CN111705084B - Construction method of mouse B cell lymphoma cell line stably expressing luciferase and knocking out mouse CD20 from human CD20 - Google Patents
Construction method of mouse B cell lymphoma cell line stably expressing luciferase and knocking out mouse CD20 from human CD20 Download PDFInfo
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Abstract
The invention relates to a method for constructing a mouse B cell lymphoma A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line stably expressing Luciferase and human-derived CD 20. The cell line utilizes electric transformation to co-transform a transgenic plasmid with Luciferase Luciferase and human CD20 genes and sgRNA knocked out by mouse CD20 genes into an A20 cell line, so that an A20 cell line which stably expresses human CD20 and Luciferase and simultaneously knocks out the mouse-derived CD20 genes of the cell line is obtained. Through screening Neo resistance elements on transgenic plasmids, stable passaged monoclonals are obtained, stable expression of luciferase and human CD20 genes is realized, and mouse CD20 genes are knocked out. The cell strain provides a measurable and visualized model for the in-vivo efficacy evaluation of the CD 20-targeted drug in mice.
Description
Technical Field
The invention belongs to the technical field of cell engineering, and relates to construction of a mouse B-cell lymphoma A20 cell line, in particular to a construction method of a mouse B-cell lymphoma A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line stably expressing Luciferase and knocking out mouse CD20 by human CD20 based on a gene editing technology.
Background
Lymphoma is one of the most common ten malignant tumors occurring in the world of the lymphatic system, and the incidence rate of lymphoma also shows a situation rising year by year in China, which is ten times higher than the incidence rate of malignant tumors, and the mortality rate is higher than that of prostate. At present, the clinical lymphomas have the characteristics of complex classification and high heterogeneity, are mainly classified into non-Hodgkin lymphoma (diffuse B cell lymphoma, mantle cell lymphoma and indolent lymphoma) and Hodgkin lymphoma, and the clinical treatment strategies of different types of lymphomas are different, wherein CD20 is used as a transmembrane protein expressed on the surface of malignant B lymphocytes and participates in the activation and differentiation of the B cells. Because CD20 is not internalized or down-regulated upon antibody binding, and is not expressed in precursor B cells and antibody-producing plasma cells, it does not affect normal B cell function, and thus is an ideal target for the clinical treatment of B cell non-hodgkin's lymphoma.
The monoclonal antibody rituximab targeting CD20 was the first human murine chimeric antibody approved for clinical B-cell non-hodgkin's lymphoma treatment, used alone or in combination with chemotherapy, opening a new model for lymphoma immunotherapy. It exerts a tumor killing effect mainly through antibody-dependent cytotoxicity ADCC, complement-dependent cytotoxicity CDC, etc. via NK cells and complement pathways. Meanwhile, most of the second-generation targeting CD20 antibodies such as ofatumumab, ocrelizumab, veltuzumab and the like are in the clinical research and early development stages, and the higher-level humanized structure (90-100%) of the second-generation targeting CD20 antibody reduces the toxic and side effects caused by higher immunogenicity of the first-generation antibody, and has wide application prospects in clinical treatment of lymphoma. Bispecific antibodies based on CD3 and CD20 are also an important direction for the development of CD20 targets, mosonetuzumab and CD20-TCB, which are currently in clinical evaluation, show phenotypically good efficacy in the treatment of relapsed and refractory B-cell non-hodgkin lymphoma, while also increasing the clinical patient response to CAR-T therapy. The bispecific antibody based on CD3-CD20 can be used as a tie to recognize CD3 molecules expressed on the surface of T cells and recognize CD20 antigen molecules expressed on the surface of B cell lymphoma cells, and stimulate the specific killing effect of the T cells on CD20 positive lymphoma cells.
The existing preclinical animal tumor modeling model cannot meet the requirement of having a healthy immune system and can directly evaluate the anti-tumor effect of a CD20 human antibody acting on human CD20 protein. A20 cell model which stably expresses human CD20 knockout mouse CD20 gene modification is constructed, and a BALB/c wild-type mouse and a BALB/c humanized hCD3E mouse are combined to be used for drug development of a targeted CD20 antibody and a CD3-CD20 bispecific antibody. In addition, by over-expressing luciferase in A20 cells stably expressing human CD20 knockout mouse CD20 gene modification, the growth and metastasis conditions of tumor cells in animals can be observed by virtue of a living body imaging system, and a visual model is provided for the anti-tumor effect evaluation of the medicine. Therefore, the construction of a20 cell line based on the mouse B cell lymphoma A20-hCD20(Tg) -mCD20(KO) -Luciferase stably expressing Luciferase and a human CD20 knockout murine CD20 combined with a humanized mouse can make up for the deletion of a preclinical evaluation model of a bispecific antibody drug targeting CD20 and CD3-CD20 at home and abroad.
Disclosure of Invention
In order to solve the defects of animal tumor models before clinic in the prior art, the application provides a model for constructing an A20 cell model which stably expresses human CD20 and luciferase and is modified by knocking out mouse CD20 gene, the cell model can be combined with a BALB/c wild type mouse and a BALB/c humanized hCD3E mouse to be used for evaluating the drug effect of a targeting CD20 antibody and a CD3-CD20 bispecific antibody, and meanwhile, the growth transfer condition of tumor cells in an animal body can be observed by virtue of a living body imaging system, so that a model which can be used for subcutaneous measurement and in-vivo transfer visualization is provided for evaluating the drug effect in the mouse body of a drug taking CD20 as a target spot.
In order to solve the technical problems, the invention provides a preparation method of a mouse B cell lymphoma A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line stably expressing Luciferase and human CD20 knockout mouse CD20, which is characterized by comprising the following steps of: a transgenic plasmid with Luciferase Luciferase and human CD20 gene and sgRNA knocked out by mouse CD20 gene are co-transformed into A20 cells by electric transformation, so that an A20 cell line which stably expresses human CD20 and Luciferase and simultaneously knocks out the mouse CD20 gene of the cell line is obtained.
Further, the preparation method of the A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line also comprises a cell line identification step; still further, the cell line identification step comprises Luciferase activity identification and/or mRNA detection of Luciferase.
Preferably, in the preparation method of the A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line, a Neo element is added into the transgenic plasmid and used for cell drug screening.
In one embodiment, the method for preparing a cell line a20-hCD20(Tg) -mCD20(KO) -Luciferase of the present invention specifically comprises the following steps:
1) constructing a transgenic plasmid for stably expressing Luciferase and human-derived CD 20;
2) sgRNA was constructed for murine CD20 knockdown: designing sgRNAs at the 5 'end of exon1 and the 3' end of exon7 of CD20 gene of A20 cells;
3) A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line construction: co-transforming the transgenic plasmid obtained in the step 1) and the sgRNA obtained in the step 2) into an A20 cell line;
4) A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line identification screening.
Further, in step 1), a CDs sequence of the human CD20 gene (SEQ ID NO: 1) and the Luciferase gene (SEQ ID NO: 2) constructing transgenic plasmid.
Further, in the step 1), transgenic plasmids which stably express Luciferase Luciferase and human-derived CD20 are constructed and screened through HindIII + ASC1 enzyme digestion, high-fidelity enzyme PCR amplification, sLIC ligation, plasmid PCR identification and Not1 linearization enzyme digestion identification.
Preferably, in step 1), the high-fidelity enzymatic PCR amplification primers are:
GTAAAACGACGGCCAGTGCCTAGGTCCCTCGAGGGGATCCACGAATTCCTGCAGCCTAATGTGTGGGCGTGGGGACCCCTAAGTATAGT（GPT000292-3-pro-F, SEQ ID NO：3）；
GGTGGCGGCCGGAGTTCAGTGGGTGCAGT
（GPT000292-3-pro-R, SEQ ID NO：4）；
ACTGAACTCCGGCCGCCACCATGACAACACCCAGAAATTC（GPT000292-3-CD20-F, SEQ ID NO：5）；
GCAGCCTGCACCTGAGGAGTTCACAATTTGGACTTTCCGC（GPT000292-3-CD20-R, SEQ ID NO：6）。
preferably, in step 1), the plasmid PCR identification primers are:
GCCAGGGTTTTCCCAGTCACGA（M13F，SEQ ID NO：7）；
GGTGCATAGATCCCTGCTGG（GPT000292-3-seqR1，SEQ ID NO：8）；
CGAAGGTTGTGGATCTGGAT（GPT000292-3-seqF1，SEQ ID NO：9）；
ATGTTGCCAAACTCTAAACC（GPT000292-3-seqR2，SEQ ID NO：10）。
in a specific embodiment, the step 2) comprises synthesizing an upstream primer and a downstream primer of the sgRNA, annealing to form a double strand, connecting and transforming with a Bsa I digested pUC57kan-T7-delG vector, identifying by PCR, further sequencing the PCR positive single clone, PCR amplifying a DNA product transcribed by the sgRNA by taking a correctly sequenced clone as a template, and transcribing the sgRNA by taking the DNA product as a template.
Further, in step 2), the upstream and downstream primers of the sgRNA are selected from:
GPT000292-3-CD20-5S1：
ATAGTAGGCTCTGCTGGGAA（GPT000292-3-CD20-5S1-F，SEQ ID NO：11）；
AAACTTCCCAGCAGAGCCTA（GPT000292-3-CD20-5S1-R，SEQ ID NO：12）；
GPT000292-3-CD20-5S2：
ATAGGAGCAGGTTGCATGGCG（GPT000292-3-CD20-5S2-F，SEQ ID NO：13）；
AAACCGCCATGCAACCTGCTC（GPT000292-3-CD20-5S2-R，SEQ ID NO：14）；
GPT000292-3-CD20-3S1：
ATAGGAGCGATCTCATTTTCCAC（GPT000292-3-CD20-3S1-F，SEQ ID NO：15）；
AAACGTGGAAAATGAGATCGCTC（GPT000292-3-CD20-3S1-R，SEQ ID NO：16）；
GPT000292-3-CD20-3S2：
ATAGGCAAGGATTCCTGCTCTT（GPT000292-3-CD20-3S2-F，SEQ ID NO：17）；
AAACAAGAGCAGGAATCCTTGC（GPT000292-3-CD20-3S2-R，SEQ ID NO：18）。
further, in step 2), the sequence of the PCR identification and sequencing primer is as follows: TTGTACTGAGAGTGCACCATATG (pUC 57-T7-F, SEQ ID NO: 19);
further, in step 2), in PCR amplifying the DNA product transcribed from the sgRNA using the clone with correct sequencing as a template, the PCR amplification primer is TCTCGCGCGTTTCGGTGATGACGG (sgRNA-universal-PCR-F, SEQ ID NO: 20) (ii) a AAAAAAAGCACCGACTCGGTGCCACTTTTTC (sgRNA-univarial-PCR-R, SEQ ID NO: 21).
Further, step 3) also comprises Neo-resistant G418 pressure screening and monoclonal selection.
Preferably, the working concentration for Neo-resistant G418 pressure screening is 300-500. mu.g/mL, more preferably 400. mu.g/mL.
In one embodiment, the step 4) a20-hCD20(Tg) -mCD20(KO) -Luciferase cell line identification includes Luciferase activity identification and mRNA detection.
In a specific embodiment, in the step 4) of mRNA detection, the primer sequences for identifying the human CD20 and luciferase are:
5’connector：
F1: CTGTCACCTGATGTCTATCAGCG（hCD20-tF1，SEQ ID NO：22）；
R1: GCCCATTCATAATCTGGACAGC（GPT000292-1-hCD20-tR1，SEQ ID NO：23）。
3’connector：
F2: AAGAGATCGTGGATTACGTCGC（GPT000292-3-luciferase-tF1，SEQ ID NO：24）；
R2：CTTTGTTCATGGCAGCCAGC（Rabbit-PloyA-TR，SEQ ID NO：25）。
internal reference:
CTAGGCCACAGAATTGAAAGATCT（42，SEQ ID NO：26）；
GTAGGTGGAAATTCTAGCATCATCC（43，SEQ ID NO：27）。
in a specific embodiment, in the step 4) of detecting mRNA, the primer sequence for identifying the knockout murine CD20 gene is as follows:
KO：
F1：ATGTGCCCTTGCCTGAGATAG（GPT000292-3-mCD20-KO-tF1，SEQ ID NO：28）；
R1：GCGTTTCTGCCAGTGTAAGACTC（GPT000292-3-mCD20-KO-tR1，SEQ ID NO：29）。
WT：
F2：CGAGACATACAGCCACTTTCTCAG（GPT000292-3-mCD20-wt-tF1，SEQ ID NO：30）；
R2：ATGCAACCCAAGCATCTGGAC（GPT000292-3-mCD20-wt-tR1，SEQ ID NO：31）。
in a preferred embodiment, in step 4) mRNA detection, the sequencing primer sequence is ATGTGCCCTTGCCTGAGATAG (SEQ ID NO: 32).
The invention also provides a mouse B cell lymphoma A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line which is prepared by the method and can stably express Luciferase and human-derived CD20 and knock out mouse-derived CD 20.
The invention also provides a method for evaluating the drug effect of a bispecific antibody targeting CD20 or CD3-CD20, which is characterized in that the drug effect of the drug is evaluated by the A20-hCD20(Tg) -mCD20(KO) -Luciferase A20 cells constructed by the method.
The invention also provides a method for screening bispecific antibody drugs targeting CD20 or CD3-CD20, which is characterized in that the A20-hCD20(Tg) -mCD20(KO) -Luciferase A20 cells constructed by the method are used for screening the drugs.
Compared with the prior art, the invention has the beneficial effects that:
1) the animal tumor model used in the prior art before clinic can not meet the requirements of having a healthy immune system and directly evaluating the anti-tumor effect of a CD20 human antibody acting on human CD20 protein, but the mouse cell model constructed by the method of the invention is adopted: can stably express human CD20 and knock out mouse CD20 gene;
2) the method of the invention determines the suitable plasmid PCR amplification primer and identification primer, obtains excellent specificity, and can efficiently screen and identify the plasmid; and a proper sgRNA for knocking out mouse-derived CD20 is constructed, so that the knocking-out rate of mouse-derived CD20 is improved;
3) a20 cell model which is stably expressed and is constructed by the method and is knockout of mouse-derived CD20 gene modification is combined with a BALB/c wild-type mouse and a BALB/c humanized hCD3E mouse, so that the model can be used for evaluating the drug effect of drugs targeting a CD20 antibody and a CD3-CD20 bispecific antibody;
4) by over-expressing luciferase in A20 cells stably expressing human-derived CD20 knockout mouse-derived CD20 gene modification, the growth and metastasis conditions of tumor cells in animal bodies can be observed by means of a living body imaging system, and a visual model is provided for the evaluation of the anti-tumor effect of the medicine.
Description of the drawings:
FIG. 1 construction strategy of plasmid for stably expressing luciferase and human-derived CD20
FIG. 2 GPT000292-3-dsDNA-hCD20-TG plasmid map
FIG. 3 identification of the GPT000292-3-dsDNA-hCD20-TG plasmid running gel map
FIG. 4 shows the running gel map of the restriction of plasmid GPT000292-3-dsDNA-hCD20-TG
FIG. 5 murine CD20 knockout strategy
FIG. 6A 20-hCD20(Tg) -mCD20(KO) -Luciferase cellular fluorescence imaging
FIG. 7A 20-hCD20(Tg) -mCD20(KO) -Luciferase cell monoclonal electrophoretogram
Detailed Description
The technical solution of the present invention is further illustrated by the following examples.
Example 1 construction of stably expressed luciferase and human-derived CD20 transgenic plasmid
A human CD20 gene CDS sequence (SEQ ID NO: 1) and a Luciferase gene (SEQ ID NO: 2) are selected to construct a transgenic vector GPT000292-3-dsDNA-hCD20-TG according to the strategy shown in figure 1, and the plasmid map is shown in figure 2.
The specific operation steps are as follows:
(1) the V42# (5170 bp/1831 bp) is cut by HindIII + ASC1 enzyme, and a 5170bp fragment is recovered;
(2) GPT000292-3-pro and GPT000292-3-CD20 fragments were amplified by high fidelity enzymatic PCR, the corresponding templates are shown in Table 1
TABLE 1 amplification primers
(3) GPT000292-3-pro, GPT000292-3-CD20 and HindIII + ASC1 digested V42# were subjected to sLIC ligation, plasmids were identified according to the primers shown in Table 2, and the plasmid identification gel images are shown in FIG. 3. The successfully identified vector is named as GPT000292-3-dsDNA-hCD 20-TG;
TABLE 2 GPT000292-3-dsDNA-hCD20-TG plasmid identification primers
(4) Not1 linearized plasmid GPT000292-3-dsDNA-hCD20-TG was digested, 8297bp fragment was recovered for electrotransfer, the digestion identification protocol of GPT000292-3-dsDNA-hCD20-TG is shown in Table 3, and the gel running map is shown in FIG. 4.
TABLE 3 restriction enzyme identification protocol for GPT000292-3-dsDNA-hCD20-TG plasmid
Example 2 construction of sgRNA for murine CD20 knock-out
In order to prevent homology regions of human CD20 and CD20 of a mouse cell line, sgRNA is designed to avoid exons of genes, and to improve knockout efficiency, the sgRNA cuts donor, the sgRNA is designed at the 5 'end of exon1 and the 3' end of exon7, and cell clones with frame shift mutation are screened, wherein a murine CD20 knockout strategy is shown in FIG. 5.
According to the CRISPR Cas9 technology, 2 sgRNAs are designed at the 5 'end of murine CD20 exon1 and the 3' end of exon7 respectively. The method comprises the following specific steps:
(1) respectively synthesizing an upstream primer and a downstream primer of the sgRNA, wherein the purification mode of the primers is PAGE, and the sequences of the primers are shown in a table 4;
TABLE 4 sgRNA construction primer Table
(2) Respectively diluting the upstream and downstream primers of the sgRNA to 100 mu mol/mu L, uniformly mixing according to the proportion of 1:1, reacting at 95 ℃ for 5min, and automatically and slowly annealing at room temperature; (3) connecting the double chains formed by annealing with a pUC57Kan-T7-delG vector digested by Bsa I for 2h, transforming and coating a Kan + plate;
(4) selecting a monoclonal, and carrying out PCR identification, wherein the PCR identification scheme is shown in Table 5;
table 5 sgRNA PCR identification protocol
(5) The PCR positive single clone is further sequenced and confirmed, and the sequencing primer is pUC 57-T7-F;
(6) using a clone with correct sequencing as a template, and performing PCR amplification on a DNA product transcribed by the sgRNA by using a primer of table 6;
table 6 sgRNA transcription template PCR preparation
(7) And (3) transcribing the sgRNA by using a DNA product transcribed by the sgRNA as a template according to the instruction of a transcription kit, and further purifying.
Example 3A 20-hCD20(Tg) -mCD20(KO) -Luciferase cell line construction and identification
1. Construction of A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line
(1) Determination of optimal working concentration for G418 resistance screening
A20 cell culture conditions were 1640 medium containing 10% fetal bovine serum, 37 deg.C, 5% CO2(ii) a Taking A20 cells in logarithmic growth phase, adjusting cell concentration to 1 × 105/mL，1×105Each cell was inoculated into 24-well plates and cultured overnight, and Neo-resistant G418 diluted at a multiple ratio to give 9 gradients of 0-800. mu.g/mL were added to 24-well plates for resistance pressure screening, with 1 replicate well for each concentration. The growth of A20 cells was then observed, and the lowest G418 concentration that caused total cell death within 5-7 days was the optimal working concentration of 400. mu.g/mL for screening transfected A20 cells.
(2) A20 cell transfection
Taking A20 cells in logarithmic phase, inoculating the cells in 10-cm dish 1 day before transfection, and carrying out transfection when the cell fusion degree reaches 60-80% in 24 hours; transfection the transgenic plasmid with Luciferase and human CD20 gene and sgRNA knocked out from mouse CD20 gene were transfected into A20 cells according to the instructions of LONZA electrotransformation apparatus (I/II/2 b); after transfection, placing the cell in a cell culture box for continuous culture;
(3) a20 transfected cell resistance stress screening and monoclonal selection
Selecting untransfected A20 cells with the same passage density as a control 48 hours after transfection of the plasmid and the sgRNA, adding Neo resistant G418, and periodically changing the liquid; after the untransfected A20 cells all died, the A20 cells with successful transfection were selected. Resuspending the A20 cells subjected to resistance screening into a single cell suspension, obtaining single clone cells in 96 wells by a limiting dilution method, and continuing amplification culture to obtain the mouse B cell lymphoma A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line stably expressing Luciferase and human-derived CD 20.
2. Identification of A20-hCD20(Tg) -mCD20(KO) -Luciferase cell line
(1) And (3) luciferase activity identification:
the cell concentrations of A20-hCD20(Tg) -mCD20(KO) -Luciferase were 5.0X 106Each 1 mL of the solution is added into a white board with 24 holes, and each group is provided with 2 compound holes; adding 10 μ L of 0.15 mg/mL fluorescein substrate into each well, incubating at 37 deg.C for 7min, and measuring fluorescence value with a fluorometer; the fluorescence intensity of each clone was compared, and the correlation between the fluorescence intensity and the number of cells was analyzed. The optimal working concentration for G418 resistance to the A20 cell line was 400. mu.g/mL. The cell clone markers are shown in Table 7, and the results of measuring the fluorescence intensity are shown in Table 8 and FIG. 7. The 2E12, E11, F8, G8 and 2H7 cell monoclonals are selected according to fluorescence intensity.
TABLE 7A 20-hCD20(Tg) -mCD20(KO) -Luciferase cell monoclonal plating
TABLE 8A 20-hCD20(Tg) -mCD20(KO) -Luciferase cellular fluorescence imaging readings
(2) Stably expressing human CD20 and luciferase in monoclonal cells are further screened through mRNA detection, and meanwhile, the clone of mouse CD20 gene is knocked out, and specific identification primer information is shown in tables 9 and 10; the sequencing primer information was ATGTGCCCTTGCCTGAGATAG (SEQ ID NO: 32).
Table 9 identification of the primer sequences for expressing human CD20 and luciferase
TABLE 10 identification of knockout mouse self CD20 Gene primer sequences
2F10, E12, F10, 3F10, 3G8, H7, 3E12, E10, 2G9, 2G8, 2E12, G11, 3E11, G10, G9, D12, 2E11, G8, E11, H10 and F8 are positive and the rest are negative through PCR and sequencing. The result of detecting mCD20 KO shows that 2F10, 3F10, 3E12, 3E11, G10 and E11 clone KO is successful, and the rest are negative. Cell monoclonal identification electrophoresis results are shown in fig. 7, and it can be determined that clones 2F10, 3F10, 3E12, 3E11, G10 and E11 can be used in subsequent experiments. The E11 clone can be used for the subsequent in vivo efficacy evaluation of the CD20 and CD3-CD20 bispecific antibody.
From the identification, the invention successfully constructs an A20 cell model capable of stably expressing luciferase and a human-derived CD20 gene knockout murine CD20 gene, can be combined with a BALB/c wild-type mouse and a BALB/c humanized hCD3E mouse, can be used for drug development of a targeted CD20 antibody and a CD3-CD20 bispecific antibody, and can be used as a model for subcutaneous measurable and in vivo metastasis visualization.
In addition, the present invention is not limited to the embodiments disclosed in the specification and the embodiments, and any combination may be made between various embodiments of the present invention as long as it does not depart from the spirit of the present invention, which should also be construed as disclosed in the present invention, and equivalent embodiments that can be directly obtained by those skilled in the art from the embodiments disclosed in the specification are also within the present invention.
Sequence listing
<110> Jiangsu Jiejiaokang Biotech limited
<120> method for constructing mouse B cell lymphoma cell line stably expressing luciferase and human CD20 knockout mouse CD20
<141> 2020-08-02
<160> 32
<170> SIPOSequenceListing 1.0
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acgcaaagct tcttcatgag ggaatctaag actttggggg ctgtccagat tatgaatggg 180
ctcttccaca ttgccctggg gggtcttctg atgatcccag cagggatcta tgcacccatc 240
tgtgtgactg tgtggtaccc tctctgggga ggcattatgt atattatttc cggatcactc 300
ctggcagcaa cggagaaaaa ctccaggaag tgtttggtca aaggaaaaat gataatgaat 360
tcattgagcc tctttgctgc catttctgga atgattcttt caatcatgga catacttaat 420
attaaaattt cccatttttt aaaaatggag agtctgaatt ttattagagc tcacacacca 480
tatattaaca tatacaactg tgaaccagct aatccctctg agaaaaactc cccatctacc 540
caatactgtt acagcataca atctctgttc ttgggcattt tgtcagtgat gctgatcttt 600
gccttcttcc aggaacttgt aatagctggc atcgttgaga atgaatggaa aagaacgtgc 660
tccagaccca aatctaacat agttctcctg tcagcagaag aaaaaaaaga acagactatt 720
gaaataaaag aagaagtggt tgggctaact gaaacatctt cccaaccaaa gaatgaagaa 780
gacattgaaa ttattccaat ccaagaagag gaagaagaag aaacagagac gaactttcca 840
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agtgaaaact ctcttcaatt ctttatgccg gtgttgggcg cgttatttat cggagttgca 300
gttgcgcccg cgaacgacat ttataatgaa cgtgaattgc tcaacagtat gaacatttcg 360
cagcctaccg tagtgtttgt ttccaaaaag gggttgcaaa aaattttgaa cgtgcaaaaa 420
aaattaccaa taatccagaa aattattatc atggattcta aaacggatta ccagggattt 480
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gtaccagagt cctttgatcg tgacaaaaca attgcactga taatgaattc ctctggatct 600
actgggttac ctaagggtgt ggcccttccg catagaactg cctgcgtcag attctcgcat 660
gccagagatc ctatttttgg caatcaaatc attccggata ctgcgatttt aagtgttgtt 720
ccattccatc acggttttgg aatgtttact acactcggat atttgatatg tggatttcga 780
gtcgtcttaa tgtatagatt tgaagaagag ctgtttttac gatcccttca ggattacaaa 840
attcaaagtg cgttgctagt accaacccta ttttcattct tcgccaaaag cactctgatt 900
gacaaatacg atttatctaa tttacacgaa attgcttctg ggggcgcacc tctttcgaaa 960
gaagtcgggg aagcggttgc aaaacgcttc catcttccag ggatacgaca aggatatggg 1020
ctcactgaga ctacatcagc tattctgatt acacccgagg gggatgataa accgggcgcg 1080
gtcggtaaag ttgttccatt ttttgaagcg aaggttgtgg atctggatac cgggaaaacg 1140
ctgggcgtta atcagagagg cgaattatgt gtcagaggac ctatgattat gtccggttat 1200
gtaaacaatc cggaagcgac caacgccttg attgacaagg atggatggct acattctgga 1260
gacatagctt actgggacga agacgaacac ttcttcatag ttgaccgctt gaagtcttta 1320
attaaataca aaggatatca ggtggccccc gctgaattgg aatcgatatt gttacaacac 1380
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gtcgccagtc aagtaacaac cgcgaaaaag ttgcgcggag gagttgtgtt tgtggacgaa 1560
gtaccgaaag gtcttaccgg aaaactcgac gcaagaaaaa tcagagagat cctcataaag 1620
gccaagaagg gcggaaagtc caaattgtga 1650
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gtaaaacgac ggccagtgcc taggtccctc gaggggatcc acgaattcct gcagcctaat 60
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aaacttccca gcagagccta 20
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<213> Artificial Sequence (Artificial Sequence)
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aaacgtggaa aatgagatcg ctc 23
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<213> Artificial Sequence (Artificial Sequence)
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ataggcaagg attcctgctc tt 22
<210> 18
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
aaacaagagc aggaatcctt gc 22
<210> 19
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ttgtactgag agtgcaccat atg 23
<210> 20
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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tctcgcgcgt ttcggtgatg acgg 24
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<213> Artificial Sequence (Artificial Sequence)
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aaaaaaagca ccgactcggt gccacttttt c 31
<210> 22
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ctgtcacctg atgtctatca gcg 23
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
gcccattcat aatctggaca gc 22
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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aagagatcgt ggattacgtc gc 22
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<213> Artificial Sequence (Artificial Sequence)
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ctttgttcat ggcagccagc 20
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Claims (5)
1. A preparation method of a mouse B cell lymphoma A20-hCD20 Tg-mCD 20 KO-Luciferase cell line stably expressing Luciferase and a human-derived CD20 knockout mouse-derived CD20 comprises the following steps:
1) constructing a transgenic plasmid for stably expressing Luciferase and human-derived CD 20;
2) sgRNA was constructed for murine CD20 knockdown: designing sgRNAs at the 5 'end of exon1 and the 3' end of exon7 of CD20 gene of A20 cells;
3) A20-hCD20 Tg-mCD 20 KO-Luciferase cell line construction: co-transforming the transgenic plasmid obtained in the step 1) and the sgRNA obtained in the step 2) into an A20 cell line;
4) A20-hCD20 Tg-mCD 20 KO-Luciferase cell line identification screening;
wherein the content of the first and second substances,
in the step 1), a CDS sequence of a human CD20 gene is selected: SEQ ID NO: 1 and the Luciferase gene: SEQ ID NO: 2 constructing the transgenic plasmid; adding Neo elements into the transgenic plasmids for cell drug screening;
the specific process of the step 2) is as follows:
a. respectively synthesizing an upstream primer and a downstream primer of the sgRNA, wherein the purification mode of the primers is PAGE, and the sequences of the primers are as follows:
the 5' end is: GPT000292-3-CD20-5S1 and GPT000292-3-CD20-5S 2; wherein the primer pair of GPT000292-3-CD20-5S1 consists of GPT000292-3-CD20-5S 1-F: SEQ ID NO: 11, and GPT000292-3-CD20-5S 1-R: SEQ ID NO: 12; the primer pair of GPT000292-3-CD20-5S2 consists of GPT000292-3-CD20-5S 2-F: SEQ ID NO: 13, and GPT000292-3-CD20-5S 2-R: SEQ ID NO: 14, the composition is as follows;
the 3' end is: GPT000292-3-CD20-3S1 and GPT000292-3-CD20-3S 2; wherein the primer pair GPT000292-3-CD20-3S1 consists of GPT000292-3-CD20-3S 1-F: SEQ ID NO: 15, and GPT000292-3-CD20-3S 1-R: SEQ ID NO: 16; the primer pair GPT000292-3-CD20-3S2 consists of GPT000292-3-CD20-3S 2-F: SEQ ID NO: 17, GPT000292-3-CD20-3S 2-R: SEQ ID NO: 18;
b. respectively diluting the upstream and downstream primers of the sgRNA to 100 mu mol/mu L, uniformly mixing according to the proportion of 1:1, reacting at 95 ℃ for 5min, and automatically and slowly annealing at room temperature;
c. connecting the double chains formed by annealing with a pUC57Kan-T7-delG vector digested by Bsa I for 2h, transforming and coating a Kan + plate;
d. selecting a monoclonal, and carrying out PCR identification, wherein the PCR identification scheme is as follows:
e. the PCR positive single clone is further sequenced and confirmed, and the sequencing primer is pUC 57-T7-F;
f. using a clone with correct sequencing as a template, and performing PCR amplification on a DNA product transcribed by the sgRNA by using the following primers;
g. transcribing the sgRNA by taking a DNA product transcribed by the sgRNA as a template according to the instruction of a transcription kit, and further purifying;
constructing and screening transgenic plasmids stably expressing Luciferase and humanized CD20 by HindIII + ASC1 enzyme digestion, high fidelity enzyme PCR amplification, sLIC connection, plasmid PCR identification and Not1 linearized enzyme digestion identification in the step 1); wherein, the high-fidelity enzyme PCR amplification primer is GPT 000292-3-pro-F: SEQ ID NO: 3, GPT 000292-3-pro-R: SEQ ID NO: 4; and GPT000292-3-CD 20-F: SEQ ID NO: 5, GPT000292-3-CD 20-R: SEQ ID NO: 6.
2. the method of claim 1, wherein the cell line identification step comprises identification of Luciferase activity by Luciferase and mRNA detection.
3. The method for preparing a cell line according to any one of claims 1 or 2, wherein the method further comprises performing Neo-resistant G418 stress screening and monoclonal selection on the cotransformed A20 cells in step 3); the working concentration of the Neo-resistant G418 pressure screen was 400. mu.g/mL.
4. The method for preparing a cell line according to claim 2, wherein the sequences of primers for identifying the expression of the human CD20 and the luciferase in the mRNA detection in the step 4) are as follows:
5' end: hCD20-tF 1: SEQ ID NO: 22, GPT000292-1-hCD20-tR 1: SEQ ID NO: 23;
3' end: GPT000292-3-luciferase-tF 1: SEQ ID NO: 24, Rabbit-PloyA-TR: SEQ ID NO: 25.
5. the method for preparing the cell line according to claim 2, wherein in the step 4) of detecting mRNA, the primer sequence for identifying the knockout murine CD20 gene is as follows: GPT000292-3-mCD20-KO-tF 1: SEQ ID NO: 28, GPT000292-3-mCD20-KO-tR 1: SEQ ID NO: 29.
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Address after: No.12 Xuefu Road, Pukou high tech Industrial Development Zone, Nanjing, Jiangsu 210032 Patentee after: Jiangsu Jicui Yaokang Biotechnology Co., Ltd Address before: No.12 Xuefu Road, Pukou high tech Industrial Development Zone, Nanjing, Jiangsu 210032 Patentee before: GEMPHARMATECH Co.,Ltd. |
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