CN102643347A - Leukemia stem cell targeting soluble protein TrxHis-hSIRP alpha - Google Patents
Leukemia stem cell targeting soluble protein TrxHis-hSIRP alpha Download PDFInfo
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Abstract
The invention relates to a soluble protein, particularly to a leukemia stem cell targeting soluble protein TrxHis-hSIRP alpha, which is composed of the extracellular domain of a human CD47 receptor SIRP alpha, and a TrxHis expression tag fused with the amino terminal of the receptor SIRP alpha, and is characterized by being composed of the extracellular domain of the human SIRP alpha protein and the expression tag fused with TrxHis, wherein the extracellular domain of the hSIRP alpha protein is the 26th-370th amino acids of the hSIRP alpha; the 26th-370th amino acids of the hSIRP alpha contain the IgV-like structural domain bound with the CD47; and the fused TrxHis expression tag is the amino terminal of the soluble protein. The TrxHis-hSIRP alpha can be produced on a large scale in vitro via genetic engineering, and bound with the high-expression CD47 on a leukemia cell or an LSC (leukemia stem cell) to prevent the binding of the inhibitory receptor SIRP alpha on a macrophage and the CD47 in case of being applied in vivo, thus promoting the phagocytosis of the macrophage on the leukemia cell and the LSC, and having important significance for leukemia targeting therapy and minimal residual disease elimination.
Description
Technical field
The present invention relates to a kind of soluble proteins; It is the extracellular fragment by people CD47 acceptor SIRP α; The TrxHis that merges with its N-terminal expresses a kind of leukemic stem cells targeting soluble albumen TrxHis-hSIRP α that label is formed, and belongs to the medical biotechnology field.
Background technology
Signal adjusting protein alpha (Signal-regulatory protein α, SIRP α) is called SHPS-1 again, SIRPA, p84 and BIT, contactin member.It mainly is expressed in scavenger cell, neurone, dendritic cell and neutrophil leucocyte.This transmembrane protein comprises the tyrosine phosphorylation site of three immunoglobulin like domain and the cytoplasmic domain of extracellular region; Its cytoplasmic domain is rat; High conservative between mouse and the people; Contain tyrosine residues and form the inhibitory motifs ITIM of inhibition acceptor, thereby activate Protein-tyrosine-phosphatase SHP-1 and the SHP-2 that comprises the SH2 structural domain based on tyrosine.SHP-1 mainly is expressed in hematopoietic cell and epithelial cell, but negativity is regulated many functions of these cells, and SHP-2 expresses extensively, can bring into play forward regulatory functions such as promoting cell growth, migration and differentiation through regulating little gtp binding protein Ras and Rho.Therefore, just outer stimulation produces reaction to SIRP α as a docking protein cell membrane, and brings into play its special biological function through raising SHP-1 or SHP-2.In addition, SIRP α also can connect Src kinase-associated protein SCAP2 (being also referred to as SKAP55hom/R) and Fyn connection albumen FYB (being also referred to as SLAP-130) and cause obvious suppression property signal.In recent years discover that SIRP α is bringing into play important effect as the outer acceptor of the born of the same parents of CD47 in the scavenging process of scavenger cell to tumour cell.
CD47 claims that again (integrin-associated protein IAP), also belongs to the member of Tegeline (Ig) superfamily to integrin associated protein.At first from people's placenta and integrin alpha V β 3 copurifications and be familiar with by people.It is a kind of antigen of wide expression, be present on the different cells in a organized way.This transmembrane protein comprises V type Ig appearance ectodomain, strides film district and cytoplasmic domain highly hydrophobic five times.CD47 is that the albumen test of pair cell epimatrix is regulated and played a role as the white corpuscle of integrating plain dependence at first.The most classical definite CD47 regulate phagocytotic source of evidence in: CD47 is knocked out removed from blood by scavenger cell very soon after red corpuscle (RBCs) is implanted into the normal mouse body in, the CD47 that shows the RBC surface combines with the SIRPa on scavenger cell surface and possibly have vital role in self cell removing cushioning scavenger cell.
The phagolysis of scavenger cell is the important adjusting factor that tumour immunity is kept watch on.Nearest research shows; In acute myelocytic leukemia (AML), acute lymphoblastic leukemia (ALL) and non_hodgkin lymphoma pernicious hematological system tumor diseases such as (NHL); A large amount of leukemia cells and leukemic stem cells (LSC) CD47 expresses significantly and raises; And can through and SIRPa between interaction, stop the leukemia cell to be removed by host's innate immune system.This also is one of major reason of drug-resistant leukemia, recurrence.And, can promote the phagolysis of scavenger cell, and can not reduce normal hemopoietic stem cell the important clinical meaning is arranged for leukemic treatment to leukemia cell and LSC through the effect between the CD47 monoclonal antibody blocking-up CD47-SIRP α of target property.
The present invention is based on the vital role of CD47-SIRP α in adjusting is engulfed, expressed the SIRP α extracellular fragment albumen of solubility, combines the leukemia cell to go up CD47 with SIRP α on scavenger cell competition, thereby the promotion scavenger cell is to the phagolysis of tumour cell.
Summary of the invention
The object of the invention provides a kind of leukemic stem cells targeting soluble albumen TrxHis-hSIRP α, and this albumen can be incorporated into leukemia cell and leukemic stem cells surface, strengthens the phagolysis of scavenger cell to leukemic stem cells effectively.
Technical scheme of the present invention is: a kind of leukemic stem cells targeting soluble albumen TrxHis-hSIRP α is characterized in that: it is made up of people SIRP α albumen extracellular fragment and the expression label that merges TrxHis; Wherein hSIRP α albumen extracellular fragment is hSIRP α the 26th ~ 370 amino acids.
Described hSIRP α the 26th ~ 370 amino acids comprises and CD47 bonded IgV spline structure territory; It is the N-terminal of this soluble proteins that the TrxHis of described fusion expresses label.Wherein, aminoacid sequence is No1, and by the nucleic acid sequence encoding of No2.
The aminoacid sequence of No1:TrxHis-hSIRP α
msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvaklnidqnpgtapkygirgiptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamadigvageeelqviqpdksvlvaagetatlrctatslipvgpiqwfrgagpgreliynqkeghfprvttvsdltkrnnmdfsirignitpadagtyycvkfrkgspddvefksgagtelsvrakpsapvvsgpaaratpqhtvsftceshgfsprditlkwfkngnelsdfqtnvdpvgesvsysihstakvvltredvhsqvicevahvtlqgdplrgtanlsetirvpptlevtqqpvraenqvnvtcqvrkfypqrlqltwlengnvsrtetastvtenkdgtynwmswllvnvsahrddvkltcqvehdgqpavskshdlkvsahpkeqgsntaaentgsner。
The nucleotide sequence of No2:TrxHis-hSIRP α
atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctgatatcggagtggcgggtgaggaggagctgcaggtgattcagcctgacaagtccgtgttggttgcagctggagagacagccactctgcgctgcactgcgacctctctgatccctgtggggcccatccagtggttcagaggagctggaccaggccgggaattaatctacaatcaaaaagaaggccacttcccccgggtaacaactgtttcagacctcacaaagagaaacaacatggacttttccatccgcatcggtaacatcaccccagcagatgccggcacctactactgtgtgaagttccggaaagggagccccgatgacgtggagtttaagtctggagcaggcactgagctgtctgtgcgcgccaaaccctctgcccccgtggtatcgggccctgcggcgagggccacacctcagcacacagtgagcttcacctgcgagtcccacggcttctcacccagagacatcaccctgaaatggttcaaaaatgggaatgagctctcagacttccagaccaacgtggaccccgtaggagagagcgtgtcctacagcatccacagcacagccaaggtggtgctgacccgcgaggacgttcactctcaagtcatctgcgaggtggcccacgtcaccttgcagggggaccctcttcgtgggactgccaacttgtctgagaccatccgagttccacccaccttggaggttactcaacagcccgtgagggcagagaaccaggtgaatgtcacctgccaggtgaggaagttctacccccagagactacagctgacctggttggagaatggaaacgtgtcccggacagaaacggcctcaaccgttacagagaacaaggatggtacctacaactggatgagctggctcctggtgaatgtatctgcccacagggatgatgtgaagctcacctgccaggtggagcatgacgggcagccagcggtcagcaaaagccatgacctgaaggtctcagcccacccgaaggagcagggctcaaataccgccgctgagaacactggatctaatgaacgg。
Described leukemic stem cells targeting soluble albumen TrxHis-hSIRP α has the aminoacid sequence of No3, and by the nucleic acid sequence encoding of No4:
The aminoacid sequence of No3:hSIRP α extracellular fragment
gvageeelqviqpdksvlvaagetatlrctatslipvgpiqwfrgagpgreliynqkeghfprvttvsdltkrnnmdfsirignitpadagtyycvkfrkgspddvefksgagtelsvrakpsapvvsgpaaratpqhtvsftceshgfsprditlkwfkngnelsdfqtnvdpvgesvsysihstakvvltredvhsqvicevahvtlqgdplrgtanlsetirvpptlevtqqpvraenqvnvtcqvrkfypqrlqltwlengnvsrtetastvtenkdgtynwmswllvnvsahrddvkltcqvehdgqpavskshdlkvsahpkeqgsntaaentgsner。
The nucleotide sequence of No4:hSIRP α extracellular fragment
ggagtggcgggtgaggaggagctgcaggtgattcagcctgacaagtccgtgttggttgcagctggagagacagccactctgcgctgcactgcgacctctctgatccctgtggggcccatccagtggttcagaggagctggaccaggccgggaattaatctacaatcaaaaagaaggccacttcccccgggtaacaactgtttcagacctcacaaagagaaacaacatggacttttccatccgcatcggtaacatcaccccagcagatgccggcacctactactgtgtgaagttccggaaagggagccccgatgacgtggagtttaagtctggagcaggcactgagctgtctgtgcgcgccaaaccctctgcccccgtggtatcgggccctgcggcgagggccacacctcagcacacagtgagcttcacctgcgagtcccacggcttctcacccagagacatcaccctgaaatggttcaaaaatgggaatgagctctcagacttccagaccaacgtggaccccgtaggagagagcgtgtcctacagcatccacagcacagccaaggtggtgctgacccgcgaggacgttcactctcaagtcatctgcgaggtggcccacgtcaccttgcagggggaccctcttcgtgggactgccaacttgtctgagaccatccgagttccacccaccttggaggttactcaacagcccgtgagggcagagaaccaggtgaatgtcacctgccaggtgaggaagttctacccccagagactacagctgacctggttggagaatggaaacgtgtcccggacagaaacggcctcaaccgttacagagaacaaggatggtacctacaactggatgagctggctcctggtgaatgtatctgcccacagggatgatgtgaagctcacctgccaggtggagcatgacgggcagccagcggtcagcaaaagccatgacctgaaggtctcagcccacccgaaggagcagggctcaaataccgccgctgagaacactggatctaatgaacgg。
Said N-terminal merges TrxHis expresses label, comprises the aminoacid sequence of No5, and by the nucleic acid sequence encoding of No6:
The aminoacid sequence of No5:TrxHis
msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvaklnidqnpgtapkygirgiptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamadi。
The nucleotide sequence of No6:TrxHis
atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctgatatc。
Said leukemic stem cells targeting soluble albumen TrxHis-hSIRP α realizes solubility expression in e. coli bl21, and with the nickel metal-chelating column purification to the His label, obtains pure TrxHis-hSIRP α albumen behind the protein cleavage.
Characteristics of the present invention are: TrxHis-hSIRP α can carry out mass production through genetically engineered external; Can combine with the CD47 of leukemia cell and the last high expression level of LSC when being applied in the body; Inhibition acceptor SIRP α combines with CD47 on the prevention scavenger cell; Thereby promote the phagolysis of scavenger cell, significance is arranged for leukemic targeted therapy and removing microresidual disease to leukemia cell and LSC.
Description of drawings
Fig. 1, the proteic structure of hSIRP α extracellular fragment, structure and binding mode figure;
The agarose gel electrophoresis of the pcr amplification product of Fig. 2, goal gene (hSIRP α);
The enzyme of Fig. 3, expression vector pET32a-hSIRP α is cut evaluation figure;
The SDS-PAGE protein electrophoresis figure that Fig. 4, TrxHis-hSIRP alpha fusion protein are expressed;
Electrophorogram behind Fig. 5, the TrxHis-hSIRP alpha fusion protein purifying; Western blot qualitative detection
Fig. 6, people U937 cell induce differentiation;
The avidity of Fig. 7, TrxHis-hSIRP alpha fusion protein and tumour cell detects;
Fig. 8, TrxHis-hSIRP alpha fusion protein promote the phagolysis of scavenger cell.
Fig. 9, TrxHis-hSIRP alpha fusion protein promote the phagolysis of scavenger cell.
Embodiment
Below combine embodiment that the present invention is done detailed explanation through accompanying drawing.
1, construction of expression vector.Sequences Design primer P1:5 '-GATATCATGGGAGTGGCGGGTGAGGAG-3 ' according to hSIRP α; P2:5 '-GCTCGAGCCGTTCATTAGAT CCAGTGT-3 '.With people's lymphoglandula cDNA is template; The PCR amplification coding hSIRP amino acid whose polynucleotide sequence of α extracellular fragment (Fig. 2); 1.5% agarose electrophoresis reclaims the back and is connected 2 hours for 16 ℃ with the pMD18-T carrier, heat-shocked transformed into escherichia coli XL10, after the amplification with purpose fragment subclone to expression vector pET32a (+); Make up pET32a-hSIRP α (Fig. 3), restriction enzyme digestion (Fig. 4), order-checking are identified.
Fig. 1, the proteic structure of hSIRP α extracellular fragment, structure and binding mode figure.
The agarose gel electrophoresis of the pcr amplification product of Fig. 2, goal gene (hSIRP α), 1 road (M) are molecular weight sign (DL2000), and the arrow indication is an amplified fragments.
Fig. 3, cut evaluation figure for the enzyme of expression vector pET32a-hSIRP α; (plasmid is used restriction enzyme EcoRV+XhoI and Sac I+Bgl II double digestion, agarose gel electrophoresis observations respectively.The M road is marker DL2000).
2, abduction delivering fusion rotein.Respectively with heat-shocked method transformed into escherichia coli BL21, it is dull and stereotyped that coating contains 100 μ g/ml penbritin LB with expression vector pET32a (+), pET32a-hSIRP α, cultivates after 12 hours at random the picking mono-clonal for 37 ℃ and be inoculated into and contain 100 μ g/ml penbritin LB liquid nutrient mediums; 220rpm cultivates 12h, is transferred in fresh LB (+) substratum with 1% for 37 ℃; 220rpm, 37 ℃ be cultured to 3h after, add the IPTG of final concentration 1.0mM; 200rpm cultivates 5h for 37 ℃.The centrifugal 10min of 6500rpm collects bacterium, and the resuspended bacterium of 200 μ l/ml substratum PBS is ultrasonicly split bacterium; Add 1%Triton-X100; Mixing, 4 ℃ left standstill 30 minutes, cleer and peaceful deposition in 4 ℃ of centrifugal collections; SDS-PAGE detects discovery, and TrxHis-hSIRP α albumen mainly is present in (Fig. 4) in the deposition with the form of inclusion body.
Fig. 4, be TrxHis-hSIRP alpha fusion protein expressed proteins electrophorogram.With plasmid pET32a (+), pET32a-hSIRP α difference transformed into escherichia coli BL21, add IPTG and induce Expression of Fusion Protein.Get full bacterial lysate, cracking supernatant (soluble component) and cracking deposition (being inclusion body) and carry out the SDS-PAGE analysis respectively.1-3 is the bacterial lysate of Trx, last cleer and peaceful deposition, and 4-6 is the bacterial lysate of TrxHis-hSIRP α, last cleer and peaceful deposition, and M is the molecular weight of albumen sign.
3, the purifying of recombinant protein.PET32a (+) empty carrier albumen TrxHis purifying from bacterium cracking supernatant, TrxHis-hSIRP α is with nickel ion chelate column (Invitrogen ProBondTM) purifying inclusion body protein, and all purification steps are operated with ProBondTM purifying handbook.Albumen behind the purifying carries out SDS-PAGE, carries out Western blot with anti-His tag antibody then, it is thus clear that can detect the correct protein band (Fig. 5) of size at TrxHis-hSIRP α.
Fig. 5, be that purifying and the Western blot of TrxHis-hSIRP alpha fusion protein detects.The M road is the molecular weight of albumen sign.Figure below is empty carrier albumen and fusion rotein Western blot detected result, one anti-be anti-His tag antibody.
4, scavenger cell induces differentiation.With human monocyte cell line U937 cell with 2 * 10
5The density in/hole is inoculated in 24 orifice plates, adds acetate Semen Myristicae Buddhist ripple ester (PMA) 160nM simultaneously and induces 3-5 days, and cell becomes adherent toxoplasma gondii appearance by circular suspended state and changes (Fig. 6).
Human monocyte cell line U937 cell induction is divided into human macrophage under Fig. 6, the phase microscope.
5, the avidity of fusion rotein and tumour cell detects.With human T lymphocyte leukemia cell is that the TrxHis-hSIRP α albumen (10 μ g/ml) of Jurkat cell and purifying was hatched 2 hours under 37 ℃ of conditions altogether, PBS, Trx albumen and hCD47 albumen (patent publication No.: CN102070719A) as contrast.Streaming detects the shared ratio of poly-Histidine positive cell.One anti-is poly-Histidine, and two anti-ly are anti-mouse IgG-FITC (Fig. 7).
Fig. 7, be Flow cytometry TrxHis-hSIRP α albumen and tumour cell avidity.
6, protein function is measured: with people U937 cell with 2 * 10
5The density in/hole is inoculated in 24 orifice plates, gives PMA160nM and induces 3-5 days.Afterwards with the Jurkat cell of DiI mark with 2 * 10
4The density in/hole adds to inoculate in advance to be had in 24 orifice plates of scavenger cell, adds hSIRP α extracellular fragment albumen (10 μ g/ml) simultaneously, and PBS, Trx albumen and hCD47 albumen are as contrast.Treatment group and control group are established three multiple holes respectively.The albumen effect was counted to engulf under the fluorescent microscope after 2 hours has the scavenger cell of Jurkat cell number, counts the dozens of visual field respectively, and the result is carried out statistical analysis.(Fig. 8, Fig. 9)
Fig. 8, observe down control group and the treatment group scavenger cell situation of engulfing to the Jurkat cell for fluorescent microscope, the arrow indication is a scavenger cell of having engulfed the Jurkat cell.
Fig. 9, for fluorescent microscope down counting swallow the scavenger cell number of Jurkat cell, count the dozens of visual field respectively, afterwards data are carried out the result of statistical analysis, Student ' s t check, * P 0.05, * * P 0.01, statistical significance is arranged.
By above this leukemic stem cells targeting soluble of the present invention albumen TrxHis-hSIRP α for preparing, it is made up of the TrxHis expression label of people SIRP α albumen extracellular fragment and fusion; Wherein hSIRP α albumen extracellular fragment is hSIRP α the 26th ~ 370 amino acids.
Described hSIRP α the 26th ~ 370 amino acids comprises and CD47 bonded IgV spline structure territory; It is the N-terminal of this soluble proteins that the TrxHis of described fusion expresses label.Wherein, aminoacid sequence is No1, and by the nucleic acid sequence encoding of No2.
The aminoacid sequence of No1:TrxHis-hSIRP α
msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvaklnidqnpgtapkygirgiptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamadigvageeelqviqpdksvlvaagetatlrctatslipvgpiqwfrgagpgreliynqkeghfprvttvsdltkrnnmdfsirignitpadagtyycvkfrkgspddvefksgagtelsvrakpsapvvsgpaaratpqhtvsftceshgfsprditlkwfkngnelsdfqtnvdpvgesvsysihstakvvltredvhsqvicevahvtlqgdplrgtanlsetirvpptlevtqqpvraenqvnvtcqvrkfypqrlqltwlengnvsrtetastvtenkdgtynwmswllvnvsahrddvkltcqvehdgqpavskshdlkvsahpkeqgsntaaentgsner。
The nucleotide sequence of No2:TrxHis-hSIRP α
atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctgatatcggagtggcgggtgaggaggagctgcaggtgattcagcctgacaagtccgtgttggttgcagctggagagacagccactctgcgctgcactgcgacctctctgatccctgtggggcccatccagtggttcagaggagctggaccaggccgggaattaatctacaatcaaaaagaaggccacttcccccgggtaacaactgtttcagacctcacaaagagaaacaacatggacttttccatccgcatcggtaacatcaccccagcagatgccggcacctactactgtgtgaagttccggaaagggagccccgatgacgtggagtttaagtctggagcaggcactgagctgtctgtgcgcgccaaaccctctgcccccgtggtatcgggccctgcggcgagggccacacctcagcacacagtgagcttcacctgcgagtcccacggcttctcacccagagacatcaccctgaaatggttcaaaaatgggaatgagctctcagacttccagaccaacgtggaccccgtaggagagagcgtgtcctacagcatccacagcacagccaaggtggtgctgacccgcgaggacgttcactctcaagtcatctgcgaggtggcccacgtcaccttgcagggggaccctcttcgtgggactgccaacttgtctgagaccatccgagttccacccaccttggaggttactcaacagcccgtgagggcagagaaccaggtgaatgtcacctgccaggtgaggaagttctacccccagagactacagctgacctggttggagaatggaaacgtgtcccggacagaaacggcctcaaccgttacagagaacaaggatggtacctacaactggatgagctggctcctggtgaatgtatctgcccacagggatgatgtgaagctcacctgccaggtggagcatgacgggcagccagcggtcagcaaaagccatgacctgaaggtctcagcccacccgaaggagcagggctcaaataccgccgctgagaacactggatctaatgaacgg。
Described leukemic stem cells targeting soluble albumen TrxHis-hSIRP α has the aminoacid sequence of No3, and by the nucleic acid sequence encoding of No4:
The aminoacid sequence of No3:hSIRP α extracellular fragment
gvageeelqviqpdksvlvaagetatlrctatslipvgpiqwfrgagpgreliynqkeghfprvttvsdltkrnnmdfsirignitpadagtyycvkfrkgspddvefksgagtelsvrakpsapvvsgpaaratpqhtvsftceshgfsprditlkwfkngnelsdfqtnvdpvgesvsysihstakvvltredvhsqvicevahvtlqgdplrgtanlsetirvpptlevtqqpvraenqvnvtcqvrkfypqrlqltwlengnvsrtetastvtenkdgtynwmswllvnvsahrddvkltcqvehdgqpavskshdlkvsahpkeqgsntaaentgsner。
The nucleotide sequence of No4:hSIRP α extracellular fragment
ggagtggcgggtgaggaggagctgcaggtgattcagcctgacaagtccgtgttggttgcagctggagagacagccactctgcgctgcactgcgacctctctgatccctgtggggcccatccagtggttcagaggagctggaccaggccgggaattaatctacaatcaaaaagaaggccacttcccccgggtaacaactgtttcagacctcacaaagagaaacaacatggacttttccatccgcatcggtaacatcaccccagcagatgccggcacctactactgtgtgaagttccggaaagggagccccgatgacgtggagtttaagtctggagcaggcactgagctgtctgtgcgcgccaaaccctctgcccccgtggtatcgggccctgcggcgagggccacacctcagcacacagtgagcttcacctgcgagtcccacggcttctcacccagagacatcaccctgaaatggttcaaaaatgggaatgagctctcagacttccagaccaacgtggaccccgtaggagagagcgtgtcctacagcatccacagcacagccaaggtggtgctgacccgcgaggacgttcactctcaagtcatctgcgaggtggcccacgtcaccttgcagggggaccctcttcgtgggactgccaacttgtctgagaccatccgagttccacccaccttggaggttactcaacagcccgtgagggcagagaaccaggtgaatgtcacctgccaggtgaggaagttctacccccagagactacagctgacctggttggagaatggaaacgtgtcccggacagaaacggcctcaaccgttacagagaacaaggatggtacctacaactggatgagctggctcctggtgaatgtatctgcccacagggatgatgtgaagctcacctgccaggtggagcatgacgggcagccagcggtcagcaaaagccatgacctgaaggtctcagcccacccgaaggagcagggctcaaataccgccgctgagaacactggatctaatgaacgg。
Said N-terminal merges TrxHis expresses label, comprises the aminoacid sequence of No5, and by the nucleic acid sequence encoding of No6:
The aminoacid sequence of No5:TrxHis
msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvaklnidqnpgtapkygirgiptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamadi。
The nucleotide sequence of No6:TrxHis
atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcga?tttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctgatatc。
Said leukemic stem cells targeting soluble albumen TrxHis-hSIRP α realizes solubility expression in e. coli bl21, and with the nickel metal-chelating column purification to the His label, obtains pure TrxHis-hSIRP α albumen behind the protein cleavage.
Characteristics of the present invention are: TrxHis-hSIRP α can carry out mass production through genetically engineered external; Can combine with the CD47 of leukemia cell and the last high expression level of LSC when being applied in the body; Inhibition acceptor SIRP α combines with CD47 on the prevention scavenger cell; Thereby promote the phagolysis of scavenger cell, significance is arranged for leukemic targeted therapy and removing microresidual disease to leukemia cell and LSC.
Sequence table:
The aminoacid sequence of No1:TrxHis-hSIRP α
msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvaklnidqnpgtapkygirgiptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamadigvageeelqviqpdksvlvaagetatlrctatslipvgpiqwfrgagpgreliynqkeghfprvttvsdltkrnnmdfsirignitpadagtyycvkfrkgspddvefksgagtelsvrakpsapvvsgpaaratpqhtvsftceshgfsprditlkwfkngnelsdfqtnvdpvgesvsysihstakvvltredvhsqvicevahvtlqgdplrgtanlsetirvpptlevtqqpvraenqvnvtcqvrkfypqrlqltwlengnvsrtetastvtenkdgtynwmswllvnvsahrddvkltcqvehdgqpavskshdlkvsahpkeqgsntaaentgsner。
The nucleotide sequence of No2:TrxHis-hSIRP α
atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctgatatcggagtggcgggtgaggaggagctgcaggtgattcagcctgacaagtccgtgttggttgcagctggagagacagccactctgcgctgcactgcgacctctctgatccctgtggggcccatccagtggttcagaggagctggaccaggccgggaattaatctacaatcaaaaagaaggccacttcccccgggtaacaactgtttcagacctcacaaagagaaacaacatggacttttccatccgcatcggtaacatcaccccagcagatgccggcacctactactgtgtgaagttccggaaagggagccccgatgacgtggagtttaagtctggagcaggcactgagctgtctgtgcgcgccaaaccctctgcccccgtggtatcgggccctgcggcgagggccacacctcagcacacagtgagcttcacctgcgagtcccacggcttctcacccagagacatcaccctgaaatggttcaaaaatgggaatgagctctcagacttccagaccaacgtggaccccgtaggagagagcgtgtcctacagcatccacagcacagccaaggtggtgctgacccgcgaggacgttcactctcaagtcatctgcgaggtggcccacgtcaccttgcagggggaccctcttcgtgggactgccaacttgtctgagaccatccgagttccacccaccttggaggttactcaacagcccgtgagggcagagaaccaggtgaatgtcacctgccaggtgaggaagttctacccccagagactacagctgacctggttggagaatggaaacgtgtcccggacagaaacggcctcaaccgttacagagaacaaggatggtacctacaactggatgagctggctcctggtgaatgtatctgcccacagggatgatgtgaagctcacctgccaggtggagcatgacgggcagccagcggtcagcaaaagccatgacctgaaggtctcagcccacccgaaggagcagggctcaaataccgccgctgagaacactggatctaatgaacgg。
The aminoacid sequence of No3:hSIRP α extracellular fragment
gvageeelqviqpdksvlvaagetatlrctatslipvgpiqwfrgagpgreliynqkeghfprvttvsdltkrnnmdfsirignitpadagtyycvkfrkgspddvefksgagtelsvrakpsapvvsgpaaratpqhtvsftceshgfsprditlkwfkngnelsdfqtnvdpvgesvsysihstakvvltredvhsqvicevahvtlqgdplrgtanlsetirvpptlevtqqpvraenqvnvtcqvrkfypqrlqltwlengnvsrtetastvtenkdgtynwmswllvnvsahrddvkltcqvehdgqpavskshdlkvsahpkeqgsntaaentgsner。
The nucleotide sequence of No4:hSIRP α extracellular fragment
ggagtggcgggtgaggaggagctgcaggtgattcagcctgacaagtccgtgttggttgcagctggagagacagccactctgcgctgcactgcgacctctctgatccctgtggggcccatccagtggttcagaggagctggaccaggccgggaattaatctacaatcaaaaagaaggccacttcccccgggtaacaactgtttcagacctcacaaagagaaacaacatggacttttccatccgcatcggtaacatcaccccagcagatgccggcacctactactgtgtgaagttccggaaagggagccccgatgacgtggagtttaagtctggagcaggcactgagctgtctgtgcgcgccaaaccctctgcccccgtggtatcgggccctgcggcgagggccacacctcagcacacagtgagcttcacctgcgagtcccacggcttctcacccagagacatcaccctgaaatggttcaaaaatgggaatgagctctcagacttccagaccaacgtggaccccgtaggagagagcgtgtcctacagcatccacagcacagccaaggtggtgctgacccgcgaggacgttcactctcaagtcatctgcgaggtggcccacgtcaccttgcagggggaccctcttcgtgggactgccaacttgtctgagaccatccgagttccacccaccttggaggttactcaacagcccgtgagggcagagaaccaggtgaatgtcacctgccaggtgaggaagttctacccccagagactacagctgacctggttggagaatggaaacgtgtcccggacagaaacggcctcaaccgttacagagaacaaggatggtacctacaactggatgagctggctcctggtgaatgtatctgcccacagggatgatgtgaagctcacctgccaggtggagcatgacgggcagccagcggtcagcaaaagccatgacctgaaggtctcagcccacccgaaggagcagggctcaaataccgccgctgagaacactggatctaatgaacgg
The aminoacid sequence of No5:TrxHis
msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvaklnidqnpgtapkygirgiptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamadi。
The nucleotide sequence of No6:TrxHis
atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctgatatc。
Claims (5)
1. leukemic stem cells targeting soluble albumen TrxHis-hSIRP α is characterized in that: it is made up of people SIRP α albumen extracellular fragment and the expression label that merges TrxHis; Wherein hSIRP α albumen extracellular fragment is hSIRP α the 26th ~ 370 amino acids.
2. leukemic stem cells targeting soluble albumen TrxHis-hSIRP α according to claim 1 is characterized in that: described hSIRP α the 26th ~ 370 amino acids comprises and CD47 bonded IgV spline structure territory; It is the N-terminal of this soluble proteins that the TrxHis of described fusion expresses label; Wherein, aminoacid sequence is No1, and by the nucleic acid sequence encoding of No2;
The aminoacid sequence of No1:TrxHis-hSIRP α
Msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvaklnidqnpgtapkygirgiptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamadigvageeelqviqpdksvlvaagetatlrctatslipvgpiqwfrgagpgreliynqkeghfprvttvsdltkrnnmdfsirignitpadagtyycvkfrkgspddvefksgagtelsvrakpsapvvsgpaaratpqhtvsftceshgfsprditlkwfkngnelsdfqtnvdpvgesvsysihstakvvltredvhsqvicevahvtlqgdplrgtanlsetirvpptlevtqqpvraenqvnvtcqvrkfypqrlqltwlengnvsrtetastvtenkdgtynwmswllvnvsahrddvkltcqvehdgqpavskshdlkvsahpkeqgsntaaentgsner；
The nucleotide sequence of No2:TrxHis-hSIRP α
atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctgatatcggagtggcgggtgaggaggagctgcaggtgattcagcctgacaagtccgtgttggttgcagctggagagacagccactctgcgctgcactgcgacctctctgatccctgtggggcccatccagtggttcagaggagctggaccaggccgggaattaatctacaatcaaaaagaaggccacttcccccgggtaacaactgtttcagacctcacaaagagaaacaacatggacttttccatccgcatcggtaacatcaccccagcagatgccggcacctactactgtgtgaagttccggaaagggagccccgatgacgtggagtttaagtctggagcaggcactgagctgtctgtgcgcgccaaaccctctgcccccgtggtatcgggccctgcggcgagggccacacctcagcacacagtgagcttcacctgcgagtcccacggcttctcacccagagacatcaccctgaaatggttcaaaaatgggaatgagctctcagacttccagaccaacgtggaccccgtaggagagagcgtgtcctacagcatccacagcacagccaaggtggtgctgacccgcgaggacgttcactctcaagtcatctgcgaggtggcccacgtcaccttgcagggggaccctcttcgtgggactgccaacttgtctgagaccatccgagttccacccaccttggaggttactcaacagcccgtgagggcagagaaccaggtgaatgtcacctgccaggtgaggaagttctacccccagagactacagctgacctggttggagaatggaaacgtgtcccggacagaaacggcctcaaccgttacagagaacaaggatggtacctacaactggatgagctggctcctggtgaatgtatctgcccacagggatgatgtgaagctcacctgccaggtggagcatgacgggcagccagcggtcagcaaaagccatgacctgaaggtctcagcccacccgaaggagcagggctcaaataccgccgctgagaacactggatctaatgaacgg。
3. leukemic stem cells targeting soluble albumen TrxHis-hSIRP α according to claim 1; It is characterized in that: described leukemic stem cells targeting soluble albumen TrxHis-hSIRP α has the aminoacid sequence of No3, and by the nucleic acid sequence encoding of No4:
The aminoacid sequence of No3:hSIRP α extracellular fragment
Gvageeelqviqpdksvlvaagetatlrctatslipvgpiqwfrgagpgreliynqkeghfprvttvsdltkrnnmdfsirignitpadagtyycvkfrkgspddvefksgagtelsvrakpsapvvsgpaaratpqhtvsftceshgfsprditlkwfkngnelsdfqtnvdpvgesvsysihstakvvltredvhsqvicevahvtlqgdplrgtanlsetirvpptlevtqqpvraenqvnvtcqvrkfypqrlqltwlengnvsrtetastvtenkdgtynwmswllvnvsahrddvkltcqvehdgqpavskshdlkvsahpkeqgsntaaentgsner；
The nucleotide sequence of No4:hSIRP α extracellular fragment
ggagtggcgggtgaggaggagctgcaggtgattcagcctgacaagtccgtgttggttgcagctggagagacagccactctgcgctgcactgcgacctctctgatccctgtggggcccatccagtggttcagaggagctggaccaggccgggaattaatctacaatcaaaaagaaggccacttcccccgggtaacaactgtttcagacctcacaaagagaaacaacatggacttttccatccgcatcggtaacatcaccccagcagatgccggcacctactactgtgtgaagttccggaaagggagccccgatgacgtggagtttaagtctggagcaggcactgagctgtctgtgcgcgccaaaccctctgcccccgtggtatcgggccctgcggcgagggccacacctcagcacacagtgagcttcacctgcgagtcccacggcttctcacccagagacatcaccctgaaatggttcaaaaatgggaatgagctctcagacttccagaccaacgtggaccccgtaggagagagcgtgtcctacagcatccacagcacagccaaggtggtgctgacccgcgaggacgttcactctcaagtcatctgcgaggtggcccacgtcaccttgcagggggaccctcttcgtgggactgccaacttgtctgagaccatccgagttccacccaccttggaggttactcaacagcccgtgagggcagagaaccaggtgaatgtcacctgccaggtgaggaagttctacccccagagactacagctgacctggttggagaatggaaacgtgtcccggacagaaacggcctcaaccgttacagagaacaaggatggtacctacaactggatgagctggctcctggtgaatgtatctgcccacagggatgatgtgaagctcacctgccaggtggagcatgacgggcagccagcggtcagcaaaagccatgacctgaaggtctcagcccacccgaaggagcagggctcaaataccgccgctgagaacactggatctaatgaacgg。
4. leukemic stem cells targeting soluble albumen TrxHis-hSIRP α according to claim 2 is characterized in that: said N-terminal merges TrxHis expresses label, comprises the aminoacid sequence of No5, and by the nucleic acid sequence encoding of No6:
The aminoacid sequence of No5:TrxHis
Msdkiihltddsfdtdvlkadgailvdfwaewcgpckmiapildeiadeyqgkltvaklnidqnpgtapkygirgiptlllfkngevaatkvgalskgqlkefldanlagsgsghmhhhhhhssglvprgsgmketaaakferqhmdspdlgtddddkamad；
The nucleotide sequence of No6:TrxHis
atgagcgataaaattattcacctgactgacgacagttttgacacggatgtactcaaagcggacggggcgatcctcgtcgatttctgggcagagtggtgcggtccgtgcaaaatgatcgccccgattctggatgaaatcgctgacgaatatcagggcaaactgaccgttgcaaaactgaacatcgatcaaaaccctggcactgcgccgaaatatggcatccgtggtatcccgactctgctgctgttcaaaaacggtgaagtggcggcaaccaaagtgggtgcactgtctaaaggtcagttgaaagagttcctcgacgctaacctggccggttctggttctggccatatgcaccatcatcatcatcattcttctggtctggtgccacgcggttctggtatgaaagaaaccgctgctgctaaattcgaacgccagcacatggacagcccagatctgggtaccgacgacgacgacaaggccatggctgatatc。
5. leukemic stem cells targeting soluble albumen TrxHis-hSIRP α according to claim 1; It is characterized in that: said leukemic stem cells targeting soluble albumen TrxHis-hSIRP α realizes solubility expression in e. coli bl21; And, obtain pure TrxHis-hSIRP α albumen behind the protein cleavage with nickel metal-chelating column purification to the His label.
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| WO2019061012A1 (en) * | 2017-09-26 | 2019-04-04 | 南京凯地生物科技有限公司 | Preparation of chimeric antigen receptor t-cell specifically targeting cd47 and application thereof |
| CN111303295A (en) * | 2018-12-11 | 2020-06-19 | 宜明昂科生物医药技术（上海）有限公司 | Recombinant chimeric membrane protein cell strain and application thereof |
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| WO2010130053A1 (en) * | 2009-05-15 | 2010-11-18 | University Health Network | COMPOSITIONS AND METHODS FOR TREATING HEMATOLOGIC CANCERS TARGETING THE SIRPα - CD47 INTERACTION |
| CN102070719A (en) * | 2010-11-24 | 2011-05-25 | 中国人民解放军第四军医大学 | Soluble leukemia stem cell targeting proteins TrxHis-hCD47 |
| CN102257001A (en) * | 2008-12-19 | 2011-11-23 | 诺瓦提斯公司 | soluble polypeptides for use in treating autoimmune and inflammatory disorders |
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| CN102257001A (en) * | 2008-12-19 | 2011-11-23 | 诺瓦提斯公司 | soluble polypeptides for use in treating autoimmune and inflammatory disorders |
| WO2010130053A1 (en) * | 2009-05-15 | 2010-11-18 | University Health Network | COMPOSITIONS AND METHODS FOR TREATING HEMATOLOGIC CANCERS TARGETING THE SIRPα - CD47 INTERACTION |
| CN102070719A (en) * | 2010-11-24 | 2011-05-25 | 中国人民解放军第四军医大学 | Soluble leukemia stem cell targeting proteins TrxHis-hCD47 |
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| WO2019061012A1 (en) * | 2017-09-26 | 2019-04-04 | 南京凯地生物科技有限公司 | Preparation of chimeric antigen receptor t-cell specifically targeting cd47 and application thereof |
| CN111303295A (en) * | 2018-12-11 | 2020-06-19 | 宜明昂科生物医药技术（上海）有限公司 | Recombinant chimeric membrane protein cell strain and application thereof |
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