CN113005146A - Recombinant plasmid, construction method thereof, recombinant image system and application - Google Patents

Abstract

The invention discloses a recombinant plasmid, a construction method thereof, a recombinant image system and application. The present invention discusses the qualitative and quantitative reliability of the bio-optical-fluorescence resonance energy transfer technique and the luciferase separation complementation system and the optimization of the luciferase separation complementation system based on the protein-protein interaction of MSI1/AGO 2. Through testing connecting peptides (linker peptides) with different lengths and elasticity/charge distribution, a group of recombinant image systems of the luciferase (Gaussia luciferase, GLuc) with the brightness which is thousands times higher than that of firefly luciferase (FLuc) are successfully constructed; also, firefly luciferase will serve as a normalization signal for quantitative analysis. The invention is mainly applied to cell experiments and animal experiments before clinic, and can measure and quantify the treatment effect of the brain tumor in real time and non-invasively.

Description

Recombinant plasmid, construction method thereof, recombinant image system and application

Technical Field

The invention relates to the technical field of biomedicine, in particular to a recombinant plasmid, a construction method thereof, a recombinant image system and application.

Background

Visualization of malignant tumors has taken great popularity in biomedical research projects today, both in animal models, pre-drug development, pharmacokinetics, and even in decisions of sequential treatment programs, to the 21 st century. Molecular imaging is a study of molecular mechanism visualization, visualization and quantification. Various molecular design and imaging platforms are used to achieve non-invasive analysis. Since the 60 s of the 20 th century, green fluorescent protein was isolated and defined from marine life as the undergraduate, , japan, and various imaging techniques using optical principles have been developed. With the ever-increasing development and development of green fluorescent protein by the American scientists, the bioluminescent images have occupied a status of being indelible in the current research and development. To date, many scientific research projects using fluorescence, bioluminescence, or even nuclear medicine, are not enumerated. Molecular imaging studies directed to protein-protein interactions have been quite historical. In the early proteomics field, the Yeast double hybrid system (Yeast-2-hybrid system) has been well established, however, most of it is applied to eukaryotes, and the effect in mammalian cells is not significant. In the field of biochemistry, co-immunoprecipitation is widely used as a primary tool for verifying protein-protein interactions, however, this technique is not applicable to cells at all, let alone in vivo applications. With the extensive study of fluorescent proteins and bio-optics, energy transfer using the overlapping divergence and excitation spectra of two donor and acceptor chromophores (chromophores) can be used as evidence that specific molecules are sufficiently close, a technique known as Fluorescence Resonance Energy Transfer (FRET). This technique is also used extensively today in studies of dynamic observation of molecular interactions, as the confocal microscopy imaging technique matures. Compared with the prior art, the application of FRET in cell physiology is visible, and the molecular information is visualized due to the quantification and tracking functions, which is a great progress; however, this technique relies on laser activation, which limits its in vivo applications. A bio-optical-fluorescence resonance energy transfer (BRET) technique instead of excitation light has been developed. The technology uses luciferase as an excitation light source, and because the luciferase only needs energy and substrate for luminescence, the technology is not influenced by energy attenuation of the excitation light source penetrating tissues, and can effectively express the interaction by optical signals in real time. Recently, another type of protein-protein interaction imaging system has been developed and made available, and a luciferase isolation complementation system (split luciferase complementation system) is known as a representative design. Conceptually, the luciferase is first divided into two segments, and the two test proteins that are likely to bind to each other are each tagged with one of the luciferase segments. If the two test proteins are sufficiently close together due to protein-protein interaction, it is possible for the two luciferase fragments or structures thereof to recombine and reactivate luciferase activity, giving rise to a light signal. To increase the efficiency of luciferase reactivation, many modified versions of the design also appeared as spring shoots after rain, as Ozawa et al published that intein (intein) sequences from Synechocystis added to one end of luciferase increased the chance of coincidence and efficiently detected light signals non-invasively in animals.

According to Inouye et al, the protein structure of luciferase (Gaussia luciferase) isolated from marine organisms of the radius and foot has two catalytic domains (catalytic domains) with obvious functions, and the existence of the catalytic domains influences the capacity of the luciferase to emit light through the interaction with coelenterazine. Gaussia luciferase acts with coelenterazine and gives off nearly thousand-fold brightness without the need for energy (ATP) as compared to the common firefly luciferase. Indiscriminately, Renilla luciferase is also associated with coelenterazineIn effect, although the emitted light signal is lower than that of Gaussia, a key factor is that Gaussia luciferase, which is an secreted protein, is transported to the outside of the cell after cytoplasmic production, resulting in errors in the quantification effect. In contrast, Renilla luciferase is retained intracellularly, so that the emitted light signal is a true reflection of the number of events occurring (e.g., protein-protein interactions). Paulmugen et al have established protein-protein interaction imaging systems using the Renilla Luciferase isolation Complementation technique (Split Renillia Luciferase Complementation) and further validated this development in small animals. In the previous study, Paulmurugan and Gambrir et al performed a number of different protein truncation assays based on the catalytic domain position of Renilla, and used a common neutral linker peptide (GGGGS) between the truncated Renilla luciferase and the test protein MyoD₂Carrying out serial connection; however, whether the presence of the linker peptide affects the complementation of luciferases remains to be investigated. A review by Chen et al contains several commonly used linker peptides, preferably glycine-rich and serine-alternating combinations with improved flexibility, as described previously (GGGGS)_nIt is often used for various fusion fluorescent proteins, etc. to avoid the physical resonance phenomenon affecting chromophores. In addition, there are also harder linker peptides such as (EAAAK)_nOr (XP)_nAnd so on, to be discussed. On the other hand, Gaussia luciferase (approximately 20 kilodaltons) is a more efficient and smaller luciferase compared to Renilla luciferase (36 kilodaltons) or firefly luciferase (approximately 61 kilodaltons). This means that the development of a contrast platform for Gaussia luciferase is a very potential and efficient approach. Earlier, Remy et al also targeted a significantly higher photoprotective signal of Gaussia luciferase as a validation tool for protein binding in a 2006 study. One study by nevu et al in 2012 applied the technology developed by Remy as described above to visualize the viral infection process as a high throughput screening platform.

Disclosure of Invention

The present invention is a drug screening platform using molecular imaging, which is applied to planning and designing different disease modes. The establishment of animal models plays a central role in the early development of drugs. Before the clinical test is taken, the safety test, the maximum tolerated dose test, the toxicological pathological test, the pharmacokinetics and the like are all the disadvantages. However, differences between individual animals often contribute to uncertainty in experimental results; meanwhile, the demand of a large number of experimental animals is contrary to the world trend. Therefore, the molecular imaging technology is applied to animal model development, the demand on the number of experimental animals is effectively reduced, the difference between individuals is minimized, the early development time of new drugs in the scientific research field can be effectively shortened, and the technology can be extended to effective feedback aiming at the arrangement of sequential treatment.

The present invention aims to create a high throughput drug screening platform for MSI1/AGO2 protein-protein interaction deconstruction, not only for the cell level, but more scalable to animal models. The method is also a qualitative, quantitative, high-throughput and stable image platform, and provides powerful information for the growth rate, the malignancy and the drug resistance of the glioblastoma multiforme so as to develop drugs.

Specifically, the technical scheme of the invention is as follows:

a method for constructing a recombinant plasmid, comprising the steps of:

implanting AGO2 into restriction endonuclease sites HindIII and BamHI by using polymerase chain reaction, and then carrying out amplification to obtain a HindIII-AGO2-BamHI fragment;

introducing a HindIII-AGO2-BamHI fragment into a multiple cloning site of a pcDNA vector to obtain pcDNA-AGO 2-C1;

the C-terminal protein of the radial-foot luciferase GLuc was amplified using the polymerase chain reaction, the restriction sites BamHI and XbaI were added, and (GGGGS) was added₂Linker peptide to obtain (GGGGS)₂-gluc (c) fragment;

will (GGGGS)₂The gluc (C) fragment was loaded into pcDNA-AGO2-C1 using cloning techniques to obtain pcDNA-AGO2-gluc (C) recombinant plasmid.

A recombinant plasmid constructed by the method of the present invention.

A method for constructing a recombinant plasmid, comprising the steps of:

adding (GGGGS) to the N-terminus of the radial-foot luciferase GLuc by using polymerase chain reaction with pCMV-GLuc1 plasmid as a vector₂After connecting peptide and restriction endonuclease cutting sites BamHI and XbaI, cloning to prepare pCMV-gluc (N);

MSI1 was amplified and purified by PCR amplification and restriction sites HindIII and BamHI were added, and MSI1 was loaded into pCMV-gluc (N) by cloning to give pCMV-MSI1-gluc (N).

A recombinant plasmid constructed by the method of the present invention.

A fusion protein obtained by fusing two recombinant plasmids as described above.

A recombinant imaging system comprises two recombinant plasmids described above.

The fusion protein is applied to or used for preparing products with tracer properties.

The fusion protein is applied to preparing a brain tumor treatment drug.

Has the advantages that: the invention successfully constructs a group of recombinant image systems of the radius-foot luciferase (Gaussia luciferase, GLuc) with the brightness which is thousands of times higher than that of firefly luciferase (firefly luciferase, FLuc) based on the protein-protein interaction of MSI1/AGO2, a biological optical-fluorescence resonance energy transfer technology and a luciferase separation and complementation system; also, firefly luciferase will serve as a normalization signal for quantitative analysis. The invention is mainly applied to cell experiments and animal experiments before clinic, and can measure and quantify the treatment effect of the brain tumor in real time and non-invasively.

Drawings

FIG. 1 shows the structure of pcDNA-AGO2-C1 and pCMV-gluc (N).

FIG. 2 shows the structures of pcDNA-AGO2-gluc (C) and pCMV-MSI1-gluc (N).

FIG. 3 is a schematic representation of the fusion protein product of MSI1-gluc (N) and AGO2-CGluc (C).

FIG. 4 is a schematic diagram of the operation of the reconstructed image system according to the present invention.

FIG. 5 is a recombinant plasmid of several different linker peptides made by cloning.

FIG. 6 is a graph showing the normal expression of different fragments of luciferase.

FIG. 7 is a schematic diagram of luminescence variation under normal and pressure conditions.

FIG. 8 is a graph showing the results of different pressure-induced relative luminescence measurements.

FIG. 9 is a schematic image of a mouse subcutaneously implanted with a brain tumor before and after treatment (Temozolomide) using the imaging system of this embodiment.

Detailed Description

The present invention provides a recombinant plasmid, a construction method thereof, a recombinant image system and an application thereof, and the present invention is further described in detail below in order to make the objects, technical schemes and effects of the present invention clearer and clearer. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

The Argonaute-2(AGO2) protein is a major member of the RNA-induced silencing complex and is involved in the regulation of non-compiled ribonucleic acids. The Musashi1(MSI1) protein is a known stem cell biomarker and is reported to inhibit gene translation via ribonucleic acid targets. Previous studies found that there was a high correlation between the MSI1 expression and drug resistance in glioblastoma specimens, and 1) the high MSI1 expression significantly increased drug resistance in glioma-associated malignant brain tumors; 2) it was further observed that MSI1 was concentrated into the cytoplasm; 3) MSI1 apparently causes nuclear MSI1 to migrate toward cytoplasm under anti-cancer drug stimulation, and AGO2 and MSI1 synergistically enhance the molecular mechanism of drug resistance and recurrence of glioblastoma multiforme (GBM).

Based on this, the present invention was made in view of the protein-protein interaction of MSI1/AGO2, and examined on the qualitative and quantitative reliability of the bio-optical-fluorescence resonance energy transfer technique and the luciferase isolation complementation system, and the optimization of the luciferase isolation complementation system. Through testing connecting peptides (linker peptides) with different lengths and elasticity/charge distribution, a group of recombinant image systems of the luciferase (Gaussia luciferase, GLuc) with the brightness higher than that of firefly luciferase (firefly luciferase, Fluc) by thousand times are successfully constructed; also, firefly luciferase will serve as a normalization signal for quantitative analysis. The invention is mainly applied to cell experiments and animal experiments before clinic, and can measure and quantify the treatment effect of the brain tumor in real time and non-invasively. The image system has the following advantages:

firstly, the recombined imaging system can immediately visualize the response of brain tumor cells to pressure;

the recombination image system is composed of two independent plasmids and has high stability;

thirdly, the luminescence signal is more than thousand times of that of the common firefly luminescence enzyme;

and fourthly, the luminescence signal fades in 3 to 5 minutes, and can be used for continuous image observation.

Specifically, the two recombinant plasmids provided by the invention have the structures shown in FIGS. 1-2, and the construction method specifically comprises the following steps:

1. pcDNA-AGO2-gluc (C) recombinant plasmid

AGO2 was amplified using polymerase chain reaction after removal of the stop codon and implantation of the restriction sites HindIII and BamHI to give a HindIII-AGO2-BamHI fragment. Then, taking a pcDNA plasmid as a vector, introducing a HindIII-AGO2-BamHI fragment into a multiple cloning site of the pcDNA vector, and constructing a first intermediate pcDNA-AGO2-C1 which has a nucleotide sequence shown by SEQ ID No.1 in a sequence table;

the C-terminal protein of the radial-foot luciferase (GLuc) was amplified by polymerase chain reaction using pCMV-GLuc1 plasmid as a vector (107-185aa and stop codon), BamHI and XbaI restriction sites were added, and (GGGGS) was added₂A linker peptide. Will (GGGGS)₂The gluc (C) fragment was loaded into the aforementioned pcDNA-AGO2-C1 intermediate using cloning techniques to prepare a product pcDNA-AGO2-gluc (C) having the nucleotide sequence shown in SEQ ID No.2 of the sequence Listing.

2. pCMV-MSI1-gluc (N) recombinant plasmid

Adding N-terminal (1-106aa) of the radial leg luciferase (GLuc) by using polymerase chain reaction with pCMV-GLuc1 plasmid as a vector(GGGGS)₂After connecting peptide and restriction endonuclease cutting sites BamHI and XbaI, cloning to prepare a second intermediate pCMV-gluc (N) which has a nucleotide sequence shown by SEQ ID No.3 in a sequence table;

MSI1 was amplified and purified by PCR amplification and restriction sites HindIII and BamHI were added, and MSI1 was cloned into pCMV-gluc (N), a second intermediate, pCMV-MSI1-gluc (N), having the nucleotide sequence shown in SEQ ID No.4 of the sequence Listing.

The fusion protein provided by the invention is shown in figure 3 and is obtained by fusing the two recombinant plasmids.

FIG. 4 is a schematic diagram of the operation of the recombinant imaging system of the present invention, wherein the C-terminal and N-terminal of GLuc are cleaved at amino acid 106, and the C-side fragment (106 amino acids in length) and N-side fragment (78 amino acids in length) are fused to the C-side of AGO2 and MSI1 proteins by molecular cloning, respectively, through a linker peptide (GGGGS)₂To reduce the influence of luciferase function possibly caused by the main protein. MSI1 undergoes intracellular trafficking, moving from the nucleus to the cytosol, and protein interaction with cytosolic AGO2 after cancer cells are subjected to stress, particularly anticancer drug therapy. The present invention takes advantage of this feature by the coincidence of two luciferase fragments cloned in MSI1 and AGO2, thereby generating a bioluminescent signal. The images are captured by an image capturing system such as a disk-type cold light instrument (in vitro) or an IVIS (in vivo) and then quantified.

The invention is further illustrated by the following specific examples.

Referring to FIG. 5, this example tests different linker peptides to try to compare whether different length and elasticity of linker peptides affect the effect of luminescence coincidence. Mainly two types (GGGGS) of elastic type were compared with (PGPGP) of harder type. Also, single linker peptides or double linker peptides (GGGGS-GGGGS; denoted as (GGGGS)₂) The effect of (1). The cloning mode is the same as the construction of the plasmid, and is mainly achieved by putting connecting peptide gene sequences with different lengths into the primer design. The research finds that: in the absence of the linker peptide, the luminescence signal was minimal. Adopt the two kindsThe linker peptide in its form was tested to clarify the most suitable design by the optical effect after protein-protein interaction and the basis value before interaction.

The application and test modes are as follows:

1. materials:

purified and spectrophotometrically purified plasmid (concentration 1 ug/ul; 260/280 ratio between 1.8-1.9);

brain tumor cell line DBTRG-05MG (CRL-2020, ATCC, USA);

six-well cell culture plates (NUNC, Thermo Fisher, usa);

cell culture media (high-glucose DMEM + 10% fetal bovine serum + 1% synthetic antibiotics);

cell transfection reagents (JetPrime, Polyplus, france);

a disk cold light (Victor3 Multilabel Plate Reader, PerkinElmer, usa);

in Vivo optical Imaging System Spectrum CT (PerkinElmer, usa);

dual Reporter optical suite (Dual Reporter Assay System, Promega, usa).

2. The operation steps are as follows:

day 0 (ready):

after the brain tumor DBTRG-05MG cells are subcultured for at least 3 times, 1x10 is planted in each well of a 6-well cell plate⁵A cell. Put into a cell culture box for overnight (at least 16 hours).

Plasmid extraction, quantification and quality assurance.

Day 1 (transfection):

taking out the brain tumor cells, replacing with 1 ml of fresh culture medium, and then putting back to the incubator for later use.

A total of 2. mu.g of plasmid per well was mixed with 200. mu.l of transfection buffer, shaken for 10 seconds and then briefly centrifuged for use, as indicated by the transfection reagents.

2 micrograms if transfected with a single plasmid; if the two plasmids are subjected to combined transfection, the proportion is maintained to be 1:1, namely, the two plasmids are added into each hole by 1 microgram/hole; for example, the mass ratio is 0.6:1.2:1.2 in the case of the three-plasmid combination (including the fluorescence enzyme for controlling firefly for quantification).

4 microliter transfection reagent is added to the plasmid mixing buffer, concussion and simple centrifugation, and then placed at room temperature for reaction for about 10 minutes.

All the mixed solution is added dropwise into the designated wells to start transfection.

Day 2 (recovery):

after 24 hours of adding transfection reagent, the medium was replaced with fresh medium, and the plates were returned to the chamber for continuous culture.

Day 3 (quantification analysis):

according to the description, the optical signal acquisition is performed by using a double-reporter optical kit, using an automatic injection system of a disk-type luminescence apparatus to perform the injection and termination reactions of the luminescence substrates D-luciferin and Coelenterazine.

If Living body Image system is used, the luminescence material is added manually and put into the machine, and the Living Image 4.0 software is used to automatically capture luminescence Image. And setting a threshold value of the quantization numerical value to be 20% for ROI selection and calculation.

FIG. 6 shows background values for different fragments of the luciferase, which may be partially functional due to truncation and truncation. The test shows that most of the truncated luciferase has no normal expression amount of more than 10%. The relative cold light quantity is divided by the GLuc luminescence reading by the FLuc luminescence reading; all cells were stable clonal cells of FLuc.

FIG. 7 shows changes in the amount of cold light in normal and stressed environments, and shows that both plasmids (GGGGS)₂In the case of the linker peptide (i.e., bGS), the most effective reporter function is provided, and the recombinant effect of the linker peptide is optimal.

FIG. 8 is a graph of relative cold light measurements with different pressure-induced responses, showing that multiple pressure pathways are perceived by the imaging system.

FIG. 9 is a live animal test, in which the same subcutaneous brain tumor-implanted mouse is imaged before and after treatment (Temozolomide) using the imaging system. The recombined image system can effectively observe the treatment state.

It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.

Sequence listing

<110> Shenzhen institute of hong Kong Ringworker university

<120> recombinant plasmid, construction method thereof, recombinant image system and application

<160> 4

<210> 1

<211> 8952

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 1

acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat 60

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ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt 180

ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca 240

ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg 300

tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 360

tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa 420

tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct tatgtactcg 480

ggagccggcc ccgcacttgc acctcctgcg ccgccgcccc ccatccaagg atatgccttc 540

aagcctccac ctagacccga ctttgggacc tccgggagaa caatcaaatt acaggccaat 600

ttcttcgaaa tggacatccc caaaattgac atctatcatt atgaattgga tatcaagcca 660

gagaagtgcc cgaggagagt taacagggaa atcgtggaac acatggtcca gcactttaaa 720

acacagatct ttggggatcg gaagcccgtg tttgacggca ggaagaatct atacacagcc 780

atgccccttc cgattgggag ggacaaggtg gagctggagg tcacgctgcc aggagaaggc 840

aaggatcgca tcttcaaggt gtccatcaag tgggtgtcct gcgtgagctt gcaggcgtta 900

cacgatgcac tttcagggcg gctgcccagc gtcccttttg agacgatcca ggccctggac 960

gtggtcatga ggcacttgcc atccatgagg tacacccccg tgggccgctc cttcttcacc 1020

gcgtccgaag gctgctctaa ccctcttggc gggggccgag aagtgtggtt tggcttccat 1080

cagtccgtcc ggccttctct ctggaaaatg atgctgaata ttgatgtgtc agcaacagcg 1140

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gaagaacaac aaaaacctct gacagattcc caaagggtaa agtttaccaa agaaattaaa 1260

ggtctaaagg tggagataac gcactgtggg cagatgaaga ggaagtaccg tgtctgcaat 1320

gtgacccggc ggcccgccag tcaccaaaca ttcccgctgc agcaggagag cgggcagacg 1380

gtggagtgca cggtggccca gtatttcaag gacaggcaca agttggttct gcgctacccc 1440

cacctcccat gtttacaagt cggacaggag cagaaacaca cctaccttcc cctggaggtc 1500

tgtaacattg tggcaggaca aagatgtatt aaaaaattaa cggacaatca gacctcaacc 1560

atgatcagag cgactgctag gtcggcgccc gatcggcaag aagagattag caaattgatg 1620

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gagatgacag acgtgactgg gcgggtgctg cagccgccct ccatcctcta cgggggcagg 1740

aataaagcta ttgcgacccc tgtccagggc gtctgggaca tgcggaacaa gcagttccac 1800

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gaagtccatc tgaagtcctt cacagagcag ctcagaaaga tctcgagaga cgccggcatg 1920

cccatccagg gccagccgtg cttctgcaaa tacgcgcagg gggcggacag cgtggagccc 1980

atgttccggc acctgaagaa cacgtatgcg ggcctgcagc tggtggtggt catcctgccc 2040

ggcaagacgc ccgtgtacgc cgaggtcaag cgcgtgggag acacggtgct ggggatggcc 2100

acgcagtgcg tgcagatgaa gaacgtgcag aggaccacgc cacagaccct gtccaacctc 2160

tgcctgaaga tcaacgtcaa gctgggaggc gtgaacaaca tcctgctgcc ccagggcagg 2220

ccgccggtgt tccagcagcc cgtcatcttt ctgggagcag acgtcactca cccccccgcc 2280

ggggatggga agaagccctc cattgccgcc gtggtgggca gcatggacgc ccaccccaat 2340

cgctactgcg ccaccgtgcg cgtgcagcag caccggcagg agatcataca agacctggcc 2400

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ttgctggcca tccgtgaggc ctgtatcaag ctagaaaaag actaccagcc cgggatcacc 2580

ttcatcgtgg tgcagaagag gcaccacacc cggctcttct gcactgacaa gaacgagcgg 2640

gttgggaaaa gtggaaacat tccagcaggc acgactgtgg acacgaaaat cacccacccc 2700

accgagttcg acttctacct gtgtagtcac gctggcatcc aggggacaag caggccttcg 2760

cactatcacg tcctctggga cgacaatcgt ttctcctctg atgagctgca gatcctaacc 2820

taccagctgt gtcacaccta cgtgcgctgc acacgctccg tgtccatccc agcgccagca 2880

tactacgctc acctggtggc cttccgggcc aggtaccacc tggtggataa ggaacatgac 2940

agtgctgaag gaagccatac ctctgggcag agtaacgggc gagaccacca agcactggcc 3000

aaggcggtcc aggttcacca agacactctg cgcaccatgt actttgctgg tggaggcggt 3060

tcaggtggag gcggatcctc gagatggtga gcaagggcga ggagctgttc accggggtgg 3120

tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc gtccgcggcg 3180

agggcgaggg cgatgccacc aacggcaagc tgaccctgaa gttcatctgc accaccggca 3240

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acgtccagga gcgcaccatc tctttcaagg acgacggtac ctacaagacc cgcgccgagg 3420

tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc gacttcaagg 3480

aggacggcaa catcctgggg cacaagctgg agtacaactt caacagccac aacgtctata 3540

tcacggccga caagcagaag aacggcatca aggctaactt caagatccgc cacaacgttg 3600

aggacggcag cgtgcagctc gccgaccact accagcagaa cacccccatc ggcgacggcc 3660

ccgtgctgct gcccgacaac cactacctga gccatcagtc cgccctgagc aaagacccca 3720

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gcatggacga gctgtacaag tctagagggc ccgtttaaac ccgctgatca gcctcgactg 3840

tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg 3900

aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga 3960

gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg 4020

aagacaatag caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa 4080

ccagctgggg ctctaggggg tatccccacg cgccctgtag cggcgcatta agcgcggcgg 4140

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tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 4260

gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 4320

attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga 4380

cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc 4440

ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa 4500

aaaatgagct gatttaacaa aaatttaacg cgaattaatt ctgtggaatg tgtgtcagtt 4560

agggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa 4620

ttagtcagca accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag 4680

catgcatctc aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct 4740

aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc 4800

agaggccgag gccgcctctg cctctgagct attccagaag tagtgaggag gcttttttgg 4860

aggcctaggc ttttgcaaaa agctcccggg agcttgtata tccattttcg gatctgatca 4920

gcacgtgatg aaaaagcctg aactcaccgc gacgtctgtc gagaagtttc tgatcgaaaa 4980

gttcgacagc gtctccgacc tgatgcagct ctcggagggc gaagaatctc gtgctttcag 5040

cttcgatgta ggagggcgtg gatatgtcct gcgggtaaat agctgcgccg atggtttcta 5100

caaagatcgt tatgtttatc ggcactttgc atcggccgcg ctcccgattc cggaagtgct 5160

tgacattggg gaattcagcg agagcctgac ctattgcatc tcccgccgtg cacagggtgt 5220

cacgttgcaa gacctgcctg aaaccgaact gcccgctgtt ctgcagccgg tcgcggaggc 5280

catggatgcg atcgctgcgg ccgatcttag ccagacgagc gggttcggcc cattcggacc 5340

gcaaggaatc ggtcaataca ctacatggcg tgatttcata tgcgcgattg ctgatcccca 5400

tgtgtatcac tggcaaactg tgatggacga caccgtcagt gcgtccgtcg cgcaggctct 5460

cgatgagctg atgctttggg ccgaggactg ccccgaagtc cggcacctcg tgcacgcgga 5520

tttcggctcc aacaatgtcc tgacggacaa tggccgcata acagcggtca ttgactggag 5580

cgaggcgatg ttcggggatt cccaatacga ggtcgccaac atcttcttct ggaggccgtg 5640

gttggcttgt atggagcagc agacgcgcta cttcgagcgg aggcatccgg agcttgcagg 5700

atcgccgcgg ctccgggcgt atatgctccg cattggtctt gaccaactct atcagagctt 5760

ggttgacggc aatttcgatg atgcagcttg ggcgcagggt cgatgcgacg caatcgtccg 5820

atccggagcc gggactgtcg ggcgtacaca aatcgcccgc agaagcgcgg ccgtctggac 5880

cgatggctgt gtagaagtac tcgccgatag tggaaaccga cgccccagca ctcgtccgag 5940

ggcaaaggaa tagcacgtgc tacgagattt cgattccacc gccgccttct atgaaaggtt 6000

gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg gggatctcat 6060

gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt acaaataaag 6120

caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 6180

gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta gctagagctt 6240

ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca 6300

caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact 6360

cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct 6420

gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc 6480

ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca 6540

ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg 6600

agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca 6660

taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa 6720

cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc 6780

tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc 6840

gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct 6900

gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg 6960

tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag 7020

gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta 7080

cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg 7140

aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtttttttgt 7200

ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 7260

tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt 7320

atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta 7380

aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat 7440

ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac 7500

tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg 7560

ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag 7620

tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt 7680

aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt 7740

gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt 7800

tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt 7860

cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct 7920

tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt 7980

ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac 8040

cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa 8100

actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa 8160

ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca 8220

aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct 8280

ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga 8340

atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc 8400

tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc 8460

tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag 8520

tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag 8580

aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt 8640

tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 8800

ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 8860

aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 8920

actttccatt gacgtcaatg ggtggagtat tt 8952

<210> 2

<211> 8670

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 2

acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat 60

tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga 120

ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt 180

ttggcagtac atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca 240

ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg 300

tcgtaacaac tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta 360

tataagcaga gctctctggc taactagaga acccactgct tactggctta tcgaaattaa 720

tacgactcac tatagggaga cccaagctgg ctagcgttta aacttaagct tatgtactcg 780

ggagccggcc ccgcacttgc acctcctgcg ccgccgcccc ccatccaagg atatgccttc 840

aagcctccac ctagacccga ctttgggacc tccgggagaa caatcaaatt acaggccaat 900

ttcttcgaaa tggacatccc caaaattgac atctatcatt atgaattgga tatcaagcca 960

gagaagtgcc cgaggagagt taacagggaa atcgtggaac acatggtcca gcactttaaa 1020

acacagatct ttggggatcg gaagcccgtg tttgacggca ggaagaatct atacacagcc 1080

atgccccttc cgattgggag ggacaaggtg gagctggagg tcacgctgcc aggagaaggc 1140

aaggatcgca tcttcaaggt gtccatcaag tgggtgtcct gcgtgagctt gcaggcgtta 1200

cacgatgcac tttcagggcg gctgcccagc gtcccttttg agacgatcca ggccctggac 1260

gtggtcatga ggcacttgcc atccatgagg tacacccccg tgggccgctc cttcttcacc 1320

gcgtccgaag gctgctctaa ccctcttggc gggggccgag aagtgtggtt tggcttccat 1380

cagtccgtcc ggccttctct ctggaaaatg atgctgaata ttgatgtgtc agcaacagcg 1440

ttttacaagg cacagccagt aatcgagttt gtttgtgaag ttttggattt taaaagtatt 1500

gaagaacaac aaaaacctct gacagattcc caaagggtaa agtttaccaa agaaattaaa 1560

ggtctaaagg tggagataac gcactgtggg cagatgaaga ggaagtaccg tgtctgcaat 1620

gtgacccggc ggcccgccag tcaccaaaca ttcccgctgc agcaggagag cgggcagacg 1680

gtggagtgca cggtggccca gtatttcaag gacaggcaca agttggttct gcgctacccc 1740

cacctcccat gtttacaagt cggacaggag cagaaacaca cctaccttcc cctggaggtc 1800

tgtaacattg tggcaggaca aagatgtatt aaaaaattaa cggacaatca gacctcaacc 1860

atgatcagag cgactgctag gtcggcgccc gatcggcaag aagagattag caaattgatg 1920

cgaagtgcaa gtttcaacac agatccatac gtccgtgaat ttggaatcat ggtcaaagat 1980

gagatgacag acgtgactgg gcgggtgctg cagccgccct ccatcctcta cgggggcagg 2040

aataaagcta ttgcgacccc tgtccagggc gtctgggaca tgcggaacaa gcagttccac 2100

acgggcatcg agatcaaggt gtgggccatt gcgtgcttcg ccccccagcg ccagtgcacg 2160

gaagtccatc tgaagtcctt cacagagcag ctcagaaaga tctcgagaga cgccggcatg 2220

cccatccagg gccagccgtg cttctgcaaa tacgcgcagg gggcggacag cgtggagccc 2280

atgttccggc acctgaagaa cacgtatgcg ggcctgcagc tggtggtggt catcctgccc 2340

ggcaagacgc ccgtgtacgc cgaggtcaag cgcgtgggag acacggtgct ggggatggcc 2400

acgcagtgcg tgcagatgaa gaacgtgcag aggaccacgc cacagaccct gtccaacctc 2460

tgcctgaaga tcaacgtcaa gctgggaggc gtgaacaaca tcctgctgcc ccagggcagg 2520

ccgccggtgt tccagcagcc cgtcatcttt ctgggagcag acgtcactca cccccccgcc 2580

ggggatggga agaagccctc cattgccgcc gtggtgggca gcatggacgc ccaccccaat 2640

cgctactgcg ccaccgtgcg cgtgcagcag caccggcagg agatcataca agacctggcc 2700

gccatggtcc gcgagctcct catccagttc tacaagtcca cgcgcttcaa gcccacccgc 2760

atcatcttct accgcgacgg tgtctctgaa ggccagttcc agcaggttct ccaccacgag 2820

ttgctggcca tccgtgaggc ctgtatcaag ctagaaaaag actaccagcc cgggatcacc 2880

ttcatcgtgg tgcagaagag gcaccacacc cggctcttct gcactgacaa gaacgagcgg 2940

gttgggaaaa gtggaaacat tccagcaggc acgactgtgg acacgaaaat cacccacccc 3000

accgagttcg acttctacct gtgtagtcac gctggcatcc aggggacaag caggccttcg 3060

cactatcacg tcctctggga cgacaatcgt ttctcctctg atgagctgca gatcctaacc 3120

taccagctgt gtcacaccta cgtgcgctgc acacgctccg tgtccatccc agcgccagca 3180

tactacgctc acctggtggc cttccgggcc aggtaccacc tggtggataa ggaacatgac 3240

agtgctgaag gaagccatac ctctgggcag agtaacgggc gagaccacca agcactggcc 3300

aaggcggtcc aggttcacca agacactctg cgcaccatgt actttgctgg tggaggcggt 3360

tcaggtggag gcggatccgg cataggcgag gcgatcgtcg acattcctga gattcctggg 3420

ttcaaggact tggagcccat ggagcagttc atcgcacagg tcgatctgtg tgtggactgc 3480

acaactggct gcctcaaagg gcttgccaac gtgcagtgtt ctgacctgct caagaagtgg 3540

ctgccgcaac gctgtgcgac ctttgccagc aagatccagg gccaggtgga caagatcaag 3600

ggggccggtg gtgactaatc tagagggccc gtttaaaccc gctgatcagc ctcgactgtg 3660

ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa 3720

ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt 3780

aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa 3840

gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc 3900

agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt 3960

gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc 4020

gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg 4080

gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat 4140

tagggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg 4200

ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct 4260

atctcggtct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa 4320

aatgagctga tttaacaaaa atttaacgcg aattaattct gtggaatgtg tgtcagttag 4380

ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt 4440

agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt atgcaaagca 4500

tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc ccgcccctaa 4560

ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt atttatgcag 4620

aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc ttttttggag 4680

gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga tctgatcagc 4740

acgtgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt 4800

tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct 4860

tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca 4920

aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg 4980

acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca 5040

cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca 5100

tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc 5160

aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg 5220

tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg 5280

atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt 5340

tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg 5400

aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt 5460

tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat 5520

cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg 5580

ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat 5640

ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg 5700

atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg 5760

caaaggaata gcacgtgcta cgagatttcg attccaccgc cgccttctat gaaaggttgg 5820

gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc 5880

tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca 5940

atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt 6000

ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg 6060

cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca 6120

acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca 6180

cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc 6240

attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt 6300

cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact 6360

caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag 6420

caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata 6480

ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc 6540

cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg 6600

ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc 6660

tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg 6720

gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc 6780

ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga 6840

ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg 6900

gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa 6960

aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ttttttgttt 7020

gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 7080

cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 7140

caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 7200

gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 7260

cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 7320

cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 7380

caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 7440

gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 7500

gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 7560

cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 7620

catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 7680

gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta 7740

ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct 7800

gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg 7860

cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 7920

tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 7980

gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa 8040

atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt 8100

ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat 8160

gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg 8220

acgtcgacgg atcgggagat ctcccgatcc cctatggtgc actctcagta caatctgctc 8280

tgatgccgca tagttaagcc agtatctgct ccctgcttgt gtgttggagg tcgctgagta 8340

gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg accgacaatt gcatgaagaa 8400

tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg 8460

acattgatta ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc 8520

atatatggag ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa 8580

cgacccccgc ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac 8640

tttccattga cgtcaatggg tggagtattt 8670

<210> 3

<211> 5801

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 3

gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 720

attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 780

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 840

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 900

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 960

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 1020

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 1080

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 1140

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc 1200

gagctcggat ccatgggagt caaagttctg tttgccctga tctgcatcgc tgtggccgag 1260

gccaagccca ccgagaacaa cgaagacttc aacatcgtgg ccgtggccag caacttcgcg 1320

accacggatc tcgatgctga ccgcgggaag ttgcccggca agaagctgcc gctggaggtg 1380

ctcaaagaga tggaagccaa tgcccggaaa gctggctgca ccaggggctg tctgatctgc 1440

ctgtcccaca tcaagtgcac gcccaagatg aagaagttca tcccaggacg ctgccacacc 1500

tacgaaggcg acaaagagtc cgcacagggc tgatctagaa ataattctta ctgtcatgcc 1560

aagtaagatg cttttctgtg ctgcaatagc aggcatgctg gggatgcggt gggctctatg 1620

gcttctgagg cggaaagaac cagctggggc tctagggggt atccccacgc gccctgtagc 1680

ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc 1740

gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt 1800

ccccgtcaag ctctaaatcg gggcatccct ttagggttcc gatttagtgc tttacggcac 1860

ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag 1920

acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa 1980

actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgggg 2040

atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattaattc 2100

tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccaggc aggcagaagt 2160

atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtcccc aggctcccca 2220

gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagt cccgccccta 2280

actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga 2340

ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagct attccagaag 2400

tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccggg agcttgtata 2460

tccattttcg gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat 2520

ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca 2580

caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg 2640

gttctttttg tcaagaccga cctgtccggt gccctgaatg aactgcagga cgaggcagcg 2700

cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact 2760

gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct 2820

caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg 2880

cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt 2940

actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc 3000

gcgccagccg aactgttcgc caggctcaag gcgcgcatgc ccgacggcga ggatctcgtc 3060

gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga 3120

ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc 3180

cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt 3240

atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga 3300

gcgggactct ggggttcgaa atgaccgacc aagcgacgcc caacctgcca tcacgagatt 3360

tcgattccac cgccgccttc tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg 3420

gctggatgat cctccagcgc ggggatctca tgctggagtt cttcgcccac cccaacttgt 3480

ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag 3540

catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg 3600

tctgtatacc gtcgacctct agctagagct tggcgtaatc atggtcatag ctgtttcctg 3660

tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta 3720

aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg 3780

ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga 3840

gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 3900

tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 3960

aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 4020

gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 4080

aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt 4140

ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc 4200

tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 4260

tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 4320

ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact 4380

tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 4440

ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta 4500

tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca 4560

aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa 4620

aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg 4680

aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 4740

ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 4800

acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat 4860

ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg 4920

gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa 4980

taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 5040

tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 5100

gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 5160

cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa 5220

aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat 5280

cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct 5340

tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga 5400

gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 5460

tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 5520

gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca 5580

ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg 5640

cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc 5700

agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 5760

gggttccgcg cacatttccc cgaaaagtgc cacctgacgt c 5801

<210> 4

<211> 6899

<212> DNA

<213> Artificial sequence (rengongxulie)

<400> 4

gacggatcgg gagatctccc gatcccctat ggtcgactct cagtacaatc tgctctgatg 60

ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120

cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180

ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240

gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300

tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360

cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 720

attgacgtca atgggtggac tatttacggt aaactgccca cttggcagta catcaagtgt 780

atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 840

atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 900

tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 960

actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 1020

aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 1080

gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 1140

ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttatggag 1200

actgacgcgc cccagcccgg cctcgcctcc ccggactcgc cgcacgaccc ctgcaagatg 1260

ttcatcgggg gactcagttg gcagactacg caggaagggc tgcgcgaata cttcggccag 1320

ttcggggagg tgaaggagtg tctggtgatg cgggaccccc tgaccaagag atccaggggt 1380

ttcggcttcg tcactttcat ggaccaggcg ggggtggata aagtgctggc gcaatcgcgg 1440

cacgagctcg actccaaaac aattgaccct aaggtggcct tccctcggcg agcacagccc 1500

aagatggtga ctcgaacgaa gaagatcttt gtgggggggc tgtcggtgaa caccacggtg 1560

gaggacgtga agcaatattt tgagcagttt gggaaggtgg acgacgccat gctgatgttt 1620

gacaaaacca ccaaccggca ccgagggttc gggtttgtca cgtttgagag tgaggacatc 1680

gtggagaaag tgtgtgaaat tcattttcat gaaatcaaca acaaaatggt ggaatgtaag 1740

aaagctcagc caaaggaggt gatgtcgcca acgggctcag cccgggggag gtctcgagtc 1800

atgccctacg gaatggacgc cttcatgctg ggcatcggca tgctgggtta cccaggtttc 1860

caagccacaa cctacgccag ccggagttat acaggcctcg cccctggcta cacctaccag 1920

ttccccgaat tccgtgtaga gcggacccct ctcccgagcg ccccagtcct ccccgagctt 1980

acagccattc ctctcactgc ctacggacca atggcggcgg cagcggcggc agcggctgtg 2040

gttcgaggga caggctctca cccctggacg atggctcccc ctccaggttc gactcccagc 2100

cgcacagggg gcttcctggg gaccaccagc cccggcccca tggccgagct ctacggggcg 2160

gccaaccagg actcgggggt cagcagttac atcagcgccg ccagccctgc ccccagcacc 2220

ggcttcggcc acagtcttgg gggccctttg attgccacag ccttcaccaa tgggtaccac 2280

ggtggaggcg gttcaggtgg aggcggatcc atgggagtca aagttctgtt tgccctgatc 2340

tgcatcgctg tggccgaggc caagcccacc gagaacaacg aagacttcaa catcgtggcc 2400

gtggccagca acttcgcgac cacggatctc gatgctgacc gcgggaagtt gcccggcaag 2460

aagctgccgc tggaggtgct caaagagatg gaagccaatg cccggaaagc tggctgcacc 2520

aggggctgtc tgatctgcct gtcccacatc aagtgcacgc ccaagatgaa gaagttcatc 2580

ccaggacgct gccacaccta cgaaggcgac aaagagtccg cacagggctg atctagaaat 2640

aattcttact gtcatgccaa gtaagatgct tttctgtgct gcaatagcag gcatgctggg 2700

gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat 2760

ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg 2820

accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc 2880

gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg gcatcccttt agggttccga 2940

tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt 3000

gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat 3060

agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat 3120

ttataaggga ttttggggat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa 3180

tttaacgcga attaattctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct 3240

ccccaggcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga 3300

aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca 3360

accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat 3420

tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc 3480

tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag 3540

ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt 3600

cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta 3660

ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg 3720

tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa 3780

ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct 3840

gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg 3900

caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca 3960

atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat 4020

cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac 4080

gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc 4140

gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa 4200

aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag 4260

gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc 4320

ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt 4380

cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca 4440

acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa 4500

tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct 4560

tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca 4620

caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca 4680

tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat 4740

ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag 4800

ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg 4860

cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa 4920

tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca 4980

ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 5040

taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 5100

agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 5160

cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 5220

tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 5280

tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcaat 5340

gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 5400

acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 5460

acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5520

cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5580

gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5640

gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 5700

agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5760

ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa 5820

ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat 5880

atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga 5940

tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac 6000

gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg 6060

ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg 6120

caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt 6180

cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct 6240

cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat 6300

cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta 6360

agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca 6420

tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat 6480

agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac 6540

atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa 6600

ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt 6660

cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg 6720

caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat 6780

attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt 6840

agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc 6899

Claims (8)

1. A method for constructing a recombinant plasmid, comprising the steps of:

implanting AGO2 into restriction endonuclease sites HindIII and BamHI by using polymerase chain reaction, and then carrying out amplification to obtain a HindIII-AGO2-BamHI fragment;

introducing a HindIII-AGO2-BamHI fragment into a multiple cloning site of a pcDNA vector to obtain pcDNA-AGO 2-C1;

the C-terminal protein of the radial-foot luciferase GLuc was amplified using the polymerase chain reaction, the restriction sites BamHI and XbaI were added, and (GGGGS) was added₂Linker peptide to obtain (GGGGS)₂-gluc (c) fragment;

will (GGGGS)₂The gluc (C) fragment was loaded into pcDNA-AGO2-C1 using cloning techniques to obtain pcDNA-AGO2-gluc (C) recombinant plasmid.

2. A recombinant plasmid constructed by the method of claim 1.

3. A method for constructing a recombinant plasmid, comprising the steps of:

adding (GGGGS) to the N-terminus of the radial-foot luciferase GLuc by using polymerase chain reaction with pCMV-GLuc1 plasmid as a vector₂After connecting peptide and restriction endonuclease cutting sites BamHI and XbaI, cloning to prepare pCMV-gluc (N);

MSI1 was amplified and purified by PCR amplification and restriction sites HindIII and BamHI were added, and MSI1 was loaded into pCMV-gluc (N) by cloning to give pCMV-MSI1-gluc (N).

4. A recombinant plasmid constructed by the method of claim 3.

5. A fusion protein obtained by fusing the recombinant plasmid according to claim 2 and the recombinant plasmid according to claim 4.

6. A recombinant imaging system comprising the recombinant plasmid of claim 2 and the recombinant plasmid of claim 4.

7. Use of the fusion protein according to claim 5 as or in the preparation of a product with tracer properties.

8. Use of the fusion protein of claim 5 for the preparation of a medicament for the treatment of brain tumors.

Images