CN116102627B - Expression of human papilloma virus HPV35L1 protein and viroid particle and preparation method thereof - Google Patents
Expression of human papilloma virus HPV35L1 protein and viroid particle and preparation method thereof Download PDFInfo
- Publication number
- CN116102627B CN116102627B CN202211702926.6A CN202211702926A CN116102627B CN 116102627 B CN116102627 B CN 116102627B CN 202211702926 A CN202211702926 A CN 202211702926A CN 116102627 B CN116102627 B CN 116102627B
- Authority
- CN
- China
- Prior art keywords
- protein
- buffer
- expression
- sequence
- hpv35l1
- Prior art date
- Legal status (The legal status 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 status listed.)
- Active
Links
- 230000014509 gene expression Effects 0.000 title claims abstract description 34
- 239000002245 particle Substances 0.000 title abstract description 9
- 238000002360 preparation method Methods 0.000 title abstract description 5
- 101000781698 Human papillomavirus 35 Major capsid protein L1 Proteins 0.000 title description 15
- 241000701806 Human papillomavirus Species 0.000 title description 5
- 241000726445 Viroids Species 0.000 title description 2
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 53
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 42
- 239000013604 expression vector Substances 0.000 claims abstract description 15
- 239000002773 nucleotide Substances 0.000 claims abstract description 9
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 9
- 230000009465 prokaryotic expression Effects 0.000 claims abstract description 8
- 238000000746 purification Methods 0.000 claims abstract description 7
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000013598 vector Substances 0.000 claims description 23
- 239000000872 buffer Substances 0.000 claims description 19
- 238000000034 method Methods 0.000 claims description 11
- 239000000243 solution Substances 0.000 claims description 8
- 239000006228 supernatant Substances 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 7
- 102000039446 nucleic acids Human genes 0.000 claims description 7
- 150000007523 nucleic acids Chemical class 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- 150000003839 salts Chemical class 0.000 claims description 6
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000002808 molecular sieve Substances 0.000 claims description 5
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 claims description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 241001052560 Thallis Species 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 3
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 3
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 3
- 238000011068 loading method Methods 0.000 claims description 3
- 239000008363 phosphate buffer Substances 0.000 claims description 3
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 claims description 2
- 239000007995 HEPES buffer Substances 0.000 claims description 2
- 239000007993 MOPS buffer Substances 0.000 claims description 2
- 239000007983 Tris buffer Substances 0.000 claims description 2
- 239000008351 acetate buffer Substances 0.000 claims description 2
- 238000005571 anion exchange chromatography Methods 0.000 claims description 2
- 238000004587 chromatography analysis Methods 0.000 claims description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 2
- 239000007788 liquid Substances 0.000 claims description 2
- 239000011780 sodium chloride Substances 0.000 claims description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical group OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 2
- 101000936049 Vibrio cholerae serotype O1 (strain ATCC 39315 / El Tor Inaba N16961) Outer membrane lipoprotein Blc Proteins 0.000 claims 3
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims 1
- 239000007979 citrate buffer Substances 0.000 claims 1
- 238000010828 elution Methods 0.000 claims 1
- 238000001556 precipitation Methods 0.000 claims 1
- 241000588724 Escherichia coli Species 0.000 abstract description 12
- 108020004705 Codon Proteins 0.000 abstract description 5
- 230000006872 improvement Effects 0.000 abstract description 4
- 238000005457 optimization Methods 0.000 abstract description 4
- 108700005307 Human papillomavirus HPV L1 Proteins 0.000 abstract description 2
- 208000022361 Human papillomavirus infectious disease Diseases 0.000 description 14
- 238000012163 sequencing technique Methods 0.000 description 13
- 150000001413 amino acids Chemical group 0.000 description 11
- 239000012634 fragment Substances 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 238000006243 chemical reaction Methods 0.000 description 8
- 230000003321 amplification Effects 0.000 description 7
- 210000004027 cell Anatomy 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 102000012410 DNA Ligases Human genes 0.000 description 5
- 108010061982 DNA Ligases Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000002296 dynamic light scattering Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 3
- 241000320412 Ogataea angusta Species 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 230000001351 cycling effect Effects 0.000 description 3
- 102000037865 fusion proteins Human genes 0.000 description 3
- 108020001507 fusion proteins Proteins 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 230000003902 lesion Effects 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 206010059313 Anogenital warts Diseases 0.000 description 2
- 108090000565 Capsid Proteins Proteins 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 101150066516 GST gene Proteins 0.000 description 2
- 101710125418 Major capsid protein Proteins 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000004899 c-terminal region Anatomy 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 239000012145 high-salt buffer Substances 0.000 description 2
- 239000002054 inoculum Substances 0.000 description 2
- 230000003211 malignant effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 210000004400 mucous membrane Anatomy 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- 101710192393 Attachment protein G3P Proteins 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 101710169873 Capsid protein G8P Proteins 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 108010070675 Glutathione transferase Proteins 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000341655 Human papillomavirus type 16 Species 0.000 description 1
- 241000701827 Human papillomavirus type 35 Species 0.000 description 1
- 101150075239 L1 gene Proteins 0.000 description 1
- 101710156564 Major tail protein Gp23 Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 208000009608 Papillomavirus Infections Diseases 0.000 description 1
- 241001631648 Polyomaviridae Species 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 208000007313 Reproductive Tract Infections Diseases 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000001339 epidermal cell Anatomy 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 206010020718 hyperplasia Diseases 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 208000024312 invasive carcinoma Diseases 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 230000036210 malignancy Effects 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- 230000003472 neutralizing effect Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007363 ring formation reaction Methods 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 230000010415 tropism Effects 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
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
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- C12N2710/20011—Papillomaviridae
- C12N2710/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- 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
- C12N2710/20011—Papillomaviridae
- C12N2710/20023—Virus like particles [VLP]
-
- 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
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Virology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the field of medical biology, in particular to expression of human papilloma virus L1 protein and viroid-like particles and a preparation method thereof. Truncating the amino acid sequence of HPV35 type L1 protein, carrying out codon optimization on the truncated protein coding nucleotide sequence to obtain an optimized coding nucleotide sequence, and finally matching with an unlabeled expression vector containing a specific SD sequence to realize unlabeled expression purification. The invention can obtain higher protein expression quantity in a prokaryotic expression system such as an escherichia coli expression system and obtain VLP with more uniform quality through the improvement.
Description
Technical Field
The invention relates to the field of medical biology, in particular to expression of human papilloma virus L1 protein and viroid-like particles and a preparation method thereof. More particularly relates to the construction and expression of human papillomavirus HPV35L1 protein VLP (virus-like particle).
Background
Human papillomavirus (human papilloma virus, HPV) is a non-enveloped, closed-loop, double-stranded DNA virus belonging to the subfamily polyomaviridae of papovaviridae, which mainly invades the epithelial mucosal tissue of the human body, thereby inducing various benign and malignant proliferative lesions. Over 200 types of HPV have been identified at present, HPV infection has obvious tissue specificity, different types of HPV have different tropisms to skin and mucous membrane, different papillary lesions can be induced, about 30 types of HPV are associated with genital tract infection, and about 20 types of HPV are associated with tumors.
HPV can be broadly divided into two categories, depending on the benign or malignant nature of the HPV-induced lesions: 1) High risk types (e.g., HPV16, HPV18, HPV31, HPV33, HPV35, HPV39, HPV45, HPV51, HPV52, HPV56, HPV58, HPV59, HPV35, etc.): high-risk HPV is closely related to human multiple tissue malignancies, mainly causing severe atypical hyperplasia and invasive carcinoma; 2) Low risk types (e.g., HPV6, HPV11, HPV40, HPV42, HPV43, HPV44, HPV54, HPV72, HPV81, etc.): low-risk HPV can cause benign proliferative diseases of epidermal cells, such as condyloma acuminatum and condyloma plana. HPV is mainly composed of viral envelope and genomic DNA. The genome is about 7900bp long, and 8 viral protein coding genes exist. Among them, 6 proteins encoded by ORFs are expressed in the early stages of viral replication, called early proteins; the 2 ORF-encoded proteins are expressed in the late stages of viral replication, termed late stage proteins. Late proteins include major coat protein L1 and minor coat protein L2, and are involved in the formation of viral coat. The HPV viral coat protein can be self-assembled, and in the patent literature, a yeast expression system or an insect expression system or an L1 protein expressed independently in a mammalian cell expression system or an L1 protein and an L2 protein are co-expressed to form a virus-like particle (VLP), and after immunization with VLP produced by an exogenous expression system, a neutralizing antibody can be induced in vivo, so that a good immune protection effect is obtained. However, the use of eukaryotic expression systems for direct expression of assembled VLPs in vivo is not very uniform in the nature of VLP production and the cost of eukaryotic expression systems is high and is not conducive to industrialization.
At present, aiming at HPV type 35, reports are made in CN202110442669.6, wherein an HPV35L1 protein is produced by using a Hansenula polymorpha expression system, the Hansenula polymorpha expression system is a eukaryotic expression system, VLP is directly assembled in vivo, and whether a qualified standard protein can be normally expressed in an escherichia coli prokaryotic expression system is not suggested in the patent, and the escherichia coli prokaryotic expression system does not have the functions of post-translational modification and the like of the Hansenula polymorpha expression system, so that the expression of HPV35L1 in the prokaryotic expression system is difficult to a certain extent. Thus, there is a need to address the problem of difficulties in expressing HPV35L1 protein within prokaryotic expression systems to obtain more uniform VLPs and lower costs for industrial applications.
Disclosure of Invention
The inventor aims at expressing HPV35L1 protein in a prokaryotic expression system based on the cost of vaccine finished products, and solves the problem that the HPV35L1 protein is difficult to express in the prokaryotic expression system. The method is realized by the following improvement: truncating the amino acid sequence of HPV35 type L1 protein, carrying out codon optimization on the truncated protein coding nucleotide sequence to obtain an optimized coding nucleotide sequence, and finally matching with a label-free expression vector containing a specific SD sequence to realize efficient expression and purification.
Firstly, the amino acid sequence (shown as SEQ ID NO: 3) of HPV35L1 protein is subjected to N/C end truncation treatment, so as to obtain better protein expression rate. The N-terminal truncation is no more than 10 amino acids, preferably 4 amino acids. The C-terminal truncation is not more than 30 amino acids, preferably 28 amino acids, and the specific truncated amino acids are set forth in SEQ ID NO:1. after N/C-terminal truncation treatment, the protein and VLP of higher quality are expressed and obtained on the unlabeled expression vector.
Wherein SEQ ID NO:1 as follows:
1 MSNEATVYLP PVSVSKVVST DEYVTRTNIY YHAGSSRLLA VGHPYYAIKK
51 QDSNKIAVPK VSGLQYRVFR VKLPDPNKFG FPDTSFYDPA SQRLVWACTG
101 VEVGRGQPLG VGISGHPLLN KLDDTENSNK YVGNSGTDNR ECISMDYKQT
151 QLCLIGCRPP IGEHWGKGTP CNANQVKAGE CPPLELLNTV LQDGDMVDTG
201 FGAMDFTTLQ ANKSDVPLDI CSSICKYPDY LKMVSEPYGD MLFFYLRREQ
251 MFVRHLFNRA GTVGETVPAD LYIKGTTGTL PSTSYFPTPS GSMVTSDAQI
301 FNKPYWLQRA QGHNNGICWS NQLFVTVVDT TRSTNMSVCS AVSTSDSTYK
351 NDNFKEYLRH GEEYDLQFIF QLCKITLTAD VMTYIHSMNP SILEDWNFGL
401 TPPPSGTLED TYRYVTSQAV TCQKPSAPKP KDDPLKNYTF WEVDLKEKFS
451 ADLDQFPLGR KFLLQAGLKA。
secondly, in order to efficiently express HPV35L1 protein using the escherichia coli system, the inventors have identified the sequence according to SEQ ID NO:1, and codon optimization of the nucleotide sequence is performed on an escherichia coli system. The optimization principle comprises the following steps: a) Selecting codons with highest or higher use frequency according to a use frequency table of the escherichia coli genetic code; b) The usual restriction enzyme recognition sites are eliminated. The optimized nucleotide sequence is obtained through the principle and multiple screening, and the optimized nucleotide sequence is shown as SEQ ID NO:2, and further provides expression cassettes, expression vectors and recombinant host cells containing said coding nucleic acids. Preferably, it is E.coli.
Finally, the invention provides a label-free expression vector of a specific SD sequence. For the expression vector, the vector pGEX for expressing the fusion protein is characterized in that a 26kDa glutathione S-transferase Gene (GST) is connected to the vector, and compared with other fusion vectors, the vector has the characteristics of mild purification condition, simple steps, no addition of denaturing agents and capability of maximally maintaining the spatial conformation and immunogenicity of the purified protein; the GST fusion protein tag has good application value, but the GST fusion protein tag coded by the vector pGEX can increase the potential safety hazard of medicinal protein products. In contrast, the GST tag of the vector is removed, and the SD sequence capable of efficiently expressing the HPV35 type L1 protein is replaced, so that a novel expression vector suitable for the HPV35L1 protein is formed. Among them, the SD sequence is preferably AGGAGATATA (5 'to 3').
The invention also provides a method for preparing the HPV35 type L1 VLP, which is characterized by comprising the following steps: according to the step of HPV35 type L1 protein obtained by the method, the pH and salt concentration of a buffer solution in which the HPV35 type L1 protein is positioned are regulated, so that the HPV35 type L1 protein is self-assembled to form VLP.
Preferably, the buffer includes, but is not limited to, tris buffer, phosphate buffer, acetate buffer, HEPES buffer, MOPS buffer, citric acid buffer, histidine buffer, boric acid buffer, preferably phosphate buffer;
the pH of the buffer is 4.75-5.25, the salt concentration is 2.0-4.0M, preferably pH4.75, pH5.0, pH5.25; wherein the salt concentration is between 2.0 and 4.0M, preferably 2.0M,2.5M,3.0M,3.5M,4.0M;
the invention can obtain higher protein expression in a prokaryotic, such as escherichia coli expression system and obtain VLPs with more uniform quality through the improvement.
Drawings
FIG. 1 XA90 pKL1-HPV35L1 vial expression electrophoresis detection results. Wherein M is marker; XA90pKL1 negative control; XA90 pKL1-HPV35L1-1 whole bacteria; XA90 pKL1-HPV35L1-1 supernatant; XA90 pKL1-HPV35L1-1 precipitate; 5.HPV18L1;6.XA90 pKL1-HPV35L1-2 whole bacteria; XA90 pKL1-HPV35L1-2 supernatant; XA90 pKL1-HPV35L1-2 precipitate.
FIG. 2 shows the result of HPV35L1 pentamer electrophoresis detection obtained by purification. Wherein M is marker; hpv35l1 pentamer.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Embodiment one: construction of a Label-free expression vector containing specific SD sequences
1. NdeI cleavage site was introduced into pGEX-6P-2 plasmid by mutation PCR:
the PCR primer names and sequences were as follows:
forward primer: 6p1-Ndeimut-F (5 'to 3'):
ATTTCA CACAGG AAACAG TACATA TGTCCC CTATAC TAGGTT ATTGGA AAATTA AG;
reverse primer: 6p1-NdeIMut-R sequence (5 'to 3'):
ATAACC TAGTAT AGGGGA CATATG TACTGT TTCCTG TGTGAA ATTGTT ATCC。
the PCR reaction system is as follows: 5 Xphusion HF buffer 10. Mu.L, ddH 2 O30.5. Mu.L, 10mM dNTP 2. Mu.L, 6 PNE-SDm-F1. Mu.L, 6 PNE-SDm-R1. Mu.L, pGEX-6P-2 (20-fold dilution) 5. Mu.L, phusion HF Enzyme 0.5.5. Mu.L.
PCR reaction program setting: 3min at 95 ℃;95℃1min,55℃1min,72℃10 min; cycling for 20 times; 15min at 72 ℃.
The PCR product was digested with DpnI and transformed into DH 5. Alpha. Host bacteria, and cultured overnight to obtain a monoclonal colony. And (3) performing amplification culture on the monoclonal colony, sequencing a vector sequence in the monoclonal colony by a professional gene sequencing company, selecting a clone with a correct sequencing result, and performing cloning amplification and plasmid extraction on the clone to obtain a vector successfully introduced into NdeI restriction enzyme sites.
2. Designing mutation PCR primer for replacing SD sequence, and replacing SD sequence of original carrier by PCR method
Primer information is as follows:
6PNE-SDm-F(5'to3'):CAATTTCACACAGGAGATATACATATGTCCCCTATACTAGG
6PNE-SDm-R(5'to3'):GTATAGGGGACATATGTATATCTCCTGTGTGAAATTGTTATCC
the PCR reaction system is as follows: 10. Mu.L of 5 Xphusion HF buffer,ddH 2 O30.5. Mu.L, 10mM dNTP 2. Mu.L, 6 PNE-SDm-F1. Mu.L, 6 PNE-SDm-R1. Mu.L, 5. Mu.L of plasmid obtained in step 1.1, phusion HF Enzyme 0.5.5. Mu.L.
PCR reaction program setting: 3min at 95 ℃;95 ℃ for 1min,55 ℃ for 1min and 72 ℃ for 10 min; cycling for 20 times; 15min at 72 ℃.
The PCR product was digested with DpnI and transformed into E.coli DH 5. Alpha. And cultured overnight to obtain monoclonal colonies. And (3) performing amplification culture on the monoclonal colony, sequencing the vector sequence in the monoclonal colony by a professional gene sequencing company, selecting a clone with a correct sequencing result, and performing cloning and propagation on the clone and extracting plasmids from the clone to obtain the vector with the SD sequence replaced successfully. The SD sequence after substitution was AGGAGATATA (5 'to 3').
3. Vector was digested with NdeI and BamHI to remove GST gene
The enzyme digestion system is as follows: cutsmart buffer 3. Mu.l, ddH 2 O3. Mu.l, 1.2. Mu.l of the obtained vector, ndeI 2. Mu.l, bamHI 2. Mu.l.
Enzyme cutting at 37 ℃ for 2 hours; 0.8% agarose gel electrophoresis, 120V,1h; and (3) cutting gel to obtain a carrier fragment with GST genes removed, corresponding to the electrophoresis band, and preserving at 4 ℃.
The carrier fragment was recovered by using an agarose gel recovery kit, and 3. Mu.l of the obtained carrier fragment was subjected to electrophoresis detection and recovery. Then the double enzyme cutting product is used for filling the sticky end by DNA polymerase I, and the reaction system is as follows: 10 XT 4 DNA ligase buffer 2.5. Mu.l, ddH 2 O1.8. Mu.l, gel recovered enzyme-cleaved vector fragment 20. Mu.l, 10mM dNTP 0.2. Mu.l, DNA polymerase I0.5. Mu.l, reacted at 25℃for 15min, EDTA (final concentration of EDTA 10 mM) was added and heated at 75℃for 20min to terminate the reaction.
And (3) carrying out religation cyclization on the carrier subjected to enzyme digestion and end-complemented, wherein a connection system is as follows: 2. Mu.l of T4 DNA ligase buffer, 16. Mu.l of linear blunt end vector fragment, 2. Mu.l of T4 DNA ligase, and 4h at 16 ℃.
The ligation product was digested and transformed into E.coli DH 5. Alpha. And cultured overnight to obtain monoclonal colonies. And (3) performing amplification culture on the monoclonal colony, sequencing a vector sequence in the monoclonal colony by a professional gene sequencing company, selecting a clone with a correct sequencing result, performing cloning amplification and plasmid extraction on the clone, and obtaining a vector for successfully replacing the SD sequence and removing the GST gene.
PCR amplified plasmid, reintroduced NdeI and BamHI cleavage sites
The PCR primers were as follows:
6PNE-SDm-noG-F (5'to3'):CAGGAGATATACATATGGGATCCCCGGAATTCCCG
6PNE-SDm-noG-R (5'to3'):GAATTCCGGGGATCCCATATGTATATCTCCTGTGTG
the PCR reaction system is as follows: 10. Mu.L of 5 Xphusion HF buffer, 30.5. Mu.L of ddH2O, 2. Mu.L of 10mM dNTP, 6 PNE-SDm-noG-F1. Mu.L, 6 PNE-SDm-noG-R1. Mu.L, 5. Mu.L of template plasmid, phusion HF Enzyme 0.5.5. Mu.L.
PCR reaction program setting: 3min at 95 ℃;95 ℃ for 1min,55 ℃ for 1min and 72 ℃ for 10 min; cycling for 20 times; 15min at 72 ℃.
The PCR product was digested with DpnI and transformed into E.coli DH 5. Alpha. And cultured overnight to obtain monoclonal colonies. The monoclonal colony is subjected to amplification culture, then the vector sequence in the monoclonal colony is sequenced by a professional gene sequencing company, clones with correct sequencing results are selected, then the clones are amplified and plasmids are extracted from the clones, the SD sequence is successfully replaced, GST genes are removed, and NdeI and BamHI vectors are reintroduced. Thus, the vector pKL1 was constructed.
Embodiment two: construction of expression vector containing codon optimized HPV35L1 Gene
The truncated sequence of human papillomavirus type 35 coat protein L1 (HPV 35L 1) gene is synthesized artificially, the sequence is shown as SEQ ID No.2 (the coded amino acid sequence is shown as SEQ ID NO.1, the N end of the truncated sequence is truncated by 4 amino acids and the C end of the truncated sequence is truncated by 28 amino acids relative to the wild type amino acid sequence shown as SEQ ID NO. 3), the DNA fragment of HPV35L1 is amplified by PCR, the L1 gene PCR fragment containing NdeI and Xho1 cleavage sites and the recombinant vector are respectively subjected to NdeI/Xho1 double cleavage, and then the recovered gene fragment and pKL1 containing the corresponding cohesive end are subjected to ligation reaction by using T4 DNA ligase, and the temperature is 10-15 h. The connection system is as follows: 6 μl of pKL1 vector fragment, HPV35L1 gene fragment2. Mu.l, 1. Mu.l of T4 DNA ligase buffer. Conversion of ligation products to ligation products after ligation reactionsE. coli Screening of recombinants was performed in DH 5. Alpha. And (3) performing amplification culture on the screened monoclonal colony, extracting plasmids, and then performing sequencing verification to obtain a recombinant expression vector pKL1-HPV35L1.
Embodiment III: expression of HPV35L1 protein
The recombinant vector pKL1-HPV35L1 with correct sequencing result in the second example is transformed into E.coli XA90 host cell, and is used as engineering bacteria for expressing recombinant protein to express HPV L1 protein. 0.05% of the inoculum size was inoculated into LB medium (Amp+) and cultured at 37℃and 220rpm for 16 hours for activation. Inoculating the activated bacterial liquid into a 2YT culture medium according to the inoculum size of 0.5%, culturing at 30 ℃ for 7h at 220rpm, adding IPTG with the final concentration of 0.2mM, performing induction culture at 30 ℃ for 16h at 220rpm, ending fermentation, and centrifugally collecting bacterial bodies for expression amount detection and purification experiments. From the result of SDS-PAGE, the protein after technical scheme improvement is soluble and efficiently expressed, the molecular weight of HPV35L1 protein is about 52.49 kDa, and the specific result is shown in FIG. 1.
As can be seen in FIG. 1, XA90pKL1 is a negative control. The test sample lanes showed a distinct expression band at a position corresponding to the theoretical molecular weight size of the protein of interest (about 52.49 kDa) compared to the negative control. The "-1" and "-2" after the sample names represent parallel 1 and parallel 2. The darkest color and the thickest band in the figure are the target protein bands. The expression level of the target protein unit cell is about 0.4mg/g wet cell.
Embodiment four: HPV35L1 protein VLP purification and assembly
Taking a proper amount of thalli according to the mass volume ratio of 1:10, fully re-suspending the bacterial cells by using a bacteria breaking buffer (20 mM PB,20 mM DTT,pH8.0), and then crushing the bacterial cells by using a high-pressure homogenizer under the following conditions: 800 bar,3 times. The cell disruption solution was then centrifuged at high speed (4 ℃,12000 rpm,60 min) to collect the supernatant. The supernatant was further precipitated by ammonium sulfate with a saturation of 30%, and the precipitate was collected by centrifugation (4 ℃,12000 rpm,60 min) at a mass to volume ratio of 1:10, and re-centrifuging (at 4 ℃ C., 12000 rpm,60 min) after fully re-dissolving with a re-dissolving buffer (20 mM PB,20 mM DTT,pH8.0), and collecting the supernatant to obtain a crude pure solution. The crude pure solution is firstly loaded to carry out Superdex200 molecular sieve chromatography, and the molecular sieve buffer solution (20 mM PB,20 mM DTT,pH8.0) collects the components of the L1 target protein according to the peak position. And then loading the molecular sieve collected sample to perform Source15Q anion exchange chromatography (SQ low-salt buffer solution: 5 mM PB,10 mM DTT,pH8.0,SQ high-salt buffer solution: 5 mM PB,1M NaCl,10 mM DTT,pH8.0), and linearly eluting and collecting components of the L1 target protein by 10 column volumes through 0-20% high-salt buffer solution, wherein the components are the purified L1 protein. The mass of the L1 pentamer was determined by Dynamic Light Scattering (DLS). Finally, the pH and salt concentration of the buffer solution in which the L1 protein is positioned are adjusted to self-assemble to form VLPs, and the preparation of the VLPs is completed. Finally, the quality of VLPs was determined by DLS.
TABLE 1 DLS detection results before and after HPV35L1 protein Assembly
As can be seen from the table, HPV35L1 has a good pentameric state of PdI.ltoreq.0.1. Particle size of VLP: 45 The particle size of nm is less than or equal to 75 nm, the PdI is less than or equal to 0.1, and the VLPs with good assembly and formation states can be effectively assembled.
Fifth embodiment: long-term stability investigation
The HPV35L1 protein VLP prepared in example 4 above was taken and examined for long-term stability data at-70 ℃ as follows.
It can be seen that after long-term examination for 9 months, the appearance, pH value, VLP average particle size and dispersion coefficient, purity, in vitro potency and the like of the antigen protein have no obvious change, and the antigen protein is quite stable (equivalent to a control in 0 month).
Claims (15)
1. A truncated HPV35 type L1 protein is characterized in that the amino acid sequence of the truncated HPV35 type L1 protein is shown in SEQ ID NO. 1.
2. A nucleic acid encoding the truncated HPV type 35L1 protein of claim 1.
3. The nucleic acid of claim 2, wherein the nucleotide sequence is set forth in SEQ ID No. 2.
4. A nucleic acid consisting of an SD sequence and a nucleic acid according to claim 2 or 3, the nucleotide sequence of said SD sequence being 5 '-AGGAGGAATTA-3'.
5. An expression cassette or expression vector comprising the nucleic acid of claim 4.
6. The expression cassette or expression vector of claim 5, which is a prokaryotic expression vector.
7. The expression cassette or expression vector of claim 6, wherein the GST tag sequence is removed based on the vector pGEX and the nucleic acid of claim 4 is integrated.
8. A recombinant host cell comprising the expression cassette or expression vector of any one of claims 5 to 7.
9. The recombinant host cell of claim 8, which is e.
10. A method of expressing the truncated HPV type 35L1 protein of claim 1, wherein the recombinant host cell of claim 8 or 9 is cultured to produce HPV type 35L1 protein, and the purification step.
11. The method of claim 10, wherein the purifying step is: taking thalli of the recombinant host cells, fully re-suspending the thalli by using a bacteria breaking buffer solution, then crushing the thalli at high pressure by using a high-pressure homogenizer, and centrifugally collecting supernatant; the supernatant is further precipitated by ammonium sulfate, the final saturation of ammonium sulfate is 30%, and the supernatant is centrifugally collected again after precipitation and redissolution to obtain crude pure liquid;
loading the crude pure solution to perform Superdex200 molecular sieve chromatography, and collecting the components of the L1 target protein according to the peak position of the L1 target protein;
and then loading a molecular sieve collected sample to perform Source15Q anion exchange chromatography, and collecting components of the L1 target protein by NaCl linear elution to obtain HPV35 type L1 protein.
12. A method for preparing HPV type 35L1 protein VLPs, comprising the steps of: the process of HPV type 35L1 protein obtained according to claim 10 or 11, wherein the pH of the buffer is adjusted to between 4.75 and 5.25 and the salt concentration is between 2.0 and 4.0M to self-assemble the HPV type 35L1 protein VLP.
13. The method of claim 12, wherein the buffer is selected from Tris buffer, phosphate buffer, acetate buffer, HEPES buffer, MOPS buffer, citrate buffer, histidine buffer, borate buffer.
14. The method of claim 13, wherein the buffer has a pH of 4.75, pH5.0, pH5.25; the salt concentration was 2.0M,2.5M,3.0M,3.5M and 4.0M.
15. The method of claim 14, further comprising the step of purifying the resulting HPV type 35L1 protein VLPs.
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211702926.6A CN116102627B (en) | 2022-12-28 | 2022-12-28 | Expression of human papilloma virus HPV35L1 protein and viroid particle and preparation method thereof |
PCT/CN2023/138110 WO2024140161A1 (en) | 2022-12-28 | 2023-12-12 | Expression of human papilloma virus hpv35 l1 protein, virus-like particles, and preparation method therefor |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202211702926.6A CN116102627B (en) | 2022-12-28 | 2022-12-28 | Expression of human papilloma virus HPV35L1 protein and viroid particle and preparation method thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN116102627A CN116102627A (en) | 2023-05-12 |
CN116102627B true CN116102627B (en) | 2023-10-24 |
Family
ID=86257408
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202211702926.6A Active CN116102627B (en) | 2022-12-28 | 2022-12-28 | Expression of human papilloma virus HPV35L1 protein and viroid particle and preparation method thereof |
Country Status (2)
Country | Link |
---|---|
CN (1) | CN116102627B (en) |
WO (1) | WO2024140161A1 (en) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116102627B (en) * | 2022-12-28 | 2023-10-24 | 北京康乐卫士生物技术股份有限公司 | Expression of human papilloma virus HPV35L1 protein and viroid particle and preparation method thereof |
CN116200416B (en) * | 2023-02-15 | 2024-03-12 | 北京康乐卫士生物技术股份有限公司 | Construction and application of plasmid expression vector based on Tac promoter |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106795518A (en) * | 2014-06-19 | 2017-05-31 | 科罗拉多大学董事会,法人 | Human papilloma virus's construct |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101245099A (en) * | 2007-02-14 | 2008-08-20 | 马润林 | Amino acid sequence of recombined human papilloma virus L1 capsid protein and uses thereof |
CN109251236B (en) * | 2017-07-14 | 2021-09-28 | 厦门大学 | Mutant of human papilloma virus 35 type L1 protein |
WO2021013070A1 (en) * | 2019-07-19 | 2021-01-28 | 神州细胞工程有限公司 | Chimeric human papillomavirus 35 type l1 protein |
CN112680462B (en) * | 2020-12-29 | 2022-02-22 | 上海博唯生物科技有限公司 | Human papilloma virus 35/HPV 35 type L1/L2 and preparation and application thereof |
CN113106107A (en) * | 2021-04-23 | 2021-07-13 | 重庆博唯佰泰生物制药有限公司 | Polynucleotide for expressing HPV35L1, expression vector, host cell and application thereof |
CN116102627B (en) * | 2022-12-28 | 2023-10-24 | 北京康乐卫士生物技术股份有限公司 | Expression of human papilloma virus HPV35L1 protein and viroid particle and preparation method thereof |
-
2022
- 2022-12-28 CN CN202211702926.6A patent/CN116102627B/en active Active
-
2023
- 2023-12-12 WO PCT/CN2023/138110 patent/WO2024140161A1/en unknown
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106795518A (en) * | 2014-06-19 | 2017-05-31 | 科罗拉多大学董事会,法人 | Human papilloma virus's construct |
Also Published As
Publication number | Publication date |
---|---|
CN116102627A (en) | 2023-05-12 |
WO2024140161A1 (en) | 2024-07-04 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN115960178B (en) | Expression of human papilloma virus HPV59L1 protein, viroid particle and preparation method thereof | |
CN116102627B (en) | Expression of human papilloma virus HPV35L1 protein and viroid particle and preparation method thereof | |
CN116041444B (en) | Expression of human papilloma virus HPV39L1 protein, viroid particle and preparation method thereof | |
WO2024088087A1 (en) | Expression of human papillomavirus (hpv) 68 l1 protein, and virus-like particles and preparation method therefor | |
US7744892B2 (en) | Optimized expression of HPV 52 L1 in yeast | |
US10537629B2 (en) | Truncated L1 protein of human papillomavirus type 11 | |
JP2007507207A5 (en) | ||
CA2519112A1 (en) | Optimized expression of hpv 31 l1 in yeast | |
CA2543928A1 (en) | Optimized expression of hpv 58 l1 in yeast | |
MXPA06003457A (en) | Optimized expression of hpv 45 l1 in yeast. | |
US9745351B2 (en) | Truncated L1 protein of human papillomavirus type 6 | |
US9738691B2 (en) | Truncated L1 protein of human papillomavirus type 58 | |
WO2024146613A1 (en) | Human papillomavirus 56-type l1 protein mutant, method for reducing recombinant protein degradation, and application | |
CA2339324A1 (en) | Protein delivery system using human papillomavirus virus-like particles | |
CN104045696A (en) | Recombinant human papilloma virus 16L1 protein and its use | |
CN116200416B (en) | Construction and application of plasmid expression vector based on Tac promoter | |
RU2546242C1 (en) | RECOMBINANT STRAIN OF YEAST Hansenula polymorpha - PRODUCER OF MAJOR CAPSID PROTEIN L1 OF HUMAN PAPILLOMAVIRUS OF TYPE 18 | |
CN104045695A (en) | Recombinant human papilloma virus 18L1 protein and its use | |
RU2546241C1 (en) | RECOMBINANT STRAIN OF YEAST Hansenula polymorpha - PRODUCER OF MAJOR CAPSID PROTEIN L1 OF HUMAN PAPILLOMAVIRUS OF TYPE 16 | |
RU2546240C1 (en) | RECOMBINANT STRAIN OF YEAST Hansenula polymorpha - PRODUCER OF MAJOR CAPSID PROTEIN L1 OF HUMAN PAPILLOMAVIRUS OF TYPE 56 | |
RU2681174C1 (en) | Method of obtaining a recombinant vaccine for prevention of humanpapilloma virus infection, recombinant vaccine | |
RU2675471C1 (en) | Recombinant yeast strain hansenula polymorpha producing the main capsid protein l1 of human papillomavirus type 6 | |
TH108752A (en) | Optimized expression of HPV 52 L1 in yeast. | |
TH82139A (en) | Optimized Expression of HPV 45L1 in Yeast | |
TH72490B (en) | Optimized Expression of HPV 45L1 in Yeast |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |