CN103305546B - 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 8003 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 divisional application on April 20th, 2011.
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 current existing carrier for expression of eukaryon mainly contains:
(1) pCMVp-NEO-BAN carrier, this carrier can stably be expressed external source goal gene by high level in most of eukaryotic cells, and can utilize G418 to screen.
(2) pEGFP carrier, as U.S.Patent Nos.5625048issued on April29,1997.The feature of pEGFP carrier is to express green fluorescent protein, excite and can send green fluorescence by the light wave of fixed wave length, this carrier can be expressed an albumen, make it to form fusion rotein with green fluorescence, for determining the location of foreign gene at cell, meanwhile, this carrier can screen with G418.
Above-mentioned carrier has weak point, and pCMVp-NEO-BAN carrier can well be expressed foreign aid's albumen, but the expression amount of screening and cloning and qualification albumen is more difficult, is unfavorable for screening.PEGFP carrier can be fairly simple for screening and detect the expression amount of albumen, but due to will with green fluorescent protein merge form fusion rotein, target protein conformation, following process and distribution are had to impact.PGreen Max1 carrier has well solved these problems.It can first carry out 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, do not affect completely for target protein conformation and following process.
Summary of the invention
The object 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 high yielding cell sarain screening stable screening mark be provided.
The present invention is achieved by the following technical solutions:
The pGreen Max1 carrier for expression of eukaryon the present invention relates to, obtain sequence 1 by Mex, IRES-EGFP fragment and carrier for expression of eukaryon restructuring, sequence 1 amounts to 8003bp, 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 the present invention relates to, by design primer, the method clone by PCR from plasmid obtains Mex, and PCR product is inserted in expression vector through SmaI is site-directed, and through BamHI and NotI double digestion, IRES-EGFP is inserted in carrier, build 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 Mex primers, upstream primer adds PmeI site, and primer sequence is as follows:
Mex?F：GATATC
GTTT?AAACGCTCTT?CCTGCATCAC?GGGAGAT
Mex?R：TGAGGCCTGA?GTTCAGACCG?GT
The clone of B, object fragment: taking the plasmid with Mex as template, carry out pcr amplification: template 1 μ l, the 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 DEG C of sex change 2 minutes; Then 30 seconds, 72 DEG C of 94 DEG C of sex change 30 seconds, 55-65 DEG C annealing are extended 50 seconds, carry out 30-40 circulation; Last 72 DEG C are extended 7-10 minute;
C, amplified production purifying: PCR product is after 1% agarose gel electrophoresis separates, and the amplified band of a visible treaty 647bp, cuts object band, carry 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 to enzyme with SmaI enzyme and cut, add 10 times of damping fluids, 2 μ l, SmaI1 μ l when enzyme is cut in reaction system, carrier 5 μ l, deionized water 12 μ l, 37 DEG C 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 T4 ligase enzyme;
D-3, conversion intestinal bacteria: the product of connection is proceeded in competent escherichia coli cell, containing 37 DEG C of cultivation 12-16 hour on the LB substratum of ammonia benzyl mycin 100 μ l/ml;
The positive colony of D-4, screening recombinant eukaryon expression vector: by bacterium colony amplification culture, purification plasmid;
D-5, utilize SpeI to carry out enzyme to cut qualification and order-checking, confirm to obtain the pMex eukaryon expression plasmid of sequence 1;
E, obtain IRES-EGFP sequence: in plasmid, utilize BamHI and NotI double digestion to obtain IRES-EGFP sequence, and after 1% agarose gel electrophoresis separates, the band of a visible treaty 1300bp, object band is cut, 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: by BamHI and NotI double digestion for carrier, when cutting, enzyme adds 10 times of damping fluids, 2 μ l in reaction system, BamHI1 μ l, NotI μ l, pMex carrier 5 μ l, deionized water 12 μ l, 37 DEG C 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: the IRES-EGFP fragment of recovery and carrier carry out external ligation with T4 ligase enzyme;
F-3, conversion intestinal bacteria: the product of connection is proceeded in competent escherichia coli cell, containing 37 DEG C of cultivation 12-16 hour on the LB substratum of ammonia benzyl mycin 100 μ l/ml;
The positive colony of F-4, screening recombinant eukaryon expression vector: by bacterium colony amplification culture, purification plasmid;
F-5, utilize HindIII to carry out enzyme to cut qualification and order-checking, confirm to obtain the pGreen Max1 eukaryon expression plasmid of sequence 1.
PCR product described in step C, its purification process step is as follows:
1. from sepharose, PCR product D NA band is cut, proceed in centrifuge tube, add the binding buffer liquid of 2 times of volumes, temperature is bathed and within 7 minutes, is made gel dissolve at 55 DEG C;
2. the gelating soln dissolving in centrifuge tube is transferred in the DNA adsorption column that test kit carries, 13000 revs/min centrifugal 2 minutes;
3. in DNA adsorption column, add 700 μ l rinsing liquids, 13000 revs/min centrifugal 2 minutes; Discard rinsing liquid, then add 500 μ l rinsing liquids, 13000 revs/min centrifugal 2 minutes; Discard after rinsing liquid, 13000 revs/min centrifugal 2 minutes, room temperature place 5 minutes;
4. DNA adsorption column is added in a new centrifuge tube, adds the elution buffer 70 μ l of 65 DEG C of preheatings, 13000 revs/min centrifugal 2 minutes, the solution at the bottom of centrifuge tube is the PCR product D NA fragment of recovery.
Connection described in step D-2, its method of attachment is as follows: in reaction system, add PCR product D NA fragment 8 μ l and the carrier segments 2 μ l of recovery, 10 times of damping fluid 2 μ l, T4DNA ligase enzyme 1 μ l, deionized water 7 μ l, 16 DEG C of reaction 10h.Method of attachment described in step F-2 is: in reaction system, add DNA fragmentation 8 μ l and the carrier segments 2 μ l of recovery, 10 times of damping fluid 2 μ l, T4DNA ligase enzyme 1 μ l, deionized water 7 μ l, 16 DEG C of reaction 10h.
Conversion intestinal bacteria described in step D-3, it is as follows that it transforms intestinal bacteria method steps:
1. get connection product 10 μ L and be added in the competent cell of 50 μ L, shake up gently ice bath 30min.
2. 42 DEG C of heat shock 90s, forward ice bath 2-3min fast to, note not shake pipe.
3. to the LB substratum that adds 500 μ L in pipe, 150rmp, 37 DEG C, 45min.
4. bacterium liquid is all coated onto on LB flat board, containing 100ug/ml Amp.
5. flat board is just being put until liquid all absorbs.
6. be inverted flat board, cultivate 12-16h. in 37 DEG C
Screening recombinant eukaryon expression vector positive colony described in step D-4, its screening recombinant eukaryon expression vector positive colony method steps is as follows:
1. the single bacterium colony of picking after transforming, is inoculated in the LB substratum that 2ml contains penbritin (100 μ g/ml), and 37 DEG C, 150rpm, shaking table is cultivated 12～16hr;
2. get 1-1.5ml culture and pour in 1.5ml centrifuge tube, 4 DEG C, the centrifugal 30s of 13000rpm, abandons supernatant, and then of short duration centrifugal, a small amount of centrifuge tube bottom raffinate sucking-off is discarded with rifle head.4 DEG C 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 precoolings, whirlpool concussion, makes bacterial suspension even;
4. in the resuspended liquid of bacterium, add 200 μ l alkaline lysis liquid II, spin upside down centrifuge tube 5 times, make the abundant cracking of thalline, until form bright solution, be placed on ice (<5min);
5. the alkaline lysis liquid III that adds 150 μ l ice precoolings, spins upside down centrifuge tube 8 times, and solution III is uniformly dispersed in the bacterial lysate of thickness, and centrifuge tube is placed in to 1～3min on ice, 4 DEG C, 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 mixes organic phase and water, and the centrifugal 2min of 13000rpm, gets supernatant in new centrifuge tube;
7. (850 μ are precipitation at room temperature nucleic acid l), mixes, and room temperature leaves standstill 2min to use the ethanol of 2～2.5 volumes.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 1ml left and right 70% ethanol in precipitation, put upside down centrifuge tube for several times, washing tube wall and precipitation, need the centrifugal 1min of 13000rpm as precipitation departs from the pipe end.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 centrifuge tube with rifle head);
9. open centrifuge tube, room temperature leaves standstill 3～5min, and ethanol is fully volatilized, until there is no visible liquid in centrifuge tube;
10. add 50 μ l TE damping fluids (containing 20 μ g/ml RNase A) in centrifuge tube, leave standstill 5min, gentle concussion mixes, and is stored in-20 DEG C.
Enzyme described in step D-5 cut qualification and order-checking, its enzyme cut qualification and sequence measurement as follows:
In reaction system, add the recombinant eukaryon expression vector 5 μ l of extraction, 10 times of damping fluid 2 μ l, restriction enzyme SpeI1 μ l, deionized water 13 μ l, 37 DEG C of reaction 2h; After 1% agarose gel electrophoresis separates, identify the object band of pMex, SpeI enzyme can cut out two fragments of about 1.3kb and 5.3kb after cutting, conform to expection, shows in insertion vector that object fragment is correct;
Enzyme in step F-5 cut qualification and order-checking, its enzyme cut qualification and sequence measurement as follows:
In reaction system, add the recombinant eukaryon expression vector 5 μ l of extraction, 10 times of damping fluid 2 μ l, restriction enzyme HindIII1 μ l, deionized water 13 μ l, 37 DEG C of reaction 2h; After 1% agarose gel electrophoresis separates, identify the object band of pGreen Max1, HindIII enzyme can cut out three fragments of about 4.9kb, 2.9kb and 230bp after cutting, conform to expection, shows in insertion vector that object fragment is correct.
What the present invention applied is EF1 α promotor, is the promotor of high expression level albumen in human body, can avoid the detrimentally affect of exogenous promoter to cell.Simultaneously this carrier can utilize green fluorescence luminous come the expression of profiling protein in cell, be convenient to the screening of cell strain.Can provide in the screening of high yield mammalian cell strain the mark of protein expression and screening, for the high efficient expression of albumen provides good carrier, also for the screening of cell strain provides a kind of new method and facility.
Brief description of the drawings
Fig. 1 is the electrophoresis photo of PCR of the present invention;
Wherein: can see that 500bp is to there being an obvious band between 750bp, the 647bp of stripe size and expection is roughly equal, conforms to expection, show Mex sequence PCR success.
Fig. 2 is the electrophoresis picture that the enzyme of pMex plasmid of the present invention is cut qualification;
Wherein: after 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 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, carrier size is 6.6kb, and IRES-EGFP sequence is 1.3kb, conform to expection size.
Fig. 4 is pGreen Max1 plasmid enzyme restriction photo;
Wherein, cutting through HindIII enzyme, can cut out three bands, is respectively the band of 4.9kb, 2.9kb and 230bp, conforms to expection band, shows that IRES-EGFP sequence has correctly been inserted in the middle of pMex carrier.
Fig. 5 the has been transfection cell of albumin A recombinant eukaryon expression vector
Wherein, after transfection, in cell, can express green fluorescent protein, can be for the screening of cell strain.
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 transfection, the expression of albumin A in the supernatant of cell, can be detected.Show vector construction success and can apply.
Embodiment
Tissue-engineering fiber support below in conjunction with accompanying drawing to protein of the present invention and preparation thereof and extracorporeal releasing experiment are implemented to elaborate: following examples are implemented under taking 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: special primer is synthetic by the raw work in Shanghai; Restriction enzyme BamHI, the NotI, pfu enzyme and the equal Fermentas of the T4 ligase enzyme company product (Sangon Biotech (Shanghai) Co., Ltd., 021-37772180) that in vector construction, use; The DNA purification kit using in vector construction is Suo Laibao company (Suo Bao bio tech ltd, Shanghai, Jiang Lu admires in Xuhui District of Shanghai) product, and by specification carries out; Carrier transfection (PEI is sigma company product) and secreting, expressing detection reagent are commercial reagent.The experimental technique of unreceipted actual conditions, conventionally 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 Mex primers, upstream primer adds PmeI site, and primer sequence is as follows:
Mex?F：GATATC
GTTT?AAACGCTCTT?CCTGCATCAC?GGGAGAT
Mex?R：TGAGGCCTGA?GTTCAGACCG?GT
The clone of B, object fragment:
Taking the plasmid with Mex as template, carry out pcr amplification: template 1 μ l, the 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 DEG C of sex change 2 minutes; Then 30 seconds, 72 DEG C of 94 DEG C of sex change 30 seconds, 55-65 DEG C annealing are extended 50 seconds, carry out 30-40 circulation; Last 72 DEG C are extended 7-10 minute;
C, expansion product purification:
PCR product is after 1% agarose electrophoresis separates, and the amplified band of a visible treaty 647bp, cuts object band, carries out purifying with company's sepharose purification kit, and method is:
C-1, from sepharose, PCR product D NA band is cut, proceed in centrifuge tube, add the binding buffer liquid of 2 times of volumes, temperature is bathed and within 7 minutes, is made gel dissolve at 55 DEG C;
C-2, the gelating soln dissolving in centrifuge tube is transferred in the DNA adsorption column that test kit carries, 13000 revs/min centrifugal 2 minutes;
C-3, in DNA adsorption column, add 700 μ l rinsing liquids, 13000 revs/min centrifugal 2 minutes; Discard rinsing liquid, then add 500 μ l rinsing liquids, 13000 revs/min centrifugal 2 minutes; Discard after rinsing liquid, 13000 revs/min centrifugal 2 minutes, room temperature place 5 minutes;
C-4, DNA adsorption column is added in a new centrifuge tube, adds the elution buffer 70 μ l of 65 DEG C of preheatings, 13000 revs/min centrifugal 2 minutes, the solution at the bottom of centrifuge tube is the PCR product D NA fragment of recovery.
As shown in Figure 1, can see that 500bp is to there being an obvious band between 750bp, the 647bp of stripe size and expection is roughly equal, conforms to expection, shows Mex sequence PCR success.
The structure of D, pMex carrier for expression of eukaryon:
D-1, enzyme are cut: carrier is cut with SmaI enzyme, the fragment that enzyme is cut is cut glue recovery after 1% agarose gel electrophoresis separates method with carrier is: in reaction system, add 10 times of damping fluids, 2 μ l, SmaI1 μ l, carrier 5 μ l, deionized water 12 μ l, 37 DEG C of water-baths 4 hours.
D-2, connection: the Mex fragment of recovery and carrier are connected US and Russia with T4 to carry out external ligation method and be: the PCR product D NA fragment 8 μ l and the carrier segments 2 μ l that in reaction system, add recovery, 10 times of damping fluid 2 μ l, T4DNA ligase enzyme 1 μ l, deionized water 7 μ l, 16 DEG C of reaction 10h.
D-3, conversion intestinal bacteria: connection product is transformed in competent escherichia coli cell, on the LB substratum that contains penbritin 100 μ l/ml, cultivates 12-16 hour for 37 DEG C, method is:
A, get and connect product 10 μ L and be added in the competent cell of 50 μ L, shake up gently ice bath 30min.
B, 42 DEG C of heat shock 90s, forward ice bath 2-3min fast to, notes not shake pipe.
C, Xiang Guanzhong add the LB substratum of 500 μ L, 150rmp, 37 DEG C, 45min.
D, bacterium liquid is all coated onto on LB flat board, containing 100ug/ml Amp.
E, flat board is just being put until liquid all absorbs.
F, inversion flat board, cultivate 12-16h. in 37 DEG C
The positive colony of D-4, screening recombinant eukaryon expression vector: by bacterium colony amplification culture, purification plasmid, method is:
A. the single bacterium colony of picking after transforming, is inoculated in the LB substratum that 2ml contains penbritin (100 μ g/ml), and 37 DEG C, 150rpm, shaking table is cultivated 12～16hr.
B. get 1-1.5ml culture and pour in 1.5ml centrifuge tube, 4 DEG C, the centrifugal 30s of 13000rpm, abandons supernatant, and then of short duration centrifugal, a small amount of centrifuge tube bottom raffinate sucking-off is discarded with rifle head.4 DEG C 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 precoolings, whirlpool concussion, makes bacterial suspension even.
D. in the resuspended liquid of bacterium, add 200 μ l alkaline lysis liquid II, spin upside down centrifuge tube 5 times, make the abundant cracking of thalline, until form bright solution, be placed on ice (<5min).
E. the alkaline lysis liquid III that adds 150 μ l ice precoolings, spins upside down centrifuge tube 8 times, and solution III is uniformly dispersed in the bacterial lysate of thickness, and centrifuge tube is placed in to 1～3min on ice, 4 DEG C, 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 mixes organic phase and water, and the centrifugal 2min of 13000rpm, gets supernatant in new centrifuge tube.
G. (850 μ are precipitation at room temperature nucleic acid l), mixes, and room temperature leaves standstill 2min to use the ethanol of 2～2.5 volumes.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 1ml left and right 70% ethanol in precipitation, put upside down centrifuge tube for several times, washing tube wall and precipitation, need the centrifugal 1min of 13000rpm as precipitation departs from the pipe end.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 centrifuge tube with rifle head).
I. open centrifuge tube, room temperature leaves standstill 3～5min, and ethanol is fully volatilized, until there is no visible liquid in centrifuge tube.
J. add 50 μ l TE damping fluids (containing 20 μ g/ml RNase A) in centrifuge tube, leave standstill 5min, gentle concussion mixes.Be stored in-20 DEG C.
D-5, enzyme are cut qualification and order-checking, confirm that the sequence obtaining is eukaryon expression plasmid, and method is:
In reaction system, add the recombinant eukaryon expression vector 5 μ l of extraction, 10 times of damping fluid 2 μ l, restriction enzyme SpeI1 μ l, deionized water 13 μ l, 37 DEG C of reaction 2h;
As shown in Figure 2, after 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 carrier of Mex fragment;
E, obtain IRES-EGFP sequence:
In plasmid, utilize BamHI and NotI double digestion to obtain IRES-EGFP sequence, and after 1% agarose gel electrophoresis separates, the band of a visible treaty 1300bp, cuts object band, carry out purifying with Gel Extraction Kit DNA purification kit
As shown in Figure 3, cut through BamHI and NotI enzyme, carrier size is 6.6kb, and IRES-EGFP sequence is 1.3kb, conforms to expection size.
The structure of F, pGreen Max carrier for expression of eukaryon:
F-1, enzyme are cut: by BamHI and NotI double digestion for carrier, the carrier segments after enzyme is cut is cut glue and reclaimed after 1% agarose gel electrophoresis separates.Method is: in reaction system, add 10 times of damping fluids, 2 μ l, BamHI1 μ l, NotI μ l, pMex carrier 5 μ l, deionized water 12 μ l, 37 DEG C of water-baths 4 hours.
F-2, connection: the IRES-EGFP fragment of recovery and carrier carry out external ligation with T4 ligase enzyme, method is: in reaction system, add DNA fragmentation 8 μ l and the carrier segments 2 μ l of recovery, 10 times of damping fluid 2 μ l, T4DNA ligase enzyme 1 μ l, deionized water 7 μ l, 16 DEG C of reaction 10h.
F-3, conversion intestinal bacteria: the product of connection is proceeded in competent escherichia coli cell, containing 37 DEG C of cultivation 12-16 hour on the LB substratum of ammonia benzyl mycin 100 μ l/ml;
The positive colony of F-4, screening recombinant eukaryon expression vector: by bacterium colony amplification culture, purification plasmid;
F-5, utilize HindIII to carry out enzyme to cut qualification and order-checking, confirm to obtain the pMex eukaryon expression plasmid of sequence 1.Method is: in reaction system, add the recombinant eukaryon expression vector 5 μ l of extraction, 10 times of damping fluid 2 μ l, restriction enzyme HindIII1 μ l, deionized water 13 μ l, 37 DEG C of reaction 2h;
As shown in Figure 4, the object band of qualification pGreen Max1 after separating with 1% agarose gel electrophoresis, cut through HindIII enzyme, can cut out three bands, it is respectively the band of 4.9kb, 2.9kb and 230bp, conform to expection band, show that IRES-EGFP sequence has correctly been inserted in the middle of 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 and has successfully built called after pGreen Max1 carrier for expression of eukaryon, as shown in sequence 1.
Embodiment 2
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 the recombinant vectors that contains 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 the recombinant vectors that contains albumin A and antibody B.
The extraction and purification of A-2, recombinant expression vector
Carry out extraction and purification for the pGreen Max1 recombinant protein A of transfection and the plasmid of pGreen Max1 recombinant antibodies B by the method for plasmid a small amount of extracting, concrete operation step is as follows:
A. the single bacterium colony of picking after transforming, is inoculated in the LB substratum that 2ml contains penbritin (100 μ g/ml), and 37 DEG C, 150rpm, shaking table is cultivated 12～16hr.
B. get 1-1.5ml culture and pour in 1.5ml centrifuge tube, 4 DEG C, the centrifugal 30s of 13000rpm, abandons supernatant, and then of short duration centrifugal, a small amount of centrifuge tube bottom raffinate sucking-off is discarded with rifle head.4 DEG C 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 precoolings, whirlpool concussion, makes bacterial suspension even.
D. in the resuspended liquid of bacterium, add 200 μ l alkaline lysis liquid II, spin upside down centrifuge tube 5 times, make the abundant cracking of thalline, until form bright solution, be placed on ice (<5min).
E. the alkaline lysis liquid III that adds 150 μ l ice precoolings, spins upside down centrifuge tube 8 times, and solution III is uniformly dispersed in the bacterial lysate of thickness, and centrifuge tube is placed in to 1～3min on ice, 4 DEG C, 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 mixes organic phase and water, and the centrifugal 2min of 13000rpm, gets supernatant in new centrifuge tube.
G. (850 μ are precipitation at room temperature nucleic acid l), mixes, and room temperature leaves standstill 2min to use the ethanol of 2～2.5 volumes.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 1ml left and right 70% ethanol in precipitation, put upside down centrifuge tube for several times, washing tube wall and precipitation, need the centrifugal 1min of 13000rpm as precipitation departs from the pipe end.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 centrifuge tube with rifle head).
I. open centrifuge tube, room temperature leaves standstill 3～5min, and ethanol is fully volatilized, until there is no visible liquid in centrifuge tube.
J. add 50 μ l TE damping fluids (containing 20 μ g/ml RNase A) in centrifuge tube, leave standstill 5min, gentle concussion mixes.Be stored in-20 DEG C.
B, albumin A recombinant eukaryon expression vector and the secreting, expressing of antibody B recombinant eukaryon expression vector in 293T cell
People's renal epithelial cell of transfection adenovirus e1a gene be 293T cell cultures with the DMEM complete culture solution that contains 10%FCS (foetal calf serum) in, before transfection, cell is layered in 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 in the DMEM nutrient solution 1ml of antibiotic-free, serum-free and mix, then two liquid are mixed into transfection liquid, room temperature is placed 10-20 minute;
2, inhale the nutrient solution of abandoning in 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 culture dish, be placed in 5% carbonic acid gas, 37 DEG C of incubators were cultivated after 4-6 hour, displacement serum free medium continued to cultivate after 24-48 hour, mobile phone cells and supernatant;
4, get transfection supernatant, the polyacrylamide gel that is 10% by concentration carries out electrophoresis, detects the secreting, expressing situation of albumin A and antibody B by the immune marking (Western Blot).
As shown in Figure 5, after transfection, in cell, can express green fluorescent protein, can be for the screening of cell strain.
As shown in Figure 6, after transfection, the expression of albumin A in the supernatant of cell, can be detected.Show vector construction success and can apply.
As shown in Figure 7, albumen is the albumen of antibody B.Albumen size is 19kD left and right.Can well find out and in cell conditioned medium, contain this antibody and can well detect this albumen by picture, show that this carrier can apply.
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 for the screening of cell strain provides a kind of new method and facility.
Claims (4)
1. a pGreen Max1 carrier for expression of eukaryon, is characterized in that, the nucleotide sequence of described carrier, as shown in SEQ ID NO:1, obtains sequence 1 by Mex, IRES-EGFP fragment and carrier for expression of eukaryon restructuring, and sequence 1 amounts to 8003bp.
2. the preparation method of a pGreen Max1 carrier for expression of eukaryon according to claim 1, it is characterized in that, by design primer, the method clone by PCR from plasmid obtains Mex, and by the site-directed expression vector that is inserted into of PCR product process SmaI, and through BamHI and NotI double digestion, IRES-EGFP is inserted in carrier, build the pGreen Max1 carrier for expression of eukaryon of restructuring.
3. 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 provides the application of the mark of protein expression and screening for the screening of high yield mammalian cell strain in eukaryotic cell.
4. application according to claim 3, is characterized in that, described target protein is antibody.
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