CN107827987B - 一种促黄体激素类似物及其制备方法 - Google Patents
一种促黄体激素类似物及其制备方法 Download PDFInfo
- Publication number
- CN107827987B CN107827987B CN201710494736.2A CN201710494736A CN107827987B CN 107827987 B CN107827987 B CN 107827987B CN 201710494736 A CN201710494736 A CN 201710494736A CN 107827987 B CN107827987 B CN 107827987B
- Authority
- CN
- China
- Prior art keywords
- pro
- cys
- leu
- thr
- gly
- 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
- 108010073521 Luteinizing Hormone Proteins 0.000 title claims abstract description 93
- 102000009151 Luteinizing Hormone Human genes 0.000 title claims abstract description 93
- 229940040129 luteinizing hormone Drugs 0.000 title claims abstract description 93
- 238000002360 preparation method Methods 0.000 title claims abstract description 17
- 238000006206 glycosylation reaction Methods 0.000 claims abstract description 24
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000013604 expression vector Substances 0.000 claims description 41
- 238000001976 enzyme digestion Methods 0.000 claims description 32
- 230000014509 gene expression Effects 0.000 claims description 28
- 108091026890 Coding region Proteins 0.000 claims description 24
- 108020004705 Codon Proteins 0.000 claims description 23
- 230000015572 biosynthetic process Effects 0.000 claims description 23
- 238000003786 synthesis reaction Methods 0.000 claims description 23
- 239000013598 vector Substances 0.000 claims description 18
- 238000001262 western blot Methods 0.000 claims description 18
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 claims description 15
- 230000000694 effects Effects 0.000 claims description 15
- 230000004071 biological effect Effects 0.000 claims description 14
- 238000001514 detection method Methods 0.000 claims description 14
- 238000000034 method Methods 0.000 claims description 14
- 230000004988 N-glycosylation Effects 0.000 claims description 13
- 150000001413 amino acids Chemical group 0.000 claims description 12
- 108010066058 beta Subunit Luteinizing Hormone Proteins 0.000 claims description 12
- 238000003259 recombinant expression Methods 0.000 claims description 12
- 239000006228 supernatant Substances 0.000 claims description 12
- 108091008146 restriction endonucleases Proteins 0.000 claims description 11
- 108020004414 DNA Proteins 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 9
- 108090000790 Enzymes Proteins 0.000 claims description 9
- 108091081024 Start codon Proteins 0.000 claims description 8
- 238000005520 cutting process Methods 0.000 claims description 8
- 241000283690 Bos taurus Species 0.000 claims description 7
- 108700010070 Codon Usage Proteins 0.000 claims description 7
- 239000012634 fragment Substances 0.000 claims description 7
- 102000012410 DNA Ligases Human genes 0.000 claims description 6
- 108010061982 DNA Ligases Proteins 0.000 claims description 6
- 108010076504 Protein Sorting Signals Proteins 0.000 claims description 6
- 102000053602 DNA Human genes 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 108020004682 Single-Stranded DNA Proteins 0.000 claims description 4
- 230000003321 amplification Effects 0.000 claims description 4
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 4
- LSQZJLSUYDQPKJ-NJBDSQKTSA-N amoxicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=C(O)C=C1 LSQZJLSUYDQPKJ-NJBDSQKTSA-N 0.000 claims description 3
- 229960003022 amoxicillin Drugs 0.000 claims description 3
- 239000003668 hormone analog Substances 0.000 claims description 3
- 238000001638 lipofection Methods 0.000 claims description 3
- LSQZJLSUYDQPKJ-UHFFFAOYSA-N p-Hydroxyampicillin Natural products O=C1N2C(C(O)=O)C(C)(C)SC2C1NC(=O)C(N)C1=CC=C(O)C=C1 LSQZJLSUYDQPKJ-UHFFFAOYSA-N 0.000 claims description 3
- 238000000746 purification Methods 0.000 claims description 3
- 238000012217 deletion Methods 0.000 claims description 2
- 230000037430 deletion Effects 0.000 claims description 2
- 239000013600 plasmid vector Substances 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 230000013595 glycosylation Effects 0.000 abstract description 17
- 230000032683 aging Effects 0.000 abstract description 3
- 229920001184 polypeptide Polymers 0.000 abstract description 3
- 108090000765 processed proteins & peptides Proteins 0.000 abstract description 3
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 3
- 210000004899 c-terminal region Anatomy 0.000 abstract description 2
- 210000004027 cell Anatomy 0.000 description 62
- 239000000523 sample Substances 0.000 description 43
- 210000001625 seminal vesicle Anatomy 0.000 description 29
- 201000010653 vesiculitis Diseases 0.000 description 29
- 238000012360 testing method Methods 0.000 description 22
- 238000004113 cell culture Methods 0.000 description 20
- 239000002609 medium Substances 0.000 description 18
- 241001465754 Metazoa Species 0.000 description 16
- UEGIPZAXNBYCCP-NKWVEPMBSA-N Gly-Cys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CS)NC(=O)CN)C(=O)O UEGIPZAXNBYCCP-NKWVEPMBSA-N 0.000 description 14
- 241000700159 Rattus Species 0.000 description 14
- 239000012228 culture supernatant Substances 0.000 description 14
- 239000012528 membrane Substances 0.000 description 14
- 108090000623 proteins and genes Proteins 0.000 description 14
- 108010014614 prolyl-glycyl-proline Proteins 0.000 description 13
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 12
- 230000037396 body weight Effects 0.000 description 12
- 239000000047 product Substances 0.000 description 12
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 description 11
- 102000012673 Follicle Stimulating Hormone Human genes 0.000 description 11
- 229940088598 enzyme Drugs 0.000 description 11
- 238000002347 injection Methods 0.000 description 11
- 239000007924 injection Substances 0.000 description 11
- YLTKNGYYPIWKHZ-ACZMJKKPSA-N Ala-Ala-Glu Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O YLTKNGYYPIWKHZ-ACZMJKKPSA-N 0.000 description 10
- NIZKGBJVCMRDKO-KWQFWETISA-N Ala-Gly-Tyr Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NIZKGBJVCMRDKO-KWQFWETISA-N 0.000 description 10
- SMCGQGDVTPFXKB-XPUUQOCRSA-N Ala-Gly-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N SMCGQGDVTPFXKB-XPUUQOCRSA-N 0.000 description 10
- IOFVWPYSRSCWHI-JXUBOQSCSA-N Ala-Thr-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](C)N IOFVWPYSRSCWHI-JXUBOQSCSA-N 0.000 description 10
- ZATRYQNPUHGXCU-DTWKUNHWSA-N Arg-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ZATRYQNPUHGXCU-DTWKUNHWSA-N 0.000 description 10
- LYJXHXGPWDTLKW-HJGDQZAQSA-N Arg-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N)O LYJXHXGPWDTLKW-HJGDQZAQSA-N 0.000 description 10
- LGGHQRZIJSYRHA-GUBZILKMSA-N Asp-Pro-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC(=O)O)N LGGHQRZIJSYRHA-GUBZILKMSA-N 0.000 description 10
- NIPJKKSXHSBEMX-CIUDSAMLSA-N Cys-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](CS)N NIPJKKSXHSBEMX-CIUDSAMLSA-N 0.000 description 10
- MRVSLWQRNWEROS-SVSWQMSJSA-N Cys-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CS)N MRVSLWQRNWEROS-SVSWQMSJSA-N 0.000 description 10
- SWJYSDXMTPMBHO-FXQIFTODSA-N Cys-Pro-Ser Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O SWJYSDXMTPMBHO-FXQIFTODSA-N 0.000 description 10
- XBELMDARIGXDKY-GUBZILKMSA-N Cys-Pro-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CS)N XBELMDARIGXDKY-GUBZILKMSA-N 0.000 description 10
- BUEFQXUHTUZXHR-LURJTMIESA-N Gly-Gly-Pro zwitterion Chemical compound NCC(=O)NCC(=O)N1CCC[C@H]1C(O)=O BUEFQXUHTUZXHR-LURJTMIESA-N 0.000 description 10
- OHOXVDFVRDGFND-YUMQZZPRSA-N His-Cys-Gly Chemical compound N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CS)C(=O)NCC(O)=O OHOXVDFVRDGFND-YUMQZZPRSA-N 0.000 description 10
- IBMVEYRWAWIOTN-UHFFFAOYSA-N L-Leucyl-L-Arginyl-L-Proline Natural products CC(C)CC(N)C(=O)NC(CCCN=C(N)N)C(=O)N1CCCC1C(O)=O IBMVEYRWAWIOTN-UHFFFAOYSA-N 0.000 description 10
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 10
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 10
- 241000880493 Leptailurus serval Species 0.000 description 10
- IBMVEYRWAWIOTN-RWMBFGLXSA-N Leu-Arg-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1CCC[C@@H]1C(O)=O IBMVEYRWAWIOTN-RWMBFGLXSA-N 0.000 description 10
- QKIBIXAQKAFZGL-GUBZILKMSA-N Leu-Cys-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(O)=O QKIBIXAQKAFZGL-GUBZILKMSA-N 0.000 description 10
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 10
- FOBUGKUBUJOWAD-IHPCNDPISA-N Leu-Leu-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 FOBUGKUBUJOWAD-IHPCNDPISA-N 0.000 description 10
- 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 10
- VTJUNIYRYIAIHF-IUCAKERBSA-N Leu-Pro Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(O)=O VTJUNIYRYIAIHF-IUCAKERBSA-N 0.000 description 10
- KIZIOFNVSOSKJI-CIUDSAMLSA-N Leu-Ser-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)O)N KIZIOFNVSOSKJI-CIUDSAMLSA-N 0.000 description 10
- 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 10
- GJJQCBVRWDGLMQ-GUBZILKMSA-N Lys-Glu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O GJJQCBVRWDGLMQ-GUBZILKMSA-N 0.000 description 10
- RAAVFTFEAUAVIY-DCAQKATOSA-N Met-Glu-Met Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCSC)C(=O)O)N RAAVFTFEAUAVIY-DCAQKATOSA-N 0.000 description 10
- BEZJTLKUMFMITF-AVGNSLFASA-N Met-Lys-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CCCNC(N)=N BEZJTLKUMFMITF-AVGNSLFASA-N 0.000 description 10
- 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 10
- 108010066427 N-valyltryptophan Proteins 0.000 description 10
- UHRNIXJAGGLKHP-DLOVCJGASA-N Phe-Ala-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O UHRNIXJAGGLKHP-DLOVCJGASA-N 0.000 description 10
- LLGTYVHITPVGKR-RYUDHWBXSA-N Phe-Gln-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O LLGTYVHITPVGKR-RYUDHWBXSA-N 0.000 description 10
- YFXXRYFWJFQAFW-JHYOHUSXSA-N Phe-Thr-Thr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O YFXXRYFWJFQAFW-JHYOHUSXSA-N 0.000 description 10
- GDXZRWYXJSGWIV-GMOBBJLQSA-N Pro-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 GDXZRWYXJSGWIV-GMOBBJLQSA-N 0.000 description 10
- AIZVVCMAFRREQS-GUBZILKMSA-N Pro-Cys-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AIZVVCMAFRREQS-GUBZILKMSA-N 0.000 description 10
- LSIWVWRUTKPXDS-DCAQKATOSA-N Pro-Gln-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O LSIWVWRUTKPXDS-DCAQKATOSA-N 0.000 description 10
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 description 10
- BBFRBZYKHIKFBX-GMOBBJLQSA-N Pro-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@@H]1CCCN1 BBFRBZYKHIKFBX-GMOBBJLQSA-N 0.000 description 10
- FMLRRBDLBJLJIK-DCAQKATOSA-N Pro-Leu-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FMLRRBDLBJLJIK-DCAQKATOSA-N 0.000 description 10
- CGSOWZUPLOKYOR-AVGNSLFASA-N Pro-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 CGSOWZUPLOKYOR-AVGNSLFASA-N 0.000 description 10
- QAAYIXYLEMRULP-SRVKXCTJSA-N Pro-Pro-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 QAAYIXYLEMRULP-SRVKXCTJSA-N 0.000 description 10
- XDKKMRPRRCOELJ-GUBZILKMSA-N Pro-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 XDKKMRPRRCOELJ-GUBZILKMSA-N 0.000 description 10
- DLPXTCTVNDTYGJ-JBDRJPRFSA-N Ser-Ile-Cys Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CS)C(O)=O DLPXTCTVNDTYGJ-JBDRJPRFSA-N 0.000 description 10
- NLJKZUGAIIRWJN-LKXGYXEUSA-N Thr-Asp-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N)O NLJKZUGAIIRWJN-LKXGYXEUSA-N 0.000 description 10
- AVYVKJMBNLPWRX-WFBYXXMGSA-N Trp-Ala-Ser Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O)=CNC2=C1 AVYVKJMBNLPWRX-WFBYXXMGSA-N 0.000 description 10
- GFJXBLSZOFWHAW-JYJNAYRXSA-N Tyr-His-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O GFJXBLSZOFWHAW-JYJNAYRXSA-N 0.000 description 10
- IRLYZKKNBFPQBW-XGEHTFHBSA-N Val-Cys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](C(C)C)N)O IRLYZKKNBFPQBW-XGEHTFHBSA-N 0.000 description 10
- FTKXYXACXYOHND-XUXIUFHCSA-N Val-Ile-Leu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O FTKXYXACXYOHND-XUXIUFHCSA-N 0.000 description 10
- ZHQWPWQNVRCXAX-XQQFMLRXSA-N Val-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N ZHQWPWQNVRCXAX-XQQFMLRXSA-N 0.000 description 10
- QZKVWWIUSQGWMY-IHRRRGAJSA-N Val-Ser-Phe Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 QZKVWWIUSQGWMY-IHRRRGAJSA-N 0.000 description 10
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 10
- 229940028334 follicle stimulating hormone Drugs 0.000 description 10
- JYPCXBJRLBHWME-UHFFFAOYSA-N glycyl-L-prolyl-L-arginine Natural products NCC(=O)N1CCCC1C(=O)NC(CCCN=C(N)N)C(O)=O JYPCXBJRLBHWME-UHFFFAOYSA-N 0.000 description 10
- 108010051307 glycyl-glycyl-proline Proteins 0.000 description 10
- 108010050848 glycylleucine Proteins 0.000 description 10
- 108010085325 histidylproline Proteins 0.000 description 10
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 10
- 108010057821 leucylproline Proteins 0.000 description 10
- 108010068488 methionylphenylalanine Proteins 0.000 description 10
- 108010080629 tryptophan-leucine Proteins 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- SUEIIIFUBHDCCS-PBCZWWQYSA-N Asn-His-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SUEIIIFUBHDCCS-PBCZWWQYSA-N 0.000 description 8
- 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 8
- ABPRMMYHROQBLY-NKWVEPMBSA-N Gly-Ser-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CO)NC(=O)CN)C(=O)O ABPRMMYHROQBLY-NKWVEPMBSA-N 0.000 description 8
- 239000002033 PVDF binder Substances 0.000 description 8
- 230000029087 digestion Effects 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 108010037389 glutamyl-cysteinyl-lysine Proteins 0.000 description 8
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 8
- 102000004169 proteins and genes Human genes 0.000 description 8
- 238000001890 transfection Methods 0.000 description 8
- 238000000246 agarose gel electrophoresis Methods 0.000 description 7
- 238000013461 design Methods 0.000 description 7
- 230000004927 fusion Effects 0.000 description 7
- 239000001963 growth medium Substances 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- 108010005942 methionylglycine Proteins 0.000 description 7
- BRPMXFSTKXXNHF-IUCAKERBSA-N (2s)-1-[2-[[(2s)-pyrrolidine-2-carbonyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)CNC(=O)[C@H]1NCCC1 BRPMXFSTKXXNHF-IUCAKERBSA-N 0.000 description 6
- ZXKNLCPUNZPFGY-LEWSCRJBSA-N Ala-Tyr-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N ZXKNLCPUNZPFGY-LEWSCRJBSA-N 0.000 description 6
- JOTRDIXZHNQYGP-DCAQKATOSA-N Arg-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N JOTRDIXZHNQYGP-DCAQKATOSA-N 0.000 description 6
- ULBHWNVWSCJLCO-NHCYSSNCSA-N Arg-Val-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCCN=C(N)N ULBHWNVWSCJLCO-NHCYSSNCSA-N 0.000 description 6
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 6
- HNNGTYHNYDOSKV-FXQIFTODSA-N Cys-Cys-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)N HNNGTYHNYDOSKV-FXQIFTODSA-N 0.000 description 6
- WTMPKZWHRCMMMT-KZVJFYERSA-N Thr-Pro-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WTMPKZWHRCMMMT-KZVJFYERSA-N 0.000 description 6
- MXDOAJQRJBMGMO-FJXKBIBVSA-N Thr-Pro-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O MXDOAJQRJBMGMO-FJXKBIBVSA-N 0.000 description 6
- 238000003556 assay Methods 0.000 description 6
- 229940088597 hormone Drugs 0.000 description 6
- 239000005556 hormone Substances 0.000 description 6
- 108010054155 lysyllysine Proteins 0.000 description 6
- 239000003550 marker Substances 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- XMBSYZWANAQXEV-UHFFFAOYSA-N N-alpha-L-glutamyl-L-phenylalanine Natural products OC(=O)CCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XMBSYZWANAQXEV-UHFFFAOYSA-N 0.000 description 5
- 230000004989 O-glycosylation Effects 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 206010042573 Superovulation Diseases 0.000 description 5
- 238000006243 chemical reaction Methods 0.000 description 5
- 238000001962 electrophoresis Methods 0.000 description 5
- 108010028295 histidylhistidine Proteins 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 239000012160 loading buffer Substances 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 238000005406 washing Methods 0.000 description 5
- 230000004584 weight gain Effects 0.000 description 5
- 235000019786 weight gain Nutrition 0.000 description 5
- WQLDNOCHHRISMS-NAKRPEOUSA-N Ala-Pro-Ile Chemical compound [H]N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WQLDNOCHHRISMS-NAKRPEOUSA-N 0.000 description 4
- YMIYZAOBQDRCPP-UHFFFAOYSA-N Ala-Thr-Cys-Cys Chemical compound CC(N)C(=O)NC(C(O)C)C(=O)NC(CS)C(=O)NC(CS)C(O)=O YMIYZAOBQDRCPP-UHFFFAOYSA-N 0.000 description 4
- NTWOPSIUJBMNRI-KKUMJFAQSA-N Asn-Lys-Tyr Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NTWOPSIUJBMNRI-KKUMJFAQSA-N 0.000 description 4
- VIRHEUMYXXLCBF-WDSKDSINSA-N Asp-Gly-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O VIRHEUMYXXLCBF-WDSKDSINSA-N 0.000 description 4
- CHRCKSPMGYDLIA-SRVKXCTJSA-N Cys-Phe-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O CHRCKSPMGYDLIA-SRVKXCTJSA-N 0.000 description 4
- DQGIAOGALAQBGK-BWBBJGPYSA-N Cys-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CS)N)O DQGIAOGALAQBGK-BWBBJGPYSA-N 0.000 description 4
- ZKAUCGZIIXXWJQ-BZSNNMDCSA-N Cys-Tyr-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)NC(=O)[C@H](CS)N)O ZKAUCGZIIXXWJQ-BZSNNMDCSA-N 0.000 description 4
- APWLZZSLCXLDCF-CIUDSAMLSA-N Gln-Cys-Met Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(O)=O APWLZZSLCXLDCF-CIUDSAMLSA-N 0.000 description 4
- HVQCEQTUSWWFOS-WDSKDSINSA-N Gln-Gly-Cys Chemical compound C(CC(=O)N)[C@@H](C(=O)NCC(=O)N[C@@H](CS)C(=O)O)N HVQCEQTUSWWFOS-WDSKDSINSA-N 0.000 description 4
- FYBSCGZLICNOBA-XQXXSGGOSA-N Glu-Ala-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O FYBSCGZLICNOBA-XQXXSGGOSA-N 0.000 description 4
- RJONUNZIMUXUOI-GUBZILKMSA-N Glu-Asn-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N RJONUNZIMUXUOI-GUBZILKMSA-N 0.000 description 4
- PNAOVYHADQRJQU-GUBZILKMSA-N Glu-Cys-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CCC(=O)O)N PNAOVYHADQRJQU-GUBZILKMSA-N 0.000 description 4
- ISXJHXGYMJKXOI-GUBZILKMSA-N Glu-Cys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCC(O)=O ISXJHXGYMJKXOI-GUBZILKMSA-N 0.000 description 4
- 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 4
- GRIRDMVMJJDZKV-RCOVLWMOSA-N Gly-Asn-Val Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O GRIRDMVMJJDZKV-RCOVLWMOSA-N 0.000 description 4
- NMROINAYXCACKF-WHFBIAKZSA-N Gly-Cys-Cys Chemical compound NCC(=O)N[C@@H](CS)C(=O)N[C@@H](CS)C(O)=O NMROINAYXCACKF-WHFBIAKZSA-N 0.000 description 4
- LHRXAHLCRMQBGJ-RYUDHWBXSA-N Gly-Glu-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)CN LHRXAHLCRMQBGJ-RYUDHWBXSA-N 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 239000000579 Gonadotropin-Releasing Hormone Substances 0.000 description 4
- LBCAQRFTWMMWRR-CIUDSAMLSA-N His-Cys-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O LBCAQRFTWMMWRR-CIUDSAMLSA-N 0.000 description 4
- STOOMQFEJUVAKR-KKUMJFAQSA-N His-His-His Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC=1N=CNC=1)C(O)=O)C1=CNC=N1 STOOMQFEJUVAKR-KKUMJFAQSA-N 0.000 description 4
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 4
- CKRJBQJIGOEKMC-SRVKXCTJSA-N His-Lys-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O CKRJBQJIGOEKMC-SRVKXCTJSA-N 0.000 description 4
- WCNWGAUZWWSYDG-SVSWQMSJSA-N Ile-Thr-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)O)N WCNWGAUZWWSYDG-SVSWQMSJSA-N 0.000 description 4
- HVHRPWQEQHIQJF-AVGNSLFASA-N Leu-Lys-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O HVHRPWQEQHIQJF-AVGNSLFASA-N 0.000 description 4
- FDBTVENULFNTAL-XQQFMLRXSA-N Leu-Val-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N FDBTVENULFNTAL-XQQFMLRXSA-N 0.000 description 4
- 108091036060 Linker DNA Proteins 0.000 description 4
- DEFGUIIUYAUEDU-ZPFDUUQYSA-N Lys-Asn-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DEFGUIIUYAUEDU-ZPFDUUQYSA-N 0.000 description 4
- RBEATVHTWHTHTJ-KKUMJFAQSA-N Lys-Leu-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O RBEATVHTWHTHTJ-KKUMJFAQSA-N 0.000 description 4
- WGILOYIKJVQUPT-DCAQKATOSA-N Lys-Pro-Asp Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O WGILOYIKJVQUPT-DCAQKATOSA-N 0.000 description 4
- WAAZECNCPVGPIV-RHYQMDGZSA-N Lys-Thr-Met Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(O)=O WAAZECNCPVGPIV-RHYQMDGZSA-N 0.000 description 4
- STLBOMUOQNIALW-BQBZGAKWSA-N Met-Gly-Cys Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H](CS)C(O)=O STLBOMUOQNIALW-BQBZGAKWSA-N 0.000 description 4
- USBFEVBHEQBWDD-AVGNSLFASA-N Met-Leu-Val Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O USBFEVBHEQBWDD-AVGNSLFASA-N 0.000 description 4
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 4
- AAERWTUHZKLDLC-IHRRRGAJSA-N Phe-Pro-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O AAERWTUHZKLDLC-IHRRRGAJSA-N 0.000 description 4
- WEDZFLRYSIDIRX-IHRRRGAJSA-N Phe-Ser-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CC=CC=C1 WEDZFLRYSIDIRX-IHRRRGAJSA-N 0.000 description 4
- IPFXYNKCXYGSSV-KKUMJFAQSA-N Phe-Ser-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N IPFXYNKCXYGSSV-KKUMJFAQSA-N 0.000 description 4
- OLZVAVSJEUAOHI-UNQGMJICSA-N Phe-Thr-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O OLZVAVSJEUAOHI-UNQGMJICSA-N 0.000 description 4
- UTAUEDINXUMHLG-FXQIFTODSA-N Pro-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@@H]1CCCN1 UTAUEDINXUMHLG-FXQIFTODSA-N 0.000 description 4
- QCARZLHECSFOGG-CIUDSAMLSA-N Pro-Glu-Cys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O QCARZLHECSFOGG-CIUDSAMLSA-N 0.000 description 4
- JUJGNDZIKKQMDJ-IHRRRGAJSA-N Pro-His-His Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(O)=O JUJGNDZIKKQMDJ-IHRRRGAJSA-N 0.000 description 4
- RYJRPPUATSKNAY-STECZYCISA-N Pro-Ile-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@@H]2CCCN2 RYJRPPUATSKNAY-STECZYCISA-N 0.000 description 4
- 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 4
- AIOWVDNPESPXRB-YTWAJWBKSA-N Pro-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2)O AIOWVDNPESPXRB-YTWAJWBKSA-N 0.000 description 4
- 101000857870 Squalus acanthias Gonadoliberin Proteins 0.000 description 4
- 239000006180 TBST buffer Substances 0.000 description 4
- VEWZSFGRQDUAJM-YJRXYDGGSA-N Thr-Cys-Tyr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N)O VEWZSFGRQDUAJM-YJRXYDGGSA-N 0.000 description 4
- PUEWAXRPXOEQOW-HJGDQZAQSA-N Thr-Met-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(N)=O)C(O)=O PUEWAXRPXOEQOW-HJGDQZAQSA-N 0.000 description 4
- XHWCDRUPDNSDAZ-XKBZYTNZSA-N Thr-Ser-Glu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N)O XHWCDRUPDNSDAZ-XKBZYTNZSA-N 0.000 description 4
- CWQZAUYFWRLITN-AVGNSLFASA-N Tyr-Gln-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CS)C(=O)O)N)O CWQZAUYFWRLITN-AVGNSLFASA-N 0.000 description 4
- WVGKPKDWYQXWLU-BZSNNMDCSA-N Tyr-His-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)N[C@@H](CCCCN)C(=O)O)N)O WVGKPKDWYQXWLU-BZSNNMDCSA-N 0.000 description 4
- NVZVJIUDICCMHZ-BZSNNMDCSA-N Tyr-Phe-Ser Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O NVZVJIUDICCMHZ-BZSNNMDCSA-N 0.000 description 4
- RUCNAYOMFXRIKJ-DCAQKATOSA-N Val-Ala-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN RUCNAYOMFXRIKJ-DCAQKATOSA-N 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 108010047857 aspartylglycine Proteins 0.000 description 4
- 229910002092 carbon dioxide Inorganic materials 0.000 description 4
- 230000001079 digestive effect Effects 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- XLXSAKCOAKORKW-AQJXLSMYSA-N gonadorelin Chemical compound C([C@@H](C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)NCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)C1=CC=C(O)C=C1 XLXSAKCOAKORKW-AQJXLSMYSA-N 0.000 description 4
- 229940035638 gonadotropin-releasing hormone Drugs 0.000 description 4
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 4
- 238000005457 optimization Methods 0.000 description 4
- 230000016087 ovulation Effects 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- 238000007789 sealing Methods 0.000 description 4
- 210000002966 serum Anatomy 0.000 description 4
- 239000012679 serum free medium Substances 0.000 description 4
- 239000012086 standard solution Substances 0.000 description 4
- 230000010474 transient expression Effects 0.000 description 4
- QUIGLPSHIFPEOV-CIUDSAMLSA-N Ala-Lys-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O QUIGLPSHIFPEOV-CIUDSAMLSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- SBYQHZCMVSPQCS-RCWTZXSCSA-N Thr-Val-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCSC)C(O)=O SBYQHZCMVSPQCS-RCWTZXSCSA-N 0.000 description 3
- 108010087924 alanylproline Proteins 0.000 description 3
- 238000000540 analysis of variance Methods 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 239000001569 carbon dioxide Substances 0.000 description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 3
- 239000006143 cell culture medium Substances 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000008217 follicular development Effects 0.000 description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 108010049041 glutamylalanine Proteins 0.000 description 3
- 238000000691 measurement method Methods 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 210000002307 prostate Anatomy 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 230000001850 reproductive effect Effects 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- IGXNPQWXIRIGBF-KEOOTSPTSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoyl]amino]-3-(1h-imidazol-5-yl)propanoic acid Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 IGXNPQWXIRIGBF-KEOOTSPTSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- TTXMOJWKNRJWQJ-FXQIFTODSA-N Ala-Arg-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N TTXMOJWKNRJWQJ-FXQIFTODSA-N 0.000 description 2
- CYBJZLQSUJEMAS-LFSVMHDDSA-N Ala-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](C)N)O CYBJZLQSUJEMAS-LFSVMHDDSA-N 0.000 description 2
- AAWLEICNDUHIJM-MBLNEYKQSA-N Ala-Thr-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C)N)O AAWLEICNDUHIJM-MBLNEYKQSA-N 0.000 description 2
- IETUUAHKCHOQHP-KZVJFYERSA-N Ala-Thr-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@H](C)N)[C@@H](C)O)C(O)=O IETUUAHKCHOQHP-KZVJFYERSA-N 0.000 description 2
- VKCOHFFSTKCXEQ-OLHMAJIHSA-N Asn-Asn-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O VKCOHFFSTKCXEQ-OLHMAJIHSA-N 0.000 description 2
- IBLAOXSULLECQZ-IUKAMOBKSA-N Asn-Ile-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CC(N)=O IBLAOXSULLECQZ-IUKAMOBKSA-N 0.000 description 2
- XPGVTUBABLRGHY-BIIVOSGPSA-N Asp-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N XPGVTUBABLRGHY-BIIVOSGPSA-N 0.000 description 2
- 241000283707 Capra Species 0.000 description 2
- QJUDRFBUWAGUSG-SRVKXCTJSA-N Cys-Cys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)N QJUDRFBUWAGUSG-SRVKXCTJSA-N 0.000 description 2
- XELISBQUZZAPQK-CIUDSAMLSA-N Cys-His-Cys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CS)N XELISBQUZZAPQK-CIUDSAMLSA-N 0.000 description 2
- IDFVDSBJNMPBSX-SRVKXCTJSA-N Cys-Lys-Leu Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O IDFVDSBJNMPBSX-SRVKXCTJSA-N 0.000 description 2
- KVGPYKUIHZJWGA-BQBZGAKWSA-N Cys-Met-Gly Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)NCC(O)=O KVGPYKUIHZJWGA-BQBZGAKWSA-N 0.000 description 2
- MBRWOKXNHTUJMB-CIUDSAMLSA-N Cys-Pro-Glu Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O MBRWOKXNHTUJMB-CIUDSAMLSA-N 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- SVZIKUHLRKVZIF-GUBZILKMSA-N Glu-Asn-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N SVZIKUHLRKVZIF-GUBZILKMSA-N 0.000 description 2
- ITVBKCZZLJUUHI-HTUGSXCWSA-N Glu-Phe-Thr 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)O)C(O)=O ITVBKCZZLJUUHI-HTUGSXCWSA-N 0.000 description 2
- HAOUOFNNJJLVNS-BQBZGAKWSA-N Gly-Pro-Ser Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O HAOUOFNNJJLVNS-BQBZGAKWSA-N 0.000 description 2
- SOEGEPHNZOISMT-BYPYZUCNSA-N Gly-Ser-Gly Chemical compound NCC(=O)N[C@@H](CO)C(=O)NCC(O)=O SOEGEPHNZOISMT-BYPYZUCNSA-N 0.000 description 2
- MKIAPEZXQDILRR-YUMQZZPRSA-N Gly-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)CN MKIAPEZXQDILRR-YUMQZZPRSA-N 0.000 description 2
- LCRDMSSAKLTKBU-ZDLURKLDSA-N Gly-Ser-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN LCRDMSSAKLTKBU-ZDLURKLDSA-N 0.000 description 2
- CTGZVVQVIBSOBB-AVGNSLFASA-N His-His-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O CTGZVVQVIBSOBB-AVGNSLFASA-N 0.000 description 2
- DQZCEKQPSOBNMJ-NKIYYHGXSA-N His-Thr-Glu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O DQZCEKQPSOBNMJ-NKIYYHGXSA-N 0.000 description 2
- MDOBWSFNSNPENN-PMVVWTBXSA-N His-Thr-Gly Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O MDOBWSFNSNPENN-PMVVWTBXSA-N 0.000 description 2
- GNXGAVNTVNOCLL-SIUGBPQLSA-N Ile-Tyr-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N GNXGAVNTVNOCLL-SIUGBPQLSA-N 0.000 description 2
- 108010065920 Insulin Lispro Proteins 0.000 description 2
- 239000012097 Lipofectamine 2000 Substances 0.000 description 2
- KNKHAVVBVXKOGX-JXUBOQSCSA-N Lys-Ala-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KNKHAVVBVXKOGX-JXUBOQSCSA-N 0.000 description 2
- ZXEUFAVXODIPHC-GUBZILKMSA-N Lys-Glu-Asn Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O ZXEUFAVXODIPHC-GUBZILKMSA-N 0.000 description 2
- PLDJDCJLRCYPJB-VOAKCMCISA-N Lys-Lys-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PLDJDCJLRCYPJB-VOAKCMCISA-N 0.000 description 2
- LMMBAXJRYSXCOQ-ACRUOGEOSA-N Lys-Tyr-Phe Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O LMMBAXJRYSXCOQ-ACRUOGEOSA-N 0.000 description 2
- 238000000585 Mann–Whitney U test Methods 0.000 description 2
- UOENBSHXYCHSAU-YUMQZZPRSA-N Met-Gln-Gly Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O UOENBSHXYCHSAU-YUMQZZPRSA-N 0.000 description 2
- WWWGMQHQSAUXBU-BQBZGAKWSA-N Met-Gly-Asn Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(N)=O WWWGMQHQSAUXBU-BQBZGAKWSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 241000283973 Oryctolagus cuniculus Species 0.000 description 2
- PTDAGKJHZBGDKD-OEAJRASXSA-N Phe-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N)O PTDAGKJHZBGDKD-OEAJRASXSA-N 0.000 description 2
- SGCZFWSQERRKBD-BQBZGAKWSA-N Pro-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]1CCCN1 SGCZFWSQERRKBD-BQBZGAKWSA-N 0.000 description 2
- RCYUBVHMVUHEBM-RCWTZXSCSA-N Pro-Pro-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O RCYUBVHMVUHEBM-RCWTZXSCSA-N 0.000 description 2
- 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 2
- ZMLRZBWCXPQADC-TUAOUCFPSA-N Pro-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 ZMLRZBWCXPQADC-TUAOUCFPSA-N 0.000 description 2
- ZXLUWXWISXIFIX-ACZMJKKPSA-N Ser-Asn-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O ZXLUWXWISXIFIX-ACZMJKKPSA-N 0.000 description 2
- SMIDBHKWSYUBRZ-ACZMJKKPSA-N Ser-Glu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O SMIDBHKWSYUBRZ-ACZMJKKPSA-N 0.000 description 2
- IOVBCLGAJJXOHK-SRVKXCTJSA-N Ser-His-His Chemical compound C([C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CN=CN1 IOVBCLGAJJXOHK-SRVKXCTJSA-N 0.000 description 2
- PTWIYDNFWPXQSD-GARJFASQSA-N Ser-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CO)N)C(=O)O PTWIYDNFWPXQSD-GARJFASQSA-N 0.000 description 2
- FLONGDPORFIVQW-XGEHTFHBSA-N Ser-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO FLONGDPORFIVQW-XGEHTFHBSA-N 0.000 description 2
- SOACHCFYJMCMHC-BWBBJGPYSA-N Ser-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CO)N)O SOACHCFYJMCMHC-BWBBJGPYSA-N 0.000 description 2
- VOHWDZNIESHTFW-XKBZYTNZSA-N Thr-Glu-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N)O VOHWDZNIESHTFW-XKBZYTNZSA-N 0.000 description 2
- KRDSCBLRHORMRK-JXUBOQSCSA-N Thr-Lys-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O KRDSCBLRHORMRK-JXUBOQSCSA-N 0.000 description 2
- KERCOYANYUPLHJ-XGEHTFHBSA-N Thr-Pro-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O KERCOYANYUPLHJ-XGEHTFHBSA-N 0.000 description 2
- STUAPCLEDMKXKL-LKXGYXEUSA-N Thr-Ser-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O STUAPCLEDMKXKL-LKXGYXEUSA-N 0.000 description 2
- AFWXOGHZEKARFH-ACRUOGEOSA-N Tyr-Tyr-His Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CC=1NC=NC=1)C(O)=O)C1=CC=C(O)C=C1 AFWXOGHZEKARFH-ACRUOGEOSA-N 0.000 description 2
- RYQUMYBMOJYYDK-NHCYSSNCSA-N Val-Pro-Glu Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)O)N RYQUMYBMOJYYDK-NHCYSSNCSA-N 0.000 description 2
- BGXVHVMJZCSOCA-AVGNSLFASA-N Val-Pro-Lys Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)O)N BGXVHVMJZCSOCA-AVGNSLFASA-N 0.000 description 2
- 230000001464 adherent effect Effects 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 238000004166 bioassay Methods 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000013599 cloning vector Substances 0.000 description 2
- 108010004073 cysteinylcysteine Proteins 0.000 description 2
- 108010060199 cysteinylproline Proteins 0.000 description 2
- 108010069495 cysteinyltyrosine Proteins 0.000 description 2
- 230000001419 dependent effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000003203 everyday effect Effects 0.000 description 2
- 230000004720 fertilization Effects 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 238000001502 gel electrophoresis Methods 0.000 description 2
- 108010078144 glutaminyl-glycine Proteins 0.000 description 2
- 108010077515 glycylproline Proteins 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 108010034529 leucyl-lysine Proteins 0.000 description 2
- 244000144972 livestock Species 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 210000001672 ovary Anatomy 0.000 description 2
- 108010051242 phenylalanylserine Proteins 0.000 description 2
- 238000002264 polyacrylamide gel electrophoresis Methods 0.000 description 2
- 239000011148 porous material Substances 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- CBUWTGCATVNMJE-UHFFFAOYSA-N 6,6-dimethylheptanal Chemical compound CC(C)(C)CCCCC=O CBUWTGCATVNMJE-UHFFFAOYSA-N 0.000 description 1
- LSMDIAAALJJLRO-XQXXSGGOSA-N Ala-Thr-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O LSMDIAAALJJLRO-XQXXSGGOSA-N 0.000 description 1
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 1
- IARGXWMWRFOQPG-GCJQMDKQSA-N Asn-Ala-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IARGXWMWRFOQPG-GCJQMDKQSA-N 0.000 description 1
- XLDMSQYOYXINSZ-QXEWZRGKSA-N Asn-Val-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N XLDMSQYOYXINSZ-QXEWZRGKSA-N 0.000 description 1
- 108091062157 Cis-regulatory element Proteins 0.000 description 1
- 241000207892 Convolvulus Species 0.000 description 1
- 230000007067 DNA methylation Effects 0.000 description 1
- 238000012270 DNA recombination Methods 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- IRJWAYCXIYUHQE-WHFBIAKZSA-N Gly-Ser-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)CN IRJWAYCXIYUHQE-WHFBIAKZSA-N 0.000 description 1
- 102000006771 Gonadotropins Human genes 0.000 description 1
- 108010086677 Gonadotropins Proteins 0.000 description 1
- WEIYKCOEVBUJQC-JYJNAYRXSA-N His-Glu-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC2=CN=CN2)N WEIYKCOEVBUJQC-JYJNAYRXSA-N 0.000 description 1
- FYTCLUIYTYFGPT-YUMQZZPRSA-N His-Gly-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FYTCLUIYTYFGPT-YUMQZZPRSA-N 0.000 description 1
- JIUYRPFQJJRSJB-QWRGUYRKSA-N His-His-Gly Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)NCC(O)=O)C1=CN=CN1 JIUYRPFQJJRSJB-QWRGUYRKSA-N 0.000 description 1
- JBSLJUPMTYLLFH-MELADBBJSA-N His-His-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CN=CN2)NC(=O)[C@H](CC3=CN=CN3)N)C(=O)O JBSLJUPMTYLLFH-MELADBBJSA-N 0.000 description 1
- OWYIDJCNRWRSJY-QTKMDUPCSA-N His-Pro-Thr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O OWYIDJCNRWRSJY-QTKMDUPCSA-N 0.000 description 1
- 108010057021 Menotropins Proteins 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- VXCHGLYSIOOZIS-GUBZILKMSA-N Pro-Ala-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 VXCHGLYSIOOZIS-GUBZILKMSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 241000282849 Ruminantia Species 0.000 description 1
- CGNLCCVKSWNSDG-UHFFFAOYSA-N SYBR Green I Chemical compound CN(C)CCCN(CCC)C1=CC(C=C2N(C3=CC=CC=C3S2)C)=C2C=CC=CC2=[N+]1C1=CC=CC=C1 CGNLCCVKSWNSDG-UHFFFAOYSA-N 0.000 description 1
- GXXTUIUYTWGPMV-FXQIFTODSA-N Ser-Arg-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O GXXTUIUYTWGPMV-FXQIFTODSA-N 0.000 description 1
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 1
- CRJZZXMAADSBBQ-SRVKXCTJSA-N Ser-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO CRJZZXMAADSBBQ-SRVKXCTJSA-N 0.000 description 1
- BECPPKYKPSRKCP-ZDLURKLDSA-N Thr-Glu Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O BECPPKYKPSRKCP-ZDLURKLDSA-N 0.000 description 1
- OQCXTUQTKQFDCX-HTUGSXCWSA-N Thr-Glu-Phe Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N)O OQCXTUQTKQFDCX-HTUGSXCWSA-N 0.000 description 1
- DJDSEDOKJTZBAR-ZDLURKLDSA-N Thr-Gly-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O DJDSEDOKJTZBAR-ZDLURKLDSA-N 0.000 description 1
- XNTVWRJTUIOGQO-RHYQMDGZSA-N Thr-Met-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O XNTVWRJTUIOGQO-RHYQMDGZSA-N 0.000 description 1
- XGZBEGGGAUQBMB-KJEVXHAQSA-N Tyr-Pro-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC2=CC=C(C=C2)O)N)O XGZBEGGGAUQBMB-KJEVXHAQSA-N 0.000 description 1
- CVIXTAITYJQMPE-LAEOZQHASA-N Val-Glu-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CVIXTAITYJQMPE-LAEOZQHASA-N 0.000 description 1
- JVGHIFMSFBZDHH-WPRPVWTQSA-N Val-Met-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)O)N JVGHIFMSFBZDHH-WPRPVWTQSA-N 0.000 description 1
- 108091060592 XDNA Proteins 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 238000004026 adhesive bonding Methods 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 210000003651 basophil Anatomy 0.000 description 1
- 238000010009 beating Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000035 biogenic effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 239000012888 bovine serum Substances 0.000 description 1
- 244000309466 calf Species 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 210000004246 corpus luteum Anatomy 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 238000003113 dilution method Methods 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 210000002257 embryonic structure Anatomy 0.000 description 1
- 230000012173 estrus Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000012737 fresh medium Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 108010082286 glycyl-seryl-alanine Proteins 0.000 description 1
- 239000002622 gonadotropin Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000009027 insemination Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000002394 ovarian follicle Anatomy 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 230000001817 pituitary effect Effects 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 239000012146 running buffer Substances 0.000 description 1
- 239000012898 sample dilution Substances 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000004065 semiconductor Substances 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 230000036299 sexual function Effects 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 238000003153 stable transfection Methods 0.000 description 1
- 239000012899 standard injection Substances 0.000 description 1
- 230000035882 stress Effects 0.000 description 1
- 210000001550 testis Anatomy 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000003146 transient transfection Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 239000002699 waste material Substances 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
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
-
- 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
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Endocrinology (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Physics & Mathematics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Plant Pathology (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- Reproductive Health (AREA)
- Peptides Or Proteins (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了一种促黄体激素(luteinizing hormone,LH)类似物及其制备方法，所述促黄体激素类似物以LHβ‑接头序列‑LHα的顺序从N末端到C‑末端连接起来，设计的接头序列带有不同的长度、可折叠性、疏水性、不同数目和类型的糖基化位点和组氨酸标签，分别与天然促黄体激素的α和β亚基融合而成具有不同时效的功能性单链多肽，具有比天然促黄体激素更优越的实用性。
Description
【技术领域】
本发明属于生物技术领域，尤其涉及一种兽用促黄体激素类似物 (luteinizinghormone,LH)及其制备方法。
【背景技术】
黄体激素(luteinizing hormone,LH)是由垂体前叶嗜碱性粒细胞 分泌的一种异二聚体糖蛋白，由LHα和LHβ两个亚基以非共价键的 方式结合形成。LH和促卵泡激素(follicle-stimulating hormone,FSH) 在雌性生物的卵巢和雄性生物的睾丸的功能调控过程中协同发挥作 用。FSH主要促使卵泡发育。LH主要促进卵泡成熟排卵，同时可 促进黄体生成。在雌性动物生殖周期中，血液中LH和FSH的浓度 周期变化曲线不同。在排卵时需要一个短暂的LH高峰，而在整个生 殖周期需要持续保持较低浓度的LH以维持动物的性功能。
优质家畜品种的扩繁通常是通过超数排卵(superovulation)和胚 胎移植来实现。超数排卵是应用外源激素诱导卵巢多个卵泡同时发 育，并排出具有受精能力的卵子的方法。孕马血清促性腺激素 (Pregnant Mare Serum Gonadotropin，PMSG)和促性腺激素释放激素 (Gonadotropin-releasing hormone，GnRH)是目前超数排卵中应用广 泛的激素。但是PMSG同一分子同时具有LH和FSH两种促性腺激 素的活性。LH和FSH又都是GnRH的下游激素，GnRH能够促使内 源性LH和FSH两种激素的合成和释放。卵巢存在着大量处于不同 阶段的卵泡。卵泡发育的不同阶段对于激素的需求是不尽相同的，发 育后期的卵泡从FSH依赖转变为LH依赖。单用PMSG或GnRH 促进超数排卵，由于LH和FSH两种激素的作用同时施加在对处于 不同阶段的卵泡，导致卵泡成熟不同步，排卵时间不一致。卵子先后 从卵泡排出之后继续发育，而处在不同发育阶段的卵子在同一时间人 工授精的条件下受精能力不一样，影响了可移植胚胎数。所以在实践 中，先采用FSH促使卵泡发育，再配合短效LH促进成熟卵子同时 排出，可使反刍动物的超排率，尤其是可移植胚胎产量明显提高。
除了通过短效LH在超排方面的应用，如果将长效LH与长效FSH 两者配合，在减少注射次数、降低应激的前提下，可促使母畜早期发 情，缩短空怀时间，提高繁殖率。
因此，短效和长效的LH在辅助生殖技术中均具实际应用的需求。
天然提取的LH具有纯度低、产量低和成本高等缺点，更重要的 是携带生物源性传染物的潜在危险，不利于在畜牧业上的推广应用。
目前专业技术人员利用DNA重组技术制备LH类似物可以避免 以上缺点。已知的重组技术包括：设计合理的目的蛋白编码序列、设 计合理的连接序列、将LHα和LHβ两个亚基基因通过连接序列融合 成为单链序列、选择合适的表达载体和表达系统。但是，目前对于“合 理的蛋白编码序列”和“合理的连接序列”仅限于在理论上的认识， 并无现成的普适序列。本发明利用宿主细胞内密码子偏爱性的知识对 LH的密码子进行重组、对含不同氨基酸的连接序列(蕴含不同的长 度、可折叠性、疏水性、电荷效应以及糖基化位点)进行重组、并对 相应的各种候选序列的表达物进行功能性比较、优化而发明了现予以 公开的序列。
本发明在现有技术的基础上，通过实验探索、试错验证，总结出 一套制备功能性LH类似物的方法，所述的功能性LH类似物是一种 新的功能性重组蛋白(recombinantprotein)。本发明公开了功能性重组 蛋白中连接序列的设计和验证方法，选择合适的连接序列、使得连接 的长度、可折叠性、疏水性、电荷效应以及糖基化位点等结构特点最 终能够体现重组蛋白所需的生物功能，符合药典对LH生物活性的要 求，所公开的制备方法具有可重复性。
【发明内容】
本发明提供一种促黄体激素类似物及其制备方法，针对现有的技 术问题，本发明根据连接序列的长度、可折叠性、疏水性、电荷效应 来设计合适的连接序列从而得到不同时效的一系列兽用促黄体激类 似物。所述的促黄体激素类似物是一种新的重组蛋白。
为达到上述目的，本发明的技术方案为：
一种促黄体激素类似物为携带有起始密码子和信号肽编码序列 但删除终止密码子的LHβ亚基编码序列与缺少起始密码子和信号肽 编码序列但携带终止密码子的LHα亚基编码序列与接头序列按照 LHβ-接头序列-LHα的顺序从N末端到C-末端连接起来的单链多肽；
所述接头序列的氨基酸序列为Seq ID 1至Seq ID 10中的任一种；
对应所述接头序列的促黄体激素类似物的氨基酸序列为Seq ID 11至Seq ID 20。
进一步地，所述促黄体激素类似物的制备方法，包括以下步骤：
步骤1、从Genebank获得牛(bovine)LHα亚基编码序列和LHβ 亚基编码序列，删除LHβ的终止密码子和LHα的起始密码子；根据 CHO-K1表达系统偏爱的密码子丰度，将野生型LH的编码序列密码 子进行重组；设计接头序列，根据接头序列的长度、可折叠性、疏水 性、电荷效应设计不同长度的带有0个、1个、2个和4个O-糖基化 和N-糖基化位点的接头序列10个，在接头序列上添加组氨酸标签(HHHHHH)，接头序列两端分别添加酶切位点BamH I和EcoR I， 以引物的形式全序列合成，纯化方式PAGE，合成量1OD，合成的接 头序列单链DNA-20℃保存备用；
步骤2、将含有全序列合成的野生型LH的重组载体pMD19-T Simple-LH9、含有全序列合成的密码子偏爱型LH的重组载体 pMD19-T Simple-LH10和空表达载体pcDNA3.1(+)分别经限制性内切 酶双酶切，回收双酶切线性化的pcDNA3.1(+)和LH(含接头序列) 片段，经DNA连接酶连接，分别形成重组表达载体pcDNA3.1(+)-LH9 和pcDNA3.1(+)-LH10，构建的重组表达载体转染感受细胞E.coli DH5α，选取抗阿莫西林阳性克隆菌落扩大培养，提取重组载体并保 存菌种，提取的质粒载体限制性内切酶双酶切鉴定；
步骤3、分别置换连接序列，共构建20个含10中不同连接序列 的野生型和密码子偏爱型的bLH表达载体；
步骤4、采用脂质体转染法，将上述构建的含有不同接头序列的 bLH表达载体转入CHO-K1细胞中，取上清液做Western Blotting检 测，选取表达量高且携带组氨酸的重组bLH表达载体，线性化后转 染CHO-K1细胞，通过G418筛选，上清液通过Western Blotting检测；
步骤5、重组bLH表达株的单克隆化，取bLH表达株细胞放大 培养作为种子库；
步骤6、通过对相应的各种候选序列的表达物的生物活性进行比 较而确定长短效序列。
进一步地，所述促黄体激素类似物的制备方法步骤2中，双酶切 的限制性内切酶为Hind III和Xba I。
进一步地，所述促黄体激素类似物的制备方法步骤2中，DNA 连接酶为T4 DNALigase。
进一步地，所述促黄体激素类似物的制备方法步骤4中G418筛 选浓度为150-350μg/mL，最优浓度为250μg/mL。
通过上述技术方案，本发明所制得的促黄体激素类似物为具有不 同时效的功能性单链多肽，比天然促黄体激素具有列优越的实用性和 商业价值。
【附图说明】
图1.1为单链融合LH表达基因的设计图；
图1.2为重组载体pMD19-T Simple-LH9和pMD19-T Simple-LH10以及空表达载体pcDNA3.1(+)分别经限制性内切酶Hind III和Xba I双酶切产物1.5％琼脂糖凝胶电泳图；
图1.3为重组表达载体的酶切鉴定
图1.4为重组载体pcDNA3.1(+)-LH10和pcDNA3.1(+)-LH9分别 经限制性内切酶双酶切产物1％琼脂糖凝胶电泳图；
图1.5为接头序列双酶切20％丙烯酰胺凝胶电泳图；
图1.6为含有不同接头序列的bLH瞬时表达结果；
图1.7为含有不同接头序列的bLH经限制性内切酶Acl I酶切的 1.5％琼脂糖凝胶电泳图；
图1.8为经G418筛选bLH表达细胞系；
图1.9为转染细胞株上清液Western Blotting检测结果图，显示糖 基化类型及数目对糖蛋白分子量的影响；
图1.10为接头序列中无糖基化bLH表达株Western Blotting检测 结果图；
图1.11为接头序列中两个N-糖基化bLH表达株Western Blotting 检测结果图；
图2为携带不同接头序列的bLH通过Western Blotting检测蛋白 相对含量的结果图。
【具体实施方式】
下面结合具体实施例与附图对本发明方案做进一步详细说明：
实施例1、不同糖基化类型的促黄体激素类似物的制备
以牛(bovine，b)的LHα和LHβ基因序列为模板，根据促黄体激 素结构与功能的关系，将LH的两个亚基按照NH2-β-linker–α-COOH 的顺序连接形成单链融合LH。根据结构特点，诸如氨基酸序列的长 度、可折叠性、疏水性、电荷效应和糖基化程度设计了10条不同接头(linker)序列，并分别与野生型和密码子偏爱型bLHα和bLHβ连 接形成20条不同序列的融合蛋白。
1.1重组bLH单链融合蛋白的设计
从NCBI的基因库Genebank获得牛(bovine)LHα亚基编码序列 (NM_173901.3)和LHβ亚基编码序列(M11506.1)，将携带有起始 密码子和信号肽编码序列但删除终止密码子的LHβ亚基编码序列与 缺少起始密码子和信号肽编码序列但携带终止密码子的LHα亚基编码序列与接头序列(1)按照LHβ-接头序列-LHα的顺序从N末端到 C-末端连接起来，如图1.1为单链融合LH表达基因的设计图。
1.1.1 接头序列的设计
综合考虑接头序列(linker)的结构特点，包括氨基酸序列的长度、 可折叠性、疏水性、电荷效应以及糖基化程度，设计了10条接头序 列。在结构允许的情况下含有组氨酸标签(HHHHHH)以利下游的 纯化工作。O-糖基化位点序列氨基酸为PTPGP，N-糖基化位点氨基 酸序列为NXT/S(X为除脯氨酸外的任一氨基酸)。接头序列两端分 别添加酶切位点BamHI和EcoR I。Linker以引物的形式合成，纯化 方式PAG，合成量1OD。Linker的编号、其氨基酸序列及单链DNA 合成序列详细信息见表1.1。
表1.1 Linker的氨基酸序列及与其对应的所合成的DNA序列
将合成的接头序列单链DNA-20℃保存备用。
1.1.2 单链融合bLH编码序列
1.1.2.1 定制全序列合成的pMd-19Tsimpe-LH9，即在克隆载体中 含野生型bLH，其中单链融合bLH是按照NH2-β-linker9–α-COOH的 顺序连接形成，序列如下：
ATGGAGATGTTCCAGGGACTGCTGCTGTGGCTGCTGCTGGGCG TGGCCGGGGTGTGGGCTTCCAGGGGGCCACTGCGGCCGCTGTG CCAGCCCATCAACGCCACCCTGGCGGCTGAGAAGGAGGCCTGC CCTGTCTGTATCACTTTCACCACCAGCATCTGCGCCGGCTACTG CCCCAGCATGAAGCGGGTGCTGCCTGTCATCCTGCCGCCCATGCCCCAGCGGGTGTGCACCTACCATGAGCTGCGCTTCGCCTCCGTT CGGCTCCCCGGCTGCCCACCTGGAGTGGACCCAATGGTCTCCT TCCCCGTGGCCCTCAGCTGTCACTGTGGACCCTGCCGCCTCAG CAGCACTGACTGCGGGGGTCCCAGAACCCAACCCTTGGCCTGT GACCACCCCCCGCTCCCAGACATCCTCTTCCTCGGATCCCCTAC CCCTGGCCCCCACCACCATCACCATCATCCTACCCCCGGACCTG AATTCCCTGATGGAGAGTTTACAATGCAGGGCTGTCCTGAATGC AAGCTAAAAGAAAACAAATACTTCTCCAAGCCAGATGCTCCAA TCTATCAGTGCATGGGGTGCTGCTTCTCCAGGGCATACCCCACT CCAGCGAGGTCTAAGAAGACAATGTTGGTCCCCAAGAACATCA CCTCGGAAGCTACATGCTGTGTGGCCAAAGCATTTACCAAGGC CACAGTGATGGGAAATGTCAGAGTGGAGAACCACACCGAGTGCCACTGCAGCACTTGTTATTATCACAAATCCTAA
起始密码子：ATG；终止密码子：TGA
1.1.2.2 定制全序列合成的pMd-19Tsimpe-LH10，即在克隆载体中 含密码子偏爱型bLH，其中单链融合bLH是按照 NH2-β-linker10–α-COOH的顺序连接形成，序列如下：
ATGGAaATGTTCCAGGGCCTGCTGCTGTGGCTGCTGCTGGGAGT GGCTGGCGTGTGGGCTAGTAGAGGACCTCTGAGGCCTCTGTGC CAGCCCATCAATGCCACCCTGGCCGCCGAGAAAGAGGCCTGCC CTGTGTGCATCACCTTCACCACCTCTATCTGCGCCGGCTACTGC CCCTCCATGAAGAGGGTGCTGCCCGTGATCCTGCCCCCCATGCC TCAGAGAGTGTGCACCTACCACGAGCTGAGATTCGCCTCCGTG CGGCTGCCTGGATGTCCTCCTGGCGTGGACCCTATGGTGTCCTT CCCTGTGGCCCTGTCTTGCCACTGCGGCCCTTGCAGACTGTCCT CTACCGATTGTGGCGGCCCTCGGACACAGCCTCTGGCCTGTGAT CATCCCCCCCTGCCCGATATCCTGTTCCTGGGATCCAACCACAC CGGCTCCCACCATCACCATCACCATGGCTCCAACCACACAGAAT TCCCCGACGGCGAGTTTACAATGCAGGGCTGCCCCGAGTGCAA GCTGAAAGAGAACAAGTATTTCTCCAAGCCCGACGCCCCCATC TACCAGTGCATGGGCTGCTGTTTCTCCCGGGCCTACCCTACCCC AGCCCGGTCCAAGAAAACCATGCTGGTGCCCAAGAACATCACC TCCGAGGCCACCTGTTGCGTGGCCAAGGCCTTTACCAAGGCCA CCGTGATGGGCAACGTGCGGGTGGAAAACCACACCGAGTGCC ACTGCTCTACCTGCTACTACCACAAGTCCTGA
起始密码子：ATG；终止密码子：TAA
1.2 野生型和密码子偏爱型bLH表达载体的构建
1.2.1 表达载体的重组
将全序列合成的重组载体pMD19-T Simple-LH9、pMD19-T Simple-LH10和空表达载体pcDNA3.1(+)分别经限制性内切酶Hind III和Xba I双酶切，酶切体系见表1.2。
表1.2 克隆载体及表达载体的酶切
备注：质粒即载体
将上述酶切体系混匀后，37℃水浴4h。酶切产物进行1.5％琼脂 糖凝胶电泳，实验结果见图1.2，图中M：marker；1：未经酶切的 pcDNA3.1(+)；2、3和4：均为pcDNA3.1(+)经Hind III和Xba I 双酶切的产物；5：未经酶切的pMd-19Tsimpe-LH9；6、7和8：均为pMd-19Tsimpe-LH9经Hind III和Xba I双酶切的产物；9：未经酶切 的pMd-19Tsimpe-LH10；10、11和12：均为pMd-19Tsimpe-LH10经 Hind III和Xba I双酶切的产物。
采用行业内技术人员已知的方法，按照TAKARA琼脂糖凝胶回 收试剂盒说明书，分别回收双酶切线性化的pcDNA3.1(+)、双酶切 pMd-19Tsimpe-LH9释放出来的野生型bLH(含Linker 9)片段和双 酶切pMd-19Tsimpe-LH10释放出来的密码子偏爱型bLH(含Linker 10)片段，分别经DNA连接酶T4 DNA Ligase连接，形成重组表达 载体pcDNA3.1(+)-LH9和pcDNA3.1(+)-LH10。再采用行业内技术人 员已知的方法将构建的重组表达载体分别转染感受态E.coli DH5α， 选取阿莫西林抗性阳性的克隆菌落扩大培养，提取重组表达载体并保 存菌种，将提取的重组表达载体分别进行限制性内切酶Hind III和 Xba I双酶切和DNA测序鉴定。
1.2.2 重组表达载体的鉴定
所构建的重组表达载体pcDNA3.1(+)-LH9和pcDNA3.1(+)-LH10 分别经Hind III和Xba I、Hind III和BamH I和EcoR I和Xba I双酶 切结果如图1.3(1.5％琼脂糖凝胶电泳图)所示，图中：M:marker；1： 未经酶切的pcDNA3.1(+)空质粒；2、3和4依次为pcDNA3.1(+)经过Hind III和Xba I、Hind III和BamH I和EcoR I和Xba I双酶切 的产物；5：未经酶切的pcDNA3.1(+)-LH9表达质粒；6、7和8： 依次为Hind III和Xba I、Hind III和BamH I和EcoRI和Xba I双酶 切pcDNA3.1(+)-LH9表达质粒；9：未经酶切的pcDNA3.1(+)-LH10 表达质粒；10、11和12：依次为Hind III和Xba I、Hind III和BamH I和EcoR I和Xba I双酶切的pcDNA3.1(+)-LH10表达质粒。
由图可知，pcDNA3.1(+)-LH9和pcDNA3.1(+)-LH10经过双酶切 得到的产物分别与预期的DNA长度相符(由Hind III和Xba I双酶 切释放790bp的LHβ-linker-LHα、由HindIII和BamH I双酶切释放 437bp的LHβ和由EcoR I和Xba I双酶切释放297bp的LHα)，结果表明构建的表达载体正确。再经DNA测序证实，5’端和3’端序列表 明与1.1.2所设计的单链融合bLH编码序列一致。
1.2.3 重组LH所含linker的置换
1.2.3.1 带有粘性末端不含linker的线性重组载体序列的制备
重组载体pcDNA3.1(+)-LH10和pcDNA3.1(+)-LH9分别经限制性 内切酶BamH I和EcoR I双酶切，获得缺失linker的重组载体序列， 酶切产物加入loading buffer，1％琼脂糖凝胶电泳见图1.4，其中：M： marker；1：未经酶切的环状pcDNA3.1(+)-LH10；2：经BamH I和 EcoR I双酶切的线性pcDNA3.1(+)-LH10，酶切下来的Linker10因分 子量过小未在电泳图中体现；3：未经酶切的环状pcDNA3.1(+)-LH9； 4：经BamH I和EcoR I双酶切的线性pcDNA3.1(+)-LH9，酶切下来 的Linker 9因分子量过小未在电泳图中体现。按照TAKARA琼脂糖 凝胶回收试剂盒说明书，分别回收获得不含linker的线性重组载体序 列pcDNA3.1(+)-LH10和pcDNA3.1(+)-LH9。
1.2.3.2 候选Linker的双酶切
将合成的linker DNA正负链各5μL,100nmol/mL混匀，95℃变 性5分钟后缓慢退火至室温。退火后形成双链的linker DNA序列由 BamH I和EcoR I双酶切，5’端和3’端均形成粘性末端。20％丙烯酰 胺凝胶电泳，90V，180分钟，实验结果如图1.5所示，M：marker； 1：未经酶切的linker1；2：经BamH I和EcoR I双酶切的linker1；3： 未经酶切的linker2；4：经BamH I和EcoR I双酶切的linker2；5：未 经酶切的linker3；6：经BamH I和EcoR I双酶切的linker3。
行业内技术人员已知双链DNA经双酶切后会提高其电泳迁移 率。由图1.5可知，linker DNA片段成功进行了双酶切。凝胶回收试 剂盒直接回收酶切后的产物，获得带有粘性末端的linker DNA片段。 针对于linkers 4-8的操作如同以上所述linkers 1-3的方法。
1.2.3.3 候选Linker分别与载体pcDNA3.1(+)-LH连接
将上述回收的不含linker的pcDNA3.1(+)-LH10和 pcDNA3.1(+)-LH9依次分别与不同的linker经DNA连接酶T4 DNA Ligase连接形成携带不同linker的野生型和密码子偏爱型的bLH(连 接体系见表1.3)。
表1.3 Linker与表达载体连接体系
连接产物成功转染大肠杆菌感受态，挑取单克隆，扩大培养，保 存菌种、提取质粒后送测序。
1.3 重组LH在CHO-K1细胞中的表达
1.3.1 CHO-K1细胞的培养
1.3.1.1 CHO-K1细胞的复苏
取一株冻存的CHO-K1细胞，37℃水浴锅中迅速融化，接种在 含10mL 10％血清Ham’s F12细胞培养基的10cm细胞培养板中，待 细胞贴壁后换取新鲜的培养基，继续培养至细胞汇合度90％。
1.3.1.2 CHO-K1贴壁细胞的传代
按业内技术人员已知的技术，在超净工作台内，将复苏的细胞旧 培养基按规定丢弃，5mL PBS洗三遍，加入1mL细胞消化液，左右 倾斜至消化液铺满底面，按规定丢弃消化液，放入5％二氧化碳培养 箱2min至细胞全部脱落，加入培养基吹打均匀，分装在细胞培养瓶 中培养。
1.3.2 携带不同linker的bLH在CHO-K1细胞中的瞬时表达
采用脂质体转染法，将上述构建的含有不同linker的bLH表达 载体转入CHO-K1细胞中，取上清液做Western blotting检测。
1.3.2.1 瞬时转染CHO-K1细胞
转染前一天，取长满细胞的10cm细胞培养皿，吸去旧培养基， PBS洗三次，胰酶消化1分钟，5mL含10％血清F12培养基吹打均 匀后计数，将细胞稀释到1xl06个细胞/mL培养基，0.5mL/孔铺24 孔板。
步骤1、1μg DNA溶于25μL Ham’s F12无血清培养基中。
步骤2、1.5μL lipofectamine 2000溶于25μL Ham’s F12无血清培 养基中。
步骤3、将上述溶液混合均匀，室温静置20min。
步骤4、混合液加入到铺有细胞的24孔板中，轻轻吹打混匀。
步骤5、24孔板置于37℃5％二氧化碳培养箱中培养，6h后换 为新鲜的培养基。
步骤6、细胞培养6d后取上清液50μL做western blotting检测。
1.3.2.2 Western blotting检测bLH的瞬时表达
步骤1、细胞培养上清液40μL加入10μL 5×loading buffer煮沸。
步骤2、在电泳槽中加入约270mL电泳缓冲液，每孔上样40μL。
步骤3、电泳：5％浓缩胶，60V，30min；12％分离胶100V，120min。
步骤4、PVDF膜甲醇激活2min，胶在负极，膜在正极，250mA 转膜120min。
步骤5、转膜结束后将PVDF膜用封闭液封闭2h。
步骤6、兔抗LHα多克隆抗体(1：400稀释)4℃孵育12h；TBST 洗膜三次。
步骤7、山羊抗兔IgG-HRP(1：5000稀释)室温孵育2h，TBST 洗膜三次。
步骤8、ECL显影。
含有不同linker的bLH瞬时表达结果，如图1.6所示：
图a为野生型bLH在CHO-K1细胞表达上清液Western Blotting 鉴定结果。
图b为密码子偏爱型的bLH分别在CHO-K1细胞表达上清液的 Western Blotting鉴定结果。
P：阳性对照；
N：转染了pcDNA3.1(+)的细胞培养上清液；
1：转染了linker为GSGPVPGPSPGPVPE的重组bLH细胞培养 上清液；
2：转染了linker为GSHHHHHHE的重组bLH细胞培养上清液；
3：转染了linker为GSNATGSGSNATE的重组bLH细胞培养上 清液；
4：转染了linker为GSNATHHHHHHE的重组bLH细胞培养上 清液；
5：转染了linker为GSNTSNGSTNNTSNE的重组bLH细胞培养 上清液；
6：转染了linker为GSPTPGPHHHHHHE的重组bLH细胞培养 上清液；
7：转染了linker为GSPTPGPPTPGPE的重组bLH细胞培养上清 液；
8：转染了linker为GSPTPSPTPSPTPSPTE的重组bLH细胞培养 上清液；
9：转染了linker为GSPTPGPHHHHHHPTPGPE的重组bLH细 胞培养上清液；
10：转染了linker为GSNHTGSHHHHHHGSNHTE的重组bLH 细胞培养上清液。
由实验结果可知：①重组蛋白的表达量与linker的长度不呈线性 关系。②糖基化位点超过2个糖蛋白的分子量几乎不再增加。③密码 子偏爱型的bLH与野生型bLH在CHO-K1细胞中的表达无显著性差 异，说明蛋白表达量除了与密码子优化及GC含量有关外还受其他因 素的影响，有可能密码子的优化造成的蛋白序列的改变导致了转录及 翻译有关调控原件的改变。另外，密码子优化同样造成了低频率使用 的tRNA的资源浪费。因此密码子优化并不是密码子适应指数越高蛋 白表达量就越高，除了需要考虑GC含量外还应该综合考虑各种顺式 作用原件、DNA甲基化和核苷酸序列对翻译的影响。
1.3.3 携带不同linker的bLH在CHO-K1细胞中的稳定表达
选取表达量高且携带组氨酸标签的重组bLH表达载体稳定转染 CHO-K1细胞。将携带不同linker的bLH表达载体线性化，采用脂质 体包裹表达载体DNA进入CHO-K1细胞，通过G418筛选获得稳定 表达重组bLH的细胞系。
1.3.3.1 表达载体的线性化
将空载体pcDNA3.1(+)和含有不同linker的pcDNA3.1(+)-bLH表 达载体线性化(酶切体系见表1.4)。
表1.4 表达载体的线性化体系
上述体系混匀后，37℃水浴锅中酶切4h。
将酶切过的表达载体加入SYBR green I和6×DNA loading buffer 1.5％琼脂糖凝胶电泳，见图1.7，图中：M:marker；1：未经酶切的 pcDNA3.1(+)；2和3：均为经Acl I单酶双切的pcDNA3.1(+)；4： 未经酶切的pcDNA3.1(+)-LH10；5和6：均为经Acl I单酶双切的pcDNA3.1(+)-LH10；7：未经酶切的pcDNA3.1(+)-LH 9；8和 9：均为经Acl I单酶双切的pcDNA3.1(+)-LH9。pcDNA3.1(+) 表达载体有两个Acl I酶切位点均存在Amp抗性基因上，Acl I酶切载 体后产生373bp的小片段，由图1.7可知，表达载体线性化成功。
待电泳结束后，采用行业内技术人员已知的方法，切取含有线性 化bLH表达载体的凝胶，回收线性化bLH表达载体，测定回收产物 的DNA浓度。
1.3.3.2 细胞筛选G418浓度的选择
在24孔板中接种CHO细胞(1xl05个细胞每孔)，待细胞长到 50％汇合度时，换成含有G418的培养基，使培养基G418的终浓度 分别为0、50、100、150、200、250、300、350、400、450、500、 550、600、650和700μg/mL，每个浓度设3复孔。每隔三天换液， 10天后，选取使CHO-K1细胞完全死亡的最小G418浓度作为CHO 细胞的G418筛选浓度。
结果表明G418浓度大于200μg/mL的细胞培养基均可以使 CHO-K1细胞完全死亡，因此细胞筛选培养基的G418浓度选择250 μg/mL。
1.3.3.3 稳定转染CHO-K1细胞
转染前一天，从CO2培养箱中取一皿CHO-K1细胞放进超净工 作台中，5mL移液器吸取旧培养基并丢弃，加入5mL PBS，左右倾 斜细胞培养皿，丢弃PBS，重复三次。加入1mL细胞消化液将细胞 在37℃CO2培养箱消化2min。加入5mL含10％新生牛血清的Ham’s F12培养基，用移液器将细胞吹打均匀。将细胞稀释后滴加在血球计 数板上，盖上盖玻片，在显微镜下进行细胞计数，将细胞用含10％ 新生牛血清的Ham’s F12培养基稀释到1xl06细胞/mL，0.5mL/孔铺 24孔板。
重组LH表达载体转染CHO-K1细胞方法如下：
1、将1μg DNA溶于25ul Ham’s F12无血清培养基中，得A液。
2、将1.5μL Lipofectamine 2000溶于25μL Ham’s F12无血清培 养基中。得B液。
3、将A B两管液体混合得C液，将C液室温放置20min。
4、将C液加入到长有细胞的24孔板中，轻轻晃动使C液与培 养基混合混匀。
5、将24孔板置于二氧化碳培养箱中培养7h，丢弃就培养基， 加入0.5mL新鲜的培养基。
6、培养24小时后换为含250μg/mL G418的筛选培养基。
7、每两天换液一次(含G418的筛选培养基)，待无质粒转染组 细胞完全死亡后传代至6孔板。
8、用筛选培养基将转染后的细胞传代培养20天。
9、取细胞培养上清液40μL做Western blotting检测。
转染CHO-K1后经G418加压筛选第8天，无质粒转染组细胞完全死 亡，转染组细胞不再死亡，结果如图1.8所示，其中：a：无质粒转 染组；b：pcDNA3.1(+)转染组；c：pcDNA3.1(+)-LH转染组。
1.3.4 Linker中的糖基化类型及数目对bLH分子量的影响
稳定转染细胞株上清液Western Blotting检测结果如图1.9所示， M：marker；1：linker无糖基化位点；2：linker中1个N-糖基化位 点；3：linker中两个N-糖基化位点；4：linker无糖基化位点；5：linker 中1个O-糖基化位点；6：linker中2个O-糖基化位点。
由图1.9可知，随着N-糖基化和O-糖基化位点的增多，糖蛋白 bLH的分子量显著提高，且单个N-糖基化位点的糖链分子量比O-糖 基化位点的分子量大。但是，表达量并不随分子量提高而增加。
1.4 重组LH表达株的单克隆化
选取两个细胞株进行单克隆化，分别为linker中无糖基化和linker 中携带两个N-糖基化的bLH表达株。取一瓶长满的bLH表达株细胞， 细胞消化液处理后加蛋白表达培养基稀释至10个细胞/mL，100μL/ 孔接种于96孔板，显微镜下观察单克隆孔并标记，培养15d，取上 清液20μL做Western Blotting检测，实验结果见图1.10和1.11。
由图1.10可知，7号细胞株表达linker中无糖基化的bLH，选取 该细胞株放大培养，作为种子库。
由图1.11可知，8号和9号细胞株均表达linker中携带两个N- 糖基化的bLH，但是9号细胞株表达量较高，选取该细胞株放大培养， 作为种子库。
实施例2、携带不同接头序列的重组LH相对生物活性的测定
将接头序列中携带0、1和2个N-糖基化位点和O-糖基化位点的 重组bLH，按照药典中黄体生成素生物测定法测定生物活性。
2.1 实验材料的准备
2.1.1 pcDNA3.1(+)转染CHO-K1细胞培养上清液和携带不同 linker的bLH转染细胞上清液(见表2.1)。
表2.1 各bLH蛋白样品性质
2.1.2 10×溶媒的配制
500mgBSA，4.5gNaCl，加蒸馏水至40mL，氢氧化钠调节pH 至7.2，定容50mL，震荡混匀。
2.1.3 标准品注射液的配置：
实验当日，取尿促性素标准品，割开安瓿，用1×溶媒将标准品 干粉洗出，按高、中、低剂量组(SH、SM和SL)制成3种浓度的 标准品溶液各13mL，相邻两浓度之比值(r)为1:0.5。高浓度标准 品溶液每lm l中含12单位。标准品溶液置4℃贮存，可供4日使用。
供试品溶液的配置
根据Western blotting测定的各种bLH的相对浓度，最低浓度样 品加入1/9体积的10×溶媒，其他样品用10×溶媒和1×溶媒制成蛋白 含量相等的bLH，成为高剂量的供试品溶液(TH)。按照相邻浓度的 比值1：0.5的依次稀释成中剂量组和低剂量组溶液(TM和TL)，4℃ 保存，配制量供4日使用。
2.1.4 不同重组bLH相对含量的测定
测定不同bLH样品的相对浓度，将不同的样品调整至相同的浓 度，测定各个样品对大鼠精囊增重的显著性。选取对大鼠精囊显著增 重的bLH，以尿促性素标准品为对照，测定bLH的生物活性。
依次取6、5.5、5、4.5、4、3.5μL样品1A，20μL样品37，23μL 样品24，11μL样品16和18μL样品56。分别加入1/4体积的5×loading buffer，补加1×loadingbuffer至30μL，混匀后煮沸5min，上样，12％ 聚丙烯酰胺凝胶电泳，电泳结束后将蛋白转移到PVDF膜上，封闭液封闭PVDF膜，将PVDF膜转移到1：400稀释的兔抗LHα亚基多克 隆抗体中，4℃孵育过夜，TBST洗膜三次，放入1:5000稀释的山羊 抗兔IgG-HRP中，常温孵育2h，TBST洗膜三次，ECL显影。
不同bLH的Western blotting检测结果如图2所示，1、2、3、4、 5和6泳道依次为6、5.5、5、4.5、4、3.5μL样品1A；7和8为20μL 样品37；9和10为23μL样品24；11和12为11μL样品16；13和 14为18μL样品56。
分析每个泳道的灰度，减去背景灰度，然后以不同上样量的1A 为X轴、灰度为Y轴制作标准曲线。将其他样品的灰度代入标准曲 线，得到相对于样品1A的量，并计算出相对样品1A的bLH浓度， 分析结果见表2.2。
表2.2 各样品相对1A的浓度
由表2.2可知，样品1A的LH含量最高，样品24的LH含量最 低。
2.1.5 不同重组bLH对大鼠精囊增重的影响
根据各样品相对于样品1A的浓度，将bLH浓度最低的样品24 加入1/9体积的10×溶媒，其他各样品试用10×溶媒和1×溶媒稀释至 相同的浓度。各样品稀释方法见表2.3。
表2.3 不同样品注射液的配制方法
实验当日，将动物编号、称重，根据体重将所有动物按照体重随 机分到6个组中。将配好的供试品溶液在注射前取出，放置至室温。 按照下表将不同的bLH注射给每组大鼠。每日于相同时间分别给每 鼠皮下注射一种供试品0.5mL，每日一次，连续注入四次，于最后一 次注入24小时后，将动物处死，称重，解剖，摘出整个前列腺，由 前叶和精囊交界处剥离出精囊，去除附着的组织，用滤纸吸去周围的 液体，称取两个精囊的重量(天平精密度0.1mg)，换算成每10g体 重的精囊重，通过两独立样本非参数检验(Mann-Whitney U检验)，检验不同给药组相对于不给药组动物精囊重量差异的显著性。
表2.4 不同bLH对大鼠精囊增重的显著性影响
说明：显著水平0.05下否定无效假设的可靠程度为95％。 设
H0：相对于对照组，实验组每10g体重精囊重无显著差异
H1：相对于对照组，实验组每10g体重精囊重有显著差异
由表2.4中两独立样本非参数检验Mann-Whitney U检验结果可 知：2、3和4组大鼠每10g体重精囊重相对于对照组(1组)有显 著差异(sig≤0.05说明H0不成立，接受H1)，第4组差异最显著。 从以上数据分析发现，在bLH上增加N-糖基化可以显著提高bLH的 生物活性，而bLH增加O-糖基化位点并没有提高bLH的生物活性。
2.2 按照药典对重组LH的生物活性测定
选取重组LH样品37(接头序列中无糖基化位点)和1A(接头 序列中有两个N-糖基化位点)，按照中华人民共和国药典1217黄体 生成素生物测定法测定重组LH的生物活性，按照药典通则1431中 量反应平行线测定法对实验数据进行方差分析和可靠性测验并计算重组LH效价。
每日于相同的时间，按照表2.5的设计分别给每组大鼠皮下注入 同样浓度的标准品溶液或样品溶液0.5mL，每日1次，连续注射4 次，于最后1次注射24小时后，按药物注射顺序将动物处死，称重， 解剖，摘出整个前列腺，由前叶和精囊交界处剥离出精囊，去除附着的组织，用滤纸吸去周围的液体，称重(天平精密度0.l mg)并换算 成每10g体重的精囊重。
表2.5 不同实验组动物的处理
2.2.1 接头序列中无糖基化的重组LH(样品37)生物活性测算
将反应值(每10g体重精囊重)按标准品(S)和重组LH样品37 的剂量分组列成方阵(见表2.6)，按照药典中生物活性测定法的量反 应平行测定法处理数据。本次实验命名为T1。
表2.6 T1动物实验结果
∑y(k)：各剂量组内求和；∑ym：各区组内求和
按照药典中量反应平行线测定法处理数据，计算供试品相对于标 准品的方差及方差的可靠性，并计算供试品的效价，数据处理结果见 表2.7，效价计算结果见表2.8。
表2.7 T1方差分析及可靠性检测
由表2.7显著性检测实验结果可知，试品间差异性显著，回归非 常显著，偏离平行、二次曲线和反向二次曲线均不显著，符合药典要 求。
表2.8 T1效价测定
样品37效价测定加过见表2.8，可信限率24.54％符合药典要求， 本次实验结果可信。
2.2.2 接头序列携带两个N-糖基化的重组LH(样品1A)生物活 性的测算
将反应值(每10g体重精囊重)按标准品(S)和重组LH样品1A 的剂量分组列成方阵(见表2.9)。
表2.9 T2动物实验结果
∑y(k)：各剂量组内求和；∑ym：各区组内求和
按照药典中量反应平行线测定法处理数据，计算供试品相对于标 准品的方差及方差的可靠性，并计算供试品的效价，数据处理结果见 表2.10，效价计算结果见表2.11。
表2.10 T2方差分析及可靠性检测
由表2.10显著性检测实验结果可知，试品间差异性不显著，回 归非常显著，偏离平行、二次曲线和反向二次曲线均不显著，符合药 典要求。
表2.11 T2效价测定
样品1A效价测定加过见表2.11，可信限率27.44％符合药典要求， 本次实验结果可信。
2.3 单次注射重组LH的生物活性测定
将出生19-21日体重40-50g的雄性SD大鼠，按照体重随机分为 10组，每组6只。将接头序列中无糖基化位点的重组LH(样品37) 作为供试品1(T1)、接头序列中携带两个N-糖基化位点的重组LH (样品1A)作为供试品2(T2)和标准品(S)按照组间剂量比1： 0.6用样品稀释液配制成高剂量组(H)、中剂量组(M)和低剂量组 (L)。将标准品及供试品高中低剂量组溶液按照表2.12的设计方案 对每组大鼠注射0.5mL。样品37和样品1A只在第一天注射一次， 标准品每天注射一次，连续注射4天，于最后一次注入24小时后， 将动物处死，称重，解剖，摘出整个前列腺，由前叶和精囊交界处剥 离出精囊，去除附着的组织，用滤纸吸去周围的液体，称取两个精囊 的重量(天平精密度0.1mg)。
表2.12 不同组大鼠的给药情况
将精囊重换算成每10g体重的精囊重，将反应值(每10g体重精 囊重)按标准品(S)、供试品1(T1，样品37)和供试品2(T2，样 品1A)分组列成方阵(见表2.4)。通过两独立样本非参数检验U检 验，检验不同给药组相对于不给药组动物精囊重量差异的显著性。
表2.13 不同给药组动物精囊增重差异显著性分析
说明：显著水平0.05下否定无效假设的可靠程度为95％。 设
H0：相对于对照组，实验组每10g体重精囊重无显著差异
H1：相对于对照组，实验组每10g体重精囊重有显著差异
由表2.13可知，给动物单次注射相同剂量的样品37(接头序列 中无糖基化位点)和样品1A(接头序列中携带2个N-糖基化位点) 对大鼠增囊增重的药效不同。单次注射样品1A能够显著提高大鼠的 精囊重，而单次注射相同剂量的样品37对大鼠的精囊重量无显著性 影响。
按照药典中量反应平行线测定法处理数据，计算供试品相对于标 准品的方差及方差的可靠性，并计算供试品的效价，数据处理结果见 表2.14，效价计算结果见表2.15。没有表2.15
表2.14 单次注射1A的方差分析及可靠性检测
由表2.10显著性检测实验结果可知，试品间差异性显著，回归 非常显著，偏离平行、二次曲线和反向二次曲线均不显著，符合药典 要求。
根据2.2.和2.3的生物活性测定，证明接头序列中不含糖基化位 点的LHβ-接头序列-LHα的表达物为短效LH类似物；接头序列中携 带2个N-糖基化位点的LHβ-接头序列-LHα的表达物为长效LH类似 物。
虽然以上描述了本发明的具体实施方式，但是熟悉本技术领域的 技术人员应当理解，本发明所描述的具体实施例只是说明性的，而不 是用于对本发明的范围的限定，熟悉本领域的技术人员在依照本发明 的精神所作的等效的修饰以及变化，都应当涵盖在本发明的权利要求 所保护的范围内。
核苷酸和/或氨基酸序列表
<110>华侨大学；太平洋贯连有限公司
<120>一种促黄体激素类似物及其制备方法
<160>20
<210>Seq ID 1
<211>15
<212>PRT
<213>人工合成
<400>
Gly Ser Gly Pro Val Pro Gly Pro Ser Pro Gly Pro Val Pro Glu
1 15
<210>Seq ID 2
<211>9
<212>PRT
<213>人工合成
<400>
Gly Ser His His His His His His Glu
1 9
<210>Seq ID 3
<211>12
<212>PRT
<213>人工合成
<400>
Gly Ser Ala Thr Gly Ser Gly Ser Asn Ala Thr Glu
1 12
<210>Seq ID 4
<211>12
<212>PRT
<213>人工合成
<400>
Gly Ser Asn Ala Thr His His His His His His Glu
1 12
<210>Seq ID 5
<211>15
<212>PRT
<213>人工合成
<400>
Gly Ser Asn Thr Ser Asn Gly Ser Thr Asn Asn Thr Ser Asn Glu
1 15
<210>Seq ID 6
<211>14
<212>PRT
<213>人工合成
<400>
Gly Ser Pro Thr Pro Gly Pro His His His His His His Glu
1 14
<210>Seq ID 7
<211>13
<212>PRT
<213>人工合成
<400>
Gly Ser Pro Thr Pro Gly Pro Pro Thr Pro Gly Pro Glu
1 13
<210>Seq ID 8
<211>17
<212>PRT
<213>人工合成
<400>
Gly Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro Thr Pro Ser Pro
1 15
Thr Glu
17
<210>Seq ID 9
<211>19
<212>PRT
<213>人工合成
<400>
Gly Ser Pro Thr Pro Gly Pro His His His His His His Pro Thr
1 15
Pro Gly Pro Glu
19
<210>Seq ID 10
<211>19
<212>PRT
<213>人工合成
<400>
Gly Ser Asn His Thr Gly Ser His His His His His His Gly Ser
1 15
Asn His Thr Glu
16 19
<210>Seq ID 11
<211>252
<212>PRT
<213>人工合成
<400>
Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly Val
1 5 10 15
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln
20 25 30
Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val
35 40 45
Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser
50 55 60
Met Lys Arg Val Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg
65 70 75
Val Cys Thr Tyr His Glu Leu Rro Phe Ala Ser Val Rro Leu Pro
80 85 90
Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala
95 100 105
Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
110 115 120
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu
125 130 135
Pro Asp Ile Leu Phe Leu Gly Ser Gly Pro Val Pro Gly Pro Ser
140 145 150
Pro Gly Pro Val Pro Glu Phe Pro Asp Gly Glu Phe Thr Met Gln
155 160 165
Gly Cys Pro Glu Cys Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys
170 175 180
Pro Asp Ala Pro Ile Tyr Gln Cys Met Gly Cys Cys Phe Ser Arg
185 190 195
Ala Tyr Pro Thr Pro Ala Arg Ser Lys Lys Thr Met Leu Val Pro
200 205 210
Lys Asn Ile Thr Ser Glu Ala Thr Cys Cys Val Ala Lys Ala Phe
215 220 225
Thr Lys Ala Thr Val Met Gly Asn Val Arg Val Glu Asn His Thr
230 235 240
Glu Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser*
245 250 252
<210>Seq ID 12
<211>246
<212>PRT
<213>人工合成
<400>
Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly Val
1 5 10 15
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln
20 25 30
Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val
35 40 45
Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser
50 55 60
Met Lys Arg Val Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg
65 70 75
Val Cys Thr Tyr His Glu Leu Rro Phe Ala Ser Val Rro Leu Pro
80 85 90
Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala
95 100 105
Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
110 115 120
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu
125 130 135
Pro Asp Ile Leu Phe Leu Gly Ser His His His His His His Glu
140 145 150
Phe Pro Asp Gly Glu Phe Thr Met Gln Gly Cys Pro Glu Cys Lys
155 160 165
Leu Lys Glu Asn Lys Tyr Phe Ser Lys Pro Asp Ala Pro Ile Tyr
170 175 180
Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala
185 190 195
Arg Ser Lys Lys Thr Met Leu Val Pro Lys Asn Ile Thr Ser Glu
200 205 210
Ala Thr Cys Cys Val Ala Lys Ala Phe Thr Lys Ala Thr Val Met
215 220 225
Gly Asn Val Arg Val Glu Asn His Thr Glu Cys His Cys Ser Thr
230 235 240
Cys Tyr Tyr His Lys Ser*
245 246
<210>Seq ID 13
<211>250
<212>PRT
<213>人工合成
<400>
Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly Val
1 5 10 15
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln
20 25 30
Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val
35 40 45
Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser
50 55 60
Met Lys Arg Val Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg
65 70 75
Val Cys Thr Tyr His Glu Leu Rro Phe Ala Ser Val Rro Leu Pro
80 85 90
Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala
95 100 105
Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
110 115 120
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu
125 130 135
Pro Asp Ile Leu Phe Leu Gly Ser Asn Ala Thr Gly Ser Gly Ser
140 145 150
Asn Ala Thr Glu Phe Pro Asp Gly Glu Phe Thr Met Gln Gly Cys
155 160 165
Pro Glu Cys Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Pro Asp
170 175 180
Ala Pro Ile Tyr Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr
185 190 195
Pro Thr Pro Ala Arg Ser Lys Lys Thr Met Leu Val Pro Lys Asn
200 205 210
Ile Thr Ser Glu Ala Thr Cys Cys Val Ala Lys Ala Phe Thr Lys
215 220 225
Ala Thr Val Met Gly Asn Val Arg Val Glu Asn His Thr Glu Cys
230 235 240
His Cys Ser Thr Cys Tyr Tyr His Lys Ser*
245 250
<210>Seq ID 14
<211>249
<212>PRT
<213>人工合成
<400>
Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly Val
1 5 10 15
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln
20 25 30
Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val
35 40 45
Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser
50 55 60
Met Lys Arg Val Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg
65 70 75
Val Cys Thr Tyr His Glu Leu Rro Phe Ala Ser Val Rro Leu Pro
80 85 90
Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala
95 100 105
Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
110 115 120
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu
125 130 135
Pro Asp Ile Leu Phe Leu Gly Ser Asn Ala Thr His His His His
140 145 150
His His Glu Phe Pro Asp Gly Glu Phe Thr Met Gln Gly Cys Pro
155 160 165
Glu Cys Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Pro Asp Ala
170 175 180
Pro Ile Tyr Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr Pro
185 190 195
Thr Pro Ala Arg Ser Lys Lys Thr Met Leu Val Pro Lys Asn Ile
200 205 210
Thr Ser Glu Ala Thr Cys Cys Val Ala Lys Ala Phe Thr Lys Ala
215 220 225
Thr Val Met Gly Asn Val Arg Val Glu Asn His Thr Glu Cys His
230 235 240
Cys Ser Thr Cys Tyr Tyr His Lys Ser*
245 249
<210>Seq ID 15
<211>252
<212>PRT
<213>人工合成
<400>
Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly Val
1 5 10 15
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln
20 25 30
Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val
35 40 45
Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser
50 55 60
Met Lys Arg Val Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg
65 70 75
Val Cys Thr Tyr His Glu Leu Rro Phe Ala Ser Val Rro Leu Pro
80 85 90
Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala
95 100 105
Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
110 115 120
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu
125 130 135
Pro Asp Ile Leu Phe Leu Gly Ser Asn Thr Ser Asn Gly Ser Thr
140 145 150
Asn Asn Thr Ser Asn Glu Phe Pro Asp Gly Glu Phe Thr Met Gln
155 160 165
Gly Cys Pro Glu Cys Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys
170 175 180
Pro Asp Ala Pro Ile Tyr Gln Cys Met Gly Cys Cys Phe Ser Arg
185 190 195
Ala Tyr Pro Thr Pro Ala Arg Ser Lys Lys Thr Met Leu Val Pro
200 205 210
Lys Asn Ile Thr Ser Glu Ala Thr Cys Cys Val Ala Lys Ala Phe
215 220 225
Thr Lys Ala Thr Val Met Gly Asn Val Arg Val Glu Asn His Thr
230 235 240
Glu Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser*
245 250 252
<210>Seq ID 16
<211>251
<212>PRT
<213>人工合成
<400>
Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly Val
1 5 10 15
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln
20 25 30
Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val
35 40 45
Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser
50 55 60
Met Lys Arg Val Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg
65 70 75
Val Cys Thr Tyr His Glu Leu Rro Phe Ala Ser Val Rro Leu Pro
80 85 90
Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala
95 100 105
Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
110 115 120
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu
125 130 135
Pro Asp Ile Leu Phe Leu Gly Ser Pro Thr Pro Gly Pro His His
140 145 150
His His His His Glu Phe Pro Asp Gly Glu Phe Thr Met Gln Gly
155 160 165
Cys Pro Glu Cys Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Pro
170 175 180
Asp Ala Pro Ile Tyr Gln Cys Met Gly Cys Cys Phe Ser Arg Ala
185 190 195
Tyr Pro Thr Pro Ala Arg Ser Lys Lys Thr Met Leu Val Pro Lys
200 205 210
Asn Ile Thr Ser Glu Ala Thr Cys Cys Val Ala Lys Ala Phe Thr
215 220 225
Lys Ala Thr Val Met Gly Asn Val Arg Val Glu Asn His Thr Glu
230 235 240
Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser*
245 250 251
<210>Seq ID 17
<211>250
<212>PRT
<213>人工合成
<400>
Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly Val
1 5 10 15
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln
20 25 30
Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val
35 40 45
Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser
50 55 60
Met Lys Arg Val Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg
65 70 75
Val Cys Thr Tyr His Glu Leu Rro Phe Ala Ser Val Rro Leu Pro
80 85 90
Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala
95 100 105
Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
110 115 120
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu
125 130 135
Pro Asp Ile Leu Phe Leu Gly Ser Pro Thr Pro Gly Pro Pro Thr
140 145 150
Pro Gly Pro Glu Phe Pro Asp Gly Glu Phe Thr Met Gln Gly Cys
155 160 165
Pro Glu Cys Lys Leu Lys Glu Asn Lys Tyr Phe Ser Lys Pro Asp
170 175 180
Ala Pro Ile Tyr Gln Cys Met Gly Cys Cys Phe Ser Arg Ala Tyr
185 190 195
Pro Thr Pro Ala Arg Ser Lys Lys Thr Met Leu Val Pro Lys Asn
200 205 210
Ile Thr Ser Glu Ala Thr Cys Cys Val Ala Lys Ala Phe Thr Lys
215 220 225
Ala Thr Val Met Gly Asn Val Arg Val Glu Asn His Thr Glu Cys
230 235 240
His Cys Ser Thr Cys Tyr Tyr His Lys Ser*
245 250
<210>Seq ID 18
<211>254
<212>PRT
<213>人工合成
<400>
Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly Val
1 5 10 15
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln
20 25 30
Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val
35 40 45
Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser
50 55 60
Met Lys Arg Val Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg
65 70 75
Val Cys Thr Tyr His Glu Leu Rro Phe Ala Ser Val Rro Leu Pro
80 85 90
Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala
95 100 105
Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
110 115 120
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu
125 130 135
Pro Asp Ile Leu Phe Leu Gly Ser Pro Thr Pro Ser Pro Thr Pro
140 145 150
Ser Pro Thr Pro Ser Pro Thr Glu Phe Pro Asp Gly Glu Phe Thr
155 160 165
Met Gln Gly Cys Pro Glu Cys Lys Leu Lys Glu Asn Lys Tyr Phe
170 175 180
Ser Lys Pro Asp Ala Pro Ile Tyr Gln Cys Met Gly Cys Cys Phe
185 190 195
Ser Arg Ala Tyr Pro Thr Pro Ala Arg Ser Lys Lys Thr Met Leu
200 205 210
Val Pro Lys Asn Ile Thr Ser Glu Ala Thr Cys Cys Val Ala Lys
215 220 225
Ala Phe Thr Lys Ala Thr Val Met Gly Asn Val Arg Val Glu Asn
230 235 240
His Thr Glu Cys His Cys Ser Thr Cys Tyr Tyr His Lys Ser*
245 250 254
<210>Seq ID 19
<211>256
<212>PRT
<213>人工合成
<400>
Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly Val
1 5 10 15
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln
20 25 30
Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val
35 40 45
Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser
50 55 60
Met Lys Arg Val Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg
65 70 75
Val Cys Thr Tyr His Glu Leu Rro Phe Ala Ser Val Rro Leu Pro
80 85 90
Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala
95 100 105
Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
110 115 120
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu
125 130 135
Pro Asp Ile Leu Phe Leu Gly Ser Pro Thr Pro Gly Pro His His
140 145 150
His His His His Pro Thr Pro Gly Pro Glu Phe Pro Asp Gly Glu
155 160 165
Phe Thr Met Gln Gly Cys Pro Glu Cys Lys Leu Lys Glu Asn Lys
170 175 180
Tyr Phe Ser Lys Pro Asp Ala Pro Ile Tyr Gln Cys Met Gly Cys
185 190 195
Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg Ser Lys Lys Thr
200 205 210
Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr Cys Cys Val
215 220 225
Ala Lys Ala Phe Thr Lys Ala Thr Val Met Gly Asn Val Arg Val
230 235 240
Glu Asn His Thr Glu Cys His Cys Ser Thr Cys Tyr Tyr His Lys
245 250 255
Ser*
256
<210>Seq ID 20
<211>256
<212>PRT
<213>人工合成
<400>
Met Glu Met Phe Gln Gly Leu Leu Leu Trp Leu Leu Leu Gly Val
1 5 10 15
Ala Gly Val Trp Ala Ser Arg Gly Pro Leu Arg Pro Leu Cys Gln
20 25 30
Pro Ile Asn Ala Thr Leu Ala Ala Glu Lys Glu Ala Cys Pro Val
35 40 45
Cys Ile Thr Phe Thr Thr Ser Ile Cys Ala Gly Tyr Cys Pro Ser
50 55 60
Met Lys Arg Val Leu Pro Val Ile Leu Pro Pro Met Pro Gln Arg
65 70 75
Val Cys Thr Tyr His Glu Leu Rro Phe Ala Ser Val Rro Leu Pro
80 85 90
Gly Cys Pro Pro Gly Val Asp Pro Met Val Ser Phe Pro Val Ala
95 100 105
Leu Ser Cys His Cys Gly Pro Cys Arg Leu Ser Ser Thr Asp Cys
110 115 120
Gly Gly Pro Arg Thr Gln Pro Leu Ala Cys Asp His Pro Pro Leu
125 130 135
Pro Asp Ile Leu Phe Leu Gly Ser Asn His Thr Gly Ser His His
140 145 150
His His His His Gly Ser Asn His Thr Glu Phe Pro Asp Gly Glu
155 160 165
Phe Thr Met Gln Gly Cys Pro Glu Cys Lys Leu Lys Glu Asn Lys
170 175 180
Tyr Phe Ser Lys Pro Asp Ala Pro Ile Tyr Gln Cys Met Gly Cys
185 190 195
Cys Phe Ser Arg Ala Tyr Pro Thr Pro Ala Arg Ser Lys Lys Thr
200 205 210
Met Leu Val Pro Lys Asn Ile Thr Ser Glu Ala Thr Cys Cys Val
215 220 225
Ala Lys Ala Phe Thr Lys Ala Thr Val Met Gly Asn Val Arg Val
230 235 240
Glu Asn His Thr Glu Cys His Cys Ser Thr Cys Tyr Tyr His Lys
245 250 255
Ser*
256
Claims (2)
1.一种促黄体激素类似物，其特征在于，所述的促黄体激素类似物的编码序列系携带有起始密码子和信号肽编码序列但删除终止密码子的LH的β亚基编码序列与缺少起始密码子和信号肽编码序列但携带终止密码子的LH的α亚基编码序列与接头序列linker按照LHβ-接头序列-LHα的顺序从N末端到C-末端连接起来；所述的接头序列为Seq ID 2或Seq ID 10中的一种，对应所述接头序列的促黄体激素类似物的氨基酸序列为Seq ID 12和Seq ID20。
2.一种权利要求1所述的促黄体激素类似物的制备方法，其特征在于，其制备方法包括以下步骤：
步骤1、从Genebank获得牛(bovine)LHα亚基编码序列和LHβ亚基编码序列，删除LHβ的终止密码子和LHα的起始密码子；根据CHO-K1表达系统偏爱的密码子丰度，将野生型LH的编码序列密码子进行重组；设计接头序列，根据接头序列的长度、可折叠性、疏水性、电荷效应设计不同长度的带有0个、1个、2个和4个O-糖基化和N-糖基化位点的接头序列10个，在接头序列上添加组氨酸标签(HHHHHH)，接头序列两端分别添加酶切位点BamH I和EcoR I，以引物的形式全序列合成，纯化方式PAGE，合成量1OD，合成的接头序列单链DNA-20℃保存备用；
步骤2、将含有全序列合成的野生型LH的重组载体pMD19-T Simple-LH9、含有全序列合成的密码子偏爱型LH的重组载体pMD19-T Simple-LH10和空表达载体pcDNA3.1(+)分别经限制性内切酶双酶切，回收双酶切线性化的pcDNA3.1(+)和LH(含接头序列)片段，经DNA连接酶连接，分别形成重组表达载体pcDNA3.1(+)-LH9和pcDNA3.1(+)-LH10，构建的重组表达载体转染感受细胞E.coli DH5α，选取抗阿莫西林阳性克隆菌落扩大培养，提取重组载体并保存菌种，提取的质粒载体限制性内切酶双酶切鉴定；
步骤3、分别置换连接序列，共构建20个含10种不同连接序列的野生型和密码子偏爱型的bLH表达载体；
步骤4、采用脂质体转染法，将上述构建的含有不同接头序列的bLH表达载体转入CHO-K1细胞中，取上清液做Western Blotting检测，选取表达量高且携带组氨酸的重组bLH表达载体，线性化后转染CHO-K1细胞，通过G418筛选，上清液通过Western Blotting检测；
步骤5、重组bLH表达株的单克隆化，取bLH表达株细胞放大培养作为种子库；
步骤6、通过对相应的各种候选序列的表达物的生物活性进行比较而确定长短效序列。
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2017100090798 | 2017-01-04 | ||
| CN201710009079 | 2017-01-04 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107827987A CN107827987A (zh) | 2018-03-23 |
| CN107827987B true CN107827987B (zh) | 2021-04-27 |
Family
ID=61643036
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710494736.2A Active CN107827987B (zh) | 2017-01-04 | 2017-06-26 | 一种促黄体激素类似物及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107827987B (zh) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115097041B (zh) * | 2022-06-28 | 2023-12-15 | 中国农业科学院北京畜牧兽医研究所 | 一种prm检测促黄体生成素的试剂盒及检测方法和应用 |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5705478A (en) * | 1989-02-21 | 1998-01-06 | Washington University | Covalently linked β subunits of the glycoprotein hormones as antagonists |
| CN1290301A (zh) * | 1997-11-17 | 2001-04-04 | 华盛顿大学 | 单链双功能的糖蛋白激素 |
| CN104152457A (zh) * | 2014-07-14 | 2014-11-19 | 林静静 | 一种兽用卵泡刺激素类似物的制备方法 |
-
2017
- 2017-06-26 CN CN201710494736.2A patent/CN107827987B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5705478A (en) * | 1989-02-21 | 1998-01-06 | Washington University | Covalently linked β subunits of the glycoprotein hormones as antagonists |
| CN1290301A (zh) * | 1997-11-17 | 2001-04-04 | 华盛顿大学 | 单链双功能的糖蛋白激素 |
| CN104152457A (zh) * | 2014-07-14 | 2014-11-19 | 林静静 | 一种兽用卵泡刺激素类似物的制备方法 |
Non-Patent Citations (4)
| Title |
|---|
| Long-Acting Follicle-Stimulating Hormone Analogs Containing N-Linked Glycosylation Exhibited Increased Bioactivity Compared with O-Linked Analogs in Female Rats;C Weenen,等;《The Journal of Clinical Endocrinology & Metabolism》;20041031;第89卷(第10期);图1 * |
| Moran Grinberg,等.The configuration of the alpha and beta subunit domains in single-chain bovine LH analogs influences the secretion and steroidogenic response.《Molecular and Cellular Endocrinology》.2008,第283卷(第1-2期), * |
| The configuration of the alpha and beta subunit domains in single-chain bovine LH analogs influences the secretion and steroidogenic response;Moran Grinberg,等;《Molecular and Cellular Endocrinology》;20080213;第283卷(第1-2期);第84-86页2.2构建单链类似物、方案1 * |
| 长效人促卵泡激素CHO细胞株的构建及其体内活性;黄晓平,等;《生物工程学报》;20140625;第30卷(第6期);摘要、1 材料与方法 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107827987A (zh) | 2018-03-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP3207416B2 (ja) | ソマトトロピン・アナログ類 | |
| Kamei et al. | Expression of a biologically active recombinant follicle stimulating hormone of Japanese eel Anguilla japonica using methylotropic yeast, Pichia pastoris | |
| US20210054041A1 (en) | Glycoprotein Hormone Long-Acting Superagonists | |
| US20220267401A1 (en) | Method for producing and purifying hybrid or non-hybrid recombinant glycoprotein hormones, hybrid or non-hybrid recombinant glycoprotein hormones, expression vectors and uses of the recombinant glycoprotein hormones | |
| CN107827987B (zh) | 一种促黄体激素类似物及其制备方法 | |
| EP3065766A1 (en) | Glycoprotein hormone long-acting superagonists | |
| CN114213523B (zh) | 一种用于重组蛋白的高糖基化修饰序列及其重组猪促卵泡激素和应用 | |
| EP0386203A1 (en) | Methods for producing gonadotropin and tsh super-agonists | |
| US10457713B2 (en) | Glycoprotein hormone long-acting superagonists | |
| Hanks et al. | Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli | |
| US20240132565A1 (en) | Process of production and purification of hybrid or non-hybrid recombinant glycoprotein hormones, hybrid or non-hybrid recombinant glycoprotein hormones, expression vectors and uses of recombinant glycoprotein hormones | |
| CN111995669A (zh) | 一种人促卵泡激素及其体外重组装方法 | |
| CN117924482A (zh) | 一种靶向INHα的纳米抗体及其应用 | |
| CN102549011A (zh) | 包含重组hcg的药物制剂 |
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 |