Vaccine based on human cytomegalovirus-coded immediate early protein IE, preparation method therefor and use thereof

Abstract

A vaccine based on a human cytomegalovirus-coded immediate early protein IE, comprising one of the following vaccines: an IE1 adenovirus vector vaccine, an IE2 adenovirus vector vaccine, an IE1 recombinant protein vaccine, and an IE2 recombinant protein vaccine, wherein the amino acid sequence of a mutant IE1-mut of a wild-type IE1 is as shown in SEQ ID NO. 1, and the amino acid sequence of a mutant IE 2-mut of a wild-type IE2 is as shown in SEQ ID NO. 2. The vaccine based on the human cytomegalovirus-coded immediate early protein IE can identify and kill brain cancer cells, and can be widely applied to the preparation of drugs for treating glioma.

Description

Vaccine based on human cytomegalovirus-encoded immediate early protein IE, preparation method and application thereof Technical Field

[0001] The present invention relates to the field of vaccine technology, in particular to an adenovirus vector vaccine based on human cytomegalovirus-encoded immediate early protein IE, and a preparation method and application thereof. Background Art

[0002] The present invention provides two adenoviral vector vaccines based on viruses encoding immediate-early (IE) proteins, as well as a subunit protein vaccine. Human cytomegalovirus (HCMV) is a ubiquitous beta-herpesvirus that infects a large portion of the global population. It is a significant cause of morbidity and mortality in immunocompromised populations, including transplant recipients and HIV / AIDS patients. HCMV can establish a lifelong latent infection, with periodic reactivation and shedding of the virus. The virus can infect various cells in the body, including epithelial cells, endothelial cells, and leukocytes, leading to a wide range of clinical manifestations, including congenital infection, hepatitis, retinitis, encephalitis, and pneumonia. The virus can also cause complications in transplant recipients, such as organ rejection and graft failure. Therefore, developing effective prophylactic and therapeutic strategies to prevent HCMV infection is crucial to improving the health outcomes of affected individuals. Recent studies have also suggested a potential link between HCMV and certain types of brain tumors, particularly glioblastoma. Glioblastoma is a particularly aggressive brain cancer, and current treatment options are limited in their efficacy. Therefore, researchers have been exploring novel treatments, including vaccines targeting HCMV proteins, particularly IE proteins. The immediate-early proteins encoded by HCMV are divided into two types: IE1 (IE1) and IE2. They play a crucial role in the survival and proliferation of cancer cells, making them attractive targets for immunotherapy. Researchers have been developing vaccines targeting IE proteins to trigger cellular immune responses that help recognize and eliminate brain cancer cells. Summary of the Invention

[0003] The purpose of the present invention is to provide a vaccine based on the immediate early protein IE encoded by human cytomegalovirus, which can identify and eliminate brain cancer cells and can be widely used in the preparation of medicines for treating gliomas.

[0004] To achieve the above objectives, the present invention provides an adenovirus vector vaccine based on human cytomegalovirus encoding immediate early protein IE, including the following:

[0005] IE1 adenovirus vector vaccine;

[0006] IE2 adenovirus vector vaccine;

[0007] IE1 recombinant protein vaccine;

[0008] IE2 recombinant protein vaccine;

[0009] The amino acid sequence of the mutant IE1-mut of the wild-type IE1 is shown in SEQ ID NO.1, and the amino acid sequence of the mutant IE2-mut of the wild-type IE2 is shown in SEQ ID NO.2.

[0010] The present invention also provides a method for preparing the above-mentioned vaccine based on the human cytomegalovirus-encoded immediate early protein IE. The method for preparing the IE1 adenovirus vector vaccine or the IE2 adenovirus vector vaccine comprises the following steps:

[0011] 1) Cell culture;

[0012] 2) Adenovirus packaging.

[0013] The present invention also provides a method for preparing the above-mentioned vaccine based on the human cytomegalovirus-encoded immediate early protein IE. The method for preparing the IE1 recombinant protein vaccine or the IE2 recombinant protein vaccine comprises the following steps:

[0014] 1) Design primers and amplify the target gene by PCR;

[0015] 2) Use BamHI-HF to digest the plasmid vector and linearize it;

[0016] 3) Homologous recombination of the target gene into the linearized vector;

[0017] 4) transformation;

[0018] 5) Select the transformants from step 4) and perform colony PCR identification;

[0019] 6) Expression of mutant human cytomegalovirus immediate early protein IE in Escherichia coli.

[0020] Preferably, the amplification primers for the target gene in step 1) are F1 and R1, F1-2 and R1-2, the sequence of F1 is shown in SEQ ID NO.3, the sequence of R1 is shown in SEQ ID NO.4, the sequence of F1-2 is shown in SEQ ID NO.7, and the sequence of R1-2 is shown in SEQ ID NO.8.

[0021] Preferably, the specific operation of step 3) is: adding the target gene fragment and the linearized vector to a test tube at a molar ratio of 2:1 for recombination reaction, mixing and incubating at 42°C for 30 minutes, then transferring to ice, standing for 2-3 minutes, and then taking 10uL of the reaction liquid to transform into competent cells.

[0022] Preferably, the specific operation of step 4) is as follows: take the competent cells on ice, add 10 μL of ligation solution after lysis, gently rotate to mix the contents, and place on ice for 30 minutes;

[0023] a. Heat shock the tube in a water bath preheated to 42°C for 90 seconds.

[0024] b. Quickly transfer the tube to an ice bath and allow the cells to cool for 2-3 minutes.

[0025] c. Add 900 µL of LB medium to each tube, then transfer the tube to a 37°C shaker and incubate for 1 hour to allow the bacteria to recover.

[0026] d. Take an appropriate amount of transformed bacterial solution and spread it on LB agar plate containing the corresponding antibiotics of the expression vector.

[0027] e. Invert the plate and incubate in a constant temperature incubator at 37°C for 16 hours.

[0028] Preferably, the primers required for colony PCR identification in step 5) are F2 and R2, F2-2 and R2-2, the sequence of F2 is shown in SEQ ID NO.5, the sequence of R2 is shown in SEQ ID NO.6, the sequence of F2-2 is shown in SEQ ID NO.9, and the sequence of R2-2 is shown in SEQ ID NO.10.

[0029] The present invention also provides the use of the above-mentioned vaccine based on the immediate early protein IE encoded by human cytomegalovirus in the preparation of medicines for treating gliomas.

[0030] The advantages and positive effects of the adenovirus vector vaccine based on human cytomegalovirus encoding immediate early protein IE, its preparation method and application according to the present invention are:

[0031] 1. The adenovirus vector and protein vaccines of the present invention can stimulate T cell activation and secrete cytokines such as IL2, IFN-γ, TNFα, IL4 and IL6.

[0032] 2. The adenovirus vector and protein vaccines of the present invention can identify and eliminate brain cancer cells and can be widely used in the preparation of drugs for treating gliomas.

[0033] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 is a map of the protein vaccine plasmid vector of the present invention;

[0035] Figure 2 is an enzyme digestion map of the present invention;

[0036] FIG3 is an agarose gel electrophoresis pattern of PCR products in the present invention;

[0037] FIG4 is a schematic diagram of colony PCR identification in the present invention;

[0038] FIG5 is a diagram showing the results of colony PCR identification in the present invention;

[0039] FIG6 is a diagram showing the sequencing results of positive clones in the present invention;

[0040] FIG7 is the SDS-PAGE detection result of the small-scale expression test of the present invention;

[0041] FIG8 is a Western blot test result of a small-scale expression test of the present invention;

[0042] FIG9 is a diagram of SDS-PAGE & Western blot analysis of large-scale expression in the present invention;

[0043] Figure 10 is a plasmid map of the adenovirus vector vaccine of the present invention;

[0044] FIG11 is a flow chart of the recombinant adenovirus of the present invention;

[0045] FIG12 is a diagram showing the packaging results of IE1 adenovirus in the present invention;

[0046] FIG13 is a diagram showing the packaging results of IE2 adenovirus in the present invention;

[0047] FIG14 shows the immune response of the IE1 adenovirus vector vaccine of the present invention;

[0048] Figure 15 shows the immune response of the IE1 recombinant protein vaccine of the present invention;

[0049] FIG16 shows the immune response of the IE2 adenovirus vector vaccine of the present invention;

[0050] Figure 17 shows the immune response of the IE2 recombinant protein vaccine of the present invention;

[0051] FIG18 is a diagram showing the therapeutic effect of the IE1 adenovirus vector vaccine of the present invention on glioma;

[0052] FIG19 is a diagram showing the therapeutic effect of the IE2 adenovirus vector vaccine of the present invention on glioma;

[0053] FIG20 shows the effects of wild-type IE and mutant IE on tumor cells in the present invention;

[0054] FIG21 shows that the mutant IE of the present invention can reverse the cell cycle arrest induced by wild-type IE. DETAILED DESCRIPTION

[0055] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0056] Unless otherwise defined, technical or scientific terms used in the present invention shall have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs.

[0057] Example 1

[0058] 1. Amino acid sequence of IE mutant

[0059] 1.1 The amino acid sequence of the IE1 mutant (IE1-mut) is shown in SEQ ID NO. 1:

[0060] MESSAKRKMDPDNPDEGPSSKVPRPETPVTKATTFLQTMLRKEVNSQLSLGDPLFPELAEESLKTFEQVTEDCNENPEKDVLAELVKQIKVRVDMVRHRIKEHMLKKYTQTEEKFTGAFNMMG GCLQNALDILDKVHEPFEEMKCIGLTMQSMYENYIVPEDKREMWMACIKELHDVSKGAANKLGGALQAKARAKKDELRRKMMYMCYRNIEFFTKNSAFPATTNGCSQAMAALQALPQCSPDEI MAYAQKIFKILDEERDKVLTHIDHIFMDILTCVETMCAEYKVTSDACMATMYGGISLLSEFCAVLCCYVLEETSVMLAKRPLITKPEVISVMKRRIEEICMKVFAQYILGADPLRVCSPSVD DLRAIAEESDEEEAIVAYTLATAGVSSSDSLVSPPESPVPATIPLSVIVAENSDQEESEQSDEEEEEGAQEEREDTVSVRSEPVSEIEEVAPEEEEDGAEEPTASGGKSTHPMVTRSKADQ.

[0061] 1.2 The amino acid sequence of the IE2 mutant (IE2-mut) is shown in SEQ ID NO. 2:

[0062] .

[0063] Example 2 Preparation of recombinant protein vaccines

[0064] The following takes IE1 recombinant protein vaccine as an example to explain its preparation method in detail.

[0065] 2. Expression of human cytomegalovirus recombinant protein in adenoviral vector

[0066] The plasmid vector map is shown in Figure 1.

[0067] 2.2 Multiple cloning site:

[0068] The plasmid vector was digested with BamHI-HF to recover a 5275 bp vector fragment. The digestion map is shown in FIG2 .

[0069] 2.3 Target gene amplification

[0070] Primers F1: CTTGGGCTGCAGGTCGACGCCACCATGGAGTCTAGCGCTAAGAGAA (SEQ ID NO. 3); R1: AGGGAGAGGGGCGGATCCTTACTGGTCGGCCTTGCTTCTGG (SEQ ID NO. 4) were used for PCR to obtain the sequence of the target gene IE-2. The PCR product was labeled b.

[0071] 2.4 PCR amplification of target gene fragments

[0072] PCR reaction system (Table 1):

[0073] Table 1

[0074] ;

[0075] PCR cycling conditions (Table 2):

[0076] Table 2

[0077]

[0078] The agarose gel electrophoresis pattern of the PCR product is shown in FIG3 . As shown in FIG3 , the size of the PCR product b is 1518 bp.

[0079] 2.5 Homologous recombination of target gene into expression vector:

[0080] Add the target DNA fragment and linearized vector to the test tube at a molar ratio of 2:1 for recombination reaction (Table 3):

[0081] Table 3

[0082]

[0083] Mix well and incubate at 42°C for 30 minutes, then transfer to ice. After 2-3 minutes, take 10 μl of the reaction solution and transform it into competent cells.

[0084] Note: For the positive control and self-ligation control, the vector and ligation group added are the same, but the gene fragment added to the positive control is the target gene (with the same homologous recombination exchange arm).

[0085] 2.6 Conversion:

[0086] Place a tube of competent cells (100 µl per tube, stored at -80°C) on ice. After thawing, add 10 µl of ligation buffer, swirl gently to mix the contents, and place on ice for 30 minutes.

[0087] 1. Place the tube in a thermostatic water bath preheated to 42°C for 90 seconds.

[0088] 2. Quickly transfer the tube to an ice bath and allow the cells to cool for 2-3 minutes.

[0089] 3. Add 900 µl of LB medium to each tube, then transfer the tube to a 37°C shaker and incubate for 1 hour to allow the bacteria to recover.

[0090] 4. Take an appropriate amount of transformed bacterial solution and spread it on LB agar plate (containing antibiotics corresponding to the expression vector).

[0091] 5. Invert the plate and incubate at 37°C in a constant temperature incubator for 16 hours.

[0092] 2.7 Identification of positive clones:

[0093] Transformants grown on the plates were picked and resuspended in 10µl of LB medium. 1µl of this medium was used as a template for colony PCR identification. A schematic diagram of colony PCR identification is shown in Figure 4. Primers F2: GGTATAAGAGGCGCGACCAG (SEQ ID NO. 5) and R2: CCTCACATTGCCAAAAGACG (SEQ ID NO. 6) were used for colony PCR identification of transformants. Positive clones yielded a 1698bp fragment, while negative clones yielded a 0bp fragment.

[0094] Colony PCR reaction system (Table 4):

[0095] Table 4

[0096] ;

[0097] Perform the reaction in a PCR instrument according to the following cycling conditions (Table 5):

[0098] Table 5

[0099]

[0100] The results of colony PCR are shown in Figure 5, where numbers 5 to 8 represent four transformants of PSE5868. Positive clones were inoculated, preserved, and aliquoted into 100 μl for sequencing. If sequencing was successful, plasmids were extracted from the clones.

[0101] 2.8 Analysis of sequencing results of positive clones:

[0102] The sequencing results of the positive clones are shown in FIG6 , which are basically consistent with the expected target gene sequence. The dark-colored markers are vector sequences, and the light-colored markers are sequences of the target gene IE1-mut.

[0103] 3. Expression of mutant human cytomegalovirus immediate early protein IE1 in E. coli

[0104] 3.1 Gene synthesis and subcloning (Table 6)

[0105] Table 6

[0106]

[0107] 3.2 Expression Evaluation

[0108] 3.2.1 Conversion

[0109] 3.2.1.1 Remove BL21(DE3) and Rosetta™ 2(DE3) competent cells from the ultra-low temperature freezer and thaw on ice;

[0110] 3.2.1.2 Add 100 ng of plasmid and mix thoroughly by pipetting gently.

[0111] 3.2.1.3 Place on ice for 30 minutes;

[0112] 3.2.1.4 Heat shock in a water bath at 42°C for 90 seconds;

[0113] 3.2.1.5 Place on ice for 3 minutes;

[0114] 3.2.1.6 Add 100 μl of room temperature LB liquid medium;

[0115] 3.2.1.7 Incubate in a shaker at 37°C, 200 rpm for 60 minutes;

[0116] 3.2.1.8 Mix the bacterial solution and apply it to a kanamycin-resistant plate;

[0117] 3.2.1.9 Invert the plate and incubate at 37°C overnight.

[0118] 3.2.2 Experimental Expression

[0119] 3.2.2.1 Pick three single clones and inoculate them into 4 ml LB tubes containing 50 μg / ml kanamycin.

[0120] 3.2.2.2 Incubate in a shaker at 37°C and 200 rpm;

[0121] 3.2.2.3 When the OD600 reaches 0.6-0.8, add 0.5 mM IPTG to two test tubes, incubate at 15°C for 16 hours and 37°C for 4 hours respectively. The last test tube is used as a negative control.

[0122] 3.2.2.4 Detect protein expression and solubility by SDS-PAGE and Western blot;

[0123] 3.3 Sample preparation

[0124] The pellet after centrifugation of 450 μl of culture medium was resuspended in 300 μl of lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 5% glycerol, pH 8.0) and lysed by sonication for 1 min;

[0125] 3.3.1 Whole-cell sample: Take 100 μl of lysis buffer, mix with 50 μl of 5x loading buffer, heat at 100°C for 10 minutes, and centrifuge at 15,000 rpm for 5 minutes;

[0126] 3.3.2 Supernatant and Inclusion Body Samples: Take the remaining 200 μl of lysate and centrifuge at 15,000 rpm for 10 minutes. Collect the supernatant and pellet separately. Mix 90 μl of 5x loading buffer with 180 μl of supernatant to prepare the supernatant sample. Resuspend the pellet in 150 μl of 5x loading buffer to prepare the inclusion body sample. Heat at 100°C for 10 minutes, centrifuge at 15,000 rpm for 5 minutes, and prepare for loading.

[0127] 3.4. Experimental results

[0128] Whole-cell, supernatant, and inclusion body samples were analyzed by SDS-PAGE and Western blot. The results are shown in Figures 7 and 8. Figure 7 shows the SDS-PAGE results, and Figure 8 shows the Western blot results.

[0129] In Figure 7: Lane M1: Protein marker;

[0130] Lane M2: Western blot Protein marker;

[0131] Lane PC1: BSA (1 μg);

[0132] Lane PC2: BSA (2 μg);

[0133] Lane NC: uninduced whole cells;

[0134] Lane 1: whole cells induced at 15°C for 16 h;

[0135] Lane 2: whole cells induced at 37°C for 4 h;

[0136] Lane NC1: uninduced cell lysate supernatant;

[0137] Lane 3: supernatant of cell lysate induced at 15°C for 16 h;

[0138] Lane 4: supernatant of cell lysis induced at 37°C for 4 h;

[0139] Lane NC2: Uninduced cell lysate pellet

[0140] Lane 5: cell lysis pellet induced at 15°C for 16 h;

[0141] Lane 6: cell lysis pellet induced at 37°C for 4 h;

[0142] The antibody used for Western blot was anti-His antibody (GenScript, Cat. No. A00186).

[0143] 3.5 Large-scale protein expression SDS-PAGE & Western blot analysis, the results are shown in Figure 9.

[0144] The preparation method of IE2 recombinant protein vaccine is the same as that of IE1 recombinant protein vaccine, with the following difference: in the preparation process of IE2 recombinant protein vaccine, the amplification primers of the target gene F1-2 are: CTTGGGCTGCAGGTCGACGCCACCATGGAGTCCAGCGCTAAGCGTA (SEQIDNO.7); R1-2: AGGGAGAGGGGCGGATCCTTACTGGCTCTTATTTCTCAGATCC (SEQIDNO.8).

[0145] Positive clone identification primers F2-2: GGTATAAGAGGCGCGACCAG (SEQ ID NO. 9); R2-2: CCTCACATTGCCAAAAGACG (SEQ ID NO. 10)

[0146] Example 3 Preparation of adenovirus vector vaccines

[0147] The following takes IE1 adenovirus vector vaccine as an example to explain its preparation method in detail.

[0148] 1. Cell lines and plasmids (Table 7):

[0149] Table 7

[0150]

[0151] 2. Main Reagents (Table 8)

[0152] Table 8 Reagents

[0153] ;

[0154] 3. Main instruments and equipment (Table 9)

[0155]

[0156] Cell culture

[0157] 1. Cell Recovery

[0158] 1) Prepare appropriate sterile culture bottles and label them with the cell line name, passage number, and date;

[0159] 2) Wearing appropriate protective equipment, remove the frozen cells from the liquid nitrogen tank and transfer them to the laboratory in a sealed container;

[0160] 3) Wearing protective clothing, remove the cells from the container and place them in a water bath at an appropriate temperature of 37°C. Submerge only the lower half of the cryovial. Thaw until only a small amount of ice remains in the vial, usually 1-2 minutes. Transfer to a biosafety cabinet.

[0161] 4) Wipe the outside of the cryovial with cotton soaked in 70% alcohol and open the cryovial in front of an alcohol lamp.

[0162] 5) Slowly pipette dropwise into the incubated culture medium to dilute the DMSO;

[0163] 6) At appropriate temperature and concentration Cultivate in

[0164] 7) Observe the cells under a microscope after 24 hours and subculture if necessary.

[0165] 2. Cell Passaging

[0166] 1) Observe the cells using an inverted microscope to assess the degree of cell confluence and confirm the absence of bacterial and fungal contamination;

[0167] 2) Remove the culture medium from the culture bottle;

[0168] 3) Wash the monolayer cells with PBS equal to half the volume of the culture medium, usually three times;

[0169] 4) Press Add 0.25% trypsin to the cell monolayer based on the surface area. Shake the flask to coat the cell monolayer with trypsin and pour off any excess trypsin.

[0170] 5) Return the culture flask to the incubator and leave it there for 2-10 minutes;

[0171] 6) Observe the cells under an inverted microscope to ensure that all cells are detached and floating. Gently tap the side of the culture flask to release any remaining adherent cells.

[0172] 7) Resuspend the cells in a small amount of culture medium containing fresh serum to inactivate trypsin and remove 100-200 μl for counting;

[0173] 8) Transfer the desired number of cells to a newly labeled flask or plate containing incubated culture medium and culture the cell line under appropriate conditions.

[0174] 9) Repeat this step according to the growth characteristics of the cell line until the cells are in good condition and free of contamination and can be plated in a six-well plate. The remaining cells can be frozen.

[0175] 3. Live Cell Count

[0176] Take a vial of passaged cells and wait for them to grow into a monolayer before use. To prepare a cell suspension: digest with 0.25% trypsin, wash with PBS, then add culture medium (or a balanced salt solution such as Hanks' solution or PBS) and pipette to create a cell suspension to be tested. Place a coverslip on the hemocytometer and prepare the cell suspension for counting: pipette an appropriate amount of cell suspension (e.g., 5 drops) into a centrifuge tube and add an equal volume of trypan blue dye (0.4%). Live cells will not be stained, but the addition of the dye allows for differentiation between live and dead cells under a microscope. If you are simply counting cells and do not consider cell viability, you can omit the trypan blue staining step; drop the cell suspension into the counting chamber, taking care not to have bubbles under the coverslip and not to let the suspension flow into the adjacent trough; count the number of cells in the four large grids, place the hemocytometer under a low-power microscope, and move the counting chamber. When you see the counting grid appear in the microscope, count the number of cells in the four large grids (each grid contains 16 middle grids) that are not stained by the dye; calculate the number of cells in the original cell suspension. Calculate the cell density according to the following formula:

[0177]

[0178] Note: The formula is divided by 4 because the number of cells in 4 large grids is counted, and the formula is multiplied by 2 because the cell suspension is diluted 1:1 with the staining solution.

[0179] 4. Prepare six-well plates

[0180] Select HEK293 cells that are in good condition and free of contamination, and use the cell passage method to evenly plate them in a six-well plate to ensure that the number of cells in each well is the same, which should reach 2×10 6 The next day, when the cells grew to about 50-80%, transfection began.

[0181] 5. Cell Cryopreservation

[0182] Observe the cells with an inverted microscope to assess the cell density and confirm that there is no bacterial and fungal contamination. Remove the culture medium in the culture flask and wash the monolayer cells with PBS equivalent to half the volume of the culture medium. Generally, three times is sufficient, at a rate of 1 mL / 25 cm 2 Add 0.25% trypsin to the surface area to digest the monolayer of cells. Shake the culture flask to allow the trypsin to cover the cell monolayer. Pour out the excess trypsin, return the culture flask to the incubator, and leave it for 2~10 minutes. Observe the cells with an inverted microscope to ensure that all cells are separated and floating. Tap the side of the culture flask to release the remaining adherent cells, and resuspend the cells in a small amount of culture medium containing fresh serum to inactivate trypsin. Take out 100~200μl for counting. In order to have a good recovery effect after freezing, the ideal cell survival rate should be more than 90%. Centrifuge the remaining cells at 150g for 5 minutes and resuspend them in freezing culture medium at 2~4×10 6 Resuspend the cells at a density of 10 cells / mL, aspirate 1 mL of the cell suspension and transfer it to a cryovial labeled with the cell line name and passage number. Place the cryovial in a freezing box and store it in a -80°C freezer overnight. Finally, transfer the frozen cryovial to the vapor phase of liquid nitrogen and record the location.

[0183] Adenovirus packaging process

[0184] 1. Introduction to the AdMax Adenovirus Packaging System

[0185] The AdMax adenoviral packaging system, developed by Professor Frank L. Graham and distributed by Microbix, a Canadian company, works by co-transfecting HEK293 cells with an adenoviral shuttle plasmid carrying the exogenous gene and a helper packaging plasmid carrying the majority of the adenoviral genome. Recombination is achieved using the Cre / loxP (or FLP / frt) recombinase system to produce recombinant adenovirus. This system is simple to operate, offers high recombination efficiency, produces high virus yields, and achieves high expression levels of the target gene.

[0186] 2. Packaging process (taking 10cm culture plate as an example):

[0187] The day before transfection, seed the cells into a 10cm cell culture dish, maintaining a cell density of 70-90%. One hour before transfection, remove the dish, discard the culture medium, add 10ml of Opti-MEM medium, and return the cells to the incubator.

[0188] Prepare the complex of transfection reagent and plasmid:

[0189] a. Take a 1.5ml Eppendorf tube, add the viral vector plasmid to be transfected, make up to 500μl with Opti-MEM medium, and mix gently;

[0190] b. Take a 1.5ml Eppendorf tube, add Trans-EZ solution, and fill up to 500μl with Opti-MEM medium. Mix gently.

[0191] c. Add the Trans-EZ diluent dropwise to the plasmid dilution, mix gently while adding, and let it stand at room temperature for 20 minutes to allow the DNA and Trans-EZ to fully combine to form a stable transfection complex.

[0192] Remove the cell culture plate and add the DNA-Trans-EZ complex obtained above to the cell culture vessel, mark it, and return it to the incubator; after 6 hours, aspirate the culture medium, wash once with PBS, and add 10 mL of fresh growth medium for culture. If a large number of cells float up, do not remove the supernatant, add 6 mL of complete culture medium, and culture overnight at 37°C. Change the medium the next day; change the medium every three days. Viral plaques will appear in about 7-15 days, and wait for purification after lesions.

[0193] 3. Packaging results

[0194] As shown in Figure 12.

[0195] 4. Harvesting of Recombinant Adenovirus

[0196] When most cells showed typical pathological changes and 50% of the cells were detached from the cell wall, the cells were collected and repeatedly frozen and thawed at -70°C and 37°C for 3 times. The viral supernatant was collected and stored at -70°C.

[0197] 5. Small-scale amplification of viruses

[0198] When the HEK293 cells reached 80-90% confluence, the culture medium was discarded, leaving a small amount to cover the cell surface; 10 μl of virus solution was added to the 10 cm 2 Dish, mix well, and place in a 37°C, 5% CO2 incubator. After 2 hours, add 10 mL of culture medium and culture in an incubator. After 4 to 5 days, the HEK293 cells become round and detach from the wall. Some cells float and their color changes from orange to yellow, indicating that the cells are diseased. They can be collected with a pipette and stored at -70°C for later use.

[0199] 6. Large-scale amplification and gradient centrifugation purification of recombinant adenovirus

[0200] Note: Many experimental studies infect cells with provirus. However, some experiments, especially animal studies, often use purified virus. We use the classic cesium chloride gradient centrifugation method described by Hitt et al. (1998). This method uses virus enrichment in a 30 × 150 mm culture dish as an example.

[0201] 1. Prepare cells:

[0202] For a 30 x 150 mm dish: When all infected cells have become rounded and partially floated, scrape the dish clean. Transfer the cells and supernatant to a 50 ml centrifuge tube and centrifuge at 750 g for 10 minutes. Resuspend in 15 ml of 0.1 M Tris-HCl (pH 8.0) and store at -80°C.

[0203] 2. Dissolve the sample by adding 1.5 ml of 5% sodium deoxycholate per 15 ml of cell lysate, mix well, and incubate at room temperature for 30 minutes. The result is a relatively clear, highly viscous suspension.

[0204] 3. Add 150 μl of magnesium chloride and 75 μl of DNase I solution to every 15 ml of cell lysate, mix well, and incubate at 37°C for 30-60 minutes, mixing every 10 minutes. The viscosity should decrease to only slightly thicker than water.

[0205] 4. Use a high-speed desktop centrifuge at 4 degrees and high speed centrifugation for 15 minutes.

[0206] 5. Meanwhile, prepare a cesium chloride gradient (using ultrapure tubes with an SW41 rotor for 5 mL of sample): add 0.5 mL of 1.5 g / cc cesium chloride solution to each tube, gently layer 3 mL of 1.35 g / cc cesium chloride solution on top, and then gently layer 3 mL of 1.25 g / cc cesium chloride solution on top. Do not disrupt the gradient after adding these solutions.

[0207] 6. Add 5 ml of the supernatant obtained in step 4 to each gradient tube.

[0208] 7. Centrifuge in SW41 rotor at 4°C, 35,000 rpm for 1 hour (acceleration and deceleration setting 1).

[0209] 8. Collect the viral band (should be between 1.25 g / cc and 1.35 g / cc).

[0210] 9. Transfer the resulting viral band to a sterile tube for an SW50.1 rotor and fill the tube to the brim with the 1.35 g / cc solution. Mix thoroughly. Centrifuge in an SW50.1 rotor at 35,000 rpm at 4°C for 16-20 hours (or in an SW41 rotor at 35,000 rpm at 10°C for 16-24 hours).

[0211] 10. Collect the virus in the smallest possible volume (usually 0.5-1 ml), transfer it to a dialysis bag, and dialyze it against 500 volumes (or more) of 10 mM Tris-HCl, pH 8.0, at 4°C for at least 24 hours, changing the solution two to three times.

[0212] 11. After dialysis, the purified virus is divided into small aliquots for storage at -80 °C.

[0213] The preparation method of IE2 adenovirus vector vaccine is the same as that of IE1 adenovirus vector vaccine. The IE2 adenovirus packaging results are shown in Figure 13.

[0214] Example 4 Vaccine Immune Response

[0215] IE1 adenovirus vector vaccine and IE1 recombinant protein vaccine induced T cell responses in the spleen.

[0216] Fourteen days after a single injection of the IE1 adenovirus vector vaccine, T cell activation in the spleen of mice was measured. The results are shown in Figure 14. Figure 14 demonstrates that the protein vaccine stimulated T cell activation and secretion of IL2, IFN-γ, TNFα, IL4, and IL6. Fourteen days after three injections of the IE1 recombinant protein vaccine, T cell activation in the spleen of mice was measured. The results are shown in Figure 15. Figure 15 demonstrates that the protein vaccine stimulated T cell activation and secretion of IL2, IFN-γ, TNFα, IL4, and IL6.

[0217] IE2 adenovirus vector vaccine and IE2 recombinant protein vaccine induced T cell responses in the spleen.

[0218] Fourteen days after a single injection of the IE2 adenovirus vector vaccine, T cell activation in the spleen of mice was measured. The results are shown in Figure 16. Figure 16 demonstrates that the protein vaccine stimulated T cell activation and secretion of IL2, IFN-γ, TNFα, IL4, and IL6. Fourteen days after three injections of the IE2 recombinant protein vaccine, T cell activation in the spleen of mice was measured. The results are shown in Figure 17. Figure 17 demonstrates that the protein vaccine stimulated T cell activation and secretion of IL2.

[0219] Example 5 The therapeutic effect of the vaccine on glioma

[0220] The therapeutic effect of IE1 adenovirus vector vaccine on glioma.

[0221] After one injection of IE1 adenovirus vector vaccine and IE1 protein vaccine, mice were subjected to intracranial tumor-bearing. The results are shown in FIG18 . FIG18 shows that the tumor volume was significantly reduced.

[0222] The therapeutic effect of IE2 adenovirus vector vaccine on glioma.

[0223] After one injection of IE2 adenovirus vector vaccine and IE2 protein vaccine, mice were subjected to intracranial tumor-bearing. The results are shown in FIG19 . FIG19 shows that the tumor volume was significantly reduced.

[0224] Example 6

[0225] Wild-type IE promotes tumor cell proliferation and inhibits apoptosis, while mutant IE reverses this trend (Figure 20). As shown in Figure 20, CCK8 proliferation assays show that wild-type IE1 and IE2 promote cell proliferation. Apoptosis assays show that wild-type IE1 and IE2 inhibit apoptosis. However, mutant IE1 and IE2 reverse the effects of wild-type IE1 and IE2 on cell proliferation and apoptosis.

[0226] Example 7

[0227] Mutant IE can reverse the cell cycle arrest induced by wild-type IE (as shown in FIG21 ).

[0228] As shown in Figure 21, cell cycle assays show that wild-type IE1 can cause G0 / G1 arrest, while wild-type IE2 can cause S and G2 / M arrest. However, mutant IE1 and IE2 can reverse the cell cycle arrest of wild-type IE1 and IE2.

[0229] Therefore, the present invention adopts the above-mentioned vaccine based on the immediate early protein IE encoded by human cytomegalovirus, which can identify and eliminate brain cancer cells and can be widely used in the preparation of drugs for treating gliomas.

[0230] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A vaccine based on human cytomegalovirus-encoded immediate early protein IE, characterized in that: Includes one of the following: IE1 adenovirus vector vaccine; IE2 adenovirus vector vaccine; IE1 recombinant protein vaccine; IE2 recombinant protein vaccine; The amino acid sequence of the mutant IE1-mut of the wild-type IE1 is shown in SEQ ID NO.1, and the amino acid sequence of the mutant IE2-mut of the wild-type IE2 is shown in SEQ ID NO.

2.

2. The method for preparing a vaccine based on human cytomegalovirus encoded immediate early protein IE according to claim 1, characterized in that: The preparation method of IE1 adenovirus vector vaccine or IE2 adenovirus vector vaccine comprises the following steps: 1) Cell culture; 2) Adenovirus packaging.

3. The method for preparing a vaccine based on human cytomegalovirus encoded immediate early protein IE according to claim 1, characterized in that: The preparation method of IE1 recombinant protein vaccine or IE2 recombinant protein vaccine comprises the following steps: 1) Design primers and amplify the target gene by PCR; 2) Use BamHI-HF to digest the plasmid vector and linearize it; 3) Homologous recombination of the target gene into the linearized vector; 4) Transformation; 5) Select the transformants in step 4) for colony PCR identification; 6) Expression of mutant immediate early protein IE of human cytomegalovirus in Escherichia coli.

4. The preparation method according to claim 3, characterized in that: In the step 1), the amplification primers for the target gene are F1 and R1, F1-2 and R1-2, the sequence of F1 is shown in SEQ ID NO.3, the sequence of R1 is shown in SEQ ID NO.4, the sequence of F1-2 is shown in SEQ ID NO.7, and the sequence of R1-2 is shown in SEQ ID NO.

8.

5. The preparation method according to claim 3, characterized in that: The specific operation of step 3) is as follows: add the target gene fragment and the linearized vector to a test tube at a molar ratio of 2:1 for recombination reaction, mix well, incubate at 42°C for 30 minutes, then transfer to ice, leave for 2-3 minutes, and then take 10uL of the reaction liquid to transform into competent cells.

6. The preparation method according to claim 3, characterized in that: The specific operation of step 4) is as follows: take the competent cells on ice, add 10 μL of ligation solution after they are dissolved, gently rotate to mix the contents, and place them on ice for 30 minutes; a. Place the tube in a constant temperature water bath preheated to 42°C for 90 seconds; b. Quickly transfer the tube to an ice bath and allow the cells to cool for 2-3 minutes. c. Add 900 µL of LB culture medium to each tube, then transfer the tube to a 37°C shaker and incubate for 1 hour to allow the bacteria to recover; d. Take an appropriate amount of the transformed bacterial solution and spread it on an LB agar plate containing the corresponding antibiotics for the expression vector; e. Invert the plate and incubate in a constant temperature incubator at 37°C for 16 hours.

7. The preparation method according to claim 3, characterized in that: The primers required for colony PCR identification in step 5) are F2 and R2, F2-2 and R2-2, the sequence of F2 is shown in SEQ ID NO.5, the sequence of R2 is shown in SEQ ID NO.6, the sequence of F2-2 is shown in SEQ ID NO.9, and the sequence of R2-2 is shown in SEQ ID NO.

10.

8. Use of the vaccine based on human cytomegalovirus-encoded immediate early protein IE as claimed in claim 1 in the preparation of medicines for treating gliomas.

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