CN107916255A - 1 gene target specific methylation epigenetic modification antibumor molecules of PDL and application thereof - Google Patents

1 gene target specific methylation epigenetic modification antibumor molecules of PDL and application thereof Download PDF

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CN107916255A
CN107916255A CN201711135791.9A CN201711135791A CN107916255A CN 107916255 A CN107916255 A CN 107916255A CN 201711135791 A CN201711135791 A CN 201711135791A CN 107916255 A CN107916255 A CN 107916255A
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CN107916255B (en
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朱乃硕
李雪
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Fudan University
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Abstract

The invention belongs to gene therapy technology field, is specially 1 gene target specific methylation epigenetic modification antibumor molecules of PDL and application thereof.The present invention is according to bioinformatics and epigenetic engineering and immunology principle,Design carries out the brand-new protein molecular PPLA of the apparent modification that methylates for people PD L1 gene promoter sequences,The intracellular of people is imported by adenovirus vector,Can high efficient expression go out for the special protein molecular PPLA of people's PD L1 gene promoter sequences,Its gene expressed can effectively suppress PD L1 genes,The molecule can produce 1 genes of PDL the chemical modification effect that methylates,Intracellular experiment confirms have the function of efficiently to block 1 gene expressions of PDL in vitro,So as to which break immune is resistant to,It is finally reached the effect for the treatment of chronic viral infection and tumour,Realize antitumor and antiviral immunotherapeutic effects,It is with a wide range of applications in terms of epigenetic engineering treatment tumour and antiviral drugs is prepared.

Description

PDL-1 gene targets specific methylation epigenetic modification antibumor molecules and its Purposes
Technical field
The invention belongs to gene therapy technology field, and in particular to PDL-1 gene target specific methylation epigenetics Modify antibumor molecules and application thereof.
Background technology
It is immune（immunity）Typically refer to resistance of the organism to infectious disease, the identification to self and non-self material And the monitoring and removing of the denaturation material to producing in vivo.It is immune to be divided into congenital immunity（innate immunity）With obtain Property is obtained to be immunized（acquired immunity）, wherein cellular immunity and humoral immunity can be subdivided into again.Immunoregulation is immune Important step.Immunoregulation（immunoregulation）Refer to organism it is horizontal to immune response process and immune response one Kind performs and Reasonable Regulation And Control, to adapt to the needs of immune defense, maintain the internal of organism to stablize（homeostasis）.Its Matter is under genetic regulation and control, the regulation process of multifactor participation.
Lymphocyte activation is the significant process among cell-mediated immune response.Antigen is captured through APC, processes, located After reason and presentation, T cell is passed information to, promotes the Th cells that T cell Proliferation, Differentiation is CD4+, Th cells can be secreted The cell factor such as IL-2, IL-4, IL-12 and IFN-γ, the propagation of the cytotoxic T cell Tc cells of further promotion CD8+, Differentiation, maturation.Tc cells specific can identify specific antigenic determinant, by being mediated with MHC- class Ⅰmolecules or non- Specific adhesion molecular action, the contact of target cell containing specific antigen peptide, targeted release cytotoxin（Proteolysis Enzyme, serine lipase etc.）, cytotoxic effect is carried out to target cell；Or by discharging TNF-α, TNF-beta, directly to target cell Carry out toxic action.
T cell activation needs several factors to act at the same time：（1）APC surfaces MHC- antigenic peptide complexes and T cell surface TCR is combined；（2）APC surfaces B7 molecules are combined with T cell surface C D28 molecules；（3）The cell factors such as IL-1, IL-2 stimulate T thin Born of the same parents activate.
Immune detection point（immune checkpoint）It is present in organism immune system, for immune system The important molecule on some paths for playing restriction effect is played function, their Immune discriminations to immune system, immune tolerance Formation and the adjusting of immune intensity there is key.The immune detection point having now been found that has very much, they are more Immunocyte surface is expressed in, plays the role of limiting immune cell function.Their intracellular sections generally contain multiple immunity receptor junket Propylhomoserin suppresses motif（Immuno-receptor tyrosine-based inhibitory motif, ITIM）, pass through ITIM bases Sequence plays the effect of negative regulation.Clearer and more definite immune detection point includes now：CTLA-4, PD-1, PD-L1 etc..
PD-1（CD279）/PD-L1（CD274）Signal path is that the important of negative regulation and tumor death is immunized in mediate T cell Signal, is that tumour cell realizes one of primary signal pathways of immune evasion.PD-1 is expressed in the T cell surface of activation, PD-L1 It is expressed in the surface of kinds of tumor cells.It has recently been demonstrated that the signal path by blocking PD-1 and PD-L1, can be effective Raising T cell reaction and its antitumor action.PD-1 is to be withered by the TasukuHonjo of Kyoto Univ Japan in 1992 On the T cell hybridoma died, the method by cutting down hybridization takes the lead in what is found, since its function and the procedural of cell are withered Die it is related, so being named as apoptosis acceptor 174.As negative regulation acceptor is immunized with other, PD-1 is expressed in T cell, Treg, B cell, mononuclearcell, DC cells, NK cells and the NKT cell surfaces of activation.The PD-1 albumen of people In on 2q37.3 chromosomes, total length 9028bp, gene order has 23% similitude with CTLA-4 for the assignment of genes gene mapping, includes outside 5 Aobvious son.PD-1 molecular sizes are 55kDa, belong to I type transmembrane protein in immunoglobulin class.Extracellular fragment contains an IgV Structural domains, have 4 N connection sites to be glycosylated；Intracellular section contains ITIM and ITSM, when receiving signal When, which can suppress cytoplasmic phosphorylation, play the function of antagonism immunization.
PD-L1 is the ligand of PD-1, it participates in the negative regulation effect in T cell activation.The human PD-L 1 assignment of genes gene mapping is in 9q24 On chromosome, total length 20065bp, there is 6 extrons, encodes 290 amino acid.PD-L1 is I type transmembrane protein, by IgV, IgC Extracellular domain, the powered intracellular region composition of hydrophobic transmembrane domain and 30 amino acid composition.The mRNA of human PD-L 1 is wide In each tissue for glowing present human body, but it is not expressed on cell membrane.Only find it in fraction cell at present（Such as liver Or the macrophage like cell in lung）And partial immunity immunoprivileged tissues（Such as eyes, uterus）Cell surface has expression.However, PD-L1 High selectivity is expressed in lung cancer, stomach cancer, colon cancer, breast cancer, cervical carcinoma, kidney, carcinoma of urinary bladder, liver cancer, spongiocytoma, black The lymphocyte and endothelial cell surface infiltrated in plain knurl and other a variety of human tumor cells and tumor microenvironment, is one Specific anti-tumor target efficiently, less toxic.
PD-L1 can be stimulated by substantial amounts of cell factor and be improved expression, as IFN-γ, TNF α, VEGE, GM-CSF and IF-10.The expression of PD-L1 molecules is had been found that on the surface of many tumour cells, and among tumor infiltrating lymphocyte （Tumor-infiltrating lymphocytes, TILs）, PD-1 can specific high expression, so as to be combined with PD-L1, Suppress its effect.When tumour cell is by immune system attack, tumour cell can start to be overexpressed PD-L1 molecules, reduce T cell Effect, thus successfully escape immune attack.
In the microenvironment of tumour, T cell can lower its immunologic cytotoxicity by PD-1/PD-L1 paths by diversified forms Effect.Now it is verified that, overexpressions of the PD-L1 in tumor cell surface so that macrophage, DCs, MDSCs etc. it is anti- Function of tumor is significantly limited.Akt, mTOR, S6 and ERK2 equimolecular of phosphorylation are important in Treg growths, atomization Molecule, PD-L1 can promote the differentiation of Treg cells, and then the function of restricted T cells by adjusting the secretions of these molecules.Separately One side PD-1 lowers the secretion of anti-apoptotic proteins Bcl-xL, and then the T of induced activation is thin by suppressing the activity of PI3K paths The apoptosis of born of the same parents.PD-1 albumen may also suppress the activity of TCR, so that the secretion of the relevant factor IL-2 of lymphocyte growth is reduced, Stop cell division.
In immunotherapy of tumors, it is resistant to by break immune, to realize killing of the specific T-cells to tumour, is reached Treat the purpose of tumour.Research shows, PD-1/PD-L1 can be reduced immune thin in the expression of lymphocyte and tumor cell surface Born of the same parents lower the response of tumour immunity to the lethal effect of tumour.Fall PD-1/PD-L1 paths using monoclonal antibody closing, treatment mouse is swollen Knurl model, hence it is evident that inhibit the growth of local tumor, show good relief of symptoms.The path is blocked at the same time, can be raised IFN γ is secreted, and is achieveed the purpose that antitumor.
The present invention is devised for human PD-L 1 base according to brand-new epigenetic technique for gene engineering and immunology principle Because of the core sequence binding proteins specific of promoter CpG II, and and methylase（Dnmts）Catalytic domain fusion, with structure The methyl chemoattractant molecule PPLA of PD-L1 genes is targeted, is packed using adenovirus, therefore by PD-L1 gene promoters Targeting methylates, and it is expected to suppress the expression of cancer cell PD-L1 genes, is resistant to break immune, reach to chronic viral infection and The therapeutic effect of tumour.And the key problem of gene therapy be select safely and effectively carrier and by therapeutic gene import target it is thin Born of the same parents.Adenovirus (Adenovirus, Ad) carrier has the advantages that host range is wide, efficient, stable, safe, easy to operate and standby Paid attention to.
The content of the invention
The purpose of the object of the invention be to provide a kind of PDL-1 gene targets specific methylation epigenetic modification resist it is swollen Knurl molecule and application thereof.
The present invention first design be directed to human PD-L 1 gene promoter, using maintain methylase Dnmt1 active domain and from The active domain fusion connection of head methylase Dnmt3a, and the brand-new protein molecular of the apparent modification that methylates is carried out, with the albumen Molecule suppresses the expression of PD-L1 genes, and finally prepares for antitumor and antiviral therapy medicine.
An aspect of of the present present invention, there is provided a kind of binding proteins specific molecule for identifying human PD-L 1 gene promoter, its ammonia Base acid sequence is shown in SEQ.ID.No.1, is named as PPLA.The present invention utilizes bioinformatics means, is given birth to first by eucaryon The promoter sequence of thing gene promoter database analysis PD-L1 genes, it is potential with CpG islands software analysis promoter core sequence Methylation sites, then using corresponding software, design is with the protein structure for DNA sequence dna specific recognition and binding function Domain, that is, obtain the binding proteins specific molecule of the identification PD-L1 gene promoters, its amino acid sequence is SEQ.ID.No.1。
Another aspect of the present invention, there is provided encode the gene of above-mentioned protein molecular PPLA, its nucleotides sequence is classified as Shown in SEQ.ID.No.2.
Another aspect of the invention, there is provided the expression vector of above-mentioned protein molecular PPLA, including selection people methylate Enzyme, recombinates to plasmid pcDNA3.1, and in the gene expression plasmid of N-terminal expressing in series PPLA, progress adenovirus packaging, structure PDL-1 P targeting specific DNA methylation enzyme domains are built, as shown in attached drawing 1.
Another aspect of the invention, by adenovirus vector import people it is intracellular, can high efficient expression go out for human PD-L 1 The special protein molecular of gene promoter sequence, and the function for the epigenetic modification that methylates, experiment can be produced to PDL-1 genes Confirm, PPLA has the function of efficiently to suppress PDL-1 gene expressions, you can effectively make PD-L1 gene promoter methylations, and The expression of PD-L1 genes can be significantly reduced, so that achieve the purpose that antitumor and antiviral lymphocyte, improve immune function, Realize antitumor and antiviral therapeutic effect, this provides new experimental basis and technology hand for the prevention of tumour and virus Section.Therefore it provides above-mentioned protein molecular PPLA, or the gene of protein molecular PPLA is encoded, or its expression vector, available for making Standby antitumor and antiviral drug.
Brief description of the drawings
Fig. 1 is PPLA adenovirus expression plasmid structure collection of illustrative plates.
Fig. 2 be PPLA expression vectors adenovirus packaging after expression map in cell.
Fig. 3 is PPLA protein expression Western blottings（Western Blot）Analyze collection of illustrative plates.
Fig. 4 is PPLA albumen and PD-L1 gene promoter DNA combination conformational maps.
Fig. 5 is that PPLA influences the methyl rate of PD-L1 genes.
Fig. 6 is PD-L1 molecule encoding mRNA expression maps.
Fig. 7 is its PD-L1 expression quantity collection of illustrative plates of the facs analysis of PPLA transfectional cells.
Fig. 8 is its PD-L1 expression quantity collection of illustrative plates of PPLA suppression tumour cells.
Embodiment
1. the design and synthesis of human PD-L 1 gene promoter binding proteins specific
Using ZF Tools Ver3.0 softwares, according to human PD-L 1 promoter region CpG sites, optimal sequence is selected, design is special Property associated proteins.The amino of the selected DNA for human PD-L 1 promoter region is combined and mediated dna methylates albumen PPLA Acid sequence is shown in SEQ.ID.No.1, corresponding each domain nucleotides sequences of DNA is classified as shown in SEQ.ID.No.2.
2. the structure and adenovirus packaging of human PD-L 1 gene promoter targeting methylase
By the DNA fragmentation clone restructuring of artificial synthesized coding PPLA to pcDNA3.1（Such as Fig. 1）On, and expression plasmid is carried out Adenovirus is packed（Such as Fig. 2）, the engineered protein PPLA of expression is obtained, confirms that its protein content exists by Western-blot 130KDa（Such as Fig. 3）, by bioinformatic analysis, space structure is simulated, the albumen of its intracellular expression can be with PD-L1 genes Promoter combines（Such as Fig. 4）.We pass through the sequencing that methylates, it was demonstrated that the albumen can induce PD-L1 gene promoters and occur specifically Property methylate phenomenon, while respectively in 24h, 48h, 72h use quantitative PCR, cell instrument of passing, and Westerblot is further analyzed Confirm that it can cause PD-L1 gene expressions significantly to decline（Such as Fig. 6,7,8）.
Sequence table
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gaacatcagc gcacccatac cggcgaaaaa ccgtataaat gcccggaatg cggcaaaagc 600
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gtgcggcacc aggggaagat catgtacgtc ggggacgtcc gcagcgtcac acagaagcat 900
atccaggagt ggggcccatt cgatctggtg attgggggca gtccctgcaa tgacctctcc 960
atcgtcaacc ctgctcgcaa gggcctctac gagggcactg gccggctctt ctttgagttc 1020
taccgcctcc tgcatgatgc gcggcccaag gagggagatg atcgcccctt cttctggctc 1080
tttgagaatg tggtggccat gggcgttagt gacaagaggg acatctcgcg atttctcgag 1140
tccaaccctg tgatgattga tgccaaagaa gtgtcagctg cacacagggc ccgctacttc 1200
tggggtaacc ttcccggtat gaacaggccg ttggcatcca ctgtgaatga taagctggag 1260
ctgcaggagt gtctggagca tggcaggata gccaagttca gcaaagtgag gaccattact 1320
acgaggtcaa actccataaa gcagggcaaa gaccagcatt ttcctgtctt catgaatgag 1380
aaagaggaca tcttatggtg cactgaaatg gaaagggtat ttggtttccc agtccactat 1440
actgacgtct ccaacatgag ccgcttggcg aggcagagac tgctgggccg gtcatggagc 1500
gtgccagtca tccgccacct cttcgctccg ctgaaggagt attttgcgtg tgtgggcggc 1560
ggcggcagcg gcggcggcgg cagcaaaatc cgggtcaaca agttctacag gcctgagaac 1620
acccacaagt ccactccagc gagctaccac gcagacatca acctgctcta ctggagcgac 1680
gaggaggccg tggtggactt caaggctgtg cagggccgct gcaccgtgga gtatggggag 1740
gacctgcccg agtgcgtcca ggtgtactcc atgggcggcc ccaaccgctt ctacttcctc 1800
gaggcctata atgcaaagag caaaagcttt gaagatcctc ccaaccatgc ccgtagccct 1860
ggaaacaaag ggaagggcaa gggaaaaggg aagggcaagc ccaagtccca agcctgtgag 1920
ccgagcgagc cagagataga gatcaagctg cccaagctgc ggaccctgga tgtgttttct 1980
ggctgcgggg ggttgtcgga gggattccac caagcaggca tctctgacac gctgtgggcc 2040
atcgagatgt gggaccctgc ggcccaggcg ttccggctga acaaccccgg ctccacagtg 2100
ttcacagagg actgcaacat cctgctgaag ctggtcatgg ctggggagac caccaactcc 2160
cgcggccagc ggctgcccca gaagggagac gtggagatgc tgtgcggcgg gccgccctgc 2220
cagggcttca gcggcatgaa ccgcttcaat tcgcgcacct actccaagtt caaaaactct 2280
ctggtggttt ccttcctcag ctactgcgac tactaccggc cccggttctt cctcctggag 2340
aatgtcagga actttgtctc cttcaagcgc tccatggtcc tgaagctcac cctccgctgc 2400
ctggtccgca tgggctatca gtgcaccttc ggcgtgctgc aggccggtca gtacggcgtg 2460
gcccagacta ggaggcgggc catcatcctg gccgcggccc ctggagagaa gctccctctg 2520
ttcccggagc cactgcacgt gtttgctccc cgggcctgcc agctgagcgt ggtggtggat 2580
gacaagaagt ttgtgagcaa cataaccagg ttgagctcgg gtcctttccg gaccatcacg 2640
gtgcgagaca cgatgtccga cctgccggag gtgcggaatg gagcctcggc actggagatc 2700
tcctacaacg gggagcctca gtcctggttc cagaggcagc tccggggcgc acagtaccag 2760
cccatcctca gggaccacat ctgtaaggac atgagtgcat tggtggctgc ccgcatgcgg 2820
cacatcccct tggccccagg gtcagactgg cgcgatctgc ccaacatcga ggtgcggctc 2880
tcagacggca ccatggccag gaagctgcgg tatacccacc atgacaggaa gaacggccgc 2940
agcagctctg gggccctccg tggggtctgc tcctgcgtgg aagccggcaa agcctgcgac 3000
cccgcagcca ggcagttcaa caccctcatc ccctggtgcc tgccccacac cgggaaccgg 3060
cacaaccact gggctggcct ctatggaagg ctcgagtggg acggcttctt cagcacaacc 3120
gtcaccaacc ccgagcccat gggcaagcag ggccgcgtgc tccacccaga gcagcaccgt 3180
gtggtgagcg tgcgggagtg tgcccgctcc cagggcttcc ctgacaccta ccggctcttc 3240
ggcaacatcc tggacaagca ccggcaggtg ggcaatgccg tgccaccgcc cctggccaaa 3300
gccattggct tggagatcaa gctttgtatg ttggccaaag cccgagagag tgcctcagct 3360
aaaataaagg aggaggaagc tgctaaggac tag 3393

Claims (4)

1. a kind of binding proteins specific molecule for identifying human PD-L 1 gene promoter, its amino acid sequence is SEQ.ID.No.1 It is shown, it is named as PPLA.
2. a kind of gene for encoding protein molecular as claimed in claim 1, its nucleotides sequence are classified as shown in SEQ.ID.No.2.
3. a kind of expression vector of gene as claimed in claim 2, including selection people's methylase, recombinate to plasmid On pcDNA3.1, and in the gene expression plasmid of N-terminal expressing in series PPLA, progress adenovirus packaging, structure PDL-1 P targetings Specific DNA methylase domain.
4. the binding proteins specific molecule of identification human PD-L 1 gene promoter as claimed in claim 1, or as right will The gene described in 2 is sought, the application in antitumor and antiviral drug is prepared.
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