CN101503699B - Cloning vector pGreen-S based on enhanced green fluorescent protein gene deletion as screen marker and construction method thereof - Google Patents
Cloning vector pGreen-S based on enhanced green fluorescent protein gene deletion as screen marker and construction method thereof Download PDFInfo
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Abstract
The invention discloses a cloning vector pGreen-S based on enhanced green fluorescent protein (EGFP) gene deletion as a selection marker, and a construction method thereof. The pGreen-S vector inserts an EGFP gene into an Xba I restriction enzyme site of a pUC18 vector, and sequentially comprises main components as follows: a replication origin sequence, an EGFP gene upstream polyclone site, an EGFP gene sequence, an EGFP gene downstream polyclone site, a lacZ gene sequence and a resistance selection gene sequence. The pGreen-S vector can be used for screening gene recombinants, is more reliable in screening results compared with a lac blue/white spot screening method, and has the characteristics of convenience, economic property, time conservation and high efficiency.
Description
Technical field
The invention belongs to gene engineering technology field, relating to a kind of specifically is the cloning vector pGreen-S and the construction process thereof of selection markers based on enhanced green fluorescent protein gene deletion.
Background technology
Beta-galactosidase gene (lacZ) is a reporter gene the most commonly used in the molecular biology, promptly is that lacZ inserts inactivation type carrier as pUC series.Its screening principle is: DNA section and coding beta-galactosidase N that carrier contains the lacZ operon hold 146 amino acid whose gene fragments (α peptide section), form multiple clone site in its coding region simultaneously, and do not influence reading frame and gene function.Chemical substance isopropyl-(IPTG) can induce this segmental synthetic.Select α peptide section defective type host bacterium (all the other the peptide sections that self can synthesize the C end), when IPTG exists, the beta-galactosidase gene of carrier and host bacterium is realized complementary (α complementation), generation has active beta-galactosidase enzymes, make substrate 5-bromo-4-chloro-3-indoles-β-D-thiogalactoside (X-gal) be decomposed into blue compound, show as and occur blue colonies on the substratum.When exogenous dna fragment is inserted into the multiple clone site of plasmid, make the encoding gene inactivation of beta-galactosidase enzymes N end fragment, can not form the α complementation, the recipient bacterium that then has recombinant plasmid presents white colony.People can screen recombinant chou from the colour-change (indigo plant/hickie colour developing) of bacterium colony.
But in the practical application, the problem that this method exists is, when the dna fragmentation (less) that inserts can not destroy the N end fragment reading frame of beta-galactosidase enzymes, the recombinant chou bacterium colony can show light blue even blue, the false negative clone occurs, and it falsely drops rate about 30%.Simultaneously, indigo plant/hickie screening mode depends on inductor IPTG and chromogenic substrate X-gal, so that make the dull and stereotyped expense (adding 0.8mg IPTG and 0.8mg X-gal in every flat board) that increases about 1.5 yuan of each screening.In addition, the dull and stereotyped termination of screening needs 4 ℃ of standing over night that color is fully manifested after the cultivation, thereby prolonged experimental period, reduced efficient.
(green fluorescent protein GFP) is natural fluoresence protein from the isolating a kind of function uniqueness of luminescent jellyfish (AequoreaVictoria) to green fluorescent protein.Behind GFP in 1994 clone, GFP and mutant thereof are marked at widespread use in the research of animal, plant, microorganism as genetic expression marker or fusion rotein.GFP fluorescence form have sensitivity, directly perceived, instant characteristics, and the generation of fluorescence do not need to add any external source substrate, enzyme or other cofactors, so GFP is well suited for the selection markers thing as genetic recombinants.
[Li Shoudong, Qi Yipeng, Hu Jianhong, Lin Hong in the reported research.The biotechnology journal, 1997,13 (3): 323-325; The king advances, Li Fuguang, Li Qianru, Elaeocarpus decipiens.Zhengzhou University's journal medicine, 2002,37 (5): 621-624; Dong Yuemei, Li Jiudi, Zhu Zhiqing.Botany Gazette, 1999,41 (5): 487-489], the carrier that forms based on the GFP expression strategy exists obviously not enough: first, foreign gene inserts the site and all is chosen in the GFP upstream region of gene, its screening strategy is to screen recombinant chou to insert the expression whether gene cause the GFP gene to be read frameing shift sign indicating number and then influence GFP, strict to inserting gene order, in case can not meet the demands then can't screen recombinant chou second, contain after the transformation in the carrier multiple clone site zone of GFP selection markers, restriction endonuclease sites is less, has reduced the handiness of gene recombination; Therefore, its practicality is subjected to significant limitation.
Summary of the invention
It is a kind of based on enhanced green fluorescence protein (enhanced greenfluorescent protein that the object of the invention is to provide, EGFP) genetically deficient is the cloning vector pGreen-S of selection markers, make the The selection result of genetic recombinants more easy to be reliable, screening process is more easy, economical, save time, efficiently.
For achieving the above object, the present invention adopts following technical scheme:
The primary member of described carrier is that replication initiation sequence-EGFP upstream region of gene multiple clone site-EGFP gene order-EGFP gene downstream multiple clone site-lacZ gene order-resistance is selected gene order.
Preferred scheme is: described EGFP upstream region of gene multiple clone site is EcoR I, Sac I, Kpn I, Sma I, Xma I, BamH I and Xba I restriction enzyme site.
More preferred scheme is: described EGFP gene downstream multiple clone site is Xba I, Sal I, PstI, Spn I and Hind III restriction enzyme site.
More preferred scheme is: it is Amp that described resistance is selected gene order
rGene order.
More preferred scheme is: a kind of host cell, contain the described cloning vector of above-mentioned arbitrary scheme.
Another object of the present invention is to provide a kind of is the construction process of selection markers cloning vector pGreen-S based on enhanced green fluorescent protein gene deletion.
For achieving the above object, the present invention adopts following technical scheme:
A kind of is the construction process of the cloning vector pGreen-S of selection markers based on enhanced green fluorescent protein gene deletion, comprises the steps:
A) design of amplification primers is that template is carried out pcr amplification EGFP gene with plasmid pEGFP-N1;
B) with the pUC18 plasmid behind Xba I linearization for enzyme restriction, be connected and transformed competence colibacillus E.coli DH5 α according to molecular biology method with the EGFP gene fragment of the pcr amplification of cutting through Xba I enzyme, coat the LB flat board of X-gal, IPTG, ammonia benzyl concentration 70 μ gmL
-1, cultivate 12h for 37 ℃;
C) picking white and green bacterium colony are transferred and are shaken 37 ℃ of incubated overnight of bottle, penbritin concentration 70 μ gmL in the LB liquid nutrient medium
-1, extract in a small amount plasmid and make Xba I enzyme and cut checking, gene sequencing.
Preferred scheme is: described amplimer is:
Upstream primer: 5 '-GCAC
TCTAGATATGGTGAGCAAGGGCG-3 ';
Downstream primer: 5 '-GCTA
TCTAGATTACTTGTACAGCTCGTCCA-3 ';
Contain Xba I restriction enzyme site in upstream primer and the downstream primer respectively.
The principal character of pGreen-S carrier of the present invention is:
The first, the EGFP gene of pGreen-S is at P
LacOverexpression under the regulation and control of promotor contains the intestinal bacteria bacterium colony of pGreen-S, can be revealed as greenly clearly under natural light, manifests more bright green under the long-wave ultra violet lamp, need not process color;
The second, pGreen-S screens recombinant chou in the green fluorescence disappearance mode of EGFP, and the disappearance that its green fluorescence disappearance is the EGFP gene causes, rather than the EGFP gene is not expressed; When genetic manipulation, with corresponding restriction endonuclease excision EGFP gene, recombinant chou makes the bacterium colony that contains recombinant plasmid manifest white because of the disappearance of EGFP gene, the visualization of color contrast obviously, thereby make the judgement of experimental result more directly perceived, if by the observation of the standing UV-light in laboratory and imaging device would observe with image data clear more convenient;
The 3rd, pGreen-S contains two groups of multiple clone site, i.e. the multiple clone site 1 (MCS1) of EGFP upstream region of gene: EcoR I, Sac I, Kpn I, Sma I, Xma I, BamH I and Xba I site; Kept the multiple clone site 2 (MCS2) that is positioned at EGFP gene downstream again: Xba I, Sal I, Pst I, Spn I and Hind III restriction enzyme site, in the actually operating, the experimenter can select a restriction enzyme site to make up respectively in above-mentioned two groups of MCS, has therefore increased the handiness of foreign gene directed cloning strategy greatly;
The 4th, pGreen-S is to be the label screening recombinant chou with the green fluorescence of EGFP disappearance, this screening method has been abandoned in the lacZ strategy dependence to X-gal, IPTG, and the instant colour developing usefulness of EGFP has also shortened experimental period greatly simultaneously, makes the screening operation economical and efficient more of recombinant chou;
The 5th, pGreen-S has kept the lacZ gene, in green/hickie screening recombinant chou, still can use indigo plant/hickie to screen as alternative approach, and the selection scheme of laboratory facilities and strategy is increased, and can bring the facility of selection to the experimenter.
Below in conjunction with the drawings and specific embodiments the present invention is described in further details:
Description of drawings
Fig. 1 is a pGreen-S carrier structure synoptic diagram of the present invention
Fig. 2 utilizes pGreen-S vector selection recombinant chou figure in the embodiment of the invention
Fig. 3 utilizes pGreen-S carrier of the present invention to carry out the screening of recombinant synoptic diagram in the embodiment of the invention.
Embodiment
Embodiment: a kind of cloning vector pGreen-S that utilizes the green fluorescence disappearance of enhanced green fluorescence protein for the label screening recombinant chou, be that (enhanced green fluorescentprotein, EGFP) forward inserts the Xba I restriction enzyme site gained of pUC18 carrier with the enhanced green fluorescence protein gene.PGreen-S vector construction process is as follows:
1, the structure of pGreen-S carrier
According to the EGFP gene order, design a pair of upstream and downstream primer, be template pcr amplification EGFP gene with plasmid pEGFP-N1 (Clontech).Primer is as follows:
Upstream primer: 5 '-GCAC
TCTAGATATGGTGAGCAAGGGCG-3 '
Downstream primer: 5 '-GCTA
TCTAGATTACTTGTACAGCTCGTCCA-3 '
All contain Xba I restriction endonuclease sites in upstream primer and the downstream primer.
Amplification obtains the 0.73kb gene fragment, after Xba I enzyme is cut, is connected under the effect of T4DNA ligase enzyme with the pUC18 plasmid that same enzyme is cut, and makes up the pGreen-S carrier.
The evaluation of 2:pGreen-S carrier
Gene connects product transformed competence colibacillus E.coli DH5 α, coats LB (the penbritin concentration 70 μ gmL of X-gal, IPTG
-1) flat board, cultivate 12～17h for 37 ℃, picking white and green bacterium colony are transferred in LB (penbritin concentration 70 μ gmL
-1) 37 ℃ of concussions of liquid nutrient medium overnight incubation, extracting plasmid carries out Xba I Restriction Enzyme spectrum analysis in a small amount.The result shows that these two kinds of bacterium colonies all contain the insertion fragment, but inserts the direction difference of gene.Dna sequence analysis shows that white colony is the bacterium colony that the EGFP gene oppositely inserts, and green bacterium colony is that EGFP gene forward inserts bacterium colony, and with the plasmid called after pGreen-S carrier that EGFP gene forward inserts, its plasmid map and multiple clone site are as shown in Figure 1.
The checking of 3:pGreen-S vector selection function
Be explanation pGreen-S vector selection recombinant chou function of the present invention, the ZZ peptide gene (0.35kb) that we choose SP inserts at the EcoR I restriction enzyme site of pGreen-S carrier MCS1 and the Pst I restriction enzyme site double digestion of MCS2.
One, ZZ peptide gene primer and amplification:
Upstream primer: 5 '-AAA
GAATTCATGGTAGACAACAAATTCAAC-3 ' contains the EcoRI restriction enzyme site; Downstream primer: 5 '-AAA
CTGCAGTTCGGCGCCTGAGCATC-3 ' contains the PstI restriction enzyme site.
With plasmid pEZZ 18 (Amersham) is template, the PCR mode ZZ peptide gene that increases.
Two, the structure of ZZ peptide gene recombinant chou:
0.35kb the goal gene amplified fragments behind EcoR I/Pst I double digestion, be cloned into the EcoR I/Pst I restriction enzyme site of pGreen-S plasmid, connect product transformed competence colibacillus E.coli DH5 α, coating LB (penbritin concentration 70 μ gmL
-1) flat board, cultivate 12～17h for 37 ℃.
Three, the screening of genetic recombinants:
Two kinds of color bacterium colonies appear on the LB flat board: white colony and green bacterium colony.Random choose white colony and green bacterium colony are transferred in 37 ℃ of concussions of LB liquid nutrient medium overnight incubation.Extract plasmid in a small amount and carry out EcoR I and the checking of Pst I double digestion, white colony is a recon, and green bacterium colony is nonrecombinant (not inserting the pGreen-S of goal gene).As shown in Figure 2, left side figure observes down for the bacterium colony natural light, and white colony is the E.coli DH5 α bacterium colony that contains recombinant chou; Green bacterium colony is for containing the E.coli DH5 α bacterium colony of pGreen-S (non-recombinant chou); Right figure observes under the bacterium colony 365nm UV-light, and green bacterium colony is for containing the E.coli DH5 α bacterium colony of pGreen-S (non-recombinant chou);
Can illustrate by this experiment, the pGreen-S carrier can utilize the fluorescent characteristic of EGFP to filter out the recombinant chou that inserts foreign gene, only need the restriction enzyme site that flexible combination can be excised the EGFP gene between MSC1, MCS2, all can utilize pGreen-S carrier of the present invention to carry out screening of recombinant.Its screening strategy as shown in Figure 3, wherein: restriction endonuclease a is arbitrary restriction endonuclease among the MCS1; Restriction endonuclease b is arbitrary restriction endonuclease among the MCS2.
In the foregoing description, the EGFP gene can also insert at other restriction enzyme site of pUC18 plasmid, reaches the purpose that embodiment makes up screening vector.
In the foregoing description, the EGFP gene can also insert the multiple clone site of pUC19 plasmid, reaches the purpose that embodiment makes up screening vector.
Above content be in conjunction with concrete preferred implementation to further describing that the present invention did, can not assert that concrete enforcement of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (3)
1. one kind is the cloning vector pGreen-S of selection markers based on enhanced green fluorescent protein gene deletion, it is characterized in that: pGreen-S carrier primary member is that replication initiation sequence-EGFP upstream region of gene multiple clone site-EGFP gene order-EGFP gene downstream multiple clone site-lacZ gene order-resistance is selected gene order;
Described EGFP upstream region of gene multiple clone site is EcoR I, Sac I, Kpn I, Sma I, Xma I, BamHI and Xba I restriction enzyme site;
Described EGFP gene downstream multiple clone site is Xba I, Sal I, Pst I, SpnI and Hind III restriction enzyme site;
The EGFP activity expression of carrier pGreen-S need not inducing of IPTG;
Respectively select a suitable restriction enzyme site in EGFP upstream region of gene multiple clone site, downstream multiple clone site, realize the disappearance of EGFP gene and the screening-gene recon.
2. as claimed in claim 1 is the cloning vector pGreen-S of selection markers based on enhanced green fluorescent protein gene deletion, it is characterized in that: it is Amp that described resistance is selected gene order
rGene order.
3. a host cell is characterized in that: contain claim 1 or 2 described cloning vectors.
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| CN102409061B (en) * | 2011-03-23 | 2014-05-28 | 江苏大学 | Preparation method of T vector based on EGFP (enhanced green fluorescent protein) |
| CN107941887B (en) * | 2017-12-29 | 2020-07-10 | 武汉艾德士生物科技有限公司 | Method for simply and rapidly evaluating efficiency of molecular cloning system |
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