that suppresses tumor-blood-vessel growth is the emphasis of current domestic and international research and development, for example employing plasmid pET22b (+) such as Colorado carries out prokaryotic expression; Zheng Qichang etc. adopt connection carrier pGEM-T, expression vector pET22b (+), and also usefulness also is prokaryotic expression system, promptly intestinal bacteria are as the expressive host bacterium; Lu Canrong etc. then adopt eukaryotic expression system, carrier for expression of eukaryon pcDNA3.1 (+); He Aibin etc., express in pichia spp as connection carrier at last with T carrier pGEM-T.These working method all are with connection and express and separately carry out that efficiency ratio is lower.
Summary of the invention
The present invention has the low shortcoming of preparation efficiency that exists in the protein process that suppresses tumor-blood-vessel growth and provides a kind of endogenetic angiogenesis inhibitive factor Arresten gene that utilizes to prepare proteic method in order to solve the Arresten gene preparation in the human body that utilizes that exists in the prior art.
The present invention realizes by the following technical solutions, utilize endogenetic angiogenesis inhibitive factor Arresten gene to prepare proteic method, step comprises: extract total RNA from human body, add the upstream and downstream primer, (RT-PCR) post transcription cloning obtains the DNA of Arresten gene, get again behind the plasmid vector double digestion with after RT-PCR amplified production Arresten dna double enzyme is cut and be connected, obtain the ligation product, at last product is transformed into host bacterium expression in escherichia coli, obtains Arresten albumen; Connection and the used plasmid of expression vector are pBV220, press the mole ratio of carrier pBV220/ purpose ArrestenDNA: 1: 1～1: 3, connect 8～10 hours at 15～18 ℃, and obtain ligation product pBV220-Arr.
The starting temperature of post transcription cloning reaction conditions is controlled at 93-95 ℃, 40sec; Annealing temperature 56-58 ℃, 40sec extends 71-73 ℃, 1min, totally 35 circulations.
Primer design and synthetic: design primer according to known Arresten mRNA gene order, primer nearly 5 ' end in upstream and downstream is introduced restriction enzyme, restriction enzyme site and a pair of initiator codon and two pairs of terminator codons respectively.
Host bacterium intestinal bacteria are E.coliJM109, DH5 α, BL21, BL21 (DE3), provide by biological chemistry teaching and research room of Mountain Western Medicine S University.
30 ℃ of intestinal bacteria culture temperature, 4 hours time, to express generally at 42 ℃, 4 hours time, optimal dissolution oxygen amount selection percentage was at 1: 5.
1, four kinds of bacterium performance screening contrast experiment data are as follows:
1.1, the expression of recombinant plasmid pBV220-Arr in JM109
(1) recombinant plasmid pBV220-Arr is transformed the JM109 competent cell
1.-70 the E.coliJM109 of ℃ preservation rules on the LB of Amp feminine gender flat board, and 37 ℃ of thermostat containers are cultivated 15h;
2. the mono-clonal bacterium colony of a diameter 2mm of picking size from flat board moves in the test tube that contains 5ml LB substratum, and 37 ℃, the strong shaking culture 8h of 220r/min;
3. the bacterium liquid 2ml that draws in the test tube is transferred in the 100ml LB triangular flask, and 37 ℃ of strong shaking culture 3h of 220r/min make cell concn reach OD
600About 0.4;
4. culture is placed 10min on ice, forward to then in two 50ml centrifuge tubes, 4000r/min, 4 ℃ of centrifugal 10min;
5. abandon supernatant, be inverted centrifuge tube 1min, flow to end remaining liq, add the 0.1mol/L CaCl of 10ml ice precooling then
2Suspension cell places 10min on ice;
6. 4000r/min, 4 ℃ of centrifugal 10min reclaim cells, abandon supernatant, add the ice-cold 0.1mol/L CaCl of 2ml respectively
2Solution, suspension cell.By every part 200 μ l packing cell, if do not transform at once, add the sterile glycerol of 50 μ l 80% respectively, put-70 ℃ frozen.
7. the pBV220 empty plasmid 4 μ l that get this chamber-70 ℃ preservation add in the 200 μ l competent cells, and gentle mixing is put 30min on ice;
8. set up contrast, the 200 μ l competent cells that do not add any plasmid are placed 30min on ice;
9. will be 6. 7. simultaneously in 42 ℃ of water-bath heat-shocked 90sec, put 2min on ice rapidly, add 800 μ lLB substratum, 37 ℃, 225r/min, shaking culture 1h allows plasmid expression antibiotics resistance albumen in the bacterium;
10. get 200 μ l transformation mixtures and be laid on LB (Amp
+) flat board, place 20min under the room temperature, treat that solution is absorbed by agar after, place plate and cultivate 15h in 37 ℃;
(2) pBV220-Arr/JM109 growth curve research
Positive colony of picking on the agar plate that transforms from success is inoculated into 5ml LB (Amp10 μ L) liquid nutrient medium, and 30 ℃, the 220r/min incubated overnight is to OD
600About=1.0, get 1 duplicate samples according to 1: 50 expanding species in 50ml (Amp50 μ L) liquid nutrient medium, 30 ℃,, under the 600nm wavelength, measure OD then respectively at 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h sampling
600, and draw the pBV220-Arr/JM109 growth curve.As schematically shown in Figure 7
(3) research of pBV220-Arr/JM109 expressing protein form
Positive colony of picking is inoculated in 5ml LB (Amp5 μ L) liquid nutrient medium, and 30 ℃, the 200r/min incubated overnight is to OD
600About=0.8, press 1: 50 expanding species again, 30 ℃, cultivate 3h to OD in 100ml LB (Amp100 μ L) liquid nutrient medium
600Be warming up to 42 ℃ of abduction deliverings about=0.6,, collect 1ml bacterium liquid, centrifugal collection bacterial sediment and supernatant respectively at 2h, 4h, 6h, 8h; The JM109 that will carry the pBV220 empty plasmid simultaneously carries out abduction delivering in contrast.With cleer and peaceful thalline SDS-PAGE gel electrophoresis on the two.Electrophoresis chamber is installed to specifications, and the separation gel of preparation 12% and 5% spacer gel are got a little sample and are added isopyknic sample-loading buffer, boil 5min in boiling water, get sample on the 20 μ L, carry out SDS-PAGE and analyze.Spacer gel voltage is 80V, adds 110V after entering separation gel, and electrophoresis 1h unloads glue and carries out coomassie brilliant blue staining 30min after finishing, and decolouring 1h takes a picture and preserves.
As schematically shown in Figure 8
1: lower molecular weight standard protein 2: express preceding 3: express back 2h 4: express back 4h 5: express back 6h 6: express back 8h
As can be seen from Figure 8, the JM109 bacterium all can be expressed Arresten albumen at 2h, 4h, 6h, 8h, its Arresten expressing quantity accounts for 20.92%, 24.65%, 21.11% and 19.08% of total tropina expression amount respectively, in conjunction with figure and expression amount as can be known recombinant plasmid pBV220-Arr in the JM109 bacterium, express 4h and reach optimum expression.
1.2, the expression of recombinant plasmid pBV220-Arr in DH5 α
(1) recombinant plasmid pBV220-Arr is transformed DH5 α competent cell, method is with 1.1 (1)
(2) pBV220-Arr/DH5 α growth curve research, method is with 1.1 (2) Fig. 9
(3) research of pBV220-Arr/DH5 alpha expression albumen form, method is with 1.1 (3) Figure 10
1: lower molecular weight standard protein 2: express preceding 3: express back 2h 4: express back 4h 5: express back 6h 6: express back 8h
As can be seen from Figure 10, DH5 α bacterium all can be expressed Arresten albumen at 2h, 4h, 6h, 8h, its Arresten expressing quantity accounts for 21.06%, 24.38%, 22.02% and 21.15% of total tropina expression amount respectively, in conjunction with figure and expression amount as can be known recombinant plasmid pBV220-Arr in DH5 α bacterium, express 4h and reach optimum expression.
1.3, the expression of recombinant plasmid pBV220-Arr in BL21
(1) recombinant plasmid pBV220-Arr is transformed the BL21 competent cell, method is with 1.1 (1)
(2) pBV220-Arr/BL21 growth curve research, method is with 1.1 (2) Figure 11
(3) research of pBV220-Arr/BL21 expressing protein form, method is with 1.1 (3) Figure 12
1: lower molecular weight standard protein 2: express preceding 3: express back 2h 4: express back 4h 5: express back 6h 6: express back 8h
As can be seen from Figure 12, the BL21 bacterium all can give expression to Arresten albumen at 2h, 4h, 6h, 8h, its Arresten expressing quantity accounts for 21.08%, 25.03%, 22.15% and 21.22% of total tropina expression amount respectively, in conjunction with figure and expression amount as can be known recombinant plasmid pBV220-Arr in the BL21 bacterium, express 4h and reach optimum expression.
1.4, the expression of recombinant plasmid pBV220-Arr in BL21 (DE3)
(1) recombinant plasmid pBV220-Arr is transformed BL21 (DE3) competent cell, method is with 1.1 (1)
(2) pBV220-Arr/BL21 (DE3) growth curve research, method is with 1.1 (2) Figure 13
(3) research of pBV220-Arr/BL21 (DE3) expressing protein form, method is with 1.1 (3) Figure 14
1: lower molecular weight standard protein 2: express preceding 3: express back 2h 4: express back 4h 5: express back 6h 6: express back 8h
As can be seen from Figure 14, BL21 (DE3) bacterium all can give expression to Arresten albumen at 2h, 4h, 6h, 8h, its Arresten expressing quantity accounts for 22.12%, 26.10%, 22.09% and 21.26% of total tropina expression amount respectively, in conjunction with figure and expression amount as can be known recombinant plasmid pBV220-Arr in BL21 (DE3) bacterium, express 4h and reach optimum expression.
1.5, the comparison expressed of recombinant plasmid pBV220-Arr different time in four kinds of bacterium
(1) recombinant plasmid pBV220-Arr expresses the SDS-PAGE analysis of 2h in four kinds of bacterium
(2) recombinant plasmid pBV220-Arr expresses SDS-PAGE analysis Figure 15 of 4h in four kinds of bacterium
1: in JM109, express 2h 2: in DH5 α, express 2h 3: in BL21, express 2 h
4: in BL21 (DE3), express 2h 5: lower molecular weight standard protein (14.4,20.1,31.0,43.0,66.2,97.4KD)
6: in JM109, express 4h 7: in DH5 α, express 4h
8: in BL21, express 4h 9: in BL21 (DE3), express 4h
As can be seen from Figure 15,2h, 4h all can give expression to Arresten albumen in four kinds of bacterium, its Arresten expressing quantity accounts for total tropina expression amount and is respectively 20.92%, 21.06%, 21.08%, 22.12% and 24.65%, 24.38%, 25.03%, 26.10% at 2h, 4h, in conjunction with figure and expression amount as can be known recombinant plasmid pBV220-Arr in BL21 (DE3) bacterium, express 4h and reach optimum expression.
(3) recombinant plasmid pBV220-Arr expresses the SDS-PAGE analysis of 6h in four kinds of bacterium
(4) recombinant plasmid pBV220-Arr expresses SDS-PAGE analysis Figure 16 of 8h in four kinds of bacterium
1: in JM109, express 6h 2: in DH5 α, express 6h 3: in BL21, express 6h
4: in BL21 (DE3), express 6h 5: lower molecular weight standard protein (14.4,20.1,31.0,43.0,66.2,97.4KD)
6: in JM109, express 8h 7: in DH5 α, express 8h 8: in BL21, express 8h 9: in BL21 (DE3), express 8h
As can be seen from Figure 16,6h, 8h all can give expression to Arresten albumen in four kinds of bacterium, and its Arresten expressing quantity accounts for total tropina expression amount and is respectively 21.11%, 22.02%, 22.15%, 22.09% and 19.08%, 21.15%, 21.22%, 21.26% at 6h, 8h.
1.6, culture temperature expresses the influence of Arresten to BL21 (DE3)
The positive substratum of LB ammonia benzyl for preparing 1: 50 expanding species of 3 bottles of 100ml simultaneously, 25 ℃, 30 ℃, 37 ℃, 200r/min, constant temperature culture is warming up to 42 ℃ abduction delivering 4hs behind the sample to logarithmic phase (about 4h) before preparation is induced, and surveys the nectar degree, back sample, totally 3 are expressed in preparation simultaneously; SDS-PAGE draws qualitative, quantitative conclusion through the gel analysis system, and collects thalline.Figure 17
3:37 ℃ of culture expression sample 4 of 2:30 ℃ of culture expression sample of 1:25 ℃ of culture expression sample: lower molecular weight standard protein 5: sample before expressing
From Figure 17 as seen, induce the expression amount of 4h all very high for 42 ℃ behind 25 ℃, 30 ℃, 37 ℃ three kinds of temperature expression 4h, be respectively 22.15%, 26.23%, 23.18%, cultivate Arresten expressing quantity maximums, secondly be 37 ℃, 25 ℃ for visible 30 ℃.
1.7, cultivate the time length and BL21 (DE3) expressed the influence of Arresten
Prepare the positive substratum of LB ammonia benzyl of 1: 50 expanding species of 3 bottles of 100ml simultaneously, 30 ℃, 200r/min, constant temperature cultivate 2h, 4h, 6h respectively, are warming up to 42 ℃ of abduction delivering 4h behind the sample before preparation is induced, and survey the nectar degree, and back sample, totally 3 are expressed in preparation simultaneously; SDS-PAGE draws qualitative, quantitative conclusion through the gel analysis system, and collects thalline.Figure 18
1: cultivate 2h and express sample 2: cultivate 4h and express sample 3: cultivate 6h and express sample 4: sample 5 before expressing: lower molecular weight standard protein
18 as seen from the graph, and Arresten albumen is respectively 23.03%, 26.23%, 23.86% at the expression amount of 2h, 4h, 6h, as seen the highest at cultivation 4h expression amount.Next is 6h and 2h.
1.8, inducing temperature expresses the influence of Arresten to BL21 (DE3)
Prepare the positive substratum of LB ammonia benzyl of 1: 50 expanding species of 3 bottles of 100ml simultaneously, 30 ℃, 200r/min, constant temperature cultivate 4h respectively, are warming up to 37 ℃, 42 ℃, 48 ℃ abduction delivering 4h behind the sample before preparation is induced, and survey the nectar degree, and back sample, totally 3 are expressed in preparation simultaneously; SDS-PAGE draws qualitative, quantitative conclusion through the gel analysis system, and collects thalline.Figure 19
Express 2:42 ℃ in sample for 1:37 ℃ and express 3:48 ℃ of expression of sample sample 4: sample 5 before expressing: lower molecular weight standard protein
19 as seen from the graph, and Arresten albumen is the highest at 42 ℃ expression amount, and 26.45%, the 48 ℃ of expression that accounts for the overall protein expression amount is taken second place, and is 18.38%, and be 0% at 37 ℃ of Arresten expressing quantities.
1.9, the inducing sustained time expresses the influence of Arresten to BL21 (DE3)
Prepare the positive substratum of LB ammonia benzyl of 1: 50 expanding species of 3 bottles of 100ml simultaneously, 30 ℃, 200r/min, constant temperature cultivate 4h respectively, are warming up to 42 ℃ of abduction delivering 4h, 6h, 8h respectively before preparation is induced behind the sample, survey the nectar degree, and back sample, totally 3 are expressed in preparation simultaneously; SDS-PAGE draws qualitative, quantitative conclusion through the gel analysis system, and collects thalline.Figure 20
1: induce 4h to express sample 2: induce 6h to express sample 3: induce 8h to express sample 4: sample 5 before expressing: lower molecular weight standard protein
20 as can be known from the graph, and abduction delivering 4h Arresten expressing quantity reaches the highest, accounts for 26.45% of overall mycoprotein, and 6h, 8h take second place, and are respectively 24.86%, 22.59%, and major cause is that the expression time long thalli growth that then can cause is old excessively, even bacterium death, albumen reduces.
1.10, dissolved oxygen content expresses the influence of Arresten to BL21 (DE3)
Add 25ml, 50ml, 100ml, the positive substratum of 200ml LB ammonia benzyl in the culturing bottle respectively at 500ml, 30 ℃, 200rpm, constant temperature is cultivated 4h respectively, is warming up to 42 ℃ of abduction delivering 4h respectively before preparation is induced behind the sample, surveys the nectar degree, back sample, totally 4 are expressed in preparation simultaneously; SDS-PAGE draws qualitative, quantitative conclusion through the gel analysis system, and collects thalline.Figure 21
1: lower molecular weight standard protein 2: sample 3:25ml LB 4:50ml LB 5:100ml LB 6:200ml LB before expressing
21 as seen from the graph, the dissolved oxygen content ratio reaches the highest at 1: 5 o'clock expression amount, account for 25.24% of overall mycoprotein, it is dress 100ml substratum in the 500ml Erlenmeyer flask, and expression amount is respectively 20.35%, 22.58% and 23.38% in 25ml LB, 50ml LB, 200ml LB, and visible dissolved oxygen content also is one of them influence factor.
1.11, the combination of optimal expression condition
In the culturing bottle of 500ml, add the positive substratum of 100ml LB ammonia benzyl, contain the BL21 (DE3) of recombinant plasmid pBV220-Arr by 1: 50 expanding species, in expressing preceding 30 ℃, 200r/min, constant temperature culture 4h, be warming up to 42 ℃, 200r/min, constant temperature culture 4h, forward and backward sample is expressed in preparation respectively, SDS-PAGE draws qualitative, quantitative conclusion through the gel analysis system, and collects thalline.
Brief summary
(1) intestinal bacteria E.coli JM109, DH5 α, BL21, BL21 (DE3) all can express Arresten albumen, and logarithmic phase is about between 0.5～0.7, SDS-PAGE analyzes and shows that expressing molecular weight of product is about 26KD, and expression product mainly exists with the inclusion body form;
(2) respectively the SDS-PAGE of four kinds of bacterium different time expression products is analyzed and target protein accounts for the analysis of total tropina expression amount and wet bacterium is heavy, it is E.coli BL21 (DE3) that finishing screen is selected the optimal expression bacterial classification, and expression amount is 26.10%;
(3) optimum culturing temperature is selected 30 ℃, and expression amount is 26.23%;
(4) best incubation time is selected 4h, and expression amount is 26.23%;
(5) the optimum expression temperature is selected 42 ℃, and expression amount is 26.45%;
(6) optimum expression time is selected 4h, and expression amount is 26.45%;
(7) optimal dissolution oxygen amount selection percentage was promptly adorned the 100ml substratum at 1: 5 in the 500ml Erlenmeyer flask, and expression amount is 25.24%;
(8) by optimization expression, the result cultivates 4h for 30 ℃ at bacterium BL21 (DE3), 42 ℃ of abduction delivering 4h, and the dissolved oxygen content ratio reaches the highest at 1: 5 o'clock expression amount, and expression amount accounts for more than 25% of whole bacterial protein.
The present invention has made up recombinant plasmid pBV220-Arr and has efficiently expressed system and obtained high expression level in E.coli JM109, DH5 α, BL21, BL21 (DE3), simultaneously expression condition is optimized from selection, culture temperature, cultivation time length, expression temperature, expression time length and six aspects of dissolved oxygen content of bacterial classification respectively, four kinds of bacterium account for 20.92%, 21.06%, 21.08%, 22.12% of total tropina expression amount respectively at 2h, 4h, 6h, 8h expressing quantity; 24.65%, 24.38%, 25.03%, 26.10%; 21.11%, 22.02%, 22.15%, 22.09%; 19.08%, 21.15%, 21.22%, 21.26%.As can be seen from the data, when expressing 4h, each bacterium expression amount reaches the highest, and the expression time prolongation then can cause thalli growth old excessively, even bacterium death, and albumen reduces.Heavy obviously greater than other bacterial classifications in conjunction with the wet bacterium of E.coliBL21 (DE3), therefore selecting BL21 (DE3) is the optimal expression bacterial classification.In like manner also be the relation of having considered between the cost benefit when selecting the optimal expression condition, select the final condition of conduct economy and high efficiency, good operation.Finally determined to add in the culturing bottle of 500ml in the positive substratum of 100ml LB ammonia benzyl, E.coli BL21 (DE3) cultivates 4h for 30 ℃, and 42 ℃ of abduction delivering 4h are the optimal expression system, and the target protein expression amount accounts for 25% of bacterial protein expression amount.Through discovering, form with inclusion body behind 42 ℃ of protein expressions exists, and for a large amount of Arresten albumen of subsequent technique production, we determine to adopt the phraseology of inclusion body, so both help the high-yield expression of target protein, helped the separation and purification in downstream again.
Arresten protein product molecular weight is about 26KD, and expression product mainly exists with the form of inclusion body.Protein with the inclusion body formal representation can prevent the degraded of proteolytic enzyme to target protein, and helps the purifying of expression product.Recombinant protein is in the partially denaturing state in the inclusion body, thereby does not have with the same biologic activity of wild albumen, so inclusion body must by renaturation, just may make Partial Protein recover its natural conception through being dissolved into monomer again, has biologic activity.
2, Arresten albumen is from endobacillary sepn process optimization experiment,
Experimental technique
2.1, the broken bacterium of ultrasonic method obtains inclusion body
(1) bacterial sediment is dissolved and 4000r/min with the STE damping fluid, 10min washing 2 times is resuspended among the STE with 1: 5 ratio, and is sub-packed in 20 1.5ml EP pipes every pipe 1ml.
(2) get 4 12000r/min, 4 ℃, centrifugal 2min abandons supernatant, adds 2M, 3M, 4M, 5M urea 1ml in every pipe respectively, after the dissolving, leaves standstill a moment, with 500W, and ultrasonic 3sec, 3sec at interval, under the condition of ice bath ultrasonic 200 times.M all refers to mol/L
(3) with ultrasonic back EP pipe with 6000r/min, 4 ℃, centrifugal 10min, it is standby to keep supernatant, precipitation adds 8mol/L urea respectively, mixing with 1ml STE washed twice, prepare supernatant and deposit sample respectively, carry out SDS-PAGE, draw qualitative, quantitative conclusion through the gel analysis system.
Arresten factor inclusion body has stronger anti-shear ability, directly use the high strength supersonic method not only to avoid the introducing of N,O-Diacetylmuramidase, and broken bacterium is effective.
2.2, the washing of inclusion body with separate
(1) optimum obtains inclusion body
Get 15 EP pipe in 2.1 (1), in 12000r/min, 4 ℃, centrifugal 2min abandons supernatant, all uses 2M urea 1ml with Eppendorf centrifuge, after the dissolving, leaves standstill a moment, with 500W, and ultrasonic 3sec, 3sec at interval, under the condition of ice bath ultrasonic 200 times.
(2) 1-5M urea washing inclusion body effect assessment
Add 2M, 3M, 4M, 5M urea 1ml respectively 2.2 deposit sample (1) is got 5, mixing leaves standstill 10min up and down, 6000r/min, 4 ℃, centrifugal 20min, it is standby to keep supernatant, washs primary sedimentation again with method, and centrifuging and taking precipitation, supernatant prepare the SDS-PAGE sample.As Figure 22
1: lower molecular weight standard protein 2: sample before expressing
The centrifugal supernatant sample of 3:2M urea washing inclusion body 4:2M urea washing inclusion body centrifugation sample
The centrifugal supernatant sample of 5:3M urea washing inclusion body 6:3M urea washing inclusion body centrifugation sample
The centrifugal supernatant sample of 7:4M urea washing inclusion body 8:4M urea washing inclusion body centrifugation sample
The centrifugal supernatant sample of 9:5M urea washing inclusion body 10:5M urea washing inclusion body centrifugation sample
Increase along with urea concentration, the per-cent that Arresten factor inclusion body accounts for the precipitation total protein increases gradually, account for 40.42%, 40.46%, 40.95%, 41.53% respectively, the impurity protein content of removing in the supernatant also increases gradually, the loss of the factor drops to minimum, but difference is little between the various concentration, therefore selects 2M urea for use.
(3) Triton X-100 washing inclusion body effect assessment
Add each 1ml of Triton X-100 solution of 1%, 2%, 4% respectively 2.2 deposit sample (1) is got 3, mixing leaves standstill 10min up and down, 6000r/min, and 4 ℃, centrifugal 20min, it is standby to keep supernatant, and centrifuging and taking precipitation, supernatant prepare the SDS-PAGE sample.Figure 23
1:1%Triton X-100 washing inclusion body centrifugation sample 2:2%Triton X-100 washing inclusion body centrifugation sample
3: sample 4:4%Triton X-100 washing inclusion body centrifugation sample before expressing
From the graph as seen, 1%, 2%, albumen accounts for 35.76%, 38.38%, 37.91% of total tropina respectively behind the 4%Triton X-100 washing inclusion body, this shows that 2%Triton X-100 washing inclusion body effect is best.
(4) synthetic method washing inclusion body effect assessment
2.2 getting 4, deposit sample (1) adds 2M urea 1ml respectively, add 1%, 2% therein in 3 respectively, 4%Triton X-100 solution, with the sucrose solution of 1M, and the EDTA of a little, mixing up and down, leave standstill 10min, 6000r/min, 4 ℃, centrifugal 20min, it is standby to keep supernatant, and centrifuging and taking precipitation, supernatant prepare the SDS-PAGE sample.Figure 24
The centrifugal supernatant sample of the 1:2M urea washing inclusion body 2:2M urea washing inclusion body centrifugation sample 3:2M urea+centrifugal supernatant sample of the 1%Triton X-100 washing inclusion body 4:2M urea+1%Triton X-100 washing inclusion body centrifugation sample 5:2M urea+centrifugal supernatant sample of the 2%Triton X-100 washing inclusion body 6:2M urea+2%Triton X-100 washing inclusion body centrifugation sample 7:2M urea+centrifugal supernatant sample of 4%Triton X-100 washing inclusion body 8:2M urea+4%Triton X-100 washing inclusion body centrifugation sample 9: sample before expressing
From the graph as seen, three kinds of difference are little, and expression amount is all very high, but 2M urea and 2%TritonX-100 washing effect are best.Expression amount accounts for 42.48% of tropina.
2.3, the purifying of inclusion body, renaturation
(1) adds in the dialysis tubing with 8M urea dissolved inclusion body, add the urea of 6M in the beaker, stir 1d with stirrer;
(2) solution in the beaker is changed to the urea of 4M, stirs 1d with stirrer;
(3) solution in the beaker is changed to the urea of 2M, stirs 1d with stirrer;
(4) solution in the beaker is changed to renaturation solution (0.6mol/l urea, 0.1mmol/l Sleep-promoting factor B, 20mmol/l TrisCl (PH8.0), 0.9mmol/l Sleep-promoting factor B), stir 1d with stirrer, repeat to change liquid and stir totally 3 times, and reclaim renaturation solution;
(5) at last solution in the beaker is changed to PBS liquid, stirs 1d, reclaim in contrast with stirrer.
(6) survey protein concentration with the Xylene Brilliant Cyanine G method.
The method of renaturing inclusion bodies has step dilution method and a gradient dilution method, and the present invention's gradient dilution method adopts dialysis process renaturation inclusion body, and the soluble protein of purifying is carried out refolding, and the while has further been improved the purity of target protein.The advantage of dialysis renaturation is the volume that does not increase purifying protein solution, can be by reducing the removal speed that the liquid concentration of passing through inside is controlled denaturing agent gradually.The annealing efficiency of inclusion body is subjected to influence of various factors, comprises proteinic renaturation concentration, the pH of buffer value, and whether the existence of the initial concentration of denaturing agent and removal speed, redox-potential, ionic strength, cosolvent and other additives waits.Before the dialysis renaturation, at first protein solution is diluted to fusion rotein concentration and is about 0.1mg/ml, urea concentration is 6M, and adjusts pH value of solution value to 7.4.Then, dialysing is reduced the urea concentration in the damping fluid step by step, in order to avoid urea descends too fast and make albumen form the aggregate precipitation.Contain cysteine residues in the target protein of expressing,, form to impel disulfide linkage so in renaturation process, added the GSH/GSSG redox system.The GSH/GSSG redox system has improved the productive rate of correct paired disulfide linkage by promoting the disulfide linkage quick exchange reaction of incorrect formation.In dialysis procedure, still have the meromixis albumen precipitation to come out.Dialysis back albumen suspension is through centrifugal layering, and supernatant liquor is the target protein solution of renaturation, and the precipitation part is the target protein that can not correctly fold.The SDS-PAGE analytical results shows that the purity of target protein reaches 40.5% in the supernatant.
2.4, Arresten albumen suppresses the chick chorioallantoic membrane experimental result of new vessel
The Arresten albumen and the positive controls of getting different concns compare
(1) chicken embryo normal development in 38-40 ℃, the incubator of 60% relative humidity.60 of chicken embryos are used in experiment altogether, dead weak smart 5, draw materials 55, total rate of fertilization 91.67%, surviving rate 90%.The blood vessel of whole C AM is the vein shape and distributes.Angiogenic growth is good under negative control group (PBS group, the blank group) carrier film, and vessel branch is moderate; The blood vessel number obviously reduces under the carrier film of 20 μ g/ml group, 40 μ g/ml sample sets and positive controls, the phenomenon that great vessels fracture, the disappearance of little blood vessel occur, the phenomenon that blood vessel detours appears in sample segment, with 20 μ g/ml experimental group, 40 μ g/ml experimental group more so.
(2) to the carrier film influence of one-level, secondary angiogenic growth on every side
With the pharmaceutical carrier film is the center, one-level, the secondary blood vessel number of counting and its side edge, compare and find relatively there was no significant difference of positive controls, 20 μ g/ml group and negative control group (PBS group, blank group), significant difference (P＜0.05) is relatively arranged with control group.The effect that Arresten factor angiogenesis inhibiting after the renaturation is described is remarkable.
The one-level of table 1:Arresten factor pair chicken embryo CAM influence, secondary vascular counts (X ± s)
| Group | N | One-level blood vessel number | Secondary blood vessel number |
| The blank Arresten 20 μ g/ml Arresten of PBS 40 μ g/ml | 6 7 12 13 12 | 2.26±0.23 2.31±0.58 1.49±0.47* 1.15±0.24* 1.39±0.22* | 10.42±3.56 10.69±4.27 8.76±4.58 7.21±2.53* 9.03±3.51 |
* P＜0.05 (with the comparison of blank group)
(3) to the carrier film influence of large, medium and small angiogenic growth on every side
The middle or small blood vessel number of control group is obviously more, with experimental group significant difference is arranged relatively, and great vessels is counted there was no significant difference, illustrates that the Arresten factor mainly suppresses the generation of middle or small blood vessel.Arresten and control group be not statistically significant relatively.
The large, medium and small vascular counts of table 2:Arresten factor pair chicken embryo CAM influence (X ± s)
| Group | N | Great vessels | Medium vessels | Little blood vessel |
| The blank Arresten 20 μ g/ml Arresten of PBS 40 μ g/ml | 6 7 12 13 12 | 5.80±2.20 5.63±2.31 4.95±3.24 5.61±1.10 5.49±2.51 | 5.50±2.18 5.38±2.54 3.14±3.26* 2.02±1.35*# 4.22±1.26 | 81.00±11.56 79.26±10.89 41.49±12.53* 38.68±10.52* 40.22±11.35* |
* #P＜0.05, P＜0.05 (comparing) (with the comparison of positive group) with the blank group
(4) to the influence of angiogenic growth under the carrier film
Fig. 1: negative control group Fig. 2: positive controls Fig. 3: Arresten group
Found out that by Fig. 1,2,3 negative control group does not suppress the effect of vasculogenesis, positive controls and Arresten group all have the effect that suppresses vasculogenesis, but the specific activity positive controls of Arresten group inhibition vasculogenesis is strong.
Above result of study shows, the recombinant human Arresten albumen of the present invention's preparation is to the propagation of extracorporeal blood vessel endotheliocyte and move inhibitedly, and can effectively suppress intravital new vessel and forms; Be that recombinant human Arresten albumen can significantly suppress vasculogenesis on cell and integral level.In a word, people Arresten recombinant protein can effectively suppress the interior vasculogenesis of propagation, migration and body of vascular endothelial cell, is demonstrating good prospects for application aspect the angiogenesis inhibitor treatment of tumour.Carrier pBV220 successfully makes up (available from Shanghai biotechnology company limited) by the Chinese research personnel, total length 3666bp, the closed loop two strands contains multiple clone site, the temperature sensitive aporepressor, positive site of Amp and replication origin, transcription pausing sequence etc.The present invention selects prokaryotic expression system for use, in prokaryotic cell prokaryocyte during efficiently expressing exogenous gene, need a regulatable promotor, because some foreign protein may be poisonous to host cell, and synthetic fast at short notice exogenous protein, can reduce of the destruction of host cell proteins enzyme to foreign protein.The pBV220 carrier is the PRPL promotor, the temperature control abduction delivering, and PR and PL promotor are the lambda particles phage promotor, be strong promoter, be subjected to the negative regulation of lambda particles phage CI gene, the CI aporepressor is a temperature sensitive albumen, in the time of 28-30 ℃, the CI aporepressor produces restraining effect, is being warming up to 42 ℃, and CI is destroyed, PR and PL promotor transcriptional start, utilize temperature control, make the host bacterium 30 ℃ of growths, 42 ℃ of abduction delivering target proteins.Temperature control inductive mode has reasonably been regulated the metabolism load and exogenous gene high-efficient expressed relation of host bacterium, has reduced the damage that a large amount of foreign proteins bring for host's bacteria growing and metabolism.
Compared with prior art, the present invention has used the plasmid pBV220 of a kind of while as connection carrier and expression vector, not only simply but also economical, provide cost savings, and expression effect is very good, has improved synthetic efficient.For required target protein synthetic provides a kind of new way.
Description of drawings
The negative control group of Fig. 1
The positive control group of Fig. 2
Fig. 3 is the Arresten protein groups
Fig. 4 is total RNA sex change gel electrophoresis figure in the human placenta
The behave RT-PCR of Arresten gene cDNA of Fig. 5
Fig. 6 cuts evaluation for the clone purification and the enzyme of PCR product
Fig. 7 is the growth curve of pBV220-Arr/JM109
Fig. 8 is that SDS-PAGE analyzed before and after recombinant plasmid pBV220-Arr expressed in the JM109 bacterium
The growth curve of Fig. 9 pBV220-Arr/DH5 α
SDS-PAGE analyzed before and after Figure 10 recombinant plasmid pBV220-Arr expressed in DH5 α bacterium
The growth curve of Figure 11 pBV220-Arr/BL21
SDS-PAGE analyzed before and after Figure 12 recombinant plasmid pBV220-Arr expressed in bacterium BL21
The growth curve of Figure 13 pBV220-Arr/BL21 (DE3)
SDS-PAGE analyzed before and after Figure 14 recombinant plasmid pBV220-Arr expressed in bacterium BL21 (DE3)
Figure 15 recombinant plasmid pBV220-Arr analyzes at the SDS-PAGE of four kinds of bacterium expressing protein 2h, 4h
Figure 16 plasmid pBV220-Arr analyzes at the SDS-PAGE of four kinds of bacterium expressing protein 6h, 8h
Figure 17 recombinant plasmid pBV220-Arr different culture temperature in bacterium BL21 (DE3) are expressed front and back SDS-PAGE and are analyzed
Figure 18 recombinant plasmid pBV220-Arr different incubation times in bacterium BL21 (DE3) are expressed front and back SDS-PAGE and are analyzed
Figure 19 recombinant plasmid pBV220-Arr different inducing temperatures in bacterium BL21 (DE3) are expressed front and back SDS-PAGE and are analyzed
Figure 20 recombinant plasmid pBV220-Arr different induction times in bacterium BL21 (DE3) are expressed front and back SDS-PAGE and are analyzed
SDS-PAGE analyzed before and after Figure 21 recombinant plasmid pBV220-Arr different dissolved oxygen scale in bacterium BL21 (DE3) reached
Figure 22 Fig. 3-1 2-5M urea washing Arresten factor inclusion body SDS-PAGE result
Figure 23 Triton X-100 washing Arresten factor inclusion body SDS-PAGE result
Figure 24 synthetic method washing Arresten factor inclusion body SDS-PAGE result
Embodiment
3, material and test method
3.1 material
3.1.1 sample
Placenta tissue is provided by Mountain Western Medicine S University's second affiliated hospital's Obstetric and Gynecologic Department.
3.1.2 plasmid and bacterial strain
Host bacterium E.coli JM109, DH5 α, BL21, BL21 (DE3) and prokaryotic expression carrier pBV220 provide by ℃ preservation of biological chemistry teaching and research room-70 of Mountain Western Medicine S University.
JM109: genotype recA1 supE44 endA1 hsdR17 gyr A96 relA1 thi Δ (lac-proAB) F ' [traD36 proAB
+Lac I
qLacZ Δ M15]
DH5 α: genotype supE44 Δ lacU169 ( 80 lacZ Δ M15) hsdR17 recA1 endA1gyrA96 thi-l relA1
BL21: genotype F
-, ompT, hsdS
B(r
B -, m
B -) gal, dem
BL21 (DE3): genotype hsdSgal (λ cI ts857indl Sam7 nin5 lacUV5-T7 gene 1) pBV220 empty plasmid carrier: total length 3666bp, amicillin resistance (Amp
+), for the temperature control inducible expression carrier, have P
RP
LStrong tandem promoter.
3.1.3 main agents
TRIzol reagent is the Invitrogen product, PCR reverse transcription test kit, 100bp DNA LadderMarker are available from precious biotechnology (Dalian) company limited, T4 dna ligase, restriction enzyme BamH I, EcoR I, Pvu II, Lambda DNA/HindIII+EcoR I Markers are all available from Huamei Bio-Engrg Co.,, UNIQ-10 pillar DNA glue reclaims test kit, agarose (Agrose), low melting-point agarose are available from Promega company, Tryptones, yeast extract are OXOID company product, and other reagent is homemade analytical pure.
3.1.4 substratum and solution
The LB substratum: the 10g/L Tryptones, 5g/L NaCl, the 10g/L yeast extract is regulated pH to 7.4, autoclaving.
LB (Amp
+) substratum: the LB substratum that contains 100mg/L Amp.
LB (Amp
+) flat board: the LB flat board that contains 100mg/L Amp.
STE：0.1mol/L NaCl，10mmol/L Tris·Cl，1mmol/L EDTA。
Solution I: 50mmol/L glucose, 25mmol/L TrisCl (pH8.0), 10mmol/L EDTA, autoclave sterilization sterilization, 4 ℃ of preservations.
Solution II: 0.2mol/L NaOH, 1%SDS (fresh preparation).
Solution III: 60ml 5mol/L KAc, 11.5ml Glacial acetic acid, 28.5ml water.
5 * tbe buffer liquid (every liter): 54g Tris alkali, 27.5g boric acid, 20ml 0.5mol/L EDTA (pH8.0)
3.1.5, design of primers and synthetic
According to the Arresten mRNA sequence (sequence number of reporting among the GenBank: AF258349), with DNAman software design primer, primer nearly 5 ' end in upstream and downstream is introduced restriction enzyme BamHI, EcoR I (line part) restriction enzyme site and a pair of initiator codon and two pairs of terminator codons (mark of emphasis part) respectively, and primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Upstream primer 5 '--GCCGAATTCATGTCTGTTGATCACGGC 3 '
Downstream primer 5 '-GCGGATCCCTATTATTATGTTCTTCTC 3 '
3.2, test method
3.2.1 extraction and the detection of total RNA in the placenta tissue
(1) from normal Chinese's placenta tissue, extracts total RNA with TRIzol reagent.
1. get the fresh placenta tissue of 100mg, grind into powder in liquid nitrogen;
2. treat that the liquid nitrogen volatilization is dried, add 1ml TRIzol immediately, after the thawing, beat with the suction of application of sample rifle, make the abundant cracking of sample, move in the 1.5ml centrifuge tube, room temperature leaves standstill 5min;
3. the chloroform that in centrifuge tube, adds the 0.2ml precooling, the vibration mixing;
4. with centrifuge tube at 4 ℃, 12000r/min, centrifugal 15min;
5. with the careful sucking-off of pipettor upper strata water (not drawing that layer of middle white), add in another centrifuge tube, add the Virahol of 0.5ml precooling, the vibration mixing;
6.-20 a ℃ precipitation is spent the night;
7. 4 ℃, 12000r/min, centrifugal 15min;
8. carefully shift out supernatant;
9. add 1ml 75% precooled ethanol washing RNA precipitation in the precipitation, 4 ℃, 12000r/min, centrifugal 5min;
10. repeat to remove supernatant with RNA precipitation of 75% precooled ethanol washing, airing a little, it is transparent that RNA shows slightly, and the water that adds 20 μ L RNase-free fully dissolves.
(2) the quantitative and total RNA of detection
1. the quantitative and detection of ultraviolet
I detects the output of RNA with 752 ultraviolet grating spectrophotometers, in the absorbancy of 260nm, and 1OD=40 μ g/ml;
Ii detects the purity of RNA, the OD of pure rna according to the light absorption value at 260nm and 280nm place
260/ OD
280Ratio should be near 2.0 (ratio be preferably between 1.9～2.1).
2. agarose gel electrophoresis detects
The sepharose of i preparation 1%
Agrose 0.25g
5 * tbe buffer liquid 2.5ml
Deionized water 22.5ml
Microwave oven heating fusion Agrose is cooled to 60 ℃ a little, adds
10mg/mL ethidium bromide 1.25 μ L
Shake up the back and fall glue, treat the glue condensation after, place electrophoresis chamber, be dipped in 1 * tbe buffer liquid balance 10 minutes, stand-by.
The sex change of ii sample
5×Loading Buffer 2μL
RNase-free water is adjusted to 10 μ L
65 ℃ of sex change 5min place cooled on ice immediately.
The iii electrophoresis
With the RNA sample of handling well, centrifugal a little, add in the well, after 60V constant voltage voltage 10min electrophoresis finishes, observations on the gel imaging instrument.
Identification and detection result:
Extract total RNA from normal people's placenta tissue, the denaturing formaldehyde electrophoresis is three bands clearly as seen, i.e. 5.8s, 18s, 28s, and 28s brightness is the twice of 18s, the results are shown in Figure 4, meets the requirements.
Quantitative and the total RNA of detection of ultraviolet:
OD
260=0.019, OD
280=0.010, OD
260/ OD
280=1.9, just in time in the range of normal value of RNA, the concentration of RNA is 0.76 μ g/ml
3.2.2, RT-PCR amplification Arresten and reclaim purifying
(1) positive control
1. reverse transcription reaction
The i reaction system is: MgCl
22 μ L
10×RT Buffer 1μL
RNase Free dH
2O 3.75μL
dNTP Mixture(10mmol/L) 1μL
RNase Inhibitor 0.25μL
AMV reversed transcriptive enzyme 0.5 μ L
Random 9 mers 0.5μL
Positive Control RNA 1μL
Total 10μL
The ii reaction conditions is: 30 ℃ of 10min
42℃ 30min
99℃ 5min
5℃ 5min
2. PCR reaction
The i reaction system is: inverse transcription reaction liquid 10 μ L
5×PCR Buffer 10μL
Sterile purified water 28.75 μ L
TaKaRa Ex Taq
TMHS 0.25μL
Control F-1 Primer 0.5μL
Control R-1 Primer 0.5uL
Total 50μL
The ii reaction conditions is: 94 ℃ of 2min 1Cycle
94℃ 30min
55℃ 30sec
72℃ 1min 30 Cycle
(2)Arresten
1. reverse transcription reaction
The i reaction system is: MgCl
22 μ L
10×RT Buffer 1μL
RNase Free dH
2O 3.75μL
dNTP Mixture(10mmol/L) 1μL
RNase Inhibitor 0.25μL
AMV reversed transcriptive enzyme 0.5 μ L
Random 9 mers 0.5μL
Total 10μL
The ii reaction conditions is: 30 ℃ of 10min
42℃ 30min
99℃ 5min
5℃ 5min
2. pcr amplification reaction system
The i reaction system is: inverse transcription reaction liquid 10 μ L
5×PCR Buffer 10μL
Sterile purified water 28.75 μ L
TaKaRa Ex Taq
TMHS 0.25μL
Control F-1 Primer 0.5μL
Control R-1 Primer 0.5μL
Total 50μL
The ii reaction conditions is: 94 ℃ of 2min 1Cycle
94℃ 40sec
57℃ 40sec
72℃ 1min 35 Cycle
72 ℃ are extended 5min
(3) get each 10 μ L of positive control and Arresten amplified production respectively, identify that with 2% agarose gel electrophoresis all the other deposit in-20 ℃ for future use.
(4) glue of cutting of PCR product reclaims purifying
1. use 1 * TBE to prepare 2% low melting-point agarose gel, and in the process of glue, use wide comb;
2. sample will all be gone up behind 100 μ L amplified productions and 20 μ L, the 6 * loading buffer mixing, 80V electrophoresis 40min, carefully be put into gel under the long-wave ultra violet lamp, irradiation time is short as far as possible, from sepharose, cut target DNA fragment with a clean sharp blade, cut gel as far as possible less, adhesive tape is transferred in the aseptic EP pipe of a 1.5mL;
3. add 1ml Binding BufferII, place 56 ℃ of water-bath 10min, glue is thoroughly melted.When adding hot melt adhesive, every 2min mixing once;
4. the sol solution that melts is transferred in the UNIQ-10 post that is mounted in the 2ml collection tube, room temperature is placed 2min, the centrifugal 1min of 8000r/min room temperature;
5. take off the UNIQ-10 post, outwell the waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, add 500 μ L Wash Solution, the centrifugal 1min of 8000r/min room temperature;
6. repeating step 5. once;
7. take off the UNIQ-10 post, outwell the waste liquid in the collection tube, the UNIQ-10 post is put into same collection tube, the centrifugal 15sec of 12000r/min room temperature;
8. the UNIQ-10 post is put into a new 1.5ml centrifuge tube, added 40 μ LElution Buffer, place 2min for 37 ℃ in pillar film central authorities;
9. the centrifugal 1min of 12000r/min room temperature puts-20 ℃ of preservations with eluted dna with the DNA that obtains;
10. get 2 μ L purified products and carry out the evaluation of 2% agarose gel electrophoresis.
Qualification result: extract total RNA from people's placenta tissue, carry out the RT-PCR amplification, amplified production is through 2.0% agarose gel electrophoresis, the results are shown in Figure 5, is the 700bp place at relative molecular weight, and the specific amplification band is arranged, for intending the target gene fragment of amplification, its molecular weight is 718bp.As accompanying drawing 5
1:100bp DNA Ladder Marker 2:RT-PCR amplified production (718bp) 3:Control RNA is as positive control (462bp)
3.2.3, the obtaining and recovery, purifying (for routine techniques) of plasmid vector pBV220
3.2.4, double digestion, the connection of PCR product and plasmid pBV220
(1) BamH I enzyme is cut
10×Buffer E 2μL
Acetylated BSA(1mg/ml) 2μL
BamH I 0.5μL
The DNA substrate 5 μ L of purifying
Aseptic deionized water 10.5 μ L
20μL
Slow suction mixes for 3 times after adding restriction endonuclease, and 37 ℃ of thermostat containers are hatched 3h
(2) dehydrated alcohol deposit D NA
20 μ L are moved in the 1.5ml EP pipe, add the dehydrated alcohol of 40 μ L ice precooling, mixing is placed 1h on ice in 4 ℃ of refrigerators, 12000r/min, and 4 ℃ of centrifugal 10min abandon supernatant, evaporation trace ethanol.
(3) EcoR I enzyme is cut
10×Buffer H 2μL
Acetylated BSA(1mg/ml) 2μL
EcoR I 0.5μL
BamH I enzyme is cut and post precipitation DNA substrate 0.5 μ L
Aseptic deionized water 15.5 μ L
20μL
Slow suction mixes for 3 times after adding restriction endonuclease, and 37 ℃ of thermostat containers are hatched 3h, and row 1% agarose gel electrophoresis is identified simultaneously.
(4) piece that digs of endonuclease bamhi reclaims and purifying
The same 3.2.2 of method (4) surveys OD simultaneously
260With OD
280, draw concentration separately
(5) the Arresten fragment of purifying and being connected of pBV220 carrier segments
Press the mole ratio of carrier/target DNA: 1: 2, then this reaction system was as follows:
The Arresten gene fragment 13 μ L of purifying
PBV220 carrier enzyme is cut gene fragment 12 μ L
T
4Dna ligase 2 μ L
10 * connection damping fluid, 3 μ L
30μL
16 ℃ of connections are spent the night and are generally 8～10 hours, obtain ligation product pBV220-Arr.
3.2.5, connect the evaluation of product
The transformation receptor bacterium also carries out alkaline lysis and extracts plasmid, through PCR and through BamH I, EcoR I, PvuII row 0.8% agarose gel electrophoresis after enzyme is cut respectively. and the results are shown in Figure 6, show that target gene fragment is successfully cloned.
1:PCR product P CR identifies 2,4: recombinant plasmid pBV220-Arr (4365 bp) 3: the pBV220 5:Lambda DNA/HindIII+EcoR I Markers (5148/4973 of purifying, 4269,3530,2027,1904,1587,1375,941,831,564) 6:100bpDNA Ladder Marker (1500,1000,900,800,700,600,500,400,300,200,100) 7: the Arresten8 of purifying: enzyme is cut evaluation (about 3645,565,150,10)
3.2.6, determined dna sequence
Get the bacterium liquid 1ml that contains recombinant plasmid, give birth to worker's genomic dna sequenator sequencing by Shanghai with the pBV220F primer.The result shows that the mRNA length of Arresten gene is 690 bp, 229 amino acid of encoding.＞0719068(JM109)PBV220+sequence exported from chromatogramfile
Order-checking shows that 229 amino acid of the non-collagen end of coding human IV type collagen α 1 chain are consistent substantially among its coded amino acid and the GenBank.(the gene number of registration of reporting among the GenBank: homology AF258349) reaches 99% but the people Arresten gene coding region cDNA sequence of reports such as Arresten gene and U.S. Colorado is extracted in this test from people's placenta, 4 gene locus differences are arranged, promptly the 132nd becomes C by A, the 148th becomes A by G, 200 become C by T, and 236 become G by A.Amino acid becomes glycine GGA by glycine GGC, and L-Ala GCC becomes Threonine ACC, and Isoleucine ATT becomes Threonine ACT, and tyrosine TAC becomes halfcystine TGC.
3.2.7, the amplification and the preservation of pBV220-Arr/JM109 bacterial strain
The correct bacterial strain of order-checking newly is laid on LB agar (Amp
+) on the flat board, be inverted for 37 ℃ and cultivate 24h, the picking positive colony is inoculated in 5ml LB (the Amp10 μ L) liquid nutrient medium, and 37 ℃, 220 rpm incubated overnight are to OD
600Be about about 0.6, get 0.2ml then and join in the sterilization EP pipe, add 50 μ L sterile glycerols ,-70 ℃ of preservations.
4, Arresten factor separation and purification
4.1 material
4.1.1 reagent
(1) urea, TritonX-100 are that worker's import packing product is given birth in Shanghai, and Sleep-promoting factor B and reduced glutathion are Beijing ancient cooking vessel state biotech development center, and other reagent herewith
(2) 60 of chicken embryos are planted chicken house by Taiyuan Xiaodian Area China provides, and 0.22 μ m filter membrane is a Sigma company product, and Arresten factor product is by this chamber preparation, and other reagent is homemade analytical pure.
4.1.2 instrument
KS-600 ultrasonic grinding machine Ningbo instrument plant
4.2, experimental technique
4.2.1 the broken bacterium of ultrasonic method obtains inclusion body
(1) bacterial sediment is dissolved and 4000r/min with STE, 10min washing 2 times is resuspended among the STE with 1: 5 ratio, and is sub-packed in 20 1.5ml EP pipes every pipe 1ml.
(2) get 4 12000r/min, 4 ℃, centrifugal 2min abandons supernatant, adds 2M (mol/L), 3M, 4M, 5M urea 1ml in every pipe respectively, after the dissolving, leaves standstill a moment, with 500W, and ultrasonic 3sec, 3sec at interval, under the condition of ice bath ultrasonic 200 times.
(3) with ultrasonic back EP pipe with 6000r/min, 4 ℃, centrifugal 10min, it is standby to keep supernatant, precipitation adds 8mol/L urea respectively, mixing with 1ml STE washed twice, prepare supernatant and deposit sample respectively, carry out SDS-PAGE, draw qualitative, quantitative conclusion through the gel analysis system.
4.2.2, the washing of inclusion body with separate
(1) optimum obtains inclusion body
Get 15 EP pipe among the 4.2.1 (1), in 12000r/min, 4 ℃, centrifugal 2min abandons supernatant, all uses 2M urea 1ml with Eppendorf centrifuge, after the dissolving, leaves standstill a moment, with 500W, and ultrasonic 3sec, 3sec at interval, under the condition of ice bath ultrasonic 200 times.
(2) 1-5M urea washing inclusion body effect assessment
Add 2M (mol/L), 3M, 4M, 5M urea 1ml respectively 4.2.2 deposit sample (1) is got 5, mixing leaves standstill 10min up and down, 6000r/min, 4 ℃, centrifugal 20min, it is standby to keep supernatant, washs primary sedimentation again with method, and centrifuging and taking precipitation, supernatant prepare the SDS-PAGE sample.
(3) Triton X-100 washing inclusion body effect assessment
Add each 1ml of Triton X-100 solution of 1%, 2%, 4% respectively 4.2.2 deposit sample (1) is got 3, mixing leaves standstill 10min up and down, 6000r/min, and 4 ℃, centrifugal 20min, it is standby to keep supernatant, and centrifuging and taking precipitation, supernatant prepare the SDS-PAGE sample.
(4) synthetic method washing inclusion body effect assessment
4.2.2 getting 4, deposit sample (1) adds 2M urea 1ml respectively, add 1%, 2% therein in 3 respectively, 4%Triton X-100 solution, with the sucrose solution of 1M, and the EDTA of a little, mixing up and down, leave standstill 10min, 6000r/min, 4 ℃, centrifugal 20min, it is standby to keep supernatant, and centrifuging and taking precipitation, supernatant prepare the SDS-PAGE sample.
4.2.3, the renaturation of inclusion body, purifying
(1) adds in the dialysis tubing with 8M urea dissolved inclusion body, add the urea of 6M in the beaker, stir 1d with stirrer;
(2) solution in the beaker is changed to the urea of 4M, stirs 1d with stirrer;
(3) solution in the beaker is changed to the urea of 2M, stirs 1d with stirrer;
(4) solution in the beaker is changed to renaturation solution (0.6mol/l urea, 0.1mmol/l Sleep-promoting factor B, 20mmol/l TrisCl (PH8.0), 0.9mmol/l Sleep-promoting factor B), stir 1d with stirrer, repeat to change liquid and stir totally 3 times, and reclaim renaturation solution;
(5) at last solution in the beaker is changed to PBS liquid, stirs 1d, reclaim in contrast with stirrer.
(6) survey protein concentration with the Xylene Brilliant Cyanine G method,
Embodiment 2:
Test method is with embodiment 1, and difference is the pcr amplification reaction system of Arresten
Reaction conditions is: 93 ℃ of 2min 1Cycle
93℃ 40sec
56℃ 40sec
71℃ 1min 35Cycle
71 ℃ are extended 5min
The Arresten fragment of purifying and being connected of pBV220 carrier segments, the mole ratio of pressing carrier/target DNA: 1: 1,15 ℃ of connections were spent the night, and the time is 10 hours, obtained ligation product pBV220-Arr.
Embodiment 3:
Above-mentioned test method is with embodiment 1, and difference is the pcr amplification reaction system of Arresten
Reaction conditions is: 95 ℃ of 2min 1Cycle
95℃ 40sec
58℃ 40sec
73℃ 1min 35Cycle
73 ℃ are extended 5min
The Arresten fragment of purifying and being connected of pBV220 carrier segments, the mole ratio of pressing carrier/target DNA: 1: 3,18 ℃ of connections were spent the night, and the time is 8 hours, obtained ligation product pBV220-Arr.
English shortenings (Abbreviation)
The English full name Chinese of english abbreviation full name
LB Luria-Bertani medium LB substratum
AMP Ampicillin penbritin
EDTA Ethyline diaminetertraaacetic acid ethylenediamine tetraacetic acid (EDTA)
Tris Tris (hydroxyrnethyl) aminomethane three (methylol) aminohexane
SDS Sodium dodecyl sulfate sodium laurylsulfonate
R/m Revolutions per minute per minute rotating speed
RNase ribonuclease RNA enzyme
EB ethidium btomide ethidium bromide
PCR prolymerase chain reaction polymerase chain reaction
OD optical density absorbance value
PBS phosphatic buffer solution phosphate buffered saline buffer
PAGE polyacrylamide gel electrophoresis polyacrylamide gel electrophoresis
DTT dithiothreitol dithiothreitol (DTT)
KD kilodaltons kilodalton
RT room temperature room temperature
The PH potential of hydrogen
SDS-PAGE sex change glue-polyacrylamide gel electrophoresis
AP ammonium persulfate Ammonium Persulfate 98.5.
Claims (5)
1, a kind of endogenetic angiogenesis inhibitive factor Arresten gene that utilizes prepares proteic method, step comprises, from human body, extract total RNA, in the adding, downstream primer, the RT-PCR post transcription cloning obtains the DNA of Arresten gene, get again behind the plasmid vector double digestion with after RT-PCR amplified production Arresten dna double enzyme is cut and be connected, obtain the ligation product, at last product is transformed in the host e. coli and expresses, obtain Arresten albumen, it is characterized in that: connection and the used plasmid of expression vector are pBV220, the mole ratio of carrier pBV220 and purpose ArrestenDNA: 1: 1～1: 3,15～18 ℃ of connections, 8～10 hours time, obtain ligation product pBV220-Arr.
2, the endogenetic angiogenesis inhibitive factor Arresten gene that utilizes according to claim 1 prepares proteic method, it is characterized in that: the selection intestinal bacteria are E.coliJM109, DH5 α, BL21, BL21 (DE3), and wherein the optimal expression bacterial classification is E.coli BL21 (DE3).
3, the endogenetic angiogenesis inhibitive factor Arresten gene that utilizes according to claim 1 and 2 prepares proteic method, it is characterized in that: e. coli expression product exists with inclusion body, the washing of inclusion body is used 2mol/L urea and 2%Triton X-100 washing with separating, and effect is best.
4, the endogenetic angiogenesis inhibitive factor Arresten gene that utilizes according to claim 1 and 2 prepares proteic method, it is characterized in that: the starting temperature of post transcription cloning reaction conditions is controlled at 93-95 ℃, 40sec; Annealing temperature is 56-58 ℃, 40sec; Elongating temperature 71-73 ℃; 1min, totally 35 circulations.
5, the endogenetic angiogenesis inhibitive factor Arresten gene that utilizes according to claim 1 and 2 prepares proteic method, it is characterized in that: 30 ℃ of intestinal bacteria culture temperature, 4 hours time, express generally at 42 ℃, 4 hours time, optimal dissolution oxygen amount selection percentage was at 1: 5.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805403A (en) * | 2010-04-07 | 2010-08-18 | 郑金平 | aurification method of prokaryotic expression Arresten protein |
| CN102212126A (en) * | 2010-04-08 | 2011-10-12 | 上海普洛康裕药物研究院有限公司 | Recombinant EDI (Endothelial Genesis Inhibitor)-8t protein with endothelial cell growth inhibiting activity |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101805403A (en) * | 2010-04-07 | 2010-08-18 | 郑金平 | aurification method of prokaryotic expression Arresten protein |
| CN101805403B (en) * | 2010-04-07 | 2013-04-03 | 郑金平 | aurification method of prokaryotic expression Arresten protein |
| CN102212126A (en) * | 2010-04-08 | 2011-10-12 | 上海普洛康裕药物研究院有限公司 | Recombinant EDI (Endothelial Genesis Inhibitor)-8t protein with endothelial cell growth inhibiting activity |
| CN102212126B (en) * | 2010-04-08 | 2013-06-19 | 上海普洛康裕药物研究院有限公司 | Recombinant EDI (Endothelial Genesis Inhibitor)-8t protein with endothelial cell growth inhibiting activity |
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