CN114262368A - 一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法 - Google Patents
一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法 Download PDFInfo
- Publication number
- CN114262368A CN114262368A CN202111608133.3A CN202111608133A CN114262368A CN 114262368 A CN114262368 A CN 114262368A CN 202111608133 A CN202111608133 A CN 202111608133A CN 114262368 A CN114262368 A CN 114262368A
- Authority
- CN
- China
- Prior art keywords
- scl2
- gly
- seq
- collagen
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 102000008186 Collagen Human genes 0.000 title claims abstract description 73
- 108010035532 Collagen Proteins 0.000 title claims abstract description 73
- 229920001436 collagen Polymers 0.000 title claims abstract description 73
- 239000000017 hydrogel Substances 0.000 title claims abstract description 32
- 238000002360 preparation method Methods 0.000 title claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 36
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 34
- 238000000034 method Methods 0.000 claims abstract description 16
- 101710137510 Saimiri transformation-associated protein Proteins 0.000 claims abstract description 12
- 230000035772 mutation Effects 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 3
- 239000002773 nucleotide Substances 0.000 claims description 36
- 125000003729 nucleotide group Chemical group 0.000 claims description 36
- 230000015572 biosynthetic process Effects 0.000 claims description 27
- 238000003786 synthesis reaction Methods 0.000 claims description 27
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims description 22
- 239000012634 fragment Substances 0.000 claims description 20
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 claims description 18
- 230000001580 bacterial effect Effects 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 239000013598 vector Substances 0.000 claims description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 229910052759 nickel Inorganic materials 0.000 claims description 11
- 235000019750 Crude protein Nutrition 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 9
- 239000012460 protein solution Substances 0.000 claims description 9
- 241000588724 Escherichia coli Species 0.000 claims description 7
- 238000001976 enzyme digestion Methods 0.000 claims description 7
- 230000005484 gravity Effects 0.000 claims description 7
- 239000007788 liquid Substances 0.000 claims description 7
- 241000193996 Streptococcus pyogenes Species 0.000 claims description 6
- 239000007983 Tris buffer Substances 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- 238000001914 filtration Methods 0.000 claims description 6
- 239000000499 gel Substances 0.000 claims description 6
- 239000011780 sodium chloride Substances 0.000 claims description 6
- 239000006228 supernatant Substances 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 5
- 239000013604 expression vector Substances 0.000 claims description 5
- 239000001963 growth medium Substances 0.000 claims description 5
- 238000003259 recombinant expression Methods 0.000 claims description 5
- 230000009466 transformation Effects 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 4
- 239000000203 mixture Substances 0.000 claims description 4
- 238000003752 polymerase chain reaction Methods 0.000 claims description 4
- 239000002244 precipitate Substances 0.000 claims description 4
- 102000012410 DNA Ligases Human genes 0.000 claims description 3
- 108010061982 DNA Ligases Proteins 0.000 claims description 3
- 102000016943 Muramidase Human genes 0.000 claims description 3
- 108010014251 Muramidase Proteins 0.000 claims description 3
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 3
- 238000012408 PCR amplification Methods 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- 239000002299 complementary DNA Substances 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 238000000502 dialysis Methods 0.000 claims description 3
- 239000004325 lysozyme Substances 0.000 claims description 3
- 229960000274 lysozyme Drugs 0.000 claims description 3
- 235000010335 lysozyme Nutrition 0.000 claims description 3
- 108091008146 restriction endonucleases Proteins 0.000 claims description 3
- 230000001131 transforming effect Effects 0.000 claims description 3
- 102000003960 Ligases Human genes 0.000 claims description 2
- 108090000364 Ligases Proteins 0.000 claims description 2
- 230000003321 amplification Effects 0.000 claims description 2
- 238000005119 centrifugation Methods 0.000 claims description 2
- 238000003199 nucleic acid amplification method Methods 0.000 claims description 2
- 230000002194 synthesizing effect Effects 0.000 claims description 2
- 238000009210 therapy by ultrasound Methods 0.000 claims description 2
- 238000011144 upstream manufacturing Methods 0.000 claims description 2
- 238000003776 cleavage reaction Methods 0.000 claims 2
- 230000007017 scission Effects 0.000 claims 2
- 238000000746 purification Methods 0.000 claims 1
- 238000001338 self-assembly Methods 0.000 abstract description 16
- 150000001413 amino acids Chemical class 0.000 abstract description 14
- 229940127554 medical product Drugs 0.000 abstract description 3
- 230000002596 correlated effect Effects 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract description 2
- 230000007935 neutral effect Effects 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 108010009111 arginyl-glycyl-glutamic acid Proteins 0.000 description 21
- LOEANKRDMMVOGZ-YUMQZZPRSA-N Gly-Lys-Asp Chemical compound NCCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O LOEANKRDMMVOGZ-YUMQZZPRSA-N 0.000 description 20
- AAORVPFVUIHEAB-YUMQZZPRSA-N Lys-Asp-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O AAORVPFVUIHEAB-YUMQZZPRSA-N 0.000 description 15
- 108010047857 aspartylglycine Proteins 0.000 description 13
- CAVKXZMMDNOZJU-UHFFFAOYSA-N Gly-Pro-Ala-Gly-Pro Natural products C1CCC(C(O)=O)N1C(=O)CNC(=O)C(C)NC(=O)C1CCCN1C(=O)CN CAVKXZMMDNOZJU-UHFFFAOYSA-N 0.000 description 12
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 10
- 108010078144 glutaminyl-glycine Proteins 0.000 description 9
- 108010029020 prolylglycine Proteins 0.000 description 9
- UUYBFNKHOCJCHT-VHSXEESVSA-N Gly-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN UUYBFNKHOCJCHT-VHSXEESVSA-N 0.000 description 8
- 108010015792 glycyllysine Proteins 0.000 description 8
- OTEWWRBKGONZBW-UHFFFAOYSA-N 2-[[2-[[2-[(2-azaniumylacetyl)amino]-4-methylpentanoyl]amino]acetyl]amino]acetate Chemical compound NCC(=O)NC(CC(C)C)C(=O)NCC(=O)NCC(O)=O OTEWWRBKGONZBW-UHFFFAOYSA-N 0.000 description 7
- NJPMYXWVWQWCSR-ACZMJKKPSA-N Ala-Glu-Asn Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O NJPMYXWVWQWCSR-ACZMJKKPSA-N 0.000 description 7
- MDNAVFBZPROEHO-DCAQKATOSA-N Ala-Lys-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O MDNAVFBZPROEHO-DCAQKATOSA-N 0.000 description 7
- MDNAVFBZPROEHO-UHFFFAOYSA-N Ala-Lys-Val Natural products CC(C)C(C(O)=O)NC(=O)C(NC(=O)C(C)N)CCCCN MDNAVFBZPROEHO-UHFFFAOYSA-N 0.000 description 7
- MZRBYBIQTIKERR-GUBZILKMSA-N Arg-Glu-Gln Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MZRBYBIQTIKERR-GUBZILKMSA-N 0.000 description 7
- AUFHLLPVPSMEOG-YUMQZZPRSA-N Arg-Gly-Glu Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AUFHLLPVPSMEOG-YUMQZZPRSA-N 0.000 description 7
- HSWYMWGDMPLTTH-FXQIFTODSA-N Asp-Glu-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HSWYMWGDMPLTTH-FXQIFTODSA-N 0.000 description 7
- PAYPSKIBMDHZPI-CIUDSAMLSA-N Asp-Leu-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O PAYPSKIBMDHZPI-CIUDSAMLSA-N 0.000 description 7
- SXIJQMBEVYWAQT-GUBZILKMSA-N Gln-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)N)N SXIJQMBEVYWAQT-GUBZILKMSA-N 0.000 description 7
- NSORZJXKUQFEKL-JGVFFNPUSA-N Gln-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCC(=O)N)N)C(=O)O NSORZJXKUQFEKL-JGVFFNPUSA-N 0.000 description 7
- LGYZYFFDELZWRS-DCAQKATOSA-N Glu-Glu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O LGYZYFFDELZWRS-DCAQKATOSA-N 0.000 description 7
- QSTLUOIOYLYLLF-WDSKDSINSA-N Gly-Asp-Glu Chemical compound [H]NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O QSTLUOIOYLYLLF-WDSKDSINSA-N 0.000 description 7
- BZKDJRSZWLPJNI-SRVKXCTJSA-N His-His-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O BZKDJRSZWLPJNI-SRVKXCTJSA-N 0.000 description 7
- DEMIXZCKUXVEBO-BWAGICSOSA-N His-Thr-Tyr Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N)O DEMIXZCKUXVEBO-BWAGICSOSA-N 0.000 description 7
- MJOZZTKJZQFKDK-GUBZILKMSA-N Leu-Ala-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(N)=O MJOZZTKJZQFKDK-GUBZILKMSA-N 0.000 description 7
- ZTLGVASZOIKNIX-DCAQKATOSA-N Leu-Gln-Glu Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZTLGVASZOIKNIX-DCAQKATOSA-N 0.000 description 7
- AUBMZAMQCOYSIC-MNXVOIDGSA-N Leu-Ile-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(N)=O)C(O)=O AUBMZAMQCOYSIC-MNXVOIDGSA-N 0.000 description 7
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 7
- YNNPKXBBRZVIRX-IHRRRGAJSA-N Lys-Arg-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O YNNPKXBBRZVIRX-IHRRRGAJSA-N 0.000 description 7
- YVSHZSUKQHNDHD-KKUMJFAQSA-N Lys-Asn-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCCN)N YVSHZSUKQHNDHD-KKUMJFAQSA-N 0.000 description 7
- DTUZCYRNEJDKSR-NHCYSSNCSA-N Lys-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN DTUZCYRNEJDKSR-NHCYSSNCSA-N 0.000 description 7
- LRALLISKBZNSKN-BQBZGAKWSA-N Met-Gly-Ser Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O LRALLISKBZNSKN-BQBZGAKWSA-N 0.000 description 7
- IHRFZLQEQVHXFA-RHYQMDGZSA-N Met-Thr-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCCCN IHRFZLQEQVHXFA-RHYQMDGZSA-N 0.000 description 7
- FDMCIBSQRKFSTJ-RHYQMDGZSA-N Pro-Thr-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O FDMCIBSQRKFSTJ-RHYQMDGZSA-N 0.000 description 7
- 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 7
- 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 7
- WMZVVNLPHFSUPA-BPUTZDHNSA-N Ser-Trp-Arg Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CO)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O)=CNC2=C1 WMZVVNLPHFSUPA-BPUTZDHNSA-N 0.000 description 7
- 108010062796 arginyllysine Proteins 0.000 description 7
- 108010054666 glycyl-leucyl-glycyl-glycine Proteins 0.000 description 7
- 108010040030 histidinoalanine Proteins 0.000 description 7
- 108010028295 histidylhistidine Proteins 0.000 description 7
- UTKUTMJSWKKHEM-WDSKDSINSA-N Glu-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O UTKUTMJSWKKHEM-WDSKDSINSA-N 0.000 description 6
- NLKVNZUFDPWPNL-YUMQZZPRSA-N Glu-Arg-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O NLKVNZUFDPWPNL-YUMQZZPRSA-N 0.000 description 6
- JMQFHZWESBGPFC-WDSKDSINSA-N Gly-Gln-Asp Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O JMQFHZWESBGPFC-WDSKDSINSA-N 0.000 description 6
- DHDOADIPGZTAHT-YUMQZZPRSA-N Gly-Glu-Arg Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N DHDOADIPGZTAHT-YUMQZZPRSA-N 0.000 description 6
- DCHQYSOGURGJST-FJXKBIBVSA-N Pro-Thr-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O DCHQYSOGURGJST-FJXKBIBVSA-N 0.000 description 6
- 108010047495 alanylglycine Proteins 0.000 description 6
- 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 6
- 108010003700 lysyl aspartic acid Proteins 0.000 description 6
- PZXPWHFYZXTFBI-YUMQZZPRSA-N Asp-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O PZXPWHFYZXTFBI-YUMQZZPRSA-N 0.000 description 5
- GPSHCSTUYOQPAI-JHEQGTHGSA-N Glu-Thr-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O GPSHCSTUYOQPAI-JHEQGTHGSA-N 0.000 description 5
- BULIVUZUDBHKKZ-WDSKDSINSA-N Gly-Gln-Asn Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O BULIVUZUDBHKKZ-WDSKDSINSA-N 0.000 description 5
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 5
- LCMWVZLBCUVDAZ-IUCAKERBSA-N Lys-Gly-Glu Chemical compound [NH3+]CCCC[C@H]([NH3+])C(=O)NCC(=O)N[C@H](C([O-])=O)CCC([O-])=O LCMWVZLBCUVDAZ-IUCAKERBSA-N 0.000 description 5
- 108010079364 N-glycylalanine Proteins 0.000 description 5
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 5
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 5
- 108010061238 threonyl-glycine Proteins 0.000 description 5
- UZSQXCMNUPKLCC-FJXKBIBVSA-N Arg-Thr-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UZSQXCMNUPKLCC-FJXKBIBVSA-N 0.000 description 4
- FGYPOQPQTUNESW-IUCAKERBSA-N Gln-Gly-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)N)N FGYPOQPQTUNESW-IUCAKERBSA-N 0.000 description 4
- PXHABOCPJVTGEK-BQBZGAKWSA-N Glu-Gln-Gly Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O PXHABOCPJVTGEK-BQBZGAKWSA-N 0.000 description 4
- DTRUBYPMMVPQPD-YUMQZZPRSA-N Gly-Gln-Arg Chemical compound [H]NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O DTRUBYPMMVPQPD-YUMQZZPRSA-N 0.000 description 4
- HFXJIZNEXNIZIJ-BQBZGAKWSA-N Gly-Glu-Gln Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HFXJIZNEXNIZIJ-BQBZGAKWSA-N 0.000 description 4
- JYPCXBJRLBHWME-IUCAKERBSA-N Gly-Pro-Arg Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O JYPCXBJRLBHWME-IUCAKERBSA-N 0.000 description 4
- PDTMWFVVNZYWTR-NHCYSSNCSA-N Ile-Gly-Lys Chemical compound CC[C@H](C)[C@H](N)C(=O)NCC(=O)N[C@@H](CCCCN)C(O)=O PDTMWFVVNZYWTR-NHCYSSNCSA-N 0.000 description 4
- DPWGZWUMUUJQDT-IUCAKERBSA-N Leu-Gln-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O DPWGZWUMUUJQDT-IUCAKERBSA-N 0.000 description 4
- UIMCLYYSUCIUJM-UWVGGRQHSA-N Pro-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 UIMCLYYSUCIUJM-UWVGGRQHSA-N 0.000 description 4
- MSIYNSBKKVMGFO-BHNWBGBOSA-N Thr-Gly-Pro Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N1CCC[C@@H]1C(=O)O)N)O MSIYNSBKKVMGFO-BHNWBGBOSA-N 0.000 description 4
- 108010025801 glycyl-prolyl-arginine Proteins 0.000 description 4
- 108010010147 glycylglutamine Proteins 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- ZVFVBBGVOILKPO-WHFBIAKZSA-N Ala-Gly-Ala Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O ZVFVBBGVOILKPO-WHFBIAKZSA-N 0.000 description 3
- BEMGNWZECGIJOI-WDSKDSINSA-N Ala-Gly-Glu Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O BEMGNWZECGIJOI-WDSKDSINSA-N 0.000 description 3
- NONSEUUPKITYQT-BQBZGAKWSA-N Arg-Asn-Gly Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N)CN=C(N)N NONSEUUPKITYQT-BQBZGAKWSA-N 0.000 description 3
- 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 3
- HAFCJCDJGIOYPW-WDSKDSINSA-N Asp-Gly-Gln Chemical compound OC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(N)=O HAFCJCDJGIOYPW-WDSKDSINSA-N 0.000 description 3
- QCVXMEHGFUMKCO-YUMQZZPRSA-N Asp-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O QCVXMEHGFUMKCO-YUMQZZPRSA-N 0.000 description 3
- ZPDVKYLJTOFQJV-WDSKDSINSA-N Gln-Asn-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O ZPDVKYLJTOFQJV-WDSKDSINSA-N 0.000 description 3
- VSXBYIJUAXPAAL-WDSKDSINSA-N Gln-Gly-Ala Chemical compound OC(=O)[C@H](C)NC(=O)CNC(=O)[C@@H](N)CCC(N)=O VSXBYIJUAXPAAL-WDSKDSINSA-N 0.000 description 3
- PAWIVEIWWYGBAM-YUMQZZPRSA-N Gly-Leu-Ala Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O PAWIVEIWWYGBAM-YUMQZZPRSA-N 0.000 description 3
- GGLIDLCEPDHEJO-BQBZGAKWSA-N Gly-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)CN GGLIDLCEPDHEJO-BQBZGAKWSA-N 0.000 description 3
- GLACUWHUYFBSPJ-FJXKBIBVSA-N Gly-Pro-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN GLACUWHUYFBSPJ-FJXKBIBVSA-N 0.000 description 3
- BMWFDYIYBAFROD-WPRPVWTQSA-N Gly-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN BMWFDYIYBAFROD-WPRPVWTQSA-N 0.000 description 3
- DFJJAVZIHDFOGQ-MNXVOIDGSA-N Ile-Glu-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O)N DFJJAVZIHDFOGQ-MNXVOIDGSA-N 0.000 description 3
- 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 3
- AFXCXDQNRXTSBD-FJXKBIBVSA-N Pro-Gly-Thr Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O AFXCXDQNRXTSBD-FJXKBIBVSA-N 0.000 description 3
- 239000003242 anti bacterial agent Substances 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 108010050848 glycylleucine Proteins 0.000 description 3
- 229930027917 kanamycin Natural products 0.000 description 3
- 229960000318 kanamycin Drugs 0.000 description 3
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 3
- 229930182823 kanamycin A Natural products 0.000 description 3
- 108010057821 leucylproline Proteins 0.000 description 3
- 108010054155 lysyllysine Proteins 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000013612 plasmid Substances 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 108010004914 prolylarginine Proteins 0.000 description 3
- IFTVANMRTIHKML-WDSKDSINSA-N Ala-Gln-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O IFTVANMRTIHKML-WDSKDSINSA-N 0.000 description 2
- SUHLZMHFRALVSY-YUMQZZPRSA-N Ala-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)NCC(O)=O SUHLZMHFRALVSY-YUMQZZPRSA-N 0.000 description 2
- 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 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- PGPJSRSLQNXBDT-YUMQZZPRSA-N Gln-Arg-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O PGPJSRSLQNXBDT-YUMQZZPRSA-N 0.000 description 2
- MFJAPSYJQJCQDN-BQBZGAKWSA-N Gln-Gly-Glu Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O MFJAPSYJQJCQDN-BQBZGAKWSA-N 0.000 description 2
- OCJRHJZKGGSPRW-IUCAKERBSA-N Glu-Lys-Gly Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O OCJRHJZKGGSPRW-IUCAKERBSA-N 0.000 description 2
- IWAXHBCACVWNHT-BQBZGAKWSA-N Gly-Asp-Arg Chemical compound NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IWAXHBCACVWNHT-BQBZGAKWSA-N 0.000 description 2
- MHHUEAIBJZWDBH-YUMQZZPRSA-N Gly-Asp-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)CN MHHUEAIBJZWDBH-YUMQZZPRSA-N 0.000 description 2
- MBOAPAXLTUSMQI-JHEQGTHGSA-N Gly-Glu-Thr Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MBOAPAXLTUSMQI-JHEQGTHGSA-N 0.000 description 2
- COVXELOAORHTND-LSJOCFKGSA-N Gly-Ile-Val Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C(C)C)C(O)=O COVXELOAORHTND-LSJOCFKGSA-N 0.000 description 2
- IEGFSKKANYKBDU-QWHCGFSZSA-N Gly-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)CN)C(=O)O IEGFSKKANYKBDU-QWHCGFSZSA-N 0.000 description 2
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 2
- XFIHDSBIPWEYJJ-YUMQZZPRSA-N Lys-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN XFIHDSBIPWEYJJ-YUMQZZPRSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- KIZQGKLMXKGDIV-BQBZGAKWSA-N Pro-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 KIZQGKLMXKGDIV-BQBZGAKWSA-N 0.000 description 2
- BNBBNGZZKQUWCD-IUCAKERBSA-N Pro-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H]1CCCN1 BNBBNGZZKQUWCD-IUCAKERBSA-N 0.000 description 2
- FKLSMYYLJHYPHH-UWVGGRQHSA-N Pro-Gly-Leu Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O FKLSMYYLJHYPHH-UWVGGRQHSA-N 0.000 description 2
- WIPAMEKBSHNFQE-IUCAKERBSA-N Pro-Met-Gly Chemical compound CSCC[C@@H](C(=O)NCC(=O)O)NC(=O)[C@@H]1CCCN1 WIPAMEKBSHNFQE-IUCAKERBSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 108010049041 glutamylalanine Proteins 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- CUVSTAMIHSSVKL-UWVGGRQHSA-N (4s)-4-[(2-aminoacetyl)amino]-5-[[(2s)-6-amino-1-(carboxymethylamino)-1-oxohexan-2-yl]amino]-5-oxopentanoic acid Chemical compound NCCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN CUVSTAMIHSSVKL-UWVGGRQHSA-N 0.000 description 1
- VWEWCZSUWOEEFM-WDSKDSINSA-N Ala-Gly-Ala-Gly Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(=O)NCC(O)=O VWEWCZSUWOEEFM-WDSKDSINSA-N 0.000 description 1
- BLIMFWGRQKRCGT-YUMQZZPRSA-N Ala-Gly-Lys Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN BLIMFWGRQKRCGT-YUMQZZPRSA-N 0.000 description 1
- MQIGTEQXYCRLGK-BQBZGAKWSA-N Ala-Gly-Pro Chemical compound C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O MQIGTEQXYCRLGK-BQBZGAKWSA-N 0.000 description 1
- CCDFBRZVTDDJNM-GUBZILKMSA-N Ala-Leu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O CCDFBRZVTDDJNM-GUBZILKMSA-N 0.000 description 1
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 1
- OQCWXQJLCDPRHV-UWVGGRQHSA-N Arg-Gly-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O OQCWXQJLCDPRHV-UWVGGRQHSA-N 0.000 description 1
- OLVIPTLKNSAYRJ-YUMQZZPRSA-N Asn-Gly-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N OLVIPTLKNSAYRJ-YUMQZZPRSA-N 0.000 description 1
- AXXCUABIFZPKPM-BQBZGAKWSA-N Asp-Arg-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O AXXCUABIFZPKPM-BQBZGAKWSA-N 0.000 description 1
- 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 1
- 238000001712 DNA sequencing Methods 0.000 description 1
- 241001198387 Escherichia coli BL21(DE3) Species 0.000 description 1
- WQWMZOIPXWSZNE-WDSKDSINSA-N Gln-Asp-Gly Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O WQWMZOIPXWSZNE-WDSKDSINSA-N 0.000 description 1
- FQCILXROGNOZON-YUMQZZPRSA-N Gln-Pro-Gly Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O FQCILXROGNOZON-YUMQZZPRSA-N 0.000 description 1
- HJARVELKOSZUEW-YUMQZZPRSA-N Gly-Pro-Gln Chemical compound [H]NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O HJARVELKOSZUEW-YUMQZZPRSA-N 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- KXUKTDGKLAOCQK-LSJOCFKGSA-N Ile-Val-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)NCC(O)=O KXUKTDGKLAOCQK-LSJOCFKGSA-N 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 1
- PDIDTSZKKFEDMB-UWVGGRQHSA-N Lys-Pro-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O PDIDTSZKKFEDMB-UWVGGRQHSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- MVBZBRKNZVJEKK-DTWKUNHWSA-N Met-Gly-Pro Chemical compound CSCC[C@@H](C(=O)NCC(=O)N1CCC[C@@H]1C(=O)O)N MVBZBRKNZVJEKK-DTWKUNHWSA-N 0.000 description 1
- MMJJFXWMCMJMQA-STQMWFEESA-N Phe-Pro-Gly Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)NCC(O)=O)C1=CC=CC=C1 MMJJFXWMCMJMQA-STQMWFEESA-N 0.000 description 1
- HFNPOYOKIPGAEI-SRVKXCTJSA-N Pro-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 HFNPOYOKIPGAEI-SRVKXCTJSA-N 0.000 description 1
- UCTIUWKCVNGEFH-OBJOEFQTSA-N Pro-Val-Gly-Pro Chemical compound N([C@@H](C(C)C)C(=O)NCC(=O)N1[C@@H](CCC1)C(O)=O)C(=O)[C@@H]1CCCN1 UCTIUWKCVNGEFH-OBJOEFQTSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 102000004079 Prolyl Hydroxylases Human genes 0.000 description 1
- 108010043005 Prolyl Hydroxylases Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- AQAMPXBRJJWPNI-JHEQGTHGSA-N Thr-Gly-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AQAMPXBRJJWPNI-JHEQGTHGSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000033444 hydroxylation Effects 0.000 description 1
- 238000005805 hydroxylation reaction Methods 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 108010064235 lysylglycine Proteins 0.000 description 1
- 239000012567 medical material Substances 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法，属于生物工程技术领域。为了解决现有重组类胶原蛋白自组装成水凝胶速度快的问题，本发明提供了一种重组Scl2类胶原蛋白，该蛋白通过在原有重组类胶原蛋白氨基酸序列基础上进行点突变获得。由本发明获得的重组类胶原蛋白成胶时间与氨基酸突变的个数呈正相关，同样具备规模化生产、批次间差异小及在中性条件下即可自组装成自支撑的水凝胶等特点。此外，该系列蛋白自组装成水凝胶时间存在显著差异，可组成一系列可变速的自组装水凝胶。利用本发明方法制备的重组类胶原蛋白不仅为其在医学用品等方面的商品化应用提供更多的选择，而且为研究类胶原蛋白自组装成胶开辟新的研究思路。
Description
技术领域
本发明属于生物工程技术领域，具体涉及一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法。
背景技术
胶原蛋白是动物结缔组织的主要成分，也是哺乳动物体内含量最多、分布最广的功能性蛋白(CN108659117A)。它约占人体总蛋白含量的三分之一，同时也是细胞外基质(ECM)的主要成分(CN101213308A)。目前，根据序列同源性及分子结构已在脊椎动物体内确定至少28种不同类型的胶原蛋白。尽管不同种类的胶原蛋白生物功能和物理特性差异巨大，但其均由三条由重复的Gly-Xaa-Yaa序列组成的多肽链以右手螺旋的形式构成。
在过去的几十年里，胶原蛋白在食品营养、化妆品、保健品、医学用品等方面发挥着重要作用(CN108057130A)(CN105709266A)(CN108472236A)。尤其作为医学材料，其在药物递送、组织移植等方面展现出的潜在价值，越来越受到人们关注(CN1210019A)。胶原蛋白主要来源于动物组织，存在着疯牛病等疾病风险，且不同批次间提取的蛋白存在差异，这使得以胶原蛋白为原料的应用受到一定的限制。此外，在酵母和植物表达系统中生产重组胶原蛋白，又必须在表达过程中引入脯氨酰4-羟化酶实现脯氨酸的羟化和重组胶原白的稳定，这大大增加规模化生产的难度(CN106256911A)。
在申请号为202110982807X的专利申请中，公开了不需要经过复杂改性方法就能自组装成一定强度水凝胶的重组细菌性类胶原蛋白。同时，其具备生物相容性良好、无免疫原性、无细胞毒性及能够在大肠杆菌中高效表达等优点。但是该重组蛋白自组装成水凝胶的时间很迅速，这限制其在生物医疗等方面的实际应用。因此，亟需开发出可变速的细菌性类胶原蛋白自组装水凝胶，使其为后续基于细菌性重组胶原蛋白的医用产品提供更多选择。
发明内容
本发明为了解决现有重组类胶原蛋白自组装成水凝胶速度快的问题，提供了一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法，具体技术方案如下：
本发明提供了一种重组Scl2类胶原蛋白，由胶原区域和非胶原区域组成，所述重组Scl2类胶原蛋白是在scl2类胶原蛋白的基础上将非胶原区域替换为如SEQ ID No.1或SEQ ID No.2或SEQ ID No.3或SEQ ID No.4或SEQ ID No.5或SEQ ID No.6或SEQ ID No.7任一所示的氨基酸序列，并将胶原区域替换为如SEQ ID No.8或SEQ ID No.9所示的氨基酸序列后获得的，所述scl2类胶原蛋白的Gene ID：110660。
本发明还提供了一种上述重组Scl2类胶原蛋白的制备方法，包括如下步骤：
(1)将化脓链球菌(Streptococcus pyogenes)Scl2.28的cDNA序列转入载体Pet28a中，获得重组载体Pet28a-Scl2，以重组载体Pet28a-Scl2为模板，根据所述重组Scl2类胶原蛋白的突变位点，设计引物，扩增DNA片段，合成重组scl2类胶原蛋白的全序列，并以合成后的全序列为模板，PCR扩增合成DNA片段，再经酶切、连接、转化大肠杆菌获得重组的Scl2类胶原蛋白重组表达载体；
(2)将步骤(1)获得的Scl2类胶原蛋白重组表达载体转化至大肠杆菌中获得阳性菌株；
(3)将步骤(2)获得的阳性菌株接种于LB培养基中培养至OD600达到0.4～0.6时，向菌液中加入IPTG，继续培养5h～6h，对菌液进行离心或过滤后，去除上清并收集菌体沉淀；
(4)向步骤(3)获得的菌体中加入蛋白提取液和溶菌酶，于冰上静置1h，获得蛋白溶液，经超声后，通过离心或过滤收集上清获得粗蛋白；
(5)利用已装镍的重力柱对步骤(4)获得的粗蛋白进行纯化，经水透析后获得重组后的类胶原蛋白。
进一步地限定，步骤(1)所述扩增DNA片段是将核苷酸序列如SEQ ID No.10所示的引物Scl2-F分别与核苷酸序列如SEQ ID No.11所示的引物Scl2-M1-1-R1、核苷酸序列如SEQ ID No.12所示的引物Scl2-M1-2-R1、核苷酸序列如SEQ ID No.13所示的引物Scl2--M1-3-R1、核苷酸序列如SEQ ID No.14所示的引物Scl2-M2-1-R1、核苷酸序列如SEQ IDNo.15所示的引物Scl2-M2-2-R1、核苷酸序列如SEQ ID No.16所示的引物Scl2-M2-3-R1、核苷酸序列如SEQ ID No.17所示的引物Scl2-M3-1-R1混合进行DNA片段扩增，并将核苷酸序列如SEQ ID No.18所示的引物Scl2-R分别与核苷酸序列如SEQ ID No.19所示的引物Scl2-M1-1-F1、核苷酸序列如SEQ ID No.20所示的引物Scl2-M1-2-F1、核苷酸序列如SEQ IDNo.21所示的引物Scl2--M1-3-F1、核苷酸序列如SEQ ID No.22所示的引物Scl2-M2-1-F1、核苷酸序列如SEQ ID No.23所示的引物Scl2-M2-2-F1、核苷酸序列如SEQ ID No.24所示的引物Scl2-M2-3-F1、核苷酸序列如SEQ ID No.25所示的引物Scl2-M3-1-F1混合进行DNA片段扩增。
进一步地限定，步骤(1)所述以合成后的全序列为模板PCR扩增合成DNA片段所用上游引物为核苷酸序列如SEQ ID No.10所示的Scl2-F，下游引物为核苷酸序列如SEQ IDNo.18所示的Scl2-R。
进一步地限定，步骤(1)所述酶切是将扩增后的DNA片段及Pet28a空载体经过NcoI和Xho I限制性内切酶进行双酶切。
进一步地限定，步骤(1)所述连接使用的连接酶为T4 DNA连接酶；所述转化利用的大肠杆菌为DH5α。
进一步地限定，步骤(5)所述利用已装镍的重力柱进行纯化的方法是将步骤(4)获得的粗蛋白溶液加入已装镍的重力柱中，待镍柱无剩余粗蛋白溶液时，加入BufferA进行杂蛋白的清洗，再加入Buffer B将目的蛋白洗脱下来。
进一步地限定，所述BufferA的组成为50mM Tris，250mM NaCl和30mM咪唑；所述Buffer B的组成为50mM Tris，250mM NaCl和400mM咪唑。
进一步地限定，步骤(5)所述透析共进行2次，每次6h。
本发明还提供了一种上述重组Scl2类胶原蛋白自组装可变速水凝胶的制备方法，是将所述重组类胶原蛋白与0.1～0.2M的盐酸或醋酸混合，制成25～50mg/mL的酸溶液，然后向酸溶液中加入2M的NaOH溶液将pH调节至6～8，室温静置即可成胶。
本发明的有益效果：
本发明在申请号为202110982807X的专利申请公开的重组类胶原蛋白的基础上，通过点突变的方式改变原有重组类胶原蛋白的氨基酸序列，进而获得了一系列自组装成水凝胶速度不同的重组类胶原蛋白。新开发的重组类胶原蛋白成胶时间与氨基酸突变的个数呈正相关。该系列蛋白同样具备规模化生产、批次间差异小及在中性条件下即可自组装成自支撑的水凝胶等特点。此外，利用本发明方法制备的重组类胶原蛋白能够采用稀醋酸溶解，自组装成水凝胶的反应条件更加温和。更加难得的是，该系列蛋白自组装成水凝胶时间存在显著差异，组成一系列可变速的自组装水凝胶。总而言之，利用本发明方法制备的重组类胶原蛋白不仅为其在医学用品等方面的商品化应用提供更多的选择，而且为研究类胶原蛋白自组装成胶开辟新的研究思路。
附图说明
图1为Scl-M1-2水凝胶扫描电镜图；
图2为Scl-M2-2水凝胶扫描电镜图；
图3为Scl-M3-1水凝胶扫描电镜图；
图4为Scl-M1-2水凝胶图；
图5为Scl-M2-2水凝胶图；
图6为Scl-M3-1水凝胶图。
具体实施方式
以下结合具体实施例和附图，对本发明作进一步的详细说明，本发明所用试剂及设备等均可通过商业化途径购买获得，涉及的PCR扩增，酶切连接、转化等分子生物学实验操作如无特殊说明，均为本领域常规实验操作或依照相应试剂的产品说明书进行。
实施例1：重组Scl2类胶原蛋白的制备方法
本发明中的重组Scl2类胶原蛋白，均由胶原区域和非胶原区域组成，非胶原区域位于胶原区域的羧基端。非胶原区域-1，非胶原区域-2，非胶原区域-3，非胶原区域-4，非胶原区域-5，非胶原区域-6，非胶原区域-7与胶原区域-1组成的蛋白分别命名为Scl2-M1-1，Scl2-M1-2，Scl2-M1-3，Scl2-M2-1，Scl2-M2-2，Scl2-M2-3和Scl2-M3-1；非胶原区域-1，非胶原区域-2，非胶原区域-3，非胶原区域-4，非胶原区域-5，非胶原区域-6，非胶原区域-7与胶原区域-2组成的蛋白分别命名为Scl2-2X-M1-1，Scl2-2X-M1-2，Scl2-2X-M1-3，Scl2-2X-M2-1，Scl2-2X-M2-2，Scl2-2X-M2-3和Scl2-2X-M3-1。
非胶原区域-1的氨基酸序列如SEQ ID No.1所示：5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTGLIQELAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’；
非胶原区域-2的氨基酸序列如SEQ ID No.2所示：5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTELIQGLAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
非胶原区域-3的氨基酸序列如SEQ ID No.3所示：5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTELIQELAQGLGGIGKKNFPTLGDEDLDHTY MTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
非胶原区域-4的氨基酸序列如SEQ ID No.4所示：5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTGLIQGLAQGLGGIEKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
非胶原区域-5的氨基酸序列如SEQ ID No.5所示：5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTGLIQELAQGLGGIGKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
非胶原区域-6的氨基酸序列如SEQ ID No.6所示：5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTELIQGLAQGLGGIGKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
非胶原区域-7的氨基酸序列如SEQ ID No.7所示：5’-MGSSHHHHHHSSGSSGDEQEEKAKVRTGLIQGLAQGLGGIGKKNFPTLGDEDLDHTYMTKLLTYLQEREQAENSWRKRLLKGIQDHALD-3’
胶原区域-1的氨基酸序列如SEQ ID No.8所示：5’-GQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGLAGQRGIVGLPGQRGERGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGDKGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPGAGAAGAGSAAALEHHHHHH-3’
胶原区域-2的氨基酸序列如SEQ ID No.9所示：5’-GQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGLAGQRGIVGLPGQRGERGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGDKGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQDGRNGERGEQGPTGPTGPAGPRGLQGLQGLQGERGEQGPTGPAGPRGFPGERGEQGPTGLAGKAGEAGAKGETGPAGPQGPRGEQGPQGLPGLAGQRGIVGLPGQRGERGEKGEAGAQGPAGPMGPAGERGEKGEPGTQGAKGDRGETGPVGPRGDKGETGERGEAGPAGKDGERGPVGPAGKDGQNGQDGLPGKDGKDGQNGKDGLPGKDGKDGQNGKDGLPGKDGKDGQDGKDGLPGKDGKDGLPGKDGKDGQPGKPLEHHHHHH-3’
重组Scl2类胶原蛋白的制备方法具体如下：
1)将化脓链球菌(Streptococcuspyogenes)Scl2.28的cDNA序列转入载体Pet28a中，获得重组载体Pet28a-Scl2，以重组载体Pet28a-Scl2为模板，将引物Scl2-F分别和引物Scl2-M1-1-R、引物Scl2-M1-2-R1、引物Scl2-M1-3-R1、引物Scl2-M2-1-R1、引物Scl2-M2-2-R1、引物Scl2-M2-3-R1、引物Scl2-M3-1-R1混合，扩增出Scl2-M1-1-1、Scl2-M1-2-1、Scl2-M1-3-1、Scl2-M2-1-1、Scl2-M2-2-1、Scl2-M2-3-1、Scl2-M3-1-1；
2)以重组载体Pet28a-Scl2为模板，分别使用引物Scl2-M1-1-F1、引物Scl2-M1-2-F1、引物Scl2-M1-3-F1、引物Scl2-M2-1-F1、引物Scl2-M2-2-F1、引物Scl2-M2-3-F1、引物Scl2-M3-1-F1和引物Scl2-R混合，扩增出Scl2-M1-1-2、Scl2-M1-2-2、Scl2-M1-3-2、Scl2-M2-1-2、Scl2-M2-2-2、Scl2-M2-3-2、Scl2-M3-1-2片段；
3)最后，再分别以片段Scl2-M1-1-1和Scl2-M1-1-2、Scl2-M1-2-1和Scl2-M1-2-2、Scl2-M1-3-1和Scl2-M1-3-2、Scl2-M2-1-1和Scl2-M2-1-2、Scl2-M2-2-1和Scl2-M2-2-2、Scl2-M2-3-1和Scl2-M2-3-2、Scl2-M3-1-1和Scl2-M3-1-2为模板，均用引物Scl2-F和Scl2-R扩增合成Scl2-M1-1、Scl2-M1-2、Scl2-M1-3、Scl2-M2-1、Scl2-M2-2、Scl2-M2-3、Scl2-M3-1全长DNA片段；
上述步骤中涉及的引物及其序列如表1所示。
表1引物及其序列
4)步骤3)扩增后的DNA片段及Pet28a空载体经过Nco I和Xho I限制性内切酶进行双酶切；
5)从步骤4)得到的酶切产物，用1％的琼脂糖凝胶电泳分离，切取目的片段；
6)在步骤5)得到的目的片段，用凝胶DNA回收试剂盒(TIANGEN)纯化；
7)将步骤6)纯化后的片段和载体在T4 DNA连接酶的作用下进行16℃过夜连接；
8)将步骤7)得到的连接产物，利用热激转化法转入大肠杆菌DH5α中，涂布至含有卡纳抗生素的固体LB培养基平板中；
9)在步骤8)得到的平板中挑取单克隆，转移至含有预先加入卡纳抗生素培养基的试管中，37℃震荡过夜培养；
10)将步骤9)得到的菌液收集，用质粒小提试剂盒(TIANGEN)制备质粒，并经DNA测序确认其序列；
11)将步骤10)测序正确的Scl2蛋白重组表达载体转化至大肠杆菌BL21(DE3)菌株中，涂布至含有卡纳抗生素的固体LB培养基平板中；
12)从步骤11)得到的平板中挑取单克隆，转移至含有预先加入卡纳抗生素培养基的试管中，37℃震荡过夜培养；
13)将步骤12)的菌液转移至预先加入卡纳抗生素的LB培养基中，37℃震荡培养至OD600达到0.4～0.6；
14)在步骤13)的菌液中加入IPTG，37℃培养5h～6h；
15)将步骤14)得到的菌液收集离心/过滤，除去上清并收集菌体沉淀；
16)向步骤15)的菌体沉淀中加入蛋白提取液和溶菌酶，冰上静止1h；
17)将步骤16)获得的蛋白溶液超声破碎，离心/过滤收集上清；
18)将步骤17)的粗蛋白溶液加入到已装镍的重力柱中；
19)待步骤18)的镍柱无剩余粗蛋白溶液时，加入BufferA(50mM Tris，250mMNaCl，30mM咪唑)进行杂蛋白的清洗；
20)在步骤19)的镍柱中加入Buffer B(50mM Tris，250mM NaCl，400mM咪唑)将目的蛋白洗脱下来；
21)将步骤20)洗脱下来的目的蛋白，直接用蒸馏水进行透析，透析时间为每次6h，共2次；
22)将步骤21)透析后的溶液进行冷冻风干即可获得重组后的细菌性类胶原蛋白。
Scl2-2X-M1-1蛋白，Scl2-2X-M1-2蛋白，Scl2-2X-M1-3蛋白，Scl2-2X-M2-1蛋白，Scl2-2X-M2-2蛋白，Scl2-2X-M2-3蛋白和Scl2-2X-M3-1蛋白的制备方法中涉及的引物与Scl2-M1-1蛋白，Scl2-M1-2蛋白，Scl2-M1-3蛋白，Scl2-M2-1蛋白，Scl2-M2-2蛋白，Scl2-M2-3蛋白和Scl2-M3-1蛋白的制备方法中涉及的引物相同，其他制备步骤也相同。
实施例2：可变速类胶原蛋白自组装水凝胶的制备方法
第一种：称取实施例1获得的重组Scl2类胶原蛋白，将其与0.1～0.2M HCl充分混合，配制成浓度为25～50mg/mL的酸溶液；之后缓慢加入2M NaOH调节溶液pH，使最终的PH在6～8；最后，室温静置即可成胶。Scl2-M1-1，Scl2-M1-2，Scl2-M1-3成胶时间为1～5min；Scl2-M2-1，Scl2-M2-2，Scl2-M2-3成胶时间为5～20min；Scl2-M3-1成胶时间为40～90min。
第二种：称取实施例1获得的重组的Scl2类胶原蛋白，将其与0.1～0.2M CH3COOH充分混合，配制成浓度为25～50mg/mL的酸溶液；之后缓慢加入2M NaOH调节溶液pH，使最终的PH在6～8；最后，室温静置即可成胶。Scl2-M1-1，Scl2-M1-2，Scl2-M1-3成胶时间为1～5min；Scl2-M2-1，Scl2-M2-2，Scl2-M2-3成胶时间为5～10min；Scl2-M3-1成胶时间为30～70min。
制备得到的Scl-M1-2水凝胶、Scl-M2-2水凝胶、Scl-M3-1水凝胶的扫描电镜图分别如图1、图2和图3所示。
由Scl-M1-2蛋白、Scl-M2-2蛋白和Scl-M3-1蛋白分别制备的水凝胶图分别如图4、图5和图6所示。由本发明制备的另外7种重组类胶原蛋白制成的水凝胶同样表现出自组装成水凝胶具有时间差异的特点。
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
SEQUENCE LISTING
<110> 中国科学院青岛生物能源与过程研究所
<120> 一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法
<160> 25
<170> PatentIn version 3.3
<210> 1
<211> 89
<212> PRT
<213> 人工合成
<400> 1
Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Ser Gly
1 5 10 15
Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Gly Leu Ile Gln Glu
20 25 30
Leu Ala Gln Gly Leu Gly Gly Ile Glu Lys Lys Asn Phe Pro Thr Leu
35 40 45
Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr Tyr
50 55 60
Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu Leu
65 70 75 80
Lys Gly Ile Gln Asp His Ala Leu Asp
85
<210> 2
<211> 89
<212> PRT
<213> 人工合成
<400> 2
Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Ser Gly
1 5 10 15
Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Glu Leu Ile Gln Gly
20 25 30
Leu Ala Gln Gly Leu Gly Gly Ile Glu Lys Lys Asn Phe Pro Thr Leu
35 40 45
Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr Tyr
50 55 60
Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu Leu
65 70 75 80
Lys Gly Ile Gln Asp His Ala Leu Asp
85
<210> 3
<211> 89
<212> PRT
<213> 人工合成
<400> 3
Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Ser Gly
1 5 10 15
Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Glu Leu Ile Gln Glu
20 25 30
Leu Ala Gln Gly Leu Gly Gly Ile Gly Lys Lys Asn Phe Pro Thr Leu
35 40 45
Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr Tyr
50 55 60
Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu Leu
65 70 75 80
Lys Gly Ile Gln Asp His Ala Leu Asp
85
<210> 4
<211> 89
<212> PRT
<213> 人工合成
<400> 4
Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Ser Gly
1 5 10 15
Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Gly Leu Ile Gln Gly
20 25 30
Leu Ala Gln Gly Leu Gly Gly Ile Glu Lys Lys Asn Phe Pro Thr Leu
35 40 45
Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr Tyr
50 55 60
Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu Leu
65 70 75 80
Lys Gly Ile Gln Asp His Ala Leu Asp
85
<210> 5
<211> 89
<212> PRT
<213> 人工合成
<400> 5
Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Ser Gly
1 5 10 15
Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Gly Leu Ile Gln Glu
20 25 30
Leu Ala Gln Gly Leu Gly Gly Ile Gly Lys Lys Asn Phe Pro Thr Leu
35 40 45
Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr Tyr
50 55 60
Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu Leu
65 70 75 80
Lys Gly Ile Gln Asp His Ala Leu Asp
85
<210> 6
<211> 89
<212> PRT
<213> 人工合成
<400> 6
Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Ser Gly
1 5 10 15
Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Glu Leu Ile Gln Gly
20 25 30
Leu Ala Gln Gly Leu Gly Gly Ile Gly Lys Lys Asn Phe Pro Thr Leu
35 40 45
Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr Tyr
50 55 60
Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu Leu
65 70 75 80
Lys Gly Ile Gln Asp His Ala Leu Asp
85
<210> 7
<211> 89
<212> PRT
<213> 人工合成
<400> 7
Met Gly Ser Ser His His His His His His Ser Ser Gly Ser Ser Gly
1 5 10 15
Asp Glu Gln Glu Glu Lys Ala Lys Val Arg Thr Gly Leu Ile Gln Gly
20 25 30
Leu Ala Gln Gly Leu Gly Gly Ile Gly Lys Lys Asn Phe Pro Thr Leu
35 40 45
Gly Asp Glu Asp Leu Asp His Thr Tyr Met Thr Lys Leu Leu Thr Tyr
50 55 60
Leu Gln Glu Arg Glu Gln Ala Glu Asn Ser Trp Arg Lys Arg Leu Leu
65 70 75 80
Lys Gly Ile Gln Asp His Ala Leu Asp
85
<210> 8
<211> 281
<212> PRT
<213> 人工合成
<400> 8
Gly Gln Asp Gly Arg Asn Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly
1 5 10 15
Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln Gly Leu Gln Gly Leu
20 25 30
Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro Ala Gly Pro Arg
35 40 45
Gly Phe Pro Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Leu Ala Gly
50 55 60
Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr Gly Pro Ala Gly Pro
65 70 75 80
Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Leu Ala
85 90 95
Gly Gln Arg Gly Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly
100 105 110
Glu Lys Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro
115 120 125
Ala Gly Glu Arg Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys
130 135 140
Gly Asp Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly Asp Lys Gly
145 150 155 160
Glu Thr Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly Glu
165 170 175
Arg Gly Pro Val Gly Pro Ala Gly Lys Asp Gly Gln Asn Gly Gln Asp
180 185 190
Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly
195 200 205
Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu
210 215 220
Pro Gly Lys Asp Gly Lys Asp Gly Gln Asp Gly Lys Asp Gly Leu Pro
225 230 235 240
Gly Lys Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly
245 250 255
Gln Pro Gly Lys Pro Gly Ala Gly Ala Ala Gly Ala Gly Ser Ala Ala
260 265 270
Ala Leu Glu His His His His His His
275 280
<210> 9
<211> 524
<212> PRT
<213> 人工合成
<400> 9
Gly Gln Asp Gly Arg Asn Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly
1 5 10 15
Pro Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln Gly Leu Gln Gly Leu
20 25 30
Gln Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro Ala Gly Pro Arg
35 40 45
Gly Phe Pro Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Leu Ala Gly
50 55 60
Lys Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr Gly Pro Ala Gly Pro
65 70 75 80
Gln Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Leu Ala
85 90 95
Gly Gln Arg Gly Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly
100 105 110
Glu Lys Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro
115 120 125
Ala Gly Glu Arg Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys
130 135 140
Gly Asp Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly Asp Lys Gly
145 150 155 160
Glu Thr Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly Glu
165 170 175
Arg Gly Pro Val Gly Pro Ala Gly Lys Asp Gly Gln Asn Gly Gln Asp
180 185 190
Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly
195 200 205
Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu
210 215 220
Pro Gly Lys Asp Gly Lys Asp Gly Gln Asp Gly Lys Asp Gly Leu Pro
225 230 235 240
Gly Lys Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly
245 250 255
Gln Asp Gly Arg Asn Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro
260 265 270
Thr Gly Pro Ala Gly Pro Arg Gly Leu Gln Gly Leu Gln Gly Leu Gln
275 280 285
Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Pro Ala Gly Pro Arg Gly
290 295 300
Phe Pro Gly Glu Arg Gly Glu Gln Gly Pro Thr Gly Leu Ala Gly Lys
305 310 315 320
Ala Gly Glu Ala Gly Ala Lys Gly Glu Thr Gly Pro Ala Gly Pro Gln
325 330 335
Gly Pro Arg Gly Glu Gln Gly Pro Gln Gly Leu Pro Gly Leu Ala Gly
340 345 350
Gln Arg Gly Ile Val Gly Leu Pro Gly Gln Arg Gly Glu Arg Gly Glu
355 360 365
Lys Gly Glu Ala Gly Ala Gln Gly Pro Ala Gly Pro Met Gly Pro Ala
370 375 380
Gly Glu Arg Gly Glu Lys Gly Glu Pro Gly Thr Gln Gly Ala Lys Gly
385 390 395 400
Asp Arg Gly Glu Thr Gly Pro Val Gly Pro Arg Gly Asp Lys Gly Glu
405 410 415
Thr Gly Glu Arg Gly Glu Ala Gly Pro Ala Gly Lys Asp Gly Glu Arg
420 425 430
Gly Pro Val Gly Pro Ala Gly Lys Asp Gly Gln Asn Gly Gln Asp Gly
435 440 445
Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu
450 455 460
Pro Gly Lys Asp Gly Lys Asp Gly Gln Asn Gly Lys Asp Gly Leu Pro
465 470 475 480
Gly Lys Asp Gly Lys Asp Gly Gln Asp Gly Lys Asp Gly Leu Pro Gly
485 490 495
Lys Asp Gly Lys Asp Gly Leu Pro Gly Lys Asp Gly Lys Asp Gly Gln
500 505 510
Pro Gly Lys Pro Leu Glu His His His His His His
515 520
<210> 10
<211> 25
<212> DNA
<213> 人工合成
<400> 10
tataccatgg gcagcagcca tcatc 25
<210> 11
<211> 28
<212> DNA
<213> 人工合成
<400> 11
accggtgcgc actttcgctt tttcttcc 28
<210> 12
<211> 25
<212> DNA
<213> 人工合成
<400> 12
accctgaatc agttcggtgc gcact 25
<210> 13
<211> 22
<212> DNA
<213> 人工合成
<400> 13
accaatgccg cccagacctt gc 22
<210> 14
<211> 25
<212> DNA
<213> 人工合成
<400> 14
ggtgcgcact ttcgcttttt cttcc 25
<210> 15
<211> 49
<212> DNA
<213> 人工合成
<400> 15
accttgcgcc agttcctgaa tcagaccggt gcgcactttc gctttttct 49
<210> 16
<211> 45
<212> DNA
<213> 人工合成
<400> 16
gcccagacct tgcgccagac cctgaatcag ttcggtgcgc acttt 45
<210> 17
<211> 49
<212> DNA
<213> 人工合成
<400> 17
accttgcgcc agaccctgaa tcagaccggt gcgcactttc gctttttct 49
<210> 18
<211> 22
<212> DNA
<213> 人工合成
<400> 18
gtgctcgagt gcggccgcgc ta 22
<210> 19
<211> 51
<212> DNA
<213> 人工合成
<400> 19
ggaagaaaaa gcgaaagtgc gcaccggtct gattcaggaa ctggcgcaag g 51
<210> 20
<211> 41
<212> DNA
<213> 人工合成
<400> 20
gcgcaccgaa ctgattcagg gtctggcgca aggtctgggc g 41
<210> 21
<211> 47
<212> DNA
<213> 人工合成
<400> 21
gcaaggtctg ggcggcattg gtaagaaaaa ctttccgacc ctgggcg 47
<210> 22
<211> 58
<212> DNA
<213> 人工合成
<400> 22
ggaagaaaaa gcgaaagtgc gcaccggtct gattcagggt ctggcgcaag gtctgggc 58
<210> 23
<211> 59
<212> DNA
<213> 人工合成
<400> 23
attcaggaac tggcgcaagg tctgggcggc attggtaaga aaaactttcc gaccctggg 59
<210> 24
<211> 55
<212> DNA
<213> 人工合成
<400> 24
ggtctggcgc aaggtctggg cggcattggt aagaaaaact ttccgaccct gggcg 55
<210> 25
<211> 59
<212> DNA
<213> 人工合成
<400> 25
attcagggtc tggcgcaagg tctgggcggc attggtaaga aaaactttcc gaccctggg 59
Claims (10)
1.一种重组Scl2类胶原蛋白，由胶原区域和非胶原区域组成，其特征在于，所述重组Scl2类胶原蛋白是在scl2类胶原蛋白的基础上将非胶原区域替换为如SEQ ID No.1或SEQID No.2或SEQ ID No.3或SEQ ID No.4或SEQ ID No.5或SEQ ID No.6或SEQ ID No.7任一所示的氨基酸序列，并将胶原区域替换为如SEQ ID No.8或SEQ ID No.9所示的氨基酸序列后获得的，所述scl2类胶原蛋白的Gene ID：110660。
2.一种权利要求1所述重组Scl2类胶原蛋白的制备方法，其特征在于，包括如下步骤：
(1)将化脓链球菌(Streptococcuspyogenes)Scl2.28的cDNA序列转入载体Pet28a中，获得重组载体Pet28a-Scl2，以重组载体Pet28a-Scl2为模板，根据所述重组Scl2类胶原蛋白的突变位点，设计引物，扩增DNA片段，合成重组scl2类胶原蛋白的全序列，并以合成后的全序列为模板，PCR扩增合成DNA片段，再经酶切、连接、转化大肠杆菌获得重组的Scl2类胶原蛋白重组表达载体；
(2)将步骤(1)获得的Scl2类胶原蛋白重组表达载体转化至大肠杆菌中获得阳性菌株；
(3)将步骤(2)获得的阳性菌株接种于LB培养基中培养至OD600达到0.4～0.6时，向菌液中加入IPTG，继续培养5h～6h，对菌液进行离心或过滤后，去除上清并收集菌体沉淀；
(4)向步骤(3)获得的菌体中加入蛋白提取液和溶菌酶，于冰上静置1h，获得蛋白溶液，经超声后，通过离心或过滤收集上清获得粗蛋白；
(5)利用已装镍的重力柱对步骤(4)获得的粗蛋白进行纯化，经水透析后获得重组后的类胶原蛋白。
3.根据权利要求2所述的制备方法，其特征在于，步骤(1)所述扩增DNA片段是将核苷酸序列如SEQ ID No.10所示的引物Scl2-F分别与核苷酸序列如SEQ ID No.11所示的引物Scl2-M1-1-R1、核苷酸序列如SEQ ID No.12所示的引物Scl2-M1-2-R1、核苷酸序列如SEQID No.13所示的引物Scl2--M1-3-R1、核苷酸序列如SEQ ID No.14所示的引物Scl2-M2-1-R1、核苷酸序列如SEQ ID No.15所示的引物Scl2-M2-2-R1、核苷酸序列如SEQ ID No.16所示的引物Scl2-M2-3-R1、核苷酸序列如SEQ ID No.17所示的引物Scl2-M3-1-R1混合进行DNA片段扩增，并将核苷酸序列如SEQ ID No.18所示的引物Scl2-R分别与核苷酸序列如SEQID No.19所示的引物Scl2-M1-1-F1、核苷酸序列如SEQ ID No.20所示的引物Scl2-M1-2-F1、核苷酸序列如SEQ ID No.21所示的引物Scl2--M1-3-F1、核苷酸序列如SEQ ID No.22所示的引物Scl2-M2-1-F1、核苷酸序列如SEQ ID No.23所示的引物Scl2-M2-2-F1、核苷酸序列如SEQ ID No.24所示的引物Scl2-M2-3-F1、核苷酸序列如SEQ ID No.25所示的引物Scl2-M3-1-F1混合进行DNA片段扩增。
4.根据权利要求2所述的制备方法，其特征在于，步骤(1)所述以合成后的全序列为模板PCR扩增合成DNA片段所用上游引物为核苷酸序列如SEQ ID No.10所示的Scl2-F，下游引物为核苷酸序列如SEQ ID No.18所示的Scl2-R。
5.根据权利要求2所述的制备方法，其特征在于，步骤(1)所述酶切是将扩增后的DNA片段及Pet28a空载体经过Nco I和Xho I限制性内切酶进行双酶切。
6.根据权利要求2所述的制备方法，其特征在于，步骤(1)所述连接使用的连接酶为T4DNA连接酶；所述转化利用的大肠杆菌为DH5α。
7.根据权利要求2所述的制备方法，其特征在于，步骤(5)所述利用已装镍的重力柱进行纯化的方法是将步骤(4)获得的粗蛋白溶液加入已装镍的重力柱中，待镍柱无剩余粗蛋白溶液时，加入BufferA进行杂蛋白的清洗，再加入Buffer B将目的蛋白洗脱下来。
8.根据权利要求7所述的制备方法，其特征在于，所述BufferA的组成为50mM Tris，250mM NaCl和30mM咪唑；所述Buffer B的组成为50mM Tris，250mM NaCl和400mM咪唑。
9.根据权利要求2所述的制备方法，其特征在于，步骤(5)所述透析共进行2次，每次6h。
10.一种权利要求1所述的重组Scl2类胶原蛋白自组装可变速水凝胶的制备方法，其特征在于，将所述重组类胶原蛋白与0.1～0.2M的盐酸或醋酸混合，制成25～50mg/mL的酸溶液，然后向酸溶液中加入2M的NaOH溶液将pH调节至6～8，室温静置即可成胶。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111608133.3A CN114262368B (zh) | 2021-12-23 | 2021-12-23 | 一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111608133.3A CN114262368B (zh) | 2021-12-23 | 2021-12-23 | 一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114262368A true CN114262368A (zh) | 2022-04-01 |
| CN114262368B CN114262368B (zh) | 2024-04-23 |
Family
ID=80830033
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111608133.3A Active CN114262368B (zh) | 2021-12-23 | 2021-12-23 | 一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114262368B (zh) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116407682A (zh) * | 2022-11-09 | 2023-07-11 | 内蒙古和讯生物科技有限公司 | 一种基因编码的重组类胶原蛋白超分子水凝胶制备方法和应用 |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060035336A1 (en) * | 2000-02-22 | 2006-02-16 | Texas A&M University System | Prokaryotic collagen-like proteins and uses thereof |
| CN111704663A (zh) * | 2020-06-29 | 2020-09-25 | 江南大学 | 一种胶原蛋白水凝胶的制备方法 |
-
2021
- 2021-12-23 CN CN202111608133.3A patent/CN114262368B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060035336A1 (en) * | 2000-02-22 | 2006-02-16 | Texas A&M University System | Prokaryotic collagen-like proteins and uses thereof |
| CN111704663A (zh) * | 2020-06-29 | 2020-09-25 | 江南大学 | 一种胶原蛋白水凝胶的制备方法 |
Non-Patent Citations (2)
| Title |
|---|
| NEUNGSEON SEO, 等: "An engineered alpha1 intergrin-binding collagenous sequence", J BIOL CHEM, vol. 285 * |
| PARESH A. PARMAR, 等: "Collagen-mimetic prptide-modifiable hydrogels for articular cartilage regeneration", BIOMATERIALS, vol. 54 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116407682A (zh) * | 2022-11-09 | 2023-07-11 | 内蒙古和讯生物科技有限公司 | 一种基因编码的重组类胶原蛋白超分子水凝胶制备方法和应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114262368B (zh) | 2024-04-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN115975047B (zh) | 一种重组融合蛋白生产多肽的方法及其应用 | |
| CN114262368B (zh) | 一种重组Scl2类胶原蛋白及其可变速水凝胶的制备方法 | |
| CN114181321B (zh) | 花鲈fgf6a、fgf6b以及fgf18重组蛋白及其制备方法和应用 | |
| CN112745385A (zh) | 重组人源化胶原蛋白及其产业化制备方法与产品应用 | |
| CN114149954A (zh) | 利用谷氨酸棒状杆菌高效分泌生产类蛛丝、类弹性蛋白并快速纯化的方法 | |
| CN107488639B (zh) | 甲苯单加氧酶及其在手性亚砜生物催化合成中的应用 | |
| CN114107353A (zh) | 一种高效表达多肽毒素的质粒及其制备方法与应用 | |
| CN110563822B (zh) | 灵芝免疫调节蛋白突变体及应用 | |
| CN113801233A (zh) | 一种索玛鲁肽的制备方法 | |
| CN110317813B (zh) | 三疣梭子蟹C型凝集素PtCLec2基因及其编码蛋白和应用 | |
| CN116554309A (zh) | 一种重组人源ⅲ型胶原蛋白及其制备方法和应用 | |
| CN113249288B9 (zh) | 一种表达glp-1类似物的重组菌及其应用 | |
| CN107936107B (zh) | 长牡蛎干扰素调节因子CgIRF-1基因重组蛋白、制备方法及应用 | |
| CN113980880B (zh) | 基因工程菌及其应用和以葡萄糖为原料生产阿洛酮糖的方法 | |
| CN112516288B (zh) | N-糖基化修饰之灵芝免疫调节蛋白的应用 | |
| CN102277371A (zh) | 脑钠肽的制备方法 | |
| CN114790474A (zh) | 一种索马鲁肽的制备方法 | |
| CN114409763A (zh) | 一种重组纤连蛋白肽的纯化方法 | |
| CN112646044A (zh) | TFF2-Fc融合蛋白及其高效表达生产方法 | |
| CN115724924A (zh) | 一种可自组装成胶的重组类胶原蛋白及其制备方法与应用 | |
| CN1448510A (zh) | 一种重组人碱性成纤维细胞生长因子的高效表达 | |
| CN113773392A (zh) | 一种甘精胰岛素的制备方法 | |
| CN113801235A (zh) | 一种赖脯胰岛素衍生物及其应用 | |
| CN114057886B (zh) | 一种索玛鲁肽衍生物及其制备方法 | |
| CN113773391B (zh) | 一种门冬胰岛素的制备方法 |
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 |