CN112301028B - SCAR marker for identifying CHO cells and construction method and application thereof - Google Patents
SCAR marker for identifying CHO cells and construction method and application thereof Download PDFInfo
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Abstract
The invention provides an SCAR marker for identifying CHO cells and a construction method and application thereof.A CHO cell genome specificity is utilized, BHK-21 cells with relatively close relativity are taken as control cells, two RAPD primers with better specificity and higher stability are screened from 60 RAPD primers, and three stable specific bands are obtained; through gene cloning, the three fragments are connected to PMD19-T and then sequenced, three sequences obtained through sequencing are compared by using an NCBI BLAST online comparison tool to obtain three sequences corresponding to Chinese hamster only (Access number: XR _004770315.1, NW _003623089.1 and XM _003515610.5), besides, other sequences with higher homology are not found, a pair of stable SCAR labeled primers are respectively designed on each sequence on the basis, and then the proper annealing temperature is obtained through searching the annealing temperature of the SCAR primers to improve the accuracy of the method. The invention has the characteristics of convenient and simple operation, stable experimental result, strong specificity, high sensitivity and the like.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to an SCAR marker for identifying CHO cells, and a construction method and application thereof.
Background
Therapeutic recombinant proteins have grown very rapidly in the pharmaceutical industry, and worldwide investment in biopharmaceutical fields such as blood products, monoclonal antibodies and hormones has reached $ 1380 million in 2010. Chinese hamster ovary Cells (CHO) were isolated from a female Chinese hamster in 1957 by the american chemist Theodore t.puck, and have been widely used in the field of biologics due to their advantages of being easy to culture in suspension and being able to produce high concentrations of therapeutic proteins. At present, CHO cells are the most predominant host cells for the production of therapeutic recombinant proteins.
Misrecognition and cross-contamination of cell lines are major problems in cell culture, greatly affecting the production of biological products. The International Conference on harmony (ICH) on drug registration technology for human use clearly indicates that cell banks such as MCB, WCB and EPC are to be subjected to cell identification. The three parts of the pharmacopoeia of the people's republic of China 2020 edition also clearly stipulate that newly established cell lines/strains, cell banks (MCB and WCB) and production end cells should be subjected to identification tests to confirm that the cells are the subject cells and that there is no cross contamination of other cells. There are many cell identification assays defined therein, including methods of cell morphology, biochemistry, immunology, cytogenetic testing, and PCR. At least one or more methods should be chosen to identify the species and cell lines of the cells and their specific characteristics to ensure the quality of the biological product.
At present, methods such as isozyme technology, karyotype analysis technology and the like are mainly adopted for identifying CHO cells, and the methods are complex in operation, long in time consumption, high in cost and low in accuracy. Random Amplified Polymorphic DNA (RAPD) technology is a molecular marker technology invented in 1990, and its principle is that it adopts shorter primer (9bp or 10bp) to make polymerase chain reaction on genome, and can make polymorphic analysis on genome with unknown sequence so as to implement the goal of species identification. The Sequence Characterized Amplified Regions (SCAR) technology is a more stable marking technology established on the basis of RAPD technology. The SCRA marking technology can realize the rapid identification of species only by a small amount of species genome DNA, and has the characteristics of simple operation, short time consumption, strong stability, high sensitivity and the like. However, so far, no relevant report of SCAR technology in CHO cell identification exists.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the SCAR marker for identifying the CHO cells of biological product production, scientific research and the like, and the construction method and the application thereof, the identification of the CHO cells can be realized only by obtaining a genome template of the CHO cells, and the SCAR marker has the characteristics of convenient and simple operation, stable experimental result, strong specificity, high sensitivity and the like, and has very important effect on the aspects of the control of the CHO cell quality of biological products, scientific research and the like.
In order to achieve the above object, the present invention provides a SCAR marker for identifying CHO cells, the SCAR marker including any one of SA-SCAR, SB-SCAR1, SB-SCAR 2;
wherein the content of the first and second substances,
the nucleotide sequence of the SA-SCAR is shown as SEQ ID No. 1;
the nucleotide sequence of the SB-SCAR1 is shown as SEQ ID No. 2;
the nucleotide sequence of the SB-SCAR2 is shown in SEQ ID No. 3.
Further, in the above SCAR marker,
the nucleotide sequence of the primer pair of the SA-SCAR is as follows:
SA-SCAR-F：TCCCGTTCAAGATGGAGGCGC；
SA-SCAR-R：AGGAAACAGCTTACATGCCGA；
the nucleotide sequence of the primer pair of the SB-SCAR1 is as follows:
SB-SCAR-F1：TCATGACAAGGAGCCAATCGG；
SB-SCAR-R1：AGATCTTATAGGGCAGCGTAAG；
the nucleotide sequence of the primer pair of the SB-SCAR2 is as follows:
SB-SCAR-F2：TTGCCCTCACCTTACCGATG；
SB-SCAR-R2：TTGCACTAACCATTGCCTC。
the invention also provides a construction method of the SCAR marker for identifying the CHO cells, which comprises the following steps:
1) separately collecting CHO cells and BHK-21 cells, and extracting DNA;
2) respectively adopting RAPD primer SA and RAPD primer SB to perform PCR amplification on the extracted DNA of the two cells;
3) respectively carrying out electrophoresis detection on the amplification products of the DNA of the two cells; compared with the cell BHK-21, a specific band appears in a product amplified by the CHO cell by the RAPD primer SA, and two specific bands appear in a product amplified by the CHO cell by the RAPD primer SB;
4) recovering and purifying the three specific bands obtained in the step 3), and sequencing to obtain nucleotide sequences of SA-SCAR, SB-SCAR1 and SB-SCAR2 respectively;
5) respectively designing three pairs of primers according to the nucleotide sequences of the SA-SCAR, the SB-SCAR1 and the SB-SCAR2 obtained in the step 4);
6) respectively adopting the three pairs of primers designed in the step 5) to carry out PCR amplification on the CHO cells and the BHK-21 cells.
Further, in step 2) of the above construction method, the nucleotide sequence of the RAPD primer SA is: GAAACGGGTG, respectively; the nucleotide sequence of the RAPD primer SB is as follows: TGGGGGACTC are provided.
Further, in step 2) of the above construction method, the PCR reaction system for RAPD amplification is: 2X Rapid taq Mix 10ul, 10uM primer 1ul, 100ng/ul template 2ul, water 7 ul.
Further, in step 2) of the above construction method, the PCR reaction conditions for RAPD amplification are: 4min at 95 ℃; ② 95 ℃ for 15 s; ③ 37.5 ℃ for 15 s; fourthly, 1min at 72 ℃; repeating the steps from the second step to the fourth step for 35 cycles; fifthly, 5min at 72 ℃; store at 16 deg.C.
Further, in step 6) of the above construction method, when the primer pairs of SA-SCAR, SB-SCAR1 and SB-SCAR2 are used for amplification, the PCR reaction conditions are as follows: 4min at 95 ℃; ② 95 ℃ for 15 s; ③ 56 ℃/58 ℃/60 ℃ for 15 s; fourthly, 15s at 72 ℃; repeating the steps from the second step to the fourth step for 35 cycles; fifthly, 5min at 72 ℃; store at 16 deg.C.
The invention also provides the application of the SCAR marker for identifying CHO cells in identifying CHO cells.
When the SCAR marker is used for identifying the CHO cells, the identification method comprises the following steps:
1) extracting DNA from a sample to be identified;
2) taking the extracted sample DNA as a template, and carrying out PCR amplification by adopting a primer pair of SA-SCAR, SB-SCAR1 or SB-SCAR 2;
3) carrying out electrophoretic analysis on the obtained PCR amplification product; if the sample to be identified is amplified by using the primers of the SA-SCAR, the SB-SCAR1 or the SCAR2, if the electrophoretic band of the amplification product is the same as that of a standard CHO cell, the sample to be identified is the CHO cell, otherwise, the sample is not.
Further, in step 2) of the above identification method, when amplification is performed using the primer set of SA-SCAR, SB-SCAR1, SB-SCAR2, PCR reaction conditions are as follows: 4min at 95 ℃; ② 95 ℃ for 15 s; ③ 60 ℃ for 15 s; fourthly, 15s at 72 ℃; repeating the steps from the second step to the fourth step for 35 cycles; fifthly, 5min at 72 ℃; store at 16 deg.C.
The invention utilizes the specificity of CHO cell genome, uses BHK-21 cells (Syria hamster cells) with close relationship as control cells, screens two RAPD primers with better specificity and higher stability from 60 RAPD primers, and obtains three stable specific bands; through gene cloning, the three fragments are connected to PMD19-T and then sequenced, three sequences obtained through sequencing are compared by using an NCBI BLAST online comparison tool to obtain three sequences corresponding to Chinese hamster only (Access number: XR _004770315.1, NW _003623089.1 and XM _003515610.5), besides, other sequences with higher homology are not found, a pair of stable SCAR labeled primers are respectively designed on each sequence on the basis, and then a proper annealing temperature is obtained through groping the annealing temperature of the SCAR primers, so that the accuracy of the identification method of the embodiment is improved.
Compared with the prior art, the invention has the following beneficial effects:
(1) compared with isoenzyme and karyotype analysis technologies, the method has the characteristics of simple operation, short consumed time, low cost and the like, so that the method can realize the rapid detection of a plurality of samples;
(2) the invention is further optimized on the basis of RAPD; the SCAR primer provided by the invention is longer than the RAPD primer, and the annealing temperature is higher than that used by RAPD, so that the SCAR marker primer provided by the invention has stronger specificity and higher stability;
(3) the template amount required by the method is extremely low, and the rapid detection of the sample can be realized under the condition that the sample is difficult to obtain;
(4) the invention has low quality requirement on DNA, and greatly reduces errors brought by operators.
Drawings
FIG. 1: polymorphism maps of RAPD primer SA on PCR amplification of CHO cells and BHK-21 cells; m: DNAmarker; lane 1: CHO cells; lane 2: BHK-21 cells;
FIG. 2: polymorphism maps of PCR amplification of CHO cells and BHK-21 cells by RAPD primers SB; m: DNAmarker; lane 1: CHO cells; lane 2: BHK-21 cells;
FIG. 3: the SA-SCAR primers are used for amplifying CHO cells and BHK-21 cells at different annealing temperatures; lanes 1, 3 and 5: amplification of CHO cells with SA-SCAR primers at annealing temperatures of 56 ℃, 58 ℃ and 60 ℃, lanes 2, 4 and 6: the SA-SCAR primer is used for amplifying the BHK-21 cells under the conditions of annealing temperatures of 56 ℃, 58 ℃ and 60 ℃;
FIG. 4: the amplification of CHO cells and BHK-21 cells under different annealing temperatures by the SB-SCAR1 primer; lanes 1, 3 and 5: amplification of CHO cells with the SB-SCAR1 primer at annealing temperatures of 56 ℃, 58 ℃ and 60 ℃, lanes 2, 4 and 6: the SB-SCAR1 primer is used for amplifying the BHK-21 cells under the conditions of annealing temperature of 56 ℃, 58 ℃ and 60 ℃;
FIG. 5: the amplification of CHO cells and BHK-21 cells under different annealing temperatures by the SB-SCAR2 primer; lanes 1, 3 and 5: amplification of CHO cells with the SB-SCAR2 primer at annealing temperatures of 56 ℃, 58 ℃ and 60 ℃, lanes 2, 4 and 6: amplification of BHK-21 cells with the SB-SCAR2 primer at annealing temperatures of 56 deg.C, 58 deg.C and 60 deg.C.
Detailed Description
The following further describes embodiments of the present invention with reference to specific examples, and reagents and equipment used in the present invention are commercially available in the conventional market unless otherwise specified. The reagents used are, unless otherwise specified, formulated according to the molecular cloning guidelines.
The construction method of SCAR marker for identifying CHO cell is as follows:
1. preparation of samples
Adding 1ml of pancreatin into a T25 culture flask, and digesting until CHO cells shed; adding 2ml of 10% FBS DMEM culture medium, uniformly mixing the cells, and transferring the cells into a 15ml centrifuge tube; centrifuging at 450g for 5min, and discarding the culture solution; after being resuspended in PBS, the PBS is transferred into a 1.5ml centrifuge tube, 450g of the PBS is centrifuged for 5min, the supernatant is discarded, and cells are collected;
2. extraction of genomic DNA of CHO cell and BHK-21 cell
1) CHO cells and BHK-21 cells were collected into 1.5ml EP tubes, approximately 10, following the above sample collection procedure5-106(ii) individual cells;
2) adding 500ul nuclear lysine solution (Promega) into the mixture, and carrying out water bath at 65 ℃ for about 30 min;
3) adding 10ul (20mg/ml) of proteinase K, and performing water bath at 55 ℃ until digestion is complete;
4) centrifuging at 14000rpm for 4-8min, and taking the supernatant to a 1.5ml centrifuge tube;
5) adding RNase solution, mixing, and standing at 37 deg.C for 15-30 min;
6) cooling to room temperature, adding 200ul protein precision solution (promega), rapidly reversing, mixing, and standing on ice for 10 min;
7) centrifuging at 14000rpm for 4min to precipitate protein;
8) sucking the supernatant (about 600ul), transferring to a new 1.5ml centrifuge tube, adding isopropanol with the same volume, slightly reversing and mixing until white linear DNA appears, and standing at 4 ℃ or-20 ℃ overnight to fully precipitate the DNA;
9) centrifuging at 14000rpm for 1min, precipitating DNA, and discarding the supernatant;
10) adding 600ul 80% ethanol, slightly reversing, mixing, washing DNA, centrifuging at 14000rpm for 1min, and repeating the steps;
11) discarding the supernatant, fully drying for about 5min, adding 50ul of TE buffer solution or sterilized water, and carrying out water bath at 65 ℃ for 1h or overnight at 4 ℃ to fully dissolve DNA;
12) measuring the DNA concentration by a spectrophotometer (NanoDrop), and detecting the quality of the extracted genome by agarose electrophoresis;
3. PCR amplification Using random primers
1) An amplification primer: and SA: GAAACGGGTG, SB: TGGGGGACTC are provided.
2) PCR reaction system
DNA polymerase: 2 rapidtaq DNA polymerase (Novezak company, cat # P222-02)
3) PCR reaction conditions
4. Electrophoretic detection
Preparing 2% agarose gel, carrying out electrophoresis at 160V for about 35min until the indicator reaches the bottom of the agarose gel;
the results are shown in fig. 1 and 2, and it can be seen that: compared with the cell BHK-21, a specific band appears in a product amplified by the CHO cell by the RAPD primer SA, and two specific bands appear in a product amplified by the CHO cell by the RAPD primer SB;
5. specific band recovery and purification
The specific fragments were recovered and purified using gel recovery kit (OMEGA), the specific procedures are described in the gel recovery kit (OMEGA).
6. Construction and sequencing of specific fragment plasmids
Connecting the purified RAPD-PCR product with PMD19-T through Solution I, and transferring the product into competent cell DH5 alpha, wherein the detailed experimental steps are shown in chapter III of molecular cloning experimental guidance; DH5 α was diluted with LB medium; the diluted DH 5. alpha. was spread on LB agarose plates containing 100ug/ml ampicillin; after 24 hours, screening the formed single colony by using an RAPD primer (the system and the program used by colony PCR are the same as the above), and sending the positive colony to a sequencing company (Kuntaili sequencing company) for sequencing to obtain three specific sequences with nucleotide sequences shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No. 3;
7. BLAST analysis of specific sequences and design of SCAR marker primers
The obtained three specific sequences were subjected to BLAST analysis in GeneBank database of NCBI (national Center of Biotechnology information), and no more homologous sequence was found except for the three sequences corresponding to Chinese hamster (Access number: XR-004770315.1, NW-003623089.1, XM-003515610.5), indicating that these three sequences are CHO cell-specific sequences and can be used as SCAR markers, respectively designated as SA-SCAR, SB-SCAR1, and SB-SCAR 2;
wherein, the nucleotide sequence SEQ ID No.1 of the SA-SCAR is:
TCCCGTTCAAGATGGAGGCGCAGGGGTTCGAGGAGGGAAGGTCCAGAACTCTGTATTTCTGAGTGGTTTCCTAAGGTGACCCTATCACTCAGCCATGGTGGAGAGCCAGAGCCCTGCTCGGCTGTATGTACTGAAGACCACCATGGCTCCTCTCTTTCCTATCCTGTCCTGGCCAATGCCAGGCAATCTCGGCATGTAAGCTGTTTCCT；
the nucleotide sequence SEQ ID No.2 of the SB-SCAR1 is:
TCATGACAAGGAGCCAATCGGAAGTTAGCTGGTGGCACTATGCTTTACGACCCTGGGTGTGCTTTACGGACAAGCGCACGACAATGACGTAGAGAGCATAGCAACCACCCTGGGAGGGCCTATGAGCCATAACAACCAGTTGACCAATCAACACAGGGCAAGCCCTCCAAGCCTGGAGGCACACCAATAGTAAGCCTGTGCGTACCCCTAGACCCTCCCCTTACGCTGCCCTATAAGATCT；
the nucleotide sequence SEQ ID No.3 of the SB-SCAR2 is:
TTGCCCTCACCTTACCGATGAAGACACTGAGCCTTAGGTGTGATAGTGACTTACAGTTTTATTTTATTTTTTAAAGATTTTATTTATTTATTATGTATACAACATTCTGCTTCCATGTATATCTGCATACCAGAAGAGGGCACCAGATCTCATAACGGATGGTTGTGAGCCACCATGTGGTTGCTGGGAATTGAACTCAGGACCTCTGGAAGAGCAGCCGGTGCTCTTAACCTCTGAGCCATCTCTCCAGCCCTACAGTTTTCTTAAAAAATCAGAACAGTGAGACATGCATAGATCTAATGAATAGATCTACCCCAGGAAGGGAAATAGACAGGATCTCCTCAGTAAATTGAGAGCATGGAGGGCGGGGGAGAGCGGAAGGGGAGAGGGGAGGAGGGAAGGGGAGAGGCAATGGTTAGTGCAA；
three pairs of primers are respectively designed according to the nucleotide sequences of SA-SCAR, SB-SCAR1 and SB-SCAR2, and are as follows:
the nucleotide sequence of the primer pair of the SA-SCAR is as follows:
SA-SCAR-F：TCCCGTTCAAGATGGAGGCGC；
SA-SCAR-R：AGGAAACAGCTTACATGCCGA；
the nucleotide sequence of the primer pair of the SB-SCAR1 is as follows:
SB-SCAR-F1：TCATGACAAGGAGCCAATCGG；
SB-SCAR-R1：AGATCTTATAGGGCAGCGTAAG；
the nucleotide sequence of the primer pair of the SB-SCAR2 is as follows:
SB-SCAR-F2：TTGCCCTCACCTTACCGATG；
SB-SCAR-R2：TTGCACTAACCATTGCCTC；
8. search for optimal annealing temperature of SCAR primer
Three pairs of SCAR primers are used for carrying out PCR amplification on CHO cells and control cells BHK-21, the specific reaction system is the same as above, and the specific PCR program is as follows:
electrophoresis detection is carried out on the amplification products of the three pairs of SCAR primers, as shown in figures 3-5, and it can be seen that when the annealing temperature is 60 ℃, compared with the BHK-21 cell, the products amplified by the CHO cell with the SCAR primers show specific bands.
When the SCAR marker is used for identifying the CHO cells, the identification method comprises the following steps:
1) extracting DNA from a sample to be identified;
2) taking the extracted sample DNA as a template, and carrying out PCR amplification by adopting a primer pair of SA-SCAR, SB-SCAR1 or SB-SCAR 2;
the PCR reaction system is the same as above;
the PCR reaction conditions are as follows: 4min at 95 ℃; ② 95 ℃ for 15 s; ③ 60 ℃ for 15 s; fourthly, 15s at 72 ℃; repeating the steps from the second step to the fourth step for 35 cycles; fifthly, 5min at 72 ℃; store at 16 deg.C;
3) carrying out electrophoretic analysis on the obtained PCR amplification product; if the sample to be identified is amplified by using the primers of the SA-SCAR, the SB-SCAR1 or the SCAR2, if the electrophoretic band of the amplification product is the same as that of a standard CHO cell, the sample to be identified is the CHO cell, otherwise, the sample is not.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Wuhan Jia Chuang Biotech shares Ltd
<120> SCAR marker for identifying CHO cells, and construction method and application thereof
<130> 2020
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gagtggtttc ctaaggtgac cctatcactc agccatggtg gagagccaga gccctgctcg 120
gctgtatgta ctgaagacca ccatggctcc tctctttcct atcctgtcct ggccaatgcc 180
aggcaatctc ggcatgtaag ctgtttcct 209
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tcatgacaag gagccaatcg gaagttagct ggtggcacta tgctttacga ccctgggtgt 60
gctttacgga caagcgcacg acaatgacgt agagagcata gcaaccaccc tgggagggcc 120
tatgagccat aacaaccagt tgaccaatca acacagggca agccctccaa gcctggaggc 180
acaccaatag taagcctgtg cgtaccccta gaccctcccc ttacgctgcc ctataagatc 240
t 241
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ttgccctcac cttaccgatg aagacactga gccttaggtg tgatagtgac ttacagtttt 60
attttatttt ttaaagattt tatttattta ttatgtatac aacattctgc ttccatgtat 120
atctgcatac cagaagaggg caccagatct cataacggat ggttgtgagc caccatgtgg 180
ttgctgggaa ttgaactcag gacctctgga agagcagccg gtgctcttaa cctctgagcc 240
atctctccag ccctacagtt ttcttaaaaa atcagaacag tgagacatgc atagatctaa 300
tgaatagatc taccccagga agggaaatag acaggatctc ctcagtaaat tgagagcatg 360
gagggcgggg gagagcggaa ggggagaggg gaggagggaa ggggagaggc aatggttagt 420
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ttgccctcac cttaccgatg 20
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Claims (10)
1. A SCAR marker for identifying CHO cells, characterized by: the SCAR marker comprises any one of SA-SCAR, SB-SCAR1 and SB-SCAR 2; wherein the content of the first and second substances,
the nucleotide sequence of the SA-SCAR is shown as SEQ ID No. 1;
the nucleotide sequence of the SB-SCAR1 is shown as SEQ ID No. 2;
the nucleotide sequence of the SB-SCAR2 is shown in SEQ ID No. 3.
2. A SCAR marker as claimed in claim 1, characterized in that:
the nucleotide sequence of the primer pair of the SA-SCAR is as follows:
SA-SCAR-F：TCCCGTTCAAGATGGAGGCGC；
SA-SCAR-R：AGGAAACAGCTTACATGCCGA；
the nucleotide sequence of the primer pair of the SB-SCAR1 is as follows:
SB-SCAR-F1：TCATGACAAGGAGCCAATCGG；
SB-SCAR-R1：AGATCTTATAGGGCAGCGTAAG；
the nucleotide sequence of the primer pair of the SB-SCAR2 is as follows:
SB-SCAR-F2：TTGCCCTCACCTTACCGATG；
SB-SCAR-R2：TTGCACTAACCATTGCCTC。
3. a method of constructing a scarr marker for the identification of CHO cells as claimed in claim 1 or 2, comprising the steps of:
1) separately collecting CHO cells and BHK-21 cells, and extracting DNA;
2) respectively adopting RAPD primer SA and RAPD primer SB to perform PCR amplification on the extracted DNA of the two cells;
3) respectively carrying out electrophoresis detection on the amplification products of the DNA of the two cells; compared with the cell BHK-21, a specific band appears in a product amplified by the CHO cell by the RAPD primer SA, and two specific bands appear in a product amplified by the CHO cell by the RAPD primer SB;
4) recovering and purifying the three specific bands obtained in the step 3), and sequencing to obtain nucleotide sequences of SA-SCAR, SB-SCAR1 and SB-SCAR2 respectively;
5) respectively designing three pairs of primers according to the nucleotide sequences of the SA-SCAR, the SB-SCAR1 and the SB-SCAR2 obtained in the step 4);
6) respectively adopting the three pairs of primers designed in the step 5) to carry out PCR amplification on the CHO cells and the BHK-21 cells.
4. The method of construction of claim 3, wherein: in the step 2), the nucleotide sequence of the RAPD primer SA is as follows: GAAACGGGTG, respectively; the nucleotide sequence of the RAPD primer SB is as follows: TGGGGGACTC are provided.
5. The method of construction of claim 3, wherein: in the step 2), the PCR reaction system of RAPD amplification is as follows: 2X Rapid taq Mix 10ul, 10uM primer 1ul, 100ng/ul template 2ul, water 7 ul.
6. The method of construction of claim 3, wherein: in the step 2), the PCR reaction conditions of RAPD amplification are as follows: 4min at 95 ℃; ② 95 ℃ for 15 s; ③ 37.5 ℃ for 15 s; fourthly, 1min at 72 ℃; repeating the steps from the second step to the fourth step for 35 cycles; fifthly, 5min at 72 ℃; store at 16 deg.C.
7. The method of construction of claim 3, wherein: in the step 6), when the primer pairs of SA-SCAR, SB-SCAR1 and SB-SCAR2 are adopted for amplification, the PCR reaction conditions are as follows: 4min at 95 ℃; ② 95 ℃ for 15 s; ③ 56 ℃/58 ℃/60 ℃ for 15 s; fourthly, 15s at 72 ℃; repeating the steps from the second step to the fourth step for 35 cycles; fifthly, 5min at 72 ℃; store at 16 deg.C.
8. Use of a SCAR marker for the identification of CHO cells according to claim 1 or 2 for the identification of CHO cells.
9. The use of claim 8, wherein the authentication method comprises the steps of:
1) extracting DNA from a sample to be identified;
2) taking the extracted sample DNA as a template, and carrying out PCR amplification by adopting a primer pair of SA-SCAR, SB-SCAR1 or SB-SCAR 2;
3) carrying out electrophoretic analysis on the obtained PCR amplification product; if the sample to be identified is amplified by using the primers of the SA-SCAR, the SB-SCAR1 or the SCAR2, if the electrophoretic band of the amplification product is the same as that of a standard CHO cell, the sample to be identified is the CHO cell, otherwise, the sample is not.
10. The use of claim 9, wherein: in the step 2), when the primer pairs of SA-SCAR, SB-SCAR1 and SB-SCAR2 are adopted for amplification, the PCR reaction conditions are as follows: 4min at 95 ℃; ② 95 ℃ for 15 s; ③ 60 ℃ for 15 s; fourthly, 15s at 72 ℃; repeating the steps from the second step to the fourth step for 35 cycles; fifthly, 5min at 72 ℃; store at 16 deg.C.
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