WO2020259558A1 - Gène ge et vecteur pour exprimer un gène - Google Patents

Gène ge et vecteur pour exprimer un gène Download PDF

Info

Publication number
WO2020259558A1
WO2020259558A1 PCT/CN2020/098017 CN2020098017W WO2020259558A1 WO 2020259558 A1 WO2020259558 A1 WO 2020259558A1 CN 2020098017 W CN2020098017 W CN 2020098017W WO 2020259558 A1 WO2020259558 A1 WO 2020259558A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
gene
cell
expression vector
vzv
Prior art date
Application number
PCT/CN2020/098017
Other languages
English (en)
Chinese (zh)
Inventor
周晨亮
何强
沈巧英
周凌云
江元翔
刘革
曾宪放
史力
莫呈钧
张智
Original Assignee
怡道生物科技(苏州)有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 怡道生物科技(苏州)有限公司 filed Critical 怡道生物科技(苏州)有限公司
Publication of WO2020259558A1 publication Critical patent/WO2020259558A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/03—Herpetoviridae, e.g. pseudorabies virus
    • C07K14/04—Varicella-zoster virus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/245—Herpetoviridae, e.g. herpes simplex virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16711—Varicellovirus, e.g. human herpesvirus 3, Varicella Zoster, pseudorabies
    • C12N2710/16722—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the invention belongs to the field of biomedicine. Specifically, the invention relates to a recombinant shingles vaccine composition and a preparation method and application thereof.
  • VZV Varicella zoster virus
  • HZ herpes zoster
  • Chickenpox is usually seen in childhood, while herpes zoster does not develop until adulthood. After the primary infection with chickenpox, the virus can be latent in the host's ganglia. With age, immune function impairment or immunosuppression, VZV can be reactivated and cause shingles.
  • herpes zoster The clinical manifestation of herpes zoster is a unilateral vesicular rash.
  • the obvious feature is that it is limited to a single skin segment, usually accompanied by radicular pain.
  • Patients may have obvious pain and discomfort. Symptoms can last for several weeks or months. In severely ill patients, they can even last for several years, resulting in a decline in the quality of life. In rare cases, shingles may not appear as a rash. Complications occur in about 25% of people with shingles, and they increase with age.
  • the most common serious complication is post-herpetic neuralgia (PHN), that is, pain that persists after the acute phase of herpes.
  • PPN post-herpetic neuralgia
  • the incidence of herpes zoster patients is 10%-30%. The pain can be It lasts for several months or even years, seriously affecting the quality of life of patients.
  • the risk factors that affect the onset of shingles are age, immune deficiency, gender
  • VZV Most primary VZV infections occur in childhood, then VZV is latent in the ganglia, and VZV can be reactivated in adulthood. Studies have shown that about 99% of Americans aged 40 and over have serological evidence of VZV infection; 90% of Europeans aged 20-29 years are seropositive for anti-VZV; in some countries in South America, Australia and Asia The primary VZV infection may occur later, but 90% of people over 40 years old will have VZV seropositivity. Therefore, on a global scale, most adults are at risk of developing shingles and its related complications.
  • the global incidence of herpes zoster is (3 ⁇ 5)/1000 person-years, and the Asia- Pacific region is (3 ⁇ 10)/1000 person-years, and it is increasing by 2.5% ⁇ 5.0% year by year, and the hospitalization rate is (2 ⁇ 25)/100,000
  • the mortality rate is (0.017 ⁇ 0.465) per 100,000 person-years, and the recurrence rate is 1% ⁇ 6%.
  • my country is in a state of high-degree aging, and the social and economic burden brought by HZ is increasing year by year.
  • HZ has brought a huge negative impact on the quality of life of patients, especially elderly patients.
  • According to data released by the National Bureau of Statistics it is estimated that the population over 40 years old in 2017 was about 650 million. If the HZ incidence rate is 2.5/1000 person-years, it is conservatively estimated that there are about 1.6 million new cases of HZ in my country each year.
  • HZ live attenuated vaccine Zostavax and HZ subunit vaccine Shingrix are on the market worldwide (HZ live attenuated vaccine Zostavax and HZ subunit vaccine Shingrix).
  • Merck’s Zostavax is a live attenuated vaccine, which contains the same virus strain as the VZV Oka strain used in the varicella vaccine.
  • the vaccine formulation uses a minimum potency of 19400 PFU. It was approved by the FDA in 2006 and has been approved in more than 60 countries.
  • One dose can be subcutaneously administered to people over 50 years of age.
  • Shingrix developed by GlaxoSmithKline is a subunit vaccine based on recombinant gE protein supplemented with a new adjuvant AS01 B.
  • a recombinant shingles vaccine composition which contains a VZV virus gE protein, which can be efficiently obtained by CHO cells.
  • the aforementioned gE protein has the amino acid sequence shown in SEQ ID NO:1.
  • a gE gene encoding the gE protein of the VZV virus is provided, the gene is a gE gene that can be expressed in CHO cells, and the gene has the nucleoside shown in SEQ ID NO: 2 Acid sequence.
  • an expression vector contains the sequence of the gE gene.
  • the expression vector contains a promoter and a cloning site for the sequence encoding the gE protein so that the promoter and the sequence are operably linked.
  • the expression vector may also have other elements, such as a signal peptide sequence (sometimes called a leader sequence), a tag sequence, a transcription termination signal, an origin of replication, and a sequence encoding an optional product.
  • the expression vector is cloned to carry the blasticidin resistance gene by introducing the 5'and 3'ends of the gE gene into the restriction endonuclease sites of XbaI and NotI, respectively.
  • the plasmid expression vector and the plasmid expression vector carrying the bleomycin resistance gene are cloned to carry the blasticidin resistance gene by introducing the 5'and 3'ends of the gE gene into the restriction endonuclease sites of XbaI and NotI, respectively.
  • the expression vector contains the aforementioned gE gene.
  • VZV gE protein is usually achieved through expression in cultured cells or through chemical synthesis.
  • Host cells frequently used and suitable for protein production include E. coli, yeast, insects, and mammals.
  • a genetically engineered cell is provided, the cell contains the expression vector, or the gE gene is integrated in its genome.
  • the aforementioned cells are CHO cells.
  • the cell is a CHO cell containing the gE gene or expression vector of the present invention, and the cell can express and produce gE protein at a high level.
  • an immunogenic protein the protein is the gE protein of the VZV virus, and the gE protein is expressed by CHO cells.
  • the immunogenic protein is prepared by the following method:
  • the use of the vaccine composition is provided to prevent or treat diseases or disorders related to herpes zoster virus infection.
  • a method for expressing the gE protein of VZV virus in CHO cells which includes the following steps:
  • step (3) Use the cell line obtained in step (3) for expression to obtain VZV protein.
  • the expression vector described in the step (1) is a plasmid expression vector carrying a blasticidin resistance gene and/or a plasmid expression vector carrying a bleomycin resistance gene .
  • the CHO cells used in the step (2) are CHO-K1 cells.
  • the protein sequence is as follows, with a total length of 546 amino acids (SEQ ID NO: 1).
  • the specifics are as follows.
  • the sequence includes The signal peptide and the main body of the antigen, but excluding the C-terminal and anchoring region in the wild-type gE protein.
  • the codons preferred by CHO cells are selected to optimize the gE gene (such as SEQ ID NO: 2), and the outsourcing company is commissioned to synthesize, as follows. It should be noted that the optimization principle is not simply to select the most frequent codon in CHO cells, but a more complex optimization scheme. There are three overall optimization principles: First, according to the degeneracy of the codons, replace the original codons with the high-frequency codons corresponding to each amino acid in CHO cells; second, to avoid excessive GC in the post-transcribed mRNA The content affects its secondary structure, which in turn affects translation efficiency. In the optimization process, try to control the GC% of the gene at 40-60%; third, avoid some commonly used restriction enzyme sites.
  • the 5'and 3'ends of the above synthesized gE gene were introduced into Xba I and Not I restriction endonuclease cleavage sites, respectively, and the fragment was cloned into the expression vectors pWX2.0 and pWX1.0 by PCR.
  • the vector pWX2.0 carries the blasticidin resistance gene
  • pWX1.0 carries the bleomycin resistance gene.
  • Both of these vectors use the cytomegalovirus (CMV) promoter/enhancer sequence to express the target gene.
  • CMV cytomegalovirus
  • the CMV promoter is a strong promoter commonly used to promote eukaryotic gene expression.
  • the corresponding expression plasmid was obtained by cloning and construction, and the sequence was verified by restriction enzyme digestion and sequencing.
  • construction methods of the above two expression vectors are conventional technical means in the field.
  • construction methods that can be referred to are:
  • the vector pWX2.0 was digested with restriction enzymes Xba I (NEB, Cat. #: R0145S) and Not I-HF (NEB, Cat. #: R3189S), and the digested product with a size of 4775 bp was recovered.
  • the above-mentioned 1660bp recovered fragment was ligated into the above 4775bp pWX2.0 vector, and the ligated product was transformed into TOP10 competent cells, and screened with a plate containing blasticidin to obtain several monoclonal positive colonies, and some of them were selected for PCR Amplification and sequencing verification. Then, pick a clone that was verified by sequencing and streak it twice on the LB plate.
  • step 2.1 using plasmid pUC57-gE as template, double enzyme digestion with Xba I and Not I-HF to obtain a 1660 bp DNA fragment. After separation by 1% agarose gel electrophoresis, the gel was tapped and recovered under UV light. 1660bp DNA fragment.
  • the vector pWX1.0 was double digested with restriction enzymes Xba I and Not I-HF, and a 4172 bp vector fragment was recovered using a gel purification kit. The 1660bp recovered fragment was ligated to the 4172bp pWX1.0 vector fragment, and the ligated product was transformed into TOP10 competent cells. Several single clones were selected and verified by PCR and sequencing. Then, pick a clone that was verified by sequencing and streak it twice on the LB plate.
  • the expression plasmid was prepared in large quantities by the method in 2 above, and linearized and stably transfected into host cell CHO-K1. In this example, a total of 6 sets of transfection experiments were performed. Subsequently, each group of transfected mini cell populations were screened by fed-batch culture, and the cell populations with higher expression levels were selected for subsequent clonal screening by limiting dilution. It can be seen that in all 6 groups of mini cell groups, the average cell expression level is between 1-1.5g/L, and the expression level of the three highest groups of cells is between 1.5-2g/L, and the highest group of cells Group protein expression can be 2.01g/L.
  • the three groups of cell populations with the highest expression levels are selected, and single clones are selected for these three groups of cell populations by limiting dilution.
  • the selected single clones are expanded during the fed-batch culture process, the supernatants of the above cloned cells are collected, and the samples are taken for western blot detection.
  • the target protein is determined according to the bands, and the cloned LDC photos, growth conditions, and expression levels are considered. , Viable cell density, viability, final lactic acid content and related product quality parameters, select the best three clones, that is, the dominant cell line.
  • the cell line obtained above is cultured and expressed in a bioreactor to obtain a cell culture supernatant containing gE protein. Similarly, the above-mentioned supernatant can be sampled for Western blot detection to determine whether it is the target protein according to the band. It has been proved that the average gE protein in the above cell lines can reach 2.5g/L.
  • the method of using CHO cell lines to stably express the VZV gE recombinant protein is a well-known method in the art.
  • the method of using CHO cell lines to stably express the VZV gE recombinant protein is a well-known method in the art.
  • the specific plasmid transfection and cell line construction methods are as follows:
  • CD CHO medium M1 to resuscitate and cultivate 1 CHO-K1 host cell as a working cell bank cell.
  • the plasmids pWX1.0-Z-gE and pWX2.0-B-gE obtained by the above 2 method were linearized, specifically, the restriction enzyme Sca I-HF was used for digestion (50 ⁇ l digestion system ), take 2 ⁇ l of the digested product and use 1% agarose gel electrophoresis to detect it. The results show that the two plasmids after Sca I-HF digestion showed a single and clear band, indicating a good linearization result.
  • the 50 ⁇ l linearized product was purified by phenol chloroform extraction and ethanol precipitation, and then dissolved in 10 mM Tris buffer.
  • the concentration of plasmid pWX1.0-Z-gE was 1191.2ng/ ⁇ l, and the concentration of pWX2.0-B-gE was 1075.8ng/ ⁇ l.
  • the above host cell CHO-K1 was inoculated into the medium M1 at a density of 7 ⁇ 10 5 cells/ml. After 24 hours, the host cells were counted, and the cells were diluted to 1.0 ⁇ 10 6 cells/ml with the pre-warmed medium M1, and then 5ml of the cell suspension was placed in a Kuhner shaker for later use.
  • the parameters of the shaker are: temperature 36.5°C, humidity 85%, carbon dioxide 6%, rotating speed 225rpm. Prepare for transfection, the specific steps are as follows:
  • the stable CHO-K1 cell line containing the optimized gE gene can be obtained by the above-mentioned transfection method.
  • media selection of mini cell populations and selection of monoclonal cells by the limiting dilution method are also well-known experimental methods. Through these methods, monoclonal cells with higher expression levels can be obtained, that is, the dominant CHO cell line.
  • the protein with a purity of 95% or more can be obtained Used as an antigen protein.
  • the above antigens and adjuvants were sucked into a recombinant shingles vaccine composition, and C57BL/6 mice were used as animal models to carry out immunization. Original research.
  • the specific method is: using gE protein as an antigen, using aluminum phosphate and CpG ODN as adjuvants to prepare a recombinant shingles vaccine composition.
  • C57BL/6 mice aged 6-8 weeks were selected to be randomly divided into groups, 10 mice in each group, the above vaccine composition was injected intramuscularly, and the vaccine group and adjuvant group were set up, 0 and 3 weeks were immunized, and the blood was collected in the 5th week.
  • the ELISA method was used to detect the antibody titer (ie total IgG) of the anti-VZV gE protein in the serum, and the ELISPOT method was used to detect the cellular immunity level in the spleen cells, mainly the expression of IFN- ⁇ .
  • the results show that the vaccine combination prepared by the gE protein obtained by the technical solution provided by the present invention has very good immunogenicity and can be used as a potential recombinant shingles vaccine candidate (specific results are shown in Table 1).
  • the immunogenicity evaluation method is a professional technical method in the field.
  • the more specific operation method is as follows:
  • C57BL/6 mice aged 6-8 weeks were selected to be randomly divided into groups, with 10 mice in each group.
  • Different doses of vaccine were injected intramuscularly (see Table 1 for the specific ratio) with an injection volume of 0.05ml; 0 and 3 weeks of immunization, blood was taken from the spleen at the 5th week, serum was separated for ELISA detection of antibodies, and splenic lymphocytes were separated for ELISPOT analysis.
  • the specific detection method can be taken as an example as follows:
  • the antigen gE stock solution is diluted with PBS to 1 ⁇ g/ml, and 100 ⁇ l of the diluted stock solution is added to each well of the ELISA plate. 4°C overnight. Washing machine.
  • Spleens were taken from each group of mice and lymphocytes were separated. The level of IFN- ⁇ expression in mouse spleen lymphocytes was measured by ELISPOT.
  • Coated ELISPOT plate (aseptic operation, performed one day before taking the spleen)
  • mice were sacrificed and immersed in 75% ethanol. Take out the mouse spleen in a clean bench. Put a burned 200-mesh copper mesh in a 35mm petri dish, add 1ml of lymphocyte separation solution, and grind with the plunger of a 1ml syringe. After filtering the suspension with spleen cells through a burned 200-mesh copper mesh, transfer to a 15ml centrifuge tube, add the lymphocyte separation solution to 4ml, and cover the surface with 0.5ml of RPMI1640 basic medium. Room temperature, 800g, speed 3, centrifugal 30min. Aspirate the lymphocyte layer, add 10ml RPMI1640 basic medium, wash, and centrifuge at 250g at room temperature for 10min. Discard the supernatant, add 2ml RPMI1640 complete medium to resuspend the cells, and count.
  • Add cells Dilute the cells with complete medium to 6 ⁇ 10 6 cells/ml according to the result of cell count, and add mAb CD28-A to the cell suspension diluted 1000 times. Add 100 ⁇ l/well to the ELISPOT plate. Positive control: 1 ⁇ l ConA stimulant was added, and the stimulation concentration was 5 ⁇ g/ml. Sample to be tested: add stimulus gE protein peptide library diluted with serum-free medium, final concentration 2 ⁇ g/ml; negative control: no ConA stimulus, nor stimulus short peptide. Incubate at 37°C with 5% CO 2 for 24 hours, during which time the culture plate cannot be moved to avoid changes in cell position and blur of ELISPOT spots.
  • Discard the cell suspension add 200 ⁇ l/well of sterile PBS and wash the plate 5 times. Take 50 ⁇ l of biotin-labeled detection antibody and add it to 10 ml of diluent (PBS+0.1% BSA), mix well, and filter with 0.2 ⁇ m filter. Add 100 ⁇ l to each well and incubate at 37°C for 2h. Discard the diluent of the biotin-labeled detection antibody, add 200 ⁇ l/well of sterile PBS and wash the plate 5 times. Dilute the antibody with diluent (PBS+0.1% BSA), take 50 ⁇ l and add 10ml of diluent, mix well, and filter with 0.2 ⁇ m filter.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Virology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Abstract

L'invention concerne une protéine gE du virus varicelle-zona immunogène, et un gène codant et un produit associé de celle-ci. L'invention concerne en outre un procédé de préparation de ladite protéine gE immunogène.
PCT/CN2020/098017 2019-06-28 2020-06-24 Gène ge et vecteur pour exprimer un gène WO2020259558A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201910574557.9A CN112142828B (zh) 2019-06-28 2019-06-28 一种gE基因及表达该基因的载体
CN201910574557.9 2019-06-28

Publications (1)

Publication Number Publication Date
WO2020259558A1 true WO2020259558A1 (fr) 2020-12-30

Family

ID=73869213

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/098017 WO2020259558A1 (fr) 2019-06-28 2020-06-24 Gène ge et vecteur pour exprimer un gène

Country Status (2)

Country Link
CN (1) CN112142828B (fr)
WO (1) WO2020259558A1 (fr)

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114703205A (zh) * 2022-03-11 2022-07-05 上海博唯生物科技有限公司 一种疱疹病毒糖蛋白gE重组蛋白、疫苗、制备方法和应用
CN115819522B (zh) * 2022-11-19 2023-08-08 广州佰芮慷生物科技有限公司 一种带状疱疹病毒疫苗、表达蛋白及重组腺病毒制备与应用
CN116942808B (zh) * 2023-07-21 2024-02-02 北京成大天和生物科技有限公司 一种重组带状疱疹疫苗组合物及其制备方法和应用

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0405867A1 (fr) * 1989-06-27 1991-01-02 SMITHKLINE BEECHAM BIOLOGICALS s.a. Composés
WO2000043527A1 (fr) * 1999-01-20 2000-07-27 Smithkline Beecham Biologicals S.A. Vaccins contre le virus varicelle-zona
CN103028113A (zh) * 2005-03-03 2013-04-10 葛兰素史密丝克莱恩生物有限公司 疫苗
CN108992667A (zh) * 2018-08-09 2018-12-14 安徽智飞龙科马生物制药有限公司 一种带状疱疹疫苗及其制备方法、应用
CN109602901A (zh) * 2019-01-08 2019-04-12 成都迈科康生物科技有限公司 一种带状疱疹病毒疫苗及其制备方法和应用
CN110035772A (zh) * 2016-11-25 2019-07-19 财团法人牧岩生命科学研究所 水痘带状疱疹病毒疫苗
WO2020063370A2 (fr) * 2018-09-27 2020-04-02 武汉博沃生物科技有限公司 Composition immunitaire, son procédé de préparation et application associée
CN110996999A (zh) * 2017-08-16 2020-04-10 株式会社车疫苗研究所 包含插入有脂肽的脂质体作为有效成分的疫苗佐剂及其用途

Patent Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0405867A1 (fr) * 1989-06-27 1991-01-02 SMITHKLINE BEECHAM BIOLOGICALS s.a. Composés
WO2000043527A1 (fr) * 1999-01-20 2000-07-27 Smithkline Beecham Biologicals S.A. Vaccins contre le virus varicelle-zona
CN103028113A (zh) * 2005-03-03 2013-04-10 葛兰素史密丝克莱恩生物有限公司 疫苗
CN110035772A (zh) * 2016-11-25 2019-07-19 财团法人牧岩生命科学研究所 水痘带状疱疹病毒疫苗
CN110996999A (zh) * 2017-08-16 2020-04-10 株式会社车疫苗研究所 包含插入有脂肽的脂质体作为有效成分的疫苗佐剂及其用途
CN108992667A (zh) * 2018-08-09 2018-12-14 安徽智飞龙科马生物制药有限公司 一种带状疱疹疫苗及其制备方法、应用
WO2020063370A2 (fr) * 2018-09-27 2020-04-02 武汉博沃生物科技有限公司 Composition immunitaire, son procédé de préparation et application associée
CN109602901A (zh) * 2019-01-08 2019-04-12 成都迈科康生物科技有限公司 一种带状疱疹病毒疫苗及其制备方法和应用

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CHEN, ZHEWEN ET AL.: "Stable Expression of Varicella-zoster Virus Glycoprotein E in CHO Cells", CHINESE JOURNAL OF BIOLOGICALS, no. 8, 20 August 2008 (2008-08-20), ISSN: 1004-5503, DOI: 20200911140919X *
HAUMONT M ET AL.: "Purification, Characterization and Immunogenicity of Recombinant Varicella-Zoster Virus Glycoprotein gE Secreted by Chinese Hamster Ovary Cells", VIRUS RESEARCH, vol. 40, no. 2, 29 February 1996 (1996-02-29), XP055600497, ISSN: 0168-1702, DOI: 20200911140742X *
HAUMONT M ET AL.: "Purification, Characterization and Immunogenicity of Recombinant Varicella-zoster Virus Glycoprotein gE Secreted by Chinese Hamster Ovary Cells", VIRUS RESEARCH, vol. 40, no. 2, 29 February 1996 (1996-02-29), XP055600497, ISSN: 0168-1702, DOI: 20200911140745Y *
LI, SHICHONG ET AL.: "New Technology of Chinese Hamster Ovary Cells Expression System", LETTERS IN BIOTECHNOLOGY, vol. 20, no. 3, 30 May 2009 (2009-05-30), ISSN: 1009-0002, DOI: 20200911141035Y *

Also Published As

Publication number Publication date
CN112142828A (zh) 2020-12-29
CN112142828B (zh) 2022-02-01

Similar Documents

Publication Publication Date Title
Routhu et al. A modified vaccinia Ankara vector-based vaccine protects macaques from SARS-CoV-2 infection, immune pathology, and dysfunction in the lungs
WO2020259559A1 (fr) Mutant de protéine ge du virus varicelle-zona et son procédé d'expression
WO2020259558A1 (fr) Gène ge et vecteur pour exprimer un gène
JP6913747B2 (ja) イヌアデノウイルスベクター
WO2021249031A1 (fr) Levure de recombinaison orale pour exprimer la protéine s d'un nouveau coronavirus, préparation à cet effet et application associée
EA022213B1 (ru) Новые конструкции белка вируса папилломы человека (hpv) и их применение в предупреждении заболевания, вызываемого hpv
CN109182380B (zh) 杆状病毒表达的猪瘟e2亚单位疫苗的制备方法及应用
WO2020259609A1 (fr) Composition de vaccin anti-zona recombinant et son application
CN112375768A (zh) Covid-19冠状病毒的假病毒及其制备方法和应用
JP2018515141A (ja) 新規のバキュロウイルスベクター及び使用の方法
Qin et al. Identification of novel T-cell epitopes on infectious bronchitis virus N protein and development of a multi-epitope vaccine
Lee et al. Interaction of allergy history and antibodies to specific varicella‐zoster virus proteins on glioma risk
Escalante et al. Four decades of prophylactic EBV vaccine research: a systematic review and historical perspective
Yao et al. The construction and immunogenicity analyses of a recombinant pseudorabies virus with porcine circovirus type 3 capsid protein co-expression
WO2021042947A1 (fr) Conception et utilisation de vaccin à adn minicercle
WO2021098521A1 (fr) Procédé de préparation rapide de vaccin épidémique et de bronchite infectieuse
CN115819522B (zh) 一种带状疱疹病毒疫苗、表达蛋白及重组腺病毒制备与应用
Cai et al. Characterization of the duck plague virus UL35 gene
KR101111998B1 (ko) 이종성 요소가 없는 ｇＭ-음성 ＥＨＶ-돌연변이체
TWI490229B (zh) 豬瘟病毒封套醣蛋白Ｅｒｎｓ之特異性單株抗體ＣＷ８１３及其於間接三明治ＥＬＩＳＡ抗體檢測之應用
CN1283788C (zh) 表达猪繁殖与呼吸综合征病毒的重组伪狂犬病毒及应用
CN115887634B (zh) 鸡传染性支气管炎二价亚单位疫苗及应用
EP2230246A1 (fr) Particules similaires au virus 5 de glycoprotéine d'artérivirus
WO2020263109A1 (fr) Anticorps monoclonal de la glucoprotéine g du virus de la laryngotrachéite infectieuse aviaire et applications de celui-ci
Li et al. The CDE region of feline Calicivirus VP1 protein is a potential candidate subunit vaccine

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20832850

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20832850

Country of ref document: EP

Kind code of ref document: A1