CN108409867A - A method of it can be used in improving fluorescent protein fluorescence signal strength - Google Patents
A method of it can be used in improving fluorescent protein fluorescence signal strength Download PDFInfo
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Abstract
The invention discloses a kind of methods that can be used in improving fluorescent protein fluorescence signal strength, the method is the expression of the polypeptide sequence guiding fluorescin of the chimeric sequences or manual simulation's design by using the signal peptide sequence of specific protein or the mutant nucleotide sequence of signal peptide sequence or signal peptide sequence, to improve fluorescence signal intensity of the fluorescin in host cell.The signal peptide of the specific protein refers specifically to the chimeric sequences of the signal peptide sequence of naturally occurring partially protein or the mutant nucleotide sequence of signal peptide sequence or signal peptide sequence or the polypeptide sequence of manual simulation's design.The present invention improves fluorescence signal intensity of the fluorescin in host cell, and this kind of fused protein can be taken as a novel complete fluorescin and use, also more efficient fluorescence signaling molecule is can be used as, for various researchs or the research and development etc. of the fluorescence labeled cell or biology of other purposes.
Description
Technical field
The present invention relates to biotechnologies, are specifically related to one kind and can be used in improving fluorescent protein fluorescence signal strength
Method.
Background technology
Fluorescin (Fluorescent proteins, FPs) is a kind of important indicator protein, according to color difference point
Class, including but not limited to following classification：Green fluorescent protein (Green fluorescent proteins), red fluorescent protein
(Red fluorescent proteins), yellow fluorescence protein (Yellow fluorescent proteins), fluorescent orange
Albumen (Orange fluorescent proteins), far infrared fluorescin (Far-red fluorescent
Proteins), cyan (blue-green) fluorescin (Cyan fluorescent proteins) and burst of ultraviolel green fluorescence egg
(UV-excitable green fluorescent proteins) (Nathan C Shaner, Paul A Steinbach in vain
and Roger Y Tsien,Nature Method,2005).Certainly, the type of fluorescin or mutant are still constantly increasing
Add.Under suitable condition, fluorescin can spontaneously form chromophore.When different fluorescins is by respective excitation
When light excites, launch different fluorescence.But fluorescin, when being expressed in external source recombinant cell, stereochemical structure is not easy just
It is really formed, what is especially showed in prokaryotic cell becomes apparent.
Secreting signal peptide in protein, alternatively referred to as signal peptide, guiding peptide etc., can instruct the polypeptide by or into
Enter the biomembrane (including the plasma membrane of prokaryotes and Eukaryotic endoplasmic reticulum) of the cell.Signal peptide is usually located at polypeptide
The ends N- are about 10-60 amino acid.In protein expression or production process, to improve the expression quantity of albumen and/or being convenient for egg
White extraction purification, people have attempted multi-signal peptide and its mutant to guide expression (the Sanjeev K.Gupta of destination protein
and Pratyoosh Shukla,Critical Reviews in Biotechnology,2015；Tim W.Overton,
Drug Discovery Today,2014；Germán L.Rosano and Eduardo A.Ceccarelli,Frontiers
In Microbiology, 2014), and obtain success.But people utilize the purpose of fluorescin, generally not obtain
Fluorescin of large-scale purification itself, but it is easy to the fluorescence signal of observation by it, mark cell or tissue or organ or life
Positioning, the detection molecules interaction etc. of object, monitoring target gene or protein expression.Therefore, efficiently turned using secreting signal peptide
Fluorescin is transported to the not common experiment purpose of outside or technological means.How the fluorescence signal intensity of fluorescin is improved
It is only the hot spot that people pay close attention to always.The screening of novel fluorescence albumen at present and the screening of fluorescin mutant are main sides
Method.For example, EGFP is on the basis of GFP, two amino acid mutation sites are introduced, are preferably folded to make EGFP have
Performance and adaptability.The present invention claims with propose a kind of method of new raising fluorescent protein fluorescence signal strength.
Invention content
The technical problem to be solved by the present invention is to provide it is a kind of can be used in improve fluorescent protein fluorescence signal strength method,
Use the embedding of the signal peptide sequence of naturally occurring partially protein or the mutant nucleotide sequence of signal peptide sequence or signal peptide sequence
The expression for closing the polypeptide sequence guiding fluorescin of sequence or manual simulation's design, to enhance fluorescin in host cell
In fluorescence signal intensity.
Technical scheme is as follows：
First, it is by making the present invention provides a kind of method that can be used in improving fluorescent protein fluorescence signal strength
With the chimeric sequences or artificial mould of the signal peptide sequence of specific protein or the mutant nucleotide sequence of signal peptide sequence or signal peptide sequence
The expression of the polypeptide sequence guiding fluorescin of meter is proposed, it is strong to improve fluorescence signal of the fluorescin in host cell
Degree.
Further, in the above scheme, the signal peptide of the specific protein refers specifically to naturally occurring Partial Protein
The signal peptide sequence of matter, including such as SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ IDNO:4、SEQ ID
NO:5、SEQ ID NO:6 or SEQ ID NO:Sequence shown in 7 etc..It is of the present invention to increase fluorescence signal intensity
The encoding polynucleotide sequence of signal peptide sequence can be according to genetic code reasoning.The Preference of codon selection also can be according to host
The difference of cell type, is adjusted.Polynucleotide sequence can pass through conventional molecular biological method known in the art
Or prepared by conventional synthesis process.
Further, in the above scheme, the chimeric sequences of the mutant nucleotide sequence of the signal peptide sequence or signal peptide sequence
It is higher than 30% with the signal peptide sequence homology, including such as SEQ ID NO:8、SEQ ID NO:9 or SEQ ID NO:10 etc.
Shown in sequence.The mutant nucleotide sequence or signal peptide sequence of the signal peptide sequence of the present invention that fluorescence signal intensity can be increased
The encoding polynucleotide sequences of chimeric sequences can be according to genetic code reasoning.The Preference of codon selection also can be according to host
The difference of cell type, is adjusted.Polynucleotide sequence can pass through conventional molecular biological method known in the art
Or prepared by conventional synthesis process.
Further, in the above scheme, the polypeptide sequence of manual simulation's design and the signal peptide sequence are homologous
Property be higher than 30%.The polypeptide sequence of manual simulation's design of the signal peptide sequence of the present invention that fluorescence signal intensity can be increased
The encoding polynucleotide sequence of row can be according to genetic code reasoning.The Preference of codon selection also can be according to host cell species
Difference, be adjusted.Polynucleotide sequence can pass through conventional molecular biological method known in the art or conventional conjunction
It is prepared at method.
Further, in the above scheme, the mutant nucleotide sequence or signal peptide of the signal peptide sequence or signal peptide sequence
The chimeric sequences of sequence or the polypeptide sequence of manual simulation's design are spliced to form with fluorescent protein sequence and merge fluorescin, described
It includes such as SEQ ID NO to merge fluorescent protein sequence:11、SEQ ID NO:12、SEQ ID NO:13 or SEQ ID NO:14、
SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19 or SEQ ID NO:
Sequence shown in 20 etc..The encoding polynucleotide sequence of fusion fluorescin of the present invention can be according to genetic code reasoning.
The Preference of codon selection can be also adjusted according to the difference of host cell species.Polynucleotide sequence can pass through this
In field prepared by well known conventional molecular biological method or conventional synthesis process.
Further, in the above scheme, in the fusion fluorescent protein sequence, signal peptide sequence or signal peptide sequence
It can be between the chimeric sequences of mutant nucleotide sequence or signal peptide sequence or the polypeptide sequence and fluorescent protein sequence of manual simulation's design
It is directly connected to, there may also be intervening sequences.
Further, the present invention also indicates that, the signal peptide sequence of above-mentioned specific protein is generally naturally occurring portion
The signal peptide sequence of point the protein either mutant nucleotide sequence of signal peptide sequence or the chimeric sequences of signal peptide sequence or artificial mould
Propose the polypeptide sequence of meter, sequence is respectively positioned on the N-terminal of fluorescin, formed fusion protein (because main group is as fluorescin,
Fluorescin is also referred to as merged herein).The signal peptide sequence or signal peptide sequence of naturally occurring partially protein
Mutant nucleotide sequence or signal peptide sequence chimeric sequences or manual simulation's design polypeptide sequence and fluorescin between can be with
It is directly connected to, there may also be intervening sequences.
In addition, the intervening sequence in the fusion fluorescin can be the polynucleotides of restriction enzyme enzyme recognition site
The amino acid sequence of sequential coding, can also be the amino acid or polypeptide sequence in other sources, usually shorter, and not influence fluorescence
The folding efficiency of albumen itself or the folding efficiency that fluorescin itself can be promoted.
The present invention also indicates that this kind of fusion fluorescin can not be secreted into extracellular or periplasmic space；It can also can
Secretion, but be completely or partially attached on the outside of cell membrane after secernment efficiency is very low or secretion or cell wall on.This kind of fusion is glimmering
If photoprotein is after the secretion of cell secretory pathway, the mutation of the signal peptide sequence or signal peptide sequence of the connection of fluorescin N-terminal
The chimeric sequences of sequence or signal peptide sequence or the polypeptide sequence of manual simulation's design can by corresponding polypeptidase shear removal,
It can also be removed by Partial Shear, removal can not also be sheared.
The present invention also provides a kind of prokaryotic cell or the expression vector of eukaryocyte, the carrier contains above-mentioned signal peptide
The polypeptide sequence of the chimeric sequences of the mutant nucleotide sequence or signal peptide sequence of sequence or signal peptide sequence or manual simulation's design, including
Such as SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ
ID NO:7、SEQ ID NO:8、SEQ ID NO:9 or SEQ ID NO:The coded polynucleotide sequence of 10 equal shown polypeptide sequences
Row, and purpose existing for the polypeptide sequence of these polynucleotide sequence codings is for improving fluorescent protein fluorescence signal strength.
Further, in the fusion fluorescent protein sequence, fluorescin C-terminal can connect any desired label sequence
Row, including such as SEQ ID NO:10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ
ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19 or SEQ ID NO:20 etc.
Shown in His-Tag sequence；Any sequence can not also be connected.
Further, the fusion fluorescin can substitute its corresponding fluorescin and be used.
The present invention also provides a kind of prokaryotic cell or the expression vector of eukaryocyte, the carrier, which contains, to be encoded by upper
The polynucleotide sequence of the fusion fluorescin of method structure is stated, including such as SEQ ID NO:11、SEQ ID NO:12、SEQ ID
NO:13 or SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ
ID NO:19 or SEQ ID NO:The encoding polynucleotide sequence of fluorescent protein sequence is merged shown in 20 etc..
Further, the fluorescin includes such as：Green fluorescent protein (Green fluorescent
Proteins), red fluorescent protein (Red fluorescent proteins), yellow fluorescence protein (Yellow
Fluorescent proteins), orange fluorescent protein (Orange fluorescent proteins), far infrared fluorescence egg
(Far-red fluorescent proteins), cyan (blue-green) fluorescin (Cyan fluorescent in vain
Proteins) and burst of ultraviolel green fluorescent protein (UV-excitable green fluorescent proteins) and its
Mutant etc..
Further, in the above scheme, the host cell includes prokaryotic cell and eukaryocyte.
The present invention this kind of fusion fluorescent protein fluorescence signal enhancing the reason of include：1) corresponding mRNA points are stabilized
Son；2) translation efficiency of protein is improved；3) protein folding efficiency is improved；4) it is whole or local fluorescin has been finely tuned
Space structure, cause chromophore transmitting fluorescence ability enhancing；5) can not be secreted into cell culture fluid (including prokaryotic cell and
Eukaryocyte) or periplasmic space (refering in particular to prokaryotic cell) or secernment efficiency it is very low and be attached on the outside of cell membrane after secreting or
On cell wall.
The beneficial effects of the invention are as follows：The present invention utilizes the signal peptide sequence that certain protein are merged in fluorescin N-terminal
Or the mutant nucleotide sequence or the chimeric sequences of signal peptide sequence or the polypeptide sequence of manual simulation's design of signal peptide sequence, to improve
Fluorescence signal intensity of the fluorescin in host cell.This kind of fused protein may be due to losing the ability of exocytosis
Or secernment efficiency it is very low and be attached on the outside of cell membrane after secreting or cell wall on, to hinder or slow down cell secretion way
Mutant nucleotide sequence or signal peptide sequence of the relevant polypeptidase of diameter to signal peptide sequence or signal peptide sequence in fusion fluorescin
Chimeric sequences or manual simulation design polypeptide sequence shearing.The signal peptide sequence or signal peptide sequence of this kind of fused protein
The mutant nucleotide sequence of row or the chimeric sequences of signal peptide sequence or the polypeptide sequence of manual simulation's design are also possible to originally would not be by
The polypeptidase of secretory pathway is sheared.Therefore this kind of fusion fluorescin, which can be taken as a novel complete fluorescin, makes
With.Individual fluorescin is compared, this kind of fusion fluorescin can be used as a kind of more efficient fluorescence signaling molecule, as other days
Right fluorescin or its mutant are equally used directly to regulation and control and expression, Molecular interaction, the destination protein of research purpose gene
Positioning and regulating and controlling sequence and protein other mechanism study, may be alternatively used for watching, study or the fluorescence of other purposes
Mark the research and development etc. of cell or biology.
Description of the drawings
Fig. 1 is pAcIGFP and pAcGFP carrier schematic diagrames；
Fig. 2 is the fluorescence letter of pAcIGFP and pAcGFP in bacillus thuringiensis (Fig. 2A) and Escherichia coli (Fig. 2 B)
Number variation diagram；
Fig. 3 is pIaIGFP and pIaGFP carrier schematic diagrames；
Fig. 4 is the fluorescence signal variation diagram of pIaIGFP and pIaGFP in bacillus thuringiensis；
Fig. 5 is p102cIGFP and p102cGFP carrier schematic diagrames；
Fig. 6 is pPIC9-IGFP and pPIC9-GFP carrier schematic diagrames；
Fig. 7 is p13-35S-IGFP and p13-35S-GFP carrier schematic diagrames.
Specific implementation mode
Technical solution for a better understanding of the present invention, by following embodiments come the present invention will be described in detail, but not
Protection scope of the present invention is limited.
Embodiment 1：Structure fusion fluorescin IGFP
The embodiment illustrates the N-terminal signal peptide and green fluorescent protein GFP of a naturally occurring protein C ry1Ia
The fusion fluorescin IGFP of splicing, amino acid sequence are SEQ ID NO:11.
The characteristics of IGFP albumen is：The N-terminal signal peptide sequence of Cry1Ia is located at the N-terminal of green fluorescent protein GFP；Cry1Ia
N-terminal signal peptide and the amino acid sequence of green fluorescent protein GFP between there is no other extra amino acid；The C-terminal of IGFP contains
There are 6 histidines, with extraction IGFP albumen when necessary.This His-Tag sequence is not that structure fusion fluorescin must
Must.The encoding polynucleotide sequence of IGFP albumen can be obtained from least three kinds of approach：1) by the way that the N-terminal of Cry1Ia will be encoded
Signal peptide is obtained with polymerase chain reaction (PCR reacts) respectively with the polynucleotide sequence of the amino acid sequence of green fluorescent protein
, then the two is stitched together by the method for over-lap PCR (Horton, R.M.et al., BioTechniques, 1990),
Form a complete fusion；2) by the way that the N-terminal signal peptide of Cry1Ia and the amino acid sequence of green fluorescent protein will be encoded
The polynucleotide sequence of row uses polymerase chain reaction (PCR reactions) to obtain respectively, is then spliced by the method for recombinant clone
At a complete fusion；3) two polynucleotide sequence splicings are directly made by artificial synthesized mode.Above-mentioned three kinds
Method is conventional molecular biological method known in the art or conventional synthesis process.
Embodiment 2：The procaryotic cell expression carrier pAcIGFP of structure fusion fluorescin IGFP
The embodiment illustrates a kind of procaryotic cell expression carrier of fusion fluorescin described herein, which can be with
Expression pattern for the promoter Pac (i.e. the promoter of cry1Ac genes) for monitoring Cry1Ac protein encoding polynucleotide chains.
By the promoter Pac, 3 ' connection cry1Ac of 5 ' connection cry1Ac genes of the coded polynucleotide chain of IGFP albumen
This expression cassette to form a complete expression cassette, and is accessed removing restrictions a property inscribe by the terminator Tac of gene
The pHT304 carriers (Olivia Arantes and Didier Lereclus, Gene, 1990) of enzyme SacI recognition sites ---
P304 Δ SacI form pAcIGFP expression vectors (Fig. 1).As a contrast, by green fluorescent protein (C-terminal carries 6 histidines)
Encoding polynucleotide sequence also connect upper Pac and Tac in the same way, form the expression cassette of GFP albumen, and access p304
The corresponding site of Δ SacI carriers forms pAcGFP expression vectors (Fig. 1).
The connection type of polynucleotide sequence described herein, at least there are three types of：1) traditional restriction enzyme and company
Connect molecular cloning associated with enzyme；2) recombinant clone method；3) method of artificial synthesized entire expression vector.The above method is this
Well known basic skills in field.Fig. 1 illustrates molecular cloning associated with a kind of traditional restriction enzyme and ligase
The carrier schematic diagram of structure, because restriction enzyme selects different or cloning process to use different, carrier, Pac sequences, Tac sequences
The sequence of splicing site between row and the encoding polynucleotide sequence of IGFP or GFP albumen can have different variations.
Embodiment 3：Applications of the expression vector pAcIGFP in bacillus thuringiensis
Expression vector pAcIGFP and pAcGFP are converted by electroporation to bacillus thuringiensis respectively
(Bacillus thuringiensis) competent cell.The monoclonal screened is being contained into 25 μ g/mL erythromycin respectively
Shaken cultivation (28.5 DEG C, 200rpm) in LB liquid medium (Luria-Bertani medium).After incubation the 9th, 12,
24,36,48,60,72 hours, cell fluorescence signal strength was monitored respectively.As a result the Su Yun gold buds containing pAcIGFP carriers are shown
Fluorescence signal intensity of the fluorescence signal intensity of spore bacilli-cell always than the Bacillus thuringiensis cell containing pAcGFP is high
About four to six times (Fig. 2A).The result absolutely proves, compared to the fluorescence that GFP, IGFP are more suitable for bacillus thuringiensis
Label uses.
Embodiment 4：Applications of the expression vector pAcIGFP in Escherichia coli
Expression vector pAcIGFP and pAcGFP are converted by Calcium Chloride Method to Escherichia coli (Escherichia respectively
Coli) TG1 competent cells.By the monoclonal screened respectively in the LB liquid medium containing 100 μ g/mL ampicillins
Shaken cultivation (37 DEG C, 200rpm) in (Luria-Bertani medium).The 8th, 12,24,36,48,60,72 is small after incubation
When, cell fluorescence signal strength is monitored respectively.As a result the fluorescence signal intensity containing pAcIGFP carrier Bacillus coli cells is shown
Always about two to seven times (Fig. 2 B) higher than the fluorescence signal intensity of the Bacillus coli cells containing pAcGFP.The result is fully said
Bright, the fluorescent marker that Escherichia coli are more suitable for compared to GFP, IGFP uses.
Embodiment 5：The procaryotic cell expression carrier pIaIGFP of structure fusion fluorescin IGFP
The embodiment illustrates a kind of procaryotic cell expression carrier of fusion fluorescin described herein, which can be with
Expression pattern for the promoter Pia (i.e. the promoter of cry1Ia genes) for monitoring Cry1Ia protein encoding polynucleotide chains.
By the promoter Pia, 3 ' connection cry1Ac of 5 ' connection cry1Ia genes of the coded polynucleotide chain of IGFP albumen
The terminator Tac of gene, to form a complete expression cassette, and this expression cassette access p304 Δ SacI carriers is corresponding
Site, formed pIaIGFP expression vectors (Fig. 3).As a contrast, by green fluorescent protein GFP (C-terminal carries 6 histidines)
Encoding polynucleotide sequence also connect upper Pia and Tac in the same way, form the expression cassette of GFP albumen, and access p304
The corresponding site of Δ SacI carriers forms pIaGFP expression vectors (Fig. 3).
The connection type of polynucleotide sequence described herein, i.e. cloning process at least there are three types of：1) traditional restricted
Molecular cloning associated with restriction endonuclease and ligase；2) recombinant clone method；3) method of artificial synthesized entire expression vector.It is above-mentioned
Method is basic experiment method known in the art.Fig. 3 illustrates a kind of traditional restriction enzyme and connects enzyme-linked
The carrier schematic diagram of molecular cloning structure, because restriction enzyme selects different or cloning process using different, carrier,
The sequence of the splicing site of the key polynucleotide sequence such as coded polynucleotide of Pia sequences, Tac sequences and IGFP or GFP albumen
Row can have different variations.
Embodiment 6：Applications of the expression vector pIaIGFP in bacillus thuringiensis
Expression vector pIaIGFP and pIaGFP are converted by electroporation respectively thin to bacillus thuringiensis competence
Born of the same parents.By the monoclonal screened respectively in the LB liquid medium (Luria-Bertani containing 25 μ g/mL erythromycin
Medium shaken cultivation (28.5 DEG C, 200rpm) in).The the 9th, 12,24,36,48,60,72 hour after incubation, monitoring was thin respectively
Born of the same parents' fluorescence signal intensity.As a result show that the Bacillus thuringiensis cell containing pIaIGFP carriers can detect at the 9th hour
Fluorescence signal, and the Bacillus thuringiensis cell containing pIaGFP is only examined in the peak expression of the promoter for (the 36th hour)
Measure faint fluorescence signal (Fig. 4).The result absolutely proves that, compared to GFP, IGFP is more suitable in bacillus thuringiensis
It is used as fluorescent marker.The result also depicts the regulation and control model of the promoter of cry1Ia genes, with forefathers to Cry1Ia eggs
The monitoring result expressed in vain is consistent (Kristy Kostichka et al., Journal of Bacteriology, 1996).Separately
Outside, result ratio Tounsi, Slim and Jaoua, Samir (FEMS Microbiology Letters, 2002) uses core
The testing result of sour marking method (northern blot) is sensitiveer.This method does not need complicated albumen or nucleic acid marking method
Operating process is sensitiveer quick.
Embodiment 7：The procaryotic cell expression carrier p102cIGFP of structure fusion fluorescin IGFP
The embodiment illustrates a kind of extensive host (Broad host range) of fusion fluorescin described herein
Procaryotic cell expression carrier, the carrier can be used for monitoring labeled bacterium.
By 5 ' connection promoter Pc (Youqiang Xu, Applied and of the coded polynucleotide chain of IGFP albumen
Environmental Microbiology, 2013), the terminator TrrnB of 3 ' connection Escherichia coli rrnB operon genes, from
And form a complete expression cassette, and by this expression cassette access pTR102 carriers (Michael Weinstein et al.,
Journal of Bacteriology, 1992) corresponding site, forms p102cIGFP expression vectors (Fig. 5).As a contrast,
The encoding polynucleotide sequence of green fluorescent protein GFP (C-terminal carries 6 histidines) is also connected in the same way and is started
The terminator TrrnB of sub- Pc and Escherichia coli rrnB operon genes, form the expression cassette of GFP albumen, and access pTR102 loads
The corresponding site of body forms p102cGFP expression vectors (Fig. 5).
The connection type of polynucleotide sequence described herein, i.e. cloning process at least there are three types of：1) traditional restricted
Molecular cloning associated with restriction endonuclease and ligase；2) recombinant clone method；3) method of artificial synthesized entire expression vector.It is above-mentioned
Method is basic experiment method known in the art.Fig. 5 illustrates a kind of traditional restriction enzyme and connects enzyme-linked
The carrier schematic diagram of molecular cloning structure, because restriction enzyme selects different or cloning process using different, carrier,
The coded polynucleotide of Pc sequences, the terminator sequence of Escherichia coli rrnB operon genes and IGFP or GFP albumen etc. is crucial
The sequence of splicing site between polynucleotide sequence can have different variations.
Embodiment 8：Applications of the expression vector p102AcIGFP in Pseudomonas alba
Expression vector p102cIGFP and p102cGFP are converted by electroporation to a kind of pseudomonas bacterium respectively
The competent cell of (Pseudomonas sp.).By the monoclonal screened respectively in the liquid LB containing 15 μ g/mL tetracyclines
Shaken cultivation (28.5 DEG C, 200rpm) in culture medium (Luria-Bertani medium).After incubation the 12nd, 24,36,48,
60,72 hours, cell fluorescence signal strength was monitored respectively.As a result the Pseudomonas alba cell containing p102cIGFP carriers is shown
Fluorescence signal intensity be consistently higher than the Pseudomonas alba cell about two containing p102cGFP arrive thirtyfold.Result explanation, phase
Compared with GFP, IGFP is more suitable for fluorescent marker use in pseudomonas bacterium.
Embodiment 9：The eukaryotic expression vector pPIC9-IGFP of structure fusion fluorescin IGFP
The embodiment illustrates a kind of eukaryotic expression vector of fusion fluorescin described herein, which can be with
For determine IGFP albumen yeast cells expression.
The encoding polynucleotide sequence of IGFP albumen is accessed into pPIC9 carriers, to make IGFP be in alcohol oxidase 1
(AOX1) under the promoter of gene and the regulation and control of terminator, and the secreting signal peptide of α-factor (α-factor), most end form are removed
At pPIC9-IGFP expression vectors (Fig. 6).As a contrast, by the coding of green fluorescent protein GFP (C-terminal carries 6 histidines)
Polynucleotide sequence also accesses pPIC9 carriers in the same way, forms pPIC9-GFP expression vectors (Fig. 6).
The connection type of polynucleotide sequence described herein, i.e. cloning process at least there are three types of：1) traditional restricted
Molecular cloning associated with restriction endonuclease and ligase；2) recombinant clone method；3) method of artificial synthesized entire expression vector.It is above-mentioned
Method is basic experiment method known in the art.Fig. 6 illustrates a kind of traditional restriction enzyme and connects enzyme-linked
The carrier schematic diagram of molecular cloning structure, because restriction enzyme selects different or cloning process to use different, carrier
There can be different variations with the sequence of the splicing site of the coded polynucleotide chain of IGFP or GFP albumen.
Embodiment 10：Applications of the eukaryotic expression vector pPIC9-IGFP in yeast
Expression vector pPIC9-IGFP and pPIC9-GFP are converted by electroporation to Pichia pastoris (Pichia respectively
Pastoris) the competent cell of GS115 and X33.Recombinant clone of the screening containing single copy exogenous protein expression frame exists respectively
100mL MGY culture mediums (Minimal Glycerol Medium, 1.34%YNB, 1%glycerol, 4 × 10-5%
Biotin shaken cultivation (30 DEG C, 250rpm) in).Wait for OD600When to 2, cell is collected by centrifugation.For Mut+Type bacterial strain, collection
Cell is resuspended with MM culture mediums (Minimal Methanol, 1.34%YNB, 4 × 10-5%biotin, 0.5%methanol)
To OD600=1.Induced expression 96 hours.Period the 24th, 48,72,96 hour, monitors cell fluorescence signal strength respectively.For
MutS type bacterial strains, MM culture mediums (Minimal Methanol, 1.34%YNB, the 4 × 10-5% of the cell 20mL of collection
Biotin, 0.5%methanol) it is resuspended.Induced expression 144 hours.Period, the 24th, 48,72,96,120,144 hour, respectively
Monitor cell fluorescence signal strength.As a result show that the fluorescence signal of the Pichia pastoris containing pPIC9-IGFP carriers is high always
In the Pichia pastoris containing pPIC9-GFP.The result absolutely proves that, compared to GFP, IGFP is more suitable for making in saccharomycete
It is used for fluorescent marker.
Embodiment 11：The plant conversion carrier p13-35S-IGFP of structure fusion fluorescin IGFP
The embodiment illustrates a kind of plant conversion carrier of fusion fluorescin described herein.The carrier can be used for
Monitor expression of the IGFP albumen in plant.
Cauliflower mosaic virus (CaMV) 35S promoter P35S is connected by the 5 ' of the coded polynucleotide chain of IGFP albumen,
3 ' connection cauliflower mosaic virus (CaMV) 35S terminator T35S, to form a complete expression cassette, and this are expressed
Frame accesses pCambia1300 carriers, forms p13-35S-IGFP carriers (Fig. 7).As a contrast, by normal green fluorescent protein
The encoding polynucleotide sequence of GFP (C-terminal carries 6 histidines) also connects upper P35S and T35S in the same way, forms GFP
The expression cassette of albumen, and the corresponding site of pCambia1300 carriers is accessed, form p13-35S-GFP carriers (Fig. 7).
The connection type of polynucleotide sequence described herein, i.e. cloning process at least there are three types of：1) traditional restricted
Molecular cloning associated with restriction endonuclease and ligase；2) recombinant clone method；3) method of artificial synthesized entire expression vector.It is above-mentioned
Method is basic experiment method known in the art.Fig. 7 illustrates a kind of traditional restriction enzyme and connects enzyme-linked
The carrier schematic diagram of molecular cloning structure, because restriction enzyme selects different or cloning process using different, carrier,
Splice bits between the key polynucleotide sequence such as coded polynucleotide of P35S sequences, T35S sequences and IGFP or GFP albumen
The sequence of point can have different variations.
Embodiment 12：Applications of the plant conversion carrier p13-35S-IGFP in tobacco
The embodiment illustrate it is a kind of it is described herein fusion fluorescin plant conversion carrier in model plant tobacco
Middle transient expression.
Carrier p13-35S-IGFP and p13-35S-GFP are converted by electroporation to agrobacterium tumefaciens respectively
(Agrobacterium tumefaciens) LBA4404 competent cells.The monoclonal screened is being contained into 50 μ g/ respectively
(every liter includes 10g yeast extract, 10g to the liquid YEP medium of mL kanamycins and 50 μ g/mL tetracyclines
Peptone, 5g NaCl, pH 7.0) in shaken cultivation (28 DEG C, 200rpm).Wait for OD600When between 0.6-1.0, it is collected by centrifugation
Agrobatcerium cell, and inducing culture (10nmol/L MES-KOH pH 5.7,10mmol/L MgCl will be deposited in2) in weight
It is outstanding.Cell is collected by centrifugation, and reuses inducing culture resuspension.Finally, cell is centrifuged and collects, with containing 200 μm of ol/L
After the inducing culture of acetosyringone is resuspended, it is placed at room temperature for 1-4 hours.Adjust OD600It is worth between 0.1-0.2, then with note
Re-suspension liquid is injected into 6-8 weeks Ben's tobacco (Nicotiana benthamiana) blade by emitter.24-72 hours after injection,
Microexamination is to significant fluorescence signal.
Claims (10)
1. a kind of method that can be used in improving fluorescent protein fluorescence signal strength, which is characterized in that be by using specific egg
The chimeric sequences or manual simulation's design of the signal peptide sequence of white matter or the mutant nucleotide sequence of signal peptide sequence or signal peptide sequence
Polypeptide sequence guides the expression of fluorescin, to improve fluorescence signal intensity of the fluorescin in host cell.
2. a kind of method that can be used in improving fluorescent protein fluorescence signal strength according to claim 1, feature exist
The signal peptide sequence or signal peptide sequence of naturally occurring partially protein are referred specifically in the signal peptide of, the specific protein
The polypeptide sequence of the chimeric sequences or manual simulation's design of mutant nucleotide sequence or signal peptide sequence, including such as SEQ ID NO:1、SEQ
ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID
NO:8、SEQ ID NO:9 or SEQ ID NO:Sequence shown in 10 etc..
3. a kind of method that can be used in improving fluorescent protein fluorescence signal strength according to claim 1 or 2, feature
It is, the mutant nucleotide sequence of the signal peptide sequence or the chimeric sequences of signal peptide sequence are higher than with the signal peptide sequence homology
30%.
4. a kind of method that can be used in improving fluorescent protein fluorescence signal strength according to claim 1 or 2, feature
It is, the polypeptide sequence of manual simulation's design is higher than 30% with the signal peptide sequence homology.
5. a kind of method that can be used in improving fluorescent protein fluorescence signal strength according to claim 1, feature exist
In the chimeric sequences of the mutant nucleotide sequence or signal peptide sequence of the signal peptide sequence or signal peptide sequence or manual simulation's design
Polypeptide sequence be spliced to form with fluorescent protein sequence and merge fluorescin, the fusion fluorescent protein sequence includes such as SEQ ID
NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID
NO:17、SEQ ID NO:18、SEQ ID NO:19 or SEQ ID NO:Sequence shown in 20 etc..
6. a kind of method that can be used in improving fluorescent protein fluorescence signal strength according to claim 5, feature exist
In in the fusion fluorescent protein sequence, the mutant nucleotide sequence or signal peptide sequence of signal peptide sequence or signal peptide sequence are fitted into
It can be directly connected between sequence or the polypeptide sequence and fluorescent protein sequence of manual simulation's design, there may also be interval sequences
Row.
7. a kind of method that can be used in improving fluorescent protein fluorescence signal strength according to claim 6, feature exist
In the intervening sequence in the fusion fluorescin can be the polynucleotide sequence coding of restriction enzyme enzyme recognition site
Amino acid sequence can also be the amino acid or polypeptide sequence in other sources.In the fusion fluorescent protein sequence, signal peptide sequence
The chimeric sequences of the mutant nucleotide sequence or signal peptide sequence of row or signal peptide sequence or the polypeptide sequence and fluorescence of manual simulation's design
It can be directly connected between protein sequence, there may also be intervening sequences.
8. the expression vector of a kind of prokaryotic cell or eukaryocyte, which is characterized in that the carrier contains coding claim 2 institute
The polynucleotide sequence of fusion fluorescent protein sequence described in the signal peptide sequence or claim 5 stated.
9. a kind of method that can be used in improving fluorescent protein fluorescence signal strength according to claim 1, feature exist
In the fluorescin includes such as：Green fluorescent protein (Green fluorescent proteins), red fluorescent protein
(red fluorescent proteins), yellow fluorescence protein (Yellow fluorescent proteins), fluorescent orange
Albumen (Orange fluorescent proteins), far infrared fluorescin (Far-red fluorescent
Proteins), cyan (blue-green) fluorescin (Cyan fluorescent proteins) and burst of ultraviolel green fluorescence egg
(UV-excitable green fluorescent proteins) and its mutant etc. in vain.
10. a kind of method that can be used in improving fluorescent protein fluorescence signal strength according to claim 1, feature exist
In the host cell includes prokaryotic cell and eukaryocyte.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109734815A (en) * | 2019-02-22 | 2019-05-10 | 武汉巴菲尔生物技术服务有限公司 | A kind of enhanced fluorescin |
| CN109867726A (en) * | 2017-12-27 | 2019-06-11 | 华南师范大学 | A kind of fusion protein and its application |
| CN109971777A (en) * | 2019-03-26 | 2019-07-05 | 山西农业大学 | A kind of method of protein expression in enhancing prokaryotic system |
-
2018
- 2018-03-02 CN CN201810173307.XA patent/CN108409867A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109867726A (en) * | 2017-12-27 | 2019-06-11 | 华南师范大学 | A kind of fusion protein and its application |
| CN109734815A (en) * | 2019-02-22 | 2019-05-10 | 武汉巴菲尔生物技术服务有限公司 | A kind of enhanced fluorescin |
| CN109971777A (en) * | 2019-03-26 | 2019-07-05 | 山西农业大学 | A kind of method of protein expression in enhancing prokaryotic system |
| CN109971777B (en) * | 2019-03-26 | 2023-04-25 | 山西农业大学 | Method for enhancing protein expression in prokaryotic system |
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