CN113528448A - Construction method of human embryonic stem cells - Google Patents

Abstract

The invention relates to the field of biotechnology, in particular to a construction method of human embryonic stem cells. The invention provides a construction method of human embryonic stem cells, which comprises the following steps: integrating exogenous polynucleotide for coding B2M-HLA-G1 fusion protein into the genome of the human embryonic stem cell, wherein the B2M-HLA-G1 fusion protein comprises a first B2M segment and an HLA-G1 segment, and the integrated human embryonic stem cell does not express free B2M protein. The human embryonic stem cell provided by the inventionThe human embryonic stem cell line endogenous HLA-A, B and C proteins obtained by the construction method can not reach the surface of a cell membrane, so that the cell line can not be subjected to CD8⁺T cell recognition can effectively prevent allogeneic cell immune rejection, and the pluripotency and proliferation capacity of the embryonic stem cells are not affected, and the embryonic stem cells can still be used as a target cell source for future cell transplantation.

Description

Construction method of human embryonic stem cells

Technical Field

The invention relates to the field of biotechnology, in particular to a construction method of human embryonic stem cells.

Background

Pluripotent stem cells have the ability to proliferate by unlimited division in an in vitro culture environment and to differentiate under specific induction conditions committed to all cell types of the three germ layer origin, and are therefore expected to be used in future developments in regenerative medicine. As early as a decade ago, professors of Thomson (Thomson et al, 1998) and Reubinof (Reubinof et al, 2000) have begun to establish and differentiate human embryonic stem cell lines. Based on their research, more scientists have developed studies to apply pluripotent stem cells as a source of functional cells to repair the function of damaged tissues and treat degenerative diseases. In animal models, differentiated embryonic stem cells exhibit the ability to treat diseases such as spinal cord injury (soundarajan et al, 2007), diabetes (Liu and Lee,2012), parkinson's disease (Barberi et al, 2003), and liver failure (Soto-guillerez et al, 2006). Despite these exciting findings, the problem of immune rejection associated with pluripotent stem cell transplants has not been a good solution, which has been one of the biggest obstacles to the clinical use of future cell replacement therapies.

Disclosure of Invention

In view of the above-mentioned disadvantages of the prior art, it is an object of the present invention to provide a method for constructing human embryonic stem cells, which solves the problems of the prior art.

In order to achieve the above and other related objects, according to one aspect of the present invention, there is provided a method for constructing a human embryonic stem cell, the method comprising: integrating exogenous polynucleotide for coding B2M-HLA-G1 fusion protein into the genome of the human embryonic stem cell, wherein the B2M-HLA-G1 fusion protein comprises a first B2M segment and an HLA-G1 segment, and the integrated human embryonic stem cell does not express free B2M protein.

In some embodiments of the invention, the HLA-G1 fragment comprises:

a) polypeptide fragment with amino acid sequence shown as SEQ ID No. 1; or the like, or, alternatively,

b) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.1 and having the function of the polypeptide fragment defined in a).

In some embodiments of the invention, the first B2M fragment comprises:

c) polypeptide fragment with amino acid sequence shown as SEQ ID No. 2; or the like, or, alternatively,

d) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.2 and having the function of the polypeptide fragment defined in a).

In some embodiments of the invention, the B2M-HLA-G1 fusion protein further comprises a first flexible linker peptide between the first B2M fragment and HLA-G1 fragment;

and/or the B2M-HLA-G1 fusion protein sequentially comprises a first B2M fragment and an HLA-G1 fragment from the N end to the C end, and the B2M-HLA-G1 fusion protein comprises a polypeptide fragment with an amino acid sequence shown as SEQ ID No. 4.

In some embodiments of the invention, the method of integrating an exogenous polynucleotide encoding a B2M-HLA-G1 fusion protein into the genome of a human embryonic stem cell specifically comprises: the gene encoding the fragment of HLA-G1 is fused with the endogenous B2M gene in human embryonic stem cells, preferably by replacing the gene encoding the fragment of HLA-G1 with a stop codon located in exon 3 of the endogenous B2M gene.

In some embodiments of the invention, the construction method further comprises: integrating exogenous polynucleotide encoding B2M-HLA-G5 fusion protein into genome of human embryonic stem cell, wherein said B2M-HLA-G5 fusion protein comprises second B2M segment and HLA-G5 segment.

In some embodiments of the invention, the second B2M fragment comprises:

e) polypeptide fragment with amino acid sequence shown as SEQ ID No. 5; or the like, or, alternatively,

f) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.5 and having the function of the polypeptide fragment defined in c).

In some embodiments of the invention, the HLA-G5 fragment comprises:

g) polypeptide fragment with amino acid sequence shown as SEQ ID No. 6; or the like, or, alternatively,

h) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.6 and having the function of the polypeptide fragment defined in e).

In some embodiments of the invention, the B2M-HLA-G5 fusion protein further comprises a second flexible linker peptide between the second B2M fragment and HLA-G5 fragment;

and/or the B2M-HLA-G5 fusion protein sequentially comprises a second B2M fragment and an HLA-G5 fragment from the N end to the C end, and the B2M-HLA-G5 fusion protein comprises a polypeptide fragment with an amino acid sequence shown as SEQ ID No. 8.

In some embodiments of the invention, the method of integrating an exogenous polynucleotide encoding a B2M-HLA-G5 fusion protein into the genome of a human embryonic stem cell specifically comprises: the polynucleotide encoding the B2M-HLA-G5 fusion protein was integrated into the genome of human embryonic stem cells by a lentiviral vector.

The invention also provides a human embryonic stem cell which is obtained by the construction of the human embryonic stem cell.

Drawings

FIG. 1 shows B2M constructed for the present invention^null，B2M^mHLAG，B2M^m/sHLAGSchematic representation of hESCs cell line and related validation experiments. Wherein (A) is a stepwise construction of B2M^null，B2M^mHLAGAnd B2M^m/sHLAGSchematic representation of gene editing strategy for cell line; (B) in order to obtain a schematic diagram of a double gRNA gene editing strategy for knocking out the B2M gene, a cutting site is indicated by scissors and an arrow, a PAM sequence is purple, and the position of a designed primer for genome PCR is shown as a triangle; (C) schematic representation of genomic PCR results for 7 drug-resistant cell clones obtained in the dual gRNA gene editing protocol knock-out B2M experiment, wherein the biallelic deleted clone is marked red and the primers used are indicated in (B); (D) for a schematic of the gene editing strategy for knock-in of HLA-G1 at the B2M locus, the primers used for genomic PCR are shown as triangles and the probes designed for Southern Blot are shown as black lines; (E) schematic representation of the genomic DNAPCR results of 5 cell clones obtained in HLA-G1 knock-in experiments, the homozygous knock-in cell lines are marked red and the primers used are indicated in (D); (F) as a result of Southern Blot, it was confirmed that HLA-G1 was protected in the cell clone 4 shown in (E)Precise integration into the B2M gene and no integration was found at other sites in the genome, the probes used being indicated in (D).

FIG. 2 shows B2M constructed for the present invention^nullAnd B2M^mHLAGThe result of the cell line off-target phenomenon is shown schematically. Wherein (A) is B2M^nullhESCs results are schematic, in which no DNA mutations were found at 5 predicted off-target sites for each of gRNA1 and gRNA2 targeting B2M; (B) is B2M^mHLAGhESCs results are a schematic in which no DNA mutations were found at all 5 predicted off-target sites of gRNA3 targeting B2M.

FIG. 3 is a schematic representation of the expression of B2M and HLA I proteins in the WT, B2Mnull, B2MmHLAG and B2Mm/sHLAG cell lines of the present invention. Wherein (a) is a representation of B2M, HLA-G, HLA-a 03 (present in WT H9 cells) and HLA-BC protein expression using flow cytometry with (orange) or without (light blue) IFN- γ stimulation, wherein the false positive control group is red and each hESCs cell line is trypsinized to form a single cell suspension for flow cytometry; (B) as a result schematic diagram of Western Blot experiment, it is confirmed that B2Mm/sHLAG hESCs can express and secrete HLA-G5 protein.

FIG. 4 is a diagram showing the expression of HLA I and myocardial-specific genes in cardiomyocytes derived from the differentiation of the WT, B2Mnull, B2MmHLAG and B2Mm/sHLAG cell lines of the present invention. Wherein (A) is a schematic representation of the expression of cardiomyocyte central muscle specific genes derived from WT, B2Mnull, B2MmHLAG and B2Mm/sHLAG hESCs. (B) A schematic representation of TNNT2 expression in WT, B2Mnull, B2MmHLAG and B2Mm/sHLAG hESCs derived cardiomyocytes is shown for immunostaining. (C) The results of flow cytometry are shown to show the expression of B2M, HLA-G, HLA-A03 and HLA-BC in cardiomyocytes derived from different hESCs.

FIG. 5 is a graph showing the results of the immune response of the present invention against different genotypes of hESCs and their differentiated cells. Wherein (A) is a schematic representation of the results of flow cytometry analysis of CFSE signals in allogeneic PBMCs co-cultured with cardiomyocytes differentiated from WT, B2Mnull, B2MmHLAG and B2Mm/sHLAG hESCs, PBMCs co-cultured with PHA being used as positive control, and IFN- γ addition means that cardiomyocytes were treated with IFN- γ for 48 hours prior to MLR; (B) to collect the supernatant of PBMCs co-cultured with cardiomyocytes differentiated from WT, B2Mnull, B2MmHLAG and B2Mm/sHLAG hESCs for determination of IFN-. gamma.concentration in the supernatant, the IFN-. gamma.group was added, meaning that the cardiomyocytes were treated with IFN-. gamma.for 48 hours prior to co-culturing with PBMCs; data are presented as mean ± SEM of three independent experiments. P <0.01, two terminal Student's t test, ND, not detected; (C) for the collection of NK-92 supernatants co-cultured with WT, B2Mnull, B2MmHLAG and B2Mm/shla g hESCs for the determination of IFN- γ concentration in the supernatants, data are presented as mean ± SEM of three independent experiments, ± p <0.05, ± p <0.01 for comparison of the indicated genotypes hESCs with the wild type hESCs group, $ p <0.05 for comparison of B2MmHLAG hESCs with the B2Mm/shla g hESCs group, both-terminal Student's t test; (D) for co-culture of NK-92 and WT hESCs in B2Mm/sHLAG hESCs conditioned medium with RPMI-1640, the concentration of IFN-. gamma.in the supernatant was determined and the data are presented as mean. + -. SEM for three independent experiments,. beta.p <0.01, two-terminal Student's t assay.

FIG. 6 shows the invention from B2M^mHLAGAnd B2M^m/sHLAGTeratomas differentiated from hescs are not affected by allogeneic immune rejection in vivo. Wherein (a) is a schematic representation of an in vivo protocol for inducing allogeneic PBMC immune rejection against HLA antigens; (B) to WT, B2M^null，B2M^mHLAGAnd B2M^m/sHLAGhESCs cells were injected subcutaneously into M-NSG mice (approximately 2X 10 per site)⁶Individual cells). One month later, half of the number of mice were transplanted with activated allogeneic PBMCs. After two weeks, teratomas were recovered and stained with the corresponding antibodies. Identification of T cells by anti-CD 3 antibody and CD3^-CD56⁺Cells were identified as NK cells. Nuclei were stained with DAPI. Teratomas injected with the same volume of PBS in M-NSG mice were stained using the same conditions as a negative control. Scale bar, 50 μm. Each group n is 3; (C) teratoma-infiltrating T cells and NK cells were counted. For CD3 in three randomly selected fields of view⁺And CD3^-CD56⁺The cells were counted. Data are presented as three independent experimentsMean ± SEM. P<0.05, t-test for unpaired two students; (D) for analysis of WT (n-3) harvested on day 14 post PBMC injection by qPCR, B2M^null(n＝3)，B2M^mHLAG(n-4) and B2M^m/sHLAG(n-4) the expression level of IL-2mRNA in teratomas. Data are presented as mean ± SEM of three independent experiments. P<0.05, unpaired two students t-test.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments, and other advantages and effects of the present invention will be apparent to those skilled in the art from the disclosure of the present specification.

The inventor of the invention has found that after the embryonic stem cell is integrated with the fragment of HLA-G1 and/or the fragment of HLA-G5, the cell can be protected from CD8⁺T cell recognition, avoidance of CD8⁺T rejection can also inhibit immune rejection of NK cells, antigen presenting cells and other lymphocytes, so that allogeneic immune rejection can be inhibited more efficiently, and the invention is completed on the basis.

The invention provides a method for constructing human embryonic stem cells, which comprises the following steps: integrating exogenous polynucleotide for coding B2M-HLA-G1 fusion protein into the genome of the human embryonic stem cell, wherein the B2M-HLA-G1 fusion protein comprises a B2M fragment and an HLA-G1 fragment, and the integrated human embryonic stem cell does not express free B2M protein. After the embryonic stem cells are integrated with the HLA-G1 fragment, the B2M-HLA-G1 fusion protein can be successfully expressed, free B2M protein (namely, the B2M protein which is not fused independently) is not expressed, the B2M-HLA-G1 fusion protein can reach the surface of a cell membrane, and the expression of the protein is regulated by an endogenous B2M gene promoter, so that the cells can be prevented from being infected by CD8⁺T cell recognition, avoidance of CD8⁺T rejection, thereby effectively preventing allogeneic cell immunological rejection.

Human embryonic stem cells used in the present application may be isolated or obtained from human embryos within 14 days of fertilization that have not undergone in vivo development, and may be, for example, the commercial H9 hESC cell line.

In the construction method provided by the present invention, the coding gene of the HLA-G1 fragment may include a coding sequence of a heavy chain reading frame of HLA-G1, and the HLA-G1 fragment may include:

a) polypeptide fragment with amino acid sequence shown as SEQ ID No. 1; or the like, or, alternatively,

b) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.1 and having the function of the polypeptide fragment defined in a). Specifically, the amino acid sequence in b) specifically refers to: the polypeptide fragment is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids to the amino acid sequence shown in SEQ ID No.1, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids to the N-terminal and/or C-terminal of the amino acid sequence shown in SEQ ID No.1, and has the function of the polypeptide fragment shown in SEQ ID No. 1. For example, the HLA-G1 fragment generally has an intact alpha heavy chain structure, and, unlike classical HLA I molecules, has a mainly immunosuppressive function (e.g., can bind to inhibitory receptors that mainly include ILT2/CD85j/LILRB1, ILT4/CD85d/LILRB2, KIR2DL4/CD158d, and the like, and thus can modulate immune responses mediated by B cells, T cells, NK cells, and APC cells, and the like). The amino acid Sequence in b) may have more than 90%, 93%, 95%, 97%, or 99% identity (Sequence identity) with SEQ ID No. 1. The HLA-G1 fragment is typically of human origin.

In this application, sequence identity refers to the percentage of identical residues in the sequences that are involved in the alignment. Sequence identity of two or more sequences of interest can be calculated using computational software known in the art, such as that available from NCBI.

In the construction method provided by the present invention, the first B2M fragment may include:

c) polypeptide fragment with amino acid sequence shown as SEQ ID No. 2; or the like, or, alternatively,

d) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.2 and having the function of the polypeptide fragment defined in c). Specifically, the amino acid sequence in d) specifically refers to: the polypeptide fragment which is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids to the amino acid sequence shown in SEQ ID No.2, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2 or 3) amino acids to the N-terminal and/or C-terminal of the amino acid sequence shown in SEQ ID No.2, and has the function of the polypeptide fragment shown in SEQ ID No. 2. For example, the first B2M fragment generally has a beta sheet-like structure that has a predominantly non-covalent binding function to a Major Histocompatibility Complex (MHC) class I heavy chain. The amino acid Sequence in d) may have more than 90%, 93%, 95%, 97%, or 99% identity (Sequence identity) with SEQ ID No. 2. The first B2M fragment is typically of human origin.

In the construction method provided by the invention, the B2M-HLA-G1 fusion protein can also comprise a first flexible connecting peptide segment, and the first flexible connecting peptide segment is generally positioned between the HLA-G1 segment and the first B2M segment. The first flexible linker segment may typically be a flexible polypeptide of glycine (G), serine (S) and/or alanine (a) of suitable length such that adjacent protein domains are free to move relative to each other, e.g. the amino acid sequence of the first flexible linker segment may comprise sequences such as (GS) n, (GGS) n, (GGSG) n, (GGGS) nA, (ggggggs) nA, (ggggggna) nA, (ggggggggg) nA, wherein n is selected from integers between 1 and 10. In a specific embodiment of the present invention, the length of the amino acid sequence of the first flexible linker peptide segment can be 5-26. In a specific embodiment of the present invention, the first flexible linker peptide segment may include a polypeptide segment with an amino acid sequence shown in SEQ ID No. 3. In another specific embodiment of the invention, the B2M-HLA-G1 fusion protein comprises a first B2M fragment and an HLA-G1 fragment in sequence from the N end to the C end, and the B2M-HLA-G1 fusion protein comprises a polypeptide fragment with an amino acid sequence shown as SEQ ID No. 4.

GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer(SEQ ID NO.3)

In the construction method provided by the invention, the method for integrating the exogenous polynucleotide encoding the B2M-HLA-G1 fusion protein into the genome of the human embryonic stem cell specifically comprises the following steps: the gene encoding fragment HLA-G1 was fused to the endogenous B2M gene in human embryonic stem cells. Specific information of the endogenous B2M gene can be found in chr15:44,715,509-44,717,171GRCh38/hg38 human assembly. In one embodiment of the invention, the gene encoding fragment HLA-G1 may be substituted for a stop codon located in exon 3 of the endogenous B2M gene.

In the construction method provided by the present invention, the construction method may further include: integrating exogenous polynucleotide encoding B2M-HLA-G5 fusion protein into genome of human embryonic stem cell, wherein said B2M-HLA-G5 fusion protein comprises second B2M segment and HLA-G5 segment. The constructed human embryonic stem cell can successfully express secretory B2M-HLA-G5 fusion protein, so that the immunological rejection of NK cells, antigen presenting cells and other lymphocytes can be inhibited while CD8+ T rejection is effectively avoided, and the allogeneic immunological rejection is more efficiently inhibited.

In the construction method provided by the present invention, the second B2M fragment may include:

e) polypeptide fragment with amino acid sequence shown as SEQ ID No. 5; or the like, or, alternatively,

f) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.5 and having the function of the polypeptide fragment defined in e). Specifically, the amino acid sequence in f) specifically refers to: the polypeptide fragment which is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the amino acid sequence shown in SEQ ID No.5, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the N-terminus and/or C-terminus, and has the function of the polypeptide fragment shown in SEQ ID No. 5. For example, the second B2M fragment generally has a beta sheet-like structure that has a predominantly non-covalent binding function to a Major Histocompatibility Complex (MHC) class I heavy chain. The amino acid Sequence in f) may have more than 90%, 93%, 95%, 97%, or 99% identity (Sequence identity) with SEQ ID No. 5. The second B2M fragment is typically of human origin.

In the construction method provided by the invention, the HLA-G5 fragment comprises:

g) polypeptide fragment with amino acid sequence shown as SEQ ID No. 6; or the like, or, alternatively,

h) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.6 and having the function of the polypeptide fragment defined in g). Specifically, the amino acid sequence in h) specifically refers to: the polypeptide fragment which is obtained by substituting, deleting or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the amino acid sequence shown in SEQ ID No.6, or adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids to the N-terminus and/or C-terminus, and has the function of the polypeptide fragment shown in SEQ ID No. 6. For example, the HLA-G5 fragment generally has an intact α heavy chain extracellular structure, and, unlike classical HLA I molecules, has a mainly immunosuppressive function (e.g., binds to inhibitory receptors that mainly include ILT2/CD85j/LILRB1, ILT4/CD85d/LILRB2, KIR2DL4/CD158d, and the like, and thus can modulate immune responses mediated by B cells, T cells, NK cells, and APC cells, and the like). The amino acid Sequence in h) may have 90%, 93%, 95%, 97%, or 99% or more identity (Sequence identity) to SEQ ID No. 6. The HLA-G5 fragment is typically of human origin.

In the construction method provided by the invention, the B2M-HLA-G5 fusion protein can also comprise a second flexible connecting peptide segment, and the second flexible connecting peptide segment is usually positioned between the HLA-G5 segment and the second B2M segment. The second flexible linker segment can be generally a flexible polypeptide composed of glycine (G), serine (S) and/or alanine (a) having a suitable length such that adjacent protein domains are freely movable with respect to each other, for example, the amino acid sequence of the second flexible linker segment can include sequences such as (GS) n, (GGS) n, (GGSG) n, (GGGS) nA, (ggggggs) nA, (ggggna) nA, (ggggggggg) nA, wherein n is selected from an integer of 1 to 10. In a specific embodiment of the present invention, the length of the amino acid sequence of the second flexible linker peptide segment can be 5-26. In a specific embodiment of the present invention, the second flexible connecting peptide segment may include a polypeptide segment with an amino acid sequence shown in SEQ ID No. 7. In another specific embodiment of the invention, the B2M-HLA-G5 fusion protein comprises a second B2M fragment and an HLA-G5 fragment in sequence from the N-terminus to the C-terminus, and the B2M-HLA-G5 fusion protein comprises a polypeptide fragment with an amino acid sequence shown as SEQ ID No. 8.

GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer(SEQ ID NO.7)

In the construction method provided by the invention, the method for integrating the exogenous polynucleotide encoding the B2M-HLA-G5 fusion protein into the genome of the human embryonic stem cell specifically comprises the following steps: the polynucleotide encoding the B2M-HLA-G5 fusion protein is integrated into the genome of human embryonic stem cells by a lentiviral vector, allowing expression of the B2M-HLA-G5 fusion protein.

The second aspect of the invention provides a human embryonic stem cell, which is obtained by the construction method of the human embryonic stem cell provided by the first aspect of the invention.

The construction method of the human embryonic stem cell provided by the invention can successfully integrate the HLA-G1 fragment sequence into two endogenous B2M sites of the human embryonic stem cell, and integrate the sequence coding the B2M-HLA-G5 fusion protein into the genome of the human embryonic stem cell through a lentiviral vector. The endogenous HLA-A, B and C proteins of the human embryonic stem cell line cannot reach the surface of a cell membrane, so that the cell line cannot be subjected to CD8⁺T cell recognition can effectively prevent allogeneic cell immunological rejection, and the pluripotency and proliferation capability of the embryonic stem cells are not influencedIn turn, can still be used as a source of target cells for future cell transplantation. In addition, the cell line can successfully express the B2M-HLA-G1 fusion protein, the protein can reach the surface of a cell membrane, and the expression of the protein is regulated by an endogenous B2M gene promoter. In addition, a cell line capable of further successfully expressing the secretory B2M-HLA-G5 fusion protein can effectively avoid CD8⁺And the T rejection can also inhibit the immune rejection of NK cells, antigen presenting cells and other lymphocytes, so that the allogeneic immune rejection can be inhibited more efficiently.

The invention of the present application is further illustrated by the following examples, which are not intended to limit the scope of the present application.

Example 1

Construction of biallelic B2M Gene deleted hESCs cell line by the Dual gRNA Gene editing System

The double gRNA gene editing strategy enables efficient, specific knock-out of double alleles, and the cleaved DNA is repaired mainly by direct end ligation without introduction of nucleotide insertions or deletions (Liu et al, 2016). To knock out the B2M gene in human embryonic stem cells (hESCs), two adjacent grnas were designed, one located upstream of exon 2 of the B2M gene and the other located downstream of exon 3 (fig. 1A and 1B), with specific sequences shown as B2M gRNA1 and B2M gRNA2 in table 1, respectively. The vast majority of mature B2M protein is encoded by this targeting sequence.

TABLE 1

Two gRNA vectors (Addgene #41824), a CAG promoter-driven Cas9-P2A-GFP vector (Addgene #44719), were co-electroporated into H9 hESC (provided by Prov. Chun. Prov. Chun, university of Wisconsin, USA) along with a vector transiently expressing puromycin (SEQ ID NO. 14). After puromycin screening, resistant cell clones were selected and amplified for genomic DNA PCR analysis, 28.6% (2/7) of the clones selected were biallelicDNA deletion (B2M)^nullhESCs), whereas 57.1% (4/7) of the selected clones were single allele DNA deletions (fig. 1C). To test the potential off-target effects induced by these two gRNAs, the DNA integrity of the potential off-target sites predicted by the on-line program designed by the professor Zhang fronter laboratories (http:// crispr. mit. edu /) was analyzed using Sanger sequencing. The results show that the five major suspected off-target site DNAs of each gRNA in B2Mnull hESC remain intact (fig. 2A).

Example 2

Construction of cell line expressing HLA-G1, HLA-G5 hESCs

Two hESCs cell lines expressing HLA-G protein were designed (FIG. 1A). In B2MmHLAG hESCs, the HLA-G1 heavy chain is made flexible (G)₄S)₄The linker peptide is fused to the endogenous B2M protein. To use the reading frame encoding HLA-G1 heavy chain and flexibility (G)₄S)₄The sequence of the linker peptide replaces the stop codon located in exon 3 of the endogenous B2M gene, and B2M gRNA3 (see table 1) spanning the B2M stop codon was designed, so that the knock-in gene site was successfully achieved to be unable to be bound by B2M gRNA3, thereby increasing the efficiency of establishing homozygous knock-in cell lines. The CAG promoter driven Cas9 vector (same as example 1), recombinant donor (SEQ ID No.9), B2M gRNA3 vector (addge #41824) were co-electroporated into H9 hESC together with the transient expression puromycin vector (same as example 1). By this method, (G)₄S)₄HLA-G1 will be ligated to the end of B2M protein and expressed under the control of the B2M promoter together with endogenous B2M (fig. 1D). After drug screening, surviving cell clones were picked and amplified for genomic DNA PCR analysis. 5 puromycin-resistant cell clones were obtained and genomic PCR analysis showed that 20.0% (1/5) of the selected cell clones were double-copy knock-ins (B2M)^mHLAG) And 40.0% (2/5) is single copy knock-in (FIG. 1E). Further analysis of the homozygous knock-in clone by Southern Blot analysis revealed that (G) was present in this cell clone₄S)₄HLA-G1 was integrated only into the predetermined B2M gene location (FIG. 1F).

In order to obtain another hESCs simultaneously expressing the membrane protein HLA-G1 and the secretory protein HLA-G5, B2M^{m/ sHLAG}hESCs, a sequence of B2M CDS without a termination codon, (G)₄S)₄The flexible linker peptide sequence and HLA-G5 heavy chain coding sequence were integrated into a lentiviral vector (pLVX-CAG-Puro plasmid with BamHI and MluI insertion sites) with puromycin resistance, and this virus was used to infect B2M^mHLAGhESCs cell lines, followed by drug screening and expansion of drug resistant hESCs (fig. 1A). And, in addition, B2M^nullSimilar observations in hESCs, B2M^mHLAGThe 5B 2M gRNA3 suspected off-target site DNA in hESCs remained intact, indicating that no off-target occurred during gene editing (fig. 2B).

Amino acid sequence of HLA-G1 heavy chain sequence (HLA-G1 fragment):

GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD*(SEQ ID No.1)

amino acid sequence of endogenous B2M protein (first B2M fragment):

MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID No.2)

flexibility (G)₄S)₄Amino acid sequence of linker peptide fragment (first flexible linker peptide fragment):

GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer(SEQ ID No.3)

amino acid sequence of B2M-HLA-G1 fusion protein:

MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWKQSSLPTIPIMGIVAGLVVLAAVVTGAAVAAVLWRKKSSD*(SEQ ID No.4)

the coding sequence of the B2M-HLA-G1 fusion protein is as follows:

wherein the endogenous B2M gene exon DNA sequence is bolded, and the intron DNA sequence is underlined

Amino acid sequence of HLA-G5 heavy chain sequence (second B2M fragment):

GSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL*(SEQ ID No.6)

amino acid sequence of the second B2M fragment:

MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDM(SEQ ID No.5)

flexibility (G)₄S)₄Amino acid sequence of linker peptide stretch (second flexible linker peptide stretch):

GlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySerGlyGlyGlyGlySer(SEQ ID No.7)

amino acid sequence of B2M-HLA-G5 fusion protein:

MSRSVALAVLALLSLSGLEAIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFSAAVSRPGRGEPRFIAMGYVDDTQFVRFDSDSACPRMEPRAPWVEQEGPEYWEEETRNTKAHAQTDRMNLQTLRGYYNQSEASSHTLQWMIGCDLGSDGRLLRGYEQYAYDGKDYLALNEDLRSWTAADTAAQISKRKCEAANVAEQRRAYLEGTCVEWLHRYLENGKEMLQRADPPKTHVTHHPVFDYEATLRCWALGFYPAEIILTWQRDGEDQTQDVELVETRPAGDGTFQKWAAVVVPSGEEQRYTCHVQHEGLPEPLMLRWSKEGDGGIMSVRESRSLSEDL*(SEQ ID No.8)

the coding sequence of the B2M-HLA-G5 fusion protein:

ATGTCTCGCtccgtggccttagctgtgctcgcgctactctctctttctggcctggaggctatccagcg tactccaaagattcaggtttactcacgtcatccagcagagaatggaaagtcaaatttcctgaattgctatgtgtct gggtttcatccatccgacattgaagttgacttactgaagaatggagagagaattgaaaaagtggagcattcagact tgtctttcagcaaggactggtctttctatctcttgtactacactgaattcacccccactgaaaaagatgagtatgc ctgccgtgtgaaccatgtgactttgtcacagcccaagATAGTTAAGTGGGATCGAGACATGGGTGGAGGTGGAAGTGGTGGAGGTGGAAGTGGTGGAGGTGGAAGTGGTGGAGGTGGaagtGGCTCCCACTCCATGAGGTATttcagcgccgccgtgtcccggcccggccgcggggagccccgcttcatcgccatgggctacgtggacgacacgcagttcgtgcggttcgacagcgactcggcgtgtccgaggatggagccgcgggcgccgtgggtggagcaggaggggccggagtattgggaagaggagacacggaacaccaaggcccacgcacagactgacagaatgaacctgcagaccctgcgcggctactacaaccagagcgaggccagttctcacaccctccagtggatgattggctgcgacctggggtccgacggacgcctcctccgcgggtatgaacagtatgcctacgatggcaaggattacctcgccctgaacgaggacctgcgctcctggaccgcagcggacactgcggctcagatctccaagcgcaagtgtgaggcggccaatgtggctgaacaaaggagagcctacctggagggcacgtgcgtggagtggctccacagatacctggagaacgggaaggagatgctgcagcgcgcggacccccccaagacacacgtgacccaccaccctgtctttgactatgaggccaccctgaggtgctgggccctgggcttctaccctgcggagatcatactgacctggcagcgggatggggaggaccagacccaggacgtggagctcgtggagaccaggcctgcaggggatggaaccttccagaagtgggcagctgtggtggtgccttctggagaggagcagagatacacgtgccatgtgcagcatgaggggctgccggagcccctcatgctgagatggagtaaggagggagatggaggcatcatgtctgttagggaaagcaggagcctctctgaagacCTTTAA(SEQ ID No.10)

example 3

Detection of HLA I protein expression in genetically modified cell lines

To investigate β 2m and HLA class I proteins in Wild Type (WT), B2M^null，B2M^mHLAGAnd B2M^m/sHLAGExpression in hESCs cell lines, Western Blot and flow cytometry (FACS) experiments were performed. Before the flow cytometry experiment, IFN-gamma is added into the hESCs cell culture solution to the final concentration of 25ng/ml, and after 48 hours of culture, the flow cytometry experiment is carried out. Wild type, B2M, in the case of IFN- γ stimulation^mHLAGAnd B2M^m/sHLAGExpression of β 2m protein on the cell membrane surface was significantly increased in hESCs upon treatment with IFN- γ addition (fig. 3A), demonstrating that integration of HLA-G1 in the B2M gene did not affect expression of endogenous B2M. However, even in the case of IFN- γ stimulation, at B2M^nullCell membrane surface expression of β 2m protein was also undetectable in hESCs (fig. 3A). Thus, it was shown that B2M has been successfully knocked out by the double gRNA gene editing strategy^nullThe B2M gene in hESCs. In addition, B2M resulted from the fusion of the HLA-G1 heavy chain reading frame with the endogenous B2M gene^mHLAGAnd B2M^m/sHLAGThe level of HLA-G1 expression in the cell line should also be affected by IFN- γ. In the presence of IFN-gamma, B2M^mHLAGAnd B2M^m/sHLAGThe cell line has increased expression level of HLA-G1 on the surface of cell membrane, and under the condition of no IFN-gamma stimulation, B2M^mHLAGAnd B2M^m/sHLAGThe level of HLA-G expression in the cell lines was low (FIG. 3A). However, in WT and B2M^nullNo cell membrane surface expression of HLA-G was detected in hESCs (FIG. 3A). As for the expression of classical HLA I molecules, even under IFN-. gamma.stimulation, B2M^null，B2M^mHLAGAnd B2M^m/sHLAGHLA-A, -B or-C was not detected on the cell membrane surface of hESCs (FIG. 3A). Indicating that there is no free β 2m protein available for canonical HLA I heavy chain ligation in these cells compared to wild type hESCs, as in B2M^mHLAGAnd B2M^m/sHLAGIn hESCs, the endogenous B2M gene locus expressed only the B2M-HLA-G1 fusion protein.

For the cell lysate group, WT, B2M^mHLAGB2M^m/sHLAGhESC in RIPA⁺The lysate was collected for Western Blot. For the Medium group, WT, B2M^mHLAGB2M^m/sHLAGhESC culture to full well plate, medium was aspirated off and fresh addedFresh DMEM/F12. After 24 hours, the supernatant of each group was collected, 1 × loading buffer was added, the protein was denatured by adding β -mercaptoethanol, and used for Western Blot to detect the antibody against HLA-G5 as 5A6G7(Abcam, ab 76869). Western blot further confirms B2M^m/sHLAGhESCs express the secreted protein HLA-G5, whereas WT or B2M^mHLAGhESCs did not express or secrete HLA-G5 protein (FIG. 3B).

Example 4

HLA-G1-expressing, HLA-G5 hESCs cell lines with low immunogenicity

Further study B2M^null，B2M^mHLAGAnd B2M^m/sHLAGAlloimmunogenicity of hESCs cell lines. Results have shown that undifferentiated hESCs do not induce PBMC proliferation when co-cultured with PBMC (Li et al, 2004; Riolobos et al, 2013). Thus, in this embodiment, WT, B2M is referred to in the method of Lian et al, 2013^null，B2M^mHLAGAnd B2M^{m /sHLAG}hESCs are directionally differentiated into cardiomyocytes by the following specific method:

1. y27632 was added to the hESCs medium one hour prior to digestion to a final concentration of 10. mu.M. The feeder MEF, which was prepared in advance to be inoculated with hESCs cells, was aspirated off the MEF medium in the 6-well plate, then washed once with 2ml of DMEM/F12, the DMEM/F12 was aspirated off, and then 2ml of hESCs medium containing Y27632 and bFGF (4ng/ml) at a final concentration of 10. mu.M was added and placed in the incubator for future use.

2. And (3) sucking away the hESCs culture solution containing Y27632, adding 1ml of 0.05% pancreatin, sucking away the pancreatin, then adding 1ml of 0.05% pancreatin preheated in a water bath kettle at 37 ℃, putting into an incubator at 37 ℃ for digestion for 3min, sucking away the pancreatin, putting into the incubator at 37 ℃ for continuous digestion for 3min, and stopping digestion when observing that free single cells begin to appear on the cloned edge of the hESCs under a microscope.

3. The cells were blown up with 5ml of DMEM/F12, repeatedly blown up with a pipette, the digested clones were blown as much as possible into single cells, and the cell suspension was transferred to a 15ml centrifuge tube.

4. Centrifuge the tube at 800rpm for 30s, leave the cell pellet at the bottom of the tube, transfer the single cell suspension to a new 15ml centrifuge tube, centrifuge at 2500rpm for 1.5 min.

5. The supernatant was aspirated off, 1ml of hESCs culture medium was added to resuspend the cells, the cells were counted, approximately 1-1.5 million cells were received per 6 well, the hESCs culture medium was added to 10ml, and centrifuged at 2500rpm for 1.5 min.

6. Cells were added to prepared 6-well plates and 1, 1.5, 2.0, 2.5, 3million cells were added to each well in sequence. Placing the mixture into an incubator at 37 ℃ for continuous culture.

7. The medium was changed normally for 3 days according to the protocol for hESCs culture.

8. The old medium was aspirated off, and 4ml of RPMI/B-27with out insulin solution containing CHIR99021 (final concentration 12. mu.M) was added to each well.

After 9.24 hours, the old medium was aspirated off and 4ml of RPMI/B-27with out insulin solution was added per well.

After 10.48 hours, 2ml of medium was aspirated from each well, an equal volume of fresh RPMI/B-27with out insulin was supplemented, and IWP2 was added to a final concentration of 5. mu.M. The remaining medium in the wells was aspirated off and the prepared medium was added.

After 11.2 days, the old medium was aspirated and 4ml of new RPMI/B-27with insulin medium was added to each well.

After 12.2 days, the old medium was aspirated and fresh RPMI/B-27 medium was added, 4ml per well. The solution was changed every 3 days. After approximately 5 days after the start of differentiation, beating cardiomyocytes were seen.

Both qPCR test for myocardial specific genes and staining results for TNNT2 showed that all of the above cell lines had the ability to differentiate efficiently into cardiomyocytes (fig. 4B and 6C).

Meanwhile, the specific method for stimulating the myocardial cells by using IFN-gamma is as follows: before the mixed culture is started, IFN-gamma is added into the culture solution of the cardiac muscle cells to the final concentration of 25ng/ml, and the cardiac muscle cells are collected and mixed for culture after 48 hours of culture. B2M only^mHLAGAnd B2M^m/sHLAGhESCs differentiated cardiomyocytes showed elevated expression of HLA-G1 under IFN- γ stimulation, while lacking expression of canonical HLA class I molecules located on the cell membrane surface, similar to undifferentiated hESCs (FIG. 1)4A)。

Peripheral Blood Mononuclear Cells (PBMCs) collected from healthy volunteers were subjected to HLA-a, -B, -C-match detection (n-4). Donor #01, #02, # 03-derived PBMCs were then co-cultured with differentiated cardiomyocytes for carboxyfluorescein succinimidyl ester (CFSE) -based cell proliferation assays. In Mixed Lymphocyte Reaction (MLR) experiments, PBMC showed proliferation when co-cultured with HLA I-mismatched WT cardiomyocytes and PHA (Phytohemagglutinin-L, invitrogen, 00-4977) (positive control), whereas PBMC was co-cultured with B2M^null，B2M^mHLAGAnd B2M^m/sHLAGThe proliferation of differentiated cardiomyocytes was significantly reduced during co-culture (fig. 5A), and the co-culture protocol was as follows:

1. take 3.5x10⁶And (3) differentiating the cardiomyocytes for more than 15 days (before part of the cardiomyocytes are subjected to the step, IFN-gamma treatment is added in the in vitro culture process for 48 hours, finally the cardiomyocytes in the + IFN-gamma group are used as target cells in an MLR experiment, namely in the experiment shown in figure 5A, the cardiomyocytes in the + IFN-gamma group are subjected to IFN-gamma treatment, the cardiomyocytes in the no IFN-gamma group are not subjected to relevant treatment), adding trypsin for digestion for 3min, adding equal volume of RPMI-5 to neutralize trypsin, blowing and beating the trypsin into single cells by a pipette, and centrifuging the cells at 2500rpm for 1.5 min.

2. And (3) sucking and removing the supernatant, adding 5ml of RPMI-5 culture solution to resuspend cells, winding a sealing film on a centrifuge tube, and then placing the centrifuge tube into an X-ray irradiator for irradiation, wherein the irradiation dose is 20 Gy.

3. The irradiated cells were washed twice with 5ml RPMI and centrifuged at 2500rpm for 1.5min after each wash.

4. Resuspending cardiomyocytes in 1ml of RPMI-5, counting, adjusting the cell density to 1X 10 by adding RPMI-5⁶cells/ml for use.

5.300g, centrifugation for 10min collected PBMC, using PBS/0.1% HSA heavy suspension PBMC, cell density adjusted to 1-10 x10⁶cells/ml。

6. The CFSE solution was diluted with PBS to a final concentration of 6. mu.M in the dark.

7. Adding equal volume of CFSE solution into the PBMC solution, mixing well, placing in a 37 ℃ incubator in the dark, and incubating for 10 min.

8. After the incubation was completed, 6 times the volume of the 4 ℃ pre-cooled RPMI-1640/10% HSA solution was added to the PBMC solution, mixed well and allowed to stand at room temperature for 5 min.

Centrifuge at 800g for 15min at 9.21 ℃.

10. Supernatant was discarded, PBMC was resuspended using RPMI-5, and cell density was adjusted to 1X 10⁶cells/ml。

11. PBMC were mixed well and added to round bottom 96 well plates in 100. mu.l/well.

12. Irradiated cardiomyocytes were added to the PBMC-added wells of a 96-well plate at 100 μ l per well, at a ratio of target to effector cells of 1: 1. mix well the cells in the well. The positive control wells were loaded with PHA at a final concentration of 1. mu.g/ml.

13. The 96-well plate was placed in an incubator at 37 ℃ and cultured for 7 days.

14. After the culture is finished, the cells in the 96-well plate are collected, 1ml of PBS is added into each well of the cells, the mixture is uniformly mixed, and the mixture is centrifuged for 10min at 300 g.

15. And (3) sucking the supernatant, adding 100 mu l of flow cytometry detection fixing solution to resuspend the cells, adding the Human Fc Block antibody with the corresponding concentration, incubating for 10min on ice, adding the anti-Human CD45 antibody with the corresponding concentration, and incubating for 30min on ice.

16. 2ml PBS was added to resuspend the cells and centrifuge at 300g for 10 min.

17. The supernatant was aspirated off, 500. mu.l PBS was added to resuspend the cells, and PBMC proliferation was examined by flow cytometry.

Further ELISA experiments were performed to detect WT, B2M^null，B2M^mHLAGAnd B2M^m/sHLAGIFN- γ secretion from PBMCs (donors #01, #02, #04) after 48 hours of in vitro co-culture of cardiomyocytes. As shown in FIG. 5B, WT cardiomyocytes stimulated PBMC to secrete large amounts of IFN- γ, especially when WT myocardium was stimulated with IFN- γ for 48h prior to IFN- γ determination, when PBMC were incubated with B2M^null，B2M^mHLAGAnd B2M^m/sHLAGWhen cardiomyocytes were co-cultured, secreted IFN-. gamma.could hardly be detected.

Since HLA-G is a ligand for inhibitory receptors of LILRB1 and KIR2DL4 expressed on NK cells, HLA-G expression should be able to inhibit NK activation. Will be provided withNK-92 cells with WT, B2M^null，B2M^mHLAGAnd B2M^m/sHLAGhESCs were incubated to assess IFN- γ release. The results show that B2M^nullhESCs could induce maximal IFN- γ release (fig. 5C), suggesting that cell surface loss of HLA class I molecules would lead to NK cell attack. And B2M^mHLAGAnd B2M^m/sHLAGNK-92 cells co-cultured with hESCs significantly reduced IFN- γ secretion, while B2M expressing HLA-G1 and HLA-G5^m/sHLAGhESCs induced IFN-gamma release even lower than B2M^mHLAGResults for hESCs (fig. 5C), which demonstrate that HLA-G expression can inhibit NK cell activation.

Since HLA-G5 is a secretable protein, further investigation of B2M^m/sHLAGImmunosuppressive properties of hESCs-derived conditioned media. B2M^m/sHLAGCells were passaged and harvested, resuspended in RPMI-1640 and adjusted to a cell density of 10⁷cells/ml, incubated at 37 ℃ for 24 hours, then harvested from B2M^m/sHLAGSupernatant of hESCs was used as conditioned medium, and NK-92 cells, WT hESCs were cultured in RPMI-1640 or B2M, respectively^m/sHLAGhESCs were co-cultured in conditioned medium to evaluate IFN- γ release in the respective environments. The experimental result shows that B2M^m/sHLAGThe IFN- γ concentration in hESCs conditioned media was much lower (FIG. 5D), indicating that B2M^m/sHLAGhESCs secrete HLA-G5 soluble protein that inhibits NK cell-mediated secretion of IFN- γ.

Taken together, the results of the decreased allogeneic MLR response induced by HLA class I-depleted cells also indicate that cells expressing HLA-G protein exhibit low immunogenicity in vitro experiments.

Testing of PBMCs mediated against B2M using in vivo models^null，B2M^mHLAG，B2M^m/sHLAGAllogeneic immune response of cells. WT and Gene edited hESCs (WT and B2M)^null，B2M^mHLAGAnd B2M^m/sHLAGhESCs) was injected subcutaneously into M-NSG mice to form teratomas (M-NSG mice purchased from shanghai square model biotechnology, inc). Human PBMC were co-cultured with embryoid bodies differentiated from wild type H9 ESC for 14 days. Then, it is injected through tail veinThe H9 ESC antigen-activated allogeneic PBMC were transferred into mice according to the following protocol: preactivated 3.4X 10⁶PBMCs were resuspended in 100 μ l PBS, cell suspension was injected into each mouse via tail vein using an insulin needle, and control mice were injected with an equal volume of PBS. After 20 days, mice were sacrificed and teratomas were collected for immunostaining (fig. 6A). Analysis of the immunofluorescent staining results showed that the cells infiltrated B2M^null，B2M^mHLAGAnd B2M^m/sHLAGhescs differentiated to give teratoma the number of human T cells was greatly reduced compared to the wild-type group (fig. 6B, fig. 6C). The same conclusion was reflected by qPCR analysis for human IL-2 in teratomas (fig. 6D), indicating B2M^null，B2M^mHLAGAnd B2M^m/sHLAGTeratomas produced by hESCs differentiation were protected from allogeneic T cell-mediated immune rejection. However, despite being in the wild type, B2M^mHLAGAnd B2M^m/sHLAGVery little NK cell infiltration was detected in hESC-derived teratomas, but in B2M-derived teratomas^nullhowever, significantly enhanced NK cell infiltration was detected in teratomas of hESCs (FIG. 6B, FIG. 6C). These results show that B2M^nullThe loss of expression of HLAI molecules on the cell membrane surface of hESC-derived teratoma triggers NK cell attack. However, B2M^mHLAGAnd B2M^m/sHLAGThe HLA-G protein expressed by the derived cell teratoma effectively blocks the rejection mediated by allogeneic NK cells.

In conclusion, the present invention effectively overcomes various disadvantages of the prior art and has high industrial utilization value.

The foregoing embodiments are merely illustrative of the principles and utilities of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or change the above-mentioned embodiments without departing from the spirit and scope of the present invention. Accordingly, it is intended that all equivalent modifications or changes which can be made by those skilled in the art without departing from the spirit and technical spirit of the present invention be covered by the claims of the present invention.

Sequence listing

<110> university of Tongji

<120> method for constructing human embryonic stem cells

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Gly Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly

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Arg Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln

20 25 30

Phe Val Arg Phe Asp Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg

35 40 45

Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr

50 55 60

Arg Asn Thr Lys Ala His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr

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Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln

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Trp Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly

100 105 110

Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu

115 120 125

Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys

130 135 140

Arg Lys Cys Glu Ala Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu

145 150 155 160

Glu Gly Thr Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys

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Glu Met Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His

180 185 190

Pro Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe

195 200 205

Tyr Pro Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln

210 215 220

Thr Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr

225 230 235 240

Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg

245 250 255

Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu

260 265 270

Arg Trp Lys Gln Ser Ser Leu Pro Thr Ile Pro Ile Met Gly Ile Val

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Ala Gly Leu Val Val Leu Ala Ala Val Val Thr Gly Ala Ala Val Ala

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Ala Val Leu Trp Arg Lys Lys Ser Ser Asp

305 310

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<213> Artificial Sequence (Artificial Sequence)

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Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

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Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg

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His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser

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Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu

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Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp

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Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp

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Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile

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Val Lys Trp Asp Arg Asp Met

115

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Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

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Gly Gly Gly Ser

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Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

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Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg

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His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser

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Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu

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Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp

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Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp

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Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile

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Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser Gly Gly Gly Gly

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Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser His Ser Met

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Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg

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Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp

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Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu

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Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala

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His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr

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Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys

225 230 235 240

Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala

245 250 255

Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp

260 265 270

Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala

275 280 285

Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val

290 295 300

Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg

305 310 315 320

Ala Asp Pro Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr

325 330 335

Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile

340 345 350

Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu

355 360 365

Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala

370 375 380

Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val

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Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Lys Gln Ser

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Ser Leu Pro Thr Ile Pro Ile Met Gly Ile Val Ala Gly Leu Val Val

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Leu Ala Ala Val Val Thr Gly Ala Ala Val Ala Ala Val Leu Trp Arg

435 440 445

Lys Lys Ser Ser Asp

450

<210> 5

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Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

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Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg

20 25 30

His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser

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Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu

50 55 60

Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp

65 70 75 80

Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp

85 90 95

Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile

100 105 110

Val Lys Trp Asp Arg Asp Met

115

<210> 6

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<213> Artificial Sequence (Artificial Sequence)

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Gly Ser His Ser Met Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly

1 5 10 15

Arg Gly Glu Pro Arg Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln

20 25 30

Phe Val Arg Phe Asp Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg

35 40 45

Ala Pro Trp Val Glu Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr

50 55 60

Arg Asn Thr Lys Ala His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr

65 70 75 80

Leu Arg Gly Tyr Tyr Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln

85 90 95

Trp Met Ile Gly Cys Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly

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Tyr Glu Gln Tyr Ala Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu

115 120 125

Asp Leu Arg Ser Trp Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys

130 135 140

Arg Lys Cys Glu Ala Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu

145 150 155 160

Glu Gly Thr Cys Val Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys

165 170 175

Glu Met Leu Gln Arg Ala Asp Pro Pro Lys Thr His Val Thr His His

180 185 190

Pro Val Phe Asp Tyr Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe

195 200 205

Tyr Pro Ala Glu Ile Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln

210 215 220

Thr Gln Asp Val Glu Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr

225 230 235 240

Phe Gln Lys Trp Ala Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg

245 250 255

Tyr Thr Cys His Val Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu

260 265 270

Arg Trp Ser Lys Glu Gly Asp Gly Gly Ile Met Ser Val Arg Glu Ser

275 280 285

Arg Ser Leu Ser Glu Asp Leu

290 295

<210> 7

<211> 20

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 7

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser

20

<210> 8

<211> 434

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 8

Met Ser Arg Ser Val Ala Leu Ala Val Leu Ala Leu Leu Ser Leu Ser

1 5 10 15

Gly Leu Glu Ala Ile Gln Arg Thr Pro Lys Ile Gln Val Tyr Ser Arg

20 25 30

His Pro Ala Glu Asn Gly Lys Ser Asn Phe Leu Asn Cys Tyr Val Ser

35 40 45

Gly Phe His Pro Ser Asp Ile Glu Val Asp Leu Leu Lys Asn Gly Glu

50 55 60

Arg Ile Glu Lys Val Glu His Ser Asp Leu Ser Phe Ser Lys Asp Trp

65 70 75 80

Ser Phe Tyr Leu Leu Tyr Tyr Thr Glu Phe Thr Pro Thr Glu Lys Asp

85 90 95

Glu Tyr Ala Cys Arg Val Asn His Val Thr Leu Ser Gln Pro Lys Ile

100 105 110

Val Lys Trp Asp Arg Asp Met Gly Gly Gly Gly Ser Gly Gly Gly Gly

115 120 125

Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Ser His Ser Met

130 135 140

Arg Tyr Phe Ser Ala Ala Val Ser Arg Pro Gly Arg Gly Glu Pro Arg

145 150 155 160

Phe Ile Ala Met Gly Tyr Val Asp Asp Thr Gln Phe Val Arg Phe Asp

165 170 175

Ser Asp Ser Ala Cys Pro Arg Met Glu Pro Arg Ala Pro Trp Val Glu

180 185 190

Gln Glu Gly Pro Glu Tyr Trp Glu Glu Glu Thr Arg Asn Thr Lys Ala

195 200 205

His Ala Gln Thr Asp Arg Met Asn Leu Gln Thr Leu Arg Gly Tyr Tyr

210 215 220

Asn Gln Ser Glu Ala Ser Ser His Thr Leu Gln Trp Met Ile Gly Cys

225 230 235 240

Asp Leu Gly Ser Asp Gly Arg Leu Leu Arg Gly Tyr Glu Gln Tyr Ala

245 250 255

Tyr Asp Gly Lys Asp Tyr Leu Ala Leu Asn Glu Asp Leu Arg Ser Trp

260 265 270

Thr Ala Ala Asp Thr Ala Ala Gln Ile Ser Lys Arg Lys Cys Glu Ala

275 280 285

Ala Asn Val Ala Glu Gln Arg Arg Ala Tyr Leu Glu Gly Thr Cys Val

290 295 300

Glu Trp Leu His Arg Tyr Leu Glu Asn Gly Lys Glu Met Leu Gln Arg

305 310 315 320

Ala Asp Pro Pro Lys Thr His Val Thr His His Pro Val Phe Asp Tyr

325 330 335

Glu Ala Thr Leu Arg Cys Trp Ala Leu Gly Phe Tyr Pro Ala Glu Ile

340 345 350

Ile Leu Thr Trp Gln Arg Asp Gly Glu Asp Gln Thr Gln Asp Val Glu

355 360 365

Leu Val Glu Thr Arg Pro Ala Gly Asp Gly Thr Phe Gln Lys Trp Ala

370 375 380

Ala Val Val Val Pro Ser Gly Glu Glu Gln Arg Tyr Thr Cys His Val

385 390 395 400

Gln His Glu Gly Leu Pro Glu Pro Leu Met Leu Arg Trp Ser Lys Glu

405 410 415

Gly Asp Gly Gly Ile Met Ser Val Arg Glu Ser Arg Ser Leu Ser Glu

420 425 430

Asp Leu

<210> 9

<211> 5798

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 60

atccagcgtg agtctctcct accctcccgc tctggtcctt cctctcccgc tctgcaccct 120

ctgtggccct cgctgtgctc tctcgctccg tgacttccct tctccaagtt ctccttggtg 180

gcccgccgtg gggctagtcc agggctggat ctcggggaag cggcggggtg gcctgggagt 240

ggggaagggg gtgcgcaccc gggacgcgcg ctacttgccc ctttcggcgg ggagcagggg 300

agacctttgg cctacggcga cgggagggtc gggacaaagt ttagggcgtc gataagcgtc 360

agagcgccga ggttggggga gggtttctct tccgctcttt cgcggggcct ctggctcccc 420

cagcgcagct ggagtggggg acgggtaggc tcgtcccaaa ggcgcggcgc tgaggtttgt 480

gaacgcgtgg aggggcgctt ggggtctggg ggaggcgtcg cccgggtaag cctgtctgct 540

gcggctctgc ttcccttaga ctggagagct gtggacttcg tctaggcgcc cgctaagttc 600

gcatgtccta gcacctctgg gtctatgtgg ggccacaccg tggggaggaa acagcacgcg 660

acgtttgtag aatgcttggc tgtgatacaa agcggtttcg aataattaac ttatttgttc 720

ccatcacatg tcacttttaa aaaattataa gaactacccg ttattgacat ctttctgtgt 780

gccaaggact ttatgtgctt tgcgtcattt aattttgaaa acagttatct tccgccatag 840

ataactacta tggttatctt ctgcctctca cagatgaaga aactaaggca ccgagatttt 900

aagaaactta attacacagg ggataaatgg cagcaatcga gattgaagtc aagcctaacc 960

agggcttttg cgggagcgca tgccttttgg ctgtaattcg tgcatttttt tttaagaaaa 1020

acgcctgcct tctgcgtgag attctccaga gcaaactggg cggcatgggc cctgtggtct 1080

tttcgtacag agggcttcct ctttggctct ttgcctggtt gtttccaaga tgtactgtgc 1140

ctcttacttt cggttttgaa aacatgaggg ggttgggcgt ggtagcttac gcctgtaatc 1200

ccagcactta gggaggccga ggcgggagga tggcttgagg tccgtagttg agaccagcct 1260

ggccaacatg gtgaagcctg gtctctacaa aaaataataa caaaaattag ccgggtgtgg 1320

tggctcgtgc ctgtggtccc agctgctccg gtggctgagg cgggaggatc tcttgagctt 1380

aggcttttga gctatcatgg cgccagtgca ctccagcgtg ggcaacagag cgagaccctg 1440

tctctcaaaa aagaaaaaaa aaaaaaaaga aagagaaaag aaaagaaaga aagaagtgaa 1500

ggtttgtcag tcaggggagc tgtaaaacca ttaataaaga taatccaaga tggttaccaa 1560

gactgttgag gacgccagag atcttgagca ctttctaagt acctggcaat acactaagcg 1620

cgctcacctt ttcctctggc aaaacatgat cgaaagcaga atgttttgat catgagaaaa 1680

ttgcatttaa tttgaataca atttatttac aacataaagg ataatgtata tatcaccacc 1740

attactggta tttgctggtt atgttagatg tcattttaaa aaataacaat ctgatattta 1800

aaaaaaaatc ttattttgaa aatttccaaa gtaatacatg ccatgcatag accatttctg 1860

gaagatacca caagaaacat gtaatgatga ttgcctctga aggtctattt tcctcctctg 1920

acctgtgtgt gggttttgtt tttgttttac tgtgggcata aattaatttt tcagttaagt 1980

tttggaagct taaataactc tccaaaagtc ataaagccag taactggttg agcccaaatt 2040

caaacccagc ctgtctgata cttgtcctct tcttagaaaa gattacagtg atgctctcac 2100

aaaatcttgc cgccttccct caaacagaga gttccaggca ggatgaatct gtgctctgat 2160

ccctgaggca tttaatatgt tcttattatt agaagctcag atgcaaagag ctctcttagc 2220

ttttaatgtt atgaaaaaaa tcaggtcttc attagattcc ccaatccacc tcttgatggg 2280

gctagtagcc tttccttaat gatagggtgt ttctagagag atatatctgg tcaaggtggc 2340

ctggtactcc tccttctccc cacagcctcc cagacaagga ggagtagctg ccttttagtg 2400

atcatgtacc ctgaatataa gtgtatttaa aagaatttta tacacatata tttagtgtca 2460

atctgtatat ttagtagcac taacacttct cttcattttc aatgaaaaat atagagttta 2520

taatattttc ttcccacttc cccatggatg gtctagtcat gcctctcatt ttggaaagta 2580

ctgtttctga aacattaggc aatatattcc caacctggct agtttacagc aatcacctgt 2640

ggatgctaat taaaacgcaa atcccactgt cacatgcatt actccatttg atcataatgg 2700

aaagtatgtt ctgtcccatt tgccatagtc ctcacctatc cctgttgtat tttatcgggt 2760

ccaactcaac catttaaggt atttgccagc tcttgtatgc atttaggttt tgtttctttg 2820

ttttttagct catgaaatta ggtacaaagt cagagagggg tctggcatat aaaacctcag 2880

cagaaataaa gaggttttgt tgtttggtaa gaacatacct tgggttggtt gggcacggtg 2940

gctcgtgcct gtaatcccaa cactttggga ggccaaggca ggctgatcac ttgaagttgg 3000

gagttcaaga ccagcctggc caacatggtg aaatcccgtc tctactgaaa atacaaaaat 3060

taaccaggca tggtggtgtg tgcctgtagt cccaggaatc acttgaaccc aggaggcgga 3120

ggttgcagtg agctgagatc tcaccactgc acactgcact ccagcctggg caatggaatg 3180

agattccatc ccaaaaaata aaaaaataaa aaaataaaga acataccttg ggttgatcca 3240

cttaggaacc tcagataata acatctgcca cgtatagagc aattgctatg tcccaggcac 3300

tctactagac acttcataca gtttagaaaa tcagatgggt gtagatcaag gcaggagcag 3360

gaaccaaaaa gaaaggcata aacataagaa aaaaaatgga aggggtggaa acagagtaca 3420

ataacatgag taatttgatg ggggctatta tgaactgaga aatgaacttt gaaaagtatc 3480

ttggggccaa atcatgtaga ctcttgagtg atgtgttaag gaatgctatg agtgctgaga 3540

gggcatcaga agtccttgag agcctccaga gaaaggctct taaaaatgca gcgcaatctc 3600

cagtgacaga agatactgct agaaatctgc tagaaaaaaa acaaaaaagg catgtataga 3660

ggaattatga gggaaagata ccaagtcacg gtttattctt caaaatggag gtggcttgtt 3720

gggaaggtgg aagctcattt ggccagagtg gaaatggaat tgggagaaat cgatgaccaa 3780

atgtaaacac ttggtgcctg atatagcttg acaccaagtt agccccaagt gaaataccct 3840

ggcaatatta atgtgtcttt tcccgatatt cctcaggtac tccaaagatt caggtttact 3900

cacgtcatcc agcagagaat ggaaagtcaa atttcctgaa ttgctatgtg tctgggtttc 3960

atccatccga cattgaagtt gacttactga agaatggaga gagaattgaa aaagtggagc 4020

attcagactt gtctttcagc aaggactggt ctttctatct cttgtactac actgaattca 4080

cccccactga aaaagatgag tatgcctgcc gtgtgaacca tgtgactttg tcacagccca 4140

agatagttaa gtggggtaag tcttacattc ttttgtaagc tgctgaaagt tgtgtatgag 4200

tagtcatatc ataaagctgc tttgatataa aaaaggtcta tggccatact accctgaatg 4260

agtcccatcc catctgatat aaacaatctg catattggga ttgtcaggga atgttcttaa 4320

agatcagatt agtggcacct gctgagatac tgatgcacag catggtttct gaaccagtag 4380

tttccctgca gttgagcagg gagcagcagc agcacttgca caaatacata tacactctta 4440

acacttctta cctactggct tcctctagct tttgtggcag cttcaggtat atttagcact 4500

gaacgaacat ctcaagaagg tataggcctt tgtttgtaag tcctgctgtc ctagcatcct 4560

ataatcctgg acttctccag tactttctgg ctggattggt atctgaggct agtaggaagg 4620

gcttgttcct gctgggtagc tctaaacaat gtattcatgg gtaggaacag cagcctattc 4680

tgccagcctt atttctaacc attttagaca tttgttagta catggtattt taaaagtaaa 4740

acttaatgtc ttcctttttt ttctccactg tctttttcat agatcgagac atgggtggag 4800

gtggaagtgg tggaggtgga agtggtggag gtggaagtgg tggaggtgga agtggctccc 4860

actccatgag gtatttcagc gccgccgtgt cccggcccgg ccgcggggag ccccgcttca 4920

tcgccatggg ctacgtggac gacacgcagt tcgtgcggtt cgacagcgac tcggcgtgtc 4980

cgaggatgga gccgcgggcg ccgtgggtgg agcaggaggg gccggagtat tgggaagagg 5040

agacacggaa caccaaggcc cacgcacaga ctgacagaat gaacctgcag accctgcgcg 5100

gctactacaa ccagagcgag gccagttctc acaccctcca gtggatgatt ggctgcgacc 5160

tggggtccga cggacgcctc ctccgcgggt atgaacagta tgcctacgat ggcaaggatt 5220

acctcgccct gaacgaggac ctgcgctcct ggaccgcagc ggacactgcg gctcagatct 5280

ccaagcgcaa gtgtgaggcg gccaatgtgg ctgaacaaag gagagcctac ctggagggca 5340

cgtgcgtgga gtggctccac agatacctgg agaacgggaa ggagatgctg cagcgcgcgg 5400

acccccccaa gacacacgtg acccaccacc ctgtctttga ctatgaggcc accctgaggt 5460

gctgggccct gggcttctac cctgcggaga tcatactgac ctggcagcgg gatggggagg 5520

accagaccca ggacgtggag ctcgtggaga ccaggcctgc aggggatgga accttccaga 5580

agtgggcagc tgtggtggtg ccttctggag aggagcagag atacacgtgc catgtgcagc 5640

atgaggggct gccggagccc ctcatgctga gatggaagca gtcttccctg cccaccatcc 5700

ccatcatggg tatcgttgct ggcctggttg tccttgcagc tgtagtcact ggagctgcgg 5760

tcgctgctgt gctgtggaga aagaagagct cagattga 5798

<210> 10

<211> 1305

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atgtctcgct ccgtggcctt agctgtgctc gcgctactct ctctttctgg cctggaggct 60

atccagcgta ctccaaagat tcaggtttac tcacgtcatc cagcagagaa tggaaagtca 120

aatttcctga attgctatgt gtctgggttt catccatccg acattgaagt tgacttactg 180

aagaatggag agagaattga aaaagtggag cattcagact tgtctttcag caaggactgg 240

tctttctatc tcttgtacta cactgaattc acccccactg aaaaagatga gtatgcctgc 300

cgtgtgaacc atgtgacttt gtcacagccc aagatagtta agtgggatcg agacatgggt 360

ggaggtggaa gtggtggagg tggaagtggt ggaggtggaa gtggtggagg tggaagtggc 420

tcccactcca tgaggtattt cagcgccgcc gtgtcccggc ccggccgcgg ggagccccgc 480

ttcatcgcca tgggctacgt ggacgacacg cagttcgtgc ggttcgacag cgactcggcg 540

tgtccgagga tggagccgcg ggcgccgtgg gtggagcagg aggggccgga gtattgggaa 600

gaggagacac ggaacaccaa ggcccacgca cagactgaca gaatgaacct gcagaccctg 660

cgcggctact acaaccagag cgaggccagt tctcacaccc tccagtggat gattggctgc 720

gacctggggt ccgacggacg cctcctccgc gggtatgaac agtatgccta cgatggcaag 780

gattacctcg ccctgaacga ggacctgcgc tcctggaccg cagcggacac tgcggctcag 840

atctccaagc gcaagtgtga ggcggccaat gtggctgaac aaaggagagc ctacctggag 900

ggcacgtgcg tggagtggct ccacagatac ctggagaacg ggaaggagat gctgcagcgc 960

gcggaccccc ccaagacaca cgtgacccac caccctgtct ttgactatga ggccaccctg 1020

aggtgctggg ccctgggctt ctaccctgcg gagatcatac tgacctggca gcgggatggg 1080

gaggaccaga cccaggacgt ggagctcgtg gagaccaggc ctgcagggga tggaaccttc 1140

cagaagtggg cagctgtggt ggtgccttct ggagaggagc agagatacac gtgccatgtg 1200

cagcatgagg ggctgccgga gcccctcatg ctgagatgga gtaaggaggg agatggaggc 1260

atcatgtctg ttagggaaag caggagcctc tctgaagacc tttaa 1305

<210> 11

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tagccccaag tgaaataccc tgg 23

<210> 12

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gctgtctata aatagtcctc agg 23

<210> 13

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

acatgtaagc agcatcatgg agg 23

<210> 14

<211> 8395

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac 60

attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa 120

aaaggaagag tatgagtatt caacatttcc gtgtcgccct tattcccttt tttgcggcat 180

tttgccttcc tgtttttgct cacccagaaa cgctggtgaa agtaaaagat gctgaagatc 240

agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag atccttgaga 300

gttttcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg ctatgtggcg 360

cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata cactattctc 420

agaatgactt ggttgagtac tcaccagtca cagaaaagca tcttacggat ggcatgacag 480

taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc aacttacttc 540

tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg ggggatcatg 600

taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac gacgagcgtg 660

acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact ggcgaactac 720

ttactctagc ttcccggcaa caattaatag actggatgga ggcggataaa gttgcaggac 780

cacttctgcg ctcggccctt ccggctggct ggtttattgc tgataaatct ggagccggtg 840

agcgtgggtc tcgcggtatc attgcagcac tggggccaga tggtaagccc tcccgtatcg 900

tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga cagatcgctg 960

agataggtgc ctcactgatt aagcattggt aactgtcaga ccaagtttac tcatatatac 1020

tttagattga tttaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg 1080

ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg 1140

tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc 1200

aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc 1260

tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt 1320

agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc 1380

taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact 1440

caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac 1500

agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag 1560

aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg 1620

gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg 1680

tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga 1740

gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt 1800

ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct 1860

ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg 1920

aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt 1980

aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta 2040

atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta 2100

tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt 2160

acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctg caagcttaat 2220

gtagtcttat gcaatactct tgtagtcttg caacatggta acgatgagtt agcaacatgc 2280

cttacaagga gagaaaaagc accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg 2340

tgccttatta ggaaggcaac agacgggtct gacatggatt ggacgaacca ctgaattgga 2400

ggcgtggcct gggcgggact ggggagtggc gagccctcag atcctgcata taagcagctg 2460

ctttttgcct gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc 2520

taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg 2580

tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg 2640

tggaaaatct ctagcagtgg cgcccgaaca gggacctgaa agcgaaaggg aaaccagagc 2700

tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac 2760

tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga 2820

gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag 2880

ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat 2940

tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc 3000

tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa 3060

ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga 3120

tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga 3180

cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta 3240

aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa 3300

aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact 3360

atgggcgcag cctcaatgac gctgacggta caggccagac aattattgtc tggtatagtg 3420

cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca 3480

gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat 3540

caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct 3600

tggaatgcta gttggagtaa taaatctctg gaacagattg gaatcacacg acctggatgg 3660

agtgggacag agaaattaac aattacacaa gcttaataca ctccttaatt gaagaatcgc 3720

aaaaccagca agaaaagaat gaacaagaat tattggaatt agataaatgg gcaagtttgt 3780

ggaattggtt taacataaca aattggctgt ggtatataaa attattcata atgatagtag 3840

gaggcttggt aggtttaaga atagtttttg ctgtactttc tatagtgaat agagttaggc 3900

agggatattc accattatcg tttcagaccc acctcccaac cccgagggga cccgacaggc 3960

ccgaaggaat agaagaagaa ggtggagaga gagacagaga cagatccatt cgattagtga 4020

acggatctcg acggtatcga tcacgagact agcctcgaca caaatggcag tattcatcca 4080

caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat 4140

aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt 4200

tcgggtttat tacagggaca gcagaaatcc actttggctc gagaagcttg atatcggctc 4260

cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt tggggggagg 4320

ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg gtaaactggg aaagtgatgt 4380

cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt 4440

cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggatc cgccaccatg 4500

gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc 4560

gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc 4620

aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc 4680

gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag 4740

cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc 4800

aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg 4860

aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag 4920

ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc 4980

atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac 5040

cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac 5100

ctgagcaccc agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg 5160

ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaagct 5220

agcacgcgtg gcgcgccgtt aacggcgcgc ccctctccct cccccccccc taacgttact 5280

ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata 5340

ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt 5400

cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa 5460

gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag 5520

cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca 5580

cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc 5640

aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat 5700

tgtatgggat ctgatctggg gcctcggtac acatgcttta catgtgttta gtcgaggtta 5760

aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga 5820

taatatggcc acaaccctct agaatgaccg agtacaagcc cacggtgcgc ctcgccaccc 5880

gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca 5940

cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct 6000

tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg 6060

tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc 6120

cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc 6180

tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg 6240

accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc 6300

gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc 6360

ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca 6420

tgacccgcaa gcccggtgcc tgagtcgaca ctagtaatca acctttggat tacaaaattt 6480

gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg 6540

ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt 6600

ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg 6660

tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc 6720

agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg 6780

cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt 6840

tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc 6900

gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg 6960

gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga 7020

tctccctttg ggccgcctcc ccgcctgccg cggaattcga gctcggtacc tttaagacca 7080

atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa 7140

gggctaattc actcccaacg aagacaagat ctgctttttg cttgtactgg gtctctctgg 7200

ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct 7260

caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt 7320

aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag tagtagttca 7380

tgtcatctta ttattcagta tttataactt gcaaagaaat gaatatcaga gagtgagagg 7440

aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca 7500

aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct 7560

tatcatgtct ggctctagct atcccgcccc taactccgcc cagttccgcc cattctccgc 7620

cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc 7680

tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcgtc gagacgtacc 7740

caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg 7800

tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc 7860

cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct 7920

gaatggcgaa tggcgcgacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt 7980

tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt 8040

cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc 8100

tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga 8160

tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc 8220

cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt 8280

ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct 8340

gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa tttcc 8395

Claims (10)

1. A method of constructing a human embryonic stem cell, the method comprising: integrating exogenous polynucleotide for coding B2M-HLA-G1 fusion protein into the genome of the human embryonic stem cell, wherein the B2M-HLA-G1 fusion protein comprises a first B2M segment and an HLA-G1 segment, and the integrated human embryonic stem cell does not express free B2M protein.

2. The method of claim 1, wherein said fragment of HLA-G1 comprises:

a) polypeptide fragment with amino acid sequence shown as SEQ ID No. 1; or the like, or, alternatively,

b) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.1 and having the function of the polypeptide fragment defined in a).

3. The method of construction of claim 1, wherein the first B2M fragment comprises:

c) polypeptide fragment with amino acid sequence shown as SEQ ID No. 2; or the like, or, alternatively,

d) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.2 and having the function of the polypeptide fragment defined in a).

4. The method of claim 1, wherein the B2M-HLA-G1 fusion protein further comprises a first flexible linker peptide between the first B2M fragment and HLA-G1 fragment;

and/or the B2M-HLA-G1 fusion protein sequentially comprises a first B2M fragment and an HLA-G1 fragment from the N end to the C end, and the B2M-HLA-G1 fusion protein comprises a polypeptide fragment with an amino acid sequence shown as SEQ ID No. 4.

5. The method of claim 1, wherein the method of integrating the exogenous polynucleotide encoding the B2M-HLA-G1 fusion protein into the genome of the human embryonic stem cell specifically comprises: the gene encoding the fragment of HLA-G1 is fused with the endogenous B2M gene in human embryonic stem cells, preferably by replacing the gene encoding the fragment of HLA-G1 with a stop codon located in exon 3 of the endogenous B2M gene.

6. The method of construction according to claim 1, further comprising: integrating exogenous polynucleotide encoding B2M-HLA-G5 fusion protein into genome of human embryonic stem cell, wherein said B2M-HLA-G5 fusion protein comprises second B2M segment and HLA-G5 segment.

7. The method of construction of claim 6 wherein the second B2M fragment comprises:

e) polypeptide fragment with amino acid sequence shown as SEQ ID No. 5; or the like, or, alternatively,

f) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.5 and having the function of the polypeptide fragment defined in c).

8. The method of claim 6, wherein said fragment of HLA-G5 comprises:

g) polypeptide fragment with amino acid sequence shown as SEQ ID No. 6; or the like, or, alternatively,

h) a polypeptide fragment having an amino acid sequence with more than 90% sequence identity to SEQ ID No.6 and having the function of the polypeptide fragment defined in e).

9. The method of claim 6, wherein the B2M-HLA-G5 fusion protein further comprises a second flexible linker between the second B2M fragment and HLA-G5 fragment;

and/or the B2M-HLA-G5 fusion protein sequentially comprises a second B2M fragment and an HLA-G5 fragment from the N end to the C end, and the B2M-HLA-G5 fusion protein comprises a polypeptide fragment with an amino acid sequence shown as SEQ ID No. 8.

And/or, the method for integrating the exogenous polynucleotide for coding the B2M-HLA-G5 fusion protein into the genome of the human embryonic stem cell specifically comprises the following steps: the polynucleotide encoding the B2M-HLA-G5 fusion protein was integrated into the genome of human embryonic stem cells by a lentiviral vector.

10. A human embryonic stem cell constructed by the method of any one of claims 1 to 9.

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