CN113801881A - Use of super enhancer gene sequence in promoting human B2M gene expression - Google Patents
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Abstract
The invention provides an application of a super enhancer gene sequence in promoting human B2M gene expression, through ChIP-Seq technology, the signal change condition of an enhancer marker H3K27ac before and after IFN-gamma stimulation is compared, an enhancer sequence is found on a human B2M gene for the first time, the enhancer has the characteristic of a super enhancer after IFN-gamma stimulation, and the expression of the human B2M gene is obviously improved. By knocking down or silencing the enhancer sequence, the expression of the human B2M gene and the membrane HLA-I protein can be obviously reduced, and a new target point is provided for constructing human cells with low immunogenicity.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of a super enhancer gene sequence in promoting expression of human B2M gene and membrane HLA-I protein.
Background
The description of the background art is intended to be merely a general description of the invention in order to facilitate an understanding of the invention and is not intended to limit the invention in any way.
Whether transplant rejection occurs or not depends mainly on the histocompatibility between donor and recipient, and the degree of matching of major histocompatibility antigens, i.e., MHC molecules, is particularly important. Human MHC molecules, also known as Human Leukocyte Antigens (HLA), are highly polymorphic, and HLA matching has been a key bottleneck problem in clinical treatment of human cells and organ transplantation, since the HLA molecular patterns are often not identical between the donor and recipient.
HLA is classified into class I antigen (HLA-I), class II antigen (HLA-II) and class III antigen (HLA-III) according to its distribution and function. Beta 2 microglobulin is also called B2M, and in human body, beta 2 microglobulin is encoded by B2M gene and is an important component in HLA-I molecules. HLA-I is used as a membrane protein, the expression level of the HLA-I directly determines the immunogenicity of cells, and B2M (Beta 2 Microglobulin, also called Beta 2M) is used as a component of HLA-I and plays an important role in fixing the HLA-I protein on the surface of a cell membrane. In the absence of B2M expression, few HLA-class I proteins were detected on the cell surface [1 ].
Despite the low immunogenicity of stem cells currently widely accepted, a large body of experimental data demonstrates that transplantation of native stem cells directly still cannot avoid the immunological rejection of Eryun [2-4 ]]. The low immunogenicity of stem cells compared to adult cells is manifested by low expression of HLA class I antigens and no expression of HLA class II antigens. However, our preliminary studies of the subject groups showed that the immunogenicity of Mesenchymal Stem Cells (MSCs) was inducible. IFN-gamma is one of the most common proinflammatory factors in immune rejection, and the HLA expression level of MSC is increased under the action of the inflammatory factor IFN-gamma, and the immunogenicity is increased along with the increase [5 ]](ii) a Compared with the unstimulated group, the MSC after IFN-gamma stimulation has obviously up-regulated total HLA-I expression quantity and membrane HLA-I expression quantity and is more easily rejected by the organism [6 ]]. After IFN-gamma stimulation, the expression level of HLA-I molecules on the surfaces of ESCs and iPSCs is increased and is enough to cause CD8 in vitro and in vivo+T cell mediated immune rejection [7]. However, the mechanism of how IFN- γ regulates HLA-I gene expression is still unclear.
Although the existing reports about the B2M gene mostly study the influence of the B2M gene on the immunogenicity of cells by means of gene knockout (knock-out). This cleavage at the gene level, which results in irreversible DNA changes, has many disadvantages such as gene off-target. However, few reports exist about the apparent regulation of B2M gene expression, and the mechanism is not yet clarified.
Disclosure of Invention
Aiming at the problems in the prior art, the invention compares the signal change conditions of the modified region of an Enhancer marker H3K27ac before and after IFN-gamma stimulation through ChIP-Seq technology from the perspective of epigenetics, finds an Enhancer sequence on the B2M gene for the first time, and the Enhancer has the characteristic of Super Enhancer (SE) after IFN-gamma stimulation and obviously improves the expression of the B2M gene. By knocking down or silencing the enhancer sequence, the expression of the B2M gene and the membrane HLA-class I protein can be obviously reduced.
The enhancer sequence regions found in the present invention are located: the chr15:45002906-45022119 has the characteristic of a super enhancer after IFN-gamma stimulation, and obviously improves the expression of the B2M gene. Conversely, if the sequence can be modified in appearance or silenced by other techniques, such as by cutting or cleaving some of the nucleotides using current gene editing techniques, such that the enhancement or reduction is not achieved, and thus the level of transcription or translation of the B2M gene is reduced, the expression of the membrane HLA class I protein is reduced, thereby reducing or eliminating rejection. This is because, in the absence or reduction of B2M expression, HLA class I proteins are rarely detected on the cell surface. The enhancer is one of the key factors influencing the expression of B2M.
The invention proves that the enhancer belongs to SE through a bioinformatics hand, and finds that an SE inhibitor JQ1 can obviously reverse the effect of IFN-gamma on up-regulating B2M expression. Further knocking down (knocking down is to reduce the activity of a certain sequence by adopting a modified gene editing technology, namely knocking down, and is different from knocking down caused by cutting the sequence) the SE through a CRISPR/Cas9 gene editing technology, so that the B2M gene transcription is obviously inhibited, and the stimulation desensitization of IFN-gamma can be realized.
On the basis, the invention constructs a universal stem cell suitable for allogeneic transplantation, wherein the core enhancer gene sequence of the B2M gene is partially knocked down or silenced, or the partial sequence is silenced or the partial sequence is knocked down.
The existing method for constructing the universal stem cell generally depends on high expression of various vector-mediated immune suppression molecules, so that exogenous DNA needs to be introduced into the genome of the stem cell, and unknown safety hazards exist. Moreover, the method usually involves gene editing for several times, and the obtained multiple engineering stem cells even have the defects of unstable karyotype and the like, and the safety and the quality control of the cells are unstable. In addition, genome editing strategies of some protocols are cumbersome and multiple genetic engineering manipulations can affect the integrity of the stem cell genome. The present invention intends to explore and develop a more safe and reliable universal stem cell from the viewpoint of this SE that we have found.
According to the invention, SE is knocked down or silenced by a CRISPR gene editing technology, so that B2M gene transcription is obviously inhibited, and IFN-gamma stimulation desensitization can be realized, thereby constructing an MSC (hereinafter referred to as hypo-MSC) capable of maintaining low immunogenicity under an inflammatory environment. hypo-MSC low expression membrane HLA-I protein, desensitization to inflammatory factor IFN-gamma stimulation, no immunoreaction of allogeneic PBMC cell, and capability of avoiding allogeneic CD8+Killing of T cells and NK cells. The novel hypo-MSC is expected to be prevented from being attacked by the immune system of a patient after being transplanted into the body of the patient without considering HLA matching, plays a role in treatment and solves the problem of immunological rejection of the immune system of a human body to allogeneic cells. Meanwhile, the feasibility and repeatability of the construction method are verified in ADSC, and the result shows that the method has universality.
In one aspect, the invention provides an enhancer, which is located near the B2M gene and is capable of forming a super enhancer upon stimulation by IFN- γ.
The invention provides a nucleic acid sequence which is an enhancer and can enhance the expression of B2M and membrane HLA-I protein; if the sequence is silenced or knocked down, the expression of B2M and membrane HLA-class I protein is significantly reduced or weakened, thereby reducing the immunogenicity and immune rejection of the cell.
In some embodiments, the invention provides nucleic acid sequences that enhance the expression level of HLA class I genes of MSCs by the action of the inflammatory factor IFN- γ.
The enhancement described in the present invention means that the above-mentioned nucleic acid sequence does not directly transcribe and translate the HLA class I gene, but can enhance the transcription of the HLA class I gene. Therefore, also called enhancer sequences, when an enhancer is present, it can enhance the expression ability of a target gene sequence, but does not directly express the target gene itself. For example, a gene that directly expresses HLA class I is the gene, i.e., the DNA sequence (including exons and introns) of the gene is transcribed into RNA, and the RNA of a portion of the exons is translated into a protein sequence, and the enhancer sequence provided by the present invention can significantly enhance the expression of the gene, i.e., regulate the expression of the gene (either enhanced or attenuated or even leave it unexpressed).
Further, the enhancer region is located in chr15:45002906-45022119, and the chr15:45002906-45022119 has the amino acid sequence shown as SEQ ID NO: 7, or a sequence shown in the figure.
The enhancer sequence provided by the invention is positioned near the B2M gene, and the vicinity of the B2M gene means that the enhancer sequence is positioned at the upstream or downstream of the B2M gene, and some enhancer sequences are introns of the B2M gene (figure 1). The enhancer region sequences found in the present invention are located: chr15:45002906-45022119, namely the initial position of the enhancer is 45002906 site of 15 chromosome, the termination position is 45022119 site of 15 chromosome, the total length is 19214bp, and the sequence is shown as SEQ ID NO: 7, or a sequence shown in the figure.
Further, the enhancer region has 6 active sequences, namely E1-E6, and E1-E6 have the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 6.
Enhancers are DNA sequences that significantly increase the transcription frequency of a gene, and are mainly present in eukaryotic genes, and may be present upstream, downstream, or as introns in the gene, and thus have multiple active sequences in the region of the enhancer, which may be introns in the gene, upstream or downstream sequences, and the like.
Introns are non-coding sequences in the gene and are spliced out during mRNA processing, so mature mRNA has no intron coding sequences. Introns are nonsensical to the structure of the translated product and do not affect gene expression.
According to a great deal of research, the first intron of B2M is also part of an enhancer, E2 and E3 are both located in the first intron of B2M, and when E2 or E3 is knocked down, knocked out or silenced, the expression of the B2M gene is remarkably reduced.
The position relationship between the region where the enhancer is located and the active sequence thereof and the B2M gene is shown in FIG. 1.
The E1-E6 are respectively positioned in chr15: 45002980-: 1 to SEQ ID NO: 6. Knockdown, knock-out, or silencing of any of the active regions reduces the expression of the B2M gene, but to a different extent.
Furthermore, the core enhancer fragment of the enhancer region is E2, and the core enhancer is the enhancer region which affects the B2M gene to the highest extent.
In another aspect, the invention provides the use of an enhancer as described above for enhancing expression of the B2M gene.
Further, the enhancer can form a super enhancer under IFN-gamma stimulation, and the super enhancer can further promote the expression of the B2M gene.
In yet another aspect, the present invention provides a method for under-expressing B2M gene, wherein all or part of the sequence of the enhancer of B2M gene is knocked down or silenced, and the region of the enhancer has the sequence as shown in SEQ ID NO: 7, or a sequence shown in the figure.
In still another aspect, the present invention provides a method for underexpressing a membrane HLA-class I protein comprising one of HLA-A, HLA-B, HLA-C and B2M protein; the whole or partial sequence of the enhancer of the B2M gene is knocked down or silenced, and the region of the enhancer has the sequence shown as SEQ ID NO: 7, or a sequence shown in the figure.
In yet another aspect, the invention provides a method of reducing the expression of a membrane HLA class I protein, primarily by knocking down or silencing all or part of the sequence of an enhancer as described above.
Further, the membrane HLA-class I protein comprises one of HLA-A, HLA-B, HLA-C and B2M protein.
Further, the method comprises the steps of:
(1) knocking down all or part of the sequence of the enhancer by a CRISPR gene editing method;
(2) cells with all or part of the enhancer sequence knocked down are selected.
According to the invention, the B2M gene expression is obviously inhibited by knocking down (the knocking down is to reduce the activity of a certain sequence by adopting a modified gene editing technology instead of causing the deletion of the certain sequence) or silencing the enhancer through the CRISPR gene editing technology.
Further, the guide nucleotide sequences of the CRISPRi are sgRNA F and sgRNA R, respectively, and the sequences of the sgRNA F and the sgRNA R are respectively as shown in SEQ ID NO: 16 and SEQ ID NO: 17 is shown; the knocked-down enhancer sequence is located at chr15:45,004,417-45,004, 436.
Theoretically, any active sequence of the enhancer can obviously promote the expression of the B2M gene, so that the knocking down of any active sequence from E1 to E6 can reduce the expression of the B2M gene.
The core enhancer fragment of the SE region is E2, and the effect of reducing the expression of the B2M gene is most obvious after the E2 region is knocked down.
The B2M gene and the membrane HLA-I protein have universality for stem cells, and are theoretically suitable for any human cells.
Further, the stem cells are MSCs or ADSCs.
In still another aspect, the present invention provides a human stem cell in which all or part of the sequence of the core enhancer of the B2M gene is knocked down or silenced.
In some embodiments, the sequence of the core enhancer is partially knocked down, or silenced, or partially silenced, or knocked out; or a partial sequence is silenced while a partial sequence is knocked down.
Further, the enhancer region is located in chr15:45002906-45022119, and the chr15:45002906-45022119 has the amino acid sequence shown as SEQ ID NO: 7, or a sequence shown in the figure.
Further, the enhancer region has 6 active sequences, namely E1-E6, and E1-E6 have the sequences shown in SEQ ID NO: 1 to SEQ ID NO: 6.
Further, the core enhancer fragment of the enhancer region is E2; the stem cells are MSC or ADSC cells. Theoretically, all nucleated human cells, not limited to stem cells, can be used to construct the low immunogenicity universal cells provided by the present invention. The stem cell type of MSC or ADSC is not therefore a limitation of the present invention.
In some embodiments, a portion of the enhancer sequence is knocked down, where the knocked down sequence is located at chr15:45,004, 417-.
In yet another aspect, the invention provides a method for producing a stem cell, primarily by knocking down or silencing all or part of the core enhancer sequence of the B2M gene.
Further, the method comprises the steps of:
(1) knocking down all or part of the fragment on the enhancer sequence located at chr15:45002906-45022119 by CRISPR;
(2) cells are selected for which all or part of the sequence segment of the enhancer is knocked down.
Further, the guide nucleotide sequences of the CRISPRi are sgRNA F and sgRNA R, respectively, and the sequences of the sgRNA F and the sgRNA R are respectively as shown in SEQ ID NO: 16 and SEQ ID NO: shown at 17.
Further, the cells in the step (2) are MSC or ADSC cells; the knocked-down sequences are located at chr15:45,004,417-45,004, 436.
Theoretically, all nucleated human cells, not limited to stem cells, can be used to construct the low immunogenicity universal cells provided by the present invention. The stem cell type of MSC or ADSC is not therefore a limitation of the present invention.
In some aspects, the method comprises the steps of: providing a cell; the core plasmid (pHR-SFFV-KRAB-dCas9-P2A-mCherry or pLV-U6-gRNA-UbC-eGFP-P2A-Bsr; packaging plasmid psPAX2 and envelope plasmid pMD2.G are co-cultured with the cells, and cells with double positive screening are obtained and used as the cells with reduced enhancer.
In yet another aspect, the present invention provides a stem cell having a sequence knockdown or silenced in whole or in part of the core enhancer region of the B2M gene, for use in suppressing an allogeneic immune rejection response.
In some embodiments, the enhancer gene of B2M gene in the stem cell is silenced or knocked down, or part of the sequence is silenced, or part of the sequence is knocked down; or a partial sequence is silenced while a partial sequence is knocked down.
Further, the enhancer region is located in chr15:45002906-45022119, and the chr15:45002906-45022119 has the amino acid sequence shown as SEQ ID NO: 7; the stem cells are MSC or ADSC cells.
The gene knockdown, silencing and cleavage of the present invention are all to reduce, lose or prevent the function of the target gene from functioning normally as an enhancer.
The invention has the beneficial effects that:
1. the enhancer sequence on the B2M gene responding to IFN-gamma stimulation is explained for the first time;
2. the sequence is found to have the characteristic of enhancer before IFN-gamma stimulation for the first time, and has the characteristic of SE after stimulation, thereby enhancing the expression of B2M gene;
3. the sequence is found to promote the expression of B2M gene under the stimulation of IFN-gamma for the first time, so as to promote the expression of membrane HLA-I protein;
4. the novel epigenetic mechanism that the inflammatory factor IFN-gamma regulates the expression of the B2M gene by inducing SE is elucidated for the first time;
5. providing a new target for constructing human cells with low immunogenicity;
6. by applying CRISPR technology, the SE is edited in the stem cells through genes, and the constructed universal stem cells can realize low immunogenicity and escape from allogeneic immune reaction;
7. when the universal stem cell is constructed, foreign DNA such as immunosuppressive molecules and the like is not required to be introduced, and multiple gene editing is not required, so that the universal stem cell is simpler and more convenient to implement;
8. the invention uses reversible appearance modification without changing DNA sequence, so the constructed general stem cell (such as hypo-MSC) is safer and more reliable;
9. the method provided by the invention has universality, is not specific to MSC (mesenchymal stem cell) cells, can be realized in ADSC (all-dielectric dependent cell) cells, and can be theoretically suitable for constructing universal cells by any human stem cells and other nucleated human cells.
Drawings
FIG. 1 is a schematic diagram showing the location of the enhancer region and its active sequence in relation to the B2M gene.
FIG. 2 is a diagram showing the results of RNA extraction and real-time fluorescent quantitative PCR detection of the expression levels of HLA-A, HLA-B, HLA-C and B2M from three groups of MSCs in example 1.
FIG. 3 is the results of the expression level detection of HLA-A, HLA-B, HLA-C and B2M by the five groups of MSCs in example 2 using q-PCR and flow cytometry, respectively, wherein 3A is the result of q-PCR detection and 3B is the result of flow cytometry detection.
FIG. 4 is a graph showing the results of H3K27ac ChIP-Seq analysis and bioinformatics analysis in example 3, wherein 4A is a graph showing the results of H3K27ac ChIP-Seq analysis and 4B is a graph showing the results of bioinformatics analysis.
FIG. 5 is a schematic diagram of the experiment process of knocking down the large fragment of super enhancer and the result of detecting the expression level of HLA-I in example 4, wherein 5A is a schematic diagram of the experiment process of knocking down a part of DNA sequence of super enhancer, and 5B is a schematic diagram of the result of detecting the expression level of HLA-I.
FIG. 6 is a schematic diagram of the results of the modified hypo-MSC cell obtained by the lentivirus-mediated CRISPR technique of example 5, wherein 6A is the hypo-MSC cell flow fluorescence sorting result; 6B is a schematic diagram of a lentivirus-mediated CRISPR technical process; 6C is a schematic diagram comparing the expression levels of B2M before and after SE modification of MSC and ADSC.
FIG. 7 is a graph showing the results of co-culture of PBMCs with hypo-MSCs or MSCs in example 6, wherein 7A and 7B are the results of flow-sampling and proliferation assay of PBMCs with hypo-MSCs or MSCs; 7C is a schematic representation of the comparison of the expression levels of B2M in human interferon gamma (IFN-. gamma.) enzyme-linked immunosorbent assay hypo-MSC or MSC.
FIG. 8 is a graph showing the co-culture results of NK cells with hypo-MSC or MSC in example 7, wherein 8A and 8B are graphs showing the results of detecting NK cell activation marker molecule (CD107 a); 8C is a schematic diagram of the results of measuring NK cell cytotoxicity (LDH).
FIG. 9 is the CD8 of example 8+Schematic representation of co-culture results of T cells with hypo-MSC or MSC, wherein 9A is detection of CD8+Activation marker for T cells (CD69) results; 9B is the detection of CD8+Schematic representation of cytotoxicity (LDH) results for T cells; 9C is the detection of CD8+The result of the secretion of the inflammatory factor IFN-gamma of the T cell is shown schematically; 9D and 9E are for detecting CD8+The results of the T cells and hypo-MSC or MSC co-culture flow-type collection detection proliferation condition are shown in the figure.
Detailed Description
The specific embodiments of the present invention are set forth merely to illustrate how the invention may be practiced and are not to be construed as limiting the invention, which is defined by the claims.
Noun english abbreviation and chinese meaning contrast: MSC: mesechymal Stem Cells, Mesenchymal Stem Cells; ADSC: adipose-derived mesensical Stem Cell, Adipose stromal Stem Cell; B2M: beta 2 Microglobulin, Beta 2 Microglobulin; ESC: embryonic Stem Cells, Embryonic Stem Cells; and (3) iPSC: induced Pluripotent Stem Cells, induced Pluripotent Stem Cells; and SE: super Enhancer; TE: typical Enhance, canonical enhancer; KD: knockdown.
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example 1: IFN-gamma upregulation of HLA-I gene expression in MSCs
This example demonstrates, through specific experiments, that IFN- γ can up-regulate the expression of HLA-I gene of MSC.
1.1 cell culture and treatment
MSC culture conditions: L-DMEM medium (containing 10% fetal calf serum), 5% CO2, and constant temperature culture at 37 ℃. 24 hours before treatment, MSC was treated at 5X 105And inoculating the cells into a 6-well plate for culture, wherein the cell fusion degree reaches 60-70% the next day.
Dividing the cultured MSC into three groups, wherein the first group is a blank control, the second group adds IFN-gamma into the culture medium according to the concentration of 100ng/ml, and continuously culturing for 24 hours; thirdly, IFN-gamma is added into the culture medium according to the concentration of 100ng/ml, and the culture is continued for 48 hours; collecting cells for subsequent RNA extraction and other treatments.
1.2 RNA extraction and fluorescent quantitative PCR (qRT-PCR) of MSC
(1) RNA extraction:
1) respectively adding 1ml of Trizol cell lysate into the three groups of collected MSC cells;
2) adding 200 mul (1/5 volume) of chloroform, vortex vibrating, and standing for 10 min;
3) and (3) precooling a centrifugal machine, centrifuging: 12000g, 4 ℃, 15 min;
4) the supernatant was transferred to a new 1.5ml RNase-free EP tube;
5) adding equivalent isopropanol, turning upside down, mixing well, and standing at room temperature for 10 min;
6) and centrifuging: 12000g, 4 ℃, 15 min;
7) removing the supernatant, adding 1ml of 75% ethanol, and slightly reversing the mixture to wash the mixture;
8) and centrifuging: 12000 Xg, 4 ℃, 10 min;
9) discarding the supernatant, and air-drying for 10 min;
10) according to the size of the precipitate, 20. mu.l of DEPC water was added, RNA was dissolved at 4 ℃, and the mixture was allowed to stand for 30min, and the concentration was measured.
(2) Real-time fluorescent quantitative PCR (qRT-PCR)
1) Using a TOYOBO reverse transcription kit (cat No.: FSQ-201) and carrying out reverse transcription on the RNA extracted in the last step to obtain cDNA, wherein the system comprises the following steps:
2) according to Takara corporation (cat #: RR420A) for real-time fluorescent quantitative PCR of cDNA:
the PCR cycle conditions of the real-time fluorescence quantitative PCR vii 7 instrument are as follows:
the primer sequences used are listed in the following table:
the contents of HLA-A, HLA-B, HLA-C and B2M in the three groups of MSCs were detected by real-time fluorescent quantitative PCR, and the results of RNA extraction and real-time fluorescent quantitative PCR of the three groups of MSCs are shown in FIG. 2.
As can be seen from the q-PCR results (FIG. 2), IFN-gamma with a concentration of 100ng/ml can significantly increase the transcriptional level (i.e., the content of mRNA) of HLA-I genes (HLA-A, HLA-B, HLA-C (also abbreviated as HLA-ABC) and B2M) of MSC; and with the prolonged action time of IFN-gamma, the transcription level of HLA-I molecules of the MSC is increased, and the transcription peak can be reached within 48 hours (h).
The experiment proves that IFN-gamma can obviously or very obviously improve the transcription level of several genes related to HLA-I family, the expression of the antigen can be obviously or very obviously improved, and the higher the expression of the antigen protein is, the higher the possibility of causing rejection reaction is (the transcription level of the direct gene) is.
Example 2: experiments with enhancer involvement in the stimulation of HLA-I expression by IFN-gamma
This example used MSC pre-treated with JQ1 (purchased from abcam; cat # ab 146612; lot # APN 15092-1-1; an enhancer inhibitor) at three concentrations of 125, 250 and 500nmol/ml for 2 hours, adding 100ng/ml IFN-gamma to treat MSC, culturing for 48 hr, collecting cell, extracting RNA, qPCR to detect HLA-I related gene expression, setting corresponding MSC blank control, and MSC blank control plus IFN-gamma treatment, comparing five groups of MSCs, respectively detecting by q-PCR and flow cytometry, the result is shown in figure 3, wherein the specific process and sequence of q-PCR are shown in example 1, and the q-PCR results are shown in FIG. 3A, which are the expression levels of HLA-A, HLA-B, HLA-C and B2M of five groups of MSCs; the flow cytometry results are shown in figure 3B.
1. q-PCR results
As can be seen from the q-PCR results (FIG. 3A), IFN- γ can significantly increase the transcriptional level of HLA-I gene (HLA-A, HLA-B, HLA-C and B2M) of MSC cells. However, this stimulation was inhibited by small molecule JQ1 (an enhancer inhibitor) and there was a dose response relationship with increased inhibition with increasing concentrations of JQ 1.
2. Flow cytometry results:
the specific process is as follows:
(1) collecting the above cultured five groups of MSC cells, counting, and collecting 1 × 10 cells6Mixing cells/100 μ L, adding 10 μ L diluted HLA-ABC antibody (NOVUS, Catalog # NB100-64775), mixing, and incubating on ice for 30 min;
(2) 1mL of PBS was added to resuspend the washed cells;
(3) centrifuging at 200 Xg for 5min, and removing supernatant;
(4) resuspend the mixed cells with 40. mu.L of diluted Anti-Mouse 488 secondary antibody (Invitrogen, Catalog # A-21202), incubate for 15min on ice in the dark;
(5) 1mL of PBS was added to resuspend the washed cells;
(6) centrifuging at 200 Xg for 5min, and removing supernatant;
(7) resuspend cells with 300. mu.L PBS, move to flow tube, and flow onto machine.
In this example, the mRNA level of HLA-I was first measured by q-PCR, and then the expression of HLA-I protein on the cell membrane was further measured by flow cytometry. The flow-type results (FIG. 3B) are consistent with the q-PCR results, when the MSC is stimulated by IFN-gamma, the membrane HLA-I expression level is increased (the peak value is shifted to the right), when the MSC pretreated by JQ1 is stimulated by IFN-gamma, although the membrane HLA-I expression level is increased to a certain extent, the peak value right shift degree is not obvious in the IFN-gamma stimulation group, namely IFN-gamma can up-regulate the HLA-I expression level on the surface of the MSC cell membrane, but JQ1 can obviously inhibit the effect of IFN-gamma, which means that when the cell enhancer is inhibited by JQ1, the effect of the IFN-gamma on stimulating HLA-I up-regulation is blocked. From these results, it can be speculated that there may be an enhancer involved in the stimulation of HLA-I expression by IFN- γ, but how this enhancer is involved in particular is demonstrated in the examples below.
Example 3 IFN-. gamma.has an effect of promoting the opening and activation of an enhancer in the vicinity of the B2M gene
To further understand whether enhancers are involved in the vicinity of the HLA-I associated gene following IFN- γ stimulation and directly correlate with expression of that gene, we performed H3K27ac ChIP-Seq analysis and bioinformatics analysis.
1. The procedure for the ChIP-Seq analysis of H3K27ac is as follows:
As a result of the H3K27ac ChIP-Seq analysis, it was found that the H3K27ac signal was significantly enhanced near the B2M gene after the MSCs were stimulated with IFN-. gamma.and a new H3K27ac signal was generated (FIG. 4A). Here, it was confirmed that the B2M gene has an enhancer sequence and is a novel sequence. In FIG. 4A, the case of the enhancer near the B2M gene before IFN- γ stimulation is shown at the top, and the case of the enhancer near the B2M gene after IFN- γ stimulation is shown at the bottom, and it can be seen that the peak signal of H3K27ac is significantly increased after IFN- γ stimulation, indicating that these enhancers are activated after IFN- γ stimulation.
2. Bioinformatic analysis of SE identification:
1) H3K27ac ChIP-Seq data preprocessing
The quality of the original sequencing data is firstly evaluated by using FastQC (v0.11.9), then low-quality bases and linker sequences existing in the sequencing data are removed by using trimmatic (v0.38, default parameters) software, finally, only sequences with the length of more than 50 bases are reserved for subsequent analysis, and the quality of the filtered sequencing data is re-evaluated by using FastQC. The filtered quality-qualified sequences were then aligned to the reference genes using second generation sequencing data alignment software bowtie2(v2.4.1, — very-sensitive). To avoid the effect of PCR duplication on subsequent analyses, markduplicates (picard) was used to label and remove redundant sequences resulting from PCR.
2) H3K27ac ChIP-Seq result visualization
To more intuitively observe the abundance distribution of the H3K27ac ChIP-Seq sequencing reads on the reference Genome, the aligned BAM formatted file was converted into bedGraph format using bamCoverage (deepTools, v3.5.1, - - -binSize 200- -smoothLength 600), and then the abundance distribution characteristics of the H3K27ac ChIP-Seq reads on the reference gene were displayed using UCSC Genome Browser (UCSC Genome Browser).
3) Finding enhancers and super enhancers
Based on the aligned BAM format files, genomic regions significantly enriched in H3K27ac ChIP-Seq reads were identified using the callpeak (MACS2, v 2.2.6) algorithm, which were considered to be the presence of H3K27ac modifications. We next used ROSE (RANK identification OF SUPER-ENHANCERS) software developed by Richard a young laboratory, the institute OF technology, ma, to find enhancers and SUPER-enhancers based on the aligned BAM files and the H3K27ac modified region file identified by MACS2, which first identified enhancer regions, then merged adjacent enhancers less than 15kb apart, and finally compared the abundance distribution OF H3K27ac reads within the merged enhancer regions to identify SUPER-enhancers. Based on the H3K27ac data for hMSC before and after IFN- γ stimulation, a emerging super enhancer in hMSC after IFN- γ stimulation can be identified.
Bioinformatic analysis revealed that these regions were typical enhancers in MSC cells that were not stimulated by IFN- γ and were characterized by a super enhancer after IFN- γ stimulation (fig. 4B). FIG. 4B is a bioinformatic analysis of the sequencing results of H3K27ac, and on the left side, the enhancer signals of all the genes of MSC before IFN- γ stimulation, it can be seen that HLA-I related genes (HLA-A, HLA-B, HLA-C and B2M) are located below the intersection of two dotted lines, i.e., the enhancer of HLA-I related genes before IFN- γ stimulation does not reach the threshold of super enhancer and does not belong to the super enhancer category; in the right case of the enhancer signals of all the genes in MSC after IFN-. gamma.stimulation, it can be seen that only the enhancer of B2M among HLA-I related genes (HLA-A, HLA-B, HLA-C and B2M) reached the threshold of super enhancer, i.e., it belongs to the super enhancer category. This also indicates that the enhancer sequence is located in the vicinity of the B2M gene and not in the vicinity of the other genes HLA-A, HLA-B, HLA-C.
Example 4 reduction of the expression level of HLA-I in Membrane after knockout of the super enhancer Large fragment
By utilizing the characteristic that paired sgRNAs in a paired sgRNA CRISPR gene editing method (paired-gRNA CRISPR-Cas9) can cause two breaks in the same gene, a large fragment knockout is carried out on a partial DNA sequence (a red arrow in a figure 5A shows that the knockout position is chr15:45004785-45006364, the length of the knockout sequence is 1579bp, the large fragment spans partial regions of E2 and E3, and a knockout cell (KO) is sorted out in a flow fluorescence cell sorting mode.
The sequence of the paired sgRNAs is as follows, sg-KO 1-F: ATTCTCCAGAGCAAACTGGG (SEQ ID NO: 19); sg-KO 1-R: CCCAGTTTGCTCTGGAGAAT (SEQ ID NO: 20); sg-KO 2-F: TAGTTTACAGCAATCACCTG (SEQ ID NO: 21); sg-KO 2-R: CAGGTGATTGCTGTAAACTA (SEQ ID NO: 22).
SE core sequence knockout experiments show that when the sequence is knocked out, the expression level of HLA-I of a membrane of a KO cell is obviously reduced compared with that of an MSC control group, and even can not be detected; and desensitizes IFN-gamma stimulation, and the expression level of HLA-I of the membrane of KO cells is obviously reduced compared with the expression level of the membrane of the KO cells after the stimulation of the MSC control group.
Fig. 5A is a schematic diagram of an experiment, that is, two sgrnas are designed at positions before and after peak 1, and DNA fragmentation is caused at the positions of the two sgrnas by using CRISPR technology, so that large fragment DNA sequence knockout of a part of an enhancer is realized. The results of flow cytometry analysis showed that, after the enhancer was knocked out (KO group), the expression level of membrane HLA-I was significantly decreased (left shift of peak) compared to the non-knocked out group (MSC group) and was almost at the level of completely non-expressed B2M gene compared to blank group, as shown in FIG. 5B; and when the KO group is stimulated by IFN-gamma (KO + IFN-gamma group), the rising degree of the expression quantity of the membrane HLA-I is not obvious in the undepressed group (MSC + IFN-gamma group), and compared with the blank group, the rising degree is almost at the level of completely not expressing the B2M gene, which indicates that the MSC after the enhancer is knocked out is completely desensitized to the IFN-gamma stimulation. It was also demonstrated that deletion of the large fragment sequence of this enhancer severely affected the expression of B2M gene, thereby also reducing the amount of membrane HLA-I expression.
Therefore, large fragment knockout easily results in almost no expression of the B2M gene, which increases the immunogenicity of MSCs (because deletion of B2M causes immune rejection of natural killer cells), and cannot be used to construct universal stem cells, further suggesting that the knockdown method is more suitable than knockout for constructing universal stem cells.
Example 5: lentivirus-mediated CRISPR technology for obtaining modified hypo-MSC cells
In order to construct the universal stem cell hypo-MSC, in this example, a part of the sequence in the core enhancer segment E2 in the enhancer is knocked down by small segments, so as to successfully construct the hypo-MSC cell.
The hypo-MSC cells provided in this example were prepared by the following steps:
(1) sgRNA design and recombinant plasmid construction:
sgRNA was designed against the enhancer sequence (SEQ NO: 1) (Benchling website:https:// www.benchling.com/crispr/). sgRNA sequences are shown in the table below:
enzyme cutting sites are respectively added at two ends of the sgRNA, CACC is added at the 5 'end of a sense strand of a sgRNA sequence, and AAAC is added at the 5' end of an antisense strand, so that a cohesive end which is complementary to pLV-U6-gRNA-UbC-eGFP-P2A-Bsr plasmid (Addgene: #83925) after being cut by Fast Digest Bbs I is formed. If the first base at the 5 ' end of the sense strand is not a G, then a G is added after the 5 ' CACC and a C is added at the 3 ' end of the corresponding antisense strand. pLV-U6-gRNA-UbC-eGFP-P2A-Bsr is an sgRNA framework expression vector containing a U6 promoter, and is provided with a GFP green fluorescent protein gene and ampicillin resistance.
Firstly, carrying out enzyme digestion on pLV-U6-gRNA-UbC-eGFP-P2A-Bsr by Fast Digest Bbs I, and recovering a linearized vector after DNA gel electrophoresis.
Phosphorylating and annealing the sgRNA sequence by using T4 PNK; the linear pLV-U6-gRNA-UbC-eGFP-P2A-Bsr plasmid vector was ligated with the annealed double-stranded sgRNA sequence for 1h at room temperature using T4 ligase, respectively. The ligation product was transformed into competent bacteria, Trans 109, in ice bath for 30min at 42 ℃ for 45s for 2min on ice. Clones were selected on ampicillin resistant LB plates. And (4) picking positive clone, shaking and sequencing. The sequencing primer was the forward primer sequence of the U6 promoter, 5'-GAGGGCCTATTTCCCATGATTCC-3' (SEQ ID NO: 18). The clone with correct sequencing is the recombinant plasmid.
(2) HEK293FT cells were plated on 10cm cell culture dishes at 2X 106/dish one day in advance, ensuring that the degree of cell fusion reached 60% effect the next day.
(3) The optimal transfection time is 12-18 hours after plating. HEK293FT cells were co-transfected with the core plasmid (pHR-SFFV-KRAB-dCas9-P2A-mCherry (Addge: #60954) or the recombinant plasmid from step 1, the packaging plasmid psPAX2 (Addge: #12260) and the envelope plasmid pMD2.G (Addge: #12259) in a ratio of 4:3:2 as described in the ViaFect transfection reagent.
(4) The virus stock was collected 3 times consecutively at 24 hours, 48 hours and 72 hours after transfection, respectively, and each collected virus liquid was filtered through a 0.45 μm filter to remove cell debris and other impurities.
(5) 1 × 10 a day in advance5MSC cells were seeded in 6-well plates per well, preferably to ensure 50% cell confluence the next day of infection.
(6) Before infection, virus stock solution is mixed with fresh culture medium in equal volume (step 3), 2 mul of polybrene with 10mg/ml (final concentration is 10 mug/ml) is added, mixed evenly and added into a six-well plate. The infection is repeated three times.
(7) 48 hours after infection, after the cell fusion degree reaches 80-90%, carrying out flow fluorescence sorting, sorting out cells with double positive of mCherry (red fluorescence) and GFP (green fluorescence), and carrying out amplification culture. Theoretically, the surviving MSC is a cell line that stably knockdown the enhancer gene.
Based on the super enhancer SE on B2M gene responding to IFN-gamma stimulation, this example uses the method of lentivirus CRISPR to apparently modify the SE, and knockdown part of the segment in the E2 active sequence of the enhancer is located in chr15:45,004,417-45,004, 436.
The result shows that the expression of the B2M gene can be obviously reduced after the appearance inhibition of the SE, and the expression of the IFN-gamma stimulated B2M gene can be greatly suppressed. We further verified the feasibility and reproducibility of this approach in primary ADSC cells, and the results obtained in ADSC were consistent with MSC results (see fig. 6A and 6B in particular). This shows that our method for constructing low immunogenicity cells is not specific to MSC and has universality. We refer to the less immunogenic MSCs constructed by this method as hypo-MSCs.
It is understood that in any other cell, modification of the SE sequence, partial gene sequence knock-down, or reverse gene silencing, etc. found in the present invention can be used to inhibit SE, and after inhibition, the expression of B2M gene is reduced, so as to reduce the activity of other similar genes, reduce immunogenicity, and thus reduce rejection. It is also understood that, in addition to the gene of the present invention in which the sequence is knocked down, the SE gene may be altered or inhibited, and at other positions, the activity or function of the gene may be significantly inhibited, thereby reducing the expression of the B2M gene.
As can be seen in fig. 6A, BF (bright field) was used to control the intensity of the fluorescence signal (red fluorescence or green fluorescence), and the gene-knocked down cells had mCherry (red fluorescence) and GFP (green fluorescence) appearance, indicating the presence of stable SE-knocked down cells.
FIG. 6C is a graph showing the comparison of B2M expression levels before and after SE modification between MSC and ADSC, wherein MSC-KRAB represents MSC stably expressing dCas9-KRAB, and MSC-sgRNA represents MSC stably expressing dCas9-KRAB and sgRNA simultaneously. Only under the guidance of sgRNA did dCas9-KRAB function. This example designs a pair of sgrnas (plus and minus two) for enhancer, which can direct dCas9-KRAB to the enhancer region to play the role of knocking down the enhancer. MSC-KRAB cells, lacking the pair of sgRNAs, failed to exert enhancer knockdown effect and were used as control cells.
From the expression level of B2M in fig. 6C, there was a significant difference in MSC expression levels for MSC cells, cells without SE modification and cells with SE sequence modification. Similarly, the two genes showed significant differences in IFN- γ stimulated B2M gene expression. This fully demonstrates that the methods of the invention can inhibit SE activity, thereby significantly reducing the expression level of the B2M gene. The same conclusion is true for ADSC cells.
However, compared with the large fragment knockout in example 4, the normal expression of B2M is not completely inhibited by the small fragment knock-down of the core enhancer fragment, so that a universal stem cell hypo-MSC can be constructed.
Example 6 hypo-MSCs do not elicit an immune response in allogeneic PBMCs
In this example, allogeneic PBMC labeled with CFSE dye was cultured with hypo-MSC (cells prepared in example 5) and MSC (control), PBMC cell proliferation (PBMC proliferation reflecting the intensity of immune rejection) was detected by flow cytometry, and secretion of inflammatory factors in immune response was detected by human interferon-gamma (IFN-. gamma.) ELISA, as follows:
1. PBMC mixed culture
(1) Diluting the blood with PBS;
(2) taking a proper amount of Ficoll in a 15mL centrifuge tube, and slowly adding diluted blood to the upper layer of the Ficoll;
(3) centrifuging at 300 Xg for 30min, putting the middle flocculent layer in a new centrifuge tube, adding PBS for cleaning, and counting;
(4) isolated PBMC (allogenic) from the reacted cells were stained at 37 ℃ for 10min according to the CFDA-SE cell proliferation and tracer detection kit instructions. Then washing for 2 times by using complete cell culture solution, centrifuging, and counting the number of the cells which are re-suspended by using the complete cell culture solution to serve as reaction cells;
(9) after MSC and hypo-MSC adhere to the wall, incubating the wall with 5ng/mL mitomycin at 37 ℃ for 15min, then washing the wall for 2 times by using complete cell culture solution, centrifuging, and counting the number of the cells which are re-suspended by using the complete cell culture solution to serve as stimulated cells;
(10) at 1 × 104cells/cell density of 100 μ L stimulated cells (i.e., both MSC and hypo-MSC stimulated cells, MSC used as a control for hypo-MSC) were seeded in U-type 96-well plates;
(11) at 1 × 105cells/cell density of 100. mu.L the reaction cells were seeded in U-shaped 96-well plates;
(12) and (3) putting the U-shaped 96-well plate back to the cell culture box, culturing for 3-5 days respectively, and collecting a sample in a flow mode to detect the proliferation condition of the reaction cells.
2. Human interferon gamma (IFN- γ) enzyme-linked immunosorbent assay:
(1) pre-calculating the required number of battens, taking out the kit 30min before the experiment, and recovering to the room temperature;
(2) adding 100 μ l of standard working solution and detection sample (if the sample concentration is higher than the detection range, sampling after diluting with standard and sample diluent), making multiple holes for the standard, sealing the plates, and incubating in an incubator at 37 deg.C for 90 min;
(3) discarding the liquid, spin-drying, adding 100 μ l biotin-labeled gamma interferon antibody working solution into each reaction well (for two treatment of co-culture, PBMC + hypo-MSC; PBMC + MSC), sealing, and incubating at 37 deg.C for 60 min;
(4) washing: discarding the liquid, spin-drying, adding 350 μ l of washing solution into each reaction hole, soaking for 1-2min, spin-drying the washing solution, and repeating for 4 times;
(5) adding 100 mu l of HRP-labeled streptavidin working solution into each reaction hole, sealing the reaction holes, and incubating in an incubator at 37 ℃ for 30 min;
(6) washing: 300 mul of washing solution was added to each reaction well, and the washing solution was spun down at intervals of 30 seconds. Repeating for 4 times;
(7) adding 90 μ l of color developing agent (in dark) into each reaction hole, sealing the reaction holes, and developing in dark at 37 deg.C for about 15 min;
(8) adding 50 μ l of stop solution into each reaction hole, and immediately measuring OD value (within 5 min) with a microplate reader at a wavelength of 450 nm;
(9) measuring OD value with 450nm wavelength of a microplate reader;
(10) calculating the average OD value of the standard substance and the sample: the OD value of zero wells should be subtracted from the OD value of each standard and sample;
(11) the concentration of the standard substance is used as the abscissa and the OD value of the absorbance is used as the ordinate, and a standard curve is drawn by software (values of blank groups are removed in drawing).
(12) If the OD value of the sample is higher than the upper limit of the standard curve, the sample is re-detected after being diluted properly, and the sample is multiplied by the dilution times when the concentration is calculated.
Allogeneic PBMCs were labeled with CFSE dye and co-cultured with hypo-MSCs (cells of example 5), MSCs (control), and FIGS. 7A and 7B are schematic graphs showing the results of flow-through assays for proliferation of PBMCs with hypo-MSCs or MSCs, where E/T is the efficiency: Target ratio (ratio of Effector cells (here PBMCs) to Target cells (here MSCs or hypo-MSC cells)), indicating that the MSC group elicited a strong immune response, that PBMCs proliferated extensively, and that PBMCs of the hypo-MSC group were almost non-proliferating (FIGS. 7A and 7B). The positive control is PBMC mixed culture from two different sources, and the source of the PBMC is different, so that immune rejection reaction is caused certainly, and the PBMC is called as the positive control; the negative control is PBMC cultured alone and not mixed with other cells, so that immune rejection reaction does not occur; the positive and negative controls were used together to demonstrate the success of the experiment without the occurrence of false positives and false negatives.
The immunogenicity changes of hypo-MSC and MSC before and after co-culture are detected in one step, and the result shows that the B2M gene is obviously up-regulated after the MSC is co-cultured with PBMC, and hypo-MSC can maintain low-level B2M gene expression before and after co-culture. hypo-MSC was shown to have low immunogenicity and could maintain low immunogenicity in inflammatory environments (fig. 7C).
Example 7 hypo-MSC is able to escape killing of NK cells
Separating NK cells (natural killer cells) in the PBMC, continuously co-culturing the NK cells with hypo-MSC and MSC according to the proportion relation of 1:1, 3:1 and 10:1 (the co-culture is to put two different types of cells into the same culture system for mixed culture, namely co-culturing the NK cells with hypo-MSC and MSC respectively, detecting the rejection of the NK cells to the two types of cells), and detecting activation marker substances marker (CD107a) and cytotoxicity (LDH) of the NK cells.
The results show that there was no difference in the proportion of hypo-MSC and MSC-activated NK cells in the case of NK cells co-cultured with hypo-MSC or MSC at three mixing ratios of 1:1, 3:1 and 10:1, respectively (FIGS. 8A and 8B), nor was there any statistical difference in LDH (FIG. 8C), indicating that hypo-MSC was not sufficiently immunogenic to activate allogeneic NK cells.
Example 8 hypo-MSC is able to escape killing of CD8+ T cells
The mixed culture with MSC and PBMC is carried out for five days in advance, CD8+ T cells are separated out, and are continuously co-cultured with hypo-MSC and MSC, and the activation marker (CD69), cytotoxicity (LDH) and inflammatory factor IFN-gamma secretion of CD8+ T cells are detected.
The results indicate that hypo-MSC showed lower CD8+ T activation (fig. 9A), lower T cell killing (fig. 9B) and lower IFN- γ secretion (fig. 9C) than MSC group; CD8+ T cell proliferation experiments also demonstrated that hypo-MSC was barely able to induce CD8+ T cell proliferation (fig. 9D and 9E). Indicating that the low immunogenicity of hypo-MSC is able to escape killing of allogeneic CD8+ T cells.
The invention shown and described herein may be practiced in the absence of any element or elements, limitation or limitations, which is specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. It should therefore be understood that although the present invention has been specifically disclosed by various embodiments and optional features, modification and variation of the concepts herein described may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.
The contents of the articles, patents, patent applications, and all other documents and electronically available information described or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.
Sequence listing
<110> Zhejiang university
<120> use of super enhancer gene sequence for promoting expression of human B2M gene
<160> 22
<170> SIPOSequenceListing 1.0
<210> 1
<211> 671
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
aaagccctag cagttactgc ttttactatt agtggtcgtt tttttctccc ccccgccccc 60
cgacaaatca acagaacaaa gaaaattacc taaacagcaa ggacataggg aggaacttct 120
tggcacagaa ctttccaaac actttttcct gaagggatac aagaagcaag aaaggtactc 180
tttcactagg accttctctg agctgtcctc aggatgcttt tgggactatt tttcttaccc 240
agagaatgga gaaaccctgc agggaattcc caagctgtag ttataaacag aagttctcct 300
tctgctaggt agcattcaaa gatcttaatc ttctgggttt ccgttttctc gaatgaaaaa 360
tgcaggtccg agcagttaac tggctggggc accattagca agtcacttag catctctggg 420
gccagtctgc aaagcgaggg ggcagcctta atgtgcctcc agcctgaagt cctagaatga 480
gcgcccggtg tcccaagctg gggcgcgcac cccagatcgg agggcgccga tgtacagaca 540
gcaaactcac ccagtctagt gcatgccttc ttaaacatca cgagactcta agaaaaggaa 600
actgaaaacg ggaaagtccc tctctctaac ctggcactgc gtcgctggct tggagacagg 660
tgacggtccc t 671
<210> 2
<211> 1551
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
cccgctctgc accctctgtg gccctcgctg tgctctctcg ctccgtgact tcccttctcc 60
aagttctcct tggtggcccg ccgtggggct agtccagggc tggatctcgg ggaagcggcg 120
gggtggcctg ggagtgggga agggggtgcg cacccgggac gcgcgctact tgcccctttc 180
ggcggggagc aggggagacc tttggcctac ggcgacggga gggtcgggac aaagtttagg 240
gcgtcgataa gcgtcagagc gccgaggttg ggggagggtt tctcttccgc tctttcgcgg 300
ggcctctggc tcccccagcg cagctggagt gggggacggg taggctcgtc ccaaaggcgc 360
ggcgctgagg tttgtgaacg cgtggagggg cgcttggggt ctgggggagg cgtcgcccgg 420
gtaagcctgt ctgctgcggc tctgcttccc ttagactgga gagctgtgga cttcgtctag 480
gcgcccgcta agttcgcatg tcctagcacc tctgggtcta tgtggggcca caccgtgggg 540
aggaaacagc acgcgacgtt tgtagaatgc ttggctgtga tacaaagcgg tttcgaataa 600
ttaacttatt tgttcccatc acatgtcact tttaaaaaat tataagaact acccgttatt 660
gacatctttc tgtgtgccaa ggactttatg tgctttgcgt catttaattt tgaaaacagt 720
tatcttccgc catagataac tactatggtt atcttctgcc tctcacagat gaagaaacta 780
aggcaccgag attttaagaa acttaattac acaggggata aatggcagca atcgagattg 840
aagtcaagcc taaccagggc ttttgcggga gcgcatgcct tttggctgta attcgtgcat 900
ttttttttaa gaaaaacgcc tgccttctgc gtgagattct ccagagcaaa ctgggcggca 960
tgggccctgt ggtcttttcg tacagagggc ttcctctttg gctctttgcc tggttgtttc 1020
caagatgtac tgtgcctctt actttcggtt ttgaaaacat gagggggttg ggcgtggtag 1080
cttacgcctg taatcccagc acttagggag gccgaggcgg gaggatggct tgaggtccgt 1140
agttgagacc agcctggcca acatggtgaa gcctggtctc tacaaaaaat aataacaaaa 1200
attagccggg tgtggtggct cgtgcctgtg gtcccagctg ctccggtggc tgaggcggga 1260
ggatctcttg agcttaggct tttgagctat catggcgcca gtgcactcca gcgtgggcaa 1320
cagagcgaga ccctgtctct caaaaaagaa aaaaaaaaaa aaagaaagag aaaagaaaag 1380
aaagaaagaa gtgaaggttt gtcagtcagg ggagctgtaa aaccattaat aaagataatc 1440
caagatggtt accaagactg ttgaggacgc cagagatctt gagcactttc taagtacctg 1500
gcaatacact aagcgcgctc accttttcct ctggcaaaac atgatcgaaa g 1551
<210> 3
<211> 2046
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
acaatctgat atttaaaaaa aaatcttatt ttgaaaattt ccaaagtaat acatgccatg 60
catagaccat ttctggaaga taccacaaga aacatgtaat gatgattgcc tctgaaggtc 120
tattttcctc ctctgacctg tgtgtgggtt ttgtttttgt tttactgtgg gcataaatta 180
atttttcagt taagttttgg aagcttaaat aactctccaa aagtcataaa gccagtaact 240
ggttgagccc aaattcaaac ccagcctgtc tgatacttgt cctcttctta gaaaagatta 300
cagtgatgct ctcacaaaat cttgccgcct tccctcaaac agagagttcc aggcaggatg 360
aatctgtgct ctgatccctg aggcatttaa tatgttctta ttattagaag ctcagatgca 420
aagagctctc ttagctttta atgttatgaa aaaaatcagg tcttcattag attccccaat 480
ccacctcttg atggggctag tagcctttcc ttaatgatag ggtgtttcta gagagatata 540
tctggtcaag gtggcctggt actcctcctt ctccccacag cctcccagac aaggaggagt 600
agctgccttt tagtgatcat gtaccctgaa tataagtgta tttaaaagaa ttttatacac 660
atatatttag tgtcaatctg tatatttagt agcactaaca cttctcttca ttttcaatga 720
aaaatataga gtttataata ttttcttccc acttccccat ggatggtcta gtcatgcctc 780
tcattttgga aagtactgtt tctgaaacat taggcaatat attcccaacc tggctagttt 840
acagcaatca cctgtggatg ctaattaaaa cgcaaatccc actgtcacat gcattactcc 900
atttgatcat aatggaaagt atgttctgtc ccatttgcca tagtcctcac ctatccctgt 960
tgtattttat cgggtccaac tcaaccattt aaggtatttg ccagctcttg tatgcattta 1020
ggttttgttt ctttgttttt tagctcatga aattaggtac aaagtcagag aggggtctgg 1080
catataaaac ctcagcagaa ataaagaggt tttgttgttt ggtaagaaca taccttgggt 1140
tggttgggca cggtggctcg tgcctgtaat cccaacactt tgggaggcca aggcaggctg 1200
atcacttgaa gttgggagtt caagaccagc ctggccaaca tggtgaaatc ccgtctctac 1260
tgaaaataca aaaattaacc aggcatggtg gtgtgtgcct gtagtcccag gaatcacttg 1320
aacccaggag gcggaggttg cagtgagctg agatctcacc actgcacact gcactccagc 1380
ctgggcaatg gaatgagatt ccatcccaaa aaataaaaaa ataaaaaaat aaagaacata 1440
ccttgggttg atccacttag gaacctcaga taataacatc tgccacgtat agagcaattg 1500
ctatgtccca ggcactctac tagacacttc atacagttta gaaaatcaga tgggtgtaga 1560
tcaaggcagg agcaggaacc aaaaagaaag gcataaacat aagaaaaaaa atggaagggg 1620
tggaaacaga gtacaataac atgagtaatt tgatgggggc tattatgaac tgagaaatga 1680
actttgaaaa gtatcttggg gccaaatcat gtagactctt gagtgatgtg ttaaggaatg 1740
ctatgagtgc tgagagggca tcagaagtcc ttgagagcct ccagagaaag gctcttaaaa 1800
atgcagcgca atctccagtg acagaagata ctgctagaaa tctgctagaa aaaaaacaaa 1860
aaaggcatgt atagaggaat tatgagggaa agataccaag tcacggttta ttcttcaaaa 1920
tggaggtggc ttgttgggaa ggtggaagct catttggcca gagtggaaat ggaattggga 1980
gaaatcgatg accaaatgta aacacttggt gcctgatata gcttgacacc aagttagccc 2040
caagtg 2046
<210> 4
<211> 5094
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ccacttggta aaaaatgtga accccttgta tatagagttt ggctcacagt gtaaagggcc 60
tcagtgattc acattttcca gattaggaat ctgatgctca aagaagttaa atggcatagt 120
tggggtgaca cagctgtcta gtgggaggcc agccttctat attttagcca gcgttctttc 180
ctgcgggcca ggtcatgagg agtatgcaga ctctaagagg gagcaaaagt atctgaagga 240
tttaatattt tagcaaggaa tagatataca atcatccctt ggtctccctg ggggattggt 300
ttcaggaccc cttcttggac accaaatcta tggatattta agtcccttct ataaaatggt 360
atagtatttg catataacct atccacatcc tcctgtatac tttaaatcat ttctagatta 420
cttgtaatac ctaatacaat gtaaatgcta tgcaaatagt tgttattgtt taaggaataa 480
tgacaagaaa aaaaagtctg tacatgctca gtaaagacac aaccatccct ttttttcccc 540
agtgtttttg atccatggtt tgctgaatcc acagatgtgg agcccctgga tacggaaggc 600
ccgctgtact ttgaatgaca aataacagat ttaaaatttt caaggcatag ttttatacct 660
gatggccagc tttgtttatt tgaccaagaa tctgagttag ctagttctag gtactgacag 720
gataaataaa acacaacact gctcccgatc ttctcagttt agcagaggga cagatatgca 780
ctcaaataat taaaatatat cctgataaga atatagcata ggtacgcgcg aagaacttgg 840
caatcgaaat tttgttgttc aggctgggcg aggtgtctca tgtctgtaat cccagcactt 900
tgggaggcca tggtaggatg atcgcttgag cccaggagtc cgagaccagc ctgggcaaca 960
taacaagacc ctgtctcaat tcaaagaatt gaattaaaaa aaacaaaaaa taattttttt 1020
aaaaaagaaa tgttgttgtt caaggaacaa caacaaaaat ctagggaggt gttagagaag 1080
ccatttgcct gagctgagag taagttgcta gtggttctct tgattggtag gtggggcctg 1140
ggtttccagg catggtagcc aggaaggaca gccacatggc aggtttgggt aattccaaac 1200
agtggaggaa gggtgtctgg gggaagactt gtaggaactc agctgaaaaa attgggggat 1260
gatactctga aagaaaaaca aagttttaaa atttctactc ttacacttaa cacataatgc 1320
ttctgtgacc ggatatttag gggttttccc cccacactct gttaggagaa aaattttaga 1380
cagattaaat ttaacagagt ttaactgagc aaaaatgatt ctcgaaccag gcagctgccg 1440
gagccagaat aggttcaaaa tgactctggg ggtgccacat ggttggatga catttaggga 1500
cagaaaaagg aaagtgatgt gcagaaaatg gaagtcaggg gcagaagcag ccagattggt 1560
tgcagttcag catttgcctc atttaaacag ggtttgaaga gttggccacc tgtgattggc 1620
tgagactctg tggtataaga gtaagttaca gtctgtttac acatccagtt aggttacagt 1680
tcactatgca gagagaaatc tttagcctga acttacacag ggaggcagtt ttatttattt 1740
atttaatttt ttttttttga gacaaggtct cactctgtca cccgggctgg agttcagtgg 1800
tatgatcatg gcttattgca gcctcgactt cctggcctca agcaatcctt ccgcctcagg 1860
ctctagagta gctgggacta caggcacatg tcagcatgcc tggctaattt tgttttttaa 1920
tttttagtag agatgaactc ctggccttgc acaattcttt cgcctcagcc tccgaaaatg 1980
ctgggattac aggtgtgagc cactgtgccc agctaaggca actttaggct aaaccttttt 2040
ttgagacaga gtttctctct tgttgtctag gctggagtgc agttgcacca tcttggctca 2100
ctgcaacctc cacctccagg gttcaagtga ttctcgttcc tcagcctccc gagtaactgg 2160
gattacaggc atgcgccacc acgcctggct aattttgtgt ttttagtaga gatggtgttt 2220
caccatgttg tccaggctgg tctcaaactc ctggcctcaa gtgatcctct ggcctcagct 2280
tcccagagta ctgagattac aggcatgagc cactgtgccc tgcctaggct aaacttaatt 2340
taacaacacc aaacaatctc cagcagacac caactgggta tcccataatt caattcgatt 2400
ttgattggat ctacctggag atggtgtcag atcccgctgg ttgagggttc agtcccacaa 2460
gactgccctc cacttcagat gccaatcaca cattgtaggt tgttacctct acttctgact 2520
gaccagctgg aaaccagaac tcccatgact gcctccttga ctttggttaa tttgctagga 2580
cagttcatat ttaccaatct attataaaag attaaaggct acagacgaat aactagatga 2640
aaagatgaat agggctatat gtagggggtt gtggtggtga cagtccatgc cctctccagg 2700
tgtatgccac cctcccagca cctccacaca ttcagcaaac aggaagctca tcgttcaaga 2760
gtttttatag agcttgatct ccagctcccc ttcaccttcc cagaggtgga tgggtggggg 2820
tggaagttcc aacacactaa tcttctcatc acttggtctt tctggtgact agcaccatcc 2880
tgaggctatc taggggccca aacctatgag tataacctca ttagcatata ctcaagggtt 2940
accaaagagg cttattatta ataataaaag acactcctat cactcaaaat tcaaaggatt 3000
ttaggaactt ctgacaggaa ctggggacaa agaccaaatg tgtttcatat tataccacac 3060
ttacctaggc cattcattaa ctcttcacct ggctgcaaac taaaattaca tgtagagttt 3120
gtaagaacaa aacaaacaca cacacagaca cacacacaca cacacacaca cacacacaca 3180
cacacacaca aaacaacaag gcaggcacgg tggctcacgc ctgtaagccc aacactttgg 3240
aaagtcaagg tgagcagatt acgaggagtt caagaccagc ctggctaaca tggtgaaacc 3300
ctgtctctac taagaataca aaaattagcc aggcatggtg gcacgtgcct ataatcccag 3360
ctactcagga ggctgaggca agagaatcgc ttgaacctcg gaggcagagg ttgcagtgag 3420
ccgagatcat gccattgcac tccagcctga gtgacagagt gagactccat ctcaaaaaca 3480
gcaacaacaa caacaacaac aacaacaaca acaacaacaa ctcaccagct tcccagactc 3540
ccatccaggc caattaaacc ataatctctc ggtatggagc ccaggtatag tgttattttt 3600
taaaaaacct ctagatagtt ttaatgtaca tccaagattg acagacccat tgagttcagt 3660
cataaagaac tctctctcca ttccatatca ttcaacagag ctattttgtg aggggctgct 3720
ctgctggctg atattacaag gtaatactat gtcaaagtca aatgaaatat agagttgaat 3780
ctctgaaatt aaaatgtttt atttgggagg aaagaattgc aactcagggc atacacacag 3840
accagctggt ctttggcatg tctgaagtac aaaaaggttt tataaaatgg gaaaatgtta 3900
cttatcgctc tgagaaaatt tcactggtgc tagtaaagtt ttgaggagct ggcaagtttt 3960
gattggcaaa tgatgacaat aaacagaact agtctcagag ttgtagcagg tcatttcagt 4020
agccattagt taaacctggt ttcagatgat agcaggcagt ttcagcttcc aggcttgcag 4080
aaaatcgcat ttttggaaca atgttttgtg ccctgagtgc ttctccctgg cttcttgact 4140
ctgttttagt tgagtaagac aaaaatgacc cagtttgatg accaactttc acagttcctt 4200
tggagcatgc cttagtttct ctttttagaa gagagcaagc aaagaggagg aatacaggaa 4260
gcttaattaa ccttattaaa tataattgtg caatgcaaat taaactgaaa agtcccaaca 4320
tcagtaatag agaagtagat cctacatact gacatggaag catacccata ctatattgct 4380
gaaacaaaaa ttagtttcta caaaatatgt gtaagaagcc actttaaaaa aacaaactag 4440
gtggggcatg gtggctcaca cctgtaatcc cagcattttt ggaggccaag gtgggaggac 4500
tgcgtgagct aagaagtttg agacaagcct gggcaacata gtgagaccca gtctctctct 4560
ctatttatgt atttatttat gtatgtacat atttattgat ttatttattt gagacggagt 4620
cttgccctgt cacccaggct ggagtgcaat gtcgtgatct cagctcactg caacctccac 4680
ctcccaggtt caaacgattc tcctgcctca gcctcctgag tagctggaat tacaggcacc 4740
cgccaccata cccagctaat tattttttgt atttttagta gagacgggat ttcatcatgt 4800
tggtcaggct ggtctcaaac tcctgacctc atgatccacc cgcctcggcc tcccaaagtg 4860
ctgggattac aggtgtgagc caccgcgccc ggccgagacc cagtctcttt aaaaaaaaat 4920
ggctaacatg gtgaaacccc atctctacta aaaatacaaa aattagctgg gtgtggtggt 4980
acatacctgt aatcccagct acttgggagg ctgaggcagg agaatcactt gaacccagga 5040
ggcggaggtt gcagtgagcg gagattgcat caccgcacac tagcctgggt gaca 5094
<210> 5
<211> 3197
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gagatggagt cttgctaggt cacccaggct ggagtgcagt ggtgcgatct cggctcactg 60
caacctctgc ctcctgggtt cacaccattc tcctgcctca gcctcccaag tagctgggac 120
tacaggtgcc tgccaccacg ccaggttaat tttttgtatt tttagtagag acagggtttc 180
accgtgttag ccaggatggt ctcgatctcc tgacctcatg atccacccat cttggcctcc 240
caaagtgctg ggattacagg catgaaccac tgcgcccggc cgcatcgcta gtttttaaaa 300
actttttgta gagacagatt cttactatgt tgccaaggct ggtctcaaac tcctggcctc 360
aagagatcct ccagtcttcg gcctcccaaa aagatgggat tacaggcatg agccacctca 420
cctggcctct tttttttgta tattacctga tctcaggtat tctgctatag caacagaaag 480
acgaagacag aatccttagc tgtctgcaag tgtgcatgcc attttcatca tctgaagagt 540
cagcgagtgt cttaggtgga gtcttgcaaa agcaggccct gagccaaaga tttggatgca 600
aatgacttgt taagaaaggg ctcttcgaga ccgtgccatt gcactccagc ccgggcaaga 660
agagtgaaac tctgtttcaa aaaaaaaaaa gtggggggct cctaggaaaa gaacagtaaa 720
ggagtggggg atgaaggaca gggaatggga agaagccaag cgagagcatg atttccgaag 780
tcctacactc agcctgatca cacgggaagc tttagaacaa agaacacacc tcagagtttt 840
tcctgcctca acacaaagga gctgggcttt ggtgctcttc atcagcctgt ctttggctat 900
ccagggttgt ggaacgaggt gaaacataaa actccgaggt acttccggct ccctccagtg 960
tctgagggta atctgcagga ctgagggtaa ttgcagatgc tagctgttag cagcaaacta 1020
tgcaaaagct gaggactggc ttataaagcc agatctgggt gagtcatgtt tcctgtcaac 1080
atcctctgct gggcccataa cacatgcaac cccaaacttc cattacaagt tcaaagtttc 1140
taaggggata gcattacagt gtgtatgata ttggactcag acctgagttt gaatcctaat 1200
tccacaaaag aaattggaaa agagtcatat tgctgacttg accctttgtc accatatcca 1260
taaaatggga taattattcc tatatcataa atttacttat ttattcactt agtcatttgt 1320
taaataaata tggagtgtct actttgtgcc gggcactctt tttagggtgg ttctgagaag 1380
gggatggcaa tgagaagggc tctctaagat gcaagactcc aggcaactgc ttttacttcc 1440
agtggttctt tattttcaca gctcattaga gcaaattacc acagcaggga gatacaggtt 1500
gagtatccct tatccgaaaa gcctggcacc agaagtgttt taaattttgg attttttttt 1560
gatttttgga atatttgtta attatcagtt gagcatctct aatgtgaaaa tctaaaatcc 1620
aaaatgcccc agtgaccctt tcctttgagc atcatgttgg tgctcaaaaa gtttgagatg 1680
ttggagcatt taggatttca gatttttgga ttaaggatac tcattttgta caatggaaaa 1740
cttcttggca ctaatctgat gaaagaagaa actacgggag aatccctttc cctaaaaggc 1800
tttcagtaac aagatggccc tgactcattc tggacagttc ctccaaaggt aggcctacag 1860
gtagaactct tttctggcag cataggggtt ttccagaatg aaggtgtatt ttttttttac 1920
tgaggccagg aaaggtgaag agaatgaagg gttcctagtg acgatgcagc aaccaagtca 1980
tttatgtaat ggagccttat ctgacagatt tctaggatag aaactcagta tccaagctca 2040
ggcttggcag actgaggtgg gcaccgtccc caggattgca gtgtggatta gaggtttcaa 2100
ccccaatcat attcaacagc tccctttccc ccaacaaccc ctttttataa caattgtttt 2160
gtggtgattc ttttactaat atgatcatga aaattaagta atttagcttg tgaatgctct 2220
agcttggctg cactgctggt ataaagaagt gccagacact tgcaactgcc taggatctct 2280
gtgaaccagc ggcatggaag actgagaggc acgctggatt gatggctcag attcaagtat 2340
ggtattgcag gttctggtgt aattttctaa aatggcgaaa atctctaggt aaaactccaa 2400
aaataaaaaa ccaacataca tcttcctttg agttacctgg tagttatatt cctggaaaat 2460
tcagtgtata tgagcctgag gtagtagatg gtcaataaat actcaagaaa taaaaaaata 2520
aaaaaatata aaaatataaa acacatttct tacatgtcct tggtgcttgt aagcccagat 2580
tatatgcagg cttttttttg gacggagtct ctgttgccca ggctggagtg aagtggcgag 2640
atctgggctc actgcaacct ccacctcctg ggttcaagcg atcctcctgc ctcagcctcc 2700
tgagtagctg ggattacagg cacgcgccac caccctcagc tgatctttgt atttttagta 2760
gagaaggggt ttcaccatgt tggccaggct gatgtagaac tcctgacctc aagtgatccg 2820
cccacctcgg cctcccaaag tgttgggatt acaggcgtga gtcacagcgc ccggctgctt 2880
ttttatacat taagtgtgct tgcagaggac tgcgacctct ggccctcggc ctcttaataa 2940
atactccaag tgacttcatt ggaacaacca ctgagaatca cttatctaga gagtgggaag 3000
ttgctgatct catcactgca tggggtgggg tgagggaagc aaccctgccc ttcccctttc 3060
tcggaaagca gctggctttg aagaaagaga aaccaggacg ggaaagtcct gatttctaat 3120
ctgaaacagc gctttttgtc cagagaccgg tgacgagcga ccctgggctc gggttttgat 3180
tgggcagttc ggaaact 3197
<210> 6
<211> 1780
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ggtgcatgca ggggagcgca gagccctaag cccttctctg gggtccgccc gttttcctgc 60
tgggcttctc atttcctcac taggttctac ggtttgccga tctaaatcca gttcgtgtcc 120
tattgtattg tgtaacattt ttggcagctt acatctttgt ttattgagat atagttcaca 180
aaccatacaa ttaacatatt taagtgtaca tttcagtggg ttttagtata ttcacagggt 240
tgtgtaacca cctcaatttt aggacatttt cgtcactccc aaaagaaatt ttgtaccagt 300
tcggtgtcac tcccatttct cccaaacccc tagtcctagg aaaccaccaa tctttctgtc 360
tctatgggtt tgcctattag tatttcatag aaacagaaac atataatatg tggtcttttg 420
taactggctt ctgttttcaa aggtcgtcat attgtagcaa ggatcagttc ttcattccta 480
tttattgatg aatattactc tactgtatag atatactaag ttttgtttat ccatctatag 540
ttaatgggca tttggattgt ttccactctt cggctactga gaataaaatg tggttatcaa 600
tattcatgta taaatttttg gtgtggacat agattttcaa ttctcttgag tatatatgca 660
ggagtggaat tgctgggtga tatggtaact ctatgtttaa tcttttaagg aactactagg 720
ctgttctcca aagctgaatg taccattgtg tatgagagtt ccaatttttc tacatcctca 780
ccaatacttt taatcttttt ttattataac cattctagtg gatacgaaat ggtatctctt 840
tatggttttg atttgtattt ccctaatgac tatgcattat tttaaaatta taagctaatt 900
aatttttaca aagatttaca tgctgtgatt ttgacaactt aacttctagg tcacatgctc 960
aaaagtcaca gagcctagac ccaaaccaag cctttctatg gctaatgctc gaactccatc 1020
ctctctttcc tagaaggcaa tctgggataa atttttcaga actccatttg ttgcctctgt 1080
tagaaagaaa acactgggca agatgaacct gcaagtagag taaatggttt atttcttatt 1140
tgcttttagg agtatgtaag gagatttttt ggtctcccgt ttttacttta taaaaaatga 1200
gtgagtgggc caagcgctgt ggctcatgcc tgtgatccca gcactttggg agaccgaggc 1260
aggcagatca cctgaggtca ggagtttgat atcagcctga ccaacatggc aaaaccccat 1320
ctctgctaaa aatacaaaaa ttagccgggt gtagtgccat gcacctgtaa ttccagctat 1380
atgggaggct gaggcatgaa cattgcttga accccgggga gacggaggtt gcagtgagcc 1440
aagattgcac cactgcactc cagcctgggt gacagagtga ggctctttct caaaaaaaaa 1500
aaaaaggaaa gagaaagctc ttaattgaag tagtaaccct ttcttgggtg ttcaatgata 1560
ttgtgcttct cttcatgtaa gtgcccctta gattcctcct cctcactacc atcaggtcat 1620
agaataggga ggggcactca gtttcaacag cagtggcctc caaattttgt tcaaatgcat 1680
aactataaaa agttttttgt atacattctc tatatatagc tatacatttt atacatacac 1740
tactatcagt cttgatttag acattactaa agcttaattt 1780
<210> 7
<211> 19214
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
acctactatg tgccagcccc tgttctaggg tggaaactaa gagaatgatg tacctagagg 60
gcgctggaag ctctaaagcc ctagcagtta ctgcttttac tattagtggt cgtttttttc 120
tcccccccgc cccccgacaa atcaacagaa caaagaaaat tacctaaaca gcaaggacat 180
agggaggaac ttcttggcac agaactttcc aaacactttt tcctgaaggg atacaagaag 240
caagaaaggt actctttcac taggaccttc tctgagctgt cctcaggatg cttttgggac 300
tatttttctt acccagagaa tggagaaacc ctgcagggaa ttcccaagct gtagttataa 360
acagaagttc tccttctgct aggtagcatt caaagatctt aatcttctgg gtttccgttt 420
tctcgaatga aaaatgcagg tccgagcagt taactggctg gggcaccatt agcaagtcac 480
ttagcatctc tggggccagt ctgcaaagcg agggggcagc cttaatgtgc ctccagcctg 540
aagtcctaga atgagcgccc ggtgtcccaa gctggggcgc gcaccccaga tcggagggcg 600
ccgatgtaca gacagcaaac tcacccagtc tagtgcatgc cttcttaaac atcacgagac 660
tctaagaaaa ggaaactgaa aacgggaaag tccctctctc taacctggca ctgcgtcgct 720
ggcttggaga caggtgacgg tccctgcggg ccttgtcctg attggctggg cacgcgttta 780
atataagtgg aggcgtcgcg ctggcgggca ttcctgaagc tgacagcatt cgggccgaga 840
tgtctcgctc cgtggcctta gctgtgctcg cgctactctc tctttctggc ctggaggcta 900
tccagcgtga gtctctccta ccctcccgct ctggtccttc ctctcccgct ctgcaccctc 960
tgtggccctc gctgtgctct ctcgctccgt gacttccctt ctccaagttc tccttggtgg 1020
cccgccgtgg ggctagtcca gggctggatc tcggggaagc ggcggggtgg cctgggagtg 1080
gggaaggggg tgcgcacccg ggacgcgcgc tacttgcccc tttcggcggg gagcagggga 1140
gacctttggc ctacggcgac gggagggtcg ggacaaagtt tagggcgtcg ataagcgtca 1200
gagcgccgag gttgggggag ggtttctctt ccgctctttc gcggggcctc tggctccccc 1260
agcgcagctg gagtggggga cgggtaggct cgtcccaaag gcgcggcgct gaggtttgtg 1320
aacgcgtgga ggggcgcttg gggtctgggg gaggcgtcgc ccgggtaagc ctgtctgctg 1380
cggctctgct tcccttagac tggagagctg tggacttcgt ctaggcgccc gctaagttcg 1440
catgtcctag cacctctggg tctatgtggg gccacaccgt ggggaggaaa cagcacgcga 1500
cgtttgtaga atgcttggct gtgatacaaa gcggtttcga ataattaact tatttgttcc 1560
catcacatgt cacttttaaa aaattataag aactacccgt tattgacatc tttctgtgtg 1620
ccaaggactt tatgtgcttt gcgtcattta attttgaaaa cagttatctt ccgccataga 1680
taactactat ggttatcttc tgcctctcac agatgaagaa actaaggcac cgagatttta 1740
agaaacttaa ttacacaggg gataaatggc agcaatcgag attgaagtca agcctaacca 1800
gggcttttgc gggagcgcat gccttttggc tgtaattcgt gcattttttt ttaagaaaaa 1860
cgcctgcctt ctgcgtgaga ttctccagag caaactgggc ggcatgggcc ctgtggtctt 1920
ttcgtacaga gggcttcctc tttggctctt tgcctggttg tttccaagat gtactgtgcc 1980
tcttactttc ggttttgaaa acatgagggg gttgggcgtg gtagcttacg cctgtaatcc 2040
cagcacttag ggaggccgag gcgggaggat ggcttgaggt ccgtagttga gaccagcctg 2100
gccaacatgg tgaagcctgg tctctacaaa aaataataac aaaaattagc cgggtgtggt 2160
ggctcgtgcc tgtggtccca gctgctccgg tggctgaggc gggaggatct cttgagctta 2220
ggcttttgag ctatcatggc gccagtgcac tccagcgtgg gcaacagagc gagaccctgt 2280
ctctcaaaaa agaaaaaaaa aaaaaaagaa agagaaaaga aaagaaagaa agaagtgaag 2340
gtttgtcagt caggggagct gtaaaaccat taataaagat aatccaagat ggttaccaag 2400
actgttgagg acgccagaga tcttgagcac tttctaagta cctggcaata cactaagcgc 2460
gctcaccttt tcctctggca aaacatgatc gaaagcagaa tgttttgatc atgagaaaat 2520
tgcatttaat ttgaatacaa tttatttaca acataaagga taatgtatat atcaccacca 2580
ttactggtat ttgctggtta tgttagatgt cattttaaaa aataacaatc tgatatttaa 2640
aaaaaaatct tattttgaaa atttccaaag taatacatgc catgcataga ccatttctgg 2700
aagataccac aagaaacatg taatgatgat tgcctctgaa ggtctatttt cctcctctga 2760
cctgtgtgtg ggttttgttt ttgttttact gtgggcataa attaattttt cagttaagtt 2820
ttggaagctt aaataactct ccaaaagtca taaagccagt aactggttga gcccaaattc 2880
aaacccagcc tgtctgatac ttgtcctctt cttagaaaag attacagtga tgctctcaca 2940
aaatcttgcc gccttccctc aaacagagag ttccaggcag gatgaatctg tgctctgatc 3000
cctgaggcat ttaatatgtt cttattatta gaagctcaga tgcaaagagc tctcttagct 3060
tttaatgtta tgaaaaaaat caggtcttca ttagattccc caatccacct cttgatgggg 3120
ctagtagcct ttccttaatg atagggtgtt tctagagaga tatatctggt caaggtggcc 3180
tggtactcct ccttctcccc acagcctccc agacaaggag gagtagctgc cttttagtga 3240
tcatgtaccc tgaatataag tgtatttaaa agaattttat acacatatat ttagtgtcaa 3300
tctgtatatt tagtagcact aacacttctc ttcattttca atgaaaaata tagagtttat 3360
aatattttct tcccacttcc ccatggatgg tctagtcatg cctctcattt tggaaagtac 3420
tgtttctgaa acattaggca atatattccc aacctggcta gtttacagca atcacctgtg 3480
gatgctaatt aaaacgcaaa tcccactgtc acatgcatta ctccatttga tcataatgga 3540
aagtatgttc tgtcccattt gccatagtcc tcacctatcc ctgttgtatt ttatcgggtc 3600
caactcaacc atttaaggta tttgccagct cttgtatgca tttaggtttt gtttctttgt 3660
tttttagctc atgaaattag gtacaaagtc agagaggggt ctggcatata aaacctcagc 3720
agaaataaag aggttttgtt gtttggtaag aacatacctt gggttggttg ggcacggtgg 3780
ctcgtgcctg taatcccaac actttgggag gccaaggcag gctgatcact tgaagttggg 3840
agttcaagac cagcctggcc aacatggtga aatcccgtct ctactgaaaa tacaaaaatt 3900
aaccaggcat ggtggtgtgt gcctgtagtc ccaggaatca cttgaaccca ggaggcggag 3960
gttgcagtga gctgagatct caccactgca cactgcactc cagcctgggc aatggaatga 4020
gattccatcc caaaaaataa aaaaataaaa aaataaagaa cataccttgg gttgatccac 4080
ttaggaacct cagataataa catctgccac gtatagagca attgctatgt cccaggcact 4140
ctactagaca cttcatacag tttagaaaat cagatgggtg tagatcaagg caggagcagg 4200
aaccaaaaag aaaggcataa acataagaaa aaaaatggaa ggggtggaaa cagagtacaa 4260
taacatgagt aatttgatgg gggctattat gaactgagaa atgaactttg aaaagtatct 4320
tggggccaaa tcatgtagac tcttgagtga tgtgttaagg aatgctatga gtgctgagag 4380
ggcatcagaa gtccttgaga gcctccagag aaaggctctt aaaaatgcag cgcaatctcc 4440
agtgacagaa gatactgcta gaaatctgct agaaaaaaaa caaaaaaggc atgtatagag 4500
gaattatgag ggaaagatac caagtcacgg tttattcttc aaaatggagg tggcttgttg 4560
ggaaggtgga agctcatttg gccagagtgg aaatggaatt gggagaaatc gatgaccaaa 4620
tgtaaacact tggtgcctga tatagcttga caccaagtta gccccaagtg aaataccctg 4680
gcaatattaa tgtgtctttt cccgatattc ctcaggtact ccaaagattc aggtttactc 4740
acgtcatcca gcagagaatg gaaagtcaaa tttcctgaat tgctatgtgt ctgggtttca 4800
tccatccgac attgaagttg acttactgaa gaatggagag agaattgaaa aagtggagca 4860
ttcagacttg tctttcagca aggactggtc tttctatctc ttgtactaca ctgaattcac 4920
ccccactgaa aaagatgagt atgcctgccg tgtgaaccat gtgactttgt cacagcccaa 4980
gatagttaag tggggtaagt cttacattct tttgtaagct gctgaaagtt gtgtatgagt 5040
agtcatatca taaagctgct ttgatataaa aaaggtctat ggccatacta ccctgaatga 5100
gtcccatccc atctgatata aacaatctgc atattgggat tgtcagggaa tgttcttaaa 5160
gatcagatta gtggcacctg ctgagatact gatgcacagc atggtttctg aaccagtagt 5220
ttccctgcag ttgagcaggg agcagcagca gcacttgcac aaatacatat acactcttaa 5280
cacttcttac ctactggctt cctctagctt ttgtggcagc ttcaggtata tttagcactg 5340
aacgaacatc tcaagaaggt ataggccttt gtttgtaagt cctgctgtcc tagcatccta 5400
taatcctgga cttctccagt actttctggc tggattggta tctgaggcta gtaggaaggg 5460
cttgttcctg ctgggtagct ctaaacaatg tattcatggg taggaacagc agcctattct 5520
gccagcctta tttctaacca ttttagacat ttgttagtac atggtatttt aaaagtaaaa 5580
cttaatgtct tccttttttt tctccactgt ctttttcata gatcgagaca tgtaagcagc 5640
atcatggagg taagtttttg accttgagaa aatgtttttg tttcactgtc ctgaggacta 5700
tttatagaca gctctaacat gataaccctc actatgtgga gaacattgac agagtaacat 5760
tttagcaggg aaagaagaat cctacagggt catgttccct tctcctgtgg agtggcatga 5820
agaaggtgta tggccccagg tatggccata ttactgaccc tctacagaga gggcaaagga 5880
actgccagta tggtattgca ggataaaggc aggtggttac ccacattacc tgcaaggctt 5940
tgatctttct tctgccattt ccacattgga catctctgct gaggagagaa aatgaaccac 6000
tcttttcctt tgtataatgt tgttttattc ttcagacaga agagaggagt tatacagctc 6060
tgcagacatc ccattcctgt atggggactg tgtttgcctc ttagaggttc ccaggccact 6120
agaggagata aagggaaaca gattgttata acttgatata atgatactat aatagatgta 6180
actacaagga gctccagaag caagagagag ggaggaactt ggacttctct gcatctttag 6240
ttggagtcca aaggcttttc aatgaaattc tactgcccag ggtacattga tgctgaaacc 6300
ccattcaaat ctcctgttat attctagaac agggaattga tttgggagag catcaggaag 6360
gtggatgatc tgcccagtca cactgttagt aaattgtaga gccaggacct gaactctaat 6420
atagtcatgt gttacttaat gacggggaca tgttctgaga aatgcttaca caaacctagg 6480
tgttgtagcc tactacacgc ataggctaca tggtatagcc tattgctcct agactacaaa 6540
cctgtacagc ctgttactgt actgaatact gtgggcagtt gtaacacaat ggtaagtatt 6600
tgtgtatcta aacatagaag ttgcagtaaa aatatgctat tttaatctta tgagaccact 6660
gtcatatata cagtccatca ttgaccaaaa catcatatca gcattttttc ttctaagatt 6720
ttgggagcac caaagggata cactaacagg atatactctt tataatgggt ttggagaact 6780
gtctgcagct acttctttta aaaaggtgat ctacacagta gaaattagac aagtttggta 6840
atgagatctg caatccaaat aaaataaatt cattgctaac ctttttcttt tcttttcagg 6900
tttgaagatg ccgcatttgg attggatgaa ttccaaattc tgcttgcttg ctttttaata 6960
ttgatatgct tatacactta cactttatgc acaaaatgta gggttataat aatgttaaca 7020
tggacatgat cttctttata attctacttt gagtgctgtc tccatgtttg atgtatctga 7080
gcaggttgct ccacaggtag ctctaggagg gctggcaact tagaggtggg gagcagagaa 7140
ttctcttatc caacatcaac atcttggtca gatttgaact cttcaatctc ttgcactcaa 7200
agcttgttaa gatagttaag cgtgcataag ttaacttcca atttacatac tctgcttaga 7260
atttggggga aaatttagaa atataattga caggattatt ggaaatttgt tataatgaat 7320
gaaacatttt gtcatataag attcatattt acttcttata catttgataa agtaaggcat 7380
ggttgtggtt aatctggttt atttttgttc cacaagttaa ataaatcata aaacttgatg 7440
tgttatctct tatatctcac tcccactatt acccctttat tttcaaacag ggaaacagtc 7500
ttcaagttcc acttggtaaa aaatgtgaac cccttgtata tagagtttgg ctcacagtgt 7560
aaagggcctc agtgattcac attttccaga ttaggaatct gatgctcaaa gaagttaaat 7620
ggcatagttg gggtgacaca gctgtctagt gggaggccag ccttctatat tttagccagc 7680
gttctttcct gcgggccagg tcatgaggag tatgcagact ctaagaggga gcaaaagtat 7740
ctgaaggatt taatatttta gcaaggaata gatatacaat catcccttgg tctccctggg 7800
ggattggttt caggacccct tcttggacac caaatctatg gatatttaag tcccttctat 7860
aaaatggtat agtatttgca tataacctat ccacatcctc ctgtatactt taaatcattt 7920
ctagattact tgtaatacct aatacaatgt aaatgctatg caaatagttg ttattgttta 7980
aggaataatg acaagaaaaa aaagtctgta catgctcagt aaagacacaa ccatcccttt 8040
ttttccccag tgtttttgat ccatggtttg ctgaatccac agatgtggag cccctggata 8100
cggaaggccc gctgtacttt gaatgacaaa taacagattt aaaattttca aggcatagtt 8160
ttatacctga tggccagctt tgtttatttg accaagaatc tgagttagct agttctaggt 8220
actgacagga taaataaaac acaacactgc tcccgatctt ctcagtttag cagagggaca 8280
gatatgcact caaataatta aaatatatcc tgataagaat atagcatagg tacgcgcgaa 8340
gaacttggca atcgaaattt tgttgttcag gctgggcgag gtgtctcatg tctgtaatcc 8400
cagcactttg ggaggccatg gtaggatgat cgcttgagcc caggagtccg agaccagcct 8460
gggcaacata acaagaccct gtctcaattc aaagaattga attaaaaaaa acaaaaaata 8520
atttttttaa aaaagaaatg ttgttgttca aggaacaaca acaaaaatct agggaggtgt 8580
tagagaagcc atttgcctga gctgagagta agttgctagt ggttctcttg attggtaggt 8640
ggggcctggg tttccaggca tggtagccag gaaggacagc cacatggcag gtttgggtaa 8700
ttccaaacag tggaggaagg gtgtctgggg gaagacttgt aggaactcag ctgaaaaaat 8760
tgggggatga tactctgaaa gaaaaacaaa gttttaaaat ttctactctt acacttaaca 8820
cataatgctt ctgtgaccgg atatttaggg gttttccccc cacactctgt taggagaaaa 8880
attttagaca gattaaattt aacagagttt aactgagcaa aaatgattct cgaaccaggc 8940
agctgccgga gccagaatag gttcaaaatg actctggggg tgccacatgg ttggatgaca 9000
tttagggaca gaaaaaggaa agtgatgtgc agaaaatgga agtcaggggc agaagcagcc 9060
agattggttg cagttcagca tttgcctcat ttaaacaggg tttgaagagt tggccacctg 9120
tgattggctg agactctgtg gtataagagt aagttacagt ctgtttacac atccagttag 9180
gttacagttc actatgcaga gagaaatctt tagcctgaac ttacacaggg aggcagtttt 9240
atttatttat ttaatttttt ttttttgaga caaggtctca ctctgtcacc cgggctggag 9300
ttcagtggta tgatcatggc ttattgcagc ctcgacttcc tggcctcaag caatccttcc 9360
gcctcaggct ctagagtagc tgggactaca ggcacatgtc agcatgcctg gctaattttg 9420
ttttttaatt tttagtagag atgaactcct ggccttgcac aattctttcg cctcagcctc 9480
cgaaaatgct gggattacag gtgtgagcca ctgtgcccag ctaaggcaac tttaggctaa 9540
accttttttt gagacagagt ttctctcttg ttgtctaggc tggagtgcag ttgcaccatc 9600
ttggctcact gcaacctcca cctccagggt tcaagtgatt ctcgttcctc agcctcccga 9660
gtaactggga ttacaggcat gcgccaccac gcctggctaa ttttgtgttt ttagtagaga 9720
tggtgtttca ccatgttgtc caggctggtc tcaaactcct ggcctcaagt gatcctctgg 9780
cctcagcttc ccagagtact gagattacag gcatgagcca ctgtgccctg cctaggctaa 9840
acttaattta acaacaccaa acaatctcca gcagacacca actgggtatc ccataattca 9900
attcgatttt gattggatct acctggagat ggtgtcagat cccgctggtt gagggttcag 9960
tcccacaaga ctgccctcca cttcagatgc caatcacaca ttgtaggttg ttacctctac 10020
ttctgactga ccagctggaa accagaactc ccatgactgc ctccttgact ttggttaatt 10080
tgctaggaca gttcatattt accaatctat tataaaagat taaaggctac agacgaataa 10140
ctagatgaaa agatgaatag ggctatatgt agggggttgt ggtggtgaca gtccatgccc 10200
tctccaggtg tatgccaccc tcccagcacc tccacacatt cagcaaacag gaagctcatc 10260
gttcaagagt ttttatagag cttgatctcc agctcccctt caccttccca gaggtggatg 10320
ggtgggggtg gaagttccaa cacactaatc ttctcatcac ttggtctttc tggtgactag 10380
caccatcctg aggctatcta ggggcccaaa cctatgagta taacctcatt agcatatact 10440
caagggttac caaagaggct tattattaat aataaaagac actcctatca ctcaaaattc 10500
aaaggatttt aggaacttct gacaggaact ggggacaaag accaaatgtg tttcatatta 10560
taccacactt acctaggcca ttcattaact cttcacctgg ctgcaaacta aaattacatg 10620
tagagtttgt aagaacaaaa caaacacaca cacagacaca cacacacaca cacacacaca 10680
cacacacaca cacacacaaa acaacaaggc aggcacggtg gctcacgcct gtaagcccaa 10740
cactttggaa agtcaaggtg agcagattac gaggagttca agaccagcct ggctaacatg 10800
gtgaaaccct gtctctacta agaatacaaa aattagccag gcatggtggc acgtgcctat 10860
aatcccagct actcaggagg ctgaggcaag agaatcgctt gaacctcgga ggcagaggtt 10920
gcagtgagcc gagatcatgc cattgcactc cagcctgagt gacagagtga gactccatct 10980
caaaaacagc aacaacaaca acaacaacaa caacaacaac aacaacaact caccagcttc 11040
ccagactccc atccaggcca attaaaccat aatctctcgg tatggagccc aggtatagtg 11100
ttatttttta aaaaacctct agatagtttt aatgtacatc caagattgac agacccattg 11160
agttcagtca taaagaactc tctctccatt ccatatcatt caacagagct attttgtgag 11220
gggctgctct gctggctgat attacaaggt aatactatgt caaagtcaaa tgaaatatag 11280
agttgaatct ctgaaattaa aatgttttat ttgggaggaa agaattgcaa ctcagggcat 11340
acacacagac cagctggtct ttggcatgtc tgaagtacaa aaaggtttta taaaatggga 11400
aaatgttact tatcgctctg agaaaatttc actggtgcta gtaaagtttt gaggagctgg 11460
caagttttga ttggcaaatg atgacaataa acagaactag tctcagagtt gtagcaggtc 11520
atttcagtag ccattagtta aacctggttt cagatgatag caggcagttt cagcttccag 11580
gcttgcagaa aatcgcattt ttggaacaat gttttgtgcc ctgagtgctt ctccctggct 11640
tcttgactct gttttagttg agtaagacaa aaatgaccca gtttgatgac caactttcac 11700
agttcctttg gagcatgcct tagtttctct ttttagaaga gagcaagcaa agaggaggaa 11760
tacaggaagc ttaattaacc ttattaaata taattgtgca atgcaaatta aactgaaaag 11820
tcccaacatc agtaatagag aagtagatcc tacatactga catggaagca tacccatact 11880
atattgctga aacaaaaatt agtttctaca aaatatgtgt aagaagccac tttaaaaaaa 11940
caaactaggt ggggcatggt ggctcacacc tgtaatccca gcatttttgg aggccaaggt 12000
gggaggactg cgtgagctaa gaagtttgag acaagcctgg gcaacatagt gagacccagt 12060
ctctctctct atttatgtat ttatttatgt atgtacatat ttattgattt atttatttga 12120
gacggagtct tgccctgtca cccaggctgg agtgcaatgt cgtgatctca gctcactgca 12180
acctccacct cccaggttca aacgattctc ctgcctcagc ctcctgagta gctggaatta 12240
caggcacccg ccaccatacc cagctaatta ttttttgtat ttttagtaga gacgggattt 12300
catcatgttg gtcaggctgg tctcaaactc ctgacctcat gatccacccg cctcggcctc 12360
ccaaagtgct gggattacag gtgtgagcca ccgcgcccgg ccgagaccca gtctctttaa 12420
aaaaaaatgg ctaacatggt gaaaccccat ctctactaaa aatacaaaaa ttagctgggt 12480
gtggtggtac atacctgtaa tcccagctac ttgggaggct gaggcaggag aatcacttga 12540
acccaggagg cggaggttgc agtgagcgga gattgcatca ccgcacacta gcctgggtga 12600
cagagcaaga ctttgtcaca cacacacaca caaaaaagtg gacgcttgca tcacttgagc 12660
ctgggaggtt gaggctgcag tgagcagaga tcaggacact acactccagc ctgggtgatg 12720
aagcaagtcc ctcaaaaagg aaaaaaaaaa aaagtagacc cttgcacccc aaatctgtat 12780
tgttcaaagg ttaactgtat cctcctttaa aaaaggggtt actgttaaat aataattttt 12840
atatcttact ttttttcatg atccctaagg aaaacatgtc acaaatggat atgtctttct 12900
agttttgtca acaaccactt tcacggtatt tttgggctgt tgctttttac ttgtcatttt 12960
tgtgttgtaa acccttgaag caaaactcaa ggtcttttct ttttttcttt tcttttcttt 13020
tctgagacag ggtattaccc tgtcacccag gcttgtgtgc agaggcacaa tcttggctca 13080
ctgcagcctc agcctcttgg actcaagcaa tcctcccatc tcagcctccc aagtagctga 13140
gaccacaggt gtgcaccacc cattcctggc taattttttt tgtgtatttt tttgtagaga 13200
tgggggtctc actatgtttc cctggctggt cttgaactct tgggttcaag cgatcctcca 13260
gtctcgcctc tcaaagtgct gggattacag agccaccaca ctcagccatt tcctataata 13320
aaaaaatatt ttgtctgtga tgtagccatc aaagttttag ggggaaaaat tggcattgtc 13380
tttccctgga gtctagacct ataggaaaaa aattgaaggc taatatttgg gttctagata 13440
acgcaaaata aataataaaa catctgtgat taccttgatt gaaaagtttg tgcatagaaa 13500
ttattctggt actttggaga gctacaagaa ggcctaacag aaaaaaaaac tgataaatac 13560
aattaaaata acagtcgagc atatattaaa gcacttacta tgcactaaac tctatatatc 13620
tcctgtcatt agttggatgc aattattaga catctttggt cctccacaac ataggtgatt 13680
aaaaagccta agaaactgaa gcgttatcta tgtctttggt aagacacaca ggcccaagtg 13740
aagcctgcaa tgatgtgcag gcttcccctc ccccacggac cacatggacc acttcctctt 13800
tttttttttt tttttttttc tttagacaga gtctcactgt atcgcctagg ctggagtgca 13860
gtggcgcaat ctcggctcac tgcaacctct gcttcctggg ttcaagtgat tctcctgcct 13920
cagcctcccg agtagctggg aatacaggca tgcaccacca tgcctgacta atttttttat 13980
gtgtgtgttt ttggtagaga tggggtttca ccatgctggc caggctggtc tcaaactcct 14040
gacctcgtga tctgctcgcc ttggcctccc aaagtgctgg gattacaggc acttcctctt 14100
tttactgttt tcacttagaa aaactgagag gacttttgct gccactgaat gacacagcat 14160
agttgaagtg gttttctcgt catttattat ggctcgaatg tttgtccctt ctgaaattca 14220
tgttgaaact taatcctggc tgggcacagt ggctcacact tctaatccca gcattttggg 14280
aggctgagac tggaggatcg catgagccca ggagtttgag accagcctgg gcaacatagt 14340
gagtcctcct ctctacaaaa tcaaacaaac aaaaattagc tgggtgtggt agcacacacc 14400
tgtggtccca gctacttgga aggcggaggc aggaggattg tttgaaccca ggaggagttt 14460
aagagcagtc tgggcaatat agtgagacct catatctgtc tatctatgta tctatgtatc 14520
tatgtatcta tgtatctatc tatctatcta tctatctaaa ataaattaaa ttaaaaaata 14580
aaaaataatg aggcaaagaa aagaaagtta atcctcaata taacagtatt aagaagtagg 14640
gcatttaaga ggtgactggg tcatgagggc tccttcatga atggattaat ggattaatgg 14700
gttatcctga gaatgggtct gttataaaag ccagtttcgc tctttctctt gtgcccctct 14760
aaatctttat ctttagccat gttatgatgc ctctctgata tctttaccca tgttatgatg 14820
cggaacaaag ccctcaccca aagcaaacca gatacagccc ctcaatgttg aacttctcag 14880
cctcttccat acatttttct tcctttcttt tttttgagat aggatcttgc tctgccattg 14940
tgccattgca gcctcaaact cttgggctca agtgatcctg ctgcttcagc atcctgagta 15000
gcggggctta cagatgcaca cccccatgca tcactaattt tttttttttt ttttgagatg 15060
gagtcttgct aggtcaccca ggctggagtg cagtggtgcg atctcggctc actgcaacct 15120
ctgcctcctg ggttcacacc attctcctgc ctcagcctcc caagtagctg ggactacagg 15180
tgcctgccac cacgccaggt taattttttg tatttttagt agagacaggg tttcaccgtg 15240
ttagccagga tggtctcgat ctcctgacct catgatccac ccatcttggc ctcccaaagt 15300
gctgggatta caggcatgaa ccactgcgcc cggccgcatc gctagttttt aaaaactttt 15360
tgtagagaca gattcttact atgttgccaa ggctggtctc aaactcctgg cctcaagaga 15420
tcctccagtc ttcggcctcc caaaaagatg ggattacagg catgagccac ctcacctggc 15480
ctcttttttt tgtatattac ctgatctcag gtattctgct atagcaacag aaagacgaag 15540
acagaatcct tagctgtctg caagtgtgca tgccattttc atcatctgaa gagtcagcga 15600
gtgtcttagg tggagtcttg caaaagcagg ccctgagcca aagatttgga tgcaaatgac 15660
ttgttaagaa agggctcttc gagaccgtgc cattgcactc cagcccgggc aagaagagtg 15720
aaactctgtt tcaaaaaaaa aaaagtgggg ggctcctagg aaaagaacag taaaggagtg 15780
ggggatgaag gacagggaat gggaagaagc caagcgagag catgatttcc gaagtcctac 15840
actcagcctg atcacacggg aagctttaga acaaagaaca cacctcagag tttttcctgc 15900
ctcaacacaa aggagctggg ctttggtgct cttcatcagc ctgtctttgg ctatccaggg 15960
ttgtggaacg aggtgaaaca taaaactccg aggtacttcc ggctccctcc agtgtctgag 16020
ggtaatctgc aggactgagg gtaattgcag atgctagctg ttagcagcaa actatgcaaa 16080
agctgaggac tggcttataa agccagatct gggtgagtca tgtttcctgt caacatcctc 16140
tgctgggccc ataacacatg caaccccaaa cttccattac aagttcaaag tttctaaggg 16200
gatagcatta cagtgtgtat gatattggac tcagacctga gtttgaatcc taattccaca 16260
aaagaaattg gaaaagagtc atattgctga cttgaccctt tgtcaccata tccataaaat 16320
gggataatta ttcctatatc ataaatttac ttatttattc acttagtcat ttgttaaata 16380
aatatggagt gtctactttg tgccgggcac tctttttagg gtggttctga gaaggggatg 16440
gcaatgagaa gggctctcta agatgcaaga ctccaggcaa ctgcttttac ttccagtggt 16500
tctttatttt cacagctcat tagagcaaat taccacagca gggagataca ggttgagtat 16560
cccttatccg aaaagcctgg caccagaagt gttttaaatt ttggattttt ttttgatttt 16620
tggaatattt gttaattatc agttgagcat ctctaatgtg aaaatctaaa atccaaaatg 16680
ccccagtgac cctttccttt gagcatcatg ttggtgctca aaaagtttga gatgttggag 16740
catttaggat ttcagatttt tggattaagg atactcattt tgtacaatgg aaaacttctt 16800
ggcactaatc tgatgaaaga agaaactacg ggagaatccc tttccctaaa aggctttcag 16860
taacaagatg gccctgactc attctggaca gttcctccaa aggtaggcct acaggtagaa 16920
ctcttttctg gcagcatagg ggttttccag aatgaaggtg tatttttttt ttactgaggc 16980
caggaaaggt gaagagaatg aagggttcct agtgacgatg cagcaaccaa gtcatttatg 17040
taatggagcc ttatctgaca gatttctagg atagaaactc agtatccaag ctcaggcttg 17100
gcagactgag gtgggcaccg tccccaggat tgcagtgtgg attagaggtt tcaaccccaa 17160
tcatattcaa cagctccctt tcccccaaca accccttttt ataacaattg ttttgtggtg 17220
attcttttac taatatgatc atgaaaatta agtaatttag cttgtgaatg ctctagcttg 17280
gctgcactgc tggtataaag aagtgccaga cacttgcaac tgcctaggat ctctgtgaac 17340
cagcggcatg gaagactgag aggcacgctg gattgatggc tcagattcaa gtatggtatt 17400
gcaggttctg gtgtaatttt ctaaaatggc gaaaatctct aggtaaaact ccaaaaataa 17460
aaaaccaaca tacatcttcc tttgagttac ctggtagtta tattcctgga aaattcagtg 17520
tatatgagcc tgaggtagta gatggtcaat aaatactcaa gaaataaaaa aataaaaaaa 17580
tataaaaata taaaacacat ttcttacatg tccttggtgc ttgtaagccc agattatatg 17640
caggcttttt tttggacgga gtctctgttg cccaggctgg agtgaagtgg cgagatctgg 17700
gctcactgca acctccacct cctgggttca agcgatcctc ctgcctcagc ctcctgagta 17760
gctgggatta caggcacgcg ccaccaccct cagctgatct ttgtattttt agtagagaag 17820
gggtttcacc atgttggcca ggctgatgta gaactcctga cctcaagtga tccgcccacc 17880
tcggcctccc aaagtgttgg gattacaggc gtgagtcaca gcgcccggct gcttttttat 17940
acattaagtg tgcttgcaga ggactgcgac ctctggccct cggcctctta ataaatactc 18000
caagtgactt cattggaaca accactgaga atcacttatc tagagagtgg gaagttgctg 18060
atctcatcac tgcatggggt ggggtgaggg aagcaaccct gcccttcccc tttctcggaa 18120
agcagctggc tttgaagaaa gagaaaccag gacgggaaag tcctgatttc taatctgaaa 18180
cagcgctttt tgtccagaga ccggtgacga gcgaccctgg gctcgggttt tgattgggca 18240
gttcggaaac tgtaaaagcg aattaaaagg gtgacaagct agtgttttag cctatccagt 18300
tccgggagtt tgcacgcaga cgctctgctt cgtgaccttg gctctgctct gtgggcgccg 18360
cccccagcct gggcgcgtcc atcgtcgagt accttctcct ctgcctcccc ctccctctgc 18420
ttctatctct ctccaattgc cctccctggc ctgcggccgc ccggtcctcc ttcccagccc 18480
agtgcagcca ggcacccggg ttcggcttgc tcaggtctct gtccgggact gggaagccac 18540
ggagggccgg gaaagtggca cactcctgga gctcaagctt cctactctct catcgctgaa 18600
taactaccgg gcaggactgg gtggaacaga cagcatattt aggtcattga tgggctgctg 18660
ggtggatgga gctggaaagt gatgggcact gctttctggg gctgcccagg ttccttccga 18720
gggctcgctt ttcctgggca gagcgggaaa gaggaggggc aggccggtcg cgaaacgcaa 18780
atagtcgaga atagcgatcc ggggagaagc aggtgtctgt ggggcccaag agaagtacca 18840
tctcggtaag taggccggtg catgcagggg agcgcagagc cctaagccct tctctggggt 18900
ccgcccgttt tcctgctggg cttctcattt cctcactagg ttctacggtt tgccgatcta 18960
aatccagttc gtgtcctatt gtattgtgta acatttttgg cagcttacat ctttgtttat 19020
tgagatatag ttcacaaacc atacaattaa catatttaag tgtacatttc agtgggtttt 19080
agtatattca cagggttgtg taaccacctc aattttagga cattttcgtc actcccaaaa 19140
gaaattttgt accagttcgg tgtcactccc atttctccca aacccctagt cctaggaaac 19200
caccaatctt tctg 19214
<210> 8
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
<210> 10
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
cctagcagtt gtggtcatcg g 21
<210> 11
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
ctccctcctt ttccacctga ac 22
<210> 12
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
tcctggttgt cctagctgtc 20
<210> 13
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
gagtgctgtc tccatgtttg atg 23
<210> 15
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
<210> 18
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gagggcctat ttcccatgat tcc 23
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
<210> 20
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
<210> 21
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
<210> 22
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Claims (10)
1. An enhancer, wherein the enhancer region is located adjacent to the human B2M gene and is capable of forming a super enhancer upon stimulation by IFN- γ.
2. The enhancer of claim 1, wherein the enhancer region is located in chr15:45002906 and 45022119, and the chr15:45002906 and 45022119 have the amino acid sequence as shown in SEQ ID NO: 7, or a sequence shown in the figure.
3. The enhancer of claim 2, wherein the enhancer region has 6 active sequences, E1-E6, E1-E6 have the sequence as set forth in SEQ ID NO: 1 to SEQ ID NO: 6.
4. The enhancer of claim 3 wherein the core enhancer segment of the enhancer region is E2.
5. Use of an enhancer as claimed in any one of claims 1 to 4 for enhancing expression of the B2M gene.
6. The use of claim 5, wherein the enhancer is capable of becoming a super enhancer upon IFN- γ stimulation, the super enhancer further promoting expression of the B2M gene.
7. A method for reducing the expression of a membrane HLA-class I protein, wherein all or part of the sequence of the enhancer of any one of claims 1 to 4 is knocked out, knocked down or silenced.
8. The method of claim 7, wherein the membrane HLA class I protein comprises one of HLA-A, HLA-B, HLA-C and B2M protein.
9. The method of claim 8, comprising the steps of:
(1) knocking down all or part of the sequence of an enhancer by a CRISPR gene editing method;
(2) cells with all or part of the enhancer sequence knocked down are selected.
10. The method according to claim 9, wherein the CRISPRi guide nucleotide sequences are sgRNA F and sgRNA R, respectively, the sequences of sgRNA F and sgRNA R are set forth in SEQ ID NO: 16 and SEQ ID NO: 17 is shown; the knocked-down enhancer sequence is located at chr15:45,004,417-45,004, 436.
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