CN108642059A - Transformation suitable for silkworm expression, which has, promotes cell proliferation factor gene and its expression vector and application - Google Patents
Transformation suitable for silkworm expression, which has, promotes cell proliferation factor gene and its expression vector and application Download PDFInfo
- Publication number
- CN108642059A CN108642059A CN201810421158.4A CN201810421158A CN108642059A CN 108642059 A CN108642059 A CN 108642059A CN 201810421158 A CN201810421158 A CN 201810421158A CN 108642059 A CN108642059 A CN 108642059A
- Authority
- CN
- China
- Prior art keywords
- expression
- gene
- silk
- silkworm
- expression vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 57
- 241000255789 Bombyx mori Species 0.000 title claims abstract description 50
- 230000014509 gene expression Effects 0.000 title claims abstract description 47
- 230000004663 cell proliferation Effects 0.000 title claims abstract description 30
- 239000013604 expression vector Substances 0.000 title claims abstract description 29
- 230000009466 transformation Effects 0.000 title claims abstract description 17
- 238000012216 screening Methods 0.000 claims abstract description 14
- 108010013296 Sericins Proteins 0.000 claims abstract description 12
- 230000001737 promoting effect Effects 0.000 claims abstract description 11
- 239000003623 enhancer Substances 0.000 claims abstract description 8
- 239000003550 marker Substances 0.000 claims abstract description 7
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 230000009261 transgenic effect Effects 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 10
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims description 9
- 210000000988 bone and bone Anatomy 0.000 claims description 5
- 230000007547 defect Effects 0.000 claims description 5
- 235000013601 eggs Nutrition 0.000 claims description 5
- 238000004519 manufacturing process Methods 0.000 claims description 5
- 239000003292 glue Substances 0.000 claims description 4
- 108091008146 restriction endonucleases Proteins 0.000 claims description 4
- 230000005058 diapause Effects 0.000 claims description 3
- 230000029087 digestion Effects 0.000 claims description 3
- 231100000252 nontoxic Toxicity 0.000 claims description 3
- 230000003000 nontoxic effect Effects 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 2
- 238000011144 upstream manufacturing Methods 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 claims 1
- 150000001413 amino acids Chemical class 0.000 abstract description 6
- 230000003248 secreting effect Effects 0.000 abstract description 6
- 239000003102 growth factor Substances 0.000 abstract description 5
- 238000011161 development Methods 0.000 abstract description 4
- 210000004907 gland Anatomy 0.000 abstract description 3
- 238000000844 transformation Methods 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 25
- 239000000243 solution Substances 0.000 description 14
- 238000001514 detection method Methods 0.000 description 11
- 210000001772 blood platelet Anatomy 0.000 description 10
- 230000026731 phosphorylation Effects 0.000 description 10
- 238000006366 phosphorylation reaction Methods 0.000 description 10
- 238000012545 processing Methods 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 230000000694 effects Effects 0.000 description 9
- 238000000605 extraction Methods 0.000 description 8
- 239000006180 TBST buffer Substances 0.000 description 7
- 239000013612 plasmid Substances 0.000 description 7
- 238000011160 research Methods 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- 101100462438 Mus musculus Otulin gene Proteins 0.000 description 6
- 108700019146 Transgenes Proteins 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 6
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 108010081589 Becaplermin Proteins 0.000 description 5
- 101000692455 Homo sapiens Platelet-derived growth factor receptor beta Proteins 0.000 description 5
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 5
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 5
- 238000009396 hybridization Methods 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 239000000463 material Substances 0.000 description 5
- 230000035755 proliferation Effects 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 108020004414 DNA Proteins 0.000 description 4
- 108010022355 Fibroins Proteins 0.000 description 4
- 102100026547 Platelet-derived growth factor receptor beta Human genes 0.000 description 4
- 108010087230 Sincalide Proteins 0.000 description 4
- 238000010609 cell counting kit-8 assay Methods 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 238000004043 dyeing Methods 0.000 description 4
- 230000002068 genetic effect Effects 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 4
- 108020003589 5' Untranslated Regions Proteins 0.000 description 3
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 108010068370 Glutens Proteins 0.000 description 3
- 239000012620 biological material Substances 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002386 leaching Methods 0.000 description 3
- 238000000520 microinjection Methods 0.000 description 3
- 108090000765 processed proteins & peptides Proteins 0.000 description 3
- 230000006798 recombination Effects 0.000 description 3
- 238000005215 recombination Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 230000029663 wound healing Effects 0.000 description 3
- 230000037303 wrinkles Effects 0.000 description 3
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 101000595923 Homo sapiens Placenta growth factor Proteins 0.000 description 2
- 101000611951 Homo sapiens Platelet-derived growth factor subunit B Proteins 0.000 description 2
- 102100035194 Placenta growth factor Human genes 0.000 description 2
- 108010005233 alanylglutamic acid Proteins 0.000 description 2
- 230000004071 biological effect Effects 0.000 description 2
- 239000003519 biomedical and dental material Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000003501 co-culture Methods 0.000 description 2
- 230000009514 concussion Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 108010025306 histidylleucine Proteins 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 230000001537 neural effect Effects 0.000 description 2
- 238000005457 optimization Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 235000019624 protein content Nutrition 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- 101150028074 2 gene Proteins 0.000 description 1
- 101150090724 3 gene Proteins 0.000 description 1
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- HHGYNJRJIINWAK-FXQIFTODSA-N Ala-Ala-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N HHGYNJRJIINWAK-FXQIFTODSA-N 0.000 description 1
- SHYYAQLDNVHPFT-DLOVCJGASA-N Ala-Asn-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SHYYAQLDNVHPFT-DLOVCJGASA-N 0.000 description 1
- XAGIMRPOEJSYER-CIUDSAMLSA-N Ala-Cys-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N XAGIMRPOEJSYER-CIUDSAMLSA-N 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- XEPSCVXTCUUHDT-AVGNSLFASA-N Arg-Arg-Leu Natural products CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CCCN=C(N)N XEPSCVXTCUUHDT-AVGNSLFASA-N 0.000 description 1
- JVMKBJNSRZWDBO-FXQIFTODSA-N Arg-Cys-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CO)C(O)=O JVMKBJNSRZWDBO-FXQIFTODSA-N 0.000 description 1
- OGUPCHKBOKJFMA-SRVKXCTJSA-N Arg-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCCN=C(N)N OGUPCHKBOKJFMA-SRVKXCTJSA-N 0.000 description 1
- DNUKXVMPARLPFN-XUXIUFHCSA-N Arg-Leu-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DNUKXVMPARLPFN-XUXIUFHCSA-N 0.000 description 1
- BNYNOWJESJJIOI-XUXIUFHCSA-N Arg-Lys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)N BNYNOWJESJJIOI-XUXIUFHCSA-N 0.000 description 1
- FVBZXNSRIDVYJS-AVGNSLFASA-N Arg-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCN=C(N)N FVBZXNSRIDVYJS-AVGNSLFASA-N 0.000 description 1
- SYFHFLGAROUHNT-VEVYYDQMSA-N Arg-Thr-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(O)=O SYFHFLGAROUHNT-VEVYYDQMSA-N 0.000 description 1
- AUZAXCPWMDBWEE-HJGDQZAQSA-N Arg-Thr-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O AUZAXCPWMDBWEE-HJGDQZAQSA-N 0.000 description 1
- HAJWYALLJIATCX-FXQIFTODSA-N Asn-Asn-Arg Chemical compound C(C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N)CN=C(N)N HAJWYALLJIATCX-FXQIFTODSA-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
- IAMNNSSEBXDJMN-CIUDSAMLSA-N Asp-Cys-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC(=O)O)N IAMNNSSEBXDJMN-CIUDSAMLSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 101100533230 Caenorhabditis elegans ser-2 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108700010070 Codon Usage Proteins 0.000 description 1
- QLCPDGRAEJSYQM-LPEHRKFASA-N Cys-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CS)N)C(=O)O QLCPDGRAEJSYQM-LPEHRKFASA-N 0.000 description 1
- BDWIZLQVVWQMTB-XKBZYTNZSA-N Cys-Glu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CS)N)O BDWIZLQVVWQMTB-XKBZYTNZSA-N 0.000 description 1
- MXZYQNJCBVJHSR-KATARQTJSA-N Cys-Lys-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CS)N)O MXZYQNJCBVJHSR-KATARQTJSA-N 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 206010048768 Dermatosis Diseases 0.000 description 1
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- XOKGKOQWADCLFQ-GARJFASQSA-N Gln-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CCC(=O)N)N)C(=O)O XOKGKOQWADCLFQ-GARJFASQSA-N 0.000 description 1
- ZFBBMCKQSNJZSN-AUTRQRHGSA-N Gln-Val-Gln Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZFBBMCKQSNJZSN-AUTRQRHGSA-N 0.000 description 1
- RTOOAKXIJADOLL-GUBZILKMSA-N Glu-Asp-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)O)N RTOOAKXIJADOLL-GUBZILKMSA-N 0.000 description 1
- INGJLBQKTRJLFO-UKJIMTQDSA-N Glu-Ile-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O INGJLBQKTRJLFO-UKJIMTQDSA-N 0.000 description 1
- QJVZSVUYZFYLFQ-CIUDSAMLSA-N Glu-Pro-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O QJVZSVUYZFYLFQ-CIUDSAMLSA-N 0.000 description 1
- RJIVPOXLQFJRTG-LURJTMIESA-N Gly-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N RJIVPOXLQFJRTG-LURJTMIESA-N 0.000 description 1
- 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 1
- WNGHUXFWEWTKAO-YUMQZZPRSA-N Gly-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)CN WNGHUXFWEWTKAO-YUMQZZPRSA-N 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- LRAUKBMYHHNADU-DKIMLUQUSA-N Ile-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@@H](C)CC)CC1=CC=CC=C1 LRAUKBMYHHNADU-DKIMLUQUSA-N 0.000 description 1
- JHNJNTMTZHEDLJ-NAKRPEOUSA-N Ile-Ser-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O JHNJNTMTZHEDLJ-NAKRPEOUSA-N 0.000 description 1
- 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 1
- 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 1
- NTXYXFDMIHXTHE-WDSOQIARSA-N Leu-Val-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 NTXYXFDMIHXTHE-WDSOQIARSA-N 0.000 description 1
- 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 1
- CLBGMWIYPYAZPR-AVGNSLFASA-N Lys-Arg-Arg Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O CLBGMWIYPYAZPR-AVGNSLFASA-N 0.000 description 1
- YXPJCVNIDDKGOE-MELADBBJSA-N Lys-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)N)C(=O)O YXPJCVNIDDKGOE-MELADBBJSA-N 0.000 description 1
- FZUNSVYYPYJYAP-NAKRPEOUSA-N Met-Ile-Ala Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O FZUNSVYYPYJYAP-NAKRPEOUSA-N 0.000 description 1
- HLZORBMOISUNIV-DCAQKATOSA-N Met-Ser-Leu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(C)C HLZORBMOISUNIV-DCAQKATOSA-N 0.000 description 1
- 235000008708 Morus alba Nutrition 0.000 description 1
- 240000000249 Morus alba Species 0.000 description 1
- 241000283973 Oryctolagus cuniculus Species 0.000 description 1
- 238000009004 PCR Kit Methods 0.000 description 1
- HWLKHNDRXWTFTN-GUBZILKMSA-N Pro-Pro-Cys Chemical compound C1C[C@H](NC1)C(=O)N2CCC[C@H]2C(=O)N[C@@H](CS)C(=O)O HWLKHNDRXWTFTN-GUBZILKMSA-N 0.000 description 1
- YDTUEBLEAVANFH-RCWTZXSCSA-N Pro-Val-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H]1CCCN1 YDTUEBLEAVANFH-RCWTZXSCSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- XOWKUMFHEZLKLT-CIQUZCHMSA-N Thr-Ile-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O XOWKUMFHEZLKLT-CIQUZCHMSA-N 0.000 description 1
- UZDHNIJRRTUKKC-DLOVCJGASA-N Val-Gln-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](C(C)C)C(=O)O)N UZDHNIJRRTUKKC-DLOVCJGASA-N 0.000 description 1
- UEHRGZCNLSWGHK-DLOVCJGASA-N Val-Glu-Val Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O UEHRGZCNLSWGHK-DLOVCJGASA-N 0.000 description 1
- YLRAFVVWZRSZQC-DZKIICNBSA-N Val-Phe-Glu Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N YLRAFVVWZRSZQC-DZKIICNBSA-N 0.000 description 1
- LCHZBEUVGAVMKS-RHYQMDGZSA-N Val-Thr-Leu Chemical compound CC(C)C[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)[C@@H](C)O)C(O)=O LCHZBEUVGAVMKS-RHYQMDGZSA-N 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000005267 amalgamation Methods 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010080488 arginyl-arginyl-leucine Proteins 0.000 description 1
- 108010068380 arginylarginine Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 230000003796 beauty Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000002759 chromosomal effect Effects 0.000 description 1
- 239000013599 cloning vector Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- NKLPQNGYXWVELD-UHFFFAOYSA-M coomassie brilliant blue Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=C1 NKLPQNGYXWVELD-UHFFFAOYSA-M 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000005014 ectopic expression Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 230000027948 extracellular matrix binding Effects 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 108010021843 fluorescent protein 583 Proteins 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000010448 genetic screening Methods 0.000 description 1
- 108010073628 glutamyl-valyl-phenylalanine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 239000010903 husk Substances 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000017448 oviposition Effects 0.000 description 1
- 108010064486 phenylalanyl-leucyl-valine Proteins 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108010015796 prolylisoleucine Proteins 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 108010054624 red fluorescent protein Proteins 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 230000000250 revascularization Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 230000009759 skin aging Effects 0.000 description 1
- 208000017520 skin disease Diseases 0.000 description 1
- 238000010129 solution processing Methods 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000010902 straw Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 230000009182 swimming Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 239000004753 textile Substances 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 108010084932 tryptophyl-proline Proteins 0.000 description 1
- 210000003462 vein Anatomy 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
- C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
-
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Physics & Mathematics (AREA)
- Insects & Arthropods (AREA)
- Tropical Medicine & Parasitology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Peptides Or Proteins (AREA)
Abstract
The present invention relates to the transformations suitable for silkworm expression to have promotion cell proliferation factor gene and its expression vector and application, the nucleotide sequence of the gene is as shown in SEQ ID NO.2, the amino acid that it is encoded is as shown in SEQ ID NO.1, transformation, which is had, promotes cell proliferation factor gene to constitute expression cassette with secreting type sericin 1 gene promoter and secreting type sericin 1 gene terminator, enhancer hr3 Enhanced expressings are used in combination, it is connected into piggyBac swivel bases arm and fluorescent screening marker gene structure expression system simultaneously, the expression system can in domestic natural silk gland the active promotion growth factor of high efficient expression, obtain the silk for having and promoting cell Proliferation, there is huge Development volue.
Description
Technical field
The invention belongs to biotechnologies, and in particular to be suitable for silkworm expression transformation have promote cell Proliferation because
Subbase is because further relating to correlative expression vector and application.
Background technology
Silkworm is also known as silkworm, originating from China, has the history of more than one thousand years domestication in China.Silkworm is to be with mulberry leaf
The economic insects that the spinning of food is cocoond, silk are the bases for constituting Silk Industry.Silk as textile material, have easy processing,
Performance of keeping humidity is good, resists a variety of advantages such as ultraviolet light；Silk has good mechanical performance, life as biomaterial simultaneously
The characteristics such as object compatibility, stronger environmental stability and degradability are gradually closed in the fields such as medicine and bioengineered tissue
Note.However for a long time, the defect of silk self performance becomes such as not easy dyeing, bad mechanical property etc. and restricts Silk Industry hair
The bottleneck of exhibition can not meet the needs of medical tissue engineering research.Therefore, innovation research is carried out to silk itself, to silk
The optimization and improvement of self performance and function are very urgent.
At the beginning of 21 century, finds in the correlative study of drosophila isotype biology and Successful utilization piggyBac transposon turns base
It because research achieves important breakthrough, is enlightened by the research, has researcher will using the method for microinjection silkworm embryos
The piggyBac swivel base expression vectors for carrying green fluorescent protein (EGFP) reporter gene import silkworm body early embryo, and in G1
For the transgenic silkworm that expression EGFP has successfully been obtained in silkworm seed.Then, Thomas etc. is by neural compound eye tissue-specific promoter
Green fluorescence protein gene genetic marker (3XP3-EGFP) be applied to silkworm transgene genetic screening in, simplify transgenosis
The preparation flow of silkworm improves the screening efficiency of transgenosis.Using the transgenic technology, establish including fibroin expression system
And a variety of expression systems including silk gum expression system, it can successful expression is transferred in silkworm silk foreign protein.It is based on
Foreign protein is imported in fibroin and obtains novel silk material by piggyBac transposon vectors, this is new silk materials
Modification and initiative provide new approach.
Silk is mainly made of silk gum and fibroin, and sericin is a kind of colloidal carrier, is wrapped in the outer layer of fibroin albumen
Form silk fiber.Sericin is highly soluble in water, by 3 gene of sericin 1 gene (Ser1), 2 gene of silk gum (Ser2) and silk gum
(Ser3) it is successively expressed in middle division of silkgland different parts, wherein Ser1 gene expression amounts highest, therefore the promoter sequence of the gene
Row are commonly used for expression foreign protein and obtain new silk material.2007, Tomita etc. utilized silkworm sericin 1 gene (Ser1)
Promoter establishes Ser1 expression systems, by 300bp Ser1 promoter regulations EGFP gene silk gum layer secreting, expressing, and
Utilize the activity of trans- controlling element IE1 and enhancer element hr3 activation Ser1 promoters from baculoviral so that EGFP
Expression increase by 10 times, account for the 0.7% of cocoon layer quality.Subsequent Iizuka etc. utilizes the ends the 5' untranslated of BmNPVpol genes
(5'-UTR) optimizes the system in area, and the translation efficiency of foreign protein EGFP is made to improve 2 times.But hr3/IE1 can be destroyed
The tissue specificity of Ser1 promoters leads to marker gene and foreign gene ectopic expression, and then causes transgenic bombyx mori dead.
2013, Wang etc. reported a kind of efficient Ser1 expression systems hSRSE, the system by Ser1 genes 528bp startup
The signal coding sequence of son, 58bp complete 5'-UTR and 87bp is constituted.To improve expression efficiency, and hr3CQ is utilized to increase
The 3'-UTR (Ser1PA) of hadron and Ser1 genes optimizes Ser1 expression systems so that recombination red fluorescent protein
(DsRed) 16 times of output increased, reaches the 9.5% of cocoon layer quality, this is also that the current expression efficiency reported is highest
Ser1 expression systems.2010, the research teams such as Adachi added hr3 enhancer amalgamation and expression people before Ser1 gene promoters
Ι collagen type α chains successfully obtain the transgenosis silk of people's Ι collagen type α chains, and content of the foreign protein in silk can
To reach 8%.The transgenosis silk can be as the silk material of cell culture, wherein the people's Ι collagen type α chains tool expressed
There is biological activity.2014, the research teams such as WangF were also with hr3 enhancers and the expression recombination of Ser1 gene promoters
Human acid fibroblast growth factor (hFGF1) obtains a kind of new silk biomaterial, and the silk is without any processing can
The proliferation of mouse embryonic fibroblasts NIH3T3 is remarkably promoted, and the mechanical performance of raw silk slightly enhances.Contain hFGF1
Transgenosis raw silk be expected to may be directly applied to medical field.
Vectors containing human platelet-derived growth is the alkaline protein being present in the granule of platelet, has tetra- kinds of A, B, C, D
Subunit forms homotype by two identical or different polypeptide chains or heterodimer plays a role.B chain monomer sizes are about
14kD, the dimer PDGF-BB formed by two B chains can play a role in a plurality of access, be to be primarily present shape in human body
Formula.PDGF has multiple efficacies, therefore all has broad application prospects in medical cosmetology etc..Medically, PDGF by
It is one good medicine for fire victim and dermatosis patient in having the function of promotion wound healing.It is a variety of when there is wound
The releasable PDGF of cell, as angiogenesis rupture can intra platelet free calcium go out a variety of growth factors, including PDGF, PDGF
Neighbouring phoirocyte can be stimulated to grow, and connective tissue is the vanguard for rebuilding damaged tissues, the wound that heals.Therefore
PDGF plays a significant role in wound healing process.Meanwhile PDGF is in the good medicine that beauty industry is also removing wrinkle and resisting aging, this be by
In the effect for the revascularization remodeling that PDGF has, PDGF can promote veins beneath the skin to be formed, repair subcutaneous blood microcirculqtory system,
Sufficient nutrition is provided for skin, the synthesis of collagen, delay skin aging can also be promoted.PDGF, which is a kind of important rush, to be had
Silk splitting factor, can promote various kinds of cell group's division growth, to keep wrinkle naturally long flat.Therefore, skill is recombinated using transgenosis
Art production PDGF-BB albumen can be used as the new sources of albumen, is expected to the application form for occurring new, has huge development prospect.
Invention content
Promote cell Proliferation in view of this, having one of the objects of the present invention is to provide the transformation suitable for silkworm expression
Factor gene；The second object of the present invention, which is to provide to have containing the transformation suitable for silkworm expression, promotes cell Proliferation
The expression vector of factor gene；The third object of the present invention is that providing the expression vector produces in silkworm with promotion carefully
Application in the silk of born of the same parents' proliferation；The fourth object of the present invention, which is to provide to produce in silkworm using the expression vector, to be had
The method of Bone Defect Repari function silk.
For achieving the above object, the present invention provides the following technical solutions：
1. the transformation suitable for silkworm expression, which has, promotes cell proliferation factor gene, the transformation, which has, promotes cell to increase
The nucleotide sequence of factor gene is grown as shown in SEQ ID NO.2.
2. there is the expression vector for promoting cell proliferation factor gene containing the transformation suitable for silkworm expression.
Preferably, the expression vector contains enhancer hr3 successively, and secreting type sericin 1 gene promoter, transformation, which have, to be promoted
Into the terminator of cell proliferation factor gene and sericin 1 gene.
Preferably, the expression vector also contains fluorescent screening marker gene expression cassette, the fluorescent screening marker gene
Expression cassette is located at the upstreams the enhancer hr3.
Preferably, expression vector sequence shown in SEQ ID NO.2 is connected into sequence shown in SEQ ID NO.3
At BamHI and NotI restriction enzyme sites, then with being connected into after AscI digestions in the pBac through identical digestion { 3xp3EGFPaf } carrier.
3. the expression vector is produced in silkworm with the application in the silk for promoting cell Proliferation.
4, the method with Bone Defect Repari silk is produced in silkworm using the expression vector, is included the following steps：
The Eggs of Silkworm that the expression vector is injected to termination of diapause, is sterilized with after nontoxic glue sealing through formaldehyde vapor, is hatched, until
The production of hybrid seeds, screening transgenic positive moth circle are selfed or are returned after adult, and the cocoon shell of positive transgenic silkworm is to be repaiied with bone
Multiple function silk.
The beneficial effects of the present invention are：Have the invention discloses the transformation suitable for silkworm expression and promotes cell Proliferation
Factor gene, by by vectors containing human platelet-derived growth albumen (HumanPlatelet Derived Growth Factor
Subunit B, hPDGFB) ripe peptide amino acid sequence, the 123-144 amino acids (PlGF- with PlGF-2 albumen
It 2123-144) merges, forms PDGF-Modified (PDGFM), 1 expression system of efficient silk gum established early period is then utilized to drive
Dynamic PDGFM prepares silk in silkworm, and silk obtained has the biological activity for promoting cell Proliferation, to make wrinkle certainly
It is so long flat.Therefore, silk obtained can be used as biomaterial, have huge development prospect.
Description of the drawings
In order to keep the purpose of the present invention, technical solution and advantageous effect clearer, the present invention provides following attached drawing and carries out
Explanation：
Fig. 1 is transgene expression vector pBac [3xp3DsRed, hSPDGF-BBMSer1PA] structure chart (3xp3DsRed tables
Show fluorescent transgenic riddled basins；Hr3CQ indicates enhancer hr3；Ser1Pro indicates secreting type sericin 1 gene promoter；
5'UTR indicates the ends 5' non-translational region；SP indicates signal peptide；PDGF-BB indicates that the human blood platelets of codon optimization design spreads out
Raw growth factor gene coded sequence；M indicates extracellular binding domain sequence；Ser1PA indicates the terminator of sericin 1 gene；ITR tables
Show piggyBac swivel base arms sequence).
Fig. 2 is fluorescent transgenic screening figure (A：Silkworm seed fluorogram B：Silkworm seed white light figure C：Moth fluorogram D：Moth white light figure).
Fig. 3 is PDGFM Cocoon glutelin SDS-PAGE electrophoresis and WesternBlot detection figures (WT：Normal cocoon shell egg
In vain；1-15：Transgenic positive individual cocoon glutelin).
Fig. 4 is that PDGF is proliferated access schematic diagram.
Fig. 5 is the variation of the intracellular PDGFR phosphorylation levels of NIH3T3 after the processing of PDGFM silks.
Fig. 6 is the variation of the intracellular MEK phosphorylation levels of NIH3T3 after the processing of PDGFM silks.
Fig. 7 is the variation of the intracellular ERK phosphorylation levels of NIH3T3 after the processing of PDGFM silks.
Fig. 8 is to turn PDGFM gene cocoon pieces to promote NIH3T3 cell Proliferation Live＆Dead coloration results.
Fig. 9 turns PDGFM gene cocoon pieces and promotes NIH3T3 cell Proliferation EdU coloration results.
Figure 10 turns PDGFM gene cocoon pieces and promotes the CCK-8 experiments of NIH3T3 cell Proliferations
Figure 11 is the release profiles (A of PDGFM albumen in transgenosis silk：Silk leaching liquor Western Blot analyses；B：
Release profiles).
Figure 12 is normal silk and PDGF transgenosis silk infrared spectrograms.
Specific implementation mode
Below in conjunction with attached drawing, the preferred embodiment of the present invention is described in detail.
(P50) is made greatly in the embodiment of the present invention for examination cultivated silkworm breed variety to be preserved by this laboratory.Larva is in 25 DEG C of artificial climates
It is raised with man-made feeds in case.Mouse embryonic fibroblasts NIH3T3 is preserved by this laboratory, and is incubated at containing 10% (v/
V) in the DMEM culture mediums of fetal calf serum (FBS, Gibco), condition of culture is 37 DEG C, 5%CO2.Plasmid vector
PSLfa1180fa, pBac { 3xp3EGFPaf }, pBac { 3xp3DsRedaf } are preserved by this laboratory.
Main agents and solution are formulated as follows：The conventional medium arrived used in the process of molecular cloning, reagent buffer
Equal references《The Molecular Cloning:A Laboratory guide third edition is translated》And《TaKaRa goods catalogue》Middle laboratory conventional reagent preparation method
Chapters and sections (S1-S11) are configured.Archaeal dna polymerase Ex-Taq, LA-Taq Kit, convenient restriction restriction endonuclease, alkaline phosphatase,
Cloning vector pMD19-T simple carriers Kit, DNA Ligation Kit Ver.2.0, PCR kit for fluorescence quantitative is sequenced
SYBR premix Ex TaqTMIt is purchased from TaKaRa companies.Conversion competent escherichia coli cell Trans1-T1, conventional matter
Grain DNA extracts kit Easypure Plasmid MiniPrep Kit are purchased from Quan Shi King Companies.Ago-Gel DNA recycling
Kit Gel Extraction Mini Kit (50) are purchased from Hua Shun biotechnologies company.The ultrapure plasmid of transgenosis injection carries
Kit QIA prep Spin Miniprep Kit (50) are taken to be purchased from QIAGEN.Total RNA Kit II (50) kit is purchased
From Omega Bio-Tec companies.People's (PDGF) protein polyclone antibody anti-rhPDGF antibody, recombination human platelet spread out
Raw growth factor protein standard items rhPDGF-std is purchased from abcam companies, and cell Proliferation detects CCK-8 kits and is purchased from green cloud
Its company, live/dead cell dyeing (Live＆Dead) kit and EdU cell proliferation reagent boxes are purchased from invitrogen companies.
The synthesis of embodiment 1, gene
To improve the extracellular matrix binding ability of PDGF-BB, vectors containing human platelet-derived growth egg will be downloaded from NCBI
(HumanPlatelet Derived Growth Factor subunit B, hPDGFB, GenBank in vain:NM_002608.3)
Ripe peptide amino acid sequence, merged with the 123-144 amino acids (PlGF-2123-144) of PlGF-2 albumen, formed
PDGF-Modified (PDGFM), amino acid sequence as shown in SEQ ID NO.1, wherein the 1st~110 be hPDGFB, the 101st
~132 are PlGF-2123-144, are optimized to PDGFM according to silkworm codon usage bias, nucleotide sequence is such as
Shown in SEQ ID NO.2, and by company's synthetic gene sequence.
The structure of embodiment 2, transgene expression vector
The PDGFM gene coded sequences being commercially synthesized are building up to psl1180 by BamHI and NotI restriction enzyme sites
In [hr3CQSer1spDsRedSer] (SEQ ID NO.3), psl1180hr3CQSer1spPDGFMSer1 is formed, then pass through
The sites AscI are building up in the sites AscI of pBac { 3xp3DsRedaf } carrier, form transgene expression vector pBac
{ 3xp3DsRed, hSPDGF-BBMSer1PA }, structure chart is as shown in Figure 1.
Embodiment 3, microinjection and fluorescent screening
Transgene expression vector is extracted using QIAGEN Plasimd Mini Kit plasmid extraction kits
Each plasmid concentration is diluted to 400ng/ μ l, and presses 1: 1 mole of score by phPDGFMSer1 and assistant carrier pHA3PIG plasmids
It is not mixed with assistant carrier pHA3PIG plasmids.By the injection of mixed plasmid, the big of termination of diapause makes body early embryo
(2~5h after oviposition), then seals injection orifice with nontoxic glue, after five minutes through the disinfection of 35% formaldehyde vapor, is placed in
25 DEG C, hatch in the environment of relative humidity 85%, the larva (G0 generations) of hatching is raised using man-made feeds, until being carried out after adult
Selfing or the backcrossing production of hybrid seeds, the G1 of acquisition is for silkworm seed (the 7th day) under macroscopical Stereo fluorescence microscope (Olypus MVX10, Japan)
Detection, red fluorescence observation use wavelength for the exciting light of 490~530nm, filter out excitated red in eyes or neural specific
The transgenic positive moth circle of fluorescence, and it is named as PDGFM, the results are shown in Figure 2.The fluorescent screening statistics of its transgenic bombyx mori is such as
Shown in table 1, wherein from 58 G1 of PDGFM for screening 13 positive moth circles, positive rate 22.4% in moth circle in total.
The fluorescent screening of 1. transgenic bombyx mori of table counts
Embodiment 4, the detection of expression for recombinating PDGFM albumen
The G1 for recombinating PDGFM transgenic bombyx moris is individually raised for the positive individuals in different positive moth circles, and
SDS-PAGE detections and the WesternBlot detections of recombinant protein, detection are carried out to the cocoon shell of 15 positive individuals after being placed on small straw bundles to spin cocoons
Method is as follows：Cocoon shell is impregnated in liquid nitrogen until become fragile, powder is ground into using pulverizer, it is dense according to the cocoon shell of 30mg/ml
Degree be dissolved in 8MUrea, 50mMTris-Cl, pH8.0, buffer solution in, 80 DEG C of water-bath 30min, at room temperature 13400rpm/min from
Heart 10min collects supernatant.Cocoon shell total protein progress 12%SDS-Page electrophoresis detections through extraction, coomassie brilliant blue staining, together
When by the total protein of extraction through 12%SDS-Page gel electrophoresis separation after, using electric transferring film method by the albumen in SDS-PAGE glue turn
It moves on pvdf membrane.Pvdf membrane is placed in the TBST buffer solutions containing 5% skimmed milk power, 4 DEG C of closings overnight.Before immuning hybridization, in
Room temperature cleans pvdf membrane 3 times using TBST, each 5min.1 is pressed using the TBST containing 5% skimmed milk power：1000 dilution configurations are anti-
Pvdf membrane is immersed shaken at room temperature incubation 2h, TBST cleaning 5 times in hybridization solution, every time by rhPDGF (abcam) primary antibody hybridization solution
10min.1 is pressed using TBST：20000 dilution ratios configure goat-anti rabbit secondary antibody (being purchased from green skies company) hybridization of HRP labels
Liquid, the pvdf membrane after TBST is cleaned are immersed in shaken at room temperature incubation 2h, TBST cleaning 5 times in secondary antibody hybridization solution, every time
10min.Pvdf membrane after cleaning is placed on clean preservative film, ECL developing solutions (Amersham Biosciences) is equal
Even to drop on PDVF film surfaces, room temperature, which is protected from light, is incubated 5min, utilizes Chemiscope Series (Clinx science
Instruments) instrument is exposed and is imaged, and the results are shown in Figure 3.As a result it shows：Transgenosis cocoon glutelin swimming lane exists
There is differential band at 14kDa molecules Marker, it is in the same size with the theory of PDGF-B.The above result shows that this research is established
Ser1 expression systems can efficient recombinant production people's PDGF albumen, and be secreted into silkworm silk.In addition, recombinant protein exists
Content in the individual of different positive moth circles has significant difference, implies and turns PDGFM gene silkworms in silk gland cell
Expression is influenced by strong chromosomal position effect.
Embodiment 5, the detection for being proliferated access correlation factor phosphorylation level
In the proliferation access that PDGF causes, since the combination of PDGF and receptor PDGFR cause receptor that phosphorylation occurs, draw
The raising of the correlation factors phosphorylation levels such as downstream MEK, ERK is played, signal is transferred in core, finally causes cell Proliferation (figure
4).By normal silk cocoon and turns PDGFM genes silk cocoon and be immersed in PBS solution with the concentration of 30mg/ml, room temperature concussion.Every 4h
Supernatant is collected in centrifugation, is added isometric new PBS solution and is continued concussion extraction.By normal silk cocoon and turn PDGFM gene silk cocoons
After all supernatant concentrations collected, NIH3T3 cells 5min is handled together with low serum DMEM culture mediums.Lead to after lytic cell
Cross the content of Western Blot detection intracellulars PDGFR, MEK, ERK and the phosphorylation level of three.By normal silk and turn
Treated that the intracellular correlation factor testing results of NIH3T3 are shown for PDGFM gene silks extracting solution, in and PDGFR is not detected
Phosphorylation level, and turn PDGFM gene silks extracting solution treated PDGFR phosphorylation levels in cell and significantly increase (Fig. 5),
Likewise, the phosphorylation level for turning PDGFM gene silks extracting solution treated MEK and ERK in cell is all apparently higher than normally
Silk extracting solution processing group (Fig. 6, Fig. 7).Result above, which further demonstrates, turns PDGFM gene silks with promotion cell increasing
The effect grown.
Embodiment 6, the detection of transgenosis silk proliferation activity
Normal silk cocoon and transgenosis silk cocoon are removed into outer layer husks and internal layer cocoon shell, the middle layer left are cut into identical circle
Shape cocoon piece is several, and size hole more each than 96 porocyte culture plates is smaller, cleans cocoon piece 3 times with distilled water, ultraviolet irradiation sterilizes overnight
For use.The low serum DMEM culture mediums that NIH3T3 cells replace medium to 0.5% (v/v) FBS carry out Nature enemy, after resuspension
96 porocyte culture plates are seeded to, density is 2 × 103A/hole, cell is adherent after cultivating 12h, after ultraviolet processing is added thereto
Normal silk cocoon and transgenosis silk cocoon cocoon piece, continue to cultivate in incubator.Use Live＆Dead staining kits afterwards for 24 hours
It is dyed, 100 μ l is added per hole and dye working solution, is protected from light and is incubated 20min, observed under red fluorescence under green fluorescence respectively
Living cells and dead cell, and take pictures.After the cocoon piece of normal silk cocoon and transgenosis silk cocoon and cell co-culture for 24 hours, EdU is carried out
Dyeing, nucleus is observed under DAPI light, the cell being newly proliferated is observed under red fluorescence, and take pictures after dyeing.Likewise, just
The cocoon piece of normal silk cocoon and transgenosis silk cocoon co-cultures every hole backward for 24 hours with cell and 10 μ l CCK-8 reagents is added, in incubator
Continue to incubate 1h, light absorption value is detected at 450nm.Live＆Dead coloration result Green fluorescence display living cells, red fluorescence are shown
Dead cell, the quantity for turning the NIH3T3 living cells of PDGFM gene cocoon piece processing groups are significantly more than control group, illustrate to obtain novel
Silk has the activity (Fig. 8) for promoting cell Proliferation.Red fluorescence indicates the cell being newly proliferated, DAPI light in EdU coloration results
Show that nucleus, the quantity for turning the NIH3T3 cells that PDGFM gene cocoon piece processing groups are newly proliferated are significantly more than control group, explanation obtains
The new silk obtained has the activity (Fig. 9) for promoting cell Proliferation.CCK-8 is the results show that turn PDGFM gene cocoon piece processing groups
The quantity of NIH3T3 cells be significantly more than control group, illustrate that the new silk obtained has the activity (figure for promoting cell Proliferation
10)。
The release of PDGFM recombinant proteins in embodiment 7, transgenosis silk
Silk leaching liquor is obtained using PBS extraction silks, Western Blot analyses are carried out to leaching liquor, as a result such as Figure 12
Shown in middle A.Illustrate that this method can successfully extract the recombinant protein in silk.Extend extraction total time, according to Western
Blot results carry out gray analysis using software Lane 1D can obtain each sample protein content, be obtained in Figure 11 in B after cumulative
Release profiles.The result shows that as extraction time extends, the PDGFM recombinant protein contents of release gradually increase.
Embodiment 8, silk performance evaluation
Using Fourier infrared spectrograph to normal silk and turn PDGFM silks carry out Secondary Structure Content measurement, knot
Fruit is as shown in figure 12.30 times measurement the results show that infrared absorption pattern of the two within the scope of 800~4000cm has no obviously
Difference.Carry out after peak-fit processing that the results are shown in Table 2 to the characteristic peak of amide I, as a result show in transgenosis silk α spirals and
The content no significant difference of β-pleated sheet structure.
The content of 2 normal silk of table and secondary structure in PDGF transgenosis silks
To sum up, expression system is optimized first in the present invention, it is anti-establishes efficient transgenic bombyx mori sericterium biology
Device expression system is answered, the efficient secretory expression of foreign gene is realized.Herein on basis, there is promotion cell Proliferation and move
The vectors containing human platelet-derived growth (PDGF) of shifting function is used as target, to vectors containing human platelet-derived growth (PDGF-BB) egg
It is recombinantly expressed in vain, the Preference engineer that is used according to domestic silkworm gene group codon, modification have simultaneously synthesized human blood platelets
The encoding gene of derivative growth factor (PDGF-BBM), structure transgene expression vector pBac { 3xp3DsRed, hSPDGF-
BBMSer1PA }, by microinjection silkworm embryos, we establish transgenic bombyx mori strain PDGF, and screening obtains 13 differences
The strain in positive moth circle source, has obtained the silk of genetic improvement.Using SDS-PAGE and Western blot detection methods,
We detect that PDGF albumen has high efficient expression in the cocoon shell of PDGFM silkworms.
We have carried out heredity to silk using the high-efficient transgenic domestic natural silk gland bioreactor expression system of foundation and have changed
It is good, and the potentiality for the silk being used as to biomedical material carry out preliminary exploration.Promote the study found that transgenosis silk has
Growth and proliferation of cell and the function of migration, and can for a long time be preserved in silk.It therefore can by the new silk of genetic improvement
It is developed further into the biomedical material for promoting wound healing function.Pass through this expressive function albumen in silk
Method can be widely applied to genetic improvement and obtain the new silk with different function and purposes, expands the application field of silk,
The market competitiveness for improving silk, creates more economic values, is that new power is injected in the development of Silk Industry.
Finally illustrate, preferred embodiment above is merely illustrative of the technical solution of the present invention and unrestricted, although logical
It crosses above preferred embodiment the present invention is described in detail, however, those skilled in the art should understand that, can be
Various changes are made to it in form and in details, without departing from claims of the present invention limited range.
Sequence table
<110>Southwestern University
<120>Transformation suitable for silkworm expression, which has, promotes cell proliferation factor gene and its expression vector and application
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 132
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Met Ser Leu Gly Ser Leu Thr Ile Ala Glu Pro Ala Met Ile Ala Glu
1 5 10 15
Cys Lys Thr Arg Thr Glu Val Phe Glu Ile Ser Arg Arg Leu Ile Asp
20 25 30
Arg Thr Asn Ala Asn Phe Leu Val Trp Pro Pro Cys Val Glu Val Gln
35 40 45
Arg Cys Ser Gly Cys Cys Asn Asn Arg Asn Val Gln Cys Arg Pro Thr
50 55 60
Gln Val Gln Leu Arg Pro Val Gln Val Arg Lys Ile Glu Ile Val Arg
65 70 75 80
Lys Lys Pro Ile Phe Lys Lys Ala Thr Val Thr Leu Glu Asp His Leu
85 90 95
Ala Cys Lys Cys Glu Thr Val Ala Ala Ala Arg Pro Val Thr Arg Arg
100 105 110
Arg Pro Lys Gly Arg Gly Lys Arg Arg Arg Glu Lys Gln Arg Pro Thr
115 120 125
Asp Cys His Leu
130
<210> 2
<211> 399
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
atgagcctgg gtagcctgac catagcggag cctgccatga ttgcggaatg taaaactcgt 60
actgaagtct ttgaaataag ccgtaggtta atagacagaa caaacgctaa tttcctggtc 120
tggcctccat gcgtggaagt tcaacgctgt tcaggttgct gtaacaatag aaacgtgcag 180
tgccgcccga cacaagttca gttgcgtccc gtccaagtaa ggaaaatcga gatagtcaga 240
aaaaagccta tcttcaagaa ggccactgta actttggaag accacttggc ctgtaaatgc 300
gaaacggttg ctgctgctcg tcctgtcacc cgtcgacgcc ctaaaggtcg tggtaaacgt 360
cgacgcgaaa aacaacgtcc tactgattgt catttataa 399
<210> 3
<211> 2036
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
ccatggcagc gtcgtgaaaa gaggcaatga caaatacaaa acgacgtatg agcagacccg 60
tcgccaagac gggtctacct ctaagatgat gtcatttgtt ttttaaaact aactcgcttt 120
acgagtagaa ttctacgtgt aaaacataat caagagatga tgtcatttgt ttttcaaaac 180
caaactcgct ttacgagtag aattctacgt gtaaaacaca atcaaaagat gatgtcattc 240
gtttttcaaa accgaattta agaaatgatg tcatttgttt ttcaaaacca aactcgcttt 300
acgagcagaa ttctacgtgt aaaacacaat caagagatga tgtcatttgt ttttcaaaac 360
tgaatgatgt catttgtttt tcaaaactaa acttgctttg cgagtagaat tctacgtgta 420
aaacacagtc aagagatgat gtcatttgtt tttcaaaact gaaccggctt tacgagtaga 480
attctacttg taaaacataa tcaagagatg atgtcatttg tttttcaaaa ctgaactggc 540
tttacgagta gaattctacg tgtaaaacat aatcaagaga tgatgtcatc attaaactga 600
tgtcatttta tacacgattg ttaacatgtt taataatgac taatttgttt ttccaaatta 660
aactcgcttt acgagtagaa ttctacttgt aacgcacgat taagtatgaa tcataagctg 720
atgtcatttg ttttcgacat aaaatgttta tacaatggaa tcttcttgta aattatccaa 780
ataatataat ttatccgatt ctacgttaca tttaaattcg ttgttatcgt acaattcttc 840
aggacacgcc atgtattggt catttttagc gtgcaaccaa cgattgtatt tgacgccgtc 900
gttggattgc gtgttcaggt tggcgtacac gtgactgggc acggcttctt tttccatggg 960
acgtcgacga aaacagcaca cacactacat accatgtatt tgacgcacac acgcatgtat 1020
actatttatt gtcaaacttt tgttcttgac gtctgtgttc aaactgagaa tagattaaat 1080
attgtttgtc tttattaata ttttttaata gtgtagtctt ggcgaaattt gtgattataa 1140
aagtataaaa tacaatcata atagtgtacg aacttacaat tccaattaat tatagtcgaa 1200
tttcgactac tgcgggacct ctagtattaa taattctctt taaaaaaaaa cagagcatca 1260
aatactgcac aaatgtcaag cgggtctcaa cgagccatga ataaattaga aatcaattaa 1320
taacataaaa taggcaaaca aaataaaacc atttacatag agaacgtttg ttgaacaaaa 1380
acaataactt gtatacattg tttgcacaaa tgtttgaagc gaaaatttat tactctctac 1440
gtaagcttga tcaaacttcg ttttcgtata aaacgcgttg gcccaaccac tttggcatag 1500
tcgtcttatc atcgggtctc taaggatcaa gcgatccaaa gaccgccaac atgcgtttcg 1560
ttctgtgctg cactttgatt gcgttggctg cgctcagcgt aaaagccttc ggtcaccacc 1620
ccggcaatcg agatacagga tccgcggccg ctacaactaa acacgacttg gagtattcct 1680
tgtagtgttt aagattttaa atcttactta atgacttcga acgattttaa cgataacttt 1740
ctctttgttt aactttaatc agcatacata aaaagccccg gttttgtatc gggaagaaaa 1800
aaaatgtaat tgtgttgcct agataataaa cgtattatca aagtgtgtgg ttttccttta 1860
ccaaagaccc ctttaagatg ggcctaatgg gcttaagtcg agtcctttcc gatgtgttaa 1920
atacacattt attacactga tgcgtcgaat gtacactttt aataggatag ctccactaaa 1980
aattatttta tttatttaat ttgttgcacc aaaactgata cattgacgaa aagctt 2036
Claims (7)
1. the transformation suitable for silkworm expression, which has, promotes cell proliferation factor gene, it is characterised in that：The transformation, which has, to be promoted
Into cell proliferation factor gene nucleotide sequence as shown in SEQ ID NO.2.
2. containing the transformation for being suitable for silkworm expression described in claim 1 there is the expression for promoting cell proliferation factor gene to carry
Body.
3. expression vector according to claim 2, it is characterised in that：The expression vector contains enhancer hr3 successively, point
Secrete type sericin 1 gene promoter, transformation has the terminator for promoting cell proliferation factor gene and sericin 1 gene.
4. expression vector according to claim 3, it is characterised in that：The expression vector also contains fluorescent screening and marks base
Because of expression cassette, the fluorescent screening marker gene expression cassette is located at the upstreams the enhancer hr3.
5. expression vector according to claim 2 or 3, it is characterised in that：The expression vector is shown in SEQ ID NO.2
Sequence is connected at BamHI the and NotI restriction enzyme sites of sequence shown in SEQ ID NO.3, then with being connected into through same enzyme after AscI digestions
In pBac { 3xp3EGFPaf } carrier cut.
6. any one of claim 2~5 expression vector is produced in silkworm with answering in the silk for promoting cell Proliferation
With.
7. the method with Bone Defect Repari silk is produced in silkworm using any one of claim 2~5 expression vector,
It is characterised in that it includes following steps：The Eggs of Silkworm that the expression vector is injected to termination of diapause, with nontoxic glue sealing
It sterilizes, hatches, until the production of hybrid seeds is selfed or be returned after adult, screening transgenic positive moth circle, the positive turns base by formaldehyde vapor
Because the cocoon shell of silkworm is to have the function of Bone Defect Repari silk.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810421158.4A CN108642059B (en) | 2018-05-04 | 2018-05-04 | Modified gene with cell proliferation promoting factor suitable for silkworm expression and expression vector and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810421158.4A CN108642059B (en) | 2018-05-04 | 2018-05-04 | Modified gene with cell proliferation promoting factor suitable for silkworm expression and expression vector and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108642059A true CN108642059A (en) | 2018-10-12 |
| CN108642059B CN108642059B (en) | 2020-07-28 |
Family
ID=63749479
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201810421158.4A Active CN108642059B (en) | 2018-05-04 | 2018-05-04 | Modified gene with cell proliferation promoting factor suitable for silkworm expression and expression vector and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108642059B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424035A (en) * | 2020-04-13 | 2020-07-17 | 西南大学 | Method for expressing human connective tissue growth factor with biological activity based on silkworm silk gland, product and application thereof |
| CN111534543A (en) * | 2020-05-07 | 2020-08-14 | 西南大学 | Eukaryotic CRISPR/Cas9 knockout system, basic vector, vector and cell line |
| CN112852876A (en) * | 2021-03-04 | 2021-05-28 | 西南大学 | Silkworm silk gland recombinant expression vector for expressing human epidermal growth factor and preparation method and application thereof |
| CN116239667A (en) * | 2022-07-21 | 2023-06-09 | 西南大学 | ehEGF recombinant protein with cell proliferation promoting activity, and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270367A (en) * | 2008-04-30 | 2008-09-24 | 苏州大学 | Construction method for domestic silkworm silk glandulae biological factory and pharmacy use |
| CN104017814A (en) * | 2014-06-20 | 2014-09-03 | 西南大学 | Chicken infectious bursal disease virus structural protein VP2 applicable to silkworm middle silk gland expression as well as expression vector and application thereof |
| CN104513821A (en) * | 2013-09-27 | 2015-04-15 | 西南大学 | Modified human acidic fibroblast growth factor gene and recombinant vector and applications thereof |
| CN105861515A (en) * | 2016-04-27 | 2016-08-17 | 西南大学 | Reconstructed human serum albumin gene suitable for cultivated silk gland expression and expression system and application thereof |
-
2018
- 2018-05-04 CN CN201810421158.4A patent/CN108642059B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101270367A (en) * | 2008-04-30 | 2008-09-24 | 苏州大学 | Construction method for domestic silkworm silk glandulae biological factory and pharmacy use |
| CN104513821A (en) * | 2013-09-27 | 2015-04-15 | 西南大学 | Modified human acidic fibroblast growth factor gene and recombinant vector and applications thereof |
| CN104017814A (en) * | 2014-06-20 | 2014-09-03 | 西南大学 | Chicken infectious bursal disease virus structural protein VP2 applicable to silkworm middle silk gland expression as well as expression vector and application thereof |
| CN105861515A (en) * | 2016-04-27 | 2016-08-17 | 西南大学 | Reconstructed human serum albumin gene suitable for cultivated silk gland expression and expression system and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| MIKAEL M.MARTINO ET AL.: ""Growth Factors Engineered for Super-Affinity to the Extracellular MatrixEnhance Tissue Healing"", 《SCIENCE》 * |
| 王晖等: ""家蚕cyp450基因家族密码子的用法分析"", 《黑龙江畜牧兽医》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111424035A (en) * | 2020-04-13 | 2020-07-17 | 西南大学 | Method for expressing human connective tissue growth factor with biological activity based on silkworm silk gland, product and application thereof |
| CN111534543A (en) * | 2020-05-07 | 2020-08-14 | 西南大学 | Eukaryotic CRISPR/Cas9 knockout system, basic vector, vector and cell line |
| CN112852876A (en) * | 2021-03-04 | 2021-05-28 | 西南大学 | Silkworm silk gland recombinant expression vector for expressing human epidermal growth factor and preparation method and application thereof |
| WO2022183952A1 (en) * | 2021-03-04 | 2022-09-09 | 西南大学 | Bombyx mori silk gland recombinant expression vector expressing human epidermal growth factor, preparation method therefor, and application thereof |
| CN116239667A (en) * | 2022-07-21 | 2023-06-09 | 西南大学 | ehEGF recombinant protein with cell proliferation promoting activity, and preparation method and application thereof |
| CN116239667B (en) * | 2022-07-21 | 2024-05-10 | 西南大学 | EhEGF recombinant protein with cell proliferation promoting activity, and preparation method and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108642059B (en) | 2020-07-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20190106467A1 (en) | Chimeric spider silk and methods of use thereof | |
| CN108642059A (en) | Transformation suitable for silkworm expression, which has, promotes cell proliferation factor gene and its expression vector and application | |
| CN103911383B (en) | Be suitable for transformation human acid fibroblast growth factor gene that domestic natural silk gland expresses and expression system and application | |
| Naskar et al. | Introduction to silk biomaterials | |
| CN102358902A (en) | Silkworm fibroin heavy-chain gene mutation sequence and mutation method and application | |
| CN104513821B (en) | The human acid fibroblast growth factor gene and its recombinant vector of transformation and application | |
| CN104313053A (en) | Method for producing human collagen type II | |
| CN111793643A (en) | Silkworm fibroin heavy chain expression system for expressing target protein distributed in silk fibroin and sericin, preparation method and application | |
| CN109844112A (en) | Genetic recombination bagworm silk | |
| CN111850039A (en) | Silkworm silk fibroin heavy chain expression system with expression protein distributed in silkworm silk glue layer and preparation method and application thereof | |
| JP6362878B2 (en) | Fusion protein containing modified peptide repeat peptide and silk fiber containing the fusion protein | |
| CN110257394A (en) | Silkworm Bmhsp19.9 gene is cultivating the application in the cultivated silkworm breed variety being resistant to extreme temperature | |
| CN105861515A (en) | Reconstructed human serum albumin gene suitable for cultivated silk gland expression and expression system and application thereof | |
| CN101423841A (en) | Method for producing cultivated silkworm novel green cocoon practical variety by using GFP protein | |
| CN101255423A (en) | Method for creating cultivated silkworm chromatic cocoon by using pigment protein | |
| CN108486153A (en) | The application and method of FGF2 and 1 fusion of TGF-β in promoting silk cell-proliferation activity and anti-inflammatory properties | |
| CN108588083A (en) | Suitable for the transformation platelet derived growth factor gene and its expression vector of silkworm expression and application | |
| JP5030610B2 (en) | Silkworms with high expression of useful proteins by antiviral protein gene knockdown and use thereof | |
| JP2007252327A (en) | Cell adhesive silk yarn and method for producing the same | |
| KR20150084152A (en) | Transgenic silkworms producing recombinant antibacterial peptide | |
| CN103865927B (en) | Utilize silkworm egg xanthan protein promoter sex and tissue specific expression foreign gene and method | |
| Kotani et al. | Development of New Biomaterials from Insect and its Virus | |
| JP2023119400A (en) | Method for making transgenic silkworm using dormant egg | |
| JP2024069712A (en) | Method for producing genetically modified cloned silkworms | |
| CN115992181A (en) | Method for producing chimeric silk by silkworm for alfalfa silver vein moth nuclear polyhedrosis virus |
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 | ||
| TR01 | Transfer of patent right |
Effective date of registration: 20240407 Address after: 402160 No. 801 Heshun Avenue, Yongchuan District, Chongqing (1st Floor, Building 3, Zone C, Yongchuan District Big Data Industrial Park) Patentee after: Chongqing century legend technology development partnership (L.P.) Country or region after: China Address before: 400715 No. 2, natural road, Beibei District, Chongqing Patentee before: SOUTHWEST University Country or region before: China |
|
| TR01 | Transfer of patent right |