CN109055425B - Xenopus laevis oocyte expression vector with yellow or red fluorescent protein label and application thereof - Google Patents

Abstract

The invention discloses a yellow or red fluorescent label-containing vector capable of being used for Xenopus laevis oocyte expression and application thereof, wherein pGH19 plasmid is modified by a molecular biological means, and the vector is specifically developed into a vector pGH19-EYFP or pGH19-mRFP which contains an enhanced yellow or monomer red fluorescent protein label and is suitable for Xenopus laevis oocyte expression. The vector can normally express a target gene, and can detect the distribution condition and the expression mode of target protein with a yellow or red fluorescent label through a fluorescent microscope or a confocal microscope, thereby providing a more visual and concise detection mode for the protein condition expressed by Xenopus laevis oocytes; the spatiotemporal dynamics of protein expression can be observed in real time; for the later extraction, purification, identification and functional analysis of the target protein, the expression condition of the target protein can be detected only by using the universal antibody, so that the experimental process is greatly simplified, and the experimental time and the economic cost are saved.

Description

Xenopus laevis oocyte expression vector with yellow or red fluorescent protein label and application thereof

Technical Field

The present invention relates to the fields of molecular biology and genetic engineering. In particular to a Xenopus laevis oocyte expression vector with a yellow or red fluorescent label and application thereof.

Background

Xenopus laevis oocytes are a heterologous expression system with strong functions and wide application range, and are widely applied to the expression of heterologous proteins such as exogenous calcium ion channels, chloride ion channels, potassium ion channels, sodium ion channels, gamma-aminobutyric acid receptors, glutamic acid receptors, acetylcholine receptors and the like and the research on the physiological and pharmacological characteristics of the heterologous proteins. It plays an important role in discussing the action mechanism of the compound, evaluating and screening the drug effect and safety of the target compound, and is a heterologous expression system which is indispensable in the research of the fields of biology, medicine and pharmacology, neurology, agriculture and pharmacology, and the like.

The yellow and red fluorescent proteins refer to proteins which can emit yellow and red fluorescence under the excitation of green light (500-565nm), and have been used as novel reporter genes in a plurality of research fields of biology. Enhanced Yellow Fluorescent Protein (eYFP), which is a mutant of Yellow Fluorescent Protein, and Monomeric Red Fluorescent Protein (mRFP) are widely applied to researches such as gene expression, cell differentiation, in vivo positioning and transferring of Protein and the like because of stable luminescence and high brightness and discrimination.

The pGH19 plasmid is a widely used protein expression vector containing multiple restriction enzyme sites, and is widely used for heterologous expression research of Xenopus oocytes because the DNA sequences of the 5 'UTR and the 3' UTR of Xenopus beta-globin are contained near the promoter and the terminator of the pGH19 plasmid, and heterologous proteins can be successfully expressed in the Xenopus oocytes.

The protein expressed in Xenopus laevis oocytes by utilizing pGH19 plasmid can be used for detecting the physiological and pharmacological properties of the protein through electrophysiological experiments, but whether the target protein is expressed or not and the time-space expression dynamic state cannot be observed under a microscope, effective subcellular localization and purification and identification of the target protein cannot be carried out, and a large amount of time and economic cost are consumed for obtaining a specific antibody of the target protein.

Disclosure of Invention

The invention aims to provide a Xenopus laevis oocyte expression vector with a yellow or red fluorescent protein label, which can successfully express active target protein in Xenopus laevis oocytes, has yellow or red fluorescence capable of being directly microscopic examined, can carry out real-time monitoring and subcellular localization on the expression of the protein, and can purify the expressed protein and detect the binding mode of different subunits by using universal antibodies anti-EYFP and mRFP, thereby saving the time and economic cost for preparing specific antibodies.

In order to achieve the purpose, the invention adopts the following technical scheme:

the Xenopus laevis oocyte expression vector with yellow or red fluorescent protein labels is characterized in that the vector is obtained by connecting EYFP gene with a modified sequence or mRFP gene with a modified sequence to pGH19 plasmid; the EYFP gene with a modified sequence or the mRFP gene with a modified sequence is specifically characterized in that a recognition base sequence of restriction enzyme A, an artificially designed enzyme cutting site base sequence and a recognition base sequence of restriction enzyme B carried by the 3 'end are added at the 5' end of the EYFP gene or the mRFP gene.

The base sequence of the artificially designed restriction enzyme site is shown as SEQ ID NO.3, the restriction enzyme site of the sequence is added, so that the problem that the target gene cannot be inserted after the pGH19 plasmid is inserted into the EYFP and mRFP genes can be solved, the activity of the EYFP and mRFP genes and the original function of the pGH19 plasmid can not be damaged after the target gene is successfully inserted, and the target protein can be provided with a yellow or red fluorescent label on the premise of ensuring the original function of the pGH19 plasmid.

The restriction enzyme A can be EcoRI restriction enzyme, XmaI restriction enzyme, SmaI restriction enzyme, BamHI restriction enzyme, BstBI restriction enzyme, XhoI restriction enzyme or EcoRV restriction enzyme.

The base recognition sequence of the restriction enzyme A can be an EcoRI restriction enzyme base recognition sequence (GAATTC, shown in SEQ ID NO. 4), an XmaI restriction enzyme base recognition sequence (CCCGGG, shown in SEQ ID NO. 5), a SmaI restriction enzyme base recognition sequence (CCCGGG, shown in SEQ ID NO. 6), a BamHI restriction enzyme base recognition sequence (GGATCC, shown in SEQ ID NO. 7), a BstBI restriction enzyme base recognition sequence (TTCGAA, shown in SEQ ID NO. 8), an XhoI restriction enzyme base recognition sequence (CTCGAG, shown in SEQ ID NO. 9) or an EcoRV restriction enzyme base recognition sequence (GATATC, shown in SEQ ID NO. 10).

The restriction enzyme B can be XbaI restriction enzyme, BsrGI restriction enzyme, PspOMI restriction enzyme, ApaI restriction enzyme, BmgBI restriction enzyme or BstPI restriction enzyme.

The base recognition sequence of the restriction endonuclease B can be an XbaI restriction endonuclease base recognition sequence (TCTAGA, shown as SEQ ID NO. 11), a BsrGI restriction endonuclease base recognition sequence (TGTACA, shown as SEQ ID NO. 12), a PspOMI restriction endonuclease base recognition sequence (GGGCCC, shown as SEQ ID NO. 13), an ApaI restriction endonuclease base recognition sequence (GGGCCC, shown as SEQ ID NO. 14), a BmgBI restriction endonuclease base recognition sequence (CACGTC, shown as SEQ ID NO. 15) or a BstPI restriction endonuclease base recognition sequence (GGTNACC, shown as SEQ ID NO. 16).

Specifically, the invention provides a Xenopus laevis oocyte expression vector pGH19-EYFP with a yellow fluorescent label, and the base sequence of the vector is shown as SEQ ID NO. 1.

Specifically, the invention also provides a Xenopus laevis oocyte expression vector pGH19-mRFP with a red fluorescent label, and the base sequence of the vector is shown as SEQ ID NO. 2.

The invention also provides the application of the vector in biological detection, in particular the application in real-time observation of protein expression dynamics or protein purification or detection.

The invention also provides a gene expression vector formed by inserting a target gene into the vector.

The invention also provides application of the gene expression vector in biological detection, in particular application in real-time observation of protein expression dynamics or protein purification or detection.

The invention also provides a construction method of the vectors pGH19-EYFP and pGH19-mRFP of the sequences shown in SEQ ID NO.1 or SEQ ID NO.2, which comprises the following steps:

1) downloading EYFP and mRFP base sequences from an NCBI website, designing primers, modifying a forward primer and a reverse primer, respectively adding restriction sites and protective bases of endonucleases EcoRI and XbaI at the 5 'ends of the forward primer and adding an artificially designed restriction site base sequence after the restriction site of the EcoRI at the 5' end of the forward primer. The primer sequence is as follows:

Forward Primer(5’-3’):

aagGAATTCGAAGCTTCTCGAGATATCATGGTGAGCAAGGGCGAGGAGC，

Reverse Primer(5’-3’):ttgTCTAGATTACTTGTACAGCTCGTCCATGCCG.

wherein, lower case letters represent protective bases, italic letters represent EcoRI and XbaI restriction enzyme recognition base sequences, and underlined letters represent artificially designed inserted enzyme cutting site base sequences.

2) Obtaining EYFP or mRFP gene products with modified sequences through PCR amplification and DNA recovery operation;

3) the pGH19 plasmid was double digested with the restriction enzymes EcoRI and XbaI. And connecting the digested plasmid and the EYFP or mRFP gene with a modified sequence by T4 ligase to obtain the Xenopus laevis oocyte expression vector pGH19-EYFP or pGH19-mRFP with yellow or red fluorescent labels.

In the present invention, the EcoRI restriction enzyme in step 3) may be replaced by XmaI restriction enzyme, SmaI restriction enzyme, BamHI restriction enzyme, BstBI restriction enzyme, XhoI restriction enzyme, EcoRV restriction enzyme or any restriction enzyme containing the above-mentioned cleavage site.

The XbaI restriction enzyme in step 3) of the present invention may be replaced by BsrGI restriction enzyme, PspOMI restriction enzyme, ApaI restriction enzyme, BmgBI restriction enzyme or BstPI restriction enzyme, or any restriction enzyme containing the above-mentioned cleavage site.

The pGH19 vector widely applied to a Xenopus laevis oocyte expression system is transformed, the pGH19 vector is connected with an EYFP or mRFP gene, and a section of artificially designed enzyme cutting site base sequence is inserted into the 5' end of the pGH19 vector to ensure the normal insertion and expression of a target gene. When the target gene is inserted into the vector, the pGH19-EYFP or pGH19-mRFP vector is subjected to enzyme digestion and then is subjected to homologous recombination or T4 connection with the target gene with the same enzyme digestion site, so that the expression vector which can express the target gene protein, has a yellow or red fluorescent label and can be applied to a Xenopus laevis oocyte expression system can be successfully constructed. The carrier can be used for normal electrophysiological experimental detection, can also be used for detecting the space-time expression dynamic of the target protein in Xenopus laevis oocytes through a fluorescence microscope, is convenient for subcellular localization and extraction and purification of the target protein, and provides an intuitive and convenient tool for biological experiments.

The pGH19 plasmid and the EYFP or mRFP gene are fused and transformed, so that the target protein can be provided with a yellow or red fluorescent label on the premise of not influencing the original function of the pGH19 plasmid; can be used for detecting the state of the expressed protein without influencing the activity and the electrophysiological detection of the target protein. The target protein expressed by the vector can be used for observing the protein expression dynamics in real time; during later detection, a specific antibody is not needed, and the target protein can be purified and the expression condition of the target protein can be detected only by using a universal antibody, so that the experimental process is greatly simplified, and the experimental time and cost are saved.

Drawings

FIG. 1 is a plasmid map of Xenopus laevis oocyte expression vector pGH19-EYFP with fluorescent tag provided by the present invention;

FIG. 2 is a plasmid map of Xenopus laevis oocyte expression vector pGH19-mRFP with fluorescent tag provided by the present invention;

FIG. 3 PCR amplification electropherograms of the EGFP and mRFP genes;

FIG. 4 restriction electrophoresis of pGH19 vector;

FIG. 5 is a PCR amplification electrophoretogram of the base sequence of AmRDL gene;

FIG. 6 restriction electrophoretogram of pGH19-EYFP and pGH19-mRFP vectors;

FIG. 7 is a plasmid map of Xenopus laevis oocyte expression vector pGH19-EYFP-AmRDL with fluorescent tag capable of expressing AmRDL gene provided by the invention;

FIG. 8 is a plasmid map of Xenopus laevis oocyte expression vector pGH19-mRFP-AmRDL with fluorescent tag capable of expressing AmRDL gene provided by the invention;

FIG. 9 electrophysiological detection of pGH19-AmRDL and pGH19-EYFP-AmRDL and pGH19-mRFP-AmRDL expression vectors;

FIG. 10 fluorescent detection of expression of pGH19-EYFP-AmRDL gene on Xenopus oocytes;

FIG. 11 fluorescent detection of expression of pGH19-mRFP-AmRDL gene on Xenopus oocytes.

Detailed Description

To facilitate understanding of the technical solutions and advantages of the present invention by those skilled in the art, the following description of the embodiments is made with reference to the accompanying drawings. It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Example 1 construction of pGH19-EYFP and pGH19-mRFP recombinant vectors

1. Experimental Material

Max DNA Polymerase kit was purchased from TaKaRa, AxyPrep DNA gel recovery kit was purchased from AXYGEN, EcoRI and XbaI restriction enzyme kit and T4 DNA Ligase kit were purchased from Thermo, and Trans-T1 E.coli competent cells were purchased from Beijing Quanjin Biotechnology, Inc.

2. Experimental procedure

2.1 PCR amplification and recovery of EYFP and mRFP sequences

1) The primers for amplifying EYFP and mRFP sequences were designed and synthesized by Nanjing Kingsler Biotech Co., Ltd, and the purification method was RPC.

2) Using TaKaRa Co

Max DNA Polymerase kit, 200 u L PCR tube adding the following components, configured into 25.0L reaction system.

3) According to

Max DNA Polymerase kit instructions set up PCR amplification reaction program.

4) Subjecting the PCR amplification product to agarose gel electrophoresis, and detecting the target band in an ultraviolet gel imaging system (FIG. 3, wherein lane 1 is the amplified EYFP gene fragment; lane 2 is amplified mRFP gene fragment). And cutting the target strip into glue and recovering the target segment.

2.2 restriction and recovery of pGH19 plasmid vector

1) The pGH19 plasmid was double-digested with the restriction enzymes EcoRI and XbaI. The following components were added to 200. mu.L of each PCR tube to prepare a 20. mu.L reaction system:

mixing the reaction systems uniformly, then preserving the heat for 2h at 37 ℃ in a PCR instrument, and heating for 5min at 80 ℃ to complete the linearization of the pGH19 plasmid;

2) the linearized pGH19 plasmid was subjected to agarose gel electrophoresis detection, and the digested target band was detected in an ultraviolet gel imaging system (FIG. 4, in which lane 1 is undigested pGH19 plasmid as negative control; lane 2 is digested pGH19 plasmid). And cutting the target strip into glue and recovering the target segment.

2.3 ligation transformation of recombinant pG19-EYFP and mRFP vectors

1) The recovered EYFP and mRFP products were ligated to pGH19 vector products using T4 DNA Ligase kit from Thermo. The following components were added to a 200. mu.L PCR tube, respectively, to prepare a 20. mu.L reaction system:

and mixing the reaction systems uniformly, and then preserving the heat for 30min at 22 ℃ in a PCR instrument to finish the connection of the recombinant pGH19-EYFP and pGH19-mRFP vectors.

2) The recombinant pGH19-EYFP and pGH19-mRFP vectors were transformed into Trans-T1 E.coli competent cells, cultured overnight and single clones were picked for sequencing.

3. Results of the experiment

Sequencing results show that the recombinant pGH19-EYFP and pGH19-mRFP vectors respectively contain complete EYFP and mRFP gene sequences and artificially designed enzyme cutting site base sequences, the construction of the vectors is successful, and plasmid maps of Xenopus laevis oocyte expression vectors pGH19-EYFP and pGH19-mRFP with fluorescent labels are respectively shown in figures 1 and 2. The base sequence of the Xenopus laevis oocyte expression vector pGH19-EYFP with a yellow fluorescent label is shown in SEQ ID NO. 1; the base sequence of the Xenopus laevis oocyte expression vector pGH19-mRFP with the red fluorescent label is shown in SEQ ID NO. 2.

Example 2 construction of bee (Apis mellifera) RDL Gene expression vectors pGH19-EYFP-AmRDL and pGH19-mRFP-AmRDL

1. Experimental Material

Reagent and restriction enzyme HindIII from Thermo, PrimeScript^TMRT Kit with gDNA Erase from TaKaRa, Cloneexpress II One Step Cloning Kit from Nanjing Novokation Biotech Co., Ltd, and the rest of the experimental materials were the same as in example 1.

2. Experimental procedure

2.1 cloning of base sequence of coding region of AmRDL Gene

1) Downloading nucleotide sequence of bee RDL gene (AmRDL) from NCBI website, designing and amplifying primer of complete coding region sequence, and synthesizing by Nanjing Kingsler Biotechnology limited company, wherein purification mode is RPC.

2) Using TaKaRa Co

Max DNA Polymerase kit, adding the following components into a 200-L PCR tube to prepare a 25.0-L reaction system:

3) according to

Setting a PCR amplification reaction program according to the instruction of a Max DNA Polymerase kit;

4) the PCR amplification products were subjected to agarose gel electrophoresis and the band of interest was detected in an ultraviolet gel imaging system (FIG. 5). And cutting the target strip into glue and recovering the target segment.

2.2 restriction and recovery of pGH19-EYFP and pGH19-mRFP vectors

1) The digestion of pGH19-EYFP or pGH19-mRFP plasmid vectors was carried out using the HindIII restriction enzyme kit from Thermo. The following components were added to 200. mu.L of each PCR tube to prepare a 20. mu.L reaction system:

mixing the reaction systems uniformly, then preserving the heat for 2h at 37 ℃ in a PCR instrument, and heating for 5min at 80 ℃ to complete the linearization of the pGH19-EYFP or pGH19-mRFP vector;

2) the linearized pGH19-EYFP or pGH19-mRFP vector is subjected to agarose gel electrophoresis detection, and a digested target band is detected in an ultraviolet gel imaging system (shown in figure 6, wherein a lane 1 is a digested and linearized pGH19-EYFP plasmid; lane 2 is the plasmid pGH19-EYFP that was not digested as a negative control; lane 3 is the enzyme-cleaved linearized pGH19mRFP plasmid; lane 4 is the undigested pGH19-mRFP plasmid, as a negative control). And cutting the target strip into glue and recovering the target segment.

2.3 ligation transformation of recombinant pGH19-EYFP-AmRDL or pGH19-mRFP-AmRDL vectors

1) The pGH19-EYFP or pGH19-mRFP vector products and the AmRDL gel recovery products obtained by recovery were ligated by Clonexpress II One Step Cloning Kit. The following components were added to a 200. mu.L PCR tube, respectively, to prepare a 10. mu.L reaction system:

and uniformly mixing the reaction systems, then preserving the heat for 30min at 37 ℃ in a PCR instrument, and then placing the mixture in a refrigerator at 4 ℃ for storage to finish the connection of the recombinant pGH19-EYFP-AmRDL and pGH19-mRFP-AmRDL vectors.

2) The recombinant pGH19-EYFP-AmRDL and pGH19-mRFP-AmRDL vectors are transformed into Trans-T1 escherichia coli competent cells, cultured overnight and a monoclonal strain is picked and sent to Nanjing Kingsry Biotech Limited for sequencing.

3. Results of the experiment

Sequencing results show that the recombinant pGH19-EYFP-AmRDL or pGH19-mRFP-AmRDL vector contains complete EYFP and mRFP gene sequences and AmRDL gene sequences, and the vector is successfully constructed (figures 7 and 8).

Example 3AmRDL expression and detection of EGFP or mRFP Gene and Gene without fluorescent tag in Xenopus oocytes

1. Experimental Material

NotI restriction enzyme and mMESSAGE mMACHINE^TMThe T7 kit was purchased from Thermo corporation, phenol chloroform (phenol: chloroform: isoamyl alcohol 25:24:1, pH>7.8) purchaseFrom Beijing Soilebao science and technology, Xenopus laevis was provided by Shanghai Life sciences research institute of Chinese academy of sciences.

2. Experimental procedure

2.1 linearization of pGH19-AmRDL, pGH19-EYFP-AmRDL and pGH19-mRFP-AmRDL vectors

1) The digestion of pGH19-AmRDL, pGH19-EYFP-AmRDL and pGH19-mRFP-AmRDL vectors was carried out using the NotI restriction enzyme kit from Thermo. The following components were added to a 200. mu.L PCR tube to prepare a 150. mu.L reaction system:

and (3) uniformly mixing the reaction systems, performing enzyme digestion at 37 ℃ for 7.5h in a PCR instrument, heating at 65 ℃ for 15min to inactivate the enzyme, and then storing in a refrigerator at 4 ℃ to finish the linearization of pGH19-AmRDL, pGH19-EYFP-AmRDL and pGH19-mRFP-AmRDL vectors.

2.2 purification of the linearized product

1) Addition of ddH to the linearized product₂O to 400 mu L, then adding 200 mu L phenol chloroform, and centrifuging for 10min at 13000 g/min;

2) sucking supernatant fluid 400 μ L, adding 200 μ L phenol chloroform, centrifuging at 13000g/min for 10 min;

3) sucking 300 μ L of supernatant, adding 800 μ L of anhydrous ethanol and 30 μ L of sodium acetate solution, and standing for 7-8 hr;

4) centrifuging at 13000g/min for 15min, discarding the supernatant, adding 1mL of precooled 75% ethanol, washing the precipitate, centrifuging at 13000g/min for 10min, and discarding the supernatant;

5) drying the precipitate in a clean bench, adding 10 μ L ddH₂Dissolving O, measuring the concentration, and storing for later use.

2.3 in vitro Synthesis of cRNA

The method adopts the Thermo company mMESSAGE mMACHINE^TMcRNA was synthesized in vitro using T7 kit and stored at-80 ℃ in an ultra low temperature freezer for further use.

2.4 Xenopus laevis oocyte retrieval and cRNA injection

Dissecting mature female Xenopus laevis and taking out proper amount of oocyte from abdomen, treating the taken-out oocyte with Type IA collagenase to remove the membrane on the periphery of the oocyte, and washing for later use. The cRNA was injected into freshly prepared xenopus oocytes with a microinjector, and each oocyte was injected with a 23nL volume of cRNA.

2.5 electrophysiological assay and fluorescence detection of Xenopus oocytes

After the cRNA-injected oocytes were cultured in a 16 ℃ incubator for 48 hours, electrophysiological experiments and fluorescence detection were started.

3. Results of the experiment

The AmRDL gene fused with the fluorescent label and the AmRDL gene not fused with the fluorescent label are detected through a double-electrode voltage clamp experiment, and the result shows that the existence of the fluorescent label has no influence on the detection result (figure 9).

The spatial-temporal expression dynamics of the AmRDL protein in Xenopus laevis oocytes is monitored in real time through a fluorescence microscope, and preliminary subcellular localization can be carried out on the AmRDL protein (shown in figure 10 and figure 11), so that the next step of extraction and purification of the AmRDL protein and deeper functional study can be facilitated.

Various other modifications and changes may be made by those skilled in the art based on the above teachings and concepts, and all such modifications and changes are intended to fall within the scope of the appended claims.

Sequence listing

<110> Nanjing university of agriculture

<120> Xenopus laevis oocyte expression vector with yellow or red fluorescent protein label and application thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 3810

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

gggcgaatta attcgagctc ggtacccagc ttgcttgttc tttttgcaga agctcagaat 60

aaacgctcaa ctttggcaga tcaattcccc ggggatccga attcgaagct tctcgagata 120

tcatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 180

acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 240

acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 300

ccctcgtgac caccttcggc tacggcctac agtgcttcgc ccgctacccc gaccacatga 360

agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 420

tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 480

tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 540

acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 600

acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 660

ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgcta cccgacaacc 720

actacctgag ctaccagtcc gccctgagca aaggccccaa cgagaagcgc gatcacatgg 780

tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacagat 840

cttaatctag agggcccgat cgccgagcat cacgtcgcgc acggcggatg tctgagccag 900

gagcttgatc tggttaccac taaaccagcc tcaagaacac ccgaatggag tctctaagct 960

acataatacc aacttacact ttacaaaatg ttgtccccca aaatgtagcc attcgtatct 1020

gctcctaata aaaagaaagt ttcttcacat tctaaaaaaa aaaaaaaaaa aaaaaaaaaa 1080

aaaaaacccc cccccccccc ctgcaggcgg ccgcctcgag gctagcttga gtattctata 1140

gtgtcaccta aatagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta 1200

tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc 1260

ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg 1320

aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg 1380

tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg 1440

gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa 1500

cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc 1560

gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc 1620

aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag 1680

ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct 1740

cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta 1800

ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc 1860

cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc 1920

agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt 1980

gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct 2040

gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc 2100

tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca 2160

agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta 2220

agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa 2280

atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg 2340

cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg 2400

actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc 2460

aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc 2520

cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa 2580

ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc 2640

cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg 2700

ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc 2760

cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat 2820

ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg 2880

tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc 2940

ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg 3000

aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat 3060

gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg 3120

gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg 3180

ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct 3240

catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac 3300

atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta 3360

taaaaatagg cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa 3420

cctctgacac atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag 3480

cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta 3540

tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag 3600

atgcgtaagg agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg 3660

ggaagggcga tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc 3720

tgcaaggcga ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac 3780

ggccagtgaa ttgtaatacg actcactata 3810

<210> 2

<211> 3765

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

gggcgaatta attcgagctc ggtacccagc ttgcttgttc tttttgcaga agctcagaat 60

aaacgctcaa ctttggcaga tcaattcccc ggggatccga attcgaagct tctcgagata 120

tcatggcctc ctccgaggac gtcatcaagg agttcatgcg cttcaaggtg cgcatggagg 180

gctccgtgaa cggccacgag ttcgagatcg agggcgaggg cgagggccgc ccctacgagg 240

gcacccagac cgccaagctg aaggtgacca agggcggccc cctgcccttc gcctgggaca 300

tcctgtcccc tcagttccag tacggctcca aggcctacgt gaagcacccc gccgacatcc 360

ccgactactt gaagctgtcc ttccccgagg gcttcaagtg ggagcgcgtg atgaacttcg 420

aggacggcgg cgtggtgacc gtgacccagg actcctccct gcaggacggc gagttcatct 480

acaaggtgaa gctgcgcggc accaacttcc cctccgacgg ccccgtaatg cagaagaaga 540

ccatgggctg ggaggcctcc accgagcgga tgtaccccga ggacggcgcc ctgaagggcg 600

agatcaagat gaggctgaag ctgaaggacg gcggccacta cgacgccgag gtcaagacca 660

cctacatggc caagaagccc gtgcagctgc ccggcgccta caagaccgac atcaagctgg 720

acatcacctc ccacaacgag gactacacca tcgtggaaca gtacgagcgc gccgagggcc 780

gccactccac cggcgcctaa tctagagggc ccgatcgccg agcatcacgt cgcgcacggc 840

ggatgtctga gccaggagct tgatctggtt accactaaac cagcctcaag aacacccgaa 900

tggagtctct aagctacata ataccaactt acactttaca aaatgttgtc ccccaaaatg 960

tagccattcg tatctgctcc taataaaaag aaagtttctt cacattctaa aaaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa accccccccc cccccctgca ggcggccgcc tcgaggctag 1080

cttgagtatt ctatagtgtc acctaaatag cttggcgtaa tcatggtcat agctgtttcc 1140

tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg 1200

taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc 1260

cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg 1320

gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc 1380

ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac 1440

agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa 1500

ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca 1560

caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc 1620

gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata 1680

cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta 1740

tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca 1800

gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga 1860

cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg 1920

tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg 1980

tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg 2040

caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag 2100

aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa 2160

cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat 2220

ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc 2280

tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc 2340

atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc 2400

tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc 2460

aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc 2520

catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt 2580

gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc 2640

ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa 2700

aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt 2760

atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg 2820

cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc 2880

gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa 2940

agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt 3000

gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt 3060

caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag 3120

ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta 3180

tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat 3240

aggggttccg cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat 3300

catgacatta acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg 3360

tgatgacggt gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta 3420

agcggatgcc gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg 3480

gggctggctt aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg 3540

tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgccatt cgccattcag 3600

gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc 3660

gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg 3720

acgttgtaaa acgacggcca gtgaattgta atacgactca ctata 3765

<210> 3

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gaagcttctc gagatatc 18

<210> 4

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gaattc 6

<210> 5

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cccggg 6

<210> 6

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cccggg 6

<210> 7

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ggatcc 6

<210> 8

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ttcgaa 6

<210> 9

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctcgag 6

<210> 10

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

gatatc 6

<210> 11

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tctaga 6

<210> 12

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

tgtaca 6

<210> 13

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gggccc 6

<210> 14

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

gggccc 6

<210> 15

<211> 6

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gacgtc 6

<210> 16

<211> 7

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

ggtnacc 7

Claims (6)

1. The Xenopus laevis oocyte expression vector with yellow or red fluorescent protein labels is characterized in that the vector is obtained by connecting EYFP gene with a modified sequence or mRFP gene with a modified sequence to pGH19 plasmid; the EYFP gene with a modified sequence or the mRFP gene with a modified sequence is specifically that an EcoRI restriction endonuclease recognition base sequence and a artificially designed enzyme cutting site base sequence are added at the 5 'end of the EYFP gene or the mRFP gene, and a XbaI restriction endonuclease recognition base sequence is carried at the 3' end; the base sequence of the artificially designed restriction enzyme site is shown as SEQ ID NO. 3; the base recognition sequence of the EcoRI restriction endonuclease is selected from SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO. 10; the base recognition sequence of the XbaI restriction endonuclease is selected from SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15 or SEQ ID NO. 16.

2. The Xenopus laevis oocyte expression vector pGH19-EYFP with a yellow fluorescent label is characterized in that the base sequence of the vector is shown as SEQ ID NO. 1.

3. The Xenopus laevis oocyte expression vector pGH19-mRFP with a red fluorescent label is characterized in that the base sequence of the vector is shown as SEQ ID NO. 2.

4. A gene expression vector comprising the expression vector of any one of claims 1 to 3 and a gene of interest inserted therein.

5. Use of the expression vector of any one of claims 1 to 3 or the gene expression vector of claim 4 in biological assays.

6. Use of the expression vector of any one of claims 1 to 3 or the gene expression vector of claim 4 for real-time observation of protein expression dynamics or protein purification or expression pattern detection.

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