CN115948468A

CN115948468A - Human cytomegalovirus recombinant vector and preparation method and application thereof

Info

Publication number: CN115948468A
Application number: CN202211102596.7A
Authority: CN
Inventors: 王斌; 江莎莎; 张现娟; 李俊; 鲁晓晴; 高青
Original assignee: Qingdao Wanming Saibo Pharmaceutical Co ltd; Qingdao University
Current assignee: Qingdao Wanming Saibo Pharmaceutical Co ltd; Qingdao University
Priority date: 2022-09-09
Filing date: 2022-09-09
Publication date: 2023-04-11
Also published as: WO2024051150A1

Abstract

The invention provides a human cytomegalovirus recombinant vector and a preparation method and application thereof, wherein the recombinant vector comprises one or more of nucleotide sequence segments coded by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4. The human cytomegalovirus recombinant vector provided by the invention can express dominant antigen epitopes of human cytomegalovirus PP65, PP150, IE1, gB and gH proteins, has good immunogenicity on mouse models and clinical population samples, and can induce organisms to generate strong cellular immune response in a short time. After 14 days of single immunization, the killer T cells and the helper T cells can be obviously activated, and 60 days of single immunization, the memory T cells are effectively activated, which shows that the vaccine can cause the high-efficiency anti-human cytomegalovirus immune response of an organism. In addition, the vaccine is fast and simple to prepare, and can be produced in a large scale in a short time.

Description

Human cytomegalovirus recombinant vector and preparation method and application thereof

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a human cytomegalovirus recombinant vector, and a preparation method and application thereof.

Background

Human Cytomegalovirus (HCMV), also known as human herpesvirus-5 (HHV-5), is one of the herpesviruses that can cause human infections. Research shows that the congenital infection rate of HCMV is higher than 1.5 percent in China, but the regional difference is large. HCMV can cause congenital infection during pregnancy, and the risk of intrauterine transmission of primary infection in the early stage of pregnancy can reach 40% -50%. About 5% to 10% of congenital infected infants present with clinical symptoms, particularly with restricted embryonic growth and birth defects such as retinopathy, neurological defects and nerve deafness, and early pregnancy infections make symptoms more severe; even if the serum antibody-positive mother is infected again, the fetus may be infected with a virus, which may cause birth defects. Until now, no specific drugs are available for the treatment of HCMV infection. It is generally accepted that prevention in advance using vaccines is more effective than treatment after infection. Since the 70's of the 20 th century, vaccine development for HCMV began to begin; in 1999, the development of prophylactic HCMV vaccines was the highest priority target by american medical research, yet vaccine research is currently still in the clinical trial phase.

Research shows that in vitro stimulation of T cells by HCMV antigen is possible to replace traditional vaccination, but the technology is difficult to popularize on a commercial scale due to the complex collection and treatment process of human Peripheral Blood Mononuclear Cells (PBMCs).

HCMV is classically classified in the herpesviridae sub-family beta herpesviridae, has a double stranded (ds) DNA genome characterized by a large genome size, and previous studies estimated more than 200 open reading framesFrame (ORF), while current ribosomal mapping and transcript analysis detects additional ORFs not previously identified, the number may reach 751. HCMV establishes a complex relationship with the host immune system, including both transmission and latent infection. The host immune system can be mediated by type I and type II Interferons (IFNs), natural killer cells (NKs), and CD8 ⁺ T cells and CD4 ⁺ T cells and the like control primary and latent HCMV infection in different ways. Meanwhile, HCMV infection can down-regulate the antigen presentation of MHC-I and MHC-II, and prevent the production of cytokines and chemokines. Cytotoxic T Cell (CTL) and Helper T cell (Th) as antigen-specific T cells can effectively induce CD8 when infected or induced by polypeptide ⁺ T cells and CD4 ⁺ T cell mediated responses, thus providing an important strategy for stimulating T cells. Therefore, these cells play a central role in inhibiting HCMV infection. Stimulation of the resulting effector T cells may also play an important role in the control and prevention of HCMV infection.

Disclosure of Invention

Therefore, the main object of the present invention is to provide a recombinant human cytomegalovirus vaccine for activating cytotoxic T cells and helper T cells, thereby inhibiting HCMV infection.

To achieve the above objects, according to one aspect of the present invention, there is provided a human cytomegalovirus recombinant vector comprising one or more of the nucleotide sequence segments encoded by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 or SEQ ID No. 4.

The inventor of the invention adopts a mode of combining the prediction of messenger software with animal and cell experiment screening to determine four dominant antigen epitope genes of the encoding HCMV protein, namely nucleotide sequence segments shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, which can express human cytomegalovirus PP65, PP150, IE1, gB and gH proteins.

The human cytomegalovirus recombinant vector provided by the invention comprises a vector adenovirus, wherein the vector adenovirus is a replication-defective human adenovirus 5 with deletion of E1 and E3. Preferably, the vector adenovirus is derived from the AdMaxTM adenovirus system.

In a typical embodiment of the invention, one or more of the nucleotide sequence segments encoded by SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 are inserted into the E1 region of an adenovirus.

According to one aspect of the invention, the application of the human cytomegalovirus recombinant vector in preparing a vaccine for preventing human cytomegalovirus infection is also provided.

In a preferred embodiment, the recombinant vector is prepared as an injection, nasal drops or spray.

In a more preferred embodiment, the recombinant vector is prepared as an intramuscular injection.

According to another aspect of the present invention, there is provided a method for preparing the human cytomegalovirus recombinant vector, comprising the steps of:

(1) Respectively constructing shuttle plasmid vectors containing dominant antigen epitope genes of the coding human cytomegalovirus protein shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and/or SEQ ID NO. 4;

(2) Transfecting the shuttle plasmid vector obtained in the step (1) and the targeting skeleton plasmid into a host cell;

(3) Transfecting the plasmid obtained in the step (2) into a virus packaging cell HEK293, and packaging, amplifying and purifying to obtain adenovirus of the recombinant human cytomegalovirus protein;

(4) And (4) carrying out amplification culture on the recombinant adenovirus obtained in the step (3).

According to the preparation method provided by the invention, the shuttle plasmid vector in the step (1) is one or more of PSE5256, PSE5257, PSE5258 and PSE5259. In a preferred embodiment of the invention, the vector of the dominant epitope gene SEQ ID No.1 is PSE5256; the carrier of the dominant antigen epitope gene SEQ ID NO.2 is PSE5257; the carrier of the dominant antigen epitope gene SEQ ID NO.3 is PSE5258; the carrier of the dominant antigen epitope gene SEQ ID NO.4 is PSE5259.

According to the preparation method provided by the present invention, the targeting backbone plasmid in step (2) can be pbhglox (delta) e1,3cre.

Wherein, the shuttle plasmid vector and the targeting skeleton plasmid both belong to AdMax adenovirus system. The shuttle plasmid vector and the targeting skeleton plasmid are jointly used for carrying out recombinant adenovirus packaging containing dominant antigen epitope genes for encoding human cytomegalovirus proteins in host cells.

According to the preparation method provided by the invention, the purification method in the step (3) is cesium chloride gradient centrifugation.

According to the preparation method provided by the invention, the expansion culture method in the step (4) is suspension culture.

The human cytomegalovirus recombinant vector provided by the invention can express dominant antigen epitopes of human cytomegalovirus PP65, PP150, IE1, gB and gH proteins, has good immunogenicity on mouse models and clinical population samples, and can induce organisms to generate strong cellular and humoral immune response in a short time. After single immunization for 14 days, the killer T cells and the helper T cells can be obviously activated, the human cytomegalovirus and the serum of the immunized mouse are incubated together, the capability of the virus to infect the human embryonic lung fibroblasts is obviously reduced, and the recombinant vector has obvious blocking effect on the infection of the human cytomegalovirus. The recombinant vector can be used for preparing human cytomegalovirus vaccines, the preparation method is quick and simple, and large-scale production for treating latent infection of human cytomegalovirus can be realized in a short period.

In addition to the objects, features and advantages described above, other objects, features and advantages of the present invention are also provided. The present invention will be described in further detail below with reference to the drawings.

Drawings

Embodiments of the invention are described in detail below with reference to the attached drawing figures, wherein:

FIG. 1A, FIG. 1B, FIG. 1C and FIG. 1D are the profiles of shuttle plasmid vectors PSE5256, PSE5257, PSE5258 and PSE5259 containing the dominant antigen epitope genes encoding human cytomegalovirus proteins shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4 in sequence; FIG. 1E is a map of the tool vector in example 2 of the present invention;

FIG. 2 is the restriction enzyme map of tandem dominant antigen epitope adenovirus expression vector of human cytomegalovirus PP65, PP150, IE1, gB, gH proteins;

FIG. 3A, FIG. 3B, FIG. 3C and FIG. 3D are agarose gel electrophoresis patterns of PCR products in example 2 of the present invention;

FIG. 4 is a schematic diagram of the PCR identification of colonies in example 2 of the present invention;

FIG. 5A, FIG. 5B, FIG. 5C and FIG. 5D are PCR identification charts of colonies in example 2 of the present invention;

FIGS. 6A, 6B, 6C, 6D and 6E are diagrams illustrating the packaging and virus-removal identification of a recombinant adenovirus in example 2 of the present invention;

FIG. 7 is a graph of the percentage of body weight detected after intramuscular injection of immunized rAd5-HCMV to mice for 14 consecutive days;

FIG. 8 is a graph of the percent of feeding assays for intramuscular injection of immunized rAd5-HCMV in mice for 14 consecutive days;

FIG. 9 shows that mice were immunized by intramuscular injection of rAd5-HCMV and induced to produce CD8 on day 14 ⁺ A T cell immune response map;

FIG. 10 shows the intramuscular injection of immune rAd5-HCMV to mice and the induction of CD4 production on day 14 ⁺ A T cell immune response map;

FIG. 11 is a graph of the activation of memory cells of different constructs of recombinant adenovirus at day 60 after intramuscular injection of immunized rAd5-HCMV in mice.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

Example 1

Screening of dominant epitope of human cytomegalovirus

1. Bioinformatics was applied to the B cell, th cell and CTL cell epitopes prediction of 5 HCMV-encoded membrane proteins. The membrane protein sequence of HCMV was derived from the protein database of the National Center for Biotechnology Information, NCBI (http:// www.ncbi.nlm.nih.gov/http:// www.ncbi.nlm.nih.gov /). Selecting five proteins of PP65, PP150, IE1, gB and gH, and respectively using DNAstar, IEDB (score > 0.5) and TMHMM software to predict B cell epitopes; predicting Th cell epitopes of HCMV membrane protein by using SYFPEITHI (score > 20), IEDB (rank < 1), netMHCII pan and NetMHCII software; the HCMV membrane protein CTL cell epitope is predicted by using IEDB, SYFPEITHI and NetMHCI software.

1.1 screening of Th epitopes

Four kinds of software SYFPEITHI, IEDB, netMHCII pan and NetMHCII are used for screening and predicting 21 membrane proteins and the set Th epitopes of virus coding proteins such as IE1, IE2, PP65, PP150 and the like, and four kinds of software are used for simultaneous prediction and selecting an overlapping result from the Th epitopes. Selecting 6 HLA alleles, including HLA-DRB1 x 01, HLA-DRB1 x 03, HLA-DRB1 x 04, HLA-DRB1 x 07, HLA-DRB1 x 01, HLA-DRB1 x 11. The screening criteria were set to SYFPEITHI scores >20, IEDB rank < -1.00, netMHC II pan rank < -10, netMHC II rank < -10. The length of the ligand was set at 15, and the other settings were defaults. 27 membrane proteins and other virus coding proteins Th epitope are screened out.

1.2 screening of epitopes for B antigens

B-cell epitope software for predicting HCMV recombinant vaccines: the software TMHMM, ABCPred, IEDB, and DNAstar were used. Given that the extramembranous protein is the precursor condition for B-cell epitopes, the TMHMM software was used to predict the non-transmembrane region of the 17 HCMV virus-encoded membrane glycoproteins as segments of the screening epitope. ABCpred as a second HCMV virus encoding B cell epitope prediction software with parameters set to: the length of the peptide segment is 16 amino acids, the threshold value is selected to be more than or equal to 0.8 min, the obtained peptide segment is used as the result predicted by the software, and the peptide segment which is not overlapped with the predicted result of other software is excluded by taking the result as the reference and integrating other software to carry out screening. Predicting by a Bepipred method in the on-line software IEDB, wherein the parameters are default parameters, comparing the obtained result with the result predicted by the ABCPred software, and reserving peptide sections with the same prediction results of the two softwares. The method comprises the steps of predicting by using a Protean module in DNAstar, selecting parameters including beta-turn and random coil in a secondary structure, hydrophilicity, flexibility, surface accessibility and antigen index, selecting peptide fragments simultaneously meeting the four parameters which are more than or equal to the four parameters, using the peptide fragments as a prediction result of software, comparing the result with the results obtained by the three kinds of software, further excluding the epitope which is not overlapped with the software, obtaining a final prediction screening epitope, and regarding the epitope as the HCMV encoding B cell dominant antigen epitope. 21 HCMV-encoded B cell dominant epitopes of the membrane protein are screened out.

2. Further screening the overlapping parts of B cell epitope, th cell epitope and CTL cell epitope of five proteins of PP65, PP150, IE1, gB and gH.

The epitope which can be simultaneously recognized by B cells, T cells and Th cells in the five proteins of PP150, PP65, IE1, gH and gB is used as the dominant epitope. The overlapping dominant antigen epitope sequence is shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO. 3.

3. The overlapping parts of the B cell epitope, the Th cell epitope and the CTL cell epitope aiming at the PP65 and IE1 proteins are connected in series with the whole sequence of the PP150 protein, and the serial sequence is shown as SEQ ID NO. 4.

Example 2

Preparation of the human cytomegalovirus recombinant vector of the present invention

1. Vector construction and in vitro identification and expression of target protein

1.1. Vector construction

The maps of shuttle plasmid vectors PSE5256, PSE5257, PSE5258 and PSE5259 which are shown in FIGS. 1A-1D and sequentially contain dominant antigen epitope genes of the human cytomegalovirus protein and are shown in SEQ ID NO.1 (NO. 1), SEQ ID NO.2 (NO. 2), SEQ ID NO.3 (NO. 3) and SEQ ID NO.4 (NO. 4) are constructed.

1.1.1. Enzyme digestion

The tool vector was digested with BamHI-HF, the map of the tool vector is shown in FIG. 1E, and the 5275bp vector fragment was recovered, and the map of the digestion is shown in FIG. 2.

1.1.2. Amplification of target Gene

1.1.2.1 primer design Synthesis:

1)Primer 1(+)

CTGCAGGTCGACTCTAGAGCCACCATGGGAAGCGCCTTTAGCAG

description of the primers: SEQ ID NO.5, sequence of the gene NO.1 for PCR fishing, PCR fragment mark a

2)Primer 2(－)AGGGAGAGGGGCGGATCCTTAGGTGAACTTCTCCTCGGTC

Description of the primers: SEQ ID NO.6, sequence of the gene NO.1 for PCR fishing, PCR fragment mark a

3)Primer ID(+)GGTATAAGAGGCGCGACCAG

Description of the primers: SEQ ID NO.7, used for colony PCR to identify transformants.

4)Primer ID(－)CCTCACATTGCCAAAAGACG

Description of the primers: SEQ ID NO.8, used for colony PCR to identify transformants.

1.1.2.2PCR amplification of target Gene fragments

TABLE 1 PCR reaction System

Note: the steps 2-4 are repeated for 30 times.

1.1.2.3 agarose gel electrophoresis pattern of PCR product, as shown in FIG. 3: SEQ ID NO.1 (FIG. 3A), SEQ ID NO.2 (FIG. 3B), SEQ ID NO.3 (FIG. 3C) and SEQ ID NO.4 (FIG. 3D).

Primer 1+2: size of PCR product a: 507bp

Dl2,000dna marker: 2kb,1.5kb,1Kb,750bp,500bp,250bp,100bp

1.1.3 homologous recombination of target Gene into expression vector

The DNA fragment of interest and the linearized vector are added to a test tube in a molar ratio of 2.

TABLE 2

Reagent	Positive control (μ l)	Self-contained control (μ l)	Connecting group (mu l)
				Target gene fragment after glue recovery	4	4	4
Linearized expression vector	1	1	1
				Seamless cloning reaction solution	15	0	15
dd H ₂ O	To 20	To 20	To 20

After mixing, incubation was carried out at 42 ℃ for 30 minutes and then transferred to ice. After standing for 2-3 minutes, 10ul of the reaction liquid was transferred to competent cells.

Description of the drawings: the positive control and the self-ligation control, the added vector and the ligation group are identical, but the gene fragment added in the positive control is the target gene (with the same homologous recombination exchange arm).

1.1.4 transformation

One competent cell (100. Mu.l per tube, stored at-80 ℃) was placed on ice, 10. Mu.l of the ligation solution was added after lysis, the contents were mixed by gentle rotation, and placed on ice for 30 minutes.

The tube was placed in a thermostatted water bath kettle pre-warmed to 42 ℃ and heat-shocked for 90 seconds.

The tubes were quickly transferred to an ice bath and the cells were allowed to cool for 2-3 minutes.

900. Mu.l of LB medium was added to each tube, and then the tubes were transferred to a 37 ℃ shaker and incubated for 1 hour to resuscitate the bacteria.

The right amount of the transformant was spread on LB agar plates (containing antibiotics corresponding to the expression vectors).

The plate was inverted and incubated at 37 ℃ for 16 hours in a constant temperature incubator.

1.1.5 Positive clone identification

Transformants grown on the plate were picked and resuspended in 10. Mu.l of LB medium, and 1. Mu.l of the medium was used as a template for colony PCR identification. The schematic diagram of colony PCR identification is shown in FIG. 4, in which linear vector represents: and (3) linearizing the carrier.

The positive clone obtains a 693bp fragment, and the negative clone obtains a 0bp fragment.

TABLE 3PCR reaction System

Note: the steps 2-5 are repeated for 30 times.

The colony PCR identification map is shown in FIGS. 5A-D: SEQ ID NO.1 (FIG. 5A), SEQ ID NO.2 (FIG. 5B), SEQ ID NO.3 (FIG. 5C) and SEQ ID NO.4 (FIG. 5D), 1 to 4 were selected 4 transformants.

Dl2,000dna marker: 2kb,1.5kb,1Kb,750bp,500bp,250bp,100bp

Positive clones were inoculated, stored and 100ul was dispensed for sequencing. The positive clone sequencing result shows that the sequence is basically consistent with the expected target gene sequence, which indicates that the vector is successfully constructed.

2. Recombinant adenovirus packaging, preparation and identification

2.1. Recombinant adenovirus packaging

The constructed vectors rd5-p1 (NO. 1), rd5-p2 (NO. 2), rd5-p3 (NO. 3) and rd5-p4 (NO. 4) AdMax adenovirus system framework plasmids pBHGlox _ E1 and 3Cre co-transfect HEK293 cells for packaging recombinant adenovirus. The process is as follows:

a) One day prior to transfection, HEK293 cells were seeded in six-well plates at 8 × 10 per well ⁵ Cells in a medium MEM +10% FBS, at 37 ℃ with 5% CO ₂ The cells were cultured in a cell incubator overnight.

b) The day of transfection, the medium was changed and the culture was continued in fresh 10% FBS-containing MEM medium. When the cells grew to 80-90% of the basal area, the backbone plasmid (pBHGlox _ E1,3 Cre) and shuttle plasmid were taken and transfected with TurboFect transfection reagent (Thermo scientific, REF, R0531) according to the instructions attached thereto. The method comprises the following specific steps:

(1) Taking 3.2 mu g of skeleton plasmid and 0.8 mu g of shuttle plasmid from each transfection hole, and mixing uniformly; the plasmid was diluted with 400. Mu.L of Opti-MEM medium.

(2) mu.L of TurboFect transfection reagent was added to the plasmid diluted in Opti-MEM medium and mixed gently.

(3) The transfection reagent and plasmid mixture was left at room temperature for 20min and then added to the cells.

c) The day after transfection, confluent cells were passaged at 25cm ² Culturing in a cell culture flask in MEM medium containing 5% FBS, observing every day, and transferring 75cm when the bottom of the flask is full of cells ² In the cell culture flasks, the cells were observed daily for signs of toxicity. The appearance of the virus is that the cells become bigger and round, become grape-shaped, and begin to appear obvious plaques. The cells are detoxified when most of them are diseased and fall off from the bottom.

d) The detoxified cells were resuspended, centrifuged at 500g for 10 minutes, the supernatant was discarded, the cells were resuspended in 2mL of PBS, and then placed in a refrigerator at-70 ℃ and a water bath at 37 ℃ for three repeated freeze thawing. The supernatant containing the virus was collected by centrifugation at 12000g for 10 minutes, and the pellet was discarded.

2.2 identification of recombinant adenovirus

2.2.1 recombinant adenovirus culture

The day before transfection, cells were seeded into 10cm cell culture vessels, and the density of cells at transfection was controlled to 70-90%.

Taking out the cell culture vessel one hour before transfection, removing the original cell culture medium, adding 10ml of Opti-MEM culture medium, and returning the cells to the incubator;

preparation of complexes of transfection reagents and plasmids:

a. taking one 1.5ml Ep tube, adding a virus vector plasmid to be transfected, supplementing the virus vector plasmid to 500 mu l by using an Opti-MEM culture medium, and gently mixing the mixture;

b. taking one 1.5ml Ep tube, adding the Trans-EZ solution, supplementing the solution to 500 mu l by using an Opti-MEM culture medium, and gently mixing the solution;

c. the Trans-EZ dilution was added dropwise to the plasmid dilution, gently mixed together, and then allowed to stand at room temperature for 20 minutes to allow the DNA and Trans-EZ to bind well to form a stable transfection complex.

Taking out the cell culture plate, adding the DNA-Trans-EZ complex obtained above into a cell culture vessel, marking, and putting back into an incubator;

after 6h, absorbing the culture medium, washing with PBS once, adding 10mL of fresh growth culture medium for culture, if a large number of cells float up, not removing supernatant, adding 6mL of complete culture medium, culturing overnight at 37 ℃, and changing the culture solution the next day;

changing once every three days, generating virus plaques about 7-15 days, and waiting for purification after pathological changes.

2.2.2 recombinant adenovirus packaging identification

FIGS. 6A-E are graphs showing the packaging and identification of recombinant adenovirus of this example, wherein FIG. 6A is a graph showing the packaging and identification of rAd5-p1 (pSE 5256); FIG. 6B is a graph of packaging and identification of rAd5-p2 (pSE 5257); FIG. 6C is a graph of packaging and identification of rAd5-p3 (pSE 5258); FIG. 6D is a graph of packaging and identification of rAd5-p4 (pSE 5259); FIG. 6E is a diagram of packaging and virus identification of Ad5 (pSB 126). As can be seen from FIGS. 6A-E, when 293T cells infected with rAd5-p1, rAd5-p2, rAd5-p3 and rAd5-p4 are observed under an inverted fluorescence microscope, the green fluorescence is obvious, and the virus packaging is proved to be successful.

2.2.3 harvesting of recombinant adenovirus

After most cells have typical lesions and 50% of the cells are detached from the wall, the cells are collected, frozen and thawed repeatedly at minus 80 ℃ and 37 ℃ for 3 times, and virus supernatant is collected and stored at minus 80 ℃.

2.2.4 amplification of Virus in Small quantities

When the cultured HEK293 cells reach 80-90% confluence, removing the culture solution, and leaving a little to cover the cell surface; adding 10 μ l of the above virus solution into 10cm by using a pipette ² Mixing in a petri dish, adding at 37 deg.C, 5% ₂ An incubator; after 2h, adding 10mL of culture solution, and putting into an incubator for culture; after 4-5 days, HEK293 to be cultured becomes round, becomes detached, some cells float, and the color of the cells changes from orange to yellow, which indicates that the cells are diseased, and the cells can be collected by a pipette and stored at-70 ℃ for later use.

2.3 Mass amplification and gradient centrifugation purification of recombinant adenovirus

Preparing cells:

for a 30 × 150mm dish: when all infected cells were rounded and partially floating, the plates were scraped and the cells and supernatant were transferred to 50ml centrifuge tubes, 750g, and centrifuged for 10 min. Resuspend with 15ml of 0.1M Tris (hydroxymethyl) aminomethane-HCl (pH 8.0). Storing at-80 deg.C.

The sample was dissolved, 1.5ml of 5% sodium deoxycholate was added per 15ml of cell lysate, mixed well and incubated at room temperature for 30 minutes. The result is a relatively clear, high viscosity suspension.

150ul of magnesium chloride and 75ul of DNase I solution were added to each 15ml of cell lysate, mixed well, incubated at 37 ℃ for 30-60 minutes, and mixed well every 10 minutes. The viscosity should be reduced to be only slightly more viscous than water.

A high speed bench top centrifuge was used, 4 degrees, high speed centrifugation for 15 minutes.

Simultaneously, a cesium chloride gradient (ultrapure tubes with SW41 rotor, for 5ml of sample) was prepared: 0.5ml of a 1.5g/cc solution of cesium chloride was added to each tube, 3ml of a 1.35g/cc solution of cesium chloride was gently layered thereon, and a 3ml layer of a 1.25g/cc solution of cesium chloride was gently layered thereon. The gradient may not be disturbed after addition.

5ml of the supernatant obtained in step 4 was added to each gradient tube.

Centrifugation with SW41 rotor at 4 degrees, 35000rpm, 1 hour (acceleration and deceleration setting 1)

Viral bands were collected (should be between 1.25g/cc and 1.35 g/cc).

The resulting viral bands were transferred to sterile tubes with SW50.1 rotor, the tubes were filled with 1.35g/cc of solution), and mixed well. Centrifuge for 16-20 hours with SW50.1 rotor, 4 degree, 35000 rpm. (alternatively with SW41 rotor, 10 degrees, 35000rpm, centrifugation 16-24 hours)

The virus (usually 0.5-1 ml) is collected in as small a volume as possible and transferred to a dialysis bag for dialysis at 4 degrees, 500 volumes (or more) against 10Mm Tris-HCl, PH8.0, for at least 24 hours, with two-three solution changes between.

After dialysis, the purified virus was aliquoted into small portions-80 for storage.

The instrument comprises the following steps: beckman's SW41 rotor, SW50.1 rotor, each rotor corresponds to a sterile high-speed centrifuge tube.

Description of the solution:

1. 0.1M Tris-HCl (pH 8.0), pH 8.0:1.2g of Tris was dissolved in 80ml of deionized water. The pH is adjusted to 8.0 with hydrochloric acid, dissolved in 100ml and autoclaved.

2. 5% sodium deoxycholate: 5g sodium deoxycholate was dissolved in 100ml deionized water.

3. 2M magnesium chloride: 40.6g of anhydrous magnesium chloride are dissolved in 100ml of deionized water and filter sterilized.

4. DNase I solution: 100mg of DNase I was dissolved in 10ml of 20mM Tris-HCl, pH7.4, 50mM NaCl, 1mM dithiothreitol, 0.1mg/ml bovine serum albumin, and 50% glycerol was added. Subpackaging into small portions at-20 ℃ for storage.

5. Cesium chloride solution:

tris (hydroxymethyl) aminomethane (10 mM) was added to the specified amount of cesium chloride and dissolved with stirring, and the density was verified by weighing 1ml of each solution. Filtering, sterilizing, and storing at room temperature.

6. Dialyzate: tris (hydroxymethyl) aminomethane 10mM, pH8.0,4 ℃ was used.

2.4 titer determination of recombinant adenovirus

2.4.1 Experimental procedures

(1) HEK293 cells in good condition were selected and suspended to 5.0X 10 cells by using complete culture medium ⁵ Cell suspension of individual/ml, 1ml of cells were seeded into each well of 24-well plate, 37 ℃,5% CO ₂ And (5) culturing.

(2) Preparing 10-fold gradient diluted virus samples [ 7 sterile Ep tubes were prepared, 990. Mu.l of complete medium was added to the first Ep tube, and 900. Mu.l of complete medium was added to each of the remaining 6 tubes; diluting virus liquid to be tested: mu.l of adenovirus stock was added to 990. Mu.l of Ep tube for 1 ^-2 ) (ii) a Starting from this, 100. Mu.l of the dilution was then added to 900. Mu.l of Ep tube for 1 ^-3 ) Until it is diluted to 10 ^-7 Then sequentially mix 10 ^-4 To 10 ^-7 The diluted virus solution was added to 24-well plates in 100. Mu.l per well, one well per dilution.

(3)37℃、5％CO ₂ Infection was carried out for 48 hours.

(4) The medium was gently removed and 0.5ml of pre-cooled methanol was slowly added along the side wall of the 24-well plate and fixed at-20 ℃ for 20min (the tip did not touch the cells).

(5) Cells were gently washed 3 times 5min each with PBS.

(6) Add 0.2ml1% BSA for 1 h at 37 ℃.

(7) 0.2ml of primary antibody solution was added to each well and incubated at 37 ℃ for 1 hour.

(8) Cells were gently washed 3 times 5min each with PBS.

(9) 0.2ml of secondary antibody was added to each well and incubated at 37 ℃ for 1 hour.

(10) Cells were gently washed 3 times 5min each with PBS.

(11) Add 0.2ml of company's working solution to each well and incubate at room temperature for 5-10min (incubation time does not exceed 10 min).

(12) Discard the working solution, wash 2 times with PBS, and add 1ml PBS per well.

(13) The number of positive cells was counted using an optical microscope at 10 × objective lens with 5 fields randomly selected per well.

(14) The average number of positive cells per well and the virus titer were calculated.

2.4.2 results calculation

(1) The average number of positive cells in the field under the microscope was calculated. A gradient was selected with 5-50 positive cells in the field, and at least 5 regions were randomly selected for counting.

(2) The number of fields per well in the 24-well plate was calculated. For most microscopes, the diameter of the field of view observed by a standard 10 × eyepiece with a 10 × objective is 1.8mm, so the area of each field of view =3.14 × (D/2) ² ＝3.14x 0.92＝2.54mm ² For a standard 24-well plate, the culture area was 2.0cm ² Thus, the number of fields per well =2.0cm ² /2.54mm ² ＝2.0cm ² /2.54x 10 ^-2 cm ² =79, if the diameter of the field of view observed by the objective lens cannot be determined or if a 10 × objective lens is used, the diameter of the field of view can be determined using a hemocytometer.

(3) Calculating the titre

Viral titer (ifu/mL) = (mean number of positive cells in field × 79 × dilution factor)/(0.1 mL)

(3.1) pSE5256 Titer results

The average number of positive cells calculated in 5 fields under the microscope in the experiment is 5.4, and the virus in the hole is diluted by 10 ⁸ Fold, titer =5.4 × 79 × 10 according to the above formula ⁸ ＝4.27×10 ¹⁰ (ifu/ml)

(3.2) pSE5257 Titer results

The average number of positive cells in the experiment calculated in 5 visual fields under the microscope is 13, and the virus in the hole is diluted by 10 ⁸ Fold, titer =13 × 79 × 10 according to the above formula ⁸ ＝1.03×10 ¹¹ (ifu/ml)

(3.3) pSE5258 Titer results

The average number of positive cells in the experiment calculated in 5 visual fields under the microscope is 21.1, and the virus in the hole is diluted by 10 ⁸ Fold, titer =21.1 × 79 × 10 according to the above formula ⁹ ＝1.67×10 ¹¹ (ifu/ml)

(3.4) pSE5259 Titer results

The average number of positive cells calculated in 5 fields under the microscope in the experiment is 17.2, and the virus in the hole is diluted by 10 ⁸ Fold, titer =17.2 × 79 × 10 according to the above formula ⁸ ＝1.36×10 ¹¹ (ifu/ml)

(3.5) pSB126 Titer results

The average number of positive cells in the experiment calculated in 5 visual fields under the microscope is 5.2, and the virus in the hole is diluted by 10 ⁹ Fold, titer =5.2 × 79 × 10 according to the above formula ⁹ ＝4.12×10 ¹¹ (ifu/ml)

Example 3

Immunological evaluation of different constructed recombinant adenoviruses on mouse models

1. Vaccine safety assessment

1.1 mouse weight detection

After the mice were immunized, the change in body weight of the mice was monitored continuously for one week, and the body weight of the mice injected with the recombinant adenovirus did not significantly decrease compared to the negative control group, and the results are shown in FIG. 7.

1.2 mouse feeding assays

After immunizing mice, the mice were monitored for a week continuously for changes in food intake, and the food intake of the mice injected with the recombinant adenovirus did not significantly decrease compared to the negative control group, and the results are shown in fig. 8.

The weight gain percentage and the food intake monitoring of the mice can be preliminarily evaluated, and the recombinant adenovirus vaccine has no obvious toxicity to the mice and has better safety.

2. Vaccine cellular immune response detection

2.1 vaccine induces cellular immune response

With a titre of 1X 10 ⁸ PFU recombinant adenovirus vaccine rAd5-P1. Injecting rAd5-P2, rAd5-P3 and rAd5-P4 into immunized mice, 14 days after the first immunization, taking spleens of the mice to separate lymphocytes, preparing single cell suspension, and detecting T lymphocyte immunoreaction by flow. The results are shown in FIGS. 9 and 10.

Experimental results show that the rAd5-p1, rAd5-p2, rAd5-p3 and rAd5-p4 can activate T lymphocytes and induce cytokine secretion to different degrees compared with PBS group and Ad5 vector control group.

2.2 vaccine-induced immune memory response

The titer of the solution is 1X 10 ⁸ PFU recombinant adenovirus vaccines rAd5-P1, rAd5-P2, rAd5-P3 and rAd5-P4 are respectively injected into an immunized mouse through muscles, peripheral blood mononuclear cells and drainage lymph nodes of the mouse are taken 60 days after the first immunization, and the activation conditions of central memory T cells, effector memory T cells and tissue resident memory T cells are detected by flow. The results are shown in FIG. 11.

Experimental results show that after 60 days of first immunization, rAd5-p1, rAd5-p2, rAd5-p3 and rAd5-p4 can activate central memory T cells, effector memory T cells and tissue resident memory T cells to different degrees compared with PBS group and Ad5 vector control group.

The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The human cytomegalovirus recombinant vector is characterized by comprising SEQ ID NO.1 and SEQ ID NO.

2. One or more of the nucleotide sequence fragments encoded by SEQ ID No.3 and SEQ ID No. 4.

2. The recombinant human cytomegalovirus vector of claim 1, wherein the recombinant vector comprises a vector adenovirus, the vector adenovirus is replication-defective human adenovirus type 5 with E1 and E3 deleted, and preferably, one or more of the nucleotide sequence segments encoded by SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 are inserted into E1 region of the adenovirus.

3. The recombinant human cytomegalovirus vector of claim 1 or 2, wherein the vector adenovirus is derived from the admax (tm) adenovirus system.

4. Use of the recombinant human cytomegalovirus vector of any one of claims 1 to 3 for the preparation of a vaccine for the prevention of human cytomegalovirus infection.

5. The use of claim 4, wherein the human cytomegalovirus recombinant vector is prepared as an injection, nasal drops or spray.

6. The use of claim 4, wherein the human cytomegalovirus recombinant vector is prepared as an intramuscular injection.

7. A method for producing the human cytomegalovirus recombinant vector of any one of claims 1 to 3, comprising the steps of:

(1) Respectively constructing shuttle plasmid vectors containing dominant antigen epitope genes of the coding human cytomegalovirus protein shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and/or SEQ ID NO. 4;

(3) Transfecting the plasmid obtained in the step (2) into a virus packaging cell HEK293 to carry out packaging, amplification and purification so as to obtain adenovirus of the recombinant human cytomegalovirus protein;

8. The preparation method according to claim 7, wherein the shuttle plasmid vector in step (1) is one or more of PSE5256, PSE5257, PSE5258 and PSE 5259; preferably, the vector of the dominant epitope gene SEQ ID NO.1 is PSE5256; the carrier of the dominant antigen epitope gene SEQ ID NO.2 is PSE5257; the carrier of the dominant antigen epitope gene SEQ ID NO.3 is PSE5258; the vector of the dominant epitope gene SEQ ID NO.4 is PSE5259.

9. The method of claim 7 wherein the targeting backbone plasmid of step (2) is pbhglox (delta) e1,3cre.

10. The production method according to any one of claims 7 to 9, wherein the purification method in step (3) is cesium chloride gradient centrifugation; preferably, the expansion culture method described in step (4) is suspension culture.