CN103305546A - pGreen Max1 eukaryotic expression vector and preparation method thereof - Google Patents
pGreen Max1 eukaryotic expression vector and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a pGreen Max1 eukaryotic expression vector and a preparation method thereof in the technical field of molecular biology. Mex and IRES-EGFP segments are recombined with the eukaryotic expression vector to obtain a sequence 1; and the total length of the sequence 1 is 7976 bp, wherein a Mex segment of 647 bp is inserted between the 176th bp and the 2415th bp and an IRES-EGFP sequence of 1309 bp is inserted between the 3120th bp and the 4428th bp; and the preparation method comprises the following steps of: obtaining Mex by cloning from a plasmid by a PCR (Polymerase chain reaction) method by virtue of a designed primer, and directionally inserting the PCR product into the expression vector through a Sma I site, and inserting IRES-EGFP into the vector through BamHi and NotI double digestion, thereby establishing the recombinant pGreen Max1 eukaryotic expression vector. The vector provided by the invention is applied to obtaining a target protein or antibody from an eukaryotic cell, and provides protein expression for screening of high-yield mammalian cell lines and a mark for screening.
Description
The present invention by number of patent application is: 201110099371.6, name of patent application is: " pGreen Max1 carrier for expression of eukaryon and preparation method thereof ", patent application is artificial: Shanghai Communications University, patent application day is: the patent on April 20th, 2011 is divided an application.
Technical field
What the present invention relates to is carrier of a kind of field of molecular biotechnology and preparation method thereof, particularly be a kind of pGreen Max1 carrier for expression of eukaryon and preparation method thereof.
Background technology
Carrier for expression of eukaryon is mainly used in some specific albumen of great expression in cell, and present existing carrier for expression of eukaryon mainly contains:
(1) pCMVp-NEO-BAN carrier, this carrier can stably be expressed the external source goal gene by high level in most of eukaryotic cells, and can utilize G418 to screen.
(2) pEGFP carrier is such as U.S.Patent Nos.5625048issued on April29,1997.The characteristics of pEGFP carrier are to express green fluorescent protein, light wave by fixed wave length excites and can send green fluorescence, this carrier can be expressed an albumen, make it to form fusion rotein with green fluorescence, be used for determining that foreign gene is in the location of cell, simultaneously, this carrier can screen with G418.
Above-mentioned carrier has weak point, and the pCMVp-NEO-BAN carrier can well be expressed foreign aid's albumen, but screening and cloning and evaluation protein expression amount are relatively more difficult, are unfavorable for screening.The pEGFP carrier can be fairly simple be used for screening and detect the protein expression amount, but owing to will merge with green fluorescent protein and form fusion rotein, influential to target protein conformation, following process and distribution meeting.PGreen Max1 carrier has well solved this several problems.It can carry out first preliminary screening and cloning by G418, then can judge according to the expression amount of cell green fluorescence the expression amount of the target protein of stable cell line, and green fluorescent protein and target protein are not to form fusion rotein, but form respectively two albumen, for target protein conformation and fully not impact of following process.
Summary of the invention
The objective of the invention is for the deficiencies in the prior art, a kind of pGreen Max1 carrier for expression of eukaryon and preparation method thereof is provided, can be in the mammalian cell efficient expressing protein of the present invention or antibody realize that the high yielding cell sarain screening provides the stable screening mark.
The present invention is achieved by the following technical solutions:
The pGreen Max1 carrier for expression of eukaryon that the present invention relates to, obtain sequence 1 by Mex, IRES-EGFP fragment and carrier for expression of eukaryon restructuring, sequence 1 amounts to 7976bp, wherein between 1769bp-2415bp, insert the Mex fragment that contains 647bp, between 3120bp-4428bp, inserted the IRES-EGFP sequence that contains 1309bp.
The preparation method of the above-mentioned pGreen Max1 carrier for expression of eukaryon that the present invention relates to, the method clone by PCR from plasmid obtains Mex by the design primer, and with the site-directed pMex eukaryotic vector that is inserted into of PCR product process SmaI, and through BamHI and NotI double digestion IRES-EGFP is inserted in the carrier, make up the pGreen Max carrier for expression of eukaryon of restructuring.
The preparation method of described pGreen Max1 carrier for expression of eukaryon comprises the steps:
A, design primer: according to the Mex primers, upstream primer adds the PmeI site, and primer sequence is as follows:
Mex?F：GATATC
GTTT?AAACGCTCTT?CCTGCATCAC?GGGAGAT
Mex?R：TGAGGCCTGA?GTTCAGACCG?GT
The clone of B, purpose fragment: take with the plasmid of Mex as template, carry out pcr amplification: template 1 μ l, each 1 μ l of upstream and downstream primer 10 μ M, the dNTP substrate 4 μ l of 2.5mM, 10 times of damping fluid 5 μ l, pfu polysaccharase 1 μ l, distilled water 37 μ l; 95 ℃ of sex change 2 minutes; Then 30 seconds, 72 ℃ of 94 ℃ of sex change 30 seconds, 55-65 ℃ annealing were extended 50 seconds, carried out 30-40 circulation; Last 72 ℃ were extended 7-10 minute;
C, amplified production purifying: the PCR product is after 1% agarose gel electrophoresis separates, and the amplified band of a visible treaty 647bp downcuts the purpose band, carries out purifying with Gel Extraction Kit DNA purification kit;
The structure of D, pMex eukaryotic vector:
D-1, enzyme are cut: carrier is carried out enzyme with the SmaI enzyme cut, add 10 times of damping fluid 2 μ l, SmaI1 μ l when enzyme is cut in reaction system, carrier 5 μ l, deionized water 12 μ l, 37 ℃ of water-baths 4 hours, the carrier segments after enzyme is cut is cut glue and is reclaimed after 1% agarose gel electrophoresis separates;
D-2, connection: the fragment of the Mex of recovery and carrier carry out external ligation with the T4 ligase enzyme;
D-3, conversion intestinal bacteria: the product that connects is changed in the competent escherichia coli cell, on the LB substratum that contains ammonia benzyl mycin 100 μ l/ml, cultivated 12-16 hour for 37 ℃;
The positive colony of D-4, screening recombinant eukaryon expression vector: with the bacterium colony amplification culture, the purification plasmid;
D-5, utilize SpeI to carry out enzyme to cut and identify and order-checking, confirm to obtain the pMex eukaryon expression plasmid of sequence 1;
E, obtain the IRES-EGFP sequence: in plasmid, utilize BamHI and NotI double digestion to obtain the IRES-EGFP sequence, and after 1% agarose gel electrophoresis separation, as seen the band of a treaty 1300bp, the purpose band is downcut, carry out purifying with Gel Extraction Kit DNA purification kit;
The structure of F, pGreen Max1 carrier for expression of eukaryon:
F-1, enzyme are cut: with carrier BamHI and NotI double digestion, when cutting, enzyme in reaction system, adds 10 times of damping fluid 2 μ l, BamHI1 μ l, NotI μ l, pMex carrier 5 μ l, deionized water 12 μ l, 37 ℃ of water-baths 4 hours, the carrier segments after enzyme is cut is cut glue and is reclaimed after 1% agarose gel electrophoresis separates;
F-2, connection: IRES-EGFP fragment and the carrier of recovery carry out external ligation with the T4 ligase enzyme;
F-3, conversion intestinal bacteria: the product that connects is changed in the competent escherichia coli cell, on the LB substratum that contains ammonia benzyl mycin 100 μ l/ml, cultivated 12-16 hour for 37 ℃;
The positive colony of F-4, screening recombinant eukaryon expression vector: with the bacterium colony amplification culture, the purification plasmid;
F-5, utilize HindIII to carry out enzyme to cut and identify and order-checking, confirm to obtain the pGreen Max1 eukaryon expression plasmid of sequence 1.
PCR product described in the step C, its purification process step is as follows:
1. from sepharose PCR product D NA band is downcut, change in the centrifuge tube, add the binding buffer liquid of 2 times of volumes, 55 ℃ of lower temperature were bathed 7 minutes so that the gel dissolving;
2. the gelating soln that dissolves in the centrifuge tube is transferred in the DNA adsorption column that test kit carries, 13000 rev/mins centrifugal 2 minutes;
3. add 700 μ l rinsing liquids in the DNA adsorption column, 13000 rev/mins centrifugal 2 minutes; Discard rinsing liquid, add 500 μ l rinsing liquids again, 13000 rev/mins centrifugal 2 minutes; After discarding rinsing liquid, 13000 rev/mins centrifugal 2 minutes, room temperature was placed 5 minutes;
4. the DNA adsorption column is added in the new centrifuge tube, adds the elution buffer 70 μ l of 65 ℃ of preheatings, 13000 rev/mins centrifugal 2 minutes, the solution at the bottom of the centrifuge tube is the PCR product D NA fragment of recovery.
Connection described in the step D-2, its method of attachment is as follows: add PCR product D NA fragment 8 μ l and the carrier segments 2 μ l that reclaim in reaction system, 10 times of damping fluid 2 μ l, T4DNA ligase enzyme 1 μ l, deionized water 7 μ l, 16 ℃ of reaction 10h.The described method of attachment of step F-2 is: add dna fragmentation 8 μ l and the carrier segments 2 μ l that reclaim in reaction system, 10 times of damping fluid 2 μ l, T4DNA ligase enzyme 1 μ l, deionized water 7 μ l, 16 ℃ of reaction 10h.
The described conversion of step D-3 intestinal bacteria, it is as follows that it transforms the intestinal bacteria method steps:
1. get connection product 10ul and be added in the competent cell of 50ul, shake up gently ice bath 30min.
2. 42 ℃ of heat shock 90s forward ice bath 2-3min fast to, note not shaking pipe.
3. the LB substratum that adds 500ul in the pipe, 150rmp, 37 ℃, 45min.
4. bacterium liquid all is coated onto on the LB flat board, contains 100ug/ml Amp.
5. flat board is just being put until liquid all absorbs.
6. be inverted flat board, cultivate 12-16h. in 37 ℃
Screening recombinant eukaryon expression vector positive colony described in the step D-4, its screening recombinant eukaryon expression vector positive colony method steps is as follows:
1. the single bacterium colony after picking transforms is inoculated in the LB substratum that 2ml contains penbritin (100 μ g/ml), and 37 ℃, 150rpm, shaking table is cultivated 12～16hr;
2. get the 1-1.5ml culture and pour in the 1.5ml centrifuge tube, 4 ℃, the centrifugal 30s of 13000rpm abandons supernatant, and then of short duration centrifugal, with the rifle head a small amount of raffinate sucking-off in centrifuge tube bottom is discarded.4 ℃ of preservations of residue bacterium liquid;
3. after the concussion of cell precipitation whirlpool being broken up, be resuspended in the alkaline lysis liquid I of 100 μ l ice precooling, the whirlpool concussion makes bacterial suspension even;
4. add 200 μ l alkaline lysis liquid II in the resuspended liquid of bacterium, spin upside down centrifuge tube 5 times, make the abundant cracking of thalline, until form bright solution, place on ice (＜5min);
5. add the alkaline lysis liquid III of 150 μ l ice precooling, spin upside down centrifuge tube 8 times, the solution III is uniformly dispersed in the bacterial lysate of thickness, and centrifuge tube placed 1～3min on ice, 4 ℃, the centrifugal 5min of 13000rpm is transferred to the supernatant of～400 μ l in another centrifuge tube;
6. add 400 μ l phenol: chloroform (V:V=1:1), concussion fully mix organic phase and water, and the centrifugal 2min of 13000rpm gets supernatant in new centrifuge tube;
7. use ethanol (850 μ l) the precipitation at room temperature nucleic acid of 2～2.5 volumes, mix, room temperature leaves standstill 2min.The centrifugal 5min of 13000rpm.Careful outwells supernatant; And then of short duration centrifugal, discard with a small amount of careful sucking-off of raffinate in the first centrifuge tube of rifle bottom;
8. add about 1ml 70% ethanol in precipitation, put upside down centrifuge tube for several times, washing tube wall and precipitation, departing from the pipe end such as precipitation needs the centrifugal 1min of 13000rpm.Careful outwells supernatant, and then of short duration centrifugal, with the careful sucking-off of a small amount of raffinate in the first centrifuge tube of rifle bottom (if precipitate floating, be placed on bottom the centrifuge tube with the rifle head);
9. open centrifuge tube, room temperature leaves standstill 3～5min, and ethanol is fully volatilized, until there is not visible liquid to exist in the centrifuge tube;
10. add 50 μ l TE damping fluids (containing 20 μ g/ml RNase A) and in centrifuge tube, leave standstill 5min, gentle concussion mixing is stored in-20 ℃.
Enzyme described in the step D-5 is cut and is identified and order-checking, its enzyme cut identify and sequence measurement as follows:
In reaction system, add the recombinant eukaryon expression vector 5 μ l that extract, 10 times of damping fluid 2 μ l, restriction enzyme SpeI1 μ l, deionized water 13 μ l, 37 ℃ of reaction 2h; After 1% agarose gel electrophoresis separates, identify the purpose band of pMex, can cut out approximately two fragments of 1.3kb and 5.3kb after the SpeI enzyme is cut, conform to expection, show in the correct insertion vector of purpose fragment;
Enzyme in the step F-5 is cut and is identified and order-checking, its enzyme cut identify and sequence measurement as follows:
In reaction system, add the recombinant eukaryon expression vector 5 μ l that extract, 10 times of damping fluid 2 μ l, restriction enzyme HindIII1 μ l, deionized water 13 μ l, 37 ℃ of reaction 2h; After 1% agarose gel electrophoresis separates, identify the purpose band of pGreen Max1, can cut out approximately three fragments of 4.9kb, 2.9kb and 230bp after the HindIII enzyme is cut, conform to expection, show in the correct insertion vector of purpose fragment.
What the present invention used is EF1 α promotor, is the promotor of high expression level albumen in the human body, can avoid exogenous promoter to the detrimentally affect of cell.What this carrier can utilize green fluorescence simultaneously luminously comes the expression of profiling protein in cell, is convenient to the screening of cell strain.The mark of protein expression and screening can be provided in the screening of high yield mammalian cell strain, and for the high efficient expression of albumen provides good carrier, also the screening for cell strain provides a kind of new method and facility.
Description of drawings
Fig. 1 is the electrophoresis photo of PCR of the present invention;
Wherein: can see 500bp to an obvious band is arranged between the 750bp, the 647bp of stripe size and expection conforms to expection about equally, shows Mex sequence PCR success.
Fig. 2 is the electrophoresis picture that the enzyme of pMex plasmid of the present invention is cut evaluation;
Wherein: after process SpeI enzyme is cut, can cut out two bands of 1.3kb and 5.3kb, conform to expection, show correct being inserted in the middle of the carrier of Mex fragment.
Fig. 3 is the restriction enzyme digestion and electrophoresis photo of pMex and IRES-EGFP;
Wherein: cut through BamHI and NotI enzyme, the carrier size is 6.6kb, and the IRES-EGFP sequence is 1.3kb, conforms to the expection size.
Fig. 4 is pGreen Max1 plasmid enzyme restriction photo;
Wherein, cutting through the HindIII enzyme, can cut out three bands, is respectively the band of 4.9kb, 2.9kb and 230bp, conforms to the expection band, shows that the IRES-EGFP sequence correctly is inserted in the middle of the pMex carrier.
Fig. 5 the has been transfection cell of albumin A recombinant eukaryon expression vector
Wherein, after the transfection, green fluorescent protein can be expressed in the cell, the screening of cell strain can be used for.
The photo of the supernatant immunofluorescence of the cell of Fig. 6 is transfection albumin A recombinant eukaryon expression vector.
Fig. 7 is antibody B protein electrophoresis photo.
Wherein, after the transfection, in the supernatant of cell, can detect the expression of albumin A.Show vector construction success and can use.
Embodiment
Below in conjunction with accompanying drawing the tissue-engineering fiber support of protein of the present invention and preparation thereof and extracorporeal releasing experiment are implemented to elaborate: following examples are implemented under take technical solution of the present invention as prerequisite; provided detailed embodiment and process, but protection scope of the present invention is not limited to following embodiment.
Carrier and gene source explanation in following examples: it is synthetic that special primer is given birth to the worker by Shanghai; Restriction enzyme BamHI, the NotI that uses in the vector construction, pfu enzyme and the equal Fermentas of T4 ligase enzyme company product (Sangon Biotech (Shanghai) Co., Ltd., 021-37772180); The DNA purification kit that uses in the vector construction is Suo Laibao company (the precious bio tech ltd of Shanghai rope, Xuhui District of Shanghai admire Jiang Lu) product, and by specification carries out; Carrier transfection (PEI is sigma company product) and secreting, expressing detection reagent are the commercial reagent.The experimental technique of unreceipted actual conditions, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
PGreen Max1 construction of eukaryotic expression vector method
A, design primer:
According to the Mex primers, upstream primer adds the PmeI site, and primer sequence is as follows:
Mex?F：GATATC
GTTT?AAACGCTCTT?CCTGCATCAC?GGGAGAT
Mex?R：TGAGGCCTGA?GTTCAGACCG?GT
The clone of B, purpose fragment:
Take with the plasmid of Mex as template, carry out pcr amplification: template 1 μ l, each 1 μ l of upstream and downstream primer 10 μ M, the dNTP substrate 4 μ l of 2.5mM, 10 times of damping fluid 5 μ l, pfu polysaccharase 1 μ l, distilled water 37 μ l; 95 ℃ of sex change 2 minutes; Then 30 seconds, 72 ℃ of 94 ℃ of sex change 30 seconds, 55-65 ℃ annealing were extended 50 seconds, carried out 30-40 circulation; Last 72 ℃ were extended 7-10 minute;
C, expansion product purification:
The PCR product is after 1% agarose electrophoresis is separated, and the amplified band of a visible treaty 647bp downcuts the purpose band, carries out purifying with company's sepharose purification kit, and method is:
C-1, from sepharose PCR product D NA band is downcut, change in the centrifuge tube, add the binding buffer liquid of 2 times of volumes, 55 ℃ of lower temperature were bathed 7 minutes so that the gel dissolving;
C-2, the gelating soln that dissolves in the centrifuge tube is transferred in the DNA adsorption column that test kit carries, 13000 rev/mins centrifugal 2 minutes;
C-3, add 700 μ l rinsing liquids in the DNA adsorption column, 13000 rev/mins centrifugal 2 minutes; Discard rinsing liquid, add 500 μ l rinsing liquids again, 13000 rev/mins centrifugal 2 minutes; After discarding rinsing liquid, 13000 rev/mins centrifugal 2 minutes, room temperature was placed 5 minutes;
C-4, the DNA adsorption column is added in the new centrifuge tube, adds the elution buffer 70 μ l of 65 ℃ of preheatings, 13000 rev/mins centrifugal 2 minutes, the solution at the bottom of the centrifuge tube is the PCR product D NA fragment of recovery.
As shown in Figure 1, can see 500bp to an obvious band is arranged between the 750bp, the 647bp of stripe size and expection conforms to expection about equally, shows Mex sequence PCR success.
The structure of D, pMex carrier for expression of eukaryon:
D-1, enzyme are cut: carrier is cut with the SmaI enzyme, the fragment that enzyme is cut with carrier is cut the glue recovery after 1% agarose gel electrophoresis separates method is: add 10 times of damping fluid 2 μ l, SmaI1 μ l, carrier 5 μ l in reaction system, deionized water 12 μ l, 37 ℃ of water-baths 4 hours.
D-2, connection: the Mex fragment of recovery is connected T4 to connect US and Russia to carry out external ligation method and be with carrier: add PCR product D NA fragment 8 μ l and the carrier segments 2 μ l that reclaim in reaction system, 10 times of damping fluid 2 μ l, T4DNA ligase enzyme 1 μ l, deionized water 7 μ l, 16 ℃ of reaction 10h.
D-3, conversion intestinal bacteria: will connect product and be transformed in the competent escherichia coli cell, and cultivate 12-16 hour for 37 ℃ on the LB substratum that contains penbritin 100 μ l/ml, method is:
A, get and connect product 10ul and be added in the competent cell of 50ul, shake up gently ice bath 30min.
B, 42 ℃ of heat shock 90s forward ice bath 2-3min fast to, note not shaking pipe.
C, Xiang Guanzhong add the LB substratum of 500ul, 150rmp, 37 ℃, 45min.
D, bacterium liquid all is coated onto on the LB flat board, contains 100ug/ml Amp.
E, flat board is just being put until liquid all absorbs.
F, inversion flat board are cultivated 12-16h. in 37 ℃
The positive colony of D-4, screening recombinant eukaryon expression vector: with the bacterium colony amplification culture, the purification plasmid, method is:
A. the single bacterium colony after picking transforms is inoculated in the LB substratum that 2ml contains penbritin (100 μ g/ml), and 37 ℃, 150rpm, shaking table is cultivated 12～16hr.
B. get the 1-1.5ml culture and pour in the 1.5ml centrifuge tube, 4 ℃, the centrifugal 30s of 13000rpm abandons supernatant, and then of short duration centrifugal, with the rifle head a small amount of raffinate sucking-off in centrifuge tube bottom is discarded.4 ℃ of preservations of residue bacterium liquid.
C. after the concussion of cell precipitation whirlpool being broken up, be resuspended in the alkaline lysis liquid I of 100 μ l ice precooling, the whirlpool concussion makes bacterial suspension even.
D. add 200 μ l alkaline lysis liquid II in the resuspended liquid of bacterium, spin upside down centrifuge tube 5 times, make the abundant cracking of thalline, until form bright solution, place on ice (＜5min).
E. add the alkaline lysis liquid III of 150 μ l ice precooling, spin upside down centrifuge tube 8 times, the solution III is uniformly dispersed in the bacterial lysate of thickness, and centrifuge tube placed 1～3min on ice, 4 ℃, the centrifugal 5min of 13000rpm is transferred to the supernatant of～400 μ l in another centrifuge tube.
F. add 400 μ l phenol: chloroform (V:V=1:1), concussion fully mix organic phase and water, and the centrifugal 2min of 13000rpm gets supernatant in new centrifuge tube.
G. use ethanol (850 μ l) the precipitation at room temperature nucleic acid of 2～2.5 volumes, mix, room temperature leaves standstill 2min.The centrifugal 5min of 13000rpm.Careful outwells supernatant; And then of short duration centrifugal, discard with a small amount of careful sucking-off of raffinate in the first centrifuge tube of rifle bottom.
H. add about 1ml 70% ethanol in precipitation, put upside down centrifuge tube for several times, washing tube wall and precipitation, departing from the pipe end such as precipitation needs the centrifugal 1min of 13000rpm.Careful outwells supernatant, and then of short duration centrifugal, with the careful sucking-off of a small amount of raffinate in the first centrifuge tube of rifle bottom (if precipitate floating, be placed on bottom the centrifuge tube with the rifle head).
I. open centrifuge tube, room temperature leaves standstill 3～5min, and ethanol is fully volatilized, until there is not visible liquid to exist in the centrifuge tube.
J. add 50 μ l TE damping fluids (containing 20 μ g/ml RNase A) and in centrifuge tube, leave standstill 5min, gentle concussion mixing.Be stored in-20 ℃.
D-5, enzyme are cut and are identified and order-checking, confirm that the sequence that obtains is eukaryon expression plasmid, and method is:
In reaction system, add the recombinant eukaryon expression vector 5 μ l that extract, 10 times of damping fluid 2 μ l, restriction enzyme SpeI1 μ l, deionized water 13 μ l, 37 ℃ of reaction 2h;
As shown in Figure 2, after process SpeI enzyme is cut, can cut out two bands of 1.3kb and 5.3kb, conform to expection, show correct being inserted in the middle of the carrier of Mex fragment;
E, obtain the IRES-EGFP sequence:
In plasmid, utilize BamHI and NotI double digestion to obtain the IRES-EGFP sequence, and after 1% agarose gel electrophoresis separation, the band of a visible treaty 1300bp downcut the purpose band, carries out purifying with Gel Extraction Kit DNA purification kit
As shown in Figure 3, cut through BamHI and NotI enzyme, the carrier size is 6.6kb, and the IRES-EGFP sequence is 1.3kb, conforms to the expection size.
The structure of F, pGreen Max carrier for expression of eukaryon:
F-1, enzyme are cut: with carrier BamHI and NotI double digestion, the carrier segments after enzyme is cut is cut glue and is reclaimed after 1% agarose gel electrophoresis separates.Method is: add 10 times of damping fluid 2 μ l in reaction system, BamHI1 μ l, NotI μ l, pMex carrier 5 μ l, deionized water 12 μ l, 37 ℃ of water-baths 4 hours.
F-2, connection: IRES-EGFP fragment and the carrier of recovery carry out external ligation with the T4 ligase enzyme, method is: add dna fragmentation 8 μ l and the carrier segments 2 μ l that reclaim, 10 times of damping fluid 2 μ l, T4DNA ligase enzyme 1 μ l in reaction system, deionized water 7 μ l, 16 ℃ of reaction 10h.
F-3, conversion intestinal bacteria: the product that connects is changed in the competent escherichia coli cell, on the LB substratum that contains ammonia benzyl mycin 100 μ l/ml, cultivated 12-16 hour for 37 ℃;
The positive colony of F-4, screening recombinant eukaryon expression vector: with the bacterium colony amplification culture, the purification plasmid;
F-5, utilize HindIII to carry out enzyme to cut and identify and order-checking, confirm to obtain the pMex eukaryon expression plasmid of sequence 1.Method is: add the recombinant eukaryon expression vector 5 μ l that extract in reaction system, 10 times of damping fluid 2 μ l, restriction enzyme HindIII1 μ l, deionized water 13 μ l, 37 ℃ of reaction 2h;
As shown in Figure 4, the purpose band that separates the rear pGreen of evaluation Max1 with 1% agarose gel electrophoresis, cut through the HindIII enzyme, can cut out three bands, it is respectively the band of 4.9kb, 2.9kb and 230bp, conform to the expection band, show that the IRES-EGFP sequence correctly is inserted in the middle of the pMex carrier.
Carry out the order-checking of recombinant eukaryon expression vector, sequencing result shows that sequence 1 size of this recombinant eukaryon expression vector is, in full accord with Mex, IRES-EGFP sequence, illustrates successfully to have made up called after pGreen Max1 carrier for expression of eukaryon, shown in sequence 1.
The expressing protein of carrier for expression of eukaryon
The acquisition of A, recombinant eukaryon expression vector
Describe as an example of albumin A and protein B example.
A-1, obtain containing the recombinant vectors of albumin A and antibody B
Obtain the sequence of albumin A and antibody B by technology such as PCR, utilize the external connection of T4DNA ligase enzyme, obtain containing the recombinant vectors of albumin A and antibody B.
The extraction and purification of A-2, recombinant expression vector
Be used for the pGreen Max1 recombinant protein A of transfection and the plasmid of pGreen Max1 recombinant antibodies B and carry out extraction and purification with the method for plasmid a small amount of extracting, concrete operation step is as follows:
A. the single bacterium colony after picking transforms is inoculated in the LB substratum that 2ml contains penbritin (100 μ g/ml), and 37 ℃, 150rpm, shaking table is cultivated 12～16hr.
B. get the 1-1.5ml culture and pour in the 1.5ml centrifuge tube, 4 ℃, the centrifugal 30s of 13000rpm abandons supernatant, and then of short duration centrifugal, with the rifle head a small amount of raffinate sucking-off in centrifuge tube bottom is discarded.4 ℃ of preservations of residue bacterium liquid.
C. after the concussion of cell precipitation whirlpool being broken up, be resuspended in the alkaline lysis liquid I of 100 μ l ice precooling, the whirlpool concussion makes bacterial suspension even.
D. add 200 μ l alkaline lysis liquid II in the resuspended liquid of bacterium, spin upside down centrifuge tube 5 times, make the abundant cracking of thalline, until form bright solution, place on ice (＜5min).
E. add the alkaline lysis liquid III of 150 μ l ice precooling, spin upside down centrifuge tube 8 times, the solution III is uniformly dispersed in the bacterial lysate of thickness, and centrifuge tube placed 1～3min on ice, 4 ℃, the centrifugal 5min of 13000rpm is transferred to the supernatant of～400 μ l in another centrifuge tube.
F. add 400 μ l phenol: chloroform (V:V=1:1), concussion fully mix organic phase and water, and the centrifugal 2min of 13000rpm gets supernatant in new centrifuge tube.
G. use ethanol (850 μ l) the precipitation at room temperature nucleic acid of 2～2.5 volumes, mix, room temperature leaves standstill 2min.The centrifugal 5min of 13000rpm.Careful outwells supernatant; And then of short duration centrifugal, discard with a small amount of careful sucking-off of raffinate in the first centrifuge tube of rifle bottom.
H. add about 1ml 70% ethanol in precipitation, put upside down centrifuge tube for several times, washing tube wall and precipitation, departing from the pipe end such as precipitation needs the centrifugal 1min of 13000rpm.Careful outwells supernatant, and then of short duration centrifugal, with the careful sucking-off of a small amount of raffinate in the first centrifuge tube of rifle bottom (if precipitate floating, be placed on bottom the centrifuge tube with the rifle head).
I. open centrifuge tube, room temperature leaves standstill 3～5min, and ethanol is fully volatilized, until there is not visible liquid to exist in the centrifuge tube.
J. add 50 μ l TE damping fluids (containing 20 μ g/ml RNase A) and in centrifuge tube, leave standstill 5min, gentle concussion mixing.Be stored in-20 ℃.
B, albumin A recombinant eukaryon expression vector and the secreting, expressing of antibody B recombinant eukaryon expression vector in the 293T cell
People's renal epithelial cell of transfection adenovirus e1a gene is the 293T cell cultures and contain the 10%FCS(foetal calf serum) the DMEM complete culture solution in, before the transfection cell is layered in the 10cm culture dish uniformly, concrete steps are carried out according to the cell transfecting experiment of PEI mediation:
1, respectively DNA and PEI are added mixing among the DMEM nutrient solution 1ml of antibiotic-free, serum-free, then two liquid are mixed into transfection liquid, room temperature was placed 10-20 minute;
2, inhale the nutrient solution of abandoning in the culture dish, wash gently twice with the DMEM nutrient solution of antibiotic-free serum-free, add the DMEM nutrient solution of 3ml antibiotic-free serum-free;
3, press albumin A recombinant expression vector 10 μ g/ wares, transfection liquid is dropwise added in the culture dish, place 5% carbonic acid gas, 37 ℃ of incubators to cultivate after 4-6 hour, the displacement serum free medium continued to cultivate after 24-48 hour, the mobile phone cells and supernatant;
4, getting the transfection supernatant, is that 10% polyacrylamide gel carries out electrophoresis with concentration, detects the secreting, expressing situation of albumin A and antibody B by the immune marking (Western Blot).
As shown in Figure 5, after the transfection, green fluorescent protein can be expressed in the cell, the screening of cell strain can be used for.
As shown in Figure 6, after the transfection, in the supernatant of cell, can detect the expression of albumin A.Show vector construction success and can use.
As shown in Figure 7, albumen is the albumen of antibody B.The albumen size is about 19kD.Can well find out by picture and to contain this antibody in the cell conditioned medium and can well detect this albumen, show that this carrier can use.
Above-described embodiment pGreen Max1 carrier for expression of eukaryon is as the application of obtaining target protein or antibody in eukaryotic cell, for the preparation of a large amount of required target proteins to carry out preclinical study, and provide the mark of protein expression and screening for the screening of high yield mammalian cell strain, for the high efficient expression of albumen provides good carrier, also the screening for cell strain provides a kind of new method and facility.
Claims (11)
1. pGreen Max1 carrier for expression of eukaryon, it is characterized in that, the nucleotide sequence of described carrier is shown in SEQ ID NO:1, obtain sequence 1 by Mex, IRES-EGFP fragment and carrier for expression of eukaryon restructuring, sequence 1 amounts to 7976bp, wherein between 1769bp-2415bp, insert the Mex fragment that contains 647bp, between 3120bp-4428bp, inserted the IRES-EGFP sequence that contains 1309bp.
2. the preparation method of a pGreen Max1 carrier for expression of eukaryon according to claim 1, it is characterized in that, the method clone by PCR from plasmid obtains Mex by the design primer, and with the site-directed pMex eukaryotic vector that is inserted into of PCR product process SmaI, and through BamHI and NotI double digestion IRES-EGFP is inserted in the carrier, make up the pGreen Max carrier for expression of eukaryon of restructuring.
3. method according to claim 3 is characterized in that, the method specifically comprises the steps:
A, design primer: according to the Mex primers, upstream primer adds the PmeI site, and primer sequence is as follows:
Mex?F：GATATC
GTTT?AAACGCTCTT?CCTGCATCAC?GGGAGAT
Mex?R：TGAGGCCTGA?GTTCAGACCG?GT
The clone of B, purpose fragment: take with the plasmid of Mex as template, carry out pcr amplification: template 1 μ L, each 1 μ L of upstream and downstream primer 10 μ M, the dNTP substrate 4 μ L of 2.5mM, 10 times of damping fluid 5 μ L, pfu polysaccharase 1 μ L, distilled water 37 μ L; 95 ℃ of sex change 2 minutes; Then 30 seconds, 72 ℃ of 94 ℃ of sex change 30 seconds, 55-65 ℃ annealing were extended 50 seconds, carried out 30-40 circulation; Last 72 ℃ were extended 7-10 minute;
C, amplified production purifying: the PCR product with the amplification purpose band cutting-out of a treaty 647bp, carries out purifying with Gel Extraction Kit DNA purification kit after 1% agarose gel electrophoresis separates;
The structure of D, pMex eukaryotic vector:
D-1, enzyme are cut: carrier is carried out enzyme with the SmaI enzyme cut, add 10 times of damping fluid 2 μ L, SmaI1 μ L when enzyme is cut in reaction system, carrier 5 μ L, deionized water 12 μ L, 37 ℃ of water-baths 4 hours, the carrier segments after enzyme is cut is cut glue and is reclaimed after 1% agarose gel electrophoresis separates;
D-2, connection: the fragment of the Mex of recovery and carrier carry out external ligation with the T4 ligase enzyme;
D-3, conversion intestinal bacteria: the product that connects is changed in the competent escherichia coli cell, on the LB substratum that contains ammonia benzyl mycin 100 μ L/mL, cultivated 12-16 hour for 37 ℃;
The positive colony of D-4, screening recombinant eukaryon expression vector: with the bacterium colony amplification culture, the purification plasmid;
D-5, utilize SpeI to carry out enzyme to cut and identify and order-checking, confirm to obtain the pMex eukaryon expression plasmid of sequence 1;
E, obtain the IRES-EGFP sequence: in plasmid, utilize BamHI and NotI double digestion to obtain the IRES-EGFP sequence, and after 1% agarose gel electrophoresis separation, the purpose band of one treaty 1300bp is downcut, carry out purifying with Gel Extraction Kit DNA purification kit;
The structure of F, pGreen Max1 carrier for expression of eukaryon:
F-1, enzyme are cut: with carrier BamHI and NotI double digestion, when cutting, enzyme in reaction system, adds 10 times of damping fluid 2 μ L, BamHI1 μ L, NotI μ L, pMex carrier 5 μ L, deionized water 12 μ L, 37 ℃ of water-baths 4 hours, the carrier segments after enzyme is cut is cut glue and is reclaimed after 1% agarose gel electrophoresis separates;
F-2, connection: IRES-EGFP fragment and the carrier of recovery carry out external ligation with the T4 ligase enzyme;
F-3, conversion intestinal bacteria: the product that connects is changed in the competent escherichia coli cell, on the LB substratum that contains ammonia benzyl mycin 100 μ L/mL, cultivated 12-16 hour for 37 ℃;
The positive colony of F-4, screening recombinant eukaryon expression vector: with the bacterium colony amplification culture, the purification plasmid;
F-5, utilize HindIII to carry out enzyme to cut and identify and order-checking, confirm to obtain the pGreen Max1 eukaryon expression plasmid of sequence 1.
4. method according to claim 3 is characterized in that, the PCR product described in the step C, and its purification process step is as follows:
1. from sepharose PCR product D NA band is downcut, change in the centrifuge tube, add the binding buffer liquid of 2 times of volumes, 55 ℃ of lower temperature were bathed 7 minutes so that the gel dissolving;
2. the gelating soln that dissolves in the centrifuge tube is transferred in the DNA adsorption column that test kit carries, 13000 rev/mins centrifugal 2 minutes;
3. add 700 μ L rinsing liquids in the DNA adsorption column, 13000 rev/mins centrifugal 2 minutes; Discard rinsing liquid, add 500 μ L rinsing liquids again, 13000 rev/mins centrifugal 2 minutes; After discarding rinsing liquid, 13000 rev/mins centrifugal 2 minutes, room temperature was placed 5 minutes;
4. the DNA adsorption column is added in the new centrifuge tube, adds the elution buffer 70 μ L of 65 ℃ of preheatings, 13000 rev/mins centrifugal 2 minutes, the solution at the bottom of the centrifuge tube is the PCR product D NA fragment of recovery.
5. method according to claim 3, it is characterized in that, connection described in the step D-2, its method of attachment is as follows: add PCR product D NA fragment 8 μ L and the carrier segments 2 μ L that reclaim in reaction system, 10 times of damping fluid 2 μ L, T4DNA ligase enzyme 1 μ L, deionized water 7 μ L, 16 ℃ of reaction 10h; The described method of attachment of step F-2 is: add dna fragmentation 8 μ L and the carrier segments 2 μ L that reclaim in reaction system, 10 times of damping fluid 2 μ L, T4DNA ligase enzyme 1 μ L, deionized water 7 μ L, 16 ℃ of reaction 10h.
6. method according to claim 3 is characterized in that, the described conversion of step D-3 intestinal bacteria, and it is as follows that it transforms the intestinal bacteria method steps:
1. get connection product 10ul and be added in the competent cell of 50ul, shake up gently ice bath 30min.;
2. 42 ℃ of heat shock 90s forward ice bath 2-3min fast to, note not shaking pipe;
3. the LB substratum that adds 500ul in the pipe, 150rmp, 37 ℃, 45min.;
4. bacterium liquid all is coated onto on the LB flat board, contains 100 μ g/mL Amp.;
5. flat board is just being put until liquid all absorbs;
6. be inverted flat board, cultivate 12～16h. in 37 ℃.
7. method according to claim 3 is characterized in that, the screening recombinant eukaryon expression vector positive colony described in the step D-4, and its screening recombinant eukaryon expression vector positive colony method steps is as follows:
1. the single bacterium colony after picking transforms is inoculated in the LB substratum that 2mL contains 100 μ g/mL penbritins, and 37 ℃, 150rpm, shaking table is cultivated 12～16hr;
2. get the 1-1.5mL culture and pour in the 1.5mL centrifuge tube, 4 ℃, the centrifugal 30s of 13000rpm abandons supernatant, and then of short duration centrifugal, a small amount of raffinate sucking-off in centrifuge tube bottom is discarded 4 ℃ of preservations of residue bacterium liquid with the rifle head;
3. after the concussion of cell precipitation whirlpool being broken up, be resuspended in the alkaline lysis liquid I of 100 μ L ice precooling, the whirlpool concussion makes bacterial suspension even;
4. add 200 μ L alkaline lysis liquid II in the resuspended liquid of bacterium, spin upside down centrifuge tube 5 times, make the abundant cracking of thalline, until form bright solution, place on ice＜5min;
5. add the alkaline lysis liquid III of 150 μ L ice precooling, spin upside down centrifuge tube 8 times, the solution III is uniformly dispersed in the bacterial lysate of thickness, and centrifuge tube placed 1～3min on ice, 4 ℃, the centrifugal 5min of 13000rpm is transferred to the supernatant of～400 μ L in another centrifuge tube;
6. adding 400 μ L volume ratios is: the phenol of 1:1: chloroform, concussion fully mix organic phase and water, and the centrifugal 2min of 13000rpm gets supernatant in new centrifuge tube;
7. use the ethanol 850 μ L precipitation at room temperature nucleic acid of 2～2.5 volumes, mix, room temperature leaves standstill 2min, the centrifugal 5min of 13000rpm, and careful outwells supernatant; And then of short duration centrifugal, discard with a small amount of careful sucking-off of raffinate in the first centrifuge tube of rifle bottom;
8. add about 1mL 70% ethanol in precipitation, put upside down centrifuge tube for several times, washing tube wall and precipitation, depart from the centrifugal 1min of pipe end need 13000rpm such as precipitation, careful outwells supernatant, and then of short duration centrifugal, with a small amount of careful sucking-off of raffinate in the first centrifuge tube of rifle bottom, if precipitate floatingly, be placed on centrifuge tube bottom with the rifle head;
9. open centrifuge tube, room temperature leaves standstill 3～5min, and ethanol is fully volatilized, until there is not visible liquid to exist in the centrifuge tube;
10. add contain 20 μ g/mL RNase A 50 μ L TE damping fluids in centrifuge tube, leave standstill 5min, gentle concussion mixing is stored in-20 ℃.
8. method according to claim 3 is characterized in that, the enzyme described in the step D-5 is cut and identified and order-checking, its enzyme cut identify and sequence measurement as follows:
In reaction system, add the recombinant eukaryon expression vector 5 μ L that extract, 10 times of damping fluid 2 μ L, restriction enzyme SpeI1 μ L, deionized water 13 μ L, 37 ℃ of reaction 2h; After 1% agarose gel electrophoresis separates, identify the purpose band of pMex, can cut out approximately two fragments of 1.3kb and 5.3kb after the SpeI enzyme is cut, show in the correct insertion vector of purpose fragment.
9. method according to claim 3 is characterized in that, the enzyme in the step F-5 is cut and identified and order-checking, its enzyme cut identify and sequence measurement as follows:
In reaction system, add the recombinant eukaryon expression vector 5 μ L that extract, 10 times of damping fluid 2 μ L, restriction enzyme HindIII1 μ L, deionized water 13 μ L, 37 ℃ of reaction 2h; After 1% agarose gel electrophoresis separates, identify the purpose band of pGreen Max1, can cut out approximately three fragments of 4.9kb, 2.9kb and 230bp after the HindIII enzyme is cut, show in the correct insertion vector of purpose fragment.
10. the application of a pGreen Max1 carrier for expression of eukaryon according to claim 1, it is characterized in that, pGreen Max1 carrier for expression of eukaryon obtains target protein and the application of the mark of protein expression and screening is provided for the screening of high yield mammalian cell strain in eukaryotic cell.
11. application according to claim 10 is characterized in that, described target protein is antibody.
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| CN110747229A (en) * | 2019-11-22 | 2020-02-04 | 河南省医药科学研究院 | Eukaryotic expression plasmid for expressing THBS4 protein, construction method and application |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5625048A (en) * | 1994-11-10 | 1997-04-29 | The Regents Of The University Of California | Modified green fluorescent proteins |
| CN1952158A (en) * | 2005-10-19 | 2007-04-25 | 吉林农业大学 | Pig A group rotavirus Vp4 whole gene eukaryotic expression plasmid |
| CN101285054A (en) * | 2008-04-23 | 2008-10-15 | 中国农业科学院哈尔滨兽医研究所 | ST cell lines for stably expressing T7 RNA polyase, constructing process and applications thereof |
| CN101509008A (en) * | 2009-03-11 | 2009-08-19 | 李君武 | Amalgamation gene of hepatitis B virus L gene and hGM-CSF, construction and uses of its expression vector |
| CN101979509A (en) * | 2010-09-27 | 2011-02-23 | 吉林大学 | Mammal cell line for stably expressing thaumatin gene and construction method |
-
2011
- 2011-04-20 CN CN201310268113.5A patent/CN103305546B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5625048A (en) * | 1994-11-10 | 1997-04-29 | The Regents Of The University Of California | Modified green fluorescent proteins |
| CN1952158A (en) * | 2005-10-19 | 2007-04-25 | 吉林农业大学 | Pig A group rotavirus Vp4 whole gene eukaryotic expression plasmid |
| CN101285054A (en) * | 2008-04-23 | 2008-10-15 | 中国农业科学院哈尔滨兽医研究所 | ST cell lines for stably expressing T7 RNA polyase, constructing process and applications thereof |
| CN101509008A (en) * | 2009-03-11 | 2009-08-19 | 李君武 | Amalgamation gene of hepatitis B virus L gene and hGM-CSF, construction and uses of its expression vector |
| CN101979509A (en) * | 2010-09-27 | 2011-02-23 | 吉林大学 | Mammal cell line for stably expressing thaumatin gene and construction method |
Non-Patent Citations (2)
| Title |
|---|
| MIEN V.HOANG 等: ""Rho activity critically and selectively regulates endothelial cell organization during angiogenesis"", 《PNAS》 * |
| ROGER P.HELLENS 等: ""pGreen：a versatile and flexible binary Ti vector for Agrobacterium-mediated plant transformation"", 《PLANT MOLECULAR BIOLOGY》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110747229A (en) * | 2019-11-22 | 2020-02-04 | 河南省医药科学研究院 | Eukaryotic expression plasmid for expressing THBS4 protein, construction method and application |
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