A protein composition capable of improving the proliferative activity of cells infected with lentivirus and application thereof

Abstract

This invention provides a protein composition that can enhance the proliferative activity of lentivirus-infected cells and its application. The protein composition includes at least one of protein A, protein B, and protein C; the amino acid sequence of protein A is selected from one of SEQ ID NO: 1-6; the amino acid sequence of protein B is selected from one of SEQ ID NO: 7-11; and the amino acid sequence of protein C is selected from one of SEQ ID NO: 12-13. By synthesizing a nucleic acid molecule encoding the protein composition provided by this invention, integrating it into a recombinant vector, and transfecting cells using a lentivirus transfection system, the lentivirus-infected cells can express the above-mentioned protein composition, thereby enhancing the proliferative activity of lentivirus-infected cells, reducing the risk of mass cell death after lentivirus infection, and allowing lentivirus-infected cells to survive well after a period of culture, thus improving the infection efficiency of lentivirus on cells.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, specifically to a protein composition that can enhance the proliferative activity of lentivirus-infected cells and its applications. Background Technology

[0002] Lentivirals are a type of retrovirus that can integrate a target gene into the host's genome and achieve stable and persistent expression. They are highly safe and have higher titers and a wider host range than ordinary retroviruses, and can infect both dividing and non-dividing cells.

[0003] Lentiviral vectors are gene therapy vectors developed based on human immunodeficiency virus type 1 (HIV1). Compared with transient expression vectors, lentiviral vectors have extremely strong infectivity, especially in primary cells. They can not only replace transient expression vectors, but also integrate into the genome of target cells to ensure the persistent expression of the target gene, thereby enabling it to exert its function. Therefore, they have extremely broad application prospects in gene therapy.

[0004] During the process of using lentiviruses to infect cells, residual impurities such as proteins, endotoxins, and host cell DNA in the lentivirus can cause certain toxicity to the cells, leading to a deterioration in cell condition, reduced proliferation activity of lentivirus-infected cells, and consequently, a large number of lentivirus-infected cells die. This will seriously affect the infection efficiency of lentiviruses on cells. Summary of the Invention

[0005] To address the problem of massive cell death during lentivirus infection and to improve the proliferative activity of lentivirus-infected cells, this invention provides a protein composition that can enhance the proliferative activity of lentivirus-infected cells and its application.

[0006] According to a first aspect of the present invention, a protein composition is provided that can enhance the proliferative activity of lentivirally infected cells, the protein composition comprising at least one of protein A, protein B, and protein C; the nucleotide sequence of protein A is selected from one of SEQ ID NO: 1 to 6; the nucleotide sequence of protein B is selected from one of SEQ ID NO: 7 to 11; and the nucleotide sequence of protein C is selected from one of SEQ ID NO: 12 to 13.

[0007] The protein composition provided by this invention includes at least one of protein A, protein B, and protein C. The inventors successfully constructed a recombinant vector by synthesizing a nucleic acid molecule encoding the above-mentioned protein composition and integrating it into a vector. After transfecting and culturing tool cells using a lentiviral transfection system containing the recombinant vector, lentiviral fluid was obtained. Subsequently, the lentiviral fluid was used to infect cells. Studies have shown that cell proliferation activity is improved after lentiviral infection. After culturing lentivirally infected cells for a period of time, most cells can still proliferate and survive well, reducing the risk of mass cell death due to lentiviral infection. This can improve the infection efficiency of lentiviral cells. This is mainly because lentivirally infected cells can express the above-mentioned protein composition. The above results demonstrate that the protein composition provided by this invention can improve the proliferation activity of lentivirally infected cells.

[0008] Preferably, the protein composition comprises protein A, protein B, and protein C.

[0009] Preferably, the cells mentioned above include, but are not limited to, NK cells, T cells, iPS cells, hematopoietic stem cells, and HEK293 cells.

[0010] According to a second aspect of the present invention, a nucleic acid molecule is provided comprising a nucleotide sequence encoding the protein composition described above that can enhance the proliferative activity of lentiviral infected cells.

[0011] This method successfully constructs a recombinant vector by integrating the aforementioned nucleic acid molecules into the vector. The lentivirus transfection system containing this recombinant vector is used to transfect and culture tool cells to obtain lentivirus solution. The lentivirus solution is then used to infect the cells. Studies have shown that lentivirus infection enhances cell proliferation activity. After a period of culture, most lentivirus-infected cells can still proliferate and survive well, reducing the risk of mass cell death after lentivirus infection. This improves the efficiency of lentivirus infection. Therefore, integrating the nucleic acid molecules provided in this invention into the recombinant vector and transfecting cells using the lentivirus transfection system can enhance the proliferation activity of lentivirus-infected cells.

[0012] Preferably, the above-mentioned nucleic acid molecules include at least one of nucleic acid molecule A, nucleic acid molecule B, and nucleic acid molecule C; the nucleotide sequence of nucleic acid molecule A is selected from one of SEQ ID NO: 14 to 19; the nucleotide sequence of nucleic acid molecule B is selected from one of SEQ ID NO: 20 to 24; and the nucleotide sequence of nucleic acid molecule C is selected from one of SEQ ID NO: 25 to 26.

[0013] Preferably, the nucleotides of the above-mentioned nucleic acid molecules include nucleic acid molecule A, nucleic acid molecule B, and nucleic acid molecule C.

[0014] By integrating the nucleotide sequences from the above combinations into vectors, recombinant vectors can be successfully constructed. After transfecting and culturing tool cells with lentiviral transfection systems containing the above recombinant vectors, three types of lentiviruses are obtained. Then, the cells are infected with these three types of lentiviral solutions. The proliferation activity of the cells infected with lentiviruses is greatly improved, thereby significantly improving the transfection efficiency of lentiviruses.

[0015] According to a third aspect of the present invention, a recombinant vector is provided, the recombinant vector containing the above-described nucleic acid molecules.

[0016] According to a fourth aspect of the present invention, a recombinant virus is provided, which is obtained by co-transfecting cells with the above-described recombinant vector and a lentiviral packaging plasmid.

[0017] Preferably, the lentiviral packaging plasmids include at least two of psPAX2, VSV-pMD2.G, BaEV-pMD2.G, pLP1, pLP2, pLP / VSVG, and BaEV.

[0018] According to a fifth aspect of the present invention, a cell is provided that contains the above-described recombinant vector.

[0019] According to a sixth aspect of the present invention, a method for enhancing the proliferative activity of lentivirus-infected cells is provided, comprising the following steps:

[0020] S1. Construct a target plasmid containing the above-mentioned nucleic acid molecules;

[0021] S2. The above target plasmid is mixed with the lentivirus packaging plasmid to obtain a plasmid mixture;

[0022] S3. Mix the above plasmid mixture with the transfection reagent and add it to the tool cells, transfect and culture for 6-9 hours to obtain lentivirus solution;

[0023] S4. Add lentivirus solution and co-infectant to the target cells to infect them, and continue culturing the target cells.

[0024] By constructing a target plasmid containing the nucleic acid molecule of the present invention, mixing the target plasmid with a lentiviral packaging plasmid, adding a transfection reagent to transfect tool cells, and culturing them to obtain a lentiviral solution, and then infecting and culturing the target cells with the assistance of an infection aid, the proliferation activity of lentivirally infected cells and the lentiviral transfection efficiency can be improved.

[0025] Preferably, in S2, the mass ratio of the target plasmid to the lentivirus packaging plasmid is 2-23:2-10.

[0026] Preferably, in S2, the lentiviral packaging plasmid includes a first packaging plasmid and a second packaging plasmid, wherein the first packaging plasmid is a psPAX2 plasmid, and the second packaging plasmid is a VSV-pMD2.G plasmid or a BaEV-pMD2.G plasmid, and the mass ratio of the first packaging plasmid, the second packaging plasmid, and the target plasmid is 1-3:1-5:2-8. Alternatively, the lentiviral packaging plasmid includes a first packaging plasmid, a second packaging plasmid, and a third packaging plasmid, wherein the first packaging plasmid is a pLP1 plasmid, the second packaging plasmid is a pLP2 plasmid, and the third packaging plasmid is a pLP / VSVG plasmid or a BaEV plasmid, and the mass ratio of the first packaging plasmid, the second packaging plasmid, the third packaging plasmid, and the target plasmid is 1.5-7.5:1.2-5.8:3.2-9.3:6.8-10.

[0027] Preferably, the target plasmid is an expression vector containing pLenti.

[0028] Preferably, in S3, the mass ratio of the transfection reagent to the plasmid mixture is 1 to 10:1.

[0029] Preferably, in S3, the transfection reagent includes polyethyleneimine.

[0030] Preferably, in S3, the tool cells are HEK 293T cells.

[0031] Preferably, step S3 further includes filtering and concentrating the lentivirus solution. The specific steps are as follows: the supernatant after culture is filtered through a 0.45 μm filter membrane, the obtained filtrate is mixed with a concentrated solution containing polyethylene glycol 6000 and NaCl at 4°C and then placed at 4°C overnight. After that, it is centrifuged at 4000–6000 g for 10–40 min, lentivirus lysate is added, and the mixture is mixed evenly to obtain the lentivirus solution.

[0032] Preferably, in S4, the co-infectant includes at least one of protamine sulfate and polybrene.

[0033] Preferably, in S4, the target cells include, but are not limited to, hematopoietic stem cells isolated from PBMCs or CBMCs, iPS-induced hematopoietic stem cells, NK cells derived from peripheral blood, NK cells derived from umbilical cord blood, T cells derived from peripheral blood, T cells derived from umbilical cord blood, and HEK293 cells.

[0034] Preferably, in S4, the lentivirus is added to the target cells according to the multiplicity of infection (MOI) of 1 to 100. Attached Figure Description

[0035] Figure 1 The image shows the results of nucleic acid electrophoresis provided in Example 5.

[0036] Figure 2This is a graph showing the growth of peripheral blood NK cells infected with a lentivirus containing three nucleic acid molecular genes, as provided in Example 6, after 100 days of culture.

[0037] Figure 3 This image shows the growth of peripheral blood NK cells infected with the lentivirus transfected with the blank vector provided in Example 6 after 40 days of culture.

[0038] Figure 4 This is a graph showing the growth of hematopoietic stem cells induced by lentivirus infection of iPS cells provided in Example 7. Detailed Implementation

[0039] The technical features of the technical solution provided by the present invention will be further clearly and completely described below with reference to specific embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0040] Example 1

[0041] This embodiment provides a protein composition comprising at least one of protein A, protein B, and protein C. The amino acid sequence of protein A is selected from one of SEQ ID NO: 1 to 6, the amino acid sequence of protein B is selected from one of SEQ ID NO: 7 to 11, and the amino acid sequence of protein C is selected from one of SEQ ID NO: 12 to 13. The specific amino acid sequences are shown in Table 1.

[0042] Table 1. Amino acid sequence of the protein composition provided in Example 1

[0043]

[0044]

[0045] Example 2

[0046] This embodiment provides a nucleic acid molecule that encodes the protein in the protein composition provided in Example 1, serving as the target gene for subsequent recombinant vector or target plasmid construction. The nucleic acid molecule includes at least one of nucleic acid molecule A, nucleic acid molecule B, and nucleic acid molecule C. The nucleotide sequence of nucleic acid molecule A is selected from one of SEQ ID NO: 14-19, the nucleotide sequence of nucleic acid molecule B is selected from one of SEQ ID NO: 20-24, and the nucleotide sequence of nucleic acid molecule C is selected from one of SEQ ID NO: 25-26. The nucleotide sequences of the nucleic acid molecule provided in this embodiment are shown in Table 2, wherein the nucleotide sequences shown in SEQ ID NO: 14-26 respectively encode the proteins shown in SEQ ID NO: 1-13.

[0047] Table 2 shows the nucleotide sequences of the nucleic acid molecules provided in Example 2.

[0048]

[0049] Example 3

[0050] This embodiment aims to integrate one of the nucleic acid molecules A, B, and C provided in Example 2 into a vector to obtain a recombinant vector (i.e., construct a target plasmid containing the target gene / nucleic acid molecule of Example 2). The nucleic acid molecules (or genes) contained in the recombinant vector are shown in Table 3. The tool cells were transfected and cultured using lentiviral transfection systems containing the above recombinant vectors to obtain lentiviral solution. Then, the lentiviral solution was used to infect the target cells, and the cells were cultured and the status of the lentivirally infected cells was continuously observed. The specific operation steps are as follows:

[0051] S1. Synthesize the nucleic acid molecules of groups 1 to 13 shown in Table 3 and integrate them into the vector to construct the recombinant vector (i.e., the target plasmid);

[0052] Table 3. Nucleotide sequences of nucleic acid molecules contained in the recombinant vectors of each transfection system in Example 3.

[0053]

[0054]

[0055] S2. The target plasmids and lentiviral packaging plasmids of the above groups were added to serum-free DMEM medium and mixed to obtain a plasmid mixture. The lentiviral packaging plasmids included a first packaging plasmid and a second packaging plasmid. The first packaging plasmid was psPAX2 plasmid, and the second packaging plasmid was VSV-pMD2.G plasmid. The mass ratio of the first packaging plasmid, the second packaging plasmid, and the target plasmid was 2:3:5.

[0056] S3. Add polyethyleneimine to serum-free DMEM medium, mix well, and then incubate at room temperature for 1–10 min to obtain the transfection reagent. Add the transfection reagent dropwise to the plasmid mixture, mix well, and incubate at room temperature for 5–40 min to obtain a mixed solution containing polyethyleneimine and plasmid. 6 HEK 293T cells were passaged and seeded into DMEM medium containing 10 vol% fetal bovine serum and 1 vol% penicillin-streptomycin and cultured overnight. When the cell density reached 50-90%, the supernatant was discarded, and the cells were starved for 1 hour by adding DMEM medium containing 0.1-1% fetal bovine serum to obtain HEK 293T cells for lentivirus packaging. A mixed solution containing polyethyleneimine and plasmids was added dropwise to HEK 293T cells. Mix thoroughly with the supernatant of 293T cells in a culture flask, transfect and culture for 6-9 hours. Discard the supernatant, add DMEM medium containing 0.1-5 vol% fetal bovine serum, and continue culturing. Collect the supernatant after lentivirus transfection every 24 hours for a total of 1-3 times. Filter the collected supernatant after lentivirus transfection through a 0.45 μm filter membrane. Add 5-10 mL of concentrated solution containing PEG-6000 and NaCl to every 10-80 mL of the filtered filtrate, incubate at 4°C, and mix well every 20-30 min for a total of 3-5 times. Incubate overnight at 4°C, then centrifuge at 4000-6000g for 10-40 min, discard the supernatant, let stand for 1-2 min, remove the mixed liquid, add an appropriate amount of lentivirus lysis buffer (PBS buffer) to dissolve the lentivirus precipitate, mix well, and aliquot to obtain the lentivirus solution.

[0057] S4. Add the target cells to a 12-well plate at a rate of 500,000 to 3,000,000 cells / well (1,000,000 cells / well in this example), and incubate for 16 to 48 hours. Preheat the plate using a constant temperature centrifuge beforehand. Remove the 12-well plate from the incubator, discard part of the supernatant, and add lentiviral solution to the target cells (peripheral blood NK cells) at a multiplicity of infection (MOI) of 1 to 100. Then add 6 to 20 μg / mL of protamine sulfate as a co-infectant, and add serum-free NK medium to a total volume of 3 mL. Centrifuge, transfer the 12-well plate back to the incubator, and continue incubation for 24 hours. Discard most of the supernatant, add fresh NK medium, and continue incubation. Change the medium every other day and continuously observe the status of lentivir-infected cells.

[0058] The difference between the groups in Table 3 is that the target plasmids used to infect the target cells using the above steps contain different nucleic acid molecules, and each group transfects one nucleic acid molecule. The other operation steps and reagents used are the same.

[0059] Table 4. Culture results of peripheral blood NK cells after lentivirus transfection in Example 3.

[0060]

[0061]

[0062] The culture results of peripheral blood NK cells transfected with lentivirus are shown in Table 4. As shown in Table 4, compared to the control group peripheral blood NK cells transfected with an empty vector (i.e., a recombinant vector containing a nucleic acid molecule with a nucleotide sequence as shown in SEQ ID NO: 14-26), constructing a target plasmid into a recombinant vector, transfecting the target cells with a lentivirus transfection system containing this target plasmid, and then infecting peripheral blood NK cells with this lentivirus and culturing them for a period of time significantly increased the proliferation activity of peripheral blood NK cells. This was mainly reflected in a significant increase in cell number and fold expansion compared to the control group, and a significant increase in cell survival time.

[0063] Example 4

[0064] This embodiment aims to integrate two of the nucleic acid molecules A, B, and C provided in Example 2 into vectors to obtain recombinant vectors (i.e., constructing target plasmids containing the target gene / nucleic acid molecule of Example 2). Two nucleic acid molecules from groups 1 to 52 shown in Table 5 are synthesized and integrated into vectors to construct recombinant vectors (i.e., target plasmids). The tool cells are transfected and cultured using the lentiviruses containing the two recombinant vectors in each group (the viral load ratio of the two is 1 to 5: 1 to 5, and the specific ratio used in this embodiment is 1: 1) transfection system to obtain lentivirus solution. Then, the lentivirus solution is used to infect the target cells, and the cells are cultured and the state of lentivirus-infected cells is continuously observed. The specific operation steps are as described in Example 3.

[0065] Table 5. Nucleotide sequences of nucleic acid molecules contained in the recombinant vectors of each transfection system in Example 4.

[0066]

[0067]

[0068] Table 6. Culture results of peripheral blood NK cells after lentivirus transfection in Example 4.

[0069]

[0070]

[0071] The culture results of peripheral blood NK cells transfected with lentivirus are shown in Table 6. As shown in Table 6, compared to the control group peripheral blood NK cells transfected with an empty vector (i.e., a recombinant vector containing any of the nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO: 14-26), constructing two target plasmids into recombinant vectors using two of the nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO: 14-26 respectively, transfecting and culturing the target cells with a lentivirus transfection system containing these two target plasmids, and then infecting peripheral blood NK cells with this lentivirus and culturing them for a period of time, significantly increased the proliferation activity of peripheral blood NK cells. This was mainly reflected in a significant increase in cell number and fold expansion compared to the control group, and a significant increase in cell survival time.

[0072] Example 5

[0073] This embodiment aims to integrate nucleic acid molecules A, B, and C from the nucleic acid molecules provided in Example 2 into vectors to obtain recombinant vectors (i.e., constructing target plasmids containing the target gene / nucleic acid molecule from Example 2). Three nucleic acid molecules from groups No. 1 to No. 60 shown in Table 7 were synthesized and integrated into vectors to construct recombinant vectors (i.e., target plasmids). The tool cells were transfected and cultured using the lentiviruses containing the three recombinant vectors in each group (the viral load ratio of the three was 1-5:1-5:1-5, but the specific ratio used in this embodiment was 1:1:1). The lentivirus solution was then used to infect the target cells, and the cells were cultured and the state of lentivirus-infected cells was continuously observed. Specific operating steps are referred to in Example 3.

[0074] Table 7. Nucleotide sequences of nucleic acid molecules contained in the recombinant vectors of each transfection system in Example 5.

[0075]

[0076]

[0077]

[0078] Table 8. Culture results of peripheral blood NK cells after transfection in Example 5.

[0079]

[0080]

[0081] After culturing peripheral blood NK cells infected with the aforementioned lentivirus for 7 days, genomic DNA was extracted from the cells, and PCR primers for amplifying nucleic acid molecules A, B, and C were designed using primer design software. The PCR primer pairs were used to amplify nucleic acid molecules A, B, and C from the cellular genomic DNA, respectively. The PCR amplification products were separated and verified by electrophoresis. The electrophoresis results are shown below. Figure 1 As shown, where, Figure 1 A through C represent the electrophoresis results of nucleic acid molecules A, B, and C, respectively. It should be noted that... Figure 1 The banded lanes only show the electrophoresis results of PCR amplification products from a portion of the groups after amplification of nucleic acid molecules A, B, and C in the cellular genomic DNA. Figure 1 The electrophoresis results demonstrate that the corresponding target plasmid can be successfully transfected into the target cells.

[0082] The culture results of peripheral blood NK cells transfected with lentivirus are shown in Table 8. As shown in Table 8, compared to the control group peripheral blood NK cells transfected with an empty vector (i.e., a recombinant vector containing any of the nucleic acid molecules shown in SEQ ID NO: 14–26), constructing three target plasmids into recombinant vectors using three of the nucleic acid molecules shown in SEQ ID NO: 14–19, SEQ ID NO: 20–24, and SEQ ID NO: 25–26 respectively, and then transfecting and culturing the target cells with a lentivirus transfection system containing these three target plasmids, yielded lentiviruses. Subsequently, after infecting and culturing peripheral blood NK cells with these lentiviruses for a period of time, the proliferation activity of peripheral blood NK cells was significantly increased, mainly reflected in a significant increase in cell number and fold expansion compared to the control group, and a significant increase in cell survival time.

[0083] Example 6

[0084] Peripheral blood NK cells generally survive for approximately 28 days in vitro. Following the experimental procedures of Example 3, peripheral blood NK cells were used as the target cells. Nine clones (No. 1, No. 8, No. 12, No. 24, No. 31, No. 40, No. 47, No. 52, and No. 56) from Example 5 were cultured for 100 days in a serum-free medium containing lipid concentrate at 37°C with 5% carbon dioxide. The medium was changed daily. The culture results are shown in Table 9. Figure 2 As shown, where Figure 2A through I represent the growth results of peripheral blood NK cells cultured for 100 days in groups No.1, No.8, No.12, No.24, No.31, No.40, No.47, No.52, and No.56, respectively. Simultaneously, peripheral blood NK cells transfected with lentiviruses containing target plasmids (nucleic acid molecules A, B, and C) (i.e., peripheral blood NK cells transfected with the blank vector) served as a control group. The experimental procedures and reagents used in the control group were the same as those in the aforementioned groups. The growth results of peripheral blood NK cells cultured for 40 days in the control group are shown below. Figure 3 As shown, Figure 3 Figures A through C show the observation results from different viewpoints.

[0085] Table 9. Results of peripheral blood NK cell culture

[0086]

[0087] From Table 9 and Figure 2 It can be seen that clones No.1, No.8, No.12, No.24, No.31, No.40, No.47, No.52 and No.56 still had a relatively large number of surviving NK cells after 100 days of culture.

[0088] By comparison Figure 2 and Figure 3 It can be seen that after the peripheral blood NK cells in the control group were infected with lentivirus, there were basically no viable NK cells left after 40 days of culture, while clones No.1, No.8, No.12, No.24, No.31, No.40, No.47, No.52 and No.56 still had a relatively large number of viable NK cells after 100 days of culture.

[0089] Example 7

[0090] The lifespan of iPS-induced hematopoietic stem cells is generally around 20 days, making long-term culture difficult. Following the experimental steps of Example 3, iPS-induced hematopoietic stem cells were used as the target cells. A control group and an experimental group were set up. The control group consisted of iPS-induced hematopoietic stem cells transfected with a blank vector. In the experimental group, nucleic acid molecules (No. 09) combining SEQ ID NO: 14, SEQ ID NO: 23, and SEQ ID NO: 26 were transfected into iPS-induced hematopoietic stem cells according to the method in Example 3, and the cells were cultured. Cell status was observed daily, and cell proliferation was recorded. The results are shown in Table 10. Figure 4 As shown.

[0091] Table 10. Results of IPS-induced hematopoietic stem cell culture

[0092]

[0093] From Table 10 and Figure 4 It can be seen that the control group cells were almost completely dead by day 25. However, when the cells transduced with the above three plasmid combinations (the three plasmids contain nucleic acid molecules with nucleotide sequences as shown in SEQ ID NO: 14, SEQ ID NO: 23, and SEQ ID NO: 2, respectively) were cultured for 103 days, a large number of cells still survived and were in good growth condition. Based on the calculation of the cell expansion fold, the cell proliferation rate of the transfected plasmids decreased, the survival time was prolonged, and the total expansion fold increased.

[0094] Based on the above experimental results, it can be seen that by synthesizing nucleic acid molecules encoding the protein composition provided by the present invention and integrating them into a recombinant vector (target plasmid), and then transfecting cells using a lentivirus transfection system, the obtained lentivirus solution can infect the cells. The lentivirus-infected cells can express the above-mentioned protein composition, thereby improving the proliferation activity of lentivirus-infected cells, reducing the risk of mass cell death after lentivirus infection, and enabling lentivirus-infected cells to continue to proliferate and survive well after a period of culture, thus significantly improving the transfection efficiency of lentivirus.

[0095] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention, but such modifications or substitutions are all within the scope of protection of the present invention.

Claims

1. A protein composition capable of increasing the proliferative activity of a cell infected with a lentivirus, characterized by: The protein composition includes at least one of protein A, protein B, and protein C; The amino acid sequence of protein A is selected from one of SEQ ID NO: 1 to 6; The amino acid sequence of protein B is selected from one of SEQ ID NO: 7 to 11; The amino acid sequence of protein C is selected from one of SEQ ID NO: 12-13.

2. The protein composition as described in claim 1 that can enhance the proliferative activity of lentivirus-infected cells, characterized in that: The protein composition includes protein A, protein B, and protein C.

3. The protein composition as described in claim 1, which enhances the proliferative activity of lentivirally infected cells, characterized in that: The cells include at least one of NK cells, T cells, iPS cells, hematopoietic stem cells, and HEK293 cells.

4. A nucleic acid molecule, characterized in that: The nucleic acid molecule includes a nucleotide sequence encoding a protein composition as described in any one of claims 1 to 3 that can enhance the proliferative activity of lentivirus-infected cells.

5. The nucleic acid molecule as described in claim 4, characterized in that: The nucleic acid molecule includes at least one of nucleic acid molecule A, nucleic acid molecule B, and nucleic acid molecule C; The nucleotide sequence of the nucleic acid molecule A is selected from one of SEQ ID NO: 14 to 19; The nucleotide sequence of the nucleic acid molecule B is selected from one of SEQ ID NO: 20-24; The nucleotide sequence of the nucleic acid molecule C is selected from one of SEQ ID NO: 25-26.

6. The nucleic acid molecule as described in claim 5, characterized in that: The nucleic acid molecules include nucleic acid molecule A, nucleic acid molecule B, and nucleic acid molecule C.

7. A recombinant vector, characterized in that: The recombinant vector contains the nucleic acid molecule as described in claim 4.

8. A recombinant virus, characterized in that: The recombinant virus is obtained by co-transfecting cells with the recombinant vector as described in claim 7 and the lentiviral packaging plasmid.

9. A cell, characterized in that: The cells contain the recombinant vector as described in claim 8.

10. A method for enhancing the proliferative activity of lentivirus-infected cells, characterized in that, Includes the following steps: S1. Construct a target plasmid containing the nucleic acid molecule as described in claim 4; S2. The target plasmid is mixed with the lentivirus packaging plasmid to obtain a plasmid mixture; S3. Mix the plasmid mixture with the transfection reagent and add it to the tool cells, transfect and culture for 6-9 hours to obtain lentivirus solution; S4. Add the lentivirus solution and the co-infectant to the target cells to infect the target cells, and continue to culture the target cells.

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