Envelope protein with wide cell tropism and efficient infection efficiency and application thereof

Abstract

The invention discloses an envelope protein with wide cell tropism and high infection efficiency and application of the envelope protein. The invention 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: truncation of a cytoplasm tail region is carried out, the capability of assembling and forming virus particles is improved, and the infection capability is improved; four amino acid residues are inserted, so that the fusion genicity is reduced, and the damage of the envelope protein to target cells can be effectively reduced. The invention also protects a nucleic acid molecule for coding 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 the delivery vector, the efficiency of substance delivery to the human primary cells can be effectively improved, and the product has great application value for modification of the human primary cells and has profound social significance.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an envelope protein with broad cell tropism and high infection efficiency and application thereof. Background Art

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

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

[0004] The envelope protein of baboon endogenous retrovirus (BaEV) is a γ-retrovirus-like endogenous retrovirus, and its recognized receptors are sodium-dependent neutral amino acid transporters (ASCT-1 and ASCT-2). ASCT-1 and ASCT-2 are widely distributed in various cells, including human primary cells. After constructing them into viral vectors, they can achieve high-efficiency delivery to human primary cells. Summary of the invention

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

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

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

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

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

[0010] Exemplarily, the other segment may be a protein tag used for purification and / or identification.

[0011] The protein tags are specifically shown in Table 1.

[0012] Table 1 Tag sequences

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

[0014] Compared with the wild-type BaEV envelope protein, the B17M mutant protein was modified as follows: the cytoplasmic tail region was truncated to improve its ability to assemble into viral particles and its infection ability; four amino acid residues were inserted to reduce its fusogenicity, which can effectively reduce the damage of the envelope protein to the target cells.

[0015] The present invention also protects nucleic acid molecules encoding B17M mutant proteins.

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

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

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

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

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

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

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

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

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

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

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

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

[0028] The present invention also protects the use of a B17M mutant protein or a nucleic acid molecule encoding a B17M mutant protein or any of the above recombinant plasmids or any of the above recombinant viruses in preparing a delivery vector.

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

[0030] The present invention also protects a delivery vector comprising the B17M mutant protein.

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

[0032] The present invention also protects a delivery vector, which comprises any of the above-mentioned recombinant viruses.

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

[0034] The present invention also protects the use of any of the above-mentioned recombinant viruses as a delivery vector.

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

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

[0037] Specifically, any of the above delivery vectors is a vector for delivery to mammalian cells.

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

[0039] Exemplarily, the cell is a primary cell.

[0040] Exemplarily, the cell is a T cell or a hematopoietic stem cell.

[0041] After constructing the B17M mutant protein onto the delivery vector, the efficiency of delivering substances (nucleic acids, proteins, small molecule drugs, etc.) into human primary cells can be effectively improved. This product has great application value for the transformation of human primary cells (gene editing, protein overexpression, etc.) and will have far-reaching social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 This is the result of Example 3.

[0043] Figure 2This is the result of Example 4.

[0044] Figure 3 This is the result of step 4 of Example 5.

[0045] Figure 4 This is the result of step five of Example 5. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0047] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources. Unless otherwise specified, the quantitative tests in the following examples are set up for three repeated experiments, and the results are averaged. Unless otherwise specified, cell culture is carried out in a 37°C, 5% CO2 incubator. Human peripheral blood mononuclear cells (PBMC): isolated from human peripheral blood in vitro.

[0048] psPAX2 plasmid (lentiviral backbone plasmid): Addgene product, catalog number #12260. pLVX-IRES-ZsGreen1 plasmid (plasmid for delivering GFP reporter gene): TaKaRa product, catalog number 632187. Backbone plasmid pNL4-3R-E-luciferase, described in the following literature (i.e., the 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: Miltenyi Biotec, catalog number 130-093-387. CD28 antibody: Miltenyi Biotec, catalog number 130-093-375. Luciferase reporter gene assay: Novozymes, catalog number DD1204. Lentiviral titer ELISA kit: BioAolong, catalog number BF06203. Fluorescent dye targeting CD3 (i.e. PE / Cyanine7 anti-human CD3 Antibody): BioLegend, catalog number 317334. Fluorescent dye targeting Lineage (i.e. APC anti-human Lineage Cocktail): BioLegend, catalog number 348703. Fluorescent dye targeting CD45 (i.e. Alexa Fluor 700anti-humanCD45 Antibody): BioLegend product, catalog number 368514. Fluorescent dye for CD34 (i.e. PE / Cyanine7 anti-human CD34Antibody): BioLegend product, catalog number 343616. CD34MicroBead Kit: Miltenyi Biotec product, catalog number 130-046-702. PEI reagent: Polyscience product, catalog number 24765-1; prepare 1 mg / ml PEI solution with water when using. Opti-MEM culture medium: Gibco product, catalog number 31985-070. Stem cell culture medium (StemSpan TM SFEMII): Product of STEMCELL Technologies, catalog number 09655. X-vivo medium: Product of Lonza, catalog number 04-418Q.

[0050] Example 1. Obtaining B17M mutant protein

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

[0052] The inventors of the present invention obtained a large number of mutant proteins based on the wild-type BaEV envelope protein by truncating different lengths and regions of the cytoplasmic tail region, as well as adding and / or replacing and / or deleting different amino acid residues. Through comparison, analysis, preparation and effect verification, a mutant protein with excellent performance was screened from the large number of mutant proteins mentioned above, and it was named B17M mutant protein.

[0053] The B17M mutant protein is shown in SEQ ID NO: 4. In SEQ ID NO: 4, amino acid residues 1-19 constitute a signal peptide (guiding protein secretion outside the cell), amino acid residues 20-514 constitute an extracellular domain, amino acid residues 515-524 constitute a transmembrane domain, and amino acid residues 525-550 are a cytoplasmic tail region (compared with this segment of the wild-type BaEV envelope protein, the C-terminal 17 amino acid residues were truncated, and four amino acid residues were added to the C-terminal, and the added 4 amino acid residues are located at positions 547-550 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 by the inventors after humanized codon optimization.

[0054] Example 2: Construction of recombinant plasmid

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

[0056] Compared with the recombinant plasmid pcDNA3.1(+)-B17M, the difference of the recombinant plasmid BaEV is only that the nucleotides 923-2575 in SEQ ID NO: 6 are replaced by 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 difference of the recombinant plasmid BaEV-Rless is that the nucleotides 923-2575 in SEQ ID NO: 6 are replaced by the DNA molecule shown in SEQ ID NO: 7 (encoding a 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 complete sequence of the plasmid is shown in SEQ ID NO: 8. In SEQ ID NO: 8, nucleotides 1450-2985 encode VSV-G.

[0059] Example 3. Comparison of the efficiency of pseudotyped lentivirus delivery of luciferase gene to 293T cells

[0060] 1. Preparation of B17M pseudotyped lentivirus

[0061] 1. Culture 293T cells in a culture dish with a diameter of 15 cm 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 to mix, and let stand for 15 minutes. This is the transfection product for one culture dish.

[0063] 3. Add the transfectant prepared in step 2 to the culture dish that completed step 1 and culture for 6 hours.

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

[0065] 2. Preparation of BaEV pseudotyped lentivirus

[0066] Recombinant plasmid BaEV was used instead of recombinant plasmid pcDNA3.1(+)-B17M, and the operation was performed according to step 1 to obtain the virus liquid of wild-type BaEV envelope protein pseudotyped lentivirus, referred to as BaEV virus liquid, which was packaged and stored.

[0067] 3. Preparation of BaEV-Rless pseudotyped lentivirus

[0068] Recombinant plasmid BaEV-Rless was used instead of recombinant plasmid pcDNA3.1(+)-B17M, and the operation was performed according to step 1 to obtain the virus liquid of truncated protein pseudotype lentivirus, referred to as BaEV-Rless virus liquid, which was packaged and stored.

[0069] IV. Pseudovirus p24 ELISA quantification and infection of 293T cells

[0070] Test virus solution: B17M virus solution prepared in step 1, BaEV virus solution prepared in step 2, or BaEV-Rless virus solution prepared in step 3.

[0071] 1. Take the test virus solution, dilute it to 10,000 times the volume with PBS buffer, and then use the lentivirus titer ELISA detection kit to detect the P24 protein content (in pg / ml).

[0072] 2. Take the test virus solution and adjust the virus content with DMEM culture medium containing 2% fetal bovine serum according to the test results of step 1.

[0073] 3. Take a 96-well plate, add the virus solution prepared in step 2 (the amount of P24 protein in each well is 20 ng) and 293T cells (the amount of cells added to each well is 20,000), mix well, and culture for 48 hours in DMEM medium containing 2% fetal bovine serum. Set up a control well in which the virus solution is replaced with an equal volume of DMEM medium containing 2% fetal bovine serum, which is the NC well.

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

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

[0076] Results 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: Comparison of fusogenicity

[0078] Test plasmids: 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 them in DMEM medium containing 2% fetal bovine serum until the cell density reaches 70%-80%.

[0080] 2. Add 5 μg of the test plasmid to 200 μl of Opti-MEM medium, then add 15 μl of 1 mg / ml PEI solution, shake thoroughly, and then let stand at room temperature for 15 minutes. This 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 (evenly drop it on the 293T cells and gently shake it), and then culture for 24 hours or 48 hours.

[0082] 4. After completing step 3, observe under a microscope to see if syncytia are formed.

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

[0084] Results Figure 2 . Figure 2 A is a photo taken after 24 hours of culture; 24 hours after transfection, there was no membrane fusion between the wild-type BaEV envelope protein and the B17M mutant protein, while the truncated protein underwent strong membrane fusion to form a giant syncytium. Figure 2 B is a photo taken after 48 hours of culture. After 48 hours of transfection, the wild-type BaEV envelope protein had no membrane fusion phenomenon, the B17M mutant protein had slight membrane fusion, and the truncated protein had strong membrane fusion while the cells died and floated. The order of 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 lentiviruses

[0086] 1. 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 to mix and let stand for 15 minutes. This is the transfection product for one culture dish.

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

[0090] 4. After completing step 3, discard the supernatant, add DMEM medium containing 2% fetal bovine serum, culture for 48 hours, then transfer the entire culture system to a centrifuge tube, centrifuge at 4°C and 4000 rpm for 30 minutes, 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 4°C and 22000 rpm for 2 h, discard the supernatant, suspend the precipitate with 200 μl PBS buffer, and then centrifuge at 4°C and 8000 rpm for 5 min, discard the precipitate, collect the supernatant, which is the virus liquid of the B17M mutant protein pseudotype lentivirus, referred to as B17M virus liquid, and store it in aliquots.

[0093] 2. Preparation of VSV pseudotyped lentivirus

[0094] Recombinant plasmid pMD2.G-VSV-G was used instead of recombinant plasmid pcDNA3.1(+)-B17M, and the operation was performed according to step 1 to obtain the virus liquid of VSVG pseudotype lentivirus, referred to as VSV virus liquid, which was packaged and stored.

[0095] 3. Lentivirus titer determination

[0096] The test virus solution is: the B17M virus solution prepared in step 1 or the VSV virus solution prepared in step 2.

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

[0098] 2. Take a 96-well plate, inoculate 20,000 293T cells per well, then add 100 μl of virus dilution to each well, and then culture for 48 hours. Set up 3-5 replicate wells for each virus dilution.

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

[0100] 4. Take the front and back wells with a fluorescence ratio of 1% to calculate the virus titer. The well with a fluorescence ratio greater than 1% is well A (the fluorescence ratio of well A is expressed as A%), and the well with a fluorescence ratio less than 1% is well B (the fluorescence ratio of well B is expressed as 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%×20000+3×B%×20000) / (2×X).

[0102] IV. Pseudotyped lentivirus delivery to primary T cells

[0103] 1. Obtain and stimulate primary T cells

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

[0105] Take a 6-well plate, add PBS buffer containing 5 μg / ml CD3 antibody and 5 μg / ml CD28 antibody, incubate at 4°C for 12 hours, and then discard the supernatant.

[0106] (2) Enrichment of T cells from PBMCs

[0107] The PBMCs were cultured for 30 min, the suspended cells in the supernatant were collected, and the cells were resuspended in X-vivo medium containing 1000 IU / ml IL-2 to obtain a cell concentration of 1×10 6 cells / ml of cell suspension.

[0108] (3) Stimulation of T cells

[0109] Take the 6-well plate that has completed step (1), add the cell suspension obtained in step (2) (3 ml / well), and culture for 24 hours.

[0110] 2. Infection of primary T cells with pseudotyped lentivirus

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

[0112] (2) Take a 96-well plate, add 100 μl of the cell suspension prepared in step (1) to each well, and then add 100 μl of the test virus solution (MOI=5) to each well, and culture for 48 hours.

[0113] The test virus solution is: B17M virus solution prepared in step 1 or VSV virus solution prepared in step 2, and the virus titer is adjusted with X-vivo medium containing 1000 IU / ml IL-2. Set up NC wells in which the test virus solution is replaced with an equal volume of X-vivo medium containing 1000 IU / ml IL-2.

[0114] 3. Delivery efficiency detection

[0115] After completing step 2, the cells are collected, stained with a fluorescent dye targeting CD3, and then flow cytometry analysis is performed to count the proportion of cells with GFP fluorescence among CD3-positive cells.

[0116] A higher proportion of cells with GFP fluorescence indicates a higher delivery efficiency.

[0117] Results Figure 3The ratio of cells with GFP fluorescence in CD3 positive cells is from high to low: B17M virus liquid > VSV virus liquid. That is, the delivery efficiency is from high to low: 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] 5. Pseudotyped lentivirus delivery to human CD34+ cells

[0119] 1. Stimulation of CD34+ cells

[0120] (1) CD34+ cells were sorted from peripheral blood (Peripheral blood was obtained from lymphoma patients in Peking University Cancer Hospital after injection of stem cell mobilization agent) using CD34 magnetic beads.

[0121] (2) The CD34+ cells obtained in step (1) were cultured for 24 h in a stem cell culture 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.

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

[0123] (1) After completing step 1, collect and count the cells, and adjust the cell concentration with stem cell culture medium to obtain a cell concentration of 0.2×10 6 cells / ml of cell suspension.

[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 hours.

[0125] The test virus solution is: the B17M virus solution prepared in step 1 or the VSV virus solution prepared in step 2, and the virus titer is adjusted with stem cell culture medium. Set up NC wells in which the test virus solution is replaced with an equal volume of stem cell culture medium.

[0126] 3. Delivery efficiency detection

[0127] After completing step 2, collect the cells, stain them with fluorescent dyes for lineage, CD45, and CD34 at the same time, and then perform flow cytometry analysis to count the proportion of cells with GFP fluorescence in the target cells. Target cells refer to cells that are lineage negative, CD45 positive, and CD34 positive.

[0128] A higher proportion of cells with GFP fluorescence indicates a higher delivery efficiency.

[0129] Results Figure 4 The ratio of cells with GFP fluorescence in the target cells is from high to low: B17M virus liquid > VSV virus liquid. That is, the delivery efficiency is from high to low: B17M mutant protein > VSV envelope protein. The results show that the delivery efficiency of B17M mutant protein to hematopoietic stem cells is higher than that of VSV envelope protein in the prior art.

[0130] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Protein, which is (a) or (b) as follows: (a) a protein, designated as B17M mutant protein, as shown in SEQ ID NO: 4; (b) A fusion protein with the B17M mutant protein.

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

3. A recombinant plasmid having the nucleic acid molecule according to claim 2.

4. A recombinant virus having the nucleic acid molecule according to claim 2.

5. A recombinant virus having the protein according to claim 1.

6. Use of the protein according to claim 1, the nucleic acid molecule according to claim 2, the recombinant plasmid according to claim 3, the recombinant virus according to claim 4, or the recombinant virus according to claim 5 in preparing a delivery vector.

7. A delivery vector comprising the protein of claim 1.

8. A delivery vector comprising the recombinant virus according to claim 4 or 5.

9. Use of the recombinant virus according to claim 4 or 5 as a delivery vector.

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

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