CN106191216A - A kind of method of copy number of foreign gene in quantitative PCR detection transgenic cell - Google Patents
A kind of method of copy number of foreign gene in quantitative PCR detection transgenic cell Download PDFInfo
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Abstract
The present invention relates to the quantitative detecting method of the weight chain gene copy number of antibody molecule in a kind of transgenetic animal cell CHO, use SYBR Green quantifying PCR method, it is achieved the light chain gene copy number of antagonist molecule and heavy chain gene copies number carry out detection quantitative, accurate, high-throughout.The method detection sensitivity height of the present invention and range of linearity width, reliable results, can be used for studying the stability of antibody cell strain, filter out the through engineering approaches cell strain of stable high expressed.
Description
Technical field
The invention belongs to technical field of molecular biology, relate to the weight chain of antibody molecule in a kind of transgenetic animal cell
The quantitative detecting method of gene copy number.
Background technology
Zooblast has been widely used in production various bioactive substances such as vaccine, somatomedin, antigen, Dan Ke
The recombinant protein medicines such as grand antibody, these zooblasts include 293 cells, Vero cell, bhk cell, PER.C6
Cell, SP2/0 cell, NS0 cell, Chinese hamster ovary celI etc., be wherein most widely used surely belongs to Chinese hamster ovary celI.
The most recombinant expressed antibody drug ratified through U.S. FDA just reaches twenties kinds, and the annual market value created is the most several
10000000000 dollars.Along with the development of technique for gene engineering, increasing foreign protein such as antibody passes through transgene method
Obtain.
But for transgenic cell, exogenous gene is non-endogenic material, cell during growth and breeding,
The situation that exogenous gene is lost can be produced, here it is the hereditary stability of gene.Transgenic cell generally also exists base
Because of unstable situation, for the cell of industrialized production, especially for pharmacy corporation, stablizing of cell strain
Property seems particular importance, and it is stablized for product quality and serves very important effect (Barnes LM etc.
Biotechnol.Bioeng.,2003,81(6):631-9.).So being highly desirable to from molecular level, from the angle of heredity
Analyse in depth the stability of the exogenous gene being integrated into cell strain.At present, the stability of Study of Exogenous gene is mainly
By detection gene copy number change analyze (Chusainow J etc., Biotechnol.Bioeng., 2009,
102(4):1182-1196.)。
Southern Blotting (Kaneko Y etc., Journal of Bioscience are commonly used in the detection of gene copy number at present
And Bioengineering, 2010,109 (3): 274 280), Real-time PCR (Lattenmayer C etc., J
Biotechnol.2007,128 (4): 716-725) method such as, and use the method for Real-time PCR to eliminate
The loaded down with trivial details step of Southern Blotting method, can the simple and quick copy number detecting genome exactly.Real
Time PCR (real-time PCR) is again Q-PCR (quantitative PCR, quantitative PCR, real-time quantitative PCR),
Refer to add fluorophor (such as SYBR-Green, TaqMan fluorescent probe etc.), profit in PCR reaction system
Monitoring whole PCR process in real time with the accumulation of fluorescence signal, unknown template is entered by the change finally by fluorescence intensity
The method of row quantitative analysis.Quantitative PCR according to the difference of fluorescent material, be broadly divided into SYBR-Green quantitative PCR,
TaqMan quantifying PCR method.
TaqMan quantifying PCR method is on the basis of original pair of primers, needs to recombine one with positive and negative
The specific probe that target sequence between primer combines, fluorophor is connected to the 5' end of probe, and cancellation base
Group is then at 3' end.When probe matches with target sequence, the fluorescence that fluorophor is launched is because connecing with the quenching group of 3' end
Closely it is quenched；During PCR amplification, probe enzyme action is degraded, is made reporter fluorescence by the 5'-3' 5 prime excision enzyme activity of Taq enzyme
Group separates with quencher fluorophor, thus fluorescence monitoring system can receive fluorescence signal.
In the PCR reaction system that SYBR-Green is fluorescent dye, add excess SYBR-Green dyestuff,
SYBR-Green dyestuff non-specifically mixes double-stranded DNA rather than after single stranded DNA, transmitting fluorescence signal, and not
The SYBR dye molecule mixed in chain will not launch fluorescence signal, thus ensures that fluorescence signal is along with the increasing of PCR primer
Add and synchronize to increase.Compared with TaqMan quantifying PCR method, SYBR-Green quantifying PCR method has to be made
With simply, DNA dyestuff is relatively inexpensive, and cost is relatively low, it is not necessary to adds specific probe, and is applicable to any sequence,
Versatility is good.
During standard PCR amplification, the rule of exponential amplification, N are typically followed in the accumulation of productn=N0(1+e)n(Nn
For the product amount after amplification n wheel, N0For starting template amount, e is amplification efficiency, and n is period).But expand to PCR
In the later stage, along with being continuously increased of product, amplified production is the most no longer exponentially increased, the end-product amount of PCR and initial mould
There is no linear relationship between plate amount, initial DNA copy number cannot be calculated according to final PCR primer amount.And quantitatively
In PCR reaction, can detect the fluorescence intensity that product produces in real time, exponentially expand the stage at fluorescence signal, PCR produces
There is linear relationship between logarithm value and the starting template amount of thing amount, therefore can carry out quantitative analysis.
During quantitative PCR, each PCR sample has a Ct threshold value (Cycle threshold value), Ct
Value is meant that: the period that the fluorescence signal in each reaction tube is experienced when arriving the threshold value set, it is however generally that,
The fluorescence signal of front 15 circulations of PCR reaction is as autofluorescent background signal, and the default setting of fluorescence threshold is 3-15
10 times of the standard deviation of the fluorescence signal of circulation, it may be assumed that Ct=10 × SDcycle (3-15).Mass data research shows,
There is linear relationship with the logarithm of the starting copy number of this template in the Ct value of each template, starting copy number is the most, and Ct value is more
Little.
The standard substance utilizing known starting copy number can make standard curve, and wherein abscissa represents the logarithm of starting copy number,
Vertical coordinate represents Ct value.Therefore, as long as obtaining the Ct value of unknown sample, this sample can be calculated from standard curve
Starting copy number.Along with the progress of genome plan in recent years, substantial amounts of species gene group has checked order, including
People, mice, rat, Chinese hamster, machin, fruit bat, nematicide, cattle, Brachydanio rerio, Rhesus Macacus, silkworm, chicken,
The biologies such as orangutan, goat, macaque, pig, horse, panda, in succession illustrate the accurate size of genomic DNA, then can push away
Calculate DNA molecular number contained in the cellular genome of unit mass, according to the starting copy number of sample, and then can be absolute
The quantitatively number of copies of the exogenous gene in cell.
Summary of the invention
First purpose of the present invention is to provide and a kind of detects antibody molecule heavy chain gene copies number in transgenetic animal cell
Quantitative detecting method, the heavy chain gene forward primer used in the method is JRTH3 and heavy chain gene reverse primer is
JRTH3R, wherein
The sequence of JRTH3 is: 5'-CAAGCTGACCGTGGATAA-3'(is as shown in SEQ ID No.1),
The sequence of JRTH3R is: 5'-GACAGGCTCTTCTGAGTG-3'(is as shown in SEQ ID No.2).
To achieve these goals, the present invention uses the steps:
A) synthesis is for the specific primer JHc-1F (DNA as shown in SEQ ID No.7 of antibody molecule heavy chain gene
Molecule) and JHc-1R (DNA molecular as shown in SEQ ID No.8), make with the heavy chain DNA of complete antibody molecule
Carry out regular-PCR amplification for template, prepare the plasmid DNA with heavy chain fragment, thus prepare heavy chain plasmid DNA
Standard substance.
B) with JRTH3 and JRTH3R as forward primer and reverse primer, using the plasmid DNA in step a) as template
Carry out quantitative PCR reaction, calculate the copy number of plasmid DNA.Thus Criterion curve.
C) genomic DNA and the cell lines of untransfected antibody molecule of the cell lines of transfection antibody molecule are extracted
Genomic DNA as template, with JRTH3 and JRTH3R as forward primer with reverse primer, carry out quantitative PCR
Reaction, simultaneously with aseptic ultra-pure water as blank.Quantitative PCR reaction carries out the test of solubility curve after terminating, obtain
Obtain Ct value, calculate the heavy chain gene copies number of antibody molecule in zooblast according to the standard curve in Ct value and step b).
Above-mentioned steps b) and c) in, when carrying out quantitative pcr amplification, PCR reaction cumulative volume be 30 μ l:SYBR Green
PCR Mix (SYBR Green PCR mixed liquor) 15 μ l；Forward primer (10 μMs) 0.5 μ l；Reverse primer (10
μM)0.5μl；DNA profiling 10 μ l；Aseptic ultra-pure water 4 μ l.Reaction condition is 95 DEG C of 10min；95℃30
S, 60 DEG C of 30s, 72 DEG C of 30s, totally 40 circulations.
Above-mentioned antibody can be such as humanization anti-tnf-alpha monoclonal antibody, it is also possible to is people-Mus mosaic anti-tnf-alpha
Monoclonal antibody, it is also possible to be anti-HER 2 humanized monoclonal antibody, it is also possible to be that anti-vegf Humanized monoclonal resists
Body etc..
Second object of the present invention is to provide and a kind of detects antibody molecule light chain gene copy number in transgenetic animal cell
Quantitative detecting method, the light chain gene forward primer used in the method is JRTL1F and light chain gene reverse primer
JRTL1R, wherein
The sequence of JRTL1F is: 5'-GCAGTGGAAGGTGGATAA-3'(is as shown in SEQ ID No.3),
The sequence of JRTL1R is: 5'-TTTGCTCAGTGTCAGGGT-3'(is as shown in SEQ ID No.4).
To achieve these goals, the present invention uses the steps:
A) synthesis is for the specific primer JLc-1F (DNA as shown in SEQ ID No.5 of antibody molecule light chain gene
Molecule) and JLc-1R (DNA molecular as shown in SEQ ID No.6), make with the light chain DNA of complete antibody molecule
Carry out regular-PCR amplification for template, prepare the plasmid DNA with light chain segments, thus prepare light chain plasmids DNA
Standard substance.
B) with JRTL1F and JRTL1R as forward primer and reverse primer, using the plasmid DNA in step a) as mould
Plate carries out quantitative PCR reaction, obtains Ct value and calculates the copy number of plasmid DNA, thus Criterion curve.
C) genomic DNA and the cell lines of untransfected antibody molecule of the cell lines of transfection antibody molecule are extracted
Genomic DNA as template, with JRTL1F and JRTL1R as forward primer and reverse primer to carry out quantitative PCR anti-
Should, simultaneously with aseptic ultra-pure water as blank.Quantitative PCR reaction carries out the test of solubility curve after terminating, it is thus achieved that
Ct value, calculates the light chain gene copy number of antibody molecule in zooblast according to the standard curve in Ct value and step b).
Above-mentioned steps b) and c) in, when carrying out quantitative pcr amplification, PCR reaction cumulative volume be 30 μ l:SYBR Green
PCR Mix (SYBR Green PCR mixed liquor) 15 μ l；Forward primer (10 μMs) 0.5 μ l；Reverse primer (10
μM)0.5μl；DNA profiling 10 μ l；Aseptic ultra-pure water 4 μ l.Reaction condition is 95 DEG C of 10min；95℃30
S, 60 DEG C of 30s, 72 DEG C of 30s, totally 40 circulations.
Above-mentioned antibody can be such as humanization anti-tnf-alpha monoclonal antibody, it is also possible to is people-Mus mosaic anti-tnf-alpha
Monoclonal antibody, it is also possible to be anti-HER 2 humanized monoclonal antibody, it is also possible to be that anti-vegf Humanized monoclonal resists
Body etc..
The present invention uses SYBR-Green Real-time PCR method, can be for the light chain base of antibody molecule in zooblast
The copy number of cause and/or heavy chain gene measures individually or simultaneously, and is absolute quantitation so that different experiments, difference are thin
The testing result of intercellular can be compared to each other；Can be used for studying the stability of antibody cell strain, filter out stable high expressed
Through engineering approaches cell strain.
The method detection sensitivity height of the present invention and range of linearity width, minimum can detecting in cell contains only 1 gene
The sample of copy number, maximum can reach 1500 with last copy number；Linear relationship is good, and R2 is all higher than 0.99, foot
With the needs of gene copy number detection in competent sample.
The reliable results of the method detection of the present invention, the standard deviation ＜ 0.3 of the mensuration Ct value of sample.
The method of the present invention, PCR detection atopic is high, and the melting curve of quantitative PCR product only has single absworption peak,
Specific amplification signal nothing but.
The inventive method versatility is good, can be by changing design specific primer, for the light chain gene of different antibodies molecule
Or heavy chain gene carries out the detection of gene copy number；Can measure the gene copy number in various kinds of cell, these cells can simultaneously
To include 293 cells, Vero cell, bhk cell, PER.C6 cell, SP2/0 cell, NS0 cell, CHO
Cell, DG44 cell etc..
The method of the present invention can be with the gene copy number of the multiple sample of high throughput assay.
The method of the present invention uses SYBR-Green to be fluorescent dye, and the advantage with simple cheap was reacted at PCR
Cheng Zhongke replaces the TaqMan fluorescent probe of costliness completely, reaches the purpose of Accurate Determining gene copy number simultaneously.
Accompanying drawing explanation
Fig. 1. the quantitative pcr amplification curve of light chain standard substance, wherein abscissa is period, and vertical coordinate is fluorescence intensity.
In figure, 6 curves are the quantitative pcr amplification curves of light chain plasmids DNA standard substance of variable concentrations from left to right.
Fig. 2. light chain gene standard curve, wherein abscissa is the logarithm of light chain plasmids DNA standard substance plasmid copy number,
Vertical coordinate is the Ct value that quantitative PCR apparatus software Automatic Optimal obtains.
Fig. 3. the quantitative pcr amplification curve of heavy chain standard substance, wherein abscissa is period, and vertical coordinate is fluorescence intensity.
In figure, 6 curves are the quantitative pcr amplification curves of heavy chain plasmid DNA standard substance of variable concentrations from left to right.
Fig. 4. heavy chain gene standard curve, wherein abscissa is the logarithm of light chain plasmids DNA standard substance plasmid copy number,
Vertical coordinate is the Ct value that quantitative PCR apparatus software Automatic Optimal obtains.
Fig. 5. the quantitative pcr amplification curve of light chain gene, wherein abscissa is period, and vertical coordinate is fluorescence intensity.
In figure, curve is genomic DNA and the cell lines of untransfected antibody molecule of the cell lines of transfection antibody molecule
The quantitative pcr amplification curve of genomic DNA, the curve of baseline is the blank curve of aseptic ultra-pure water.
Fig. 6. the quantitative PCR melting curve of light chain gene, wherein abscissa is temperature；Vertical coordinate is fluorescence relative time
Rate of change.In figure, curve is genomic DNA and the animal of untransfected antibody molecule of the cell lines of transfection antibody molecule
The PCR melting curve of the genomic DNA of cell strain, the curve of baseline is the blank curve of aseptic ultra-pure water.
Fig. 7. the quantitative pcr amplification curve of heavy chain gene, wherein abscissa is period, and vertical coordinate is fluorescence intensity.
In figure, curve is genomic DNA and the cell lines of untransfected antibody molecule of the cell lines of transfection antibody molecule
The quantitative pcr amplification curve of genomic DNA, the curve of baseline is the blank curve of aseptic ultra-pure water.
Fig. 8. the quantitative PCR melting curve of heavy chain gene, wherein abscissa is temperature, and vertical coordinate is fluorescence relative time
Rate of change.In figure, curve is genomic DNA and the animal of untransfected antibody molecule of the cell lines of transfection antibody molecule
The PCR melting curve of the genomic DNA of cell strain, the curve of baseline is the blank curve of aseptic ultra-pure water.
Detailed description of the invention
Below with specific embodiment, being described further technical scheme, wherein zooblast choosing is the most normal
Chinese hamster ovary celI as example, but the present invention is not limited to these embodiments.
Technical scheme includes standard plasmid DNA and the preparation of genomic DNA, the foundation of standard curve, light
The detection of the copy number of heavy chain gene, the process such as calculating of gene copy number.Technical scheme includes following experiment
Flow process:
The quantitative PCR detecting method of embodiment 1 light chain gene copy number
1.1, the acquisition of plasmid DNA:
A) design is for the specific primer of light chain gene: the nucleotides sequence of specific forward primer JLc1F is classified as:
5'-AGGACAGTGGCTGCACCA-3'(is as shown in SEQ ID No.5)；The core of specific reverse primers JLc1R
Nucleotide sequence is: 5'-TCAACACTCTCCTCTGTT-3'(is as shown in SEQ ID No.6).
B) light chain pLc plasmid strain is obtained: with the light chain DNA of complete antibody molecule as template, carry out regular-PCR expansion
Increase, it is thus achieved that antibody molecule light chain Lc fragment, be then inserted on p simple-19T carrier, convert DH5 α competence thin
Born of the same parents, it is thus achieved that the pLc plasmid strain containing antibody light chain gene.
C) through sequencing, it is thus achieved that molecular size is the pLc plasmid strain of 3016bp.
1.2, the preparation of light chain plasmids DNA standard substance
By the pLc plasmid strain containing antibody light chain gene, it is inoculated in the LB fluid medium of 10ml, 37 DEG C of shaking tables
Overnight incubation, takes 1ml culture fluid, and the centrifugal supernatant of abandoning of 10000rpm, bacterium solution precipitation presses Axygen company " plasmid DNA
Extraction agent box in a small amount " method extracting plasmid DNA in description, finally it is dissolved in the aseptic ultra-pure water of 60ml.Obtain
The plasmid nucleic acid-protein analyser (SmartSpec of Bio-Rad companyTMPlus Spectrophotometer) detection carry
The DNA mass taken.With aseptic ultra-pure water, light chain plasmids is diluted to 0.002ng/ml, then uses 5 times of doubling dilutions,
Prepare light chain plasmids DNA standard substance.
1.3, the foundation of standard curve
Take the light chain plasmids DNA standard substance prepared in 10 μ l steps 1.2 as DNA profiling, forward primer JRTL1F 0.5
μ l (concentration 10 μMs), positive anti-primer JRTL1R 0.5 μ l (concentration 10 μMs), SYBR Green PCR Mix 15 μ l
(purchased from Applied Biosystems company) and aseptic ultra-pure water 4 μ l, carry out quantitative PCR reaction.Wherein use
The iQ5Real-Time PCR Detection System z system that real-time PCR is Bio-Rad company.Quantitative PCR
Reaction condition is 95 DEG C of 10min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, totally 40 circulations.Light chain
Quantitative pcr amplification curve is shown in Fig. 1.
Wherein, the nucleotides sequence of forward primer JRTL1F is classified as: 5'-GCAGTGGAAGGTGGATAA-3'(such as SEQ
Shown in ID No.3)；The nucleotides sequence of reverse primer JRTL1R is classified as: 5'-TTTGCTCAGTGTCAGGGT-3'(is such as
Shown in SEQ ID No.4).
According to below equation calculating plasmid DNA copies number:
It is calculated: the copy number that 1ng light chain plasmids contains is 3.02 × 108Individual, then the copy of light chain plasmids DNA standard substance
Number is respectively as follows: 6.04 × 106,1.21×106,2.42×105,4.83×104,9.66×103,1.93×103。
The Ct value obtained using quantitative PCR apparatus software (iQ5 that instrument carries analyze software) Automatic Optimal as vertical coordinate,
The logarithm of starting template Plays plasmid copy number generates standard curve as abscissa and sees the amplification efficiency of Fig. 2, PCR
91.6%, the curve correlation coefficient R2=0.999 of the two, the regression equation of standard curve is: Y=-3.542X+38.459.
Establishing criteria curve, the copy number of lowest detection is 1.93 × 103, i.e. 1.93 × 103/ 3723=0.5, namely can examine
Measure 1 copy number, it is possible to the needs of detection in competent transgenic cell；The copy number of maximum detection is 6.04 × 106,
I.e. 6.04 × 106/ 3723=1622, namely maximum can detect 1622 copy numbers.
1.4, cell is cultivated
Recover a Chinese hamster ovary celI strain having transfected antibody light chain gene, be inoculated in serum-free medium, 37 DEG C, 5%CO2
Cultivating in shaking table, when cell viability reaches more than 98%, be inoculated in respectively in other two bottles of shaking flasks, one bottle adds MTX
(methotrexate, Methotrexate), another bottle, without adding entering MTX, starts to cultivate continuously under the same conditions, respectively
Cultivate the 10th, 20,30,35,40 generation time, take cell and be centrifuged, precipitation be stored in-80 DEG C of refrigerators.
1.5, the preparation of cell genomic dna
According to QIAmp DNA Mini Kit description, Chinese hamster ovary celI and the transfection of extracting untransfected antibody molecule respectively are anti-
The genomic DNA of the Chinese hamster ovary celI strain of body light chain gene, is dissolved in aseptic ultra-pure water.The genomic DNA core obtained
Acid albumin analyser (the SmartSpec of Bio-Rad companyTMPlus Spectrophotometer) the DNA matter of Detection and Extraction
Amount, and genomic DNA is diluted to 1ng/ μ l, as the negative control of quantitative PCR detection with aseptic ultra-pure water respectively
With sample, and with aseptic ultra-pure water as blank.
1.6, the detection of the copy number of light chain gene:
Respectively taking genomic DNA 10 μ l as PCR reaction template, JRTL1F/JRTL1R is respectively positive anti-primer, carries out
Quantitative PCR reacts, and carries out the test of melting curve, and result is shown in Fig. 5 and 6.
Real-time quantitative PCR reaction system is as follows, and the condition of quantitative PCR reaction is 95 DEG C of 10min；95℃30s、60℃
30s, 72 DEG C of 30s, totally 40 circulations.
The test of melting curve is arranged: for product from the beginning of 55 DEG C, heat up with 0.5 DEG C for interval, to 95 DEG C,
Each temperature keeps 30s and detects fluorescence.Reaction terminate after, iQ5 analyze software automatically with fluorescence with temperature rate of change with
The relation of temperature is drawn, and obtains melting curve.The standard substance of variable concentrations, sample gene group DNA, negative control gene
Group DNA and blank, expand simultaneously, and test sample sets multiple multiple hole and carries out Parallel testing (>=3).
From Fig. 5,6 it can be seen that sample P CR amplified reaction is good, and the CHO of water and untransfected antibody molecule is thin
The genome of born of the same parents does not has any amplification, all of sample to be all only at about 81 DEG C and a single absworption peak occurs, and PCR is described
The specificity of amplification is good.Meanwhile, the Ct value of all of sample all falls within the scope of standard curve.After reaction terminates,
Analyze software and Automatic Optimal can obtain Ct value, calculate 10ng according to the standard curve of Ct value with the detection of light chain gene copy number
Light chain gene copy number contained by sample DNA.
1.7, the calculating of gene copy number:
According to the progress of CHO genome research, the Genome Size of Chinese hamster ovary celI is 2.45Gb (Xu X etc., Nat
Biotechnol, 2011,29 (8): 735-741), then the genomic DNA of 10ng contains 3723 DNA moleculars, permissible
Light chain gene copy number by containing in following equation calculating transgenic CHO cell genome:
In above-mentioned formula, the Genome Size of zooblast Chinese hamster ovary celI is 2.45Gb.
The results are shown in Table 1.For obtaining higher antibody expression, the Chinese hamster ovary celI of transfection antibody needs through long-term continuous
MTX increase stressed process, this process be actually antibody light chain gene in cellular genome copy number increase
The process (the .Biotechnology Advances.2010 such as Cacciatore JJ, (28): 673 681) added.In table 1 also
Can be seen that and use MTX to increase stressed light chain gene copy number more than the light chain gene copy number being added without MTX.
Bubner B et.al. thinks, the standard deviation of the Ct value of the amplified reaction of general single sample is less than 0.3, it is possible to
It is regarded to accurately measure the copy number (Bubner B etc., Plant Cell Rep.2004,23 (5): 263-271) of sample.By
Table 1 is visible, and the standard deviation of the Ct value of this method each sample amplification reaction is respectively less than 0.3, illustrates that our method is
Accurately and reliably.
The copy number of table 1 quantitative PCR detection light chain gene
Note: in table 1 ,+MTX refers to that adding MTX ,-MTX in shaking flask refers to be added without in shaking flask MTX.
The detection method of embodiment 2 heavy chain gene copies number
2.1, the acquisition of plasmid DNA:
A) design is for the forward primer JHc-1F and reverse primer JHc-1R of heavy chain gene:
The nucleotides sequence of forward primer JHc-1F is classified as: 5'-
ATGCTAGCAAGCTTCCATGGGCGTCGACAAAGGGACCATCTGTGT-3'(such as SEQ ID No.
Shown in 7)；The nucleotides sequence of reverse primer JHc-1R is classified as: 5'-
TAGGTACCTCGAGTCATTTACCAGGAGACAG-3'(is as shown in SEQ ID No.8).
B) heavy chain pHc plasmid strain is obtained
With the heavy chain DNA of complete antibody molecule as template, carry out regular-PCR amplification, it is thus achieved that antibody molecule heavy chain Hc
Fragment, is then inserted in carrier T, converts DH5 α competent cell, it is thus achieved that heavy chain pLc plasmid strain.
C) through sequencing, it is thus achieved that molecular size is the pHc plasmid strain of 3718bp.
2.2, the preparation of heavy chain standard plasmid DNA
By the pHc plasmid strain containing antibody heavy chain gene, it is inoculated in the LB fluid medium of 10ml, 37 DEG C of shaking tables
Overnight incubation, takes 1ml culture fluid, and the centrifugal supernatant of abandoning of 10000rpm, bacterium solution precipitation is by by Axygen company " plasmid
DNA in a small amount extraction agent box " the method extracting plasmid DNA of description, finally it is dissolved in the aseptic ultra-pure water of 60ml.Obtain
The plasmid nucleic acid-protein analyser (SmartSpec of Bio-Rad company obtainedTMPlus Spectrophotometer) detection
The DNA mass extracted.With aseptic ultra-pure water, light chain plasmids is diluted to 0.002ng/ml, then uses 5 times of doubling dilutions,
Prepare heavy chain plasmid DNA standard substance.
2.3, the foundation of standard curve
Taking heavy chain plasmid DNA standard substance prepared by 10 μ l as DNA profiling, positive anti-primer JRTH3F 0.5 μ l is (dense
Spend 10 μMs) and reverse primer JRTH3R 0.5 μ l (concentration 10 μMs), SYBR Green PCR Mix 15 μ l (be purchased from
Applied Biosystems company) and aseptic ultra-pure water 4 μ l, carry out quantitative PCR reaction.Wherein use is real-time
Quantitative PCR apparatus is the iQ5Real-Time PCR Detection System z system of Bio-Rad company.Quantitative PCR reacts
Condition is 95 DEG C of 10min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, totally 40 circulations.Heavy chain quantitative
PCR amplification curve is shown in Fig. 3.
Wherein, the nucleotides sequence of forward primer JRTH3F is classified as: 5'-CAAGCTGACCGTGGATAA-3'(such as SEQ
Shown in ID No.1)；The nucleotides sequence of reverse primer JRTH3R is classified as: 5'-GACAGGCTCTTCTGAGTG-3'
(as shown in SEQ ID No.2).
According to below equation calculating plasmid DNA copies number:
It is calculated: the copy number that 1ng heavy chain plasmid contains is 2.45 × 108Individual, then the copy of heavy chain plasmid DNA standard substance
Number is respectively as follows: 4.90 × 106,9.80×105,1.96×105,3.92×104,7.84×103,1.57×103。
The Ct value obtained using quantitative PCR apparatus software (iQ5 that instrument carries analyze software) Automatic Optimal as vertical coordinate,
The logarithm of starting template Plays plasmid copy number generates standard curve as abscissa and sees the amplification efficiency of Fig. 4, PCR
91.8%, the curve correlation coefficient R2=1.000 of the two, the regression equation of standard curve is: Y=-3.536X+38.388.
Establishing criteria curve, the copy number of lowest detection is 1.57 × 103, i.e. 1.57 × 103/ 3723=0.42, namely can
A copy number detected, it is possible to the needs of detection in competent transgenic cell；The copy number of maximum detection is 4.96 × 106,
I.e. 4.96 × 106/ 3723=1332, namely maximum can detect 1332 copy numbers.In view of light chain heavy chain concordance and
The good linear of detection curve, it should 1500 copy numbers can also be measured, if but strictly describe, can be with 1300
For maximum detected value.
2.4, cell is cultivated
Recover a Chinese hamster ovary celI strain having transfected antibody heavy chain gene, be inoculated in serum-free medium, 37 DEG C, 5%CO2
Cultivating in shaking table, when cell viability reaches more than 98%, be inoculated in respectively in other two bottles of shaking flasks, one bottle adds MTX
(methotrexate, amethopterin), another bottle, without adding entering MTX, starts to cultivate continuously under the same conditions, respectively
Cultivate the 10th, 20,30,35,40 generation time, take cell and be centrifuged, precipitation be stored in-80 DEG C of refrigerators.
2.5, the preparation of cell genomic dna
According to QIAmp DNA Mini Kit description, Chinese hamster ovary celI and the transfection of extracting untransfected antibody molecule respectively are anti-
The genomic DNA of the Chinese hamster ovary celI strain of body light chain gene, is dissolved in aseptic ultra-pure water.The genomic DNA core obtained
Acid albumin analyser (the SmartSpec of Bio-Rad companyTMPlus Spectrophotometer) the DNA matter of Detection and Extraction
Amount, and with aseptic ultra-pure water, genomic DNA is diluted to 1ng/ml respectively, as the negative control of quantitative PCR detection
With sample, and with aseptic ultra-pure water as blank.
2.6, the detection of the copy number of heavy chain gene:
Respectively taking genomic DNA 10 μ l as PCR reaction template, JRTH3F/JRTH3R is respectively positive anti-primer, carries out
Quantitative PCR reacts, and carries out the test of melting curve, and result is shown in Fig. 7 and 8.
Real-time quantitative PCR reaction system is as follows, and the condition of quantitative PCR reaction is 95 DEG C of 10min；95℃30s、60℃
30s, 72 DEG C of 30s, totally 40 circulations.
The test of melting curve is arranged: for product from the beginning of 55 DEG C, heat up with 0.5 DEG C for interval, to 95 DEG C,
Each temperature keeps 30s and detects fluorescence.Reaction terminate after, iQ5 analyze software automatically with fluorescence with temperature rate of change with
The relation of temperature is drawn, and obtains melting curve.The standard substance of variable concentrations, sample gene group DNA, negative control gene
Group DNA and blank, expand simultaneously, and test sample sets multiple multiple hole and carries out Parallel testing (>=3).
It can be seen that sample P CR amplified reaction is good from 7,8, and water and the Chinese hamster ovary celI of untransfected antibody molecule
Genome do not have any amplification, all of sample be all only at about 81 DEG C occur a single absworption peak, illustrate that PCR expands
The specificity increased is good.Meanwhile, the Ct value of all of sample all falls within the scope of standard curve.After reaction terminates, point
Analysis software Automatic Optimal can obtain Ct value, calculates 10ng according to the standard curve of Ct value with the detection of light chain gene copy number
Light chain gene copy number contained by sample DNA.
2.7, the calculating of gene copy number:
According to the progress of CHO genome research, the Genome Size of Chinese hamster ovary celI is 2.45Gb (Xu X et.al., Nat
Biotechnol, 2011,29 (8): 735-741), then the genomic DNA of 10ng contains 3723 DNA moleculars, permissible
Heavy chain gene copies number by containing in following equation calculating transgenic CHO cell genome:
The results are shown in Table 2.For obtaining higher antibody expression, the Chinese hamster ovary celI of transfection antibody needs through long-term continuous
MTX increase stressed process, this process be actually antibody heavy chain gene in cellular genome copy number increase
The process (the .Biotechnology Advances.2010 such as Cacciatore JJ, (28): 673 681) added.In table 2 also
Can be seen that and use MTX to increase stressed heavy chain gene copies number more than the heavy chain gene copies number being added without MTX.
Bubner B et.al. thinks, the standard deviation of the Ct value of the amplified reaction of general single sample is less than 0.3, it is possible to
It is regarded to accurately measure the copy number (Bubner B etc., Plant Cell Rep.2004,23 (5): 263-271) of sample.By
Table 2 is visible, and the standard deviation of the Ct value of this method each sample amplification reaction is respectively less than 0.3, illustrates that our method is
Accurately and reliably.
The copy number of table 2 quantitative PCR detection heavy chain gene
Note: in table 2 ,+MTX refers to that adding MTX ,-MTX in shaking flask refers to be added without in shaking flask MTX.
The expression of recombinant antibodies depends on the coordinate expression of light chain and heavy chain gene, there is therebetween a conjunction
Suitable light chain heavy chain ratio (Chusainow J etc., Biotechnol Bioeng.2009,102 (4): 1182-1196.；Ho
SC1 etc., J Biotechnol.2013,165 (3-4): 157-166).
The method using the present invention carries out dynamic monitoring to the copy number of light chain gene and heavy chain gene, can filter out light chain and weight
The copy number of chain gene relatively, the overexpression cell line of the suitable scope of the ratio that is in, as business-like stable
Expression cell line, the constant product quality for business-like recombinant antibodies has very important effect.
Claims (8)
1. antibody heavy chain gene copy number in a SYBR-Green quantifying PCR method detection transgenic cell
Method, it is characterised in that the forward primer used and reverse primer are SEQ ID No.1 and SEQ respectively
DNA molecular shown in ID No.2.
2. heavy chain in SYBR-Green quantifying PCR method detection transgenic cell as claimed in claim 1
The method of gene copy number, it is characterised in that specifically include following steps:
A) the specific forward primer JHc-1F for antibody heavy chain gene and specific reverse primers are synthesized
JHc-1R, carries out regular-PCR amplification using the heavy chain DNA of complete antibody as template, prepares with weight
The plasmid DNA of chain fragment, thus prepare heavy chain plasmid DNA standard substance；The sequence of described JHc-1F such as SEQ
ID No.7, the sequence of described JHc-1R is as shown in SEQ ID No.8；
B) with the forward primer shown in SEQ ID No.1 and the reverse primer shown in SEQ ID No.2, with step
The plasmid DNA standard substance with heavy chain fragment in a) carry out quantitative PCR reaction as template, calculate matter
The copy number of grain DNA, sets up PCR reaction normal curve；
C) genomic DNA of the cell lines of transfection antibody molecule and the dynamic of untransfected antibody molecule are extracted
The genomic DNA of thing cell strain is as template, shown in employing SEQ ID No.1 and SEQ ID No.2 just
To primer and reaction primer, carry out quantitative PCR reaction, simultaneously with aseptic ultra-pure water as blank；Fixed
Amount PCR reacts and obtains Ct value after terminating, and carries out the test of solubility curve, according in Ct value and step b)
Standard curve calculate the heavy chain gene copies number of antibody molecule in zooblast；
Above-mentioned steps b) and c) in, when carrying out quantitative pcr amplification, its quantitative PCR reaction cumulative volume be
30 μ l:SYBR Green PCR Mix 15 μ l, the forward primer 0.5 μ l of 10 μMs of concentration, 10 μMs of concentration
Reverse primer 0.5 μ l, DNA profiling 10 μ l, aseptic ultra-pure water, 4 μ l；
Its quantitative PCR reaction condition is: first 95 DEG C of 10min, the most successively 95 DEG C of 30s, 60 DEG C 30
S, 72 DEG C of 30s, totally 40 circulations.
3. in SYBR-Green quantifying PCR method detection transgenic cell as claimed in claim 1 or 2
The method of heavy chain gene copies number, it is characterised in that described transgenic cell is selected from Chinese hamster ovary celI.
4. in a SYBR-Green quantifying PCR method detection transgenic cell, antibody molecule light chain gene is copied
The method of shellfish number, it is characterised in that the light chain gene primer used is SEQ ID No.3 and SEQ ID No.
DNA molecular shown in 4.
5. heavy chain in SYBR-Green quantifying PCR method detection transgenic cell as claimed in claim 4
The method of gene copy number, it is characterised in that specifically include following steps:
A) the specific forward primer JLc1F for antibody light chain gene and specific reverse primers are synthesized
JLc1R, carries out regular-PCR amplification using the light chain DNA of complete antibody as template, prepares with light chain
The plasmid DNA of fragment, thus prepare light chain plasmids DNA standard substance；The sequence of described JLc1F such as SEQ ID
Shown in No.5, the sequence of described JLc1R is as shown in SEQ ID No.6；
B) with the forward primer shown in SEQ ID No.3 and SEQ ID No.4 and reverse primer, with step a)
In plasmid DNA standard substance carry out quantitative PCR reaction as template, calculate the copy number of plasmid DNA,
Set up PCR reaction normal curve；
C) genomic DNA of the cell lines of transfection antibody molecule and the dynamic of untransfected antibody molecule are extracted
The genomic DNA of thing cell strain is as template, shown in employing SEQ ID No.3 and SEQ ID No.4 just
To primer and reaction primer, carry out quantitative PCR reaction, simultaneously with aseptic ultra-pure water as blank；Fixed
Amount PCR reacts and obtains Ct value after terminating, and carries out the test of solubility curve, according in Ct value and step b)
Standard curve calculate the heavy chain gene copies number of antibody molecule in zooblast；
Above-mentioned steps b) and c) in, when carrying out quantitative pcr amplification, its quantitative PCR reaction cumulative volume be
30 μ l:SYBR Green PCR Mix 15 μ l, the forward primer 0.5ml of 10 μMs of concentration, 10 μMs of concentration
Reverse primer 0.5 μ l, DNA profiling 10ml, aseptic ultra-pure water 4ml；
Its quantitative PCR reaction condition is: first 95 DEG C of 10min, the most successively 95 DEG C of 30s, 60 DEG C 30
S, 72 DEG C of 30s, totally 40 circulations.
6. in the SYBR-Green quantifying PCR method detection transgenic cell as described in claim 4 or 5
The method of heavy chain gene copies number, it is characterised in that described transgenic cell is selected from Chinese hamster ovary celI.
7. the SYBR-Green quantifying PCR method detection transgenic as described in arbitrary in claim 1-3 is thin
The method of heavy chain gene copies number application in the through engineering approaches cell strain of high expressed is stablized in screening in born of the same parents.
8. the SYBR-Green quantifying PCR method detection transgenic as described in arbitrary in claim 4-6 is thin
The method of light chain gene copy number application in the through engineering approaches cell strain of high expressed is stablized in screening in born of the same parents.
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