CN112105724A - Construction method of antigen presenting cell line without endogenous HLA gene background, antigen presenting cell line and application thereof - Google Patents
Construction method of antigen presenting cell line without endogenous HLA gene background, antigen presenting cell line and application thereof Download PDFInfo
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- C12N2510/00—Genetically modified cells
Abstract
Provided is a method for constructing an antigen presenting cell line without an endogenous HLA gene background, which comprises the following steps: 3 performing 3 endogenous 3 HLA 3- 3 C 3 gene 3 knockout 3 on 3 an 3 HLA 3- 3 typed 3 single 3 C 3 1 3 R 3 antigen 3 presenting 3 cell 3 by 3 using 3 a 3 CRISPR 3 / 3 Cas 3 9 3 gene 3 editing 3 system 3, 3 wherein 3 the 3 C 3 1 3 R 3 antigen 3 presenting 3 cell 3 comprises 3 HLA 3- 3 A 3, 3 HLA 3- 3 B 3 and 3 HLA 3- 3 C 3 genes 3, 3 HLA 3- 3 A 3 is 3 not 3 expressed 3, 3 and 3 HLA 3- 3 B 3 is 3 not 3 substantially 3 expressed 3; 3 And antigen presenting cell lines obtained by the method without the background of endogenous HLA genes.
Description
The invention relates to the technical field of immunity, in particular to the technical field of antigen presenting cells, and specifically relates to a construction method of an antigen presenting cell line without an endogenous HLA gene background, the antigen presenting cell line and application thereof.
In the world today, the incidence of cancer is rising year by year, and immunotherapy is considered as one of the most likely means for curing tumors. The tumor specific immune target is the key to the safety and effectiveness of immunotherapy. Traditional antibody and CAR-T therapy is directed against tumor cell surface proteins that account for only 20-25% of all possible targets of solid tumors, whereas human cell surface HLA molecules present all intracellular antigens for T cell recognition, thus targeting epitopes presented by HLA molecules can expand the target available for immunotherapy to 4-fold that of traditional approaches. Therefore, specific typing of HLA antigen presenting cells is of great importance for research of novel antigens, and how to reduce interference of other HLA molecules becomes an important issue.
The antigen presenting tool cells commonly used at present are K562 cells, and usually directly over-express the HLA molecules required to be typed in K562. The prior art has the defects that the K562 cells contain self HLA molecules and are directly over-expressed in the K562 cells, and when the K562 cells are used for antigen presentation, background interference exists, and results are false positive and the like.
Disclosure of Invention
The invention solves the problems of high background and false positive of antigen presenting cells, and provides a tool cell for the expression of background-free HLA molecules for researches related to antigen presentation and the like.
According to a first aspect, there is provided in one embodiment a method of constructing an antigen presenting cell line free of a background of endogenous HLA genes, comprising: and (3) knocking out endogenous HLA-C genes of the single HLA-typed C1R antigen presenting cell by using a CRISPR/Cas9 gene editing system, wherein the C1R antigen presenting cell contains HLA-A, HLA-B and HLA-C genes, HLA-A is not expressed, and HLA-B is not substantially expressed.
Preferably, endogenous HLA-C gene knockout is achieved by introducing a Cas9-sgRNA protein complex (RNP) into the above-described C1R antigen presenting cell, wherein the sgRNA comprises a recognition sequence GTGAACCTGCGGAAACTGCG (SEQ ID NO: 1).
Preferably, the sgRNA is obtained by in vitro transcription from an in vitro transcription template.
Preferably, the in vitro transcription template is obtained by amplifying a plasmid containing a gRNA framework by PCR, wherein primers used for PCR amplification include the recognition sequence SEQ ID NO: 1 and T7 promoter sequences.
Preferably, the above method comprises:
(1) obtaining an in vitro transcription template of sgRNA of a target HLA-C gene by PCR amplification, wherein primers amplified by the PCR comprise:
HLA-C0401 gRNA FW：
TAATACGACTCACTATAGTGAACCTGCGGAAACTGCGGTTTTAGAGCTAGAAATAGC (SEQ ID NO: 2); and
gRNA scafford RV：AGCACCGACTCGGTGCCACT(SEQ ID NO：3)；
the template used for PCR amplification comprises a gRNA framework;
(2) carrying out in vitro transcription on the in vitro transcription template obtained in the step (1) by using T7RNA polymerase to synthesize sgRNA;
(3) embedding the sgRNA obtained in the step (2) and Cas9 protein into a Cas9-sgRNA protein complex (RNP), introducing the Cas9-sgRNA protein complex into the C1R antigen presenting cell, and culturing and sorting to obtain the antigen presenting cell line without the endogenous HLA gene background, wherein the HLA-C gene is knocked out.
Preferably, the template used for PCR amplification in step (1) above is a pMD 19-T plasmid containing a gRNA backbone.
Preferably, the Cas9-sgRNA protein complex is introduced into the C1R antigen presenting cell by means of electrical transformation in the step (3).
Preferably, the antigen presenting cell line without the background of the endogenous HLA gene is sorted using a flow cytometer in step (3) above.
According to a second aspect, there is provided in one embodiment an antigen presenting cell line without a background of endogenous HLA genes constructed according to the method of the first aspect.
Preferably, the HLA-C gene in the above antigen presenting cell line is subjected to frame shift mutation due to insertion or deletion.
According to a third aspect, there is provided in one embodiment the use of the antigen presenting cell line of the second aspect without the background of endogenous HLA genes as a tool cell in antigen research or immunotherapy.
The antigen presenting cell line without the background of the endogenous HLA gene obtained by the method removes the interference of the endogenous HLA molecule in the antigen presenting cell C1R, and can effectively reduce the background, improve the accuracy and obtain a credible result in the research of new antigens and the like. In addition, on the basis of the antigen presenting cell line, HLA molecules needing typing can be over-expressed, and target cells can be rapidly obtained.
FIG. 1 is a schematic diagram of the strategy for constructing an antigen presenting cell line without the background of endogenous HLA genes in the example of the present invention;
fig. 2 is a schematic diagram of a template plasmid map and an in vitro transcription template of sgRNA targeting HLA-C0401 molecules obtained by PCR in the embodiment of the present invention, and an in vitro transcription template of the sgRNA of HLA-C gene is synthesized by specific primer PCR, wherein the upstream of the product contains a T7 promoter, and the downstream thereof contains a gRNA backbone (scaffold);
FIG. 3 is a diagram showing the result of electrophoresis of T7E1 for detecting editing efficiency after C1R cells are knocked out by transferring Cas9-sgRNA protein complex (RNP) in the example of the present invention, wherein the lanes from left to right are a DNA marker, a control group without sgRNA (3. mu.g of Cas9 protein only), and an experimental group (3. mu.g of Cas9 protein plus 2. mu.g of sgRNA);
FIG. 4 is a diagram showing the sequencing results of sanger sequencing detection editing efficiency after C1R cells are knocked out by electrotransfer RNP in the example of the present invention, and shows the sequencing results of three clones of control, 8-base deletion and 11-base deletion;
FIG. 5 is a graph showing the results of various insertion deletions (indels) of various clones of C1R cells after editing their HLA sites in the present example;
FIG. 6 is a graph showing HLA-A, B, C antibody staining results after HLA-C knockout of C1R cells of different clones in examples of the present invention;
FIG. 7 is a graph showing HLA-C knockdown results of different clones of C1R cells using HLA-C antibody in the present example.
The present invention will be described in further detail with reference to the following detailed description and accompanying drawings. In the following description, numerous details are set forth in order to provide a better understanding of the present application. However, those skilled in the art will readily recognize that some of the features may be omitted or replaced by other materials, methods, or the like in various circumstances.
Referring to FIG. 1, in one embodiment of the present invention, the antigen presenting cell line without the background of endogenous HLA genes is constructed by HLA-typed single C1R antigen presenting cells containing HLA-A, HLA-B and HLA-C genes, wherein HLA-A is not expressed and HLA-B is not substantially expressed, i.e., no HLA-B expression is detected by serology. In the strategy of the invention, an HLA-C0401 (wherein 0401 represents specific typing of HLA-C molecules) molecule in C1R cells is knocked out by using a CRISPR/Cas9 gene editing system, and then flow sorting is carried out to obtain C1R-H-null (non-expression) monoclonal cells.
The C1R antigen presenting cells used in the present invention can be obtained from American Type Culture Collection (ATCC) under the number CRL-1993. Meanwhile, the cell has been reported in the literature (Storkus WJ, et al. reverse of natural kit exhibiting specificity in target cells expressed transformed class I HLA genes. Proc. Natl. Acad. Sci. USA 86:2361-2364,1989.PubMed: 2784569).
There are several different variations of the CRISPR/Cas9 gene editing system, the principle of which is the same. The invention verifies that endogenous HLA-C gene knockout is realized by introducing Cas9-sgRNA protein complex (RNP) into C1R antigen presenting cells. On the basis of the verification and effectiveness of the invention, the technicians in the field can make some appropriate modifications on the basis of the spirit of the invention, so that different CRISPR/Cas9 gene editing systems can effectively knock out HLA-C genes in C1R antigen presenting cells.
The embodiment of the invention proves that sgRNA (single guide RNA) which is very effective for knocking out HLA-C gene is provided, wherein the sgRNA comprises a recognition sequence GTGAACCTGCGGAAACTGCG (SEQ ID NO: 1). Thus, a polypeptide comprising SEQ ID NO: the sgRNA of 1 is preferred in the present invention.
The sgRNA in the embodiment of the present invention may be obtained by amplifying a plasmid containing a gRNA framework by PCR to obtain an amplification product as an in vitro transcription template, and then transcribing the in vitro transcription template in vitro to obtain the sgRNA. As a preferred embodiment, consider SEQ ID NO: 1, and the primers used in the process of generating in vitro transcription templates through PCR amplification comprise the recognition sequences of SEQ ID NO: 1, and also a T7 promoter sequence is included in the primer in consideration of the promoter required for transcription.
As a most preferred embodiment, the process of the present invention comprises the steps of:
(1) obtaining an in vitro transcription template of sgRNA of a target HLA-C gene by PCR amplification, wherein primers amplified by the PCR comprise:
HLA-C0401 gRNA FW：
TAATACGACTCACTATAGTGAACCTGCGGAAACTGCGGTTTTAGAGCTAGAAATAGC (SEQ ID NO: 2); and
gRNA scafford RV：AGCACCGACTCGGTGCCACT(SEQ ID NO：3)；
the template used for PCR amplification comprises a gRNA framework;
(2) carrying out in vitro transcription on the in vitro transcription template obtained in the step (1) by using T7RNA polymerase to synthesize sgRNA;
(3) embedding the sgRNA obtained in the step (2) and Cas9 protein into a Cas9-sgRNA protein complex (RNP), introducing the Cas9-sgRNA protein complex into the C1R antigen presenting cell, and culturing and sorting to obtain the antigen presenting cell line without the endogenous HLA gene background, wherein the HLA-C gene is knocked out.
It should be noted that the template used in the PCR amplification in step (1) can be provided by a variety of template plasmids, such as the commonly used pMD 19-T plasmid (FIG. 2), which contains a gRNA backbone. Such plasmids also include the P1123 plasmid supplied by Fenghui Bio Inc., and the like.
The technical solutions of the present invention are described in detail below by way of examples, and it should be understood that the examples are only illustrative and should not be construed as limiting the scope of the present invention.
Examples
1. In vitro transcription template of sgRNA targeting HLA-C0401 molecule obtained by PCR
Primer sequences were designed and synthesized as follows:
HLA-C0401 gRNA FW：
TAATACGACTCACTATAGTGAACCTGCGGAAACTGCGGTTTTAGAGCTAGAAATAGC(SEQ ID NO：2)；
gRNA scafford RV：AGCACCGACTCGGTGCCACT(SEQ ID NO：3)。
the PCR reaction system is shown in Table 1 below:
TABLE 1
The reaction procedure is shown in table 2 below:
TABLE 2
After PCR reaction, the PCR product is cut and recovered to obtain the in vitro transcription template.
2. In vitro transcription synthesis of sgRNA
(1) The reaction mixtures shown in table 3 below (reagent MMESSAGE MMACHINE T7 KIT, Invitrogen, AM1344) were prepared in EP tubes:
TABLE 3
(2) Uniformly mixing the reaction system, instantly centrifuging, and reacting at 37 ℃ for 3-4 h;
(3) adding 1 μ L of TUBRO DNase, mixing, centrifuging instantly, and reacting at 37 deg.C for 15 min;
(4) the reaction was stopped by adding 115. mu.L of Nuclease-free Water (Nuclear-free Water) and 15. mu.L of hypnone (Anmonieum Aeoleate);
(5) adding phenol-chloroform (pH4.5) with equal volume, shaking vigorously, mixing, centrifuging at 14000rpm at 4 deg.C for 15 min;
(6) transferring the upper aqueous phase solution into a new 1.5mL EP tube, adding equal volume of chloroform, shaking vigorously, mixing, centrifuging at 14000rpm at 4 deg.C for 15 min;
(7) transferring the upper aqueous phase solution into a new 1.5mL EP tube, adding isopropanol with the same volume, shaking and uniformly mixing, and standing at room temperature for 10min or overnight at-20 ℃;
(8) centrifuging at 14000rpm for 15min at 4 deg.C, discarding supernatant (carefully sucking to prevent RNA from being sucked away), adding 500 μ L75% ethanol, and centrifuging at 14000rpm for 15min at 4 deg.C;
(9) the supernatant was discarded (carefully aspirated to prevent aspiration of RNA pellet), centrifuged at 14000rpm for 3min at 4 ℃ and the supernatant was dried by pipetting with a small gun without RNase free;
(10) air drying at room temperature, volatilizing ethanol, adding 20 μ L water containing no RNase free, standing at room temperature for 5min, and standing on ice for 20min to dissolve completely;
(11) diluting the 1 μ L of mRNA solution by 3 times, then respectively taking 1 μ L of the diluted mRNA to detect OD 260/230nm value and OD 260/280nm value, adding another 1 μ L of the diluted mRNA solution into 15.5 μ L of RNA denaturation buffer solution, uniformly mixing, placing on ice quickly after denaturation at 65 ℃ for 20min, then carrying out agarose gel electrophoresis, and estimating the concentration and degradation degree of the synthesized mRNA by combining the OD value, or directly carrying out electrophoresis without denaturation.
(12) The synthesized mRNA is divided into small tubes according to the requirement, marked and stored in a refrigerator at minus 80 ℃.
3. HLA molecule with electricity-transferred Cas9-RNP knockout of C1R
(1) Culturing C1R cells with good growth state in a 24-well plate by 2x105Each was resuspended in 100. mu.L of Opti-MEM (Invitrogen, 11058-021).
(2) RNP complexes were prepared with the components of table 4 below:
TABLE 4
After incubation for 15min at room temperature, the formed RNP complex can exist stably for 2 hours at room temperature.
(3) Uniformly mixing the two components in the steps (1) and (2), adding the mixture into an electric shock cup, and carrying out electric conversion (710V, 30ms, CELETRIX electric conversion instrument);
(4) the electroporated C1R cells were cultured in 10% FBS IMDM medium at 37 ℃ in 5% CO2The culture was carried out for 48 hours.
4. C1R cell editing efficiency test
After 48 hours of electroporation of C1R cells, the cells were harvested and genomic DNA was extracted, and the desired fragment was amplified by PCR. T7 nuclease I (T7E1) and sanger sequencing were performed for detection.
(1) T7E1 analysis
PCR was performed on the edited genomic DNA of the cells using the following primers:
C0401FW：gcttcatcgcagtgggctac(SEQ ID NO：4)；
C0401RV：cgggagatctacgggagatgg(SEQ ID NO：5)。
the PCR product was gel recovered and reacted according to the following Table 5 components:
TABLE 5
| Components | Content (wt.) |
| PCR recovery of products | 200ng |
| 10XNEBuffer 2 | 2μL |
| DNase/RNase-free water | Make up to 19. mu.L |
The denaturing annealing reaction was performed in a PCR apparatus under the following reaction conditions shown in Table 6:
TABLE 6
To 19. mu.L of the product after the annealing, 1. mu. L T7E1 enzyme (NEB, MO3O2S) was added for digestion. The reaction was carried out at 37 ℃ for 15min and detected by 1% agarose electrophoresis.
As a result, as shown in FIG. 3, the group was significantly cleaved by T7 nuclease 1(T7E1 enzyme), demonstrating successful editing knock-out.
(2) sanger sequencing detection of C1R editing efficiency
After PCR of the C1R cell genome, the gel recovery product was analyzed by sanger sequencing, and fig. 4 shows a control, and two edited clone sequencing results, showing 8 base deletions and 11 base deletions. Sequencing and verifying that the target gene locus generates knockout.
FIG. 5 shows that various insertion deletions (indels) are generated after editing the HLA molecule sites of the C1R cells of different clones, which indicates that the target gene sites generate frame shift mutations and stop codons are formed in advance, indicating that the HLA-C0401 gene knockout is successful.
5. Monoclonal screening
After electrotransformation, the C1R cells were subjected to flow sorting and monoclonality, as follows:
(1) take 2x10 respectively6Electrically transformed C1R cells, placed in three flow tubes;
(2) washing twice with 1mL PBS, centrifuging, removing supernatant, and resuspending with 200 μ L PBS;
(3) mu.L of HLA-A, B, C (w6/32) (Biolegend, 311402) antibody was added to one tube, 5. mu.L of HLA-C (Biolegend, 373302) antibody was added to one tube, and no treatment was added to one tube as a control;
(4) incubating for half an hour on ice in the dark, washing twice with PBS, removing supernatant, and resuspending with 200 μ L PBS;
(5) adding 2 mu L of DAPI (1mg/mL) 5 minutes before the flow type machine loading, and loading the machine after mixing uniformly;
(6) removing dead cells by flow machine, sorting HLA-A, B, C and HLA-C double negative cells, culturing each cell;
(7) the single clones were cultured.
Flow results are shown in FIGS. 6 and 7, and FIG. 6 shows the result of staining with HLA-A, B, C antibody after the C1R cells of different clones knock out HLA-C, wherein the flow analysis shows that HLA-A, B, C is negative in the selected monoclonal 1(clone1) and clone 5(clone5), thus proving that the knockout of HLA-C0401 is successful.
FIG. 7 shows the result of HLA-C antibody staining after HLA-C knock-out by C1R cells of different clones, wherein the selected monoclonal 1(clone1) and clone 5(clone5) were negative for HLA-C by flow analysis, further demonstrating the success of HLA-C0401 knock-out.
The present invention has been described in terms of specific examples, which are provided to aid understanding of the invention and are not intended to be limiting. For a person skilled in the art to which the invention pertains, several simple deductions, modifications or substitutions may be made according to the idea of the invention.
Claims (10)
- A method of constructing an antigen presenting cell line free of endogenous HLA gene background, the method comprising: and (3) knocking out an endogenous HLA-C gene of an HLA typing single C1R antigen presenting cell by using a CRISPR/Cas9 gene editing system, wherein the C1R antigen presenting cell comprises HLA-A, HLA-B and HLA-C genes, wherein HLA-A is not expressed, and HLA-B is not substantially expressed.
- The method for constructing an antigen presenting cell line without an endogenous HLA gene background, according to claim 1, wherein the endogenous HLA-C gene knockout is realized by introducing Cas9-sgRNA protein complex (RNP) into the C1R antigen presenting cell, wherein the sgRNA comprises a recognition sequence GTGAACCTGCGGAAACTGCG (SEQ ID NO: 1).
- The method of claim 2, wherein the sgRNA is obtained by in vitro transcription from an in vitro transcription template.
- The method for constructing an antigen presenting cell line without the background of endogenous HLA genes, according to claim 3, wherein the in vitro transcription template is obtained by PCR amplification of a plasmid containing a gRNA backbone, wherein primers used for PCR amplification comprise the recognition sequences SEQ ID NO: 1 and T7 promoter sequences.
- The method of constructing an antigen presenting cell line without a background of endogenous HLA genes according to any one of claims 1 to 4, comprising:(1) obtaining an in vitro transcription template of sgRNA targeting HLA-C gene by PCR amplification, wherein the primers for the PCR amplification comprise:HLA-C0401 gRNA FW：TAATACGACTCACTATAGTGAACCTGCGGAAACTGCGGTTTTAGAGCTAGAAATAGC (SEQ ID NO: 2); andgRNA scafford RV：AGCACCGACTCGGTGCCACT(SEQ ID NO：3)；the template used for PCR amplification comprises a gRNA framework;(2) in-vitro transcription is carried out on the in-vitro transcription template obtained in the step (1) by using T7RNA polymerase to synthesize sgRNA;(3) embedding the sgRNA obtained in the step (2) and Cas9 protein into a Cas9-sgRNA protein complex (RNP), introducing the Cas9-sgRNA protein complex into the C1R antigen presenting cell, and culturing and sorting to obtain the antigen presenting cell line without the endogenous HLA gene background, wherein the HLA-C gene is knocked out.
- The method for constructing an antigen presenting cell line without the background of endogenous HLA genes according to claim 5, wherein the template used for PCR amplification in the step (1) is pMD 19-T plasmid containing gRNA backbone; in the step (3), the Cas9-sgRNA protein complex is introduced into the C1R antigen presenting cell by means of electrotransformation.
- The method for constructing an antigen presenting cell line without the background of endogenous HLA genes as claimed in claim 5, wherein said step (3) comprises sorting the antigen presenting cell line without the background of endogenous HLA genes by flow cytometry.
- An antigen presenting cell line without a background of endogenous HLA genes constructed according to the method of any one of claims 1 to 7.
- The antigen presenting cell line of claim 8, wherein the HLA-C gene in the antigen presenting cell line is a frameshift mutation due to an insertion or deletion.
- Use of the antigen presenting cell line without the background of endogenous HLA genes of claim 8 or 9 as a tool cell in antigen research or immunotherapy.
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