CN116790675A - Recombinant lentiviral vector and application thereof in mammalian T cells - Google Patents

Abstract

The present application provides a recombinant lentiviral vector based on a plvx eukaryotic lentiviral expression vector comprising the elements of (a) an elongated Gag element replacing the Gag element in the plvx vector, downstream of the 5' LTR, the Gag element comprising the amino acid sequence as shown in SEQ ID NO: 6; (b) A murine stem cell virus MSCV promoter in a plvx vector for expressing a promoter of a gene sequence of interest, said MSCV promoter comprising a sequence as set forth in SEQ ID NO:3, a sequence shown in 3; (c) Replacing a C-terminal truncated WPRE element of a WPRE of an enhanced expression element in a plvx vector, said C-terminal truncated WPRE element comprising the amino acid sequence as set forth in SEQ ID NO: 4; (d) A bGH poly a tail replacing the SV40poly a tail in a plvx vector downstream of the truncated WPRE element, the bGH poly a tail comprising the amino acid sequence as set forth in SEQ ID NO: 5.

Description

Recombinant lentiviral vector and application thereof in mammalian T cells

Technical Field

The application relates to the field of biotechnology, and belongs to a novel lentivirus expression vector. The method provides an important tool for protein function research, cell line construction, gene and cell therapy and drug screening.

Background

Gene therapy and cell therapy are currently very popular treatments for body diseases internationally. The gene therapy mainly compensates, replaces and repairs defective genes in organisms by an exogenous gene introduction mode, so that the aim of treating diseases is fulfilled. Cell therapy is mainly to modify immune cells of a patient by genetic engineering and cell engineering means to enable the immune cells to more specifically identify target cells such as tumor cells, so that the effect of specifically killing the tumor cells is realized. Depending on the mode of gene delivery, gene therapy and cell therapy can be divided into viral and non-viral modes, with viral mode being the currently predominant mode of operation.

Lentiviruses are a class of retroviruses, one of the most common of which is derived from the HIV virus. The chemical nature of lentiviruses is single stranded RNA viruses whose genome comprises Gag expressing the coat protein and Pol expressing integrase and reverse transcriptase. Lentiviruses have a broad infectivity, can infect most mammalian cells, can obtain cDNA by reverse transcription after infecting the cells, and then randomly integrate the genome into the host genome to form a stably expressed cell line. At the same time, unlike gamma retroviruses, which can only infect dividing cells, lentiviruses can infect dividing and non-dividing cells at the same time, and thus lentiviruses have become a very important tool for gene therapy and cell therapy.

In order to continuously increase the safety of lentiviral vectors, lentiviral vectors have been seen to date for three generations. Currently, the second generation lentivirus system and the third generation lentivirus system are more commonly used. The second-generation lentiviral system is a three-plasmid system comprising an expression plasmid for expressing a target gene, a packaging plasmid for expressing tet-rev and an envelope plasmid for expressing envelope proteins. The third generation lentivirus system is further improved on the basis of the second generation, and the third generation system is independent of the Tet protein by deleting the U3 region at the 5' LTR end and fusing a promoter upstream. In addition, the removal of U3 at the 3' LTR end portion reduces its ability to self-replicate, while dividing Gag-pol and Rev into two plasmid expressions, further reducing the risk of wild-type virus formation.

Currently, the main vectors of the third generation of three plasmid lentivirus preparation systems comprise two helper plasmids expressing Gag-pol and VSVG envelope proteins, and expression plasmids containing genes of interest, such as GFP, luciferases, CD19 CAR, etc. Examples of promoters that are commonly used are EF1a, MSCV, PGK, etc. For human T lymphocytes, EF1a has highest efficiency, and generally can detect positive rate of 38% -63%, but culture screening is required for more than 15 days, and gene expression is low, so that the requirements of cell therapy and protein expression research cannot be met (Lentivirus-Mediated Gene Transfer and Expression in Established Human Tumor Antigen-Specific Cytotoxic T Cells and Primary Unstimulated T Cells, 2003).

To meet the market demand for high-expression lentiviral vectors, we have developed a novel lentiviral expression vector. The vector is characterized in that the infection efficiency of the lentiviral vector to primary human T lymphocytes can be further improved by increasing the length of Gag protein, replacing a new promoter, truncating WPRE, increasing termination signals at the downstream of 3' LTR and the like, so that the expression of a target gene can be further enhanced. The optimized vector can better meet the requirements of the drug research and development market on lentiviral vectors with high expression efficiency aiming at primary T lymphocytes.

Disclosure of Invention

The application designs a new recombinant lentiviral vector through the improvement and combination of elements. Aiming at the problem of low expression efficiency of the traditional lentiviral vector on the humanized T lymphocyte, the application can promote the expression of the lentiviral vector in the humanized T lymphocyte, thereby meeting the requirements of the cell and gene therapy field on the efficient lentiviral vector, further realizing the high expression of the target gene, and in addition, the application can reduce the use amount of the lentivirus under the condition of equal expression amount, further reduce the drug development cost and promote the safety. Specifically, the present application includes the following aspects:

1. a recombinant lentiviral vector based on a plvx eukaryotic cell lentiviral expression vector comprising the following elements:

(a) An extended Gag element, located at the 5' LTR, replacing the Gag element in the plvx vector

Downstream, the Gag element comprises the amino acid sequence as set forth in SEQ ID NO: 6;

(b) A murine stem cell virus MSCV promoter in a plvx vector for expressing a promoter of a gene sequence of interest, said MSCV promoter comprising a sequence as set forth in SEQ ID NO:

3, a sequence shown in 3;

(c) Substitution of C-terminal truncated WPRE of enhanced expression element WPRE in plvx vector

An element, said C-terminally truncated WPRE element comprising the amino acid sequence as set forth in SEQ ID NO: 4;

(d) A bGH poly a tail replacing the SV40poly a tail in a plvx vector downstream of the truncated WPRE element, the bGH poly a tail comprising the amino acid sequence as set forth in SEQ ID

NO: 5.

2. The recombinant lentiviral vector of item 1, further comprising:

(e) An optional gene sequence of interest, downstream of the MSCV promoter.

3. The recombinant lentiviral vector of item 1 or 2, constructed by a method comprising the steps of:

(i) Replacing the Gag element in the plvx vector with an elongated Gag element comprising an amino acid sequence as set forth in SEQ ID NO: 6;

(ii) Replacing the promoter for expressing the gene sequence of interest in the plvx vector with a murine stem cell virus MSCV promoter comprising the amino acid sequence as set forth in SEQ ID NO:

3, a sequence shown in 3;

(iii) Replacing the WPRE of the enhanced expression element in the plvx vector with a shorter C-terminal truncated WPRE comprising the amino acid sequence as set forth in SEQ ID NO:

4;

(iv) Replacement of the SV40poly A tail in the plvx vector with a bGH poly A tail comprising the amino acid sequence as set forth in SEQ ID NO: 5.

4. The recombinant lentiviral vector of item 3, wherein the method further comprises the steps of:

(v) An optional gene sequence of interest is inserted downstream of the MSCV promoter.

5. The recombinant lentiviral vector of any one of claims 1 to 4, wherein the plvx eukaryotic lentiviral expression vector comprises the sequence set forth in SEQ ID NO:1, and a sequence shown in 1.

6. The recombinant lentiviral vector of any one of claims 1 to 5, having a structure as shown in FIG. 1.

7. The recombinant lentiviral vector of any one of claims 1 to 6, wherein the recombinant lentiviral vector comprises a sequence as set forth in SEQ ID NO:2, and a sequence shown in seq id no.

8. A lentiviral particle comprising the recombinant lentiviral vector of any one of claims 1-7.

9. The lentiviral particle of claim 7, packaged by a 293T packaged lentiviral system.

10. A recombinant cell obtained by transfecting a host cell with the recombinant lentiviral vector of any one of claims 1 to 7.

11. The recombinant cell of claim 10, which is obtained by transfecting a host cell selected from the group consisting of: primary T cells, immortalized T cells, T isolated from tumor tissue

Cells, congenital lymphocytes (ILC).

12. A pharmaceutical composition comprising the recombinant lentiviral vector of any one of claims 1-7 and/or the recombinant cell of claim 10 or 11.

13. The pharmaceutical composition of claim 13, further comprising a pharmaceutically acceptable additive.

14. A kit comprising the recombinant lentiviral vector of any one of items 1 to 7 and +.

Or the recombinant cell of item 10 or 11 and/or the pharmaceutical composition of item 12 or 13.

15. A method of transfecting a cell comprising the steps of:

(1) Contacting the cell with the recombinant lentiviral vector of any one of claims 1 to 7, and transfecting the cell.

16. The method of item 15, wherein the cell is selected from the group consisting of: primary T cells, immortals

Biochemical T cells, T cells isolated from tumor tissue, and congenital lymphocytes (ILCs).

17. A method of constructing a recombinant lentiviral vector comprising the steps of:

(i) Replacing the Gag element in the plvx vector with an elongated Gag element comprising an amino acid sequence as set forth in SEQ ID NO: 6;

(ii) Replacing the promoter for expressing the gene sequence of interest in the plvx vector with a murine stem cell virus MSCV promoter comprising the amino acid sequence as set forth in SEQ ID NO:

3, a sequence shown in 3;

(iii) Replacing the WPRE of the enhanced expression element in the plvx vector with a shorter C-terminal truncated WPRE comprising the amino acid sequence as set forth in SEQ ID NO:

4;

(iv) Replacement of the SV40poly A tail in the plvx vector with a bGH poly A tail comprising the amino acid sequence as set forth in SEQ ID NO: 5.

18. The method of item 17, further comprising the step of:

(v) An optional gene sequence of interest is inserted downstream of the MSCV promoter.

Drawings

FIG. 1 is a schematic representation of an expression vector, wherein a is wild-type vector WT-112 and b is optimized vector Mut-114 of the application.

FIG. 2 is a gradient dilution map of lentivirus WT-112.

FIG. 3 is a graph of post-optimization lentiviral Mut-114 gradient dilutions.

FIG. 4 is a statistical chart of the Luciferase assay 72h after T cell virus infection.

FIG. 5 is a statistical chart of the Luciferase assay 96h after T cell virus infection.

FIG. 6 is a schematic diagram of a carrier of one embodiment of a generic carrier of the present application.

Detailed Description

The practice of the present application will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, organic chemistry, molecular biology, microbiology, recombinant DNA technology, genetics, immunology and cell biology, which are within the skill of the art. A description of these methods can be found, for example, in Sambrook et al, molecular Cloning: A Laboratory Manual (3 rd edition, 2001); sambrook et al, molecular Cloning: A Laboratory Manual (2 nd edition, 1989); maniatis et al, molecular Cloning: A Laboratory Manual (1982); ausubel et al Current Protocols in Molecular Biology (John Wiley and Sons, updated 7 in 2008); short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols in Molecular Biology Greene Pub.associates and Wiley-Interscience; glover, DNA Cloning: A Practical Approach, vol.I & II (IRL Press, oxford, 1985); anand, techniques for the Analysis of Complex Genomes, (Academic Press, new York, 1992); transcription and Translation (b.hames & s.higgins, eds., 1984); perbal, A Practical Guide to Molecular Cloning (1984); harlow and Lane, antibodies, (Cold Spring Harbor Laboratory Press, cold Spring Harbor, n.y., 1998) Current Protocols in Immunology Q.E.Coligan, A.M.Kruisbeek, D.H.Margulies, E.M.Shevach and w.strober, eds., 1991); annual Review of Immunology; and journal monographs such as Advances in Immunology.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of the present application, the following terms are defined below.

In the present application, the term "lentiviral vector" refers to a vector comprising structural and functional genetic elements that are predominantly derived from the outside of the LTR of a lentivirus. Lentiviral vectors are capable of providing efficient delivery, integration and long-term expression of transgenes into non-dividing cells in vitro and in vivo. Various lentiviral vectors are known in the art, see Naldini et al (1996 a,1996b and 1998); zufferey et al, (1997); dull et al, 1998, U.S. Pat. Nos. 6,013,516 and 5,994,136.

In the present application, the term "lentiviral particle" is intended to mean a viral particle comprising an envelope, having one or more characteristics of a lentivirus, and capable of invading a target host cell. Such characteristics include, for example, infection of a non-dividing host cell; transducing a non-dividing host cell; infecting or transducing a host immune cell; a lentiviral virion comprising one or more of Gag structural polypeptides p7, p24 and p 17; a lentiviral envelope comprising one or more of the glycoproteins p41, p120 and p160 encoded by env; a genome comprising one or more lentivirus cis-acting sequences that act on replication, proviral integration or transcription; a genome comprising a gene encoding a lentiviral protease, a reverse transcriptase or an integrase; or contains a genome encoding a regulatory activity, such as Tat or Rev. The transfer plasmid may comprise a cPPT sequence as described in us patent No. 8,093,042.

In the present application, the term "nucleotide" refers herein to a monomeric unit of DNA or RNA consisting of a sugar moiety (pentose), a phosphate and a nitrogen-containing heterocyclic base. The base is linked to the sugar moiety via a glycosidic carbon (the 1' carbon of the pentose) and this combination of base and sugar is a nucleoside. When a nucleoside contains a phosphate group bonded to the 3 'or 5' position of pentose, it is referred to as a nucleotide. The sequence of polymeric operably linked nucleotides is generally referred to herein as a "base sequence", "nucleotide sequence" or a nucleic acid or polynucleotide "strand" and is represented herein by its chemical formula in the conventional direction from left to right, from the 5 'end to the 3' end, which refer to the terminal 5 'phosphate group and the terminal 3' hydroxyl group at the "5'" and "3'" ends of the polymer sequence, respectively.

In the present application, the terms "oligonucleotide", "polynucleotide" and "nucleic acid" refer herein to a molecule comprising two or more, preferably more than three, deoxyribonucleotides and/or ribonucleotides. Its exact size depends on many factors, which in turn depend on the final function or use of the oligonucleotide. Oligonucleotides may be synthesized or obtained by cloning or from natural (e.g., genomic) sources. As used herein, the term "polynucleotide" refers to a polymer molecule composed of nucleotide monomers covalently bonded in chain form. DNA (deoxyribonucleic acid) and RNA (ribonucleic acid) are examples of polynucleotides.

In the present application, the term "primer" refers herein to an oligonucleotide, whether occurring naturally or synthetically produced, which is capable of acting as a starting point for nucleic acid synthesis when placed under conditions that induce synthesis of primer extension products complementary to a nucleic acid strand, e.g., in the presence of four different nucleotide triphosphates and a polymerase, e.g., a thermostable enzyme, in an appropriate buffer ("buffer" includes pH, ionic strength, cofactor, etc.) and at an appropriate temperature. For maximum efficiency in amplification, the primer is preferably single stranded, but alternatively double stranded. If double-stranded, the primer is first treated to separate its strand and then used to prepare an extension product. Preferably, the primer is an oligodeoxynucleotide. The primer must be long enough to initiate synthesis of the extension product in the presence of a polymerase, such as a thermostable polymerase. The exact length of the primer depends on many factors, including temperature, primer source, and method of use. For example, an oligonucleotide primer typically contains 15-25 nucleotides, depending on the complexity of the target sequence, but it may contain more or fewer nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybridization complexes with the template.

As used herein, the term "recombinant lentiviral vector" or "recombinant LV") refers to an artificially created polynucleotide vector that is assembled from LV and a plurality of additional segments due to human intervention and manipulation.

In this document, the expression "comprising" includes the meaning of "comprising" and "consisting of …".

Various aspects of the application are described in detail below. The following description is not intended to be limiting, and those skilled in the art can make any modifications and substitutions based on the technical knowledge in the art without significantly impeding the technical effects of the present application.

The present application provides a recombinant lentiviral vector based on a plvx eukaryotic lentiviral expression vector, characterized in that the corresponding pro-element in the plvx vector is replaced with an improved element selected from the group consisting of (a) an elongated Gag element replacing the Gag element in the plvx vector, downstream of the 5' LTR, said Gag element comprising an amino acid sequence as shown in SEQ ID NO: 6; (b) A murine stem cell virus MSCV promoter in a plvx vector for expressing a promoter of a gene sequence of interest, said MSCV promoter comprising a sequence as set forth in SEQ ID NO:3, a sequence shown in 3; (c) Replacing a C-terminal truncated WPRE element of a WPRE of an enhanced expression element in a plvx vector, said C-terminal truncated WPRE element comprising the amino acid sequence as set forth in SEQ ID NO: 4; (d) A bGH poly a tail replacing the SV40poly a tail in a plvx vector downstream of the truncated WPRE element, the bGH poly a tail comprising the amino acid sequence as set forth in SEQ ID NO: 5. It should be noted that "replacing" herein refers to replacing the original element with the modified element, and it is not necessary to completely and accurately replace the entire sequence of the original element with the sequence of the modified element, for example, when the sequence of the original element is removed, a certain length of sequence before and after the removal may be removed, as long as the functions of the upstream and downstream elements are not affected. The definition and specific sequence of primordial elements are well known to those skilled in the art, and reference may be made, for example, to the specifications of various commercial pLVX vectors (e.g., the TAKARA company pLVX-Puro vector), which are incorporated herein by reference. In one embodiment, the recombinant lentiviral vector is a universal vector tool for expressing any gene sequence of interest, which may include a region downstream of the MSCV promoter for insertion of any gene sequence of interest. In one embodiment, the region for insertion of any gene sequence of interest has any number of restriction sites. In one embodiment, it further comprises: (e) An optional gene sequence of interest, which is located downstream of the MSCV promoter, is not subject to any restrictions and can be selected according to the needs of those skilled in the art. In one embodiment the gene sequence of interest is a gene sequence encoding a protein. The target gene sequence is inserted downstream of the MSCV promoter so as to be expressed in a cell under the control of the MSCV promoter. In one embodiment, the recombinant lentiviral vector may further comprise a desired element such as a resistance gene sequence, a marker gene sequence, etc., which may be selected according to the actual situation for the person skilled in the art, for convenience of operation, without affecting the expression function. For example, the resistance gene sequences may be as follows: puromycin. In the present application, "pLVX eukaryotic lentiviral expression vector" or "pLVX vector" refers to a lentiviral expression vector type commonly used in the art, having a general backbone, as is well known in the art, and reference may be made, for example, to the specification of the TAKARA company pLVX-Puro vector, which specification is incorporated herein by reference. In one embodiment, the generic skeleton has the following features: 1, comprising an RSV or CMV promoter in the 5'ltr region, downstream of the 5' ltr a truncated Gag sequence linked to the lentiviral packaging signal HIV-1 ψ;2, the REV response sequence RRE is arranged at the downstream, which has the effect of promoting the expression of exogenous genes, and the cPPT/CTS sequence is connected at the rear, so that the transduction efficiency is improved and the transgene expression is promoted; 3, optionally followed by a promoter for expression of the gene of interest (typically a mammalian promoter such as EF1a, etc.) and optionally the gene of interest; 4 having a full length WPRE sequence downstream of the promoter and gene of interest; 5, downstream of WPRE is LTR from which part of the U3 region was removed, and also contains a SV40 termination sequence. "plvx eukaryotic lentiviral expression vector" or "plvx vector" comprises any series of vectors derived from the backbone, for example: pLVX-M-Puro from adedge (Plasmid # 125839), pLVX-Puro vector from NOVOPro company (V010426 #). In one embodiment, the plvx eukaryotic lentiviral expression vector comprises the sequence set forth in SEQ ID NO:1, and a sequence shown in 1. In one embodiment, the recombinant lentiviral vector has a structure as shown in FIG. 1. In a preferred embodiment, the recombinant lentiviral vector comprises a sequence as set forth in SEQ ID NO:2, and a sequence shown in seq id no.

In one aspect, the application also provides a lentiviral particle comprising a recombinant lentiviral vector of the application. The lentiviral particles may be packaged by any lentiviral packaging system without particular limitation, and may be selected as desired by one skilled in the art. In one embodiment, the packaging system may be, for example, a 293T packaging system, packaged with PMD2.G and PSPAX2 helper plasmids or other third generation helper plasmids.

In one aspect, the application provides a recombinant cell obtained by transfecting a host cell with a recombinant lentiviral vector of the application. The host cell is not particularly limited, and any cell derived from various organisms may be used. From the viewpoint of the technical effects of the recombinant lentiviral vector of the present application that have been verified, it is preferable that the host cell is a T cell. In one embodiment, the recombinant cell is obtained by infecting a cell selected from the group consisting of: primary T cells, immortalized T cells, T cells isolated from tumor tissue, and Innate Lymphocytes (ILCs). Here, primary T cells refer to: t cells isolated from animal tissue, which have not undergone any gene editing, cannot proliferate indefinitely in vitro; herein are such cells as used in the examples; immortalized T cells refer to: transforming primary T cells in vitro by viral transduction or other means to obtain cells with in vitro unlimited proliferation capacity; t cells isolated from tumor tissue refer to: culturing tumor tissue in vitro, allowing proliferation of T cells, and rapidly amplifying obtained T cells by trophoblast such as PBMC; the innate lymphocytes refer to: also known as innate immune cells, are a subset of lymphocytes, distinct from T cells and B cells, located in the intestinal mucosal surface, enhance immune responses, maintain mucosal integrity and promote lymphoid organogenesis.

In one aspect, the application also provides a method of transfecting a cell comprising the steps of: the cells are transfected by contacting the cells with the recombinant lentiviral vector of the application. Other specific transfection methods are well known to those skilled in the art and may be arbitrarily selected as desired.

In one aspect, the application also provides a pharmaceutical composition comprising a recombinant lentiviral vector of the application and/or a cell of the application. In one embodiment, the pharmaceutical composition may further comprise pharmaceutically acceptable additives. In one embodiment, the application provides a composition that may comprise a recombinant lentiviral vector of the application and/or a cell of the application, and a pharmaceutically acceptable additive. The pharmaceutically acceptable additives may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and, or nonionic surfactants, and the like. In one embodiment of the application, the pharmaceutical composition may be formulated for oral administration, intravenous administration (e.g., intravenous injection, i.v.), intramuscular administration (e.g., intramuscular injection, i.m.), in situ administration at the tumor site, inhalation, rectal administration, vaginal administration, transdermal administration, or administration through a subcutaneous depot.

In one embodiment, the application also provides a kit comprising the recombinant lentiviral vector of the application and/or the recombinant cell of the application and/or the pharmaceutical composition of the application.

In one aspect, the present application also provides a method for constructing the recombinant lentiviral vector of the present application, comprising the steps of:

(i) Replacing the Gag element in the plvx vector with an elongated Gag element comprising an amino acid sequence as set forth in SEQ ID NO: 6;

(ii) Replacing the promoter for expressing the gene sequence of interest in the plvx vector with a murine stem cell virus MSCV promoter comprising the amino acid sequence as set forth in SEQ ID NO:3, a sequence shown in 3;

(iii) Replacing the WPRE of the enhanced expression element in the plvx vector with a shorter C-terminal truncated WPRE comprising the amino acid sequence as set forth in SEQ ID NO: 4;

(iv) Replacement of the SV40poly A tail in the plvx vector with a bGH poly A tail comprising the amino acid sequence as set forth in SEQ ID NO: 5.

In one embodiment, the construction method further comprises the steps of:

(v) An optional gene sequence of interest is inserted downstream of the MSCV promoter.

The method may be a sequence design method or a method of modifying an existing vector. There is no necessary limitation in order between the steps, and the steps may be arbitrarily arranged as needed.

Examples

The materials used in the examples are as follows:

experimental materials

2.2 experimental facility:

example 1: vector construction

WT-112 vector construction:

the use of a suitable expression vector is critical for successful expression of the exogenous gene, and it is necessary to provide, as a mammalian cell expression vector capable of high expression suitable for industrial scale applications: strong promoters and efficient transcription termination sequences; introns or elements that enhance expression. The control vector of the application is to clone the designed gene luciferase (luciferase) onto a universal plvx eukaryotic cell lentiviral expression vector (shown as SEQ ID NO:1, purchased from Boteng Bio-pharmaceutical Co., ltd.) to obtain a vector shown as SEQ ID NO: WT-112 vector shown in FIG. 7.

Mut-114 vector construction

The inventors of the present application have modified the above-described vector in order to obtain a mammalian cell expression vector capable of high expression suitable for industrial scale application. The vector of the application is used for modifying the control lentiviral vector, and is technically characterized by comprising the following steps:

(a) An elongated Gag element downstream of the 5' LTR that replaces the Gag element in plvx-WT-112, said Gag element comprising the amino acid sequence as set forth in SEQ ID NO: 6;

(b) A murine stem cell virus MSCV promoter replacing the human extended growth factor promoter EF1a in plvx-WT-112, said MSCV promoter comprising the amino acid sequence as set forth in SEQ ID NO:

3, a sequence shown in 3;

(c) Replacing a C-terminal truncated WPRE element of the enhanced expression element WPRE in plvx-WT-112, said C-terminal truncated WPRE element comprising the amino acid sequence as set forth in SEQ ID NO:

4;

(d) A bGH poly a tail containing substitution of SV40poly a tail in plvx-WT-112 downstream of the truncated WPRE element, said bGH poly a tail comprising a sequence as set forth in seq id NO: 5.

The pattern of the vector Mut-114 is shown in FIG. 1B, and the sequence of the vector Mut-114 is shown in SEQ ID NO:8 (FIG. 6 shows a map of one embodiment of Mut-114 without luciferases inserted, one embodiment of which is shown in SEQ ID NO: 2). The vector uses whole-gene chemical synthesis, entrusts the whole-gene chemical synthesis of the Sonchus biological pharmaceutical Co., ltd according to the designed sequence, and is verified to be correct by sequencing.

Virus preparation and titer detection

Virus preparation, purification and titre detection were carried out by Sonchus Boteng Biopharmaceutical Co.

Example 2: preparation of virus-infected T cells

Recovery and activation of CD3T cells

CD3T cells (Miaoshun, PB 03-N-2C) were purchased from Miaoshun (Shanghai) Biotechnology Inc.

The water bath is opened in advance, and the temperature is adjusted to be 37 ℃ for preheating.

2 CD3T cells are taken out of a liquid nitrogen tank, transferred into a foam box containing dry ice, sterilized and then transferred into a cell-cell transfer window.

CD3T cells were rapidly thawed in a pre-heated 37 ℃ water bath and transferred to a clean 15ml centrifuge tube containing 10ml T cell complete media () and mixed well.

Centrifuge at room temperature, 400g,10min.

The supernatant was discarded and the cell pellet was resuspended in 8ml T cell complete medium and counted.

Cell density was adjusted to 1X10 with T cell complete medium ⁶ And each ml.

Mu.l of activating beads (gold Style, L00899) were added according to 1E6/ml, gently mixed, and the cells were plated in 6-well plates at 5.0ml per well.

Cells were incubated in an incubator at 37℃with 5% CO2 for 48h.

Lentivirus infection of T cells

Cell counting, collecting cells in a 15ml centrifuge tube, centrifuging 500g for 6min;

respectively adjusting the cell density to 2E6 cells/ml, adding polybrene, adjusting the final concentration to 8 mug/ml, adding 200 μl of cell suspension (4E 5 cells/well) to each well, and adding 5 groups of time points of 6 gradients in total +4=64 wells;

moi=2, moi=5, moi=10, moi=20, moi=30 infection of CD3T cells, respectively, viral gradient dilutions are shown in fig. 2, fig. 3;

200 μl of virus dilution was added per well;

centrifuging at 32 ℃ at 1500rpm for 90min;

after centrifugation, the cells were incubated in an incubator at 37℃with 5% CO 2.

24h liquid change for virus infection

After 24h of virus infection, the virus-infected cell plates were centrifuged at 400g for 6min;

gently aspirate 300 μl of supernatant with a pipette and discard;

complete medium was supplemented with 500 μ l T cells per well.

Cells were incubated in an incubator at 37℃with 5% CO 2.

Luciferase assay

Counting each group of cells, taking 5.5E5 cells from each group into a new 96-well plate, and adjusting the cell density to be consistent by using a T cell complete culture medium;

cells were assayed by Luciferase at 72h and 96h after virus infection, 1.5E5 cells were taken from each group into a white plate, 3 replicates of each group, and an equal volume of BIO-Glo was added, incubated at room temperature for 10min in the absence of light, and assayed by a multifunctional microplate reader (Thermo, varioskan LUX). The magnitude of the Luciferase value represents the magnitude of the ability of the virus to infect CD3T cells. The group with moi=0 served as a non-viral infection control group. 72h,96h Luciferase detection experiments are shown in FIG. 4 and FIG. 5, respectively.

From the experimental results, it can be concluded that the engineered mutant (mutant) lentiviral vector of the application has higher expression level of the target gene in primary human T lymphocytes.

The sequences used in the present application are as follows:

SEQ ID NO：1

agcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgc

cttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttatt

aggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattg

tatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctct

ggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgccc

gtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtg

gcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggct

tgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactag

cggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcg

atgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaa

gcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatac

tgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaac

cctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaag

agcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagat

atgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcac

ccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttcct

tgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccaga

caattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgtt

gcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaag

gatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatg

ctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaa

ttaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaaca

agaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatat

aaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaata

gagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacagg

cccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacgga

tctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaat

agtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaatttta

tcgatcacgagactagcctcgagaagcttgatatcgaattccaccgtgaggctccggtgcccgtcagtg

ggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtg

cctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgag

ggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgcc

agaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtg

ccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggt

gggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggc

gctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctag

ccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaaga

tctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacat

gttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggc

cggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggccc

ggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatg

gaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgt

cctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcga

gcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgg

gtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggat

cttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaggatccgc

cacctgaagcggccgcgtcgactaaaatcaacctctggattacaaaatttgtgaaagattgactggtatt

cttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgta

tggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggc

aacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtc

agctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcc

cgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcc

tttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggcc

ctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttc

gccctcagacgagtcggatctccctttgggccgcctccccgcctggaattcgagctcggtacctttaag

accaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggct

aattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagc

ctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaa

gtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaa

aatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcagaga

gtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaata

aagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagct

atcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggct

gactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgagga

ggcttttttggaggcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggc

cgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccc

cctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcct

gaatggcgaatgggaggtggcacttttcggggaaatgtgtgcagctctggcccgtgtctcaaaatctct

gatgttacattgcacaagataaaaatatatcatcatgaacaataaaactgtctgcttacataaacagtaata

caaggggtgttatgagccatattcaacgggaaacgtcgaggccgcgattaaattccaacatggatgctg

atttatatgggtataaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgcttgtatggg

aagcccgatgcgccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgag

atggtcagactaaactggctgacggaatttatgccacttccgaccatcaagcattttatccgtactcctgat

gatgcatggttactcaccactgcgatccccggaaaaacagcgttccaggtattagaagaatatcctgatt

caggtgaaaatattgttgatgcgctggcagtgttcctgcgccggttgcactcgattcctgtttgtaattgtc

cttttaacagcgatcgcgtatttcgcctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcg

agtgattttgatgacgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgcc

attctcaccggattcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaatta

ataggttgtattgatgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaact

gcctcggtgagttttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaat

aaattgcagtttcatttgatgctcgatgagtttttctaactgtcagaccaagtttactcatatatactttagatt

gatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatccct

taacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttt

tttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatca

agagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctag

tgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctg

ttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccg

gataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgac

ctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaag

gcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggg

gaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctc

gtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctg

gccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgag

ctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagag

cgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggttt

cccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcacccca

ggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaa

cagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgc

a

SEQ ID NO：2

agcttaagacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagccca

tatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgc

ccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggt

ggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattg

acgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttgg

cagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg

atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcacc

aaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgt

gtacggtgggaggtctatataagcaggggtctctctggttagaccagatctgagcctgggagctctctg

gctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgt

ctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtgg

cgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggctt

gctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagc

ggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcga

tgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaag

cagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatact

gggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaac

cctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaag

agcaaaacaaaagtaagaaaaaagcacagcaagcagcagctgacacaggacacagcagccaggtc

agccaaaattaccctatagtgcagaacatccaggggcaaatggtacatcaggccatatcacctagaact

ttaaatgcatgggtaaaagtagtagaagagaaggctttcagcccagaagtgatacccatgttttcagcatt

atcagaaggagccaccccacaagatttaaacaccatgctaaacacagtggggggacatcaagcagcc

atgcaaatgttaaaagagaccatcaatgaggaagctgcagaatgggatagagtgcatccagtgcatgc

agggcctattgcaccaggccagatgagagaaccaaggggaagtgacatagcaggaactactagtacc

aagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcag

cgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgct

gagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaa

gaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaac

tcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcaca

cgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatc

gcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattg

gtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaaga

atagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacct

cccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacaga

gacagatccattcgattagtgaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattg

gggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattaca

aaaacaaattacaaaaattcaaaattttatcgatcacgagactagcctcgagaagcttgatatcgaattcc

accgtgagtgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatg

gaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgg

gccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccag

atgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaa

tgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgag

ctcaataaaagagcccacaacccctcactcggcgcgccagtcctccgatagactgcgtcgcccgggta

cccgtattcccaataaagcctcttgctgtttgcatccgaatcgtggactcgctgatccttgggagggtctc

ctcagattgattgactgcccacctcgggggtctttcattggatccgccacctgaagcggccgcgtcgact

aaaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgcta

tgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtata

aatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtg

tttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgcttt

ccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggc

tgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctgtgtt

gccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttcctt

cccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccact

ttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttg

tactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgctt

aagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactag

agatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgtcatcttattattcagt

atttataacttgcaaagaaatgaatatcagagagtgagaggctgtgccttctagttgccagccatctgttgt

ttgcccctcccccgtgccttccttgaccctggaaggtgccactcccactgtcctttcctaataaaatgagg

aaattgcatcgcattgtctgagtaggtgtcattctattctggggggtggggtggggcaggacagcaagg

gggaggattgggaagagaatagcaggcatgctggggatcatgtctggctctagctatcccgcccctaa

ctccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaatttttttta

tttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggag

gcctagggacgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaa

cgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagct

ggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatg

ggaggtggcacttttcggggaaatgtgtgcagctctggcccgtgtctcaaaatctctgatgttacattgca

caagataaaaatatatcatcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttat

gagccatattcaacgggaaacgtcgaggccgcgattaaattccaacatggatgctgatttatatgggtat

aaatgggctcgcgataatgtcgggcaatcaggtgcgacaatctatcgcttgtatgggaagcccgatgc

gccagagttgtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagacta

aactggctgacggaatttatgccacttccgaccatcaagcattttatccgtactcctgatgatgcatggtta

ctcaccactgcgatccccggaaaaacagcgttccaggtattagaagaatatcctgattcaggtgaaaata

ttgttgatgcgctggcagtgttcctgcgccggttgcactcgattcctgtttgtaattgtccttttaacagcga

tcgcgtatttcgcctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatg

acgagcgtaatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccattctcaccgga

ttcagtcgtcactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattg

atgttggacgagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagt

tttctccttcattacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttc

atttgatgctcgatgagtttttctaactgtcagaccaagtttactcatatatactttagattgatttaaaacttca

tttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttc

gttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaat

ctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaac

tctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagtt

aggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggct

gctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgca

gcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaact

gagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggta

tccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggt

atctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggc

ggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcac

atgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctc

gccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacg

caaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaa

agcgggcagtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacacttt

atgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgacc

atgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgca

SEQ ID NO：3

tgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatggaaa

atacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgggccaa

acaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccagatgcgg

tcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaatgaccc

tgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgagctcaat

aaaagagcccacaacccctcactcggcgcgccagtcctccgatagactgcgtcgcccgggtacccgt

attcccaataaagcctcttgctgtttgcatccgaatcgtggactcgctgatccttgggagggtctcctcag

attgattgactgcccacctcgggggtctttcatt

SEQ ID NO：4

aatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacg

ctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgt

ataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcact

gtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcg

ctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctc

ggctgttgggcactgacaattccgtggtgttgtcggggaagctgacgtcctttccatggctgctcgcctg

tgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttc

cttcccgc

SEQ ID NO：5

ctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccctggaaggt

gccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtcattctattc

tggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggcatgctgg

gga

SEQ ID NO：6

atgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggtt

aaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacga

ttcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccat

cccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaa

aggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaag

aaaaaagcacagcaagcagcagctgacacaggacacagcagccaggtcagccaaaattaccctata

gtgcagaacatccaggggcaaatggtacatcaggccatatcacctagaactttaaatgcatgggtaaaa

gtagtagaagagaaggctttcagcccagaagtgatacccatgttttcagcattatcagaaggagccacc

ccacaagatttaaacaccatgctaaacacagtggggggacatcaagcagccatgcaaatgttaaaaga

gaccatcaatgaggaagctgcagaatgggatagagtgcatccagtgcatgcagggcctattgcaccag

gccagatgagagaaccaaggggaagtgacatagcaggaactactagtacc

SEQ ID NO：7

agcttaatgtagtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgc

cttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttatt

aggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcattgcagagatattg

tatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctct

ggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgccc

gtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtg

gcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggct

tgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactag

cggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcg

atgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaa

gcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatac

tgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaac

cctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaag

agcaaaacaaaagtaagaccaccgcacagcaagcggccgctgatcttcagacctggaggaggagat

atgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcac

ccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttcct

tgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccaga

caattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgtt

gcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaag

gatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatg

ctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaa

ttaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaaca

agaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatat

aaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaata

gagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacagg

cccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacgga

tctcgacggtatcggttaacttttaaaagaaaaggggggattggggggtacagtgcaggggaaagaat

agtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaatttta

tcgatcacgagactagcctcgagaagcttgatatcgaattccaccgtgaggctccggtgcccgtcagtg

ggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgaaccggtg

cctagagaaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgag

ggtgggggagaaccgtatataagtgcagtagtcgccgtgaacgttctttttcgcaacgggtttgccgcc

agaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacgggttatggcccttgcgtg

ccttgaattacttccacgcccctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggt

gggagagttcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcttgggc

gctggggccgccgcgtgcgaatctggtggcaccttcgcgcctgtctcgctgctttcgataagtctctag

ccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtcttgtaaatgcgggccaaga

tctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacat

gttcggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggc

cggcctgctctggtgcctggcctcgcgccgccgtgtatcgccccgccctgggcggcaaggctggccc

ggtcggcaccagttgcgtgagcggaaagatggccgcttcccggccctgctgcagggagctcaaaatg

gaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccgt

cctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcga

gcttttggagtacgtcgtctttaggttggggggaggggttttatgcgatggagtttccccacactgagtgg

gtggagactgaagttaggccagcttggcacttgatgtaattctccttggaatttgccctttttgagtttggat

cttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgaggatccgc

cacctgaagcgccaccatggaagacgccaaaaacataaagaaaggcccggcgccattctatccgctg

gaagatggaaccgctggagagcaactgcataaggctatgaagagatacgccctggttcctggaacaat

tgcttttacagatgcacatatcgaggtggacatcacttacgctgagtacttcgaaatgtccgttcggttgg

cagaagctatgaaacgatatgggctgaatacaaatcacagaatcgtcgtatgcagtgaaaactctcttca

attctttatgccggtgttgggcgcgttatttatcggagttgcagttgcgcccgcgaacgacatttataatga

acgtgaattgctcaacagtatgggcatttcgcagcctaccgtggtgttcgtttccaaaaaggggttgcaa

aaaattttgaacgtgcaaaaaaagctcccaatcatccaaaaaattattatcatggattctaaaacggattac

cagggatttcagtcgatgtacacgttcgtcacatctcatctacctcccggttttaatgaatacgattttgtgc

cagagtccttcgatagggacaagacaattgcactgatcatgaactcctctggatctactggtctgcctaa

aggtgtcgctctgcctcatagaactgcctgcgtgagattctcgcatgccagagatcctatttttggcaatc

aaatcattccggatactgcgattttaagtgttgttccattccatcacggttttggaatgtttactacactcgga

tatttgatatgtggatttcgagtcgtcttaatgtatagatttgaagaagagctgtttctgaggagccttcagg

attacaagattcaaagtgcgctgctggtgccaaccctattctccttcttcgccaaaagcactctgattgaca

aatacgatttatctaatttacacgaaattgcttctggtggcgctcccctctctaaggaagtcggggaagcg

gttgccaagaggttccatctgccaggtatcaggcaaggatatgggctcactgagactacatcagctattc

tgattacacccgagggggatgataaaccgggcgcggtcggtaaagttgttccattttttgaagcgaagg

ttgtggatctggataccgggaaaacgctgggcgttaatcaaagaggcgaactgtgtgtgagaggtccta

tgattatgtccggttatgtaaacaatccggaagcgaccaacgccttgattgacaaggatggatggctaca

ttctggagacatagcttactgggacgaagacgaacacttcttcatcgttgaccgcctgaagtctctgatta

agtacaaaggctatcaggtggctcccgctgaattggaatccatcttgctccaacaccccaacatcttcga

cgcaggtgtcgcaggtcttcccgacgatgacgccggtgaacttcccgccgccgttgttgttttggagca

cggaaagacgatgacggaaaaagagatcgtggattacgtcgccagtcaagtaacaaccgcgaaaaa

gttgcgcggaggagttgtgtttgtggacgaagtaccgaaaggtcttaccggaaaactcgacgcaagaa

aaatcagagagatcctcataaaggccaagaagggcggaaagatcgccgtgtaaggccgcgtcgacta

aaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctat

gtggatacgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataa

atcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgt

ttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttc

cccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggct

gttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgc

cacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcc

cgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcggatctcc

ctttgggccgcctccccgcctggaattcgagctcggtacctttaagaccaatgacttacaaggcagctgt

agatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaag

atctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactag

ggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgt

gactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtagtagttcatgt

catcttattattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcag

cttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagt

tgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcccgcccctaactccgcccat

cccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcaga

ggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggga

cgtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgact

gggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaata

gcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggaggtgg

cacttttcggggaaatgtgtgcagctctggcccgtgtctcaaaatctctgatgttacattgcacaagataa

aaatatatcatcatgaacaataaaactgtctgcttacataaacagtaatacaaggggtgttatgagccatat

tcaacgggaaacgtcgaggccgcgattaaattccaacatggatgctgatttatatgggtataaatgggct

cgcgataatgtcgggcaatcaggtgcgacaatctatcgcttgtatgggaagcccgatgcgccagagtt

gtttctgaaacatggcaaaggtagcgttgccaatgatgttacagatgagatggtcagactaaactggctg

acggaatttatgccacttccgaccatcaagcattttatccgtactcctgatgatgcatggttactcaccact

gcgatccccggaaaaacagcgttccaggtattagaagaatatcctgattcaggtgaaaatattgttgatg

cgctggcagtgttcctgcgccggttgcactcgattcctgtttgtaattgtccttttaacagcgatcgcgtatt

tcgcctcgctcaggcgcaatcacgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgt

aatggctggcctgttgaacaagtctggaaagaaatgcataaacttttgccattctcaccggattcagtcgt

cactcatggtgatttctcacttgataaccttatttttgacgaggggaaattaataggttgtattgatgttggac

gagtcggaatcgcagaccgataccaggatcttgccatcctatggaactgcctcggtgagttttctccttca

ttacagaaacggctttttcaaaaatatggtattgataatcctgatatgaataaattgcagtttcatttgatgct

cgatgagtttttctaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattta

aaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactg

agcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgctt

gcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccg

aaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccacc

acttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagt

ggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcggg

ctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagataccta

cagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagc

ggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtc

ctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatg

gaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcct

gcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagcc

gaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcct

ctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcag

tgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccgg

ctcgtatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgcc

aagcgcgcaattaaccctcactaaagggaacaaaagctggagctgca

SEQ ID NO：8

agcttaagacattgattattgactagttattaatagtaatcaattacggggtcattagttcatagccca

tatatggagttccgcgttacataacttacggtaaatggcccgcctggctgaccgcccaacgacccccgc

ccattgacgtcaataatgacgtatgttcccatagtaacgccaatagggactttccattgacgtcaatgggt

ggagtatttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattg

acgtcaatgacggtaaatggcccgcctggcattatgcccagtacatgaccttatgggactttcctacttgg

cagtacatctacgtattagtcatcgctattaccatggtgatgcggttttggcagtacatcaatgggcgtgg

atagcggtttgactcacggggatttccaagtctccaccccattgacgtcaatgggagtttgttttggcacc

aaaatcaacgggactttccaaaatgtcgtaacaactccgccccattgacgcaaatgggcggtaggcgt

gtacggtgggaggtctatataagcaggggtctctctggttagaccagatctgagcctgggagctctctg

gctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgt

ctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtgg

cgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggctt

gctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagc

ggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcga

tgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaag

cagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatact

gggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaac

cctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaag

agcaaaacaaaagtaagaaaaaagcacagcaagcagcagctgacacaggacacagcagccaggtc

agccaaaattaccctatagtgcagaacatccaggggcaaatggtacatcaggccatatcacctagaact

ttaaatgcatgggtaaaagtagtagaagagaaggctttcagcccagaagtgatacccatgttttcagcatt

atcagaaggagccaccccacaagatttaaacaccatgctaaacacagtggggggacatcaagcagcc

atgcaaatgttaaaagagaccatcaatgaggaagctgcagaatgggatagagtgcatccagtgcatgc

agggcctattgcaccaggccagatgagagaaccaaggggaagtgacatagcaggaactactagtacc

aagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcag

cgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgct

gagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaa

gaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaac

tcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcaca

cgacctggatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatc

gcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtggaattg

gtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaaga

atagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacct

cccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacaga

gacagatccattcgattagtgaacggatctcgacggtatcggttaacttttaaaagaaaaggggggattg

gggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattaca

aaaacaaattacaaaaattcaaaattttatcgatcacgagactagcctcgagaagcttgatatcgaattcc

accgtgagtgaaagaccccacctgtaggtttggcaagctagcttaagtaacgccattttgcaaggcatg

gaaaatacataactgagaatagagaagttcagatcaaggttaggaacagagagacagcagaatatgg

gccaaacaggatatctgtggtaagcagttcctgccccggctcagggccaagaacagatggtccccag

atgcggtcccgccctcagcagtttctagagaaccatcagatgtttccagggtgccccaaggacctgaaa

tgaccctgtgccttatttgaactaaccaatcagttcgcttctcgcttctgttcgcgcgcttctgctccccgag

ctcaataaaagagcccacaacccctcactcggcgcgccagtcctccgatagactgcgtcgcccgggta

cccgtattcccaataaagcctcttgctgtttgcatccgaatcgtggactcgctgatccttgggagggtctc

ctcagattgattgactgcccacctcgggggtctttcattggatccgccaccatggaagacgccaaaaac

ataaagaaaggcccggcgccattctatccgctggaagatggaaccgctggagagcaactgcataagg

ctatgaagagatacgccctggttcctggaacaattgcttttacagatgcacatatcgaggtggacatcact

tacgctgagtacttcgaaatgtccgttcggttggcagaagctatgaaacgatatgggctgaatacaaatc

acagaatcgtcgtatgcagtgaaaactctcttcaattctttatgccggtgttgggcgcgttatttatcggag

ttgcagttgcgcccgcgaacgacatttataatgaacgtgaattgctcaacagtatgggcatttcgcagcct

accgtggtgttcgtttccaaaaaggggttgcaaaaaattttgaacgtgcaaaaaaagctcccaatcatcc

aaaaaattattatcatggattctaaaacggattaccagggatttcagtcgatgtacacgttcgtcacatctc

atctacctcccggttttaatgaatacgattttgtgccagagtccttcgatagggacaagacaattgcactg

atcatgaactcctctggatctactggtctgcctaaaggtgtcgctctgcctcatagaactgcctgcgtgag

attctcgcatgccagagatcctatttttggcaatcaaatcattccggatactgcgattttaagtgttgttccat

tccatcacggttttggaatgtttactacactcggatatttgatatgtggatttcgagtcgtcttaatgtataga

tttgaagaagagctgtttctgaggagccttcaggattacaagattcaaagtgcgctgctggtgccaaccc

tattctccttcttcgccaaaagcactctgattgacaaatacgatttatctaatttacacgaaattgcttctggt

ggcgctcccctctctaaggaagtcggggaagcggttgccaagaggttccatctgccaggtatcaggca

aggatatgggctcactgagactacatcagctattctgattacacccgagggggatgataaaccgggcg

cggtcggtaaagttgttccattttttgaagcgaaggttgtggatctggataccgggaaaacgctgggcgt

taatcaaagaggcgaactgtgtgtgagaggtcctatgattatgtccggttatgtaaacaatccggaagcg

accaacgccttgattgacaaggatggatggctacattctggagacatagcttactgggacgaagacgaa

cacttcttcatcgttgaccgcctgaagtctctgattaagtacaaaggctatcaggtggctcccgctgaatt

ggaatccatcttgctccaacaccccaacatcttcgacgcaggtgtcgcaggtcttcccgacgatgacgc

cggtgaacttcccgccgccgttgttgttttggagcacggaaagacgatgacggaaaaagagatcgtgg

attacgtcgccagtcaagtaacaaccgcgaaaaagttgcgcggaggagttgtgtttgtggacgaagtac

cgaaaggtcttaccggaaaactcgacgcaagaaaaatcagagagatcctcataaaggccaagaaggg

cggaaagatcgccgtgtaatgaagcggccgcgtcgactaaaatcaacctctggattacaaaatttgtga

aagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatca

tgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttg

tggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggc

attgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcatc

gccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcg

gggaagctgacgtcctttccatggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttct

gctacgtcccttcggccctcaatccagcggaccttccttcccgcctggaattcgagctcggtacctttaa

gaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaaggg

ctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctga

gcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttc

aagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtgg

aaaatctctagcagtagtagttcatgtcatcttattattcagtatttataacttgcaaagaaatgaatatcaga

gagtgagaggctgtgccttctagttgccagccatctgttgtttgcccctcccccgtgccttccttgaccct

ggaaggtgccactcccactgtcctttcctaataaaatgaggaaattgcatcgcattgtctgagtaggtgtc

attctattctggggggtggggtggggcaggacagcaagggggaggattgggaagagaatagcaggc

atgctggggatcatgtctggctctagctatcccgcccctaactccgcccatcccgcccctaactccgccc

agttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggc

ctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctata

gtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttac

ccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccg

atcgcccttcccaacagttgcgcagcctgaatggcgaatgggaggtggcacttttcggggaaatgtgtg

cagctctggcccgtgtctcaaaatctctgatgttacattgcacaagataaaaatatatcatcatgaacaata

aaactgtctgcttacataaacagtaatacaaggggtgttatgagccatattcaacgggaaacgtcgagg

ccgcgattaaattccaacatggatgctgatttatatgggtataaatgggctcgcgataatgtcgggcaatc

aggtgcgacaatctatcgcttgtatgggaagcccgatgcgccagagttgtttctgaaacatggcaaagg

tagcgttgccaatgatgttacagatgagatggtcagactaaactggctgacggaatttatgccacttccg

accatcaagcattttatccgtactcctgatgatgcatggttactcaccactgcgatccccggaaaaacagc

gttccaggtattagaagaatatcctgattcaggtgaaaatattgttgatgcgctggcagtgttcctgcgcc

ggttgcactcgattcctgtttgtaattgtccttttaacagcgatcgcgtatttcgcctcgctcaggcgcaatc

acgaatgaataacggtttggttgatgcgagtgattttgatgacgagcgtaatggctggcctgttgaacaa

gtctggaaagaaatgcataaacttttgccattctcaccggattcagtcgtcactcatggtgatttctcacttg

ataaccttatttttgacgaggggaaattaataggttgtattgatgttggacgagtcggaatcgcagaccga

taccaggatcttgccatcctatggaactgcctcggtgagttttctccttcattacagaaacggctttttcaaa

aatatggtattgataatcctgatatgaataaattgcagtttcatttgatgctcgatgagtttttctaactgtcag

accaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcc

tttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaa

agatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgc

taccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcaga

gcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcacc

gcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccg

ggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcac

acagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagc

gccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggaga

gcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctg

acttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcgg

cctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtgga

taaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagt

cagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattc

attaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtg

agttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtga

gcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcact

aaagggaacaaaagctggagctgca

Claims (18)

1. a recombinant lentiviral vector based on a plvx eukaryotic cell lentiviral expression vector comprising the following elements:

(a) An elongated Gag element downstream of the 5' ltr replacing the Gag element in the plvx vector, said Gag element comprising an amino acid sequence as set forth in SEQ ID NO: 6;

(b) A murine stem cell virus MSCV promoter in a plvx vector for expressing a promoter of a gene sequence of interest, said MSCV promoter comprising a sequence as set forth in SEQ ID NO:3, a sequence shown in 3;

(c) Replacing a C-terminal truncated WPRE element of a WPRE of an enhanced expression element in a plvx vector, said C-terminal truncated WPRE element comprising the amino acid sequence as set forth in SEQ ID NO: 4;

(d) A bGH poly a tail replacing the SV40poly a tail in a plvx vector downstream of the truncated WPRE element, the bGH poly a tail comprising the amino acid sequence as set forth in SEQ ID NO: 5.

2. The recombinant lentiviral vector of claim 1, further comprising:

(e) An optional gene sequence of interest, downstream of the MSCV promoter.

3. The recombinant lentiviral vector of claim 1 or 2, constructed by a method comprising the steps of:

(i) Replacing the Gag element in the plvx vector with an elongated Gag element comprising an amino acid sequence as set forth in SEQ ID NO: 6;

(ii) Replacing the promoter for expressing the gene sequence of interest in the plvx vector with a murine stem cell virus MSCV promoter comprising the amino acid sequence as set forth in SEQ ID NO:3, a sequence shown in 3;

(iii) Replacing the WPRE of the enhanced expression element in the plvx vector with a shorter C-terminal truncated WPRE comprising the amino acid sequence as set forth in SEQ ID NO: 4;

(iv) Replacement of the SV40poly A tail in the plvx vector with a bGH poly A tail comprising the amino acid sequence as set forth in SEQ ID NO: 5.

4. The recombinant lentiviral vector of claim 3, wherein the method further comprises the steps of:

(v) An optional gene sequence of interest is inserted downstream of the MSCV promoter.

5. The recombinant lentiviral vector of any one of claims 1-4, wherein the plvx eukaryotic lentiviral expression vector comprises the sequence set forth in SEQ ID NO:1, and a sequence shown in 1.

6. The recombinant lentiviral vector of any one of claims 1-5, having a structure as shown in figure 1.

7. The recombinant lentiviral vector of any one of claims 1-6, wherein the recombinant lentiviral vector comprises the sequence set forth in SEQ ID NO:2, and a sequence shown in seq id no.

8. A lentiviral particle comprising the recombinant lentiviral vector of any one of claims 1-7.

9. The lentiviral particle of claim 7, packaged by a 293T packaging lentiviral system.

10. A recombinant cell obtained by transfecting a host cell with the recombinant lentiviral vector of any one of claims 1 to 7.

11. The recombinant cell of claim 10, obtained by transfection of a host cell selected from the group consisting of: primary T cells, immortalized T cells, T cells isolated from tumor tissue, and Innate Lymphocytes (ILCs).

12. A pharmaceutical composition comprising the recombinant lentiviral vector of any one of claims 1-7 and/or the recombinant cell of claim 10 or 11.

13. The pharmaceutical composition of claim 13, further comprising a pharmaceutically acceptable additive.

14. A kit comprising the recombinant lentiviral vector of any one of claims 1 to 7 and/or the recombinant cell of claim 10 or 11 and/or the pharmaceutical composition of claim 12 or 13.

15. A method of transfecting a cell comprising the steps of:

(1) Contacting a cell with the recombinant lentiviral vector of any one of claims 1 to 7, and transfecting the cell.

16. The method of claim 15, wherein the cell is selected from the group consisting of: primary T cells, immortalized T cells, T cells isolated from tumor tissue, and Innate Lymphocytes (ILCs).

17. A method of constructing a recombinant lentiviral vector comprising the steps of:

(i) Replacing the Gag element in the plvx vector with an elongated Gag element comprising an amino acid sequence as set forth in SEQ ID NO: 6;

(ii) Replacing the promoter for expressing the gene sequence of interest in the plvx vector with a murine stem cell virus MSCV promoter comprising the amino acid sequence as set forth in SEQ ID NO:3, a sequence shown in 3;

(iii) Replacing the WPRE of the enhanced expression element in the plvx vector with a shorter C-terminal truncated WPRE comprising the amino acid sequence as set forth in SEQ ID NO: 4;

(iv) Replacement of the SV40poly A tail in the plvx vector with a bGH poly A tail comprising the amino acid sequence as set forth in SEQ ID NO: 5.

18. The method of claim 17, further comprising the step of:

(v) An optional gene sequence of interest is inserted downstream of the MSCV promoter.