CN116478895B - Recombinant salmonella typhimurium genetically engineered bacterium HCS1, microbial inoculum and application thereof - Google Patents
Recombinant salmonella typhimurium genetically engineered bacterium HCS1, microbial inoculum and application thereof Download PDFInfo
- Publication number
- CN116478895B CN116478895B CN202310320028.2A CN202310320028A CN116478895B CN 116478895 B CN116478895 B CN 116478895B CN 202310320028 A CN202310320028 A CN 202310320028A CN 116478895 B CN116478895 B CN 116478895B
- Authority
- CN
- China
- Prior art keywords
- salmonella typhimurium
- hcs1
- gene
- genetically engineered
- homology arm
- 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
- 241000293869 Salmonella enterica subsp. enterica serovar Typhimurium Species 0.000 title claims abstract description 110
- 101150093275 hcs1 gene Proteins 0.000 title claims abstract description 92
- 241000894006 Bacteria Species 0.000 title claims abstract description 89
- 239000002068 microbial inoculum Substances 0.000 title claims abstract description 24
- 230000002238 attenuated effect Effects 0.000 claims abstract description 18
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 15
- 230000002441 reversible effect Effects 0.000 claims description 54
- 101150085542 relA gene Proteins 0.000 claims description 45
- 239000002773 nucleotide Substances 0.000 claims description 40
- 125000003729 nucleotide group Chemical group 0.000 claims description 40
- 101150076547 spoT gene Proteins 0.000 claims description 31
- 238000012408 PCR amplification Methods 0.000 claims description 30
- 238000011144 upstream manufacturing Methods 0.000 claims description 24
- 239000013612 plasmid Substances 0.000 claims description 23
- 230000003321 amplification Effects 0.000 claims description 21
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 18
- 230000006801 homologous recombination Effects 0.000 claims description 12
- 238000002744 homologous recombination Methods 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 11
- 238000002360 preparation method Methods 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 238000012217 deletion Methods 0.000 claims description 7
- 230000037430 deletion Effects 0.000 claims description 7
- 101150025220 sacB gene Proteins 0.000 claims description 6
- 238000006276 transfer reaction Methods 0.000 claims description 6
- 239000004480 active ingredient Substances 0.000 claims description 5
- 208000029742 colonic neoplasm Diseases 0.000 claims description 5
- 230000021615 conjugation Effects 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 229920000858 Cyclodextrin Polymers 0.000 claims description 2
- 229920000168 Microcrystalline cellulose Polymers 0.000 claims description 2
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 2
- 229920002472 Starch Polymers 0.000 claims description 2
- 101100309436 Streptococcus mutans serotype c (strain ATCC 700610 / UA159) ftf gene Proteins 0.000 claims description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 claims description 2
- 235000010216 calcium carbonate Nutrition 0.000 claims description 2
- 239000001506 calcium phosphate Substances 0.000 claims description 2
- 229910000389 calcium phosphate Inorganic materials 0.000 claims description 2
- 235000011010 calcium phosphates Nutrition 0.000 claims description 2
- 229940110456 cocoa butter Drugs 0.000 claims description 2
- 235000019868 cocoa butter Nutrition 0.000 claims description 2
- 235000019813 microcrystalline cellulose Nutrition 0.000 claims description 2
- 239000008108 microcrystalline cellulose Substances 0.000 claims description 2
- 229940016286 microcrystalline cellulose Drugs 0.000 claims description 2
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 2
- 235000019422 polyvinyl alcohol Nutrition 0.000 claims description 2
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 claims description 2
- 239000008107 starch Substances 0.000 claims description 2
- 235000019698 starch Nutrition 0.000 claims description 2
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 claims description 2
- 238000004321 preservation Methods 0.000 claims 1
- 206010028980 Neoplasm Diseases 0.000 abstract description 62
- 241000699670 Mus sp. Species 0.000 abstract description 27
- 231100000419 toxicity Toxicity 0.000 abstract description 4
- 230000001988 toxicity Effects 0.000 abstract description 4
- 238000001815 biotherapy Methods 0.000 abstract description 2
- 230000001580 bacterial effect Effects 0.000 description 31
- 239000002609 medium Substances 0.000 description 23
- 239000000047 product Substances 0.000 description 20
- 239000007787 solid Substances 0.000 description 17
- 239000007788 liquid Substances 0.000 description 16
- 239000012228 culture supernatant Substances 0.000 description 14
- 238000002347 injection Methods 0.000 description 14
- 239000007924 injection Substances 0.000 description 14
- 241000607142 Salmonella Species 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 13
- 210000004881 tumor cell Anatomy 0.000 description 11
- 201000011510 cancer Diseases 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 9
- 238000011282 treatment Methods 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 7
- 230000002829 reductive effect Effects 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 102000003855 L-lactate dehydrogenase Human genes 0.000 description 6
- 108700023483 L-lactate dehydrogenases Proteins 0.000 description 6
- 239000012880 LB liquid culture medium Substances 0.000 description 6
- 229960005091 chloramphenicol Drugs 0.000 description 6
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 230000002147 killing effect Effects 0.000 description 6
- 239000000523 sample Substances 0.000 description 6
- 239000003795 chemical substances by application Substances 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 108090000340 Transaminases Proteins 0.000 description 4
- 102000003929 Transaminases Human genes 0.000 description 4
- 230000000259 anti-tumor effect Effects 0.000 description 4
- 239000000872 buffer Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 230000009545 invasion Effects 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 230000008685 targeting Effects 0.000 description 4
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 3
- 108010082126 Alanine transaminase Proteins 0.000 description 3
- 108010003415 Aspartate Aminotransferases Proteins 0.000 description 3
- 102000004625 Aspartate Aminotransferases Human genes 0.000 description 3
- 238000011740 C57BL/6 mouse Methods 0.000 description 3
- 206010021143 Hypoxia Diseases 0.000 description 3
- 239000002246 antineoplastic agent Substances 0.000 description 3
- 229940041181 antineoplastic drug Drugs 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000010253 intravenous injection Methods 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 230000000813 microbial effect Effects 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- 238000012546 transfer Methods 0.000 description 3
- 230000004614 tumor growth Effects 0.000 description 3
- 208000024172 Cardiovascular disease Diseases 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 2
- 229930182566 Gentamicin Natural products 0.000 description 2
- 231100000111 LD50 Toxicity 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 208000026106 cerebrovascular disease Diseases 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 201000011024 colonic benign neoplasm Diseases 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 230000002526 effect on cardiovascular system Effects 0.000 description 2
- 239000013613 expression plasmid Substances 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 230000001146 hypoxic effect Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 208000028774 intestinal disease Diseases 0.000 description 2
- 231100000636 lethal dose Toxicity 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000011275 oncology therapy Methods 0.000 description 2
- 102000005962 receptors Human genes 0.000 description 2
- 108020003175 receptors Proteins 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 230000001225 therapeutic effect Effects 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 102100032814 ATP-dependent zinc metalloprotease YME1L1 Human genes 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 108050009160 DNA polymerase 1 Proteins 0.000 description 1
- 206010052804 Drug tolerance Diseases 0.000 description 1
- 108010040721 Flagellin Proteins 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- 241000186781 Listeria Species 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 239000006137 Luria-Bertani broth Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 101800000795 Proadrenomedullin N-20 terminal peptide Proteins 0.000 description 1
- 208000015634 Rectal Neoplasms Diseases 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000033289 adaptive immune response Effects 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 208000022362 bacterial infectious disease Diseases 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 229940093761 bile salts Drugs 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 239000002158 endotoxin Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003495 flagella Anatomy 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 210000004211 gastric acid Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000028709 inflammatory response Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229920006008 lipopolysaccharide Polymers 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000013011 mating Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 102000007863 pattern recognition receptors Human genes 0.000 description 1
- 108010089193 pattern recognition receptors Proteins 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000001959 radiotherapy Methods 0.000 description 1
- 208000013718 rectal benign neoplasm Diseases 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 230000001018 virulence Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000001262 western blot 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
- C07K14/195—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
- C07K14/255—Salmonella (G)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
-
- 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
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
-
- 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
- C12R2001/42—Salmonella
-
- 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)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Public Health (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Molecular Biology (AREA)
- Diabetes (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Gastroenterology & Hepatology (AREA)
- Epidemiology (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Emergency Medicine (AREA)
- Endocrinology (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to the technical field of tumor biotherapy, and discloses recombinant salmonella typhimurium genetically engineered bacteria HCS1, a microbial inoculum and application thereof. The genetically engineered bacterium HCS1 is attenuated salmonella typhimurium which lacks relA genes and spoT genes, and the attenuated salmonella typhimurium is salmonella typhimurium VNP20009. The genetically engineered bacterium HCS1 not only can normally grow, but also can target to tumor sites and proliferate after being infected by tumor-bearing mice, and obviously reduces the toxicity of the genetically engineered bacterium HCS1 to organisms.
Description
Technical Field
The invention relates to the technical field of tumor biotherapy, in particular to recombinant salmonella typhimurium genetically engineered bacteria HCS1, a microbial inoculum and application thereof.
Background
Nowadays, cancer of all malignant tumors is generally pointed out, and cancer cells have characteristics of endogenous property, heterogeneity, mutation resistance and the like, so that the cancer cells become important and difficult in the field of the current biomedical development. Cancer is one of the major factors threatening human life health in the 21 st century, and therefore the search for therapeutic methods thereof is becoming increasingly important.
At present, the main treatment mode aiming at the tumor is the traditional comprehensive treatment of operation, radiotherapy and chemotherapy, and although the pain of a patient can be relieved to a certain extent and the tumor volume is reduced, the methods have obvious limitations, including causing tumor recurrence, drug tolerance and toxic and side effects of organisms.
In recent years, cancer researchers have developed a new type of cancer therapy, namely, bacterial-mediated oncology therapy, to address the problems of the traditional treatment methods in the clinic.
Tumor microenvironment refers to the cellular environment in which a tumor exists, which largely determines the survival and progression of the tumor. Because of the unique microenvironment such as hypoxia, immunosuppression, rich nutrition and the like caused by irregular vascular system in tumor tissues, facultative anaerobes can specifically colonise and reproduce, thereby showing the high targeting specificity of bacteria to tumors. Thus, a significant advantage of bacterial-mediated cancer treatment is that bacteria can selectively accumulate at tumor sites, where their concentration in the tumor can reach one thousand times, even more than ten thousand times, that of normal tissue. Bacteria which are widely studied at present comprise salmonella, listeria, escherichia coli and the like, and have remarkable anti-tumor activity.
During bacterial targeting of a tumor, a variety of Pathogen-associated molecular patterns (Pathosen-associated molecular patterns, PAMPs) present on the surface of bacterial cells, such as lipopolysaccharide, flagellin, nucleic acids, etc., can be recognized by pattern recognition receptors of the body, thereby activating corresponding inflammatory signaling pathways, recruiting immune cells, eliciting a strong inflammatory response, and killing tumor cells by further activating the body's innate and adaptive immune responses.
However, salmonella, which grow flagellum, can move, can evade the host's immune system in various ways, and can continuously infect the host, thus itself can cause toxicity to the body, creating a safety problem in bacterial treatment. On the other hand, if the ability of engineered bacteria to resist gastric acid and bile salts of a host is impaired when certain anticancer drugs are delivered to a tumor site, the ability to colonize the body is affected, and the effect of the tumor therapeutic drugs is greatly affected at this time.
Attenuated salmonella typhimurium is a facultative anaerobe, and has many hypoxic regions in tumor tissues, salmonella can be enriched in the hypoxic regions of the tumor, and the colony count in the tumor is far greater than that in other tissues. Although in preclinical experiments, attenuated salmonella VNP20009 has a very good anti-tumor effect, clinical first-stage research results indicate that VNP20009 has poor anti-tumor effect in humans, and is mainly expressed as follows: can not be effectively enriched in tumor tissues, and has insignificant anti-tumor effect.
Therefore, it is necessary to construct a bacterial strain that has a good tumor-inhibiting effect and is attenuated in virulence.
Disclosure of Invention
The invention aims to provide a bacterial strain which can reduce toxicity and damage to normal tissues of a body in the process of targeting a tumor by bacteria, so that the safety of the tumor targeted treatment by bacteria is improved.
In order to achieve the above object, a first aspect of the present invention provides a recombinant salmonella typhimurium genetically engineered bacterium HCS1, wherein the genetically engineered bacterium HCS1 is an attenuated salmonella typhimurium deleted of relA gene and spoT gene, and the attenuated salmonella typhimurium is salmonella typhimurium VNP20009;
the nucleotide sequence of the relA gene is shown as SEQ ID NO.1, and the nucleotide sequence of the spoT gene is shown as SEQ ID NO. 2.
In a second aspect, the present invention provides a microbial inoculum comprising the recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the first aspect.
In a third aspect, the present invention provides an application of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the first aspect or the microbial inoculum or the culture supernatant thereof described in the second aspect in preparing a targeted antitumor drug.
In a fourth aspect, the present invention provides the use of a recombinant salmonella typhimurium genetically engineered bacterium HCS1 as described in the first aspect or a bacterial agent as described in the second aspect or a culture supernatant thereof in the manufacture of a medicament for the treatment of an intestinal disorder.
In a fifth aspect, the present invention provides an application of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the first aspect or the microbial inoculum or the culture supernatant thereof described in the second aspect in preparing a medicament for treating cardiovascular and cerebrovascular diseases.
In a sixth aspect, the present invention provides a recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the foregoing first aspect or a bacterial agent described in the foregoing second aspect or a culture supernatant thereof for use in the preparation of a medicament for treating diabetes.
In a seventh aspect, the present invention provides an expression vector, which is the recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the first aspect.
An eighth aspect of the present invention provides a medicament comprising an active ingredient and an auxiliary material, wherein the active ingredient comprises the microbial inoculum or culture supernatant thereof described in the second aspect.
Compared with the prior art, the invention has at least the following advantages:
(1) The invention takes the attenuated salmonella typhimurium strain VNP20009 with high tumor targeting specificity and good cancer inhibiting effect as an original strain, carries out genetic engineering on the basis of the original strain, and obtains the genetically engineered bacterium HCS1 after knockout of the gene relA and spoT, wherein the genetically engineered bacterium HCS1 can still normally grow, and can target to tumor sites and proliferate after infection of tumor-bearing mice, and obviously reduce the toxicity of the genetically engineered bacterium HCS1 to organisms.
Drawings
FIG. 1 shows the Salmonella typhimurium VNP20009relA according to the present invention - Is a PCR electrophoresis identification map of (2);
fig. 2 and 3 are PCR electrophoresis identification diagrams of recombinant salmonella typhimurium genetically engineered bacteria HCS1 provided by the present invention;
FIG. 4 is a graph showing the growth of recombinant Salmonella typhimurium genetically engineered bacteria HCS1, control strain I and control strain II;
FIG. 5 is a graph showing the invasion results of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 and the control strain II provided by the invention on tumor cells MC38 and CT26 in vitro.
FIG. 6 is a graph showing the result of killing tumor cells MC38 and CT26 in vitro by the recombinant Salmonella typhimurium genetically engineered bacterium HCS1 and the control strain II provided by the invention.
FIG. 7 is a graph showing colony count results of colonic tumors colonised by recombinant salmonella typhimurium genetically engineered bacteria HCS1 injected at different doses and control strain II;
FIG. 8 is a graph showing the results of the change of serum transaminase levels of tumor-bearing mice after injection of recombinant salmonella typhimurium genetically engineered bacteria HCS1 and control strain II at different doses;
FIG. 9 is a graph showing the growth of tumor-bearing mice and tumors thereof after injection of recombinant Salmonella typhimurium genetically engineered bacteria HCS1 and control strain II at different doses, wherein A in FIG. 9 is a graph showing the change of tumor volume of the tumor-bearing mice over time, B in FIG. 9 is a graph showing the change of body weight of the tumor-bearing mice over time, and C in FIG. 9 is a graph showing the change of survival rate of the tumor-bearing mice over time;
FIG. 10 shows the protein expression detection patterns of the recombinant salmonella typhimurium genetically engineered bacteria HCS1/pBAD-ClyA and the control strain IIVNP 20009/pBAD-ClyA.
Detailed Description
The endpoints and any values of the ranges disclosed herein are not limited to the precise range or value, and are understood to encompass values approaching those ranges or values. For numerical ranges, one or more new numerical ranges may be found between the endpoints of each range, between the endpoint of each range and the individual point value, and between the individual point value, in combination with each other, and are to be considered as specifically disclosed herein.
As described above, the first aspect of the present invention provides a recombinant salmonella typhimurium genetically engineered bacterium HCS1, wherein the genetically engineered bacterium HCS1 is an attenuated salmonella typhimurium deleted of relA gene and spoT gene, and the attenuated salmonella typhimurium is salmonella typhimurium VNP20009;
the nucleotide sequence of the relA gene is shown as SEQ ID NO.1, and the nucleotide sequence of the spoT gene is shown as SEQ ID NO. 2.
According to a preferred embodiment, the genetically engineered bacterium HCS1 is constructed by a method comprising the steps of:
(1) Carrying out PCR amplification by adopting a forward primer I-1 and a reverse primer I-1 and taking a salmonella typhimurium strain VNP20009 genome as a template to obtain a relA gene upstream homology arm; adopting a forward primer I-2 and a reverse primer I-2, and carrying out PCR amplification by taking a salmonella typhimurium VNP20009 genome as a template to obtain a relA gene downstream homology arm; carrying out PCR amplification by using the forward primer I-1 and the reverse primer I-2 and using the upstream homology arm of the relA gene and the downstream homology arm of the relA gene as templates to obtain an amplification product I connected with the upstream homology arm of the relA gene and the downstream homology arm of the relA gene; and
carrying out PCR amplification by using a forward primer II-1 and a reverse primer II-1 and a salmonella typhimurium strain VNP20009 genome as a template to obtain a spoT gene upstream homology arm; carrying out PCR amplification by adopting a forward primer II-2 and a reverse primer II-2 and taking a salmonella typhimurium VNP20009 genome as a template to obtain a downstream homology arm of the spoT gene; carrying out PCR amplification by using the forward primer II-1 and the reverse primer II-2 and using the upstream homology arm of the spoT gene and the downstream homology arm of the spoT gene as templates to obtain an amplification product II connected with the upstream homology arm of the spoT gene and the downstream homology arm of the spoT gene;
(2) Ligating the amplification product I to a pDM4 plasmid to obtain a pDM4-relA plasmid; and ligating the amplification product II to a pDM4 plasmid to obtain a pDM4-spoT plasmid;
(3) Under the condition of conjugation transfer reaction, carrying out homologous recombination I-1 by adopting escherichia coli SM10 lambda pir transformed with the pDM4-relA plasmid as donor bacteria and salmonella typhimurium VNP20009 as acceptor bacteria to obtain salmonella typhimurium VNP20009-I; homologous recombination I-2 is carried out on the salmonella typhimurium VNP20009-I through reverse selection of sacB genes to obtain salmonella typhimurium VNP20009relA lacking relA genes - ；
(4) Under the condition of conjugal transfer reaction, using colibacillus SM10 lambda pir transformed with pDM4-spoT plasmid as donor bacteria and using Salmonella typhimurium VNP20009relA of deletion relA gene as receptor bacteria - Homologous recombination II-1 is carried out to obtain the salmonella typhimurium VNP20009relA - -I; the Salmonella typhimurium VNP20009relA was selected by reverse direction of sacB gene - -I homologous recombination II-2.
In the present invention, the salmonella typhimurium VNP20009 is deposited under accession number ATCC202165, commercially available from the american type biological resource collection (ATCC).
The method of PCR amplification is not particularly limited by the present invention, and those skilled in the art can select it according to the prior art means known in the art, and the following description of the present invention will exemplarily provide a preferred embodiment, and those skilled in the art should not understand the limitation of the present invention.
The method of transformation and conjugation transfer according to the present invention is not particularly limited, and those skilled in the art may select according to prior art means known in the art, and the transformation may be exemplified by a chemical transformation method, and the conjugation transfer may be performed by referring to the method of Protocols of Conjugative Plasmid Transfer in Salmonella:plate, broth, and Filter Mating Approaches published by khajachi BK et al.
Preferably, in the step (1), the nucleotide sequence of the forward primer I-1 is shown as SEQ ID NO.3, the nucleotide sequence of the reverse primer I-1 is shown as SEQ ID NO.4, the nucleotide sequence of the forward primer I-2 is shown as SEQ ID NO.5, and the nucleotide sequence of the reverse primer I-2 is shown as SEQ ID NO. 6.
Preferably, in the step (1), the nucleotide sequence of the forward primer II-1 is shown as SEQ ID NO.7, the nucleotide sequence of the reverse primer II-1 is shown as SEQ ID NO.8, the nucleotide sequence of the forward primer II-2 is shown as SEQ ID NO.9, and the nucleotide sequence of the reverse primer II-2 is shown as SEQ ID NO. 10.
The recombinant salmonella typhimurium genetically engineered bacterium HCS1 provided by the invention can generate a large number of living thalli of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 through liquid culture, and the culture method has no special requirement, so long as the recombinant salmonella typhimurium genetically engineered bacterium HCS1 can be proliferated, for example, the recombinant salmonella typhimurium genetically engineered bacterium HCS1 can be proliferated according to the volume ratio of 1:100 inoculum size the live bacteria of the recombinant salmonella typhimurium genetically engineered bacteria HCS1 are inoculated into a salmonella culture medium and cultured for 3-5 hours at 37 ℃ to obtain a culture solution. The salmonella media may be various media suitable for salmonella culture known in the art, for example, LB media.
The present invention may further isolate the live bacterial cells of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 in the above-mentioned culture solution, and the isolation method is not particularly limited as long as the bacterial cells can be enriched from the culture solution, for example, by centrifugation and/or filtration, and the conditions of the centrifugation and the filtration may be known conditions, and the present invention will not be described herein.
As described above, the second aspect of the present invention provides a microbial agent comprising the recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the first aspect.
In the present invention, the form of the microbial inoculum may be a form of microbial inoculum conventional in the art, for example, may be a solid, liquid or semisolid form.
Preferably, the microbial inoculum contains living bacteria of the recombinant salmonella typhimurium genetically engineered bacteria HCS1.
In the present invention, the number of living cells in the microbial inoculum may be selected within a wide range, and may be, for example, 10 as long as the requirements of the relevant standards are satisfied 8 cfu/g microbial inoculum above.
In the present invention, the preparation method of the microbial inoculum can refer to the conventional preparation method in the field, and will not be described herein.
As described above, the third aspect of the present invention provides an application of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the foregoing first aspect or the microbial inoculum or the culture supernatant thereof described in the foregoing second aspect in preparing a targeted antitumor drug.
As described above, the fourth aspect of the present invention provides the use of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the foregoing first aspect or the microbial inoculum or culture supernatant thereof described in the foregoing second aspect in the preparation of a medicament for treating an intestinal disease.
As described above, the fifth aspect of the present invention provides the use of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the foregoing first aspect or the microbial inoculum or the culture supernatant thereof described in the foregoing second aspect in the preparation of a medicament for treating cardiovascular and cerebrovascular diseases.
As described above, the sixth aspect of the present invention provides the use of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the foregoing first aspect or the microbial agent described in the foregoing second aspect or the culture supernatant thereof in the preparation of a medicament for treating diabetes.
As described above, the seventh aspect of the present invention provides an expression vector which is the recombinant salmonella typhimurium genetically engineered bacterium HCS1 described in the first aspect.
Preferably, the tumor is at least one of a colon tumor and a rectal tumor.
In the present invention, the preparation method of the culture supernatant may be selected conventionally in the art, and may be, for example: inoculating the strain or the microbial inoculum into a salmonella culture medium for culture to obtain a fermentation liquor, centrifuging the fermentation liquor to obtain a bacterial precipitate, washing with PBS buffer solution, and then resuspending to obtain a culture supernatant.
As described above, according to an eighth aspect of the present invention, there is provided a medicament comprising an active ingredient comprising the microbial agent of the second aspect or a culture supernatant thereof and an auxiliary material.
Preferably, the auxiliary material is at least one selected from microcrystalline cellulose, cyclodextrin, starch, polyvinyl alcohol, cocoa butter, calcium phosphate and calcium carbonate.
The invention will be described in detail below by way of examples.
In the examples below, unless otherwise indicated, the methods are conventional in the art and the reagents are commercially available.
Experimental strains:
coli SM10 λpir: available from BCCM company under the number LMBP 3889;
control strain I: wild salmonella typhimurium 14028s: purchased from ATCC company under deposit number ATCC 14028S;
control strain II: attenuated salmonella typhimurium VNP20009: purchased from ATCC company under deposit number ATCC202165;
control strain III: the wild type Salmonella typhimurium 14028s lacking the relA gene and spoT gene is from the Jung-Joon Min professor task group of the national university of south China, and the details can be referred to the Genetically engineered Salmonella typhimurium as an imageable therapeutic probe for cancer paper published by Vu H Nguyen et al.
Culture medium:
LB solid medium: weighing 25g of LB culture medium powder and 15g of agar powder, dissolving in 1000mL of double distilled water, sterilizing at 121 ℃ for 20min under high pressure, pouring a proper amount of the culture medium into a sterilized culture dish after the temperature of the culture medium is reduced to 55+/-5 ℃, and preserving at 4 ℃ for later use after solidification;
LB liquid medium: weighing 25g of LB culture medium powder, dissolving in 1000mL of double distilled water, sterilizing at 121 ℃ for 20min under high pressure, cooling, and preserving at 4 ℃ for later use;
PBS buffer: 8g of NaCl, 200mg of KCl and 1.44g of Na are weighed 2 HPO 4 240mg KH 2 PO 4 Dissolving in 800mL double distilled water, regulating pH to 7.4 with KCl, constant volume to 1L, autoclaving at 121deg.C for 20min, cooling, and preserving at 4deg.C.
Experimental mice:
6-8 week old C57BL/6 mice: purchased from Jiangsu Jizhikang biotechnology Co.
Construction of a subcutaneous tumor model of MC38 mice (tumor-bearing mice):
6-8 weeks after shaving, C57BL/6 females were anesthetized in an anesthesia cassette containing 2vol% isoflurane, and 50. Mu.L of MC38 cell suspension (containing 1X 10) was injected subcutaneously on the right side of each mouse using a 0.5mL syringe 6 Individual cells).
The construction of the inducible expression plasmid pBAD-ClyA can be carried out by reference to the method of Genetically engineered Salmonella typhimurium as an imageable therapeutic probe for cancer issued to Vu H Nguyen et al.
Construction of Strain HCS 1:
(1) Carrying out PCR amplification by adopting a forward primer I-1 and a reverse primer I-1 and taking a salmonella typhimurium strain VNP20009 genome as a template to obtain a relA gene upstream homology arm; adopting a forward primer I-2 and a reverse primer I-2, and carrying out PCR amplification by taking a salmonella typhimurium VNP20009 genome as a template to obtain a relA gene downstream homology arm;
the nucleotide sequence of the forward primer I-1 is shown as SEQ ID NO.3, the nucleotide sequence of the reverse primer I-1 is shown as SEQ ID NO.4, the nucleotide sequence of the forward primer I-2 is shown as SEQ ID NO.5, and the nucleotide sequence of the reverse primer I-2 is shown as SEQ ID NO. 6;
and
Carrying out PCR amplification by using a forward primer II-1 and a reverse primer II-1 and a salmonella typhimurium strain VNP20009 genome as a template to obtain a spoT gene upstream homology arm; carrying out PCR amplification by adopting a forward primer II-2 and a reverse primer II-2 and taking a salmonella typhimurium VNP20009 genome as a template to obtain a downstream homology arm of the spoT gene; carrying out PCR amplification by using the forward primer I-1 and the reverse primer I-2 and using the upstream homology arm of the relA gene and the downstream homology arm of the relA gene as templates to obtain an amplification product I connected with the upstream homology arm of the relA gene and the downstream homology arm of the relA gene;
the nucleotide sequence of the forward primer II-1 is shown as SEQ ID NO.7, the nucleotide sequence of the reverse primer II-1 is shown as SEQ ID NO.8, the nucleotide sequence of the forward primer II-2 is shown as SEQ ID NO.9, and the nucleotide sequence of the reverse primer II-2 is shown as SEQ ID NO. 10;
the procedure for PCR amplification of the upstream homology arm of the relA gene was: preheating at 95 ℃ for 5min to enable the template to be fully denatured, and then entering an amplification cycle; in each amplification cycle, the template is denatured by holding at 95 ℃ for 15 seconds, then the temperature is reduced to 62 ℃ for 30 seconds to enable the primer and the template to be fully annealed; maintaining at 72 deg.c for 1min to make primer extend on the template to synthesize DNA and complete one amplification cycle, and repeating the amplification cycle for 30 times; finally, keeping at 72 ℃ for 5min to enable the product to extend completely, and preserving at 4 ℃ to obtain a relA gene upstream homology arm;
the procedure for PCR amplification of the homology arm upstream of spoT gene was identical to that of the homology arm upstream of relA gene;
the procedure for PCR amplification of the downstream homology arm of the relA gene and the downstream homology arm of the spoT gene was the same and similar to that of the upstream homology arm of the relA gene, except that:
after the primer and the template are fully annealed, the temperature is maintained at 72 ℃ for 30 seconds, so that the primer extends on the template to synthesize DNA, and one amplification cycle is completed;
the procedure for PCR amplification of amplification product I and amplification product II was the same and similar to that for PCR amplification of the homology arm upstream of the relA gene, except that:
after the primer and the template are fully annealed, the temperature is kept at 72 ℃ for 90 seconds, so that the primer extends on the template to synthesize DNA, and one amplification cycle is completed;
the reaction systems in the above PCR amplifications are all the same and are shown in Table 1:
TABLE 1
| Component (A) | Volume (mu L) |
| 2×Phanta Max Buffer | 25 |
| Forward primer (10. Mu.M) | 2 |
| Reverse primer (10. Mu.M) | 2 |
| dNTP Mix | 1 |
| DNA polymerase | 1 |
| Double distilled water | 19 |
The upstream homology arm (nucleotide sequence shown as SEQ ID NO. 11) of the relA gene, the downstream homology arm (nucleotide sequence shown as SEQ ID NO. 12) of the relA gene, the upstream homology arm (nucleotide sequence shown as SEQ ID NO. 13) of the spoT gene, the downstream homology arm (nucleotide sequence shown as SEQ ID NO. 14) of the spoT gene, the amplified product I (nucleotide sequence shown as SEQ ID NO. 1) and the amplified product II (nucleotide sequence shown as SEQ ID NO. 2) are respectively quantified by agarose gel electrophoresis, and cut off from gel for separation and purification;
(2) Respectively enzyme cutting the amplified product I, the amplified product II and the pDM4 plasmid, and respectively connecting the enzyme-cut amplified product I and the enzyme-cut amplified product II to the enzyme-cut pDM4-relA plasmid; respectively obtaining pDM4-relA plasmid and pDM4-spoT plasmid;
(3) Under the condition of conjugation transfer reaction, carrying out homologous recombination I-1 by adopting escherichia coli SM10 lambda pir transformed with the pDM4-relA plasmid as donor bacteria and salmonella typhimurium VNP20009 as acceptor bacteria, and screening in LB solid medium containing 0.01wt% of chloramphenicol to obtain salmonella typhimurium VNP20009-I containing the pDM4-relA plasmid;
selecting 10-15 bacterial colonies, and performing amplification culture in an LB liquid culture medium for 5 hours to obtain bacterial liquid; the bacterial liquid is diluted to different concentration gradients (10 respectively) by LB liquid culture medium -5 cfu/mL、10 -6 cfu/mL、10 -7 cfu/mL), plated onto LB solid medium containing 10wt% sucrose, cultured overnight at 37 ℃; homologous recombination I-2 is carried out on salmonella typhimurium VNP20009-I by reverse selection of sacB gene in pDM4-relA plasmid, 100 single colonies are selected from LB solid medium containing 10wt% of sucrose after 24 hours, streaked on LB solid medium and LB solid medium containing 0.01wt% of chloramphenicol respectively, cultured overnight at 37 ℃, colonies incapable of growing on LB solid medium containing 0.01wt% of chloramphenicol are selected, and PCR identification is carried out by adopting forward primer I-1 and reverse primer I-2;
picking a colony capable of obtaining an amplified product I after PCR amplification, streaking on an LB solid medium for 24 hours, and selecting a single colony to respectively carry out PCR identification by adopting a forward primer I-1, a reverse primer I-2 and a forward primer III and a reverse primer III for amplifying a relA gene, wherein the forward primer III and the reverse primer III are used for amplifying a relA gene, and the specific reference is shown in figure 1;
the nucleotide sequence of the forward primer III is shown as SEQ ID NO.15, and the nucleotide sequence of the reverse primer III is shown as SEQ ID NO. 16;
wherein, the forward primer I-1 and the reverse primer I-2 can obtain an amplified product I after PCR amplification, and the forward primer III and the reverse primer III for amplifying the relA gene can not obtain a colony of the amplified product I after PCR amplification, namely, salmonella typhimurium VNP20009relA with deleted relA gene - ；
Selecting single bacterial colony, and performing amplification culture in LB liquid medium to obtain Salmonella typhimurium VNP20009relA - Bacterial liquid; adding 50vol% of glycerol, and freezing at-80 ℃ for later use;
(4) Under the condition of conjugal transfer reaction, using colibacillus SM10 lambda pir transformed with pDM4-spoT plasmid as donor bacteria and using Salmonella typhimurium VNP20009relA of deletion relA gene as receptor bacteria - Homologous recombination II-1 is carried out, and salmonella typhimurium VNP20009relA containing pDM4-spoT plasmid is obtained by screening in LB solid medium containing 0.01wt% chloramphenicol - -I；
Selecting 10-15 bacterial colonies, and performing amplification culture in an LB liquid culture medium for 5 hours to obtain bacterial liquid; the bacterial liquid is diluted to different concentration gradients (10 respectively) by LB liquid culture medium -5 cfu/mL、10 -6 cfu/mL、10 -7 cfu/mL), plated onto LB solid medium containing 10wt% sucrose, cultured overnight at 37 ℃; salmonella typhimurium VNP20009relA by reverse selection of sacB gene in pDM4-spoT plasmid - After homologous recombination II-2 is carried out for 24 hours, 100 single colonies are selected from LB solid medium containing 10wt% of sucrose, streaked on LB solid medium and LB solid medium containing 0.01wt% of chloramphenicol respectively, cultured overnight at 37 ℃, colonies which cannot grow on LB solid medium containing 0.01wt% of chloramphenicol are selected, and PCR identification is carried out by using forward primer II-1 and reverse primer II-2;
picking a colony capable of obtaining an amplified product II after PCR amplification, streaking on an LB solid medium for 24 hours, and selecting a single colony to respectively carry out PCR identification by adopting a forward primer II-1 and a reverse primer II-2 and a forward primer IV and a reverse primer IV for amplifying spoT genes, wherein the forward primer IV and the reverse primer IV are used for amplifying spoT genes, and the specific reference is shown in figures 2 and 3;
the nucleotide sequence of the forward primer IV is shown as SEQ ID NO.17, and the nucleotide sequence of the reverse primer IV is shown as SEQ ID NO. 18;
the forward primer II-1 and the reverse primer II-2 can obtain an amplified product II after PCR amplification, and the forward primer IV and the reverse primer IV used for amplifying the spoT gene can not obtain a colony of the amplified product II after PCR amplification, namely, attenuated salmonella typhimurium (namely HCS 1) with the relA gene and the spoT gene deleted;
selecting single bacterial colony, and performing amplification culture in LB liquid culture medium to obtain recombinant salmonella typhimurium genetically engineered bacterium HCS1 bacterial liquid; and 50vol% of glycerol is added, and the mixture is frozen at the temperature of-80 ℃ for standby.
FIG. 1 shows the Salmonella typhimurium VNP20009relA according to the present invention - Is a PCR electrophoresis identification map of (C). In FIG. 1, M is an indicator band of DNA molecular weight, lanes 1-9 are all stabilized and passaged Salmonella typhimurium VNP20009relA - PCR of colonies, lanes 10-11 are PCR of colonies of attenuated Salmonella ΔppGpp strain (i.e., strain obtained after deletion of relA gene and spoT gene of control strain I), lanes 12-13 are colony PCR of control strain II, and primers used in lanes 1-13 are forward primer I-1 and reverse primer I-2; lanes 14-22 are Salmonella typhimurium VNP20009relA corresponding to lanes 1-9 - Colony PCR,23 is colony PCR of attenuated salmonella ΔppGpp strain, 24 is colony PCR of control strain II, and primers used in lanes 14-23 are forward primer III and reverse primer III for amplifying relA gene.
Fig. 2 and 3 are PCR electrophoresis identification diagrams of recombinant salmonella typhimurium genetically engineered bacteria HCS1 provided by the present invention. In FIG. 2, M is an indicator strip of DNA molecular standard weight, lanes 1-7 are colony PCR of recombinant salmonella typhimurium engineering bacteria HCS1 after stable passage, lane 8 is colony PCR of attenuated salmonella ΔppGpp strain (i.e. strain obtained after deletion of relA gene and spoT gene of control strain I), lane 9 is colony PCR of control strain II, and primers used in lanes 1-9 are forward primer II-1 and reverse primer II-2;
in FIG. 3, M is an indicator band of DNA molecular weight, lanes 1 to 7 are colony PCR of recombinant Salmonella typhimurium engineering bacteria HCS1 after stable passage, lane 8 is colony PCR of attenuated Salmonella ΔppGpp strain (i.e., strain obtained after deletion of relA gene and spoT gene by control strain I), lane 9 is colony PCR of control strain II, and primers used in lanes 1 to 9 are forward primer IV and reverse primer IV for amplifying spoT gene.
Example 1
This example was used to determine the growth curves of strain HCS1 as well as the control strain.
Respectively picking single colonies of the strain HCS1, the control strain I and the control strain II, inoculating the single colonies into an LB liquid culture medium, and shaking overnight by adopting a shaking table at a rotating speed of 200rpm to obtain bacterial liquid;
inoculating the above bacterial solutions into 30mL of LB liquid medium to obtain initial concentration of 1×10 7 cfu/mL of test bacterial liquid is taken for 1h, 2h, 4h, 6h, 8h, 10h, 12h and 24h after inoculation, and OD is measured 600 Values, a graph of the growth of each bacterium was obtained, see in particular fig. 4.
Fig. 4 is a growth curve diagram of the recombinant salmonella typhimurium genetically engineered bacteria HCS1, the control strain I and the control strain II provided by the invention. As can be seen from fig. 4, the knockout of the genes relA and spoT did not affect the entry of the obtained genetically engineered bacteria HCS1 into the logarithmic growth phase and subsequent growth compared to the control strain II.
Example 2
This example was used to determine the invasion of the tumor cells by the strain HCS1 and the control strain in vitro.
Co-culturing HCS1 and control strain II with CT26 and MC38 tumor cells respectively at MOI=100 infection complex number in cell incubator for 1 hr, then culturing with gentamycin culture medium containing 150 μg/mL for 30min, removing culture medium, cleaning, and diluting and spotting the lysed cells with dilution gradient of 10 0 、10 -1 、10 -2 、10 -3 The number of bacteria invading into cells under the same conditions was counted after culturing, and the specific results are shown in FIG. 5.
FIG. 5 is a graph showing the invasion results of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 and the control strain II provided by the invention on tumor cells MC38 and CT26 in vitro. As can be seen from fig. 5, at a multiplicity of infection with moi=100, the number of invasion of the recombinant salmonella typhimurium genetically engineered bacteria HCS1 to both tumor cells was significantly less than that of the control strain II.
Example 3
This example was used to determine the killing capacity of the strain HCS1 and the control strain against tumor cells in vitro.
Lactate Dehydrogenase (LDH) is an extremely stable cytoplasmic enzyme, but once the cell membrane is broken, LDH is released outside the cell. Thus, the effect of bacteria on cell integrity can be reflected by measuring the level of LDH in the cell culture supernatant after bacterial infection, thus exploring the killing effect of bacteria on cells.
Bacterial strain HCS1 and control bacterial strain II were co-cultured with CT26 and MC38 tumor cells, respectively, in a cell culture incubator at a multiplicity of infection of moi=100 for 6h, the medium was removed and washed, then further cultured with a medium containing 150 μg/mL gentamicin for 12h, and the cell culture supernatant was collected to determine the release amount of LDH therein, and the specific results are shown in fig. 6.
FIG. 6 is a graph showing the result of killing tumor cells MC38 and CT26 in vitro by the recombinant Salmonella typhimurium genetically engineered bacterium HCS1 and the control strain II provided by the invention. As can be seen from fig. 6, infection with the strain HCS1 significantly reduced the LDH content in the tumor cell MC38 and CT26 culture compared to the control strain II, indicating that the killing effect of the strain HCS1 on cytotoxicity is significantly smaller than the control strain II under in vitro conditions.
Example 4
This example was used to determine the colonisation of tumor-bearing mice with bacterial strain HCS1 and control strains.
Intravenous injection of 1.0X10 to the tail of tumor-bearing mice 7 CFUs、5.0×10 7 CFUs、1.0×10 8 Strains of CFUs HCS1, 1.0X10 7 Control strain II of CFUs and PBS buffer; on day 3 after injection, spleen, liver, tumor, etc. tissues of mice were taken.
The tissues after weight measurement were placed in PBS bufferHomogenizing to obtain liquid to be tested, and adopting 10-time gradient dilution method to make the concentration gradient of liver and spleen respectively be 10 -1 、10 -2 、10 -3 Tumor concentration gradients of 10 respectively -3 、10 -4 、10 -5 The test solution (B) was spread on LB solid medium, cultured overnight at 37℃and then subjected to viable colony counting, and the concentration of bacteria (CFUs/g) in the corresponding tissue was calculated from the weight of the above tissue, and the specific results are shown in FIG. 7.
FIG. 7 is a graph showing colony count results of colonic tumors colonised by recombinant Salmonella typhimurium genetically engineered bacteria HCS1 injected at different doses and control strain II. As can be seen from fig. 7, the colonization of the liver and spleen tissue by the strain HCS1 was significantly reduced compared to the control strain II, while the colonization of the tumor site by the high dose of the strain HCS1 was not significantly affected.
Example 5
This example was used to determine the serum transaminase changes of tumor-bearing mice on day 7 after injection of strain HCS1 and control strain.
Intravenous injection of 1.0X10 to the tail of tumor-bearing mice 7 CFUs、5.0×10 7 CFUs、1.0×10 8 Strains of CFUs HCS1, 1.0X10 7 Control strain II of CFUs and PBS buffer; on day 7 after injection, heart was bled and serum was isolated, and then hospitals were assigned to examine the level of transaminase (including glutamic-pyruvic transaminase and aspartate transaminase) in serum, and the specific results are shown in fig. 8.
FIG. 8 is a graph showing the results of the changes in serum transaminase levels of tumor-bearing mice after injection of different doses of recombinant Salmonella typhimurium genetically engineered bacteria HCS1 and control strain II. As can be seen from fig. 8, compared with the control strain II, the alanine Aminotransferase (ALT) and aspartate Aminotransferase (AST) contents in the blood of tumor-bearing mice treated with the strain HCS1 were significantly reduced, indicating that the strain HCS1 was able to significantly attenuate liver damage to the body after bacterial treatment.
Example 6
This example was used to determine the inhibition of tumors in tumor-bearing mice by strain HCS1 and control strains.
Respectively to the chargesIntravenous injection of 1.0X10 to the tail of tumor mice 7 CFUs、5.0×10 7 CFUs、1.0×10 8 Strains of CFUs HCS1, 1.0X10 7 Control strain II of CFUs and PBS buffer; tumor volumes and body weights of mice were recorded at 3d, 6d, 9d, 12d, 15d post injection, and survival rates of mice over injection time were recorded, with specific results shown in fig. 8.
FIG. 9 is a graph showing the growth of tumor-bearing mice and tumors thereof after injection of recombinant Salmonella typhimurium genetically engineered bacteria HCS1 and control strain II at different doses. As can be seen from fig. 8, compared with the control strain II, the strain HCS1 has significantly reduced weight impact on tumor-bearing mice and significantly improved survival rate of tumor-bearing mice, and furthermore, tumor growth of tumor-bearing mice is significantly retarded or even eliminated after treatment with high dose of strain HCS1.
Example 7
This example was used to test the ability of HCS1 strains as protein expression delivery vehicles.
The strain HCS1 and the control strain II are respectively transferred into an induced expression plasmid pBAD-ClyA, and the recombinant strains HCS1/pBAD-ClyA and VNP20009/pBAD-ClyA are obtained after culture.
Single colonies were picked and cultured overnight in liquid medium at a volume ratio of 1:100 were inoculated into a new liquid medium for continuous culture for 4 hours, then were induced to culture with arabinose at a final concentration of 0.2wt% for 6 hours, and the bacterial liquid was collected. Western-Blot immunoblotting method is used for detecting expression of ClyA protein, and the loading amount of each hole is 5 multiplied by 10 7 CFUs, the specific results are shown in fig. 10.
FIG. 10 shows the protein expression detection patterns of the recombinant salmonella typhimurium genetically engineered bacteria HCS1/pBAD-ClyA and the control strain IIVNP 20009/pBAD-ClyA. Wherein M represents a standard protein molecule, sample 1 is non-induced VNP20009/pBAD-ClyA, sample 2 is induced VNP20009/pBAD-ClyA, sample 3 is non-induced HCS1/pBAD-ClyA, sample 4 is induced HCS1/pBAD-ClyA, and the ClyA protein size is 34kDa. As can be seen from fig. 9, the protein expression ability of the strain HCS1 was not affected compared to the control strain II.
Example 8
This example was used to determine the median lethal dose of the strain HCS1 and the control strain.
6-8 week old C57BL/6 mice were divided into 6 groups (16 mice per group), 2 of which were given tail intravenous injection 10 8 CFUs、10 9 Strain of CFUs HCS1, group 2 tail vein injection 10 7 CFUs、10 8 Control strain II of CFUs, group 2 tail vein injection 10 8 CFUs、10 9 Control strain III of CFUs; mice were continuously monitored for mortality after injection of different doses of bacteria for 7 days post injection and recorded, and half the lethal dose was calculated, with the specific results shown in table 1.
Half lethal dose: represents the minimum bacterial dose required to cause half of a given animal of a given weight or age to die within a prescribed period of time by specifying the route of infection.
Table 1:
as can be calculated from the results in Table 1, the median lethal doses of the strain HCS1, the control strain II and the control strain III were 4.72X10, respectively 8 CFUs、6.54×10 6 CFUs、1.72×10 8 CFUs。
The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, a number of simple variants of the technical solution of the invention are possible, including combinations of the individual technical features in any other suitable way, which simple variants and combinations should likewise be regarded as being disclosed by the invention, all falling within the scope of protection of the invention.
Claims (9)
1. The recombinant salmonella typhimurium genetically engineered bacterium HCS1 is characterized in that the genetically engineered bacterium HCS1 is attenuated salmonella typhimurium deleted of relA gene and spoT gene, the attenuated salmonella typhimurium is salmonella typhimurium VNP20009, and the preservation number is ATCC202165;
the nucleotide sequence of the relA gene is shown as SEQ ID NO.1, and the nucleotide sequence of the spoT gene is shown as SEQ ID NO. 2.
2. A method for constructing the recombinant salmonella typhimurium genetically engineered bacterium HCS1 of claim 1, comprising the steps of:
(1) Carrying out PCR amplification by adopting a forward primer I-1 and a reverse primer I-1 and taking a salmonella typhimurium strain VNP20009 genome as a template to obtain a relA gene upstream homology arm; adopting a forward primer I-2 and a reverse primer I-2, and carrying out PCR amplification by taking a salmonella typhimurium VNP20009 genome as a template to obtain a relA gene downstream homology arm; carrying out PCR amplification by using the forward primer I-1 and the reverse primer I-2 and using the upstream homology arm of the relA gene and the downstream homology arm of the relA gene as templates to obtain an amplification product I connected with the upstream homology arm of the relA gene and the downstream homology arm of the relA gene; and
carrying out PCR amplification by using a forward primer II-1 and a reverse primer II-1 and a salmonella typhimurium strain VNP20009 genome as a template to obtain a spoT gene upstream homology arm; carrying out PCR amplification by adopting a forward primer II-2 and a reverse primer II-2 and taking a salmonella typhimurium VNP20009 genome as a template to obtain a downstream homology arm of the spoT gene; carrying out PCR amplification by using the forward primer II-1 and the reverse primer II-2 and using the upstream homology arm of the spoT gene and the downstream homology arm of the spoT gene as templates to obtain an amplification product II connected with the upstream homology arm of the spoT gene and the downstream homology arm of the spoT gene;
(2) Ligating the amplification product I to a pDM4 plasmid to obtain a pDM4-relA plasmid; and ligating the amplification product II to a pDM4 plasmid to obtain a pDM4-spoT plasmid;
(3) Under the condition of conjugation transfer reaction, carrying out homologous recombination I-1 by adopting escherichia coli SM10 lambda pir transformed with the pDM4-relA plasmid as donor bacteria and salmonella typhimurium VNP20009 as acceptor bacteria to obtain salmonella typhimurium VNP20009-I; homologous recombination I-2 is carried out on the salmonella typhimurium VNP20009-I through reverse selection of sacB genes to obtain a deletion relA groupSalmonella typhimurium VNP20009relA - ；
(4) Under the condition of conjugal transfer reaction, using colibacillus SM10 lambda pir transformed with pDM4-spoT plasmid as donor bacteria and using Salmonella typhimurium VNP20009relA of deletion relA gene as receptor bacteria - Homologous recombination II-1 is carried out to obtain the salmonella typhimurium VNP20009relA - -I; the Salmonella typhimurium VNP20009relA was selected by reverse direction of sacB gene - And (3) carrying out homologous recombination II-2 to obtain the recombinant salmonella typhimurium genetically engineered bacterium HCS1.
3. The method according to claim 2, wherein in step (1), the nucleotide sequence of the forward primer I-1 is shown as SEQ ID NO.3, the nucleotide sequence of the reverse primer I-1 is shown as SEQ ID NO.4, the nucleotide sequence of the forward primer I-2 is shown as SEQ ID NO.5, and the nucleotide sequence of the reverse primer I-2 is shown as SEQ ID NO. 6.
4. A method according to claim 2 or 3, wherein in step (1), the nucleotide sequence of the forward primer II-1 is shown in SEQ ID No.7, the nucleotide sequence of the reverse primer II-1 is shown in SEQ ID No.8, the nucleotide sequence of the forward primer II-2 is shown in SEQ ID No.9, and the nucleotide sequence of the reverse primer II-2 is shown in SEQ ID No. 10.
5. A microbial inoculum, characterized in that the microbial inoculum contains the recombinant salmonella typhimurium genetically engineered bacterium HCS1 of claim 1.
6. The microbial inoculum according to claim 5, wherein the microbial inoculum contains viable cells of the recombinant salmonella typhimurium genetically engineered bacterium HCS1.
7. The use of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 of claim 1 or the microbial inoculum of claim 5 or 6 in the preparation of a medicament for treating colon tumor.
8. The use of the recombinant salmonella typhimurium genetically engineered bacterium HCS1 of claim 1 in protein expression delivery.
9. A medicament for treating colon tumor, which is characterized in that the medicament contains an active ingredient and auxiliary materials, wherein the active ingredient contains the microbial inoculum of claim 5 or 6; and/or the number of the groups of groups,
the auxiliary material is at least one selected from microcrystalline cellulose, cyclodextrin, starch, polyvinyl alcohol, cocoa butter, calcium phosphate and calcium carbonate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310320028.2A CN116478895B (en) | 2023-03-28 | 2023-03-28 | Recombinant salmonella typhimurium genetically engineered bacterium HCS1, microbial inoculum and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310320028.2A CN116478895B (en) | 2023-03-28 | 2023-03-28 | Recombinant salmonella typhimurium genetically engineered bacterium HCS1, microbial inoculum and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116478895A CN116478895A (en) | 2023-07-25 |
| CN116478895B true CN116478895B (en) | 2024-03-19 |
Family
ID=87222323
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310320028.2A Active CN116478895B (en) | 2023-03-28 | 2023-03-28 | Recombinant salmonella typhimurium genetically engineered bacterium HCS1, microbial inoculum and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116478895B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20060103971A (en) * | 2005-03-29 | 2006-10-09 | 주식회사 미래바이오 | Ppgpp-defective mutant salmonella and the live vaccine containing the same |
| KR20130102668A (en) * | 2012-03-07 | 2013-09-23 | 전남대학교산학협력단 | Salmonellae having improved inducible gene expression and use thereof |
| CN105462907A (en) * | 2015-11-02 | 2016-04-06 | 四川农业大学 | Attenuation salmonella typhimurium and construction method and application |
| KR20160062289A (en) * | 2014-11-24 | 2016-06-02 | 한국원자력연구원 | Novel Salmonella for treatment of cancer, and use thereof |
| KR20160137903A (en) * | 2016-11-22 | 2016-12-01 | 한국원자력연구원 | Novel Salmonella for treatment of cancer, and use thereof |
| CN110747155A (en) * | 2019-11-12 | 2020-02-04 | 湖南大学 | Attenuated recombinant engineering bacterium, preparation method and application thereof, and tumor targeted drug |
| CN115717120A (en) * | 2021-08-26 | 2023-02-28 | 湖南大学 | Controllable growth engineering bacterium and construction method and application thereof |
-
2023
- 2023-03-28 CN CN202310320028.2A patent/CN116478895B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20060103971A (en) * | 2005-03-29 | 2006-10-09 | 주식회사 미래바이오 | Ppgpp-defective mutant salmonella and the live vaccine containing the same |
| KR20130102668A (en) * | 2012-03-07 | 2013-09-23 | 전남대학교산학협력단 | Salmonellae having improved inducible gene expression and use thereof |
| KR20160062289A (en) * | 2014-11-24 | 2016-06-02 | 한국원자력연구원 | Novel Salmonella for treatment of cancer, and use thereof |
| CN105462907A (en) * | 2015-11-02 | 2016-04-06 | 四川农业大学 | Attenuation salmonella typhimurium and construction method and application |
| KR20160137903A (en) * | 2016-11-22 | 2016-12-01 | 한국원자력연구원 | Novel Salmonella for treatment of cancer, and use thereof |
| CN110747155A (en) * | 2019-11-12 | 2020-02-04 | 湖南大学 | Attenuated recombinant engineering bacterium, preparation method and application thereof, and tumor targeted drug |
| CN115717120A (en) * | 2021-08-26 | 2023-02-28 | 湖南大学 | Controllable growth engineering bacterium and construction method and application thereof |
Non-Patent Citations (8)
| Title |
|---|
| Comparison of anticancer activities and biosafety between Salmonella enterica Serovar Typhimurium ΔppGpp and VNP20009 in a murine cancer model;Liu X等;《Frontiers in Microbiology》;第13卷;Article 914575 * |
| Effect of Salmonella treatment on an implanted tumor (CT26) in a mouse model;Yun M等;《The Journal of Microbiology》;第50卷(第3期);第502–510页 * |
| Engineered oncolytic bacteria HCS1 exerts high immune stimulation and safety profiles for cancer therapy;Yujie S等;《Theranostics》;第13卷(第15期);第5546-5560页 * |
| Immune response induced by Salmonella typhimurium defective in ppGpp synthesis;Na HS等;《Vaccine》;第24卷(第12期);第2027-2034页 * |
| Phase I study of the intravenous administration of attenuated Salmonella typhimurium to patients with metastatic melanoma;Toso JF等;《Journal of Clinical Oncology》;第20卷(第1期);第142-152页 * |
| ppGpp-dependent stationary phase induction of genes on Salmonella pathogenicity island 1;Song M等;《The Journal of Biological Chemistry》;第279卷(第33期);第34183-34190页 * |
| Targeted cancer therapy using engineered Salmonella typhimurium;Zheng JH等;《Chonnam Medical Journal》;第52卷(第3期);第173-184页 * |
| 减毒沙门氏菌在抗肿瘤治疗中的应用;蒋如如等;《基础医学与临床》;第39卷(第9期);第1356-1360页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116478895A (en) | 2023-07-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101155911B (en) | 5 FU 5 fluorouracil tolerance bacterium and preparation method thereof | |
| CN102046190B (en) | Therapeutic agent for anaerobic diseases | |
| CA2342040C (en) | Anaerobic bacterium as a drug for cancer gene therapy | |
| CN111100844B (en) | Separation and application of salmonella bacteriophage RDP-SA-17118 | |
| CN114574390B (en) | Bifidobacterium longum subspecies infantis for relieving colonitis and application thereof | |
| CN112662635B (en) | High-temperature-resistant Escherichia coli bacteriophage RDP-EC-20031 and application thereof | |
| CN102741407A (en) | Transformation plasmid | |
| US20210079405A1 (en) | Nucleic Acid Systems That Enable Bacteria to Specifically Target Solid Tumors via Glucose-Dependent Viability | |
| CN108441462A (en) | One bacillus subtilis and preparation method thereof | |
| CN113559134B (en) | Medicine for treating tumor | |
| CN116478895B (en) | Recombinant salmonella typhimurium genetically engineered bacterium HCS1, microbial inoculum and application thereof | |
| CN113512559B (en) | Mycoplasma bovis Mbov _0701 mutant gene and mutant strain and application thereof | |
| CN114438111B (en) | Construction of PP2 strict anaerobic salmonella strain and application thereof in tumor treatment | |
| CN115717120A (en) | Controllable growth engineering bacterium and construction method and application thereof | |
| CN109593761B (en) | Small RNA related to Brucella virulence and application thereof in preparation of attenuated Brucella | |
| CN113046384A (en) | Construction method of broad-spectrum antiviral recombinant salmonella | |
| KR101660294B1 (en) | Novel bacterial vector system based on glmS | |
| CN112501093B (en) | Riemerella anatipestifer phoR gene partial fragment deletion attenuated strain and application thereof | |
| CN114456992B (en) | Salmonella strain capable of being automatically cracked, preparation method thereof and application thereof in tumor treatment | |
| CN107513516B (en) | Non-hemolytic streptococcus agalactiae WC1535 delta cyl and construction and application thereof | |
| CN110951659B (en) | Application of deletion of acetokinase ackA gene | |
| CN108220217B (en) | Attenuated listeria monocytogenes for delivering and expressing exogenous antigen and application thereof | |
| CN114438106B (en) | Rv2779c gene and application of expression product thereof as target spot for resisting mycobacterium tuberculosis | |
| WO2022006748A1 (en) | Genetically engineered live bacteria and methods of constructing the same | |
| CN103243098B (en) | RelA cut and TLR7 active sequence modified locked nucleic acid deoxyribozyme for targeted therapy of tuberculosis 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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |