CN105624192A - Preparation of mammary cancer cell strain capable of stably secreting near-infrared fluorescence labeled exosomes - Google Patents
Preparation of mammary cancer cell strain capable of stably secreting near-infrared fluorescence labeled exosomes Download PDFInfo
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Abstract
The invention discloses a preparation method of a mammary cancer cell strain capable of stably secreting near-infrared fluorescence labeled exosomes. The preparation method comprises the following steps: establishing a plamid vector capable of fused expression of near-infrared fluorescence iRFP682 protein and CD63 protein, co-infecting human mammary cancer MDA-MB-231 cells by using a plasmid-packaged recombinant adeno-associated virus (rAAV) and a helper virus in a certain proportion, screening to obtain the human mammary cancer MDA-MB-231 cells capable of stably expressing near-infrared fluorescence protein iRFP682 labeled exosomes, separating and purifying the near-infrared fluorescence protein iRFP682 labeled exosomes by multi-step centrifugation, and carrying out identification. The method finally obtains a novel biomarker which can be used for mammary cancer tumor microenvironment in-vivo in-vitro study, thereby providing a powerful tool for further studying the intercellular material transfer mechanism, and also providing a new support and chance for cancer diagnosis and therapeutic schemes.
Description
Technical field
The present invention relates to biomarker field. It is more particularly related to secrete the preparation of the breast carcinoma cell strain of body outside a kind of stably excreting near-infrared fluorescent labelling.
Background technology
1986, have in the sheep red blood cell (SRBC) supernatant that scholar cultivates in vitro and be found that a kind of vesicles having membrane structure, be referred to as and outer secrete body. to 1996, in the human B cell that Epstein-Barr virus converts, some have the vesicles surface of membrane structure can express major histocompatibility complex (majorhistocompatibilitycomplex to have scholar to find, MHC) class ��molecule, T cell can be activated, and secrete the forming process of body to intra-erythrocyte and discharge approach is similar (referring to VanNielG, Porto-CarreiroI, SimoesS, RaposoG.Exosomes:acommonpathwayforaspecializedfunction.J Biochem2006, 140:13��21), since then, people start externally to secrete body and launch to study widely. secreting outward body is that the film vesicle that diameter is 30��120nm deriving from late endosome (also referred to as many vesicles body) secreted by living cells is (referring to StoorvogelW, KleijmeerMJ, GeuzeHJ, RaposoG.Thebiogenesisandfunctionsofexosomes.Traffic2002,3:321��330). current research direction mainly has stem cell, immunity, microRNA and target administration. within 2013, Nobel's biology/Medicine is granted by three scientists that contribution is prominent in cell vesicle, promotes the outer research secreting body to reach new high.
Various different types of cells, tissue, organ and system is mutually coordinated, the complicated body that jointly maintains us properly functioning (referring to J.Skog, T.W �� rdinger, S.vanRijn, D.H.Meijer, L.Gainche, M.Sena-Esteves, W.T.CurryJr., B.S.Carter, A.M.Krichevsky, X.O.Breakefield, GlioblastomamicrovesiclestransportRNAandproteinsthatprom otetumourgrowthandprovidediagnosticbiomarkers, Nat.CellBiol.2008,10:1470��1476). Information transmission between this and cell is closely-related, and the information transmission between cell, main is that some particular chemicals being manufactured by cell oneself and discharging realizes, for instance secrete outward body. When body is in abnormal state, for instance when disease (cancer) occurs, some semiochemicalses will be discharged to regulate physical function and adapt to change. At this moment, secrete outward body as a member in these semiochemicalses, just played very big effect (referring to mericanCancerSociety, GlobalCancerFacts&Figures, 2ndEditionAmericanCancerSociety, Atlanta, 2011). Secrete outward body and contain protein, mRNA, the signaling molecules such as miRNA, the physiological status of they reflection secretory cells and functional status, even also can comprise the molecular information that cytopathic is relevant, thus providing abundant potential biomarker molecular source (referring to YuS, CaoH, ShenB, FengJTumor-derivedexosomesincancerprogressionandtreatmen tfailure.Oncotarget.2015,7:10��18). About secreting outward body as signal transmission one of study hotspot having become cytobiology, molecular biology and medical domain to study between cell of biomarker. the generation of multiple disease, development and the preventions such as cell signal transmission and tumor are directly related (referring to SimonsM, RaposoG, Exosomes vesicularcarriersforintercellularcommunication.CurrOpinC ellBiol2009,21:575��581). Therefore, the research secreting outward body will play a significant role in fields such as the diagnosis of cancer and treatments.
Near-infrared fluorescent albumen (Near-infraredfluorescentprotein, iRFP) it is a class fluorescin of green fluorescent protein homology, possesses the luminous advantage of green fluorescent protein (referring to MikhailBaloban, DariaM.Shcherbakova, VladislavV.Verkhusha, Two-ColorImagingusingSpectralVariantsofiRFP670andiRFP682 Near-InfraredFluorescentProteins, BiophysicalJournal, 2015,108:624-631). but, compared to GFP, its exciting light and wavelength of transmitted light are longer, it is positioned at near-infrared region (650��900nm), in animal tissue, light absorbs relatively low with light scattering, there is higher penetrance, it is more suitable for the imaging deep of animal live soma, it is that living imaging more preferably fluorescent tag molecule is (referring to GrigoryS.Filonov, VladislavV.Verkhusha, ANear-InfraredBiFCReporterforInVivoImagingofProtein-Prot einInteractionsOriginalResearchArticleChemistry&Biology, 2013, 20:1078-1086). the exciting light of near-infrared fluorescent albumen iRFP682 and wavelength of transmitted light respectively 663nm and 682nm, be monomer fluorescence protein molecular, and its luminosity is relatively stable, and longer spectrum characteristic makes its more potentiality in the application of living animal imaging of tissue.
Thus, if the superiority of the unique function and the external spike in vivo of near-infrared fluorescent albumen of secreting outward body can be combined, can for studying intercellular substance metastasis offer powerful further.
Summary of the invention
It is an object of the invention to solve at least the above and/or defect, and the advantage that at least will be described later is provided.
The preparation method that it is a still further object of the present invention to provide the breast carcinoma cell strain secreting body outside a kind of stably excreting near-infrared fluorescent labelling, it is by by coding near-infrared fluorescent albumen iRFP682 with secrete outward the gene integration of body CD63 protein fusion expression in breast cancer cell genome, thus the outer breast carcinoma cell strain secreting body of stably excreting near-infrared fluorescent labelling can be obtained, finally give a new biomarkers that can be used for the research of breast cancer tumour microenvironment vivo and vitro, powerful is provided for studying intercellular substance metastasis further, the scheme that diagnoses and treats simultaneously also giving cancer provides new support and opportunity.
In order to realize these purposes according to the present invention and further advantage, it is provided that the preparation method secreting the breast carcinoma cell strain of body outside a kind of stably excreting near-infrared fluorescent labelling, it comprises the steps:
1) the outer acquisition secreting body tag PROTEIN C D63 gene of coding:
Extracting total serum IgE from human breast carcinoma MDA-MB-231 cell to carry out reverse transcription and obtain cDNA, with band XbaI and ClaI restriction enzyme site, and the primer of Linker sequence carries out pcr amplification and obtains genes of interest fragment CD63-Linker;
2) structure of recombinant plasmid vector:
Step 1) the described CD63-Linker that obtains and plasmid backbone pAAV-iRFP682 obtains recombinant plasmid vector pAAV-CD63-Linker-iRFP682 through T4DNA ligase effect;
3) preparation of recombinant adeno-associated virus:
Use described recombinant plasmid vector pAAV-CD63-Linker-iRFP682 and the external cotransfection AAV-293 cell of helper plasmid, pack recombinant adeno-associated virus rAAV-CD63-Linker-iRFP682;
4) preparation of the breast carcinoma cell strain of body is secreted outside stably excreting near-infrared fluorescent protein labeling:
Use described recombinant adeno-associated virus rAAV-CD63-Linker-iRFP682 and auxiliary adeno-associated virus coinfection human breast carcinoma MDA-MB-231 cell, obtained the outer human breast carcinoma MDA-MB-231 cell strain secreting body of stably excreting near-infrared fluorescent protein labeling by fluorescent screening;
5) the outer body of secreting of near-infrared fluorescent labelling human breast carcinoma MDA-MB-231 cell extracts and identifies
Adopt Centrifugation method DNA from step 4) labelling successful MDA-MB-231 cell culture supernatant extracts the outer of iRFP682 labelling secrete body and identify.
Preferably, wherein, described step 5) in the step of Centrifugation method DNA be:
First described cell culture supernatant is removed cell debris through 2000g centrifugal 15min, 5000g centrifugal 30min of centrifugal 15min and 12000g successively under 4 DEG C of conditions, obtain the first supernatant;
Secondly after described first supernatant being used the membrane filtration that aperture is not more than 0.2 ��m, in 4 DEG C, 12000g when centrifugal 120min, obtain outer secreting body and slightly precipitating;
Finally described with PBS solution is resuspended outer secrete body and slightly precipitate, after mix homogeneously when 4 DEG C and 12000g centrifugal 90min, obtain purifying and outer secrete body, use that PBS solution is resuspended to be placed on-80 DEG C of conditions and save backup by secreting body outside described purification.
Secreting in the purification process of body outside, secrete outward the cup-shaped membrane structure vesicle that body is 30��100nm, be subject to ambient pressure and temperature impact, so no matter being extraction process or defrosting use procedure, all needing to control temperature at about 4 DEG C. Thaw and need to carry out on ice. In addition for avoid protease externally to secrete the interference of body, also should adding protease inhibitor before frozen, 4 DEG C can preserve 3 weeks, and-80 DEG C can preserve 2-3.
Preferably, wherein, described step 5) in iRFP682 labelling outer secrete the method that body identifies and be: use westernblot to identify the described outer Membrane surface proteins secreting body, by the structure secreting body knot outer described in transmission electron microscope observing, and observe the outer fluorescent labeling secreting body by SIM Structured Illumination super-resolution fluorescence microscope.
Preferably, wherein, described Linker sequence is: ggtggaggcggttcaggcggaggtggctctggcggtggcggatcg, and carry out stable with secreting outward surface mark CD63 fusion is expressed both to have can guarantee that near-infrared fluorescent albumen iRFP682, again without influence on both respective biologic activity.
Preferably, wherein, step 3) described helper plasmid be pDG plasmid, wherein said recombinant plasmid vector pAAV-CD63-Linker-iRFP682 can provide ITR sequence necessary to AAV-293 virus, helper plasmid pDG can provide necessary Rep and the Cap gene such as AAV-293 recombinant replication, packaging virus shell, thus packaging carries recombinant adeno-associated virus rAAV-CD63-Linker-iRFP682.
Preferably, wherein, described auxiliary adeno-associated virus is rAAVSVAV2, to improve the integration efficiency of exogenous gene.
Preferably, wherein, step 2) molar ratio of described CD63-Linker and plasmid backbone pAAV-iRFP682 is 2��5, is beneficial to the formation of recombinant plasmid vector.
Preferably, wherein, step 4) ratio of described recombinant adeno-associated virus and described human breast carcinoma MDA-MB-231 cell number is 3000��6000, to ensure the efficiency of infection.
The present invention at least includes following beneficial effect:
Genetically engineered breast carcinoma cell strain prepared by the present invention can secrete body by the outer of stably excreting near-infrared fluorescent albumen iRFP682 labelling, and carried out effective purification by the centrifugal method of substep to secreting body outside near-infrared labelling, the unique function of body and the superiority of near-infrared fluorescent albumen external spike in vivo is secreted outside utilizing, can be used as the new biomarker thing of breast cancer tumour microenvironment vivo and vitro research, provide powerful for studying intercellular substance metastasis further, also diagnosing and treating of cancer, scheme has important using value simultaneously.
Part is embodied by the further advantage of the present invention, target and feature by description below, and part is also by by being understood by those skilled in the art the research of the present invention and practice.
Accompanying drawing explanation
Fig. 1 is the SIM Structured Illumination super-resolution fluorescence micro-imaging of the breast carcinoma cell strain secreting body outside the stably excreting near-infrared fluorescent labelling that the present invention prepares;
Fig. 2 is that after the present invention purifies, the outer Westernblot secreting body film surface marker albumen detects collection of illustrative plates;
Fig. 3 is that the present invention purifies the outer TEM high-resolution-ration transmission electric-lens figure secreting body;
Fig. 4 is that the present invention purifies the outer SIM Structured Illumination super-resolution fluorescence micro-imaging secreting body;
The centrifugal extraction process of substep that the successful MDA-MB-231 extracellular of labelling secretes body that what Fig. 5 illustrated is.
Detailed description of the invention
Below in conjunction with accompanying drawing, the present invention is described in further detail, to make those skilled in the art can implement according to this with reference to description word.
Should be appreciated that used herein such as " have ", existence or the interpolation of other elements one or more or its combination do not allotted in " comprising " and " including " term.
In a kind of way of realization of the present invention, it is provided that the preparation method secreting the breast carcinoma cell strain of body outside stably excreting near-infrared fluorescent labelling, it comprises the steps:
1) acquisition of body tag PROTEIN C D63 gene is secreted outside
MDA-MB-231 cell attachment is cultivated in the L-15 culture medium containing 10% hyclone, is placed in 37 DEG C, in the saturated humidity incubator of 5%CO2. When cell density reaches more than 80% (less than 1 �� 107), PBS twice, with tryptic digestive juice (0.25%Trypsin, 0.02%EDTA) digest 2��3min, 900r/min is centrifuged 5min, collect cell precipitation, total serum IgE is extracted according to guanidinium isothiocyanate/phynol method (TRIZOL) method, carry out reverse transcription again, reaction system: 10 �� RTMix2 �� l, 1 �� SuperpuredNTPs (2.5mMeach) 2 �� l, Oligo-(dT) 152 �� l, QuantReverseTranscriptase1 �� l, template ribonucleic acid 1 �� l, add RNase-Free water to 20 �� l. Amplification condition: 37 DEG C, 60min. Glue reclaims cDNA, with cDNA for template, utilize the gene upstream and downstream primer amplification CD63 gene containing restricted enzyme XbaIClaI site and linker sequence, glue reclaims product, it is connected overnight with 16 DEG C of pMD-18T carrier, convert TG1 competence bacteria, choose positive monoclonal bacterium in the LB culture medium containing ammonia benzyl, 37 DEG C of constant-temperature tables (200r/min) cultivate 16h, extracting plasmid, after XbaI and ClaI enzyme action is identified, deliver the order-checking comparison of Shanghai Sheng Gong biotechnology company limited.
2) structure of recombination, amalgamation and expression plasmid vector and qualification
PAAV-CD63-Linker-iRFP682 recombiant plasmid is built according to the molecular cloning method of standard. Taking and identify correct pMD-18T-CD63 plasmid, through Cla I and XbaI double digestion, glue reclaims the fragment of 776bp; Activation amplification pAAV-iRFP682 plasmid, convert escherichia coli SURE2 competence, be coated with amicillin resistance flat board, choose positive monoclonal bacterium in the LB culture fluid of amicillin resistance, 37 DEG C of constant-temperature table 200r/min cultivate 16 hours, and extracting plasmid is standby. Identifying that correct pAAV-iRFP682 plasmid backbone is through Cla I and XbaI double digestion, glue reclaims 5559bp fragment; Plasmid backbone pAAV-iRFP682 (5559bp) and CD63-Linker (776bp) genes of interest fragment (mol ratio is 1:3) are under T4DNA ligase effect, 16 DEG C of coupled reaction 4h, it is transformed in competence Sure-2, picking positive colony expands, extract and plasmid purification, obtain recombiant plasmid, carry out electrophoresis after enzyme action and order-checking is identified, it is thus achieved that pAAV-CD63-Linker-iRFP682 recombiant plasmid.
3) preparation of recombinant adeno-associated virus
Inoculation AAV-293 cell adhere-wall culture in the DMEM culture medium containing 10%FBS, it is placed in 37 DEG C, in the incubator of 5%CO2, cell density is allowed to reach 80% in 48 hours, with twice cell surface of PBS, with tryptic digestive juice (0.25%Trypsin, 0.02%EDTA) digest 3min, 900rpm is centrifuged 5min, add appropriate culture medium resuspended after, go down to posterity with the ratio of 1:3 and continue to cultivate, trophophase cell dissociation of taking the logarithm is passaged in 15cm culture dish and continues cultivation, cultivate 24h cell density and reach more than 70%, with PEI transfection reagent cotransfection pAAV-CD63-Linker-iRFP682 recombiant plasmid and pDG plasmid, packaging recombinant adeno-associated virus rAAV-CD63-Linker-iRFP682. adopting chloroform sedimentation method purification concentrating virus, it is 8 �� 1010vg/mL that quantitative fluorescent PCR measures rAAV-CD63-Linker-iRFP682 recombinant virus titre. reaction system: SybrGreenqPCRMasterMix (2 ��) 10uL, ForwardPrimer (10mol/L) 1uL, Reverseprimer (10mol/L) 1uL, Template1uL, add ddH2O to 20uL. amplification condition (three-step approach): 1. 94 DEG C, 5min, 2. 94 DEG C, 10s, 3. 60 DEG C, 30s, 4. 72 DEG C, 30s, 2.��4. 30cycles.
4) rAAV viral infection human breast carcinoma MDA-MB-231 cell and fluorescent screening
MDA-MB-231 cell attachment is cultivated in the L-15 culture medium containing 10% hyclone, is placed in 37 DEG C, without CO2Saturated humidity incubator in. when cell density reaches more than 80%, carry out had digestive transfer culture, by count with in every hole 105Individual cell is inoculated in 24 orifice plates, cultivates 24h to completely adherent for 37 DEG C. Under the assosting effect of auxiliary adeno-associated virus rAAVSVAV2, infect above-mentioned adherent MDA-MB-231 cell with the ratio that recombinant adeno-associated virus copy number/number of cells is 5000, and on inverted fluorescence microscope, detect the expression of fluorescin. By single cell clone to 96 orifice plates, filter out the MDA-MB-231 cell strain of stably express iRFP682. With reference to Fig. 1, the successful MDA-MB-231 cell membrane of labelling sends out near-infrared fluorescent, and secretes outward body and produce or also there is near-infrared fluorescent in intensive place.
5) extraction of body is secreted in the successful MDA-MB-231 extracellular of labelling
With reference to Fig. 5, Centrifugation method DNA is adopted to secrete body outside extracting. Secrete outward body substep centrifugal method for extracting as follows: first described cell culture supernatant is removed cell debris through 2000g centrifugal 15min, 5000g centrifugal 30min of centrifugal 15min and 12000g successively under 4 DEG C of conditions, obtain the first supernatant;
Secondly, after described first supernatant being used the membrane filtration that aperture is not more than 0.2 ��m, when 4 DEG C and 12000g, centrifugal 120min, secretes body outside obtaining and slightly precipitates;
Finally described with 1 �� PBS solution is resuspended outer secrete body and slightly precipitate, after mix homogeneously when 4 DEG C and 12000g centrifugal 90min, obtain purifying and outer secrete body, use that 1 �� PBS solution is resuspended to be placed on-80 DEG C of conditions and save backup by secreting body outside described purification.
6) qualification of body is secreted in the successful MDA-MB-231 extracellular of labelling
With reference to Fig. 2, take 10 �� g and 5 �� g respectively through step 5) purify after body secretion, body film surface marker albumen is secreted outside after purifying with the anti-human CD63 monoclonal antibody detection of rabbit in Westernblot tests, do positive control with the successful MDA-MB-231 cell pyrolysis liquid of labelling simultaneously, detecting special CD63 protein band, wherein the respectively described positive control of (a), (b) and (c) in figure, 10 �� g and 5 �� g secrete, outside purifying, the band that body is corresponding.
With reference to Fig. 3, observed by TEM high-resolution-ration transmission electric-lens and purify the outer sacculus rotundus secreting body discovery 30-150nm size, be evenly distributed on copper mesh.
With reference to Fig. 4, observe the outer body of secreting of purification by SIM Structured Illumination super-resolution fluorescence microscope and find that the outer body of secreting after purifying sends strong near-infrared fluorescent signal.
As mentioned above, genetically engineered breast carcinoma cell strain prepared by the present invention can secrete body by the outer of stably excreting near-infrared fluorescent albumen iRFP682 labelling, then westernblot is utilized, extraction is closely secreted body outward and is identified by TEM high-resolution-ration transmission electric-lens and SIM Structured Illumination super-resolution fluorescence microscope, illustrate that the centrifugal method of substep can efficiently separate purification to secreting body outside near-infrared labelling, the unique function of body and the superiority of near-infrared fluorescent albumen external spike in vivo is secreted outside utilizing, can be used as the new biomarker thing of breast cancer tumour microenvironment vivo and vitro research, powerful is provided for studying intercellular substance metastasis further, also diagnosing and treating of cancer, scheme has important using value simultaneously.
Number of devices described herein and treatment scale are used to the explanation of the simplification present invention. To secreting the application of preparation of breast carcinoma cell strain of body outside the stably excreting near-infrared fluorescent labelling of the present invention, modifications and variations will be readily apparent to persons skilled in the art.
Although embodiment of the present invention are disclosed as above, but it is not restricted in description and embodiment listed utilization. It can be applied to various applicable the field of the invention completely. For those skilled in the art, it is easily achieved other amendment. Therefore, under the general concept limited without departing substantially from claim and equivalency range, the present invention is not limited to specific details and shown here as the legend with description.
Claims (8)
1. the preparation method secreting the breast carcinoma cell strain of body outside a stably excreting near-infrared fluorescent labelling, it is characterised in that comprise the steps:
1) the outer acquisition secreting body tag PROTEIN C D63 gene of coding:
Extracting total serum IgE from human breast carcinoma MDA-MB-231 cell to carry out reverse transcription and obtain cDNA, with band XbaI and ClaI restriction enzyme site, and the primer of Linker sequence carries out pcr amplification and obtains genes of interest fragment CD63-Linker;
2) structure of recombinant plasmid vector:
Step 1) the described CD63-Linker that obtains and plasmid backbone pAAV-iRFP682 obtains recombinant plasmid vector pAAV-CD63-Linker-iRFP682 through T4DNA ligase effect;
3) preparation of recombinant adeno-associated virus:
Use described recombinant plasmid vector pAAV-CD63-Linker-iRFP682 and the external cotransfection AAV-293 cell of helper plasmid, pack recombinant adeno-associated virus rAAV-CD63-Linker-iRFP682;
4) preparation of the breast carcinoma cell strain of body is secreted outside stably excreting near-infrared fluorescent protein labeling:
Use described recombinant adeno-associated virus rAAV-CD63-Linker-iRFP682 and auxiliary adeno-associated virus coinfection human breast carcinoma MDA-MB-231 cell, obtained the human breast carcinoma MDA-MB-231 cell strain secreting body outside stably excreting near-infrared fluorescent protein labeling by fluorescent screening;
5) the outer body of secreting of near-infrared fluorescent labelling human breast carcinoma MDA-MB-231 cell extracts and identifies:
Adopt Centrifugation method DNA from step 4) labelling successful MDA-MB-231 cell culture supernatant extracts the outer of iRFP682 labelling secrete body and identify.
2. the preparation method secreting the breast carcinoma cell strain of body outside stably excreting near-infrared fluorescent labelling as claimed in claim 1, it is characterised in that described step 5) in the step of Centrifugation method DNA be:
First described cell culture supernatant is removed cell debris through 2000g centrifugal 15min, 5000g centrifugal 30min of centrifugal 15min and 12000g successively under 4 DEG C of conditions, obtain the first supernatant;
Then after using aperture to be not more than the membrane filtration of 0.2 ��m described first supernatant, 4 DEG C, 12000g when centrifugal 120min, obtain outer secreting body and slightly precipitating;
Finally described with PBS solution is resuspended outer secrete body and slightly precipitate, after mix homogeneously in 4 DEG C, 12000g when centrifugal 90min, obtain purifying and outer secrete body, use that PBS solution is resuspended to be placed on-80 DEG C of conditions and save backup by secreting body outside described purification.
3. the preparation method secreting the breast carcinoma cell strain of body outside stably excreting near-infrared fluorescent labelling as claimed in claim 1, it is characterized in that, described step 5) in iRFP682 labelling outer secrete the method that body identifies and be: use westernblot to identify the described outer Membrane surface proteins secreting body, by the structure secreting body outer described in transmission electron microscope observing, and observe the outer fluorescent labeling secreting body by SIM Structured Illumination super-resolution fluorescence microscope.
4. the preparation method secreting the breast carcinoma cell strain of body outside stably excreting near-infrared fluorescent labelling as claimed in claim 1, it is characterised in that described Linker sequence is: ggtggaggcggttcaggcggaggtggctctggcggtggcggatcg.
5. the preparation method secreting the breast carcinoma cell strain of body outside stably excreting near-infrared fluorescent labelling as claimed in claim 1, it is characterised in that step 3) described helper plasmid be pDG plasmid.
6. the preparation method secreting the breast carcinoma cell strain of body outside stably excreting near-infrared fluorescent labelling as claimed in claim 1, it is characterised in that described auxiliary adeno-associated virus is rAAVSVAV2.
7. the preparation method secreting the breast carcinoma cell strain of body outside stably excreting near-infrared fluorescent labelling as claimed in claim 1, it is characterised in that step 2) molar ratio of described CD63-Linker and plasmid backbone pAAV-iRFP682 is 2��5.
8. the preparation method secreting the breast carcinoma cell strain of body outside stably excreting near-infrared fluorescent labelling as claimed in claim 1, it is characterized in that, step 4) ratio of described recombinant adeno-associated virus and described human breast carcinoma MDA-MB-231 cell number is 3000��6000.
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