An envelope protein with broad cell tropism and high efficiency of infection and uses thereof

Abstract

The application discloses an envelope protein with wide cell tropism and high infection efficiency and application thereof. The application provides a B17M mutant protein as shown in SEQ ID NO: 4. Compared with a wild-type BaEV envelope protein, the B17M mutant protein is modified as follows: the cytoplasmic tail region is truncated to improve the ability of the B17M mutant protein to assemble into virus particles and improve the infection capacity of the B17M mutant protein; four amino acid residues are inserted to reduce the fusogenicity of the B17M mutant protein, so that the damage of the envelope protein to target cells can be effectively reduced. The application also protects a nucleic acid molecule encoding the B17M mutant protein, a recombinant plasmid with the nucleic acid molecule and a recombinant virus with the nucleic acid molecule. After the B17M mutant protein is constructed on a delivery vector, the efficiency of material delivery into human primary cells can be effectively improved, the product has great application value for the modification of human primary cells, and will have far-reaching social significance.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to an envelope protein with wide cell tropism and high infection efficiency and application thereof. BACKGROUND

[0002] Delivering substances (nucleic acids, proteins, small molecule drugs, etc.) into cells is a common experimental method in the field of biotechnology, and different delivery vectors will affect the delivery efficiency. At present, common delivery vectors include water blister virus glycoprotein (VSV-G) pseudotyped lentiviral vectors, adenovirus vectors, adeno-associated virus vectors, and liposome vectors. Among them, the VSV-G pseudotyped lentiviral vector is also one of the commonly used delivery vectors in clinical practice.

[0003] The VSV-G pseudotyped lentiviral delivery vector is modified from human immunodeficiency virus (HIV), and the original HIV envelope protein is replaced by VSV-G. The receptor recognized by VSV-G is low-density lipoprotein (LDL). LDL is a protein highly expressed on the surface of various cells, so VSV-G pseudotyped lentivirus has wide cell tropism and high delivery efficiency. However, the expression level of LDL on the surface of human primary cells in the resting state is very low, so the delivery efficiency of VSV-G viral vectors for human primary cells in the resting state is relatively low.

[0004] The baboon endogenous retrovirus (BaEV) envelope protein is a gamma-like endogenous retrovirus, and its recognized receptor is sodium-dependent neutral amino acid transporter (ASCT-1 and ASCT-2). ASCT-1 and ASCT-2 are widely distributed in various cells, including human primary cells, and can achieve high-efficiency delivery to human primary cells after being constructed on the viral vector. SUMMARY

[0005] The purpose of the present application is to provide an envelope protein with wide cell tropism and high infection efficiency and application thereof.

[0006] The present application provides a protein named B17M mutant protein, as shown in SEQ ID NO: 4.

[0007] The present application also protects a fusion protein with the B17M mutant protein.

[0008] The fusion protein includes the B17M mutant protein.

[0009] The fusion protein can also include other segments.

[0010] Illustratively, the other segments can be protein tags for purification and / or identification.

[0011] The protein tag can be specifically as shown in Table 1.

[0012] Table 1 Sequence of tag

[0013] Tag Residue Sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

[0014] Compared with the wild type BaEV envelope protein, the B17M mutant protein is modified as follows: the cytoplasmic tail is truncated to improve the ability of assembling into virus particles and improve the infection ability; four amino acid residues are inserted to reduce the fusogenicity and effectively reduce the damage of the envelope protein to the target cells.

[0015] The present application also protects a nucleic acid molecule encoding the B17M mutant protein.

[0016] Specifically, the nucleic acid molecule is a DNA molecule as shown in SEQ ID NO: 5.

[0017] The present application also protects a recombinant plasmid having the nucleic acid molecule.

[0018] Specifically, the recombinant plasmid is a recombinant plasmid pcDNA3.1(+)-B17M.

[0019] The present application also protects a recombinant virus having the nucleic acid molecule.

[0020] Specifically, the recombinant virus is a recombinant lentivirus.

[0021] The present application also protects a recombinant virus having the protein.

[0022] Specifically, the recombinant virus is a recombinant lentivirus.

[0023] Specifically, the recombinant virus is a HIV skeleton lentivirus with the B17M mutant protein as the envelope protein.

[0024] Specifically, the recombinant virus is a recombinant virus obtained by co-transfecting a mammalian cell (such as 293T cell) with the recombinant plasmid pcDNA3.1(+)-B17M and the skeleton plasmid pNL4-3R-E-luciferase, and then culturing the cell.

[0025] Specifically, the recombinant virus is a recombinant virus obtained by co-transfecting a mammalian cell (such as 293T cell) with the recombinant plasmid pcDNA3.1(+)-B17M, the psPAX2 plasmid and the pLVX-IRES-ZsGreen1 plasmid, and then culturing the cell.

[0026] The recombinant plasmid pcDNA3.1(+)-B17M has a nucleic acid molecule encoding the B17M mutant protein.

[0027] Specifically, the recombinant plasmid pcDNA3.1(+)-B17M is shown as SEQ ID NO: 6.

[0028] The application also protects the use of the B17M mutant protein or the nucleic acid molecule encoding the B17M mutant protein or any of the above-mentioned recombinant plasmids or any of the above-mentioned recombinant viruses in the preparation of a delivery carrier.

[0029] The delivery carrier is a viral delivery carrier or a liposome delivery carrier.

[0030] The application also protects a delivery carrier comprising the B17M mutant protein.

[0031] The delivery carrier is a viral delivery carrier or a liposome delivery carrier.

[0032] The application also protects a delivery carrier comprising any of the above-mentioned recombinant viruses.

[0033] The delivery carrier is a viral delivery carrier.

[0034] The application also protects the use of any of the above-mentioned recombinant viruses as a delivery carrier.

[0035] The application also protects a kit for preparing a delivery carrier, comprising the B17M mutant protein or the nucleic acid molecule encoding the B17M mutant protein or any of the above-mentioned recombinant plasmids or any of the above-mentioned recombinant viruses.

[0036] The delivery carrier is a viral delivery carrier or a liposome delivery carrier.

[0037] Specifically, any of the above-mentioned delivery carriers is a carrier for delivery to mammalian cells.

[0038] Specifically, any of the above-mentioned delivery carriers is a carrier for delivery to human cells.

[0039] Illustratively, the cells are primary cells.

[0040] Illustratively, the cells are T cells or hematopoietic stem cells.

[0041] After the B17M mutant protein is constructed on the delivery carrier, the efficiency of delivering substances (nucleic acids, proteins, small molecule drugs, etc.) to human primary cells can be effectively improved, and the product has great application value for the modification (gene editing, protein overexpression, etc.) of human primary cells, which will have far-reaching social significance. BRIEF DESCRIPTION OF DRAWINGS

[0042] Figure 1 Results of Example 3.

[0043] Figure 2Result of Step Four of Example 5.

[0044] Figure 3 Result of Step Four of Example 5.

[0045] Figure 4 Result of Step Five of Example 5. DETAILED DESCRIPTION

[0046] The present application will be further described in conjunction with the specific embodiments, which are presented only for the purpose of illustrating the present application and not for the purpose of limiting the same. The following examples are provided as a guide to further improve the present application for those skilled in the art, and do not in any way constitute a limitation on the present application.

[0047] The experimental methods in the following examples are all routine methods, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified. The quantitative tests in the following examples are all set up in triplicate, and the results are averaged, unless otherwise specified. The cell culture in the following examples is all carried out in a 37°C, 5% CO2 incubator, unless otherwise specified. Human peripheral blood mononuclear cells (PBMCs): isolated from ex vivo human peripheral blood.

[0048] psPAX2 plasmid (lentivirus backbone plasmid): Addgene product, product catalog number #12260. pLVX-IRES-ZsGreen1 plasmid (plasmid for delivering GFP reporter gene): TaKaRa product, product catalog number 632187. Backbone plasmid pNL4-3R-E-luciferase, described in the following literature (i.e. vector with the luciferase gene containing backbone pNL4-3R-E in the literature): A Single Substitution in gp41 Modulates the Neutralization Profile of SHIV during In Vivo Adaptation; "Wang Q, Liu L, Ren W, Gettie A, Wang H, Liang Q, Shi X, Montefiori DC, Zhou T, Zhang L; Cell Reports 27, 2593-2607. 293T cells: ATCC, number CRL-3216.

[0049] CD3 antibody: Biolegend product, catalog number 130-093-387. CD28 antibody: Biolegend product, catalog number 130-093-375. Luciferase reporter gene detection reagent: Promega product, catalog number DD1204. Lentivirus titer ELISA detection kit: Biovision product, catalog number BF06203. Fluorescent dye against CD3 (i.e. PE / Cyanine7 anti-human CD3 Antibody): BioLegend product, catalog number 317334. Fluorescent dye against Lineage (i.e. APC anti-human Lineage Cocktail): BioLegend product, catalog number 348703. Fluorescent dye against CD45 (i.e. Alexa 700anti-human CD45 Antibody): BioLegend product, catalog number 368514. Fluorescent dye against CD34 (i.e. PE / Cyanine7 anti-human CD34 Antibody): BioLegend product, catalog number 343616. CD34 MicroBead Kit: Miltenyi Biotec product, catalog number 130-046-702. PEI reagent: Polyscience product, catalog number 24765-1; used at 1 mg / ml PEI solution in water. Opti-MEM medium: Gibco product, catalog number 31985-070. stem cell medium (StemSpan TM SFEMII): STEMCELL Technologies product, catalog number 09655. X-vivo medium: Lonza product, catalog number 04-418Q.

[0050] Example 1, obtaining of B17M mutant proteins

[0051] The wild type BaEV envelope protein is shown in SEQ ID NO: 1. The coding sequence of the BaEV envelope protein in the BaEV genomic DNA is shown in SEQ ID NO: 2. Based on SEQ ID NO: 2, the inventors performed humanization codon optimization, and the optimized sequence is shown in SEQ ID NO: 3.

[0052] The inventors of the present application, based on the wild-type BaEV envelope protein, made truncations of different lengths and regions of the cytoplasmic tail, and additions and / or substitutions and / or deletions of different amino acid residues, to obtain a large number of mutant proteins. From the large number of mutant proteins, a mutant protein with excellent performance was screened by comparison, analysis, preparation and effect verification, and was named B17M mutant protein.

[0053] The B17M mutant protein is shown in SEQ ID NO: 4. In SEQ ID NO: 4, the first to 19th amino acid residues constitute a signal peptide (guiding the protein to be secreted outside the cell), the 20th to 514th amino acid residues constitute an extracellular domain, the 515th to 524th amino acid residues constitute a transmembrane domain, and the 525th to 550th amino acid residues are a cytoplasmic tail (compared with the segment of the wild-type BaEV envelope protein, the C-terminal 17 amino acid residues are truncated, and four amino acid residues are added at the C-terminal, and the four added amino acid residues are located at the 547th to 550th positions of SEQ ID NO: 4). The double-stranded DNA molecule shown in SEQ ID NO: 5 encodes the B17M mutant protein shown in SEQ ID NO: 4, and is a DNA molecule obtained after codon optimization for humanization by the inventors.

[0054] Example 2, construction of a recombinant plasmid

[0055] The recombinant plasmid pcDNA3.1(+)-B17M is a circular double-stranded DNA molecule, and the full sequence of the plasmid is shown in SEQ ID NO: 6. In SEQ ID NO: 6, the 923rd to 2575th nucleotides encode the B17M mutant protein.

[0056] Compared with the recombinant plasmid pcDNA3.1(+)-B17M, the recombinant plasmid BaEV differs only in that the 923rd to 2575th nucleotides in SEQ ID NO: 6 are replaced with the DNA molecule shown in SEQ ID NO: 3 (encoding the wild-type BaEV envelope protein).

[0057] Compared with the recombinant plasmid pcDNA3.1(+)-B17M, the recombinant plasmid BaEV-Rless differs only in that the 923rd to 2575th nucleotides in SEQ ID NO: 6 are replaced with the DNA molecule shown in SEQ ID NO: 7 (encoding the truncated protein of the wild-type BaEV envelope protein).

[0058] The recombinant plasmid pMD2.G-VSV-G is a circular double-stranded DNA molecule, and the full sequence of the plasmid is shown in SEQ ID NO: 8. In SEQ ID NO: 8, the 1450th to 2985th nucleotides encode VSV-G.

[0059] Example 3, Comparison of efficiency of lentivirus pseudotyped with B17M to deliver luciferase gene into 293T cells

[0060] I. Preparation of lentivirus pseudotyped with B17M

[0061] 1. Culture 293T cells in a 15 cm diameter culture dish until the cell density reaches about 80%.

[0062] 2. Take 3 ml of Opti-MEM medium, add 6 μg of recombinant plasmid pcDNA3.1(+)-B17M and 24 μg of backbone plasmid pNL4-3R-E-luciferase, then add 90 μl of 1 mg / ml PEI solution, shake well and stand for 15 min. This is one culture dish of transfection mixture.

[0063] 3. Add the transfection mixture prepared in step 2 to the culture dish prepared in step 1 and incubate for 6 hours.

[0064] 4. After step 3, discard the supernatant, add DMEM medium containing 2% fetal bovine serum and incubate for 48 hours. Then transfer the entire culture system to a centrifuge tube, centrifuge at 4°C and 4000 rpm for 30 min, collect the supernatant, which is the virus solution of lentivirus pseudotyped with B17M mutant protein, referred to as B17M virus solution, and store it in aliquots.

[0065] II. Preparation of lentivirus pseudotyped with BaEV

[0066] Use recombinant plasmid BaEV instead of recombinant plasmid pcDNA3.1(+)-B17M, follow the procedure in step I to obtain the virus solution of lentivirus pseudotyped with wild-type BaEV envelope protein, referred to as BaEV virus solution, and store it in aliquots.

[0067] III. Preparation of lentivirus pseudotyped with BaEV-Rless

[0068] Use recombinant plasmid BaEV-Rless instead of recombinant plasmid pcDNA3.1(+)-B17M, follow the procedure in step I to obtain the virus solution of lentivirus pseudotyped with truncated protein, referred to as BaEV-Rless virus solution, and store it in aliquots.

[0069] IV. Quantification of pseudovirus by p24 ELISA and infection of 293T cells

[0070] Test virus solution: B17M virus solution prepared in step I or BaEV virus solution prepared in step II or BaEV-Rless virus solution prepared in step III.

[0071] 1. Take the test virus liquid, dilute it to 10000 times the volume with PBS buffer, then use the lentivirus titer ELISA detection kit to detect the P24 protein content (unit: pg / ml).

[0072] 2. Take the test virus liquid, adjust the virus content according to the detection results of step 1 using DMEM medium containing 2% fetal bovine serum.

[0073] 3. Take a 96-well plate, add the virus liquid prepared in step 2 (the amount of P24 protein in the virus liquid added to each well is 20 ng) and 293T cells (the amount of cells added to each well is 20000), mix well, and culture with DMEM medium containing 2% fetal bovine serum for 48h. Set up a control well with the same volume of DMEM medium containing 2% fetal bovine serum instead of the virus liquid, which is the NC well.

[0074] 4. After completing step 3, discard the supernatant, add 55μl luciferase reporter gene detection reagent to each well, react at room temperature for 2min, then measure the relative fluorescence intensity (RLU).

[0075] RLU can reflect the delivery efficiency of different envelope proteins, and the higher the relative fluorescence intensity, the higher the delivery efficiency.

[0076] The results are shown in Figure 1 . The relative fluorescence intensity from high to low is: B17M virus liquid > BaEV-Rless virus liquid > BaEV virus liquid. That is, the delivery efficiency from high to low is: B17M mutant protein > truncated protein > wild type BaEV envelope protein.

[0077] Example 4, fusion originality comparison

[0078] Test plasmid: recombinant plasmid pcDNA3.1(+)-B17M or recombinant plasmid BaEV or recombinant plasmid BaEV-Rless.

[0079] 1. Take a 6-well plate, inoculate 293T cells, and culture with DMEM medium containing 2% fetal bovine serum until the cell density is 70%-80%.

[0080] 2. Add 5μg of test plasmid to 200μl of Opti-MEM medium, then add 15μl of 1mg / ml PEI solution, shake well, then incubate at room temperature for 15min, which is the transfection material for one transfection well.

[0081] 3. After completing step 1, take the 6-well plate, add the transfection material prepared in step 2 (uniformly drop onto the 293T cells and gently shake), then culture for 24h or 48h.

[0082] 4. After step 3, observe whether syncytia is formed under microscope.

[0083] The more syncytia is formed, the stronger the fusogenicity is, and the stronger the fusogenicity is, the greater the cell damage is.

[0084] The results are shown in Figure 2 . Figure 2 A of FIG. 1 is a photo taken after 24 hours of culture; after 24 hours of transfection, no membrane fusion phenomenon occurs for the wild type BaEV envelope protein and the B17M mutant protein, and strong membrane fusion occurs for the truncated protein to form huge syncytia. Figure 2 B of FIG. 1 is a photo taken after 48 hours of culture; after 48 hours of transfection, no membrane fusion phenomenon occurs for the wild type BaEV envelope protein, slight membrane fusion occurs for the B17M mutant protein, and strong membrane fusion occurs for the truncated protein, and the cells die and float. The fusogenicity from low to high is: wild type BaEV envelope protein < B17M mutant protein < truncated protein.

[0085] Example 5, Comparison of assembly and delivery efficiency of pseudotyped lentivirus

[0086] I. Preparation of B17M pseudotyped lentivirus

[0087] 1. Culture 293T cells in a culture dish with a diameter of 15 cm until the cell density reaches about 80%.

[0088] 2. Take 3 ml of Opti-MEM medium, add 5 μg of recombinant plasmid pcDNA3.1(+)-B17M, 10 μg of psPAX2 plasmid and 15 μg of pLVX-IRES-ZsGreen1 plasmid, then add 90 μl of 1 mg / ml PEI solution, shake well and stand for 15 min, which is a culture dish portion of transfection.

[0089] 3. Add the transfection prepared in step 2 to the culture dish completed in step 1 and culture for 6 hours.

[0090] 4. After step 3, discard the supernatant, add DMEM medium containing 2% fetal bovine serum, and culture for 48 hours, then transfer the entire culture system to a centrifuge tube, centrifuge at 4°C and 4000 rpm for 30 min, and collect the supernatant.

[0091] 5. Take the supernatant obtained in step 4, filter it using a filter membrane with a pore size of 0.45 μm, and collect the filtrate.

[0092] 6. Take the filtrate obtained in step 5, centrifuge at 22000 rpm for 2 h at 4°C, discard the supernatant, suspend the precipitate with 200 μl PBS buffer, then centrifuge at 8000 rpm for 5 min at 4°C, discard the precipitate, and collect the supernatant, which is the virus solution of the B17M mutant protein pseudotyped lentivirus, referred to as B17M virus solution, and store in aliquots.

[0093] II. Preparation of VSV pseudotyped lentivirus

[0094] Replace the recombinant plasmid pcDNA3.1(+)-B17M with the recombinant plasmid pMD2.G-VSV-G, and follow the procedures in step I to obtain the virus solution of the VSVG pseudotyped lentivirus, referred to as VSV virus solution, and store in aliquots.

[0095] III. Determination of lentivirus titer

[0096] The test virus solution is the B17M virus solution prepared in step I or the VSV virus solution prepared in step II.

[0097] 1. Take the test virus solution, and perform 3-fold gradient dilution using DMEM medium containing 2% fetal bovine serum, for a total of 9 gradients, to obtain virus dilutions.

[0098] 2. Take 96-well plates, inoculate each well with 20000 293T cells, then add 100 μl of virus dilution to each well, and then incubate for 48 h. For each virus dilution, set 3-5 replicate wells.

[0099] 3. After step 2 is completed, collect cells from each well, and perform flow cytometry analysis to determine the fluorescence proportion (i.e., the percentage of cells showing green fluorescence in the total number of cells).

[0100] 4. Take the two wells with a fluorescence proportion of 1% to calculate the virus titer. The well with a fluorescence proportion greater than 1% is well A (the fluorescence proportion of well A is represented by A%), and the well with a fluorescence proportion less than 1% is well B (the fluorescence proportion of well B is represented by B%). The volume of the test virus solution corresponding to the virus dilution added to well A is X (μl).

[0101] Virus titer (Tu / μl) = (A% x 20000 + 3 x B% x 20000) / (2 x X).

[0102] IV. Delivery of pseudotyped lentivirus to primary T cells

[0103] 1. Obtain and stimulate primary T cells

[0104] (1) CD3 antibody / CD28 antibody pre-coating

[0105] Take 6-hole plate, add 5 μg / ml CD3 antibody and 5 μg / ml CD28 antibody in PBS buffer, 4℃ incubate 12 hours, then aspirate supernatant.

[0106] (2) Enrich T cells from PBMC

[0107] Incubate PBMC for 30 min, collect suspended cells in supernatant, resuspend cells with X-vivo medium containing 1000 IU / ml IL-2, to obtain cell suspension with cell concentration of 1×10 6 cells / ml.

[0108] (3) Stimulate T cells

[0109] Take 6-hole plate of step (1), add cell suspension of step (2) (3 ml / hole), incubate for 24 hours.

[0110] 2, Lentivirus infection of primary T cells

[0111] (1) After completion of step 1, collect cells and count, adjust cell concentration with X-vivo medium containing 1000 IU / ml IL-2, to obtain cell suspension with cell concentration of 0.2×10 6 cells / ml.

[0112] (2) Take 96-hole plate, add 100 μl cell suspension prepared in step (1) to each hole, then add 100 μl test virus liquid (MOI=5) to each hole, incubate for 48 hours.

[0113] Test virus liquid: B17M virus liquid prepared in step one or VSV virus liquid prepared in step two, adjust virus titer with X-vivo medium containing 1000 IU / ml IL-2. Set NC hole with equal volume of X-vivo medium containing 1000 IU / ml IL-2 instead of test virus liquid.

[0114] 3, Delivery efficiency detection

[0115] After completion of step 2, collect cells, stain cells with fluorescent dye against CD3, then perform flow fluorescence analysis, and count the proportion of cells with GFP fluorescence in CD3 positive cells.

[0116] The higher the proportion of cells with GFP fluorescence, the higher the delivery efficiency.

[0117] Results are shown in Figure 3The proportion of cells with GFP fluorescence in CD3 positive cells from high to low is: B17M virus solution > VSV virus solution. That is, the delivery efficiency from high to low is: B17M mutant protein > VSV envelope protein. The results show that the delivery efficiency of B17M mutant protein to T cells is higher than that of VSV envelope protein in the prior art.

[0118] V. Pseudotyped lentivirus delivery to human CD34+ cells

[0119] 1. Stimulation of CD34+ cells

[0120] (1) CD34+ cells were sorted from peripheral blood (the peripheral blood was obtained from a lymphoma patient in Peking University Cancer Hospital, and was collected after the patient was injected with a stem cell mobilizing agent) using CD34 magnetic beads.

[0121] (2) The CD34+ cells obtained in step (1) were cultured in stem cell medium containing 100 ng / ml SCF, 100 ng / ml Flt3-L, 20 ng / ml TPO, 20 ng / ml IL-3, and 20 ng / ml IL-6 for 24 h.

[0122] 2. Pseudotyped lentivirus infection of primary CD34+ cells

[0123] (1) After step 1 was completed, the cells were collected and counted, and the cell concentration was adjusted with stem cell medium to obtain a cell suspension with a cell concentration of 0.2 x 10 6 cells / ml.

[0124] (2) Take a 96-well plate, add 100 μl of the cell suspension prepared in step (1) to each well, then add 100 μl of the test virus solution (MOI = 5) to each well, and culture for 48 h.

[0125] The test virus solution is: B17M virus solution prepared in step one or VSV virus solution prepared in step two, and the virus titer is adjusted with stem cell medium. Set NC wells with the same volume of stem cell medium instead of the test virus solution.

[0126] 3. Delivery efficiency detection

[0127] After step 2 was completed, the cells were collected and stained with fluorescent dyes against Lineage, fluorescent dyes against CD45, and fluorescent dyes against CD34 at the same time, and then flow cytometry analysis was performed to count the proportion of cells with GFP fluorescence in target cells. Target cells refer to cells that are Lineage negative, CD45 positive, and CD34 positive.

[0128] The higher the proportion of cells with GFP fluorescence, the higher the delivery efficiency.

[0129] The results are shown in Table 1. Figure 4 The proportion of cells with GFP fluorescence in the target cells from high to low is as follows: B17M virus solution > VSV virus solution. That is, the delivery efficiency from high to low is as follows: B17M mutant protein > VSV envelope protein. The results show that the delivery efficiency of the B17M mutant protein for hematopoietic stem cells is higher than that of the VSV envelope protein in the prior art.

[0130] The application has been described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although specific examples are given in the application, it should be understood that further improvements can be made to the application. In summary, according to the principle of the application, the present application is intended to include any changes, uses or improvements of the application, including changes made by conventional techniques known in the art, which are outside the scope disclosed in the present application. Some basic features can be applied within the scope of the following claims.

Claims

1. Protein, named B17M mutant protein, as shown in SEQ ID NO:

4.

2. A fusion protein comprising the protein of claim 1 and a protein tag for purification.

3. A nucleic acid molecule encoding the protein of claim 1 or the fusion protein of claim 2.

4. A recombinant plasmid having the nucleic acid molecule of claim 3.

5. A recombinant virus having the nucleic acid molecule of claim 3.

6. A recombinant virus having the protein of claim 1 or the fusion protein of claim 2.

7. The use of the protein of claim 1, the fusion protein of claim 2, the nucleic acid molecule of claim 3, the recombinant plasmid of claim 4, the recombinant virus of claim 5, or the recombinant virus of claim 6 in the preparation of a delivery vector.

8. A delivery vector comprising the protein of claim 1 or the fusion protein of claim 2.

9. A delivery vector comprising the recombinant virus of claim 5 or 6.

10. A kit for preparing a delivery vector, comprising the protein of claim 1, the fusion protein of claim 2, the nucleic acid molecule of claim 3, the recombinant plasmid of claim 4, the recombinant virus of claim 5, or the recombinant virus of claim 6.

Images