CN102618548B

CN102618548B - Green fluorescent mutant protein probe of hypochloric acid and coding sequence thereof

Info

Publication number: CN102618548B
Application number: CN 201110029564
Authority: CN
Inventors: 杨弋; 朱青; 徐磊
Original assignee: East China University of Science and Technology
Current assignee: East China University of Science and Technology
Priority date: 2011-01-27
Filing date: 2011-01-27
Publication date: 2013-06-19
Anticipated expiration: 2031-01-27
Also published as: CN102618548A

Abstract

The invention provides a coding gene of a green fluorescent mutant protein cpSFGFP having high hypochloric acid selectivity, a recombinant plasmid carrier containing the coding gene, a host cell transformed by the recombinant plasmid carrier, and the green fluorescent mutant protein cpSFGFP which is expressed by the host cell, is coded by the coding gene and has high hypochloric acid selectivity. The recombined green fluorescent mutant protein cpSFGFP can be used as a hypochloric acid detection probe. The response of the novel green fluorescent mutant protein probe to a concentration change of hypochloric acid is superior to that of a wild type green fluorescence protein (GFP) or a mutant protein such as SFGFP of the wild type GFP in sensitivity, stability and selectivity.

Description

A kind of hypochlorous novel green fluorescence misfolded proteins probe and encoding sequence thereof

Technical field

The present invention relates to biological chemistry, protein chemistry, molecular biology.Particularly, the present invention relates to a kind of novel green Fluorescence Mutation of A albumen and coding nucleotide sequence thereof that can detect in high sensitivity hypochlorous acid level in cell.The invention still further relates to preparation method and the purposes of this mutain and nucleotide sequence thereof.

Background technology

Traditional hypochlorous acid (HOCI) probe is the organic chemistry fluorescent probe, although this class probe sensitivity is very high, specificity is good, but exist poorly soluble, easily occur fluorescent quenching, the shortcomings such as cytotoxicity are arranged, can not be used for viable bacteria and viable cell detection, can not be used for the hypochlorous acid level of different organoids in the detection and localization cell, and problem of environmental pollution is arranged.

Green fluorescent protein (Green Fluorescent Protein, be called for short GFP) is that molecular weight is 27KDa approximately from the isolated a kind of natural green fluorescin of jellyfish, the single chain polypeptide that is comprised of 239 amino-acid residues.The chromophore of GFP is by 3 amino acid that are positioned at the 65-67 position: the para hydroxybenzene imidazolone (4-p-hydroxybene-5-imidazo-linone) of Serine one dehydration tyrosine one Formation of glycine consists of.Its main fluorescence exciting wavelength is 395nm, and the emission wavelength that excites GFP to produce is mainly 508nm fluorescence.The support of wild-type GFP molecule comprises many folding β lamellas, by a plurality of little rings, these lamellas is connected.Natural GFP is folding unstablely often can produce false folding at expression in escherichia coli, poorly soluble, and often become after merging with target protein insoluble, a little less than fluorescence, the sex change tolerance that when extracting renaturation, chemical treatment and temperature is caused is poor, thereby is difficult to widespread use.Some scholars have carried out molecular gene engineering science transformation to wild-type GFP, be mainly to introduce to strengthen folding rite-directed mutagenesis and random mutation, therefrom screening obtained folding stronger, fluorescence is brighter, the better GFP misfolded proteins of solvability.For example, the mutant serine of the 65th is (S65T) after Threonine, and the maximum excitation light of this GFP variant is 485nm, and fluorescence emission is 528nm, and albumen self folds very fast and stablizes, and fluorescence intensity has improved 5 times.In addition, enhancement type GFP (enGFP) [Crameri etc. have also been prepared; Nat.Biotechnol.14; 315-319,1996], folded form report Protein G FP[Waldo etc.; NatBiotechnol.17:691-695,1999] and ring is reset and/or the green fluorescent protein variant [Topell.S etc. of acceptor insertion; Methodr Mol Biol.183:31-48,2002 and Topell.S etc.; FFBS Lett.457:283-289,1999 and, Baird.G.S etc.; Proc Natl Acad Sci USA.96:11241-11246,1999].These variants are compared with GFP, folding strength and stability, fluorescence intensity and solvability, and the sex change tolerance that chemical reagent and temperature are caused etc. makes moderate progress.Novel super folding green fluorescent protein (Superfolder green fluorescent protein is abbreviated as SFGFP) [the Pedelacq J-D etc. that particularly in recent years prepare; Nature biotechnolegy.24 (1): 79-88,2006] excellent performance, folding strength and stability, fluorescence intensity and solvability, the sex change tolerance that chemical reagent and temperature are caused improves greatly, during renaturation refolding is fast and active good, though with the chaperone of its fusion whether false folding or insoluble these performances that do not affect SFGFP.

GFP and improvement variant thereof are as the detected report molecule that does not need enzyme substrates or cofactor, be widely used in various types of cells and biological genetic expression and the various researchs of targeting proteins [Tsien.R.Y etc., Annu Rev Biochem.67:509-549,1998], for example, merge with target protein (or be called add the green fluorescent protein tail) as probe at cell inner expression, available fluorescent microscope direct viewing is followed the trail of target protein in intracellular distribution position and dynamic change.In addition, the sensing peptide (as the calmodulin peptide) that some can be had biology, chemistry, electricity and physics characteristic is inserted in GFP, the construction that forms can be used for detecting this sensing peptide to the reaction of inside and outside environmental stimulus (as redox potentials, intracellular ion concentration etc.) [the US Patent No. 7 of Roger.Y.Tsien and Geoffrey Baird, 060,793].

It is strong and weak that thereby the chromophore of discovered in recent years GFP can be changed its fluorescence by the HOCl chlorination, and some scholars utilize this principle to study the neutrophil leucocyte bactericidal mechanism with it.As [Biochemistry.44:6910-6919 such as Palazzolo.A.M, 2005] with the bactericidal properties hypochlorous acid biochemical reaction in the intestinal bacteria research neutrophil leucocyte phagosome that has transformed GFP protein-based thereby demonstration green fluorescence, propose at the H that exists nadph oxidase to produce ₂O ₂Under, the GFP fluorescence that the hypochlorous acid that myeloperoxidase (MPO) chlorine monoxide produces can the cancellation escherichia coli expression, but other oxygenants that neutrophil leucocyte produces can not cancellation GFP.And the viable bacteria green fluorescence of expressing GFP can by the hypochlorous acid cancellation, can not be reached the oxyradical cancellation that is produced by its O-O cracking by peroxynitrite.When making the bacterium contact human neutrophil that is subjected to the conditioning of serum Opsonin, fluorescence microscope is to the bacterium green fluorescence completely dissolve of being engulfed by neutrophil leucocyte, and the bacterial fluorescence outside the neutrophil leucocyte cytolemma is still very strong; When adding MPO inhibitor sodium azide in nutrient solution, this fluorescent quenching phenomenon disappears, and Palazzolo.A.M etc. think that the required hypochlorous acid amount of cancellation bacterial fluorescence is than the high order of magnitude level of bacteria growing inhibiting amount.The hypochlorous acid that these results suggest human neutrophils can produce q.s comes the kill microorganisms cause of disease.[the J Immunol.183 (4): 2632-2641 such as Schwartz.J, 2009] also attempt studying its destiny in the human neutrophil with the streptococcus aureus of expressing GFP fluorescence, find that GFP can be used as a kind of high-sensitive experiment and monitors relevant biochemical reaction in phagosome, the streptococcus aureus subpopulation of identifying tolerance neutrophil leucocyte anti-cell toxic action with probe.Report that separately not only hypochlorous acid can change its fluorescence power by modifying the GFP chromophore, other active oxygen/active nitrogens (ROS/RNS) material is as peroxynitrite [Espey MG etc.; Proc Natl Acad Sci U S A..99 (6): 3481-3486,2002], hydroxyl radical free radical [Chartchalerm etc.; EXCLI Journal.8:89-96,2009] fluorescence that etc. also can cancellation GFP.But that they adopt is all wild-type GFP, and its poor stability a little less than fluorescence, detects hypochlorous specificity in cell, insufficient sensitivity is good.We expect that the performance of SFGFP is better than wild-type GFP greatly, reset super folding green fluorescent protein (circularly permuted Superfolder green fluorescent protein if prepare more kinds of rings, be abbreviated as cpSFGFP), might therefrom select hypochlorous acid is had more selectivity and more highly sensitive novel hypochlorous acid cpSFGFP probe.

We are at [Nature Biotechnolegy.24 (1): 79-88 such as Pedelacq J-D, 2006] on the basis of preparation SFGFP technology, SFGFP is carried out ring transformation rearrangement and the dual transformation of truncated mutant, successfully obtained a plurality of new cpSFGFP mutant.And filter out good, the highly sensitive a plurality of cpSFGFP fluorescent probes of HOCl selectivity, thereby completed the present invention from prepared numerous mutant.Before the present invention, still there is no document openly or reported special hypochlorous acid green fluorescence mutain probe involved in the present invention.

Summary of the invention

First purpose of the present invention is to provide a class and hypochlorous acid is had the encoding gene of the highly sensitive novel green Fluorescence Mutation of A of highly selective albumen.

Second purpose of the present invention is to provide the plasmid vector that comprises this class row novel green Fluorescence Mutation of A protein coding gene.

The 3rd purpose of the present invention is to provide a class hypochlorous acid had the highly sensitive novel green Fluorescence Mutation of A of highly selective albumen.

The 4th purpose of the present invention is to provide the method for utilizing molecular biology method to produce and select above-mentioned green fluorescence mutain and nucleic acid sequence encoding thereof.

The 5th purpose of the present invention is to provide with this class green fluorescence mutain or its encoding gene as the hypochlorous Preliminary Applications of probe in detecting.

The encoding gene that hypochlorous acid is had the highly sensitive novel green Fluorescence Mutation of A of highly selective albumen provided by the invention is that nucleotide sequence is reset the encoding gene of the cpSFGFP that forms by two SFGFP gene orders 1 that a flexible small peptide joint encoding sequence is connected by ring.

this encoding gene is selected from the encoding gene of cpSFGFP39/40 shown in sequence 3, the encoding gene of cpSFGFP91/92 shown in sequence 5, the encoding gene of cpSFGFP129/130 shown in sequence 7, the encoding gene of cpSFGFP140/141 shown in sequence 9, the encoding gene of cpSFGFP145/146 shown in sequence 11, the encoding gene of cpSFGFP189/190 shown in sequence 13, the encoding gene of cpSFGFP214/215 shown in the encoding gene of cpSFGFP195/196 shown in sequence 15 and sequence 17.

this encoding gene can be the encoding gene of the rearrangement cpSFGFP145/146 of N-end brachymemma, for example, the encoding gene of the rearrangement cpSFGFP145/146 (N146) of N-end brachymemma, the encoding gene of the rearrangement cpSFGFP145/146 (N147) of N-end brachymemma, the encoding gene of the rearrangement cpSFGFP145/146 (N148) of N-end brachymemma, the encoding gene of the rearrangement cpSFGFP145/146 (N149) of N-end brachymemma, the encoding gene of the rearrangement cpSFGFP145/146 (N152) of the encoding gene of the rearrangement cpSFGFP145/146 (N151) of N-end brachymemma and the brachymemma of N-end.

this encoding gene can be the encoding gene of the rearrangement cpSFGFP145/146 of C-end brachymemma, for example, the encoding gene of the rearrangement cpSFGFP145/146 (C143) of C-end brachymemma, the encoding gene of the rearrangement cpSFGFP145/146 (C142) of C-end brachymemma, the encoding gene of the rearrangement cpSFGFP145/146 (C141) of C-end brachymemma, the encoding gene of the rearrangement cpSFGFP145/146 (C140) of C-end brachymemma, reset the encoding gene of cpSFGFP145/146 (C139) and the encoding gene that C-holds the rearrangement cpSFGFP145/146 (C138) of brachymemma.

The present invention also provides a kind of recombinant plasmid vector, comprises two SFGFP gene orders 1 that are connected by a flexible small peptide joint encoding sequence that hypochlorous acid is had a highly selective and resets the green fluorescence cpSFGFP mutain encoding gene that forms by ring.

The present invention also provides the host cell that is transformed by above-mentioned recombinant plasmid vector.

The present invention further provides by above-mentioned encoding gene coding, the green fluorescence cpSFGFP mutain that hypochlorous acid is had highly selective by transforming that the intestinal bacteria abduction delivering produces and obtaining by purifying.

The present invention further provides above-mentioned green fluorescence cpSFGFP mutain as the purposes of hypochlorous acid detection probes.

Detailed Description Of The Invention

A little less than original wild-type GFP fluorescence, after merging with target protein, fluorescence is more weak, solvability is poorer, some scholars have carried out genetically engineered (sudden change) transformation to it in succession for many years for this reason, obtained by selecting the GFP that performance makes moderate progress, as the enhancement type GFP (enGFP) of Crameri etc., the green fluorescent protein that ring is reset and/or acceptor inserts of the folded form of Waldo etc. report Protein G FP and Topell.S etc. and Baird.G.S etc. etc.In recent years PedelacqJ-D etc. [2] has prepared the more excellent novel super folding green fluorescent protein (SFGFP) of performance, sees Fig. 1.

Term used herein " super folding green fluorescent protein " refers to that Pedelacq J-D etc. is on " enhancement type GFP (enGFP) " basis of Waldo etc. " folded form report Protein G FP " and Crameri etc., take turns DNA reorganization through 4, further introducing S30R, Y39N, N105T, Y145F, I171V, six new mutants of A206V and ring resets, screening obtains folding and is fused to preferably the super folded formation of a kind of green fluorescent protein of prototype with two green fluorescent proteins, is called super folding green fluorescent protein (SFGFP).SFGFP is much more stable than folded form report Protein G FP and enGFP, and folding intensity is high, fluorescence is strong, solubility good, the tolerance to the Urea treatment sex change during recombinant expressed the extraction is high, refolding fast (fast 3.5 times), active good during renaturation.The Bullfrog RBC ferritin is insoluble protein at expression in escherichia coli separately, and the bacterium colony fluorescence intensity of expressing it is very weak; After Bullfrog RBC ferritin and SFGFP fusion, obtain the good hyperfluorescenceCeng Yongminggaoyingguang fusion rotein of solubility.The fluorescence of Bacillus coli cells of expressing this fusion rotein is stronger 50 times than the bacterial cell of expressing the folded form report Protein G FP that merges with this ferritin, only expresses the bacterial fluorescence of SFGFP also than strong approximately 2 times of the bacterium of only expressing folding report agent GFP.Therefore with its called after " super folding GFP is abbreviated as SFGFP " [Pedelacq J-D etc.; Naturebiotechnolegy.24 (1): 79-88,2006].

Term used herein " ring is reset (or ring transformation) " refers to adopt genetic engineering technique, as polymerase chain reaction (PCR), β lamella in GFP be connected the amino acid whose coding nucleotide sequence of the selected specific site of little ring (except avtive spot) and increase and cause ring to be reset, do not change the basic structure of resetting albumen and biologic activity and may improve its stability although cause producing new C-end and N-end.With this technology to the nucleic acid sequence encoding of 20 multiple protein heavily the row of ring [Rojas.A. etc., Biologia.54:255-277,1999; The .Nat StructBiol.7:580-585 such as Iwakura, 2000], comprise nucleic acid sequence encoding to fluorescin encircle rearrangement [Nagai.T etc., Proc Natl Acad Sci USA 98:3147-3202,2001; Roger.Y.Tsien and Geoffrey Baird.US 7,060,793; And Jason Jui-Hsuan Chiang, Isaac Li and Kevin Truong., Biotech Letters 28:471-475,2006].The present invention adopts Roger.Y.Tsien and Geoffrey Baird US Patent No. 7,060,793.2006 described ring is reset technology, when being fixed a point to increase, the SFGFP encoding gene (sees embodiment 1-2,3), avoid 65-67 amino acids chromophore, locate to increase at 39, or 91 or 129 amino acids codons etc. respectively, cause ring to be reset.Such as take the SFGFP of two fused in tandem as the basis the 39th amino acids codon etc. at them locate to increase, SFGFP is encircled rearrangement, and the mutant sequence that obtains is reset the cpSFGFP (seeing embodiment 1-1) of structure for " the 1-39 amino acids of the 40-239 amino acids+flexible joint of first SFGFP molecule+second SFGFP molecule " forms ring (garden bucket) shape.Pick out numerous protein mutants of obtaining after displacement again and keep former albumen fluorescence intensity or higher and to the reactive better cpSFGFP of hypochlorous acid.

Term used herein " flexible joint " refers to connect the small peptide between two SFGFP albumen in fusion rotein, does not affect the correct folding of two albumen (as SFGFP1 and SFGFP2) but flexibility refers to flexing on space structure.For example, the aminoacid sequence of a short circuit head peptide is GGGS, and its coding nucleotide sequence is GGTGGCGGATCC.

Term used herein " plasmid " or " plasmid vector " refer to usually carry the extra-chromosomal element of gene, and it is not the part of cell centre metabolism, normally the circular double stranded DNA molecular form.This element can be derived from the strand in any source or double-stranded DNA or RNA, autonomously replicating sequence, genome integration sequence, phage or the nucleotide sequence of linear, ring-type or superhelix shape, wherein many nucleotide sequences connect or are recombined in the unique design thing, and this construction can be introduced cell together with 3 ' suitable non-translated sequence with promoter fragment and the DNA sequence dna of selected gene product.

Term used herein " truncated mutant " refers to lack 1 of SFGFP aminoacid sequence N-end or C-end to several amino acid whose sudden changes, and the transformation of corresponding nucleotide sequence.Adopt rite-directed mutagenesis reagent (Site-Directed Mutagenesis) box of TAKARA company in the present invention, build brachymemma clone (seeing Fig. 2) by the method that provides in box.A pair of 5 ' the end of the method design is in abutting connection with the primer of, importing catastrophe point that 3 ' extreme direction is opposite, carry out pcr amplification with high-fidelity DNA polymerase (Pyrobest archaeal dna polymerase), in same system, the PCR product was carried out end smoothing and 5 ' phosphorylation reaction 10 minutes, then carry out self and connect (cyclisation) 0.5-1 hour with the efficient reagent Ligation Solution I that connects.Then select varient with the gained plasmid transformation escherichia coli.

The present invention builds the major technique of various cpSFGFP, that this area is from known " polymerase chain reaction (PCR) ", press J. Pehanorm Brooker, D.W. Russell work, Huang Peitang etc. translate " molecular cloning experiment guide ", the third edition, 597-632 page, in August, 2002, Science Press publishes, Beijing) carry out.Wherein adopt high-fidelity DNA polymerase PFU, take the fundamental chain DNA sequence dna as template, take according to the specific primer of stencil design as extending starting point, by steps such as sex change, amplification, annealing, extensions, external massive duplication goes out the subchain DNA product with the complementation of fundamental chain template DNA.

One aspect of the present invention, provide a class hypochlorous acid to be had the encoding gene of the highly sensitive novel green fluorescin of highly selective mutant cpSFGFP, in some embodiments, the nucleotide sequence of this encoding gene is encircled rearrangement and got by two SFGFP gene orders that flexible joint is connected.Preferably, by genetic engineering technique, first SFGFP albumen the 39th amino acids codon nucleotide sequence of encoding is increased to any site between the nucleotide sequence of second SFGFP albumen the 214th amino acids codon of coding, cause ring to be reset; For example, from the nucleotide sequence of the 39th or 91 or 129 or 140 or 145 or 189 or 195 or 214 amino acids codons of first SFGFP albumen of encoding, the centre comprises a small peptide joint encoding sequence, increase till the nucleotide sequence of the 40th or 92 or 130 or 141 or 146 or 190 or 196 or 215 amino acids codons of second SFGFP albumen of coding, cause ring to be reset, select to obtain the hypochlorous acid change in concentration is had the encoding gene of a plurality of cpSFGFP mutains of sound response.In one embodiment, the encoding gene of the cpSFGFP of described rearrangement is the encoding gene of cpSFGFP39/40.In another embodiment, the encoding gene of the cpSFGFP of described rearrangement is the encoding gene of cpSFGFP91/92.In another embodiment, the encoding gene of the cpSFGFP of described rearrangement is the encoding gene of cpSFGFP129/130.In another embodiment, the encoding gene of the cpSFGFP of described rearrangement is the encoding gene of cpSFGFP140/141.In another embodiment, the encoding gene of the cpSFGFP of described rearrangement is the encoding gene of cpSFGFP145/146.In another embodiment, the encoding gene of the cpSFGFP of described rearrangement is the encoding gene of cpSFGFP189/190.In another embodiment, the encoding gene of the cpSFGFP of described rearrangement is the encoding gene of cpSFGFP195/196.In also having an embodiment, the encoding gene of the cpSFGFP of described rearrangement is the encoding gene of cpSFGFP214/215.

Another aspect of the present invention, provide by genetic engineering technique further to above-mentioned through ring reset select to hypochlorous acid selectivity and remolding sensitivity cpSFGFP preferably, cpSFGFP145/146 albumen for example,

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1 or 2 or 3 or 4 or 5 or 6 or 7 amino acid of its C-end or N-end, selection obtains the hypochlorous acid change in concentration is had a plurality of cpSFGFP mutains of sound response, the invention provides the encoding gene of this class cpSFGFP mutain.In a class embodiment, provide the encoding gene of the rearrangement cpSFGFP145/146 of N-end brachymemma.The encoding gene of the rearrangement cpSFGFP145/146 (N146) of N-end brachymemma is provided in one embodiment.The encoding gene of the rearrangement cpSFGFP145/146 (N147) of N-end brachymemma is provided in another embodiment.The encoding gene of the rearrangement cpSFGFP145/146 (N148) of N-end brachymemma is provided in another embodiment.The encoding gene of the rearrangement cpSFGFP145/146 (N149) of N-end brachymemma is provided in another embodiment.The encoding gene of the rearrangement cpSFGFP145/146 (N150) of N-end brachymemma is provided in another embodiment.The encoding gene of the rearrangement cpSFGFP145/146 (N151) of N-end brachymemma is provided in another embodiment.In also having an embodiment, provide the encoding gene of the rearrangement cpSFGFP145/146 (N152) of N-end brachymemma.

In another kind of embodiment, provide the encoding gene of the rearrangement cpSFGFP145/146 of C-end brachymemma.The encoding gene of the rearrangement cpSFGFP145/146 (C143) of C-end brachymemma is provided in one embodiment.The encoding gene of the rearrangement cpSFGFP145/146 (C142) of C-end brachymemma is provided in another embodiment.The encoding gene of the rearrangement cpSFGFP145/146 (C141) of C-end brachymemma is provided in another embodiment.The encoding gene of the rearrangement cpSFGFP145/146 (C140) of C-end brachymemma is provided in another embodiment.The encoding gene of the rearrangement cpSFGFP145/146 (C139) of C-end brachymemma is provided in another embodiment.In also having an embodiment, provide the encoding gene of the rearrangement cpSFGFP145/146 (C138) of C-end brachymemma.

Another aspect of the present invention provides a class to comprise the above-mentioned recombinant plasmid vector that hypochlorous acid is had the encoding gene of the highly sensitive novel green fluorescin of highly selective mutant cpSFGFP.In some embodiments, described plasmid vector is prokaryotic expression plasmid, as pET28a plasmid and pRSETb plasmid.In other embodiments, described plasmid vector can be eukaryon expression plasmid, as the PVAX-1 plasmid.For example, in some embodiments, the invention provides pRSETb-cpSFGFP39/40, pRSETb-cpSFGFP91/92, pRSETb-cpSFGFP129/130, pRSETb-cpSFGFP140/141, pRSETb-cpSFGFP145/146, pRSETb-cpSFGFP189/190, pRSETb-cpSFGFP195/196, pRSETb-cpSFGFP214/215 recombinant plasmid.In other embodiments, the invention provides pRSETb-cpSFGFP145/146 (N146), pRSETb-cpSFGFP145/146 (N147), pRSETb-cpSFGFP145/146 (N148), pRSETb-cpSFGFP145/146 (N149), pRSETb-cpSFGFP145/146 (N150), pRSETb-cpSFGFP145/146 (N151) and pRSETb-cpSFGFP145/146 (N152) recombinant plasmid.In other embodiments, the invention provides pRSETb-cpSFGFP145/146 (C143), pRSETb-cpSFGFP145/146 (C142), pRSETb-cpSFGFP145/146 (C141), pRSETb-cpSFGFP145/146 (C140), pRSETb-cpSFGFP145/146 (C139) and pRSETb-cpSFGFP145/146 (C138) recombinant plasmid.

Another aspect of the present invention provides a kind of use host cell that above-mentioned recombinant plasmid vector transforms.In some embodiments, host cell is Bacillus coli cells.In other embodiments, host cell can be mammalian cell.Host cell comprises the cell of people, non-human primate, mouse, ox, pig, sheep, horse, rat, rabbit, dog, cat and cavy.

In another aspect of this invention, provide a class express to produce with above-mentioned recombinant plasmid vector transformed host cell (for example Bacillus coli cells) and purified above-mentioned hypochlorous acid had the highly sensitive novel green fluorescence of highly selective cpSFGFP mutain.The mutain of this class purifying comprises the aminoacid sequence of the SFGFP polypeptide that encircles rearrangement and chromophore wherein, has the highly sensitive activity of hypochlorous acid highly selective.

The present invention also provides with the cpSFGFP mutain and detects hypochlorous purposes, comprising:

1.cpSFGFP when reacting with hypochlorous acid, the fluorescence intensity of cpSFGFP can change along with the variation of hypochlorous acid level, namely reduces along with the rising of hypochlorous acid level, thereby can carry out quantitatively or half-quantitative detection hypochlorous concentration;

2. can cpSFGFP gene and the gene fusion of coding associated protein be passed through to be positioned different subcellular organelles by fusion partner in the plasmid transfered cell again by genetic engineering technique the variation of hypochlorous acid level in the position monitor cell in real time.And traditional hypochlorous acid organic chemistry probe has cytotoxicity, and solubility and poor stability can not be used for cell detection.

Experiment showed, with employing wild-type GFP or its modified version misfolded proteins (as SFGFP) detection hypochlorous acid of bibliographical information and compare, cpSFGFP probe provided by the present invention is sensitiveer to the reaction of hypochlorous acid change in concentration, more stable, and selectivity is better.

CpSFGFP hypochlorous acid probe of the present invention is protein, organism do not had the such cytotoxicity of organic chemistry probe, in the future can consider to utilize prokaryotic cell prokaryocyte or eukaryotic cell expression system, from relevant protein fusion localization and expression in different subcellular organelle in cell, intracellular HOCI level is done monitoring in real time.Hypochlorous acid is relevant to many physiology and pathological state as a kind of special active oxygen (ROS) material, estimating that optimistically this probe that the present invention develops not only can be from now on HOCl research a kind of good, stable, detection probes and method fast are provided, can be also that the exploitation of medicine provides the theory and practice foundation by the pass of further investigation HOCl physiological function and disease.

Brief description of drawings

Fig. 1 is SFGFP structure stand schematic diagram.SFGFP comprises the folding enhancing sudden change F64L of enGFP and sudden change F99S, M153T, V163A (black round spot) and 6 new sudden change: S30R, Y39N, N105T, Y145F and the A206V (white round spot) of S65T, folded form report Protein G FP.

Fig. 2 is the schematic diagram of TaKaRa company each step of rite-directed mutagenesis method.

Fig. 3 Ni ²⁺The SDS-PAGE electrophorogram of the SFGFP that column purification obtains and cpSFGFP91/92 protein sample.The purity that shows these two kinds of albumen all reaches more than 95% (sees respectively 1-200 and 2-200 band in figure).

Fig. 4--Fig. 7 shows the reaction to the hypochlorous acid change in concentration with the several representative cpSFGFP of hypochlorous acid burette test detection, and compares with contrast GFP, mCherry, SFGFP.CPxx in figure, Nxxx and Cxxx see respectively

embodiment

1,2 and 3 for writing a Chinese character in simplified form.

Fig. 8 is the peroxynitrite burette test, shows that each cpSFGFP is to the not significantly reaction of variation of peroxynitrite concentration.

Fig. 9 is that superoxide anion detects test, shows that superoxide anion has no significant effect the fluorescence intensity of SFGFP and cpSFGFP91/92, thereby can not detect superoxide anion with them.

Figure 10 is hydroxyl radical free radical detection test, shows that the hydroxyl radical free radical of higher concentration just affects to some extent on the fluorescence intensity of SFGFP and cpSFGFP91/92, thereby can not use the low levels hydroxyl radical free radical in SFGFP and cpSFGFP91/92 detection cell.

Figure 11 is that singlet oxygen detects test, shows that SFGFP and cpSFGFP91/92 are to the reactivity of singlet oxygen; CpSFGFP91/92 is very responsive to HOCl, and to HOCl and H ₂O ₂The common singlet oxygen that produces that exists is insensitive.

Figure 12 shows with the hypochlorous result of cpSFGFP91/92-mCherry fusion rotein detection, show that the mCherry in this fusion rotein does not still react the hypochlorous acid change in concentration, and cpSFGFP91/92 wherein is fairly obvious to the reaction of hypochlorous acid change in concentration, thereby can more advantageously be used as the highly sensitive detection probes of hypochlorous highly selective with mCherry as negative control.

Embodiment

Below the present invention is further elaborated with embodiment.These embodiment only are used for illustrating the present invention, and scope of the present invention are not consisted of any restriction.The main genetically engineered molecular biology cloning process that adopts routine in embodiment, these methods are well known to those of ordinary skill in the art, such as: " the molecular biology experiment reference manual " of Jian Luosikaimusi etc. and J. Pehanorm Brooker, D.W. the Russell is outstanding, Huang Peitang etc. translate: " molecular cloning experiment guide " (third edition, in August, 2002, Science Press publishes, Beijing) in relevant chapters and sections.Those of ordinary skills are according to following examples, the slightly modified and conversion be not difficult as the case may be and successfully implement the present invention, and these are revised and conversion all drops in the scope of the application's claim.

PET28a plasmid vector used in embodiment is available from Novagen company, the pRSETb plasmid vector is available from Invitrogen company, be prokaryotic expression carrier, contain His label, ampicillin resistance gene, T7 promotor and lacI gene, IP available TG (isopropylthiogalactoside) abduction delivering target protein in prokaryotic organism.

The primer of the PCR that is useful on is synthetic by Shanghai living work biotechnology company limited, purifying and correct through the mass spectroscopy evaluation.The restriction enzyme such as NdeI, BamHI, T4 ligase enzyme are accompanied with 10 * Tango available from Fermatas company during purchase ^TMDamping fluid etc.The T4 ligase enzyme is divided into lower concentration enzyme (1U/mL) and high-concentration enzyme (5U/mL), and the former is used for sticky end and connects, and the latter is used for flat end and connects.Chemical reagent is all available from Solution on Chemical Reagents in Shanghai company of traditional Chinese medicines groups.

Embodiment 1.pRSETb-cpSFGFP cloned plasmids Vector construction

1.SFGFP construction of recombinant plasmid

Pass through computer internet Http:// www.ncbi.nlm.nih.gov/protein/2B3P ARetrieval obtains SFGFP coding gene sequence (seeing sequence table).For better expressing in prokaryotic organism, KOZACK sequence (GTG) is inserted in design after the 3rd base of former sequence, then synthesizes this sequence as template by the full gene of Nanjing Genscript Biotechnology Co., Ltd..Design primer P1 (forward, TATT CATATGATGGTGAGCAAGGGCGAGGAG) and P2 (oppositely, TATT GGATCCGCCACCCTTGTACAGCTCGTCCATGCC), see the primer list.P1 contains the NdeI restriction enzyme site, and P2 contains joint (GGTGGCGGATCC), comprising the BamHI restriction enzyme site.Take the SFGFP encoding gene as template, pcr amplification SFGFP nucleotide sequence.

PFU is available from TIANGEN Biotech (Beijing) Co., Ltd., and is subsidiary when 10 * damping fluid, dNTP are the PFU purchase.

The PCR product separates purpose amplicon DNA band by 1% agarose gel electrophoresis, reclaims this amplicon with the DNA gel recovery test kit of Canadian BBI company.Amplicon with NdeI, BamHI difference double digestion plasmid vector pET28a and purifying.

After reaction finishes, reclaim test kit with U.S. Axygen cleaning, by its specification sheets operation, reclaim the amplicon enzyme and cut product; Reclaim test kit with the DNA gel of Canadian BBI company simultaneously and reclaim digested plasmid after 1% agarose gel electrophoresis.To both carry out ligation, obtain pET28a-SFGFP1-joint recombinant plasmid.

2.pET28a-SFGFP 1-SFGFP2 construction of recombinant plasmid

Design primer P3 (forward, TATT GGATCCATGGTGAGCAAGGGCGAGGAG) and P4 (oppositely, TATT CTCGAGTTAGCCACCCTTGTACAGCTCGTCCATGCC), see the primer list.P3 contains the BamHI restriction enzyme site, and P4 contains the XhoI restriction enzyme site.Take the SFGFP coding gene sequence as template, same as above, carry out pcr amplification SFGFP nucleotide sequence, the amplicon DNA in separation and recovery product; PET28a-SFGFP1-joint recombinant plasmid with BamHI, XhoI difference this amplicon of double digestion and the present embodiment 1 preparation, reclaiming separately, enzyme carries out ligation after cutting product, SFGFP is inserted in above-mentioned pET28a-SFGFP1-joint recombinant plasmid, obtains pET28a-SFGFP1-joint-SFGFP2 recombinant plasmid (SFGFP1 is identical with SFGFP2).The nucleotides sequence of the present embodiment center tap is classified GGTGGCGGATCC as, and its encoding amino acid sequence is GGGS.

3.pRSETb-cpSFGFP serial construction of recombinant plasmid

Take cpSFGFP39/40 as example.

Take pET28a-SFGFP1-joint-SFGFP2 as template, begin (forward, GGGG to the design of this section nucleotide sequence between SFGFP2 the 117th (corresponding to 40 amino acids) primer P5 according to the 118th bit base (corresponding to 39 amino acids) of SFGFP1 AGATCTAATGGCAAGCTGACCCTGAAG) and P6 (oppositely, GGGG GAATTCGGTGGCATCGCCCTCGC), see the primer list.P5 contains the BglII restriction enzyme site, and P6 contains the EcoRI restriction enzyme site, and is same as above, carries out pcr amplification, the amplicon in separation and recovery product; With BglII, EcoRI difference this amplicon of double digestion and pRSETb plasmid, reclaiming separately, enzyme carries out ligation after cutting product, connect and insert in the pRSETb carrier, obtain pRSETb-cpSFGFP39/40 plasmid (wherein the 39/40th amino acids of 39/40 finger SFGFP).

In the same way, built the plasmid vector of other cpSFGFP genes that contain following ring rearrangement, restriction enzyme site used is identical with structure pRSETb-cpSFGFP39/40 (being abbreviated as CP39), list in but the primer is different following (the former is forward primer, and the latter is reverse primer):

PRSETb-cpSFGFP91/92 (being abbreviated as CP91):

Primer P7 (sequence 49): GGGG AGATCTGGCTACGTCCAGGAGCG,

Primer P8 (sequence 50): GGGG GAATTCTTCGGGCATGGCGGACTTG;

PRSETb-cpSFGFP129/130 (being abbreviated as CP129):

Primer P9 (sequence 51): GGGG AGATCTGACTTCAAGGAGGAC,

Primer P10 (sequence 52): GGGG GAATTCGATGCCCTTCAGCTC;

PRSETb-cpSFGFP140/141 (being abbreviated as CP140):

Primer P11 (sequence 53): GGGG AGATCTAAGCTGGAGTACAACTTCAAC),

Primer P12 (sequence 54): GGGG GAATTCGTGCCCCAGGATGTTGCC);

PRSETb-cpSFGFP145/146 (being abbreviated as CP145):

Primer P13 (sequence 55): GGGG AGATCTTTCAACAGCCACAACGTCTATATC),

Primer P14 (sequence 56): GGGG GAATTCGTTGTACTCCAGCTTGTGC);

PRSETb-cpSFGFP189/190 (being abbreviated as CP189):

Primer P15 (sequence 57): GGGG AGATCTGGCGACGGCCCCGTGCTGCTG),

Primer P16 (sequence 58): GGGG GAATTCGATGGGGGTGTTCTGCTG);

PRSETb-cpSFGFP195/196 (being abbreviated as CP195):

Primer P17 (sequence 59): GGGG AGATCTCTGCCCGACAACCAC),

Primer P18 (sequence 60): GGGG GAATTCCAGCACGGGGCCGTCG);

PRSETb-cpSFGFP214/215 (being abbreviated as CP214):

Primer P19 (sequence 61): GGGG AGATCTAAGCGCGATCACATGGTC),

Primer P20 (sequence 62): GGGG GAATTCCTCGTTGGGGTCTTTGCTCAGC).

Embodiment 2.pRSETb-cpSFGFP145/146N-end truncated mutant series cloned plasmids Vector construction

Employing TaKaRa company rite-directed mutagenesis test kit is introduced sudden change in primer, contain the linearized vector of the cpSFGFP gene of truncated mutant by pcr amplification, the linearized vector recirculation is connected to obtain mutant plasmid (seeing Fig. 2) at last.The method of TAKARA company is according to [Methods Enzymol.1987.154.367-382 and Proc Natl Acad Sci U S be (2): 488-492 A.1985.82.] such as Kunkel TA and Shaner NC etc., Nat Biotechnol.22 (12): 1567-1572,2004] report method slightly make improvements (seeing Fig. 2).

Begin to carry out amino acid truncated mutant one by one from second amino acid of the N of pRSETb-cpSFGFP145/146 end.For example to clip first amino acid of the N end of cpSFGFP145/146, i.e. the 144th amino acids of SFGFP.Design primer P21 (forward primer contains deletion segment, AACAGCCACAACGTCTATATC) and P22 (reverse, GATATGTATATCTCCTTCTTA).Carry out pcr amplification take pRSETb-cpSFGFP 145/146 as template, then the PCR product is carried out 5 ' phosphorylation reaction, then recirculation ligation, obtain pRSETb-cpSFGFP145/146 (N146).

Phosphorylation reaction refers to 5 ' end base of PCR product is carried out the phosphate group addition reaction.The plasmid that extracts in microorganism or genome end all contain phosphate group, and the PCR product does not have.Only have and contain endways the phosphate group DNA molecular ligation could occur.The recirculation ligation refers to 3 ' end and 5 ' the end ligation of linearized vector.

PrimeSTAR is high-fidelity DNA polymerase, and its fidelity is also higher than PFU, the overlength fragment that is mainly used in increasing (as carrier or exceed the Long fragment gene of PFU amplification scope).PrimeSTAR is accompanied with 5 * PrimeSTAR damping fluid available from TAKARA company.

T4PNK is writing a Chinese character in simplified form of T4 polynueleotide kinase, is used for the addition reaction to 5 ' end phosphate group of DNA molecular.T4PNK attaches 10 * T4PNK damping fluid available from NEB company.

In the same way, contain N end brachymemma clone's plasmid vector below having built, but the primer is different, lists in respectively thereafter (the former is forward primer, and the latter is reverse primer):

pRSETb-cpSFGFP145/146(N147)：

Primer P23 (sequence 65): AGCCACAACGTCTATATCACG,

Primer P22 (sequence 64): GATATGTATATCTCCTTCTTA;

pRSETb-cpSFGFP145/146(N148)：

Primer P24 (sequence 66): CACAACGTCTATATCACGGCC,

Primer P22 (sequence 64): GATATGTATATCTCCTTCTTA;

pRSETb-cpSFGFP145/146(N149)：

Primer P25 (sequence 67): AACGTCTATATCACGGCCGAC,

Primer P22 (sequence 64): GATATGTATATCTCCTTCTTA;

pRSETb-cpSFGFP145/146(N150)：

Primer P26 (sequence 68): GTCTATATCACGGCCGACAAG,

Primer P22 (sequence 64): GATATGTATATCTCCTTCTTA;

pRSETb-cpSFGFP145/146(N151)：

Primer P27 (sequence 69): TATATCACGGCCGACAAGCAG,

Primer P22 (sequence 64): GATATGTATATCTCCTTCTTA;

pRSETb-cpSFGFP145/146(N152)：

Primer P28 (sequence 70): ATCACGGCCGACAAGCAGAAG,

Primer P22 (sequence 64): GATATGTATATCTCCTTCTTA.

Embodiment 3.pRSETb-cpSFGFP145/146C end truncated mutant series cloned plasmids Vector construction method and step are with embodiment 2.

Begin to carry out amino acid truncated mutant one by one from second amino acid of the C of pRSETb-cpSFGFP145/146 end.For example to clip first amino acid of the C end of cpSFGFP145/146, i.e. the 143rd amino acids of SFGFP.Design primer P29 (forward, TGATCCGGCTGCTAACAAAGCCCG) and P30 (reverse primer contains deletion segment, GTACTCCAGCTTGTGCCCCAGG).Carry out pcr amplification take pRSETb-cpSFGFP 145/146 as template, then the PCR product is carried out 5 ' phosphorylation reaction, then recirculation ligation, obtain pRSETb-cpSFGFP 145/146 (C 143).

In the same way, contain C end brachymemma clone's plasmid vector below having built, but the primer is different, lists in respectively thereafter (the former is forward primer, and the latter is reverse primer):

pRSETb-cpSFGFP145/146(C142)：

Primer P29 (sequence 71): TGATCCGGCTGCTAACAAAGCCCG,

Primer P31 (sequence 73): CTCCAGCTTGTGCCCCAGGATG;

pRSETb-cpSFGFP145/146(C141)：

Primer P29 (sequence 71): TGATCCGGCTGCTAACAAAGCCCG,

Primer P32 (sequence 74): CAGCTTGTGCCCCAGGATGTTG;

pRSETb-cpSFGFP145/146(C140)：

Primer P29 (sequence 71): TGATCCGGCTGCTAACAAAGCCCG,

Primer P33 (sequence 75): CTTGTGCCCCAGGATGTTGCCG;

pRSETb-cpSFGFP145/146(C139)：

Primer P29 (sequence 71): TGATCCGGCTGCTAACAAAGCCCG,

Primer P34 (sequence 76): GTGCCCCAGGATGTTGCCGTCC;

pRSETb-cpSFGFP145/146(C138)：

Primer P29 (sequence 71): TGATCCGGCTGCTAACAAAGCCCG,

Primer P35 (sequence 77): CCCCAGGATGTTGCCGTCCTCC.

The structure of embodiment 4.pRSETb-mCherry recombinant plasmid vector

MCherry is the cherry-red fluorescin, be coral polyp red fluorescent protein DsRed a mutant [Shaner etc., Nat Biotechnol..22 (12): 1567-1572.2004].Its maximum excitation, emission wavelength are respectively 587nm, 610nm.The fluorescin mCherry that introduces a different colours in bacterium and the experiment of viable cell microscope can highlight cpSFGFP to hypochlorous reactivity as negative control, and the present invention has built this plasmid.

MCherry encoding gene (double chain DNA molecule) is synthetic by the full gene of Nanjing Genscript Biotechnology Co., Ltd..Take the mCherry encoding gene as template, design contains forward primer P36 (sequence 78, the TATT of BamHI restriction enzyme site GGATCCGGCCACCATGGTGAGCAAG), the reverse primer P37 (sequence 79, the CTTT that contain the EcoRI restriction enzyme site GAATTCCTACTTGTACAGCTCGTCCATG).Press the identical method of embodiment 1-1 and step, carry out pcr amplification, product connects in the pRSETb carrier with BamHI, EcoRI double digestion, obtains the pRSETb-mCherry recombinant plasmid.

The structure of embodiment 5.pRSETb-cpSFGFP91/92-mCherry clone recombinant plasmid vector

MCherry with mCherry and cpSFGFP91/92 amalgamation and expression, detects fusion rotein to hypochlorous reaction to not reaction of hypochlorous acid.Express this fusion rotein and can guarantee that both concentration is consistent in bacterium and viable cell, mcherry can be used as cpSFGFP91/92 to the negative control of hypochlorous acid reaction, more is conducive to microscope direct observing.

Owing to not having suitable restriction enzyme site to use on the pRSETb-cpSFGFP91/92 recombinant plasmid, need to introduce respectively suitable restriction enzyme site with pcr amplification at this carrier two ends.

1) design primer P38 (sequence 80, forward, TATT GTCGACGCCACC ATGGTGAGCAAGG), P39 (sequence 81, oppositely, TTAT GGTACCCTTGTACAGCTCGTCCATGCC), wherein forward primer P38 contains the SalI restriction enzyme site, reverse primer P39 contains the KpnI restriction enzyme site, press the identical method of embodiment 1-1 and step, carry out pcr amplification as template with archaeal dna polymerase PFU take mCherry encoding gene (Jin Sirui company is synthetic), obtain containing the mCherry gene order of restriction enzyme site.

2) design primer P40 (sequence 82, forward, TATT GGTACCTGATCCGGC TGCTAACAAAGC), P41 (sequence 83, oppositely, TTAT GTCGACAGCTTCGAATTCTTCGGGCATG), wherein forward primer P40 contains the KpnI restriction enzyme site, reverse primer P41 contains the SalI restriction enzyme site, press the identical method of embodiment 2 and step, carry out pcr amplification take pRSETb-cpSFGFP91/92 as template with the PrimeSTARDNA polysaccharase, obtain containing the carrier pRSETb-cpSFGFP91/92 of restriction enzyme site.

The mCherry gene order that 3) will contain restriction enzyme site is passed through respectively KpnI, SalI double digestion with the carrier pRSETb-cpSFGFP91/92 that contains restriction enzyme site, connects, and obtains the pRSETb-cpSFGFP91/92-mCherry recombinant plasmid.Concrete steps are with embodiment 1-1.

Each protein expression of embodiment 6.cpSFGFP, purifying and Purity

1. according to the Pehanorm Brooker etc., " molecular cloning experiment guide ", chapter 1, scheme 1.25 routine operations, preparation competence e. coli jm109 is with above-mentioned different recombinant plasmid transformed.The single positive bacterium colony of picking moves to the 5mLLB substratum from the recombinant plasmid transformed flat board, 37 ℃ of overnight incubation.Second day is got and is cultivated bacterium switching kind of a 100mLLB substratum Erlenmeyer flask, 37 ℃ shaking culture 2-3 hour.Bacterium liquid OD600 adds the IPTG abduction delivering of final concentration 0.7mM when increasing to about 0.6-0.8, cultivated 22-23 hour for 18 ℃.4000rpm collected bacterial sediment ,-20 ℃ of preservations in centrifugal 30 minutes.

2. the centrifuge tube that will contain above-mentioned thalline suspension is placed on ice, ice-bath ultrasonic ripple smudge cells, and ultrasonic apparatus is set: probe Φ 15, power 50%, work 1s, interval 4s, total working 300s.4 ℃, the thalline suspension after the 10000rpm centrifugal breaking 30 minutes, separation of supernatant and precipitation.

3. use self-chambering Ni ²⁺Affinity chromatography column purification desired recombinant protein

Gel media: the Ni2+ chelating SepharoseFastFlow of General Corporation (GEHealthcare), 6mL post (GEHealthcare) and 0.22um filter membrane pad.By the instructions book operation, add Ni2+ affinity chromatography gel dress post;

Level pad: 20mM sodium phosphate, 500mMNaCl, 10mM imidazoles, pH7.4

Lavation buffer solution: 20mM sodium phosphate, 500mMNaCl, 50mM imidazoles, pH7.4

Elution buffer: 20mM sodium phosphate, 500mMNaCl, 200mM imidazoles, pH7.4

Cleaning buffer: 20mM sodium phosphate, 500mMNaCl, 500mM imidazoles, pH7.4

Adopt the run by gravity mode, first with the degassed washed with de-ionized water post of the suction filtration of 5-10 times of column volume.Then with the level pad balance columns of 5-10 times of column volume.Get the thalline supernatant liquor upper prop of separation, collect stream and wear liquid.With the level pad balance pillar of 20 times of column volumes, then remove unconjugated foreign protein with the lavation buffer solution of 5 times of column volumes after end of the sample; Use again the elution buffer wash-out of 5 times of column volumes, collect the desirable proteins liquid of wash-out.Clean post with residual removing in conjunction with foreign protein by force with 10 times of cleaning buffers.Using ultrapure water instead cleans to pH neutral.If temporarily need not add 20% ethanol 4 degree preserves.

With Ni ²⁺Each albumen dress dialysis tubing of column purification is placed in the PBS damping fluid of 2L precooling, and 4 ℃ are stirred dialysis.Changed one time the PBS damping fluid in every 4-6 hour, collect protein liquid after 4 times and preserve.

4.Ni ²⁺After column purification, the conventional SDS-PAGE electrophoresis of protein sample is identified

Get 10 μ g samples and carry out Purity, with the QuantityOne software analysis of U.S. Bio-Rad company, the purity of the SFGFP in sample and cpSFGFP91/92 albumen all reaches (Fig. 3,1-200 and 2-200 band) more than 95%.Fig. 3 left side half is SFGFP, and right side half is cpSFGFP91/92.AS=bacteria lysis supernatant liquor, the precipitation after the AP=bacteria lysis, Ni on the FT=sample ²⁺The stream of collecting during post is worn liquid, Ni on the Ba=sample ²⁺The component that post back balance liquid is collected, 50=washings foreign protein component, 200 SFGFP or the cpSFGFP91/92 components that obtain for wash-out, the M=Fermentas #SM0431 of company molecular weight of albumen standard substance (7 μ L).Other sample applied sample amounts are 10 μ g.

The SFGFP molecular weight that pRSETb-SFGFP expresses is 30.6KDa, and each cpSFGFP molecular weight of albumen that pRSETb-cpSFGFP expresses is 30～32KDa.

The fluorescence intensity of several cpSFGFP fluorescins of embodiment 7. and SFGFP relatively

Green fluorescent protein excitation wavelength in the present invention is that 488nm, emission wavelength are 507nm.

Getting 200 several albumen of μ L0.1 μ M adds respectively in the different holes of 384 hole black fluorescent plates (GreinerBioOne company), with the multi-functional microplate reader of Synergy2 (U.S. BioTek company), 485/20nm filter disc exciting light, the 528/20nm filter disc is surveyed emitting fluorescence (U.S. BioTek company), measures fluorescence intensity.

485/20nm refers to wavelength centered by 485nm, the 20nm bandwidth, and namely the excitation wavelength that produces of this filter disc is 465～505nm.528/20nm refers to wavelength centered by 528nm, the 20nm bandwidth, and namely to survey wavelength be the fluorescence of 508～548nm to this filter disc.

Table 1

Get relatively fluorescence of four kinds of cpSFGFP and SFGFP, the results are shown in Table 1, show that the fluorescence of cpSFGFP91/92, cpSFGFP145/146 and cpSFGFP214/215 is more intense, roughly equal or higher with SFGFP.Can see below two kinds of cpSFGFP brighter because of folding stronger fluorescence, be suitable for the horizontal direct-detection of cell and live body and observe.

The fluorometric titration of the different fluorescins of embodiment 7. hypochlorous acid cancellation detects

According to Palazzolo[Biochemistry.2005.44.6910-6919], Schwartz.J[JImmunol.183 (4): 2632-2641,2009] report HOCl titration experiments method carry out.

Replace hypochlorous acid with clorox, clorox is available from Shanghai Ling Feng chemical reagent company limited, chemical pure; Measure the absorption value under 302nm before each titration, hypochlorous acid has characteristic absorbance under 302nm, and molar extinction coefficient is 1670M ^-1m ^-1

Each fluorescin diluted sample to 0.1 μ M with 100mM sodium phosphate pH7.4 damping fluid after with purifying gets 200 μ L and adds in 96 hole black fluorescent plates, and 37 ℃ of preheatings are 10 minutes in the multi-functional microplate reader of Synergy2.

With 1mMNaOH, NaOCl evenly is diluted to 425 μ M.

After protein sample is pre-thermally-stabilised, the beginning titration experiments.Each every hole adds 3 μ L425 μ MNaOCl with Manual liquid transfering device, after the mixing 5s that vibrates in microplate reader, with the 528/20nm fluorescent value of 485/20nm optical excitation, mensuration emission.And then add 3 μ L425 μ MNaOCl, and measure the emitting fluorescence value with method, so progressively improve hypochlorous acid concentration duplicate detection 10 times.

As get GFP, mCherry, SFGFP, four kinds of albumen of cpSFGFP91/92 carry out above-mentioned titration experiments (wherein GFP is this research department's purification storage), the results are shown in Figure 4.As seen cpSFGFP91/92 is better than contrast GFP, mCherry, SFGFP to hypochlorous reaction.When the accumulation of hypochlorous acid concentration reaches 50 μ M, the fluorescent quenching of GFP, mCherry, SFGFP only drops to respectively 81%, 98%, 62% of initial fluorescence, and the fluorescent quenching of cpSFGFP91/92 drops to below 10%, shows highly sensitive to hypochlorous acid change in concentration reaction of cpSFGFP91/92.

Fig. 5 shows, when the accumulation of HOCl concentration reaches 50 μ M, the fluorescent quenching of contrast GFP, mCherry, SFGFP only drops to respectively 84%, 61% of initial fluorescence, and the fluorescent quenching of cpSFGFP214/215 drops to below 10%, shows highly sensitive to hypochlorous acid change in concentration reaction of cpSFGFP214/215.

The left figure of Fig. 6 shows that each cpSFGFP (non-brachymemma) is faster than SFGFP to the speed of response of hypochlorous acid change in concentration.Right figure shows, when HOCl concentration accumulation is increased when reaching 50 μ M, the fluorescent quenching of contrast GFP, SFGFP only drops to respectively 84%, 61% of initial fluorescence, and the fluorescent quenching of cpSFGFP145/146 drops to below 10%, shows highly sensitive to hypochlorous acid change in concentration reaction of cpSFGFP145/146.And the fluorescence intensity of cpSFGFP145/146 is also more intense, and we select cpSFGFP145/146 is carried out carrying out hypochlorous acid titration detection after N end and C end brachymemma transformation again for this reason.

Fig. 7 shows, a series of SFGFP mutant such as cpSFGFP145/146C end truncated protein, N end truncated protein higher than SFGFP, show that a plurality of mutant of one of these cpSFGFP145/146 two ends brachymemma are all very responsive to the hypochlorous acid change in concentration to the significant reaction of hypochlorous acid change in concentration.

Embodiment 9. hypochlorous acid cpSFGFP probes detect the selectivity of reactive oxygen species

In organism, reactive oxygen species is a lot, as peroxynitrite, superoxide anion, hydroxyl radical free radical etc.

1. peroxynitrite (Peroxynitrite) titration is detected

Carry out according to the described peroxynitrite titration experiments method of the report such as Espey MG [Proc Natl Acad Sci USA.99 (6): 3481-3486.2002].

From-20 ℃ or-80 ℃ of NaOH solution that take out peroxynitrite, be placed on ice until it dissolves fully.Each cpSFGFP albumen with 100mM sodium phosphate pH7.4 damping fluid after with purifying is diluted to 0.1 μ M, gets respectively 200 μ L and adds in each hole of 96 hole enzyme plates, and 37 ℃ of preheatings are 10 minutes in microplate reader.

1mMNaOH with precooling is diluted to 750 μ M with peroxynitrite.After protein sample is pre-thermally-stabilised, the beginning titration experiments.Each every hole adds 5 μ L750 μ MPeroxynitrite with Manual liquid transfering device, after the mixing 5s that vibrates in microplate reader, with the 528/20nm fluorescent value of 485/20nm optical excitation, mensuration emission.And then add 5 μ L750 μ M peroxynitrites, and measure the emitting fluorescence value with method, so progressively improve peroxynitrite concentration duplicate detection 10 times.The results are shown in Figure 8.

The albumen of all tests to the reacting phase of peroxynitrite change in concentration is seemingly done their speed of reaction of power exponent decay match with SigmaPlot and is only fluctuated approximately 1～2 times, not significantly reaction.

2. to superoxide anion (Superoxide, O ₂ ^-) detection

According to report [Biochim Biophys Acta.1760 (11): 1690-1695 such as Bou-AbdallahF, 2006] superoxide anion detection method is carried out, and generates superoxide anion with XOD (XanthineOxidase) catalyzed oxidation xanthoglobulin (Hyperoxanthine)

Take the cpSFGFP91/92 albumen of purifying as example, with 100mM sodium phosphate pH7.4 damping fluid, it is made into 0.1 μ M, getting 50 μ L adds in 384 enzyme plate holes, hole, add successively again 0.5 μ L20mM xanthoglobulin solution and 0.5 μ L10U/mL XOD, with pipettor mixing gently, the standing 5min of room temperature detects the 485/528nm fluorescent value in microplate reader.Two control groups are set simultaneously: 1) only have cpSFGFP91/92 albumen, without xanthoglobulin, without XOD; 2) cpSFGFP91/92 albumen+xanthoglobulin, and without XOD.The results are shown in Figure 9.

Superoxide anion has no significant effect the fluorescence intensity of SFGFP and cpSFGFP91/92, thereby can not detect superoxide anion with them.This has confirmed that also the GFP of bibliographical information has the physiological function of superoxide-dismutase, can consume or the disproportionation of catalysis superoxide anion, but do not affect the self property [Bou-AbdallahF etc., Biochim Biophys Acta.1760 (11): 1690-1695,2006] of superoxide anion.

3. to the detection of hydroxyl radical free radical (Hydroxylradical, OH)

Hydroxyl radical free radical detection method according to the report such as Chartchalerm Isarankura-Na-Ayudhya N [EXCLI Journal.8.89-96.2009] is carried out [9].Under the environment of relatively reduction, hydrogen peroxide can generate hydroxyl radical free radical with cupric ion, ferrous ion reaction.

Get respectively the 200 different fluorescins of μ L0.1 μ M and add in each 1.5mL light-proof centrifuge tube, then add respectively 3 μ L3.8mMCuSO ₄Solution, 7 μ L8.05mMH ₂O ₂, 2.1 μ

L

10 or 100 or 1000 μ M xitix, the vortex mixing to be producing hydroxyl radical free radical, after the standing 5min of room temperature, moves into respectively in 96 hole enzyme plate each holes, with the 485nm optical excitation, measures the intensity of 528nm emitting fluorescence.The results are shown in Figure 10.

The Analysis of test results demonstration, cpSFGFP91/92 is slightly higher than GFP, SFGFP to the reaction of hydroxyl radical free radical.Under the xitix of 0～100 μ M concentration range existed, cpSFGFP91/92 changed within 20% the fluorescence intensity of hydroxyl radical reaction.When ascorbic acid concentrations reaches 1mM, cpSFGFP91/92 fluorescence descends and reaches 57%, and GFP, SFGFP fluorescence are declined by less than 40%, but this concentration considerably beyond cell produce to the hydroxyl radical free radical level, therefore can not detect with cpSFGFP91/92 the hydroxyl radical free radical level of cell sample.With the reacting phase ratio to HOCl, CPSFGFP91/92 is also very poor to the reaction that hydroxy free radical concentration changes.

4. to singlet oxygen (Singlet Oxygen, O ₂( ¹Δ g)) detection

Singlet oxygen detection method according to the report such as Miyamoto S [Proc Natl Acad Sci U S is (2): 293-298.2006 A.103] is carried out.The HOCl that concentration is identical and H ₂O ₂Reaction can generate singlet oxygen.

Get respectively 200 each cpSFGFP albumen of μ L0.1 μ M and add in 96 hole enzyme plate each holes, add respectively HOCl and the H of 0,5,10,20,40,80,100 μ M ₂O ₂Each 100 μ L, mixing after the standing 5min of room temperature, with the 485nm optical excitation, is measured the intensity of 528nm emitting fluorescence to produce singlet oxygen.Three control groups are set simultaneously: 1) cpSFGFP albumen only, without HOCl, without H ₂O ₂2) cpSFGFP albumen+HOCl is without H ₂O ₂3) cpSFGFP albumen+H ₂O ₂, without HOCl.The results are shown in Figure 11.

When concentration ratio is lower, when being 5 μ M and 10 μ M as the concentration of HOCl, the fluorescence intensity of each albumen remains unchanged substantially.When the concentration of HOCl reached 20 μ M, cpSFGFP91/92 began to occur reaction, descended 10% in the fluorescence intensity of front 2 minutes cpSFGFP91/92 of kinetic measurement, and is substantially constant afterwards.When the concentration of HOCl reached 40 μ M, the initial fluorescence value of cpSFGFP91/92 obviously reduced, and keeps slow decreasing in dynamic process, and during only to the HOCl Enrichment its fluorescence decay speed obviously greater than HOCl and H ₂O ₂When jointly existing.As seen cpSFGFP91/92 is very responsive to HOCl, and to HOCl and H ₂O ₂The common singlet oxygen that produces that exists is insensitive.

In sum, ROS/RNS is divided into two classes to the effect of cpSFGFP91/92.One class such as superoxide anion there is no effect to it; Another kind ofly just can affect its fluorescence intensity when only having their concentration to reach higher as peroxynitrite and hydroxyl radical free radical, make cpSFGFP91/92 very high and insensitive to their detection lower limit.But add nmole level ROS Promote cell's growth during cell cultures, micromole's level ROS can cause apoptosis, and mmole level ROS causes necrocytosis.Active oxygen physiological concentration in viable cell is very low, and cpSFGFP can not detect it, just better to the HOCl selectivity, detection sensitivity is high, thereby cpSFGFP of the present invention can be used as the highly sensitive detection probes of hypochlorous highly selective.

Embodiment 10. use cpSFGFP91/92-mCherry fusion roteins detect hypochlorous acid

CpSFGFP91/92-mCherry fusion rotein with 100mM sodium phosphate pH7.4 damping fluid after with purifying is diluted to 0.1 μ M, get 200 μ L and add in 96 hole black fluorescent plates (GreinerBioOne company), 37 ℃ of preheatings are 10 minutes in the multi-functional microplate reader of Synergy2 (U.S. BioTek company).

With 1mMNaOH, NaOCl evenly is diluted to 425 μ M.

After protein sample is pre-thermally-stabilised, the beginning titration experiments.Each every hole adds 3 μ L425 μ MNaOCl with Manual liquid transfering device, after vibration mixing 5s, with 485/528 fluorescent value, then measures 590/645nm fluorescent value (mCherry 590/10nm optical excitation detects the 645/40nm fluorescence of launching) in microplate reader.And then add 3 μ L425 μ MNaOCl, and measure the emitting fluorescence value with method, so progressively improve hypochlorous acid concentration duplicate detection 10 times.The results are shown in Figure 12.

The 590/645nm fluorescence intensity that the left figure of Figure 12 shows the cpSFGFP91/92-mCherry fusion rotein does not increase progressively with HOCl concentration and changes, and right figure shows that its 485/528nm fluorescence intensity increases progressively with HOCl concentration and is changed significantly.Show that the mCherry in this fusion rotein does not still react the hypochlorous acid change in concentration, and cpSFGFP91/92 wherein is fairly obvious to the reaction of hypochlorous acid change in concentration.Cell expressing cpSFGFP can guarantee that with the mCherry fusion rotein both concentration is consistent, can be with mCherry as the negative control of cpSFGFP91/92 to the hypochlorous acid reaction, be conducive to microscope direct observing, thereby the cpSFGFP91/92-mCherry fusion rotein can be more favourable as the highly sensitive detection probes of hypochlorous highly selective.

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Claims

1. encoding gene that hypochlorous acid is had the green fluorescence mutain cpSFGFP of highly selective, two SFGFP gene orders 1 that the nucleotide sequence of described encoding gene is connected by a flexible small peptide joint encoding sequence are reset by ring and are formed, and wherein said encoding gene is selected from:

the encoding gene of the encoding gene of the encoding gene of cpSFGFP91/92 shown in the encoding gene of cpSFGFP39/40 shown in sequence 3, sequence 5, the encoding gene of the cpSFGFP129/130 shown in sequence 7, the cpSFGFP140/141 shown in sequence 9, the encoding gene of the cpSFGFP145/146 shown in sequence 11, the cpSFGFP189/190 shown in sequence 13, the encoding gene of the cpSFGFP195/196 shown in sequence 15 and the encoding gene of the cpSFGFP214/215 shown in sequence 17, the encoding gene of the rearrangement cpSFGFP145/146 of the N-end brachymemma shown in sequence 19, the encoding gene of the rearrangement cpSFGFP145/146 of the N-end brachymemma shown in sequence 21, the encoding gene of the rearrangement cpSFGFP145/146 of the N-end brachymemma shown in sequence 23, the encoding gene of the rearrangement cpSFGFP145/146 of the N-end brachymemma shown in sequence 25, the encoding gene of the encoding gene of the rearrangement cpSFGFP145/146 of the N-end brachymemma shown in sequence 27 and the rearrangement cpSFGFP145/146 of the end of the N-shown in sequence 29 brachymemma, the encoding gene of the rearrangement cpSFGFP145/146 of the C-end brachymemma shown in sequence 31, the encoding gene of rearrangement cpSFGFP145/146 shown in sequence 33, the encoding gene of the rearrangement cpSFGFP145/146 of the C-end brachymemma shown in sequence 35, the encoding gene of the rearrangement cpSFGFP145/146 of the C-end brachymemma shown in sequence 37, the encoding gene of the encoding gene of the rearrangement cpSFGFP145/146 of the C-end brachymemma shown in sequence 39 and the rearrangement cpSFGFP145/146 of the end of the C-shown in sequence 41 brachymemma.

2. a recombinant plasmid vector, comprise the described encoding gene of claim 1.

3. host cell that is transformed by the described recombinant plasmid vector of claim 2.

4. one kind by the described encoding gene of claim 1 coding, the green fluorescence cpSFGFP mutain that hypochlorous acid is had highly selective by transforming that the intestinal bacteria abduction delivering produces and purifying gets.

5. the described cpSFGFP mutain of claim 4 is as the purposes of hypochlorous acid detection probes.