CN110054678B - Membrane-bound mFLT3LG protein and application thereof - Google Patents
Membrane-bound mFLT3LG protein and application thereof Download PDFInfo
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Abstract
The invention provides a membrane-bound mFLT3LG protein and application thereof, wherein a protein sequence of FLT3LG is newly designed originally, enzyme cutting sites of the protein sequence are removed, anchoring on the surface of a membrane is ensured, a brand-new chimeric protein sequence of membrane-bound mFLT3LG (membrane FLT3 LG) is obtained, ORF of the brand-new membrane-bound mFLT3LG is cloned to a vector, and the vector and VSVG are placed on the same expression plasmid and are started by double promoters, so that the infection efficiency of lentivirus to HSC is obviously improved, and the protein has wide application prospect and great market value.
Description
Technical Field
The invention belongs to the technical field of biology, and relates to a membrane-bound mFLT3LG protein and application thereof.
Background
Gene Therapy (Gene Therapy) refers to the introduction of exogenous DNA fragments into target cells to perform targeted intervention on defective and abnormal genes in the ways of correction, repair, replacement, compensation or silencing, etc., in order to restore healthy genetic information and finally achieve the purpose of treatment or even clinical cure. How to introduce exogenous DNA segment into target cell nucleus efficiently and safely is the core problem of gene operation.
Viral-mediated presentation of DNA fragments, whether in vivo or in vitro, is a key technology for gene manipulation. At present, a plurality of natural viruses are developed into virus presentation systems by reference, such as adenovirus, adeno-associated virus, retrovirus, lentivirus and the like, and the virus systems are widely applied in the fields of life science and clinical medicine research and play a vital role in promoting the development of current scientific research.
The lentivirus system developed based on the HIV virus is currently the most widely used gene manipulation technique because of its significant advantages. The lentivirus system can infect cells in a division phase and also can infect cells in a resting phase, which provides an excellent way for gene manipulation in nerve cells or cardiac muscle cells; the payload of the lentivirus system can reach 5kb at most, which provides possibility for presentation of large-fragment DNA; in addition, the long-term stable expression characteristic of the lentivirus after integration, based on the pantropic infection capacity of the VSVG envelope protein, endows the lentivirus system with strong gene perturbation capacity and wide compatibility. As a result, the lentivirus system has become a major technological means for gene manipulation in the field of life medicine. The repair or compensation of defective genes or even the introduction of new genes based on lentiviral systems has been a major research direction in gene therapy. For example, in the field of immunotherapy, CAR-T cells infect T cells through lentivirus-packaged antibody genes, and the T cells are incubated to have brand new antigen recognition capability, so that tumor cells are specially recognized to achieve a killing effect.
Lentivirus systems have evolved primarily in three generations, with third generation systems currently in widespread use and most likely being manufactured on the GMP level in clinical studies. The third generation of slow virus system consists of four vectors, including exogenous gene expressing plasmid, structural protein gag-pol expressing plasmid essential for HIV virus replication and assembly, viral genome RNA nucleoprotein Rev expressing plasmid, envelope protein VSVG expressing plasmid, etc. Among them, the envelope protein VSVG is derived from Vesicular stomatitis virus (Vesicular stomatis virus), and RNA virus enveloped by the envelope protein can infect cells of various animals such as mice, horses, cows, pigs, primates, and the like, and thus has a pan-tropic infection ability, is widely used in the field of current biomedical research, and has become the most important envelope protein for packaging lentiviruses. The third generation lentivirus is stable as a whole, but needs to be further improved in some important fields, and still has great optimization and promotion space. Lentiviruses exhibit strong transduction efficiency in infecting adherent cells, such as 293T cells, with an MOI of 1, which effectively infects 293T cells. However, infection of suspended blood cells has always been shown to be weak, especially for hematopoietic stem cells, MOI values of even 100 are required to achieve effective infection. Hematopoietic stem cells are often the ultimate target cells for gene therapy, and a considerable number of blood diseases can be treated stably for a long time only by performing gene manipulation in hematopoietic stem cells.
However, the VSVG envelope protein also has obvious disadvantages, for example, the cell membrane receptor of the VSVG protein is not very clear, the cell membrane receptor is reported to be a ceramide molecule on the surface of a cell membrane in the literature, and the LDL receptor is found to be a binding protein of VSVG in the literature. Because the abundance of VSVG receptors expressed on the surfaces of different types of cells is different, some cells are easily infected by VSVG-coated lentiviruses, such as 293T cells, HT1080 cells and the like; however, the expression abundance of LDL receptors on the surface of many blood cells, such as T cells, B cells, HSCs (Hematopoietic stem cells) and the like, is very low, which makes them difficult to be infected by VSVG-coated lentiviruses, thus bringing technical difficulties for gene therapy of lentivirus infection of blood cells, especially HSCs. Clinical application grade HSCs often require 100 million-scale cells to perform genetic manipulation, while lower infection efficiency puts very high demands on the yield and quality of lentiviruses, significantly increasing the cost of the assay with half the effort.
Therefore, how to improve the infection and transduction ability of lentivirus to hematopoietic stem cells is a key concern of technologists in the field of blood research, and further targeted optimization and improvement are needed in a lentivirus system using VSVG as an envelope protein to realize efficient infection of HSC.
Disclosure of Invention
The invention provides a membrane-bound mFLT3LG protein and application thereof, wherein a protein sequence of FLT3LG is newly and creatively designed, enzyme cutting sites of the protein sequence are eliminated, anchoring on the surface of a membrane is ensured, and a brand-new chimeric protein sequence of membrane-bound mFLT3LG (membrane FLT3 LG) is obtained, so that the infection efficiency of lentivirus on HSC is remarkably improved, and the membrane-bound mFLT3LG protein has wide application prospect and great market value.
In order to realize the purpose, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a membrane-bound mFLT3LG protein comprising an active extracellular region of FLT3LG, a linking member, and an anchoring member.
Wherein the amino acid sequence of the active extracellular region of FLT3LG is shown in SEQ ID NO. 1.
The SEQ ID NO.1 is as follows:
MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPP.
preferably, the linking moiety comprises a linking peptide.
Preferably, the linker peptide comprises a flexible linker peptide.
Preferably, the amino acid sequence of the flexible connecting peptide is shown in SEQ ID NO. 2.
The SEQ ID NO.2 is as follows:
GSSGGSSGGSSG.
preferably, the anchoring means comprises the transmembrane region of a single-pass transmembrane protein.
Preferably, the transmembrane region of the single-transmembrane protein comprises any one of or a combination of at least two of the transmembrane region of SCF, the transmembrane region of membrane-type IL6 or the transmembrane region of VSVG, preferably the transmembrane region of SCF.
Preferably, the amino acid sequence of the transmembrane region of the SCF is shown as SEQ ID NO. 3.
The SEQ ID NO.3 is as follows:
SSLHWAAMALPALFSLIIGFAFGALYW
wherein the polypeptide segment of the C-terminal transmembrane domain of membrane SCF (mSCF) is as follows:
NPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV.
the amino acid sequence of the native FLT3LG protein is shown in SEQ ID NO.7 (NM _001459.3, NP 001450.2, full length 235aa, complete transmembrane protein 209aa after removal of 26aa signal peptide from the N-terminus, but is hydrolyzed to 155aa soluble ligand):
MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHWQRTRRRTPRPGEQVPPVPSPQDLLLVEH.
preferably, the membrane-bound mFLT3LG protein comprises an active extracellular region of FLT3LG with an amino acid sequence shown in SEQ ID No.1, a flexible connecting peptide with an amino acid sequence shown in SEQ ID No.2 and a transmembrane region of SCF with an amino acid sequence shown in SEQ ID No. 3.
Preferably, the amino acid sequence of the membrane-bound mFLT3LG protein is shown as SEQ ID No. 4;
the SEQ ID NO.4 is as follows:
MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPGSSGGSSGGSSGNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV.
in the invention, the inventor creatively utilizes the pairing and combining relationship between the cytokine receptor and the cytokine on the surface of the HSC cell to construct the membrane-bound FLT3LG cytokine, and the lentiviral particle coated by the membrane-bound cytokine can be combined with the FLT3 receptor on the surface of the HSC, so that the physical distance between the lentiviral particle and the HSC is directly shortened, and the virus infection process is realized through endocytosis.
Many cytokine receptors exist on the surface of hematopoietic stem cells, and these cytokine receptors can sense cytokines in the microenvironment, thereby controlling cell fates such as hematopoietic stem cell proliferation, self-renewal, and differentiation. Among the most important cytokine receptors on HSC surfaces are c-KIT (SCF receptor), c-MPL (THPO receptor), FLT3 (FLT 3LG receptor), IL6R (IL-6 receptor), and the like. The invention artificially creates the pairing combination relationship of the cytokine receptors and the cytokines so as to enhance the infection efficiency of lentivirus on the hematopoietic stem cells.
The invention mainly aims at the artificial optimization and reconstruction of FLT3LG, the FLT3 serving as a transmembrane receptor existing on the surface of hematopoietic stem cells can not be changed, and is not described any more here, and the signal transduction pathway of the FLT3-FLT3LG is not the core investigation content of the invention and is not developed deeply; the patent mainly aims at the expression and membrane anchoring of FLT3LG to carry out artificial optimization design.
So far, there are 4 major subtypes of mRNA reference sequences registered by FLT3LG in NCBI or UCSC database websites, among which transcript variant 3 with mRNA number NM _001459.3 (protein accession number NP _ 001450.2) is the most predominantly expressed subtype. The FLT3LG protein encodes 235 amino acids, wherein 1-26 are secretion signal peptides which can be cut off by enzyme; 27-184 is an outer membrane region; 185-205 are transmembrane regions; 206-235 are inner regions of the membrane. Although natural FLT3LG is first a membrane-bound cytokine, it is cleaved by TACE protease and is detached from the cell membrane to become a free soluble cytokine. Two molecules of FLT3LG form a homodimer and bind to two FLT3 receptors to form a tetramer, which is involved in the process of cell signaling. It can be seen that the natural FLT3LG molecule cannot be used as an effective envelope protein for lentiviral packaging due to its presence of a restriction digestion site.
Therefore, the invention firstly carries out original design on the protein sequence of FLT3LG again, eliminates the enzyme cutting site and ensures that the protein sequence is anchored on the surface of a membrane, reserves an extracellular effective active region which comprises a 1-168aa polypeptide sequence and is fused with the transmembrane region of the stem cell factor SCF with the known enzyme cutting site eliminated, and adopts flexible linking peptide consisting of 12aa to connect the extracellular active region and the transmembrane region to obtain a brand-new chimeric protein sequence of the membrane-bound mFLT3LG (membrane FLT3 LG). The protein sequence comprises 248aa, the isoelectric point PI is close to 5, and the molecular weight is 27kD.
At present, the restriction enzyme digestion site of the natural FLT3LG is not clear, so the restriction enzyme digestion site cannot be eliminated temporarily in an amino acid missense mutation mode, and the direct replacement of the restriction enzyme digestion site by a known transmembrane region which cannot be restricted by enzyme digestion is stronger in operability.
In addition to using the transmembrane domain of SCF to anchor mFLT3LG to the cell membrane, other cytokine transmembrane domains, such as the transmembrane domain of membrane-type IL6, the transmembrane domain of VSVG itself, or other single-transmembrane protein transmembrane domains, can be used to anchor the cell membrane.
In a second aspect, the invention provides an ORF reading frame of a membrane-bound mFLT3LG protein, wherein the ORF reading frame is designed in reverse for the highest usage frequency codon of human cells based on the protein of the first aspect.
Preferably, the nucleotide sequence of the ORF reading frame is shown in SEQ ID NO. 5.
The SEQ ID NO.5 is as follows:
Modified membrane FLT3LG(mmFLT3LG)ORF seq using human-optimized codon:
ATGACCGTGCTGGCCCCCGCCTGGAGCCCCACCACCTACCTGCTGCTGCTGCTGCTGCTGAGCAGCGGCCTGAGCGGCACCCAGGACTGCAGCTTCCAGCACAGCCCCATCAGCAGCGACTTCGCCGTGAAGATCAGGGAGCTGAGCGACTACCTGCTGCAGGACTACCCCGTGACCGTGGCCAGCAACCTGCAGGACGAGGAGCTGTGCGGCGGCCTGTGGAGGCTGGTGCTGGCCCAGAGGTGGATGGAGAGGCTGAAGACCGTGGCCGGCAGCAAGATGCAGGGCCTGCTGGAGAGGGTGAACACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCCCCCCCCAGCTGCCTGAGGTTCGTGCAGACCAACATCAGCAGGCTGCTGCAGGAGACCAGCGAGCAGCTGGTGGCCCTGAAGCCCTGGATCACCAGGCAGAACTTCAGCAGGTGCCTGGAGCTGCAGTGCCAGCCCGACAGCAGCACCCTGCCCCCCCCCGGCAGCAGCGGCGGCAGCAGCGGCGGCAGCAGCGGCAACCCCCCCGGCGACAGCAGCCTGCACTGGGCCGCCATGGCCCTGCCCGCCCTGTTCAGCCTGATCATCGGCTTCGCCTTCGGCGCCCTGTACTGGAAGAAGAGGCAGCCCAGCCTGACCAGGGCCGTGGAGAACATCCAGATCAACGAGGAGGACAACGAGATCAGCATGCTGCAGGAGAAGGAGAGGGAGTTCCAGGAGGTGTGATAA.
meanwhile, the ORF reading frame does not refer to a natural gene sequence, but is optimized according to the codon with the highest use frequency of human cells to obtain a brand-new ORF reading frame of the chimeric mFLT3 LG.
In a third aspect, the present invention provides a recombinant plasmid comprising the ORF reading frame of the membrane-bound mFLT3LG protein of the second aspect.
Preferably, the plasmid further comprises the nucleotide sequence of the VSVG envelope protein.
Preferably, the VSVG envelope protein and the membrane-bound mFLT3LG protein are each driven by two different promoters.
Of course, the use of FLT3L alone may not independently significantly improve packaging efficiency, as the VSVG protein is also critical for the release of lentiviral particles. Therefore, the inventors cloned a completely new ORF of membrane-bound mFLT3LG onto a vector and placed it on the same expression plasmid as VSVG. In this case, 293T cells transiently transfected with multiple plasmids simultaneously express VSVG and mFLT3LG proteins, and this innovative optimization improvement significantly increases the infection efficiency of lentiviruses on HSC. It should be noted that the lentivirus recombinant vector of the present invention not only can enhance the ability of infecting HSC hematopoietic stem cells for gene therapy, but also can enhance the ability of infecting some dendritic cells, T cells and NK cells, and all the cells with FLT3 receptors on the cell surface are sensitive to the infection of the lentivirus recombinant vector of the present invention, and have important significance in immunotherapy.
In a fourth aspect, the present invention provides a lentivirus obtainable by co-packaging the plasmid of the third aspect with a helper packaging plasmid.
In a fifth aspect, the present invention provides a use of the protein of the first aspect, the reading frame of the second aspect, the recombinant plasmid of the third aspect, or the lentivirus of the fourth aspect for the preparation of a medicament and/or a kit for gene therapy and/or immunotherapy.
Compared with the prior art, the invention has the following advantages:
the protein provided by the invention is subjected to artificial optimization design aiming at the expression and membrane anchoring of FLT3LG, a membrane-bound FLT3LG cytokine is constructed, and an original chimeric membrane-bound mFLT3LG protein sequence is redesigned, so that the protein cannot fall off from a cell membrane due to enzymolysis and digestion of protease; the lentivirus particles coated by the membrane type cytokine can be combined with an FLT3 receptor on the surface of the HSC, so that the physical distance between the lentivirus particles and the HSC is directly shortened, the virus infection process is realized through endocytosis, and the infection efficiency of lentivirus on hematopoietic stem cells is enhanced; meanwhile, a brand-new ORF of membrane-bound mFLT3LG is cloned to a vector and placed on the same expression plasmid with VSVG, and transcription and translation are driven by double promoters, so that under the condition of ensuring expression of the VSVG, the mFLT3LG is expressed simultaneously, 293T cells transiently transferred to multiple plasmids can simultaneously express VSVG and mFLT3LG proteins, and the infection efficiency of lentiviruses to HSCs is obviously improved through the original optimization and improvement.
Drawings
FIG. 1 is a schematic diagram showing the construction of a VSVG-mFLT3LG vector in the example of the present invention;
FIG. 2 is a graph showing the results of a lentivirus infected HSC cell assay.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solution of the present invention with reference to the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.
Examples
1. Determining the active extracellular region according to the sequence of the natural FLT3LG protein;
2. connecting the active extracellular region of FLT3LG with the transmembrane region of SCF through a flexible linking peptide of 12aa to form a chimeric membrane-bound mFLT3LG;
3. and (3) reversely designing an ORF reading frame of the mFLT3LG through the maximum use frequency codon data of a person. The reading frame does not take a natural sequence as a template, and has high originality;
4. cloning the VSVG envelope protein and the mFLT3G protein on the same expression plasmid, and respectively driving by two different promoters;
5. according to the conventional operation scheme, an exogenous gene expression plasmid, a gag-pol plasmid, a Rev plasmid and a VSVG-mFLT3LG plasmid are simultaneously transiently transferred to 293T cells, lentiviruses are packaged, and the viruses are concentrated through high-speed centrifugation, and finally HSC cells are infected.
The specific scheme is as follows:
firstly, the protein sequence of FLT3LG is designed again, the enzyme cutting site is removed, and the anchoring on the surface of a membrane is ensured; an extracellular effective activity region is reserved, the extracellular effective activity region comprises a 1-168aa polypeptide sequence, and is fused with a transmembrane region of a stem cell factor SCF (short-chain cytokine-like factor) with a known enzyme cutting site removed, and a flexible linking peptide consisting of 12aa is adopted in the middle to connect the extracellular activity region and the transmembrane region, so that a brand-new protein sequence of the chimeric membrane-bound mFLT3LG (membrane FLT3 LG) is obtained; the protein sequence comprises 248aa, the isoelectric point PI is close to 5, and the molecular weight is 27kD. Meanwhile, the ORF reading frame does not refer to a natural gene sequence, but is optimized according to the codon with the highest use frequency of human cells, so that a brand-new ORF reading frame of the chimeric mFLT3LG is obtained.
Secondly, cloning a brand-new ORF of membrane-bound mFLT3LG to a vector, placing the vector and VSVG on the same expression plasmid, and obtaining a VSVG-mFLT3LG plasmid, wherein the vector construction schematic diagram is shown in FIG. 1, and the VSVG and the mFLT3LG are respectively transcribed by two promoters.
Lentiviral infection HSC cell assay
According to the conventional operation scheme, a foreign gene expression plasmid, a gag-pol plasmid, a Rev plasmid and a VSVG-mFLT3LG plasmid are simultaneously transiently transferred to 293T cells, lentiviruses are packaged, and the viruses are concentrated through high-speed centrifugation, and finally HSC cells are infected. The VSVG plasmid without mFLT3LG was used as a control group to test the positive rate of lentivirus infected HSC cells in both cases, and the results are shown in FIG. 2.
As can be seen from FIG. 2, the optimized VSVG + mFLT3LG vector has higher packaging titer, and the positive rate of infected HSC cells is as high as 65%, which is significantly higher than 38% of the control group. 293T cells transiently transformed with multiple plasmids simultaneously express VSVG and mFLT3LG proteins, and the original optimization and improvement remarkably improves the infection efficiency of lentiviruses on HSC.
The amino acid sequence of the native FLT3LG protein is shown in SEQ ID NO.7 (NM _001459.3, NP 001450.2, full length 235aa, complete transmembrane protein 209aa after removal of 26aa signal peptide from the N-terminus, but is hydrolyzed to 155aa soluble ligand):
MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLLLPVGLLLLAAAWCLHWQRTRRRTPRPGEQVPPVPSPQDLLLVEH.
the amino acid sequence of the polypeptide fragment of the C-terminal transmembrane domain of membrane SCF (mSCF) is SEQ ID NO.6 as follows:
NPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV.
the amino acid sequence of Modified membrane FLT3LG (mmFLT 3 LG) protein (a chimeric cytokine consisting of the transregional regions of FLT3LG and SCF) is shown in SEQ ID NO.4 as follows:
MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPGSSGGSSGGSSGNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV.
the nucleotide sequence of Modified membrane FLT3LG (mmFLT 3 LG) ORF using human-optimized codon is SEQ ID NO.5 as follows:
ATGACCGTGCTGGCCCCCGCCTGGAGCCCCACCACCTACCTGCTGCTGCTGCTGCTGCTGAGCAGCGGCCTGAGCGGCACCCAGGACTGCAGCTTCCAGCACAGCCCCATCAGCAGCGACTTCGCCGTGAAGATCAGGGAGCTGAGCGACTACCTGCTGCAGGACTACCCCGTGACCGTGGCCAGCAACCTGCAGGACGAGGAGCTGTGCGGCGGCCTGTGGAGGCTGGTGCTGGCCCAGAGGTGGATGGAGAGGCTGAAGACCGTGGCCGGCAGCAAGATGCAGGGCCTGCTGGAGAGGGTGAACACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCCCCCCCCAGCTGCCTGAGGTTCGTGCAGACCAACATCAGCAGGCTGCTGCAGGAGACCAGCGAGCAGCTGGTGGCCCTGAAGCCCTGGATCACCAGGCAGAACTTCAGCAGGTGCCTGGAGCTGCAGTGCCAGCCCGACAGCAGCACCCTGCCCCCCCCCGGCAGCAGCGGCGGCAGCAGCGGCGGCAGCAGCGGCAACCCCCCCGGCGACAGCAGCCTGCACTGGGCCGCCATGGCCCTGCCCGCCCTGTTCAGCCTGATCATCGGCTTCGCCTTCGGCGCCCTGTACTGGAAGAAGAGGCAGCCCAGCCTGACCAGGGCCGTGGAGAACATCCAGATCAACGAGGAGGACAACGAGATCAGCATGCTGCAGGAGAAGGAGAGGGAGTTCCAGGAGGTGTGATAA.
in conclusion, the invention provides a membrane-bound mFLT3LG protein and application thereof, wherein a brand-new chimeric membrane-bound mFLT3LG (membrane FLT3 LG) protein sequence is obtained by carrying out original design on a FLT3LG protein sequence again, eliminating the enzyme cutting site and ensuring anchoring on the surface of a membrane, the ORF of the brand-new membrane-bound mFLT3LG is cloned to a vector, and is placed on the same expression plasmid with VSVG, and the start is carried out by using a double promoter, so that the HSC infection efficiency of lentiviruses is remarkably improved, and the membrane-bound mFLT3LG protein has wide application prospect and great market value.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
SEQUENCE LISTING
<110> hematological disease hospital of Chinese academy of medical sciences (hematology institute)
<120> membrane-bound mFLT3LG protein and application thereof
<130> 2019
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 168
<212> PRT
<213> artificially synthesized sequence
<400> 1
Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe
20 25 30
Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu
35 40 45
Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu
50 55 60
Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln
65 70 75 80
Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly
85 90 95
Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala
100 105 110
Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser
115 120 125
Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp
130 135 140
Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro
145 150 155 160
Asp Ser Ser Thr Leu Pro Pro Pro
165
<210> 2
<211> 12
<212> PRT
<213> artificially synthesized sequence
<400> 2
Gly Ser Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly
1 5 10
<210> 3
<211> 27
<212> PRT
<213> artificially synthesized sequence
<400> 3
Ser Ser Leu His Trp Ala Ala Met Ala Leu Pro Ala Leu Phe Ser Leu
1 5 10 15
Ile Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp
20 25
<210> 4
<211> 248
<212> PRT
<213> artificially synthesized sequence
<400> 4
Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe
20 25 30
Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu
35 40 45
Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu
50 55 60
Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln
65 70 75 80
Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly
85 90 95
Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala
100 105 110
Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser
115 120 125
Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp
130 135 140
Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro
145 150 155 160
Asp Ser Ser Thr Leu Pro Pro Pro Gly Ser Ser Gly Gly Ser Ser Gly
165 170 175
Gly Ser Ser Gly Asn Pro Pro Gly Asp Ser Ser Leu His Trp Ala Ala
180 185 190
Met Ala Leu Pro Ala Leu Phe Ser Leu Ile Ile Gly Phe Ala Phe Gly
195 200 205
Ala Leu Tyr Trp Lys Lys Arg Gln Pro Ser Leu Thr Arg Ala Val Glu
210 215 220
Asn Ile Gln Ile Asn Glu Glu Asp Asn Glu Ile Ser Met Leu Gln Glu
225 230 235 240
Lys Glu Arg Glu Phe Gln Glu Val
245
<210> 5
<211> 750
<212> DNA
<213> artificially synthesized sequence
<400> 5
atgaccgtgc tggcccccgc ctggagcccc accacctacc tgctgctgct gctgctgctg 60
agcagcggcc tgagcggcac ccaggactgc agcttccagc acagccccat cagcagcgac 120
ttcgccgtga agatcaggga gctgagcgac tacctgctgc aggactaccc cgtgaccgtg 180
gccagcaacc tgcaggacga ggagctgtgc ggcggcctgt ggaggctggt gctggcccag 240
aggtggatgg agaggctgaa gaccgtggcc ggcagcaaga tgcagggcct gctggagagg 300
gtgaacaccg agatccactt cgtgaccaag tgcgccttcc agcccccccc cagctgcctg 360
aggttcgtgc agaccaacat cagcaggctg ctgcaggaga ccagcgagca gctggtggcc 420
ctgaagccct ggatcaccag gcagaacttc agcaggtgcc tggagctgca gtgccagccc 480
gacagcagca ccctgccccc ccccggcagc agcggcggca gcagcggcgg cagcagcggc 540
aacccccccg gcgacagcag cctgcactgg gccgccatgg ccctgcccgc cctgttcagc 600
ctgatcatcg gcttcgcctt cggcgccctg tactggaaga agaggcagcc cagcctgacc 660
agggccgtgg agaacatcca gatcaacgag gaggacaacg agatcagcat gctgcaggag 720
aaggagaggg agttccagga ggtgtgataa 750
<210> 6
<211> 68
<212> PRT
<213> artificially synthesized sequence
<400> 6
Asn Pro Pro Gly Asp Ser Ser Leu His Trp Ala Ala Met Ala Leu Pro
1 5 10 15
Ala Leu Phe Ser Leu Ile Ile Gly Phe Ala Phe Gly Ala Leu Tyr Trp
20 25 30
Lys Lys Arg Gln Pro Ser Leu Thr Arg Ala Val Glu Asn Ile Gln Ile
35 40 45
Asn Glu Glu Asp Asn Glu Ile Ser Met Leu Gln Glu Lys Glu Arg Glu
50 55 60
Phe Gln Glu Val
65
<210> 7
<211> 235
<212> PRT
<213> artificially synthesized sequence
<400> 7
Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu
1 5 10 15
Leu Leu Leu Leu Ser Ser Gly Leu Ser Gly Thr Gln Asp Cys Ser Phe
20 25 30
Gln His Ser Pro Ile Ser Ser Asp Phe Ala Val Lys Ile Arg Glu Leu
35 40 45
Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val Thr Val Ala Ser Asn Leu
50 55 60
Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp Arg Leu Val Leu Ala Gln
65 70 75 80
Arg Trp Met Glu Arg Leu Lys Thr Val Ala Gly Ser Lys Met Gln Gly
85 90 95
Leu Leu Glu Arg Val Asn Thr Glu Ile His Phe Val Thr Lys Cys Ala
100 105 110
Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe Val Gln Thr Asn Ile Ser
115 120 125
Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu Val Ala Leu Lys Pro Trp
130 135 140
Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu Glu Leu Gln Cys Gln Pro
145 150 155 160
Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser Pro Arg Pro Leu Glu Ala
165 170 175
Thr Ala Pro Thr Ala Pro Gln Pro Pro Leu Leu Leu Leu Leu Leu Leu
180 185 190
Pro Val Gly Leu Leu Leu Leu Ala Ala Ala Trp Cys Leu His Trp Gln
195 200 205
Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly Glu Gln Val Pro Pro Val
210 215 220
Pro Ser Pro Gln Asp Leu Leu Leu Val Glu His
225 230 235
Claims (11)
1. A membrane-bound mFLT3LG protein, which consists of an active extracellular region of FLT3LG, a linking member and an anchoring member;
wherein the amino acid sequence of the active extracellular region of FLT3LG is shown in SEQ ID NO. 1;
the connecting component is flexible connecting peptide;
the anchoring part is a transmembrane region of a single transmembrane protein;
the transmembrane region of the single-pass transmembrane protein is the transmembrane region of the SCF;
the amino acid sequence of the membrane-bound mFLT3LG protein is sequentially an active extracellular region of FLT3LG, a flexible connecting peptide and a transmembrane region of SCF from N end to C end.
2. The membrane-bound mFLT3LG protein according to claim 1, wherein the amino acid sequence of the flexible linker peptide is shown in SEQ ID No. 2.
3. The membrane-bound mFLT3LG protein according to claim 1, wherein the amino acid sequence of the transmembrane region of the SCF is represented by SEQ ID No. 3.
4. The membrane-bound mFLT3LG protein according to claim 1, wherein the amino acid sequence of the membrane-bound mFLT3LG protein is shown in SEQ ID No. 4.
5. An ORF reading frame polynucleotide of membrane-bound mFLT3LG protein, wherein the ORF reading frame polynucleotide is designed against the protein of any one of claims 1-4.
6. An ORF reading frame polynucleotide according to claim 5, wherein the sequence of the ORF reading frame polynucleotide is shown in SEQ ID No. 5.
7. A recombinant plasmid comprising an ORF reading frame polynucleotide of the membrane-bound mFLT3LG protein of claim 5 or 6.
8. The recombinant plasmid of claim 7, wherein the plasmid further comprises a nucleotide sequence of a VSVG envelope protein.
9. The recombinant plasmid of claim 8, wherein the VSVG envelope protein and the membrane-bound mFLT3LG protein are each driven by two different promoters.
10. A lentivirus, wherein the lentivirus is co-packaged with the plasmid of any one of claims 7 to 9 and a helper packaging plasmid.
11. Use of a protein according to any one of claims 1 to 4, a reading frame polynucleotide according to claim 5 or 6, or a recombinant plasmid according to any one of claims 7 to 9 or a lentivirus according to claim 10 for the manufacture of a medicament and/or kit for gene therapy and/or immunotherapy.
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| CN105378074A (en) * | 2013-03-15 | 2016-03-02 | 费城儿童医院 | Scalable manufacturing process to produce recombinant lentiviral vectors in serum-free suspension cell culture system |
| WO2017091786A1 (en) * | 2015-11-23 | 2017-06-01 | Novartis Ag | Optimized lentiviral transfer vectors and uses thereof |
| CN109312309A (en) * | 2016-01-08 | 2019-02-05 | 雷普利穆内有限公司 | The virus of engineering |
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