CN102181465A - Fluorescent clone screening vector and preparation and application thereof - Google Patents
Fluorescent clone screening vector and preparation and application thereof Download PDFInfo
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Abstract
The invention relates to the field of molecular biology and microbiology, and discloses a fluorescent screening vector, the construction and application thereof. Polynucleotide and/or a complementary strand of the polynucleotide which encodes an enhanced green fluorescent protein are taken as a positive clone selection marker of the fluorescent screening vector; and an amino acid sequence of the enhanced green fluorescent protein is SEQ ID NO.5. The vector can be widely applied to the screening of positive clone in gene clone.
Description
Technical field
The present invention relates to molecular biology and microbiology field.Concretely, the present invention relates to a kind of fluorescent screening carrier, relate to this construction of carrier simultaneously, and this carrier is in the application of accurately screening in the positive for bacteria bacterium colony.
Background technology
After the seventies, as new milestone, indicate the human new period of deeply being familiar with life quintessence and can transforming life with the appearance of genetic engineering technique, significant achievement therebetween comprises the foundation and the development of recombinant DNA technology.Recombinant DNA (Recombinant DNA) technology is the core technology of genetic engineering, also is human technology of operating in gene and dna molecular level.It comprises following step: the 1) structure of recombinant DNA molecules; 2) with the recombinant DNA molecules transformed host cell; 3) recombinant screen and propagation; 4) culturing cell after the results amplification extracts recombinant DNA molecules (plasmid), and purifying, obtains a large amount of copies of a certain gene or dna fragmentation.
Blue hickie screening is a kind of method of recombinant screen, is the hereditary feature screening recon according to carrier, as α-Hu Bu, antibiotic resistance gene etc.The many carriers that use all have the short section of a colibacillary DNA now, and regulating and controlling sequence and preceding 146 amino acid whose coded messages of beta-galactosidase gene (lacZ) are wherein arranged.In this coding region, inserted a multiple clone site (MCS), it does not destroy frame, but can make a few aminoacid insertion to the aminoterminal of beta-galactosidase enzymes and do not influence function, this carrier is applicable to the host cell of codified beta-galactosidase enzymes C end parts sequence.Therefore, though host and plasmid-encoded fragment all do not have enzymic activity, when they exist simultaneously, can form protein with the enzyme activity.Like this, the lacZ gene is at the host cell that lacks nearly operator gene section and have between the plasmid of complete nearly operator gene section and realized complementation, is called α-Hu Bu.The LacZ+ bacterium that is produced by α-Hu Bu produces blue colonies when chromogenic substrate X-Gal exists under the effect of inductor IPTG, thereby be easy to identification.Yet, be inserted into the multiple clone site of plasmid when foreign DNA after, almost invariably cause not having the n terminal fragment of α-Hu Bu ability, make the bacterium that has recombinant plasmid form white colony.The screening of this recon is called blue hickie screening again.After then 12-16hr is cultivated in 37 ℃ of incubators inversions of the calcification bacterium flat board through connecting the product conversion with blue hickie screening, there is the bacterium of recombinant plasmid to form white colony.
The method of blue hickie screening recon is used till today always, can satisfy the experiment needs substantially.But we sum up in long-term a large amount of experiments and find that dull and stereotyped sometimes growth still can't be told blue hickie more than 15 hours, perhaps all was locus coeruleus; Be summarized as follows: 1) some special genes can influence bacterium and normally grows up, and causes bacterial growth slow, and bacterium colony can't grow into a certain size in the conventional time, to such an extent as to naked eyes can't be differentiated its color; 2) insert that gene fragment is too little does not destroy encoder block, to such an extent as to expressed proteins still can produce the α-Hu Bu effect; 3) the not corresponding lacZ genetic flaw of engineering strain.
Green fluorescent protein (Green Fluorescent Portein, GFP) be in a kind of medusa that lives in cold waters, North Pacific, to find, the monomer of its 27k Da is made of 238 amino acid, it itself is exactly a bio-luminescence system, subsidiary have can emitting biological fluorescence after exciting luminous color base, its luminescence process is different from other noclilucence tissue, does not need luciferase to participate in, also without any need for cofactor, substrate etc.When expressing and be subjected to blue light or ultraviolet light irradiation, GFP just can send bright green fluorescence in protokaryon or eukaryotic cell.
GFP fluorescence is extremely stable, and under excitation light irradiation, the anti-photobleaching of GFP (Photobleaching) energy force rate fluorescein (fluorescein) is strong, and is more stable under 450～490nm blue light wavelength especially.GFP need produce fluorescence under the state of oxidation, strong reductant can make GFP change non-fluorescence form into, but in case be exposed in the air or oxygen again, GFP fluorescence just is restored immediately. and some weak reductants do not influence GFP fluorescence. and the moderate oxidation agent is also little to the influence of GFP fluorescence, fixing as biomaterial, dewatering agent pentanedioic acid or formaldehyde etc.The fluorescence sensitivity of GFP fusion rotein is more than fluorescein-labeled fluorescence antibody height, and anti-photobleaching ability is strong, therefore more is applicable to quantitative assay and analysis.
The columnar structure that the crystalline structure of GFP is made up of 11 beta sheets, the about 3nm of diameter is about 4nm.There is an alpha-helix (Fig. 1) in post central authorities, chromophoric group on alpha-helix, very near the center of post (Fig. 1, Fig. 2).Proteic secondary structure major part is alpha-helix and beta sheet.
The GFP color base is made of the cyclisation tripeptides that Serine tyrosine glycine (SerTyrGly) forms, and have only color base to be coated in the complete GFP albumen and just can send fluorescence, cut GFP (even a few amino acid of C-terminal) also can cause GFP to lose luminous power.The crystalline structure of GFP and GFP S65T studies show that color base closely is coated in the β box that is surrounded by βZhe Die.This structure provides the microenvironment of color base emitting fluorescence, and this environment has been got rid of matrix and the oxygen influence luminous to color base.This protection is essential for emitting fluorescence.Once chromophoric group absorbs a photon, the activated water molecules will be captured its energy usually.Release energy but change the low slightly photon of emitted energy into, make it obtain protection at protein interior.Chromophoric group is by three amino acid on the protein chain: glycine, the spontaneous formation of tyrosine and Threonine (or Serine).Only possessing primary structure GFP can not be luminous, and the formation of functional color base is behind transcription and translation, and experiences the oxidation step of a cyclization and a part oxygen.
RedShifted (red skew) GFP mutantion line (EGFP ﹠amp; GFP S65T) aminoacid replacement has taken place in color base, and λ max (Excitation) is offset near the 490nm.The maximum excitation peak of red skew mutantion line all drops in the wavelength region of colour filter commonly used, so the fluorescent signal that obtains is much brighter than wtGFP.Equally, the emission wavelength of the argon ion light source of FACS and confocal microscope is 488nm, to the launching efficiency of RedShifted mutantion line also apparently higher than wtGFP.The bis-amino acid replacement has taken place in EGFP, and Leu (leucine) replaces Phe64 (phenylalanine), and Thr (Threonine) replaces Ser65 (Serine).Based on the proteolytic spectroscopic analysis of equivalent, because the increase of Em (optical extinction coefficient) and the high-level efficiency of color base configuration, EGFP excites the back light fluorescence intensity at the 488nm place be 35 times of wtGFP.(489nm) records EGFP to get Em is 53 under the same conditions, 000cm-1M-1, and wtGFP is 9,500CM-1M-1, GFPS65T are 55,000cm-1M-1.The color base configuration of EGFP is at 37 ℃ higher than wtGFP or GFPS65T luminous efficiency, and the EGFP soluble proteins 95% of expressing under this temperature is effective color base.GFPS65T is the mutant that Thr replaces Ser65, and under the similarity condition, its fluorescence intensity is better than wtGFP4～6 times, and its unique Redshifted excitation peak is positioned at 490nm, but the efficient that color base forms 37 ℃ the time is not as EGFP.
Summary of the invention
The objective of the invention is to be to overcome defective of the prior art, a kind of fluorescent screening cloning vector and preparation thereof and application are provided.
Carrier of the present invention has been replaced the α-Hu Bu screening of the lacZ of original carrier, and adopts green fluorescent protein as screening criteria.Green fluorescence protein gene is finished expression under the Lac promotor starts, as long as the generation of this functional protein is arranged, promptly can detect the existence of positive colony quickly and accurately.
One aspect of the present invention provides a kind of fluorescent screening cloning vector, with the coding enhanced green fluorescence protein polynucleotide and/or its complementary strand be its positive colony selection markers, the aminoacid sequence of described enhanced green fluorescence protein is SEQ ID NO.5.
Preferable, the polynucleotide sequence of described coding enhanced green fluorescence protein is SEQ ID NO.4.
Preferable, the polynucleotide sequence of described fluorescent screening cloning vector is SEQ ID NO.1.Its structure iron such as Figure 11.
Second aspect present invention provides the preparation method of described fluorescent screening cloning vector, and for polynucleotide and/or its complementary strand of the enhanced green fluorescence protein of will encoding is cloned into carrier, the aminoacid sequence of described green fluorescent protein is SEQ ID NO.5.
Further, the nucleotides sequence of the polynucleotide of described encoding green fluorescent protein is classified SEQ ID NO.4 as.
The polynucleotide of described encoding green fluorescent protein can adopt the mode of chemosynthesis to obtain, and obtain as the design primer and with the splicing of overlap PCR method.
Enumerate as embodiment, the carrier that is cloned into can be pUC18.Described fluorescent screening cloning vector can be and will obtain between the polynucleotide of coding enhanced green fluorescence protein and EcoRI that its complementary strand is cloned into pUC18 and the NdeI site.
Enumerate as embodiment, the preparation of fluorescent screening cloning vector can may further comprise the steps:
A. obtain the total length muEGFP gene that two ends have EcoRI and NdeI restriction enzyme site by synthetic primer and overlap PCR method, the encoding sequence of described total length muEGFP gene is shown in SEQ ID NO.4;
B. cut total length muEGFP gene and the carrier pUC18 that digestion step A obtains with EcoRI and NdeI enzyme, obtain the fluorescent screening cloning vector after digestion fragment is connected.
Because positive-selecting mark provided by the invention contains multiple clone site, in case foreign gene is inserted into multiple clone site, then can have influence on the expression of green fluorescent protein, thereby make the positive colony that is cloned into foreign gene under the 488nm rayed, not send green fluorescence, reach the screening purpose to distinguish mutually with the clone who does not insert foreign gene.
Fluorescent screening cloning vector of the present invention can be used for bacterium colony screening, the particularly application on the automatization bacterium colony screening instrument.
Living body fluorescent imaging system (in vivo bioluminescence imaging) is a molecule getting up of development in recent years, the analyzing and testing system of genetic expression.It is made up of the CCD of sensitivity and the luciferase (luciferase) and the fluorescein (luciferin) of analysis software and conduct report thereof.It has non-invasive, can repeatedly repeat in plurality of advantages such as different time points detection, fast scan imagings.And the fluorescence sensitivity of GFP fusion rotein is more than fluorescein-labeled fluorescence antibody height, and anti-photobleaching ability is strong, therefore more is applicable to quantitative assay and analysis.Present all automatic bacterium colony picking instrument, as the automatic bacterium colony picking of Microtec PM-2S type instrument, the automatic bacterium colony of CP7200 CP0600 select instrument etc. all with the colored CCD photographic means as the pattern recognition mode, be applicable to blue hickie screening class cloning vector, as what talk about previously, blue hickie screening has problems to affect the colour developing result, cause this kind mode can't make screening accurately, and the pUC-muEGFP carrier that makes up among employing the present invention is finished the clone, bacterium colony can show beautiful blue-greenish colour on the blue-greenish colour led light source, the clone result can access perfect embodiment.Be that the empty plasmid clone sends bright green light, and positive colony is not luminous.
Description of drawings
The fluorescence chromophoric group that the 65th～67 amino acids residue (Ser-Tyr-Gly) forms in Fig. 1 GFP primary structure (in the square frame).
The quaternary structure of Fig. 2 GFP.Columnar structure central authorities are for having the alpha-helix of chromophoric group.
The quaternary structure of Fig. 3 GFP.Columnar structure central authorities are for having the alpha-helix of chromophoric group.
Fig. 4 GFP aminoacid sequence is adjusted comparison result.The second behavior original series, the third line is for revising the back sequence.
Fig. 5 PCR obtains two gene fragments of muEGFP, and the first swimming lane muEGFP-1 is the chemosynthesis fragment, and the second swimming lane muEGFP-2 is for going up the fragment that P gets from pEGFP.The 3rd swimming lane is DNA marker
Fig. 6 PCR obtains the full-length gene of muEGFP.The first swimming lane muEGFP, second swimming lane are DNA marker
After Fig. 7 muEGFP was cloned into carrier pUC18, dull and stereotyped growth ultraviolet was taken pictures, the positive clone of light tone (shown in the circle).
Fig. 8 pUC-muEGFP transforms, incubated overnight, and all bacterium colonies all fluoresce on the flat board.
After inserting the purpose fragment among Fig. 9 pUC-muEGFP, dull and stereotyped top bacterium colony luminous (being positioned at the frame of top); Part bacterium colony not luminous (being positioned at the square frame of below).
Figure 10 picking positive colony detects, and proves conclusively 8 bacterium samples and is positive findings.
Figure 11 carrier pUC-muEGFP structure iron.
Embodiment
Below enumerate specific embodiment with further elaboration the present invention, should be understood that example is not to be used to limit protection scope of the present invention.
Among the embodiment, conversion is a calcium chloride transformation with escherichia coli DH5a (being not limited to lacZ gene defection type intestinal bacteria after the popularization) employing; Used restriction enzyme, ligase enzyme etc. are all available from Fermentas, and carrier pUC57 is the Fermentas product, and pEGFP-N1 is a BD Biosciences Clonetech product.
Adopt following design to prepare the fluorescent screening cloning vector among the embodiment:
A. design the sequences Design of its encoding sequence green fluorescence protein gene according to aminoacid sequence shown in the SEQ ID NO.5: with the EGFP among the pEGFP-N1 is female parent, revise the part base under the prerequisite that does not change the albumen higher structure, its sequence is an aminoacid sequence shown in nucleotide sequence shown in the SEQ ID NO.4 and the SEQ ID NO.5;
B. the foundation of implementation sequence, synthetic: use chemical synthesis process to finish the synthetic of implementation sequence, according to the particular case of gene DNA works software design primer, and chemosynthesis, synthetic good primer is water-soluble to reach suitable concentration, when first round PCR, get each 1ul of primer and mix, add that 48 degrees centigrade of annealing of downstream primer obtain the total length template as template; Getting first round PCR product 1ul is template, adds that downstream primer 55 degree annealing obtain the capacity full-length gene order.Use the overlapPCR method that the EGFP gene fragment splicing that obtains is obtained full length sequence muEGFP.
C. the structure of fluorescent screening cloning vector: cut sequence muEGFP and the carrier pUC18 that designs and synthesizes in the digestion claim 2 with the EcoRI/NdeI enzyme, connect the clone, obtain pUC-muEGFP
The structure and the checking of embodiment 1 fluorescent screening cloning vector
The mentality of designing and the technological line that make up the fluorescent screening cloning vector are as follows:
1, the sequences Design of enhanced green fluorescence protein gene.Green fluorescence protein gene among the carrier pEGFP-N1 is EGFP, and its detailed sequence is SEQ ID NO.2, and corresponding amino acid sequence is SEQ ID NO.3, and structure iron is seen Fig. 3.The columnar structure that the crystalline structure of GFP is made up of 11 beta sheets, GFP color base tyg closely are coated in the β box that is surrounded by βZhe Die, and it is protected to avoid the influence of surrounding environment.The present invention is under the situation that does not change its protein structure, nucleotide sequence has been carried out suitable modification, in this modification process, introduced restriction enzyme site commonly used, having revised 4 amino acid simultaneously, is respectively G20V, V29G, A37L, V55G (Fig. 4), revising the back nucleotide sequence is SEQ IDNO.4, corresponding amino acid sequence is SEQ ID NO.5.The preceding 243bp of SEQ ID NO.4 sequence obtains with chemical synthesis process, and has increased more restriction enzyme site under the prerequisite that does not produce frameshit, has increased sites such as EcoRI, XbaI, XhoI in EGFP sequence upstream.
2, the foundation of implementation sequence, synthetic.With particular case design primer (the oligonucleotide strand) 12 of DNA works software according to gene, chemical process is synthesized 12 single stranded DNA sequence 10D, sequence is SEQID NO.6～SEQ ID NO.17, and each primer adds the aqua sterilisa (about 6umol/ul) of 400ul, and the PCR reaction is as follows:
The first round: reaction system
SEQ?ID?NO.6 1ul
SEQ?ID?NO.17 1ul
dNTP 1ul(10mM)
10×PFU?Buffer 5ul(200mM?TrisHCl，pH8.8；100mM?KCl；20mM
MgSO4；160mM(NH4)2HSO4；1％Triton?and
1mg/mlBSA)
SEQ?ID?NO.6～17?12×0.2ul
Pfu 0.25(5U/ul)
Add water to 50ul
(all reagent all make a living worker's biological product)
Reaction parameter
98 ℃ of 3min; 94 ℃ of 18sec, 48 ℃ of 18sec, 72 ℃ of 25sec, 20 circulations;
72 ℃ are extended 5min
Second takes turns: reaction system
SEQ?ID?NO.6 1ul
SEQ?ID?NO.17 1ul
dNTP 1ul
10×PFU?Buffer 5ul
First round reaction product 1ul
Pfu 0.25ul
Add water to 50ul
Reaction parameter
95 ℃ of 3min; 94 ℃ of 19sec, 55 ℃ of 20sec, 72 ℃ of 25sec, 22 circulations;
72 ℃ are extended 3min
Second takes turns reaction solution 1% agarose gel electrophoresis, the visible specific band muEGFP-1 (Fig. 5) clearly at about 265bp place, and size conforms to expection, and glue recovery PCR product is stand-by.
Being template with pEGFP-N1 simultaneously, is the upstream and downstream primer with SEQ ID NO.16 and SEQ ID NO.18, and PCR obtains 530bp product muEGFP-2 (Fig. 5).Size conforms to expection, and glue reclaims the PCR product, and is stand-by.
Getting above 265bp product 1ul and 530bp product 1ul, is primer with SEQ ID NO.6 and SEQ ID NO.18, and PCR obtains total length muEGFP sequence SEQ ID NO.19, electrophoresis result such as Fig. 6.
3, the structure of fluorescent screening cloning vector.Total length muEGFP sequence is cut with the EcoRI/NdeI enzyme; Digest pUC18 with same enzyme simultaneously, electrophoresis reclaims the 2400bp band, remove the LacZ sequence of 270bp, connect the big fragment of pUC18 and the muEGFP that reclaim, connect product and be transformed into intestinal bacteria E.coli DH5a competent cell, 37 ℃ of incubated overnight of LB ammonia benzyl resistant panel, after 16 hours, take pictures under the ultraviolet lamp, see the green fluorescence (Fig. 7) that most of cell is sent out bright-coloured, be positive colony.12 bacterium colonies that have bright-coloured green of picking, 37 ℃ of incubated overnight of LB ammonia benzyl liquid nutrient medium, a small amount of extracting plasmid, enzyme are cut checking and are truly had the muEGFP about the long 740bp of being to be cloned on the carrier, select a positive colony.Through sequence verification, sequence results and design are in full accord.Called after pUC-muEGFP
4, the conversion of empty fluorescent screening cloning vector checking.The right-on recombinant plasmid pUC-muEGFP that will check order transforms incubated overnight.Can see clearly that under ultraviolet lamp whole bacterium colonies all send bright-coloured green fluorescence, taking pictures obtains Fig. 8, proves that the fluorescent screening cloning vector pUC-muEGFP that makes up can send fluorescence, meets the requirements substantially.
5, insert purpose fragment checking luminous.Chemically synthetic at random one section sequence SEQ ID NO.20, building-up process can be with reference to the method for technological line 2, and it is the fragment of 255bp that electrophoresis reclaims total length.Use the flush end cloning process to finish and connect the clone; Be cloned among the pUC-muEGFP according to following experimental program:
T4?DNA?Ligase?Buffer 2ul
Yellow?Buffer 2ul
PEG4000 1ul
pUC-muEGFP 2ul
PCR product 4ul
T4DNA?Ligase 1ul
SmaI 1ul
Add water to 20ul
(all reagent all make a living worker's biological product)
22 ℃ connect 1 hour, transform the dull and stereotyped last 37 ℃ of incubated overnight of LB.
Flat board is taken pictures under ultraviolet lamp, check, obviously can see having most of bacterium colony can't present bright green, and the small part bacterium colony sends the light (under the 488nm blue green light, color distinction is more remarkable) of bright green, detail as per Fig. 9.
6, inserting the fragment positive colony detects.8 non-luminous bacterium samples of picking, incubated overnight and extracting go out plasmid.With gene fragment Auele Specific Primer PCR, electrophoresis all can view the band that length is 255bp (Figure 10).
7, the application of fluorescent screening cloning vector.Proof that can be strong from above-mentioned example can use EGFP as a kind of novel positive colony selection markers fully.
Claims (10)
1. fluorescent screening cloning vector is its positive colony selection markers with polynucleotide and/or its complementary strand of coding enhanced green fluorescence protein, and the aminoacid sequence of described enhanced green fluorescence protein is SEQ ID NO.5.
2. fluorescent screening cloning vector according to claim 1 is characterized in that the sequence of the polynucleotide of described coding enhanced green fluorescence protein is SEQ ID NO.4.
3. fluorescent screening cloning vector according to claim 1 is characterized in that, obtains between EcoRI that described fluorescent screening cloning vector is cloned into pUC18 for polynucleotide and its complementary strand with described coding enhanced green fluorescence protein and the NdeI site.
4. fluorescent screening cloning vector according to claim 1 is characterized in that the polynucleotide sequence of described fluorescent screening cloning vector is SEQ ID NO.1.
5. the preparation method of a fluorescent screening cloning vector, for the polynucleotide of the enhanced green fluorescence protein of will encoding and/or its complementary strand obtain described fluorescent screening cloning vector after being cloned into carrier, the aminoacid sequence of described enhanced green fluorescence protein is SEQ ID NO.5.
6. as the preparation method of fluorescent screening cloning vector as described in the claim 5, it is characterized in that the polynucleotide sequence of described coding enhanced green fluorescence protein is SEQ ID NO.4.
7. as the preparation method of fluorescent screening cloning vector as described in the claim 5, the back obtains described fluorescent screening cloning vector between EcoRI that is cloned into carrier pUC18 for polynucleotide and its complementary strand of the enhanced green fluorescence protein of will encoding and the NdeI site.
8. as the preparation method of fluorescent screening cloning vector as described in the claim 7, it is characterized in that, specifically may further comprise the steps:
A. obtain the total length muEGFP gene that two ends have EcoRI and NdeI restriction enzyme site by synthetic primer and overlap PCR method, the encoding sequence of described total length muEGFP gene is shown in SEQ ID NO.4;
B. cut total length muEGFP gene and the carrier pUC18 that digestion step A obtains with EcoRI and NdeI enzyme, obtain the fluorescent screening cloning vector after digestion fragment is connected.
9. the purposes that is used for the bacterium colony screening as fluorescent screening cloning vector as described in the arbitrary claim of claim 1-4.
10. purposes as claimed in claim 9 is characterized in that, described fluorescent screening cloning vector is used for automatization bacterium colony screening instrument.
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| WO2022257114A1 (en) * | 2021-06-11 | 2022-12-15 | 中国科学院深圳先进技术研究院 | Picking needle automatic reset mechanism, clone picking workstation and clone picking method |
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| CN101503699A (en) * | 2009-02-20 | 2009-08-12 | 唐金宝 | Cloning vector pGreen-S based on enhanced green fluorescent protein gene deletion as screen marker and construction method thereof |
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| CN101503699A (en) * | 2009-02-20 | 2009-08-12 | 唐金宝 | Cloning vector pGreen-S based on enhanced green fluorescent protein gene deletion as screen marker and construction method thereof |
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| 《中国海洋药物杂志》 20090430 唐金宝 利用增强型绿色荧光蛋白基因作筛选标记克隆载体的构建及鉴定 p28-31 1-10 第28卷, 第2期 * |
| 《生物工程学报》 19970331 李寿东 用绿色荧光蛋白基因作为筛选标记的新型克隆载体的构建 p323～325 1-10 第13卷, 第3期 * |
| 《郑州大学学报(医学版)》 20020930 王 进 含绿色荧光蛋白报告基因pUC18 筛选载体的构建 p621-624 1-10 第37卷, 第5期 * |
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| WO2022257114A1 (en) * | 2021-06-11 | 2022-12-15 | 中国科学院深圳先进技术研究院 | Picking needle automatic reset mechanism, clone picking workstation and clone picking method |
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