CN110054678B - Membrane-bound mFLT3LG protein and application thereof - Google Patents

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

A membrane-bound mFLT3LG protein and its application

technical field

The invention belongs to the field of biotechnology, and relates to a membrane-bound mFLT3LG protein and its application.

Background technique

Gene therapy (Gene Therapy) refers to the introduction of exogenous DNA fragments into target cells, and targeted intervention on defective and abnormal genes in the form of correction, repair, replacement, compensation or silencing, in order to restore healthy genetic information, and finally achieve therapeutic or even purpose of clinical cure. How to efficiently and safely introduce exogenous DNA fragments into the nucleus of target cells is the core issue of gene manipulation.

Virus-mediated presentation of DNA fragments is a key technique for genetic manipulation, both in vivo and in vitro. At present, a variety of natural viruses have been developed into virus delivery systems for reference, such as adenovirus, adeno-associated virus, retrovirus, and lentivirus. These virus systems have been widely used in the fields of life sciences and clinical medicine research. The development of scientific research has played a vital role in promoting.

The lentiviral system developed based on the HIV virus has become the most widely used gene manipulation technology due to its remarkable advantages. The lentiviral system can infect not only dividing cells, but also quiescent cells, which provides an excellent way for gene manipulation in nerve cells or cardiomyocytes; the payload of the lentiviral system can reach up to 5kb, which is a good way for large fragments The presentation of DNA provides the possibility; in addition, the long-term stable expression characteristics of the lentivirus after integration, and the pantropic infection ability based on the VSVG envelope protein, all endow the lentivirus system with strong gene perturbation ability and wide compatibility. Therefore, the lentiviral system has become the main technical means of gene manipulation in the field of life medicine. Repairing or compensating for defective genes or even introducing new genes based on the lentiviral system has become a key research direction of gene therapy. For example, CAR-T cells in the field of immunotherapy infect T cells through lentivirus packaging antibody genes, incubate T cells with new antigen recognition capabilities, and specifically recognize tumor cells to achieve killing effects.

The evolution of the lentiviral system is mainly divided into three generations. The third-generation system is currently widely used and most likely to be produced in accordance with the GMP level in clinical research. The third-generation lentiviral system is mainly composed of four vectors: exogenous gene expression plasmid, structural protein gag-pol expression plasmid necessary for HIV virus replication and assembly, virus genome RNA nuclear transport protein Rev expression plasmid, and envelope protein VSVG expression plasmid. . Among them, the envelope protein VSVG comes from vesicular stomatitis virus (Vesicular stomatitis virus). The RNA virus wrapped by the envelope protein can infect various animal cells such as mice, horses, cattle, pigs and primates, so it has pantropic properties. Infection ability is widely used in the current life medical research field, and has become the most important envelope protein for lentivirus packaging. The third-generation lentivirus is relatively stable overall, but it still needs further improvement in some important areas, and there is still a lot of room for optimization and improvement. Lentivirus shows strong transduction efficiency in infecting adherent cells such as 293T cells, and the MOI value of 1 can effectively infect 293T cells. However, the infection of suspended blood cells has always been weak, especially for the infection of hematopoietic stem cells, and the MOI value even needs to reach 100 to achieve effective infection. However, hematopoietic stem cells are often the ultimate target cells for gene therapy. For quite a few blood diseases, only genetic manipulation in hematopoietic stem cells can have long-term and stable treatment effects.

However, the VSVG envelope protein also has obvious disadvantages. For example, the cell membrane receptor of the VSVG protein is not yet clear. It is reported in the literature that it is a ceramide molecule on the surface of the cell membrane, and some literature has found that the LDL receptor is the binding protein of VSVG. Due to the different abundance of VSVG receptors expressed on the surface of different types of cells, some cells are easily infected by lentivirus coated with VSVG, such as 293T cells, HT1080 cells, etc.; and the abundance of LDL receptors on the surface of many blood cells is very low. Low, such as T cells, B cells, HSC (Hematopoietic stem cells, hematopoietic stem cells), etc., making it difficult for them to be infected by the lentivirus coated with VSVG, which brings great benefits to the gene therapy of blood cells, especially HSC, for lentivirus infection technical difficulty. HSCs of the clinical application level often require 100 million cells to achieve genetic manipulation, and the low infection efficiency puts forward very high requirements on the yield and quality of lentivirus, which significantly increases the cost of the test and achieves twice the result with half the effort.

Therefore, how to improve the ability of lentivirus to infect and transduce hematopoietic stem cells is a key issue of concern to scientific and technological workers in the field of blood research. The lentivirus system with VSVG as the envelope protein urgently needs further targeted optimization and improvement in order to achieve HSC efficient infection.

Contents of the invention

The present invention provides a membrane-bound mFLT3LG protein and its application. By re-originally designing the protein sequence of FLT3LG, removing its enzyme cleavage site and ensuring anchoring on the membrane surface, a new chimeric membrane-bound protein is obtained. The protein sequence of mFLT3LG (membrane FLT3LG) can significantly improve the infection efficiency of lentivirus to HSC, and has broad application prospects and huge market value.

To achieve the above object, the present invention adopts the following technical solutions:

In a first aspect, the present invention provides a membrane-bound mFLT3LG protein, which includes an active extracellular region of FLT3LG, a connecting part and an anchoring part.

Wherein, the amino acid sequence of the active extracellular region of the FLT3LG is shown in SEQ ID NO.1.

Said SEQ ID NO.1 is as follows:

MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPP.

Preferably, the linking means comprises a linking peptide.

Preferably, the connecting peptide comprises a flexible connecting peptide.

Preferably, the amino acid sequence of the flexible connecting peptide is shown in SEQ ID NO.2.

Said SEQ ID NO.2 is as follows:

GSSGGSSGGSSG.

Preferably, the anchoring member comprises the transmembrane region of a single-spanning protein.

Preferably, the transmembrane region of the single-spanning protein includes any one 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 of SCF transmembrane region.

Preferably, the amino acid sequence of the transmembrane region of the SCF is shown in SEQ ID NO.3.

Said SEQ ID NO.3 is as follows:

SSLHWAAMALPALFSLIGFAFGALYW

Among them, the polypeptide segment of membrane SCF (mSCF) C-terminal transmembrane region is SEQ ID NO.6 as follows:

NPPGDSSLHWAAMALPALFSLIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV.

The amino acid sequence of the natural FLT3LG protein is SEQ ID NO.7 as follows (NM_001459.3, NP_001450.2, full-length 235aa, after removing the N-terminal 26aa signal peptide, the complete transmembrane protein is 209aa, but it will be hydrolyzed and cut into 155aa soluble Ligand):

MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLL LPVGLLLLAAAWCLHWQRTRRRTPRPGEQVPPVPSPQDLLLVEH.

Preferably, the membrane-bound mFLT3LG protein includes an active extracellular region of FLT3LG with an amino acid sequence such as SEQ ID NO.1, a flexible linker peptide with an amino acid sequence such as SEQ ID NO.2, and an amino acid sequence such as SEQ ID NO. .3 The transmembrane region of the SCF shown.

Preferably, the amino acid sequence of the membrane-bound mFLT3LG protein is shown in SEQ ID NO.4;

Said SEQ ID NO.4 is as follows:

MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPGSSGGSSGGSSGNPPGDSSL HWAAMALPALFSLIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV.

In the present invention, the inventors creatively use the paired binding relationship between cytokine receptors and cytokines on the surface of HSC cells to construct a membrane-bound FLT3LG cytokine, and the lentiviral particles coated by this membrane-type cytokine can be combined with The FLT3 receptor on the surface of HSC binds, thereby directly shortening the physical distance between lentiviral particles and HSC, and realizing the virus infection process through endocytosis.

There are many cytokine receptors on the surface of hematopoietic stem cells, and these cytokine receptors can sense cytokines in the microenvironment, thereby controlling cell fates such as proliferation, self-renewal, and differentiation of hematopoietic stem cells. Among them, the most important cytokine receptors on HSC surface are c-KIT (SCF receptor), c-MPL (THPO receptor), FLT3 (FLT3LG receptor), IL6R (IL-6 receptor) and so on. The present invention artificially creates the paired binding relationship between these cytokine receptors and cytokines to enhance the infection efficiency of lentivirus to hematopoietic stem cells.

The present invention is mainly aimed at the artificial optimization and transformation of FLT3LG. FLT3, as a transmembrane receptor existing on the surface of hematopoietic stem cells, will not be altered in any way, and will not be described here. The signal transduction pathway of FLT3-FLT3LG is not the core content of the present invention. , and will not be further developed here; this patent mainly focuses on the artificial optimization design of the expression and membrane anchoring of FLT3LG.

So far, the mRNA reference sequences of FLT3LG registered in the NCBI or UCSC database website mainly have four subtypes, among which transcript variant 3 with the mRNA number NM_001459.3 (protein accession number NP_001450.2) is the most mainly expressed subtype type. The FLT3LG protein encodes 235 amino acids, of which 1-26 is a secretion signal peptide and will be cut off by enzymes; 27-184 is an extramembrane region; 185-205 is a transmembrane region; and 206-235 is an inner region. Although the natural FLT3LG will first become a membrane-bound cytokine, it will be cleaved by TACE protease, fall off from the cell membrane, and become a free soluble cytokine. Two FLT3LG molecules form a homodimer, and combine with two FLT3 receptors to form a tetramer, which participates in the process of cell signal transduction. It can be seen that the natural FLT3LG molecule cannot be used as an effective envelope protein for lentiviral packaging due to the presence of enzyme digestion sites.

Therefore, the present invention first re-originally designs the protein sequence of FLT3LG, removes its enzyme cleavage site and ensures anchoring on the membrane surface, retains the effective extracellular active region, including the 1-168aa polypeptide sequence, and is compatible with known The transmembrane region of the stem cell factor SCF that has removed the enzyme cleavage site is fused, and a flexible link peptide composed of 12aa is used in the middle to connect the extracellular active region and the transmembrane region to obtain a new chimeric membrane-bound mFLT3LG (membrane FLT3LG) protein sequence. The protein sequence contains 248aa, the isoelectric point PI is close to 5, and the molecular weight is 27kD.

At present, the enzyme digestion site of natural FLT3LG is not very clear, so it is temporarily impossible to eliminate the enzyme digestion site by amino acid missense mutation, and it is more operable to directly replace it with a known transmembrane region that cannot be digested by enzyme digestion .

In addition to using the transmembrane region of SCF to anchor mFLT3LG to the cell membrane, the transmembrane region of other cytokines, such as the transmembrane region of membrane-type IL6, the transmembrane region of VSVG itself, or other single transmembrane regions can also be used. The transmembrane region of membrane proteins can be used to anchor the cell membrane.

In a second aspect, the present invention provides an ORF reading frame of a membrane-bound mFLT3LG protein, the ORF reading frame is based on the protein described in the first aspect, and is reverse-designed for the most frequently used codons in human cells.

Preferably, the nucleotide sequence of the ORF reading frame is shown in SEQ ID NO.5.

Said SEQ ID NO.5 is as follows:

Modified membrane FLT3LG(mmFLT3LG) ORF seq using human-optimized codon:

ATGACCGTGCTGGCCCCCGCCTGGAGCCCCACCACCTACCTGCTGCTGCTGCTGCTGCTGAGCAGCGGCCTGAGCGGCACCCAGGACTGCAGCTTCCAGCACAGCCCCATCAGCAGCGACTTCGCCGTGAAGATCAGGGAGCTGAGCGACTACCTGCTGCAGGACTACCCCGTGACCGTGGCCAGCAACCTGCAGGACGAGGAGCTGTGCGGCGGC CTGTGGAGGCTGGTGCTGGCCCAGAGGTGGATGGAGAGGCTGAAGACCGTGGCCGGCAGCAAGATGCAGGGCCTGCTGGAGAGGGTGAACACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCCCCCCAGCTGCCTGAGGTTCGTGCAGACCAACATCAGCAGGCTGCTGCAGGAGACCAGCGAGCAGCTGGTGGCCCTGAAGCCCTGGA TCACCAGGCAGAACTTCAGCAGGTGCCTGGAGCTGCAGTGCCAGCCCGACAGCAGCACCCTGCCCCCCCCCGGCAGCAGCGGCGGCAGCAGCGGCGGCAGCAGCGGCAACCCCCCCGGCGACAGCAGCCTGCACTGGGCCGCCATGGCCCTGCCCGCCCTGTTCAGCCTGATCATCGGCTTCGCCTTCGGCGCCCTGTACTGGAAGAAGAGGCAG CCCAGCCTGACCAGGGCCGTGGAGAACATCCAGATCAACGAGGAGGACAACGAGATCAGCATGCTGCAGGAGAAGGAGAGGGAGTTCCAGGAGGTGTGATAA.

At the same time, the ORF reading frame does not refer to the natural gene sequence, but is optimized for the most frequently used codons in human cells to obtain a new chimeric mFLT3LG ORF reading frame.

In a third aspect, the present invention provides a recombinant plasmid, which includes the ORF reading frame of the membrane-bound mFLT3LG protein described in the second aspect.

Preferably, the plasmid further includes the nucleotide sequence of the VSVG envelope protein.

Preferably, the VSVG envelope protein and the membrane-bound mFLT3LG protein are respectively driven by two different promoters.

Of course, using FLT3L alone may not significantly improve packaging efficiency independently, because the VSVG protein is also critical for the release of lentiviral particles. Therefore, the inventors cloned the ORF of the brand-new membrane-bound mFLT3LG into the vector, and placed it on the same expression plasmid as VSVG. In this case, 293T cells transiently transfected with multiple plasmids at the same time will express VSVG and mFLT3LG proteins at the same time. This original optimization improvement significantly improves the infection efficiency of lentivirus to HSC. It should be noted that the lentiviral recombinant vector of the present invention can not only enhance the infection ability of HSC hematopoietic stem cells for gene therapy, but also can enhance the infection of some dendritic cells, T cells and NK cells, and those with FLT3 receptors on the cell surface The cells are all sensitive to the infection of the lentiviral recombinant vector of the present invention, which is of great significance in immunotherapy.

In a fourth aspect, the present invention provides a lentivirus obtained by co-packaging the plasmid described in the third aspect and an auxiliary packaging plasmid.

In the fifth aspect, the present invention provides a protein as described in the first aspect, the reading frame as described in the second aspect, the recombinant plasmid as described in the third aspect or the lentivirus as described in the fourth aspect for the preparation of gene therapy and / or application of immunotherapeutic drugs and / or kits.

Compared with prior art, the present invention has following advantage:

The protein provided by the invention is artificially optimized and designed for the expression and membrane anchoring of FLT3LG to construct a membrane-bound FLT3LG cytokine, and the redesigned original chimeric membrane-bound mFLT3LG protein sequence will not be digested by proteases. Shedding from the cell membrane; the lentiviral particles coated with this membrane-type cytokine can bind to the FLT3 receptor on the surface of HSC, thereby directly shortening the physical distance between lentiviral particles and HSC, and realizing the virus infection process through endocytosis, Enhance the infection efficiency of lentivirus to hematopoietic stem cells; at the same time, clone the ORF of the new membrane-bound mFLT3LG into the vector, and place it on the same expression plasmid as VSVG, and the dual promoters drive transcription and translation to ensure the expression of VSVG , mFLT3LG is also expressed at the same time, and 293T cells transiently transfected with multiple plasmids at the same time will express VSVG and mFLT3LG proteins at the same time. This original optimization improvement significantly improves the infection efficiency of lentivirus to HSC.

Description of drawings

Figure 1 is a schematic diagram of the construction of the VSVG-mFLT3LG vector in the embodiment of the present invention;

Fig. 2 is a graph showing the results of lentivirus infection of HSC cells.

Detailed ways

In order to further illustrate the technical means adopted by the present invention and its effects, the technical solutions of the present invention will be further described below in conjunction with preferred embodiments of the present invention, but the present invention is not limited within the scope of the embodiments.

Example

1. According to the natural FLT3LG protein sequence, determine its active extracellular region;

2. Connect the active extracellular region of FLT3LG to the transmembrane region of SCF through a 12aa flexible linker peptide to form a chimeric membrane-bound mFLT3LG;

3. Reversely design the ORF reading frame of mFLT3LG based on the data of the most frequently used codons in humans. The reading frame does not use the natural sequence as a template and is highly original;

4. Cloning the VSVG envelope protein and mFLT3G protein on the same expression plasmid, driven by two different promoters respectively;

5. According to the routine operation plan, the exogenous gene expression plasmid, gag-pol plasmid, Rev plasmid, and VSVG-mFLT3LG plasmid were transiently transfected into 293T cells at the same time, the lentivirus was packaged, and the virus was concentrated by high-speed centrifugation, and finally infected with HSC cells.

The specific plan is as follows:

Firstly, the original protein sequence of FLT3LG was redesigned to remove its enzyme cleavage site and ensure anchoring on the membrane surface; the effective extracellular active region was retained, including the 1-168aa polypeptide sequence, and it was compatible with known enzymes The transmembrane region of the stem cell factor SCF at the cleavage site is fused, and a flexible link peptide composed of 12aa is used in the middle to connect the extracellular active region and the transmembrane region to obtain a new chimeric protein sequence of membrane-bound mFLT3LG (membrane FLT3LG); The protein sequence contains 248aa, the isoelectric point PI is close to 5, and the molecular weight is 27kD. At the same time, the ORF reading frame does not refer to the natural gene sequence, but is optimized for the most frequently used codons in human cells to obtain a new chimeric mFLT3LG ORF reading frame.

Secondly, the ORF of the brand-new membrane-bound mFLT3LG was cloned into the vector, and placed on the same expression plasmid as VSVG. See Figure 1 for the schematic diagram of vector construction. VSVG and mFLT3LG were separately transcribed by two promoters to obtain VSVG-mFLT3LG plasmid.

Lentivirus Infected HSC Cell Test

According to the routine operation protocol, the exogenous gene expression plasmid, gag-pol plasmid, Rev plasmid, and VSVG-mFLT3LG plasmid were transiently transfected into 293T cells at the same time, the lentivirus was packaged, and the virus was concentrated by high-speed centrifugation, and finally infected HSC cells. Using the VSVG plasmid without mFLT3LG as the control group, the positive rate of HSC cells infected with lentivirus in the two cases was tested, and the results are shown in Figure 2.

It can be seen from Figure 2 that the optimized VSVG+mFLT3LG vector has a higher packaging titer, and the positive rate of infecting HSC cells is as high as 65%, which is significantly higher than the 38% of the control group. 293T cells transiently transfected with multiple plasmids at the same time will express VSVG and mFLT3LG proteins at the same time. This original optimization improvement significantly improves the infection efficiency of lentivirus to HSC.

The amino acid sequence of the natural FLT3LG protein is SEQ ID NO.7 as follows (NM_001459.3, NP_001450.2, full-length 235aa, after removing the N-terminal 26aa signal peptide, the complete transmembrane protein is 209aa, but it will be hydrolyzed and cut into 155aa soluble Ligand):

MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPQPPLLLLLL LPVGLLLLAAAWCLHWQRTRRRTPRPGEQVPPVPSPQDLLLVEH.

The amino acid sequence of the polypeptide segment of the C-terminal transmembrane region of membrane SCF (mSCF) is SEQ ID NO.6 as follows:

NPPGDSSLHWAAMALPALFSLIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV.

The amino acid sequence of Modified membrane FLT3LG (mmFLT3LG) protein (a chimeric cytokine composed of a cross-region of FLT3LG and SCF) is SEQ ID NO.4 as follows:

MTVLAPAWSPTTYLLLLLLLSSGLSGTQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPGSSGGSSGGSSGNPPGDSSL HWAAMALPALFSLIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV.

The nucleotide sequence of Modified membrane FLT3LG (mmFLT3LG) ORF using human-optimized codon is SEQ ID NO.5 as follows:

ATGACCGTGCTGGCCCCCGCCTGGAGCCCCACCACCTACCTGCTGCTGCTGCTGCTGCTGAGCAGCGGCCTGAGCGGCACCCAGGACTGCAGCTTCCAGCACAGCCCCATCAGCAGCGACTTCGCCGTGAAGATCAGGGAGCTGAGCGACTACCTGCTGCAGGACTACCCCGTGACCGTGGCCAGCAACCTGCAGGACGAGGAGCTGTGCGGCGGC CTGTGGAGGCTGGTGCTGGCCCAGAGGTGGATGGAGAGGCTGAAGACCGTGGCCGGCAGCAAGATGCAGGGCCTGCTGGAGAGGGTGAACACCGAGATCCACTTCGTGACCAAGTGCGCCTTCCAGCCCCCCCAGCTGCCTGAGGTTCGTGCAGACCAACATCAGCAGGCTGCTGCAGGAGACCAGCGAGCAGCTGGTGGCCCTGAAGCCCTGGA TCACCAGGCAGAACTTCAGCAGGTGCCTGGAGCTGCAGTGCCAGCCCGACAGCAGCACCCTGCCCCCCCCCGGCAGCAGCGGCGGCAGCAGCGGCGGCAGCAGCGGCAACCCCCCCGGCGACAGCAGCCTGCACTGGGCCGCCATGGCCCTGCCCGCCCTGTTCAGCCTGATCATCGGCTTCGCCTTCGGCGCCCTGTACTGGAAGAAGAGGCAG CCCAGCCTGACCAGGGCCGTGGAGAACATCCAGATCAACGAGGAGGACAACGAGATCAGCATGCTGCAGGAGAAGGAGAGGGAGTTCCAGGAGGTGTGATAA.

In summary, the present invention provides a membrane-bound mFLT3LG protein and its application. By re-originally designing the protein sequence of FLT3LG, removing its enzyme cleavage site and ensuring its anchoring on the membrane surface, a new embedded protein can be obtained. The protein sequence of the combined membrane-bound mFLT3LG (membrane FLT3LG), cloned the ORF of the brand-new membrane-bound mFLT3LG into the vector, and placed it on the same expression plasmid as VSVG, and started it with a double promoter, which significantly improved the lentiviral reaction. The infection efficiency of HSC has broad application prospects and huge market value.

The applicant declares that the present invention illustrates the detailed methods of the present invention through the above-mentioned examples, but the present invention is not limited to the above-mentioned detailed methods, that is, it does not mean that the present invention must rely on the above-mentioned detailed methods to be implemented. Those skilled in the art should understand that any improvement of the present invention, the equivalent replacement of each raw material of the product of the present invention, the addition of auxiliary components, the selection of specific methods, etc., all fall within the scope of protection and disclosure of the present invention.

SEQUENCE LISTING

<110> Hematology Hospital, Chinese Academy of Medical Sciences (Institute of Hematology)

<120> A membrane-bound mFLT3LG protein and its application

<130> 2019

<160> 7

<170> PatentIn version 3.3

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Met Thr Val Leu Ala Pro Ala Trp Ser Pro Thr Thr Tyr Leu Leu Leu

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atgaccgtgc tggcccccgc ctggagcccc accactacc tgctgctgct gctgctgctg 60

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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

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aaggagggg agttccagga ggtgtgataa 750

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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, characterized in that the protein is composed of an active extracellular region of FLT3LG, a connecting part and an anchoring part; Wherein, the amino acid sequence of the active extracellular region of the FLT3LG is shown in SEQ ID NO.1; The connecting part is a flexible connecting peptide; The anchor component is a transmembrane region of a single-pass transmembrane protein; The transmembrane region of the single transmembrane protein is the transmembrane region of SCF; The amino acid sequence of the membrane-bound mFLT3LG protein is the active extracellular region of FLT3LG, the flexible linker peptide and the transmembrane region of SCF from the N-terminus to the C-terminus. 2. The membrane-bound mFLT3LG protein according to claim 1, wherein the amino acid sequence of the flexible linker peptide is as 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 as shown in 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 as shown in SEQ ID NO.4. 5. An ORF reading frame polynucleotide of a membrane-bound mFLT3LG protein, characterized in that the ORF reading frame polynucleotide is designed for the protein according to any one of claims 1-4. 6. The ORF reading frame polynucleotide according to claim 5, characterized in that, the sequence of the ORF reading frame polynucleotide is as shown in SEQ ID NO.5. 7. A recombinant plasmid, characterized in that the plasmid comprises the ORF reading frame polynucleotide of the membrane-bound mFLT3LG protein according to claim 5 or 6. 8. The recombinant plasmid according to claim 7, characterized in that, the plasmid further comprises the nucleotide sequence of the VSVG envelope protein. 9. The recombinant plasmid according to claim 8, wherein the VSVG envelope protein and the membrane-bound mFLT3LG protein are respectively driven by two different promoters. 10. A lentivirus, characterized in that, the lentivirus is obtained by co-packaging the plasmid according to any one of claims 7-9 and an auxiliary packaging plasmid. 11. A protein according to any one of claims 1-4, a reading frame polynucleotide according to claim 5 or 6, or a recombinant plasmid according to any one of claims 7-9 or a claim Application of the lentivirus described in 10 in the preparation of medicines and/or kits for gene therapy and/or immunotherapy.

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