CN111235151A - shRNA of CXCR4 gene and application thereof - Google Patents

Abstract

The invention provides shRNA of CXCR4 gene and application thereof, wherein the shRNA comprises siRNA of CXCR4 gene and inverted repeat sequence of the siRNA; the constructed lentiviral vector realizes the heritable silencing of CXCR4 gene in T cells, the expression level of CXCR4 in the edited T cells is obviously reduced, and a new method and a new thought are provided for the treatment of tumors and immune diseases.

Description

shRNA of CXCR4 gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to shRNA of a CXCR4 gene and application thereof.

Background

T cells are the main immune cells of human body and have important regulation function on immune system. T cells are the primary target of Human Immunodeficiency Virus (HIV), which causes a reduction in the number of T cells by mediating apoptosis and inducing an autoimmune response, triggering the loss of cellular immunity, humoral immune response, and acquired immune dysfunction syndrome. HIV enters host cells by binding of viral envelope proteins to CCR5 or C-X-C motif chemokine receptor 4(CXCR4) of CD4+ T cells. In most cases, HIV enters host cells through CCR5, initiating primary infection; mutations in the envelope protein confer viral tropism to CXCR4 later in infection.

The use of gene editing techniques for the treatment of HIV is feasible. Knockout of the CCR5 gene in autologous uninfected CD4+ T cells or CD34+ hematopoietic stem cells demonstrated good T cell proliferation and anti-HIV effects in vitro and in immunodeficient mice. Infusion of genetically modified CD4+ T cells significantly reduced the viral load in HIV-infected peripheral blood, increasing the number of CD4+ T cells compared to infusion of unmodified T cells. Studies have shown that infusion of CD4+ T cells knocked out of CCR5 can reconstitute the patient's immune system, resulting in a long lasting and potent anti-HIV effect.

The C-X-C motif chemokine receptor 4(CXCR4) is another important co-receptor in the later stages of HIV infection, the CXCR4 gene in knockout T cells is favorable for acquiring resistance to HIV infection, CD4+ T cells after CXCR4 knockout have normal proliferative functions, and show effective resistance to viruses utilizing the CXCR4 receptor.

The prior art shows that gene editing of T cells provides a new means for treating cancer, AIDS and autoimmune diseases. At present, gene editing technology has been applied to the editing of various cell genomes, but has certain difficulty on human T cells.

Disclosure of Invention

Aiming at the defects and actual requirements of the prior art, the invention provides the shRNA of the CXCR4 gene and the application thereof, the shRNA and the constructed lentiviral vector thereof realize the silencing of the CXCR4 gene in the T cell, the expression level of the CXCR4 in the T cell after editing is obviously reduced, and a new method and a new thought are provided for the treatment of tumors and immune diseases.

In order to achieve the purpose, the invention adopts the following technical scheme:

in a first aspect, the invention provides siRNA of a CXCR4 gene, which comprises a nucleic acid sequence shown as SEQ ID NO. 1-6;

SEQ ID NO:1：gcaaggcagtccatgtcatct；

SEQ ID NO:2：ggatcagcatcgactccttca；

SEQ ID NO:3：gcacatcatggttggccttat；

SEQ ID NO:4：agataactacaccgaggaaat；

SEQ ID NO:5：cctgttcttaagacgtgattt；

SEQ ID NO:6：tcctgtcctgctattgcatta。

in the invention, the full-length mRNA of CXCR4 gene is analyzed, and specific mRNA is obtained by Blast comparison, so that 6 CXCR4 gene siRNAs shown in SEQ ID NO: 1-6 are obtained by designing, wherein the target mRNA position of the siRNA shown in SEQ ID NO:1 is 421-441, the target mRNA position of the siRNA shown in SEQ ID NO:2 is 868-888, the target mRNA position of the siRNA shown in SEQ ID NO:3 is 701-721, the target mRNA position of the siRNA shown in SEQ ID NO:4 is 122-142, the target mRNA position of the siRNA shown in SEQ ID NO:5 is 1512-1532, and the target mRNA position of the siRNA shown in SEQ ID NO:6 is 739-759.

In a second aspect, the invention provides a shRNA comprising a stem-loop linked inverted repeat sequence;

the inverted repeat sequence includes an siRNA of the CXCR4 gene and an inverted repeat sequence of the siRNA.

In the present invention, shRNA sequences are designed to comprise sirnas of the CXCR4 gene as described in the first aspect, which facilitate integration into the genome, thereby allowing silencing of the CXCR4 gene to be inherited.

Preferably, the shRNA comprises a sense strand and an antisense strand.

Preferably, the stem loop of the shRNA comprises a nucleic acid sequence shown as SEQ ID NO. 7-8;

SEQ ID NO:7：TTCAAGAGA；

SEQ ID NO:8：TCTCTTGAA。

in the invention, the nucleic acid sequence shown in SEQ ID NO. 7 is a stem loop of a shRNA sense strand, and the nucleic acid sequence shown in SEQ ID NO. 8 is a stem loop of a shRNA antisense strand.

Preferably, the shRNA comprises a nucleic acid sequence shown as SEQ ID NO. 9-20;

SEQ ID NO:9：

TCGAGgcaaggcagtccatgtcatctTTCAAGAGAagatgacatggactgccttgcTTTTTTGATAT；

SEQ ID NO:10：

ACTAAAAAAgcaaggcagtccatgtcatctTCTCTTGAAagatgacatggactgccttgcC；

SEQ ID NO:11：

TCGAGggatcagcatcgactccttcaTTCAAGAGAtgaaggagtcgatgctgatccTTTTTTGATAT；

SEQ ID NO:12：

ACTAAAAAAggatcagcatcgactccttcaTCTCTTGAAtgaaggagtcgatgctgatccC；

SEQ ID NO:13：

TCGAGgcacatcatggttggccttatTTCAAGAGAataaggccaaccatgatgtgcTTTTTTGAT；

SEQ ID NO:14：

ACTAAAAAAgcacatcatggttggccttatTCTCTTGAAataaggccaaccatgatgtgcC；

SEQ ID NO:15：

TCGAGagataactacaccgaggaaatTTCAAGAGAatttcctcggtgtagttatctTTTTTTGAT；

SEQ ID NO:16：

ACTAAAAAAagataactacaccgaggaaatTCTCTTGAAatttcctcggtgtagttatctC；

SEQ ID NO:17：

TCGAGcctgttcttaagacgtgatttTTCAAGAGAaaatcacgtcttaagaacaggTTTTTTGAT；

SEQ ID NO:18：

ACTAAAAAAcctgttcttaagacgtgatttTCTCTTGAAaaatcacgtcttaagaacaggC；

SEQ ID NO:19：

TCGAGtcctgtcctgctattgcattaTTCAAGAGAtaatgcaatagcaggacaggaTTTTTTGAT；

SEQ ID NO:20：

ACTAAAAAAtcctgtcctgctattgcattaTCTCTTGAAtaatgcaatagcaggacaggaC。

in the invention, 9-10 SEQ ID NOS are positive and negative strands, 11-12 SEQ ID NOS are positive and negative strands, 13-14 SEQ ID NOS are positive and negative strands, 15-16 SEQ ID NOS are positive and negative strands, 17-18 SEQ ID NOS are positive and negative strands, 19-20 SEQ ID NOS are positive and negative strands, and the lowercase letters in each positive strand represent siRNA and a reverse repeat sequence of the CXCR4 gene.

In a third aspect, the present invention provides an shRNA expression vector comprising the shRNA according to the second aspect.

Preferably, the expression vector comprises a viral vector.

Preferably, the viral vector comprises any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector, preferably a lentiviral vector.

In a fourth aspect, the present invention provides a method for preparing the shRNA expression vector according to the third aspect, wherein the method comprises the following steps:

(1) designing shRNA according to siRNA of CXCR4 gene;

(2) and inserting shRNA into an expression vector to obtain the shRNA expression vector.

Preferably, the shRNA is inserted into the XhoI and EcoRV cleavage sites of the expression vector.

In a fifth aspect, the present invention provides a recombinant lentivirus prepared by co-transfecting a mammalian cell with the expression vector of the third aspect and a packaging helper plasmid.

Preferably, the mammalian cells comprise any one or a combination of at least two of 293 cells, 293T cells or 293F cells, preferably 293T cells.

In a sixth aspect, the present invention provides a host cell transfected with any one of or a combination of at least two of the siRNA of the first aspect, the shRNA of the second aspect, the sgRNA expression vector of the third aspect, or the recombinant lentivirus of the fifth aspect.

Preferably, the host cell comprises an immune cell.

Preferably, the immune cells comprise T cells.

In a seventh aspect, the invention provides a method of silencing a CXCR4 gene, the method comprising:

transfecting an siRNA according to the first aspect, an shRNA according to the second aspect, an shRNA expression vector according to the third aspect or a recombinant lentivirus according to the fifth aspect into a host cell for silencing of the CXCR4 gene.

In an eighth aspect, the present invention provides a CXCR4 inhibitor, wherein the CXCR4 inhibitor comprises any one of or a combination of at least two of the siRNA of the first aspect, the shRNA of the second aspect, the shRNA expression vector of the third aspect, or the recombinant lentivirus of the fifth aspect.

In a ninth aspect, the present invention provides a pharmaceutical composition comprising any one of or a combination of at least two of the siRNA of the first aspect, the shRNA of the second aspect, the shRNA expression vector of the third aspect, the recombinant lentivirus of the fifth aspect, the host cell of the sixth aspect or the CXCR4 inhibitor of the eighth aspect.

Preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

In a tenth aspect, the present invention provides a use of the siRNA of the first aspect, the shRNA of the second aspect, the shRNA expression vector of the third aspect, the recombinant lentivirus of the fifth aspect, the host cell of the sixth aspect, the CXCR4 inhibitor of the eighth aspect, or the pharmaceutical composition of the ninth aspect for the manufacture of a medicament for the treatment of a disease.

Preferably, the disease comprises a malignancy and/or an immunodeficiency disease.

Compared with the prior art, the invention has the following beneficial effects:

(1) the shRNA sequence screened by the invention contains siRNA of CXCR4 gene, and is beneficial to being integrated into T cell genome after packaging and constructing a lentiviral vector, so that the silencing of CXCR4 gene can be inherited;

(2) the shRNA lentivirus realizes the editing function of CXCR4 gene in T cells, the expression of CXCR4 in the edited T cells is obviously reduced, and a new method and thought are provided for the treatment of tumors and immune diseases.

Drawings

FIG. 1 shows a vector map of pCDH-shRNA and polyclonal restriction enzyme sites;

FIG. 2(A) shows the sequencing result of pCDH-shCXCR4-1, FIG. 2(B) shows the sequencing result of pCDH-shCXCR4-2, FIG. 2(C) shows the sequencing result of pCDH-shCXCR4-3, FIG. 2(D) shows the sequencing result of pCDH-shCXCR4-4, FIG. 2(E) shows the sequencing result of pCDH-shCXCR4-5, and FIG. 2(F) shows the sequencing result of pCDH-shCXCR 4-6;

FIG. 3 is the expression level of CXCR4 in T cells transfected with pCDH-shCXCR 4;

figure 4 shows expression of CXCR4 on the surface of T cells.

Detailed Description

To further illustrate the technical means adopted by the present invention and the effects thereof, the present invention is further described below with reference to the embodiments and the accompanying drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.

The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.

Example 1 siRNA and shRNA sequence design of CXCR4 Gene

The full-length mRNA sequence (NM-003467.2) of CXCR4 gene disclosed by GenBank is analyzed, specific mRNA is obtained through Blast comparison, siRNA of CXCR4 mRNA is designed, the target position of each siRNA is shown in figure 1, and the specific sequence information is shown in Table 1.

TABLE 16 specific siRNAs to CXCR4 Gene

Serial number	Target nucleotide sequence	Target point position
			SEQ ID NO:1	gcaaggcagtccatgtcatct	421～441
SEQ ID NO:2	ggatcagcatcgactccttca	868～888
			SEQ ID NO:3	gcacatcatggttggccttat	701～721
SEQ ID NO:4	agataactacaccgaggaaat	122～142
			SEQ ID NO:5	cctgttcttaagacgtgattt	1512～1532
SEQ ID NO:6	tcctgtcctgctattgcatta	739～759

Designing corresponding shRNA according to the siRNA sequence, wherein the shRNA comprises a Sense strand (S) and an Antisense strand (A), the shRNA sequence consists of Sense (underlined) + stem loop (loop) + Antisense + termination sequence, and the specific sequence is shown in Table 2.

TABLE 2 shRNA of CXCR4 Gene

Example 2 construction of shRNA Lentiviral expression vector of CXCR4 Gene

Carrying out double enzyme digestion on the pCHD vector by adopting XhoI/EcoRV, cutting the gel and recovering; designing a specific primer according to shRNA to complete primer annealing; and recombining the shRNA and the vector recovered by enzyme digestion to form an annular segment, transferring the annular segment into a prepared bacterial competent cell, selecting a monoclonal colony for sequencing identification, and obtaining the successfully constructed shRNA silencing vector by comparing the correct clone.

The method comprises the following specific steps:

(1) double digestion of vectors

① the clone strain containing pCDH empty vector was cultured overnight, 5mL of fresh bacterial liquid was extracted plasmid using QIAGEN plasmid miniprep kit;

② mu.g of the extracted pCDH was digested with the corresponding restriction enzymes at 37 ℃ for about 1h, the digestion system is shown in Table 3;

TABLE 3

pCDH	2μg
		Buffer	5μL
XhoI	1μL
		EcoRV	1μL
ddH2O	Make up to 50 μ L

③ subjecting the enzyme digestion product to agarose gel electrophoresis, and recovering gel after electrophoresis, wherein the method comprises cutting off gel strip containing target segment under ultraviolet lamp irradiation, weighing total weight with balance and subtracting empty tube weight to calculate gel weight, calculating gel volume according to 100mg ═ 100 μ L, adding Binding Solution (Binding Solution) 1 times of gel volume, melting gel thoroughly in 65 deg.C water bath, and shaking EP tube to accelerate gel dissolution;

④ transferring all the above liquid into a filter column, centrifuging at 13000rpm for 30s (repeating once), then discarding the liquid in the tube, adding 500 μ L of WAsolution into the column, centrifuging at 13000rpm for 30s, discarding the liquid in the tube, adding 500 μ L of Wash Solution into the column, centrifuging at 13000rpm for 30s (repeating once), and then emptying for 3 min;

the column was placed in a new 1.5mL EP tube, air dried at room temperature, and 35. mu.L of ddH was added to the column₂O, standing for 5min, centrifuging at 13000rpm for 1.5min, adding the dissolved DNA into the column again for centrifuging for one minute, discarding the column to obtain the recovered vector fragment, and determining the concentration.

(2) Annealing of ShRNA

① the synthesized primers were diluted to a stock solution with a final concentration of 100nmol/L and annealed according to the system shown in Table 4;

TABLE 4

Primer-F	2μL
		Primer-R	2μL
Nacl	2μL
		ddH2O	Make up to 20 mu L

② mixing the above materials, centrifuging instantly, placing into a PCR instrument, annealing and extending at 90 deg.C for 4min, 70 deg.C for 10min, 55 deg.C for 10min, 37 deg.C for 5min, 25 deg.C for 10min, and storing at 4 deg.C.

(3) T4 ligation reaction

Because the annealing product is matched with the cohesive end of the vector, the recovered fragment is connected with the linearized pCDH vector by using T4 ligase, the recombination process of the recovered target gene and the vector is completed, and the pCDH-shRNA lentiviral vector shown in figure 1 is constructed.

(4) Transformation of

① placing the competent cells on ice (4 deg.C) to be naturally thawed, adding 10 μ l of ligation product into the competent cells, and placing on ice (4 deg.C) for 30 min;

② is heated and shocked for 90s in 42 ℃ water bath, and is quickly placed on ice (4 ℃) for 2-3 min;

③ adding 600 μ L of SOC culture medium without antibiotic, and culturing at 37 deg.C and 200rpm for 45min under shaking;

④ 4000 centrifuging at 4000rpm for 3min, discarding 600 μ L of supernatant, blowing the bacterial liquid at the bottom of the tube, adding into a culture plate containing resistance, spreading with a sterilized spreader, and culturing overnight in a 37 deg.C incubator.

(5) Cloning and identification

And selecting a plurality of cloned transformants in the plate for sequencing identification, and storing strains.

As shown in FIG. 2(A), FIG. 2(B), FIG. 2(C), FIG. 2(D), FIG. 2(E) and FIG. 2(F), the sequencing results of pCDH-shCXCR4-1, pCDH-shCXCR4-2, pCDH-shCXCR4-3, pCDH-shCXCR4-4, pCDH-shCXCR4-5 and pCDH-shCXCR4-6, respectively, revealed that the base sequence in the vector agreed with the predicted sequence, confirming the successful construction of the lentiviral vector.

Example 3 Lentiviral packaging

The lentiviral vector constructed in example 2 was packaged into lentiviral vector using a four plasmid system, comprising the following steps:

(1) the four-plasmid system comprises gag/pol, Rev, VSV-G and a lentiviral vector pCDH-shCXCR 4-1-6 required by packaging a lentiviral vector or negative control plasmid, the plasmids are added into serum-free DMEM medium with a certain volume, and the serum-free DMEM medium is uniformly mixed and placed for 15 min;

(2) adding the above mixture into T75 culture flask paved with 293T cells, mixing, transiently transfecting 293T cells, and culturing at 37 deg.C with 5% CO₂Culturing for 6h in a cell culture box;

(3) after 6h, the culture medium is replaced by fresh medium, the culture is continued, 10mM sodium butyrate solution is added, and culture supernatant is collected after 72h for lentivirus purification detection.

Example 4 Lentivirus transduction of human Primary T cells

Mixing the collected peripheral blood with physiological saline according to the proportion of 1:1, adding Ficoll into a centrifuge tube, slowly adding the diluted peripheral blood, centrifuging at 1500rpm for 30min, slightly sucking the PBMC layer and adding into another centrifuge tube;

washing PBMC with physiological saline, transferring into X-VIVO culture medium (containing 50ng/mL OKT3, and 300IU/mL IL-2), and culturing; PBMC were isolated and activated with X-VIVO medium (50 ng/mL OKT3, 300IU/mL IL-2) and 2 days later with lentiviral infection;

adding T cell and lentivirus solution into 6-well plate pre-coated with retroNectin, centrifuging at 2000g for 2h, and centrifuging at 37 deg.C and 5% CO₂Culturing in a cell culture box with certain humidity, collecting cells after 4 days, and detecting CXKnockdown efficiency of CR 4.

Example 5 fluorescent quantitative PCR detection of CXCR4 Gene expression level

(1) RNA extraction

Adding 200 mu L of chloroform into a 1.5mL EP tube containing Trizol, shaking and uniformly mixing, standing for 5min, and centrifuging at 12000rpm and 4 ℃ for 10 min;

taking out the 1.5mL EP tube from the centrifuge, sucking the colorless and transparent water phase layer at the upper layer into another clean 1.5mL EP tube (the sample is divided into three layers, namely the lower layer is an organic phase layer, the middle layer and the upper layer are water phase layers, and RNA is positioned in the water phase at the upper layer), adding isopropanol with the same volume, slightly reversing the mixture from top to bottom, uniformly mixing, standing for 10min, and centrifuging at 12000rpm and 4 ℃ for 10 min;

carefully abandoning the supernatant, washing the RNA precipitate with 1mL of 75% ethanol, centrifuging at 7000rpm and 4 ℃ for 5min, and removing the supernatant as clean as possible;

standing and drying at room temperature for 5-10 min, adding 25 mu L DEPC water, blowing and beating for several times by using a gun head to fully dissolve RNA, and storing at-80 ℃;

the concentration of the extracted RNA was measured using a nucleic acid protein detector.

As a result, the RNA extracted was high in purity and free from contamination, as shown in Table 5.

TABLE 5

Sample name	Purity of	Concentration (ng/. mu.L)
			control group	1.80	682.25
shNC group	1.81	788.39
			Group shCXCR4-1	1.73	620.08
shCXCR4-2 group	1.77	443.38
			shCXCR4-3 group	1.83	623.08
shCXCR4-4 group	1.76	544.57
			shCXCR4-5 group	1.79	630.99
shCXCR4-6 group	1.79	430.89

(2) Reverse transcription

① the reaction solution was prepared according to Table 6, incubated at 72 ℃ for 5min, and chilled on ice;

TABLE 6

Total RNA	5μg
		Oligo dT(5μM)	2.5μL
DEPC H2O	Make up to 12.5 mu L

② adding reverse transcription reagent into the reaction solution to prepare reverse transcription system shown in Table 7, incubating at 42 deg.C for 60min, incubating at 72 deg.C for 10min, and performing reverse transcription to obtain cDNA;

TABLE 7

(3) qPCR for CXCR4

Designing CXCR4 and GAPDH primers SEQ ID NO: 21-24, preparing a qPCR system according to table 8, wherein the reaction conditions are shown in table 9, and the melting process is shown in table 10;

hCXCR4-188-F(SEQ ID NO:21)：CCGTGGCAAACTGGTACTT；

hCXCR4-188-R(SEQ ID NO:22)：GACGCCAACATAGACCACCT；

hGAPDH-127F(SEQ ID NO:23)：CCAGGTGGTCTCCTCTGA；

hGAPDH-127R(SEQ ID NO:24)：GCTGTAGCCAAATCGTTGT；

TABLE 8

Composition (I)	Volume of
		SYBR Green I mix(2×)	5μL
F(10μM)	0.3μL
		R(10μM)	0.3μL
cDNA	0.2μL
		Sterilized distilled water	Make up to 10 mu L

TABLE 9

Watch 10

The data were analyzed using an IQ5 fluorescent quantitative PCR analysis system, using 2^-△△CTThe relative gene expression was analyzed by the method, and the expression level of normally cultured human T cells was set to 1 using GAPDH as an internal reference, and the relative expression level of each transfection group was calculated.

The results are shown in FIG. 3, the silencing efficiency of shRNA-1-6 targeting CXCR4 reaches 59.41%, 86.5%, 69.5%, 28.3%, 49.7% and 55.5%, respectively, wherein the silencing efficiency of shRNA-2 is the highest.

Example 6 flow assay of CXCR4 protein expression

The expression of CXCR4 on the surface of T cells was detected by flow cytometry, and after incubating PE-labeled anti-CXCR 4 antibody (BioLegend; cat. No. 306506) with T cells for 30min, PBS was washed 3 times for flow detection.

The results are shown in fig. 4, where shCXCR 4-group 2 CXCR4 was expressed at the lowest, 3.2%.

In conclusion, the invention successfully finishes the editing of the gene target of the CXCR4 protein which is a common receptor of HIV virus in T cells by screening the shRNA of the target CXCR4, packaging a lentiviral vector and transferring the shRNA into the T cells to silence the CXCR4 gene, obviously reduces the expression of CXCR4 in the edited T cells and provides a new method and thought for treating tumors and immune diseases.

The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.

SEQUENCE LISTING

<110> Guangzhou Bai-and-Gen-Tech Co Ltd

<120> shRNA of CXCR4 gene and application thereof

<130>20200312

<160>24

<170>PatentIn version 3.3

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Claims (10)

1. siRNA of CXCR4 gene is characterized in that the siRNA comprises a nucleic acid sequence shown as SEQ ID NO 1-6.

2. An shRNA comprising a stem-loop linked inverted repeat sequence;

the inverted repeat sequence comprises an siRNA of the CXCR4 gene and an inverted repeat sequence of the siRNA;

preferably, the shRNA comprises a sense strand and an antisense strand;

preferably, the stem loop of the shRNA comprises a nucleic acid sequence shown as SEQ ID NO. 7-8;

preferably, the shRNA comprises a nucleic acid sequence shown as SEQ ID NO. 9-20.

3. An shRNA expression vector comprising the shRNA of claim 2;

preferably, the expression vector comprises a viral vector;

preferably, the viral vector comprises any one of a lentiviral vector, a retroviral vector or an adeno-associated viral vector, preferably a lentiviral vector.

4. A method for preparing a shRNA expression vector according to claim 3, comprising the steps of:

(1) designing shRNA according to siRNA of CXCR4 gene;

(2) inserting shRNA into an expression vector to obtain the shRNA expression vector;

preferably, the shRNA is inserted into the XhoI and EcoRV cleavage sites of the expression vector.

5. A recombinant lentivirus prepared by co-transfecting a mammalian cell with the expression vector of claim 3 and a packaging helper plasmid;

preferably, the mammalian cells comprise any one or a combination of at least two of 293 cells, 293T cells or 293F cells, preferably 293T cells.

6. A host cell transfected with any one of the siRNA of claim 1, the shRNA of claim 2, the sgRNA expression vector of claim 3, or the recombinant lentivirus of claim 5, or a combination of at least two thereof;

preferably, the host cell comprises an immune cell;

preferably, the immune cells comprise T cells.

7. A method of silencing a CXCR4 gene, the method comprising:

transfecting the siRNA of claim 1, the shRNA of claim 2, the shRNA expression vector of claim 3 or the recombinant lentivirus of claim 5 into a host cell for silencing of the CXCR4 gene.

8. A CXCR4 inhibitor wherein the CXCR4 inhibitor comprises any one of or a combination of at least two of the siRNA of claim 1, the shRNA of claim 2, the shRNA expression vector of claim 3 or the recombinant lentivirus of claim 5.

9. A pharmaceutical composition comprising any one of or a combination of at least two of the siRNA of claim 1, the shRNA of claim 2, the shRNA expression vector of claim 3, the recombinant lentivirus of claim 5, the host cell of claim 6 or the CXCR4 inhibitor of claim 8;

preferably, the pharmaceutical composition further comprises any one or a combination of at least two of a pharmaceutically acceptable carrier, excipient or diluent.

10. Use of the siRNA of claim 1, the shRNA of claim 2, the shRNA expression vector of claim 3, the recombinant lentivirus of claim 5, the host cell of claim 6, the CXCR4 inhibitor of claim 8, or the pharmaceutical composition of claim 9 in the manufacture of a medicament for the treatment of a disease;

preferably, the disease comprises a malignancy and/or an immunodeficiency disease.

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