CN103773770A - Green fluorescent protein capable of being secreted out of cells and application thereof in screening of recombinant protein high-yield expression cell strain - Google Patents
Green fluorescent protein capable of being secreted out of cells and application thereof in screening of recombinant protein high-yield expression cell strain Download PDFInfo
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Abstract
The invention discloses a green fluorescent protein (SEGFP) capable of being secreted out of the cells. The invention also provides a construction method of the protein, and determines the protein's characteristic of capability of being secreted out of the cells. The invention utilizes the SEGFP characteristic of capability of being secreted out of the cells, thus the fluorescent intensity can be rapidly and conveniently detected by a multifunctional ELIASA (enzyme-linked immunosorbent assay), so the SEGFP protein can be rapidly screened and cloned in the early stage of the cell screening process. The invention also provides a method for conveniently comparing the cell yield and the cell clone expression stability by utilizing the SEGFP protein. The method can replace the conventional GFP flow analysis method, greatly reduces the experiment cost and the experiment complexity, and has the advantage of high throughput screening. The invention also discloses a convenient application of the SEGFP protein in flow cell sorting. The invention also discloses the DNA coding sequence of the SEGFP protein.
Description
Technical field:
The present invention relates to molecular biology and cytobiology field, its technology belongs to related experimental methods and the technology of molecular biology and cytobiology, particularly a kind of secretor type green fluorescent protein that can be used for stablizing high expressing cell system structure.
Background technology:
Green fluorescent protein (Green Fluorescent Protein, be called for short GFP) be a kind of a kind of protein (Morin finding in the jellyfish for Aequorea victoria at formal name used at school, J.G.J.Cell Physiol. (1971) 77,305-318).Comprise in 238 amino acid whose sequences at it, the 65th to 67 amino acid (Serine-tyrosine-glycine) residue, can spontaneously form a kind of fluorescent chromophore, therefore can autofluorescence under the irradiation that has exciting light, and need to be by substrate or other the acting in conjunction factor (Tsien, R.Y.Annu.Rev.Biochem. (1998) .67:509-544.) along with the progress of biotechnology, GFP reporter gene is also used to the expression regulation analysis of genome range, real-time monitored (Journal of Endocrinology. (2001) .170 such as J M Tavar é that Subcellular Localization research in vivo of protein and protein move in cell, 297-306).GFP can be on bacterium, and yeast is used as reporter gene in insect expression system and genetically modified animals and plants, constructs experimental model (Chalfie, the Science such as M. (1994) 263, the 802-805 of high expression level reporter gene; (1996) Gene173:83-87 such as Plautz JD; (2006) the J Biotechnol126:463-474 such as Suzuki J; Plant Sci (1999) 142:93-99 such as Blumenthal A).
But along with the development of biological-pharmacy, to the increasing demand of recombinant protein, make to build the high expression level Cells for production of stablizing as far as possible obtaining and be tied to form the direction and goal into biotech development within the short as far as possible time.No matter and utilize the expression of GFP indication target protein or indicate the site of high efficient expression, searching out and expressing enough high and stable clone is all committed step (Qiao wherein, J. wait Molecular Biology (2009) 390 (4): 579-59) this need to build and screen a large amount of cell clones, and then their output relatively fast, and require to screen and express high cell and will keep stable expression level.
Although GFP has the advantage of its high flux screening, still there are some negative impacts in GFP on clone builds.The effect of GFP aspect cell growth inhibiting and cell death inducing on the one hand, make in many cells, be difficult for building clone (the Biochemical and Biophysical Research Communications (1999) 260 (3) such as Hsiao-Sheng Liu: 712-717), this has restricted to screen expresses the sufficiently high cell clone of GFP of stably express GFP.GFP is as intracellular protein on the other hand, and the height of more different cell clone output needs lysing cell to detect or carries out flow cytometer showed, and this has reduced the analysis throughput of cell clone, and is not easy to the stability of comparison cell clone.Because flow cytometer showed method cost is high, operation steps complexity, can only pass judgment on cell stability by the variation of more single generation in practice in addition.And single generation relatively may there is certain error, and cannot accurately reflect cell yield variation tendency.Therefore the how variation of simple and convenient testing goal gene expression dose and accurately judge the committed step that stable cell clone is also industrial screening high expressing cell system.
Therefore improve GFP, retaining its fluorescent characteristic reduces the negative impact of its cell growth simultaneously and is not easy to the character detecting, develop can fast and convenient detection fluorescin, can provide new reporter gene and development tool for the industrial cell construction that carries out.
Summary of the invention:
The object of the invention is the deficiency existing in order to solve above-mentioned background technology, build green fluorescent protein---the SEGFP that obtains a kind of secretor type, the engineering cell system stable for screening high expression level provides new thinking.
Object of the present invention realizes by following proposal: according to the sequence of original GFP albumen, add the signal peptide sequence of the preceding paragraph human IgG kappa chain at its N end, build a kind of secretor type green fluorescent protein.Transfection secretor type green fluorescent protein, to mammalian cell CHO, and detects the secretion of this albumen in extracellular.Utilize this albumen to build the clone of stably express, and this albumen can be used for the comparison of expression level and the expression stability of clone.Secretor type fluorescin can also be used for the sorting of flow cytometer.
Outstanding advantages of the present invention is that SEGFP can be secreted into extracellular feature, can make it convenient for cystic cancer cell line, and protein expression level that can rapid detection cell clone and analysis of cells clone's expression stability, save in a large number experimental cost, provide new instrument for filtering out the engineering cell strain of high expression level.
Accompanying drawing explanation:
Fig. 1 .Western Blotting detects the expression level of secretor type green fluorescent protein.L: cell pyrolysis liquid; S: nutrient solution supernatant
Fig. 2. the analysis of secretor type green fluorescent protein cell clone expression level
The comparison of unicellular fluorescence intensity of odd-numbered day (QFI) between Fig. 3 .SEGFP cell clone.
Embodiment:
The structure of embodiment 1, secretor type green fluorescent protein (SEGFP)
About the structure of secretor type green fluorescent protein (SEGFP), it is the 5 ' end by the signal peptide of human IgG kappa chain being added in to GFP protein gene in design of primers.Primer sequence is as follows:
SEGFP upstream primer:
5 ' atggacatga gggtccccgc tcagctcctg gggctcctgc tactctggct ccgaggtgcc agatgtatgg tgagcaaggg-3 ' SEGFP downstream primer:
5′-tcagcggtttgggcccgcggtaccgtcgactgcagaattcgaagcttgagct-3′
By standard round pcr, clone obtains GFP albumen, signal peptide sequence and GFP protein sequence is building up in same sequence simultaneously.The purifying that carries out DNA fragmentation after this sequence enzyme being cut by MluI and two restriction enzyme sites of XhoI, is then connected into this fragment in mammalian expression vector, identifies the positive colony that is connected with this fragment.Obtain secretor type green fluorescent protein thereby build.The sequence of determining secretor type green fluorescent protein through order-checking is as shown in appendix.
The intensity detection of the transfection of embodiment 2, secretor type green fluorescent protein and the fluorescin of cell exocrine and WesternBlotting detect
Transfection secretor type green fluorescent protein (SEGFP) in mammalian cell CHO, and the cell after transfection is cultivated in shaking flask.The cell of transfection after 72 hours, is got 1ml cell in shaking flask, 1000rpm, and centrifugal 5min, separates cell with nutrient solution, and nutrient solution, according to the centrifugal 5min of 12000rpm, is left and taken supernatant to be measured.Cell adds 1ml1 × TXWSB lysate (TXSWB; 1%Triton X-100,100mM Tris-HCl pH8,100mMNaCl, 10mM) EDTA cracking 15min on ice, 12000rpm, centrifugal 5min, obtains lysate.Take fresh medium and 1 × TXWSB as contrast, with the fluorescence intensity (Ex:485 of multi-functional microplate reader detection nutrient solution and lysate; Em:525).Calculate born of the same parents outer with the interior fluorescence intensity ratio of born of the same parents, can find SEGFP in extracellular fluorescence intensity apparently higher than intracellular fluorescence intensity.Along with the increase of cultivated days, SEGFP is the fluorescence intensity apparently higher than the 3rd day at the 6th day fluorescence intensity level, illustrates that the SEGFP albumen that adds signal peptide can continue fluorescin to be secreted into extracellular.
Chinese hamster ovary celI to transfection SEGFP after tri-days, collects cleer and peaceful cell in also centrifugation, cell is used to 1*TXWSB lysing cell, collecting cell lysate.The cell pyrolysis liquid obtaining and nutrient solution supernatant are carried out to SDS-PAGE gel electrophoresis, protein band is transferred on pvdf membrane by transferring film process.Then primary antibodie is used the antibody (1:1000, health is century) of the anti-GFP of rabbit to hatch 1 hour, and TBST cleans 3 times, each 10 minutes; The antibody (1:2000, Zhong Shan Golden Bridge) of the goat anti-rabbit igg of two anti-use HRP marks is hatched 1 hour, and TBST cleans 3 times, each 10 minutes; Finally use DAB staining examine to detect SEGFP albumen expression level in upper cleer and peaceful lysate.Western Blotting detected result shows that most secretor type green fluorescent protein is secreted into extracellular (Fig. 1).
By means of location and the secretion of signal peptide, GFP albumen can be secreted into extracellular, can detect the GFP albumen of emiocytosis by the method for simple and fast more, has improved greatly the operation efficiency of experiment.The secretion effect of SEGFP has reduced the accumulation of fluorescin in cell simultaneously, has avoided the apoptosis that may cause in intracellular a large amount of accumulation due to GFP.
Embodiment 3, secretor type green fluorescent protein (SEGFP) are conducive to the structure of stable cell lines compared with GFP albumen
In Chinese hamster ovary celI, electrotransfection GFP and SEGFP expression vector respectively, with 5 × 10
6cell carries out transfection, and it is 10ug DNA that electricity turns condition, 350V, 30ms.After transfection 24 hours, inoculate 96 orifice plates according to 1000 cells/well, carry out stabilized cell clone's screening with the substratum of the G418 that contains 0.6mg/ml.The cell growing out at fluorescence microscopy Microscopic observation after 2 weeks, carries out statistic of classification to the fluorescent protein expression level of cell, carries out the statistical study of cell clone.The positive colony rate of SEGFP is 62.50% as shown in Figure 2,61.82% of a little higher than GFP; Further analyze positive monoclonal ratio and show that SEGFP has the positive mono-clonal of positive colony that exceedes 75%, GFP is less than 40%.Statistical study shows that SEGFP becomes cloning efficiency at cell, particularly forms in the ratio of positive monoclonal and has significant advantage (p < 0.05) compared with GFP.See that from clone's number SEGFP has 18 positive monoclonals, GFP only has 13, shows that SEGFP will obviously be better than GFP in the screening of stablizing high expressing cell clone.
Expand the unicellular positive colony screening to 24 orifice plates and 6 orifice plates and respectively at cultivating collecting cell culture supernatant 3 days from 96 orifice plates respectively.Detect the fluorescence intensity in supernatant according to the method described in embodiment 2, and utilize relatively cellular expression levels of fluorescence intensity in supernatant.Along with the increase of volume of culture, fluorescence intensity strengthens gradually, and the cell clone that in 96 orifice plates, fluorescent value is high also has higher fluorescent value in 24 orifice plates, and regression analysis shows that both fluorescence intensities have marked positive correlation.Further, after enlarged culturing, cell is positively correlated with the fluorescent value of 24 orifice plates in the fluorescence intensity of 6 orifice plates.As shown in Figure 2 in the distribution of the different cell clone luciferase expression of 24 orifice plate levels levels, most cell clone expression amounts are very low, the distribution of cloning along with the increase of fluorescence intensity reduces rapidly, fluorescence intensity has accounted for 80% left and right lower than 200 clone, even there is the cell clone of 65% left and right almost there is no the expression of GFP, and fluorescence intensity is in clone's less than 10% of 500-1000, expresses the highest cell clone fluorescence intensity and be greater than 900.
4 stability analyses that positive monoclonal is grown that GFP and SEGFP expression amount are the highest are further selected.According to 0.5 × 10
6/ ml density, carries out cell inoculation according to the volume of culture 50ml shaking flask of 10mi/ bottle, at CO
2in incubator 37 ℃, 5%CO
2, 150rpm cultivates.While going down to posterity, carry out cell counting at every turn, and calculate the cell colony doubling time (PDT) of each generation.In the passage process that approaches 40 days, GFP and SEGFP cell clone all can normal growths.Although but the average population doubling time (PDT) of analysis of cells population doubling time discovery 8 strain cells was distributed between 18-30 hour, but wherein SEGFP4 clone's PDT is 21.01～23.69 hours, and the mean value of GFP clone PDT is 22.51～29.46 hours.The PDT average of cell clone is carried out to statistical study and also show that SEGFP clone's PDT is significantly less than GFP clone (p < 0.05), illustrate that SEGFP4 strain cell is compared the 4 strain clones multiplications of GFP faster, a little less than the impact of cell growth.Further observing GFP has 3 strain clones to exceed 24 hours, even has 2 deviations to be greater than 10 hours.Show that GFP has show in two processes that are cloned in Growth of Cells very unstable.Therefore visible GFP has negative impact in the certain cell growth of intracellular expression.Not significantly (p=0.47) of the variance difference that the variance analysis that different GFP cell clone is separately carried out to PDT also shows PDT between SEGFP clone, and between the variance of the PDT of the 4 strain cell clones of GFP, there is significant differences (p < 0.01), illustrate that between the 4 strain cell clones of SEGFP, growth conditions is more consistent, cell expressing height has no significant effect growth.Can illustrate and utilize signal peptide that fluorescin is secreted into extracellular thus, reduce it in intracellular concentration, alleviate the disadvantageous effect of GFP albumen at thin intracellular accumulation, be conducive to the growth that guarantees that cell is more stable.
SEGFP can be by continuous release in the nutrient solution of cell, and in nutrient solution, still keeps excitability, under the exciting light of 488nm, can nutrient solution be detected in fluorescence intensity.Can improve like this complicacy of the detection method of GFP in kytoplasm, in screening cell clone, directly utilize substratum in the height of fluorescence intensity of GFP pick out and express the high cell clone of GFP.Result of study has also disclosed in the process of cystic cancer cell line, also has higher fluorescence intensity at the higher cell clone of 96 orifice plate expression in period after enlarged culturing, and the fluorescence intensity of cell clone keeps good consistence at different cultivation stages.Therefore the fluorescence intensity level that utilizes SEGFP to be secreted into outside born of the same parents can be selected the cell clone that GFP expression level is high easily.We also observe the most positive mono-clonals of the cell clone that directly utilizes fluorescence intensity to screen high expression level, screening correct ratio will be higher than the result judging by fluorescence microscope, the positive colony of GFP albumen need to be chosen at fluorescence microscopy Microscopic observation, and therefore SEGFP is obviously better than GFP albumen on colony screening.So utilize SEGFP albumen can increase the flux of cell screening, the early screening process of cell clone with regard to picking the clone to high expression level.
Embodiment 4, secretor type green fluorescent protein are convenient to the comparison of cell clone output
By the 4 strain SEGFP cell clones that the screen cultivation of going down to posterity, 3 times weekly.While going down to posterity at every turn according to 0.5 × 10
6/ ml density, carries out cell inoculation according to the volume of culture 50ml shaking flask of 10ml/ bottle, at CO
2in incubator 37 ℃, 5%CO
2, 150rpm cultivates.The cell clone culture screening more than 6 weeks, is used to trypan blue staining when each passage, counting cell number anyway on blood counting chamber.Detect the fluorescence intensity in cells and supernatant, the fluorescence intensity detecting shows that the fluorescence intensity between 4 strain cells all fluctuates between 3000-12000 simultaneously, and mean value has different as seen.
We are by the concept of the monocell expressing output of secretory protein, built one can quantization cell expression level index---the fluorescence intensity (QFI) in unicellular odd-numbered day, this index can have been deducted the impact of cell culture density and generation, reflects the GFP ability to express of the individual cells of logarithmic phase in different cell clones.The QFI that calculates four strain cell clones shows that its QFI average value ranges is in 1.5-3.0 × 10
-3fI/cell/Day left and right, statistical study shows that between different cell clones, expressing production Q FI has notable difference.Wherein SEG-3A3 expression level is significantly higher than SEG-3G4 and SEG-4D1 (p < 0.001), and SEG-1B1 clone's a little higher than SEG-3G4 of output and SEG-4D1 (p < 0.05) are (Fig. 3).And SEG-3G4 and SEG-4D1 can find that relatively going up of fluorescence intensity the average fluorescent strength of SEG-3G4 still will be higher than the value of SEG-4D1, substantially there is no difference but the analysis of QFI just shows the mean value of SEG-3G4 and SEG-4D1, illustrate that both expression levels are suitable.
Therefore utilize SEGFP can make GFP be secreted into extracellular, the fluorescence intensity outside born of the same parents of utilizing that in early days can be fast and convenient in cystic cancer cell line process filters out the cell clone that expression level is high, and further in shaking flask when enlarged culturing, replace fluorescence intensity by calculating unicellular production Q FI, eliminate the impact that cell density brings, can reflect more really the ability to express of cell clone, comparatively simply distinguish the height of clonal expression level.
Embodiment 5, secretor type green fluorescent protein can be used for the analysis of cell clone expression stability
For the SEGFP cell clone of screening, according to the cultivation of going down to posterity for 3 times weekly.Use 50ml shaking flask, according to the volume of culture of 10ml/ bottle, 37 ℃, 5%CO2,150rpm cultivates.The cell clone screening is cultivated in 50ml shaking flask more than 6 weeks, when each passage, used trypan blue staining, counting cell number anyway on blood counting chamber.Collect the cell culture fluid supernatant of each generation simultaneously, detect fluorescence intensity in supernatant, calculate the fluorescence intensity (QFI) in unicellular odd-numbered day according to viable cell density, QFI by more different cell clones changes the stability that can judge that cell clone is expressed, thereby filters out the cell clone of high expression level continually and steadily.
For the cell clone that utilizes GFP to build, the method of analysis of cells clone stability is mainly the population analysis that carries out cell clone by flow cytometer, according to the ratio (Qiao of different generation GFP positive cells in flow cytometer showed, J. wait Molecular Biology (2009) 390 (4): 579-59) and weighted mean fluorescence intensity variation (Bailey, the Biotechnology and Bioengineering (2002) 80 (6) such as C.G: whether the expression that 670-676) reflects cell clone stable.The SEGFP that we use also can detect the height of fluorescence intensity in cell, after utilizing the cell in above-mentioned 4 the 4th generations of strain cell clone of flow cytometry analysis and the 13rd generation, find that SEG-4D1 colony is obviously offset to the direction yielding poorly really, and twice flow cytometer showed of other 3 strain cell clones almost do not change, show that these cell clone stability are better.
Meanwhile, we have detected the fluorescence intensity in above-mentioned cell clone culture supernatant, calculate the variation of QFI in the process that goes down to posterity between different cell clones, reflect the variation of the expression level of cell clone.Result shows the comparable situation of 4 strain cell clones at the QFI in the 4th generation and the 13rd generation, consistent with the result of flow cytometer showed, and the QFI of SEG-4D1 is from 3.14 × 10 of the 4th generation
-3fI/cell/day drops to 1.99 × 10 of the 13rd generation
-3fI/cell/day, reduced about 35%, and output between two generations of statistical study there were significant differences (p < 0.05).Other 3 strain clones are similar to flow cytometer showed result, and QFI does not have noticeable change, can think that these are cloned in maintenance in the process of going down to posterity and stablize.
Because flow cytometer showed method cost is high, operation steps complexity, can only pass judgment on cell stability by the variation of more single generation in practice.And single generation relatively may there is certain error, and cannot accurately reflect cell yield variation tendency.Therefore the how variation of simple and convenient testing goal gene expression dose and accurately judge the committed step that stable cell clone is also industrial screening high expressing cell system.For SEGFP albumen, we can be in the fluorescence intensity in detection supernatant after cultivation that goes down to posterity at every turn, and calculate the value of QFI, QFI value according to each generation is carried out regression analysis, can obtain the conclusion identical with the above results, and monitor at any time the changing conditions of cell yield, reach the object of Early judgement cell stability.But due to the error between generation, make QFI there will be difference just in the result in per generation, be difficult for reflecting intuitively the stability (not showing result) of cell clone.So we represent the ability to express of cell with the QFI mean value of multiple generations, along with the increase of passage number, should be able to tend to gradually steady for the average of stable clone QFI.And the unsettled cell of expression level, due to the unhomogeneity of colony, the average of the QFI of cell clone can show obvious downtrending.
For engineering cell system, except growing stable requirement, no less important be exactly the stable of output.Whether stablize although can compare the expression of cell clone for the analysis of intracellular GFP flow cytometer, method depends on expensive equipment, and instrument exists the error of system in the time that different time points detects, and is difficult to carry out careful comparison.SEGFP is as the GFP of secretor type, and its detection method is easy, and error is little, can monitor at any time the variation of cell yield.The QFI calculating in the culturing process that goes down to posterity also can be directly used in the analysis of cell clone expression stability.After each passage, can use multi-functional microplate reader carry out the detection of fluorescence intensity and calculate QFI, often obtain after new data, just can carry out in conjunction with these data the analysis of QFI mean value, express stable cell strain thereby filter out in early days.
Embodiment 6, secretor type green fluorescent protein are used for the cell sorting of flow cytometer
According to the method for embodiment 2, cleer and peaceful cell pyrolysis liquid on collecting cell nutrient solution.First the relatively dependency of the inside and outside fluorescence intensity of SEGFP cell clone born of the same parents, to determine whether the expression height inside and outside SEGFP cell has consistence.Result shows the very significantly (R of dependency of the inside and outside fluorescence intensity of SEGFP cell clone born of the same parents
2value=0.866).In the cell clone of SEGFP, in born of the same parents, high its fluorescin being secreted into outside born of the same parents of cell clone of fluorescence intensity also has corresponding high-caliber fluorescence intensity, show that the inside and outside fluorescence intensity dependency of the cell of SEGFP cell clone is fine, for the high cell of screening secretion fluorescence intensity that utilizes airflow classification provides experiment basis.And fluorescence intensity outside different SEGFP cell clone born of the same parents can reach 600-1300, not only apparently higher than negative control, and fluorescence intensity between clone has the difference of 700 left and right, guarantee that enough large differentiation interval can be so that the high cell clone of screening GFP expression level.
According to Kaufman, W.L. the method waiting. (Nucleic Acids Research36 (17): e111-e111 (2008)), the cell of the 3rd day after collection transient transfection, after using 2ml substratum resuspended, proceed in the cell sorting collection tube of BD, detect the fluorescence intensity of GFP using 488nm as exciting light, use FACS Calibur (BD Biosciences, San Jose, CA, USA) collect SEGFP and express the highest 10% cell.The cell that the cell of collection is added to 0.6mg/ml G418 screening stably express, carries out cell sorting again after 7 days, then the cell of collecting is cultivated gradually to expand to obtain cell mass.Cell before collecting and cell colony after 2 enrichments are carried out to flow cytometer showed according to unified condition and show, along with the obviously skew to the right of fluorescence curve of the increase flow cytometer showed of sorting number of times.The per-cent of positive cell only has 34.37% before sorting, is increased to 73.9% through a positive ratio of FACS, and after secondary FACS, positive cell has reached 95.28%, and the weighted mean of its fluorescence intensity is also 5 times before sorting.
We have also compared the height of the single cell clone output of the SEGFP positive cell screening after cell clone and the sorting that traditional limiting dilution assay obtains.The distribution of the clone's who relatively screens the outer fluorescence intensity of born of the same parents, most cell clone expression amounts that wherein traditional limiting dilution assay obtains are very low, and the distribution of cloning along with the increase of fluorescence intensity reduces rapidly.And utilize fluorescence intensity in the cell clone that FACS obtains to be no more than 15% lower than clone's ratio of 200 (FI=0-200), be approximately 55% and fluorescence intensity exceedes 500 cell clone ratio, and screen the cell clone that fluorescence intensity is greater than 900.But the cell colony that utilizes GFP after FACS enrichment is carrying out in the process of mono-clonal, because GFP is in intracellular expression, for clone's the judgement of expression level and the analysis of stability, need to be by flow cytometer or fluorescent microscope, this has just affected the flux of screening.And due to the high consistency of fluorescence intensity inside and outside SEGFP albuminous cell, can first use it on flow cytometer, to carry out cell sorting, then by detecting the cell clone after the screening sorting of the fluorescence intensity fast high-flux in substratum.What therefore SEGFP can be very easy be applied to FACS and can be in subclone subsequently high-throughout screening high yield single cell clone.
Claims (7)
1. one kind includes the secretor type green fluorescent protein (SEGFP) of sequence shown in appendix.
2. the signal peptide that secretor type green fluorescent protein as claimed in claim 1 contains human IgG kappa chain.
3. secretor type green fluorescent protein as claimed in claim 1 can be secreted in cell culture fluid in mammalian cell.
4. the application in secretor type green fluorescent protein as claimed in claim 1 cell clone rapid screening in early days.
5. secretor type green fluorescent protein as claimed in claim 1 is in the application of the comparison of stably express cell clone expression level.
6. secretor type green fluorescent protein can be for the comparison of stable cell lines expression level stability as claimed in claim 1.
7. secretor type green fluorescent protein can be for the cell sorting of flow cytometer as claimed in claim 1.
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| CN112037853A (en) * | 2019-10-30 | 2020-12-04 | 东莞太力生物工程有限公司 | Method for screening cell strain capable of expressing expected product |
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