CN110295173A - Isolated carp antiviral protein Rhbdd3 and its antiviral activity - Google Patents
Isolated carp antiviral protein Rhbdd3 and its antiviral activity Download PDFInfo
- Publication number
- CN110295173A CN110295173A CN201910576228.8A CN201910576228A CN110295173A CN 110295173 A CN110295173 A CN 110295173A CN 201910576228 A CN201910576228 A CN 201910576228A CN 110295173 A CN110295173 A CN 110295173A
- Authority
- CN
- China
- Prior art keywords
- rhbdd3
- carp
- gene
- primer
- virus
- 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.)
- Granted
Links
- 101150098975 Rhbdd3 gene Proteins 0.000 title claims abstract description 85
- 241000252233 Cyprinus carpio Species 0.000 title claims abstract description 46
- 230000000840 anti-viral effect Effects 0.000 title claims abstract description 27
- 241000251468 Actinopterygii Species 0.000 claims abstract description 24
- 241000700605 Viruses Species 0.000 claims abstract description 22
- 206010058874 Viraemia Diseases 0.000 claims abstract description 9
- 208000009663 Acute Necrotizing Pancreatitis Diseases 0.000 claims abstract description 8
- 206010058096 Pancreatic necrosis Diseases 0.000 claims abstract description 8
- 230000002155 anti-virotic effect Effects 0.000 claims abstract description 8
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
- 239000003814 drug Substances 0.000 claims abstract description 6
- 230000014509 gene expression Effects 0.000 claims abstract description 5
- 229940079593 drug Drugs 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000013612 plasmid Substances 0.000 claims description 31
- 239000002299 complementary DNA Substances 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 13
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 12
- 230000003321 amplification Effects 0.000 claims description 12
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 12
- 238000002360 preparation method Methods 0.000 claims description 12
- 239000012634 fragment Substances 0.000 claims description 9
- 238000010839 reverse transcription Methods 0.000 claims description 7
- 238000013461 design Methods 0.000 claims description 6
- 238000001400 expression cloning Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000008194 pharmaceutical composition Substances 0.000 claims description 6
- 108091008146 restriction endonucleases Proteins 0.000 claims description 6
- 238000012163 sequencing technique Methods 0.000 claims description 6
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 5
- 238000011144 upstream manufacturing Methods 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 4
- 241000588724 Escherichia coli Species 0.000 claims description 3
- 238000010369 molecular cloning Methods 0.000 claims description 2
- 230000000694 effects Effects 0.000 abstract description 19
- 108090000623 proteins and genes Proteins 0.000 abstract description 18
- 208000015181 infectious disease Diseases 0.000 abstract description 17
- 238000005516 engineering process Methods 0.000 abstract description 8
- 230000035755 proliferation Effects 0.000 abstract description 6
- 101150014408 MINDY3 gene Proteins 0.000 description 31
- 101100005010 Mus musculus Ca8 gene Proteins 0.000 description 31
- 101150111716 ankrd1 gene Proteins 0.000 description 31
- 210000004027 cell Anatomy 0.000 description 25
- 235000019688 fish Nutrition 0.000 description 21
- 239000007788 liquid Substances 0.000 description 16
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 15
- 238000001514 detection method Methods 0.000 description 12
- 201000010099 disease Diseases 0.000 description 12
- 230000002018 overexpression Effects 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 12
- 239000000243 solution Substances 0.000 description 11
- 241000972773 Aulopiformes Species 0.000 description 10
- 235000019515 salmon Nutrition 0.000 description 9
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 8
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- 238000001890 transfection Methods 0.000 description 7
- 238000011534 incubation Methods 0.000 description 6
- 241000894006 Bacteria Species 0.000 description 5
- 108091026890 Coding region Proteins 0.000 description 5
- 108020004414 DNA Proteins 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 239000000975 dye Substances 0.000 description 5
- 238000003119 immunoblot Methods 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 102000035195 Peptidases Human genes 0.000 description 4
- 108091005804 Peptidases Proteins 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 235000019833 protease Nutrition 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- 238000003753 real-time PCR Methods 0.000 description 4
- 230000009385 viral infection Effects 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 101900217743 Infectious pancreatic necrosis virus Capsid protein VP2 Proteins 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 239000012124 Opti-MEM Substances 0.000 description 3
- 101001039859 Spring viremia of carp virus Matrix protein Proteins 0.000 description 3
- 101150036700 VP3 gene Proteins 0.000 description 3
- 208000014117 bile duct papillary neoplasm Diseases 0.000 description 3
- 238000010804 cDNA synthesis Methods 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000007541 cellular toxicity Effects 0.000 description 3
- 230000004087 circulation Effects 0.000 description 3
- 230000034994 death Effects 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 239000013589 supplement Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 231100000419 toxicity Toxicity 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000283074 Equus asinus Species 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- 108010087230 Sincalide Proteins 0.000 description 2
- 210000001015 abdomen Anatomy 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000001154 acute effect Effects 0.000 description 2
- 230000000740 bleeding effect Effects 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 238000010609 cell counting kit-8 assay Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 208000001936 exophthalmos Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000000799 fluorescence microscopy Methods 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 108010050848 glycylleucine Proteins 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000015788 innate immune response Effects 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 238000011880 melting curve analysis Methods 0.000 description 2
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 2
- 210000000822 natural killer cell Anatomy 0.000 description 2
- 230000000149 penetrating effect Effects 0.000 description 2
- 230000001376 precipitating effect Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 230000002062 proliferating effect Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 238000004064 recycling Methods 0.000 description 2
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 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 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 230000029812 viral genome replication Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- MASUWVVNWALEEM-UHFFFAOYSA-M 1-methoxy-5-methylphenazin-5-ium;methyl sulfate Chemical compound COS([O-])(=O)=O.C1=CC=C2N=C3C(OC)=CC=CC3=[N+](C)C2=C1 MASUWVVNWALEEM-UHFFFAOYSA-M 0.000 description 1
- PEZMQPADLFXCJJ-ZETCQYMHSA-N 2-[[2-[[(2s)-1-(2-aminoacetyl)pyrrolidine-2-carbonyl]amino]acetyl]amino]acetic acid Chemical compound NCC(=O)N1CCC[C@H]1C(=O)NCC(=O)NCC(O)=O PEZMQPADLFXCJJ-ZETCQYMHSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- QCVGEOXPDFCNHA-UHFFFAOYSA-N 5,5-dimethyl-2,4-dioxo-1,3-oxazolidine-3-carboxamide Chemical compound CC1(C)OC(=O)N(C(N)=O)C1=O QCVGEOXPDFCNHA-UHFFFAOYSA-N 0.000 description 1
- OILNWMNBLIHXQK-ZLUOBGJFSA-N Ala-Cys-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O OILNWMNBLIHXQK-ZLUOBGJFSA-N 0.000 description 1
- LMFXXZPPZDCPTA-ZKWXMUAHSA-N Ala-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N LMFXXZPPZDCPTA-ZKWXMUAHSA-N 0.000 description 1
- VNYMOTCMNHJGTG-JBDRJPRFSA-N Ala-Ile-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O VNYMOTCMNHJGTG-JBDRJPRFSA-N 0.000 description 1
- SOBIAADAMRHGKH-CIUDSAMLSA-N Ala-Leu-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O SOBIAADAMRHGKH-CIUDSAMLSA-N 0.000 description 1
- HOVPGJUNRLMIOZ-CIUDSAMLSA-N Ala-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N HOVPGJUNRLMIOZ-CIUDSAMLSA-N 0.000 description 1
- IEAUDUOCWNPZBR-LKTVYLICSA-N Ala-Trp-Gln Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N IEAUDUOCWNPZBR-LKTVYLICSA-N 0.000 description 1
- 239000012103 Alexa Fluor 488 Substances 0.000 description 1
- 239000012110 Alexa Fluor 594 Substances 0.000 description 1
- 208000031295 Animal disease Diseases 0.000 description 1
- YBZMTKUDWXZLIX-UWVGGRQHSA-N Arg-Leu-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YBZMTKUDWXZLIX-UWVGGRQHSA-N 0.000 description 1
- 206010003445 Ascites Diseases 0.000 description 1
- FRSGNOZCTWDVFZ-ACZMJKKPSA-N Asp-Asp-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O FRSGNOZCTWDVFZ-ACZMJKKPSA-N 0.000 description 1
- VAWNQIGQPUOPQW-ACZMJKKPSA-N Asp-Glu-Ala Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O VAWNQIGQPUOPQW-ACZMJKKPSA-N 0.000 description 1
- PCJOFZYFFMBZKC-PCBIJLKTSA-N Asp-Phe-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O PCJOFZYFFMBZKC-PCBIJLKTSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241001609213 Carassius carassius Species 0.000 description 1
- 244000247747 Coptis groenlandica Species 0.000 description 1
- 235000002991 Coptis groenlandica Nutrition 0.000 description 1
- 241000252230 Ctenopharyngodon idella Species 0.000 description 1
- UPURLDIGQGTUPJ-ZKWXMUAHSA-N Cys-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CS)N UPURLDIGQGTUPJ-ZKWXMUAHSA-N 0.000 description 1
- HBHMVBGGHDMPBF-GARJFASQSA-N Cys-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CS)N HBHMVBGGHDMPBF-GARJFASQSA-N 0.000 description 1
- NXQCSPVUPLUTJH-WHFBIAKZSA-N Cys-Ser-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O NXQCSPVUPLUTJH-WHFBIAKZSA-N 0.000 description 1
- 101710088194 Dehydrogenase Proteins 0.000 description 1
- 108010005054 Deoxyribonuclease BamHI Proteins 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 102000002322 Egg Proteins Human genes 0.000 description 1
- 108010000912 Egg Proteins Proteins 0.000 description 1
- 108091006149 Electron carriers Proteins 0.000 description 1
- 241000283070 Equus zebra Species 0.000 description 1
- HXOLDXKNWKLDMM-YVNDNENWSA-N Gln-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N HXOLDXKNWKLDMM-YVNDNENWSA-N 0.000 description 1
- XFAUJGNLHIGXET-AVGNSLFASA-N Gln-Leu-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XFAUJGNLHIGXET-AVGNSLFASA-N 0.000 description 1
- WZZSKAJIHTUUSG-ACZMJKKPSA-N Glu-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O WZZSKAJIHTUUSG-ACZMJKKPSA-N 0.000 description 1
- WOMUDRVDJMHTCV-DCAQKATOSA-N Glu-Arg-Arg Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O WOMUDRVDJMHTCV-DCAQKATOSA-N 0.000 description 1
- RTOOAKXIJADOLL-GUBZILKMSA-N Glu-Asp-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)N RTOOAKXIJADOLL-GUBZILKMSA-N 0.000 description 1
- HNVFSTLPVJWIDV-CIUDSAMLSA-N Glu-Glu-Gln Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HNVFSTLPVJWIDV-CIUDSAMLSA-N 0.000 description 1
- QXDXIXFSFHUYAX-MNXVOIDGSA-N Glu-Ile-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O QXDXIXFSFHUYAX-MNXVOIDGSA-N 0.000 description 1
- NJCALAAIGREHDR-WDCWCFNPSA-N Glu-Leu-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NJCALAAIGREHDR-WDCWCFNPSA-N 0.000 description 1
- JZJGEKDPWVJOLD-QEWYBTABSA-N Glu-Phe-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JZJGEKDPWVJOLD-QEWYBTABSA-N 0.000 description 1
- CBWKURKPYSLMJV-SOUVJXGZSA-N Glu-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CCC(=O)O)N)C(=O)O CBWKURKPYSLMJV-SOUVJXGZSA-N 0.000 description 1
- BIYNPVYAZOUVFQ-CIUDSAMLSA-N Glu-Pro-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O BIYNPVYAZOUVFQ-CIUDSAMLSA-N 0.000 description 1
- IUZGUFAJDBHQQV-YUMQZZPRSA-N Gly-Leu-Asn Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IUZGUFAJDBHQQV-YUMQZZPRSA-N 0.000 description 1
- QAMMIGULQSIRCD-IRXDYDNUSA-N Gly-Phe-Tyr Chemical compound C([C@H](NC(=O)C[NH3+])C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C([O-])=O)C1=CC=CC=C1 QAMMIGULQSIRCD-IRXDYDNUSA-N 0.000 description 1
- IALQAMYQJBZNSK-WHFBIAKZSA-N Gly-Ser-Asn Chemical compound [H]NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O IALQAMYQJBZNSK-WHFBIAKZSA-N 0.000 description 1
- 101710088172 HTH-type transcriptional regulator RipA Proteins 0.000 description 1
- 101710113864 Heat shock protein 90 Proteins 0.000 description 1
- 102100034051 Heat shock protein HSP 90-alpha Human genes 0.000 description 1
- DZMVESFTHXSSPZ-XVYDVKMFSA-N His-Ala-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DZMVESFTHXSSPZ-XVYDVKMFSA-N 0.000 description 1
- DFHVLUKTTVTCKY-PBCZWWQYSA-N His-Asn-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CN=CN1)N)O DFHVLUKTTVTCKY-PBCZWWQYSA-N 0.000 description 1
- VOEGKUNRHYKYSU-XVYDVKMFSA-N His-Asp-Ala Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O VOEGKUNRHYKYSU-XVYDVKMFSA-N 0.000 description 1
- SWSVTNGMKBDTBM-DCAQKATOSA-N His-Gln-Glu Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N SWSVTNGMKBDTBM-DCAQKATOSA-N 0.000 description 1
- ZSKJIISDJXJQPV-BZSNNMDCSA-N His-Leu-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CN=CN1 ZSKJIISDJXJQPV-BZSNNMDCSA-N 0.000 description 1
- 101001091385 Homo sapiens Kallikrein-6 Proteins 0.000 description 1
- 241000252234 Hypophthalmichthys nobilis Species 0.000 description 1
- DMHGKBGOUAJRHU-RVMXOQNASA-N Ile-Arg-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(=O)O)N DMHGKBGOUAJRHU-RVMXOQNASA-N 0.000 description 1
- DMHGKBGOUAJRHU-UHFFFAOYSA-N Ile-Arg-Pro Natural products CCC(C)C(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O DMHGKBGOUAJRHU-UHFFFAOYSA-N 0.000 description 1
- PPSQSIDMOVPKPI-BJDJZHNGSA-N Ile-Cys-Leu Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(C)C)C(=O)O PPSQSIDMOVPKPI-BJDJZHNGSA-N 0.000 description 1
- LPFBXFILACZHIB-LAEOZQHASA-N Ile-Gly-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CCC(=O)O)C(=O)O)N LPFBXFILACZHIB-LAEOZQHASA-N 0.000 description 1
- MLSUZXHSNRBDCI-CYDGBPFRSA-N Ile-Pro-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)O)N MLSUZXHSNRBDCI-CYDGBPFRSA-N 0.000 description 1
- PRTZQMBYUZFSFA-XEGUGMAKSA-N Ile-Tyr-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)NCC(=O)O)N PRTZQMBYUZFSFA-XEGUGMAKSA-N 0.000 description 1
- DLEBSGAVWRPTIX-PEDHHIEDSA-N Ile-Val-Ile Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)[C@@H](C)CC DLEBSGAVWRPTIX-PEDHHIEDSA-N 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical class CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 102100034866 Kallikrein-6 Human genes 0.000 description 1
- 241000755266 Kathetostoma giganteum Species 0.000 description 1
- ULXYQAJWJGLCNR-YUMQZZPRSA-N Leu-Asp-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O ULXYQAJWJGLCNR-YUMQZZPRSA-N 0.000 description 1
- KAFOIVJDVSZUMD-DCAQKATOSA-N Leu-Gln-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-DCAQKATOSA-N 0.000 description 1
- KAFOIVJDVSZUMD-UHFFFAOYSA-N Leu-Gln-Gln Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)NC(CCC(N)=O)C(O)=O KAFOIVJDVSZUMD-UHFFFAOYSA-N 0.000 description 1
- HQUXQAMSWFIRET-AVGNSLFASA-N Leu-Glu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HQUXQAMSWFIRET-AVGNSLFASA-N 0.000 description 1
- FAELBUXXFQLUAX-AJNGGQMLSA-N Leu-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C FAELBUXXFQLUAX-AJNGGQMLSA-N 0.000 description 1
- XVZCXCTYGHPNEM-UHFFFAOYSA-N Leu-Leu-Pro Natural products CC(C)CC(N)C(=O)NC(CC(C)C)C(=O)N1CCCC1C(O)=O XVZCXCTYGHPNEM-UHFFFAOYSA-N 0.000 description 1
- DRWMRVFCKKXHCH-BZSNNMDCSA-N Leu-Phe-Leu Chemical compound CC(C)C[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C([O-])=O)CC1=CC=CC=C1 DRWMRVFCKKXHCH-BZSNNMDCSA-N 0.000 description 1
- DPURXCQCHSQPAN-AVGNSLFASA-N Leu-Pro-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 DPURXCQCHSQPAN-AVGNSLFASA-N 0.000 description 1
- XOWMDXHFSBCAKQ-SRVKXCTJSA-N Leu-Ser-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C XOWMDXHFSBCAKQ-SRVKXCTJSA-N 0.000 description 1
- YLMIDMSLKLRNHX-HSCHXYMDSA-N Leu-Trp-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YLMIDMSLKLRNHX-HSCHXYMDSA-N 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- NKKFVJRLCCUJNA-QWRGUYRKSA-N Lys-Gly-Lys Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN NKKFVJRLCCUJNA-QWRGUYRKSA-N 0.000 description 1
- YTJFXEDRUOQGSP-DCAQKATOSA-N Lys-Pro-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O YTJFXEDRUOQGSP-DCAQKATOSA-N 0.000 description 1
- CNXOBMMOYZPPGS-NUTKFTJISA-N Lys-Trp-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](C)C(O)=O CNXOBMMOYZPPGS-NUTKFTJISA-N 0.000 description 1
- UGCIQUYEJIEHKX-GVXVVHGQSA-N Lys-Val-Glu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O UGCIQUYEJIEHKX-GVXVVHGQSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- PZUUMQPMHBJJKE-AVGNSLFASA-N Met-Leu-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCNC(N)=N PZUUMQPMHBJJKE-AVGNSLFASA-N 0.000 description 1
- UROWNMBTQGGTHB-DCAQKATOSA-N Met-Leu-Asp Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O UROWNMBTQGGTHB-DCAQKATOSA-N 0.000 description 1
- HMEVNCOJHJTLNB-BVSLBCMMSA-N Met-Trp-Phe Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O)N HMEVNCOJHJTLNB-BVSLBCMMSA-N 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 206010028851 Necrosis Diseases 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- DVOCGBNHAUHKHJ-DKIMLUQUSA-N Phe-Ile-Leu Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O DVOCGBNHAUHKHJ-DKIMLUQUSA-N 0.000 description 1
- CWFGECHCRMGPPT-MXAVVETBSA-N Phe-Ile-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O CWFGECHCRMGPPT-MXAVVETBSA-N 0.000 description 1
- YMIZSYUAZJSOFL-SRVKXCTJSA-N Phe-Ser-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O YMIZSYUAZJSOFL-SRVKXCTJSA-N 0.000 description 1
- QTDBZORPVYTRJU-KKXDTOCCSA-N Phe-Tyr-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](C)C(O)=O QTDBZORPVYTRJU-KKXDTOCCSA-N 0.000 description 1
- VDTYRPWRWRCROL-UFYCRDLUSA-N Phe-Val-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 VDTYRPWRWRCROL-UFYCRDLUSA-N 0.000 description 1
- DIZLUAZLNDFDPR-CIUDSAMLSA-N Pro-Cys-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H]1CCCN1 DIZLUAZLNDFDPR-CIUDSAMLSA-N 0.000 description 1
- NMELOOXSGDRBRU-YUMQZZPRSA-N Pro-Glu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CCC(=O)O)NC(=O)[C@@H]1CCCN1 NMELOOXSGDRBRU-YUMQZZPRSA-N 0.000 description 1
- QEWBZBLXDKIQPS-STQMWFEESA-N Pro-Gly-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O QEWBZBLXDKIQPS-STQMWFEESA-N 0.000 description 1
- XYSXOCIWCPFOCG-IHRRRGAJSA-N Pro-Leu-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XYSXOCIWCPFOCG-IHRRRGAJSA-N 0.000 description 1
- MHHQQZIFLWFZGR-DCAQKATOSA-N Pro-Lys-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O MHHQQZIFLWFZGR-DCAQKATOSA-N 0.000 description 1
- YHUBAXGAAYULJY-ULQDDVLXSA-N Pro-Tyr-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O YHUBAXGAAYULJY-ULQDDVLXSA-N 0.000 description 1
- PGSWNLRYYONGPE-JYJNAYRXSA-N Pro-Val-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O PGSWNLRYYONGPE-JYJNAYRXSA-N 0.000 description 1
- 102100037471 Rhomboid domain-containing protein 3 Human genes 0.000 description 1
- 101710160469 Rhomboid domain-containing protein 3 Proteins 0.000 description 1
- 241000277263 Salmo Species 0.000 description 1
- JPIDMRXXNMIVKY-VZFHVOOUSA-N Ser-Ala-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JPIDMRXXNMIVKY-VZFHVOOUSA-N 0.000 description 1
- IDCKUIWEIZYVSO-WFBYXXMGSA-N Ser-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C)C(O)=O)=CNC2=C1 IDCKUIWEIZYVSO-WFBYXXMGSA-N 0.000 description 1
- HBOABDXGTMMDSE-GUBZILKMSA-N Ser-Arg-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O HBOABDXGTMMDSE-GUBZILKMSA-N 0.000 description 1
- OJPHFSOMBZKQKQ-GUBZILKMSA-N Ser-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CO OJPHFSOMBZKQKQ-GUBZILKMSA-N 0.000 description 1
- BRIZMMZEYSAKJX-QEJZJMRPSA-N Ser-Glu-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N BRIZMMZEYSAKJX-QEJZJMRPSA-N 0.000 description 1
- OQPNSDWGAMFJNU-QWRGUYRKSA-N Ser-Gly-Tyr Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 OQPNSDWGAMFJNU-QWRGUYRKSA-N 0.000 description 1
- QYSFWUIXDFJUDW-DCAQKATOSA-N Ser-Leu-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QYSFWUIXDFJUDW-DCAQKATOSA-N 0.000 description 1
- UPLYXVPQLJVWMM-KKUMJFAQSA-N Ser-Phe-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(C)C)C(O)=O UPLYXVPQLJVWMM-KKUMJFAQSA-N 0.000 description 1
- JGUWRQWULDWNCM-FXQIFTODSA-N Ser-Val-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O JGUWRQWULDWNCM-FXQIFTODSA-N 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- OHAJHDJOCKKJLV-LKXGYXEUSA-N Thr-Asp-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O OHAJHDJOCKKJLV-LKXGYXEUSA-N 0.000 description 1
- MEJHFIOYJHTWMK-VOAKCMCISA-N Thr-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)[C@@H](C)O MEJHFIOYJHTWMK-VOAKCMCISA-N 0.000 description 1
- NWECYMJLJGCBOD-UNQGMJICSA-N Thr-Phe-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O NWECYMJLJGCBOD-UNQGMJICSA-N 0.000 description 1
- PRTHQBSMXILLPC-XGEHTFHBSA-N Thr-Ser-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PRTHQBSMXILLPC-XGEHTFHBSA-N 0.000 description 1
- BCYUHPXBHCUYBA-CUJWVEQBSA-N Thr-Ser-His Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O BCYUHPXBHCUYBA-CUJWVEQBSA-N 0.000 description 1
- NLWDSYKZUPRMBJ-IEGACIPQSA-N Thr-Trp-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(C)C)C(=O)O)N)O NLWDSYKZUPRMBJ-IEGACIPQSA-N 0.000 description 1
- BKVICMPZWRNWOC-RHYQMDGZSA-N Thr-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)[C@@H](C)O BKVICMPZWRNWOC-RHYQMDGZSA-N 0.000 description 1
- OGXQLUCMJZSJPW-LYSGOOTNSA-N Trp-Gly-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC1=CNC2=CC=CC=C21)N)O OGXQLUCMJZSJPW-LYSGOOTNSA-N 0.000 description 1
- PZXUIGWOEWWFQM-SRVKXCTJSA-N Tyr-Asn-Asn Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O PZXUIGWOEWWFQM-SRVKXCTJSA-N 0.000 description 1
- MTEQZJFSEMXXRK-CFMVVWHZSA-N Tyr-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CC=C(C=C1)O)N MTEQZJFSEMXXRK-CFMVVWHZSA-N 0.000 description 1
- TYFLVOUZHQUBGM-IHRRRGAJSA-N Tyr-Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 TYFLVOUZHQUBGM-IHRRRGAJSA-N 0.000 description 1
- OVBMCNDKCWAXMZ-NAKRPEOUSA-N Val-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](C(C)C)N OVBMCNDKCWAXMZ-NAKRPEOUSA-N 0.000 description 1
- YQYFYUSYEDNLSD-YEPSODPASA-N Val-Thr-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O YQYFYUSYEDNLSD-YEPSODPASA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 210000000436 anus Anatomy 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 108010043240 arginyl-leucyl-glycine Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000001109 blastomere Anatomy 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 210000004443 dendritic cell Anatomy 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 235000014103 egg white Nutrition 0.000 description 1
- 210000000969 egg white Anatomy 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 210000000981 epithelium Anatomy 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 108010066198 glycyl-leucyl-phenylalanine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 108010018006 histidylserine Proteins 0.000 description 1
- 239000011229 interlayer Substances 0.000 description 1
- 230000000366 juvenile effect Effects 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- LQNUZADURLCDLV-UHFFFAOYSA-N nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC=C1 LQNUZADURLCDLV-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 210000004923 pancreatic tissue Anatomy 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 108010073101 phenylalanylleucine Proteins 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 229940115272 polyinosinic:polycytidylic acid Drugs 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 108010029020 prolylglycine Proteins 0.000 description 1
- 238000000197 pyrolysis Methods 0.000 description 1
- 238000010814 radioimmunoprecipitation assay Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 108091092562 ribozyme Proteins 0.000 description 1
- 235000002020 sage Nutrition 0.000 description 1
- VSIVTUIKYVGDCX-UHFFFAOYSA-M sodium;4-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].COC1=CC([N+]([O-])=O)=CC=C1[N+]1=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=NN1C1=CC=C([N+]([O-])=O)C=C1 VSIVTUIKYVGDCX-UHFFFAOYSA-M 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- 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
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- 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
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/461—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from fish
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/40—Fish
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2267/00—Animals characterised by purpose
- A01K2267/02—Animal zootechnically ameliorated
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- 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
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
-
- 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
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Environmental Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Virology (AREA)
- Veterinary Medicine (AREA)
- Microbiology (AREA)
- Oncology (AREA)
- Gastroenterology & Hepatology (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Communicable Diseases (AREA)
- Pharmacology & Pharmacy (AREA)
- Toxicology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Public Health (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to fish gene field of engineering technology, disclose a kind of isolated carp antiviral protein Rhbdd3 and its antiviral activity, the nucleotide sequence of carp antiviral protein Rhbdd3 encoding gene is sequence shown in 1-1050bp in SEQ ID NO:1, and the amino acid sequence of coding is sequence shown in SEQ ID NO:2.The expression for the Rhbdd3 albumen that the present invention clones can not only inhibit the proliferation of huichun viremia virus (SVCV), but also can inhibit the proliferation of infectivity pancreatic necrosis virus (IPNV).Rhbdd3 albumen plays an important role in the course of infection that cell resists SVCV and IPNV, while not influencing the normal activity of cell.Gene and albumen of the invention provides new target spot to prepare anti-SVCV and IPNV drug, also provides important technical basis for the cultivation of fish novel anti-virus kind.
Description
Technical field
The present invention relates to freshwater fish gene engineering technology field more particularly to a kind of isolated carp antiviral proteins
Rhbdd3 and its antiviral activity.
Background technique
Along with the continuous development of China's culture fishery, the disease incidence of aquatic animal disease especially viral disease is close
Apparent ascendant trend is presented over year.Viral Diseases of Aquatic Animal rapid onset, the death rate be high, without effective vaccine or is directed to mostly
Property drug, once morbidity be difficult to cure, bring huge economic loss, seriously restrict China's culture fishery health and
Sustainable development.Therefore, exploitation broad-spectrum antiviral medicament and cultivation fish novel anti-virus kind have highly important practical valence
Value.
The innate immunity is the first line of defence that body resists virus attack, current research discovery, Rhomboid family member
Rhbdd3 (Rhomboid domain-containing protein 3) has played important work in the mammal innate immunity
With.Rhomboid family in drosophila most prior to finding, with transmembrane structure and proteinase activity.Similitude based on sequence,
Sharing 14 kinds of albumen is attributed to Rhomboid family at present, wherein 5 kinds have proteinase activity, 9 kinds then lose proteinase activity.
The study found that Rhbdd3 is distributed mainly on intracellular and does not have proteinase activity in mouse, poly (I:C) processing can be induced
The up-regulation of NK cells in mice Rhbdd3, the activity of Rhbdd3 adjustable NK cell and Dendritic Cells in turn.However, at present
There is no fish Rhbdd3 gene in public database, sequencing arranges really, and the function of fish Rhbdd3 albumen is also there is not yet any report
Road.First passage PCR amplification of the present invention obtains the nucleotide sequence of carp Rhbdd3 genetic fragment, and constructs on this basis
Rhbdd3 eukaryon expression plasmid has verified Rhbdd3 after transfecting carp epithelium oncocyte EPC and salmon blastomere CHSE-214
Protein overexpression fishes virus-the huichun viremia virus (SVCV) and infectious pancreas necrosis virus great to harm
(IPNV) influence being proliferated.
Spring viremia (SVC) is a kind of Acute Fulminant's bleeding as caused by huichun viremia virus (SVCV)
Disease.Normal outbreak of epidemic when spring is at 8~20 DEG C of water temperature, especially 13~15 DEG C of the disease.Carp, fancy carp, grass carp, crucian carp and its change
A variety of cyprinid fish such as kind, silver carp, flathead can be infected.Sick fish is often gathered in water outlet, body colour nigrescence, exophthalmos, anus
Red and swollen, abdomen expands and serious ascites, slow respiration, often disequilibrium and sidestream.Anxious, the sick fish death rate of disease morbidity can be high
Up to 90% or so, seriously endangers fish production and cause crushing blow.Because its is highly pathogenic, SVC is by world animal health group
Knit OIE be classified as must notifiable epidemic disease and China's fish port quarantine first kind quarantine object.2008, the Ministry of Agriculture
No. 1125 bulletins are also classified as " People's Republic of China (PRC) enter the territory animal one kind infectious disease " for this its, and only one is arranged so far
For the fish epidemic disease of a kind of infectious disease.Currently, still lacking the drug and method of effectively control SVC, current control is arranged in the world
Applying is the monitoring for carrying out SVCV, finds early and is isolated and is slaughtered.
Infectivity pancreatic necrosis virus (IPNV) is salmon fishes highly contagious disease-infectious pancreatic necrosis disease
(IPN) cause of disease.IPN disease is often in acute prevalence, and the infection juvenile fish death rate is more than 90%.Sick fish body color nigrescence, exophthalmos,
Abdomen expands, skin and fin ray bleeding, the visible pancreatic tissue of histotomy are downright bad.The normal prevalence at 10~15 DEG C of water temperature of the disease,
Survival fish becomes IPNV carrier after the onset, and can be can be discharged into environment virus by urine, excrement, fish-egg, sperm etc.
Continue to propagate.IPN most has found early in northern US, then in the multinational happening and prevelence such as Europe, Japan, and in 80 years 20th century
In generation, is passed to China, breaks out the farm for being concentrated mainly on China's Rainbow Trout In Northeast China and Atlantic salmon, and have in recent years
The gradually gesture of sprawling constitutes grave danger to China's culture fishery.
Therefore, those skilled in the art, which is dedicated to developing, a kind of has broad anti-viral activity and not influence cell normal
Active antiviral protein, the prevention and treatment for the biggish fishes infectious cause of disease SVCV and IPNV of harmfulness provide new medicine
Object target spot also provides important technical basis for the cultivation of fish novel anti-virus kind.
Summary of the invention
In view of the above drawbacks of the prior art, technical problem to be solved by the invention is to provide a kind of carp antivirus proteins
White (Rhbdd3) has the antiviral work for huichun viremia virus (SVCV) and infectivity pancreatic necrosis virus (IPNV)
Property, can reduce that SVCV and IPNV be infectious, suppressing virus replication, while not influencing the normal activity of cell again, be SVCV and
The prevention and treatment of IPNV provide new drug target, overcome the deficiencies in the prior art.
To achieve the above object, described the present invention provides a kind of isolated carp antiviral protein Rhbdd3 gene
The gene coding region Rhbdd3 nucleotide sequence is sequence shown in 1-1050bp in SEQ ID NO:1.
The present invention also provides a kind of isolated carp antiviral protein Rhbdd3, the amino acid of the albumen Rhbdd3 coding
Sequence is sequence shown in SEQ ID NO:2.
The present invention also provides a kind of recombinant plasmids comprising the gene coding region Rhbdd3 nucleotide sequence.
The present invention also provides a kind of preparation methods for preparing the recombinant plasmid, the described method comprises the following steps:
Step 1: extracting the RNA of carp, RNA described in reverse transcription obtains carp cDNA；
Step 2: the upstream primer and downstream primer of design Rhbdd3 gene order conserved region, with step 1 acquisition
The carp cDNA be template amplification, obtain amplified fragments, be purified and recycle and be connected on pMD18-T carrier and connected
Product carries out two-way sequence survey after connection product conversion DH5 α competent escherichia coli cell and screening positive clone
It is fixed, obtain Rhbdd3 gene order；
Step 3: the Rhbdd3 gene order obtained according to the step 2, the design gene coding region Rhbdd3
Expression cloning forward primer and reverse primer, the carp cDNA obtained using the step 1 obtain amplification piece as template amplification
Section, and the amplified fragments are subjected to molecular cloning, obtain recombinant plasmid.
Further, the step 3 further include before amplification, need to be in the expression cloning forward primer and reverse primer
It is upper to introduce Nhe I, EcoR I restriction enzyme site respectively.
Further, the upstream primer and downstream primer are respectively pMD18-Rhbdd3-F and pMD18-Rhbdd3-R,
Wherein the DNA sequence dna of the pMD18-Rhbdd3-F is ACCTCAATATGACAGCGTCGTAAAC, the pMD18-Rhbdd3-R
DNA sequence dna be TATCCCTGAGCTATGACGCTGA.
Further, the expression cloning forward primer and reverse primer are respectively pCI-Rhbdd3-F and pCI-
Rhbdd3-R, wherein the DNA sequence dna of the pCI-Rhbdd3-F is TTAGCTAGCCACCATGCTCGATCATCTTTTTTCAGC
The DNA sequence dna of AT, the pCI-Rhbdd3-R are CGGAATTCCTATGACGCTGATGGCTTCTTCC。
The present invention also provides a kind of carp antiviral protein Rhbdd3 genes as described above to prepare anti-spring viremia of carp virus blood
Application in syndrome virus pharmaceutical composition or infectivity resistant pancreatic necrosis virus pharmaceutical composition.
The present invention also provides a kind of carp antiviral protein Rhbdd3 as described above to prepare anti-spring viremia disease
Application in poison compositions or infectivity resistant pancreatic necrosis virus pharmaceutical composition.
The present invention also provides a kind of carp antiviral protein Rhbdd3 genes as described above to cultivate fish novel anti-virus
Application in kind.
Compared with prior art, the present invention at least has technical effect beneficial below:
(1) expression of carp antiviral protein Rhbdd3 of the invention can inhibit the duplication of SVCV and IPNV virus, reduce
The infectivity of SVCV and IPNV virus；
(2) overexpression of carp antiviral protein Rhbdd3 of the invention does not influence the normal activity of cell；
(3) Rhbdd3 protein gene of the invention and Rhbdd3 albumen height are conservative, for the antiviral drugs for preparing wide spectrum
Provide new target spot；
(4) Rhbdd3 protein gene of the invention and Rhbdd3 albumen provide again for the cultivation of fish novel anti-virus kind
The technical basis wanted.
It is described further below with reference to technical effect of the attached drawing to design of the invention, specific structure and generation, with
It is fully understood from the purpose of the present invention, feature and effect.
Detailed description of the invention
Fig. 1 is the amplification schematic diagram of the gene coding region carp Rhbdd3 of a preferred embodiment of the invention；
Fig. 2 is the amplification schematic diagram of the gene coding region salmon Rhbdd3 of a preferred embodiment of the invention；
Fig. 3 is the extension increasing sequence comparison result signal of the gene coding region salmon Rhbdd3 of a preferred embodiment of the invention
Figure；
Fig. 4 is the recombinant plasmid pCI-Rhbdd3 restriction enzymes double zyme of the building of a preferred embodiment of the invention
The result schematic diagram cut；
Fig. 5 is that the result of a preferred embodiment of the invention being overexpressed using Western blot detection Rhbdd3 is illustrated
Figure；
Fig. 6 is utilization cell Proliferation/toxicity detection reagent C CK-8 detection Rhbdd3 of a preferred embodiment of the invention
It is overexpressed the result data figure influenced on EPC cell activity；
Fig. 7 is utilization cell Proliferation/toxicity detection reagent C CK-8 detection Rhbdd3 of a preferred embodiment of the invention
It is overexpressed the result data figure influenced on CHSE-214 cell activity；
Fig. 8 is the utilization indirect immunofluorescence detection Rhbdd3 protein overexpression pair of a preferred embodiment of the invention
The fluorescence imaging result schematic diagram that SVCV infection influences；
Fig. 9 is that being overexpressed using Western blot detection Rhbdd3 for a preferred embodiment of the invention infects SVCV
The immunoblot results schematic diagram of influence；
Figure 10 is that the utilization fluorescence quantitative PCR method of a preferred embodiment of the invention detects Rhbdd3 protein overexpression pair
The result data figure of SVCV proliferative effect；
Figure 11 is the utilization indirect immunofluorescence detection Rhbdd3 protein overexpression of a preferred embodiment of the invention
The fluorescence imaging result schematic diagram that IPNV infection is influenced；
Figure 12 is that being overexpressed using Western blot detection Rhbdd3 for a preferred embodiment of the invention feels IPNV
Contaminate the immunoblot results schematic diagram influenced；
Figure 13 is being overexpressed using fluorescence quantitative PCR method detection Rhbdd3 to IPNV for a preferred embodiment of the invention
The result data figure of proliferative effect；
Figure 14 is the structural representation map of the pCI-neo empty carrier of the application of a preferred embodiment of the invention；
Figure 15 is the structural representation map of the recombinant plasmid pCI-Rhbdd3 of the preparation of a preferred embodiment of the invention.
Specific embodiment
Multiple preferred embodiments of the invention are introduced below with reference to Figure of description, keep its technology contents more clear and just
In understanding.The present invention can be emerged from by many various forms of embodiments, and protection scope of the present invention not only limits
The embodiment that Yu Wenzhong is mentioned.
(1) extraction of carp RNA
Sample total serum IgE is extracted using classical Trizol method.Mortar is directly placed into after taking carp tissue block to be shredded with eye scissors
In, a small amount of liquid nitrogen is added, grinds rapidly, is transferred to centrifuge tube after grinding sufficiently, takes 50-100mg tissue that 1mL Trizol is added, mixes
It is even, it is placed at room temperature for 10min, then 4 DEG C, 12000 × g, is centrifuged 5min.
Take supernatant that 200 μ L chloroforms are added, oscillation is placed at room temperature for 15min, 4 DEG C, 12000 × g, is centrifuged 15min after mixing.
It takes upper strata aqueous phase to another new centrifuge tube, 500 μ L isopropanols is added, place 10min on ice, 4 DEG C, 12000 ×
G is centrifuged 15min.
Supernatant is abandoned, the 75% ethyl alcohol 1mL washing precipitating being pre-chilled on ice is added, 4 DEG C, 7500 × g, is centrifuged 10min.
Supernatant is abandoned, room temperature is dried, and is dissolved RNA precipitate with the water of no RNA enzyme, is carried out reverse transcription immediately.
(2) synthesis of carp cDNA
Using the reverse transcription reagent box of Invitrogen company, operating procedure carries out cDNA synthesis to specifications, specifically
Steps are as follows:
The RNA, 1 μ L random hexamers, supplement of 1 μ g said extracted are sequentially added in PCR reaction tube without RNA
The water of enzyme is to 12 μ L of total volume；It in 65 DEG C of reaction 5min, is immediately placed on ice, 45 × Reaction of μ L is then added
Buffer, 1 μ L RiboLock RNase Inhibitor, 2 μ L 10mM dNTP Mix and 1 μ L RevertAid M-MuLV
RT, 25 DEG C of reaction 5min, 42 DEG C of heat preservation 45min obtain cDNA later.
(3) amplification of carp Rhbdd3 gene
First with 7.0 software of Primer Express 5.0 and BioEdit, according to carp, crucian, zebra in GenBank
Rhbdd3 gene order conserved regions design the primer pMD18-Rhbdd3-F and pMD18-Rhbdd3-R of the predictions such as fish, gold thread Barb,
PCR amplification, primers DNA sequences are carried out by template of the carp cDNA are as follows:
PMD18-Rhbdd3-F:ACCTCAATATGACAGCGTCGTAAAC；
PMD18-Rhbdd3-R:TATCCCTGAGCTATGACGCTGA.
PCR reaction system is as follows: 30.5 μ L of ddH2O, 10 × LA Buffer II (Mg2+plus) 5 μ L, dNTP
8 μ L of Mixture (2.5mmol/L), each 2 μ L of upstream and downstream primer (10 μm of ol/L), LA Taq Polymerase (5U/ μ L) 0.5 μ
L, 2 μ L of cDNA template, total volume are 50 μ L.
PCR reaction condition are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 2min, altogether
35 circulations；72 DEG C of extension 10min；4 DEG C of heat preservations.
Pcr amplification product is connected on pMD18-T carrier after cutting glue purification recycling through 1% agarose gel electrophoresis.Connection
Product converts DH5 α competent escherichia coli cell, and bacterium solution PCR screening positive clone simultaneously send raw work bioengineering (Shanghai) share
Co., Ltd carries out two-way sequencing.
(4) building of recombinant plasmid pCI-Rhbdd3
Rhbdd3 gene is designed according to Rhbdd3 gene order in (3) using 5.0 software of Primer Express
The expression cloning forward primer pCI-Rhbdd3-F and reverse primer pCI-Rhbdd3-R of code area, and draw respectively in the forward direction
Nhe I, EcoR I restriction enzyme site are introduced on object and the reverse primer, carry out Rhbdd3 gene volume by template of the carp cDNA
The PCR amplification in code area, primers DNA sequences are as follows:
PCI-Rhbdd3-F:TTAGCTAGCCACCATGCTCGATCATCTTTTTTCAGCAT；
PCI-Rhbdd3-R:CGGAATTCCTATGACGCTGATGGCTTCTTCC。
For pcr amplification reaction system with described in (3), amplification is as shown in Figure 1.Pcr amplified fragment is cut glue purification to return
Receive, then respectively by the segment of recycling and pCI-neo carrier (being purchased from Promega company, map is as shown in figure 14) with Nhe I and
37 DEG C of digestion 3h of EcoR I restriction enzyme, endonuclease bamhi purification and recovery connected under T4DNA connection enzyme effect in 16 DEG C
Night.After conversion utilize bacterium solution PCR and double digestion screening positive clone, and send Sangon Biotech (Shanghai) Co., Ltd. into
The two-way sequencing of row, recombinant plasmid are named as pCI-Rhbdd3 (map is as shown in figure 15).
In addition, the present invention has also carried out the extraction of salmon total serum IgE using salmon tissue, reverse transcription obtains salmon cDNA, and carries out
The amplification (Fig. 2) of the gene coding region salmon Rhbdd3, method is the same as described in (1) to (4).Sequencing discovery, the salmon of amplification
The gene coding region Rhbdd3 nucleotide sequence and carp Rhbdd3 coding sequence similitude are up to 100% (Fig. 3), show
Rhbdd3 gene is very conservative in carp and salmon, and embodying Rhbdd3 gene and Rhbdd3 albumen of the invention can answer in fish
Popularity.
(5) preparation of recombinant plasmid pCI-Rhbdd3 and empty carrier pCI-neo
Correct monoclonal bacterium solution is sequenced in 30 μ L and is inoculated into the LB liquid medium that 3mL contains 50 μ g/mL ampicillins
In, 37 DEG C, 220 revs/min, shaking table culture is overnight, described in plasmid extraction kit (being purchased from Invitrogen company) extraction
The recombinant plasmid pCI-Rhbdd3 and empty carrier plasmid pCI-neo, the specific steps are as follows:
The bacterium solution of overnight incubation is placed in 1.5mL centrifuge tube, room temperature, 12000 × g are centrifuged 1min, abandon supernatant to the greatest extent, repeat
The above operation is until the centrifugation of all bacterium solutions finishes；
250 μ L Resuspension Solution suspension precipitating is added in bacterial precipitation；
250 μ L Lysis Solution are added mildly and fully spinning upside down 4-6 times cracks thallus sufficiently, until
Bright solution is formed, this step is no more than 5min；
350 μ L Neutralization Solution are added and fully spin upside down 4-6 times, room temperature, 12000 × g,
It is centrifuged 5min；
It draws supernatant and is transferred to adsorption column, room temperature, 12000 × g are centrifuged 1min, abandon filtrate；
It is washed twice with 500 μ L Wash Solution, abandons filtrate；
Room temperature, 12000 × g are centrifuged void column 1min；
Adsorption column moves into new 1.5mL centrifuge tube, adds 50 μ L Eluent Solution, room temperature in adsorption column film center
2min is stood, 12000 × g is centrifuged 1min；
With spectrophotometric determination plasmid concentration.
(6) the double digestion identification of recombinant plasmid pCI-Rhbdd3
By the pCI-Rhbdd3 recombinant plasmid of building Nhe I and EcoR I 37 DEG C of digestion 3h of restriction enzyme, gel
Electrophoresis result shows, recombinant plasmid obtains two specific fragments of size about 5460bp and 1050bp respectively after digestion
(result is as shown in Figure 4), in the same size with target fragment, further sequencing is showing constructed recombinant plasmid reading frame just
It is really errorless.
(7) recombinant plasmid pCI-Rhbdd3 and empty carrier pCI-neo transfects EPC cell
Specific step is as follows (by taking 24 porocyte culture plates as an example):
The day before transfection, spreads 24 porocyte culture plates, and transfection when cell density reaches about 70%-90% in second day turns
Every hole prepares following mixed liquor when dye:
A pipe: 0.8 μ g plasmid is dissolved in 50 μ L Opti-MEM culture mediums；
B pipe: 2 μ L Lipofectamine 2000 (being purchased from Invitrogen company) are dissolved in 50 μ L Opti-MEM culture
In base, mixes, be placed at room temperature for 5min；
Then two pipe of AB is mixed, is placed at room temperature for 20min, during which washes cell twice with Opti-MEM；
It feeds the mixture into corresponding aperture, mixes gently, change the complete medium containing 10%FBS into after cultivating 4-6h.
(8) Rhbdd3 is overexpressed the detection influenced on cell activity
Rhbdd3 protein overexpression pair is detected using cell Proliferation/toxicity detection reagent C CK-8 (being purchased from Dojindo company)
Cell activity influences.Its basic principle is to contain 2- (2- methoxyl group -4- nitrobenzophenone) -3- (4- nitrobenzene in CCK-8 reagent
Base) -5- (2,4- disulfonic acid benzene) -2H- tetrazolium monosodium salt (WST-8), its energy under the action of electron carrier 1-Methoxy PMS
Water-soluble orange-yellow formazan is reduced to by intracellular dehydrogenase, the amount for generating formazan is directly proportional to cell number, can survey indirectly
Determine living cells quantity.Specific step is as follows:
The day before transfection is inoculated with 100 μ L EPC or CHSE-214 cell suspensions into 96 porocyte culture plates, cultivated
Night transfects recombinant plasmid pCI-Rhbdd3 and empty carrier by method described in (7) when cell density reaches about 70%-90%
pCI-neo；
The CCK-8 of 10 μ L is added into every hole by 12,36 and 72h after transfection；
By culture plate in being incubated for 1h in incubator, the absorbance at microplate reader measurement 450nm wavelength is utilized.
As a result as shown in Figure 6 and Figure 7, show that the overexpression of Rhbdd3 has no effect on the activity of EPC and CHSE-214 cell.
(9) immunoblot experiment (western blot)
Specific step is as follows:
After the recombinant plasmid pCI-Rhbdd3 and empty carrier pCI-neo transfects EPC cell for 24 hours, supernatant is abandoned, is used
PBS is washed twice, each 3min, be added RIPA lysate, with rifle piping and druming it is several under, come into full contact with lysate and cell, incubate on ice
10min is educated, cell pyrolysis liquid is transferred in 1.5mL EP pipe, 12000 × g, 5min is centrifuged, 20 μ L is taken to be centrifuged 5 μ L of supernatant
5 × albumen sample-loading buffer mix, 95 DEG C are boiled 5min, carry out SDS-PAGE.10%SDS-PAGE gel uses SDS-PAGE
Gel reagent preparation box prepares (being purchased from Shanghai Yi Sheng Biotechnology Co., Ltd)；
The filter paper and pvdf membrane to fit like a glove with separation gel size is cut, 15min is balanced in transfering buffering liquid；
After gel electrophoresis, separation gel balances 10min in appropriate transfering buffering liquid；
Assembling transfers interlayer, at constant pressure transfer protein 20V, 40min；
Transferring film finishes, and takes the film out, and is put into hybridizing box, adds 10mL 5%PBST- bovine serum albumin(BSA) (BSA) confining liquid,
37 DEG C of incubation 2h；
Outwell confining liquid, be added this laboratory preparation Rhbdd3 antibody (primary antibody, it is dilute with volume ratio 1:1000 with confining liquid
Release), 37 DEG C of incubation 2h；
PBST is washed 3 times, each 10min, and the goat anti-rabbit IgG antibody that HRP is marked is added, and (it is raw to be purchased from Shanghai assist sage for secondary antibody
Object Science and Technology Ltd., with volume ratio 1:10000 dilution), 37 DEG C of incubation 1h；
Secondary antibody is outwelled, PBST is washed 3 times, each 10min；
ECL chromophoric substrate (being purchased from TIANGEN Biotech (Beijing) Co., Ltd.) is added, band develops the color in 5-10min.
After immunoblot results are as shown in figure 5, show pCI-Rhbdd3 transfection, the expression of Rhbdd3 albumen is obtained
It significantly improves.
The immunoblotting steps of virus infected cell are identical as above-mentioned steps, only with the diluted rabbit-anti of volume ratio 1:1000
SVCV M protein antibodies (preparation of this laboratory), rabbit-anti IPNV VP2 protein antibodies (preparation of this laboratory) or internal reference HSP90 egg
White rabbit resists (being purchased from Cell Signaling Technology company) to be used as primary antibody.
As a result as shown in figs. 9 and 12, after Rhbdd3 protein overexpression, SVCV M albumen and IPNV VP2 protein expression water
It is flat to be substantially reduced, prompt the infection of virus to be inhibited.
(10) indirect immunofluorescence (IF)
After the recombinant plasmid pCI-Rhbdd3 and empty carrier pCI-neo transfects EPC or CHSE-214 cell for 24 hours,
SVCV is inoculated with MOI=1 or IPNV is inoculated with MOI=0.5, different time is washed cell 2 times, each 3min with PBS after infection,
300 μ L, 4% paraformaldehyde, the fixed 30min of room temperature is added in every hole；
Fixer is absorbed, PBS is washed cell 3 times, and the PBS that 300 μ L X-100 containing 0.2%Triton are added in every hole is penetrating
15min；
Penetrating liquid is absorbed, PBS is washed 3 times, and the confining liquid of 4%BSA, 37 DEG C of closing 2h are added；
Confining liquid is absorbed, is added with the diluted primary antibody of confining liquid (preparation of this laboratory, rabbit-anti SVCV M protein antibodies or rabbit
Anti- IPNV VP2 protein antibodies, with volume ratio 1:800 dilution), 37 DEG C of incubation 2h；
Absorb primary antibody, PBS washes 3 times, each 3min, be added fluorescence secondary antibody (donkey anti-rabbit that Alexa Fluor 488 is marked or
The donkey anti-rabbit that Alexa Fluor 594 is marked, is purchased from Invitrogen company, with volume ratio 1:2000 dilution), 37 DEG C of incubations
1h；
Secondary antibody is absorbed, PBS is washed 3 times, each 3min, addition DAPI dye karyolymph (it is purchased from Roche company, with volume ratio 1:
2000 dilutions), room temperature dyes 10min, and PBS is washed 3 times, each 3min；
500 μ L PBS are added in every hole, are placed under inverted fluorescence microscope and observe and take pictures.
As a result as shown in Figure 8 and Figure 11, it is infected cell number after Rhbdd3 is overexpressed to significantly reduce, shows Rhbdd3 egg
The white proliferation that can not only inhibit SVCV, can also inhibit the proliferation of IPNV.
(11) influence that Rhbdd3 protein overexpression is proliferated SVCV
The recombinant plasmid pCI-Rhbdd3 (experimental group) and the empty carrier pCI-neo (control group) transfect EPC cell
After for 24 hours, SVCV is inoculated with MOI=1,12,24 and 36h collects the virus infection liquid of experimental group and control group respectively after infection,
Carry out the measurement of SVCV N gene copy number.Specific steps are as follows:
The recombinant plasmid pCI-Rhbdd3 and empty carrier pCI-neo transfects EPC cell；
After transfection for 24 hours, it is washed twice with serum-free M199, SVCV is inoculated with MOI=1, is changed into after 20 DEG C of absorption 1h containing 2%
The M199 culture medium of FBS；
12,24 and 36h collects the virus infection liquid of experimental group and control group respectively after infection, extracts RNA with Trizol method；
CDNA synthesis is carried out using reverse transcription reagent box (being purchased from Invitrogen company)；
SVCV N gene copy number is carried out using SYBR Green fluorescence quantifying PCR method to measure.
Fluorescence quantification PCR primer DNA sequence dna is as follows:
Forward primer: N-TF1:ATCAGGCCGATTATCCTTCCA；
Reverse primer: N-TR1:AGATAAGCATTCACATGCTGTAT.
10 μ L 2 × SYBR Premix Ex Taq are sequentially added in 200 μ L Fluorescence PCR pipes (is purchased from Takara public affairs
Department), 10 μm of ol/L forward primers of concentration, each 0.4 μ L of reverse primer, 0.4 μ L ROX reference dye II and sample to be tested
2 μ L of cDNA, supplement distilled water to 20 μ L, is placed in 7500Fast fluorescence quantitative PCR instrument and is reacted, and reaction condition is 95 DEG C
30s；95 DEG C of 5s, 60 DEG C of 30s 40 circulations later, after, melting curve analysis is carried out by instrument immediately, finally by ABI
7500 2.0.6 softwares calculate amplified production Tm value；By control group, to be set as 1 for statistical analysis for N gene copy number for 24 hours.
The results are shown in Figure 10, shows that Rhbdd3 is overexpressed the proliferation that can significantly inhibit SVCV.
(12) influence that Rhbdd3 protein overexpression is proliferated IPNV
The recombinant plasmid pCI-Rhbdd3 (experimental group) and the empty carrier pCI-neo (control group) transfect CHSE-214
After cell for 24 hours, IPNV is inoculated with MOI=0.5,18h collects the virus infection liquid of experimental group and control group after infection, carries out
The measurement of IPNV VP3 gene copy number.Specific steps are as follows:
The recombinant plasmid pCI-Rhbdd3 and empty carrier pCI-neo transfects CHSE-214 cell；
After transfection for 24 hours, it is washed twice with serum-free DMEM, IPNV is inoculated with MOI=0.5, is changed into after 20 DEG C of absorption 1h containing 2%
The DMEM culture medium of FBS；
18h collects the virus infection liquid of experimental group and control group after infection, extracts RNA with Trizol method；
CDNA synthesis is carried out using reverse transcription reagent box (being purchased from Invitrogen company)；
IPNV VP3 gene copy number is carried out using SYBR Green fluorescence quantifying PCR method to measure.
Fluorescence quantification PCR primer DNA sequence dna is as follows:
Forward primer: VP3-F1:CGACCGACATGAACAAAATCA；
Reverse primer: VP3-R1:AGTTGCAGCTGTATTCGCACA.
10 μ L 2 × SYBR Premix Ex Taq are sequentially added in 200 μ L Fluorescence PCR pipes (is purchased from Takara public affairs
Department), 10 μm of ol/L forward primers of concentration, each 0.4 μ L of reverse primer, 0.4 μ L ROX reference dye II and sample to be tested
2 μ L of cDNA, supplement distilled water to 20 μ L, is placed in 7500Fast fluorescence quantitative PCR instrument and is reacted, and reaction condition is 95 DEG C
30s；95 DEG C of 5s, 60 DEG C of 30s 40 circulations later, after, melting curve analysis is carried out by instrument immediately, finally by ABI
7500 2.0.6 softwares calculate amplified production Tm value；It is for statistical analysis that control group VP3 gene copy number is set as 1.
As a result as shown in figure 13, show that the overexpression of Rhbdd3 can significantly inhibit the proliferation of IPNV.
The present invention isolates Rhbdd3 gene by gene clone technology from carp, passes through a series of verification experimental verification, table
The Rhbdd3 albumen that the bright present invention obtains has excellent ntiviral characteristic, can significantly reduce infectivity, the suppression of SVCV and IPNV
Virus replication processed plays an important role in the course of infection that cell resists SVCV and IPNV.In addition, the overexpression of Rhbdd3
The normal activity of cell is not influenced.Rhbdd3 albumen and its gene of the invention provides newly to prepare anti-SVCV and IPNV drug
Target spot, also provide important technical basis for the cultivation of fish novel anti-virus kind.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Sequence table
<110>Shanghai Aquatic Product Institute
<120>the carp antiviral protein Rhbdd3 and its antiviral activity separated
<130> 20190619
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1050
<212> DNA
<213>carp (Cyprinus carpio)
<400> 1
atgctcgatc atcttttttc agcatggatg tggtttggat caaaccggcc aggatattgt 60
agtggaacat ttgtaatagt catattcatt ttattgactt ggctctgtgg tattcacgcc 120
agcctaagtt taggaccagg tggggaattt cctggatttt atgactttat cttctacgcc 180
ttcagtaatg aagagttgac ctcatttctc tacaacatca ccctgctgct gtggatcggg 240
ccctgtcagg agagaagatg gggcactttt gtcttcctag ctctctcttt tatctccact 300
gtgcttcttc ctcctatcta tggccttttt ctctttgtta ccggggatga agccagcagg 360
gttagtggat actcagccac ccaccttgcc ctgctcacag ctcagtgtcg gcaagtgaag 420
cagagacggg tcctgcgttt tgttcctgtc tggttcttgc cttggctctt cttgctcctc 480
gacctctttc tgcttcccag tgctccagga ctgctgcatt tttatgccat ttgtctggga 540
ctcaactaca ataatgagtt tattgaaata ctggagcaga tagagagact gggtgcatgt 600
tcctgcttgc caaaatgggc ctacatccct gttacctctc atccatacct gccagtttac 660
cacaacacct caataagacc agagccttca accagtagat cccagttgga ggatcattcg 720
gcatggcaat attctgtgtc agagtggcat caggagcctt tgttggcaac agattcacag 780
ttgttagagg agcagatgct gagagcaggg atattagcat cactccacga tgcaccagaa 840
ggtgaagcgg acaaagtgga agtacccaag tcctcagtgt cttcattaag gttgcaacag 900
ctggagaaga tgggcttccc cacagagaaa gctgttgtgg ctctggccgc tacagggcag 960
ttggatggag cgatctcttt gcttattgat gatcagattg gggaggaggc tgtggtcatc 1020
tctaaaggga agaagccatc agcgtcatag 1050
<210> 2
<211> 349
<212> PRT
<213>carp (Cyprinus carpio)
<400> 2
Met Leu Asp His Leu Phe Ser Ala Trp Met Trp Phe Gly Ser Asn Arg
1 5 10 15
Pro Gly Tyr Cys Ser Gly Thr Phe Val Ile Val Ile Phe Ile Leu Leu
20 25 30
Thr Trp Leu Cys Gly Ile His Ala Ser Leu Ser Leu Gly Pro Gly Gly
35 40 45
Glu Phe Pro Gly Phe Tyr Asp Phe Ile Phe Tyr Ala Phe Ser Asn Glu
50 55 60
Glu Leu Thr Ser Phe Leu Tyr Asn Ile Thr Leu Leu Leu Trp Ile Gly
65 70 75 80
Pro Cys Gln Glu Arg Arg Trp Gly Thr Phe Val Phe Leu Ala Leu Ser
85 90 95
Phe Ile Ser Thr Val Leu Leu Pro Pro Ile Tyr Gly Leu Phe Leu Phe
100 105 110
Val Thr Gly Asp Glu Ala Ser Arg Val Ser Gly Tyr Ser Ala Thr His
115 120 125
Leu Ala Leu Leu Thr Ala Gln Cys Arg Gln Val Lys Gln Arg Arg Val
130 135 140
Leu Arg Phe Val Pro Val Trp Phe Leu Pro Trp Leu Phe Leu Leu Leu
145 150 155 160
Asp Leu Phe Leu Leu Pro Ser Ala Pro Gly Leu Leu His Phe Tyr Ala
165 170 175
Ile Cys Leu Gly Leu Asn Tyr Asn Asn Glu Phe Ile Glu Ile Leu Glu
180 185 190
Gln Ile Glu Arg Leu Gly Ala Cys Ser Cys Leu Pro Lys Trp Ala Tyr
195 200 205
Ile Pro Val Thr Ser His Pro Tyr Leu Pro Val Tyr His Asn Thr Ser
210 215 220
Ile Arg Pro Glu Pro Ser Thr Ser Arg Ser Gln Leu Glu Asp His Ser
225 230 235 240
Ala Trp Gln Tyr Ser Val Ser Glu Trp His Gln Glu Pro Leu Leu Ala
245 250 255
Thr Asp Ser Gln Leu Leu Glu Glu Gln Met Leu Arg Ala Gly Ile Leu
260 265 270
Ala Ser Leu His Asp Ala Pro Glu Gly Glu Ala Asp Lys Val Glu Val
275 280 285
Pro Lys Ser Ser Val Ser Ser Leu Arg Leu Gln Gln Leu Glu Lys Met
290 295 300
Gly Phe Pro Thr Glu Lys Ala Val Val Ala Leu Ala Ala Thr Gly Gln
305 310 315 320
Leu Asp Gly Ala Ile Ser Leu Leu Ile Asp Asp Gln Ile Gly Glu Glu
325 330 335
Ala Val Val Ile Ser Lys Gly Lys Lys Pro Ser Ala Ser
340 345
<210> 3
<211> 25
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 3
acctcaatat gacagcgtcg taaac 25
<210> 4
<211> 22
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 4
tatccctgag ctatgacgct ga 22
<210> 5
<211> 38
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 5
ttagctagcc accatgctcg atcatctttt ttcagcat 38
<210> 6
<211> 31
<212> DNA
<213>artificial sequence (Artificial sequence)
<400> 6
cggaattcct atgacgctga tggcttcttc c 31
Claims (10)
1. a kind of isolated carp antiviral protein Rhbdd3 gene, which is characterized in that the gene coding region Rhbdd3 nucleotide
Sequence is sequence shown in 1-1050bp in SEQ ID NO:1.
2. a kind of isolated carp antiviral protein Rhbdd3, which is characterized in that the amino acid sequence of the albumen Rhbdd3 coding
It is sequence shown in SEQ ID NO:2.
3. a kind of recombinant plasmid comprising the gene coding region Rhbdd3 as described in claim 1 nucleotide sequence.
4. a kind of preparation method for preparing recombinant plasmid as claimed in claim 3, which is characterized in that the method includes following
Step:
Step 1: extracting the RNA of carp, RNA described in reverse transcription obtains carp cDNA；
Step 2: the upstream primer and downstream primer of design Rhbdd3 gene order conserved region, the institute obtained with the step 1
State carp cDNA be template amplification, obtain amplified fragments, be purified recycle and be connected on pMD18-T carrier obtain connection produce
Object carries out two-way sequencing after connection product conversion DH5 α competent escherichia coli cell and screening positive clone,
Obtain Rhbdd3 gene order；
Step 3: the Rhbdd3 gene order obtained according to the step 2, designs the expression of the gene coding region Rhbdd3
Forward primer and reverse primer are cloned, amplified fragments are obtained as template amplification using the carp cDNA that the step 1 obtains, and
The amplified fragments are subjected to molecular cloning, obtain recombinant plasmid.
5. the preparation method of recombinant plasmid as claimed in claim 4, which is characterized in that the step 3 further includes expanding
Before, Nhe I, EcoR I restriction enzyme site need to be introduced respectively on the expression cloning forward primer and reverse primer.
6. the preparation method of recombinant plasmid as claimed in claim 4, which is characterized in that the upstream primer and downstream primer point
Not Wei pMD18-Rhbdd3-F and pMD18-Rhbdd3-R, wherein the DNA sequence dna of the pMD18-Rhbdd3-F is
The DNA sequence dna of ACCTCAATATGACAGCGTCGTAAAC, the pMD18-Rhbdd3-R is
TATCCCTGAGCTATGACGCTGA。
7. the preparation method of recombinant plasmid as claimed in claim 4, which is characterized in that the expression cloning forward primer and anti-
It is respectively pCI-Rhbdd3-F and pCI-Rhbdd3-R to primer, wherein the DNA sequence dna of the pCI-Rhbdd3-F is TTAGCT
The DNA sequence dna of AGCCACCATGCTCGATCATCTTTTTTCAGCAT, the pCI-Rhbdd3-R is
CGGAATTCCTATGACGCTGATGGCTTCTTCC。
8. a kind of carp antiviral protein Rhbdd3 gene as described in claim 1 is preparing anti-huichun viremia virus drug
Application in composition or infectivity resistant pancreatic necrosis virus pharmaceutical composition.
9. a kind of carp antiviral protein Rhbdd3 as claimed in claim 2 is preparing anti-huichun viremia virus pharmaceutical composition
Application in object or infectivity resistant pancreatic necrosis virus pharmaceutical composition.
10. a kind of carp antiviral protein Rhbdd3 gene as described in claim 1 is in cultivating fish novel anti-virus kind
Using.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910576228.8A CN110295173B (en) | 2019-06-28 | 2019-06-28 | Isolated carp antiviral protein Rhbdd3 and antiviral activity thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910576228.8A CN110295173B (en) | 2019-06-28 | 2019-06-28 | Isolated carp antiviral protein Rhbdd3 and antiviral activity thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110295173A true CN110295173A (en) | 2019-10-01 |
| CN110295173B CN110295173B (en) | 2022-10-21 |
Family
ID=68029350
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910576228.8A Active CN110295173B (en) | 2019-06-28 | 2019-06-28 | Isolated carp antiviral protein Rhbdd3 and antiviral activity thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110295173B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113754750A (en) * | 2021-09-30 | 2021-12-07 | 华中农业大学 | Antibacterial peptide and application thereof in aquaculture |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100173972A1 (en) * | 2007-04-12 | 2010-07-08 | Medical Research Council | Methods and uses related to rhbdl4 |
| CN105079796A (en) * | 2014-05-07 | 2015-11-25 | 中国人民解放军第二军医大学 | Anti-inflammation effect and application of immuno-regulatory molecule Rhbdd3 |
| WO2017180587A2 (en) * | 2016-04-11 | 2017-10-19 | Obsidian Therapeutics, Inc. | Regulated biocircuit systems |
-
2019
- 2019-06-28 CN CN201910576228.8A patent/CN110295173B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100173972A1 (en) * | 2007-04-12 | 2010-07-08 | Medical Research Council | Methods and uses related to rhbdl4 |
| CN105079796A (en) * | 2014-05-07 | 2015-11-25 | 中国人民解放军第二军医大学 | Anti-inflammation effect and application of immuno-regulatory molecule Rhbdd3 |
| WO2017180587A2 (en) * | 2016-04-11 | 2017-10-19 | Obsidian Therapeutics, Inc. | Regulated biocircuit systems |
Non-Patent Citations (3)
| Title |
|---|
| LIU JUAN 等: "Rhbdd3 controls autoimmunity by suppressing the production of IL-6 by dendritic cells via K27-linked ubiquitination of the regulator NEMO", 《NAT IMMUNOL.》 * |
| 张海强 等: "鲤春病毒血症病毒核蛋白、磷蛋白与基质蛋白的表达、抗体制备及免疫原性比较", 《水产学报》 * |
| 无: "XP_018960245.1，"PREDICTED: LOW QUALITY PROTEIN: rhomboid domain-containing protein 3-like [Cyprinus carpio]"", 《GENBANK》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113754750A (en) * | 2021-09-30 | 2021-12-07 | 华中农业大学 | Antibacterial peptide and application thereof in aquaculture |
| CN113754750B (en) * | 2021-09-30 | 2023-08-25 | 华中农业大学 | Antibacterial peptide and application thereof in aquaculture |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110295173B (en) | 2022-10-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN111560354B (en) | Recombinant novel coronavirus, preparation method and application thereof | |
| CN106282216B (en) | A kind of preparation method of recombinant long-acting chicken interferon α | |
| CN102695522B (en) | New Human reoviruslike agent strain and vaccine | |
| CN101918440B (en) | Use of avian cytokines and genetic sequences encoding the avian cytokines | |
| CN107841507A (en) | A kind of porcine circovirus 2 type Cap cell-penetrating peptides antigen-4 fusion protein gene of high efficient expression and its application | |
| CN112353939B (en) | Application of GTPBP4 protein as immunosuppressant and construction of cell line for knocking down or over expressing GTPBP4 | |
| CN107987136A (en) | ZIKV-NS1 albumen and its application in zika virus transmission_blocking vaccine is prepared | |
| CN102558313A (en) | Enterovirus 71 type specific recombinant protein antigen and application thereof | |
| CN111925994B (en) | Recombinant African swine fever virus with DP71L gene deleted and preparation method and application thereof | |
| CN114524862B (en) | Construction and application of avian influenza (H5 + H7) trivalent DNA vaccine | |
| Peng et al. | Identification of a TRIM32 from Penaeus monodon is involved in autophagy and innate immunity during white spot syndrome virus infection | |
| CN113862284B (en) | Gene, virus-like particle, vaccine and preparation and application for encoding recombinant avian influenza virus HA protein | |
| CN110295173A (en) | Isolated carp antiviral protein Rhbdd3 and its antiviral activity | |
| CN112587660B (en) | Application of Seneca valley virus 3D protein in preparation of immune response inducer or adjuvant | |
| Wessel et al. | Piscine orthoreovirus (PRV) ơ3 protein binds dsRNA | |
| CN104419686B (en) | recombinant PRRS virus HV-nsp9 and application thereof | |
| CN111057682B (en) | Avian H9N2 subtype avian influenza strain separation identification and application | |
| CN102516394B (en) | CMG 2mutant and Fc fusion protein, its encoding gene and its application | |
| Chen et al. | Vector mosquito ecology and Japanese encephalitis virus genotype III strain detection from Culex tritaeniorhynchus and pig in Huaihua, China | |
| CN110092823A (en) | Isolated carp antiviral protein Pdcd6ip and antiviral activity | |
| CN113827714B (en) | H7N9 subtype avian influenza virus-like particle vaccine preparation, preparation and application | |
| CN109867713A (en) | A kind of canine distemper genetic engineering subunit vaccine | |
| CN109266623A (en) | One strain vaccine strain rSHA- △ 200 and its construction method and application | |
| CN104152477A (en) | Schistosoma japonicum recombinant antigen SjPDI (schistosoma japonicum protein disulfide isomerase) and preparation method and application thereof | |
| CN113855795A (en) | Avian hepatitis E virus ORF2 subunit vaccine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 200433 No. 265, Jiamusi Road, Shanghai, Yangpu District Patentee after: SHANGHAI FISHERIES Research Institute SHANGHAI FISHERIES TECHNICAL EXTENSION STATION Address before: 200433 No. 265, Jiamusi Road, Shanghai, Yangpu District Patentee before: SHANGHAI FISHERIES Research Institute |