CN104017772A - Cell strain for expressing HER2 gene, and construction method and application thereof - Google Patents
Cell strain for expressing HER2 gene, and construction method and application thereof Download PDFInfo
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Abstract
The invention provides a cell strain for expressing HER2 gene, and a construction method and application thereof. The method comprises the following steps: 1) inserting HER2 gene into the polyclone site of the expression vector to obtain a recombinant expression vector; and 2) transfecting a cell strain by using the recombinant expression vector to obtain the monoclonal cell strain for expressing HER2 gene. The cell strain can efficiently and stably express the HER2 gene, has resistance to the existing HER2 targeted antibody drug trastuzumab, can successfully construct a human breast cancer transplantation tumor model in the nude mouse body, and can be used for screening novel breast cancer drugs.
Description
Technical field
The present invention relates to cell strain and the application thereof of a kind of HER2 of expression.
Background technology
HER2/ErbB2 protein receptor belongs to EGF-R ELISA (epidermal growth factor receptor, EGFR) family, it is the transmembrane glycoprotein with receptor tyrosine kinase (RTK) activity by c-erbB2 (her2/neu) genes encoding, full-length molecule amount is 185kDa, it is bad that high expression level in the malignant tumours such as mammary cancer, cancer of the stomach and ovarian cancer often indicates that Metastasis and prognosis easily occurs tumour, and resist height correlation with chemicotherapy, endocrine therapy.Because HER2 is expressed in the surface of tumour cell, and in adult normal tissue, do not express or only have extremely low expression, making it become a good drug target.The Humanized monoclonal antibodies medicine Herceptin (trastuzumab of first anti-HER2, trade(brand)name Herceptin) since listing in 1998, in mammary cancer clinical treatment, show unique curative effect, but also existing the efficient low and patient of single medicine to produce the phenomenon of resistance, this makes development of new HER2 target therapeutic agent become active demand.
The preclinical study of HER2 targeted drug need to be evaluated the biologic activity of medicine in multiple HER2 high expression level transplanted tumor model.But existing HER2 high expression tumour cell strain limited amount, and be subject to into the restriction of knurl ability, can be used for setting up the very limited of transplanted tumor model, therefore need artificial constructed efficient, stably express HER2 and become the good tumor cell line of knurl ability.2009 disclosed patent " a kind of HER2 stable expression cell line and the application in drug screening thereof " (publication number CN101343633A) by pEGFPN3/1 carrier to transfection HER2 full-length gene in multiple host cell, and screening obtains the clone of stably express HER2, but how still not solve the problem of high efficient expression HER2 in cell.
Zooblast protein expression technology is mainly used DHFR (Tetrahydrofolate dehydrogenase) and GS (glutamine synthetase) gene copy amplification method.Because gene amplification needs 6 to 12 months, the protein expression result of this method can not be predicted in early days, is the rate-limiting step of expression technology.In recent years, developing disposable expression technology abroad, but these new technologies need high-flux cell colony screening just can draw the production clone of medium level conventionally always.
The present invention is applied to GC-rich gene high expression carrier technique the transfection of HER2 gene, has obtained efficient, stably express HER2 and has become the good human breast cancer cell strain of knurl ability, can be used for the screening of novel HER2 targeted drug.
Summary of the invention
The object of this invention is to provide a kind of application of cell strain and described cell strain of efficiently, stably expressing HER2 gene.
The present invention realizes object of the present invention by following technical solution:
A cell strain of 1, expressing HER2, is characterized in that:
Described cell strain is that preserving number is the cell strain of CCTCC NO:C201421.
2, the cell strain as described in technical scheme 1, is characterized in that:
Described cell strain shows resistance to HER2 targeting antibodies medicine Herceptin; And/or
In nude mouse, construct cancer transplanted tumor model.
3, a method of setting up the cell strain of expressing HER2, is characterized in that:
Said method comprising the steps of:
1) by the multiple clone site of HER2 gene insertion expression vector, obtain recombinant expression vector; With
2) utilize described recombinant expression vector transfection cell strain, obtain expressing the monoclonal cell strain of HER2.
4, according to the method described in technical scheme 3, it is characterized in that:
Described expression vector is GC-rich expression vector.
5, according to the method described in technical scheme 3 or 4, it is characterized in that:
Described HER2 gene is full-length gene or its active fragments; More preferably, described HER2 gene is full-length gene.
6, according to the method described in any one in technical scheme 3 to 5, it is characterized in that:
Described cell strain shows resistance to HER2 targeting antibodies medicine Herceptin; And/or
In nude mouse, construct cancer transplanted tumor model.
7, the method as described in any one in technical scheme 3 to 6, is characterized in that:
Described cell strain is human breast cancer cell MCF-7, and more preferably, described cell strain is that preserving number is the cell strain of CCTCC NO:C201421; And/or
Described cell strain constructs people's mammary gland transplanted tumor model in nude mouse;
8, the cell strain as described in technical scheme 1 or the cell strain set up by method as described in any one in technical scheme 3 to 7 application in screening cancer drug.
9, the application as described in technical scheme 7, is characterized in that:
Described cancer is malignant tumour.
10, the application as described in technical scheme 8, is characterized in that:
Described cancer is mammary cancer, cancer of the stomach or ovarian cancer; More preferably, described cancer is mammary cancer.
The cell strain of setting up by method of the present invention can efficiently, stably be expressed HER2, and preferred described cell strain shows resistance to existing HER2 targeting antibodies medicine Herceptin, can in nude mouse, successfully build the especially transplanted tumor model of human breast carcinoma etc. of cancer, can be used for the screening of the medicine of novel for example mammary cancer for the treatment of cancer.
Brief description of the drawings
Fig. 1. two kinds of Her2 plasmid transient transfection 293T cell results;
Fig. 2 .Western trace detects pcDNA3.1/Her2 plasmid stable transfection MCF-7 cell result;
Fig. 3 .Western trace detects GC rich/Her2 plasmid stable transfection MCF-7 cell result;
Fig. 4 .FACS detects pcDNA3.1/Her2 plasmid stable transfection MCF-7 cell result;
Fig. 5 .FACS detects GC rich/Her2 plasmid stable transfection MCF-7 cell result;
The in-vitro multiplication of Fig. 6 .MCF-7/HER2 cell suppresses experiment; With
In the body of Fig. 7 MCF-7/HER2 cell, become knurl experiment.
Microbial preservation
The information of the described microbial preservation relating to is as follows:
Classification And Nomenclature: human breast cancer cell MCF-7/HER2
Latin name: Homo sapiens
Depositary institution's title: Chinese Typical Representative culture collection center (CCTCC)
Preservation title address: Luo Jia Shan, wuchang, wuhan
Preservation date: on January 25th, 2014
Deposit number: CCTCC NO:C201421
Embodiment
As mentioned above, the object of this invention is to provide and can stablize, express efficiently cell strain of HER2 gene and uses thereof.
In a first aspect of the present invention, provide the cell strain of a kind of HER2 of expression.In some embodiments, described cell strain can show resistance to HER2 targeting antibodies medicine Herceptin.In some embodiments, described cell strain can also construct cancer transplanted tumor model in nude mouse.
Some preferred embodiment in, described cell strain is to express the MCF-7 cell strain (being below sometimes referred to as MCF-7/HER2 cell strain) of HER2; More preferably, described cell strain is that the deposit number that is deposited in Chinese Typical Representative culture collection center is the cell strain of CCTCC NO:C201421.
In a second aspect of the present invention, a kind of method of setting up the cell strain of expressing HER2 is provided, it is characterized in that, said method comprising the steps of:
1) by the multiple clone site of HER2 gene insertion expression vector, obtain recombinant expression vector; With
2) utilize described recombinant expression vector transfection cell strain, obtain expressing the monoclonal cell strain of HER2.
In some embodiments, described cell strain can show resistance to HER2 targeting antibodies medicine Herceptin.In some embodiments, described cell strain can also construct cancer transplanted tumor model in nude mouse.
Some preferred embodiment in, described cell strain is human breast cancer cell MCF-7; More preferably, described cell strain is that preserving number is the cell strain of CCTCC NO:C201421.
In some embodiments, the described HER2 gene of insertion is full-length gene or its active fragments.In some embodiments, does is described HER2 gene that (full-length gene order is open, can be from http://www.ncbi.nlm.nih.gov/gene/ for full-length gene? term=2064 obtains, and is for No. ID 2064).In some embodiments, described HER2 gene can be its active fragments.Described active fragments has the general sense that those skilled in the art understands, and, is had the basic function of HER2 by the coded aminoacid sequence of described active fragments that is, for example, have receptor tyrosine kinase (RTK) activity.For example described active fragments comprises those sequences of described full-length gene or comprises those functional domain of carrying out its basic function).
Some preferred embodiment in, described expression vector is the expression vector of GC-rich (being rich in GC).The inventor is surprised to find that, the expression vector of GC-rich can make HER2 be cloned into cell strain be especially cloned into human breast cancer cell MCF-7 in after can effectively and stably express.The expression vector of described GC-rich can be for example from the Hangzhou general biological company limited of peace (Amprotein-China) commercially available (also can referring to PCT/US2007/14488).Do not wish to be bound by theory, the inventor thinks that the expression vector of GC-rich may be different from other expression vectors on protein expression regulatory mechanism, its reason may be that the DNA toughness of super high GC content is higher, not foldable, makes transcription factor easily approach genetic transcription and regulates sequence.
The technique means that the present invention adopts transfection is not particularly limited.Described transfection can by or preferably carry out (Lipofectamine2000, invitrogen company) by liposome.
In some embodiments, after described transfection, also comprise the step of dilution and/or resistance screening.Described dilution is generally limiting dilution
In some embodiments, described cell strain constructs cancer transplanted tumor model in nude mouse.The present invention has no particular limits cancer, and for example described cancer can be mammary cancer, cancer of the stomach or ovarian cancer.Some preferred embodiment in, described cancer is human breast carcinoma.
In a third aspect of the present invention, the invention provides cell strain as above or the application of the cell strain set up by method as above.In some embodiments, described application comprises to be got rid of taking lived human body or animal body as objective for implementation, and any any application that can belong to patent right authorized object outside being applied in the diagnosis of disease and methods for the treatment of.For example,,, be applied as the application in screening cancer drug.
In some embodiments, described cancer is malignant tumour, for example solid malignant; More preferably, described cancer is mammary cancer, cancer of the stomach or ovarian cancer; Further preferably, described cancer is mammary cancer.
In a fourth aspect of the present invention, the invention provides any host who comprises cell strain described above.Preferably, described host is mammalian hosts, for example mouse (as nude mice).
Below the form by embodiment is further detailed the present invention, but these embodiment only for purpose of explanation, is not to be construed as limiting the scope of the invention.
Embodiment
The material using in embodiment and source thereof are as follows:
PBabe/Her2 plasmid is presented by Mark professor I.Greene of medical college of the University of Pennsylvania, and the plasmid that contains Her2cDNA full length sequence also can be buied from OriGene Technologies; GC-Rich expression vector is purchased from Hangzhou An Pu company; Lipofetamine2000 is purchased from Invitrogen company; Anti-Her2 antibody A b-1 is purchased from NeoMarkers company.
1.1Her2 expression plasmid builds
Taking pBabe/Her2 plasmid as template, design primer amplification people source Her2 (gene ID2064) full-length gene, through EcoRI+NotI double digestion, be connected with pCDNA3.1 and GC-Rich expression vector respectively, transform XL-10 competent cell, select positive colony, through EcoRI+NotI enzyme cut with sequence verification after, obtain a strain pCDNA3.1/Her2 plasmid clone and four strain GC-Rich/Her2 plasmid clones (numbering is respectively 2-3,2-4,2-5 and 2-6).By harvested cell lysate after these plasmids difference transient transfections 293T cell, western trace detects Her2 expression of results as shown in Figure 1.Using the 293T cell of untransfected as negative control (NC swimming lane), using the SKBR3 cell of high expression level Her2 as positive control (SKBR swimming lane), GC-Rich/Her2 plasmid 2-3,2-5 and 2-6 clone all can be in 293T cell high efficient expression Her2 albumen, and expression level will be higher than pCDNA3.1/Her2 plasmid; Wherein GC-Rich/Her2 plasmid 2-3 clone and pCDNA3.1/Her2 plasmid are for the transfection of step 1.2 MCF-7 cell.
1.2MCF-7 cell transfecting and screening
The 1st day: MCF-7 cell is passaged to T25 bottle to 50% full scale in normal substratum (DMEM+10%FBS+ is dual anti-).
The 2nd day: 5ug is mixed in 1ml Opti-MEM serum free medium through plasmid and the 10ulLipofectamine2000 of PvuI linearization for enzyme restriction, and room temperature leaves standstill 30 minutes, and transfectional cell spends the night.
The 3rd day: inhale and abandon transfection media, change normal substratum.
The 4th day: the whole cells of trysinization, be passaged to 10 96 orifice plates, every hole 100ul selects substratum (normal substratum+1.2mg/ml G418).Within later every 3-4 days, renew fresh selection substratum.
The 3rd week: visible 96 orifice plates have cell clone to grow, select growth conditions and cellular form is better cloned, trypsin digestion cell is transferred to 24 orifice plates.
The 4th week: after 24 orifice plate cells cover with, be passaged to 6 orifice plates, be passaged to two T25 bottles after 6 orifice plates cover with, one bottle is used as Her2 expression identification, and another bottle is as cell conservation.
1.3Western trace qualification Her2 expresses
Add 0.5-1ml RIPA lysate to be used as in the T25 bottle of Her2 expression identification in step 1.2, collecting cell lysate also uses BCA method to measure total protein concentration; Electrophoresis applied sample amount is adjusted to every hole 10ug total protein, carry out going to pvdf membrane after 8%SDS-PAGE electrophoresis, add the sealing of TPBS+5% skim-milk, add anti-Her2 antibody A b-1 as primary antibodie room temperature incubation 1 hour, add again the anti-room temperature incubation of HRP mark sheep anti mouse two after 1 hour, carry out ECL colour developing.Fig. 2 and Fig. 3 gather in the crops the Her2 protein expression situation of cloning after showing respectively pCDNA3.1/Her2 plasmid and GC-Rich/Her2 plasmid transfection MCF-7 cell.Using the MCF-7 cell pyrolysis liquid without transfection as negative control (NC swimming lane), using SKBR3 or BT474 cell pyrolysis liquid as positive control (PC swimming lane or SKBR swimming lane, BT474 swimming lane).In the cell clone that pCDNA3.1/Her2 plasmid transfection obtains, only have a small amount of expression Her2 albumen, and expression level is not high.After GC-Rich/Her2 plasmid transfection, obtain the higher cell clone of multiple Her2 protein expression levels, comprised 3-1,5-3,6-4,8-2 etc.
1.4FACS detects Her2 and expresses
Be used as from step 1.2 with 0.25% pancreatin in the T25 bottle of Her2 expression identification and digest collecting cell, be resuspended in FACS damping fluid (PBS+1%BSA), in sealing 30 minutes on ice, add subsequently anti-Her2 antibody in hatching 30 minutes on ice, add again FITC mark two to resist in hatching 30 minutes on ice, detect with flow cytometer.Using MCF-7 cell as negative control, SKBR3 cell is as positive control.
Fig. 4 shows that clone 3-2,5-2, the 6-1 surface of cell membrane Her2 expression level of pcDNA3.1/Her2 transfection MCF-7 cell acquisition is not high, and contrasting with the MCF-7 of untransfected to compare does not have obvious gap.Fig. 5 shows the clone 3-1 that GC-Rich/Her2 transfection MCF-7 cell obtains, and 5-3 and 6-4 surface of cell membrane Her2 expression level are higher, very approaching with positive control SKBR3 cell; Wherein clone 6-4 and be named as MCF-7/HER2, and be stored in Chinese Typical Representative culture collection center (CCTCC).
The susceptibility of 1.5MCF-7/HER2 cell to Herceptin
Collect MCF-7/HER2 cell with 0.25% trysinization, after counting, be inoculated in 96 orifice plates.With adding in 96 orifice plates and process cell after substratum dilution Herceptin, and set up the contrast that does not add antibody.Continue to cultivate after four days, add CCK-8 reagent incubation 1 hour, measure 490nm place absorbancy by microplate reader, and index using this reading as cell viability, experimental result is as shown in Figure 6.Compare with not adding contrasting of antibody, in the time that Herceptin concentration increases to 10 μ tg/ml, MCF-7/HER2 cell proliferation is not still subject to remarkably influenced, illustrates that MCF-7/HER2 cell shows resistance to Herceptin in testing in vitro.
The one-tenth knurl of 1.6MCF/HER2 cell in nude mouse
MCF-7/HER2 cell in logarithmic phase, with after trysinization, is inoculated in advance and in the second breast pad, is designated as inoculation first day next day on the left of the nude mice of neck subdermal implantation 17 β estradiol slow releasing tablet.Twice use vernier caliper measurement tumour major diameter (L) and minor axis (W) weekly after inoculation, the calculation formula of gross tumor volume (TV) is: TV=0.5 × L × W × W.MCF-7/HER2 cell was inoculated after one week, and visible tumour is grown steadily, and tumor formation rate reaches 100%, and gross tumor volume rapid development, as shown in Figure 7, illustrate that MCF-7/HER2 cell successfully constructs transplanted human breast carcinoma model in nude mouse, can be used for the screening of novel HER2 targeted drug.
Claims (10)
1. a cell strain of expressing HER2, is characterized in that:
Described cell strain is that preserving number is the cell strain of CCTCC NO:C201421.
2. cell strain as claimed in claim 1, is characterized in that:
Described cell strain shows resistance to HER2 targeting antibodies medicine Herceptin; And/or
In nude mouse, construct cancer transplanted tumor model.
3. a method of setting up the cell strain of expressing HER2, is characterized in that:
Said method comprising the steps of:
1) by the multiple clone site of HER2 gene insertion expression vector, obtain recombinant expression vector; With
2) utilize described recombinant expression vector transfection cell strain, obtain expressing the monoclonal cell strain of HER2.
4. method according to claim 3, is characterized in that:
Described expression vector is GC-rich expression vector.
5. according to the method described in claim 3 or 4, it is characterized in that:
Described HER2 gene is full-length gene or its active fragments; More preferably, described HER2 gene is full-length gene.
6. according to the method described in any one in claim 3 to 5, it is characterized in that:
Described cell strain shows resistance to HER2 targeting antibodies medicine Herceptin; And/or
In nude mouse, construct cancer transplanted tumor model.
7. the method as described in any one in claim 3 to 6, is characterized in that:
Described cell strain is human breast cancer cell MCF-7, and more preferably, described cell strain is that preserving number is the cell strain of CCTCC NO:C201421; And/or
Described cell strain constructs people's mammary gland transplanted tumor model in nude mouse.
8. cell strain as claimed in claim 1 or the cell strain set up by method described in any one in claim 3 to 7 application in screening cancer drug.
9. application as claimed in claim 7, is characterized in that:
Described cancer is malignant tumour.
10. application as claimed in claim 8, is characterized in that:
Described cancer is mammary cancer, cancer of the stomach or ovarian cancer; More preferably, described cancer is mammary cancer.
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN108471732A (en) * | 2015-10-30 | 2018-08-31 | 杰克逊实验室 | With the relevant composition of tumor analysis and method |
CN109734799A (en) * | 2018-07-23 | 2019-05-10 | 深圳市伯劳特生物制品有限公司 | A kind of application of expression and the method and Her2 full-length proteins that purify Her2 full-length proteins |
CN111088287A (en) * | 2019-12-20 | 2020-05-01 | 江苏浦珠生物医药科技有限公司 | HER2 gene humanized mouse tumor cell model, construction method and application |
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CN102124019A (en) * | 2007-01-25 | 2011-07-13 | 安普罗泰恩公司 | Use of chick beta actin gene intron-1 |
Non-Patent Citations (1)
Title |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108471732A (en) * | 2015-10-30 | 2018-08-31 | 杰克逊实验室 | With the relevant composition of tumor analysis and method |
CN109734799A (en) * | 2018-07-23 | 2019-05-10 | 深圳市伯劳特生物制品有限公司 | A kind of application of expression and the method and Her2 full-length proteins that purify Her2 full-length proteins |
CN111088287A (en) * | 2019-12-20 | 2020-05-01 | 江苏浦珠生物医药科技有限公司 | HER2 gene humanized mouse tumor cell model, construction method and application |
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