CN116445310A - Double-adhesion-antibacterial peptide co-expression recombinant yeast and application thereof - Google Patents
Double-adhesion-antibacterial peptide co-expression recombinant yeast and application thereof Download PDFInfo
- Publication number
- CN116445310A CN116445310A CN202310336046.XA CN202310336046A CN116445310A CN 116445310 A CN116445310 A CN 116445310A CN 202310336046 A CN202310336046 A CN 202310336046A CN 116445310 A CN116445310 A CN 116445310A
- Authority
- CN
- China
- Prior art keywords
- faeg
- fanc
- recombinant yeast
- antibacterial peptide
- adhesion
- 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.)
- Pending
Links
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 65
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 65
- 230000004186 co-expression Effects 0.000 title claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 49
- 210000004027 cell Anatomy 0.000 claims abstract description 38
- 239000013612 plasmid Substances 0.000 claims abstract description 33
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 28
- 230000014509 gene expression Effects 0.000 claims abstract description 24
- 241000235058 Komagataella pastoris Species 0.000 claims abstract description 16
- 238000000855 fermentation Methods 0.000 claims abstract description 13
- 230000004151 fermentation Effects 0.000 claims abstract description 13
- 238000004873 anchoring Methods 0.000 claims abstract description 12
- 238000010276 construction Methods 0.000 claims abstract description 12
- 238000000034 method Methods 0.000 claims abstract description 12
- 241001506991 Komagataella phaffii GS115 Species 0.000 claims abstract description 8
- 230000003248 secreting effect Effects 0.000 claims abstract description 8
- 238000000338 in vitro Methods 0.000 claims abstract description 5
- 210000002421 cell wall Anatomy 0.000 claims abstract description 4
- 230000008569 process Effects 0.000 claims abstract description 3
- 241000894006 Bacteria Species 0.000 claims description 29
- 235000018102 proteins Nutrition 0.000 claims description 21
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 11
- 101000798100 Bos taurus Lactotransferrin Proteins 0.000 claims description 10
- 229940072440 bovine lactoferrin Drugs 0.000 claims description 10
- 238000012258 culturing Methods 0.000 claims description 8
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 7
- 230000004927 fusion Effects 0.000 claims description 6
- 230000028327 secretion Effects 0.000 claims description 6
- 108700042778 Antimicrobial Peptides Proteins 0.000 claims description 5
- 102000044503 Antimicrobial Peptides Human genes 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 5
- 108010084455 Zeocin Proteins 0.000 claims description 5
- 230000000688 enterotoxigenic effect Effects 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 claims description 5
- 230000008827 biological function Effects 0.000 claims description 4
- 238000010367 cloning Methods 0.000 claims description 4
- 239000003814 drug Substances 0.000 claims description 3
- 238000001976 enzyme digestion Methods 0.000 claims description 3
- 108020004705 Codon Proteins 0.000 claims description 2
- 230000030609 dephosphorylation Effects 0.000 claims description 2
- 238000006209 dephosphorylation reaction Methods 0.000 claims description 2
- 230000029087 digestion Effects 0.000 claims description 2
- 238000005457 optimization Methods 0.000 claims description 2
- 230000000968 intestinal effect Effects 0.000 abstract description 10
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 9
- 108010037896 heparin-binding hemagglutinin Proteins 0.000 abstract description 7
- 230000001131 transforming effect Effects 0.000 abstract 1
- 239000000725 suspension Substances 0.000 description 24
- 230000001580 bacterial effect Effects 0.000 description 22
- 241000699670 Mus sp. Species 0.000 description 17
- 239000000243 solution Substances 0.000 description 17
- 206010012735 Diarrhoea Diseases 0.000 description 12
- 239000006228 supernatant Substances 0.000 description 12
- 208000015181 infectious disease Diseases 0.000 description 11
- 241000699666 Mus <mouse, genus> Species 0.000 description 9
- 239000001963 growth medium Substances 0.000 description 8
- 239000003651 drinking water Substances 0.000 description 7
- 235000020188 drinking water Nutrition 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000003242 anti bacterial agent Substances 0.000 description 6
- 229940088710 antibiotic agent Drugs 0.000 description 6
- 244000052616 bacterial pathogen Species 0.000 description 6
- 239000000203 mixture Substances 0.000 description 6
- 239000013642 negative control Substances 0.000 description 6
- 239000013641 positive control Substances 0.000 description 6
- 241001465754 Metazoa Species 0.000 description 5
- 210000003608 fece Anatomy 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 210000001630 jejunum Anatomy 0.000 description 5
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 4
- 102000010911 Enzyme Precursors Human genes 0.000 description 4
- 108010062466 Enzyme Precursors Proteins 0.000 description 4
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000600 sorbitol Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 210000005253 yeast cell Anatomy 0.000 description 4
- BRZYSWJRSDMWLG-DJWUNRQOSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2s,3r,4r,5s,6r)-3-amino-4,5-dihydroxy-6-[(1r)-1-hydroxyethyl]oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H]([C@@H](C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-DJWUNRQOSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 108010019160 Pancreatin Proteins 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000006285 cell suspension Substances 0.000 description 3
- 230000002354 daily effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 229940088598 enzyme Drugs 0.000 description 3
- 244000144972 livestock Species 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 229940055695 pancreatin Drugs 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 229960005486 vaccine Drugs 0.000 description 3
- 230000004580 weight loss Effects 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108700010070 Codon Usage Proteins 0.000 description 2
- 206010059866 Drug resistance Diseases 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 238000011049 filling Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000007358 intestinal barrier function Effects 0.000 description 2
- 230000007413 intestinal health Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 102000004196 processed proteins & peptides Human genes 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 210000000813 small intestine Anatomy 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 208000024891 symptom Diseases 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 1
- 238000011725 BALB/c mouse Methods 0.000 description 1
- 102000012983 Fanconi Anemia Complementation Group Proteins Human genes 0.000 description 1
- 108010079804 Fanconi Anemia Complementation Group Proteins Proteins 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- 102000012750 Membrane Glycoproteins Human genes 0.000 description 1
- 108010090054 Membrane Glycoproteins Proteins 0.000 description 1
- 241000283903 Ovis aries Species 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 206010034133 Pathogen resistance Diseases 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 101150045735 cinB gene Proteins 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000009194 climbing Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000003235 crystal violet staining Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000000556 factor analysis Methods 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000010820 immunofluorescence microscopy Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 210000004347 intestinal mucosa Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000000155 melt Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000004080 punching Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 208000026775 severe diarrhea Diseases 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Classifications
-
- 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
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
-
- 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
- A61K39/02—Bacterial antigens
- A61K39/025—Enterobacteriales, e.g. Enterobacter
- A61K39/0258—Escherichia
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- 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
- A61P31/04—Antibacterial agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
- C07K14/245—Escherichia (G)
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
-
- 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
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- 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
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
-
- 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)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Animal Behavior & Ethology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Mycology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Epidemiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Toxicology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the field of biotechnology, in particular to a double-adhesion-antibacterial peptide co-expression recombinant yeast and application thereof, wherein the construction process of the recombinant yeast is to carry out secretory expression of antibacterial peptide Lfcin B in Pichia pastoris GS115 to obtain recombinant yeast GS115/LF of secretory expression antibacterial peptide; taking pichia pastoris cell wall protein Gcw21 as an anchoring protein, taking adhesion factors FaeG and FanC as target proteins, and constructing recombinant plasmids in vitro; and (3) transforming the recombinant plasmid into recombinant yeast GS115/LF to obtain the double-adhesion-antibacterial peptide co-expression recombinant yeast. The recombinant yeast can obtain two different exogenous proteins of the adhesin and the antibacterial peptide through one-time fermentation, not only can anchor the adhesin FaeG and FanC on the cell surface and ensure that the adhesin FaeG and FanC have the capability of competitively adhering to intestinal cell specific receptors with ETEC, but also can secrete and express the antibacterial peptide Lfcin B with a bactericidal effect on the ETEC.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a double-adhesion-antibacterial peptide co-expression recombinant yeast and application thereof.
Background
Enterotoxigenic escherichia coli (EnterotoxigenicEscherichiacoli, ETEC) is an important pathogenic bacterium causing diarrhea in young animals (newborn piglets, weaned piglets, lambs, etc.). The use of antibiotics to treat ETEC infections is a more-used method, but in recent years, due to the abuse of antibiotics, a series of problems such as drug residues, bacterial resistance and the like are brought about, so that development of antibiotic-reducing and antibiotic-replacing products is required.
The antibacterial peptide is a kind of small molecular polypeptide existing in the immune system of organisms, has the advantages of broad-spectrum antibacterial activity, difficult generation of drug resistance, small toxic and side effects, no immunogenicity and the like, and is considered as one of the most promising antibiotic substitutes. The antibacterial peptide is added into feed or drinking water, and animals can directly kill pathogenic bacteria in organisms by feeding or drinking water, reduce the number of intestinal pathogenic bacteria, improve intestinal flora composition, improve intestinal barrier function, and play a role in treatment or prevention.
Adhesins are specific protein structures on the surface of bacteria, called adhesin antigens (F antigens), which can cause bacteria to adsorb on intestinal cells, and are a prerequisite for ETEC pathogenesis. The adhesins are classified into pilus adhesins and non-pilus adhesins, wherein pilus adhesins mainly include F4 (K88), F5 (K99), F6 (987P), F41, F42, F165, F17, F18, etc., and F4 (K88) and F5 (K99) are main antigens causing diarrhea in newborn piglets, and wherein main forming subunits of F4 (K88) and F5 (K99) are FaeG and FanC, respectively. The development of vaccines based on anti-adhesin immunity based on monovalent or multivalent adhesin antigens provides different preventive or therapeutic approaches to address the diseases caused by ETEC infection.
Disclosure of Invention
According to the invention, two adhesins are anchored on the yeast cell wall by using a pichia pastoris surface display technology, meanwhile, the antimicrobial peptide is expressed by using a secretion expression system, and a double adhesin-antimicrobial peptide co-expression pichia pastoris engineering bacteria GS115/LF-FanC-FaeG is constructed and obtained, so that the recombinant yeast can obtain two exogenous proteins with different biological functions of the adhesins and the antimicrobial peptide through one-time fermentation without being influenced by each other. The adhesin on the surface of the recombinant yeast cell can competitively adhere to a specific receptor of an intestinal cell with ETEC, inhibit adhesion of pathogenic bacteria, play a role in space occupation protection, and meanwhile, the antibacterial peptide generated in the recombinant yeast fermentation supernatant can kill the ETEC, so that intestinal health of animals in vivo can be effectively protected under the double effects, and a theoretical basis is provided for preparation of the compound microecological preparation.
The technical scheme is that the recombinant yeast GS115/LF-FanC-FaeG co-expresses adhesins FaeG and FanC derived from ETEC on the cell surface, and simultaneously the recombinant yeast also secretly expresses the antimicrobial peptide bovine lactoferrin Lfcin B;
the construction process of the recombinant yeast comprises the steps of carrying out secretory expression on the antibacterial peptide bovine lactoferrin Lfcin B in Pichia pastoris GS115 to obtain recombinant yeast GS115/LF; taking pichia pastoris cell wall protein Gcw21 as an anchoring protein, taking adhesion agents FaeG and FanC derived from enterotoxigenic escherichia coli as target proteins, and constructing two adhesion agent tandem expression recombinant plasmids pPICZ alpha A/(FanC-Gcw 21) - (FaeG-Gcw 21) in vitro; and (3) converting the recombinant plasmid pPICZalpha A/(FanC-Gcw 21) - (FaeG-Gcw 21) into recombinant yeast GS115/LF, and screening to obtain the double-adhesion-antibacterial peptide co-expression recombinant yeast GS115/LF-FanC-FaeG.
The double-adhesion-antibacterial peptide co-expression recombinant yeast GS115/LF-FanC-FaeG comprises the following specific construction steps:
step 1, construction of recombinant yeast for secretory expression of antibacterial peptide bovine lactoferrin Lfcin B: inserting a gene sequence of bovine lactoferrin Lfcin B subjected to codon optimization into a multiple cloning site of a secretion expression vector pPIC9K to obtain a recombinant plasmid pPIC9K/Lfcin B, converting the recombinant plasmid into pichia pastoris GS115, and screening by using a G418 resistance flat plate to obtain recombinant yeast GS115/LF for secretion expression of the antibacterial peptide; wherein the sequence of Lfcin B is shown as SEQ ID NO. 1;
step 2, constructing recombinant plasmids for expressing two adhesins FaeG and FanC in a co-display manner: the gene sequences of the adhesion agent FaeG and the anchoring protein Gcw21 are respectively inserted into the multiple cloning sites of an expression vector pPICZ alpha A to form fusion genes, so that a recombinant plasmid pPICZ alpha A/FaeG-Gcw21 for displaying and expressing FaeG by taking the Gcw21 as the anchoring protein is obtained, and the recombinant plasmid pPICZ alpha A/FanC-Gcw21 for displaying and expressing FanC by taking the Gcw21 as the anchoring protein is obtained by adopting the same method; double digestion is carried out on pPICZ alpha A/FaeG-Gcw21 by using BamHI and Bgl II to obtain an expression cassette containing FaeG-Gcw21 fusion genes; the pPICZalpha A/FanC-Gcw21 is subjected to single enzyme digestion by BamHI and dephosphorylation treatment; connecting an expression cassette containing FaeG-Gcw21 fusion genes into BamHI single-digested pPICZalpha A/FanC-Gcw21 to obtain recombinant plasmids pPICZalpha A/(FanC-Gcw 21) - (FaeG-Gcw 21) for co-display expression of two adhesins; wherein the FaeG sequence is shown as SEQ ID NO.2, the FanC sequence is shown as SEQ ID NO.3, and the Gcw21 sequence is shown as SEQ ID NO. 4;
step 3, constructing coexpression double-adhesion agent-antibacterial peptide recombinant yeast: recombinant yeast GS115/LF for secreting and expressing antibacterial peptide is used as host bacteria, the host bacteria are prepared into competent cells, then recombinant plasmids pPICZ alpha A/(FanC-Gcw 21) - (FaeG-Gcw 21) for co-displaying and expressing two kinds of adhesion are transformed into the competent cells, and double adhesion-antibacterial peptide co-expression recombinant yeast GS115/LF-FanC-FaeG is obtained by screening and culturing by using a Zeocin resistance plate.
And the application of the double-adhesion-antibacterial peptide co-expression recombinant yeast GS115/LF-FanC-FaeG in preparing medicines for protecting intestinal tracts.
And the application of the double-adhesion-antibacterial peptide co-expression recombinant yeast GS115/LF-FanC-FaeG in preparing microecological preparation for protecting intestinal tracts.
In addition, the recombinant yeast can obtain two exogenous proteins with different biological functions, namely an adhesion agent and an antibacterial peptide through one-time fermentation, wherein the adhesion agent is FaeG and FanC, and the antibacterial peptide is bovine lactoferrin Lfcin B.
Compared with the prior art, the beneficial effect of this technical scheme lies in:
(1) The main method for preventing or treating ETEC caused diseases is to use antibiotics or vaccines, and the two methods are usually used separately. The invention provides a double-adhesion-antibacterial peptide co-expression recombinant yeast, which can obtain two foreign proteins with different biological functions of adhesion and antibacterial peptide through one-time fermentation without being influenced by each other.
(2) The recombinant yeast strain provided by the invention anchors the adhesion agents FaeG and FanC on the cell surface, so that the recombinant yeast strain has the capability of competitively adhering intestinal cell specific receptors with ETEC, thereby inhibiting adhesion of pathogenic bacteria and playing a role in site-occupying protection; meanwhile, the antibacterial peptide Lfcin B secreted and expressed by the strain has a good bactericidal effect on ETEC; the microecological preparation based on coexpression of the double-adhesion-antibacterial peptide engineering bacteria organically combines the drug treatment and the vaccine prevention, and compared with the antibiotic treatment, the problem of drug resistance is not easy to cause.
(3) The co-expression double-adhesion-antibacterial peptide engineering bacteria can be used as a microecological preparation, and the double functions of adhesion and antibacterial peptide are utilized to reduce the number of intestinal pathogenic bacteria ETEC, improve the intestinal flora composition and improve the intestinal barrier function, so that the immunity of livestock and poultry is improved, the physique of the livestock and poultry is enhanced, the growth speed of the livestock and poultry is accelerated, and the feeding benefit is improved.
Drawings
FIG. 1 is a schematic diagram showing the construction of recombinant plasmids pPICZαA/(FanC-Gcw 21) - (FaeG-Gcw 21).
FIG. 2 is a flow chart of the construction of a recombinant yeast GS115/LF-FanC-FaeG co-expressed as a dual-adhesin-antibacterial peptide.
FIG. 3 shows the bacteriostatic activity of the recombinant yeast fermentation supernatant co-expressed with the double-adhesion agent-antibacterial peptide on enterotoxigenic E.coli.
FIG. 4 is a flow cytometry analysis of a double-adhesin-antibacterial peptide co-expression recombinant yeast cell surface display expressing two adhesins.
FIG. 5 shows the results of an adhesion experiment of recombinant yeast co-expressed with a double-adhesion element and an antibacterial peptide to pig small intestine epithelial cells IPEC-J2.
FIG. 6 is a schematic diagram of an animal experiment scheme of the intestinal tract protection effect of a microecological preparation based on recombinant yeast co-expressed with a double-adhesin-antibacterial peptide on ETEC infected mice.
FIG. 7 shows the results of GS115/LF-FanC-FaeG bacterial suspension pretreatment to ameliorate the clinical symptoms caused by ETEC infection; wherein (A) percent weight loss; (B) diarrhea score; (C) survival rate; (D) counts of K88ab-GFP in feces; (E) count of K99-mCherry in feces.
FIG. 8 shows the effect of GS115/LF-FanC-FaeG bacterial suspension pretreatment on empty intestine pathology; wherein (a) mucosal morphology H & E staining; (B) fluff height; (C) crypt depth; (D) ratio of fluff height to recess depth.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples, which are not intended to limit the scope of the invention.
Example 1: construction of recombinant Yeast GS115/LF for secretion expression of antibacterial peptide Lfcin B
The protein sequence of the antibacterial peptide Lfcin B is shown as SEQ ID NO:1, the gene sequence is optimized according to the codon preference of Pichia pastoris, then the Pichia pastoris is delivered to Wohano biological science and technology Co, complete gene synthesis is carried out, enzyme cutting sites of EcoRI and NotI are added at the 5 'end and the 3' end of the antibacterial peptide Lfcin B respectively, and the antibacterial peptide Lfcin B is connected to the plasmid pPIC9K polyclonal site to obtain the recombinant plasmid pPIC9K/Lfcin B.
And (3) carrying out single enzyme digestion on pPIC9K/Lfcin B by using restriction enzyme SacI to linearize the pPIC9K/Lfcin B, converting the pPIC9K/Lfcin B into competent Pichia pastoris GS115 cells by using an electrotransformation method, coating the pPIC9K/Lfcin B on an MD plate with the final concentration of the antibiotic G418 of 1mg/mL, culturing the pPIC9K/Lfcin B at 30 ℃ until a single colony grows, then inoculating the pPIC9K/Lfcin B on the MD plate with the final concentration of the antibiotic G418 of 2mg/mL, culturing the pPIC9K/Lfcin B at 30 ℃ until a colony grows, and then inoculating the pPIC9K/Lfcin B on the MD plate with the final concentration of the antibiotic G418 of 4mg/mL, and gradually increasing the screening pressure of the antibiotics to obtain multicopy transformants. Recombinant yeast which secretively expresses the antibacterial peptide Lfcin B is obtained after colony PCR identification and is named as GS115/LF.
Example 2: construction of recombinant plasmid pPICZαA/(FanC-Gcw 21) - (FaeG-Gcw 21) co-displaying and expressing two adhesins
NCBI search to obtain FaeG protein sequence shown in SEQ ID NO:2, and FanC protein sequence shown in SEQ ID NO: 3. The His tag is carried on the N-end of FaeG, and the FLAG tag is carried on the N-end of FanC, so that the method can be used for detecting flow cytometry or immunofluorescence microscopy and subsequently checking whether the adhesion FaeG and FanC are successfully displayed and expressed on the surface of Pichia pastoris cells.
Optimizing the gene sequences of FaeG and FanC according to the codon preference of Pichia pastoris, respectively adding the gene sequences of His tag and FLAG tag at the 5' end, carrying out total gene synthesis by Wuhan engine biotechnology limited company, respectively adding enzyme cutting sites of EcoRI and KpnI at the 5' end and 3' end, respectively connecting to the multicloning sites of plasmid pPICZ alpha A, and obtaining recombinant plasmids pPICZ alpha A/FaeG and pPICZ alpha A/FanC.
Pichia pastoris endogenous GPI cell wall protein Gcw21 is selected as an anchoring protein to display and express two adhesins, and the protein sequence of the Gcw21 is shown as SEQ ID NO: 4. The gene sequence of the anchoring protein Gcw21 with the signal peptide removed is obtained by taking the genome of Pichia pastoris GS115 as a template through PCR amplification, the restriction sites of KpnI and NotI are respectively introduced at the 5 'end and the 3' end of the gene sequence, the KpnI and the NotI are used for double restriction after purification and recovery, simultaneously double restriction is carried out on recombinant plasmids pPICZ alpha A/FaeG and pPICZ alpha A/FanC, then the recombinant plasmids are respectively connected, are transformed into E.coliTop10 competent cells by a chemical method, are coated on LBLZ resistant plates (the final concentration of the antibiotics Zeocin is 25 mu g/mL), after single colony is selected to be grown up, the single colony is selected to LBLZ liquid medium (the final concentration of the antibiotics Zeocin is 25 mu g/mL), the plasmid is extracted at 37 ℃ and is cultivated at 200rpm, and the recombinant plasmids pPICZ alpha/FaeG-FaeG 21 and FacW-GcW 21 are successfully obtained through double restriction identification and sequencing verification of KpnI and NotI.
TABLE 1 primer for PCR amplification to obtain Gcw21 gene sequence
Note that: the underlined sites are the cleavage sites for KpnI and NotI, respectively.
According to the pichia pastoris expression vector pPICZ alpha A operation manual, an in vitro multi-expression cassette tandem technology is adopted to construct two adhesin co-display expression recombinant plasmids pPICZ alpha A/(FanC-Gcw 21) - (FaeG-Gcw 21). The pPICZ alpha A/FaeG-Gcw21 was double digested with BglII and BamHI, and the expression cassette containing the FaeG-Gcw21 gene was recovered by cut-out, and further the pPICZ alpha A/FanC-Gcw21 was single digested with BamHI and dephosphorylated, and the expression cassette containing the FaeG-Gcw21 gene was ligated with the single digested and dephosphorylated pPICZ alpha A/FanC-Gcw21, and the recombinant plasmid pPICZ alpha A/(FanC-Gcw 21) - (FaeG-Gcw 21) was obtained by ligating the expression cassette containing the FaeG-Gcw21 gene to the BamHI site of the single digested and dephosphorylated pPICZ alpha A/FanC-Gcw21, as BglII and BamHI were co-tail enzymes, and had the feature of being end-to-end ligatable, as shown in FIG. 1.
Example 3: construction of double-adhesion-antibacterial peptide co-expression recombinant yeast GS115/LF-FanC-FaeG and shake flask fermentation culture
Electrotransformed competent cells were prepared using recombinant yeast GS115/LF as host bacteria. GS115/LF was inoculated into 100mLYPD liquid medium and incubated at 30℃in a 250rpm shaker to OD 600 The value is between 1.3 and 1.5, bacterial suspension is averagely split into two 50mL sterile centrifuge tubes, centrifuged at room temperature and 5000rpm for 5min, 40mL of freshly prepared sterile LDST solution is added into each tube after supernatant is discarded, the bacteria are re-suspended and incubated at 30 ℃ for 30min, then centrifuged at room temperature and 5000rpm for 5min, 1mL of pre-cooled 1mol/L sorbitol solution is added after supernatant is discarded to re-suspend the bacteria, and the bacteria are totally transferred into a new 1.5mL sterile centrifuge tube, and then the bacteria are washed 3 times by the pre-cooled 1mol/L sorbitol solution, and finally 400 mu L of pre-cooled 1mol/L sorbitol solution is added for re-suspensionThe cells are obtained and packaged according to 80 mu L/tube, and are stored in a refrigerator at-80 ℃ for standby.
Placing the extracted recombinant plasmid pPICZ alpha A/(FanC-Gcw 21) - (FaeG-Gcw 21) solution in a vacuum concentrator to concentrate to a concentration of about 1 mug/mu L, taking 10 mu L, adding the solution into a GS115/LF competent cell solution thawed on ice in advance, slightly mixing the solution, transferring the solution into a 0.2cm electric rotating cup subjected to advanced sterilization and ice bath treatment, placing the electric rotating cup on ice for incubation for 5min, performing electric shock in an electroporation instrument, setting the electric shock voltage to be 1.5KV, enabling the electric shock time to be about 5ms, rapidly adding 1mL of ice-cold sorbitol after the electric shock is finished, slowly and uniformly blowing the solution by a pipetting gun, transferring the solution to a 1.5mL sterile centrifuge tube, incubating the solution for 1.5h at 30 ℃, coating the solution on a YPDZ resistant plate (Zeocin final concentration is 100 mug/mL), culturing the solution at 30 ℃ until a single colony is grown, and obtaining the recombinant yeast GS/(cinB) - (FanG-Gcw 21) - (FaeG-Gcw 21) through colony PCR method identification.
Inoculating recombinant yeast GS115/LF-FanC-FaeG into BMGY culture medium, culturing at 28deg.C and 250rpm for 24 hr, collecting thallus, re-suspending in 250mL triangular flask containing 50mL MMY culture medium, and controlling initial inoculation OD 600 The culture was carried out at 28℃and 250rpm for 120 hours, with 4% (v/v) methanol being added every 24 hours.
Example 4: analysis of antimicrobial peptides produced by recombinant yeast co-expressed with double adhesin-antimicrobial peptides
In-vitro antibacterial activity analysis is carried out by adopting an agar hole diffusion method, gram negative bacteria ETECK88 is used as indicator bacteria, LB liquid culture medium is used for culturing the indicator bacteria to logarithmic phase, 100 mu L of bacterial suspension of the indicator bacteria is added when the LB solid culture medium is cooled to about 50 ℃, the indicator bacteria are poured into a flat plate after being uniformly mixed, a sterile puncher (with the diameter of 5 mm) is used for punching, 100 mu L of fermentation supernatant of a recombinant yeast sample is added into the hole, the culture is carried out for 12 hours at the temperature of 37 ℃, fermentation supernatant of host bacteria Pichia pastoris GS115 is used as a control, and the diameter of a bacteriostasis ring is observed and measured by using a vernier caliper.
The recombinant yeast GS115/LF-FanC-FaeG is sampled at intervals of 24 hours for 0.5mL, centrifuged at room temperature and 5000rpm for 2min, the supernatant is collected, the antibacterial activity of the supernatant on ETECK88 in different fermentation time is measured, the result is shown in figure 3, the diameter of the antibacterial ring of the ETECK88 is gradually increased along with the extension of the fermentation time, the maximum antibacterial ring is reached at 72 hours, and the diameter of the antibacterial ring is 16mm. The result shows that the fermentation supernatant of GS115/LF-FanC-FaeG has better antibacterial activity on enterotoxigenic escherichia coli.
Example 5: analysis of adhesion produced by double adhesion element-antibacterial peptide co-expression recombinant yeast
Expression of FaeG and FacC was demonstrated using the anchoring protein Gcw21 with a His tag at the N-terminus of FaeG and a FLAG tag at the N-terminus of FanC, so that flow cytometry was used to analyze expression of FaeG and FacC on the cell surface. Taking recombinant yeast GS115/LF-FanC-FaeG fermented 72h sample, centrifuging at 5000rpm at room temperature for 3min, collecting thallus, washing with sterile PBS three times, re-suspending thallus with 1mLPBS, and adjusting OD 600 About equal to 5, a step of; taking 200 mu L of resuspended bacterial liquid, centrifuging at 6000rpm for 3min at room temperature, discarding supernatant, adding 100 mu L of His tag or FLAG tag primary suspension bacterial body diluted by PBS containing 1% BSA respectively, and incubating at room temperature for 2h; after centrifugation, the cells were washed three times with sterile PBS, the supernatant was discarded, 100. Mu.L of secondary antibody Dyight 488, goatAnti-MouseigG diluted with PBS containing 1% BSA was added and incubated at room temperature for 1h in the absence of light; after centrifugation, the cells were washed three times with sterile PBS, suspended with 1.5mLPBS solution, and filtered with a melt cloth to disperse the cell mass; sample detection was performed on a flow cytometer with reference to a flow cytometer operation manual, the number of cells collected and measured was 10000, the fluorescence excitation light wavelength was 488nm, and the FL1 channel signal was recorded. The results of flow cytometry analysis are shown in FIG. 4, compared with the control bacteria GS115, the peak images of the flow cytometry histograms of recombinant bacteria GS115/LF-FanC-FaeG bacteria have obvious drift to the right on the X axis no matter whether the recombinant bacteria GS115/LF-FanC-FaeG bacteria are treated by His tag antibody or FLAG tag antibody, which shows that the bacteria surfaces of the GS115/LF-FanC-FaeG successfully express the adhesion factors FaeG and FanC.
The interaction of pig small intestine epithelial cells IPEC-J2 and recombinant Pichia pastoris is selected to simulate the specific adhesion condition of intestinal epithelium and recombinant Pichia pastoris. The frozen IPEC-J2 cell cryopreservation tube was removed from the liquid nitrogen, rapidly thawed in a 37℃water bath, and centrifuged at 1200r/min for 5min.And sucking out the frozen solution in the frozen tube from the other side of the cell sediment in the ultra-clean workbench, adding 1mLDEME-H culture medium into the frozen tube, and re-suspending the cell sediment. And sucking the resuspended cell suspension, adding the cell suspension into a cell culture bottle, supplementing the culture medium to 5mL, and placing the culture bottle into an incubator for culturing until the logarithmic phase of growth. The culture medium in the flask was poured off and washed 3 times with PBS. Adding 1.5mL of pancreatin, putting the cell bottle into an incubator, digesting for 4-6min, taking out, observing under a microscope, discarding most pancreatin when gaps exist between cells, continuing digesting the rest pancreatin for 1-2min, adding 5mL of medium of DMEM-H, and repeatedly blowing the cells to form cell suspension. Filling culture flask with 5mL of LDEME-H culture medium, inoculating appropriate amount of IPEC-J2 cells into 24-well plate with cell climbing sheet, filling 1mL with DEME-H culture medium, and placing at 37deg.C and 5% CO 2 Is cultured overnight in a cell incubator.
Taking 2mL of bacterial suspension fermented for 72h, centrifuging at 8000r/min for 2min, separating bacterial cells in an ultra-clean workbench, washing with a proper amount of PBS for three times, re-suspending, and adjusting the bacterial suspension concentration to be about 5 multiplied by 10 7 CFU/mL. The bacterial suspension is centrifuged at 8000r/min for 5min, and resuspended with 1mL of nonreactive DEME-H for later use. IPEC-J2 cells in 24-well plates were washed 3 times with 1mL of sterile PBS, and then the resuspended bacterial suspension was inoculated into 24-well plates and incubated in a 37℃incubator for 3h. After incubation, the slide is taken out, placed on a glass slide, slowly washed 3 times with 1mL of sterile PBS, the non-adhered recombinant Pichia pastoris is washed off, a proper amount of 4% glutaraldehyde is dripped on the slide, the slide is fixed for 15min, the slide is slowly washed 3 times with sterile PBS, after the excessive moisture is absorbed by filter paper, a proper amount of 0.05% crystal violet solution is dripped on the slide for 3min, the slide is slowly washed to be colorless with sterile water, and the slide is observed and recorded by a microscope.
Under the condition of crystal violet staining, the yeast cells are in a dot-shaped particle shape, the IPEC-J2 cells are in an oval cake shape, the observation result is shown in figure 5, and the control yeast in the left figure has certain adhesiveness to the IPEC-J2 cells, probably that the yeast is non-specifically bound to the cells through surface glycoprotein and other appendages, and belongs to physical adhesion; in the right graph, the aggregation degree of GS115/LF-FanC-FaeG is relatively high, and the adhesion sites of IPEC-J2 cells are identified through the specificity of FaeG and FanC on the surface of Pichia pastoris, so that the adhered recombinant Pichia pastoris is obviously increased, and the adhesion is changed from physical adhesion to specific adhesion. The experimental results show that GS115/LF-FanC-FaeG has certain capability of competing with ETEC for adhesion.
Example 6: research experiment of intestinal protection effect of microecological preparation based on recombinant yeast co-expressed by double-adhesin-antibacterial peptide on ETEC infected mice
An animal experimental scheme of the effect of the microecological preparation based on recombinant yeast co-expressed with the double-adhesin-antibacterial peptide on protecting intestinal tracts of ETEC infected mice is shown in FIG. 6. 40 male BALB/c mice with similar weight and SPF grade of 6 weeks are selected, randomly divided into 4 groups, each group comprises 2 mouse cages, 5 mice are placed in the same environment, the temperature of a mouse room is controlled to be about 25 ℃, and the sunshine time is 7:00 to 19:00. After 3 days of adaptation, a pretreatment period of 14 days is carried out, and a group A and a negative control group (NegativeControl, NC) are added with 2.5 percent of zymogen suspension of host bacteria GS115 in drinking water; group B, positive control group (PositiveControl, PC), adding 2.5% zymogen suspension of host bacteria GS115 into drinking water; group C, double-adhesion-antibacterial peptide group, adding 2.5% GS115/LF-FanC-FaeG zymogen suspension into drinking water; group D, double-adhesion group, adding 2.5% GS115/FanC-FaeG zymogen suspension into drinking water. Infection with ETEC was started on day 14, and each mouse of group a (NC group) was perfused with 0.2 mlfbs; each mouse of group B (PC group), group C (double-adhesin-antibacterial peptide group) and group D (double-adhesin group) was perfused with 0.2mLPBS containing 5×10 9 ETEC marker strains K99-mCherry and 5X 10 9 ETEC marker strain K88ab-GFP; infection treatment was continued for 3 days. The 4 groups of mice were fed regular diet during the whole experiment, and were fed and drinking water freely.
Each mouse was weighed daily from the first day of infection treatment, and monitored for weight changes; observing the living state of each mouse every day, and monitoring the death condition; each mouse was observed daily for diarrhea, and diarrhea was scored, with severity of diarrhea scored according to the following criteria, score 0, normal (normal stool); 1 minute, mild diarrhea (slight wet and soft, no coloration around the anus is seen); 2 minutes, moderate diarrhea (wet stool in muddy form and light or moderate perianal coloration); 3 minutes, severe diarrhea (watery stool or mucoid with severe perianal staining).
Faecal samples of each mouse were collected 24h, 48h and 72h of infection, the mice were fixed at the time of sampling, their tails were lifted, the lower abdomen of the mice was gently pressed with a finger, fresh faeces were collected in a 2mL centrifuge tube weighing the corresponding number, and weighed again. According to the weight of the sample, a proper amount of sterilized normal saline is added for 10-time dilution, and the mixture is mixed with glass beads to fully homogenize the mixture. The supernatant was aspirated for 10-fold gradient dilution after 2-5min rest, then plated on Amp-resistant LB plates, incubated overnight at 37 ℃, and counted by irradiation with an excitation light source.
Mice were sacrificed 3 days after infection treatment by cervical dislocation, dissected, and 1-2cm long chymal free jejunum tissue was taken from each mouse, fixed in 4% paraformaldehyde, paraffin embedded, cut into 5 μm thick sections, stained with hematoxylin-eosin (H & E), the sections were observed under an optical microscope (nikoneclipse 100) and photographed, and the villus height and crypt depth were recorded.
Data statistical analysis: all data are expressed as mean ± standard error, single factor analysis of variance (One-wayANOVA) using graphpadprism8.0, significance of differences between groups using Tukey' smultipleompa rison, P <0.05 was significant, and plot using prism 8.0.
The mice were infected with a mixture of K88ab-GFP and K99-mCherry bacteria after 14 days of pretreatment, and their weights were weighed daily to observe diarrhea and death. The results showed that the positive control mice had significantly increased weight loss and diarrhea scores after ETEC infection (p < 0.001) compared to the negative control mice, but the addition of GS115/LF-FanC-FaeG bacterial suspension or GS115/FanC-FaeG bacterial suspension at pretreatment significantly reduced the clinical diarrhea symptoms of the ETEC infected mice, as evidenced by significantly decreased weight loss and diarrhea scores (p < 0.05) (fig. 7A and 7B). Furthermore, there was no significant difference in survival between groups (fig. 7C).
Mice were infected with a mixture of K88ab-GFP and K99-mCherry after 14 days of pretreatment, stool samples were collected and counted at 24h, 48h and 72h, respectively, and after 3 days of ETEC in the stomach of mice in the positive control group compared to the negative control group, the numbers of K88ab-GFP and K99-mCherry in the stool were significantly increased (p < 0.0001), but the addition of GS115/LF-FanC-FaeG bacterial suspension or GS115/FanC-FaeG bacterial suspension during pretreatment significantly reduced the colonisation of the intestinal tract with K88ab-GFP and K99-mCherry, and the addition of GS115/LF-FanC-FaeG bacterial suspension during pretreatment was less than the numbers of K88ab-GFP and K99-mCherry in the feces at 72h compared to the GS115/FanC-FaeG bacterial suspension (FIGS. 7D and 7E).
Mice were sacrificed 3 days after infection and jejunal tissues were dissected for villus morphology and histopathological observation. As shown in fig. 8A, the morphology of the jejunum villus of the mice of the negative control group showed that the intestinal epithelial cells were ordered with intact mucus layers. However, infection with ETEC in the positive control group resulted in mucosal morphology disruption (fig. 8A), decrease in jejunum villus height (V), increase in crypt depth (C), decrease in V/C (fig. 8B-D) compared to the negative control group (p < 0.05). However, compared with the positive control group, the GS115/LF-FanC-FaeG bacterial suspension or the GS115/FanC-FaeG bacterial suspension is added during pretreatment, so that morphological damage of jejunum mucosa caused by ETEC is inhibited (figure 8A), V and V/C of jejunum of mice are obviously increased, C is reduced (figure 8B-D) (p is less than 0.05), and the GS115/LF-FanC-FaeG bacterial suspension is added during pretreatment, so that the effect is better than that of the GS115/FanC-FaeG bacterial suspension. The results show that the pretreatment of GS115/LF-FanC-FaeG bacterial suspension can relieve intestinal inflammation induced by ETEC, thereby protecting intestinal health.
Claims (5)
1. A double-adhesion-antibacterial peptide co-expression recombinant yeast GS115/LF-FanC-FaeG is characterized in that: the recombinant yeast co-displays and expresses adhesion factors FaeG and FanC derived from ETEC on the cell surface, and simultaneously the recombinant yeast also secretively expresses antimicrobial peptide bovine lactoferrin Lfcin B;
the construction process of the recombinant yeast comprises the steps of carrying out secretory expression on the antibacterial peptide bovine lactoferrin Lfcin B in Pichia pastoris GS115 to obtain recombinant yeast GS115/LF; taking pichia pastoris cell wall protein Gcw21 as an anchoring protein, taking adhesion agents FaeG and FanC derived from enterotoxigenic escherichia coli as target proteins, and constructing two adhesion agent tandem expression recombinant plasmids pPICZ alpha A/(FanC-Gcw 21) - (FaeG-Gcw 21) in vitro; and (3) converting the recombinant plasmid pPICZalpha A/(FanC-Gcw 21) - (FaeG-Gcw 21) into recombinant yeast GS115/LF, and screening to obtain the double-adhesion-antibacterial peptide co-expression recombinant yeast GS115/LF-FanC-FaeG.
2. The double-adhesion-antibacterial peptide co-expression recombinant yeast GS115/LF-FanC-FaeG according to claim 1, which is characterized by comprising the following specific construction steps:
step 1, construction of recombinant yeast for secretory expression of antibacterial peptide bovine lactoferrin Lfcin B: inserting a gene sequence of bovine lactoferrin Lfcin B subjected to codon optimization into a multiple cloning site of a secretion expression vector pPIC9K to obtain a recombinant plasmid pPIC9K/Lfcin B, converting the recombinant plasmid into pichia pastoris GS115, and screening by using a G418 resistance flat plate to obtain recombinant yeast GS115/LF for secretion expression of the antibacterial peptide; wherein the sequence of Lfcin B is shown as SEQ ID NO. 1;
step 2, constructing recombinant plasmids for expressing two adhesins FaeG and FanC in a co-display manner: the gene sequences of the adhesion agent FaeG and the anchoring protein Gcw21 are respectively inserted into the multiple cloning sites of an expression vector pPICZ alpha A to form fusion genes, so that a recombinant plasmid pPICZ alpha A/FaeG-Gcw21 for displaying and expressing FaeG by taking the Gcw21 as the anchoring protein is obtained, and the recombinant plasmid pPICZ alpha A/FanC-Gcw21 for displaying and expressing FanC by taking the Gcw21 as the anchoring protein is obtained by adopting the same method; double digestion is carried out on pPICZ alpha A/FaeG-Gcw21 by using BamHI and Bgl II to obtain an expression cassette containing FaeG-Gcw21 fusion genes; the pPICZalpha A/FanC-Gcw21 is subjected to single enzyme digestion by BamHI and dephosphorylation treatment; connecting an expression cassette containing FaeG-Gcw21 fusion genes into BamHI single-digested pPICZalpha A/FanC-Gcw21 to obtain recombinant plasmids pPICZalpha A/(FanC-Gcw 21) - (FaeG-Gcw 21) for co-display expression of two adhesins; wherein the FaeG sequence is shown as SEQ ID NO.2, the FanC sequence is shown as SEQ ID NO.3, and the Gcw21 sequence is shown as SEQ ID NO. 4;
step 3, constructing coexpression double-adhesion agent-antibacterial peptide recombinant yeast: recombinant yeast GS115/LF for secreting and expressing antibacterial peptide is used as host bacteria, the host bacteria are prepared into competent cells, then recombinant plasmids pPICZ alpha A/(FanC-Gcw 21) - (FaeG-Gcw 21) for co-displaying and expressing two kinds of adhesion are transformed into the competent cells, and double adhesion-antibacterial peptide co-expression recombinant yeast GS115/LF-FanC-FaeG is obtained by screening and culturing by using a Zeocin resistance plate.
3. Use of a recombinant yeast GS115/LF-FanC-FaeG co-expressed with a dual-adhesin-antibacterial peptide according to claim 1 or 2 for the preparation of a medicament for protecting the intestinal tract.
4. Use of a recombinant yeast GS115/LF-FanC-FaeG co-expressed with a dual-adhesin-antibacterial peptide according to claim 1 or 2 for the preparation of a microecological preparation for protecting the intestinal tract.
5. A double-adhesin-antibacterial-peptide co-expression recombinant yeast GS115/LF-FanC-FaeG according to claim 1 or 2, characterized in that: the recombinant yeast can obtain two foreign proteins with different biological functions, namely an adhesion agent and an antibacterial peptide through one-time fermentation, wherein the adhesion agent is FaeG and FanC, and the antibacterial peptide is bovine lactoferrin Lfcin B.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310336046.XA CN116445310A (en) | 2023-03-31 | 2023-03-31 | Double-adhesion-antibacterial peptide co-expression recombinant yeast and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310336046.XA CN116445310A (en) | 2023-03-31 | 2023-03-31 | Double-adhesion-antibacterial peptide co-expression recombinant yeast and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116445310A true CN116445310A (en) | 2023-07-18 |
Family
ID=87129556
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310336046.XA Pending CN116445310A (en) | 2023-03-31 | 2023-03-31 | Double-adhesion-antibacterial peptide co-expression recombinant yeast and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116445310A (en) |
-
2023
- 2023-03-31 CN CN202310336046.XA patent/CN116445310A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101473028B (en) | Recombinant lactobacillus and use of the same | |
| KR20190110605A (en) | Swine Coronavirus Vaccine | |
| CN112300976B (en) | Recombinant lactobacillus plantarum for expressing newcastle disease virus antigen gene and fermentation process and application thereof | |
| CN108251378B (en) | A kind of interstital stem cell excretion body and its preparation method and application being overexpressed PTGDS gene | |
| CN116396974B (en) | African swine fever virus antigen protein recombinant expression vector, recombinant plant lactobacillus, and preparation method and application thereof | |
| CN107474142A (en) | Promote polypeptide and its relevant biological material and the application of destination protein secretion | |
| CN112625096B (en) | Avian infectious bronchitis virus-like particle and preparation method and application thereof | |
| CN110172433B (en) | Recombinant bacillus subtilis engineering bacterium for producing porcine epidermal growth factor and application thereof | |
| CN116445310A (en) | Double-adhesion-antibacterial peptide co-expression recombinant yeast and application thereof | |
| CN104418945A (en) | Preparation method of peptide and application of peptide in preparation of medicine and feed additive | |
| CN109608541B (en) | Yolk antibody for resisting swine enterotoxigenic escherichia coli and preparation method thereof | |
| CN116903753B (en) | Broad-spectrum antigen worm polypeptide, and preparation and application thereof | |
| JPH0825898B2 (en) | Composition for immunization against urethral infection caused by Escherichia coli | |
| CN111978380A (en) | Wild type clostridium emphysema cytotoxin A as well as preparation method, application and vaccine thereof | |
| CN112048459A (en) | Salmonella choleraesuis, vaccine and application thereof | |
| CN111171144A (en) | Preparation and application of antibody for resisting porcine epidemic diarrhea virus | |
| JP2001526043A (en) | TGC-methods for inducing targeted somatic cell transgenesis | |
| CN110305225A (en) | SVA-PCV2 fusion protein and preparation method thereof, gene, biomaterial, application and vaccine | |
| CN115992116B (en) | Multifunctional dairy cow lysozyme and preparation method and application thereof | |
| CN114164159B (en) | Bivalent vaccine for preventing and treating salmonicida and Edwardsiella tarda infection of fish, and preparation method and application thereof | |
| CN113372453B (en) | Fusion protein and porcine epidemic diarrhea virus S1 protein recombinant subunit vaccine | |
| CN116903755B (en) | Fusion protein and nucleic acid molecule for pig interleukin3, 7 and 15 co-expression, and substitution biological material and application thereof | |
| CN115044524B (en) | Genetically engineered recombinant lactobacillus and application thereof in resisting vibrio mimicus infection | |
| CN114836356B (en) | Lawsonia intracellularis attenuated vaccine strain, vaccine and application thereof | |
| CN117535333B (en) | Method for promoting secretion of outer membrane vesicles of gonococcus, gonococcus knockout strain, preparation method and application thereof |
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 |