CN115044615A - An AAV vector targeting and infecting breast cancer cells and its application - Google Patents

Abstract

The invention discloses a chimeric AAV2 capsid targeting breast cancer cells, a method for constructing an AAV2 vector targeting breast cancer cells infecting cells, a method for targeting breast cancer cells using the vector, and a method for carrying a suicide gene to treat breast cancer. In the present invention, the EC1 protein is inserted into the surface of the AAV2 capsid and the R585 and R588 sites of the mutated VP1 protein through genetic engineering technology to obtain an AAV virus vector (AAV2M-EC1). Compared with the wild-type AAV2, AAV2M-EC1 has a targeted The characteristics of breast cancer cells, and greatly reduce the ability to infect the liver and muscle tissue. Using AAV2M‑EC1 as a vector, carrying the suicide gene HSV‑TK, and injected into the tumor mice through the tail vein, it can significantly inhibit the proliferation of mouse breast cancer cells.

Description

An AAV vector targeting and infecting breast cancer cells and its application

technical field

The invention belongs to the field of biotechnology, and in particular relates to an AAV virus targeting breast cancer cells and its application.

Background technique

Breast cancer is a malignant tumor that occurs in the breast epithelial tissue and is one of the most common malignant tumors in women. According to research reports, the number of new cases and deaths of breast cancer in China each year account for 12.2% and 9.6% of the world's total, and there is a trend of increasing year by year. Due to the complex pathological classification of breast cancer, its treatment is poorly targeted and the response varies greatly. With the deepening of the understanding of breast cancer and the development of the current concept of personalized medicine, the treatment of breast cancer has entered the era of comprehensive treatment, and a treatment model of local and systemic treatment of breast cancer has been formed. At present, according to the stage of the tumor, various methods such as surgical resection, radiotherapy and chemotherapy, endocrine therapy, biological targeted therapy and adjuvant therapy of traditional Chinese medicine are often used in clinical practice. At present, the toxic and side effects of chemotherapeutic drugs and the generation of tumor resistance are urgent clinical problems to be solved. Tumor-targeted therapy is a hot spot in anti-tumor research in recent years. Compared with chemotherapy, molecular-targeted therapy has obvious advantages in targeting and toxic side effects. treatment has important clinical significance.

Adeno-associated virus (AAV), as a vector for gene delivery, has the advantages of good safety, low immunogenicity and stable expression, and has been widely used in gene therapy. In recent years, AAV-mediated tumor gene therapy has attracted much attention. AAV has been successfully used to deliver and transduce various tumor therapeutic genes (suicide genes, anti-angiogenic genes, immune-related genes, etc.) to inhibit tumor occurrence, development and metastasis . How to eliminate the original tissue tropism (off-target effect) of AAV2 and confer new targeting capabilities is the key to the development of breast cancer-targeting vectors. Since the capsid protein of AAV determines the tissue cell specificity of AAV, the modification and transformation of AAV capsid protein can improve the targeting ability of AAV. Epithelial cell adhesion molecule (EpCAM) is a type I transmembrane glycoprotein, which is mainly involved in various physiological activities such as cell proliferation, differentiation, migration and tumor cell immune escape. Studies have reported that the expression of EpCAM in primary breast cancer tissues is more than 100 times higher than that in normal tissues, so EpCAM can be used as a potential targeting molecule for breast cancer, and it can be introduced into AAV viral vectors to enhance the targeting of AAV viruses. .

SUMMARY OF THE INVENTION

The object of the present invention is to provide a novel AAV viral vector that can target infecting breast cancer cells, which is mainly realized by a packaging method of a novel AAV viral vector, and the specific steps are as follows:

(1) Using the AAV2 capsid plasmid pRC as a template, the pRVP1/3 plasmid expressing VP1 and VP3 was obtained by mutating the T138 site of the VP1 protein as A, and the capsid pRVP1/3 DNA sequence was SEQ ID NO: 1;

(2) Using the AAV2 capsid plasmid pRC as a template, the VP2 protein M1 and M203 sites of the pRC plasmid were subjected to site-directed mutagenesis to obtain a plasmid pVP2, which provided the capsid protein VP2, and its DNA sequence was SEQ ID NO: 2;

(3) combining the plasmid pRVP1/3 obtained in step (1), the plasmid pVP2 obtained in step (2), pHelper, and pAAV to construct an AAV four-plasmid packaging system;

(4) packaging the AAV virus with the packaging system obtained in step (3).

In step (1), plasmid RVP1/3 is a PCR template, and arginine at position 585 of capsid protein VP1 is mutated to alanine, and arginine at position 588 is mutated to alanine to obtain pRVP1/3M plasmid , the capsid pRVP1/3M plasmid DNA sequence is SEQ ID NO:3.

The pRVP1/3M plasmid, the plasmid pVP2 obtained in step (2), pHelper, and pAAV were combined to construct an AAV four-plasmid packaging system, and packaged into an AAV virus.

In step (2), on the basis of the pVP2 plasmid, the EC1 protein sequence is inserted into the amino terminus of the VP2 protein to obtain the pVP2-EC1 plasmid.

The EC1 protein sequence described in step (2) was screened by Nikolas Stefan et al. through phage display technology, the DNA sequence is SEQ ID NO:5, and the amino acid sequence is SEQ ID NO:6.

The pRVP1/3M plasmid or pRVP1/3 plasmid, pVP2-EC1 plasmid, pHelper and pAAV were combined to construct an AAV four-plasmid packaging system, and packaged into AAV virus.

The plasmid pAAV described in step (3) is any one of pAAV-eGFP, pAAV-Luciferase and pAAV-TK expression plasmids.

As a preferred version, taking a 10cm dish as an example, the virus packaged in the present invention includes the following:

8μg pVP2, 8μg pRVP1/3, 10μg pHelper and 6μg pAAV-luciferase were used to package AAV2-LUC virus,

8μg pVP2, 8μg pRVP1/3, 10μg pHelper and 6μg pAAV-TK were used to package AAV2-TK virus,

8μg pVP2, 8μg pRVP1/3M, 10μg pHelper and 6μg pAAV-luciferase were used to package AAV2M-LUC virus,

8 μg pVP2-EC1, 8 μg pRVP1/3M, 10 μg pHelper and 6 μg pAAV-luciferase were used to package AAV2M-EC1-LUC virus,

8 μg pVP2-EC1, 8 μg pRVP1/3M, 10 μg pHelper and 6 μg pAAV-TK were used to package AAV2M-EC1-TK virus.

The packaged AAV virus vector was purified by centrifugation, and then Western Blotting was used to detect the expression of three components in the AAV virus capsid: VP1, VP2, and VP3, so as to evaluate the transformation of the AAV virus.

The present invention applies the obtained AAV virus vector in the preparation of a drug for targeted treatment of breast cancer.

The application of the AAV virus vector carrying the suicide gene HSV-TK in the preparation of a drug for inhibiting the proliferation of breast cancer cells. The breast cancer cells are 4T1 breast cancer cells.

The distribution of AAV2-LUC, AAV2M-LUC and AAV2M-EC1-LUC viral vectors in mouse tissues and organs was evaluated by targeted infection of mouse xenografts by injecting AAV virus carrying luciferase through the tail vein of mice , Small animal in vivo imaging to detect the distribution of AAV virus in mice; take various organs of mice to image and detect the distribution of AAV virus.

The invention also provides an application of the AAV2-EC1 virus obtained by the packaging method based on the AAV virus vector. After injection into tumor-bearing mice, the targeting effect of AAV2M-EC1-TK on mouse breast cancer cells was evaluated by detecting the body weight, tumor volume, and mouse survival rate of tumor-bearing mice.

beneficial effect

In the present invention, the pRC plasmid is transformed into pRVP1/3 and pVP2 plasmids, and the obtained two types of plasmids, pAAV plasmid and pHelper plasmid are constructed into a four-plasmid packaging system, and the AAV2 virus is successfully packaged through the four-plasmid packaging system.

In addition, by further mutating pRVP1/3 to obtain pRVP1/3M plasmid, inserting EC1 sequence into pVP2 to obtain pVP2-EC1 plasmid, then two plasmids, pAAV plasmid and pHelper plasmid were obtained to construct a four-plasmid packaging system, and AAV was carried out by this system. packaging to obtain the AAV2M-EC1 viral vector. Compared with the wild-type AAV2 virus vector and the modified AAV2M-EC1 virus vector, the AAV2-EC1 virus vector has the characteristics of targeting breast cancer cells, high infection efficiency, strong infection ability, and the ability to infect liver and muscle tissue is greatly reduced , and has broad prospects in the preparation of drugs for the treatment of breast cancer.

Description of drawings

Figure 1 is a schematic diagram of AAV2 capsid protein transformation and plasmid digestion identification diagram, wherein A is a schematic diagram of the transformation, B is an enzyme digestion identification diagram, wherein M: DNA Marker; 1: pR-EC1-VP2 plasmid; 2: pR-EC1- VP2 plasmid digested by endonuclease Nco I and EcoRV; 3: pR-VP2 plasmid; 4: pR-VP2 plasmid digested by endonuclease Nco I and EcoRV; 5: pR-VP1,3 plasmid; 6 : The pR-VP1,3 plasmid was digested with endonucleases Nco I and EcoR V.

Figure 2 shows the detection of AAV virus capsid proteins VP1, VP2 and VP3 by Western blot.

Figure 3 is an in vivo imaging image of AAV2M-EC1 virus targeting 4T1 xenografts.

Figure 4 is an image of main tissues and organs after AAV2M-EC1 virus infects 4T1 xenografted mice.

Figure 5 is a graph of luciferase enzyme activity in various tissues and organs of mice.

Figure 6 shows the identification map of pAAV-TK plasmid digestion, which was double-digested by EcoR1+Hind111.

Fig. 7 is a flow chart of experimental animals for AAV2M-EC1-TK gene therapy for breast cancer.

Figure 8 is the body weight growth curve of mice after AAV2M-EC1-TK gene therapy for breast cancer.

Figure 9 shows the survival curve of mice after AAV2M-EC1-TK gene therapy for breast cancer.

Figure 10 is a graph showing the change of tumor volume in mice after AAV2M-EC1-TK gene therapy for breast cancer.

Figure 11 is a visual diagram of the tumor in vitro after AAV2M-EC1-TK gene therapy for breast cancer.

Figure 12 is a statistical graph of tumor mass in mice after AAV2M-EC1-TK gene therapy for breast cancer.

Detailed ways

The present invention will be further described below in conjunction with the embodiments, but the claimed scope of the present invention is not limited to the scope expressed by the embodiments.

Example 1 Construction of pRVP1/3 plasmid

(1) Design mutation primers

According to the DNA sequence of AAV2 capsid, a mutation primer was designed at the start codon of VP2 to mutate the threonine T at the 138th amino acid position of VP1 to alanine A, and the designed primer sequence was sent to GeneWise Biosciences Technology Co., Ltd. Synthesis.

T138-F: GAGGAACCTGTTAAGACCCTCCGGGAAAAAAGAGGCCGGT

T138-R: ACCGGCCTCTTTTTTCCCGGAGGGTCTTAACAGGTTCCTC

(2) PCR site-directed mutation of the T138 site

Using the AAV2 capsid plasmid pRC (gifted by Professor Zhang Ye of Peking Union Medical College) as a template, using Prime STAR ^@ HS DNA Polymerase purchased from TAKARA, and combining the primers obtained in step (1), PCR amplification was carried out. The PCR reaction system See Table 1, and the reaction program is shown in Table 2:

Table 1 PCR reaction system

Table 2 PCR reaction program

After the PCR reaction, the PCR product was digested with DpnⅠ for 2 hours, the template plasmid was digested, and 5 μL of the digested product was transferred into DH5α Escherichia coli competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) /mL ampicillin on the LB plate medium, cultured overnight at 37°C at a constant temperature to obtain positive colonies of the mutated plasmid pRVP1/3.

(3) Plasmid extraction and identification

The positive colonies obtained in step (3) were inoculated into LB medium containing 100 mg/mL ampicillin, and cultured overnight at 37°C on a shaker. The bacterial liquid cultured overnight was centrifuged at 4000 rpm for 5 min to collect the precipitate, and the endotoxin-free plasmid extraction kit purchased from Tiangen Biotechnology Co., Ltd. was used to extract the plasmid, and the restriction enzymes Nde I and Xba I (purchased from Takara Company) were used to extract the plasmid. Double-enzyme digestion (Table 3) was used for detection, and DNA sequencing (Jinweizhi Biotechnology Co., Ltd.) was used for identification. The results are shown in Figure 1B. After double-enzyme digestion, two bands of 1000bp and 6200bp appeared, indicating that the pRVP1/3 plasmid was successfully constructed. .

Table 3 Double enzyme digestion detection system

Example 2 Construction of pVP2 plasmid

The steps and methods are the same as those in Example 1, except that the site-directed mutagenesis primers, mutation sites and plasmids are changed.

The methionine at the 1st and 203rd amino acid positions of the VP2 protein was site-directed to alanine, and the mutation primers were:

M1-F: AATGATTTAAATCAGGTCTGGCTGCCGATGGTTAT

M1-R: ATAACCATCGGCAGCCAGACCTGATTTAAATCATT

M203-F: GTCTGGGAACTAATACGCTGGCTACAGGCAGTGGC

M203-R: GCCACTGCCTGTAGCCAGCGTATTAGTTCCCAGAC

The results are shown in Figure 1B. After double digestion, two bands of 1000bp and 6200bp appeared. The methionine at the 1st and 203rd amino acid positions was identified by DNA sequencing as site-directed mutation to alanine, indicating that the pVP2 plasmid was successfully constructed.

Example 3 Construction of pVP2-EC1 plasmid

(1) Design homologous recombination primers:

AAV2-F: CATAGGTTCCTCAACCAGCTT

AAV2-R: GCTCCGGGAAAAAAGAGGCCGPCR

(2) PCR amplification of plasmid pVP2:

The pVP2 plasmid obtained in Example 2 was used as a template, and the primers designed in step (1) were used for PCR amplification to obtain a linearized pVP2 plasmid. The PCR amplification system was the same as that in Example 1.

(3) Synthesis of EC1 sequence and its primers

The DNA sequence of EC1 is SEQ ID NO: 1, and the amino acid sequence is SEQ ID NO: 2, and primers are designed according to the sequence of EC1. Both the EC1 sequence and the primer sequence are synthesized by Golden Vision Biotechnology Co., Ltd.:

EC1-F: GTTAAGCTGGTTGAGGAA

EC1-R: TACCGGCCTCTTTTTTCC

And adopt PCR technology to amplify to obtain EC1 gene, the reaction system and procedure are the same as Example 1.

(4) PCR product purification and recombination

The PCR products obtained in step (2) and step (3) were separated by gel electrophoresis, then the gel pieces containing the target band were cut out, and the DNA gel kits (purchased from Tiangen Biochemical Technology Co., Ltd.) were used for recovery respectively. , the recovered product pVP2 and EC1 are mixed according to the molar ratio of 1:3, and homologous to the same group is carried out. The homologous recombination kit was purchased from Beijing Quanshijin Biotechnology Co., Ltd. The reaction system is as follows:

Gently mix the above reaction system and place it at 50 °C for 15 min. After the reaction, the centrifuge tube was placed on ice to cool for a few seconds, after which the recombinant product was used for transformation amplification.

Add 5 μL of the above recombinant product to DH5α competent cells (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), mix gently, place on ice for 15 min, heat shock at 42°C for 60 s and quickly place on ice for 2 min, add 400 μL of room temperature LB medium was incubated at 37°C for 1 h on a shaker, and 100 μL of cells were evenly spread on the plate, incubated at 37°C on a shaker overnight, positive clones were picked, and the plasmids were extracted for sequencing and double-enzyme digestion verification. The results are shown in Figure 1B. After digestion, two bands of 1000bp and 6600bp appeared, indicating that the pVP2-EC1 plasmid was successfully constructed.

Example 4 Construction of pRVP1/3M capsid expression plasmid

The method steps are the same as in Example 1, except that the template is changed to the plasmid pRVP1/3 obtained in Example 1, and the arginine R at the 585th and 588th amino acid positions of the capsid protein VP1 is mutated to alanine A by designing primers. , the primer sequence is:

M-F: TCTGTATCTACCAACCTCCAGGCAGGCAACGCACAAGCAGCTACCGCAGATGTC

M-R: GACATCTGCGGTAGCTGCTTGTGCGTTGCCTGCCTGGAGGTTGGTAGATACAGA;

After PCR site-directed mutagenesis, the plasmids were identified by DNA sequencing, and pRVP1/3M plasmids with R585 and R588 site mutations were successfully constructed.

Example 5 Packaging and purification of recombinant adeno-associated virus

(1) HEK293T cells were cultured in DMEM medium (purchased from Thermo Fisher Scientific) with a concentration of 10% fetal bovine serum (purchased from Biological Industries), and cultured in a cell incubator at 37°C, 5% CO ₂ until the Growth phases were counted, cells were collected and counted. Inoculate in a 10cm diameter cell culture dish with an inoculation amount of 4×10 ⁶ cells, and continue to culture for 20h until the cell density is 80-90%; 4h before virus packaging, fresh cell culture medium needs to be replaced.

(2) Virus packaging

8μg pVP2, 8μg pRVP1/3, 10μg pHelper and 6μg pAAV-luciferase were used to package AAV2-LUC virus,

8μg pVP2, 8μg pRVP1/3M, 10μg pHelper and 6μg pAAV-luciferase were used to package AAV2M-LUC virus,

8 μg pVP2-EC1, 8 μg pRVP1/3M, 10 μg pHelper and 6 μg pAAV-luciferase were used to package AAV2M-EC1-LUC virus,

Wherein pVP2 is prepared in Example 2, pRVP1/3 is prepared in Example 1, pVP2-EC1 is prepared in Example 3, pRVP1/3M is prepared in Example 4, pHelper is purchased from the addgene website, pAAV-luciferase is from Beijing Donated by Professor Zhang Ye of Union Medical College.

Add the above plasmids to 500 μL of serum-free DMEM medium (liquid A), mix gently and let stand for 5 minutes;

(3) Add 13 μL of transfection reagent Imafect (purchased from Beijing Keyin Technology Co., Ltd.) to 500 μL of serum-free DMEM medium (liquid B), mix gently, and let stand for 5 minutes. Gently add droplet B to solution A and let it stand for a few seconds, then mix gently, and let the mixture stand for 15 minutes.

(4) Add the mixture dropwise to the cell culture dish and mix gently, and replace the fresh cell culture medium after 6 hours; after the cell culture medium turns yellow, the medium is changed and the supernatant is collected. After transfection, 600 μL of PBS is used for 72 hours of culture. (Weigh 8.0g NaCl, 0.2g KCl, 1.44g Na ₂ HPO ₄ , 0.24g KH ₂ PO ₄ and dissolve them in 800 mL of distilled water, adjust the solution to 7.4 with HCl, and finally add distilled water to dilute to 1L, autoclave at 4°C Storage) Gently blow down the adherent cells in the dish and store in a -80°C refrigerator for later use.

(5) Take out the packaged AAV virus obtained in step (2) and freeze and thaw three times between -80°C and 37°C, and vortex the precipitation for each freeze-thaw time, so that the virus can better escape from the cells. release;

(6) Use 100U totipotent nuclease (purchased from Shanghai Yisheng Biotechnology Co., Ltd.) to incubate the freeze-thawed cells for 3 times at 37°C for 2 hours, and then centrifuge at 10,000 rpm for 10 minutes to collect the supernatant;

(7) Gently add 15% (3mL), 25% (2mL), 40% (2mL) and 60% (1mL) density gradient iodixanol (Table 4) in a 10mL ultracentrifuge tube from top to bottom, After each layer was stabilized, the supernatant obtained in step (6) was slowly added to the uppermost layer, and centrifuged at 40,000 rpm for 4h;

(8) The target virus exists in the position of 40% of iodixanol, and the liquid of the layer (40% iodixanol) of the target virus is drawn into the filter tube of 10000kDa, and the filter tube is filled with PBS and centrifuged at 3000rpm for 4min. Repeated centrifugal filtration to remove iodixanol and concentrated the target virus; the concentrated virus was stored in a refrigerator at -80°C for later use.

Table 4 Iodixanol solution formulations of different concentrations

60% of the iodixanol stock solution was purchased from Stem cell;

10×PBS-MK: 10×100mL PBS solution+0.2g MgCl ₂ ·6H ₂ O+0.19g KCl, dissolve;

0.5% phenol red: Weigh 0.1 g of phenol red powder into 20 mL of 50% ethanol, and heat to dissolve.

Example 6 Western Blotting detection of protein expression AAV virus

(1) Take 20 μL of the virus obtained in Example 5, add 5 μL of protein preparation buffer (purchased from Thermo Fisher Scientific), boil the sample in boiling water at 100°C for 7 minutes, and quickly put it on ice to obtain an AAV virus sample. The sample is stored at -20°C save;

(2) Take 20 μL of the AAV sample obtained in step (1) and run it through 10% SDS-PAGE 80V constant voltage electrophoresis, take out the PAGE gel after electrophoresis, and transfer it to the membrane with a constant current of 300mA for 90min;

(3) The PVDF membrane (purchased from Merck Millipore) after the protein sample was transferred to the membrane was placed in 1×TBST (2.42g Tris-base, 8.80g NaCl was dissolved in 900mL ddH2O, 500μL Tween-20 was added, and the pH was adjusted. To 7.40, dilute to 5% milk prepared in 1000 mL) and seal it on a shaker at room temperature for 1 hour. After the seal is complete, discard the milk and add TBST several times on a shaker at room temperature for 7 minutes to wash off the residual milk;

(4) The PVDF membrane was placed in anti-CAP antibody (1:1000) prepared with 3% BSA (purchased from American Research Products), incubated at 4°C on a shaker overnight; the primary antibody was removed by multiple rinsing with TBST the next day. The PVDF membrane was placed in anti-mouse IgG (1:2000) prepared with 3% BSA (purchased from proteintech) and incubated on a shaker at room temperature for 1 h, washed with TBST for three times, and the results were observed after ECL color development.

The results are shown in Figure 2. Like the unmodified AAV, the modified AAV2-EC1 virus has obvious capsid protein bands, but because the EC1 protein is inserted into VP2 in the experimental design, the position of the VP2-EC1 protein occurs. The change is located above VP1. This result fully demonstrates that the present invention successfully inserts the EC1 sequence into VP2, and the VP2 inserted into the EC1 sequence together with VP1 and VP3 can be assembled into AAV virus in cells.

Example 9

(1) 4T1 cells were passaged one day before subcutaneous tumor bearing, and 1/2 of the original dish was retained;

(2) On the second day, when the cell confluence is about 80%, trypsinize the adherent cells, add DMEM cell culture medium containing serum to terminate the digestion, centrifuge at 800 rpm for 3 min, discard the supernatant and add PBS to fully resuspend into a single cell cell;

(3) Count the cells using a cell counting plate, inoculate 2.5×10 ⁵ cells subcutaneously on the back of each mouse, with a total volume of 75 μL, carry out tumor loading, and put the mice back into the mouse cage after the tumor loading is completed. Normal feeding for about a week;

(4) The tumor-bearing model mice were subjected to tumor volume measurement, and when the tumor diameter was about 3 mm, local skin depilation was performed on the tumor;

(5) 18 mice were randomly divided into 3 groups, namely AAV2-LUC group, AAV2M-LUC group and AAV2M-EC1-LUC group, with 6 mice in each group;

(6) 1×10 ¹¹ vg of virus was injected into tumor-bearing mice by tail vein injection. After 7 days, each mouse was intraperitoneally injected with Luciferin, the luciferase substrate. The injection volume of Luciferin was 1.5 mg/10 g. , that is, 20 g mice were intraperitoneally injected with 100 μL of 30 mg/mL Luciferase enzyme substrate working solution, and the Luciferase enzyme substrate working solution was purchased from Shanghai Yisheng Biotechnology Co., Ltd.;

(7) Within 10-12 minutes after luciferase substrate injection, the distribution of AAV virus in mice was observed using an animal in vivo imager. The results are shown in Figure 4. The fluorescence of mice in the AAV2-LUC group was mainly distributed in the liver; AAV2M - The fluorescence of mice in the LUC group is mainly distributed in the back muscles, and the fluorescence signal of the liver is significantly weakened; the fluorescence signal of the mice in the AAV2M-EC1-LUC group is mainly distributed in the tumor site. The targeting has been significantly enhanced, and the AAV2M-EC1 virus can well target breast cancer cells in mice;

(8) Immediately after obtaining the in vivo imaging results of the individual small animals, the mice are dissected, and the individual organs and tissues are subjected to in vivo imaging. The time from substrate injection to the end of imaging did not exceed 25 min. The imaging results of the main tissues and organs of the mice are shown in Figure 5. The results show that the fluorescence of the mice in the AAV2-LUC group is mainly distributed in the liver; the fluorescence of the mice in the AAV2M-LUC group is slightly enriched in tumor and muscle tissue; AAV2M-EC1-LUC The fluorescence signals of the mice in the group were mainly distributed in the tumor site, and the fluorescence signals could hardly be detected in other tissues and organs. This result shows that the AAV2M-EC1 virus designed in the present invention has the strongest ability to infect breast cancer, and the AAV2M-EC1 virus is in vivo in mice. Can target breast cancer cells well;

(9) After imaging, take about 100 mg of tissue, put each tissue into an EP tube containing 1 mL of PBS for washing, centrifuge at 3000 rpm for 3 min, and add the pellet to a lysate containing 400 μL of RIPA (purchased from Beijing Soleibao Technology). Co., Ltd.), and put 6 small ceramic beads into the electric homogenizer for homogenization: 10s for homogenization, 10s for rest, a total of 3 times. If there are still large pieces of tissue after homogenization for 3 times, continue 1-2 rounds of homogenization, incubate the homogenized suspension on ice for 10 min, and mix with a shaker for a total of three times. 12000rpm, centrifuge for 10min to obtain supernatant;

(10) Transfer the supernatant obtained in step (9) to a new EP tube, and then adjust the concentration of mouse tissue total protein to the same, 25 μg/μL, a total of 20 μL, and add it to the assay tube to prepare fresh Luciferase substrate working solution: Dilute Luciferin substrate 50 times and mix in detection buffer, store in the dark; turn on the fluorescence detector in a dark room. Add 50 μL of the substrate reaction solution to each tube, mix quickly, and run the machine for detection after 10 s to obtain the enzyme activity value of Luciferase in each tissue and organ. The results are shown in Figure 6. The results show that: AAV2-LUC is distributed in tumor, liver and muscle tissue, but mainly in liver tissue; AAV2M-LUC due to the mutation of R585 and R588 sites in the viral capsid protein, the virus The ability to infect various tissues in mice was weakened; while AAV2M-EC1-LUC mainly infects tumor cells due to the insertion of EC1 protein, and the infection ability to liver and muscle tissue is significantly lower than the other two viruses, so AAV2M-EC1- The LUC virus was best at targeting breast cancer cells.

Example 10 Construction of pAAV-TK plasmid

The TK coding sequence was synthesized by Goldwisdom Biological Company and constructed directly into the pAAV vector. The 5'-end restriction site was EcoR1, and the 3'-end restriction site was Hind111. The pAAV-TK plasmid was digested with EcoR1+Hind111 and separated by 1% agarose gel electrophoresis. Two restriction fragments of 1200 bp and 5000 bp were found (Fig. 6). The results were consistent with expectations. The recombinant plasmid identified as positive by enzyme digestion was sequenced by DNA, and it was confirmed that the inserted sequence in the plasmid was correct, and the reading frame docking was correct.

Example 11 Packaging of AAV virus containing suicide gene TK

The steps and methods are the same as those in Example 5, except that the plasmids for packaging are changed, wherein pVP2 is prepared in Example 2, pVP2-EC1 is prepared in Example 3, pRVP1/3 is prepared in Example 1, and pRVP1/ 3M is prepared in Example 4, and pAAV-TK is prepared in Example 10:

pVP2, pRVP1/3, pHelper and pAAV-TK were used to package AAV2-TK virus;

pVP2, pRVP1/3M, pHelper and pAAV-TK were used to package AAV2M-TK virus;

pVP2-EC1, pRVP1/3M, pHelper and pAAV-TK were used to package the AAV2M-EC1-TK virus.

Example 12 AAV virus mediates suicide gene TK in mice to treat tumors

(1) The tumor-bearing model mice were subjected to tumor volume measurement, and when the tumor size was about 3 mm, local skin depilation was performed on the tumor;

(2) The 18 mice were divided into 3 groups by random grouping, namely AAV2-TK group, AAV2M-TK group and AAV2M-EC1-TK group, with 6 mice in each group.

(3) 1×10 ¹¹ vg of virus was injected into tumor-bearing mice by tail vein injection, and 100 mg/kg of ganciclovir GCV (purchased from Selleck Company) was intraperitoneally injected into each mouse after 48 hours. The design is shown in Figure 7;

(4) Within 12 days after the first injection of GCV, intraperitoneal injection of 100 mg/kg of GCV was performed every 24 hours; and the body weight of the mice was recorded. , AAV2M-TK group and AAV2M-EC1-TK group of mice body weight no significant difference. The long diameter a and short diameter b of the mouse tumor were recorded regularly, and the tumor volume was calculated according to the formula V=ab ² /2. The results are shown in Figure 10. The tumor volume of the mice in the AAV2-TK group and AAV2M-TK group gradually increased over time, and the subordinate tumor volume in the AAV2M-EC1-TK group was significantly smaller than that in the above two groups, indicating that the AAV2M-EC1-TK injection was very effective after injection. Well inhibited proliferation of breast cancer cells.

According to the mouse survival curve in Figure 9, the mice in the AAV2-TK group died on the 6th day after the virus injection, and the AAV2M-TK group on the 5th and 6th days after the injection. This may be due to the weak ability of AAV2-TK and AAV2M-TK viruses to target tumors. After the virus enters the mouse, it mainly infects its main organs and tissues, resulting in an off-target effect, thus causing the suicide gene to kill normal cells. Death occurred; however, the mice in the AAV2M-EC1-TK group never died. This experiment further shows that the AAV2M-EC1-TK virus has a stronger targeting ability to breast cancer cells and less damage to the mice.

After continuous intraperitoneal injection of GCV for 10 days, the tumor proliferation of the mice in the AAV2M-EC1-TK group was significantly inhibited. On the 12th day of GCV injection, the tumors of the mice in each group were surgically removed, and the tumors were measured and weighed. The results are shown in Figure 10, Figure 11 and Figure 12, the tumor diameter of the AAV2-TK group was 10-12 mm, the diameter of the tumor in the AAV2M-TK group was 10-12 mm, and the tumor diameter of the mice in the AAV2M-EC1-TK group was smaller, with an average diameter of 5-7 mm; AAV2-TK The tumor mass of the AAV2M-TK group was 0.5 g, the tumor mass of the AAV2M-TK group was 0.3 g, and the tumor mass of the mice in the AAV2M-EC1-TK group was about 0.15 g, which were statistically different, indicating that after the injection of AAV2M-EC1-TK, mouse breast cancer Cell proliferation was significantly inhibited.

sequence listing

<110> Three Gorges University

<120> An AAV vector targeting and infecting breast cancer cells and its application

<160> Total number 6

<210>1

<211>2208

<212> DNA

<213> Artificial sequences

<223>VP1/3 DNA

<400> SEQ ID NO: 1

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGGCGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCA ATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGC TTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA

<210>2

<211>2208

<212> DNA

<213> Artificial sequences

<223>pVP2

<400> SEQ ID NO: 2

CTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGCTGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCA ATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGC TTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA

<210>3

<211>2207

<212> DNA

<213> Artificial sequences

<223>VP1/3M

<400> SEQ ID NO: 3

ATGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGGCGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACTCCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGATGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAA TAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGTATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGGCAGGCAACGCACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCT TCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA

<210>4

<211>2745

<212> DNA

<213> Artificial sequences

<223>VP2-EC1

<400> SEQ ID NO: 4

ATGGACCTGGGCAAGAAGCTGCTGGAGGCCGCTAGAGCCGGCCAAGACGACGAGGTGAGAATCCTGGTGGCCAACGGCGCCGACGTCAACGCCTACTTCGGTACCACACCGCTACACCTTGCGGCTGCTCACGGCAGACTTGAGATAGTGGAAGTTCTGTTGAAGAATGGAGCTGACGTCAACGCGCAAGACGTGTGGGGCATCACGCCCCTGCACTTAGCCGCATACAATGGACACCTGGAGATCGTGGAAGTCCTGCTGAAGTATGGAGCCGACGTAAACGCACACGACACAAGAGGCTGGACTCCCCTGCACCTGGCCGCCATCAACGGCCACCTGGAAATAGTTGAGGTGCTGCTCAAGAATGTAGCCGACGTAAATGCGCAAGACAGAAGCGGCAAGACCCCCTTCGACCTGGCCATCGACAACGGCAACGAGGACATCGCCGAGGTGCTGCAGAAGGCCGCCAAGCTGAACGGCGGCGGCGGCAGCGGCGGCGGCGGATCCGGCGGCGGCGGATCCGGCGGCGGCGGATCCCTGGCTGCCGATGGTTATCTTCCAGATTGGCTCGAGGACACTCTCTCTGAAGGAATAAGACAGTGGTGGAAGCTCAAACCTGGCCCACCACCACCAAAGCCCGCAGAGCGGCATAAGGACGACAGCAGGGGTCTTGTGCTTCCTGGGTACAAGTACCTCGGACCCTTCAACGGACTCGACAAGGGAGAGCCGGTCAACGAGGCAGACGCCGCGGCCCTCGAGCACGACAAAGCCTACGACCGGCAGCTCGACAGCGGAGACAACCCGTACCTCAAGTACAACCACGCCGACGCGGAGTTTCAGGAGCGCCTTAAAGAAGATACGTCTTTTGGGGGCAACCTCGGACGAGCAGTCTTCCAGGCGAAAAAGAGGGTTCTTGAACCTCTGGGCCTGGTTGAGGAACCTGTTAAGACGGCTCCGGGAAAAAAGAGGCCGGTAGAGCACTCTCCTGTGGAGCCAGACT CCTCCTCGGGAACCGGAAAGGCGGGCCAGCAGCCTGCAAGAAAAAGATTGAATTTTGGTCAGACTGGAGACGCAGACTCAGTACCTGACCCCCAGCCTCTCGGACAGCCACCAGCAGCCCCCTCTGGTCTGGGAACTAATACGCTGGCTACAGGCAGTGGCGCACCAATGGCAGACAATAACGAGGGCGCCGACGGAGTGGGTAATTCCTCGGGAAATTGGCATTGCGATTCCACATGGATGGGCGACAGAGTCATCACCACCAGCACCCGAACCTGGGCCCTGCCCACCTACAACAACCACCTCTACAAACAAATTTCCAGCCAATCAGGAGCCTCGAACGACAATCACTACTTTGGCTACAGCACCCCTTGGGGGTATTTTGACTTCAACAGATTCCACTGCCACTTTTCACCACGTGACTGGCAAAGACTCATCAACAACAACTGGGGATTCCGACCCAAGAGACTCAACTTCAAGCTCTTTAACATTCAAGTCAAAGAGGTCACGCAGAATGACGGTACGACGACGATTGCCAATAACCTTACCAGCACGGTTCAGGTGTTTACTGACTCGGAGTACCAGCTCCCGTACGTCCTCGGCTCGGCGCATCAAGGATGCCTCCCGCCGTTCCCAGCAGACGTCTTCATGGTGCCACAGTATGGATACCTCACCCTGAACAACGGGAGTCAGGCAGTAGGACGCTCTTCATTTTACTGCCTGGAGTACTTTCCTTCTCAGATGCTGCGTACCGGAAACAACTTTACCTTCAGCTACACTTTTGAGGACGTTCCTTTCCACAGCAGCTACGCTCACAGCCAGAGTCTGGACCGTCTCATGAATCCTCTCATCGACCAGTACCTGTATTACTTGAGCAGAACAAACACTCCAAGTGGAACCACCACGCAGTCAAGGCTTCAGTTTTCTCAGGCCGGAGCGAGTGACATTCGGGACCAGTCTAGGAACTGGCTTCCTGGACCCTGTTACCGCCAGCAGCGAGT ATCAAAGACATCTGCGGATAACAACAACAGTGAATACTCGTGGACTGGAGCTACCAAGTACCACCTCAATGGCAGAGACTCTCTGGTGAATCCGGGCCCGGCCATGGCAAGCCACAAGGACGATGAAGAAAAGTTTTTTCCTCAGAGCGGGGTTCTCATCTTTGGGAAGCAAGGCTCAGAGAAAACAAATGTGGACATTGAAAAGGTCATGATTACAGACGAAGAGGAAATCAGGACAACCAATCCCGTGGCTACGGAGCAGTATGGTTCTGTATCTACCAACCTCCAGAGAGGCAACAGACAAGCAGCTACCGCAGATGTCAACACACAAGGCGTTCTTCCAGGCATGGTCTGGCAGGACAGAGATGTGTACCTTCAGGGGCCCATCTGGGCAAAGATTCCACACACGGACGGACATTTTCACCCCTCTCCCCTCATGGGTGGATTCGGACTTAAACACCCTCCTCCACAGATTCTCATCAAGAACACCCCGGTACCTGCGAATCCTTCGACCACCTTCAGTGCGGCAAAGTTTGCTTCCTTCATCACACAGTACTCCACGGGACAGGTCAGCGTGGAGATCGAGTGGGAGCTGCAGAAGGAAAACAGCAAACGCTGGAATCCCGAAATTCAGTACACTTCCAACTACAACAAGTCTGTTAATGTGGACTTTACTGTGGACACTAATGGCGTGTATTCAGAGCCTCGCCCCATTGGCACCAGATACCTGACTCGTAATCTGTAA

<210>5

<211>537

<212> DNA

<213> Artificial sequences

<223>EC1

<400> SEQ ID NO: 5

ATGGACCTGGGCAAGAAGCTGCTGGAGGCCGCTAGAGCCGGCCAAGACGACGAGGTGAGAATCCTGGTGGCCAACGGCGCCGACGTCAACGCCTACTTCGGTACCACACCGCTACACCTTGCGGCTGCTCACGGCAGACTTGAGATAGTGGAAGTTCTGTTGAAGAATGGAGCTGACGTCAACGCGCAAGACGTGTGGGGCATCACGCCCCTGCACTTAGCCGCATACAATGGACACCTGGAGATCGTGGAAGTCCTGCTGAAGTATGGAGCCGACGTAAACGCACACGACACAAGAGGCTGGACTCCCCTGCACCTGGCCGCCATCAACGGCCACCTGGAAATAGTTGAGGTGCTGCTCAAGAATGTAGCCGACGTAAATGCGCAAGACAGAAGCGGCAAGACCCCCTTCGACCTGGCCATCGACAACGGCAACGAGGACATCGCCGAGGTGCTGCAGAAGGCCGCCAAGCTGAACGGCGGCGGCGGCAGC GGCGGCGGCGGATCC GGCGGCGGCGGATCCGGCGGCGGCGGATCC

<210>6

<211>179

<212> amino acid sequence

<213> Artificial sequences

<223>EC1

<400> SEQ ID NO: 6

MDLGKKLLEAARAGQDDEVRILVANGADVNAYFGTTPLHLAAAHGRLEIVEVLLKNGADVNAQDVWGITPLHLAAYNGHLEIVEVLLKYGADVNAHDTRGWTPLHLAAINGHLEIVEVLLKNVADVNAQDRSGKTPFDLAIDNGNEDIAEVLQKAAKLNGGGGSGGGSGGGGSGGGGS

Claims (10)

1. A packaging method of an AAV viral vector is characterized by specifically comprising the following steps:

(1) the AAV2 capsid plasmid pRC is taken as a template, and the mutant VP1 protein T138 site is taken as A to obtain pRVP1/3 plasmid for expressing VP1 and VP3, wherein the capsid VP1/3DNA sequence is SEQ ID NO: 1;

(2) the AAV2 capsid plasmid pRC is taken as a template, and the plasmid pVP2 is obtained by performing site-directed mutagenesis on VP2 protein M1 and M203 sites of the pRC plasmid, provides capsid protein VP2, and has a DNA sequence of SEQ ID NO: 2;

(3) combining the plasmid pRVP1/3 obtained in the step (1) and the plasmid pVP2 obtained in the step (2), pHelper and pAAV to construct an AAV four-plasmid packaging system;

(4) packaging the AAV virus by using the packaging system obtained in the step (3).

2. The method for packaging an AAV viral vector according to claim 1, wherein in step (1), plasmid RVP1/3 is used as PCR template, arginine at position 585 and arginine at position 588 of capsid protein VP1 are mutated to alanine, and pRVP1/3M plasmid is obtained, wherein the DNA sequence of capsid VP1/3M is SEQ ID NO: 3.

3. the method of claim 2, wherein the plasmid pRVP1/3M and the plasmid pVP2, pHelper and pAAV obtained in step (2) are combined to construct an AAV four-plasmid packaging system, which is packaged into AAV.

4. The method of claim 3, wherein the plasmid pVP2-EC1 is obtained by inserting the EC1 protein sequence into the amino terminus of VP2 protein based on the plasmid pVP2 in step (2), and the VP2-EC1 DNA sequence is SEQ ID NO. 4.

5. The method of claim 4, wherein the EC1 of step (2) has the DNA sequence of SEQ ID NO. 5 and the amino acid sequence of SEQ ID NO. 6.

6. The method of claim 5, wherein the plasmid pRVP1/3M or pRVP1/3 plasmid, pVP2-EC1 plasmid, pHelper, and pAAV are combined to construct an AAV four-plasmid packaging system, and packaged into AAV virus.

7. The method according to claim 6, wherein the plasmid pAAV of step (3) is any one of pAAV-Luciferase and pAAV-TK expression plasmids.

8. Use of an AAV viral vector produced by the method for packaging an AAV viral vector according to any of claims 1-7 for the preparation of a medicament for the targeted treatment of breast cancer.

9. The use of claim 8, wherein the AAV viral vector carries the suicide gene HSV-TK in the preparation of a medicament for inhibiting proliferation of breast cancer cells.

10. The use of claim 9, wherein said breast cancer cells are 4T1 breast cancer cells.

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