CN112625118B - 一种金钱鱼促生殖细胞成熟基因igf3及其应用 - Google Patents
一种金钱鱼促生殖细胞成熟基因igf3及其应用 Download PDFInfo
- Publication number
- CN112625118B CN112625118B CN202011353432.2A CN202011353432A CN112625118B CN 112625118 B CN112625118 B CN 112625118B CN 202011353432 A CN202011353432 A CN 202011353432A CN 112625118 B CN112625118 B CN 112625118B
- Authority
- CN
- China
- Prior art keywords
- igf3
- gene
- protein
- seq
- recombinant protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 101100286583 Xenopus laevis igf3 gene Proteins 0.000 title claims abstract description 91
- 230000011748 cell maturation Effects 0.000 title claims abstract description 17
- 241001596950 Larimichthys crocea Species 0.000 title claims abstract description 8
- 230000001737 promoting effect Effects 0.000 title abstract description 14
- 230000012010 growth Effects 0.000 title abstract description 9
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 40
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 40
- 241001274197 Scatophagus argus Species 0.000 claims abstract description 35
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 29
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 25
- 239000002299 complementary DNA Substances 0.000 claims abstract description 17
- 230000035800 maturation Effects 0.000 claims abstract description 15
- 239000013604 expression vector Substances 0.000 claims abstract description 10
- 210000000287 oocyte Anatomy 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract description 10
- 239000002773 nucleotide Substances 0.000 claims abstract description 6
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 6
- 239000003814 drug Substances 0.000 claims abstract description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 8
- 229920001184 polypeptide Polymers 0.000 claims description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 6
- 210000004027 cell Anatomy 0.000 claims description 5
- 230000001131 transforming effect Effects 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 4
- 238000012258 culturing Methods 0.000 claims description 3
- 241000861914 Plecoglossus altivelis Species 0.000 claims description 2
- 241000251468 Actinopterygii Species 0.000 abstract description 15
- 238000011161 development Methods 0.000 abstract description 6
- 230000018109 developmental process Effects 0.000 abstract description 6
- 230000014509 gene expression Effects 0.000 description 14
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 12
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 description 9
- 102000013275 Somatomedins Human genes 0.000 description 9
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 101150012716 CDK1 gene Proteins 0.000 description 5
- 241000252212 Danio rerio Species 0.000 description 5
- 101100059559 Emericella nidulans (strain FGSC A4 / ATCC 38163 / CBS 112.46 / NRRL 194 / M139) nimX gene Proteins 0.000 description 5
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 5
- 101100273808 Xenopus laevis cdk1-b gene Proteins 0.000 description 5
- 150000001413 amino acids Chemical group 0.000 description 5
- 239000003480 eluent Substances 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 230000002611 ovarian Effects 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 4
- 241000894006 Bacteria Species 0.000 description 4
- 241001052560 Thallis Species 0.000 description 4
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 4
- 101001076308 Xenopus laevis Insulin-like growth factor III Proteins 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 210000004602 germ cell Anatomy 0.000 description 4
- 238000001819 mass spectrum Methods 0.000 description 4
- 229910052759 nickel Inorganic materials 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000009465 prokaryotic expression Effects 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 108700005075 Regulator Genes Proteins 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 230000033228 biological regulation Effects 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 230000001939 inductive effect Effects 0.000 description 3
- 238000001294 liquid chromatography-tandem mass spectrometry Methods 0.000 description 3
- 108020004999 messenger RNA Proteins 0.000 description 3
- 210000001672 ovary Anatomy 0.000 description 3
- 230000035790 physiological processes and functions Effects 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 230000005070 ripening Effects 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 238000013518 transcription Methods 0.000 description 3
- 230000035897 transcription Effects 0.000 description 3
- UCIYCBSJBQGDGM-LPEHRKFASA-N Ala-Arg-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(=O)O)N UCIYCBSJBQGDGM-LPEHRKFASA-N 0.000 description 2
- VQAVBBCZFQAAED-FXQIFTODSA-N Ala-Pro-Asn Chemical compound C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(=O)N)C(=O)O)N VQAVBBCZFQAAED-FXQIFTODSA-N 0.000 description 2
- HPKSHFSEXICTLI-CIUDSAMLSA-N Arg-Glu-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O HPKSHFSEXICTLI-CIUDSAMLSA-N 0.000 description 2
- PHHRSPBBQUFULD-UWVGGRQHSA-N Arg-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCCN=C(N)N)N PHHRSPBBQUFULD-UWVGGRQHSA-N 0.000 description 2
- LVMUGODRNHFGRA-AVGNSLFASA-N Arg-Leu-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O LVMUGODRNHFGRA-AVGNSLFASA-N 0.000 description 2
- NPAVRDPEFVKELR-DCAQKATOSA-N Arg-Lys-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O NPAVRDPEFVKELR-DCAQKATOSA-N 0.000 description 2
- BSYKSCBTTQKOJG-GUBZILKMSA-N Arg-Pro-Ala Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O BSYKSCBTTQKOJG-GUBZILKMSA-N 0.000 description 2
- ICRHGPYYXMWHIE-LPEHRKFASA-N Arg-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ICRHGPYYXMWHIE-LPEHRKFASA-N 0.000 description 2
- DCJNIJAWIRPPBB-CIUDSAMLSA-N Cys-Ala-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CS)N DCJNIJAWIRPPBB-CIUDSAMLSA-N 0.000 description 2
- XTHUKRLJRUVVBF-WHFBIAKZSA-N Cys-Gly-Ser Chemical compound SC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O XTHUKRLJRUVVBF-WHFBIAKZSA-N 0.000 description 2
- 102000002322 Egg Proteins Human genes 0.000 description 2
- 108010000912 Egg Proteins Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- JSYULGSPLTZDHM-NRPADANISA-N Gln-Ala-Val Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O JSYULGSPLTZDHM-NRPADANISA-N 0.000 description 2
- GNDJOCGXGLNCKY-ACZMJKKPSA-N Gln-Cys-Cys Chemical compound N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O GNDJOCGXGLNCKY-ACZMJKKPSA-N 0.000 description 2
- QYKBTDOAMKORGL-FXQIFTODSA-N Gln-Gln-Asp Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N QYKBTDOAMKORGL-FXQIFTODSA-N 0.000 description 2
- CAXXTYYGFYTBPV-IUCAKERBSA-N Gln-Leu-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O CAXXTYYGFYTBPV-IUCAKERBSA-N 0.000 description 2
- RUFHOVYUYSNDNY-ACZMJKKPSA-N Glu-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O RUFHOVYUYSNDNY-ACZMJKKPSA-N 0.000 description 2
- XXGQRGQPGFYECI-WDSKDSINSA-N Gly-Cys-Glu Chemical compound NCC(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CCC(O)=O XXGQRGQPGFYECI-WDSKDSINSA-N 0.000 description 2
- HQSKKSLNLSTONK-JTQLQIEISA-N Gly-Tyr-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 HQSKKSLNLSTONK-JTQLQIEISA-N 0.000 description 2
- 101001076292 Homo sapiens Insulin-like growth factor II Proteins 0.000 description 2
- 108010065920 Insulin Lispro Proteins 0.000 description 2
- 102100025947 Insulin-like growth factor II Human genes 0.000 description 2
- SITWEMZOJNKJCH-UHFFFAOYSA-N L-alanine-L-arginine Natural products CC(N)C(=O)NC(C(O)=O)CCCNC(N)=N SITWEMZOJNKJCH-UHFFFAOYSA-N 0.000 description 2
- BOFAFKVZQUMTID-AVGNSLFASA-N Leu-Gln-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N BOFAFKVZQUMTID-AVGNSLFASA-N 0.000 description 2
- QNBVTHNJGCOVFA-AVGNSLFASA-N Leu-Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O QNBVTHNJGCOVFA-AVGNSLFASA-N 0.000 description 2
- BRTVHXHCUSXYRI-CIUDSAMLSA-N Leu-Ser-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O BRTVHXHCUSXYRI-CIUDSAMLSA-N 0.000 description 2
- QQXJROOJCMIHIV-AVGNSLFASA-N Leu-Val-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCSC)C(O)=O QQXJROOJCMIHIV-AVGNSLFASA-N 0.000 description 2
- PRSBSVAVOQOAMI-BJDJZHNGSA-N Lys-Ile-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCCCN PRSBSVAVOQOAMI-BJDJZHNGSA-N 0.000 description 2
- GAHJXEMYXKLZRQ-AJNGGQMLSA-N Lys-Lys-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O GAHJXEMYXKLZRQ-AJNGGQMLSA-N 0.000 description 2
- DNWBUCHHMRQWCZ-GUBZILKMSA-N Lys-Ser-Gln Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O DNWBUCHHMRQWCZ-GUBZILKMSA-N 0.000 description 2
- WZVSHTFTCYOFPL-GARJFASQSA-N Lys-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)[C@H](CCCCN)N)C(=O)O WZVSHTFTCYOFPL-GARJFASQSA-N 0.000 description 2
- LQMHZERGCQJKAH-STQMWFEESA-N Met-Gly-Phe Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 LQMHZERGCQJKAH-STQMWFEESA-N 0.000 description 2
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 2
- WYPVCIACUMJRIB-JYJNAYRXSA-N Phe-Gln-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N WYPVCIACUMJRIB-JYJNAYRXSA-N 0.000 description 2
- SXMSEHDMNIUTSP-DCAQKATOSA-N Pro-Lys-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O SXMSEHDMNIUTSP-DCAQKATOSA-N 0.000 description 2
- YQHZVYJAGWMHES-ZLUOBGJFSA-N Ser-Ala-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YQHZVYJAGWMHES-ZLUOBGJFSA-N 0.000 description 2
- JEHPKECJCALLRW-CUJWVEQBSA-N Ser-His-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JEHPKECJCALLRW-CUJWVEQBSA-N 0.000 description 2
- UBRMZSHOOIVJPW-SRVKXCTJSA-N Ser-Leu-Lys Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O UBRMZSHOOIVJPW-SRVKXCTJSA-N 0.000 description 2
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 2
- MFMGPEKYBXFIRF-SUSMZKCASA-N Thr-Thr-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MFMGPEKYBXFIRF-SUSMZKCASA-N 0.000 description 2
- IJKNKFJZOJCKRR-GBALPHGKSA-N Thr-Trp-Ser Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CO)C(O)=O)=CNC2=C1 IJKNKFJZOJCKRR-GBALPHGKSA-N 0.000 description 2
- 241000276707 Tilapia Species 0.000 description 2
- GFZQWWDXJVGEMW-ULQDDVLXSA-N Tyr-Arg-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O GFZQWWDXJVGEMW-ULQDDVLXSA-N 0.000 description 2
- 108010047495 alanylglycine Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 108010066058 beta Subunit Luteinizing Hormone Proteins 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- 108010045126 glycyl-tyrosyl-glycine Proteins 0.000 description 2
- 210000002149 gonad Anatomy 0.000 description 2
- 230000002710 gonadal effect Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 2
- 102000028416 insulin-like growth factor binding Human genes 0.000 description 2
- 108091022911 insulin-like growth factor binding Proteins 0.000 description 2
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 2
- 239000012160 loading buffer Substances 0.000 description 2
- 108010054155 lysyllysine Proteins 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 210000004681 ovum Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000001850 reproductive effect Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000013535 sea water Substances 0.000 description 2
- 108010007375 seryl-seryl-seryl-arginine Proteins 0.000 description 2
- 239000013598 vector Substances 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- KQFRUSHJPKXBMB-BHDSKKPTSA-N Ala-Ala-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C)NC(=O)[C@@H](N)C)C(O)=O)=CNC2=C1 KQFRUSHJPKXBMB-BHDSKKPTSA-N 0.000 description 1
- RXTBLQVXNIECFP-FXQIFTODSA-N Ala-Gln-Gln Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O RXTBLQVXNIECFP-FXQIFTODSA-N 0.000 description 1
- AWZKCUCQJNTBAD-SRVKXCTJSA-N Ala-Leu-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN AWZKCUCQJNTBAD-SRVKXCTJSA-N 0.000 description 1
- RIPMDCIXRYWXSH-KNXALSJPSA-N Ala-Trp-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N3CCC[C@@H]3C(=O)O)N RIPMDCIXRYWXSH-KNXALSJPSA-N 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- DNBMCNQKNOKOSD-DCAQKATOSA-N Arg-Pro-Gln Chemical compound NC(N)=NCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O DNBMCNQKNOKOSD-DCAQKATOSA-N 0.000 description 1
- FTMRPIVPSDVGCC-GUBZILKMSA-N Arg-Val-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FTMRPIVPSDVGCC-GUBZILKMSA-N 0.000 description 1
- LTDGPJKGJDIBQD-LAEOZQHASA-N Asn-Val-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O LTDGPJKGJDIBQD-LAEOZQHASA-N 0.000 description 1
- TZBJAXGYGSIUHQ-XUXIUFHCSA-N Asp-Leu-Leu-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O TZBJAXGYGSIUHQ-XUXIUFHCSA-N 0.000 description 1
- 102100037182 Cation-independent mannose-6-phosphate receptor Human genes 0.000 description 1
- 241000252230 Ctenopharyngodon idella Species 0.000 description 1
- 241000723298 Dicentrarchus labrax Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- KJRXLVZYJJLUCV-DCAQKATOSA-N Gln-Arg-Met Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(O)=O KJRXLVZYJJLUCV-DCAQKATOSA-N 0.000 description 1
- LVNILKSSFHCSJZ-IHRRRGAJSA-N Gln-Gln-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N LVNILKSSFHCSJZ-IHRRRGAJSA-N 0.000 description 1
- ZTVGZOIBLRPQNR-KKUMJFAQSA-N Glu-Met-Tyr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ZTVGZOIBLRPQNR-KKUMJFAQSA-N 0.000 description 1
- COVXELOAORHTND-LSJOCFKGSA-N Gly-Ile-Val Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O COVXELOAORHTND-LSJOCFKGSA-N 0.000 description 1
- PDUHNKAFQXQNLH-ZETCQYMHSA-N Gly-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)NCC(O)=O PDUHNKAFQXQNLH-ZETCQYMHSA-N 0.000 description 1
- 101001028831 Homo sapiens Cation-independent mannose-6-phosphate receptor Proteins 0.000 description 1
- 101001034652 Homo sapiens Insulin-like growth factor 1 receptor Proteins 0.000 description 1
- BCISUQVFDGYZBO-QSFUFRPTSA-N Ile-Val-Asp 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)CC(O)=O BCISUQVFDGYZBO-QSFUFRPTSA-N 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 102100039688 Insulin-like growth factor 1 receptor Human genes 0.000 description 1
- 108010011942 LH Receptors Proteins 0.000 description 1
- PPTAQBNUFKTJKA-BJDJZHNGSA-N Leu-Cys-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O PPTAQBNUFKTJKA-BJDJZHNGSA-N 0.000 description 1
- VPKIQULSKFVCSM-SRVKXCTJSA-N Leu-Gln-Arg Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O VPKIQULSKFVCSM-SRVKXCTJSA-N 0.000 description 1
- PRZVBIAOPFGAQF-SRVKXCTJSA-N Leu-Glu-Met Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(O)=O PRZVBIAOPFGAQF-SRVKXCTJSA-N 0.000 description 1
- PTYVBBNIAQWUFV-DCAQKATOSA-N Met-Cys-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCSC)N PTYVBBNIAQWUFV-DCAQKATOSA-N 0.000 description 1
- BKIFWLQFOOKUCA-DCAQKATOSA-N Met-His-Ser Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CO)C(=O)O)N BKIFWLQFOOKUCA-DCAQKATOSA-N 0.000 description 1
- PESQCPHRXOFIPX-UHFFFAOYSA-N N-L-methionyl-L-tyrosine Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 PESQCPHRXOFIPX-UHFFFAOYSA-N 0.000 description 1
- 241001327682 Oncorhynchus mykiss irideus Species 0.000 description 1
- 241000276569 Oryzias latipes Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 241000276618 Perciformes Species 0.000 description 1
- HFZNNDWPHBRNPV-KZVJFYERSA-N Pro-Ala-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O HFZNNDWPHBRNPV-KZVJFYERSA-N 0.000 description 1
- ZUZINZIJHJFJRN-UBHSHLNASA-N Pro-Phe-Ala Chemical compound C([C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H]1NCCC1)C1=CC=CC=C1 ZUZINZIJHJFJRN-UBHSHLNASA-N 0.000 description 1
- SNGZLPOXVRTNMB-LPEHRKFASA-N Pro-Ser-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N2CCC[C@@H]2C(=O)O SNGZLPOXVRTNMB-LPEHRKFASA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 241001138501 Salmonella enterica Species 0.000 description 1
- COLJZWUVZIXSSS-CIUDSAMLSA-N Ser-Cys-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CO)N COLJZWUVZIXSSS-CIUDSAMLSA-N 0.000 description 1
- ADJDNJCSPNFFPI-FXQIFTODSA-N Ser-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO ADJDNJCSPNFFPI-FXQIFTODSA-N 0.000 description 1
- NUEHQDHDLDXCRU-GUBZILKMSA-N Ser-Pro-Arg Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NUEHQDHDLDXCRU-GUBZILKMSA-N 0.000 description 1
- 241001417495 Serranidae Species 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 241000347391 Umbrina cirrosa Species 0.000 description 1
- OUUBKKIJQIAPRI-LAEOZQHASA-N Val-Gln-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O OUUBKKIJQIAPRI-LAEOZQHASA-N 0.000 description 1
- KISFXYYRKKNLOP-IHRRRGAJSA-N Val-Phe-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(=O)O)N KISFXYYRKKNLOP-IHRRRGAJSA-N 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 238000005804 alkylation reaction Methods 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 108700021031 cdc Genes Proteins 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 102000014650 luteinizing hormone receptor activity proteins Human genes 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 108010085203 methionylmethionine Proteins 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 108010012581 phenylalanylglutamate Proteins 0.000 description 1
- 239000003016 pheromone Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 108010004914 prolylarginine Proteins 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 239000012487 rinsing solution Substances 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 238000002864 sequence alignment Methods 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 235000019640 taste Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 108010084932 tryptophyl-proline Proteins 0.000 description 1
Images
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
- C07K14/575—Hormones
- C07K14/65—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/22—Hormones
- A61K38/30—Insulin-like growth factors, i.e. somatomedins, e.g. IGF-1, IGF-2
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Molecular Biology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Endocrinology (AREA)
- Biotechnology (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Diabetes (AREA)
- Epidemiology (AREA)
- Reproductive Health (AREA)
- Wood Science & Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Plant Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pregnancy & Childbirth (AREA)
- Gynecology & Obstetrics (AREA)
- Microbiology (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
本发明公开了一种金钱鱼促生殖细胞成熟基因IGF3，它的核苷酸序列如SEQ IDNO：3所示。还公开了编码所述基因IGF3的蛋白，它的氨基酸序列如SEQ IDNO：4所示。以及所述金钱鱼促生殖细胞成熟基因IGF3的cDNA，它的核苷酸序列如SEQ IDNO：1所示。和编码所述基因IGF3的cDNA的蛋白，它的氨基酸序列如SEQ IDNO：2所示。以及包括所述基因IGF3的表达载体、金钱鱼IGF3重组蛋白及其制备方法，和上述基因IGF3、编码所述基因IGF3的蛋白或所述的重组蛋白在制备对金钱鱼卵母细胞具有促成熟作用的药物方面的应用，开发的IGF3重组蛋白有望应用于研发新型鱼类催熟催产制剂。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种金钱鱼促生殖细胞成熟基因IGF3及其应用。
技术背景
繁殖和生长是生物最基本的特征；它们之间有着密切的联系，但又是相互区别的。胰岛素样生长因子(IGFs)是调节生长和生殖轴的关键因素之一。IGFs由3种配体(IGF1、IGF2和最新发现的IGF3)、2种受体(IGF1R、IGF2R)和6种IGF结合蛋白(IGFBP)组成。在鱼类中，IGF1和IGF2主要是发挥促进生长的生理功能，但自从在鱼类卵巢中发现特异表达的IGF3以来，IGFs在鱼类性腺发育中的作用受到了广泛的关注。
IGF3是鱼类特有的IGF类型，最先是在斑马鱼和罗非鱼中克隆鉴定出来，并发现IGF3在性腺中特异性表达。随后，在欧洲鲈、虹鳟鱼、黄姑鱼、草鱼、青鳉、南方鲶以及石斑鱼等鱼类同样证实了IGF3的存在，并表明IGF3与性腺的发育相关，提示了IGF3在鱼类性腺发育中具有重要的生理功能。进一步研究发现，IGF3重组蛋白能在体外诱导斑马鱼卵母细胞成熟。最终证实了IGF3在诱导卵母细胞成熟过程中的重要生理功能，是一个诱导卵母细胞成熟的新型关键因子，由此开辟了研究鱼类卵子成熟的新通路。
随后，在探讨IGF3诱导卵母细胞成熟的机理相继展开。目前研究发现IGF3和LH具有显著的关联性。在斑马鱼的研究中发现，LH能正调控IGF3的表达，相关的研究在罗非鱼中同样发现LH激活的PKA通路同样能激活IGF3的启动子，并激活IGF3的表达。此外，借助基因敲除技术在斑马鱼中敲除LHβ基因后，IGF3的表达量显著下降，并且IGF3重组蛋白能够挽救LHβ基因敲除后导致的卵子不能成熟。同时，研究表明IGF3在斑马鱼卵巢发育过程中的表达模式和LH受体的表达模式相似，进而提示了IGF3是介导LH信号诱导鱼类卵母细胞成熟的重要衔接因子。
金钱鱼(Scatophagus argus)隶属鲈形目、金钱鱼科、金钱鱼属，俗称金鼓。作为南方名特优名贵海水经济鱼类，金钱鱼是名贵的水族观赏鱼类之一，同时金钱鱼味道鲜美，因此广受消费者青睐。2015年本申请所在团队突破金钱鱼全人工繁殖技术，但实际生产中发现金钱鱼和其他海水鱼类一样，传统的人工催熟效果不明显，极大限制了大规模化苗种的培育。因此，如何突破高效的人工催熟催产是海水鱼类面临的巨大挑战。IGF3作为基因表达的蛋白产物，将具有更便利的应用空间，具有传统的激素诱导卵母细胞成熟无可比拟的优势。
发明内容
本发明的目的在于提供一种金钱鱼促生殖细胞成熟基因IGF3，其编码的蛋白，cDNA、所述cDNA编码的蛋白以及包含所述基因IGF3的表达载体。
本发明的目的还在于提供一种金钱鱼IGF3重组蛋白及其制备方法。
本发明的最后一个目的在于提供上述基因IGF3，其编码的蛋白或所述重组蛋白在制备对金钱鱼卵母细胞具有促成熟作用的药物方面的应用。
本发明的上述第一个目的可以通过以下技术方案来实现：一种金钱鱼促生殖细胞成熟基因IGF3，它的核苷酸序列如SEQ IDNO：3所示。
编码所述基因IGF3的蛋白，它的氨基酸序列如SEQ IDNO：4所示。
所述金钱鱼促生殖细胞成熟基因IGF3的cDNA，它的核苷酸序列如SEQ IDNO：1所示。
本发明利用反转录PCR技术从金钱鱼中克隆得到新型胰岛素类生长因子(Insulin-ike growth factor 3，IGF3)的cDNA序列。
编码所述基因IGF3的cDNA的蛋白，它的氨基酸序列如SEQ IDNO：2所示。
金钱鱼IGF3基因的cDNA序列如SEQ ID NO：1所示，全长594bp，其编码蛋白共198个氨基酸，序列如SEQ ID NO：2所示，获得的金钱鱼促生殖细胞成熟基因IGF3，其不含有cDNA中的信息肽部分，扩增片段大小为507bp，序列如SEQ ID NO：3所示，其编码的蛋白共169个氨基酸，序列如SEQ ID NO：4所示。
本发明还提供了一种表达载体，包括所述基因IGF3。
本发明的上述第二个目的可以通过以下技术方案来实现：一种金钱鱼IGF3重组蛋白的制备方法，包括用所述的表达载体转化寄主细胞，培养转化体，获得重组的金钱鱼IGF3重组蛋白。
本发明还提供了一种金钱鱼IGF3重组蛋白，包括用所述的酯酶表达载体转化寄主细胞，培养转化体，从培养物中获得重组蛋白的步骤的方法制备。
本发明利用反转录PCR技术从金钱鱼中克隆到了促生殖细胞成熟基因IGF3，并构建了IGF3的原核表达载体，可大量表达获得金钱鱼促生殖细胞成熟基因IGF3所编码的重组蛋白。
本发明的上述最后一个目的可以通过以下技术方案来实现：所述基因IGF3、编码所述基因IGF3的蛋白或所述的重组蛋白在制备对金钱鱼卵母细胞具有促成熟作用的药物方面的应用。
本发明具有如下优点：本发明利用反转录PCR技术从金钱鱼中克隆得到新型胰岛素类生长因子(insulin-like growth factor 3，IGF3)的cDNA序列，并根据实验结果说明了IGF3可能参与调控金钱鱼卵母细胞成熟的生理过程，为深入了解鱼类卵母细胞成熟的生殖内分泌调控机制提供一定的理论依据和重要线索。开发的金钱鱼IGF3重组蛋白有望应用于研发新型鱼类催熟催产制剂。
附图说明
图1是实施例1中PCR扩增金钱鱼IGF3基因电泳图；
图2是实施例1中金钱鱼IGF3重组蛋白表达与纯化，M：marker，1：过柱前，2：流穿，3-6：50mM浓度的咪唑洗脱液洗涤，7-10：250mM浓度的咪唑洗脱液洗脱；
图3是实施例1中金钱鱼IGF3重组蛋白SDS-PAGE检测，M：marker，1-2：洗涤液，3-4：洗脱液；
图4是实施例2中金钱鱼IGF3重组蛋白质谱结果，图中显示为下划线部分可信度在95％以上，显示为黑体字部分可认为完全不可信，完全忽略，IGF3重组蛋白质谱结果覆盖率为84.02％，确定重组蛋白为目的蛋白；
图5是实施例3中不同浓度金钱鱼IGF3重组蛋白对细胞周期基因在mRNA转录水平上的表达调节，A表示IGF3重组蛋白对cdc2表达的影响，B表示IGF3重组蛋白对cyclinB表达的影响。
具体实施实例
实施例1 IGF3重组蛋白的制备
1、金钱鱼RNA的提取
取健康金钱鱼全鱼，以冰浴麻醉约2min后，杀鱼取样，分离卵巢组织，采用Trizol试剂法抽提获得金钱鱼卵巢组织总RNA。
2、IGF3基因的克隆
第一链cDNA的合成：
使用TaKaRa公司的PrimeScriptTM RT reagent Kit with gDNA Eraser(PerfectReal Time)试剂盒，先除去金钱鱼卵巢组织总RNA中的基因组DNA，然后将总RNA反转录得到第一链的单链cDNA，于-20℃冰箱保存备用。
以cDNA为模板的IGF3基因克隆：
根据生物信息学中相关的序列比对和结构分析结果，在基因IGF3的cDNA序列(如SEQ ID NO：1所示，其编码的蛋白质序列如SEQ ID NO：2所示)中去除编码29个氨基酸的信号肽的序列，以剩下的序列在两端设计特异性引物，如下：
IGF3-RT-F：5’-tgggatccggtaccaagcttGCCTGGCCGCTCTCCTCTGA-3’(SEQ ID NO：5)
IGF3-RT-R：5’-tggtggtggtggtgctcgagTTATGATTCAAATCCCATCATTC-3’(SEQ IDNO：6)
以第一链cDNA为模板，采用引物IGF3-RT-F和IGF3-RT-R进行PCR扩增，获得不含信息肽的IGF3基因，扩增片段大小为507bp，如图1所示，扩增序列为如SEQ IDNO：3所示，其编码的蛋白质序列如SEQ ID NO：4所示。电泳分离DNA片段，切胶回收目的产物。将纯化后的目的产物连接至pMBP载体，转化大肠杆菌DH5α，挑选阳性克隆测序，测序结果经BLAST比对分析，比对结果表明目的产物确为基因IGF3。
3、基因IGF3原核表达载体的构建
挑选阳性克隆的大肠杆菌DH5α，使用质粒小抽试剂盒抽提质粒，然后转化至原核表达工程菌rosetta2，将获得的阳性单克隆菌落进行菌液PCR验证，PCR扩增的片段大小与目的片段大小相似，约507bp，并将该阳性单菌落进行测序验证，测序结果与目片段的序列一致，说明成功构建了IGF3的原核重组表达载体。
4、基因IGF3的原核表达及纯化
重组蛋白的诱导表达：
将含有重组表达载体(pMBP-IGF3)的工程菌rosetta2先经过LB培养基的扩大培养，当菌液OD600值介于0.6～0.8之间时，加入IPTG(0.5mM，37℃，4h)进行诱导培养，诱导培养后收集菌液，离心获得菌体，加入含0.5M氯化钠的10mM Tris-HCl(pH 8.0)溶液重悬菌体，菌体重悬液超声波破碎，然后入蛋白Loading buffer，混匀，沸水浴中煮5分钟，进行SDS-PAGE电泳检测。
重组蛋白的纯化、脱盐、酶切及冻干：
将诱导表达后的菌体超声破碎，离心，弃沉淀，将上清经孔径0.45微米的滤膜过滤备用。
用去离子水洗镍柱，至pH 7.0，往柱子内加入约50mL含0.5M氯化钠的10mM Tris-HCl(pH 8.0)溶液平衡镍柱，稀释后的样品液过镍柱，收集穿过液，再重复过镍柱两次。重组蛋白挂柱后，先用含0.5M氯化钠的10mM Tris-HCl(pH 8.0)漂洗液漂洗，再分别以50mM、250mM浓度的咪唑洗脱液洗脱，对不同浓度咪唑洗脱液洗脱下来的物质进行SDS-PAGE检测(图2)，获得纯化的重组蛋白。
将纯化后的重组蛋白进行脱盐，先用0.02M的PB缓冲液进行清洗和平衡柱子。加入样品，收集穿过液。待样品完全进入下端柱子后，再加入PB缓冲液洗脱，收集洗脱液，即为脱盐后的重组蛋白。
往脱盐后的重组蛋白加tev蛋白酶酶切，质量比10∶1(MBP-IGF3：MBP-tev)，4℃酶切过夜，然后电泳检测酶切效率。酶切效率超过90％以上后，过amylose resin，收集流穿重组蛋白并进行SDS-PAGE检测，IGF3重组蛋白的分子量约为19kDa(图3)。
将酶切后的重组蛋白进行冻存：将含有目的蛋白的收集液置于合适大小的干净管子中，用封口膜封好，在膜上扎几个针眼，以便冻存时水分的散失。管子竖直放于-20度冰箱约2小时以上，待全部冻成冰状，迅速放于-80度冰箱。第二天，将-80度保存的管子置于调试完好的冻存机挂瓶内，开始冻存。冻存时间视水分散失快慢而定，一般8小时以上或者过夜。将冻存完成的、含有目的蛋白的管子取下迅速放于-80度冰箱保存，以便下一步的实验。
实施例2 IGF3重组蛋白的质谱检验
首先是蛋白质的还原烷基化，加入终浓度10mM二硫苏糖醇(DTT)还原IGF3重组蛋白，接着加入终浓度55mM碘乙酰胺(IAM)，最后加入1μg的Trypsin酶，过夜酶解8h～16h。酶解产生的多肽用C18柱子除盐，已经除盐的多肽抽干后用15μL Loading Buffer(0.1％甲酸，3％乙腈)溶解多肽。多肽上LC-MS/MS(ekspertTMnanoLC；AB Sciex TripleTOF 5600-plus)仪器进行分析，然后对结果进行评估。LC-MS/MS下机后，将原始下机数据直接提交到与AB SCIEX Triple TOFTM 5600plus质谱仪连接的Proteinpilot软件中进行数据库检索。
质谱结果(如图4所示)序列显示为下划线部分可信度在95％以上，显示为黑体字部分可认为完全不可信，完全忽略。但是如果序列处于-COOH端，即使是黑体字部分也不见得是错误的，原因在于-COOH极易被LC-MS/MS过滤掉。
图4显示IGF3重组蛋白的质谱结果可信度在95％以上部分覆盖度为84.02％，确定该蛋白为目的蛋白。
实施例3 IGF3重组蛋白调节生殖细胞成熟调控基因的表达
从湛江市场购买获得金钱鱼为研究对象，以特异表达基因IGF3的卵巢组织为材料，用IGF3重组蛋白离体孵育卵巢碎片，研究IGF3重组蛋白对相关生殖细胞成熟调控基因cdc2和cyclinB在mRNA转录水平表达的调节情况。
分别以浓度0、1、100、10000nM的IGF3重组蛋白(实施例1中制备)孵育卵巢碎片3h，然后使用实时荧光定量技术分别检测生殖细胞成熟调控基因cdc2和cyclinB在mRNA转录水平上的表达情况，检测结果如图5所示。金钱鱼cdc2和cyclinB基因序列由转录组数据库中获得，进而设计引物进行实时荧光定量实验。
从图5的检测结果可获知，不同浓度的IGF3重组蛋白对cdc2和cyclinB均起促进作用，说明IGF3重组蛋白对金钱鱼卵母细胞具有显著的促成熟作用。
以上列举具体实施例对本发明进行补充说明，需要指出的是，以上实施例只用于对本发明作进一步说明，不代表本发明的保护范围，其他人根据本发明的提示做出的非本质的修改和调整，仍属于本发明的保护范围。
序列表
<110> 广东海洋大学
中山大学
<120> 一种金钱鱼促生殖细胞成熟基因IGF3及其应用
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 594
<212> DNA
<213> 新型胰岛素类生长因子 IGF3(insulin-like growth factor 3)
<400> 1
atgcactcct catgccaccc ttttgccaga ccgcaagcac tgaaggtgtt gtgcattcgg 60
gtgtgtgtgt tctccatgtg tctggcagcc tggccgctct cctctgaggc agccagactc 120
cgctgtgggt ctgatctcct aagtgacctc atatttgtgt gtggggatcg tggaatctat 180
ttaggtaaag gtacatggtc tggttatgga gctcggccca gagggaaggg gatagtggac 240
cagtgctgtc ggccagccgg ctgtgaactt caacatttag agatgtactg tgctaaacca 300
aagaaccagc aggacaccac gacttcccca gccacaacca cagcatctca cactacgacg 360
cagctggtca tggcacagca gtttcaagca gtatttcaga aaagactttt agagcaactg 420
ggggctccca acagtctgaa gagggaagct tataggaaga aatcacagcc ttcaccacga 480
cgaaaaagca aaatttcatc atcacgcaag aagattaatg tacagaatac aactaccagg 540
tcaccttcag cctcaaaaag tccccttcaa agaatgatgg gatttgaatc atga 594
<210> 2
<211> 197
<212> PRT
<213> 新型胰岛素类生长因子 IGF3(insulin-like growth factor 3)
<400> 2
Met His Ser Ser Cys His Pro Phe Ala Arg Pro Gln Ala Leu Lys Val
1 5 10 15
Leu Cys Ile Arg Val Cys Val Phe Ser Met Cys Leu Ala Ala Trp Pro
20 25 30
Leu Ser Ser Glu Ala Ala Arg Leu Arg Cys Gly Ser Asp Leu Leu Ser
35 40 45
Asp Leu Ile Phe Val Cys Gly Asp Arg Gly Ile Tyr Leu Gly Lys Gly
50 55 60
Thr Trp Ser Gly Tyr Gly Ala Arg Pro Arg Gly Lys Gly Ile Val Asp
65 70 75 80
Gln Cys Cys Arg Pro Ala Gly Cys Glu Leu Gln His Leu Glu Met Tyr
85 90 95
Cys Ala Lys Pro Lys Asn Gln Gln Asp Thr Thr Thr Ser Pro Ala Thr
100 105 110
Thr Thr Ala Ser His Thr Thr Thr Gln Leu Val Met Ala Gln Gln Phe
115 120 125
Gln Ala Val Phe Gln Lys Arg Leu Leu Glu Gln Leu Gly Ala Pro Asn
130 135 140
Ser Leu Lys Arg Glu Ala Tyr Arg Lys Lys Ser Gln Pro Ser Pro Arg
145 150 155 160
Arg Lys Ser Lys Ile Ser Ser Ser Arg Lys Lys Ile Asn Val Gln Asn
165 170 175
Thr Thr Thr Arg Ser Pro Ser Ala Ser Lys Ser Pro Leu Gln Arg Met
180 185 190
Met Gly Phe Glu Ser
195
<210> 3
<211> 507
<212> DNA
<213> 新型胰岛素类生长因子 IGF3(insulin-like growth factor 3)
<400> 3
gcctggccgc tctcctctga ggcagccaga ctccgctgtg ggtctgatct cctaagtgac 60
ctcatatttg tgtgtgggga tcgtggaatc tatttaggta aaggtacatg gtctggttat 120
ggagctcggc ccagagggaa ggggatagtg gaccagtgct gtcggccagc cggctgtgaa 180
cttcaacatt tagagatgta ctgtgctaaa ccaaagaacc agcaggacac cacgacttcc 240
ccagccacaa ccacagcatc tcacactacg acgcagctgg tcatggcaca gcagtttcaa 300
gcagtatttc agaaaagact tttagagcaa ctgggggctc ccaacagtct gaagagggaa 360
gcttatagga agaaatcaca gccttcacca cgacgaaaaa gcaaaatttc atcatcacgc 420
aagaagatta atgtacagaa tacaactacc aggtcacctt cagcctcaaa aagtcccctt 480
caaagaatga tgggatttga atcatga 507
<210> 4
<211> 168
<212> PRT
<213> 新型胰岛素类生长因子 IGF3(insulin-like growth factor 3)
<400> 4
Ala Trp Pro Leu Ser Ser Glu Ala Ala Arg Leu Arg Cys Gly Ser Asp
1 5 10 15
Leu Leu Ser Asp Leu Ile Phe Val Cys Gly Asp Arg Gly Ile Tyr Leu
20 25 30
Gly Lys Gly Thr Trp Ser Gly Tyr Gly Ala Arg Pro Arg Gly Lys Gly
35 40 45
Ile Val Asp Gln Cys Cys Arg Pro Ala Gly Cys Glu Leu Gln His Leu
50 55 60
Glu Met Tyr Cys Ala Lys Pro Lys Asn Gln Gln Asp Thr Thr Thr Ser
65 70 75 80
Pro Ala Thr Thr Thr Ala Ser His Thr Thr Thr Gln Leu Val Met Ala
85 90 95
Gln Gln Phe Gln Ala Val Phe Gln Lys Arg Leu Leu Glu Gln Leu Gly
100 105 110
Ala Pro Asn Ser Leu Lys Arg Glu Ala Tyr Arg Lys Lys Ser Gln Pro
115 120 125
Ser Pro Arg Arg Lys Ser Lys Ile Ser Ser Ser Arg Lys Lys Ile Asn
130 135 140
Val Gln Asn Thr Thr Thr Arg Ser Pro Ser Ala Ser Lys Ser Pro Leu
145 150 155 160
Gln Arg Met Met Gly Phe Glu Ser
165
<210> 5
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tgggatccgg taccaagctt gcctggccgc tctcctctga 40
<210> 6
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tggtggtggt ggtgctcgag ttatgattca aatcccatca ttc 43
Claims (7)
1.一种金钱鱼促生殖细胞成熟基因IGF3，其特征是：它的核苷酸序列如SEQ ID NO:3所示。
2.权利要求1所述基因IGF3编码的蛋白，其特征是：它的氨基酸序列如SEQ ID NO:4所示。
3.一种金钱鱼促生殖细胞成熟基因IGF3的cDNA，其特征是：它的核苷酸序列如SEQ IDNO:1所示。
4.权利要求3所述cDNA编码的蛋白，其特征是：它的氨基酸序列如SEQ ID NO:2所示。
5.一种表达载体，其特征是包括权利要求1所述基因IGF3。
6.一种金钱鱼IGF3重组蛋白的制备方法，其特征是：包括用权利要求5所述的表达载体转化寄主细胞，培养转化体，获得重组的金钱鱼IGF3重组蛋白。
7.权利要求1所述基因IGF3或权利要求1所述基因IGF3编码的蛋白在制备对金钱鱼卵母细胞具有促成熟作用的药物方面的应用。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011353432.2A CN112625118B (zh) | 2020-11-26 | 2020-11-26 | 一种金钱鱼促生殖细胞成熟基因igf3及其应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011353432.2A CN112625118B (zh) | 2020-11-26 | 2020-11-26 | 一种金钱鱼促生殖细胞成熟基因igf3及其应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112625118A CN112625118A (zh) | 2021-04-09 |
| CN112625118B true CN112625118B (zh) | 2022-05-24 |
Family
ID=75306414
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011353432.2A Active CN112625118B (zh) | 2020-11-26 | 2020-11-26 | 一种金钱鱼促生殖细胞成熟基因igf3及其应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112625118B (zh) |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB201204280D0 (en) * | 2012-03-09 | 2012-04-25 | Univ Nordland | Methods |
| CN105296499A (zh) * | 2015-11-03 | 2016-02-03 | 中国水产科学研究院淡水渔业研究中心 | 一种福瑞鲤igf3基因及其扩增引物组和扩增方法 |
| CN109182252B (zh) * | 2018-07-31 | 2023-02-10 | 西北师范大学 | 一种斑马鱼卵母细胞催熟剂及其应用 |
| CN111018966B (zh) * | 2019-12-27 | 2022-07-29 | 河南师范大学 | Hemibarbus maculatus胰岛素样生长因子3、其蛋白、其抗体及应用 |
| CN111217902A (zh) * | 2020-01-20 | 2020-06-02 | 中国水产科学研究院淡水渔业研究中心 | 一种鲤鱼胰岛素样生长因子igf3重组蛋白的制备与应用 |
-
2020
- 2020-11-26 CN CN202011353432.2A patent/CN112625118B/zh active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112625118A (zh) | 2021-04-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2015143930A1 (zh) | 一种有活性的金鱼Tgf2转座子重组转座酶蛋白的表达方法 | |
| NO175870B (zh) | ||
| CN108048468B (zh) | 青虾视蛋白基因及其编码的蛋白和应用 | |
| CN112625118B (zh) | 一种金钱鱼促生殖细胞成熟基因igf3及其应用 | |
| CN103724415B (zh) | 斜带石斑鱼性别调控基因Rspo1及其制备方法和应用 | |
| CN101525617A (zh) | 中华绒螯蟹Crustin-1基因及体外重组表达 | |
| CN108715851B (zh) | 一种斜带石斑鱼摄食调控相关基因AgRP1及其应用 | |
| JP5531290B2 (ja) | 遺伝子 | |
| CN114736281A (zh) | 海马催产素avt和it及其在制备催产药剂中的应用 | |
| CN114380903A (zh) | 一种胰岛素或其类似物前体 | |
| CN110257409B (zh) | 一种红螯螯虾孵化酶基因及其应用 | |
| CN102277327B (zh) | 过表达RimL的大肠杆菌及其在制备N-乙酰化胸腺素α中的应用 | |
| CN102051370A (zh) | 文蛤组织蛋白酶b基因及编码蛋白和应用 | |
| CN114057897B (zh) | 一种许氏平鲉il-6重组蛋白、可溶表达方法及应用 | |
| CN111217902A (zh) | 一种鲤鱼胰岛素样生长因子igf3重组蛋白的制备与应用 | |
| CN101565703A (zh) | 中华绒螯蟹Crustin-2基因及其重组蛋白的应用 | |
| CN110592054B (zh) | 海马孵化酶基因及其编码蛋白和应用 | |
| CN114106142B (zh) | 一种黄鳝生长催乳素抗血清及其制备方法与应用 | |
| CN104073501A (zh) | 日本沼虾血红蛋白基因及其克隆方法与重组蛋白制备方法 | |
| CN111041012B (zh) | 凡纳滨对虾肌内质网Ca-ATP酶LvSERCA基因及其编码蛋白与应用 | |
| CN101481689A (zh) | 新序列人胰岛素样生长因子-ⅰ基因的克隆及其高效表达 | |
| CN111088257B (zh) | 一种脊尾白虾卵黄蛋白结合蛋白基因及其应用 | |
| CN114621959B (zh) | 一种编码牙鲆igf2可溶性蛋白的基因及蛋白重组表达方法与应用 | |
| Wang et al. | High‐level expression of cecropin CMIV in E. coli from a fusion construct containing the human tumor necrosis factor | |
| TWI476205B (zh) | 一種分離之地毯海葵色澤蛋白質 |
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 |