CN101724649B - Method for displaying heterologous proteins on surfaces of cells and product - Google Patents
Method for displaying heterologous proteins on surfaces of cells and product Download PDFInfo
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- CN101724649B CN101724649B CN2008102018945A CN200810201894A CN101724649B CN 101724649 B CN101724649 B CN 101724649B CN 2008102018945 A CN2008102018945 A CN 2008102018945A CN 200810201894 A CN200810201894 A CN 200810201894A CN 101724649 B CN101724649 B CN 101724649B
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Abstract
The invention relates to a method for displaying heterologous proteins on the surfaces of yeast cells and a product. The invention discloses a gene construction, and the recombinant expression heterologous proteins can be displayed on the surfaces of the cells after introducing the gene construction into the cells. The adoption of the gene construction and an expression system can lead the display and the expression effects of the heterologous proteins on the surfaces of the cells to be very excellent, retain good protein activity, conveniently realize the fixation of the heterologous proteins and simplify the complicated steps of protein purification.
Description
Technical field
The invention belongs to biological technical field, more specifically, the present invention relates to a kind of method and product at the cell surface display foreign protein.
Background technology
Along with the development of biotechnology, the albumen with some functionally active is applied to each field more and more.Yet albumen is as there being bioactive material, and itself is very easily in inactivation, and poor stability is preserved very difficulty, and is difficult to recycle.And, the possibility although at present proteic extensive reorganization preparation has become, however also there are many difficulties in proteic separation after expressing and purification, and purification step is complicated.Therefore those skilled in the art are devoted to seek the proteic stability of raising, simplify proteic purifying, immobilization albumen and the method that makes the albumen recycling.
In addition; As a kind of concrete example, organophosphorus or carbamate insecticides are one type of agricultural chemicals commonly used in agriculture prodn, and kind is various, insecticidal spectrum wide, lower-price characteristic because of it has; In agriculture prodn, be widely used, for grain high yield and agricultural development are given security.But because the unreasonable use of agricultural chemicals; Organophosphorus or carbamate pesticide residue exceed standard in the numerous food product; Cause the acute poisoning incident to happen occasionally; The competitive power of people's healthy and agricultural-food in the world market in serious threat, caused the extensive concern of national governments and various circles of society at present.Organophosphorus commonly used at present or carbamate chemicals for agriculture detection method have immunological detection method and enzyme to suppress method.Traditional immunology detection technology can not detect multiple different types of agricultural chemicals simultaneously because of its antibodies specific, in practical application, is restricted.And enzyme suppresses method and has mainly utilized the susceptibility of E.C. 3.1.1.7 to organophosphorus or carbamate chemicals for agriculture, has advantages such as easy, sensitivity, becomes the comparatively general Fast Determination of Pesticide Residue technology of application.E.C. 3.1.1.7 can narrow spectrumly be suppressed by organophosphorus or carbamate chemicals for agriculture; But the E.C. 3.1.1.7 poor stability of free form, inactivation very easily, and its source is limited; Purge process is complicated, and these have all limited its application in production practice widely.Therefore, this area need be found more efficiently acquisition and utilize the method for E.C. 3.1.1.7, and E.C. 3.1.1.7 stability particularly is provided, and simplifies the method for E.C. 3.1.1.7 purification technique.
Summary of the invention
The object of the present invention is to provide a kind of gene constructs, described gene constructs can be at the cell surface expression foreign protein after being imported into suitable cell.
Another object of the present invention is to provide a kind of cell, described cell surface display has foreign protein.
In first aspect of the present invention, a kind of gene constructs is provided, said gene constructs from 5 ' to 3 ' comprises the element that following operability links to each other successively:
(a) glucoamylase signal coding sequence;
(b) encoding sequence of foreign protein; With
(c) encoding sequence of yeast α agglutinin gene C end grappling signal.
In another preference, described foreign protein is selected from (but being not limited to): receptor protein, ligandin, zymoprotein or antibody protein etc.
In another preference, described foreign protein is that length is 20-1200aa; Preferably 50-1000aa; That better is 80-800aa, 100aa according to appointment, 200aa, 300aa, 500aa, 700aa.
In another preference, in the described construction, foreign protein is: E.C. 3.1.1.7.
In another preference, described E.C. 3.1.1.7 is the E.C. 3.1.1.7 of band signal peptide and grappling signal not.
In another preference, described glucoamylase signal coding sequence is the sequence of 1-75 position in the sequence shown in the SEQ ID NO:1; Or
Described E.C. 3.1.1.7 encoding sequence is the sequence of 76-1824 position in the sequence shown in the SEQ ID NO:1; Or
The encoding sequence of described yeast α agglutinin gene C end grappling signal is the sequence of 1858-2817 position in the sequence shown in the SEQ ID NO:1.
In another preference, between element (b) and element (c), comprise a label protein encoding sequence.
In another preference, described label protein is a Flag albumen.
In another preference, described element (a) and (b) and (c) between comprise or do not comprise the connection peptides encoding sequence, described connection peptides has 1-15aa; Preferably has 1-10aa; More preferably has 1-5aa.
In another preference, described E.C. 3.1.1.7 is the drosophila melanogaster E.C. 3.1.1.7.
In another preference, described gene constructs has the sequence shown in the SEQ ID NO:1.
Aspect of the present invention two, a kind of expression vector of reorganization is provided, said expression vector contains described gene constructs.
In another preference, the skeleton plasmid of described expression vector is pYES-DEST52.
In the third aspect of the invention, a kind of cell is provided, described cell is a yeast cell, contains described expression vector in the said cell; Or be integrated with described gene constructs in its genome.
In another preference, described yeast cell is selected from: yeast saccharomyces cerevisiae or pichia spp.
In another preference, in the described gene constructs, the encoding sequence of foreign protein is: the encoding sequence of E.C. 3.1.1.7.
In another preference, described yeast cell is a brewing yeast cell, and presenting and expressing is gone up on its surface has E.C. 3.1.1.7.
In fourth aspect of the present invention, provide a kind of and express the method for foreign protein at cell surface display, said method comprises:
(i) described gene constructs is imported in the yeast cell, obtain reconstitution cell;
(ii) cultivate the reconstitution cell of (i), thereby express foreign protein at cell surface display.
In another preference, in step (i), in the described gene constructs, the encoding sequence of foreign protein is: the encoding sequence of E.C. 3.1.1.7; Thereby step (ii) in, at the cell surface display E.C. 3.1.1.7.
Aspect the of the present invention the 5th, a kind of test kit that detects organophosphorus in the testing sample or carbamate chemicals for agriculture is provided, comprise in the said test kit:
Cell has a gene constructs in the described cell, the encoding sequence of foreign protein is in the described gene constructs: the encoding sequence of E.C. 3.1.1.7; Or
Solid phase carrier, and absorption or be embedded in the cell in the solid phase carrier, have a gene constructs in the described cell, the encoding sequence of foreign protein is in the described gene constructs: the encoding sequence of E.C. 3.1.1.7.
In another preference, also include, but is not limited in the described test kit: the substrate of phosphorylcholine esterase (like vagusstoff or Acetylcholine iodide), developer (like DTNB), microtiter plate (like 96 orifice plates), working instructions.
Aspect the of the present invention the 6th, a kind of method that detects organophosphorus in the testing sample or carbamate chemicals for agriculture is provided, said method comprises:
(1) with cell fixation in solid phase carrier or be suspended in the medium with said cell compatibility, form detection architecture; Have a gene constructs in the described cell, the encoding sequence of foreign protein is in the described gene constructs: the encoding sequence of E.C. 3.1.1.7;
(2) in the detection architecture of (1), add testing sample, mix; With
(3) activity of E.C. 3.1.1.7 in the system of detection (2), thus confirm whether contain organophosphorus or carbamate chemicals for agriculture or its content in the testing sample.
In another preference; In step (3); Comprise: in the detection architecture of step (2), add the substrate of E.C. 3.1.1.7, the hydrolysis situation of substrate is confirmed whether to contain in the testing sample organophosphorus or carbamate chemicals for agriculture or its content through detecting E.C. 3.1.1.7.
In another preference, the substrate of described E.C. 3.1.1.7 is vagusstoff (ATch), or Acetylcholine iodide (AtchI).
In another preference, in step (2), also comprise: control systems is set, does not add testing sample in the described control systems; In the step (3), also comprise: relatively acetylcholine esterase active in acetylcholine esterase active in the detection architecture and the control systems; If acetylcholine esterase active significantly reduces and (as reducing by 10%, preferably reduces by 20% in the detection architecture; More preferably reduce by 40% or lower); Then show and contain organophosphorus or carbamate chemicals for agriculture (quilitative method) in the testing sample.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 .pYES-S-AChE-fag-Ag α construction of recombinant plasmid flow process and collection of illustrative plates.
Fig. 2. carrier pYES-S-AchE-flag-Ag α enzyme is cut evaluation, wherein:
Swimming lane M:DNA Marker is by the λ DNA of HindIII, BamHI and EcoRI digestion;
Swimming lane 1: by the pYES-S-AchE-flag-Ag α of PvuII and Bg1II enzymic digestion.
Fig. 3. the mensuration of acetylcholinesterase enzyme activity, wherein:
Fig. 3 A. blank yeast reaction system enzyme activity determination;
Fig. 3 B. is inductive transformant yeast reaction system enzyme activity determination not;
Transformant yeast reaction system enzyme activity determination after Fig. 3 C. induces.
Embodiment
The inventor is through deep research; Design first obtains a kind of gene constructs; Described gene constructs in importing to cell after; Can make recombinant expressed cell surface display express foreign protein (like E.C. 3.1.1.7), and the presenting and expressing effect of foreign protein on cell surface is excellent especially, can keep good protein-active.Adopt gene constructs of the present invention and expression system, can realize the immobilization of foreign protein easily, and can simplify the loaded down with trivial details step of protein purification.
In optimal way of the present invention, express E.C. 3.1.1.7 at cell surface display, can directly utilize said surface display to have existence that the cell of E.C. 3.1.1.7 detects organophosphorus in the testing sample or carbamate chemicals for agriculture whether or amount.Detect with the E.C. 3.1.1.7 that adopts free form and to compare, the E.C. 3.1.1.7 character that is illustrated in cell surface is more stable, and need not loaded down with trivial details purge process and can use, and has simplified the detection step effectively, has improved detection accuracy.
As used herein, described " operability links to each other " or " operationally being connected in " refer to a kind of like this situation, and promptly the activity of same other part of linear DNA sequence can regulated or control to some part of linear DNA sequence.For example, if the transcribing of promotor control sequence, it is exactly operationally to be connected in encoding sequence so.
As used herein, described " containing ", " having " or " comprising " comprised " comprising ", " mainly by ... constitute ", " basically by ... constitute " and " by ... constitute "; " mainly by ... constitute ", " basically by ... constitute " belong to the subordinate concept of " containing ", " having " or " comprising " with " by ... formation ".
Construction
In order to realize foreign protein is illustrated in the purpose of yeast cell surface, the inventor had once attempted the several genes construction process, had obtained best gene constructs at last, thereby realized the good presenting and expressing of foreign protein.Therefore, the invention provides a kind of gene constructs, said construction from 5 ' to 3 ' comprises the element that following operability links to each other successively: (a) glucoamylase signal coding sequence; (b) encoding sequence of foreign protein; (c) encoding sequence of yeast α agglutinin gene C end grappling signal.
In the fusion rotein of described construction coding, with the signal peptide of glucoamylase signal peptide as foreign protein.The inventor discovers, the glucoamylase signal peptide has good cell surface alignment effect, can instruct the foreign protein of expressing in the kytoplasm to pass cell walls well, finally is illustrated in the surface of yeast cell.As optimal way of the present invention, described glucoamylase signal coding sequence is the sequence of 1-75 position among the SEQID NO:1; Or the sequence of its degeneracy or its varient, as long as described varient encoded protein also remains with all or part of (more than at least 50%, preferably more than at least 80%) activity or function of glucoamylase signal peptide.
In the fusion rotein of described construction coding, with the grappling signal of yeast α agglutinin gene C end grappling signal as foreign protein.When being used for when of the present invention, yeast α agglutinin gene C end grappling signal has very excellent albumen anchoring effect, makes foreign protein well, stably be anchored on cell surface, and keeps good protein-active.As optimal way of the present invention, the encoding sequence of described yeast α agglutinin gene C end grappling signal is the sequence of 1858-2817 position among the SEQ ID NO:1; Or the sequence of its degeneracy or its varient, as long as described varient encoded protein also remains with all or part of (more than at least 50%, preferably more than at least 80%) activity or function of yeast α agglutinin gene C end grappling signal.
As used herein, described " foreign protein " can be the multiple albumen that needs presenting and expressing, and for example, described foreign protein can be (but being not limited to): receptor protein, ligandin, zymoprotein or antibody protein etc.The albumen of other type also is available.
Described acceptor proteinoid for example is a nuclear receptor, like orphan nuclear receptor, steroid receptor or the like.With the ligand binding domain presenting and expressing of acceptor proteinoid or acceptor proteinoid at cell surface, thereby obtain reconstitution cell, described reconstitution cell can be applicable to fields such as drug screening, for example is used to screen the part that is complementary with this receptor.
Described zymoprotein for example is E.C. 3.1.1.7, protein kinase etc.With the zymoprotein presenting and expressing at cell surface, thereby obtain reconstitution cell, described reconstitution cell can be applicable to the enzymic catalytic reaction field.Help keeping the biological activity of zymoprotein, and help the immobilization of enzyme and the recycling of enzyme.
Each element of gene constructs of the present invention is that operability links to each other.Can directly link to each other between described each element, perhaps also can comprise the proper spacing sequence, for example have the catenation sequence of 1-45bp; The catenation sequence that preferably has 1-30bp; The catenation sequence that more preferably has 1-15bp.Described catenation sequence for example is a restriction enzyme site, or the label protein encoding sequence.
The present invention also comprises the recombinant expression vector that contains said construction, and any expression vector of yeast cell to express that is applicable to can be used for preparing expression vector of the present invention, as long as it can duplicate in yeast cell and stablize.A key character of expression vector is to contain replication orgin, promotor, marker gene and translation controlling elements usually.As optimal way of the present invention, described expression vector is selected from (but being not limited to): pYSE-DEST52 (Invitrogen), pPIC9K (Invitrogen) etc.It also is that those skilled in the art are known with the method that makes up recombinant expression vector that construction is cloned into expression vector.
The construction that contains the E.C. 3.1.1.7 encoding sequence
As the preferred especially mode of the present invention, described foreign protein is a kind of zymoprotein.Preferred, described zymoprotein is an E.C. 3.1.1.7.
Described E.C. 3.1.1.7 is the albumen that those skilled in the art know, and its encoding sox also is well known in the art.The encoding sequence of the proteic bioactive fragment of any E.C. 3.1.1.7 can be applied among the present invention.Here, the implication of the proteic bioactive fragment of E.C. 3.1.1.7 is meant as a kind of protein fragments, the proteic all or part of function of its E.C. 3.1.1.7 that still can be kept perfectly (activity as at least 80%, better at least 90% activity).E.C. 3.1.1.7 albumen and encoding sox thereof that aminoacid sequence forms through replacement, disappearance or the interpolation of one or more amino-acid residues also can be applicable among the present invention.Replacement, disappearance or the interpolation of the one or more amino-acid residues of described process and the E.C. 3.1.1.7 albumen that forms also has the proteic all or part of function of E.C. 3.1.1.7.
The source of E.C. 3.1.1.7 and encoding sox thereof is more extensive, for example can separate from fly, fish brain, mammalian etc.As a kind of optimal way of the present invention, described E.C. 3.1.1.7 is the drosophila melanogaster E.C. 3.1.1.7.The aminoacid sequence of described E.C. 3.1.1.7 can be that the sequence shown in the NM_057605 is substantially the same with the GenBank accession number.
As optimal way of the present invention, described E.C. 3.1.1.7 does not have signal peptide and the grappling signal of himself, and its encoding sequence has the sequence of 76-1824 position among the SEQ ID NO:1.
In the fusion rotein of described construction coding, the signal peptide that has replaced E.C. 3.1.1.7 to carry with the glucoamylase signal peptide.The inventor discovers; The signal peptide that the glucoamylase signal peptide carries than E.C. 3.1.1.7 has better cell surface locating effect; The glucoamylase signal peptide can instruct the E.C. 3.1.1.7 albumen of expressing in the kytoplasm to pass cell walls well, finally is illustrated in the surface of yeast cell.As optimal way of the present invention, described glucoamylase signal peptide non-coding sequence is the sequence of 1-75 position among the SEQ IDNO:1.
In the fusion rotein of described construction coding, the grappling signal that has replaced E.C. 3.1.1.7 to carry with yeast α agglutinin gene C end grappling signal.Yeast α agglutinin gene C end grappling signal has more excellent albumen anchoring effect, makes E.C. 3.1.1.7 well, stably be anchored on cell surface, and keeps good enzymic activity.As optimal way of the present invention, the encoding sequence of described yeast α agglutinin gene C end grappling signal is the sequence of 1858-2817 position among the SEQ ID NO:1.
As optimal way of the present invention, between E.C. 3.1.1.7 and yeast α agglutinin gene C end grappling signal, also comprise a label protein encoding sequence, thereby behind accurate translation, help detection of fusion proteins.Preferably some aminoacid sequences are than short label protein, and described label protein for example is a Flag albumen, and it has the encoding sequence shown in the 1825-1848 position among the SEQ ID NO:1.For example, can adopt the antibody (like anti-Flag) of anti-said label protein to detect proteic expression; Perhaps, can adopt the antibody of anti-said label protein to come purifying protein or show said proteic cell.
Cell
The present invention also provides a kind of cell of reorganization, and described cell is a yeast cell, contains described expression vector in the said cell, or is integrated with described gene constructs in its genome.Preferably, described yeast cell is selected from: yeast saccharomyces cerevisiae, pichia spp.Presenting and expressing foreign protein stably on the surface of described cell, thus can directly be used for the relevant application of said foreign protein, as be used for screening of medicaments or medicine performance katalysis or the like.
Method at cell surface display expression foreign protein of the present invention mainly comprises: (i) gene constructs of the present invention is imported in the yeast cell, obtain reconstitution cell; (ii) cultivate the reconstitution cell of (i), thereby express foreign protein at cell surface display.
As optimal way of the present invention; Presenting and expressing E.C. 3.1.1.7 stably on the surface of described cell; Thereby can be used for detecting organophosphorus or carbamate chemicals for agriculture in the testing sample, also can be used for fields such as the relevant environmental protection of E.C. 3.1.1.7, medical science, agricultural, military affairs simultaneously.On enzyme activity level; The reference test that the enzyme activity of the E.C. 3.1.1.7 that the yeast cell surface of the present invention's preparation is showed detects the finished product enzyme powder of buying with the market shows; The E.C. 3.1.1.7 vigor of yeast cell surface of the present invention is very high; Reach 3.06 μ mol/minmL, can meet or exceed the enzyme activity level of the finished product enzyme powder of buying the market fully.
The method that gene constructs is incorporated in the host cell is known in the art, for example can adopt the following method that is selected from: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.Can screen the reconstitution cell of having introduced gene constructs through resistance marker.
The reconstitution cell that obtains can adopt conventional yeast cell cultural method to cultivate, and under the condition that is suitable for the host cell growth, cultivates.Can regulate culture condition according to the suitable scale ground of cultivating, make it expressing protein well.
Described reconstitution cell can be placed in the solution, processes cell suspending liquid and uses.Preferred, described reconstitution cell can be immobilized in the solid phase carrier, thereby is convenient to stably use, and recovery and the recycling of being convenient to cell.Can pass through fixed cell, realize the immobilization of foreign protein (like enzyme), avoided the character of floating preteins unstable, very easily inactivation, can not reusable shortcoming, thereby make corresponding foreign protein to be applied better.
The method of immobilized cell is a technology known in the art, for example can adopt the technology that is selected from below (but being not limited to): (1) absorption method (comprising physisorphtion, ionic bond method etc.); (2) entrapping method (like sodium alginate gel immobilized cell method); (3) covalent cross-linking method.For yeast cell, a kind of optional fixing means is an entrapping method, and it is that cell is embedded in the imporosity of water insoluble carrier equably, and cell can not spill and substrate and product can get into and ooze out.Cell and carrier do not play any association reaction, and cell is in best physiological status.Therefore, the stability of enzyme is high, and vigor is lasting.
Cell fixation common used material (or being called solid phase carrier) for example can be selected from (but being not limited to): (1) polyose (Mierocrystalline cellulose, agar, polydextran gel, Protanal TXF 200, K-carrageenin, DEAE-Mierocrystalline cellulose etc.); (2) protein (osso-albumin, gelatin etc.); (3) inorganic carrier (aluminum oxide, gac, pottery, magnet, silicon-dioxide, kaolin, calcium phosphate gel etc.); (4) synthetic vectors (SEPIGEL 305, PS, resol etc.).
For yeast cell, preferably can adopt sodium-alginate to realize fixing.Can yeast cell be mixed with sodium-alginate, then mixed solution splashed in the calcium chloride solution, make sodiun alginate change water-insoluble calcium alginate gel into, will be embedded in wherein thus, this fixing means is that those skilled in the art are known.
Test kit
The present invention also provides a kind of test kit that detects organophosphorus in the testing sample or carbamate chemicals for agriculture, comprises in the said test kit: surface display of the present invention is expressed the cell (or cell culture) that E.C. 3.1.1.7 is arranged.Perhaps, contain in the described test kit: solid phase carrier, and absorption or the surface display that is embedded in the solid phase carrier are expressed the cell that E.C. 3.1.1.7 is arranged.
In addition, also can comprise in the described test kit being used for the active reagent of enzyme analysis, comprise: the substrate of phosphorylcholine esterase (like vagusstoff or Acetylcholine iodide), developer (like DTNB), microtiter plate (like 96 orifice plates), and working instructions etc.
In addition, when needs, also can comprise the reagent that is used for dilute sample in the described test kit; Or be used for the substratum of culturing cell.
In addition, also can comprise working instructions etc. in the described test kit, be used to instruct the testing staff correctly to use or operate.
Organophosphorus or carbamate chemicals for agriculture detection method
The detection method of described organophosphorus or carbamate chemicals for agriculture is based on following principle: E.C. 3.1.1.7 can narrow spectrumly be suppressed by organophosphorus or carbamate chemicals for agriculture, and the concentration of its inhibiting rate and agricultural chemicals is proportional.Therefore through test sample acetylcholine esterase active is changed, but the residual condition of organophosphorus or carbamate chemicals for agriculture in the rapid detection sample.The enzymic activity of E.C. 3.1.1.7 can be confirmed through the response situation of itself and substrate.
Therefore; The present invention provides a kind of method that detects organophosphorus in the testing sample or carbamate chemicals for agriculture; Said method comprises: surface display of the present invention is expressed cell fixation that E.C. 3.1.1.7 is arranged in solid phase carrier or be suspended in the medium with said cell compatibility in (1), forms detection architecture; (2) in the detection architecture of (1), add testing sample, mix; (3) activity of E.C. 3.1.1.7 in the system of detection (2), thus confirm whether contain organophosphorus or carbamate chemicals for agriculture or its content in the testing sample.
The mensuration of the enzymic activity of E.C. 3.1.1.7 is the known technology of those skilled in the art.But E.C. 3.1.1.7 catalysis nerve conduction meta-bolites (like vagusstoff) hydrolysis; Its hydrolysate can with developer (like the dithio dinitrobenzoic acid; DTNB) reaction produces substance that show color (DTNB can show yellow), is worth over time with the spectrophotometric determination absorbancy; Calculate inhibiting rate, can determine whether have organophosphorus or carbamate chemicals for agriculture and amount in the product through inhibiting rate.Preferably, can come to reflect more accurately the changing conditions of enzymic activity through control systems is set.
Described " with the medium of said cell compatibility " is meant that the enzyme to said cell and cell surface display does not all exert an influence, and the medium that does not exert an influence for the interaction of the interaction of organophosphorus or carbamate chemicals for agriculture and E.C. 3.1.1.7 and E.C. 3.1.1.7 and its substrate.Described medium can be suitable reaction buffer for example, like sodium phosphate buffer.
Through the embodiment checking; Cell surface display has the cell detection system of E.C. 3.1.1.7 when being applied to Pesticides Testing; All has very sensitive detection effect for many kinds of organic phosphates and carbamate chemicals for agriculture; Particularly for agricultural chemicals such as Propoxur 97, MTMC, furans pellet, 503nhibiting concentration (IC
50) be up to 0.00073 μ g/mL.
Major advantage of the present invention is:
(1) the present invention has found and can make the method for foreign protein presenting and expressing in yeast cell surface well, utilizes the expressive host of yeast as foreign protein; Adopt method and system of the present invention, the presenting and expressing effect of foreign protein on cell surface is excellent especially, can keep good protein-active, can realize the immobilization of foreign protein easily, and can simplify the loaded down with trivial details step of protein purification.
(2) the present invention expresses E.C. 3.1.1.7 at yeast surface; Can so that the reconstitution cell that carries E.C. 3.1.1.7 directly and agricultural chemicals interact; Avoided passing from organism or juice the complicated process of separation and purification enzyme acetylcholine, production technique is greatly easy, cost reduces.
(3) express E.C. 3.1.1.7 at yeast surface, can pass through immobilized yeast, realize the immobilization of enzyme; Avoided the character of resolvase unstable; Inactivation very easily can not reusable shortcoming, thereby can it be developed as the Detecting Pesticide equipment of sensitive more and robotization such as transmitter.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in the restriction scope of the present invention.The experimental technique of unreceipted actual conditions in the following example; Usually according to people such as normal condition such as Sambrook; Molecular cloning: lab guide (New York:Cold Spring Harbor Laboratory Press; 1989) condition described in, or the condition of advising according to manufacturer.Unless otherwise indicated, otherwise per-cent and umber calculate by weight.
Acetylcholinesterase (AChE) full length gene 1947bp is (referring to the GenBank accession number: NM_057605); 649 amino acid of codified are (referring to the GenBank accession number: NP_476953); This zymoprotein sequence N end contains signal peptide sequence (1-38aa), and its C end contains GPI grappling signal (620-649aa).
In order to make the acetylcholinesterase can be at yeast surface display, the inventor deletes the grappling signal (622-649aa) of this zymoprotein, is replaced by α agglutinin gene (Ag α) the C end grappling signal sequence (320aa) of yeast self.Between AChE gene and α agglutinin gene anchor region, add a Flag epitope simultaneously, for use in detection of fusion proteins.
In addition; In order to improve zymoprotein in the localized effect of yeast surface; The inventor has replaced AChE self signal peptide with glucoamylase (Glucoamylase) secretion signal peptide sequence; And utilize the pYES-DEST52Gateway system, and made up pYES-S-AChE-flag-Ag α recombinant plasmid, see Fig. 1.
The expression vector establishment method is specific as follows:
From the cDNA library of fruit bat and yeast saccharomyces cerevisiae, obtain AChE respectively
(1-1863bp)Gene and Ag α
(3 ' end 960bp)(α lectin, its sequence are referring to the GenBank accession number: X16861) gene, and be cloned in the PMD18-T carrier (available from Invitrogen company), obtain recombinant plasmid T-AChE
(1-1863)With T-Ag α
(3 ' end 960bp)
Through the round pcr of routine, with T-AChE
(1-1863)Be template, in 3 ' end, introduce flag label base sequence (the following aminoacid sequence of encoding: DYKDDDDK) and the NotI restriction enzyme site, obtain product A ChE
(1-1863)-flag fragment.Simultaneously with T-Ag α
(3 ' end 960bp)Be template, 5 ' end is introduced Not I restriction enzyme site, obtains product A g α
(3 ' end 960bp)Fragment.With above-mentioned two product fragments is template, utilizes overlapping pcr, is integrated into Segment A ChE
(1-1863)-flag-Ag α
(3 ' end 960bp)And continue as template; With glucoamylase (Glucoamylase) secreting signal peptide base sequence (its sequence is seen 1-75 position among the SEQ IDNO:1)) introduce in 5 ' the end primer; Replace the signal peptide base sequence (1-114bp) of AChE gene fragment 5 ' end with this, obtain S-AChE
(115-1863)-flag-Ag α
(3 ' end 960bp)Fragment.Utilize the TOPO technology with this fragment cloning to carrier pENTR/D-TOPO
In the attL1/attL2 site of (Invitrogen company), obtain recombinant plasmid pEntr-S-AChE
(115-1863)-flag-Ag α
(3 ' end 960bp), utilize Gateway
TMTechnology is with fusion gene Segment A ChE
(115-1863)-flag-Ag α
(3 ' end 960bp)Be inserted in the attL1/attL2 site of pYSE-DEST52 (Invitrogen company) expression vector, obtain purpose plasmid pYES-AChE
(115-1863)-flag-Ag α
(3 ' end 960bp)
Above-mentioned constructed recombinant vectors pYES-S-AChE-flag-Ag α identifies that with PvuII and BgIII double digestion the endonuclease bamhi size is 718bp, conforms to actual desired, sees Fig. 2.Sequencing result shows that this endonuclease bamhi is the required purpose fragment of experiment.
S-AChE
(115-1863)-flag-Ag α
(3 ' end 960bp)Nucleotide sequence shown in SEQ ID NO:1, wherein the 1-75 position is a glucoamylase secretion signal peptide-coding sequence; The 76-1824 position is the AChE encoding sequence; The 1825-1848 position is a Flag label coding sequence; The 1858-2817 position is 3 ' the end encoding sequence of Ag α.
Embodiment 2. E.C. 3.1.1.7 abduction deliverings and enzyme activity are identified
With recombinant plasmid pYES-S-AChE-flag-Ag α transformed saccharomyces cerevisiae INVSc1 (available from Invitrogen company), select to screen transformant on the flat board at SC.The mono-clonal of picking is 30 ℃ of overnight cultures in 5ml SC substratum, and subsequently 10,000rpm, centrifugal 5min collecting cell under 4 ℃ of conditions is that the SC substratum of carbon source is OD with its dilution in order to semi-lactosi (Gal)
600Value is 0.4 cell suspension, induced 4 hours for 30 ℃, and again 10,000rpm, centrifugal 5min harvested cell under 4 ℃ of conditions.
Cell is resuspended in the sodium phosphate buffer (pH7.6), makes its OD
600Value is 0.5, adds substrate Acetylcholine iodide (ATchI) and developer DTNB subsequently successively, and its final concentration is 1mM.This reaction system is placed 37 ℃, survey its OD
412Value, every later on separated 0.5min measures once METHOD FOR CONTINUOUS DETERMINATION 15min.Enzyme activity is defined as every mL cell suspension (OD
600=1) the μ mol number of every min hydrolysis substrate, enzyme activity unit is μ mol/mLmin.Concrete formula is following:
Enzyme activity=V * A/ (V
0* K * L * c) (formula 1)
In the formula: A representes absorbancy rate (min over time
-1); V
0The volume (mL) of expressed enzyme vitality test time institute obtained cell suspension, V
0=0.1; K representes optical extinction coefficient [L/ (mmolmm)], K=1.36; C representes the light absorption value of yeast cell suspension, c=1; The optical path length of solution when L representes to measure enzyme activity, i.e. cuvette light path (mm), L=2; V representes the TV (mL) of reaction system, V=0.2.
With the blank yeast of Pignus pignoris grain not, as contrast, measure the E.C. 3.1.1.7 vigor of inducing back transformant sterilised yeast suspension without Gal inductive transformant sterilised yeast suspension.Compare (Fig. 3 A, Fig. 3 B) with contrast, the transformant yeast after inducing can detect tangible enzymic activity (Fig. 3 C), and calculating its enzyme activity according to formula 1 is 3.06 μ mol/mLmin.The above results has proved that also E.C. 3.1.1.7 is illustrated in yeast cell surface, but not cell inner expression or secreting, expressing.
Embodiment 3. organophosphorus pesticide 503nhibiting concentration (IC
50) measure
Centrifugal collection behind the yeast cell abduction delivering AChE is resuspended in the sodium phosphate buffer (pH7.6) again, is inoculated in subsequently in 96 orifice plates, makes its OD
600Value is 0.5, adds final concentration and is respectively 0,10
-4, 10
-3, 10
-2, 10
-1With the organophosphorus pesticide of 1 μ g/mL, hatch 30min under 30 ℃, add substrate A TChI and developer DTNB that final concentration is 1mM then, the reaction TV is 200 μ L, 37 ℃ of reaction 15min down, every interval 0.5min detects OD in the reaction process
412Value.Use conventional method of linear interpolation to calculate the IC of agricultural chemicals respectively to AChE
50Value.
Can know that from table 1 result 12 kinds of organophosphorus pesticides are all very remarkable to the susceptibility of AChE, what wherein susceptibility was best is MTMC, its IC
50Value is 0.00073 μ g/mL, even if the SD-9129 of susceptibility difference, its IC
50Value has also reached 0.046 μ g/mL.E.C. 3.1.1.7 susceptibility is still higher after this explanation immobilization, has good using value.
Table 1 organophosphorus pesticide is to the 503nhibiting concentration IC of AChE
50
Fixing of embodiment 4. yeast cell
Take by weighing the 1.6g sodium-alginate in aseptic small beaker, add aseptic deionized water a little, the furnishing pasty state adds remaining water (total amount is 40ml) again.Heat on the fire to fusing, be cooled to about 30 ℃, the surface display that adds the above-mentioned preparation of 10ml has the yeast culture liquid of E.C. 3.1.1.7; Mix; Pour in the funnel that bottom is equipped with sebific duct and hemostatic clamp,, drip to constant speed and to fill 10%CaCl through the aperture of 1.5～3.0mm
2Process gel beads in the plate of solution.Gel beads is at CaCl
2Soak 30min in the solution so that form stable structure, afterwards gel beads is changed in the 300ml Erlenmeyer flask, add 200ml SC substratum with after the aseptic deionized water washing 3 times.
Embodiment 5. detection kit
A kind ofly detect the test kit that whether organophosphorus exists in the testing sample, contain in this test kit:
Container 2, and be loaded on the Acetylcholine iodide (ATchI) in this container;
Container 3, and be loaded on the developer DTNB in this container;
And, the working instructions of detection method are described.
All documents in that the present invention mentions are all quoted as a reference in this application, are just quoted such as a reference separately as each piece document.Should be understood that in addition after having read above-mentioned teachings of the present invention, those skilled in the art can do various changes or modification to the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
< 110>Shanghai Inst. of Life Science, CAS
< 120>a kind of method and product at the cell surface display foreign protein
<130>087048
<160>1
<170>PatentIn?version3.3
<210>1
<211>2817
<212>DNA
< 213>artificial sequence
<220>
<221>misc_feature
< 223>fusion gene
<400>1
Claims (10)
1. a gene constructs is characterized in that, said gene constructs from 5 ' to 3 ' comprises the element that following operability links to each other successively:
(a) glucoamylase signal coding sequence;
(b) encoding sequence of foreign protein; Described foreign protein is an E.C. 3.1.1.7; With
(c) encoding sequence of yeast α agglutinin gene C end grappling signal.
2. gene constructs as claimed in claim 1 is characterized in that,
Described glucoamylase signal coding sequence is the sequence of 1-75 position in the sequence shown in the SEQ ID NO:1;
Described E.C. 3.1.1.7 encoding sequence is the sequence of 76-1824 position in the sequence shown in the SEQ ID NO:1; With
The encoding sequence of described yeast α agglutinin gene C end grappling signal is the sequence of 1858-2817 position in the sequence shown in the SEQ ID NO:1.
3. gene constructs as claimed in claim 1 is characterized in that, between element (b) and element (c), comprises a label protein encoding sequence.
4. the expression vector of a reorganization is characterized in that, said expression vector contains the arbitrary described gene constructs of claim 1-3.
5. a cell is characterized in that, described cell is a yeast cell, contains the described expression vector of claim 4 in the said yeast cell; Or be integrated with the arbitrary described gene constructs of claim 1-3 in its genome.
6. cell as claimed in claim 5 is characterized in that, described yeast cell is selected from: yeast saccharomyces cerevisiae or pichia spp.
7. the method at cell surface display expression foreign protein is characterized in that; Described foreign protein is an E.C. 3.1.1.7, and said method comprises:
(i) the arbitrary described gene constructs of claim 1-3 is imported in the yeast cell, obtain reconstitution cell;
(ii) cultivate the reconstitution cell of (i), thereby express foreign protein at cell surface display.
8. a test kit that detects organophosphorus in the testing sample or carbamate chemicals for agriculture is characterized in that, comprises in the said test kit:
Claim 5 or 6 described cells.
9. method that detects organophosphorus in the testing sample or carbamate chemicals for agriculture is characterized in that said method comprises:
(1) with claim 5 or 6 described cell suspensions in the medium of said cell compatibility in, form detection architecture;
(2) in the detection architecture of (1), add testing sample, mix; With
(3) activity of E.C. 3.1.1.7 in the system of detection (2), thus confirm whether contain organophosphorus or carbamate chemicals for agriculture or its content in the testing sample.
10. method as claimed in claim 9; It is characterized in that; In step (3); Comprise: in the detection architecture of step (2), add the substrate of E.C. 3.1.1.7, the hydrolysis situation of substrate is confirmed whether to contain in the testing sample organophosphorus or carbamate chemicals for agriculture or its content through detecting E.C. 3.1.1.7.
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| CN102277308B (en) * | 2011-07-29 | 2013-01-23 | 江南大学 | Immobilized catalyst for use in production of maltose and preparation method thereof |
| CN103421757B (en) * | 2012-05-18 | 2015-09-30 | 中国科学院上海生命科学研究院 | A kind of Acetylcholine esterase mutant and application thereof |
| CN103555720B (en) * | 2013-11-19 | 2015-08-19 | 湖北工业大学 | A kind of structure with the yeast strain of production and recycled fiber element restriction endonuclease dual-use function |
| CN103789332A (en) * | 2014-03-10 | 2014-05-14 | 湖北工业大学 | Construction of yeast strain having naringinase producing and recycling functions |
| CN104830887B (en) * | 2015-05-13 | 2018-07-03 | 武汉华美生物工程有限公司 | The secreted expression carrier and its construction method of a kind of acetylcholinesterase and application |
| CN110577958B (en) * | 2018-06-11 | 2023-08-15 | 吾幫土智慧生活有限公司 | Nucleic acid, recombinant plasmid, transformant, acetylcholinesterase and preparation method thereof |
| EP3867362A4 (en) * | 2018-10-18 | 2022-08-03 | Academia Sinica | Method and kit for pesticides detection, and plasmid, baculovirus, cell and method of preparing the same for pesticides detection |
| CN109745652A (en) * | 2019-01-18 | 2019-05-14 | 扬州大学 | The method and its miniaturized devices of immobilized microorganism degrading organic phosphor and application |
| CN110484457A (en) * | 2019-08-21 | 2019-11-22 | 河南省医药科学研究院 | A kind of saccharomyces cerevisiae engineered yeast, purposes and the catalyst of cell surface display pectase |
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