CN116948933A - 一种表达锚定柔嫩艾美耳球虫ron2抗原的重组植物乳杆菌及应用 - Google Patents
一种表达锚定柔嫩艾美耳球虫ron2抗原的重组植物乳杆菌及应用 Download PDFInfo
- Publication number
- CN116948933A CN116948933A CN202310904846.7A CN202310904846A CN116948933A CN 116948933 A CN116948933 A CN 116948933A CN 202310904846 A CN202310904846 A CN 202310904846A CN 116948933 A CN116948933 A CN 116948933A
- Authority
- CN
- China
- Prior art keywords
- ron2
- lactobacillus plantarum
- dcpep
- recombinant lactobacillus
- recombinant
- 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
- 240000006024 Lactobacillus plantarum Species 0.000 title claims abstract description 108
- 235000013965 Lactobacillus plantarum Nutrition 0.000 title claims abstract description 107
- 229940072205 lactobacillus plantarum Drugs 0.000 title claims abstract description 107
- 101100454739 Arabidopsis thaliana LUG gene Proteins 0.000 title claims abstract description 59
- 101100305998 Toxoplasma gondii (strain ATCC 50611 / Me49) RON2 gene Proteins 0.000 title claims abstract description 59
- 239000000427 antigen Substances 0.000 title claims abstract description 23
- 102000036639 antigens Human genes 0.000 title claims abstract description 23
- 108091007433 antigens Proteins 0.000 title claims abstract description 23
- 241000223932 Eimeria tenella Species 0.000 title claims abstract description 21
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 48
- 230000000694 effects Effects 0.000 claims abstract description 21
- 108020001507 fusion proteins Proteins 0.000 claims abstract description 21
- 102000037865 fusion proteins Human genes 0.000 claims abstract description 21
- 239000012634 fragment Substances 0.000 claims abstract description 15
- 238000004873 anchoring Methods 0.000 claims abstract description 10
- 101100059000 Bacillus subtilis (strain 168) capA gene Proteins 0.000 claims abstract description 8
- 101100083407 Mycobacterium tuberculosis (strain ATCC 25618 / H37Rv) pgsA1 gene Proteins 0.000 claims abstract description 8
- 229940126578 oral vaccine Drugs 0.000 claims abstract description 8
- 101150076330 pgsA gene Proteins 0.000 claims abstract description 8
- 244000144977 poultry Species 0.000 claims abstract description 5
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 5
- 241000287828 Gallus gallus Species 0.000 claims description 32
- 235000013330 chicken meat Nutrition 0.000 claims description 31
- 239000013612 plasmid Substances 0.000 claims description 27
- 210000000952 spleen Anatomy 0.000 claims description 22
- 108010002350 Interleukin-2 Proteins 0.000 claims description 16
- 208000003495 Coccidiosis Diseases 0.000 claims description 14
- 206010023076 Isosporiasis Diseases 0.000 claims description 14
- 241001591005 Siga Species 0.000 claims description 14
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 12
- 239000003814 drug Substances 0.000 claims description 11
- 108010074328 Interferon-gamma Proteins 0.000 claims description 10
- 150000001413 amino acids Chemical class 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- 102000004127 Cytokines Human genes 0.000 claims description 7
- 108090000695 Cytokines Proteins 0.000 claims description 7
- 210000001986 peyer's patch Anatomy 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 230000003213 activating effect Effects 0.000 claims description 6
- 239000002773 nucleotide Substances 0.000 claims description 6
- 125000003729 nucleotide group Chemical group 0.000 claims description 6
- 108020004705 Codon Proteins 0.000 claims description 4
- -1 but not limited to Substances 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 4
- 230000004927 fusion Effects 0.000 claims description 4
- 210000002865 immune cell Anatomy 0.000 claims description 4
- 210000001165 lymph node Anatomy 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 235000013594 poultry meat Nutrition 0.000 claims description 4
- 230000002265 prevention Effects 0.000 claims description 4
- 239000002904 solvent Substances 0.000 claims description 4
- 230000004936 stimulating effect Effects 0.000 claims description 4
- 102100037850 Interferon gamma Human genes 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 238000009472 formulation Methods 0.000 claims description 3
- 229920001184 polypeptide Polymers 0.000 claims description 3
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 2
- 239000000654 additive Substances 0.000 claims description 2
- 239000003963 antioxidant agent Substances 0.000 claims description 2
- 210000003719 b-lymphocyte Anatomy 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 claims description 2
- 239000003086 colorant Substances 0.000 claims description 2
- 235000003599 food sweetener Nutrition 0.000 claims description 2
- 239000007951 isotonicity adjuster Substances 0.000 claims description 2
- 238000005457 optimization Methods 0.000 claims description 2
- 239000000375 suspending agent Substances 0.000 claims description 2
- 239000003765 sweetening agent Substances 0.000 claims description 2
- 241000272517 Anseriformes Species 0.000 claims 2
- 125000003275 alpha amino acid group Chemical group 0.000 claims 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims 1
- 239000003755 preservative agent Substances 0.000 claims 1
- 229960005486 vaccine Drugs 0.000 abstract description 29
- 210000003250 oocyst Anatomy 0.000 abstract description 24
- 230000003902 lesion Effects 0.000 abstract description 19
- 238000000034 method Methods 0.000 abstract description 19
- 239000013598 vector Substances 0.000 abstract description 19
- 230000001165 anti-coccidial effect Effects 0.000 abstract description 17
- 210000004027 cell Anatomy 0.000 abstract description 17
- 210000004534 cecum Anatomy 0.000 abstract description 15
- 230000002829 reductive effect Effects 0.000 abstract description 15
- 235000019786 weight gain Nutrition 0.000 abstract description 12
- 230000004584 weight gain Effects 0.000 abstract description 11
- 210000004443 dendritic cell Anatomy 0.000 abstract description 10
- 230000008569 process Effects 0.000 abstract description 5
- 101150097271 RON2 gene Proteins 0.000 abstract description 3
- 230000005847 immunogenicity Effects 0.000 abstract description 3
- 230000008685 targeting Effects 0.000 abstract 2
- 238000009395 breeding Methods 0.000 abstract 1
- 230000001488 breeding effect Effects 0.000 abstract 1
- 230000002103 transcriptional effect Effects 0.000 abstract 1
- 230000003053 immunization Effects 0.000 description 28
- 238000002649 immunization Methods 0.000 description 27
- 102000004169 proteins and genes Human genes 0.000 description 26
- 210000004698 lymphocyte Anatomy 0.000 description 22
- 238000001514 detection method Methods 0.000 description 18
- 210000002966 serum Anatomy 0.000 description 16
- 102000000588 Interleukin-2 Human genes 0.000 description 15
- 230000000968 intestinal effect Effects 0.000 description 14
- 239000007788 liquid Substances 0.000 description 12
- 238000003776 cleavage reaction Methods 0.000 description 11
- 230000007017 scission Effects 0.000 description 11
- 238000001976 enzyme digestion Methods 0.000 description 9
- 239000000047 product Substances 0.000 description 9
- 230000035755 proliferation Effects 0.000 description 9
- 238000012360 testing method Methods 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 241000224483 Coccidia Species 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 229940088598 enzyme Drugs 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 239000000499 gel Substances 0.000 description 6
- 230000028993 immune response Effects 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 238000001262 western blot Methods 0.000 description 6
- 238000002965 ELISA Methods 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 230000009545 invasion Effects 0.000 description 5
- 230000036285 pathological change Effects 0.000 description 5
- 231100000915 pathological change Toxicity 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- 239000001993 wax Substances 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 4
- ULGZDMOVFRHVEP-RWJQBGPGSA-N Erythromycin Chemical compound O([C@@H]1[C@@H](C)C(=O)O[C@@H]([C@@]([C@H](O)[C@@H](C)C(=O)[C@H](C)C[C@@](C)(O)[C@H](O[C@H]2[C@@H]([C@H](C[C@@H](C)O2)N(C)C)O)[C@H]1C)(C)O)CC)[C@H]1C[C@@](C)(OC)[C@@H](O)[C@H](C)O1 ULGZDMOVFRHVEP-RWJQBGPGSA-N 0.000 description 4
- 108010087230 Sincalide Proteins 0.000 description 4
- 239000002671 adjuvant Substances 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 235000021053 average weight gain Nutrition 0.000 description 4
- 238000010609 cell counting kit-8 assay Methods 0.000 description 4
- 238000010276 construction Methods 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 201000010099 disease Diseases 0.000 description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 4
- 210000003608 fece Anatomy 0.000 description 4
- 210000001035 gastrointestinal tract Anatomy 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 208000015181 infectious disease Diseases 0.000 description 4
- 230000003903 intestinal lesions Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 238000008157 ELISA kit Methods 0.000 description 3
- 241000223924 Eimeria Species 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 230000000740 bleeding effect Effects 0.000 description 3
- 230000037396 body weight Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000001900 immune effect Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 244000045947 parasite Species 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
- 238000012795 verification Methods 0.000 description 3
- 241000283707 Capra Species 0.000 description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 2
- 102000012410 DNA Ligases Human genes 0.000 description 2
- 108010061982 DNA Ligases Proteins 0.000 description 2
- 241000238631 Hexapoda Species 0.000 description 2
- 208000030852 Parasitic disease Diseases 0.000 description 2
- 101710194807 Protective antigen Proteins 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 238000009825 accumulation Methods 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000033289 adaptive immune response Effects 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 101150078331 ama-1 gene Proteins 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 238000004364 calculation method Methods 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 229910052802 copper Inorganic materials 0.000 description 2
- 239000010949 copper Substances 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000003651 drinking water Substances 0.000 description 2
- 235000020188 drinking water Nutrition 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 210000003743 erythrocyte Anatomy 0.000 description 2
- 229960003276 erythromycin Drugs 0.000 description 2
- 238000003958 fumigation Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 210000004969 inflammatory cell Anatomy 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 210000000936 intestine Anatomy 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000002184 metal Substances 0.000 description 2
- 229910052751 metal Inorganic materials 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000003071 parasitic effect Effects 0.000 description 2
- 230000007918 pathogenicity Effects 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000007170 pathology Effects 0.000 description 2
- YBYRMVIVWMBXKQ-UHFFFAOYSA-N phenylmethanesulfonyl fluoride Chemical compound FS(=O)(=O)CC1=CC=CC=C1 YBYRMVIVWMBXKQ-UHFFFAOYSA-N 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000000527 sonication Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000003053 toxin Substances 0.000 description 2
- 231100000765 toxin Toxicity 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- YRIZYWQGELRKNT-UHFFFAOYSA-N 1,3,5-trichloro-1,3,5-triazinane-2,4,6-trione Chemical compound ClN1C(=O)N(Cl)C(=O)N(Cl)C1=O YRIZYWQGELRKNT-UHFFFAOYSA-N 0.000 description 1
- 206010000050 Abdominal adhesions Diseases 0.000 description 1
- 241000272525 Anas platyrhynchos Species 0.000 description 1
- 241000272814 Anser sp. Species 0.000 description 1
- 101710203310 Apical membrane antigen 1 Proteins 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 241000271566 Aves Species 0.000 description 1
- 244000063299 Bacillus subtilis Species 0.000 description 1
- 235000014469 Bacillus subtilis Nutrition 0.000 description 1
- 108010077805 Bacterial Proteins Proteins 0.000 description 1
- 101100038180 Caenorhabditis briggsae rpb-1 gene Proteins 0.000 description 1
- 241000224484 Eimeriidae Species 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 239000006142 Luria-Bertani Agar Substances 0.000 description 1
- FSNCEEGOMTYXKY-JTQLQIEISA-N Lycoperodine 1 Natural products N1C2=CC=CC=C2C2=C1CN[C@H](C(=O)O)C2 FSNCEEGOMTYXKY-JTQLQIEISA-N 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 102000016943 Muramidase Human genes 0.000 description 1
- 108010014251 Muramidase Proteins 0.000 description 1
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 1
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 1
- 108010020346 Polyglutamic Acid Proteins 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- 229930006000 Sucrose Natural products 0.000 description 1
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 1
- 230000006044 T cell activation Effects 0.000 description 1
- 241000863480 Vinca Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003833 bile salt Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000002737 cell proliferation kit Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 230000002183 duodenal effect Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000004520 electroporation Methods 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000029578 entry into host Effects 0.000 description 1
- 230000026502 entry into host cell Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 239000008098 formaldehyde solution Substances 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 210000003736 gastrointestinal content Anatomy 0.000 description 1
- 210000002288 golgi apparatus Anatomy 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000013632 homeostatic process Effects 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000015788 innate immune response Effects 0.000 description 1
- 230000031891 intestinal absorption Effects 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229960000274 lysozyme Drugs 0.000 description 1
- 239000004325 lysozyme Substances 0.000 description 1
- 235000010335 lysozyme Nutrition 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229920002643 polyglutamic acid Polymers 0.000 description 1
- KMUONIBRACKNSN-UHFFFAOYSA-N potassium dichromate Chemical compound [K+].[K+].[O-][Cr](=O)(=O)O[Cr]([O-])(=O)=O KMUONIBRACKNSN-UHFFFAOYSA-N 0.000 description 1
- 238000012257 pre-denaturation Methods 0.000 description 1
- 239000006041 probiotic Substances 0.000 description 1
- 235000018291 probiotics Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000028070 sporulation Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000008096 xylene Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/44—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from protozoa
- C07K14/455—Eimeria
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/70—Feeding-stuffs specially adapted for particular animals for birds
- A23K50/75—Feeding-stuffs specially adapted for particular animals for birds for poultry
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/002—Protozoa antigens
- A61K39/012—Coccidia antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P33/00—Antiparasitic agents
- A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
- A61P37/02—Immunomodulators
- A61P37/04—Immunostimulants
-
- 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
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
- A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/55—Medicinal preparations containing antigens or antibodies characterised by the host/recipient, e.g. newborn with maternal antibodies
- A61K2039/552—Veterinary vaccine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/035—Fusion polypeptide containing a localisation/targetting motif containing a signal for targeting to the external surface of a cell, e.g. to the outer membrane of Gram negative bacteria, GPI- anchored eukaryote proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
- C12R2001/25—Lactobacillus plantarum
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Organic Chemistry (AREA)
- Veterinary Medicine (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Polymers & Plastics (AREA)
- Public Health (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Mycology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Food Science & Technology (AREA)
- Animal Husbandry (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Plant Pathology (AREA)
- Physiology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
本发明涉及一种表达锚定柔嫩艾美耳球虫RON2抗原的重组植物乳杆菌及应用。本发明提供的所述重组植物乳杆菌中含有RON2‑树突状细胞靶向肽(DCpep)融合蛋白转录基因,其由RON2基因片段与3个DCpep基因片段串联组成。该融合蛋白具有良好的免疫原性,可通过pgsA'载体锚定表达在植物乳杆菌表面,可用于制备口服疫苗。与市售疫苗相比,本重组植物乳杆菌免疫组雏鸡增重率和抗球虫指数明显升高,卵囊排出量和盲肠病变明显降低。以上结果证实,本发明构建的口服表面锚定柔嫩艾美耳球虫RON2抗原靶向树突状细胞的重组植物乳杆菌具有良好的免疫保护效果,为家禽养殖过程中鸡柔嫩艾美耳球虫病的防控提供新方向。
Description
技术领域
本发明属于工程微生物技术领域,具体涉及一种表达锚定柔嫩艾美耳球虫RON2抗原的重组植物乳杆菌、包含所述重组植物乳杆菌在制备防治鸡球虫病药物中的应用。
背景技术
公开该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不必然被视为承认或以任何形式暗示该信息构成已经成为本领域一般技术人员所公知的现有技术。
球虫病是一种严重的家禽寄生虫疾病。鸡球虫病(Avian Coccidiosis)是由艾美耳属球虫感染导致的一种急性肠道寄生虫病。鸡球虫在病原学分类上属于顶复器门(Apicomplaxa)、孢子虫纲(Sporozoasida)、真球虫目(Euccidiasina)、艾美耳科(Eimeriidae)、艾美耳属(Eimeria)。目前全球已有记载的十三种鸡球虫中我国发现有九种,球虫在鸡肠道寄生部位因球虫种类各异,致病力也各有差异,其中柔嫩艾美耳球虫(Eimeria tenella)主要寄生于鸡盲肠,致病力最强,引起的危害最为严重。
柔嫩艾美耳球虫属于顶复门原虫,顶复门原虫是一类专性胞内寄生性原虫,虽然不同种类的顶复门原虫寄生的宿主种类和细胞类型有所不同,但由于顶复门原虫的虫体前部均具有可分泌蛋白的顶复器细胞结构,使得它们有一套共同的宿主细胞入侵机制。入侵过程依赖于顶复器分泌的多种特殊蛋白相互协同完成。
入侵相关蛋白共同参与了顶复门原虫的入侵过程,这些入侵相关蛋白共同发挥作用介导虫体对宿主细胞的黏附和识别。其中棒状体蛋白为重要的入侵蛋白。棒状体颈部蛋白位于棒状体的颈部,由内质网和高尔基体加工合成后转运至棒状体内储存,受Ca2+信号刺激后在棒状体的颈部经蛋白酶水解加工后分泌到细胞外,包括RON1~RON8。而在棒状体颈部蛋白中RON2是更为重要的,在参与形成运动接触环(MJ)的棒状体颈部蛋白中只有RON2含有两个跨膜区,这就是RON2能够跨过宿主细胞膜与顶膜抗原-1(AMA1)相互作用的原因,从而介导入侵宿主细胞,并且AMA1与RON2相互作用在顶复门原虫中形成MJ结构的分子机制是保守存在的,因此阻断AMA1与RON2之间的相互作用,阻断MJ结构的形成也可成为抑制顶复门原虫的入侵的途径,表明棒状体颈部蛋白RON2也有可能成为新型药物或疫苗的研究方向。
柔嫩艾美耳球虫病的预防和控制主要依靠抗球虫药物和球虫活疫苗。然而,抗球虫药物的长期使用导致了耐药性虫株的出现和动物产品中药物残留的问题,给人类健康和环境带来了危害。球虫疫苗可作为防制鸡球虫病的有效策略,但球虫活疫苗价格昂贵、保存困难,又存在返毒、散毒的风险。因此,开发安全有效的球虫疫苗对防控球虫病非常重要。
发明内容
为改善现有技术的不足,本发明提供了表面锚定柔嫩艾美耳球虫RON2抗原靶向树突状细胞的重组植物乳杆菌及其初步应用,该重组菌中含有RON2-DCpep融合基因,具有良好的免疫原性,且重组植物乳杆菌能够将柔嫩艾美耳球虫融合抗原锚定在植物乳杆菌表面表达。以其对动物进行免疫,具有良好的免疫效果,能诱导派氏淋巴结DC的活化,诱导T细胞增加并分泌细胞因子IFN-γ和IL-2,促进sIgA和IgG抗体水平升高;与市售疫苗相比,本发明提供的重组植物乳杆菌免疫组雏鸡增重率和抗球虫指数明显升高,卵囊排出量和盲肠病变明显降低。
基于上述技术效果,本发明提供如下的技术方案:
本发明第一方面,提供一种表达锚定柔嫩艾美耳球虫RON2抗原的重组植物乳杆菌,与野生型相比,该重组植物乳杆菌被改造为具有RON2-Dcpep融合蛋白表达活性;
所述融合蛋白由RON2与Dcpep连接而成,Dcpep序列如SEQ ID NO.1所示。
现有研究表明,棒状体颈部蛋白RON2可能具有球虫病抗原活性,本发明设计将其作为疫苗抗原,上述方案中,所述RON2的为GenBank登录号XM_013375678C的胞外区(aa.1082~1402)。为了进一步增强疫苗的免疫原活性,本发明在RON2的基础上增加了Dcpep作为佐剂。
树突状细胞(Dendritic cell,DC)的重要作用是摄取、加工处理和递呈抗原,刺激机体产生免疫应答反应。DC是一类成熟时具有许多树突状突起的细胞,因此而命名。这类细胞成熟时能够识别、摄取和加工外源性抗原并将抗原肽提呈给初始T细胞进而诱导T细胞活化增殖,是功能最强的抗原提呈细胞。DC是机体适应性免疫应答的始动者,也是连接固有免疫应答和适应性免疫应答的“桥梁”。
发明人在研究中发现,DCpep能很好地靶向树突状细胞,从而引起机体的免疫应答。用DCpep作为佐剂,有着序列短的优势,可以解决标签表达的难题,同时也为抗原制备提供了方便。且DCpep仅由12个氨基酸组成,能够特异性靶向结合DCs,有助于DCs识别抗原,诱发免疫反应,且序列短,对免疫稳态起重要作用。然而,在本发明的一些实施方式中,发明人发现由12个氨基酸组成的DCpep,其组成使其空间构象容易被改变,DCpep的优势难以稳定发挥,发明人对此进行了研究来改善这种新发现的不利的情况,最终在研究过程中发现将DCpep做串联使用时能够改善上述问题,尤其是将3个DCpep做串联时使用既能避免空间构象的改变又能使其充分暴露,并且对DC细胞的作用更好,在发挥其优势的前提下,能够更好的协助激发机体更强的免疫应答。因此,本发明效果较好的一些实施方式中,所述融合蛋白由RON2及3个DCpep基因片段(3×DCpep)串联组成的,其密码子优化后核苷酸序列如SEQ IDNO.2所示,氨基酸序列如SEQ ID NO.3所示。另外的一些实施方式中,所述核苷酸序列还包括基于密码子简并性能够翻译得到RON2-Dcpep融合蛋白的其它序列,或在高严谨条件下可与SEQ ID NO.2所示序列杂交的核苷酸序列。
本发明针对上述融合蛋白作为抗原的能力进行了验证,结果表明RON2-DCpep相较于RON2表达得到的抗原具有更好的免疫原性,并且与植物乳杆菌内源代谢流相容性更高,在重组植物乳杆菌中更容易表达。因此,上述第一方面优选的实施方式中,所述出发菌株具体的实例如植物乳杆菌NC8。
通常来说,重组的外源蛋白通常可由质粒表达载体定向运输,根据蛋白与宿主菌的相对位置,本领域将重组外源蛋白的表达情况分为胞内分泌表达,胞外分泌表达及菌株表面锚定表达。本发明设计以上述重组植物乳杆菌作为疫苗,为了增强该工程菌的抗原性,本发明设计上述融合蛋白在重组植物乳杆菌表面锚定表达。所述锚定基团为聚谷氨酸合成酶A(pgsA),来自枯草芽孢杆菌,由1143个核苷酸构成,可编码381个氨基酸,是一段膜蛋白锚定序列,其N端存在一个跨膜区,为其表面展示创造了条件,并且pgsA'(pgsA截短为pgsA',其核苷酸序列如SEQ ID NO.4,氨基酸序列如SEQ ID NO.5所示)来作为表面单锚定表达的元件,使抗原能够在受体菌表面锚定并稳定表达。
因此,本发明又一种效果较好的实施方式中,所述重组植物乳杆菌中RON2-Dcpep融合蛋白与上述锚定元件融合表达,融合后多肽的氨基酸序列如SEQ ID NO.6所示,即所述重组植物乳杆菌相比野生型,具有SEQ ID NO.6所示多肽的表达活性。
另外,上述第一方面中,所述重组植物乳杆菌的“改造”方式为通过重组质粒构建,具体的实例如pSIP409-pgsA'质粒,同时,RON2-DCpep融合基因中还含有启动子、终止子和酶切位点基因序列,所述酶切位点为XbaI(TCTAGA)和HindIII(AAGCTT)。
第二方面,提供第一方面所述重组植物乳杆菌在制备防治鸡球虫病药物中的应用。
效果较好的一种实施方式中,所述防治鸡球虫病药物为疫苗,更优选的,为一种口服疫苗。
第三方面,提供一种口服疫苗,所述疫苗以上述第一方面中的重组植物乳杆菌作为抗原。
上述口服疫苗中还包括药学上所必须的辅料,如溶剂、增溶剂、悬浮剂、等渗剂、缓冲剂、舒缓剂、防腐剂、抗氧化剂、着色剂、甜味剂或其他制剂添加剂。由于上述重组植物乳杆菌相较于野生型具有更为优异的耐酸、耐胆盐能力,这有效降低了疫苗制备的工艺难度,拓宽了药物辅料的选择范围,还提供了更好的肠道粘附力。
第四方面,提供第一方面所述重组植物乳杆菌在制备家禽饲料中的应用。
所述家禽包括鸡、鸭或鹅,该饲料中应当包含动物生长所需的可食用物质,如碳水化合物、脂类、氨基酸、微量元素、饲料添加剂、维生素等。
第五方面,提供第一方面所述重组植物乳杆菌、第三方面所述口服疫苗在如下任意一个方面的应用:
(1)激活机体免疫细胞和/或制备激活机体免疫细胞的产品;
(2)激活派氏淋巴结DC和/或制备激活派氏淋巴结DC的产品;
(3)刺激脾和/或肠系膜淋巴结中的特异性细胞因子、T细胞、B细胞的产生和/或制备刺激脾、肠系膜淋巴结和/或派氏淋巴结中的细胞因子的产品;所述细胞因子、T淋巴细胞、CD4+、CD8+、IFN-γ、IL-2、IgG、SIgA。
以上一个或多个技术方案的有益效果在于:
本发明提供的重组植物乳杆菌具有融合蛋白RON2-Dcpep表达能力,该融合蛋白相比RON2的抗原活性显著提升,并且在重组菌株中的表达量也有所提升。本发明还在上述融合蛋白中增加了锚定元件,使上述融合蛋白能够锚定表达在重组植物乳杆菌表面。
此外,对动物进行免疫,能诱导派氏淋巴结DC的活化,诱导T细胞增加并分泌细胞因子IFN-γ和IL-2,促进sIgA和IgG抗体水平升高;与市售疫苗相比,本重组植物乳杆菌免疫组雏鸡增重率和抗球虫指数明显升高,卵囊排出量和盲肠病变明显降低。
附图说明
构成本发明的一部分的说明书附图用来提供对本发明的进一步理解,本发明的示意性实施例及其说明用于解释本发明,并不构成对本发明的不当限定。
图1为实施例1中双酶切获取合成目的基因片段;
图2为实施例1中目的基因PCR扩增结果;
图3为实施例1中胶回收双酶切载体产物;
图4为实施例1中重组质粒pSIP409-pgsA'-RON2的双酶切验证;M:DL10,000DNAMarker;1:pSIP409-pgsA'-RON2单酶切;2:pSIP409-pgsA'-RON2的双酶切验证;
图5为实施例1中重组质粒pSIP409-pgsA'-RON2-DCpep的双酶切验证;M:DL10,000DNA Marker;1:pSIP409-pgsA'-RON2-DCpep的双酶切验证;2:pSIP409-pgsA'-RON2-DCpep单酶切;
图6为实施例1中pSIP409-pgsA'-RON2的质粒图谱;
图7为实施例1中pSIP409-pgsA'-RON2-DCpep的质粒图谱;
图8为实施例1中菌株生长曲线图;
图9为实施例1中经超声破碎法处理获取的pSIP409-pgsA'-RON2蛋白在重组植物乳杆菌中表达的Western blot检测;M:蛋白Marker;1:pSIP409-pgsA';2:pSIP409-pgsA'-RON2的表达产物;
图10为实施例1中经超声破碎法处理获取的pSIP409-pgsA'-RON2-DCpep蛋白在重组植物乳杆菌中表达的Western blot检测;M:蛋白Marker;1:pSIP409-pgsA';2:pSIP409-pgsA'-RON2-DCpep的表达产物;
图11为实施例2中流式细胞术检测结果;
图12为实施例2中脾脏中淋巴细胞增殖结果;
图13为实施例2中血清中细胞因子IFN-γ水平变化;
图14为实施例2中血清中细胞因子IL-2水平变化;
图15为实施例2中血清中IgG抗体水平变化;
图16为实施例2中肠道灌洗液中SIgA抗体水平变化;
图17为实施例2中雏鸡攻虫后体重变化;
图18为实施例2中卵囊排出量;
图19为实施例2中盲肠病变评分;
图20为实施例2中攻虫后盲肠病变情况;
图21为实施例2中盲肠组织病理切片;
上述附图中,*,P<0.05;**,P<0.01***,P<0.001;****,P<0.0001。
具体实施方式
应该指出,以下详细说明都是示例性的,旨在对本发明提供进一步的说明。除非另有指明,本文使用的所有技术和科学术语具有与本发明所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是,这里所使用的术语仅是为了描述具体实施方式,而非意图限制根据本发明的示例性实施方式。如在这里所使用的,除非上下文另外明确指出,否则单数形式也意图包括复数形式,此外,还应当理解的是,当在本说明书中使用术语“包含”和/或“包括”时,其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
为了使得本领域技术人员能够更加清楚地了解本发明的技术方案,以下将结合具体的实施例详细说明本发明的技术方案。
实施例1表面锚定柔嫩艾美耳球虫RON2抗原靶向树突状细胞重组植物乳杆菌的构建及验证
通过对柔嫩艾美耳球虫的研究发现,RON2是柔嫩艾美耳球虫的一种重要入侵功能蛋白。本研究选择RON2为保护性抗原,以植物乳杆菌NC8株为初始菌株,以pSIP409为载体表达保护性抗原,以DCpep为佐剂,从而构建NC8-pSIP409-pgsA'-RON2-DCpep和NC8-pSIP409-pgsA'-RON2重组植物乳杆菌,通过Western blot检测目的蛋白的表达情况,并评价其对柔嫩艾美耳球虫感染的免疫保护效果。
1.材料与方法
菌株及质粒材料:BL21、DH5α、pSIP409-pgsA'质粒、植物乳杆菌(Lactobacillusplantarum,L.plantarum)NC8由吉林省动物微生态制剂工程研究中心实验室构建并保存。
酶与主要试验试剂:限制性内切酶(XbaI、HindⅢ、EcoRI)、T4 DNA连接酶、DNAMarker(DL-2000、DL-5000、DL-10000),Prime STAR Max Premix(2×)、SDS PAGE LoadingBuffer(5×)购于TaKaRa有限公司;普通质粒小提试剂盒、DNA纯化回收试剂盒、DNA凝胶回收试剂盒、HRP标记的山羊抗鼠IgG二抗、His标签蛋白纯化试剂盒购自于北京康为世纪有限公司;红霉素、卡那霉素、溶菌酶购于北京Solarbio科技有限公司;Bradford蛋白浓度检测试剂盒购于碧云天生物科技有限公司。
试验主要仪器设备:梯度PCR仪(Leica);凝胶成像分析系统(Universal HoodⅡ);制冷型金属浴(TANGEN);生化培养箱HERACELL240i(Thermo Scientific);电泳仪(BIO-RAD);分析天平ME204E(Mettler Toledo);电击穿孔仪(Gene Pulser Xcell TM System)。
1.1试验方法
重组植物乳杆菌的构建与鉴定:
目的基因RON2-DCpep的合成:
说明书
RON2基因序列:RON2为GenBank登录号XM_013375678C的胞外区(aa.1082~1402),RON2序列与3个DCpep串联为目的片段,并连接到pUC-57载体。
注:XbaI酶切位点基因序列:TCTAGA;HindIII酶切位点基因序列:AAGCTT;DCpep基因序列:TTTTATCCATCATATCATTCAACTCCACAACG TCCA(SEQ ID NO.1)。3×DCpep基因序列如SEQ ID NO.7所示。
引物设计:
为从pUC-57-RON2-DCpep质粒中获得RON2片段,设计了引物RON2-F和RON2-R,其序列如下:
RON2-F:5’-GCTCTAGAATGAGGAGCGCCGGCTTCC-3’(SEQ ID NO.8);
RON2-R:5’-CCAAGCTTTTAATGGTGATGGTGATGATGGAAATCT-3’(SEQ ID NO.9)。
pSIP409-pgsA'-RON2重组质粒的构建:
RON2目的片段的获取:
双酶切pUC57-RON2-DCpep质粒,使用凝胶回收试剂盒回收RON2-DCpep片段,按照试剂盒说明书中记载的方法进行。以RON2-DCpep为模版,进行目的基因RON2的PCR扩增。PCR体系如下:
表1酶切体系
表2PCR体系
PCR扩增条件如下:
95℃预变性3min,95℃变性30s,65℃退火30s,72℃延伸30s,35个循环,72℃延伸5min,4℃保存。对PCR扩增产物进行纯化。
纯化后取出2μL样品进行1%琼脂糖凝胶电泳检测。
载体的获取:
通过双酶切pSIP409-pgsA',与目的基因相同的酶切位点,酶切体系如下:
表3酶切体系
37℃酶切过夜后进行胶回收,胶回收方法按照试剂盒说明书进行。
目的片段和载体连接:
RON2-DCpep、RON2片段与pSIP409-pgsA'载体连接;
回收产物通过T4 DNA连接酶与载体连接。连接体系如下:
表4连接反应体系
连接条件为16℃过夜。
pSIP409-pgsA'和pSIP409-pgsA'-RON2重组质粒转化到DH5α感受态细胞:
通过普通转化方法将pSIP409-pgsA'-RON2-DCpep和pSIP409-pgsA'-RON2重组质粒转化到DH5α感受态。
a.取50μL冰浴上融化的感受态细胞,加入目的DNA,轻轻混匀,在冰浴中放置30分钟;
b.42℃水浴热激45秒,然后快速将EP管转移到冰浴中3分钟,该过程不要摇动离心管;
c.向每个离心管中加入700μL LB培养基,混匀后置于37℃,200rpm培养12小时,使细菌复苏;
d.吸取100μL已转化的感受态细胞加到含红霉素的LB琼脂培养基上,将细胞均匀涂开。将平板置于37℃至液体被吸收,倒置平板,37C过夜培养。
pSIP409-pgsA'-RON2-DCpep和pSIP409-pgsA'-RON2重组质粒酶切鉴定:
挑取单菌落到5mL LB液体培养基中37℃培养过夜。吸取200μL 80%甘油和800μL菌液到冻存管中,混匀,-80℃冰箱保存菌种。剩下新鲜菌液提取质粒,进行双酶切鉴定。酶切体系如下:
表5酶切体系
37℃酶切过夜后进行琼脂糖凝胶电泳,鉴定条带大小。
质粒测序:酶切及PCR鉴定正确后对质粒进行测序(吉林省库美生物科技有限公司)。
重组质粒转化到NC8感受态细胞:
NC8感受态制作:
取NC8冻存菌复苏后传代培养,获得OD600在0.2~0.3之间的菌液,清洗缓冲液(蔗糖34.21g、MgCl2 0.029g、ddH2O 100mL,pH值调至7.4)洗2遍,4℃,5000rpm离心10min,弃去上清。用电击缓冲液(Na3PO4 0.19g、MgCl2 0.009g、ddH2O 100mL,pH值调至7.4)重悬沉淀,然后以100μL每管分装,-80℃冰箱保存。
重组质粒的转化:通过电转化方法将构建完成的质粒转入NC8。条件为:
2000V,400Ω,25μF。
重组植物乳杆菌的表达鉴定:
首先进行植物乳杆菌培养条件的优化,检测生长曲线:取冻存菌液复苏后传代培养,30℃厌氧。间隔1h取2mL菌液测定OD600,OD600值在0.3左右时加诱导态SPPIP(12.5μL/5mL)。此外,通过滴板菌落计数,来计算两株重组植物乳杆菌的免疫剂量(饲喂植物乳杆菌最佳数量为0.5×109~1×109CFU)。
为检测构建的两株重组植物乳杆菌NC8-pSIP409-pgsA'-RON2-DCpep和NC8-pSIP409-pgsA'-RON2蛋白表达情况,采用超声破碎法提取重组菌蛋白进行Western blot验证,稀释好的His、Flag抗体为一抗(1:3000),HRP标记的山羊抗鼠IgG为二抗。
1.2结果
目的基因合成结果:构建得到的目的基因RON2-DCpep大小为1104bp,如图1所示。
RON2目的基因片段获取结果:通过PCR获得RON2目的片段,在996bp处可见目的基因片段,RON2基因的PCR扩增结果,如图2所示。
载体进行双酶切,在8742bp处可见载体条带,如图3所示。
酶切鉴定结果:将重组质粒pSIP409-pgsA'-RON2双酶切验证,在996bp、8742bp处可见清晰条带(图4)。将重组质粒pSIP409-pgsA'-RON2-DCpep双酶切验证,在1104bp、8742bp处可见清晰条带(图5),表明目的基因片段成功连接到目的载体。
质粒测序:质粒测序结果正确,用SnapGene软件制作了pSIP409-pgsA'-RON2和pSIP409-pgsA'-RON2-DCpep的质粒图谱,如图6、图7。
重组植物乳杆菌的表达鉴定结果:
重组植物乳杆菌培养条件优化:经过3次培养及测定OD600,NC8-pSIP409-pgsA'、NC8-pSIP409-pgsA'-RON2和NC8-pSIP409-pgsA'-RON2-DCpep的生长曲线检测结果(24h)用GraphPad Prism软件绘制,见图8。菌液转接后4h为最佳诱导时间。根据3次菌落滴板计数检测结果选择第10h(诱导后6h)为饲喂时间点,0.2mL NC8-pSIP409-pgsA'、0.22mL NC8-pSIP409-pgsA'-RON2-DCpep、0.25mL NC8Δ-pSIP409-pgsA'-RON2菌液中有1×109CFU。
Western blot技术鉴定融合蛋白表达结果:为鉴定重组植物乳杆菌的反应原性,进行Western blot检测。根据氨基酸序列预期NC8-pSIP409-pgsA'-RON2的蛋白分子量约为44kDa,NC8-pSIP409-pgsA'-RON2-DCpep的蛋白分子量约为48kDa。将诱导后的重组植物乳杆菌超声破碎后进行检测,在44kDa、48kDa处可见清晰的蛋白条带(图9、图10)。蛋白大小符合预期,说明两株重组植物乳杆菌都能表达相应蛋白。
1.4小结
本实施例成功构建表达RON2-DCpep和RON2的重组植物乳杆菌NC8-pSIP409-pgsA'-RON2-DCpep和NC8-pSIP409-pgsA'-RON2。
实施例2重组植物乳杆菌的免疫效果研究
重组植物乳杆菌疫苗有着成本低廉,便于规模化生产的优势,且能够在机体肠道内长期定植,持续发挥作用,并且植物乳杆菌自身就是益生菌,能够提高机体免疫力、促进黏膜免疫。口服的免疫方式,又使之更为方便。因此,重组植物乳杆菌疫苗有着广阔的发展前景。
植物乳杆菌可作为抗原递送载体,并且具有天然的佐剂活性,调节肠道菌群和促进肠道吸收的同时诱导机体产生黏膜免疫和全身性免疫。因此将实施例1中构建成功的重组植物乳杆菌NC8-pSIP409-pgsA'-RON2-DCpep和NC8-pSIP409-pgsA'-RON2口服免疫雏鸡,并检测肠道中SIgA,血清中IgG含量和细胞因子IFN-γ、IL-2的含量;检测脾淋巴细胞中CD4+、CD8+T细胞水平等指标。分析重组植物乳杆菌对机体的免疫调节能力。并在雏鸡口服柔嫩艾美耳球虫孢子化卵囊后,对其体重增长率、卵囊排出量、盲肠病变评分等指标进行统计并计算抗球虫指数(ACI),以评估重组植物乳杆菌对鸡柔嫩艾美耳球虫的免疫保护作用。
2.1材料与方法
试验菌株:NC8-pSIP409-pgsA'-RON2重组植物乳杆菌、NC8-pSIP409-pgsA'-RON2-DCpep重组植物乳杆菌、NC8-pSIP409-pgsA'空载体植物乳杆菌。重组植物乳杆菌按照实施例1的方法构建。
柔嫩艾美耳球虫孢子化卵囊在2.5% K2Cr2O7中,置于4℃,由吉林省动物微生态制剂工程研究中心保存。
试验动物:白羽肉鸡(1日龄)购于长春北方养殖场。动物房清洁处理采用甲醛熏蒸消毒法清除或杀灭环境中的有害微生物,料槽水槽采用干热灭菌处理,严格保证无球虫污染。饲养笼为经过甲醛熏蒸后消毒的无菌且不含有寄生虫虫卵的金属笼,饮用水皆为经过高温灭菌的蒸馏水,饲料为严格控制不含有抗球虫成分及任何抗生素的颗粒料。
试验试剂:鸡脾淋巴细胞分离液试剂盒、CCK-8细胞增殖试剂盒购于北京Solarbio科技有限公司;细胞因子(IFN-γ、IL-2)ELISA试剂盒购于江苏科特生物科技有限公司;Mouse anti Chicken CD4-PE、CD3-FITC、CD8-APC抗体购自BD Bioscience;鸡球虫三价活疫苗(DLV疫苗)购于西安斯凯达生物制品有限公司。
试验仪器:主要仪器同实施例1。
实验动物分组及免疫方案:
免疫分组:1日龄健康雏鸡,实验分6组,每组20只共120只;从1日龄开始,严格按照每日饲喂不含有抗生素和抗球虫药物的颗粒料,饮水为高温灭菌的蒸馏水,待3日龄选择个体差别不大的鸡只进行分组,分为6组每组20只雏鸡。在腿部应用腿箍进行编号,并在腿箍上进行组别和个体的编号标记。具体分组及免疫程序如下:
表6分组及免疫程序
免疫程序:第3-5天为初次免疫,第17-19天为加强免疫。加强免疫后对雏鸡状态进行观察,在第29天进行免疫效果评价,流式细胞术检测脾脏淋巴细胞的CD4+和CD8+细胞数量,使用ELISA方法检测肠道内容物中SIgA、血清中IgG、IFN-γ、IL-2含量和水平。
流式细胞术:通过流式细胞术检测检测脾淋巴细胞的CD4+、CD8+T淋巴细胞数量。攻虫前,各组随机挑选5只鸡,取脾脏,超净台中用铜网磨成单细胞悬液,按鸡脾淋巴细胞分离液试剂盒说明书分离出淋巴细胞后进行计数,各样品取1×106个单细胞用CD3+、CD4+、CD8+抗体进行共同孵育和单标样品处理,4℃避光孵育30min后PBS清洗两次。流式细胞仪检测,实验数据使用FlowJo 7.6.1分析。
检测脾脏淋巴细胞增殖情况:
攻虫前后使用CCK-8法检测脾淋巴细胞增殖能力;
超净台中用铜网将脾磨成单细胞悬液;按鸡脾淋巴细胞分离液试剂盒说明分离出淋巴细胞;用RPMI1640培养基(10%FBS、1%双抗)悬起,计数;根据计数结果每只鸡每孔铺5×105个细胞在96孔板中,加入实施例1所述的RON2蛋白(2μg/孔)进行刺激,在细胞培养箱中培养45h;每孔加入10μL CCK-8,反应3h后用酶标仪检测,并计算刺激指数(SI),SI=(实验组-空白组)/(对照组-空白组)。
ELISA检测肠道灌洗液中SIgA水平:
每组随机5只鸡,取十二指肠段,剪开肠管,用玻片刮取肠内壁,将刮取物放入含PMSF的PBS中,4000rpm离心15min,保留上清保存至-80℃备用。使用ELISA试剂盒检测检测其中SIgA水平。具体操作见说明书。
细胞因子IFN-γ和IL-2以及IgG水平的检测:
检测试验鸡体内细胞因子IFN-γ、IL-2和IgG抗体的水平,对试验鸡进行心脏采血,将血液装入EP管内,37℃培养箱静置2h后,使用离心机离心(4000rpm,15min)分离血清,-80℃保存。IFN-γ、IL-2和IgG抗体的检测步骤参照ELISA试剂盒说明书操作方法进行。
免疫保护指标检测
体重检测:
攻虫后每天按照分组给每只实验动物称量体重并记录,称重饲养时注意保证PBS空白组的绝对无球虫感染。
平均增重=处死时体重-攻虫时体重;
增重率(%)=(处死时体重-攻虫时体重)/攻虫时体重×100%;
相对增重率(%)=实验组平均增重/PBS组平均增重×100%。
卵囊排出量检测:
攻虫后第7天开始按分组收集粪便,将收集到的粪便按组各取2g放置于干净的研钵中,加入充分饱和的食盐水58mL,使粪便与食盐水均匀混合后吸取样品放于计数板的计数室内,静止2-3min后使用显微镜进行计数,卵囊值计算见表7。
表7卵囊值计算
卵囊比数(%)=实验组卵囊数/攻虫组卵囊数×100%。
肠道病变评分:
攻虫后按分组每组随机处死5只鸡,对应编号截取盲肠,逐只观察病变情况,并进行评分,具体记分标准见表8。
表8病变评分标准
H&E染色检测肠道病理变化:
攻虫后第7天处死试验鸡取盲肠中段,在4%甲醛溶液中固定48小时以上。两端修剪后进行石蜡包埋、H&E染色。步骤如下:
脱水:采用酒精梯度脱水,依组织块大小制定脱水时间,依次在70%、80%、85%、90%、95% I和95% II、100% I和100% II分别脱水;
透明:沥干酒精后置于二甲苯透明8min,透明到肉眼观察呈煮熟的肉皮样,根据组织大小掌握透明时间;
浸蜡与包埋:将组织分别放置在蜡I和蜡II和蜡Ⅲ,在55℃条件下分别浸蜡40min后将组织块置于包埋盒中进行包埋;
切片、展片、烤片及染色:每个包埋块切出厚度3μm的组织片,41℃进行展片,将载玻片置于80℃烤片1h后,进行H&E染色;
封片后,显微镜下观察病理切片结果。
抗球虫指数ACI计算
ACI=存活率+相对增重率-(卵囊值+病变值),评判标准见表9。
表9ACI评判标准
2.2结果
流式细胞术检测:
口服免疫重组植物乳杆菌后脾淋巴细胞中CD4+、CD8+T细胞比例增加;
二次免疫后,通过鸡脾淋巴细胞分离液试剂盒将淋巴细胞分离,分离得到的淋巴细胞,使用流式细胞术进行检测,经CD3、CD4和CD8抗体孵育,检测出鸡脾淋巴细胞中T淋巴细胞群分化情况结果如图11所示;RON2-DCpep组和RON2组与PBS组和Vector组相比淋巴细胞中CD4+比例均升高,显著高于PBS组(P<0.001);与PBS组相比RON2-DCpep组和RON2组淋巴细胞中CD8+比例也显著升高(P<0.001),并且RON2-DCpep组显著高于RON2组(P<0.01)。
脾脏中淋巴细胞增殖结果:
口服免疫重组植物乳杆菌后脾淋巴细胞增殖能力增强;
通过CCK-8法检测脾淋巴细胞在RON2蛋白刺激下的增殖能力,从而计算出相应的刺激指数SI(SI=(实验组-空白组)/(对照组-空白组))。结果如图12所示:经口服免疫NC8-pSIP-409-pgsA'-RON2-DCpep和NC8-pSIP-409-pgsA'-RON2重组植物乳杆菌后,脾淋巴细胞在RON2蛋白刺激后,特异性增殖能力RON2-DCpep组和RON2组显著高于Challenge组(P<0.001)。
口服免疫重组植物乳杆菌后血清中IFN-γ水平增加:
免疫后,检测口服免疫NC8-pSIP-409-pgsA'-RON2-DCpep和NC8-pSIP-409-pgsA'-RON2重组植物乳杆菌在攻虫前后血清中IFN-γ的水平。ELISA结果如图13所示,免疫后,RON2-DCpep组和RON2组表达量显著高于Challenge组与Vector组(P<0.001);攻虫后,两组重组植物乳杆菌组IFN-γ的水平仍显著高于Challenge组与Vector组(P<0.001),并且显著高于Vaccine组(P<0.001),且RON2-DCpep组水平最高。
口服免疫重组植物乳杆菌后血清中IL-2水平增加:
通过ELISA检测重组植物乳杆菌在攻虫前后对血清中IL-2的调节作用:在攻虫前,免疫重组植物乳杆菌组的细胞因子IL-2水平显著高于Challenge组(P<0.001),但显著低于Vaccine组(P<0.001);攻虫后,整体IL-2水平均有所提高,RON2-DCpep组提高明显,显著高于Challenge组和Vector组(P<0.001),与Vaccine组无差异,结果见图14。
口服免疫重组植物乳杆菌后血清中IgG水平升高:
两次免疫后,检测鸡血清攻虫前后各组IgG的含量。结果如图15所示,攻虫前重组植物乳杆菌RON2-DCpep组和RON2组血清中IgG水平高于Challenge组(P<0.001);攻虫后RON2-DCpep组和
RON2组的血清中IgG水平升高,显著高于Challenge组(P<0.001)。
口服免疫重组植物乳杆菌后肠道灌洗液中SIgA水平升高:
通过ELISA检测攻虫前后各组SIgA水平。结果如图16所示,攻虫前后RON2-DCpep组和RON2组肠道灌洗液中SIgA水平均显著高于Challenge组(P<0.001),并且攻虫后的RON2-DCpep组和RON2组SIgA水平显著高于疫苗组(P<0.001)。
口服免疫重组植物乳杆菌后平均增重升高:
检测口服NC8-pSIP-409-pgsA'RON2-DCpep和NC8-pSIP-409-pgsA'-RON2重组植物乳杆菌在攻虫后对于实验动物体重的影响,按照分组及编号每天同一时间记录雏鸡体重,并计算出平均增重以及相对增重,结果如表10所示,成长趋势如图17所示,结果表明RON2-DCpep组增重率显著高于Challenge组(P<0.001),并且同时也高于Vaccine组,RON2组的增重率略低于RON2-DCpep组,仍高于Challenge组、Vector组和Vaccine组。免疫后RON2-DCpep组和RON2组体重增长总体明显高于Challenge组(P<0.001),其中RON2-DCpep组相对增重率高达93.89%,RON2组相对增重率高达93.49%。
表10体重变化
口服免疫重组植物乳杆菌后卵囊排出量减少:
为检测口服免疫NC8-pSIP-409-pgsA'-RON2-DCpep和NC8-pSIP-409-pg sA'-RON2重组植物乳杆菌对攻虫后卵囊排出量的影响。由表11、图18可知,RON2-DCpep组和RON2组卵囊排出量明显降低,RON2-DCpep组卵囊减少率最高为60.61%,显著高于Challenge组和Vaccine组(P<0.001,P<0.01),RON2组卵囊减少率为54.33%,也显著高于Challenge组和Vaccine组(P<0.001,P<0.5)。
表11卵囊排出量
口服免疫重组植物乳杆菌后盲肠病变减轻:
为检测口服免疫NC8-pSIP-409-pgsA'-RON2-DCpep和NC8-pSIP-409-pgsA'-RON2重组植物乳杆菌对盲肠病变的影响,在攻虫后第7天处死实验动物,每组随机选取五只鸡取出盲肠,仔细观察肠道病理变化,并根据表8进行病变评分。评分结果如表12,其中RON2-DCpep组和RON2组病变值最低,Challenge组盲肠病变最为严重,Vector组其次。图19所示免疫重组植物乳杆菌有效减少了盲肠病变,RON2-DCpep组和RON2组盲肠病变显著降低,显著低于Challenge、Vector组(P<0.05)。
表12盲肠病变评分
肠道病变对比结果显示口服免疫重组植物乳杆菌后盲肠病变减轻:
如图20所示,与空白对照PBS组相比,Challenge组和Vector组,肠壁可观察到明显出血点,盲肠整体变性严重缩短;RON2-DCpep组攻虫后盲肠病变情况减轻,眼观肠壁无明显的病理变化,盲肠无明显病变;RON2组攻虫后也减轻了肠道病变,但存在少量出血点;Vaccine组肠道病变较轻,但也出现轻度萎缩的表现,肠壁存在少量出血点。
盲肠组织病理切片结果显示口服免疫重组植物乳杆菌后盲肠病变减轻:
为检测口服免疫NC8-pSIP-409-pgsA'-RON2-DCpep和NC8-pSIP-409-pgsA'-RON2重组植物乳杆菌对攻虫后盲肠病理变化的影响,采用H&E染色检测方法来直观比对肠道病理变化。其中Challenge组和Vector组可见局部肠绒毛破碎不完整,红细胞和炎性细胞浸润增多,并且在组织内可见大量球虫卵囊堆积;对比观察RON2-DCpep组、RON2组、Vaccine组的病理损伤明显减轻,可见球虫卵囊堆积情况减少,只能观察到卵囊零星出现,并且肠绒毛损伤降低,红细胞和炎性细胞浸润减少。通过对比可看出RON2-DCpep组、RON2组与Vaccine组相比病理变化更为减轻,结果如图21所示。
抗球虫指数(ACI)表明重组植物乳杆菌具有良好的抗球虫保护效果:
抗球虫指数(ACI)是检测抗球虫效果的重要指标,为检测口服免疫NC8-pSIP-409-pgsA'-RON2-DCpep和NC8-pSIP-409-pgsA'-RON2重组植物乳杆菌后其综合抗球虫效果,固计算出各组抗球虫指数(ACI),如表13所示:RON2-DCpep组抗球虫指数最高,为170.89;重组植物乳杆菌RON2组相近于RON2-DCpep组,其ACI为170.49,并且NC8-pSIP-409-pgsA'-RON2-DCpep和NC8-pSIP-409-pgsA'-RON2重组植物乳杆菌组的抗球虫效果高于商品化疫苗组。结果表明,NC8-pSIP-409-pgsA'-RON2-DCpep和NC8-pSIP-409-pgsA'-RON2重组植物乳杆菌具有良好的球虫保护效果。
表13抗球虫指数
2.3小结
本实施例试验证明了口服免疫重组植物乳杆菌NC8-pSIP-409-pgsA'-RON2-DCpep和NC8-pSIP-409-pgsA'-RON2后可增加血清中细胞因子IFN-γ、IL-2水平、提高肠道中SIgA以及血清中IgG抗体水平、增强脾淋巴细胞的增殖能力、并刺激CD4+、CD8+T淋巴细胞的比例增加。
免疫表达RON2蛋白的重组植物乳杆菌能提高增重、减少卵囊排出量,降低感染球虫后的盲肠病变,说明该重组植物乳杆菌对柔嫩艾美耳球虫具有良好的免疫保护效果,并证明了DCpep蛋白具有增强免疫反应的作用。
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (10)
1.一种表达锚定柔嫩艾美耳球虫RON2抗原的重组植物乳杆菌,其特征在于,与野生型相比,该重组植物乳杆菌被改造为具有RON2-Dcpep融合蛋白表达活性;
所述融合蛋白由RON2与Dcpep连接而成,Dcpep序列如SEQ ID NO.1所示。
2.如权利要求1所述重组植物乳杆菌,其特征在于,所述RON2的为GenBank登录号XM_013375678C的胞外区,氨基酸序号1082~1402所示的序列。
3.如权利要求1所述重组植物乳杆菌,其特征在于,所述融合蛋白由RON2及3个DCpep基因片段串联组成,其密码子优化后核苷酸序列如SEQ ID NO.2所示,氨基酸序列如SEQ IDNO.3所示。
4.如权利要求3所述重组植物乳杆菌,其特征在于,所述核苷酸序列还包括基于密码子简并性能够翻译得到RON2-Dcpep融合蛋白的其它序列,或在高严谨条件下可与SEQ IDNO.2所示序列杂交的核苷酸序列。
5.如权利要求1所述重组植物乳杆菌,其特征在于,所述重组植物乳杆菌中RON2-Dcpep融合蛋白与锚定元件pgsA'融合表达,融合后多肽的氨基酸序列如SEQ ID NO.6所示。
6.如权利要求1所述重组植物乳杆菌,其特征在于,所述重组植物乳杆菌的“改造”方式为通过pSIP409-pgsA'重组质粒构建,同时,RON2-DCpep融合基因中含有启动子、终止子和酶切位点基因序列,所述酶切位点为XbaI和HindIII。
7.权利要求1-6任一项所述重组植物乳杆菌在制备防治球虫病药物中的应用,其特征在于,所述防治球虫病药物为口服疫苗。
8.一种口服疫苗,其特征在于,所述疫苗以上述权利要求1-6中任一项的重组植物乳杆菌作为抗原,所述口服疫苗中还包括药学上所必须的辅料,包括但不限于溶剂、增溶剂、悬浮剂、等渗剂、缓冲剂、舒缓剂、防腐剂、抗氧化剂、着色剂、甜味剂或其他制剂添加剂。
9.权利要求1-6任一项所述重组植物乳杆菌在制备家禽饲料中的应用,其特征在于,所述家禽包括鸡、鸭或鹅。
10.权利要求1-6任一项所述重组植物乳杆菌、权利要求8所述口服疫苗在如下任意一个方面的应用:
(1)激活机体免疫细胞和/或制备激活机体免疫细胞的产品;
(2)激活派氏淋巴结DC和/或制备激活派氏淋巴结DC的产品;
(3)刺激脾和/或肠系膜淋巴结中的特异性细胞因子、T细胞、B细胞的产生和/或制备刺激脾、肠系膜淋巴结和/或派氏淋巴结中的细胞因子的产品;所述细胞因子、T淋巴细胞、CD4+、CD8+、IFN-γ、IL-2、IgG、SIgA。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310904846.7A CN116948933A (zh) | 2023-07-21 | 2023-07-21 | 一种表达锚定柔嫩艾美耳球虫ron2抗原的重组植物乳杆菌及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202310904846.7A CN116948933A (zh) | 2023-07-21 | 2023-07-21 | 一种表达锚定柔嫩艾美耳球虫ron2抗原的重组植物乳杆菌及应用 |
Publications (1)
Publication Number | Publication Date |
---|---|
CN116948933A true CN116948933A (zh) | 2023-10-27 |
Family
ID=88448705
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202310904846.7A Pending CN116948933A (zh) | 2023-07-21 | 2023-07-21 | 一种表达锚定柔嫩艾美耳球虫ron2抗原的重组植物乳杆菌及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN116948933A (zh) |
-
2023
- 2023-07-21 CN CN202310904846.7A patent/CN116948933A/zh active Pending
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Chu et al. | Development of a subunit vaccine containing recombinant Riemerella anatipestifer outer membrane protein A and CpG ODN adjuvant | |
Han et al. | Vaccination of goats with glyceraldehyde-3-phosphate dehydrogenase DNA vaccine induced partial protection against Haemonchus contortus | |
CN113150171B (zh) | 含分子内佐剂的非洲猪瘟病毒重组蛋白、表达载体及应用 | |
CN102276730B (zh) | 金黄色葡萄球菌IsdBid-TRAP融合蛋白制备方法及其应用 | |
CN111514284A (zh) | 鸡球虫多价重组蛋白疫苗及其制备方法和应用 | |
Liu et al. | Recombinant invasive Lactobacillus plantarum expressing the Eimeria tenella fusion gene TA4 and AMA1 induces protection against coccidiosis in chickens | |
Huang et al. | Eimeria maxima microneme protein 2 delivered as DNA vaccine and recombinant protein induces immunity against experimental homogenous challenge | |
CN114426981B (zh) | 非洲猪瘟病毒抗原蛋白重组表达载体、重组植物乳酸菌及其制备方法和应用 | |
Sizemore et al. | Live, attenuated Salmonella typhimurium vectoring Campylobacter antigens | |
Yao et al. | Live recombinant Lactococcus lactis vaccine expressing immobilization antigen (i-Ag) for protection against Ichthyophthirius multifiliis in goldfish | |
CN113943376B (zh) | 一种融合基因、其编码蛋白及其在抗非洲猪瘟中的应用 | |
Zhang et al. | Immunological evaluation of invasive Lactobacillus plantarum co-expressing EtMIC2 and chicken interleukin-18 against Eimeria tenella | |
CN114621970A (zh) | 一种融合基因、其所编码的蛋白及其在鱼类弹状病毒口服疫苗的应用 | |
Hoan et al. | Identification and immunogenicity of microneme protein 2 (EbMIC2) of Eimeria brunetti | |
Erster et al. | Molecular detection of Babesia ovis in sheep and ticks using the gene encoding B. ovis surface protein D (BoSPD) | |
CN107551264A (zh) | L3和/或l5源作为寄生虫病疫苗或诊断的用途 | |
CN116948933A (zh) | 一种表达锚定柔嫩艾美耳球虫ron2抗原的重组植物乳杆菌及应用 | |
Pan et al. | Lactobacillus plantarum surface-displayed Eimeria tenella profilin antigens with FliC flagellin elicit protection against coccidiosis in chickens | |
Zhu et al. | Identification and characterization of a cDNA clone-encoding antigen of Eimeria acervulina | |
Lange et al. | Missing the target: DNAk is a dominant epitope in the humoral immune response of channel catfish (Ictalurus punctatus) to Flavobacterium columnare | |
EP2658568A1 (en) | Canine babesiosis vaccine antigen | |
KR101928898B1 (ko) | 변형된 초유 단백질 및 그 어플리케이션 | |
CN102304185A (zh) | 预防奶牛乳房炎的融合蛋白sip-trap及制备方法和应用 | |
Zhi et al. | Combined oral immunization with probiotics Entercoccus faecalis delivering surface-anchored Eimeria tenella proteins provide protective efficacies against homologous infection in chickens | |
TWI285647B (en) | Prevention, treatment and detection of porcine progressive atrophic rhinitis |
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 |