CN117946953A - Recombinant lactococcus lactis, probiotic preparation, construction method, cECF expression method and application - Google Patents
Recombinant lactococcus lactis, probiotic preparation, construction method, cECF expression method and application Download PDFInfo
- Publication number
- CN117946953A CN117946953A CN202410155654.5A CN202410155654A CN117946953A CN 117946953 A CN117946953 A CN 117946953A CN 202410155654 A CN202410155654 A CN 202410155654A CN 117946953 A CN117946953 A CN 117946953A
- Authority
- CN
- China
- Prior art keywords
- lactococcus lactis
- recombinant
- cegf
- cecf
- pnz8149
- 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
- 235000014897 Streptococcus lactis Nutrition 0.000 title claims abstract description 130
- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 30
- 239000006041 probiotic Substances 0.000 title claims abstract description 25
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 25
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 25
- 238000010276 construction Methods 0.000 title claims abstract description 14
- 241000194035 Lactococcus lactis Species 0.000 title claims abstract 19
- 239000013612 plasmid Substances 0.000 claims abstract description 44
- 230000000968 intestinal effect Effects 0.000 claims abstract description 20
- 230000000694 effects Effects 0.000 claims abstract description 16
- 230000001262 anti-secretory effect Effects 0.000 claims abstract description 12
- 241000894006 Bacteria Species 0.000 claims description 37
- 108090000623 proteins and genes Proteins 0.000 claims description 31
- 239000012634 fragment Substances 0.000 claims description 26
- 102000004169 proteins and genes Human genes 0.000 claims description 25
- 206010012735 Diarrhoea Diseases 0.000 claims description 24
- 239000006228 supernatant Substances 0.000 claims description 21
- 239000000047 product Substances 0.000 claims description 16
- 108010053775 Nisin Proteins 0.000 claims description 14
- NVNLLIYOARQCIX-MSHCCFNRSA-N Nisin Chemical compound N1C(=O)[C@@H](CC(C)C)NC(=O)C(=C)NC(=O)[C@@H]([C@H](C)CC)NC(=O)[C@@H](NC(=O)C(=C/C)/NC(=O)[C@H](N)[C@H](C)CC)CSC[C@@H]1C(=O)N[C@@H]1C(=O)N2CCC[C@@H]2C(=O)NCC(=O)N[C@@H](C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(NCC(=O)N[C@H](C)C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCSC)C(=O)NCC(=O)N[C@H](CS[C@@H]2C)C(=O)N[C@H](CC(N)=O)C(=O)N[C@H](CCSC)C(=O)N[C@H](CCCCN)C(=O)N[C@@H]2C(N[C@H](C)C(=O)N[C@@H]3C(=O)N[C@@H](C(N[C@H](CC=4NC=NC=4)C(=O)N[C@H](CS[C@@H]3C)C(=O)N[C@H](CO)C(=O)N[C@H]([C@H](C)CC)C(=O)N[C@H](CC=3NC=NC=3)C(=O)N[C@H](C(C)C)C(=O)NC(=C)C(=O)N[C@H](CCCCN)C(O)=O)=O)CS[C@@H]2C)=O)=O)CS[C@@H]1C NVNLLIYOARQCIX-MSHCCFNRSA-N 0.000 claims description 14
- 238000012258 culturing Methods 0.000 claims description 14
- 239000001963 growth medium Substances 0.000 claims description 14
- 239000004309 nisin Substances 0.000 claims description 14
- 235000010297 nisin Nutrition 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000000203 mixture Substances 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 241000282465 Canis Species 0.000 claims description 9
- 206010061218 Inflammation Diseases 0.000 claims description 9
- 230000006698 induction Effects 0.000 claims description 9
- 230000004054 inflammatory process Effects 0.000 claims description 9
- 102000003960 Ligases Human genes 0.000 claims description 8
- 108090000364 Ligases Proteins 0.000 claims description 8
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 7
- 238000012408 PCR amplification Methods 0.000 claims description 7
- 238000009630 liquid culture Methods 0.000 claims description 7
- 230000007413 intestinal health Effects 0.000 claims description 5
- 230000003698 anagen phase Effects 0.000 claims description 4
- 238000001976 enzyme digestion Methods 0.000 claims description 4
- 238000005457 optimization Methods 0.000 claims description 4
- 108020004705 Codon Proteins 0.000 claims description 3
- 108700010070 Codon Usage Proteins 0.000 claims description 3
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 3
- 230000002401 inhibitory effect Effects 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 239000013600 plasmid vector Substances 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims 1
- 125000003729 nucleotide group Chemical group 0.000 claims 1
- 239000003833 bile salt Substances 0.000 abstract description 11
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 7
- 210000004211 gastric acid Anatomy 0.000 abstract description 7
- 230000023011 digestive tract development Effects 0.000 abstract description 5
- 230000012010 growth Effects 0.000 abstract description 5
- 230000008439 repair process Effects 0.000 abstract description 5
- 230000036039 immunity Effects 0.000 abstract description 3
- 244000052616 bacterial pathogen Species 0.000 abstract description 2
- 230000003204 osmotic effect Effects 0.000 abstract description 2
- 230000003115 biocidal effect Effects 0.000 abstract 1
- 230000002708 enhancing effect Effects 0.000 abstract 1
- 244000057717 Streptococcus lactis Species 0.000 description 111
- 238000012360 testing method Methods 0.000 description 28
- 230000001580 bacterial effect Effects 0.000 description 21
- 239000007788 liquid Substances 0.000 description 21
- 241000699670 Mus sp. Species 0.000 description 16
- 241001465754 Metazoa Species 0.000 description 14
- 241000282472 Canis lupus familiaris Species 0.000 description 13
- 238000000855 fermentation Methods 0.000 description 13
- 230000004151 fermentation Effects 0.000 description 13
- 238000002474 experimental method Methods 0.000 description 12
- 210000004027 cell Anatomy 0.000 description 11
- 238000001727 in vivo Methods 0.000 description 10
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 10
- 230000001575 pathological effect Effects 0.000 description 9
- 230000004083 survival effect Effects 0.000 description 9
- 210000001072 colon Anatomy 0.000 description 8
- 238000001514 detection method Methods 0.000 description 8
- 210000001198 duodenum Anatomy 0.000 description 8
- 239000000411 inducer Substances 0.000 description 8
- 239000000843 powder Substances 0.000 description 8
- 230000003248 secreting effect Effects 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 102000003814 Interleukin-10 Human genes 0.000 description 7
- 108090000174 Interleukin-10 Proteins 0.000 description 7
- 208000037817 intestinal injury Diseases 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 210000002966 serum Anatomy 0.000 description 7
- 238000012795 verification Methods 0.000 description 7
- 102000004127 Cytokines Human genes 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 102000004889 Interleukin-6 Human genes 0.000 description 6
- 108090001005 Interleukin-6 Proteins 0.000 description 6
- 238000001035 drying Methods 0.000 description 6
- 210000001035 gastrointestinal tract Anatomy 0.000 description 6
- 230000001105 regulatory effect Effects 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 241000588724 Escherichia coli Species 0.000 description 5
- 241000699666 Mus <mouse, genus> Species 0.000 description 5
- 102100040247 Tumor necrosis factor Human genes 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000012228 culture supernatant Substances 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 230000002496 gastric effect Effects 0.000 description 5
- 235000014655 lactic acid Nutrition 0.000 description 5
- 239000004310 lactic acid Substances 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 241000186660 Lactobacillus Species 0.000 description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 4
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 4
- 229940093761 bile salts Drugs 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 235000013351 cheese Nutrition 0.000 description 4
- 238000003776 cleavage reaction Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 239000012154 double-distilled water Substances 0.000 description 4
- 210000003608 fece Anatomy 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 4
- 230000002757 inflammatory effect Effects 0.000 description 4
- 229940039696 lactobacillus Drugs 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 230000007017 scission Effects 0.000 description 4
- 238000012216 screening Methods 0.000 description 4
- 235000020183 skimmed milk Nutrition 0.000 description 4
- 239000000725 suspension Substances 0.000 description 4
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 102000003777 Interleukin-1 beta Human genes 0.000 description 3
- 108090000193 Interleukin-1 beta Proteins 0.000 description 3
- 241000191967 Staphylococcus aureus Species 0.000 description 3
- 239000011543 agarose gel Substances 0.000 description 3
- 230000003110 anti-inflammatory effect Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 230000000770 proinflammatory effect Effects 0.000 description 3
- 230000028327 secretion Effects 0.000 description 3
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 3
- 238000001694 spray drying Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 238000010200 validation analysis Methods 0.000 description 3
- 239000013598 vector Substances 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 2
- 101800003838 Epidermal growth factor Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 241000194036 Lactococcus Species 0.000 description 2
- 102000019149 MAP kinase activity proteins Human genes 0.000 description 2
- 108040008097 MAP kinase activity proteins Proteins 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 241001138501 Salmonella enterica Species 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 240000008042 Zea mays Species 0.000 description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 235000005822 corn Nutrition 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 102000052116 epidermal growth factor receptor activity proteins Human genes 0.000 description 2
- 108700015053 epidermal growth factor receptor activity proteins Proteins 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 235000021001 fermented dairy product Nutrition 0.000 description 2
- 230000005714 functional activity Effects 0.000 description 2
- 230000015784 hyperosmotic salinity response Effects 0.000 description 2
- 210000003405 ileum Anatomy 0.000 description 2
- 238000003384 imaging method Methods 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- YOHYSYJDKVYCJI-UHFFFAOYSA-N n-[3-[[6-[3-(trifluoromethyl)anilino]pyrimidin-4-yl]amino]phenyl]cyclopropanecarboxamide Chemical compound FC(F)(F)C1=CC=CC(NC=2N=CN=C(NC=3C=C(NC(=O)C4CC4)C=CC=3)C=2)=C1 YOHYSYJDKVYCJI-UHFFFAOYSA-N 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- -1 p-AKT Proteins 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000008107 starch Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 239000008399 tap water Substances 0.000 description 2
- 235000020679 tap water Nutrition 0.000 description 2
- 238000009423 ventilation Methods 0.000 description 2
- 235000013618 yogurt Nutrition 0.000 description 2
- TVZRAEYQIKYCPH-UHFFFAOYSA-N 3-(trimethylsilyl)propane-1-sulfonic acid Chemical compound C[Si](C)(C)CCCS(O)(=O)=O TVZRAEYQIKYCPH-UHFFFAOYSA-N 0.000 description 1
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- GOZMBJCYMQQACI-UHFFFAOYSA-N 6,7-dimethyl-3-[[methyl-[2-[methyl-[[1-[3-(trifluoromethyl)phenyl]indol-3-yl]methyl]amino]ethyl]amino]methyl]chromen-4-one;dihydrochloride Chemical compound Cl.Cl.C=1OC2=CC(C)=C(C)C=C2C(=O)C=1CN(C)CCN(C)CC(C1=CC=CC=C11)=CN1C1=CC=CC(C(F)(F)F)=C1 GOZMBJCYMQQACI-UHFFFAOYSA-N 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 241000304886 Bacilli Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 244000025596 Cassia laevigata Species 0.000 description 1
- 235000006693 Cassia laevigata Nutrition 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 241001414720 Cicadellidae Species 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 102400001368 Epidermal growth factor Human genes 0.000 description 1
- 108050001049 Extracellular proteins Proteins 0.000 description 1
- 241000192125 Firmicutes Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 235000010469 Glycine max Nutrition 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 102000000589 Interleukin-1 Human genes 0.000 description 1
- 108010002352 Interleukin-1 Proteins 0.000 description 1
- 238000012449 Kunming mouse Methods 0.000 description 1
- 241001112724 Lactobacillales Species 0.000 description 1
- 241000192132 Leuconostoc Species 0.000 description 1
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 241000607142 Salmonella Species 0.000 description 1
- 241000194018 Streptococcaceae Species 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 235000021053 average weight gain Nutrition 0.000 description 1
- 235000015278 beef Nutrition 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 229940099352 cholate Drugs 0.000 description 1
- BHQCQFFYRZLCQQ-OELDTZBJSA-N cholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 235000015142 cultured sour cream Nutrition 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 239000013530 defoamer Substances 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000035622 drinking Effects 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 230000002183 duodenal effect Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 108010021310 endodeoxyribonuclease NcoI Proteins 0.000 description 1
- 108010002480 endodeoxyribonuclease SacI Proteins 0.000 description 1
- 229940116977 epidermal growth factor Drugs 0.000 description 1
- XJRPTMORGOIMMI-UHFFFAOYSA-N ethyl 2-amino-4-(trifluoromethyl)-1,3-thiazole-5-carboxylate Chemical compound CCOC(=O)C=1SC(N)=NC=1C(F)(F)F XJRPTMORGOIMMI-UHFFFAOYSA-N 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 210000004051 gastric juice Anatomy 0.000 description 1
- 230000000762 glandular Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 210000002175 goblet cell Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 210000001630 jejunum Anatomy 0.000 description 1
- 101150009839 lacF gene Proteins 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 235000020124 milk-based beverage Nutrition 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 229910001453 nickel ion Inorganic materials 0.000 description 1
- 210000004279 orbit Anatomy 0.000 description 1
- 235000019629 palatability Nutrition 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 101150064613 pepN gene Proteins 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 229960004641 rituximab Drugs 0.000 description 1
- 150000003839 salts Chemical group 0.000 description 1
- 229940124513 senna glycoside Drugs 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000004879 turbidimetry Methods 0.000 description 1
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000004018 waxing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 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
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/746—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for lactic acid bacteria (Streptococcus; Lactococcus; Lactobacillus; Pediococcus; Enterococcus; Leuconostoc; Propionibacterium; Bifidobacterium; Sporolactobacillus)
-
- 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
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
-
- 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
- A61K38/18—Growth factors; Growth regulators
- A61K38/1808—Epidermal growth factor [EGF] urogastrone
-
- 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
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- 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
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- 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
- C12R2001/46—Streptococcus ; Enterococcus; Lactococcus
-
- 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)
- Public Health (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Genetics & Genomics (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Toxicology (AREA)
- Pain & Pain Management (AREA)
- Rheumatology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Plant Pathology (AREA)
Abstract
The application discloses a recombinant lactococcus lactis, a probiotic preparation, a construction method, a cECF expression method and application. The recombinant lactococcus lactis is lactococcus lactis NZ3900 transferred with recombinant plasmid without antisecretory expression cEGF. The recombinant lactococcus lactis not only can promote intestinal development and repair without antibiotic expression cECF, but also has the effect of probiotics, and has the effect of enhancing the immunity and the growth performance of organisms. The recombinant lactococcus lactis has obvious antibacterial effect on common intestinal pathogenic bacteria, can resist gastric acid and intestinal bile salt high osmotic pressure environment, and can play a probiotic role in intestinal colonisation.
Description
Technical Field
The application relates to the technical field of lactococcus lactis, in particular to recombinant lactococcus lactis, a probiotic preparation, a construction method, a cECF expression method and application.
Background
Lactococcus lactis is one of the commonly used fermenters in the fermentation industry, especially in fermented dairy products. In addition, lactococcus lactis is one of common induced expression host bacteria, and has important research and application values. Lactococcus lactis belongs to the phylum of the phylum Leuconostoc (Firmicutes), the class of the Bacilli (Bacillli), the order of the Lactobacillus (Lactobacillales), the family of the Streptococcaceae (Streptococcus), the genus Lactococcus (Lactobacillus). Lactococcus lactis is generally considered to include three subspecies, of which milk sources are lactococcus lactis subspecies (l.lactissu sp. Lactis) and lactococcus lactis subspecies (l.lactissu sp. Cremoris), and lactococcus lactis subspecies holorii (l.lactissu sp. Hordiniae) isolated from leafhoppers.
The lactococcus lactis NZ3900 has a genotype of lacF-, pepN is nisRnisK, can grow in an M17 culture medium and added with 5g/L glucose, and is cultured at 30 ℃ under aerobic conditions, and the preservation mode is 20% glycerol at-80 ℃. Lactococcus lactis NZ3900 grows rapidly and is relatively simple to metabolize, and the decomposition is separated from anabolism; the genome is small but contains enough biological information, and intracellular and extracellular proteins are easy to separate and purify, so that the advantages make the lactococcus lactis have great research value in metabolic regulation. Lactococcus lactis NZ3900 has been widely used in dairy products as a starter for the production of dairy products such as sour cream, yogurt, soy yogurt, milk beverages, and the like. It is also a common starter for the preparation of cheese, such as cheddar cheese, cottage cheese and the like, and lactococcus lactis NZ3900 has a significant impact on the flavor of fermented dairy products such as cheese.
Disclosure of Invention
The embodiment of the application provides a recombinant lactococcus lactis capable of expressing cECF without resistance by modifying NZ 3900. The recombinant lactococcus lactis not only can express cECF without resistance, but also can promote intestinal development and repair, and enhance the immunity and growth performance of organisms. Therefore, the embodiment of the application at least discloses the following technical scheme:
The embodiment of the application provides a recombinant lactococcus lactis, namely lactococcus lactis NZ3900 transformed with recombinant plasmid without antisecretory expression cEGF.
The embodiment of the application provides a probiotic preparation for protecting intestinal health, which comprises recombinant lactococcus lactis with concentration not lower than 5 multiplied by 10 8.
The embodiment of the application provides a construction method of recombinant lactococcus lactis, wherein the recombinant lactococcus lactis is lactococcus lactis NZ3900 transferred with recombinant plasmid without antisecretory expression cEGF, and the construction method comprises the following steps:
Constructing a recombinant plasmid without antisecretory expression cEGF; and
The recombinant plasmid was transferred into lactococcus lactis NZ3900.
The embodiment of the application provides a method for expressing cECF without resistance, which comprises the following steps:
Constructing the recombinant lactococcus lactis;
Inoculating the recombinant lactococcus lactis to M17 liquid culture, and culturing for 24 hours at 30 ℃; and
Inoculating the culture into an M17 liquid culture medium according to the ratio of 1:25, continuously culturing until bacteria enter a logarithmic growth phase, adding 10-80 ng/mL of lactobacillin to induce for 5-30 hours, stopping culturing, and centrifuging at the temperature of 4 ℃ for 5min at 12000r/min to respectively collect supernatant, wherein the supernatant comprises cECF.
The embodiment of the application provides an application of the recombinant lactococcus lactis in preparation of a preparation for improving intestinal diarrhea.
The embodiment of the application provides the application of the recombinant lactococcus lactis in the first aspect in preparing the preparation related to inhibiting intestinal inflammation and repairing intestinal injury
Compared with the prior art, the application has at least one of the following beneficial effects:
(1) The recombinant strain uses a lactococcus lactis expression system, and realizes secretion-free expression for food-grade lactococcus lactis with food-grade screening marks as host bacteria.
(2) CEGF of secretory expression of the strain can promote intestinal development and repair and enhance the immunity and growth performance of organisms.
(3) In vivo experiments prove that the recombinant strain combines the intestinal repair function of cEGF with the probiotic function of lactococcus lactis, regulates the organism inflammation level and treats intestinal injury.
(4) The recombinant lactococcus lactis has obvious antibacterial effect on common intestinal pathogenic bacteria, can resist gastric acid and intestinal bile salt high osmotic pressure environment, and can play a probiotic role in intestinal colonisation.
(5) The fermentation condition and the drying process method provided by the embodiment of the application also retain the viable count of the recombinant strain to a great extent, and have little influence on the secretory expression of cEGF.
Drawings
FIG. 1 is a schematic diagram of the recombinant plasmid pTOPO-Blunt-Simple-cEGF according to an embodiment of the application.
FIG. 2 shows the result of PCR amplification detection of the target sequence of cEGF gene provided in the example of the present application; DL2000 DNA molecular weight standard; lane 1: positive control (hEGF); lane 2: cEGF.
FIG. 3 shows the result of electrotransformation of cEGF and pNZ8149 ligation products provided in the examples of the present application.
FIG. 4 shows the results of PCR amplification and identification of cEGF and pNZ8149 ligation product recombinant strains provided in the examples of the present application; DL2000 DNA molecular weight standard; lane 1: a negative control; lane 2: cEGF-pNZ8149 (recombinant strain).
FIG. 5 shows the results of double-restriction PCR amplification and identification of recombinant plasmid cEGF-pNZ8149 provided by the example of the present application; DL 5000DNA molecular weight standard; lane 1: recombinant plasmid cEGF-pNZ8149.
FIG. 6 is a graph showing Western Blot results of culture supernatants and bacterial cells of recombinant lactococcus lactis cEGF-pNZ8149 provided by the example of the present application; lane 1: a negative control; lane 2: cEGF-pNZ8149 culture supernatant; lane 3: cEGF-pNZ 8149.
FIG. 7 is a graph showing Western Blot results of culture supernatants of Nisin-induced recombinant lactococcus lactis cEGF-pNZ8149 at different concentrations provided in the examples of the present application; lane 1: a negative control; lane 2:0ng/mL; lane 3:1ng/mL lane 4:2ng/mL; lane 5:5ng/mL; lane 6:10ng/mL; lane 7:20ng/mL; lane 8:40ng/mL.
FIG. 8 is a graph showing Western Blot results of culture supernatants of recombinant lactococcus lactis cEGF-pNZ8149 induced at different times according to an example of the present application; lane 1: a negative control; lane 2:5h; lane 3:10h; lane 4:15h; lane 5:20h; lane 6:25h; lane 7: and 30h.
FIG. 9 is a diagram showing the result of recombinant lactococcus lactis cEGF-pNZ8149 culture supernatant purified Tricine-SDS-PAGE; m is protein maker; lane 1: cEGF-pNZ8149 supernatant.
FIG. 10 is a graph showing the effect of recombinant lactococcus lactis cEGF-pNZ8149 supernatant and empty vector supernatant on the number of CMT-1211 cells provided in the examples of the present application.
FIG. 11 is a diagram showing a pathological section of the duodenum of a mouse 7d subjected to in vivo validation test of recombinant lactococcus lactis cEGF-pNZ8149 provided by an embodiment of the present application; A. blank control group; B. model control group; C. empty pNZ8149 group; D. recombinant cEGF-pNZ8149 group.
FIG. 12 is a diagram showing a pathological section of the duodenum of a mouse 14d subjected to in vivo validation test of recombinant lactococcus lactis cEGF-pNZ8149 provided by an embodiment of the present application; A. blank control group; B. model control group; C. empty pNZ8149 group d. Recombinant cEGF-pNZ8149 group.
FIG. 13 is a graph of 7d colon pathology of mice tested by recombinant lactococcus lactis cEGF-pNZ8149 in vivo verification provided by the example of the present application; A. blank control group; B. model control group; C. empty pNZ8149 group; D. recombinant cEGF-pNZ8149 group.
FIG. 14 is a graph of a colon pathology section of a mouse 14d tested by recombinant lactococcus lactis cEGF-pNZ8149 in vivo verification provided by the example of the present application; A. blank control group; B. model control group; C. empty pNZ8149 group; D. recombinant cEGF-pNZ8149 group.
FIG. 15 is a graph showing the results of the in vivo validation test of recombinant lactococcus lactis cEGF-pNZ8149 against the change in the cytokine in mice provided in the examples of the present application; TNF-alpha; IL-6; IL-1 beta; IL-10.
FIG. 16 shows the WB test results of p-EGFR, EGFR, p-AKT, AKT, p-ERK1/2 and ERK1/2 in intestinal tissues of beagle dogs in the animal experiment, the diarrhea model group, the empty pNZ8149 control group and the recombinant cEGF-pNZ8149 group provided by the example of the present application.
Fig. 17 shows the results of lactic acid bacteria content in the feces of beagle dogs in the animal experiment provided by the example of the present application, the empty white control group, the diarrhea model group, the empty pNZ8149 control group and the recombinant cEGF-pNZ8149 group.
Fig. 18 is a HE staining pattern of the duodenal pathological section of beagle in the animal experiments provided by the examples of the present application, the empty white control group, the diarrhea model group, the empty pNZ8149 control group, and the recombinant cEGF-pNZ8149 group.
Fig. 19 is a HE staining pattern of colon pathological sections of beagle in animal experiments provided by the examples of the present application, a blank control group, a diarrhea model group, an empty pNZ8149 control group, and a recombinant cEGF-pNZ8149 group.
Fig. 20 is a HE staining pattern of jejunal pathological sections of beagle in animal experiments provided by the examples of the present application, a white control group, a diarrhea model group, an empty pNZ8149 control group, and a recombinant cEGF-pNZ8149 group.
Fig. 21 is a HE staining pattern of ileum pathological sections of beagle in animal experiments provided by the examples of the present application, a white control group, a diarrhea model group, an empty pNZ8149 control group, and a recombinant cEGF-pNZ8149 group.
Fig. 22 is a graph showing the results of the content of inflammatory cytokines in beagle blood in the empty white control group, diarrhea model group, empty pNZ8149 control group and recombinant cEGF-pNZ8149 group of the animal experiment provided by the embodiment of the present application. A is TNF, B is IL-6, C is IL-1β, and D is IL-10.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application will be described in further detail with reference to the following examples. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application. The reagents not specifically and individually described in the present application are all conventional reagents and are commercially available; methods which are not specifically described in detail are all routine experimental methods and are known from the prior art.
It should be noted that, the terms "first," "second," and the like in the description and the claims of the present invention and the above drawings are used for distinguishing similar objects, and are not necessarily used for describing a particular sequence or order, nor do they substantially limit the technical features that follow. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The embodiment of the application provides a recombinant lactococcus lactis, namely lactococcus lactis NZ3900 transformed with recombinant plasmid without antisecretory expression cEGF. The strain utilizes food-grade lactococcus lactis, lacF deletion strains NZ3900 and LacF as screening markers and a food-grade expression vector pNZ8149 as an expression system to construct recombinant lactococcus lactis cEGF-pNZ8149, uses a Nisin inducer to induce and express the recombinant lactococcus lactis cEGF-pNZ8149, carries out different component treatment on recombinant bacterial liquid, and is identified as successful construction of recombinant bacteria, wherein the expression level of cEGF in supernatant is highest, namely cEGF is expressed without antisecretory under the action of Nisin inducer.
The research on the optimum condition of the recombinant lactococcus lactis cEGF-pNZ8149 induced expression provided by the embodiment of the application shows that the expression quantity of cEGF is related to the concentration and the induction time of the Nisin inducer, and the expression quantity is highest when the Nisin concentration is 5ng/mL and the induction time is 20 hours.
According to the embodiment of the application, through a cell proliferation CCK8 test, the biological activity of cEGF protein secreted and expressed by recombinant lactococcus lactis cEGF-pNZ8149 is verified in vitro, and research shows that cEGF protein secreted and expressed by the bacterium has proliferation promoting effect on cells and biological activity. The animal experiment result of the embodiment of the application shows that the recombinant lactococcus lactis cEGF-pNZ8149 plays a role in repairing and proliferation of cEGF protein and repairing intestinal injury and simultaneously plays a probiotic effect.
The antibacterial capacity and stress resistance studies of the recombinant lactococcus lactis cEGF-pNZ8149 show that the recombinant lactococcus lactis cEGF-pNZ8149 has strong antibacterial capacity to escherichia coli K88, K99, O157, O189, staphylococcus aureus and salmonella, has strong cholate resistance and gastric acid resistance, and is sensitive to high-temperature environments.
According to the embodiment of the application, two methods of spray drying and freeze drying are selected to prepare the probiotic preparation respectively, and compared with the two methods, the freeze drying method of the skim milk powder with high viable count, convenient operation, safety and stability and good palatability is finally selected.
According to the embodiment of the application, animal experiments are carried out on the finally prepared probiotic preparation. Animal test results show that the preparation method of the probiotic preparation provided by the embodiment greatly maintains the intestinal tract repairing function of cEGF and is also very beneficial to the in vivo colonisation and probiotic action. To this end, embodiments of the present application provide a probiotic formulation for protecting intestinal health comprising recombinant lactococcus lactis as described above in a concentration of not less than 5 x10 8.
In some embodiments, the present application provides a method for constructing a recombinant lactococcus lactis, wherein the recombinant lactococcus lactis is lactococcus lactis NZ3900 transformed with a recombinant plasmid without antisecretory expression cEGF. The construction method comprises the following steps: constructing a recombinant plasmid without antisecretory expression cEGF; the recombinant plasmid was transferred into lactococcus lactis NZ3900. Specifically, lactococcus lactis NZ3900 is from MO BI Tec under the product number #VS-ELS03900-01.
In some embodiments, the constructing step of the recombinant plasmid comprises:
obtaining cECF target fragment; and
Carrying out a ligation reaction on the digested target fragment and the digested carrier plasmid by using T4 ligase, wherein a ligation product comprises the recombinant plasmid;
wherein the ligation reaction system comprises, in 20. Mu.l, 1.5. Mu.l of the fragment of interest, 4.0. Mu.l of the plasmid vector, 0.5. Mu. l T. Mu.4 ligase, 10 XBuffer and the balance of water, wherein the activity of the T4 ligase is 1000U.
In some embodiments, the obtaining of the fragment of interest of cECF comprises:
Obtaining a cECF protein sequence;
Performing codon optimization on the protein sequence by using the codon preference of lactococcus lactis to obtain a DNA sequence with the length of 156 bp;
Synthesizing a DNA sequence with the length of 156bp, a SPusp signal peptide sequence, a 6 XHis tag and a connecting sequence of NcoI and SacI restriction enzyme digestion sequences;
Ligating the ligation sequence with the plasmid pTOPO-Blunt-Simple to obtain a recombinant plasmid comprising the cECF fragment of interest; and
And carrying out PCR amplification on the recombinant plasmid containing the cECF target fragment to obtain the cECF target fragment.
In some embodiments, the nucleotide sequences of primer pairs for PCR amplification of recombinant plasmids comprising the cECF fragment of interest are shown in SEQ ID NO. 1-2.
In addition, the embodiment of the application also provides a method for expressing cECF without resistance, which comprises the following steps:
the step of constructing the recombinant lactococcus lactis comprises the steps of inoculating the recombinant lactococcus lactis into an M17 liquid culture medium and culturing at 30 ℃ for 24 hours;
Inoculating the culture into an M17 liquid culture medium according to the ratio of 1:25, continuously culturing until bacteria enter a logarithmic growth phase, adding 10-80 ng/mL of lactobacillin to induce for 5-30 hours, stopping culturing, and centrifuging at the temperature of 4 ℃ for 5min at 12000r/min to respectively collect supernatant, wherein the supernatant comprises cECF.
In addition, the embodiment of the application also provides the application of the recombinant lactococcus lactis in preparing the preparation for improving the intestinal diarrhea and the application of the recombinant lactococcus lactis in preparing the preparation for inhibiting the intestinal inflammation and repairing the intestinal injury.
The application will now be illustrated by the following more specific examples, which are not to be construed as limiting the embodiments of the application.
Example 1: construction of recombinant plasmids
(1) Obtaining cECF target fragment
According to the canine epidermal growth factor gene sequence published by NCBI, a cEGF protein sequence is obtained and named cEGF, then a DNA sequence with the length of 156bp (shown as SEQ ID NO. 5) is obtained after codon optimization according to the codon preference of lactococcus lactis, the sequence is connected with a SPusp signal peptide sequence, 1 XHis tag is added at the tail end of the sequence, ncoI and SacI restriction enzyme cleavage sites are respectively added at two ends of the sequence, and a target sequence with the length of 270bp (the sequence is shown as SEQ ID NO. 6) is obtained. Then, the gene is submitted to Wuhan Tian Yihui Yuan biotechnology Co., ltd for synthesis, and the synthesized gene is connected with a plasmid pTOPO-Blunt-Simple to obtain a recombinant plasmid named pTOPO-Blunt-Simple-cEGF (shown in figure 1, the sequence of which is shown in SEQ ID NO. 7).
And designing cEGF-F and cEGF-R by taking pTOPO-Blunt-Simple-cEGF as a template, and performing PCR expansion to obtain the target fragment of cECF. Wherein, the nucleotide sequences of cEGF-F and cEGF-R are shown as follows, and cEGF-F:5'-ccatggatgaaaaaaaagattatctcagct-3' is synthesized by Wohan engine biotechnology Co., ltd, and is shown as SEQ ID NO. 1. cEGF-R:5'-gagctcctagtggtggtggtg-3', as shown in SEQ ID NO. 2.
The amplified PCR reaction system is shown in Table 1. The PCR reaction procedure included: 98℃2min,98℃10s,55℃5s,72℃5min,4℃infinity, 35 cycles. The PCR product was stored at 4℃and 10. Mu.L of the PCR product was subjected to electrophoresis on a 1% agarose gel at 120V for 30min, and the result was recorded by detection using an ultraviolet imaging system, showing that the band was about 270bp, which was consistent with the expectation, and the amplification result was shown in FIG. 2, and the PCR product was purified and recovered.
TABLE 1 PCR reaction System for obtaining cECF fragment of interest
| Composition of the components | Dosage (mu L) |
| cEGF-F | 2 |
| cEGF-R | 2 |
| ptopO-Blunt-Simple-cEGF | 1 |
| PremixPrimeSTARMax | 25 |
| Double distilled water | 20 |
| Total volume of | 50 |
(2) Restriction ligation of a target Gene and plasmid
Table 2cECF NcoI/SacI double cleavage System for the fragment of interest and plasmid pNZ8149
| Composition of the components | Dosage of |
| Plasmid pNZ8149/PCR product | 3μl/12μl |
| NocⅠ | 1.5μl |
| SacⅠ | 1.5μl |
| 10×Buffer | 2μl |
| Double distilled water | Upto40μl |
The target fragment cECF and plasmid pNZ8149 were treated at 37℃for 1 hour according to the cleavage system shown in Table 2, and the cleavage product was subjected to 1% agarose gel electrophoresis, followed by purification and recovery of the target fragment.
Carrying out a ligation reaction on the digested target fragment and the digested carrier plasmid by using T4 ligase, wherein a ligation product comprises the recombinant plasmid; wherein the connection is carried out in a water bath at 22 ℃ for 1h and at 65 ℃ for 10min, and the connection reaction system is shown in Table 3.
TABLE 3 ligation reaction System for cECF fragment of interest and plasmid pNZ8149
| Composition of the components | Dosage of |
| CECF-purpose fragment | 1.5μl |
| Plasmid pNZ8149 | 4μl |
| T4 ligase | 0.5μl |
| 10×Buffer | 2μl |
| Double distilled water | Upto20μl |
Example 2: construction of recombinant lactococcus lactis cEGF-pNZ8149
Lactococcus lactis NZ3900 competent cells were prepared and thawed on ice. After thawing, the recombinant plasmid obtained in example 1 (i.e., the ligation product obtained in Table 3) was mixed and transferred into a pre-chilled 2mm electrotransformation cup for shock transformation. After electric shock, adding precooled recovery culture medium G-SGM17, standing on ice for 5min, transferring the bacterial liquid into a centrifuge tube, and standing and culturing at 30 ℃ for 2h. Centrifuging, removing supernatant, adding 100 mu l M to recover culture medium, resuspending thallus, coating the bacterial liquid after resuspension on bromocresol purple screening culture medium, screening, standing at 30deg.C for 18-24h, and electrotransformation results are shown in figure 3.
Yellow single colonies were picked and inoculated into 5mL of M17 broth overnight for cultivation to give pNZ8149-F:5'-gatttcgttcgaaggaactac-3' as shown in SEQ ID NO. 3; pNZ8149-R:5'-atcaatcaaagcaacacgtgc-3', shown as SEQ ID NO.4, is used as a primer for bacterial liquid PCR, and the bacterial liquid PCR system is shown as Table 4. The PCR reaction procedure included: 98℃2min,98℃10s,55℃5s,72℃5min,4℃infinity, 35 cycles, and PCR product 4 ℃.
TABLE 4 bacterial liquid PCR reaction system
| Composition of the components | Dosage (mu L) |
| pNZ8149-F | 2 |
| pNZ8149-R | 2 |
| Bacterial liquid | 1 |
| PremixPrimeSTARMax | 25 |
| Double distilled water | 20 |
| Total volume of | 50 |
At 120V, 10. Mu.L of the PCR product was subjected to electrophoresis on a 1% agarose gel for 30min, and the result was recorded by detection using an ultraviolet imaging system, which showed that the band was about 270bp, which was consistent with the expectation, and the amplification results were shown in FIG. 4. The recombinant plasmid of the positive strain is verified by extracting bacterial liquid PCR, double enzyme digestion is carried out by using NcoI and SacI endonucleases, and double enzyme digestion products are detected by agarose gel nucleic acid electrophoresis, the result is shown in figure 5, two bands are respectively about 2500bp and 270bp, and the expected result is consistent, so that the target gene is inserted into a pNZ8149 expression vector. The recombinant strain identified as positive is subjected to sequence identification by Wuhan qing research and new generation technology Co., ltd, the sequencing result is compared with blast software of cEGF sequence designed in the research at NCBI, and the sequencing result of the inserted fragment of the recombinant plasmid is consistent with the target gene sequence, which shows that the recombinant lactococcus lactis expressing cEGF protein is successfully constructed and is named as recombinant lactococcus lactis cEGF-pNZ8149.
EXAMPLE 3 Induction of expression and identification of recombinant lactococcus lactis cEGF-pNZ8149 expression cEGF protein
(1) Induced expression of cEGF proteins
Recombinant lactococcus lactis cEGF-pNZ8149 as positive bacteria and empty vector control bacteria pNZ8149 were prepared in a ratio of 1:100 portions of the strain are inoculated into M17 liquid culture medium, and the strain is subjected to stationary culture at 30 ℃ for 24 hours. Cultures were inoculated in M17 liquid medium at a ratio of 1:25, the culture was continued until bacteria entered into the logarithmic growth phase (OD=0.4), the culture was terminated after induction with 10ng/mL of Nisin (Nisin) for 15 hours, the supernatant and the thalli were collected by centrifugation at 12000r/min at 4℃for 5min, respectively, -80℃for storage for Westernblot detection of whether recombinant lactococcus cEGF-pNZ8149 was able to secrete cEGF protein, and the results are shown in FIG. 6, indicating that the recombinant bacteria were successful in secretory expression of cEGF protein.
(2) Condition optimization of cEGF protein expression
Positive recombinant bacteria liquid is induced by Nisin with the concentration of 0ng/mL,1ng/mL,2ng/mL,5ng/mL,10ng/mL,20ng/mL,40ng/mL and 80ng/mL respectively, after 20h induction, culture is stopped, supernatant and thalli are collected respectively after centrifugation at the temperature of 4 ℃ and 12000r/min for 5min, the result is shown in FIG. 7, and the result shows that the secretory expression quantity of cEGF is different when Nisin inducers with different concentrations are induced, the secretory expression quantity of cEGF protein is almost zero when the concentration of the inducers is low at 0ng/mL and 1ng/mL, and the secretory expression quantity of cEGF protein is not increased when the concentration of the inducers is too high, but is reduced, so that the optimal induction concentration is estimated to be 5ng/mL.
After 5ng/mL of Nisin was used to induce 5h,10h,15h,20h,25h and 30h, the culture was terminated, and the supernatant and the cells were collected by centrifugation at 12000r/min for 5min at 4℃and stored at-80℃for Westernblot detection, the results are shown in FIG. 8, which shows that the secretion amount at the pre-induction period cEGF was increased, but the secretion amount reached the peak after 20h was no longer increased, thus the optimal induction time was inferred to be 20h.
(3) Purification of cEGF proteins
Recombinant lactococcus lactis cEGF-pNZ8149 was induced and cultured with 5ng/mL Nisin for 20 hours, the supernatant was collected, concentrated by ammonium sulfate, dialyzed by dialysis bag, filtered by 0.45 μm filter to remove impurities, purified by using a nickel ion chromatography column, and Tricine-SDS-PAGE was performed, and the result is shown in FIG. 9, and the size of the target protein cEGF substantially matches the expected size.
Example 4 in vitro verification of the functional Activity of recombinant lactococcus lactis cEGF-pNZ8149 protein
Recombinant lactococcus lactis cEGF-pNZ8149 and empty vector control bacteria pNZ8149 are subjected to plate streak resuscitating culture, single colony is selected and inoculated into 5mL of M17 liquid medium, the mixture is subjected to stationary culture at 30 ℃ for 12h, then is transferred into M17 liquid medium according to the volume ratio of 1:25, the mixture is subjected to stationary culture at 30 ℃ until 0D 600 is approximately equal to 0.3 (about 2 h), nisin inducer with the final concentration of 5ng/mL is respectively added for culturing for 20h, the mixture is centrifuged at 4 ℃ for 12000r/min for 5min, and the supernatant is collected, filtered and sterilized by a 0.22 mu M sterile filter and then is stored at-80 ℃ for standby.
Resuscitates the canine breast cancer cells CMT-1211, adjusts the concentration of the subcultured cells and switches to 96-well cell culture plates, sucks the culture fluid when the cells reach 60% confluence, repeatedly washes the cells with sterile PBS for 3 times, adds DMEM without serum for starvation culture for 24 hours, washes the cells with sterile PBS for 1 time, changes DMEM without serum, groups the cells according to table 1, respectively adds 10% cck8 premix (10 μl cck8+90 μ LDMEM) after 12 hours, cultures for 1 hour at 37 ℃ and then observes the cell growth state, detects OD 450, each treatment group for 6 replicates, each replicate is one well, and 3 replicates are performed in total, and the results are shown in fig. 10. The result shows that the cEGF protein induced and expressed by the recombinant lactococcus lactis cEGF-pNZ8149 has the effect of promoting cell proliferation, which proves that the cEGF protein secreted and expressed has biological activity.
TABLE 5 recombinant lactococcus lactis cEGF-pNZ8149 cell proliferation Activity assay kit
| Grouping | Treatment mode |
| Blank control | 100 Mu L high sugar DMEM |
| No-load control bacterium pNZ8149 | 90 Μl of high sugar dmem+10μ LNisin induced empty supernatant |
| Recombinant lactococcus lactis cEGF-pNZ8149 | 90 Mu L of high-sugar DMEM+10 mu LNisin-induced recombinant lactobacillus supernatant |
Example 5: in vivo verification of the functional Activity of recombinant lactococcus lactis cEGF-pNZ8149 protein
Randomly selecting 36 four-week-old Kunming mice (Beijing veleaf rituximab, product number: 43513) with half male and female, adapting to 3d, randomly dividing into 4 groups, respectively setting a blank control group, a diarrhea model group, an empty pNZ8149 group and a recombined cEGF-pNZ8149 group, wherein 9 groups are used for keeping the conditions of temperature, feed and the like consistent, adopting a continuous drinking method, feeding tap water to the blank control group, feeding 5% DSS solution to the other groups, and after 3d feeding, the mice have diarrhea symptoms, establishing a diarrhea model, randomly selecting 3 mice in each group, taking blood from eye sockets, centrifuging at 4 ℃ for 15min at 2000r/min, separating serum, and measuring serum related indexes (intestinal inflammatory cytokines: IL-6, IL-1 beta, TNF-alpha and IL-10) of the 0 d.
The specific groups and test schemes of the formal tests are shown in Table 6, and the mice are fasted for 12 hours and drink water freely in the 7 th and 14 th test periods, 3 mice are selected randomly from each group, the eyebox is used for blood collection, centrifugation is carried out at 4 ℃ for 15min at 2000r/min, serum is separated, and serum related indexes are measured. Measurement of growth performance: weighing the mice at regular time every day during the feeding period, recording the weight of the mice, and calculating the average weight gain of the mice after the test is finished; pathological section preparation: sampling the same part of a mouse (duodenum and colon), taking a tissue sample with the size of about 1cm 2, placing the tissue sample into a universal tissue fixing solution for fixing, sending the tissue sample to a company to prepare slices, finishing, washing, dehydrating, waxing, embedding, slicing (with the thickness of 4 mu m) and other treatment steps, observing, taking a picture and photographing, observing the organ histopathological changes of the mice in each period, and analyzing the indexes such as villus length, quantity, recess depth, villus-to-recess ratio and the like; after preliminary treatment with Excel 2007 software, statistical analysis was performed with SPSS18.0 software. The statistical value of (P < 0.05) was considered to be significant for the difference significance test and multiple comparisons using ANOVA, LSD. The test results are expressed as Mean ± standard deviation (Mean ± SD).
TABLE 6 animal test grouping
| Group of | 7d | 14d |
| Blank control group | PBS | PBS |
| Diarrhea model group | PBS | PBS |
| Empty pNZ8149 control | Empty fungus suspension | Empty fungus suspension |
| Recombinant cEGF-pNZ8149 group | Recombinant bacterial suspension | Recombinant bacterial suspension |
According to the analysis of statistical results, the results of the weight influence of each treatment group on the weight of the mice are shown in Table 7, and the weight growth state of the mice in the recombinant cEGF-pNZ8149 group in the whole test period is good, and the mice have no obvious difference from a blank control group and extremely obvious difference from the mice in a diarrhea model group, so that the cEGF protein secreted and expressed by the recombinant bacterium cEGF-pNZ8149 has good effect. The difference between the empty-load pNZ8149 control group and the diarrhea model group is obvious in 7 d-14 d, which shows that the probiotics have good probiotics, but the empty-load bacteria are still obvious in difference from the recombinant bacteria, which shows that the simple probiotics have slower effects and the improvement effect is inferior to that of the recombinant bacteria.
Table 7 analysis of mice body weight of each treatment group
As shown in fig. 11, 12, 13 and 14, according to the analysis of the pathological section results of the duodenum and colon of each group 7d and 14d, the duodenum of the model control group had different degrees of inflammation and was accompanied by different degrees of tissue dissolution at 7d and 14d, the empty group had slight glandular atrophy and inflammation at 7d, and the duodenum tissue structures of the recombinant cEGF-pNZ8149 group and the blank group were complete, intestinal villi was good, and no inflammation occurred. The colon goblet cells were decreased and inflamed in the model control group 7d, slightly inflamed in the 14d, slightly inflamed in the empty groups 7d, 14d, and obviously consistent with the blank group in the recombinant cEGF-pNZ8149 group, without obvious inflammatory lesions.
From the analysis of the cytokine results in FIG. 15, at 0d, the expression of the other groups of mice were up-regulated for the pro-inflammatory factors IL-1β (P < 0.01), TNF- α (P < 0.05) and IL-6 (P < 0.05) compared with the blank group, and the expression of the anti-inflammatory factor IL-10 (P < 0.05) was significantly down-regulated, indicating that the diarrhea model was successfully established; the recombinant cEGF-pNZ8149 group is compared with a model control group in the 7d test period, the expression of pro-inflammatory factors TNF-alpha (P < 0.01) and IL-1 beta (P < 0.05) is obviously down-regulated, the expression of anti-inflammatory factors IL-10 (P < 0.05) is obviously up-regulated, and the difference between an empty-load group and the model control group is not obvious; during the 14d test period, the recombinant cEGF-pNZ8149 group still significantly up-regulates the expression of IL-6 (P < 0.05) and IL-1β (P < 0.05), and has significant differences from the empty group, but the empty group has no significant differences from the model control group except that IL-1β (P < 0.05) is significantly down-regulated; and the difference between the recombinant cEGF-pNZ8149 group and the blank group is not obvious at 7d and 14 d.
Therefore, according to animal test results, the recombinant lactococcus lactis cEGF-pNZ8149 can successfully reach the intestinal tract of a mouse and secrete cEGF, and the repair effect of the epidermal growth factor and the probiotic effect of the lactococcus lactis are simultaneously exerted in the intestinal tract, so that the intestinal injury is treated faster and better, and the balance of flora in the intestinal tract is maintained.
Example 5: determination of antibacterial and stress-resistant properties of recombinant lactococcus lactis cEGF-pNZ8149
(1) Antibacterial test
Inoculating the indicator bacteria into 5mL of LB liquid medium according to a proportion of 1% by using Bacillus coli O157, bacillus coli O139, bacillus coli K88, bacillus coli K99, salmonella Salmonella choleraesuis and Staphylococcus aureus Staphylococcus aureus as indicator bacteria, culturing for 6 hours at 37 ℃ under 200r/min, diluting the indicator bacteria culture solution to a proper concentration by using a turbidimetry method, and uniformly coating the diluted indicator bacteria culture solution on the LB solid medium by using a cotton swab; inoculating recombinant lactococcus lactis cEGF-pNZ8149 to an M17 culture medium, standing at 30 ℃ for culturing for 24 hours to prepare recombinant lactococcus lactis cEGF-pNZ8149 fermentation liquor, centrifuging the recombinant lactococcus lactis cEGF-pNZ8149 fermentation liquor, filtering and sterilizing the supernatant, adding the sterilized and filtered recombinant lactococcus lactis cEGF-pNZ8149 supernatant into oxford cups, dispersing in a refrigerator at 4 ℃ overnight, culturing in a constant temperature incubator at 30 ℃ for 12 hours, and observing a bacteriostasis ring; the bacteriostasis circle of recombinant lactococcus lactis cEGF-pNZ8149 is measured to judge the bacteriostasis capacity of recombinant lactococcus lactis cEGF-pNZ8149 on the indicator bacteria, and the method is repeated for 3 times continuously. The test results are shown in Table 8.
TABLE 8 in vitro bacteriostasis test of recombinant lactococcus lactis cEGF-pNZ8149 fermentation supernatant
| Indicator bacteria | CEGF-pNZ8149 recombinant bacterium inhibition region diameter (mm) |
| BacilluscoliO139 | 9.78±2.46 |
| BacilluscoliO157 | 6.93±1.33 |
| BacilluscoliK88 | 13.33±5.11 |
| BacilluscoliK99 | 11.33±3.81 |
| Salmonellacholeraesuis | 16.31±5.73 |
| Staphylococcusaureus | 13.72±3.75 |
(2) High temperature tolerance test
Inoculating recombinant lactococcus lactis cEGF-pNZ8149 to an M17 culture medium, standing at 30 ℃ for 24 hours to prepare recombinant lactococcus lactis cEGF-pNZ8149 fermentation liquor, respectively placing the fermentation liquor in water baths at 37, 50 and 60 ℃ for heat treatment for 5 minutes, taking the heat-treated bacterial liquor for stepwise multiple dilution, measuring the residual viable count by a pouring method, and placing the fermentation liquor at the same amount at 30 ℃ for comparison, wherein each group of tests are repeated for 3 times. The high temperature resistant results of the strain are shown in Table 9, and the recombinant lactococcus lactis cEGF-pNZ8149 has biological activity of only 11.62% and 4.91% after being treated at 50 ℃ and 60 ℃ for 5min, and the strain is sensitive to temperature, so that the high temperature environment is avoided as much as possible during the commercialized processing in the later period, and the viable bacteria rate of the strain is kept as much as possible.
TABLE 9 tolerance of recombinant lactococcus lactis cEGF-pNZ8149 to high temperatures
| Treatment temperature (. Degree. C.) | CEGF-pNZ8149 viable count | CEGF-pNZ8149 survival |
| 30 | (7.45±0.17)×107 | 100％ |
| 37 | (3.72±0.33)×107 | 49.93±0.06％ |
| 50 | (8.66±0.37)×106 | 11.62±0.07％ |
| 60 | (3.66±0.29)×106 | 4.91±0.06％ |
(3) Bile salt tolerance test
Inoculating recombinant lactococcus lactis cEGF-pNZ8149 into an M17 culture medium, performing stationary culture at 30 ℃ for 24 hours to prepare recombinant lactococcus lactis cEGF-pNZ8149 fermentation liquor, taking 0.5mL of bacterial liquid, adding the bacterial liquid into 4.5mL of 0.30% simulated bile salt, rapidly and fully mixing the bacterial liquid on a vortex machine, and placing the mixture in a 30 ℃ incubator for stationary culture. Taking out the strain at 0h and 24h for dilution, and mapping the bacterial count by using a pouring method, and placing the same bacterial liquid into sterile physiological saline for comparison. The results of the simulation of bile salt tolerance by recombinant lactococcus lactis cEGF-pNZ8149 are shown in Table 10. As can be seen from Table 10, recombinant lactococcus lactis cEGF-pNZ8149 was able to normally withstand the environment of simulated bile salts, and had approximately 79.57% survival rate after 24h treatment in simulated bile salts, and thus recombinant lactococcus lactis cEGF-pNZ8149 was able to withstand the environment of intestinal bile salts.
TABLE 10 tolerance of recombinant lactococcus lactis cEGF-pNZ8149 to bile salts
| Processing time | Simulated bile salt group | Control group | Survival rate |
| 0h | (1.33±0.11)×105 | (1.07±0.03)×105 | 100％ |
| 24h | (7.79±0.09)×104 | (9.79±0.07)×104 | 79.57±0.05％ |
(4) Gastric juice tolerance test
0.5ML of bacterial liquid is taken and added into simulated gastric fluid, and is quickly and fully mixed on a vortex instrument, and is placed in a 30 ℃ incubator for static culture. Taking out the strain at 0h, 2h and 4h for dilution, and mapping the bacterial count by using a pouring method, and placing the same bacterial liquid into sterile physiological saline for comparison. Gastric acid resistant results are shown in Table 11. As shown in Table 11, the survival rate of recombinant lactococcus lactis cEGF-pNZ8149 in simulated gastric fluid with pH value of about 2.0 is high, and the survival rates of 2h and 4h are up to 85.59% and 72.48% respectively. By combining the data, the recombinant lactococcus lactis cEGF-pNZ8149 has strong gastric acid resistance, can survive in the stomach and keep good survival rate, and plays a role in probiotics.
TABLE 11 tolerance of recombinant lactococcus lactis cEGF-pNZ8149 to gastric acid
| Processing time | Simulated gastric fluid set | Control group | Survival rate |
| 0h | (1.18±0.17)×104 | (1.33±0.25)×104 | 100％ |
| 2h | (1.01±0.37)×104 | (1.18±0.17)×104 | 85.59％ |
| 4h | (0.79±0.10)×104 | (1.09±0.05)×104 | 72.48％ |
Example 6: recombinant lactococcus lactis cEGF-pNZ8149 fermentation conditions and mode selection
Resuscitates recombinant lactococcus lactis cEGF-pNZ8149200mL, and carries out stationary culture at 30 ℃ for 24 hours to prepare seed liquid, and prepares a fermentation culture medium according to the proportion of 25% lactose, 10% peptone, 10% yeast extract, 5% beef extract, 0.08% magnesium sulfate, 0.1% potassium dihydrogen phosphate and 0.1% defoamer. The 30L tank is prepared according to the volume of 20L without adding lactose, the initial constant volume is 18L, the sterilization condition is 121 ℃ for 20min, the volume is fixed to 900ml after 500g of lactose is dissolved, the lactose is put into a feeding bottle, sterilized and cooled, and then added before inoculation, and the pH value before sterilization is about 7.2; the exhaust port is tied by 12 layers of gauze; and (5) normal ventilation and cooling after the actual elimination is completed. Adding seed liquid, and inoculating 200ml; stirring speed is 50rpm, whole-course culture temperature is 30 ℃, and ventilation is not carried out. Adding inducer (0.1 ml of 1mg/ml stock solution is taken and added into 100ml of sterile water) after fermentation for 6 hours, and sampling 1 tube to detect the number of viable bacteria according to 22 hours in a fermentation period, wherein the number of viable bacteria is 4.8X10 9.
The probiotic preparation is prepared by three methods of starch spray drying, corn cob powder adsorption drying and skimmed milk powder freeze-drying, and the viable count of different methods is measured. The results are shown in Table 12, and compared with the number of viable bacteria sampled by the fermenter, the method for drying the recombinant lactococcus lactis cEGF-pNZ8149 has the advantages of long process time, complex operation and mold pollution of the preparation, and thus, the technique of freeze-drying the skimmed milk powder is selected to process the recombinant lactococcus lactis cEGF-pNZ8149.
Table 12 survival rates of recombinant lactococcus lactis cEGF-pNZ8149 in different drying methods
| Drying method | Number of viable bacteria | Survival rate |
| Starch spray drying | 1.9×109 | 39.58％ |
| Corn cob powder adsorption drying | 5×108 | 10.4％ |
| Freeze drying of skimmed milk powder | 3.3×109 | 68.75％ |
Example 7: effect verification of recombinant lactococcus lactis cEGF-pNZ8149 preparation
After 12 healthy beagle dogs (commercially available) are randomly selected and are suitable for 3d, the conditions of temperature, dog food and the like are consistent, the grouping mode is shown in table 13, drinking water of a diarrhea model group, an empty pNZ8149 control group and a recombined cEGF-pNZ8149 group is changed into senna leaf water, tap water is normally fed to a blank control group, and the model test is carried out after three days of treatment. And (3) in a later-period empty pNZ8149 control group, a recombinant cEGF-pNZ8149 group and a test period, wherein freeze-dried bacterial powder is fed once a day. The detection indexes are mainly as follows:
(1) Intestinal development: at 14d, samples were taken from the duodenum and colon of dogs, paraffin sections were prepared and HE stained to observe the histopathological changes of the canine organs at each stage of each group.
(2) Immune index: 0d, 7d and 14d are respectively collected by vein to separate serum, and ELISA is used for measuring intestinal inflammatory cytokines: IL-6, IL-1. Beta., TNF-alpha., IL-10.
(3) Lactic acid bacteria amount detection: and (4) aseptically collecting feces of dogs in each group at 14d, and detecting the amount of lactic acid bacteria in the intestinal tract by a fluorescence quantification method.
(4) CEGF detection of downstream signals: western Blot detects cEGFR and activation of the downstream signal ERK1/2, AKT in intestinal tissue.
TABLE 13 grouping of animal experiments
| Group of | Treatment mode |
| Blank control group | Basic dog food + water |
| Diarrhea model group | Basic dog food + water |
| Empty pNZ8149 control | Basic canine food+pNZ8149 freeze-dried powder |
| Recombinant cEGF-pNZ8149 group | Basic canine food + cEGF-pNZ8149 preparation |
As shown in fig. 16, the levels of p-EGFR, p-AKT, p-ERK1/2 were significantly increased in the 8 recombinant cEGF-pNZ8149 group compared to the diarrhea model group and the empty pNZ8149 control group, indicating that cEGF expressed by recombinant secretion in the probiotic preparation activates EGFR and its downstream signals.
As shown in fig. 17, compared with the blank control group, the content of lactic acid bacteria in the canine feces of the diarrhea model group is obviously reduced, the content of lactic acid bacteria in the canine feces of the recombinant cEGF-pNZ8149 group is obviously increased compared with the diarrhea model group, and the empty pNZ8149 control group is also high. Indicating that both empty pNZ8149 and lyophilized and dried formulations of recombinant cEGF-pNZ8149 can successfully enter gastrointestinal colonisation in dogs and exert a probiotic effect.
As shown in fig. 18, 19, 20 and 21, according to the analysis of pathological section results of duodenum, colon, jejunum and ileum of each group, the intestinal injury of the recombinant cEGF-pNZ8149 group is obviously lighter than that of a diarrhea model group and an empty pNZ8149 control group, which shows that the preparation largely preserves the repairing effect of cEGF on the intestinal tract.
From the cytokine results of fig. 22, the recombinant cEGF-pNZ8149 group significantly upregulated the expression of the anti-inflammatory factor IL-10, down-regulated the expression of the pro-inflammatory factor, and also significantly different from the empty group, compared to the diarrhea model group. The recombinant probiotic preparation can effectively regulate the body inflammation level and improve the body state.
Therefore, according to the animal test results, the results of the in-vivo effect verification of dogs on cEGF-pNZ8149 preparations, namely cEGF downstream signal paths, lactobacillus content, intestinal development and immune indexes, show that the cEGF-pNZ8149 probiotic preparation provided by the embodiment effectively maintains the viable bacteria amount of lactococcus lactis, smoothly reaches the gastrointestinal internal colonisation of dogs and plays a role; and the secretory expression cEGF also has good activity, can treat intestinal injury and inflammation of dogs, and has good effect.
In summary, the embodiment of the application provides a recombinant lactococcus lactis with secretory expression cEGF, which not only realizes the non-antisecretory expression of cEGF, but also can play the probiotic effect of the lactococcus lactis to protect the intestinal health. The application also respectively verifies that the constructed recombinant lactococcus lactis cEGF-pNZ8149 can successfully secrete and express active cEGF protein through in vivo and in vitro experiments, and researches and tests prove that the bacterium has good antibacterial effect and gastric acid and bile salt resistance. Therefore, the embodiment of the application also prepares the probiotic preparation by selecting an optimal process to provide the probiotic preparation capable of protecting the intestinal health of dogs, and the verification shows that the prepared probiotic preparation well maintains the functions of cEGF-pNZ8149 lactococcus lactis and can well colonise and play a role.
The present application is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present application are intended to be included in the scope of the present application.
Claims (10)
1. A recombinant lactococcus lactis is lactococcus lactis NZ3900 transformed with recombinant plasmid without antisecretory expression cEGF.
2. The recombinant lactococcus lactis according to claim 1, wherein said recombinant lactococcus lactis is capable of being induced by Nisin to extracellular expression cECF.
3. The recombinant lactococcus lactis according to claim 2, wherein the extracellular expression cECF is highest when the Nisin concentration is 5ng/mL and the induction is performed for 20 hours.
4. A probiotic formulation for protecting canine intestinal health, characterized by comprising the recombinant lactococcus lactis according to any one of claims 1 to 3 in a concentration of not less than 5 x 10 8.
5. A method for constructing a recombinant lactococcus lactis, namely lactococcus lactis NZ3900, into which a recombinant plasmid without antisecretory expression cEGF is transferred, the method comprising:
Constructing a recombinant plasmid without antisecretory expression cEGF; and
The recombinant plasmid was transferred into lactococcus lactis NZ3900.
6. The construction method according to claim 5, wherein the construction step of the recombinant plasmid comprises:
obtaining cECF target fragment; and
Carrying out a ligation reaction on the digested cECF target fragment and the digested carrier plasmid by using T4 ligase, wherein a ligation product comprises the recombinant plasmid;
wherein the ligation reaction system comprises, in 20. Mu.l, 1.5. Mu.l of the fragment of interest, 4.0. Mu.l of the plasmid vector, 0.5. Mu. l T. Mu.4 ligase, 10 XBuffer and the balance of water, wherein the activity of the T4 ligase is 1000U.
7. The method according to claim 6, wherein the step of obtaining the target fragment cECF comprises:
Obtaining a cECF protein sequence;
Performing codon optimization on the protein sequence by using the codon preference of lactococcus lactis to obtain a DNA sequence with the length of 156 bp;
Synthesizing a DNA sequence with the length of 156bp, a SPusp signal peptide sequence, a 6 XHis tag and a connecting sequence of NcoI and SacI restriction enzyme digestion sequences;
Ligating the ligation sequence with the plasmid pTOPO-Blunt-Simple to obtain a recombinant plasmid comprising the cECF fragment of interest; and
And carrying out PCR amplification on the recombinant plasmid containing the cECF target fragment to obtain the cECF target fragment.
8. The construction method according to claim 7, wherein the nucleotide sequence of the primer pair for PCR amplification of the recombinant plasmid comprising the cECF fragment of interest is shown in SEQ ID NO. 1-2.
9. A method of antibiotic-free expression cECF, comprising:
obtaining the recombinant lactococcus lactis according to any one of claims 1 to 3 by the construction method according to any one of claims 5 to 8;
inoculating the recombinant lactococcus lactis to an M17 liquid culture medium, and culturing at 30 ℃ for 24 hours;
Inoculating the culture into an M17 liquid culture medium according to the ratio of 1:25, continuously culturing until bacteria enter a logarithmic growth phase, adding 10-80 ng/mL of lactobacillin to induce for 5-30 hours, stopping culturing, and centrifuging at the temperature of 4 ℃ for 5min at 12000r/min to respectively collect supernatant, wherein the supernatant comprises cECF.
10. Use of the recombinant lactococcus lactis according to any one of claims 1 to 3 for the preparation of a preparation for improving canine intestinal diarrhea, inhibiting intestinal inflammation, and repairing intestinal damage.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2023107183214 | 2023-06-16 | ||
| CN202310718321.4A CN116676246A (en) | 2023-06-16 | 2023-06-16 | Recombinant Lactococcus lactis, probiotic preparation, construction method, cECF expression method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117946953A true CN117946953A (en) | 2024-04-30 |
Family
ID=87790684
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310718321.4A Pending CN116676246A (en) | 2023-06-16 | 2023-06-16 | Recombinant Lactococcus lactis, probiotic preparation, construction method, cECF expression method and application |
| CN202410155654.5A Pending CN117946953A (en) | 2023-06-16 | 2024-02-04 | Recombinant lactococcus lactis, probiotic preparation, construction method, cECF expression method and application |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310718321.4A Pending CN116676246A (en) | 2023-06-16 | 2023-06-16 | Recombinant Lactococcus lactis, probiotic preparation, construction method, cECF expression method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN116676246A (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117625505B (en) * | 2023-11-27 | 2024-05-03 | 华中农业大学 | Lactococcus lactis vector oral vaccine for producing enterotoxin escherichia coli K88 genotype and application |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003250250B2 (en) * | 2002-06-19 | 2008-02-14 | Intrexon Actobiotics Nv | Methods and means to promote gut absorption |
| CN102796755B (en) * | 2012-06-28 | 2015-02-04 | 郑州大学 | Lactococcus lactis expression vector and preparation method and application thereof |
| CN110283765A (en) * | 2019-04-30 | 2019-09-27 | 华中农业大学 | With the environmentally friendly pig's epidermal growth factor Recombinant Lactococcus lactis for repairing function of intestinal canal |
-
2023
- 2023-06-16 CN CN202310718321.4A patent/CN116676246A/en active Pending
-
2024
- 2024-02-04 CN CN202410155654.5A patent/CN117946953A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CN116676246A (en) | 2023-09-01 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110144304B (en) | Lactobacillus casei strain and application thereof | |
| CN117946953A (en) | Recombinant lactococcus lactis, probiotic preparation, construction method, cECF expression method and application | |
| CN110591938B (en) | High-stress-resistance bacillus coagulans mutant strain and application thereof | |
| US8741622B2 (en) | Stress tolerant Bifidobacteria | |
| CN113151064B (en) | Pediococcus pentosaceus and application thereof | |
| CN108102987B (en) | Preparation of lactobacillus reuteri SS23-52 and dry powder leavening agent thereof and application of lactobacillus reuteri SS23-52 in pure probiotic yogurt | |
| CN112852684B (en) | Lactobacillus plantarum strain Y388 and application thereof | |
| CN112625979B (en) | Lactobacillus casei for resisting helicobacter pylori and application thereof | |
| CN116179440B (en) | Bacillus gallinarum and application thereof | |
| CN113403227A (en) | Lactobacillus plantarum and preparation method and application thereof | |
| CN101368164B (en) | Combined cholate hydrolysis enzyme gene and lactobacillus casei comprising the gene | |
| CN108179122B (en) | High-adhesion probiotic enterococcus faecium and application thereof | |
| CN112592870B (en) | Bacillus coagulans G4, fermentation liquor thereof, preparation method and application | |
| KR101613897B1 (en) | Shuttle vector comprising a promoter inducibly expressed under bile acid and composition comprising the same | |
| CN107828703B (en) | Lactobacillus reuteri Fullarton-9-35 and application thereof | |
| Johnson et al. | Selection of Lactobacillus acidophilus strains for use in “acidophilus products” | |
| KR100457879B1 (en) | Plasmid originated from Bifidobacterium, recombinant expression vector using the plasmid and transformation method | |
| CN112708577B (en) | Lactobacillus fermentum DALI02 with high intestinal adhesion and immunoregulation function and application thereof | |
| CN102382779B (en) | Manufacture method for set type additive-free yogurt and lactobacillus casei used therein | |
| JP6412865B2 (en) | Bifidobacterium breve strain specific gene | |
| CN108102976B (en) | Lactobacillus reuteri SS23-27 and application thereof in preparation of pure probiotic yogurt | |
| CN1195062C (en) | Non-antibiotics resistant shuttle plasmid expression carrier and its constructing method and uses | |
| CN108330082B (en) | Lactobacillus paracasei and application thereof | |
| KR101221587B1 (en) | Method for Preparing Mozzarella cheese using Streptococcus macedonicus LC743 | |
| CN102337231A (en) | Fermented intestine-strengthening cheese/powder production method and its applied lactobacillus casei |
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 |