Adenovirus and CCR5-delta32 mutant gene recombinant as well as preparation method and application thereof

Abstract

The invention relates to the technical field of gene engineering, in particular to an adenovirus and CCR5-delta 32 mutant gene recombinant as well as a preparation method and application thereof. The invention provides a CCR5-delta 32 mutant gene expression cassette with a sequence as shown in SEQ ID NO: 1, a recombinant constructed by an adenovirus and the expression cassette, a construction method of the recombinant and application of the recombinant in preparation of drugs for treating HIV-1 virus infection diseases, cells are transfected by the recombinant to produce virus liquid, the virus liquid can efficiently inhibit expression of CCR5 and CXCR4 molecules on the cell surface, and the CCR5-delta 32 mutant gene expression cassette can be used for preparing drugs for treating HIV-1 virus infection diseases. And the infection capability of the cells on the HIV-1 virus can be improved.

Description

Technical Field

[0001] The present invention relates to the field of gene engineering technology, and in particular to an adenovirus and CCR5-Δ32 mutant gene recombinant, and a preparation method and application thereof. Background Art

[0002] AIDS, also known as acquired immunodeficiency syndrome (AIDS), is a systemic disease caused by the human immunodeficiency virus (HIV). Common types include HIV-1 and HIV-2, of which HIV-1 is the dominant type in China. HIV mainly invades the human immune system. Untreated infected people are prone to various serious infections and malignant tumors in the late stage of the disease, which eventually leads to death.

[0003] There is currently no effective drug to cure AIDS, and it is generally believed that AIDS is incurable. The current treatment goal is to maximally and permanently inhibit viral replication in the patient's body, so that the patient can reconstruct and maintain immune function, while reducing the morbidity and mortality of HIV infection and non-AIDS related diseases. Combination therapy with multiple antiviral drugs is widely used, and general treatment is also required, that is, treatment to restore or improve immune function and treatment of opportunistic infections and malignant tumors. Therefore, how to prevent HIV-1 virus from entering host cells and inhibit viral replication in patients is one of the important problems in treating AIDS. At the same time, through research, it was found that patients who turned from HIV positive to negative after retroviral drug treatment already had CCR5 mutant genes in their bodies.

[0004] As various vectors that can be used for gene therapy, they themselves have no significance for treating any disease and have no clinical application value; as genes that can treat various diseases, due to the difficulty of introducing them into target cells and expressing them in target cells, they only have potential therapeutic value but no actual clinical value. Only by combining genes with potential therapeutic effects with vectors of transferable genes, and introducing therapeutic genes into target cells and expressing them on cells through the mediation of the vector, can a real clinical therapeutic effect be achieved. The common method for homologous recombination in eukaryotic cells is to fuse the expression cassette of the target gene with the vector, which is a time-consuming and labor-intensive process. However, the technology of homologous recombination and construction of fusion expression vectors in prokaryotic cells (Escherichia coli) can solve the above problems.

[0005] Adenoviral vectors are commonly used gene vectors in genetic engineering technology. Their greatest characteristics are relatively high transfection efficiency, good operability, the ability to carry large gene fragments and easily achieve industrial production of virus particles. At the same time, they can infect both dividing and non-dividing cells, have high safety and low pathogenicity. However, adenoviral vectors have deficiencies, mainly in the lack of infection specificity and immunogenicity. Research shows that when exogenous genes are carried in the E1 or E3 deletion regions of adenoviral vectors, long-term expression of exogenous genes can be achieved while reducing immunogenicity. Summary of the Invention

[0006] The purpose of the present invention is to provide a recombinant of adenovirus and CCR5-Δ32 mutant gene, its preparation method and application. This recombinant can induce the CCR5 protein on the host cell surface to be unable to be normally expressed on the cell membrane surface, thereby effectively preventing HIV-1 gp120 from effectively binding to the CCR5-Δ32 mutant gene, so that the HIV-1 virus cannot enter the host cell for replication, thus achieving the effect of treating AIDS.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention provides a CCR5-Δ32 mutant gene expression cassette, and the nucleotide sequence of the CCR5-Δ32 mutant gene expression cassette is as shown in SEQ ID NO.1.

[0009] The present invention also provides a recombinant of adenovirus and CCR5-Δ32 mutant gene, and the recombinant of adenovirus and CCR5-Δ32 mutant gene contains the CCR5-Δ32 mutant gene expression cassette shown in SEQ ID NO:1 and the adenovirus 5 genome sequence shown in GenBank:NC_001406.

[0010] Furthermore, the 5' end of the CCR5-Δ32 mutant gene expression cassette is connected to the 3329th base in the forward direction of the adenovirus 5 genome sequence, and the 3' end of the CCR5-Δ32 mutant gene expression cassette is connected to the 452nd base in the forward direction of the adenovirus 5 genome sequence.

[0011] The present invention also provides a construction method of the CCR5-Δ32 mutant gene expression cassette, specifically including the following steps:

[0012] (1) Using the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers, the human tumor suppressor CCR5-Δ32 gene is amplified by PCR. The amplified full-length CCR5-Δ32 gene is cloned into the prokaryotic plasmid pUC19, and sequencing verification is carried out. The plasmid with correct sequencing verification is reserved for use;

[0013] (2) Amplify the LTR sequence of RSV, the poly(A) sequence of BGH, and the adenovirus E1 region sequence by PCR respectively, introduce linker sequences on one side respectively, verify by sequencing. When performing the PCR reaction again, splice the LTR and PA sequences to the 5' and 3' ends close to the CCR5-Δ32 gene respectively to obtain the spliced product LTR-CCR5-Δ32-PA. Splice the adenovirus E1 region and its upstream sequence to the outermost side of the CCR5-Δ32 gene to construct the CCR5-Δ32 composite gene.

[0014] Further, in step (1), the reaction conditions for the PCR amplification are: denaturation at 94°C for 4 min in the first cycle, annealing at 58°C for 1 min, and extension at 72°C for 4 min; for each subsequent cycle: denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 4 min, for a total of 30 cycles.

[0015] The present invention also provides a method for constructing the recombinant of the adenovirus and the CCR5-Δ32 mutant gene, comprising the following steps:

[0016] (1) Amplify the LTR sequences on both sides of adenovirus 5 by PCR, introduce PacI restriction sites respectively, and clone the LTR sequences on both sides into the pUC18 vector to construct the recombinant vector pGT-1;

[0017] (2) Co-transfect the recombinant vector pGT-1 and wild adenovirus 5 ATCC-VR-5 into the Escherichia coli strain BJ5183 to cause homologous recombination between the adenovirus 5 genome and the recombinant vector pGT-1, and obtain the recombinant vector pGT-2 containing the whole genome of adenovirus 5;

[0018] (3) Co-transfect the recombinant vector pGT-2 and the CCR5-Δ32 composite gene into the Escherichia coli strain BJ5183 to cause homologous recombination between the two, and obtain the recombinant vector pGT-3;

[0019] (4) Linearize the recombinant vector pGT-3 by PacI digestion to remove the vector sequence derived from pUC18, and obtain the recombinant of the adenovirus and the CCR5-Δ32 mutant gene.

[0020] Further, in steps (2)-(3), the specific method for the homologous recombination is: incubate at 4°C for 30 min, shock at 42°C for 50 s, then incubate at 4°C for 1 min, add 1 ml of LB culture medium and culture for 1 h. Transfer the incubated engineered bacteria to an agar culture plate containing ampicillin. After 24 h, pick a single strain clone with a sterilized toothpick, and then place it in a clean culture flask containing LB for culture.

[0021] The present invention also provides a CCR5△32 lentivirus, which is prepared by transfecting cells with the recombinant adenovirus and CCR5-Δ32 mutant gene described above.

[0022] The present invention also provides the use of the CCR5-Δ32 mutant gene expression cassette, or the recombinant adenovirus and CCR5-Δ32 mutant gene, or the CCR5△32 lentivirus in the preparation of drugs for preventing and treating AIDS.

[0023] Furthermore, the drug inhibits the expression of CCR5 and CXCR4 molecules on the cell surface, so that HIV-1 gp120 cannot effectively bind to the CCR5-Δ32 mutant gene, and the HIV-1 virus cannot enter the host cell for replication, thereby achieving the effect of treating AIDS.

[0024] Beneficial effects:

[0025] The present invention provides a CCR5-Δ32 mutant gene expression cassette, a recombinant constructed by an adenovirus and the expression cassette, and a construction method thereof. The recombinant is transfected into cells to produce a virus solution, which can efficiently inhibit the expression of CCR5 and CXCR4 molecules on the cell surface and can improve the infectivity of cells to HIV-1 virus. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1 It is a diagram showing the inhibition rate of the expression of cell surface molecules CCR5 and CXCR4 in Jurkat cells infected with CCR5△32 lentivirus in Example 4 of the present invention;

[0028] Figure 2 It is a diagram showing the blocking effect of live cells infected with CCR5△32 lentivirus on R5-tropic HIV-1 virus in Example 5 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0029] The various exemplary embodiments of the present invention will be described in detail below. This detailed description should not be considered as a limitation of the present invention, but should be understood as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0030] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not intended to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0031] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0032] Without departing from the scope or spirit of the present invention, various modifications and variations can be made to the specific embodiments of the present invention specification, which will be obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention will be obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0033] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0034] The chemical reagents, biochemical reagents and materials used in the present invention can be obtained from commercial sources unless otherwise specified.

[0035] The present invention will be described in detail below with reference to the accompanying drawings, so that the advantages and features of the invention can be more easily understood by those skilled in the art, thereby making the protection scope of the present invention more clearly defined, but the present invention is not limited to the scope of the described embodiments. The reagents and raw materials used in the following examples are all commercially available, and the test methods without specific conditions are usually carried out under conventional conditions or according to the conditions recommended by each manufacturer. Additionally, unless otherwise stated, the experimental methods, detection methods, and preparation methods disclosed in the present invention all adopt the conventional techniques in the fields of molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field of the present invention. Example 1 Construction of the CCR5-Δ32 Mutation Gene Expression Cassette Sequence

[0036] In this example, the CCR5-Δ32 mutation gene expression cassette sequence was constructed according to the following method:

[0037] Using the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers, the CCR5-Δ32 gene was obtained by PCR amplification from the cDNA of the CCR5-Δ32 population. Reaction system: Taq DNA polymerase (5U / μl) 0.25 μl, template DNA 1 - 4 μl (total amount < 1 μg), dNTPs (2.5 mM) 4 μl, upstream primer (SEQ ID NO.3) (10 μM) 2 μl, downstream primer (SEQ ID NO.4) (10 μM) 2 μl, 10× Taq Buffer 5 μl, supplemented with ultrapure water to a final volume of 50 μl. Reaction conditions: In the first cycle, denaturation at 94 °C for 4 min, annealing at 58 °C for 1 min, extension at 72 °C for 4 min; in subsequent cycles: denaturation at 94 °C for 1 min, annealing at 58 °C for 1 min, extension at 72 °C for 4 min, for a total of 30 cycles. Analyzed by agarose gel electrophoresis and the CCR5-Δ32 gene was recovered. The amplified full-length CCR5-Δ32 gene (Gene ID: AF009962.1) (containing 5' and 3' untranslated region sequences) was cloned into the prokaryotic plasmid pUC19, sequenced for verification, and the plasmid with correct sequencing verification was reserved for use.

[0038] Subsequently, the LTR sequence (containing the promoter, positions 71 - 325) of RSV (rous sarcoma virus), the poly(A) sequence of BGH (bovine growth hormone), and the adenovirus E1 region sequence were amplified by PCR respectively, and a linker sequence was introduced on one side for sequencing verification. When performing the PCR reaction again, the LTR and PA sequences were respectively spliced to the 5' and 3' ends adjacent to the CCR5-Δ32 gene to obtain the spliced product LTR-CCR5-Δ32-PA. The adenovirus E1 region and its upstream sequence were respectively spliced to the outermost side of the CCR5-Δ32 gene to construct the CCR5-Δ32 composite gene, and the specific sequence of this CCR5-Δ32 composite gene is shown in SEQ ID NO.1.

[0039] Example 2 Construction of the recombinant of adenovirus and CCR5-Δ32 mutant gene

[0040] In this example, the construction method of the recombinant of adenovirus and CCR5-Δ32 mutant gene is as follows:

[0041] (1) The LTR sequences on both sides of adenovirus 5 were amplified by PCR, and PacI restriction enzyme sites were introduced respectively. The LTR sequences on both sides were cloned into the pUC18 vector to construct the recombinant vector pGT-1;

[0042] (2) Co-transfect the constructed pGT-1 vector with wild-type adenovirus 5 ATCC-VR-5 (adenovirus strain 75, titer: 10(6.75), TCID(50) / ml) into Escherichia coli strain BJ5183 (preserved by Cydan Biotechnology Co., Ltd., accession number: P-e012) to cause homologous recombination between the adenovirus 5 genome and pGT-1. Method: Incubate at 4°C for 30 min, shock at 42°C for 50 s, then incubate at 4°C for 1 min, add 1 ml of LB culture medium and culture for 1 h. Transfer the incubated engineered bacteria to an agar culture plate containing ampicillin. After 24 h, pick a single strain clone with a sterilized toothpick and then place it into a clean culture flask containing LB. After 24 h. The positive virus clone is amplified, screened by PCR and identified by PacI digestion to obtain the recombinant vector pGT-2 containing the entire adenovirus 5 genome.

[0043] (3) Co-transfect the constructed recombinant vector pGT-2 and the CCR5-Δ32 complex gene into Escherichia coli strain BJ5183 to cause homologous recombination between the two. As above, the positive clone is amplified, screened by PCR and identified by digestion. The recombinant vector pGT-3 is obtained, and most of the adenovirus 5 sequences are contained in this vector (the E1 region and the upstream partial sequence are replaced by the CCR5-Δ32 gene expression cassette).

[0044] (4) The recombinant vector pGT-3 is linearized by PacI digestion to remove the vector sequence derived from pUC18, and a recombinant of adenovirus and the CCR5-Δ32 mutant gene is obtained. The sequence of this recombinant of adenovirus and the CCR5-Δ32 mutant gene is composed of the left side of the adenovirus 5 genome sequence, the right side of the adenovirus 5 genome sequence and the sequence shown in SEQ ID NO.2. The sequence shown in SEQ ID NO.2 is 3990 base pairs in total. Among them, the detailed information of the above sequence is as follows:

[0045] 1) The left side of the adenovirus 5 genome sequence and the right side of the adenovirus 5 genome sequence can be seen in the complete adenovirus 5 genome sequence set (GenBank: NC_001406);

[0046] 2) The 1-70th base pairs in the sequence shown in SEQ ID NO.2 are part of the right arm sequence of adenovirus (the 70th base pair of the above sequence is located at the 3329th position in the forward direction of the adenovirus 5 genome sequence, reverse complementary), which is used to connect with the right side of the adenovirus 5 genome sequence;

[0047] 3) The 71-325th base pairs in the sequence shown in SEQ ID NO.2 are the LTR (promoter) of Rous sarcoma virus;

[0048] 4) The 326-379th base pairs in the sequence shown in SEQ ID NO.2 are the 5'-untranslated region;

[0049] 5) The base sequence at positions 380 - 1406 in the sequence shown in SEQ ID NO.2 is the coding sequence of the CCR5-Δ32 gene;

[0050] 6) The base sequence at positions 1407 - 3875 in the sequence shown in SEQ ID NO.2 is the 3'-untranslated region (where the polyadenylate tail polyA starts from position 3651);

[0051] 7) The base sequence at positions 3876 - 3990 in the sequence shown in SEQ ID NO.2 is a partial sequence of the left arm of adenovirus (the base at position 3877 of the above sequence is complementary to the 452nd base in the forward direction of the adenovirus 5 genome sequence), which is used for ligation to the left side of the adenovirus 5 genome sequence.

[0052] In addition, the base sequence at positions 71 - 3875 in the sequence shown in SEQ ID NO.2 is the CCR5-Δ32 mutant gene expression cassette sequence as shown in SEQ ID NO.1. It can be seen that the CCR5-Δ32 mutant gene expression cassette sequence prepared in Example 1 of the present invention is a characteristic sequence composed of a promoter - CCR5-Δ32 cDNA - polyadenine nucleotide.

[0053] Example 3 Preparation of virus solution from recombinant adenovirus and CCR5-Δ32 mutant gene

[0054] The specific steps for preparing the virus solution from the recombinant adenovirus and CCR5-Δ32 mutant gene obtained in this example are as follows:

[0055] Transfect the recombinant adenovirus and CCR5-Δ32 mutant gene into 293 cells (frozen by SinoCuro Group Co., Ltd., preservation number: E-393). Inoculate 293T cells into DMEM culture medium containing 10% fetal bovine serum, and culture overnight in an incubator at 37°C and 5% CO2. On the day of transfection, change to fresh DMEM medium (containing 10% fetal bovine serum) and continue culturing. When the cell density reaches 70% - 90%, perform transfection. After culturing for 7 days, collect the cells, centrifuge at 1000 rpm for 15 min, discard the supernatant, freeze-thaw the cells at 37°C / 80°C three times, then centrifuge at 4000 rpm for 30 min, take the supernatant, discard the precipitate, use the supernatant for secondary infection to amplify the virus, lyse the virus in the same way, take the supernatant and perform CsCl2 density gradient centrifugation under the conditions: 4°C, 60000 rpm, 16 h. Take the recombinant adenovirus separation band with a No. 7 injection needle. Dialyze with a Spectra MW6000 dialysis bag in N1H buffer for 4 h, and keep the whole process at 4°C. Take out the virus solution, filter and sterilize it with a 0.25 μm filter membrane, and aliquot. Store at -80°C. The final titer of the obtained recombinant lentivirus is 5×10 5 TU / ml.

[0056] In summary, the present invention packages the human CCR5-Δ32 manipulated by a promoter containing an adenovirus cis-acting sequence and LTR intracellularly, and constructs a gene recombinant with high transfection efficiency, strong operability and controlled by a single promoter based on this. Subsequently, this gene recombinant can also be used to prepare drugs for treating anti-HIV-1 virus infection.

[0057] Example 4: Effect of CCR5△32 Lentivirus on Cells

[0058] Culture the CD4+ T lymphocyte line Jurkat cells, which are target cells of HIV-1 and express CCR5 and CXCR4. Infect Jurkat cells with 1 ml of concentrated and purified virus solution (5×10 5 TU / ml), and screen with 1640 medium containing puromycin (2 μg / ml) 48 h later. Change the medium every 3 days, and select viable cells after 12 days. Use uninfected Jurkat cells as a control.

[0059] Use flow cytometry to detect the expression of CCR5 and CXCR4 molecules on the surface of viable cells, detect once every 2 days, and calculate the inhibition rate. The results are as Figure 1 shown. The inhibition rate of Jurkat cells infected with CCR5△32 lentivirus on surface CCR5 is 56.8%, and the inhibition rate on CXCR4 is 61.9%. It shows that the selected viable cells have the ability to resist HIV-1 infection.

[0060] Example 5: Effect of CCR5△32 Lentivirus on HIV-1 Virus

[0061] Inoculate the viable cells screened in Example 2 into a culture plate, and culture in an incubator at 37°C and 5% CO2 for 48 h. Then infect with 10 3 TCID 50 (50% tissue culture infective dose) of the HIV-1 R5 strain ADA. Detect the p24 protein content in the cell culture supernatant 72 h later, and compare with the blocking effect of the CCR5 antagonist Maraviroc at the same time. The results show that the viable cells infected with CCR5△32 lentivirus and the CCR5 antagonist Maraviroc have a significant blocking effect on the R5-tropic HIV-1 virus ( Figure 2 ).

[0062] As can be seen from the above examples, the present invention provides a CCR5-Δ32 mutant gene expression cassette, a recombinant constructed by an adenovirus and the expression cassette, and its construction method. The recombinant is transfected into cells to produce a virus solution, which can efficiently inhibit the expression of CCR5 and CXCR4 molecules on the cell surface and improve the ability of cells to resist HIV-1 virus infection.

[0063] The above-described embodiments merely represent several implementation manners of the present invention. The description thereof is relatively specific and detailed, but it should not be construed as a limitation to the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A CCR5-Δ32 mutant gene expression cassette, characterized in that: The nucleotide sequence of the CCR5-Δ32 mutant gene expression cassette is shown in SEQ ID NO.

1.

2. A recombinant of adenovirus and CCR5-Δ32 mutant gene, characterized in that: The adenovirus and CCR5-Δ32 mutant gene recombinant contains the CCR5-Δ32 mutant gene expression box shown in SEQ ID NO: 1 and the adenovirus 5 genome sequence shown in GenBank: NC_001406.

3. The adenovirus and CCR5-Δ32 mutant gene recombinant according to claim 2, characterized in that: The 5' end of the CCR5-Δ32 mutant gene expression cassette is connected to the forward 3329 base of the adenovirus 5 genome sequence, and the 3' end of the CCR5-Δ32 mutant gene expression cassette is connected to the forward 452 base of the adenovirus 5 genome sequence.

4. The method for constructing the CCR5-Δ32 mutant gene expression cassette according to claim 1, characterized in that: The specific steps include: (1) Using the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 as primers, the human tumor suppressor factor CCR5-Δ32 gene was amplified by PCR, and the amplified full-length CCR5-Δ32 gene was cloned into the prokaryotic plasmid pUC19, and sequenced and verified. The plasmid with the correct sequence verification was reserved for use; (2) PCR was used to amplify the RSV LTR sequence, the BGH poly(A) sequence and the adenovirus E1 region sequence, and the linker sequence was introduced on one side for sequencing verification. When the PCR reaction was performed again, the LTR and PA sequences were spliced ​​to the 5' and 3' ends of the CCR5-Δ32 gene, respectively, to obtain the spliced ​​product LTR-CCR5-Δ32-PA. The adenovirus E1 region and its upstream sequence were spliced ​​to the outermost side of the CCR5-Δ32 gene, respectively, to form the CCR5-Δ32 composite gene.

5. The construction method according to claim 4, characterized in that: In step (1), the reaction conditions of the PCR amplification are: the first cycle is denaturation at 94°C for 4 min, annealing at 58°C for 1 min, and extension at 72°C for 4 min; the subsequent cycles are denaturation at 94°C for 1 min, annealing at 58°C for 1 min, and extension at 72°C for 4 min, for a total of 30 cycles.

6. The method for constructing a recombinant of adenovirus and CCR5-Δ32 mutant gene according to any one of claims 2 to 3, characterized in that: The following steps are involved: (1) PCR amplified the LTR sequences on both sides of adenovirus 5, introduced PacI restriction sites respectively, and cloned the LTR sequences on both sides into the pUC18 vector to construct the recombinant vector pGT-1; (2) co-transfecting the recombinant vector pGT-1 and wild adenovirus 5 ATCC-VR-5 into the Escherichia coli strain BJ5183, so that the adenovirus 5 genome and the recombinant vector pGT-1 undergo homologous recombination, thereby obtaining a recombinant vector pGT-2 containing the full genome of adenovirus 5; (3) The recombinant vector pGT-2 and the CCR5-Δ32 composite gene were co-transfected into the Escherichia coli strain BJ5183 to allow homologous recombination between the two to obtain the recombinant vector pGT-3; (4) The recombinant vector pGT-3 was linearized by PacI digestion to remove the vector sequence derived from pUC18, thereby obtaining the adenovirus and CCR5-Δ32 mutant gene recombinant.

7. The construction method according to claim 6, characterized in that: In steps (2)-(3), the specific methods of homologous recombination are: incubate at 4°C for 30 minutes, shock at 42°C for 50 seconds, incubate at 4°C for 1 minute, add 1 ml of LB culture solution and culture for 1 hour, transfer the incubated engineered bacteria to an agar culture plate containing ampicillin, and after 24 hours, pick a single strain clone with a sterilized toothpick and then culture it in a clean culture bottle containing LB.

8. A CCR5△32 lentivirus, characterized in that The CCR5Δ32 lentivirus is prepared by transfecting cells with the adenovirus according to any one of claims 2 to 3 and the CCR5-Δ32 mutant gene recombinant.

9. Use of the CCR5-Δ32 mutant gene expression cassette according to claim 1, the adenovirus and CCR5-Δ32 mutant gene recombinant according to any one of claims 2 to 3, or the CCR5△32 lentivirus according to claim 8 in the preparation of drugs for preventing and treating AIDS.

10. The use according to claim 9, characterized in that The drug inhibits the expression of CCR5 and CXCR 4 molecules on the cell surface, thereby preventing HIV-1gp120 from effectively binding to the CCR5-Δ32 mutant gene, making it impossible for the HIV-1 virus to enter the host cell for replication, thereby achieving the effect of treating AIDS.

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