CN103820472A - HPV16, 18L1 recombinant DNA vaccine for preventing and treating esophagus cancers - Google Patents

Abstract

The HPV16 and 18L1 recombinant DNA vaccine used for the prevention and treatment of esophageal cancer belongs to the technical field of biomedicine. The present invention provides an optimized gene sequence encoding the main capsid protein L1 of HPV16, 18, a pVR-HPV16, 18L1 DNA vaccine containing the sequence and a method for preparing the pVR-HPV16, 18L1 DNA vaccine. The recombinant HPV16, 18L1 recombinant DNA vaccine provided by the invention can also be used to prepare vaccines for treating and preventing diseases caused by HPV, such as cervical cancer and head and neck cancer.

Description

HPV16,18L1 recombinant DNA vaccine for the prevention and treatment of esophageal cancer

technical field

The invention belongs to the field of biopharmaceuticals. The invention relates to a preparation method of a tumor DNA vaccine containing codon-optimized HPV16, 18L1 gene.

Background technique

Cervical cancer is the second largest gynecological malignancy in the world, second only to breast cancer. There are about 500,000 new cases of cervical cancer worldwide, and about 200,000 deaths from cervical cancer, of which more than 90% are from developing countries. The mortality rate is the first among all kinds of gynecological tumors. There are 131,500 newly discovered cases in my country every year, accounting for about 28.8% of the global total. In the 1970s, ZurHausen proposed that HPV is the virological cause of cervical cancer. After that, domestic and foreign scholars conducted a lot of research on the relationship between the two and proposed that more than 90% of cervical cancer is caused by HPV infection. The International Association for Cancer Research ( IARC) published a study showing that more than 2/3 of cervical cancer cases are related to HPV16 (51%) or HPV18 (16.2%) infection.

Esophageal cancer is one of the most common malignant tumors in the world. There are about 400,000 new cases in the world every year, and my country accounts for about half of them. Among them, the Huaihe River Basin and Taihang Mountains are high-incidence areas for esophageal cancer. Syrjane's research in 1982 showed that there was a correlation between HPV and esophageal squamous cell carcinoma, and many subsequent studies also confirmed this point of view (Shen ZY, Xu LY, Li EM, et a1. The multistage processofcarcinogenesis in human esophageal epithelial cells induced byhumanpapillomavirus.Oncol Rep, 2004, 11:647-54.) Among them, the research of Qu Peng and Li Jintao also showed that HPV infection may be the virological inducement of esophageal squamous cell carcinoma. 34.8%, HPV18 infection rate was 34.8%. (Comparative study on HPV infection rate of different esophageal squamous cell carcinoma specimens in Anyang area, Qu Peng, Li Jintao)

Human papillomavirus (Human papillomavirus, HPV) is an epitheliophilic virus that widely exists in nature. Its genome is divided into E region, L region and URR region. The E6 and E7 encoded in the E region are the main oncoproteins of HPV, which mainly degrade the tumor suppressor protein or cause the release of transcription factors to cause infinite cell proliferation and malignant transformation. transform. Because of the important role of E6 and E7 proteins in the cell cycle, research on therapeutic vaccines mostly focuses on E6 and E7 genes. But it is precisely because E6 and E7 integrate the host genome and can induce cancer, so the safety cannot be guaranteed.

L1 in the L region of the HPV genome, as the main capsid protein of HPV, can self-assemble into VLPs in vitro alone or together with the minor capsid protein L2. Two HPV preventive vaccines that have been marketed and widely vaccinated, Merck’s HPV6, 11, 16, 18 quadrivalent VLP Gardasil vaccine and GSK’s HPV16, 18 bivalent VLP Cervarix vaccine, are both composed of HPV L1 protein Assembled VLPs. Its mechanism of action is to use VLP3 as the target antigen to induce the body to produce specific neutralizing antibodies to prevent the infection of the corresponding type of HPV.

DNA vaccine refers to the direct injection of a recombinant eukaryotic expression vector encoding a certain protein antigen into the animal body, so that the foreign gene is expressed in vivo, and the antigen produced activates the immune system of the body, thereby inducing specific humoral immunity and cellular immunity answer. DNA vaccines are easy to prepare, have good stability, and will not produce neutralizing antibodies against DNA vectors, and can be repeatedly immunized. Studies have shown that DNA vaccines encoding HPV16, 18 types E6, and E7 proteins have anti-inflammatory effects on cervical epithelial cells in clinical trials. Patients with tumors have obvious therapeutic effects. (GARCIA F, PETRY K U, MUDERSPACH L, et al. ZYC101a for treatment of high-grade cervical intraepithelial neoplasia: arandomized controlled trial[J]. Obstetrics&Gynecology. 2004, 103(2): 317)

The L1 gene has no carcinogenicity, and the protein encoded by L1 can induce higher humoral immunity and cellular immunity, so the DNA vaccine based on HPV16 and 18 can be developed for the prevention and treatment of esophageal cancer and cervical cancer.

Contents of the invention

The invention provides a codon-optimized gene sequence encoding the main capsid protein L1 of HPV16 and 18 and a recombinant DNA vaccine containing the above-mentioned optimized gene.

A codon-optimized gene encoding the main capsid protein L1 of human papillomavirus type 16, 18 (HPV16, 18), the gene has the nucleotide sequence shown in SEQ ID NO:1.

A recombinant DNA vaccine containing the above-mentioned optimized gene carries the above-mentioned gene.

A recombinant DNA vaccine containing the above-mentioned optimized gene is achieved through the following steps:

(1) Obtain codon-optimized HPV16, 18L1 gene by substituting codons in HPV16, 18L1 gene sequence with codons frequently used by mammals;

(2) Cloning the codon-optimized HPV16, 18L1 gene obtained in step (1) into the VR-1020 plasmid to obtain a recombinant DNA vaccine carrying the codon-optimized HPV16, 18L1 gene sequence;

(3) Detect the in vitro expression of the recombinant DNA vaccine and extract the recombinant DNA plasmid using the Qiagene endofree large extraction kit, and inject Balb/c mice intramuscularly with the priming-boosting strategy to detect the immune effect.

The present invention provides a codon-optimized gene (mod. Under the condition of capsid protein L1 amino acid sequence, codons of HPV16 and 18L1 gene sequences are replaced with codons frequently used by mammals.

The invention provides a codon-optimized HPV16, 18L1 gene recombinant DNA vaccine. The optimized nucleotide sequence of the gene (mod.HPV16, 18L1) encoding the main capsid protein L1 of papillomavirus type 16, 18 (HPV16, 18) is shown in SEQ ID NO: 1, and it is constructed into The recombinant DNA vaccine was obtained on the DNA carrier. The optimized HPV16, 18L1 gene recombinant DNA vaccine was transfected into cells for protein expression. The results showed that the optimized HPV16, 18L1 gene (mod.HPV16, 18L1) could express protein efficiently.

In a preferred embodiment, said DNA vector is VR-1012 plasmid.

In a preferred embodiment, the in vitro expression cells are HEK293 cells.

On the other hand, the present invention also provides the optimized gene encoding the main capsid protein L1 of papillomavirus type 16, 18 (HPV16, 18) in the preparation of a drug or vaccine composition for preventing and treating diseases caused by HPV The use of HPV, wherein the HPV is preferably HPV16, 18 types.

On the other hand, the present invention also provides the use of the recombinant HPV16, 18L1 DNA vaccine described in the present invention in the preparation of medicines or vaccine compositions for preventing and/or treating diseases caused by HPV such as cervical cancer, esophageal cancer and head and neck cancer .

Detailed ways

Materials and methods

Escherichia coli DH5α was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd., DNA A-Tailing Kit, pMD16, and 18-T vector were purchased from Bao Bioengineering (Dalian) Co., Ltd. HEK293 cells were provided by Mr. Jia Runqing, Institute of Viral Disease Prevention and Control, Chinese Center for Disease Control and Prevention. The glycerol bacteria containing the pVR-HPV16, 18L1 plasmid and the plasmid pMK-RQ containing the codon-optimized HPV16, 18L1 gene were provided by Zhou Yubo. Restriction endonucleases Bgl II and Sal I were purchased from NewEngland Biology, and T4 DNA ligase and DNA Marker were purchased from Treasure Bioengineering (Dalian) Co., Ltd. Ordinary DNA purification kit, agarose gel recovery kit, pfu DNA polymerase, and dNTP were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. Plasmid extraction kit (Midi) and endotoxin-free plasmid extraction kit (Maxi, Mega) were purchased from QIAGEN. Prestained protein markers were purchased from Fermentas. Lipofectamine2000, a liposome transfection reagent, was purchased from Invitrogen. DMEM medium, OptiMEM I medium, fetal bovine serum (FBS), and penicillin and streptomycin double antibodies were purchased from Gibco. Mouse anti-HPV16 and 18L1 monoclonal antibodies were purchased from Abcam, and mouse anti-β-actin monoclonal antibodies were purchased from Beijing Zhongshan Jinqiao Biotechnology Company. Goat anti-mouse IgG antibody (Anti-MOUSE IgG(H&L)(GOAT) Antibody IRDye700DX) was purchased from Rockland Company. Other analytical reagents are provided by the laboratory. Lipofectamine2000, a liposome transfection reagent, was purchased from Invitrogen. QuickShuttle transfection reagent was purchased from Beijing Kangbiquan Biotechnology Co., Ltd. DMEM medium, RPMI-1640 medium, Quick Spot mouse IFN-γ ELISPOT pre-coating kit, and EZ-Sep ^TM Mouse1X easy-to-get mouse lymphocyte separation medium were purchased from Shenzhen Dakwei Biotechnology Co., Ltd. Other analytical reagents are provided by the laboratory. The peptides designed in the experiment were synthesized by Beijing Zhongke Yaguang Biotechnology Co., Ltd. The primers required for the experiment were synthesized by Shanghai Yingjun Biotechnology Co., Ltd.

Related techniques such as molecular biology and immunology involved in the present invention, such as nucleic acid manipulation techniques, protein qualitative and quantitative analysis, etc., have been fully described in the scientific literature (for example, see J. Sambrook E. F. Fritsch T. Manny Totis, A Laboratory Guide to Molecular Cloning (Second Edition)).

Embodiment 1 recombinant VR-HPV16, the construction of 18L1 DNA plasmid

1.1 PCR amplification of HPV16, 18L1-pVR gene fragment

1.1.1 The HPV16, 18L1-pVR gene fragment was amplified by PCR using pMK-RQ containing the codon-optimized HPV18L1 gene as a template and the synthetic sequence as a primer. After the completion of PCR, 1 μL of the reaction solution was taken for DNA gel (1%) electrophoresis detection. The PCR product was recovered after a good detection result was obtained.

1.1.2 Construction of HPV16L1 gene sequence: PCR amplification was carried out with PUC-HPV16L1 plasmid containing HPV16L1 gene as template and synthetic sequence as primer. dNTP8μl, Pfu2μl, ddH2O77μl, total system 100μl. After 30 cycles (denaturation at 94°C for 30s, annealing at 58°C for 30s, and extension at 72°C for 30s), the HPV16L1 gene sequence was obtained, and the PCR reaction product was purified by gel cutting.

1.2 Ligation of HPV18L1-pVR gene fragment and T vector The HPV16, 18L1-pVR gene fragment amplified by PCR was connected to pMD16, 18-T vector to preserve and ensure high enzyme cutting efficiency. Since the pfu enzyme cannot add A to the 3' end of the amplified product, it is necessary to first use the DNA A-Tailing Kit to add A to the 3' end of the PCR product. Take an appropriate amount of PCR product according to the instructions, add 5 μL of 10×A-Tailing Buffer, 4 μL of dNTP, 0.5 μL of A-Tailing Enzyme, make up water to 50 μL, place in a PCR instrument at 72°C for 20 minutes, and then stand on ice for 1 to 2 minutes. Then take an appropriate amount of the above PCR product with A added at the 3' end, add 1 μL of pMD16, 18-TVector, replenish water to 5 μL, then add 5 μL Solution I, and react at 16°C for 2 hours.

1. Transformation of 3T vector ligation products Add all the ligation products (10 μL) in 1.2 to 50 μL DH5α competent cells, and place in ice for 30 minutes. After heating at 42°C for 90 seconds, place in ice for 2 to 3 minutes. Then add 400 μL of LB medium, shake and incubate at 37°C for 45 minutes. Take 100 μL of transformed competent cells, and gently spread it on the LB agar plate medium containing X-Gal, IPTG, and Amp with an elbow glass rod. After the liquid on the plate is absorbed, place it in a 37°C incubator and incubate for 12-16h.

1.4 Small extraction and double digestion of the recombinant T vector and pVR plasmid Pick the white colony on the plate, inoculate it into 4mL of Amp-resistant LB medium, and culture it overnight at 37°C with vigorous shaking. Purify the plasmid according to the instructions of the small extraction kit, and then use the restriction endonucleases Bgl II and Sal I to double digest the recombinant T vector and pVR plasmid (HPV18 type). For the HPV16 type, the expression vector VR-1012 was double digested with Bgl I and Sal I. Enzyme digestion system: 20 μl of VR-1012 vector plasmid, 0.5 μl of BSA, 45 μl of 10×buffer, 2 μl of endonuclease Bgl I, 2 μl of Sal I, 0.5 μl of ddH2O, 50 μl of the total enzyme digestion system, digested at 37°C for 3-4 hours. The band of interest was recovered with an agarose gel recovery kit. Take 1 μL of the recovered DNA and perform 1% agarose gel electrophoresis to detect the recovery effect.

1.5 Ligation transformation of HPV16, 18L1 fragment and pVR plasmid For HPV18 type, the ligation system is: appropriate amount of HPV18L1 fragment and pVR plasmid recovered by digestion in 2.2.1.4, 10×T4 Ligase buffer 2 μL, T4 ligase 1.5 μL, water up to 20 μL . Ligation overnight at 16°C. For the HPV16 type ligation system, HPV16L 17 μl, VR-10121 μl, 10× ligation buffer 1 μl, T4 ligase 1 μl. Ligation overnight at 16°C. Add all ligation products (20 μL) to 50 μL DH5α competent cells and place on ice for 30 minutes. After heating at 42°C for 90 seconds, place in ice for 2 to 3 minutes. Then add 400 μL of LB medium, shake and incubate at 37°C for 45 minutes. Take 100 μL of transformed competent cells, and gently spread it on the LB agar plate medium containing Kana with an elbow glass rod. After the liquid on the plate is absorbed, place it in a 37°C incubator and incubate for 12-16 hours.

1.6 Small extraction and identification of recombinant plasmids Pick a single colony on the plate and inoculate it into 4 mL of Kana-resistant LB medium, and culture it overnight at 37°C with vigorous shaking. Purify the plasmid according to the instructions of the small extraction kit, and then use the restriction enzymes Bgl I and SalI to treat the HPV16 type DNA-HPV16L1 and use the restriction enzymes BglII and SalI to extract the recombinant DNA-HPV18L1 plasmid , double enzyme digestion identification. After the preliminary identification is correct, it will be sent to Beijing BGI for sequencing. The recombinant pVR plasmids with correct sequencing results were named pVR-HPV16L1 and pVR-HPV18L1.

Example 2 Detection of pVR-HPV16, 18L1 expression in vitro

2.1 Culture of HEK293 cells The medium used for culturing HEK293 cells was DMEM medium containing 10% FBS and 1% double antibody, and the culture conditions were 37°C, 5% CO ₂ constant temperature incubator.

Transfection of cells with the plasmid mentioned in 2.2

(1) One day before transfection, 293 cells were trypsinized and counted, and the cells were transferred to a six-well plate, and the density was controlled so that the density on the day of transfection was close to 90%. Cells were plated in 2 mL of serum-containing normal growth medium without antibiotics.

(2) For each well of cells, dilute 4 μg of plasmid (pVR-HPV16, 18L1) with 250 μL of OPTI-MEM I medium. Mix Lipofectamine2000 reagent before use, and dilute 10μL Lipofectamine2000 with 250μL OPTI-MEM I medium. Mix gently, let stand at room temperature for 5 minutes, then mix with diluted plasmid, and let stand at room temperature for 20 minutes.

(3) Add the complex (500 μL) dropwise into each well, shake the culture plate back and forth (or left and right), and mix gently. Place in a constant temperature incubator at 37°C, 5% CO ₂ . After 4 to 6 hours, the medium was changed to a medium for normal growth.

2.3 Western Blot detection of HPV16, 18L1 protein

(1) After 48 hours of transfection, discard the medium, wash the cells with PBS, add 80 μL of cell lysate (50mM Tris-HCl (pH8.0), 150mM NaCl, 1% Triton X-100, 100μg /mL PMSF (add just before use)), use a pipette to blow or scrape the cells with a cell scraper, collect the lysed cells into a 1.5mL Eppendorf tube, and centrifuge at 12000r/min for 10min.

(2) Take the supernatant and add 1/4 volume of 5× electrophoresis loading buffer, cook in boiling water for 5-10 minutes, take 20 μL for SDS-PAGE electrophoresis (5% for stacking gel, 10% for separating gel), 110V Constant voltage electrophoresis; after electrophoresis, transfer the protein to nitrocellulose (NC) membrane by semi-dry transfer method; place the NC membrane in PBS containing 5% skimmed milk powder at 37°C and shake slowly for 1 hour; discard the blocking solution , add 1:1000 mouse anti-HPV16, 18L1 monoclonal antibody (8.F.324) overnight at 4°C refrigerator; wash the membrane 3 times with PBS containing 0.05% Tween-20, 10min each time; add 1:5000 dilution Goat anti-mouse IgG antibody (Anti-MOUSE IgG(H&L)(GOAT) Antibody IRDye700DX) was incubated with shaking at room temperature for 45min; the membrane was washed 3 times with PBS containing 0.05% Tween-20, 10min each time; the membrane was placed in PBST , and then use the Odyssey far infrared image analyzer to detect HPV16, 18L1 protein, (see Figure 1, 2 for the results).

Example 3 Experiment of Recombinant DNA Vaccine pVR-HPV16, 18L1 Immune Mice

3.1 Preparation and immunization plan for immunized mice

Twenty healthy female BALB/cH-2Kd mice of SPF grade 4-6 weeks old obtained from commercial sources were randomly divided into 3 groups, 10 mice in each group, and immunized twice at the 0th and 3rd week by intramuscular injection into the thigh. The immune response was measured one week after the booster immunization. See Table 1 for specific immunization contents and doses.

Table 1 Mouse immunization groups and doses

3.2 Detection of HPV16, 18L1 humoral immune response in immunized mice

One week after the last booster immunization (day 28), the venous blood of the mice was collected, and the serum was separated. HPV16 and 18 pseudovirions were prepared according to the method reported in the literature (Buck, C.B., D.V. Pastrana, D.R. Lowy, et, al. J. Virol. 2004, 78:751-757.).

(1) Determination of pseudovirus titer. 1. Cell technology and loading. Take a bottle of well-grown 293FT cells with a fusion rate of about 80%, discard the stock solution, and wash with PBS twice. Add 1ml of 0.05% trypsin digestion solution to the opposite side of the cell surface of the culture flask, turn the culture flask flat to ensure that the trypsin completely covers the cell layer, and digest for 2-3 minutes. Add 5ml of neutralized DMEM culture solution to stop the digestion, use a pipette to blow and blow the surface of the cultured cells ten times repeatedly to wash down the cells on the wall of the bottle, and then use a pipette to repeatedly blow and blow the culture medium 15 times to make the cells evenly dispersed. Transfer the culture medium to a 15ml centrifuge tube and centrifuge at 200g. Discard the supernatant and resuspend in 5ml DMEM. The cell count is about 2×10 ⁷ . Cells were diluted to a density of 3×10 ⁵ cells/ml. Add the cells into the 96-well culture plate with a row gun (100ul/well). (Note: Gently mix the cells before adding each sample), put the culture plate into the cell culture incubator, 37°C overnight. Take a 96-well cell culture plate and dilute the pseudovirus with neutralized DMEM (100ul/well). 2. Pseudovirus titration. Add heparin sodium (100mg/ml heparin sodium 2ul/well) to the positive control. Add the pseudovirus and sodium heparin in the above steps into a 96-well culture plate containing 293FT cells, put the culture plate into a cell culture incubator, and incubate at 37° C. for 72 hours. (Caution: Do not shake, place gently in the incubator). Add 0.05% chaps to the 96-well ELISA plate (20ul/well), and evenly cover the bottom of the well. Add supernatant (40ul/well) after culturing for 72h, inactivate alkaline phosphatase at 65°C for 30min. Add coloring substrate (200ul/well) and let stand at room temperature for 2-3 hours. (Note: This step should be strictly protected from light). A405nm was measured with a microplate reader. Select the appropriate pseudovirus concentration for the downstream serum antibody neutralization test according to the assay results.

(2) Determination of neutralizing antibody titer. Dilute 293FT cells to 3×10 ⁵ /mL, spread 100uL/well in a 96-well cell culture plate, incubate at 37°C for 3 hours, dilute serum at 1:20, dilute SEAP-PsV16 and 18 in a certain proportion, and dilute 20ul of serum Add to 80ul diluted PSV, ice-bath for 1h, add the mixed solution to 293FT cells, and continue to culture for 72h. The wells without serum are used as negative controls. Each sample and negative control are double wells. Add 0.05% CHAPSin PBS, 20ul/well. Add cell supernatant 40ul/well, extinguish endogenous alkaline phosphatase at 65°C for 30min, add 200ul PNP reaction substrate (20ml diethanolamine Mg+Zn solution, one piece of PNP), keep room temperature away from light for 2-4 hours, Bio -Rad550 type microplate reader measures the OD value at the wavelength of 405nm. A405nm value lower than 50% of the negative control was judged as positive. The antibody titers produced by the pVR-HPV16L1 group and pVR-HPV18L1 reached 800 and 640 respectively, indicating that the recombinant DNA vaccines pVR-HPV16L1 and pVR-HPV18L1 can induce a higher humoral immune response in BALB/c mice (see Figure 3) .

3.3 Detection of PV16, 18L1 cell immune response in immunized mice

One week after the booster immunization (day 28), the mice were sacrificed by cervical dislocation and the spleen was removed, and the splenic lymphocytes of the mice were separated with lymphocyte separation medium, and the secretion of IFN-γ (γ- Interferon) T lymphocytes. Take 50 μl of 2×105/m1 mouse spleen lymphocytes and an equivalent amount of 8 μg/m1 polypeptide (use the BIMAS peptide binding software on the Internet http://www-bimas.cit.nih.gov/molbio/hla_bind/ to identify the predicted T cell recognition peptide (restricted by H2-Kd) and synthesized by conventional synthesis technology) was added to the ELISPOT plate coated with anti-mouse IFN-γ antibody and blocked, and each mouse was made with three replicate wells. In addition, each small The negative control wells of mice were only added with the same amount of cells without polypeptide, and the positive control wells were added with cells and phytohemagglutinin (PHA) with a final concentration of 20 μg/m1. The specific method is as follows:

(1) Separation of cells. Execute the mouse, soak it in 75% ethanol for 1-2 minutes, remove the mouse spleen with tweezers, and put 4-5ml of lymphocyte separation solution in the petri dish: shake the lymphocyte separation solution before use, and only point it when grinding. Press, not circle grinding. It is best to gently grind each spleen within 5 minutes. After each spleen is finished, the piston must be screwed on to prevent the liquid from evaporating, resulting in uneven density and difficult to form layers. Immediately transfer the grinding solution to a centrifuge tube, and cover the page with about 200-500 μL of easy-to-culture medium (pay attention to adding it along the tube wall when adding cell culture medium). For each mouse that is ground, immediately remove the spleen cell suspension from the culture medium. Transfer the dish to a centrifuge tube, and pay attention to tightly cover the tube cap. After all the spleens of the mice were processed, an overlay of 1640 was uniformly added. Centrifuge at 800g for 30 minutes, and the rhythm of speed reduction should be controlled. Aspirate the lymphocyte layer to a 15ml tube (pay attention to aspirating as much upper layer cells as possible when absorbing the separated lymphocyte layer), slowly add 10mL1640 medium, cover, invert 6-10 times, and centrifuge at 250g for 10 minutes. Discard the supernatant, add 1mL Lympho-SpotTM serum-free medium to resuspend, and count the cells.

(2) Cell loading and incubation. 1. Counting and calculation: Wipe the counting plate and cover slip clean, and cover the counting plate with the cover slip. Aspirate a little of the cell suspension, add it dropwise on the edge of the cover slip, make the suspension fill the gap between the cover slip and the counting plate, and let it rest for 3 minutes. Be careful not to have air bubbles under the cover slip, or allow the suspension to flow into the side tank. The total number of cells in the large grid, only the left and upper cells are counted. Then calculate according to the formula: number of cells/mL=total number of cells in the four major grids/4×10 ⁴ (note: under the microscope, there are occasional cell clusters composed of more than two cells, which should be calculated as a single cell, if the cell cluster accounts for more than 10% , indicating that the dispersion is not good, and the cell suspension needs to be prepared again). The approximate number of cells is about 1-10 x ¹⁰⁷ . When diluting, first dilute the cells to 10 ⁷ /ml, then take 300ul (the cell volume at this time is 3×10 ⁶ ) and dilute to 1ml (just add 700ul1640 medium), so the sample volume is 3×10 ⁶ /ml ml. 2. Activation of the pre-coated plate: 200 μL/well EZ-Culture ^TM serum-free medium, leave it at room temperature for 5-10 minutes, then buckle it out. 3. Add cell suspension, add the adjusted concentration of cell suspension to each experimental well, 100 μL/well3×10 ⁵ cells/well. The positive control was 200,000. Add stimuli: conA or PHA 10 μL/well. Add the peptide stimulator synthesized according to the prediction on the website (with Lympho-Spot ^TM serum-free medium to prepare a final concentration of 8ug/ml, 10μL/well) to the experimental well. Do not tap the ELISPOT plate after adding the stimulus. 3. Incubation: After adding all samples and stimuli, close the lid of the plate. Place in 37°C, 5% CO2 incubator for 16-20hr. After adding all the samples, cover the plate, put it into a carbon dioxide incubator, and incubate at 37°C for 18-24h. Collisions cause cells to shift, resulting in blurred, smeared spots. During the whole culture process, avoid moving and bumping the culture plate, and minimize the number of switching incubators.

(3) Chromogenic operation after cell culture (sterile operation is no longer required)

Pour off the cells and medium in the well. Add 200 μL of ice-cold deionized water to each well and incubate at 4°C for 10 min (cells are lysed by hypotonic method). You can also put the plate with deionized water in the -20°C refrigerator for 5 minutes, and then transfer it to the 4°C refrigerator for 5 minutes to replace the ice bath. To prevent freezing, the board should not be placed in a -20°C refrigerator for more than 5 minutes. Each well was washed 10 times with 200 μL washingbuffer. For the last time, button dry on absorbent paper. According to the concentration specified in the kit, add 100 μL of diluted biotin-labeled detection antibody to each well, and incubate at 37°C for 1 hour. Wash each well 5 times with 200 μL PBST. For the last time, button dry on absorbent paper. According to the concentration specified in the kit, add 100 μL of diluted enzyme-labeled avidin to each well, and keep at 37°C for 1 hour. Wash each well 5 times with 200 μL PBST. For the last time, button dry on absorbent paper. According to the instructions of the kit, thaw the prepared AEC chromogenic solution. Add 100 μL of chromogenic solution to each well, and let stand at room temperature for 15-45 minutes (at 20-25°C, 25 minutes for color development is more appropriate), and keep away from light. (Stay in the dark at 37°C for 12-15 minutes). After the spots grow to a suitable size, wash them twice with deionized water to terminate the color development process. Put the board upside down on the absorbent paper, pat dry the tiny water droplets, then remove the protective layer, put it in a ventilated place, and let it stand at room temperature for 10-30min to let the film dry naturally. Be careful not to put the plate in the oven to prevent the membrane from becoming brittle and cracking. Place the ELISPOT plate in the Biosys Bioreader4000PRO automatic plate reader, adjust the appropriate parameters, count the spots, and record various parameters of the spots for statistical analysis.

The results showed that the spleen lymphocytes of groups 2 and 3 secreted IFN-γ cells under the stimulation of the corresponding HPV16 and 18L1 synthetic polypeptides, which was significantly higher than that of the first group of control groups. In the HPV16 type, 1× ¹⁰⁶ spleen The number of spots in lymphocytes reached 2070 (see Figure 4), and the number of spots in 1×10 ⁶ splenic lymphocytes in HPV18 types reached more than 500 (see Figure 5), indicating that the recombinant pVR-HPV16, 18L1 vaccine can be used in BALB /CH-2Kd mice induced higher cellular immune responses in vivo.

Claims (8)

1. a coding human papilloma virus 16 for codon optimized type, the gene of 18 type Major capsid protein L1s, this gene has the nucleotide sequence shown in SEQ ID NO:1.

2. a recombinant dna plasmid, this recombinant plasmid carries gene claimed in claim 1.

3. the preparation HPV16 that recombinates described in claim 2, the method for 18L1DNA vaccine, is characterized in that, comprises the steps:

(1) replace HPV16 by the codon using with Mammals high frequency, the codon of 18L1 gene order, obtains codon optimized type HPV16,18L1 gene;

(2) codon optimized type HPV16 step (1) being obtained, 18L1 gene clone enters carrier DNA plasmid, obtains carrying codon optimized type HPV16, the recombinant dna plasmid of 18L1 gene order.

4. according to the method for claim 3, wherein said carrier DNA plasmid is plasmid VR-1012.

5. the purposes of gene in medicine or vaccine composition for the preparation of the disease that causes of control HPV according to claim 1.

6. purposes according to claim 5, HPV type is selected from HPV6,11,16,18,30,31,33,35,39,45,51,52,56,58,59 or 68 types.

7. recombinant DNA vaccine according to claim 2 is for the preparation of preventing and/or treating disease that HPV causes as the purposes in the medicine of the esophageal carcinoma or vaccine composition.

8. recombinant DNA vaccine according to claim 2 is for the preparation of preventing and/or treating other diseases that HPV causes as the purposes in the medicine of cervical cancer, head and neck cancer or vaccine composition.

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