Lentiviral expression vector, lentivirus and preparation method and application thereof

Abstract

The embodiment of the invention provides a lentivirus expression vector, a lentivirus and a preparation method and application of the lentivirus, relates to the technical field of biological medicines, and aims to improve the infection capability of the lentivirus on target cells. The lentivirus comprises BaEV envelope glycoprotein, BaEV envelope glycoprotein, BaEV envelope glycoprotein and BaEV envelope glycoprotein. Wherein the extracellular region of the BaEV envelope glycoprotein comprises at least one of the following sequences: a spacer sequence and a recognition sequence, and the spacer sequence comprises a spacer unit; the recognition sequence comprises BaEVSecSig-recognition protein-BaEVTM (BaEVSecSig-Recognition Protein); wherein the BaEV SecSig is a BaEV signal peptide, and the BaEV < TM > is a BaEV transmembrane domain. The lentivirus is used for infecting target cells.

Description

Technical Field

[0001] The present disclosure relates to the field of biomedicine technology, and in particular to a lentivirus expression vector, a lentivirus, and a preparation method and application thereof. Background Art

[0002] Lentiviral vector (LVs) is a gene therapy vector developed based on HIV-1 (human immunodeficiency virus type 1). Different from general retroviral vectors, it has the ability to infect both dividing cells and non-dividing cells. This vector can effectively integrate foreign genes into host chromosomes, thereby achieving persistent expression. In terms of infectivity, it can effectively infect various types of cells such as neurons, hepatocytes, cardiomyocytes, tumor cells, endothelial cells, stem cells, etc., thereby achieving good gene therapy effects. Summary of the invention

[0003] The purpose of the embodiments of the present disclosure is to provide a lentiviral expression vector, a lentivirus, and a preparation method and application thereof, so as to improve the infection ability of the lentivirus to target cells.

[0004] To achieve the above objectives, the embodiments of the present disclosure provide the following technical solutions:

[0005] On the one hand, a lentivirus is provided, comprising: a BaEV envelope glycoprotein; wherein the extracellular region of the BaEV envelope glycoprotein comprises at least one of the following sequences: a spacer sequence and a recognition sequence, the spacer sequence comprising: a spacer unit; the recognition sequence comprises: BaEVSecSig-recognition protein-BaEVTM; wherein the BaEVSecSig is a BaEV signal peptide, and the BaEVTM is a BaEV transmembrane domain.

[0006] In the above-mentioned lentivirus, the extracellular region of the BaEV envelope glycoprotein includes at least one of a spacer sequence and a recognition sequence, that is, by adding at least one of a spacer sequence and a recognition sequence to the extracellular region of the BaEV envelope glycoprotein, the extracellular region of the BaEV envelope glycoprotein is genetically modified. The modified BaEV envelope glycoprotein will increase the interaction between the BaEV lentivirus and the target cell surface receptors, thereby improving the lentivirus's ability to infect target cells.

[0007] In some embodiments, the Spacer unit comprises conservative amino acids.

[0008] In some embodiments, the spacer unit includes at least one of SAG, GAS, ASG, AGS, GSA and SGA.

[0009] In some embodiments, the spacer sequence comprises 9 to 27 amino acids.

[0010] In some embodiments, the spacer sequence comprises at least one of: SAGSAGSAGSAGSAG, GASGASGASGASGAS, ASGASGASGASGASG, AGSAGSAGSAGSAGSAGS, GSAGSAGSAGSAGSAGSA, and SGASGASGASGASGASGA.

[0011] In some embodiments, the insertion sites of the spacer sequence include positions 117 and 338 of the BaEV envelope glycoprotein amino acid sequence.

[0012] In some embodiments, the BaEV envelope glycoprotein comprising the spacer sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 1 to SEQ ID No. 6.

[0013] In some embodiments, the recognition protein includes: a receptor protein, an antibody, a single-chain recognition sequence modified from an antibody, a nanobody, a lectin, or a molecular chaperone protein.

[0014] In some embodiments, the recognition protein comprises: protein A or protein G.

[0015] In some embodiments, the recognition protein is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 7 and SEQ ID No. 8.

[0016] In some embodiments, the sequence of the BaEVSecSig is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of SEQ ID No.9; and / or, the sequence of the BaEVTM is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of SEQ ID No.10.

[0017] On the other hand, a lentiviral expression vector is provided, the lentiviral expression vector comprising: a nucleotide sequence expressing the lentivirus described in any one of the above embodiments.

[0018] In some embodiments, the nucleotide sequence of the spacer sequence expressing the BaEV envelope glycoprotein is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No.11 to SEQ ID No.16.

[0019] In some embodiments, the nucleotide sequence expressing the recognition sequence of the BaEV envelope glycoprotein is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 17 and SEQ ID No. 18.

[0020] The above-mentioned lentiviral expression vector is used to express the lentivirus provided in some of the above-mentioned embodiments. Therefore, the lentiviral expression vector has the beneficial technical effects of the lentiviral body provided in any of the above-mentioned embodiments, which will not be described in detail here.

[0021] In another aspect, a method for preparing a lentivirus is provided, the method comprising: inoculating packaging cells in a culture container for culture; adding a vector plasmid, a packaging plasmid and a lentivirus expression vector to a cell culture medium to obtain a first solution; adding a transfection reagent to the cell culture medium and mixing to obtain a second solution; when the confluence of the packaging cells reaches a preset value, mixing the first solution and the second solution and adding the mixture to the culture container for transfection;

[0022] After the transfection is completed, the lentivirus is obtained;

[0023] Among them, the lentivirus includes: BaEV envelope glycoprotein; the extracellular region of the BaEV envelope glycoprotein includes at least one of the following sequences: a spacer sequence and a recognition sequence; the spacer sequence includes: a spacer unit; the recognition sequence includes: BaEVSecSig-recognition protein-BaEVTM; the BaEVSecSig is the BaEV signal peptide, and the BaEVTM is the BaEV transmembrane domain.

[0024] In some embodiments, the lentiviral expression vector comprises: at least one of an amino acid sequence expressing a spacer sequence of the extracellular region of the BaEV envelope glycoprotein and an amino acid sequence expressing a recognition sequence of the extracellular region of the BaEV envelope glycoprotein.

[0025] In some embodiments, a lentiviral expression vector expressing a spacer sequence of a BaEV envelope glycoprotein is represented as a first vector; a lentiviral vector expressing a recognition sequence of a BaEV envelope glycoprotein is represented as a second vector; in the first solution, the lentiviral expression vector comprises: the first vector and the second vector, and the ratio of the mass of the first vector to the mass of the second vector ranges from 5 to 10.

[0026] In some embodiments, in the first solution, the mass ratio of the vector plasmid, the packaging plasmid and the lentiviral expression vector is (3-5):3:(1-3).

[0027] In some embodiments, before plating the packaging cells into a culture container for culture, the step further includes: modifying the culture container with coacervate microgels.

[0028] In some embodiments, the coacervate microgel comprises: folic acid and a polycationic electrolyte.

[0029] In some embodiments, the polycationic electrolyte includes at least one of protamine sulfate and polylysine.

[0030] In some embodiments, the coacervate microgel modification of the culture container includes: mixing a folic acid solution with a polycation electrolyte solution to obtain a third solution; applying the third solution to the culture container, adding a phosphate solution to induce gelation, and obtaining a coacervate microgel-modified culture container.

[0031] In yet another aspect, a use of the lentivirus as described in any of the above embodiments in transfection of primary human NK cells is provided.

[0032] The preparation method and application of the above-mentioned lentivirus have the same beneficial technical effects as the lentivirus provided in some of the above-mentioned embodiments, and will not be described in detail here. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to more clearly illustrate the technical solutions in the present disclosure, the following briefly introduces the drawings required for use in some embodiments of the present disclosure. Obviously, the drawings described below are only drawings of some embodiments of the present disclosure. For ordinary technicians in this field, other drawings can also be obtained based on these drawings. In addition, the drawings described below can be regarded as schematic diagrams, and are not limitations on the actual size of the products involved in the embodiments of the present disclosure, the actual process of the method, etc.

[0034] Figure 1 is a flow chart of a method for preparing a lentivirus according to some embodiments of the present disclosure;

[0035] Figure 2 is a structural diagram of a lentivirus according to some embodiments of the present disclosure;

[0036] Figure 3 is a graph showing the percentage of GFP-positive cells according to some embodiments of the present disclosure;

[0037] Figure 4 is another ratio diagram of GFP-positive cells according to some embodiments of the present disclosure;

[0038] Figure 5 is another ratio diagram of GFP-positive cells according to some embodiments of the present disclosure;

[0039] Figure 6 is another ratio diagram of GFP-positive cells according to some embodiments of the present disclosure;

[0040] Figure 7This is a flow cytometry fluorescence image of antibody cell labeling according to Example 1 of the present disclosure;

[0041] Figure 8 This is a flow cytometry fluorescence image of antibody cell labeling according to Example 5 of the present disclosure;

[0042] Fig. 9 This is a flow cytometry fluorescence image of antibody cell labeling according to Example 6 of the present disclosure. DETAILED DESCRIPTION

[0043] The following will be combined with the accompanying drawings to clearly and completely describe the technical solutions in some embodiments of the present disclosure. Obviously, the described embodiments are only part of the embodiments of the present disclosure, rather than all the embodiments. Based on the embodiments provided by the present disclosure, all other embodiments obtained by ordinary technicians in this field belong to the scope of protection of the present disclosure.

[0044] Unless the context requires otherwise, throughout the specification and claims, the term "comprise" and other forms thereof, such as the third person singular form "comprises" and the present participle form "comprising", are to be interpreted as open, inclusive, that is, "including, but not limited to". In the description of the specification, the terms "one embodiment", "some embodiments", "exemplary embodiments", "example", "specific example" or "some examples" and the like are intended to indicate that specific features, structures, materials or characteristics associated with the embodiment or example are included in at least one embodiment or example of the present disclosure. The schematic representation of the above terms does not necessarily refer to the same embodiment or example. In addition, the specific features, structures, materials or characteristics described may be included in any one or more embodiments or examples in any appropriate manner.

[0045] In the following, the terms "first" and "second" are used for descriptive purposes only and are not to be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of the features. In the description of the embodiments of the present disclosure, unless otherwise specified, "plurality" means two or more.

[0046] “At least one of A, B, and C” has the same meaning as “at least one of A, B, or C” and both include the following combinations of A, B, and C: A only, B only, C only, the combination of A and B, the combination of A and C, the combination of B and C, and the combination of A, B, and C.

[0047] “A and / or B” includes the following three combinations: A only, B only, and a combination of A and B.

[0048] As used herein, "about" includes the stated value and the average value that is within an acceptable range of variation for the particular value as determined by one of ordinary skill in the art taking into account the measurements in question and the errors associated with the measurement of the particular quantity (i.e., the limitations of the measurement system).

[0049] Cell and Gene Therapy (CGT) is an advanced technology that combines gene therapy and cell therapy, aiming to treat and prevent diseases by modifying genes and cells. Among them, gene therapy mainly introduces exogenous normal genes into target cells to correct or compensate for diseases caused by defects and abnormal genes. Common gene therapy technologies include gene supplementation and gene editing. The former uses vectors to introduce normal genes into patients, while the latter directly edits gene sequences through technologies such as CRISPR (clustered regularly-interspaced short palindromic repeats); cell therapy uses cells from human autologous or allogeneic sources to be manipulated in vitro and then introduced into the human body for disease treatment.

[0050] The core technical link in cell gene therapy is to introduce the target gene into the target cell through gene delivery tools, and lentivirus is the most important type of such tools. Lentiviral vectors (LVs) are retroviruses transformed from human immunodeficiency virus (HIV-1). Their genome is single-stranded positive-strand RNA, and the toxic genes have been removed and replaced by exogenous target genes, so they have high biosafety and stability.

[0051] At present, there have been many reports on the application of lentiviral vectors in the field of cell gene therapy, and have achieved relatively excellent performance. For example, vesicular stomatitis virus lentivirus (VSV-G-Lv) is widely used to encapsulate CRISPR-Cas genes and CAR (Chimeric antigen receptor) genes to achieve targeted T cell delivery. In addition, BaEV lentivirus (Baboon envelope pseudotyped lentiviral vector, BaEV-LV) can effectively transduce various types of cells, such as human B cells, T cells and NK cells, especially for CD34+ stem cells, and its performance is even better than VSV-G-LVs and RDTR-LVs.

[0052] Among them, BaEV lentivirus is a baboon envelope pseudotype lentivirus vector. NK cells (Naturekiller cells) are also called natural killer cells. RDTR is a mutant of the RD114 feline retrovirus envelope glycoprotein.

[0053] Although lentiviral vectors have achieved great application prospects and commercial value, their production process is complex and costly, especially for the production of high-titer vectors, which is still a major development challenge in the industry. Among them, the preparation of BaEV lentiviral vectors is particularly difficult.

[0054] The envelope protein of the BaEV lentivirus is the baboon endogenous retrovirus envelope glycoprotein (BaEV-G), which is expressed by the corresponding gene on the BaEV plasmid. The protein structure includes an extracellular domain, a transmembrane domain, and a cell tail domain, which is also the intracellular domain.

[0055] In some examples, the optimization of BaEV lentivirus is based on the modification of the intracellular domain, such as BaEV-Rless, which has no fusion inhibitory R peptide, and BaEV / TR, where the cell tail domain is replaced with the cell tail domain of the MLV (Murine Leukemia Virus, murine leukemia virus) envelope glycoprotein. However, the expression of BaEV-Rless in 293T cells will cause 293T cells to form a large number of syncytia, leading to massive cell death; while BaEV / TR has greatly reduced cytotoxicity and can reduce the appearance of syncytia, but the virus titer is lower than BaEV-Rless.

[0056] Among them, 293T cells are human renal epithelial cell lines derived from 293 cells and expressing SV40 large T antigen. 293T cells are widely used in transient transfection to overexpress various target proteins or to package viruses. This type of cell does not require high nutrients in the culture medium. It is an adherent cell, but its adherence is not strong, and slight interference may cause them to fall off. When replacing fresh culture medium or washing with phosphate buffered saline (PBS), new culture medium should be added carefully so that the liquid does not hit the adherent cells. The growth state of 293T cells is directly related to the efficiency of virus packaging and transfection. Cells with fewer passages and shorter total culture time should be selected as much as possible.

[0057] In other examples, the chimeric envelope glycoprotein was constructed by introducing the intracellular domain of the VSV-G (Vesicularstomatitisvius) envelope glycoprotein, which improved the BaEV envelope virus packaging efficiency to a certain extent, but it was still not obvious, and the effect of infecting NK cells and T cells was poor.

[0058] In addition, since the envelope protein of the BaEV lentivirus is structurally unstable and easily degraded and inactivated, it can easily infect basal cells and inactivate them during packaging preparation, resulting in a low lentivirus titer.

[0059] Based on this, how to systematically optimize the entire process of lentivirus preparation to obtain high-titer lentiviral vectors with better targeting and transfection efficiency is a core technical problem that urgently needs to be solved in the industry.

[0060] The embodiments of the present disclosure provide a method for preparing a lentivirus, such as Figure 1 and Figure 2 As shown, the method for preparing lentivirus includes steps: R1 to R5.

[0061] R1. Inoculate the packaging cells into the culture container 40 for culture.

[0062] Exemplarily, 293T cells are plated into a culture container 40 (eg Figure 2 ), 293T cells are packaging cells, also called basal cells.

[0063] R2. Add the vector plasmid, packaging plasmid and lentiviral expression vector into the cell culture medium to obtain a first solution.

[0064] Exemplarily, the vector plasmid, packaging plasmid and lentiviral expression vector are mixed in Opti-MEM serum-free medium to obtain a first solution.

[0065] The vector plasmid is cloned with the target gene fragment, and a polynucleotide sequence can be introduced into the host cell, the polynucleotide sequence includes the target gene sequence, so as to transform the host and promote the expression of the introduced sequence, and the expression of the sequence includes, for example, the transcription and translation of the sequence. The vector plasmid is constructed by itself, and the polynucleotide sequence therein can be a chimeric antigen receptor coding sequence, an immunoglobulin (Immunoglobulin, Ig) gene coding sequence, a cytokine gene coding sequence, etc., which can be used in the field of gene and drug therapy.

[0066] Exemplarily, the packaging plasmid includes: pCMV-ΔR8.91 plasmid, which is a second-generation lentiviral packaging helper plasmid, and the pCMV-ΔR8.91 plasmid contains viral functional genes such as gag, pol, rev and tat.

[0067] The lentiviral expression vector is, for example, a BaEV plasmid, which is a vector encoding a BaEV envelope glycoprotein (BaEV-G), which may generally include an extracellular region, a transmembrane domain, and a cell tail domain from the amino terminus to the carboxyl terminus. In enveloped viruses, the transmembrane domain passes through the viral envelope and connects the extracellular region located on the outside of the viral envelope and the cell tail domain located on the inside of the viral envelope. The extracellular region corresponds to the portion of amino acids 1-488 (including endpoints) of BaEV-G (NCBI sequence accession number YP_009109691.1). Among them, NCBI is the abbreviation of National Center for Biotechnology Information, which means the National Center for Biotechnology Information of the United States.

[0068] Opti-MEM is a basal cell culture medium consisting of the minimum concentrations of nutrients necessary for culture growth.

[0069] R3. Add the transfection reagent to the cell culture medium and mix to obtain a second solution.

[0070] Exemplarily, Lipofectamine 2000 is mixed with Opti-MEM medium to obtain a second solution.

[0071] Lipofectamine 2000 is a highly efficient transfection reagent suitable for a wide range of adherent and suspension cell lines. It has a good transfection effect and can achieve very high protein expression levels.

[0072] R4. When the confluence of the cells to be packaged reaches a preset value, the first solution and the second solution are mixed and added into a culture container for transfection.

[0073] Exemplarily, when the confluence of 293T cells reaches a preset value, the culture medium is replaced with serum-free DMEM culture medium.

[0074] Exemplarily, the preset value range of the confluence of 293T cells is 70% to 90%.

[0075] Exemplarily, the first solution and the second solution are mixed and then added dropwise to the DMEM culture medium, and gently shaken to allow the reagents to cover all cells.

[0076] Exemplarily, the culture container 40 includes: a T175 cell culture flask or a culture dish.

[0077] Cell confluence refers to the degree of adhesion and arrangement between cells when cells proliferate in the culture container 40, usually expressed as a percentage. The concept of cell confluence does not only refer to cell density, but it is a state that comprehensively reflects the cell growth process. Generally speaking, when cells grow and converge to form a monolayer, the cells become more crowded, and the degree of flatness decreases, and the surface area in contact with the culture medium is also reduced; at the same time, some nutrients in the culture medium will gradually be consumed, especially in the area close to the cells. The division of cells that form a monolayer will stop. Therefore, the judgment of confluence provides a rough reference for the time selection of the experiment and the subculture time of the culture.

[0078] DMEM (Dulbecco's modification of Eagle's medium) is a medium developed on the basis of MEM medium. It is a medium containing various amino acids and glucose. The characteristics of this medium are as follows: (1) The amino acid content is twice that of Eagle's medium, and it contains non-essential amino acids such as glycine; (2) The vitamin content is four times that of Eagle's medium; (3) It contains pyruvate, an important substance in the glycolysis pathway; (4) It contains trace amounts of iron ions.

[0079] R5. After transfection, 100 lentiviruses were obtained.

[0080] Exemplarily, after the culture container is cultured in a cell culture incubator, the culture medium is replaced with a DMEM complete medium containing FBS, and the first supernatant is collected after transfection. After the first supernatant is centrifuged, the second supernatant is collected, and the second supernatant is filtered to obtain the lentivirus.

[0081] FBS (Fetal Bovine Serum) is a light yellow, clear, non-hemolytic, foreign-free, slightly viscous liquid. Fetal bovine serum should be taken from fetal cows delivered by caesarean section. Exemplarily, the mass content of FBS in the culture medium is 10%. Exemplarily, the cell precipitate is removed by ultracentrifugation or PEG (polyethylene glycol) precipitation, and the second clear liquid is collected and filtered through a filter to obtain the lentivirus.

[0082] like Figure 2 As shown, the BaEV lentivirus 100 can be obtained through the above steps R1 to R5.

[0083] In BaEV lentivirus 100, the extracellular region of BaEV-G mediates the interaction between BaEV lentivirus 100 and the surface receptors of target cells, thereby affecting the ability of the lentivirus to infect target cells.

[0084] In some examples, the amino acid sequence of the extracellular region of BaEV-G is shown below.

[0085] SEQ ID No.19:

[0086] GFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPK

[0087] DTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTN

[0088] KLIQSPCNGIKGQSICWSTTAPIHVSDGGGGPLDTTRIKSVQRKLEEIHKALYPELQYHPLAIP

[0089] KVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKLGPPTPLAIPNFLLSYVTRSSD

[0090] NISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCG

[0091] NNMAYTYLPTNWTGLCVLATLLPDIDIIPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAA

[0092] FTTGATGLGVSVTQYTKLSNQLISDVQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP.

[0093] The nucleotide sequence of the lentiviral expression vector corresponding to the amino acid sequence of SEQ ID No. 19 is as follows.

[0094] SEQ ID No.20:

[0095] ggctttgatgatccgcgcaaagcgattgaactggtgcagaaacgctatggccgcccgtgcgattgcagcggcggccaggtgagcgaaccgcc

[0096] gagcgatcgcgtgagccaggtgacctgcagcggcaaaaccgcgtatctgatgccggatcagcgctggaaatgcaaaagcattccgaaagata

[0097] ccagcccgagcggcccgctgcaggaatgcccgtgcaacagctatcagagcagcgtgcatagcagctgctataccagctatcagcagtgccgc

[0098] agcggcaacaaaacctattataccgcgaccctgctgaaaacccagaccggcggcaccagcgatgtgcaggtgctgggcagcaccaacaaac

[0099] tgattcagagcccgtgcaacggcattaaaggccagagcatttgctggagcaccaccgcgccgattcatgtgagcgatggcggcggcccgctg

[0100] gataccacccgcattaaaagcgtgcagcgcaaactggaagaaattcataaagcgctgtatccggaactgcagtatcatccgctggcgattccga

[0101] aagtgcgcgataacctgatggtggatgcgcagaccctgaacattctgaacgcgacctataacctgctgctgatgagcaacaccagcctggtgg

[0102] atgattgctggctgtgcctgaaactgggcccgccgaccccgctggcgattccgaactttctgctgagctatgtgacccgcagcagcgataacatt

[0103] agctgcctgattattccgccgctgctggtgcagccgatgcagtttagcaacagcagctgcctgtttagcccgagctataacagcaccgaagaaat

[0104] tgatctgggccatgtggcgtttagcaactgcaccagcattaccaacgtgaccggcccgatttgcgcggtgaacggcagcgtgtttctgtgcggc

[0105] aacaacatggcgtatacctatctgccgaccaactggaccggcctgtgcgtgctggcgaccctgctgccggatattgatattattccgggcgatga

[0106] accggtgccgattccggcgattgatcattttatttatcgcccgaaacgcgcgattcagtttattccgctgctggcgggcctgggcattaccgcggc

[0107] gtttaccaccggcgcgaccggcctgggcgtgagcgtgacccagtataccaaactgagcaaccagctgattagcgatgtgcagattctgagcag

[0108] caccattcaggatctgcaggatcaggtggatagcctggcggaagtggtgctgcagaaccgccgcggcctggatctgctgaccgcggaacag

[0109] ggcggcatttgcctggcgctgcaggaaaaatgctgcttttatgtgaacaaaagcggcattgtgcgcgataaaattaaaaccctgcaggaagaactggaacgccgccgcaaagatctggcgagcaacccgctgtggaccggcctgcagggcctgctgccg.

[0110] In some examples, the gene fragment corresponding to the extracellular region of BaEV-G in the lentiviral expression vector is modified, and the structure of BaEV-G can be modified to improve the infection ability of BaEV lentivirus 100 to target cells.

[0111] The lentiviral expression vector is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No.11 to SEQ ID No.16.

[0112] SEQ ID No.11:

[0113] ggctttgatgatccgcgcaaagcgattgaactggtgcagaaacgctatggccgcccgtgcgattgcagcggcggccaggtgagcgaaccgcc

[0114] gagcgatcgcgtgagccaggtgacctgcagcggcaaaaccgcgtatctgatgccggatcagcgctggaaatgcaaaagcattccgaaagata

[0115] ccagcccgagcggcccgctgcaggaatgcccgtgcaacagctatcagagcagcgtgcatagcagctgctataccagctatcagcagtgccgc

[0116] agcggcaacaaaacctattataccgcgaccctgctgaaaacccagaccggcggcaccagcgatgtgcaggtgctgggcagcaccaacaaac

[0117] tgattcagagcccgtgcaacggcattaaaggccagagcatttgctggagcaccaccgcgccgattcatgtgagcgatggcggcggcccgctg

[0118] gataccacccgcattaaaagcgtgcagcgcaaactggaagaaattcataaagcgctgtatccggaactgcagagcgcgggcagcgcgggca

[0119] gcgcgggcagcgcgggcagcgcgggctatcatccgctggcgattccgaaagtgcgcgataacctgatggtggatgcgcagaccctgaacat

[0120] tctgaacgcgacctataacctgctgctgatgagcaacaccagcctggtggatgattgctggctgtgcctgaaactgggcccgccgaccccgctg

[0121] gcgattccgaactttctgctgagctatgtgacccgcagcagcgataacattagctgcctgattattccgccgctgctggtgcagccgatgcagttta

[0122] gcaacagcagctgcctgtttagcccgagctataacagcaccgaagaaattgatctgggccatgtggcgtttagcaactgcaccagcattaccaa

[0123] cgtgaccggcccgatttgcgcggtgaacggcagcgtgtttctgtgcggcaacaacatggcgtatacctatctgccgaccaactggaccggcct

[0124] gtgcgtgctggcgaccctgctgccggatattgatattattagcgcgggcagcgcgggcagcgcgggcagcgcgggcagcgcgggcccggg

[0125] cgatgaaccggtgccgattccggcgattgatcattttatttatcgcccgaaacgcgcgattcagtttattccgctgctggcgggcctgggcattacc

[0126] gcggcgtttaccaccggcgcgaccggcctgggcgtgagcgtgacccagtataccaaactgagcaaccagctgattagcgatgtgcagattctg

[0127] agcagcaccattcaggatctgcaggatcaggtggatagcctggcggaagtggtgctgcagaaccgccgcggcctggatctgctgaccgcgg

[0128] aacagggcggcatttgcctggcgctgcaggaaaaatgctgcttttatgtgaacaaaagcggcattgtgcgcgataaaattaaaaccctgcaggaagaactggaacgccgccgcaaagatctggcgagcaacccgctgtggaccggcctgcagggcctgctgccg。

[0129] SEQ ID No.12:

[0130]

[0131] SEQ ID No.13:

[0132] ggctttgatgatccgcgcaaagcgattgaactggtgcagaaacgctatggccgcccgtgcgattgcagcggcggccaggtgagcgaaccgcc

[0133] gagcgatcgcgtgagccaggtgacctgcagcggcaaaaccgcgtatctgatgccggatcagcgctggaaatgcaaaagcattccgaaagata

[0134] ccagcccgagcggcccgctgcaggaatgcccgtgcaacagctatcagagcagcgtgcatagcagctgctataccagctatcagcagtgccgc

[0135] agcggcaacaaaacctattataccgcgaccctgctgaaaacccagaccggcggcaccagcgatgtgcaggtgctgggcagcaccaacaaac

[0136] tgattcagagcccgtgcaacggcattaaaggccagagcatttgctggagcaccaccgcgccgattcatgtgagcgatggcggcggcccgctg

[0137] gataccacccgcattaaaagcgtgcagcgcaaactggaagaaattcataaagcgctgtatccggaactgcaggcgagcggcgcgagcggcg

[0138] cgagcggcgcgagcggcgcgagcggctatcatccgctggcgattccgaaagtgcgcgataacctgatggtggatgcgcagaccctgaacatt

[0139] ctgaacgcgacctataacctgctgctgatgagcaacaccagcctggtggatgattgctggctgtgcctgaaactgggcccgccgaccccgctg

[0140] gcgattccgaactttctgctgagctatgtgacccgcagcagcgataacattagctgcctgattattccgccgctgctggtgcagccgatgcagttta

[0141] gcaacagcagctgcctgtttagcccgagctataacagcaccgaagaaattgatctgggccatgtggcgtttagcaactgcaccagcattaccaa

[0142] cgtgaccggcccgatttgcgcggtgaacggcagcgtgtttctgtgcggcaacaacatggcgtatacctatctgccgaccaactggaccggcct

[0143] gtgcgtgctggcgaccctgctgccggatattgatattattgcgagcggcgcgagcggcgcgagcggcgcgagcggcgcgagcggcccggg

[0144] cgatgaaccggtgccgattccggcgattgatcattttatttatcgcccgaaacgcgcgattcagtttattccgctgctggcgggcctgggcattacc

[0145] gcggcgtttaccaccggcgcgaccggcctgggcgtgagcgtgacccagtataccaaactgagcaaccagctgattagcgatgtgcagattctg

[0146] agcagcaccattcaggatctgcaggatcaggtggatagcctggcggaagtggtgctgcagaaccgccgcggcctggatctgctgaccgcgg

[0147] aacagggcggcatttgcctggcgctgcaggaaaaatgctgcttttatgtgaacaaaagcggcattgtgcgcgataaaattaaaaccctgcaggaagaactggaacgccgccgcaaagatctggcgagcaacccgctgtggaccggcctgcagggcctgctgccg。

[0148] SEQ ID No.14:

[0149] ggctttgatgatccgcgcaaagcgattgaactggtgcagaaacgctatggccgcccgtgcgattgcagcggcggccaggtgagcgaaccgc

[0150] cgagcgatcgcgtgagccaggtgacctgcagcggcaaaaccgcgtatctgatgccggatcagcgctggaaatgcaaaagcattccgaaag

[0151] ataccagcccgagcggcccgctgcaggaatgcccgtgcaacagctatcagagcagcgtgcatagcagctgctataccagctatcagcagtg

[0152] ccgcagcggcaacaaaacctattataccgcgaccctgctgaaaacccagaccggcggcaccagcgatgtgcaggtgctgggcagcaccaa

[0153] caaactgattcagagcccgtgcaacggcattaaaggccagagcatttgctggagcaccaccgcgccgattcatgtgagcgatggcggcggc

[0154] ccgctggataccacccgcattaaaagcgtgcagcgcaaactggaagaaattcataaagcgctgtatccggaactgcaggcgggcagcgcgg

[0155] gcagcgcgggcagcgcgggcagcgcgggcagcgcgggcagctatcatccgctggcgattccgaaagtgcgcgataacctgatggtggat

[0156] gcgcagaccctgaacattctgaacgcgacctataacctgctgctgatgagcaacaccagcctggtggatgattgctggctgtgcctgaaactg

[0157] ggcccgccgaccccgctggcgattccgaactttctgctgagctatgtgacccgcagcagcgataacattagctgcctgattattccgccgctgc

[0158] tggtgcagccgatgcagtttagcaacagcagctgcctgtttagcccgagctataacagcaccgaagaaattgatctgggccatgtggcgtttag

[0159] caactgcaccagcattaccaacgtgaccggcccgatttgcgcggtgaacggcagcgtgtttctgtgcggcaacaacatggcgtatacctatct

[0160] gccgaccaactggaccggcctgtgcgtgctggcgaccctgctgccggatattgatattattgcgggcagcgcgggcagcgcgggcagcgc

[0161] gggcagcgcgggcagcgcgggcagcccgggcgatgaaccggtgccgattccggcgattgatcattttatttatcgcccgaaacgcgcgatt

[0162] cagtttattccgctgctggcgggcctgggcattaccgcggcgtttaccaccggcgcgaccggcctgggcgtgagcgtgacccagtataccaa

[0163] actgagcaaccagctgattagcgatgtgcagattctgagcagcaccattcaggatctgcaggatcaggtggatagcctggcggaagtggtgct

[0164] gcagaaccgccgcggcctggatctgctgaccgcggaacagggcggcatttgcctggcgctgcaggaaaaatgctgcttttatgtgaacaaaa

[0165] gcggcattgtgcgcgataaaattaaaaccctgcaggaagaactggaacgccgccgcaaagatctggcgagcaacccgctgtggaccggcctgcagggcctgctgccg。

[0166] SEQ ID No.15:

[0167] ggctttgatgatccgcgcaaagcgattgaactggtgcagaaacgctatggccgcccgtgcgattgcagcggcggccaggtgagcgaaccgc

[0168] cgagcgatcgcgtgagccaggtgacctgcagcggcaaaaccgcgtatctgatgccggatcagcgctggaaatgcaaaagcattccgaaag

[0169] ataccagcccgagcggcccgctgcaggaatgcccgtgcaacagctatcagagcagcgtgcatagcagctgctataccagctatcagcagtg

[0170] ccgcagcggcaacaaaacctattataccgcgaccctgctgaaaacccagaccggcggcaccagcgatgtgcaggtgctgggcagcaccaa

[0171] caaactgattcagagcccgtgcaacggcattaaaggccagagcatttgctggagcaccaccgcgccgattcatgtgagcgatggcggcggc

[0172] ccgctggataccacccgcattaaaagcgtgcagcgcaaactggaagaaattcataaagcgctgtatccggaactgcagggcagcgcgggca

[0173] gcgcgggcagcgcgggcagcgcgggcagcgcgggcagcgcgtatcatccgctggcgattccgaaagtgcgcgataacctgatggtggat

[0174] gcgcagaccctgaacattctgaacgcgacctataacctgctgctgatgagcaacaccagcctggtggatgattgctggctgtgcctgaaactg

[0175] ggcccgccgaccccgctggcgattccgaactttctgctgagctatgtgacccgcagcagcgataacattagctgcctgattattccgccgctgc

[0176] tggtgcagccgatgcagtttagcaacagcagctgcctgtttagcccgagctataacagcaccgaagaaattgatctgggccatgtggcgtttag

[0177] caactgcaccagcattaccaacgtgaccggcccgatttgcgcggtgaacggcagcgtgtttctgtgcggcaacaacatggcgtatacctatct

[0178] gccgaccaactggaccggcctgtgcgtgctggcgaccctgctgccggatattgatattattggcagcgcgggcagcgcgggcagcgcggg

[0179] cagcgcgggcagcgcgggcagcgcgccgggcgatgaaccggtgccgattccggcgattgatcattttatttatcgcccgaaacgcgcgattc

[0180] agtttattccgctgctggcgggcctgggcattaccgcggcgtttaccaccggcgcgaccggcctgggcgtgagcgtgacccagtataccaaa

[0181] ctgagcaaccagctgattagcgatgtgcagattctgagcagcaccattcaggatctgcaggatcaggtggatagcctggcggaagtggtgctg

[0182] cagaaccgccgcggcctggatctgctgaccgcggaacagggcggcatttgcctggcgctgcaggaaaaatgctgcttttatgtgaacaaaag

[0183] cggcattgtgcgcgataaaattaaaaccctgcaggaagaactggaacgccgccgcaaagatctggcgagcaacccgctgtggaccggcctgcagggcctgctgccg。

[0184] SEQ ID No.16:

[0185] ggctttgatgatccgcgcaaagcgattgaactggtgcagaaacgctatggccgcccgtgcgattgcagcggcggccaggtgagcgaaccgc

[0186] cgagcgatcgcgtgagccaggtgacctgcagcggcaaaaccgcgtatctgatgccggatcagcgctggaaatgcaaaagcattccgaaag

[0187] ataccagcccgagcggcccgctgcaggaatgcccgtgcaacagctatcagagcagcgtgcatagcagctgctataccagctatcagcagtg

[0188] ccgcagcggcaacaaaacctattataccgcgaccctgctgaaaacccagaccggcggcaccagcgatgtgcaggtgctgggcagcaccaa

[0189] caaactgattcagagcccgtgcaacggcattaaaggccagagcatttgctggagcaccaccgcgccgattcatgtgagcgatggcggcggc

[0190] ccgctggataccacccgcattaaaagcgtgcagcgcaaactggaagaaattcataaagcgctgtatccggaactgcagagcggcgcgagcg

[0191] gcgcgagcggcgcgagcggcgcgagcggcgcgagcggcgcgtatcatccgctggcgattccgaaagtgcgcgataacctgatggtggat

[0192] gcgcagaccctgaacattctgaacgcgacctataacctgctgctgatgagcaacaccagcctggtggatgattgctggctgtgcctgaaactg

[0193] ggcccgccgaccccgctggcgattccgaactttctgctgagctatgtgacccgcagcagcgataacattagctgcctgattattccgccgctgc

[0194] tggtgcagccgatgcagtttagcaacagcagctgcctgtttagcccgagctataacagcaccgaagaaattgatctgggccatgtggcgtttag

[0195] caactgcaccagcattaccaacgtgaccggcccgatttgcgcggtgaacggcagcgtgtttctgtgcggcaacaacatggcgtatacctatct

[0196] gccgaccaactggaccggcctgtgcgtgctggcgaccctgctgccggatattgatattattagcggcgcgagcggcgcgagcggcgcgag

[0197] cggcgcgagcggcgcgagcggcgcgccgggcgatgaaccggtgccgattccggcgattgatcattttatttatcgcccgaaacgcgcgatt

[0198] cagtttattccgctgctggcgggcctgggcattaccgcggcgtttaccaccggcgcgaccggcctgggcgtgagcgtgacccagtataccaa

[0199] actgagcaaccagctgattagcgatgtgcagattctgagcagcaccattcaggatctgcaggatcaggtggatagcctggcggaagtggtgct

[0200] gcagaaccgccgcggcctggatctgctgaccgcggaacagggcggcatttgcctggcgctgcaggaaaaatgctgcttttatgtgaacaaaa

[0201] gcggcattgtgcgcgataaaattaaaaccctgcaggaagaactggaacgccgccgcaaagatctggcgagcaacccgctgtggaccggcctgcagggcctgctgccg.

[0202] Exemplarily, after any one of SEQ ID No. 11 to SEQ ID No. 16 is synthesized, it is cloned and replaced into a lentiviral expression vector by means of restriction endonucleases to obtain a modified lentiviral expression vector, which is denoted as a first vector.

[0203] The first vector is used through the lentiviral expression vector in step R2, that is, the first vector includes a sequence of the spacer sequence 10 expressing the extracellular region of the BaEV envelope glycoprotein, and the modified BaEV-G can be obtained after the sequence of the first vector is expressed.

[0204] like Figure 2 As shown, the amino acid sequence of the modified extracellular region of BaEV-G includes at least one spacer sequence 10, and the spacer sequence 10 includes a spacer unit.

[0205] Exemplarily, the Spacer unit includes conservative amino acids.

[0206] Exemplarily, the spacer unit includes at least one of SAG, GAS, ASG, AGS, GSA and SGA.

[0207] In some examples, the number of amino acids included in the spacer sequence 10 ranges from 9 to 27. For example, the number of amino acids included in the spacer sequence 10 is 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27, etc., and there is no limitation here.

[0208] Illustratively, the spacer sequence 10 includes at least one of the following sequences.

[0209] SEQ ID No. 21: SAGSAGSAGSAGSAG; SEQ ID No. 22: GASGASGASGASGAS; SEQ ID No. 23: ASGASGASGASGASG; SEQ ID No. 24: AGSAGSAGSAGSAGSAGS; SEQ ID No. 25: GSAGSAGSAGSAGSAGSA; SEQ ID No. 26: SGASGASGASGASGA.

[0210] For example, the amino acid sequence of the modified extracellular region of BaEV-G is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 1 to SEQ ID No. 6.

[0211] SEQ ID No.1:

[0212] GFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPK

[0213] DTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTN

[0214] KLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQSAGSAGS

[0215] AGSAGSAGYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKLGPPTP

[0216] LAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITN

[0217] VTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIDIISAGSAGSAGSAGSAGPG

[0218] DEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSS

[0219] TIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP。

[0220] SEQ ID No.2:

[0221] GFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPK

[0222] DTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTN

[0223] KLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQGASGASG

[0224] ASGASGASYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKLGPPTP

[0225] LAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITN

[0226] VTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIDIIGASGASGASGASGASPG

[0227] DEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSS

[0228] TIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP。

[0229] SEQ ID No.3:

[0230] GFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPK

[0231] DTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTN

[0232] KLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQASGASGA

[0233] SGASGASGYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKLGPPTP

[0234] LAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITN

[0235] VTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIDIIASGASGASGASGASGPG

[0236] DEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSS

[0237] TIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP。

[0238] SEQ ID No.4:

[0239] GFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPK

[0240] DTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTN

[0241] KLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQAGSAGSA

[0242] GSAGSAGSAGSYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKLGP

[0243] PTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSI

[0244] TNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIDIIAGSAGSAGSAGSAGS

[0245] AGSPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISD

[0246] VQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP。

[0247] SEQ ID No.5:

[0248] GFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPK

[0249] DTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTN

[0250] KLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQGSAGSAG

[0251] CASESSAGSAYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKLGP

[0252] PTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSI

[0253] TNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIDIIGSAGSSAGS

[0254] GSAPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISD

[0255] VQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP。

[0256] SEQ ID No.6:

[0257] GFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPK

[0258] DTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTN

[0259] CLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQSGASGAS

[0260] GASGASGASGAYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKLGP

[0261] PTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSI

[0262] TNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIISGASGASGASGASGA

[0263] SGAPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISD

[0264] VQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP.

[0265] Among them, the insertion site of the spacer sequence 10 is a site with a very low prediction score according to the Parker method in the antigenic determinant prediction of the amino acid sequence (Antigenic Determinant Prediction (v1), detaibio.com). After inserting the spacer sequence 10, the prediction is re-performed, and the prediction score of this site is higher than zero, thereby generating more antigenic determinants.

[0266] Exemplarily, the insertion sites of spacer sequence 10 include positions 117 and 338 of the amino acid sequence of BaEV envelope glycoprotein.

[0267] Therefore, the modified BaEV lentivirus 100 increases the number of spacer sequences 10 to improve the recognition range between the recognition region and the receptor protein on the surface of the target cell, thereby improving the lentivirus infection efficiency and having a relatively high infection ability for the target cells.

[0268] The inventors found that BaEV lentivirus 100 transfection of target cells relies on the interaction between the BaEV-G extracellular region and the receptors on the surface of the target cells. However, for human T cells and NK cells, the expression of the corresponding receptors on their surfaces is often low, making the infection efficiency of BaEV lentivirus 100 generally low. Therefore, by using the interaction between antibodies and antigens, the type and intensity of the interaction between BaEV lentivirus 100 and target cells can be enhanced, and the transfection efficiency of BaEV lentivirus 100 can be improved.

[0269] Since the recognition protein can recognize and capture most common proteins, introducing the recognition protein into the lentiviral expression vector can improve the transfection efficiency of BaEV lentivirus 100.

[0270] Exemplarily, the recognition protein includes: a receptor protein, an antibody, a single-chain recognition sequence modified from an antibody, a nanobody, a lectin or a molecular chaperone protein.

[0271] For example, receptor proteins include hormones, neurotransmitters or growth factors, lectins include concanavalin A, and molecular chaperone proteins include heat shock protein 70 (Hsp70).

[0272] Among them, protein A and protein G are two very commonly used antibody molecules.

[0273] For example, the NCBI sequence accession number of protein A is ACH54083.1, and protein A is from Staphylococcus pseudointermedius ED99, which is a Gram-positive bacterium belonging to the genus Staphylococcus.

[0274] Exemplarily, the gene sequence of protein G (GenBank) is: CAA37410.1, and protein G comes from strptococcus, which is a Streptococcus bacterium.

[0275] Therefore, if Figure 2 As shown, the embodiments of the present disclosure introduce protein A or protein G into the backbone of the lentiviral expression vector, and then when it is packaged into the BaEV lentivirus 100, a recognition motif 20 including protein A and / or protein G is introduced on the surface of the lentiviral capsid protein. The recognition motif 20 is targeted and can mediate the recognition of the target cells by the lentivirus, thereby improving the targeting of the lentivirus and enhancing the transfection efficiency of the BaEV lentivirus 100.

[0276] Exemplarily, the lentiviral expression vector in which protein A and / or protein G are introduced into the BaEV lentivirus 100 is represented as the second vector. The second vector comprises: a sequence expressing the recognition sequence of the BaEV envelope glycoprotein.

[0277] In some examples, the BaEV lentivirus 100 after protein A and / or protein G are introduced on the surface of the lentivirus capsid protein includes a recognition sequence, which is the recognition motif 20; the recognition sequence includes: BaEVSecSig-recognition protein-BaEVTM. BaEVSecSig is a BaEV signal peptide, and BaEVTM is a BaEV transmembrane domain.

[0278] Signal peptides are short (5 to 30 amino acids in length) peptide chains that direct newly synthesized proteins to the secretory pathway.

[0279] For example, the recognition protein is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 7 and SEQ ID No. 8.

[0280] SEQ ID No.7:

[0281] MENKNFFSIRKLSIGVGSCLIASSLLVNTPSFAEEGDNNAEAQQNAFSEVVKLPNLSEEQRNGFIQSLKDDPSTSQDVLNEAKKLNDSQEGSQPAPDYSDEQQNAFYEILHLPNLTEEQRNGYIQSLKDDPSVSANILVEAKNMNVNQTPTQPAPSFDEAQQNAFYEIVNLPNLTEEQRNGFIQSLKDDPSVSKDILVEAKKLNDSQAKPDYSEAQQNAFYEILHLPNLTEEQRNGFIQSLKDDPSVSSDILAEAKKLNDSQAPKEDNNVKDNNSGENKAEDKGNKENKAEDKGSKEDKAEDKGSKEDKAEDKGSKEDKAEDKGSKEDKAEDKGSIEDKAKDKDNKEGKAADKGMDKAKDAMHVVQPGETVEKIAKANNTTVEQIAKDNHLEDKNMILPGQKLVVDNQKAMKDSQEAKANHEMKALPETGEENDMALFGTSLTGGLSLALGLYILGRGRKTN。

[0282] SEQ ID No.8:

[0283] MKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTEAVDAATAEKVFKQYANDNGVDGEWTYDDATKTFTVTEKPEVIDASELTPAVTTYKLVINGKTLKGETTTKAVDAETAEKAFKQYANDNGVDGVWTYDDATKTFTVTE。

[0284] The sequence of BaEVSecSig is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of SEQ ID No.9: MGFTTKIIFLYNLVLVYA. The sequence of BaEVTM is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of SEQ ID No.10: YLLPFLGPLLTLLLLLTIGPCIF.

[0285] By including a recognition sequence in the BaEV lentivirus 100, a recognition motif 20 of protein A or protein G is introduced on the surface of the lentivirus capsid protein. The recognition motif 20 is targeted and mediates the recognition of the target cells by the lentivirus, thereby improving the targeting of the lentivirus and thus improving the transfection efficiency of the BaEV lentivirus 100.

[0286] The nucleotide sequence of Protein A is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of SEQ ID No.27.

[0287] SEQ ID No.27:

[0288]

[0289] The nucleotide sequence of Protein G is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of SEQ ID No.28.

[0290] SEQ ID No.28:

[0291] .

[0292] Based on the nucleotide sequences of protein A and protein G described above, the nucleotide sequence of the second vector includes any one of the nucleotide sequences of protein A and protein G.

[0293] Illustratively, the second vector comprises at least 25%, 30%, 40%, 50%, 60%, 70% or 80% derived from any one of SEQ ID No.17 and SEQ ID No.18.

[0294] SEQ ID No.17:

[0295] atgggctttaccaccaaaattatttttctgtataacctggtgctggtgtatgcgatggaaaaacaaaaactttttcagcattcgtaaattatcaattggggt

[0296] gggttcatgtttaatcgcgagctctttacttgtgaatacaccaagtttcgcagaagaaggagataataacgcagaagcgcaacaaaacgctttctct

[0297] gaggtagtaaaattacctaaccttagcgaagaacaacgtaatggtttcattcaaagccttaaagatgatccaagtacaagtcaagatgtgcttaatg

[0298] aagctaaaaaattaaatgatagtcaagagggatctcaacctgctcctgattacagtgatgaacaacaaaatgcattttatgaaattttacaccttcca

[0299] aacttaactgaagaacaacgcaatggctatattcaaagtcttaaagatgacccaagtgtaagcgctaatattcttgttgaagctaaaaatatgaatgt

[0300] taaccaaacacctacacaacctgcgccaagtttcgatgaagcgcaacaaaatgcattctatgagattgtaaacttaccaaatcttactgaagagca

[0301] acgtaacggtttcatccaaagccttaaagacgatccaagtgtaagtaaagatatccttgttgaagctaaaaagttaaatgacagccaagcaaaacc

[0302] tgattacagtgaagcgcaacaaaatgcattttatgaaattttacaccttccaaacttaactgaagaacaacgtaacggtttcatccaaagccttaaag

[0303] acgatccgagtgtaagtagtgatattcttgctgaagctaagaaattaaatgacagccaagcgcctaaagaagacaacaacgtaaaagacaataat

[0304] tcaggtgaaaacaaagctgaagacaaaggcaacaaagaaaacaaagctgaagataaaggcagcaaagaagacaaagctgaagataaaggc

[0305] agcaaagaagacaaagctgaagataaaggcagcaaagaagacaaagctgaagataaaggcagcaaagaagacaaagctgaagataaagg

[0306] cagcatagaagataaagctaaagacaaagacaacaaagaaggcaaagctgcagacaaaggtatggacaaagcgaaagatgcaatgcatgtc

[0307] gttcaacctggtgaaacagtagaaaaaattgctaaagctaataacacaactgtagaacaaatcgctaaagataatcatttagaagataaaaacatg

[0308] attttaccaggtcaaaaacttgttgttgacaaccaaaaagcaatgaaagacagccaagaagctaaagcaaaccacgaaatgaaagctttacctga

[0309] aacaggtgaagaaaacgatatggcattattcggtacatcacttacaggtggtcttagcttagcattaggtttatacatcttaggacgtggcagaaaaacaaactaatatctgctgccgtttctgggcccgctgctgaccctgctgctgctgctgaccattggcccgtgcattttt。

[0310] SEQ ID No.18:

[0311] .

[0312] like Figure 2 As shown, an embodiment of the present disclosure further provides a lentivirus, which is a BaEV lentivirus 100. The extracellular region of the envelope glycoprotein of the BaEV lentivirus 100 includes at least one of the following sequences: a spacer sequence 10 and a recognition sequence.

[0313] For example, the sequence of the extracellular region of the envelope glycoprotein of the BaEV lentivirus 100 may include an amino acid sequence and a nucleic acid sequence.

[0314] Exemplarily, the spacer sequence 10 includes: a spacer unit; a recognition sequence includes: BaEVSecSig-recognition protein-BaEVTM; wherein, BaEVSecSig is a BaEV signal peptide, and BaEVTM is a BaEV transmembrane domain.

[0315] Illustratively, the extracellular region of the envelope glycoprotein of the BaEV lentivirus 100 includes the amino acid sequence expressed by the first vector, that is, the BaEV-G extracellular region has at least one spacer sequence 10. The introduction of the spacer sequence 10 refers to the above content and will not be repeated here.

[0316] For example, the amino acid sequence of the extracellular region of BaEV-G is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 1 to SEQ ID No. 6. The introduction of SEQ ID No. 1 to SEQ ID No. 6 is referred to the above content and will not be repeated here.

[0317] By deriving the envelope glycoprotein of BaEV lentivirus 100 from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No.1 to SEQ ID No.6, the recognition range of the recognition region and the target cell surface receptor protein can be increased, thereby improving the lentivirus infection efficiency and having a relatively high infection ability for target cells.

[0318] Exemplarily, the envelope glycoprotein extracellular region of the BaEV lentivirus 100 includes the amino acid sequence expressed by the second vector. The sequence is represented as: BaEVSecSig-recognition protein-BaEVTM. For the introduction of BaEVSecSig-recognition protein-BaEVTM, please refer to the above content and will not be repeated here.

[0319] By including the amino acid sequence expressed by the second vector in the extracellular region of the envelope glycoprotein of the BaEV lentivirus 100, the targeting of the lentivirus can be improved, thereby enhancing the transfection efficiency of the BaEV lentivirus 100.

[0320] The embodiments of the present disclosure also provide a use of the lentivirus as described in any of the above embodiments in transfection of primary human NK cells.

[0321] The conditions for preparing the lentivirus were further optimized as follows.

[0322] In some examples, the lentiviral expression vector in step R2 includes: a first vector and a second vector, and the ratio of the mass of the first vector to the mass of the second vector ranges from 5 to 10.

[0323] For example, the ratio of the mass of the first carrier to the mass of the second carrier is 5, 6, 7, 8, 9 or 10, etc., which is not limited here.

[0324] The transfection ability of the BaEV lentivirus 100 can be improved by setting the ratio of the mass of the first vector to the mass of the second vector to be in the range of 5 to 10. The introduction of specific experimental data refers to the subsequent content and will not be described in detail here.

[0325] In some examples, in step R2, in the first solution, the mass ratio of the vector plasmid, the packaging plasmid, and the lentiviral expression vector is (3-5):3:(1-3).

[0326] The mass fraction of the lentiviral expression vector may be the sum of the mass of the first vector and the mass of the second vector. Alternatively, the lentiviral expression vector is derived from an unmodified BaEV plasmid of the nucleotide sequence of SEQ ID No. 20.

[0327] Exemplarily, the mass ratio of the vector plasmid, packaging plasmid and lentiviral expression vector is 3:3:1, 3:3:1.5, 3:3:2, 3:3:2, 3.5:3:1, 3.5:3:1.5, 3.5:3:2, 3.5:3:3, 4:3:1, 4:3:1.5, 4:3:2, 5:3:1, 5:3:2 or 5:3:3, etc., but is not limited here.

[0328] The yield of BaEV lentivirus 100 can be increased by setting the mass ratio of vector plasmid, packaging plasmid and lentiviral expression vector to (3-4):3:(1-2). The introduction of specific experimental data refers to the subsequent content and will not be described here.

[0329] In some embodiments, before the packaging cells are plated into the culture container for culture, step R1 further includes: modifying the coacervate microgel 30 of the culture container 40 .

[0330] Microgel is a colloidal dispersion system of micron or submicron-sized gel particles with an intramolecular cross-linked structure. Coacervate microgel 30 is a special microgel system formed by agglomerating or aggregating microgel particles in a certain way (physical or chemical action) during the preparation or application process.

[0331] By modifying the culture container 40 with the condensate microgel 30, the adhesion of the basal cells to the culture substrate can be enhanced to prevent them from falling off during the toxin production process; the microgel encapsulates the culture factors and slowly releases the culture factors through external stimulation, thereby increasing the activity of the basal cells and the toxin production time.

[0332] Exemplarily, the polymer microgel comprises: folic acid and a polycationic electrolyte, for example, the polycationic electrolyte comprises at least one of protamine sulfate and polylysine.

[0333] In some examples, the modification of the coacervate microgel 30 of the culture container 40 includes the following steps: mixing a folic acid solution with a polycation electrolyte solution to obtain a third solution; applying the third solution to the culture container 40, adding a phosphate solution to induce gelation, and obtaining a culture container 40 modified with the coacervate microgel 30.

[0334] Exemplarily, a folic acid solution with a concentration range of 11 mmol / mL to 45 mmol / mL is mixed with a polycation electrolyte solution with a concentration range of 15 mmol / mL to 88 mmol / mL to obtain a third solution with a pH range of 8.5 to 10.5, wherein the ratio of the molar number of folic acid to the molar number of polycation electrolyte is in the range of 1:1.5 to 1:6. The third solution is applied to a culture container 40, and a phosphate solution with a pH range of 6.5 to 8.0 is added to initiate gelation, thereby obtaining a culture container 40 modified with a coacervate microgel 30, wherein the ratio of the volume of the phosphate solution to the volume of the folic acid solution is in the range of 1:5 to 1:20.

[0335] Illustratively, the concentration of the folic acid solution is 11 mmol / mL, 15 mmol / mL, 18 mmol / mL, 20 mmol / mL, 24 mmol / mL, 28 mmol / mL, 32 mmol / mL, 36 mmol / mL or 45 mmol / mL, etc., but is not limited thereto.

[0336] Illustratively, the concentration of the polycationic electrolyte solution is 15 mmol / mL, 20 mmol / mL, 30 mmol / mL, 35 mmol / mL, 40 mmol / mL, 55 mmol / mL, 60 mmol / mL, 80 mmol / mL or 88 mmol / mL, etc., but is not limited thereto.

[0337] Illustratively, the pH of the third solution is 8.5, 9, 9.5 or 10, etc., which is not limited here.

[0338] Illustratively, the ratio of the molar number of folic acid to the molar number of the polycationic electrolyte is 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, 1:5, 1:5.5 or 1:6, etc., but is not limited thereto.

[0339] Illustratively, the phosphate solution is 20 mmol / L sodium dihydrogen phosphate, and the pH of the phosphate solution is 6.5, 7, 7.5 or 8, etc., which is not limited here.

[0340] Illustratively, the volume ratio of the phosphate solution to the folic acid solution ranges from 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:15, 1:16, 1:18, 1:19 or 1:20, etc., but is not limited thereto.

[0341] In the culture container 40 modified by the coacervate microgel 30, there are cations + and anions - formed by polyelectrolytes, and the cations + are more than the anions -. Since the basal cells contain more negative charges, the cations + can adsorb the basal cells on the culture substrate, enhance the adhesion of the basal cells to the culture substrate, and prevent them from falling off during the toxin production process.

[0342] Therefore, the above embodiment improves the adhesion of the 293T cell growth substrate by modifying the 293T cell growth substrate, thereby effectively avoiding the produced BaEV lentivirus 100 from inducing the formation of syncytia, thereby reducing the probability of BaEV lentivirus 100 shedding and death, and increasing the yield of BaEV lentivirus 100.

[0343] According to the above-mentioned method for preparing lentivirus, the following examples are provided.

[0344] Example 1

[0345] 1. Virus packaging.

[0346] (1) 20h to 24h before transfection, 3×10 5 Cell number (cells) / 20 mL (DMEM medium containing 10% FBS) 293T cells were plated into a T175 cell culture flask (or a 15 cm culture dish without any modification or treatment).

[0347] (2) When 293T cells reached 70% to 90% confluence during transfection, the medium was replaced with serum-free DMEM (0.6 × 10 5 cells / cm 2 in 15 mL of medium, there are 0.6 x 10 5 cells, the culture dish contains 15 ml of culture medium).

[0348] (3) Take a 15 mL centrifuge tube and prepare the solution according to the following table. Gently invert to mix.

[0349]

[0350] pCMV-ΔR8.91 is the packaging plasmid.

[0351] Please refer to the table below for specific configuration.

[0352] / Plasmid concentration (μg / μL) Quality Ratio Mass (μg) Volume (μL) Vector plasmid 1.14 1 7.92 6.95 pCMV-ΔR8.91 1.048 1 7.92 7.56 BaEV plasmid 1.964 1 7.92 4.03 Enzyme-free water / / / 81.49

[0353] (4) Add the liquid in tube 1 (plasmid solution) dropwise (or insert the pipette tip into the liquid and rotate it slowly) to tube 2 (Lipofectamine mixture), gently invert and mix for 10 to 15 seconds, and let stand at room temperature for 5 minutes.

[0354] (5) Add the reagent obtained in (4) above dropwise to the cell culture dish and shake gently to cover all cells. After culturing in a cell culture incubator for 4 hours, replace the culture medium with preheated DMEM complete medium containing 10% FBS, and collect the supernatant 48 to 72 hours after transfection, about 20 mL.

[0355] (6) After collecting about 20 mL of supernatant, remove the cell precipitate by ultracentrifugation or PEG precipitation, collect the supernatant, and then filter it with a 0.45 μm filter to obtain the virus.

[0356] 2. Virus concentration.

[0357] (1) Slowly add 5 mL of 5X PEG-3000NaCl stock solution to every 20 mL of filtered virus supernatant and place at 4°C.

[0358] Here, PEG-3000 represents polyethylene glycol with a molecular weight of 3000. 5XPEG-3000NaCl mother solution refers to a solution formed by dissolving 8.766 g of NaCl and 50 g of PEG-3000 in 200 mL of pure water.

[0359] (2) Mix once every 20 to 30 minutes for 3 to 5 times; and leave at 4°C overnight.

[0360] (3) Centrifuge at 4°C and 4000 x g for 20 min to allow the lentiviral particles to accumulate at the bottom of the tube, with a clear precipitate visible.

[0361] (4) Discard the supernatant and centrifuge again at 4000 x g at 4°C for 5 min (or leave the tube alone for 1–2 min) to remove any residual liquid.

[0362] (5) Add an appropriate amount of HBSS / PBS / sterile double-distilled water to each tube. Add 200 μL of PBS to each tube corresponding to 25 mL of virus stock solution to dissolve the lentiviral precipitate and store at 4°C for 4 hours.

[0363] Among them, HBSS (Hank's Balanced Salt Solution) is Hank's balanced salt solution, which is generally used to prepare culture medium or wash cells during cell culture.

[0364] (6) Collect the precipitated virus particles, package them into 10 μL to 50 μL / tube, store them at -80°C, and use them as needed.

[0365] 3. Virus titration.

[0366] (1) 20h to 24h before transfection, 3×10 4 cells / 100 μL / well (DMEM medium containing 10% FBS) was plated into a 96-well cell culture container 40.

[0367] (2) Make 8 virus gradients: 5-fold dilution, with an initial virus volume of 3 μL / well.

[0368] ① According to the limiting dilution method, the PEG-concentrated virus stock solution was diluted 5-fold in DMEM medium containing 2% FBS and 2 μg / mL polybrene, and the dilution was continued for 8 times, and cultured at 37°C and 5% CO2.

[0369] Among them, polybrene, also known as hexadimethrinebromide, is a cationic polymer.

[0370] ② The dilution method is as follows: prepare 8 1.5mL centrifuge tubes, add 40μL complete medium to each tube; add 10μL of virus stock solution to the first tube, mix well, then pipette 10μL and add to the second tube to mix well. And so on. Then mix the diluted virus solution (15μL / well) with the above cells (50μL / well), and add to the corresponding well plate. Centrifuge at 5000rpm (revolutions / minute) for 3h.

[0371] ③After culturing at 37°C for 6-8 hours, add 200 μL of DMEM medium containing 10% FBS to facilitate cell growth, and culture overnight at 37°C and 5% CO2.

[0372] (3) Wash once with RPMI1640 medium, resuspend in 200 μL of medium, and plate in a 96-well plate for 2 to 3 days.

[0373] Among them, RPMI is the abbreviation of Roswell Park Memorial Institute, which refers to Roswell Park Memorial Institute. RPMI is a type of cell culture medium developed by the institute, and 1640 is the code for the culture medium.

[0374] (4) Detect the GFP ratio using flow cytometry, and select samples with a ratio between 10% and 30% to calculate the virus titer.

[0375] Among them, GFP is the abbreviation of Green Fluorescent Protein, which means green fluorescent protein.

[0376] The formula for calculating virus titer is: virus titer = number of cells x fluorescence percentage x 10 3 / Volume of virus stock solution (μL).

[0377] Example 2

[0378] Based on Example 1, the plasmid mixing ratio in step (3) of virus packaging was adjusted as shown in the following table.

[0379]

[0380] Please refer to the table below for specific configuration.

[0381]

[0382]

[0383] In Example 1, the mass ratio of the vector plasmid, the mass of the packaging plasmid and the mass of the BaEV plasmid is 1:1:1. In Example 2, the mass ratio of the vector plasmid, the mass of the packaging plasmid and the mass of the BaEV plasmid is 4:3:2.

[0384] Example 3

[0385] Based on Example 1, the plasmid mixing ratio in step (3) of virus packaging was adjusted as shown in the following table.

[0386]

[0387] Please refer to the table below for specific configuration.

[0388] / Plasmid concentration (μg / μL) Quality Ratio Mass (μg) Volume (μL) Vector plasmid 1.14 3 15.84 13.9 pCMV-ΔR8.91 1.048 3 15.84 15.1 BaEV plasmid 1.964 1 5.28 2.7 Enzyme-free water / / / 88.3

[0389] In Example 3, the mass ratio of the vector plasmid, the packaging plasmid and the BaEV plasmid is 3:3:1.

[0390] Example 4: The coacervate microgel 30 modifies the culture container 40.

[0391] (1) Preparation of condensate microdroplets.

[0392] 20 μL of folic acid solution (FA, 11.08 mg / ml, 25 mM, pH = 10.0) and 100 μL of protamine sulfate solution (Prot, 4.25 mg / ml, average amino acid molecular weight 170 Da, monomer concentration 25 mM, pH = 10.0) were mixed to prepare Prot / FA coacervate microdroplets.

[0393] (2) Modification of the base of the culture container 40.

[0394] The above-mentioned coacervate microdroplet solution was coated on a T175 cell culture flask (or a 15 cm culture dish), and then 2 μL of phosphate solution (20 mM sodium dihydrogen phosphate, pH = 7.0) was added thereto to induce gelation; the solution was then allowed to stand at 37° C. for 1 hour.

[0395] (3) The plasmid mixing ratio was the same as that in Example 2, and the other packaging preparation operations and steps remained unchanged.

[0396] Example 5: Modification of the extracellular region of BaEV-G.

[0397] (1) The original amino acid sequence of the extracellular region of the BaEV envelope glycoprotein is derived from SEQ ID No.19.

[0398] (2) The nucleotide sequence corresponding to the original amino acid sequence of the extracellular region of the BaEV envelope glycoprotein is derived from SEQ ID No. 20.

[0399] (3) The amino acid sequence of the modified extracellular region of the BaEV envelope glycoprotein is derived from SEQ ID No.1.

[0400] (4) The nucleotide sequence corresponding to the amino acid sequence of the modified extracellular region of the BaEV envelope glycoprotein is derived from SEQ ID No.11.

[0401] After SEQ ID No.11 was synthesized, it was cloned into the extracellular region of the BaEV envelope glycoprotein using restriction endonucleases for replacement; the plasmid mixing ratio was referred to that shown in Example 2, and the remaining packaging preparation operations and steps remained unchanged.

[0402] Embodiment 6: adding recognition element 20.

[0403] (1) Plasmid construction.

[0404] The following gene sequences were synthesized: BaEVSecSig-Protein A-BaEVTM and / or BaEVSecSig-Protein G-BaEVTM.

[0405] BaEVSecSig refers to the BaEV signal peptide; BaEVTM refers to the BaEV transmembrane domain.

[0406] The specific sequence is as follows:

[0407] BaEVSecSig:MGFTTKIIFLYNLVLVYA.

[0408] BaEVTM:YLLPFLGPLLTLLLLLTIGPCIF.

[0409] The nucleotide sequence corresponding to the gene is derived from SEQ ID No.17.

[0410] SEQ ID No. 17 was inserted into the first vector to replace the SAG region of the BaEV plasmid to obtain the second vector.

[0411] (2) The first vector and the second vector are mixed at a mass ratio of 9:1, and then added as an envelope plasmid to form a first solution.

[0412] (3) The plasmid mixing ratio was the same as that in Example 2, and the other packaging preparation operations and steps remained unchanged.

[0413] Example 7

[0414] The lentivirus prepared in Example 5 and Example 6 was used to transfect primary human NK cells to test the transfection efficiency.

[0415] The specific steps are as follows:

[0416] (1) After NK cells were revived and cultured for 48 h, lentivirus was thawed on ice and mixed by pipetting.

[0417] (2) Calculate the volume of lentiviral solution required for transfection: Number of cells exposed to lentivirus min =700 / 0.1×300=2ⅹ10 6 MOI = 10, where MOI is the abbreviation of multiplicity of infection, indicating the multiplicity of infection.

[0418] (3) Thaw Vectofusin-1 (infectious virus enhancer) at room temperature and vortex to mix.

[0419] (4) The final concentration of Vectofusin-1 in the culture medium was 2.5 μg / mL.

[0420] (5) Dilute the lentiviral solution and Vectofusin-1 with culture medium, respectively, and ensure that their final volumes are the same.

[0421] (6) Mix the two solutions and pipette to mix well.

[0422] (7) Add the above mixed solution (<10min) into the cell suspension and mix well by pipetting.

[0423] (8) Centrifuge at 400 x g for 2 h at 32°C.

[0424] (9) Culture at 37°C and 5% to 10% CO2. Wash with culture medium every 24 hours after transfection.

[0425] Monitor the culture conditions every day and culture in separate bottles according to the situation; after the 4th day of transfection, remove the clear liquid as much as possible without touching the cells. Use preheated culture medium to replenish the culture medium to the original volume. The culture medium contains: 5% AB, IL-2, IL-15; replace the culture medium every 2 to 3 days, or when the cell confluence is close to 80% and the culture medium is exhausted; culture for 12 days. Among them, IL-2 and IL-15 are cytokines, and 5% AB refers to 5% human AB type serum. Flow cytometry was then used to detect the proportion of GFP-positive cells.

[0426] The following describes the virus titer in the virus stock solution obtained by the preparation method of the lentivirus provided in Examples 1 to 6. The titer is the reciprocal of the dilution. The virus titer is the concentration of the virus suspension, also known as the titer of the virus. The unit is transfection unit / mL, i.e. TU / mL.

[0427] The calculation formula is: virus titer = number of cells x fluorescence percentage x 10 3 / volume of virus stock solution (μL) to calculate the titer of the lentivirus prepared in Examples 1 to 7. The number of cells is the cell base number used in the examples disclosed herein, and the value is 3×10 5 The volume of the virus stock solution is 100 μL.

[0428] Figure 3 The figure is the percentage of GFP positive cells. The horizontal axis represents the fluorescence intensity in au (arbitrary unit), and the vertical axis represents the number of cells in au. The blue area in the figure is the blank control data, and the pink area is the fluorescence intensity data after the lentivirus prepared in Example 1 was transfected into NK cells. Figure 3 It can be seen that the fluorescence percentage of NK cells transfected with the lentivirus prepared in Example 1 is 2.3%. Therefore, according to the formula, virus titer = number of cells x fluorescence percentage x 10 3 / The volume of the virus stock solution shows that the virus titer = 3 x 10 5 ⅹ2.3% ⅹ10 3 / 100=6.9ⅹ10 4 TU / mL, that is, the virus titer of the lentivirus prepared in Example 1 was 6.9 x 10 4 TU / mL.

[0429] Figure 4 Another ratio chart of GFP positive cells. The blue area in the figure is the blank control data, and the pink area is the fluorescence intensity data after the lentivirus prepared in Example 2 was transfected into NK cells. Figure 4 It can be seen that the fluorescence percentage of NK cells transfected with the lentivirus prepared in Example 2 is 3.4%. Therefore, according to the formula, virus titer = number of cells x fluorescence percentage x 10 3 / The volume of the virus stock solution shows that the virus titer = 3 x 10 5 ⅹ3.4% ⅹ10 3 / 100=1.02ⅹ10 5 TU / mL, that is, the virus titer of the lentivirus prepared in Example 2 is 1.02 x 10 5 Compared with Example 1, Example 2 can increase the yield of BaEV lentivirus 100 by about 2 times by setting the mass fractions of the vector plasmid to 4, the mass fractions of the packaging plasmid to 3, and the mass fractions of the BaEV plasmid to 2.

[0430] Figure 5 This is another ratio chart of GFP positive cells. The blue area in the figure is the blank control data, and the pink area is the fluorescence intensity data after the lentivirus prepared in Example 3 was transfected into NK cells. Figure 5 It can be seen that the fluorescence percentage of NK cells transfected with the lentivirus prepared in Example 3 is 4.1%. Therefore, according to the formula, virus titer = number of cells x fluorescence percentage x 10 3 / The volume of the virus stock solution shows that the virus titer = 3 x 10 5 ⅹ4.1% ⅹ10 3 / 100=1.23ⅹ10 5 TU / mL, that is, the virus titer of the lentivirus prepared in Example 3 was 1.23 x 10 5 Compared with Example 1, Example 3 can increase the yield of BaEV lentivirus 100 by more than 2 times by setting the mass fractions of the vector plasmid to 3 parts, the mass fractions of the packaging plasmid to 3 parts, and the mass fraction of the BaEV plasmid to 1 part.

[0431] Figure 6 This is another ratio chart of GFP positive cells. The blue area in the figure is the blank control data, and the pink area is the fluorescence intensity data after the lentivirus prepared in Example 4 was transfected into NK cells. Figure 6 It can be seen that the fluorescence percentage of NK cells transfected with the lentivirus prepared in Example 4 is 58.2%. Therefore, according to the formula, virus titer = number of cells x fluorescence percentage x 10 3 / The volume of the virus stock solution shows that the virus titer = 3 x 10 5 ⅹ58.2% ⅹ10 3 / 100=1.7ⅹ10 6 TU / mL, that is, the virus titer of the lentivirus prepared in Example 4 was 1.7 x 10 6 Compared with Example 1, Example 4 can increase the yield of BaEV lentivirus 100 by 1 order of magnitude by modifying the culture container 40 through the coacervate microgel 30.

[0432] Therefore, by Figure 7 to Figure 9 It can be seen that the lentivirus packaging effect and the yield of lentivirus can be improved by adjusting the mass mixing ratio of the vector plasmid, packaging plasmid and BaEV plasmid.

[0433] Figure 7 This is the antibody cell labeling flow fluorescence image of Example 1. Figure 8 This is the antibody cell labeling flow fluorescence image of Example 5, Fig. 9 It is the antibody cell marker flow fluorescence diagram of Example 6. Among them, the horizontal axis CD56-APC represents the relative intensity of the marker signal of antibody CD56. Antibody CD56 is a glycoprotein distributed on the cell surface. It is often present on the surface of human nerve cells and astrocytes. There is also a small amount of distribution on the surface of lymphocytes. Antibody CD56 is a specific marker for NK cells; the vertical axis CAR-GFP represents the relative intensity of the signal of the expressed protein. The antibody cell marker flow fluorescence diagram is divided into four areas, among which the upper right area is the area where both antibody CD56 and CAR are expressed. The larger the value of this area, the stronger the ability of the lentivirus to infect primary NK cells.

[0434] Figure 7 The value in the upper right area is 18.7%. Figure 8 The value in the upper right area is 24.1%. Fig. 9 The value of the upper right area is 31.1%, which shows that compared with Example 1, after the BaEV-G extracellular region is modified in Example 5, the infection ability of the lentivirus to primary NK cells is enhanced; after the BaEV-G extracellular region is modified in Example 6, recognition motif 20 is further added to the surface of the lentivirus capsid protein, and the infection ability of the lentivirus to primary NK cells is further enhanced.

[0435] Therefore, by Figure 7 to Figure 9 It can be seen that the BaEV-G extracellular region modification has improved the recognition range of the recognition region and the receptor protein on the surface of the target cell, thereby improving the lentivirus infection efficiency, and the lentivirus has a relatively high ability to infect target cells. The introduction of targeted recognition motif 20 on the surface of the lentivirus capsid protein mediates the recognition of the target cell by the lentivirus, thereby improving the targeting of the lentivirus and further improving the lentivirus's ability to infect target cells.

[0436] The above is only a specific embodiment of the present disclosure, but the protection scope of the present disclosure is not limited thereto. Any changes or substitutions that can be thought of by any person skilled in the art within the technical scope disclosed in the present disclosure should be included in the protection scope of the present disclosure. Therefore, the protection scope of the present disclosure should be based on the protection scope of the claims.

Claims

1. A lentivirus, characterized in that include: BaEV envelope glycoprotein; Wherein, the extracellular region of the BaEV envelope glycoprotein includes at least one of the following sequences: A spacer sequence, comprising: a spacer unit; The recognition sequence includes: BaEVSecSig-recognition protein-BaEVTM; wherein the BaEVSecSig is the BaEV signal peptide, and the BaEVTM is the BaEV transmembrane domain.

2. The lentivirus according to claim 1, characterized in that The spacer unit includes conservative amino acids.

3. The lentivirus according to claim 2, characterized in that The spacer unit includes at least one of SAG, GAS, ASG, AGS, GSA and SGA.

4. The lentivirus according to claim 3, characterized in that The number of amino acids included in the spacer sequence ranges from 9 to 27.

5. The lentivirus according to claim 4, characterized in that The spacer sequence comprises at least one of SAGSAGSAGSAGSAG, GASGASGASGASGAS, ASGASGASGASGASG, AGSAGSAGSAGSAGSAGS, GSAGSAGSAGSAGSAGSA and SGASGASGASGASGASGA.

6. The lentivirus according to claim 1, characterized in that The insertion sites of the spacer sequence include positions 117 and 338 of the BaEV envelope glycoprotein amino acid sequence.

7. The lentivirus according to claim 1, characterized in that The BaEV envelope glycoprotein comprising the spacer sequence is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 1 to SEQ ID No.

6.

8. The lentivirus according to claim 1, characterized in that The recognition protein includes: receptor protein, antibody, single-chain recognition sequence transformed from antibody, nano antibody, lectin or molecular chaperone protein.

9. The lentivirus according to claim 1, characterized in that The recognition protein includes: protein A or protein G.

10. The lentivirus according to claim 1, characterized in that The recognition protein is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 7 and SEQ ID No.

8.

11. The lentivirus according to any one of claims 1 to 10, characterized in that The sequence of the BaEVSecSig is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of SEQ ID No.9; and / or, the sequence of the BaEVTM is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of SEQ ID No.

10.

12. A lentiviral expression vector, characterized in that: include: Expressing the nucleotide sequence of the lentivirus according to any one of claims 1 to 11.

13. The lentiviral expression vector according to claim 12, characterized in that: The nucleotide sequence of the spacer sequence expressing the BaEV envelope glycoprotein is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No.11 to SEQ ID No.

16.

14. The lentiviral expression vector according to claim 12 or 13, characterized in that: The nucleotide sequence expressing the recognition sequence of the BaEV envelope glycoprotein is derived from at least 25%, 30%, 40%, 50%, 60%, 70% or 80% of any one of SEQ ID No. 17 and SEQ ID No.

18.

15. A method for preparing a lentivirus, characterized in that: include: The packaging cells are inoculated into a culture container for cultivation; adding the vector plasmid, the packaging plasmid and the lentiviral expression vector into the cell culture medium to obtain a first solution; adding a transfection reagent to a cell culture medium and mixing to obtain a second solution; When the confluence of the cells to be packaged reaches a preset value, the first solution and the second solution are mixed and added into a culture container for transfection; After the transfection is completed, the lentivirus is obtained; Among them, the lentivirus includes: BaEV envelope glycoprotein; the extracellular region of the BaEV envelope glycoprotein includes at least one of the following sequences: a spacer sequence and a recognition sequence; the spacer sequence includes: a spacer unit; the recognition sequence includes: BaEVSecSig-recognition protein-BaEVTM; the BaEVSecSig is the BaEV signal peptide, and the BaEVTM is the BaEV transmembrane domain.

16. The method for preparing a lentivirus according to claim 15, characterized in that: The lentiviral expression vector comprises at least one of an amino acid sequence expressing a spacer sequence of the BaEV envelope glycoprotein and an amino acid sequence expressing a recognition sequence of the BaEV envelope glycoprotein.

17. The method for preparing a lentivirus according to claim 16, characterized in that: The lentiviral expression vector expressing the spacer sequence of the BaEV envelope glycoprotein is denoted as the first vector; the lentiviral expression vector expressing the recognition sequence of the BaEV envelope glycoprotein is denoted as the second vector; In the first solution, the lentiviral expression vector includes: the first vector and the second vector, and the ratio of the mass of the first vector to the mass of the second vector is in the range of 5-10.

18. The method for preparing a lentivirus according to claim 15, characterized in that: In the first solution, the mass ratio of the vector plasmid, the packaging plasmid and the lentiviral expression vector is (3-5):3:(1-3).

19. The method for preparing a lentivirus according to any one of claims 15 to 18, characterized in that: Before culturing the packaging cells in the culture container, the method further includes: modifying the coacervate microgel of the culture container.

20. The method for preparing a lentivirus according to claim 19, characterized in that: The coacervate microgel comprises folic acid and polycation electrolyte.

21. The method for preparing a lentivirus according to claim 19, characterized in that: The polycation electrolyte includes at least one of protamine sulfate and polylysine.

22. The method for preparing a lentivirus according to claim 19, characterized in that: The condensate microgel modification of the culture container comprises: mixing the folic acid solution with the polycation electrolyte solution to obtain a third solution; The third solution is applied to the culture container, and a phosphate solution is added to initiate gelation to obtain a culture container modified with coacervate microgel.

23. Use of the lentivirus according to any one of claims 1 to 11 in transfection of primary human NK cells.

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