TWI379004B - A vector for expression of two proteins - Google Patents

Description

1379004 7 February 2012 J/0曰6, invention description: [Technical field to which the invention pertains] The present invention relates to a carrier capable of expressing a two-protein; in addition, the present invention relates to a fusion protein-shaped silk Method of Protein; The present invention is also directed to a method of detecting the performance of a protein. [Prior Art] In modern molecular biology, 'in order to explore protein function transfection ^ansfection' - a vector encoding a nuclear microsequence of a protein (generation (4) is a technique of taking, licking, and ubiquitous. Usually, if it is necessary to send two cells to the cells at the same time, the most common practice is to use two different vector constructs (to the cells. However, by rotating: the effect of each person is encoded as a different win. Peptide-encoded phase two vectors will result in a unique expression of each protein and the efficiency of the transfection side is also affected by the ratio of the diplasts used and the different promoters that initiate the multi-sequence encoding the nucleotide sequence. There are many methods for measuring the amount of whitening of cells, and one of them is the use of a fluorescent cursor. The carrier of egg protein f has many disadvantages, including the C-terminal end of the fusion (C_terminal). ) instead of the N-terminal, the mutated egg has its own self-adjusting egg, which is easier to purify and difficult to select (excipitation ^eocin) (ampicillin) 5 , and the existing carrier has a less convenient use of the knife. Site, not The restriction enzyme cleavage site is more commonly used. Therefore, it is indeed necessary to design a simultaneous _ protein in the second day of February 2012.财 鲜 鲜 鲜 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The peptide-encoding nucleotide sequence is simultaneously transfected into the same cell. At the same time, the vector expresses the relationship between the two different peptides encoding the acid sequence and the protein. Another advantage is that one of the two proteins can be used for For a transfection marker/screener, there is a problem with the above-mentioned knowledge, and the purpose of the present invention is to provide a message that the body of the body can express the multi-peptide encoding the nuclear sequence of the core. The body provides a method for encoding the two-segment peptide encoding the nucleotide sequence 歹4 for fusion and white matter expression. A preferred embodiment of the vector sequence is represented by the sequence identifier: 1. The carrier used in the invention can be removed from the limited lion -) Legacy A nucleic acid sequence that incorporates a sequence of a design, is transported in two different gene systems, or is expressed in a host cell. The vector of the present invention is composed of DNA, although a dirty vector can also be used. The surface includes the plastid, phagemid, bacterial or viral genome, such as adenovirus (aden〇^) or cancer virus (poxvirus). See the vector T^jr has a segment-designed DNA sequence' And this sequence can be used by restriction enzyme excision and it knot _ _ people's gift to its collocation column (zero 1 kiss 13790004 a: ~ a February 2012 # day sequences) and can be expressed in RNA transcript sequences. The expression vector can be replicated after the host cell replicates or can be inserted into the genome of the host cell, and also has a restriction enzyme cleavage site contained in one or more of the expression vectors, and is embedded in a design DN. The A sequence is used as a new recombinant vector and retains its ability to replicate in host cells. In the case of a plastid, the design sequence contained in the plastid may be replicated many times due to an increase in the copy number of the host bacteria, or may be replicated only once in the host. Taking the phage function as an example (phage), it can be actively replicated in the lytic phase or passively replicated in the lys〇genic phase. The s-vector can further have one or more marker sequences to provide a means for the practitioner to identify whether the vector has been successfully delivered to the cell after transfection or transformation. This marker sequence contains various genes, such as genes encoded as proteins with increased or decreased resistance or sensitivity to antibiotics or other compounds; genes encoding enzymes such as galactosidase (beta_galact〇sidase), luciferin The enzyme activity such as enzyme (ludfemse) or alkaline phosphatase (alkaline ph〇sphatase) can be detected by standard assays of the prior art; or the appearance of cells, hosts, colonies or viruses that are successfully transformed or transfected can be directly observed. Altered genes, such as green light proteins. - A selection marker should be able to distinguish between a host cell or a living host that has been successfully transformed with a nucleic acid sequence that has not been successfully performed under appropriate screening conditions. Some preferred but unrestricted labeling groups, including (4) resistance to antibiotics, such as the genes of kanamydn or ampicillin, or genes that produce resistance to temperature, or A gene (such as G418 resistance) that allows its host cell or living host to survive certain factors, chemicals, or food deficiencies. For the preparation of LB agar broth (agar br〇th) or agar medium _) used in most of the experiments today, the screening marker with arsenicmycin resistance is - preferred embodiment, 6 1379004 February 2012 ^ Days therefore screening markers allow for a more convenient and appropriate method to screen for successfully transformed colonies. In the present invention, a coding sequence and a regulatory sequence may be linked by a covalently linked link, and the performance of the sequence of the encoded phase may be affected by the regulatory sequence or (4). If the coding sequence is to be translated into a functional protein, the above-mentioned DNA sequence can be said to be linked together in the following cases: 5, the promoter of the end-regulatory promoter initiates the transcription of the coding phase, and The DN=linkage between the sequences is not (1) a mutation that triggers a DNA μ segment transfer (frame_swft); (7) an ability to interfere with the promoter region to direct transcription of the coding sequence; (1) an ability to interfere with the translation of the corresponding RNA into a protein. Thus, if the promoter region has the ability to affect DNA rides (4), the promoter region can be ligated to the coding sequence such that the transcripts produced can be translated into the desired protein or multi-peptide. The promoter may be a constitutive or an inducible promoter, and may also provide expression 'and can be-specific cells, tissues, organs at a specific growth stage of the host cell or the living host. Or - providing performance on a portion of a multicellular host organism. &In the mRNA expression system of squirting mammalian cells, a multi-credit encoding human Cytomegai〇vims (CMV) enhancer-promoter sequence or human elongation factor _丨α (human Elongation Factor_la; foot_1 (controlled by the 〇 promoter). The human cytomegalovirus promoter has been disclosed in U.S. Patent Nos. 5,168,062 and 5,385,839, the disclosure of which is incorporated herein by reference. It is a large double-stranded DNA genome with 24 spit. The viral genome consists of a long and short unique region, which is a repeat sequence and reverses each other in sequence. The virus contains four genes of about equal size in DNA preparation. The arrangement of the body is due to the possible reverse combination of the two gene segments. The promoter includes the typical TATA and CAAT regions (TATA b〇x md 7 1379004 · - - ... * « . *· 2〇 12 years 2 CAAT box) (Chambon et at., Annual Rev. Biochem., 50: 349-383, 1981) 'About 23 nucleotides downstream of the TATA region is the starting end of the jump synthesis, and the promoter Tune The region is defined in the upper part of the CAAT region to the _465 (tetra) acid. According to the convention, in the 5th of the main early gene of human cytomegalovirus, the end of the lining "+1", and above the _Wei_negative calculation table and standard miscellaneous The sequence of this repeat sequence and its vicinity plays an important role in the degree of expression of the downstream gene, which can be obtained by measuring the relative expression of downstream in the regulation sequence of different segments. The human elongation factor-Ια promoter can be induced to the south. The performance of the nucleotide sequence (G〇ld_LA, 1996, BioTechniques, 21: l〇l3-l〇i5; Mizushima S et al., 1990, Nuc. Acid Res” 18: 5322). The characteristics of the regulatory sequences vary depending on the species or cell type, but in a nutshell, 5, non-transcribed and 5 non-translated sequences are required to participate in the initiation of transcription or translation, respectively, to repair the TATA region, the mapping sequence, a CAAT region or analog thereof, etc. wherein the 5' non-transcriptional regulatory sequence includes a promoter region, which includes a promoter sequence for transcriptional control of a linkable gene. The regulatory sequence may also include an enhancer sequence. Upstream activator Activat〇r) sequence. The vector of the present invention may be a hybrid & 5, a sequence (leadei) or a signal sequence. In a host cell or a living host, the leader sequence has a modification that allows for post-transfer and丨 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ This leader sequence can also help to secrete the expression product from the cells described above, and the leader sequence can be any host pr〇-, pre-, or prepr〇-like sequence acting in a host cell or a living host. In some examples, the carrier of the present invention can be embedded in a signal sequence, and the 5 tiger sequence system is a region of a portion of the protein that will be removed later. The si* of the present invention includes all of the above sequences except with or without a signal sequence. 8—1379004 February 2, 2012 Other promoters, selection markers, leader sequences and other related sequences not exemplified in the present specification, which also appear or are used in the construction of the gene vector of the present invention, such as terminators, transcription Or translation facilitator or embedding factor (10) factor, the above can refer to the commonly used guide books in this technical field, such as; Sambr〇〇k, Lahu, eds, -Molecular Cloning: A Laboratory Manual^ Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989) 'FM Ausubel, et at., eds, ^Current Protocols in Molecular Biology", John Wiley & Sons, Inc., New York > WB Wood et al. /T^ nematode Caenorhabitis elegant Cold Spring Habor Laboratory

Press (1988) and D.L. Riddle et al., “d five likes //, Cold Spring

Hartor Laboratory Press (1997), or reference patent w〇 95/〇7463, w〇 96/23810 'WO 95/21191 > WO 97/11094 > WO 97/42320 > WO

98/06737, WO 98/21355, US-A-6, 207, 410, US-A-5, 693, 492, and EP 1 085 089, which may be implemented by those of ordinary skill in the art. A multi-peptide encoding nucleotide sequence may comprise the entire protein coding sequence or a region coding sequence encoded as a protein fragment. The protein fragment translated by this sequence includes an N-terminal functional block (dGmain), a functional block, or a subunit protein. In one embodiment, the position of the multi-peptide encoding nucleotide sequence can also be small interfering with ribonucleic acid (siRNA) molecule insertion replacement. The restriction endonuclease site located on the vector (encj, such as the ratio of (10)^) can be inserted into the preset 2 DNA sequence by appropriate cleavage, and then a new recombinant vector is generated after the linkage, and the vector remains The ability to replicate in host cells. The root of the present hybrid can express the carrier of the two protein f, which includes a plurality of genetically selected multiple restriction sites (_, eg e), and the multiple selection sites of the gene can record the fusion egg On the lower side of Cheng, the secret nucleus encodes the nucleus 9 - 13790004 February 2012 #曰曰 acid sequence embedded from the 5, end or 3, end. A fusion protein in which a multi-peptide peptide sequence is inserted at the 5' end or 3' end. The gene multiple selection sites may include restriction endonuclease sites such as BspEl, Sal Ϊ, BamH I, Bsm 1, Acc I, Xma ί, Sma ] _, Spe I, Sphl, Kpnl, Acc65 1, Ahdl ' Clal, BspDl, Non, Eagl, Fsel,

NgoMl ' Nae I ' BseR I ' Pme I ' Pml I ' Aat I ^ EcoR I > BseE II '

BstBl, Bsu36l, Agel, Hindlll, Hpal, Bgll, Mul, Rsrl, Nc〇 I, iVi/e I, So? I, 5 and I, I, I, I. A preferred embodiment is illustrated as a sequence identification number: 2. Sequence identification number: 3 or 2. According to the present invention, the fusion protein is expressed as a protein using a peptide bond or a peptide to link to another protein site. The multi-peptide encodes a nucleotide sequence which can be ligated to the end of the nucleotide sequence of the fusion polypeptide or to the c-terminus. A preferred embodiment of the present invention is a green fluorescent protein (GFp) or a glutathione transferase Cglutathione-SAansfemse (GST) which binds a multi-peptide encoding nucleotide sequence to a expression vector. The glutathione_S_transferase fusion egg can be a protein fragment or a complete protein. Other proteins which can be used as fusion proteins can be used but are not limited to red fluorescent protein (RFP) or flag label (FLAGtag). The green fluorescent protein label (ton) can also be exchanged with a peptidyl acid that can express the receptor (__), and the receptor protein can be used to purify the successfully transfected cells. In addition, the fusion protein utilized in the present invention can be substituted with a multi-peptide encoding nucleus sequence of other proteins for use in a single-hybrid. Traditionally, this microsurgery is a separate vector: one for the capture (stomach) protein and the other for the expression bait = white. According to the performance of the present invention, the attractant protein and the capture protein can be simultaneously expressed in the same carrier. In this respect, the table of the present invention can examine the interaction between mammalian proteins. It is known that the method can be divided into secret county, Weixian and other. For example, 'tags can be combined with or separated from the quality of the source or separated from the sisters _ while 赖 ^ 面 surface # can be directly face electron or silk density, radiation, non-radiative energy materials, etc., or miscellaneous via antibody binding, fiber antibiotics Protein-biotin (streptavidin-biotin) binding or the like is obtained. The above method of detecting the amount of labeling is a conventional technique. There are many techniques for not delivering the performance of the present invention to human cells. The technology involves transfection of calcium phosphate-diethylaminoethyl (diethylaminGe%1; deae), transfection or infection with foreign viruses, transfection of liposome medium (Qiu (10) lipofection), ballistic transformation (ba version ic transf_ati〇n), (micro) injection transfection, cell electroporation (electroporati〇n) and similar methods. Under certain conditions, it is preferred to label a vector having a nucleic acid molecule to a particular cell. In such an example, the target molecule can be attached to the vehicle using a vehicle that transports the nucleic acid molecule of the invention into the cell, such as an adenovirus or other virus and liposome. For example, a molecule such as an antibody having a surface membrane protein specificity on a corresponding target cell, or a ligand having a corresponding receptor on the cell, which can be ligated or incorporated into a carrier medium having a nucleic acid molecule The carrier of the substance is particularly preferably a monoclonal antibody. Liposome is a commercial product available from Life Technologies, such as the products lip〇fectintm and LIPOFECTACETM, which are cationic lipids such as di〇leyl〇xy) -propyl]-N,N,N-trimethyl ammonium chloride ( DOTMA ) And dimethyl dioctadecyl ammonium bromide (DDAB). The manufacturing method of the liposome has been known in the art and has been recorded in many public journals, and can also be referred to Ι3790Ό4 February yT, February 2012

Gregonadis, G,, 7 > *e destroy her c/mo/ogy, 3: 235-241 (1985). Here, the liposome is used to transport the vector having the nucleic acid molecule of the present invention, and the surface attached to the surface associated with the internal sputum can be self-contained and constructed in the human Up(10)me structure to facilitate labeling or promoting absorption of human cells. The protein species may include a structural capsid protein of certain cells, a structural shell tropical protein fragment, an antibody that can be internalized after binding to a protein, or a target intracellular location plus intracellular half-life. Eggs from f and so on. The polymeric H (pGlymerie) transport secret can also successfully deliver nucleic acid molecules to cells, and the boot portion is currently known. Other nucleic acid molecules may also allow for oral delivery. The vector can also be added to - cell-free transliteration, this system can be E y y f iron Ua _imurium, τ3 and E. coli T7 phage (Invitrogen) ^ TNT® Salmonella typhimurium SP6, tile (10) T3 or E co / z' Ding 7 engages in reticulocyte lysate (reticui〇c plus

Salmonella typhimurium SF6>E. coliT3 coli T7 coupled wheat germ extraction system (Pr〇mega). In order to produce or obtain the expression of the vector having the multi-peptide-encoding nucleotide sequence of the present invention, the host cell or the transformed host of the hybridization needs to be cultured under certain conditions. (4) The vector can be expressed or produced. Win-encoded peptide acid sequence. The conditions are adjusted according to the host of the skilled artisan in this field, or according to the regulatory sequences possessed by the body to control the performance of the polypeptide-encoding nucleotide sequence. In general, suitable conditions may include the use of a suitable medium, appropriate = or nutrients, appropriate temperature, and the presence of an appropriate inducing factor or component. The peptide sequence is encoded by the (inducing activator). This can be _f this technology can choose to adjust 12 13 * 79004 February 2012 〆 ^ 曰 whole. Furthermore, the multi-peptide encoding nuclear mini-sequence can be expressed in a sustained, transient, or only inducible form under the appropriate conditions described above. Those skilled in the art will also appreciate that the multi-success of the network of the vectors of the present invention can be produced after the subsequent translation of the modified immature form, depending on the host cell used. And the vector can be glycosylated, depending on the host cell used. The host cells and cell lines used may be prokaryotes (such as E. coli) or eukaryotic cells (such as CH0 cells, COS cells, BHK cells, HeLa cells, HEPG2 cells, 3T3L1 adipocytes, CTC12 cells and L6 muscle cells, yeast). Bacterial expression system, recombinant baculovirus expressed in insect cells). The expression vector of the present invention can be generally applied to mammalian cells such as humans, mice, hamsters, pigs, goats, primates, etc., but performs better in human cells or cell lines. The invention also provides methods of isolating two-expressing proteins. The protein may be isolated from the host that has been transfected with the expression vector of the present invention, or may be isolated from the culture medium of the co-primor cell if it is a secreted protein. The steps of purifying the protein by a thorough purification method can be performed by those skilled in the art. The invention also discloses a button set, which can be prepared by a skilled person - the preset performance is healthy. This set of table counts includes the performance of the present invention, and other components may be based on the user (4). This set may contain primers required for the polymerization of the chimeric chain reaction (PCR), by which the embedded multi-peptide nucleotide sequence can be screened. The kit of the invention allows a skilled person to detect the performance of the preset egg self-quality, which may include the detection of the filament, for example, the secret injection peptide (four) egg body, such as a single mouse mouse glutathione _S _Transferase antibody (Sigma), multiple goats face ^ 1379004 - . . . February 2012 yi-glycopeptide-S_transferase antibody (Amersham Pharmacia) and the like. In addition, such as green fluorescent white antibody, such as monoclonal mouse green fluorescent protein antibody (Sigma). The invention can be used as a whole antibody (wh〇leantjb〇dy), a monoclonal antibody, a multi-drug antibody and an antigen-binding fragment antibody (antigen_bin(jing fi*agment), which is disclosed but not limited thereto. The detection methods used are Western blotting method, enzyme immunoassay (EUSA) and radioimmunoassay (radio face-to-face; rja). The kit may include secondary antibodies depending on the user's design and needs. It is used for direct anti-primary antibodies, such as mouse antibodies or goat antibodies, and antibodies with tagged proteins. The tagged protein can be a fluorescent protein (such as green glory white or red camping protein) gold particles, Wasabi is treated with H氡rseradjsh per〇xidase; or by detecting acidase to detect protein expression. The detection method can be used by anyone skilled in the art. This set can also include restriction incision. An enzyme for cleavage of a specific position contained in a multi-gene selection site of the expression vector to embed a multi-peptide coding nucleotide sequence into the expression vector of the present invention. The kit may further comprise a link set ( Ligatum) 'can also contain one Carrying the expression vector of the present invention and a cell strain which can express the nucleotide sequence of the multi-peptide encoded by the expression vector. According to the present invention, the vector capable of expressing the two proteins may have one or more The following advantages: ~ 〇) This vector can simultaneously display the second selection gene, increasing the convenience and availability of the test; (2) The vector has two genes and multiple selections, and the gene contains multiple selection points. The cleavage site is a common species; (1) This vector has a common antibiotic resistance gene, which increases convenience during the experiment; and '1379004 20 February 2 (4) This vector can simultaneously display the second selection Colony gene, can be tested in white interactive materials. Taking a target egg [Embodiment] Constructing 21 riding 7 Γ is the carrier of Kirin's kanji protein. It^Jr/romoter)^^^^ ^e-dpromoter)^ Antibiotic (Ampidmn) resistance gene, breastfeeding The animal screening marker mover can be linked to the first-age egg* coding phase, and the towel-thirty-column includes the nuclear thief sequence of the green fluorescent protein (GFP). Cloning site). The _ _ s fluorescein (4) acid sequence upstream or lower ^: with the green threo protein under the bitter acid sequence =. The second promoter can be ligated to the second fusion protein editing sequence, and the second snail (GST) is more like the tropicle Cl- site. The second gene locus may be located upstream or under the nucleus sequence of each of the transglutaminase-transferases, or in the second case of the two-year-old _S_transferase-forming enzyme 2 The multiple selection sites of the gene are located downstream of the surface-de-sex-S daughter-in-law sequence. The present invention can be used to represent a two-shell carrier. Wherein the 'the promoter of the vector-the promoter and the second promoter group are from the cytomegalovirus promoter and the human genus seven promoters = two (four), the expression vector can be married - _ and - the restriction enzyme Cut the points ^. The domain can ensure that the two bases are observed to the same mammal 15 February 2012 animal cells. The performance of the two proteins from the same vector can be controlled. This vector contains a multi-gene selection site that provides for the insertion of a multi-peptide encoding nucleotide sequence and an ampicillin resistance selection to screen for successful colonies. In summary, this vector can be used in experiments in which dual protein expression is performed in a single mammalian cell. Experimental Materials and Methods 1. PTOM vector preparation of PTOM vector backbone by pEBG vector ((4) Jiaqing; B〇ney c M over al., 2000, Molecular Endocrinology, 14 (6): 805-813) and pEGFP vector (BD Bi〇sciences Ci〇ntech) J=_Vector to construct. Using a high-accuracy pcR (twist function fidelity PCR) kit, and]you I and Rsr II restriction enzyme sites to act on the 5, and 3' ends to synthesize a fragment of about 6 kb of pEBG health, The pEGFP vector was treated in the same step, and the DNA 4 extracted from each of the pEBG vector and the pEGFp vector was further mixed, and the hydrazine was designated as ρΤ〇Μ and sequenced. 2. Cell culture and transfection. In this example, human embryonic kidney 293 cells (ΗΕΚ 293 ) and mouse muscle fibroblasts (NIH 3Τ3) were used as test subjects, and the former provided dmem (Dulbecco's modified Eagle medium) with 1%%. Fetal bovine blood, raw (Sigma), 100 units/ml penicillin (penicilUn) and sputum "streptomycin" were cultured at 10% C〇2, 3rc. In the latter case, the serum was changed to secret bovine serum (GIBCO), and the rest of the culture conditions were the same as before. When the cells were cultured to an average concentration of 1~2_6/60 mm, add 5.3 mg of DNA blood and 15 ml of Lipofectamine to each plate of cells for 5 hours on the transfection side. Procedure Γ379004 —— - ——〉 February 2012 Tethered Reference Reagent Operation Manual (GIBCO). 3. Cell lysate (lySate) and immunoturbation analysis After 48 hours of transfection, the cells were rapidly frozen at -70 °C for use. The frozen cells of each 60 mm culture plate were given 〇6 ml of lysis buffer (to also buffe 〇 (containing 50 mM tris-based pH 7.9 (Tris-Base pH 7.9), 50 mM sodium chloride (NaCl) , 〇.l mM ethylenediaminetetraacetic acid (EDTA), 2 mM glycerol sodium phosphate (β-glycerophosphate), 1 mM dithiothreitol (DTT), 1 phenylmethylsulfonyl fluoride ; PMSF ), 25 should be treated with a discoin inhibitor (calyculin A), 0.5% surfactant (Triton X-100), 1 tablet / 50 ml proteolytic inhibitor (R) 'The cell lysate was centrifuged at 13,5 rpm for 10 minutes. Each sample was taken with the same protein concentration above the supernatant, and the protein concentration was measured by the Bradford test (Bio-Rad). A volume of 2X sodium dodecyl sulfate (SDS) sample buffer, heated at 95 ° C for 10 minutes, using sodium dodecyl sulfate-polyacrylamide gel Electrophoresis; SDS-PAGE) separation. After electrophoresis, the protein is cleaved to polydifluoro Polyvinylidene difluoride (PVDF) membrane, after the protein and its primary antibody and horseradish peroxidase-conjugated (HRP-conjugated) secondary antibody, chemical cold light method for measuring cold light intensity, operation The procedure is referred to the reagent operation manual (Pierce). 4. Cellular fluorescence microscopy observation of HEK 293 cells cultured in 60 mm culture dishes. Add 2-3 mg of pTOM vector and 15 ml of Lipofectamine to each plate for transfection ( For example, the 3rd 17 1379004 —......—one-one _ 2 〇 12 February y 及 and 4 (), in some cases can also use 6 ml of secret coffee (R〇che Or 15 ml of LiP〇feCtamine2000 (GlBC〇BRL). After 24 hours of transfection, the transfection status of the cells was directly observed by fluorescence microscope. The experimental results have specific limits _ viewpoint and gene multi-point Τ〇Μ A schematic representation of the vector is shown in Figures 1 and 2. The Τ〇Μ vector contains a two-promoter, the cytomegalovirus (CMV) promoter and the human elongation factor _1α promoter. The two promoters respectively induce the expression of the green fluorescent protein and the glutathione_s•transferase fusion protein on the vector. The inclusion of fresh t-mycin resistance gene can make it easier to prepare most of the laboratory LB agar squama (or medium) and (4). To test the performance efficiency of the pTOM vector for proteins with a molecular weight greater than 5 ,, construct green fluorescein and glutathione transferases by using the 5, 3, and 3, respectively, BamHI and Ng_ sites. After the plastid of the protein of Macn)phage 1 (MST1; about 66 kD), the plastid was transfected into the decellularized cells, and the microfibrillar was as shown in Fig. 3A. Buckle time point method

The effect of glutathione-S-transferase and green fluorescent protein antibody was confirmed in one step, as shown in the left block of Figure 3B. Another test, pT (DM system, inserts the associated association F〇tein_4 (DAP4) gene into the ρΤ〇Μ vector to make it a green & fluorescence protein and a glutathione & transferase hexagram fusion protein. The carrier of 116kD), after transfecting into 293 cells, the result of using the western blotting method to use the action of the surface glutathione-S-transferase and the green fluorescent protein antibody, as shown in the first plot. Simultaneously expressing the two proteins in the same cell, the red (four) grade from (five) 11 job reading t_ein; Qing) embedded in this vector and the addition of 2 years in February next day glutathione 4 transferase, transfected into HEK 293 Within the cell, it becomes a vector capable of expressing green fluorescent protein and glutathione transferase_red fluorescent protein fusion protein. As a result of fluorescence microscopy, as shown in Fig. 4, more than 95% of the cells showed green fluorescent protein and glutathione_s_transferase_red fluorescent protein simultaneously. It is worth noting that green fluorescence can be observed in the whole cell, and red fluorescence is observed only in the cytoplasm in the same cell. It is confirmed by the difference in fluorescence position that the two proteins are common in the same cell towel. Expression; this is intended to rule out the observed light leakage caused by poor quality of the green or red filter. Discussion The PTOM vector includes three promoters: the cytomegalovirus promoter, the human elongation factor-Ια promoter, and the monkey virus 40 (SV40) promoter. The cytomegalovirus promoter system is one of the promoters most commonly used in eukaryotic cells in today's transgenic gene technology, although there has been a performance of the age-regulated iij pressure-regulated motility (Bruening, 1998 Nucleic AcidsRes) 26, 486-489), which has a wide range of cell types and is highly potent. Human elongation factor _1α promoter (Hana〇kaetal, 丨99ι

Differentiation 48, 183-189) is a promoter commonly used in human cell lines, and it can also promote a large amount of silk filaments, and the promoter-promoted gene expression is not affected by time and cell type. It can be seen that the pT〇M vector has both a cytomegalovirus promoter and a human elongation factor _1α promoter, which ensures that the target gene can be highly expressed by the vector; see, as shown in Fig. 4, more than 95% The cells showed green fluorescent protein and glutathione-S-transferase-red fluorescent protein fusion protein expression. In addition, the Wolf's disease 毋40 promoter promotes the expression of the ampicillin resistance gene, allowing the vector and bacteria to transform to produce resistance' (10). This promoter does not use the internal ribosomal entry sites (IRES) to initiate the antibiotic resistance gene's internal ribosome entry site via the dependence of ^^丨八5, -1379004 - heart-, - — Γ-. 2〇 In February of the 12th, the existence of the RNA structure in the untransformed region (9), as a mechanism of action, used another efficient method to initiate the antibiotic resistance gene. With the skeleton of the pTOM vector, some sequences can be substituted to achieve different effects. First, the de-glycopeptide-S-transferase tag located downstream of the human elongation factor seven promoter can be easily changed to achieve the desired experimental purpose, such as if the molecular weight of the protein to be fused with the target is smaller than the label' The fusion protein may not be able to mimic the function of f (endGge_pr〇tein) due to the interference side of the labeled protein. In this case, the sequence of the glutathione-s transferase gene may be smaller by another molecular weight. The tag gene flag tag (FLAGtag; sequence ID: 8 and sequence ID: 9) is replaced, so the flag tag _ egg can be more accurate as the endogenous protein 'and the correct folding. In addition, the tag can also be constructed by embedding-segment hairpin DNA, which is combined with a promoter to carry a small interfering ribonucleic acid (siRNA), which can be used for the selection of the small pre-ribonucleic acid. It is itself a cell with a green county protein gene to increase the target gene, now. In addition, the green glory protein tag (tag) located downstream of the cytomegalovirus promoter can be used as a surrogate marker for other eggs, such as green, photoprotein tags, and -receptors ((10)_). The corresponding ligand Uigand) is used to purify successful cells, such as flow cytometry ^cs). In addition, the two standard irons in the pT〇M vector can be replaced with protein shells for use in a mammalian two-hybrid system to simplify the current steps (Dang et al., biliary M〇1). Ce is 〇1" 954 962). Nowadays, many methods have been proposed for mammalian cell screening, and each method uses the attractant ((4) gene and the prey gene into different plastids, and then uses the co-transfection to give the triplasts - In the cell, the hair _ is a (four) stimulating gene and a capture gene, respectively, in place of the green fluorescent protein in the pTOM vector and the transgenic enzyme of the scorpion scorpion, more capable of 20 February 2012 ^^ day ti丨ΐ protein and capture New Zealand is expressed in the same cell. This can rule out the situation of breastfeeding, normal folding of the material, or the need to implement the cake in the yeast, so that the implementation of Wei further explores the relationship between mammalian proteins. Interaction = = 所示 0 Μ 与 与 与 与 与 与 与 与 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 The end-loaded Γ green fluorescent protein and the glutathione-transferase gene are ^ (4) body with giomycin (Z_n) as a selection marker, W PT 〇 M ^ body of ampicillin is common, and pTOM carrier eve = can ride It is convenient and convenient, and all of the above can be the same as the death of the company. It is suitable for labeling and classification in mammalian cells. It is also suitable for marker screening and classification. It contains the beauty of """<" resistance characteristics of the drug^There is usually a knowledgeable person in this field and the W domain is wealthy. Any of the patents of the present invention are not included in the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram of a pTOM vector according to an embodiment of the present invention. 1379 (104 ～ -. . . __ 2 2 February 2nd, the second picture is a gene multi-selection point contained in the pTOM vector Fig. 3A is a result of fluorescence microscopy after transfection of HEK293 cells with pTOM vector containing green fluorescent protein and glutathione_S_transferase_MST1. Fig. 3B is Transfection of green fluorescent protein with chytrisin s_transferase-MST1 vector (left column), and transfection of pTOM vector containing green fluorescent protein and glutathione-S-transferase-DAP4 After the HEK293 cells (right bar), observe each other by Western blotting ^ Results. Figure 4 shows the results of fluorescence microscopy after transfection of HEK 293 cells with pTOM vector containing green fluorescent protein and glutathione-S_transferase-red fluorescent protein. Explanation of symbols]

S 22 1379004 ~ 一———一—— - '- • " February 2012 〆 戽 list <110> Lin Yanshou <120> can represent two protein carriers <130><140>TW 097150143 <141>2008-12-22 <160>9 <210>1 <211>9041 <212>DNA <213> Artificial sequence <220><223> pTOM vector nucleotide sequence. ≪ 400> 1 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg 60 cgttacataa cttacggtaa atggaccgcc tggctgaccg cccaacgacc cccgcccatt 120 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca 180 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc 240 aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta 300 catgacctta tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac 360 2012 February May > 0 said catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg actcacgggg 420 atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg 480 ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg gtaggcgtgt 540 acggtgggag gtctatataa gcagagctgg tttagtgaac cgtcagatcc gctagcgcta 600 ccggtcgcca ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg 660 gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc 720 gatgccacct acggcaagct gaccctgaag Ttcatctgca ccaccggcaa gctgcccgtg 780 ccctggccca ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgc tacccc 840 gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag 900 cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag 960 ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac 1020 atcctggggc acaagctgga gtacaactac aacagccaca acgtctatat catggccgac 1080 aagcagaaga acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc 1140 gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg 1200 cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc 1260 gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag 1320 ctgtacaagt ccggactcag atctcgagct caagcttcga attctgcagt cgacgttacc 1380 gcgggcccgg gttccaccgg atctagataa ctgatcataa tcagccatac cacatttgta 1440 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg 1500 aatgcaattg ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat 1560 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc 1620 aaactcatca atgtatctta acgcgtaata ggttaatgtc atgataataa tggtttctta 16 80 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta 1740 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata 1800 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc 1860 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga 1920 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct 1980 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg 2040 tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta 2100 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat 2160 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt 2220 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga 2280 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga 2340 gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat taactggcga 2400 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc 2460 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc 2520 cgg tgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg 2580 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat 2640 2 billion 12 February Houle day cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata 2700 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct 2760 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga 2820 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg 2880 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc 2940 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgttcttct 3000 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc 3060 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt 3120 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg 3180 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct 3240 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag 3300 ggtcggaaca Ggagagcgca cgagggagct tccaggggga aacgcctggt atctt tatag 3360 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg 3420 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg 3480 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac 3540 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt 3600 gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc gttggccgat 3660 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc 3720 aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta tgcttccggc 3780 tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca gctatgacca 3840 tgattacgcc aagcttaatt ccctccccag caggcagaag tatgcaaagc atgcatctca 3900 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca 3960 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg 4020 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct 4080 tttgcaaaaa gctttgcaaa gatggataaa gttttaaaca gagaggaatc tttgcagcta 4140 atggaccttc taggtcttga aaggagtggg aattggctcc ggtgcccgtc agtgggcaga 4200 gcgcacatcg cccacagtcc ccgagaagtt tggggggagg ggtcggcaat tgaaccggtg 4260 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt 4320 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc 4380 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc 4440 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacctggctg cagtacgtga 4500 ttcttgatcc cgagcttcgg gttggaagtg ggtgggagag ttcgaggcct tgcgcttaag 4560 gagccccttc gcctcgtgct tgagttgagg cctggcctgg gcgctggggc cgccgcgtgc 4620 gaatctggtg gcaccttcgc gcctgtctcg ctgctttcga taagtctcta gccatttaaa 4680 atttttgatg acctgctgcg acgctttttt tctggcaaga tagtcttgta aatgcgggcc 4740 aagatctgca cactggtatt tcggtttttg gggccgcggg cggcgacggg gcccgtgcgt 4800 cccagcgcac atgttcggcg aggcggggcc tgcgagcgcg gccaccgaga atcggacggg 4860 ggtagtctca agctggccgg cctgctctgg tgcctggcct cgcgccgccg tgtatcgccc 4920 2012 Nian 2 Yue 〆 said cgccctgggc ggcaaggctg gcccggtcgg caccagttgc gtgagcggaa agatggccgc 4980 ttcccggccc tgctgcaggg Agctcaaaat ggaggacgcg gcgctcggga gagcgggcgg 5040 gtgagtcacc cacacaaagg aaaagggcct ttccgtcctc agccgtcgct tcatgtgact 5100 ccacggagta ccgggcgccg tccaggcacc tcgattagtt ctcgagcttt tggagtacgt 5160 cgtctttagg ttggggggag gggttttatg cgatggagtt tccccacact gagtgggtgg 5220 agactgaagt taggccagct tggcacttga tgtaattctc cttggaattt gccctttttg 5280 agtttggatc ttggttcatt ctcaagcctc agacagtggt tcaaagtttt tttcttccat 5340 ttcaggtgtc gtgaggaatt ctctagagat ccctcgacct cgagatccat tgtgctggat 5400 ctacaatggc ccctatacta ggttattgga aaattaaggg ccttgtgcaa cccactcgac 5460 ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc gatgaaggtg 5520 ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt ccttattata 5580 ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata gctgacaagc 5640 acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt gaaggagcgg 5700 ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt gaaactctca 5760 aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat cgtttatgtc 5820 ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg tatga cgctc 5880 ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa ttagtttgtt 5940 ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc agcaagtata 6000 tagcatggcc tttgcagggc tggcaagcca cgtttggtgg tggcgaccat cctccaaaat 6060 cggatctggt tccgcgtgga tccactagtg gtaccatcga tgcggccgcg actctagagt 6120 gagggtcccc acctgggacc cttgagagta tcaggtctcc cacgtgggag acaagaaatc 6180 cctgtttaat atttaaacag cagtgttccc catctgggtc cttgcacccc tcactctggc 6240 ctcagccgac tgcacagcgg cccctgcatc cccttggctg tgaggcccct ggacaagcag 6300 aggtggccag agctgggagg catggccctg gggtcccacg aatttgctgg ggaatctcgt 6360 ttttcttctt aagacttttg ggacatggtt tgactcccga acatcaccga cgtgtctcct 6420 gtttttctgg gtggcctcgg gacacctgcc ctgcccccac gagggtcagg actgtgactc 6480 tttttagggc caggcaggtg cctggacatt tgccttgctg gacggggact ggggatgtgg 6540 gagggagcag acaggaggaa tcatgtcagg cctgtgtgtg aaaggaagct ccactgtcac 6600 cctccacctc ttcacccccc actcaccagt gtcccctcca ctgtcacatt gtaactgaac 6660 ttcaggataa taaagtgttt gcctccaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 6720 aaaaaagaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac 6780 ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc 6840 ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg 6900 gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag 6960 tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc 7020 gctacacttg ccagcgcctt agcgcccgct cctttcgctt tcttcccttc ctttctcgcc 7080 acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt 7140 agtgctttac ggcaccacga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg 7200 2012 Nian 2 Yue 〆 day ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt 7260 ggactcttgt tccaaactgg aacaacactc aactctatct cgggctattc ttttgattta 7320 taagggattt tgccgatttc ggtctattgg ttaaaaaatg agctgattta acaaaaattt 7380 aacgcggatt ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc 7440 tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc 7500 tgacgggctt gtctgctccc Ggcatccgct tacagacaag ctgtgaccgt ctccgggagc 7560 tgcatgtgtc agaggttggc ggaccgctat caggacatag cgttggctac ccgtgatatt 7620 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct 7680 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc 7740 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca 7800 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga 7860 tcctccagcg cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg 7920 aaacacggaa ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga 7980 ataaaacgca cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc 8040 actctgtcga taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt 8100 ccccacccca ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg 8160 caggccctgc catagcctca ggttactcat atatacttta gattgattta aaacttcatt 8220 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 8280 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 8340 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgct accag 8400 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 8460 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 8520 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 8580 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 8640 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 8700 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 8760 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 8820 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 8880 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 8940 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 9000 tatcccctga ttctgtggat aaccgtatta ccgccatgca t 9041

<210>2 <211>66 <212>DNA 1137900 2〇February 12 曰<213> Artificial sequence <220><223> pTOM vector of the first gene multiple selection point Nucleotide sequence. <400>2 tccggactca gatctcgagc tcaagcttcg aattctgcag tcgacgttac cgcgggcccg 60 ggttcc 66 <210>3 <211>45 <212>DNA<213>Artificial sequence<220><223> The nucleotide sequence of the selection point. <400>3 gttccgcgtg gatccactag tggtaccatc gatgcggccg cgact 45 <210>4 <211>660 <212>DNA<213>Artificial sequence<220> February 2012 <«*日<223> The nucleotide sequence of the glutathione-S-transferase gene. ≪ 400 > 4 atgtccccta tactaggtta ttggaaaatt aagggccttg tgcaacccac tcgacttctt 60 ttggaatatc ttgaagaaaa atatgaagag catttgtatg agcgcgatga aggtgataaa 120 tggcgaaaca aaaagtttga attgggtttg gagtttccca atcttcctta ttatattgat 180 ggtgatgtta aattaacaca gtctatggcc atcatacgtt atatagctga caagcacaac 240 atgttgggtg gttgtccaaa agagcgtgca gagatttcaa tgcttgaagg agcggttttg 300 gatattagat acggtgtttc gagaattgca tatagtaaag actttgaaac tctcaaagtt 360 gattttctta gcaagctacc tgaaatgctg aaaatgttcg aagatcgttt atgtcataaa 420 acatatttaa atggtgatca tgtaacccat cctgacttca tgttgtatga cgctcttgat 480 gttgttttat acatggaccc aatgtgcctg gatgcgttcc caaaattagt ttgttttaaa 540 aaacgtattg aagctatccc acaaattgat aagtacttga aatccagcaa gtatatagca 600 tggcctttgc agggctggca agccacgttt ggtggtggcg accatcctcc aaaatcggat 660 < 210 > 5 < 211 > 717 < 212 > DNA < 213 〉Artificial sequence. <220><223> Green fluorescent protein gene nucleotide sequence. ≪ 400 > 5 atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac 60 ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac 120 ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc 180 ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag 240 cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc 300 ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg 360 gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac 420 aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac 480 I379004 - February 2012 yi date ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc 540 gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac 600 tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc 660 ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaag 717 < 210 >6<211>27<212>DNA<213>Artificial sequence <220><223> Embedding pTOM vector sequence (EGFP fusion protein downstream). <400>6 aacgagaagc gcgatcacat ggtcctg 27 <210>7 <211>21 <212>DNA<213>Artificial sequence<220><223> Insertion of pTOM vector sequence primer (glutathione) _s-transferase gene fusion protein downstream). Ί3790Ό4 — one — ~ · — — — · \ February 2012 day* \ <400>7 gggctggcaa gccacgtttg g 21 <210>8 <211>27 <212>DNA <213>Artificial sequence<220><223> Flag tag nucleotide sequence. <400>8

Atg gac tac aag gac gac gat gac aag 27

Met Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <210>9 <211>9 <212> PRT <213> Artificial sequence <220><223><400>9

Met Asp Tyr Lys Asp Asp Asp Asp Lys

Claims (1)

1379004 February/February 2012, Patent Application Range: The performance vector 1. A performance vector whose nucleotide sequence is the sequence identification number: ^, contains: ~ '-the first-promoter' its connection-- The fusion protein coding sequence, wherein the "first fusion protein coding sequence comprises - the nucleotide sequence encoding the green fluorescent egg and the - the first gene multiple selection point; the first gene multiple selection point is located in the coding The green fluorescent protein is downstream of the nuclear phytic acid sequence, and the second-polypeptide-encoding nuclear acid sequence is selected, and the green light-light protein forms a chemical-second promoter- a second fusion protein coding sequence, wherein the turn sequence comprises a nucleotide sequence encoding a v-Glutathione; =ST) and a second gene multi-selection point; the second gene multi-k colony is located The nuclear-sequence sequence encoding the WEs-glycopeptide_s•transferase is used for the selection of the second-polypeptide-encoding nuclear-sense sequence to form a fusion with the transgenic enzyme of the face-like glycoform; a bacterial selection marker (selection marker) ), which is an ampicillin resistance gene; a mammalian selection marker; wherein, the first promoter and the second promoter are selected from the group consisting of a cytomegalovirus promoter and a human elongation factor-Ια promoter; The expression vector utilizes a Mwl and a/rII restriction enzyme cleavage site. 2. The expression vector of claim i, wherein the Chu 耽 glyco-transferase gene phase comprises a sequence number: 4 3. The performance carrier as described in the patent application ,:: 曰 gene sequence includes the order ship: S /, wenching fluorescent protein 4. I can be divided into cells, which contain any of the (4) patents The expression carrier of the item. The method of expressing the two proteins in the performance vector described in Item No. 1 to Item 3 of the patent item, the steps of which include: ^ Now the i code is - - a nucleotide sequence of a protein. The expression vector comprises a first fusion protein coding sequence: a mover, wherein the "first-fused egg self-encoding phase includes -, a fluorescent egg red (tetra) acid sequence and - - multiple selection of genes; ^ ^ Xi selection point is located in the code for The green glory protein II is the nucleus-sequence of the first protein, the light = the nuclear protein encoding the H protein (four) sequence cloning the expression vector 'the expression vector includes - the second of the second fusion protein coding sequence The coding sequence of the second fusion protein comprises: a sorghum horse as a glutathione: a S-transferase (tetra) acid sequence and a second gene multiple selection point; the second gene multiple colony is located in the coding a nucleic acid sequence of the nucleotide sequence of the glutathione transferase for culturing the nucleotide sequence encoding the second protein to form a fusion with the glutathione-S·transferase; ~ Transfer the performance carrier into a performance system. 6. The method of clause 5, wherein the expression vector is delivered to a cell via transformation or transformation. 7. The method of claim 6, wherein the cell line is a breastfeeding February 1/3 of the cells. She applied for the method described in Section 5, which was added to a cell-free transcriptional translation system. 9. A method for separating a protein, the method comprising: introducing a vector as described in claim 1 of the patent; in-cell; cultivating the cell having the vector of claim i; And separating the protein from the cell. 10. A kit for constructing a vector having a second protein comprising: a vector according to claim 1; and a primer which is used in a polymerase chain reaction for screening A presence in which one of the multiple selection sites is embedded; wherein the primer is selected from the group consisting of SEQ ID NO: 6 and SEQ ID NO: 7. 11. A kit for detecting the performance of a protein in a performance vector, comprising: the vector of claim 1; and an antibody for use in the scope of the patent application The protein represented by the vector; wherein the anti-system is selected from the group consisting of an antibody against glutathione_s_transferase and an antibody against green fluorescent protein. 12. A vector for use in a two-hybrid screening comprising: the vector of claim i, wherein the coding is a quotation of ba 〇丨 〇丨 〇丨 ba ba ba ba ba ba a nucleotide sequence replacing the green fluorescent protein nucleotide sequence 'in addition to a nuclear thief sequence encoding a prey pr〇tein to replace the glutathione transferase nucleotide sequence; or In a