CN110540601B - Recombinant PLB-hEGF fusion protein and application thereof - Google Patents
Recombinant PLB-hEGF fusion protein and application thereof Download PDFInfo
- Publication number
- CN110540601B CN110540601B CN201910699099.1A CN201910699099A CN110540601B CN 110540601 B CN110540601 B CN 110540601B CN 201910699099 A CN201910699099 A CN 201910699099A CN 110540601 B CN110540601 B CN 110540601B
- Authority
- CN
- China
- Prior art keywords
- hegf
- fusion protein
- plb
- recombinant
- protein
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 88
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 88
- 230000014509 gene expression Effects 0.000 claims abstract description 45
- 101500025419 Homo sapiens Epidermal growth factor Proteins 0.000 claims abstract description 43
- 239000013604 expression vector Substances 0.000 claims abstract description 31
- 108010026228 mRNA guanylyltransferase Proteins 0.000 claims abstract description 16
- 108090000623 proteins and genes Proteins 0.000 claims description 37
- 241000894006 Bacteria Species 0.000 claims description 12
- 239000002773 nucleotide Substances 0.000 claims description 4
- 125000003729 nucleotide group Chemical group 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 25
- 150000001413 amino acids Chemical group 0.000 abstract description 13
- 238000000034 method Methods 0.000 abstract description 13
- 230000008569 process Effects 0.000 abstract description 7
- 230000001976 improved effect Effects 0.000 abstract description 6
- 238000004153 renaturation Methods 0.000 abstract description 6
- 238000000338 in vitro Methods 0.000 abstract description 5
- 238000009776 industrial production Methods 0.000 abstract 1
- GVUGOAYIVIDWIO-UFWWTJHBSA-N nepidermin Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](NC(=O)[C@H](CS)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CS)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C(C)C)C(C)C)C1=CC=C(O)C=C1 GVUGOAYIVIDWIO-UFWWTJHBSA-N 0.000 description 35
- 229940116978 human epidermal growth factor Drugs 0.000 description 34
- 102000004169 proteins and genes Human genes 0.000 description 23
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 description 22
- 101800003838 Epidermal growth factor Proteins 0.000 description 21
- 102400001368 Epidermal growth factor Human genes 0.000 description 21
- 102100037883 Phospholipase B1, membrane-associated Human genes 0.000 description 21
- 229940116977 epidermal growth factor Drugs 0.000 description 21
- 238000003752 polymerase chain reaction Methods 0.000 description 20
- 239000013598 vector Substances 0.000 description 20
- 235000018102 proteins Nutrition 0.000 description 19
- 101001096022 Homo sapiens Phospholipase B1, membrane-associated Proteins 0.000 description 15
- 239000013612 plasmid Substances 0.000 description 14
- 108091026890 Coding region Proteins 0.000 description 13
- 108020004414 DNA Proteins 0.000 description 13
- 235000014304 histidine Nutrition 0.000 description 11
- 108091005804 Peptidases Proteins 0.000 description 10
- 239000004365 Protease Substances 0.000 description 10
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 10
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 10
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 9
- 239000013613 expression plasmid Substances 0.000 description 8
- 210000003000 inclusion body Anatomy 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 210000001236 prokaryotic cell Anatomy 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000000246 agarose gel electrophoresis Methods 0.000 description 6
- 235000001014 amino acid Nutrition 0.000 description 6
- 230000001131 transforming effect Effects 0.000 description 6
- 102000012410 DNA Ligases Human genes 0.000 description 5
- 108010061982 DNA Ligases Proteins 0.000 description 5
- 108010074860 Factor Xa Proteins 0.000 description 5
- 238000010367 cloning Methods 0.000 description 5
- 238000001976 enzyme digestion Methods 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 230000006698 induction Effects 0.000 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 description 5
- 229930027917 kanamycin Natural products 0.000 description 5
- 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 5
- 229960000318 kanamycin Drugs 0.000 description 5
- 229930182823 kanamycin A Natural products 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- 108090000765 processed proteins & peptides Proteins 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000012216 screening Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000012258 culturing Methods 0.000 description 4
- 238000013461 design Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 229940088598 enzyme Drugs 0.000 description 4
- 230000001939 inductive effect Effects 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical compound CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 3
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 3
- 238000009004 PCR Kit Methods 0.000 description 3
- 238000000137 annealing Methods 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 230000010261 cell growth Effects 0.000 description 3
- 238000003776 cleavage reaction Methods 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 230000006872 improvement Effects 0.000 description 3
- 238000005457 optimization Methods 0.000 description 3
- 238000012257 pre-denaturation Methods 0.000 description 3
- 230000001737 promoting effect Effects 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000007017 scission Effects 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 230000014621 translational initiation Effects 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- 108010076804 DNA Restriction Enzymes Proteins 0.000 description 2
- 238000001712 DNA sequencing Methods 0.000 description 2
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 108010013369 Enteropeptidase Proteins 0.000 description 2
- 102100029727 Enteropeptidase Human genes 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 238000012300 Sequence Analysis Methods 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 241001052560 Thallis Species 0.000 description 2
- 108090000190 Thrombin Proteins 0.000 description 2
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 230000036531 allelopathy Effects 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- 238000005520 cutting process Methods 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000013492 plasmid preparation Methods 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 239000000741 silica gel Substances 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 230000002194 synthesizing effect Effects 0.000 description 2
- 229960004072 thrombin Drugs 0.000 description 2
- 230000037314 wound repair Effects 0.000 description 2
- QFVHZQCOUORWEI-UHFFFAOYSA-N 4-[(4-anilino-5-sulfonaphthalen-1-yl)diazenyl]-5-hydroxynaphthalene-2,7-disulfonic acid Chemical compound C=12C(O)=CC(S(O)(=O)=O)=CC2=CC(S(O)(=O)=O)=CC=1N=NC(C1=CC=CC(=C11)S(O)(=O)=O)=CC=C1NC1=CC=CC=C1 QFVHZQCOUORWEI-UHFFFAOYSA-N 0.000 description 1
- 101800000263 Acidic protein Proteins 0.000 description 1
- 108010089921 CTCGAG-specific type II deoxyribonucleases Proteins 0.000 description 1
- 102000001301 EGF receptor Human genes 0.000 description 1
- 108060006698 EGF receptor Proteins 0.000 description 1
- 241000192016 Finegoldia magna Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101000851176 Homo sapiens Pro-epidermal growth factor Proteins 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- 108091036060 Linker DNA Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108091081024 Start codon Proteins 0.000 description 1
- 241000194017 Streptococcus Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 238000001261 affinity purification Methods 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000003196 chaotropic effect Effects 0.000 description 1
- 230000001876 chaperonelike Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- 108010021310 endodeoxyribonuclease NcoI Proteins 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 150000002411 histidines Chemical class 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000002297 mitogenic effect Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 101150008001 pl gene Proteins 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229910021487 silica fume Inorganic materials 0.000 description 1
- 229910002027 silica gel Inorganic materials 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000006257 total synthesis reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000013519 translation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/315—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Streptococcus (G), e.g. Enterococci
-
- 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
- C07K14/475—Growth factors; Growth regulators
- C07K14/485—Epidermal growth factor [EGF], i.e. urogastrone
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/50—Fusion polypeptide containing protease site
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Toxicology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention discloses a recombinant PLB-hEGF fusion protein and application thereof, wherein the recombinant PLB-hEGF fusion protein comprises a fusion protein obtained by fusing a B structure domain of a protein L and a human epidermal growth factor protein, wherein the B structure domain of the protein L comprises a sequence shown in SEQ ID NO: 1-5, wherein the amino acid sequence of the human epidermal growth factor protein is shown as SEQ ID NO: shown in fig. 8. When the recombinant PLB-hEGF fusion protein provided by the invention is constructed into a fusion protein expression vector for expressing hEGF, the soluble expression of hEGF is improved, the time-consuming process of in vitro renaturation is saved, the specific activity is obviously improved, and the recombinant PLB-hEGF fusion protein has important value for the industrial production of soluble hEGF with high specific activity.
Description
Technical Field
The invention relates to the technical field of genetic engineering and protein engineering, in particular to the technical field of construction of recombinant human epidermal growth factor fusion protein, and specifically relates to a recombinant PLB-hEGF fusion protein and application thereof.
Background
Protein L (Protein L, PL) is a Protein which was first found in 1988 on the cell wall of Streptococcus macrodigestus (Peptostreptococcus magnus), and was subsequently isolated and identified as "Protein L" because it binds to the L chain (light chain) of an antibody. PL consists of 719 amino acid residues, has a molecular weight of about 76kD, does not contain any disulfide bonds in the molecule, and does not have any disulfide-linked subunits. It is an acidic protein molecule with an isoelectric point (pI) of 4.0. It differs from protein a (protein a) and protein g (protein g) in that it does not bind to the Fc region of an antibody, but rather binds to the Kappa-type light chain (L) of an antibody.
The PL gene contains 5 coding elements: 1 signal peptide consisting of 18AA, 1 ' A ' region consisting of 79AA at the N-terminal, 5 ' B ' regions with 72-76 AA homologous repeats and a ' C ' region with 2 ' 52AA repeats at the C-terminal. The C region is followed by a hydrophilic proline-rich "W" region, which is presumed to be the "transmural region" of the bacterial cell wall, and a final hydrophobic "M" region, which should be the "anchoring region" of the bacterial cell membrane.
Epidermal Growth Factor (EGF) is a type of Growth Factor that is widely found in humans and animals. EGF may promote a variety of small mitogenic proteins for cell growth. The mature peptide contains 53 amino acids, the molecular weight is about 6kD, the mature peptide contains 3 disulfide bonds, the bioactivity of EGF extracted from natural tissues is as high as over 1000 ten thousand International Units (IU)/mg, and the international and domestic pharmaceutical standard is only more than or equal to 50 ten thousand IU/mg, and the mature peptide is 1/20 of the specific activity of natural EGF. It can be seen that the mismatch rate of 3 disulfide bonds in the molecule can reach 95% (19/20), which indicates that there is a great room for improving the correct folding rate and thus the specific activity of EGF, and has important medical industrial value for saving EGF process cost and improving the biological effect of EGF.
The specific activity of natural Human Epidermal Growth Factor (hEGF) can reach over 1000 ten thousand International Units (IU), while the standard of Human pharmacy is only equal to or more than 50 ten thousand IU, which is only 1/20 of the specific activity of natural EGF, so that the space for improving the specific activity of the recombinant hEGF is still huge.
Loss of recombinant hEGF specific activity is due primarily to inclusion body renaturation steps upon in vitro expression. The hEGF molecule has 7 cysteine residues to form 3 pairs of disulfide bonds, so that the 3-level structure of EGF is maintained to be stable, and a three-position space closely combined with EGF receptors is formed. When the inclusion body is expressed, the EGF inclusion body protein is dissolved and denatured by urea or guanidine chaotropic salt, and then in vitro renaturation is carried out to recover the EGF 3-level structure (Re-folding) so as to obtain the soluble EGF. This renaturation process is incomplete and there may be partial disulfide mismatch, thus making it impossible to satisfy the specific activity requirement of pharmaceutical raw materials of EGF even if renaturated soluble EGF, often with a specific activity of less than 50 ten thousand IU.
Since EGF has the effect of widely promoting cell growth, application research and demand of EGF in the fields of wound repair and skin care are promoted, the market demand for EGF is continuously increased, but the price of qualified EGF raw materials is still high, so that the improvement of EGF yield and specific activity has important industrial value.
Disclosure of Invention
The invention mainly aims to provide a recombinant PLB1-hEGF fusion protein and application thereof, aiming at improving the soluble expression of hEGF.
In order to achieve the above object, the present invention provides a recombinant PLB-hEGF fusion protein, comprising a fusion protein obtained by fusing a B domain of Protein L (PLB) with a human epidermal growth factor (hEGF) protein, wherein the B domain of protein L comprises a sequence as set forth in SEQ ID NO: 1-5, wherein the amino acid sequence of the human epidermal growth factor protein is shown as SEQ ID NO: shown in fig. 8.
Optionally, the B domain of the protein L is as set forth in SEQ ID NO:1, PLB1 protein.
Optionally, a translation initiation optimization sequence is added to the N terminal of the PLB1 protein to form an N terminal optimized PLB1 protein, and the amino acid sequence of the N terminal optimized PLB1 protein is shown in SEQ ID NO: shown at 7.
Optionally, the C-terminal of the N-terminal optimized PLB1 protein is added with a flexible linker, a protease recognition region, an optimized hEGF coding region and a histidine tag, wherein,
the flexible joint comprises 6-30 amino acids;
the protease recognition area comprises any one of a blood coagulation factor Xa protease recognition area, a thrombin recognition area, an enterokinase recognition area and a TEV enzyme recognition area;
the histidine tag comprises 5-9 His.
Optionally, the flexible linker comprises 8-20 amino acids;
the protease recognition region is a blood coagulation factor Xa protease recognition region;
the histidine tag comprises 6 His.
Optionally, the sequence of the flexible linker is as set forth in SEQ ID NO:14, 15;
the sequence of the blood coagulation factor Xa protease recognition region is shown in SEQ ID NO:16, 17;
the sequence of the histidine tag is shown as SEQ ID NO: 18, 19.
Alternatively, the amino acid sequence of the recombinant PLB1-hEGF fusion protein is as set forth in SEQ ID NO: shown at 10.
The invention also provides a coding gene of the recombinant PLB-hEGF fusion protein.
Optionally, the nucleotide sequence of the coding gene of the recombinant PLB-hEGF fusion protein is as set forth in SEQ ID NO: shown at 11.
The invention further provides a recombinant PLB-hEGF fusion protein expression vector or an expression engineering bacterium containing the coding gene of the recombinant PLB-hEGF fusion protein.
Optionally, the recombinant PLB-hEGF fusion protein expression vector is obtained by inserting the coding gene of the recombinant PLB-hEGF fusion protein between the NcoI and XhoI cleavage sites of the prokaryotic cell expression plasmid.
Optionally, the prokaryotic cell expression plasmid selects pET28 as a parent expression vector.
The invention also provides the application of the recombinant PLB-hEGF fusion protein expression vector in expressing the PLB-hEGF fusion protein.
The invention also provides a preparation method of the recombinant PLB-hEGF fusion protein, which comprises the following steps: and transforming the recombinant PLB-hEGF fusion protein expression vector into a competent expression strain to obtain expression engineering bacteria, and culturing the expression engineering bacteria to obtain the recombinant PLB-hEGF fusion protein.
In addition, the invention also provides a preparation method of the recombinant PLB-hEGF fusion protein expression engineering bacterium, which comprises the following steps:
a full-gene artificial synthesis method based on polymerase chain reaction synthesizes a DNA coding sequence of a fusion protein PLB-hEGF of PLB and hEGF, wherein the sequence of the fusion protein PLB-hEGF is shown as SEQ ID NO:10, and the DNA coding sequence is shown as SEQ ID NO:11 is shown in the figure;
carrying out NocI and XhoI DNA restriction enzyme digestion and agarose gel electrophoresis separation and purification on the encoding DNA of the fusion protein PLB-hEGF to obtain an insert fragment;
performing double enzyme digestion by endonuclease NocI and XhoI and separating and purifying prokaryotic cell expression plasmid by agarose gel electrophoresis to obtain a linearized empty vector;
connecting the linearized empty vector with a coding DNA fragment of the fusion protein PLB-hEGF by using T4DNA ligase, then transforming an allelopathy strain DH5 alpha, and obtaining an engineering plasmid with a correct sequence after positive cloning amplification, plasmid preparation and DNA sequencing;
and transforming BL21(DE3) competent expression strain with the engineering plasmid, and screening and performing induced expression test in the presence of kanamycin to obtain the recombinant PLB-hEGF fusion protein expression engineering strain.
According to the technical scheme provided by the invention, the B structural domain of the protein L is fused with the human epidermal growth factor protein to obtain the PLB-hEGF fusion protein, and when the fusion protein expression vector constructed by using the PLB-hEGF fusion protein is used for expressing hEGF, the soluble expression of hEGF is improved, the time-consuming process of in vitro renaturation is saved, the specific activity is obviously improved, and the method has important value for industrially producing soluble and high-specific-activity hEGF.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other related drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic diagram of an embodiment of a PLB1-hEGF fusion protein expression cassette of the fusion protein expression vector of the present invention;
FIG. 2 is a physical diagram of pET28-PLB1-hEGF expression vector prepared in example 1;
FIG. 3 is a physical diagram of pET30-hEGF control expression vector prepared in example 1;
FIG. 4 is a SDS-PAGE pattern of expression induced by the pET28-PLB1-hEGF expression vector assay in example 2;
FIG. 5 is a SDS-PAGE pattern of expression induction from pET30-hEGF control expression vector test in example 2.
The implementation, functional features and advantages of the objects of the present invention will be further explained with reference to the accompanying drawings.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
Since EGF has the effect of widely promoting cell growth, thereby promoting the application research and the demand of EGF in the fields of wound repair and skin care, the market demand for EGF is continuously increased, but the price of qualified (high specific activity) human EGF raw materials is still high, so that the improvement of the yield and the specific activity of hEGF has important industrial value.
In order to solve the technical problems, in the invention, in the process of exploring the recombinant expression of PL, the B region structural domain still has a good self-folding function in Escherichia coli after 79AA at the N-terminal are removed, even after B2 and B4 are removed, the coding regions of B1, B3 and B5 are combined, the self-folding and full-soluble fusion expression is still maintained, and the highly self-folding soluble protein has the effect of inducing the folding of other proteins to a certain extent, namely the effect of 'chaperone-like protein'. Therefore, the invention tries to fuse the molecular folding induction effect of other proteins which are not easy to fold in the research process, and the folding induction effect of downstream fusion proteins with multiple disulfide bonds or without disulfide bonds can achieve obvious effect. Based on the above, the invention provides a recombinant PLB-hEGF fusion protein, comprising a fusion protein obtained by fusing a B domain of a protein L with a human epidermal growth factor protein, wherein the B domain of the protein L comprises a sequence as shown in SEQ ID NO: 1-5, wherein the amino acid sequence of the human epidermal growth factor protein is shown as SEQ ID NO: shown in fig. 8.
According to the technical scheme provided by the invention, the B structural domain of the protein L is fused with the human epidermal growth factor protein to obtain the PLB-hEGF fusion protein, and when the fusion protein expression vector constructed by using the PLB-hEGF fusion protein is used for expressing hEGF, the soluble expression of hEGF is improved, the time-consuming process of in vitro renaturation is saved, the specific activity is obviously improved, and the method has important value for industrially producing soluble and high-specific-activity hEGF.
Any one of the B domains PLB 1-PLB 5 of the protein L can be used as an upstream fusion protein, and in a preferred embodiment of the recombinant PLB-hEGF fusion protein provided by the present invention, the B domain of the protein L is a polypeptide as set forth in SEQ ID NO:1, and the fusion protein correspondingly obtained is named as recombinant PLB1-hEGF fusion protein.
In this embodiment, the PLB1 protein is further optimized, specifically, a translation initiation optimization sequence is added to the N-terminal of the PLB1 protein to form an N-terminal optimized PLB1 protein, and the amino acid sequence of the N-terminal optimized PLB1 protein is as shown in SEQ ID NO: shown at 7.
Further, the C terminal of the N-terminal optimized PLB1 protein is added with a flexible linker, a protease recognition region, an optimized hEGF coding region and a histidine tag, so that the PLB1-FL-PRS-hEGF fusion protein shown in figure 1 is correspondingly formed. In fig. 1, RBS indicates a ribosome binding region, PLB1 indicates the B1 domain of protein L, FL indicates a flexible linker, PRS indicates a protease recognition region, hEGF indicates an optimized hEGF coding region, and 6xHis-Tag indicates a purification Tag consisting of 6 histidines.
Further, the flexible linker comprises 6-30 amino acids, preferably 8-20 amino acids, and more preferably the sequence of the flexible linker is as shown in SEQ ID NO:14, 15; the protease recognition region comprises any one of a coagulation factor Xa (FXa) protease recognition region, a thrombin recognition region, an enterokinase recognition region and a TEV enzyme recognition region, preferably the FXa protease recognition region, more preferably the protein recognition region comprises 4 amino acid residues, and the sequence of the protein recognition region is shown as SEQ ID NO:16, 17; the histidine tag comprises 5-9 His, preferably 6 His, more preferably the sequence of the histidine tag is as shown in SEQ ID NO: 18, 19.
Based on the sequence of the flexible linker, the FXa protease recognition region and the histidine tag, the recombinant PLB1-hEGF fusion protein provided by the invention is more preferably a recombinant PLB1-hEGF fusion protein with the amino acid sequence shown as SEQ ID NO: shown at 10.
The invention further provides a coding gene of the recombinant PLB-hEGF fusion protein.
As a preferred embodiment of the above coding gene, the nucleotide sequence of the coding gene of the recombinant PLB-hEGF fusion protein is shown in SEQ ID NO: shown at 11.
The invention further provides a recombinant PLG-hEGF fusion protein expression vector or an expression engineering bacterium containing the coding gene of the recombinant PLG-hEGF fusion protein, and the fermentation production of the self-folding soluble recombinant hEGF can be realized by the aid of the recombinant PLG-hEGF fusion protein expression vector.
In an embodiment of the recombinant PLB-hEGF fusion protein expression vector provided by the present invention, the recombinant PLB-hEGF fusion protein expression vector is obtained by inserting the coding gene of the recombinant PLB-hEGF fusion protein between NcoI and XhoI cleavage sites of a prokaryotic cell expression plasmid, wherein the nucleotide sequence of the coding gene of the recombinant PLB-hEGF fusion protein is as set forth in SEQ ID NO:11, the prokaryotic cell expression plasmid is preferably a pET system expression vector, and more preferably pET 28.
The recombinant PLG-hEGF fusion protein expression vector can be used for expressing the PLG-hEGF fusion protein, and has the following advantages when expressing target protein: (1) the ratio of soluble protein to inclusion body protein is more than 50%; (2) the target protein is easily obtained by means of histidine Tag (His-Tag) affinity purification; (3) the cleaved PLB may also be further purified by antibody Kappa chain ligand affinity chromatography.
Based on the provided recombinant PLB-hEGF fusion protein expression vector, the invention also provides a preparation method of the recombinant PLB-hEGF fusion protein, which comprises the following steps: and transforming the recombinant PLB-hEGF fusion protein expression vector into a competent expression strain to obtain expression engineering bacteria, and culturing the expression engineering bacteria to obtain the recombinant PLB-hEGF fusion protein.
In addition, the invention also provides a preparation method of the recombinant PLB-hEGF fusion protein expression engineering bacterium based on the provided recombinant PLB-hEGF fusion protein expression engineering bacterium, which comprises the following steps:
step S10, synthesizing a DNA coding sequence of a fusion protein PLB-hEGF of PLB and hEGF based on a Polymerase Chain Reaction (PCR) whole-gene artificial synthesis method, wherein the sequence of the fusion protein PLB-hEGF is shown as SEQ ID NO:10, and the DNA coding sequence is shown as SEQ ID NO:11 is shown in the figure;
s20, carrying out NocI and XhoI DNA restriction enzyme digestion and agarose gel electrophoresis separation and purification on the coding DNA of the fusion protein PLB-hEGF to obtain an insert fragment;
s30, separating and purifying prokaryotic cell expression plasmids by endonuclease NocI and XhoI double enzyme digestion and agarose gel electrophoresis to obtain linearized empty vectors serving as parent vectors (seeds vectors);
s40, connecting the linearized empty vector with a coding DNA fragment of the fusion protein PLB-hEGF by using T4DNA ligase, then transforming an allelopathy strain DH5 alpha, and obtaining an engineering plasmid with a correct sequence after positive clonal amplification, plasmid preparation and DNA sequencing;
and step S50, converting the engineering plasmid into BL21(DE3) competent expression strain, and screening and IPTG (Isopropyl Thiogalactoside, an inducer with extremely strong action) induced expression test in the presence of kanamycin to obtain the high-expression engineering strain of the expression plasmid of the recombinant PLB-hEGF fusion protein.
In an embodiment of the preparation method of the recombinant PLB-hEGF fusion protein expression engineering bacterium provided by the present invention, the prokaryotic cell expression plasmid may be a pET system expression vector, more preferably pET28, the vector pET28 is linked with a DNA fragment encoding the fusion protein PLB-hEGF using T4DNA ligase to transform competent bacterium DH5 α, and a positive clone pET28-PLB-hEGF with a correct sequence is obtained as the engineering plasmid, whose sequence is as shown in SEQ ID NO:32 is shown; the pET28-PLB-hEGF plasmid was then transformed into BL21(DE3) competent expression strain, and screening and inducible expression tests were performed in the presence of kanamycin to obtain expression engineered strain pET28-PLB-hEGF/BL21(DE 3).
The technical solutions of the present invention are further described in detail below with reference to specific examples and drawings, it should be understood that the following examples are merely illustrative of the present invention and are not intended to limit the present invention.
Example 1 construction of recombinant PLB1-hEGF fusion protein expression vector
Step one, artificial synthesis and optimization of recombinant PLB1 and hEGF fusion protein encoding DNA, in this example, PLB1 is taken as an example (the sequence is shown as SEQ ID NO:1, 6), and hEGF is taken as an example of a 53-amino acid mature peptide (the sequence is shown as SEQ ID NO:8, 9):
(1) reverse translation: the DNA sequences encoding PLB1 and hEGF (SEQ ID NOS: 1,6,8,9) were optimized using DNAworks software, respectively.
(2) Insert (Insert) expression cassette (ORF) assembly:
the translation initiation region-optimized sequence was added to the 5 '-end of codon-optimized PLB1 (SEQ ID NOS: 7,12,13), and the flexible linker DNA coding sequence (SEQ ID NOS: 14,15), FXa recognition region (PRS) DNA coding sequence (SEQ ID NOS: 16,17), and hEGF codon-optimized sequence (SEQ ID NOS: 8,9) were combined in order and inserted into the 3' -end of PLB 1. The stop codon of hEGF was removed and inserted directly into the XhoI site at the end of pET28MCS to obtain the 6XHis histidine tag on the vector (FIG. 1, SEQ ID NO: 11).
(3) ORF total synthesis primer design:
firstly, primer design: inputting the spliced PLB1-hEGF fusion protein ORF coding sequence (SEQ ID NO:11) into NCBI online software DNAworks design primers, wherein the design primers are shown as SEQ ID NO: 20-31;
designing recombination sites of the inserters: adding [5 ' -CC-3 ' ] to the 5 ' -end of the start code of the first primer at the 5 ' -end to form an NcoI endonuclease recognition sequence and adding four protecting bases (SEQ ID NO: 20) to the 5 ' -end of the cleavage site; an XhoI endonuclease recognition sequence and four protective bases (SEQ ID NO: 31) were added to the 5' -end of the last codon of the last primer.
(4) Overlap extension PCR (overlap PCR) method for synthesizing full-length coding DNA sequence:
firstly, respectively diluting primers to 5 mu Mole concentration by using sterile deionized and sterilized water;
adding 1 microliter of the primers from each tube into 1 clean 200 microliter PCR tube;
③ then adding 4 mu L of 10mM dNTP, 5 mu L of 10x Pyrobest PCR Buffer and 50 mu L of sterile deionized water according to the instruction of the Takara Pyrobest PCR kit;
fourthly, 0.25 mu L of Pyrobest high fidelity DNA polymerase is added finally;
PCR parameters: pre-denaturation at 95 ℃/3 min; denaturation 94 ℃/30sec, annealing 58 ℃/30sec, extension 72 ℃/1min,18 cycles; the complete extension is 72 ℃/5 min.
(5) The full-length coding DNA sequence was amplified by the conventional polymerase chain reaction (General PCR) method:
taking 1 mu L of PCR product in the previous step as a template, and adding the template into a 200 mu L clean PCR tube;
② adding 4 μ L of each of the initial primer and the terminal primer (SEQ ID NO:20, 31);
③ then adding 4 μ L of 10mM dNTP, 5 μ L of 10x Pyrobest Buffer and 50 μ L of sterile deionized water in sequence according to the instruction of Takara Pyrobest PCR kit;
fourthly, 0.5 mu L of Pyrobest high fidelity DNA polymerase is added finally;
PCR parameters: pre-denaturation at 95 deg.C/3 min, denaturation at 94 deg.C/30 sec, annealing at 60 deg.C/30 sec, extension at 72 deg.C/2 min, and 25 cycles; the complete extension is 72 ℃/5 min.
(6) Purification and enzyme digestion of PCR products: the PCR product was purified on a silica gel column and digested twice with NcoI and XhoI restriction enzymes overnight.
(7) And (3) purifying an enzyme-digested DNA product: the sticky linker inserts were recovered by 0.8% agarose gel electrophoresis using a DNA recovery kit on gel to obtain NcoI and XhoI at the ends, respectively.
Step two, preparing a prokaryotic expression vector pET 28:
(1) mu.g of pET28 vector was digested simultaneously with restriction enzymes NcoI and XhoI.
(2) And (3) carrying out electrophoresis separation on 0.8% agarose gel, and carrying out gel recovery and purification on the linearized pET28 vector.
Step three, connection and transformation:
(1) connecting the treated PLB1-hEGF insert with pET28 vector by using T4DNA ligase;
(2) transforming an escherichia coli competent strain DH5 alpha, culturing overnight in an agar culture dish containing kanamycin (Kan) antibiotic at 37 ℃, then picking out a single colony, and identifying positive clone by conventional PCR;
(3) the positive clone is sent to DNA sequence analysis, the clone with correct sequence is reserved, and DNA plasmid is prepared by the conventional micro silica gel column method;
the obtained engineering vector plasmid is named as pET28-PLB1-hEGF (the physical diagram of the pET28-PLB1-hEGF expression vector is shown in figure 2, and the sequence is shown in SEQ ID NO: 32).
Step four, construction of control vector pET30-hEGF (because the main frame elements and DNA sequences of commercial E.coli expression vector pET30 and pET28 are identical, except for differences in the insertion sequence and the insertion site of the cloning region. the cloning region of pET28 is removed with NcoI and XhoI, and the cloning region of pET30 is removed with NdeI and XhoI.NdeI to facilitate cloning of the codons beginning with the non-G base downstream of the start codon ATG. since mature hEGF starts with AAC (or AAT, encoding N amino acids), after addition of ATG, is either [5 '-ATGAAC-3' ] or [5 '-ATGAAT-3' ], the control plasmid employs NdeI [5 '-TATCAG-3' ] and XhoI sites on pET30 vector to cut the hEGF coding sequence):
(1) designing a PCR primer:
the 5 ' -end of the upstream primer is added with [5 ' -CATATG-3 ' ] and 4T base protection NdeI enzyme cutting sites, the downstream is removed with a termination code, and is added with [5 ' -CTCGAG-3 ' ] and 4T base protection XhoI enzyme cutting sites, and the downstream 6xHis-Tag sequence comes from a vector and keeps the maximum similarity with the C-end of hEGF in pET28-PLB1-hEGF (SEQ ID NO:33, 34);
hEGF forward primer [ 5'-TTTTCATATGAACTCTGACTCTGAATGCC-3' ];
hEGF downstream primer [ 5'-TTTTCTCGAGGCGCAGTTCCCACCATTTC-3' ].
(2) PCR method amplified hEGF insert with NdeI and XhoI recombination sites:
taking 10ng pET20-PLB1-hEGF plasmid as a DNA template, and respectively adding 2 mu L upstream and downstream PCR primers (SEQ ID NO:35 and 36) with the concentration of 10 mu M;
secondly, according to the instruction of a Takara Pyrobest PCR kit, sequentially adding 4 mu L of 10mM dNTP, 5 mu L of 10x Pyrobest PCR Buffer and adding 50 mu L of sterile deionized water;
thirdly, 0.25 mu L of Pyrobest high-fidelity DNA polymerase is added finally;
PCR parameters: pre-denaturation at 95 ℃/3 min; denaturation 94 ℃/30sec, annealing 58 ℃/30sec, extension 72 ℃/1min,18 cycles; the complete extension is 72 ℃/5 min.
(3) The PCR product and pET30 empty vector DNA were digested with NdeI and XhoI separately overnight, separated by 0.8% agarose gel electrophoresis, cut to recover the target band, and purified with a gel recovery kit.
(4) T4DNA ligase was ligated to the vector and insert DNA and reacted overnight at 4 ℃ in a refrigerator.
(5) The ligation product was transformed into competent BL21(DE3) competent strains, Kan screened single colonies, a small amount of plasmid was prepared and sent for DNA sequence analysis, and clones with the correct sequence were retained for expression control testing.
The control vector obtained in this step was pET30-hEGF (physical map of the pET30-hEGF control vector is shown in FIG. 3, SEQ ID NO: 37), and the expression strain was pET30-hEGF/BL21(DE 3).
Example 2 vector expression assay
Step one, constructing an expression strain pET28-PLB1-hEGF/BL21(DE 3):
(1) the obtained pET28-PLB1-hEGF expression vector DNA plasmid is transformed into an escherichia coli expression strain BL21(DE3) competent cell to obtain a kanamycin (Kan) resistant single colony.
(2) Positive clones were identified by PCR and sent to plasmid sequencing for a second positive, leaving the correct pET28-PLB1-hEGF/BL21(DE3) expressing strain for expression testing.
Step two,
(1) The pET28-PLB1-hEGF/BL21(DE3) and the control plasmid strain pET30-hEGF/BL21(DE3) obtained above were separately 1: 100-500 dilution, coating on a Kan agar-containing plate, and screening once again to reduce BL21(DE3) cells which are grown in entrainment and do not contain expression vectors to the greatest extent so as to ensure the expression effect of the expression strain.
(2) Selecting a plurality of single colonies respectively, culturing in an LB culture medium, providing screening pressure by Kan, inducing for 8 hours by IPTG 0.5mM, collecting 1mL of bacterial liquid, centrifugally collecting thalli, washing 1mL of 1 XPBS once, suspending in 0.5mL of 1 XPBS, ultrasonically crushing thalli, centrifugally taking supernatant, retaining precipitate, adding 0.25mL of 8M urea solution, shaking and suspending the precipitate, then adding 0.25mL of 1 XPBS, and mixing uniformly.
(3) mu.L of each of the supernatant and the precipitate suspension was mixed with SDS-PAGE loading buffer, denatured by heating at 95 ℃ for 10 minutes, collected by centrifugation to the bottom of the tube, and placed on ice.
(4) mu.L of each sample was subjected to 12% SDS-PAGE, stained with Coomassie blue, destained, and observed for expression of the bacterial total protein band, the soluble PLB1-hEGF fusion protein band (supernatant) and the inclusion body PLB1-hEGF fusion protein band, and the ratio therebetween. FIG. 4 is a SDS-PAGE pattern of expression induction of pET28-PLB1-hEGF expression vector tested under the following conditions: 0.5mM IPTG, 25 ℃, 200RPM, inducing for 8 hours, and obtaining a mycoprotein SDS-PAGE electrophoresis chart, wherein PLB1-hEGF fusion protein is mainly distributed in the supernatant; FIG. 5 is a SDS-PAGE pattern of pET30-hEGF control expression vector tested for induced expression under the following conditions: after 0.5mM IPTG induction at 25 deg.C and 200RPM for 8 hours, the expressed hEGF protein was mainly distributed in the upper precipitate as seen in SDS-PAGE electrophoresis.
The test results are: recombinant PLB1-hEGF, 165aa (SEQ ID NO:10), theoretical pI 5.06, theoretical molecular weight 18.8 kDa. According to SDS-PAGE electrophoresis calculation, the expression level of the PLB1-hEGF fusion protein accounts for 20-25% of the total protein of the thallus, the soluble protein accounts for 80-85% (shown in figure 4), the inclusion bodies account for 15-20%, the solubility of the target protein is better than that of pET30-hEGF direct expression, and the latter is almost the expression of the inclusion bodies (shown in figure 5).
The above is only a preferred embodiment of the present invention, and it is not intended to limit the scope of the invention, and various modifications and changes will occur to those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention shall be included in the scope of the present invention.
Claims (4)
1. The recombinant PLB-hEGF fusion protein is a fusion protein obtained by fusing a B structure domain of protein L and human epidermal growth factor protein, wherein the amino acid sequence of the recombinant PLB1-hEGF fusion protein is shown as SEQ ID NO: shown at 10.
2. A gene encoding the recombinant PLB-hEGF fusion protein of claim 1.
3. The gene encoding recombinant PLB-hEGF fusion protein of claim 2 wherein the nucleotide sequence of the gene encoding recombinant PLB-hEGF fusion protein is as set forth in SEQ ID NO: shown at 11.
4. A recombinant PLB-hEGF fusion protein expression vector or expression engineering bacterium comprising the gene encoding the recombinant PLB-hEGF fusion protein of claim 2 or 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910699099.1A CN110540601B (en) | 2019-07-29 | 2019-07-29 | Recombinant PLB-hEGF fusion protein and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910699099.1A CN110540601B (en) | 2019-07-29 | 2019-07-29 | Recombinant PLB-hEGF fusion protein and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110540601A CN110540601A (en) | 2019-12-06 |
| CN110540601B true CN110540601B (en) | 2021-06-29 |
Family
ID=68709908
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910699099.1A Active CN110540601B (en) | 2019-07-29 | 2019-07-29 | Recombinant PLB-hEGF fusion protein and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110540601B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115851653B (en) * | 2022-09-23 | 2024-05-24 | 重庆工商大学 | Protein SUS1, gene SUS1, recombinant expression vector, transformant and pharmaceutical application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013164694A1 (en) * | 2012-04-30 | 2013-11-07 | Biocon Limited | Targeted/immunomodulatory fusion proteins and methods for making same |
| CN103834664A (en) * | 2014-03-27 | 2014-06-04 | 中国人民解放军军事医学科学院军事兽医研究所 | Recombinant epidermal growth factor (EGF) and preparation method thereof |
| CN106084066A (en) * | 2016-06-08 | 2016-11-09 | 盘古基因生物工程(南京)股份有限公司 | A kind of Soluble epidermal's growth factor fusion protein expression vector and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003229392A1 (en) * | 2002-05-31 | 2003-12-19 | Scegen Pty Ltd | Self-coalescing or self-aggregating proteins derived from a membrane translocating sequence |
-
2019
- 2019-07-29 CN CN201910699099.1A patent/CN110540601B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013164694A1 (en) * | 2012-04-30 | 2013-11-07 | Biocon Limited | Targeted/immunomodulatory fusion proteins and methods for making same |
| CN103834664A (en) * | 2014-03-27 | 2014-06-04 | 中国人民解放军军事医学科学院军事兽医研究所 | Recombinant epidermal growth factor (EGF) and preparation method thereof |
| CN106084066A (en) * | 2016-06-08 | 2016-11-09 | 盘古基因生物工程(南京)股份有限公司 | A kind of Soluble epidermal's growth factor fusion protein expression vector and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110540601A (en) | 2019-12-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US10000544B2 (en) | Process for production of insulin and insulin analogues | |
| KR101026526B1 (en) | Method for the secretory production of heterologous protein in Escherichia coli | |
| CN110964096B (en) | Preparation method of recombinant human C-reactive protein | |
| CN110835366B (en) | Tag polypeptide for promoting soluble expression of protein and application thereof | |
| CN110540601B (en) | Recombinant PLB-hEGF fusion protein and application thereof | |
| US5759815A (en) | Production of platelet derived growth factor (PDGF) an muteins thereof | |
| CN112500495A (en) | Purification method and application of ELP-III type collagen | |
| CN110511950B (en) | Application of PAB protein in construction of fusion protein expression vector with chaperone-like protein effect | |
| CN114380903B (en) | Insulin or analogue precursor thereof | |
| CN107746432B (en) | A β 42 modified protein and expression and purification method thereof | |
| JPH01180900A (en) | Polypeptide having cell adhesion activity | |
| CN113774039B (en) | Recombinant DNA polymerase and application thereof | |
| KR20130141001A (en) | A novel vector system for isolation and purification of target proteins | |
| CN111233986B (en) | Engineering bacterium for expressing streptolysin mature protein and application thereof | |
| CN110498859B (en) | Recombinant PLB-hbFGF fusion protein and application thereof | |
| CN110777165B (en) | Enhancer sequence and plasmid vector, preparation method and application thereof, and transformant | |
| CN101775404A (en) | Method for highly expressing basic protein with prokaryotic expression system | |
| EP1981978A2 (en) | Affinity polypeptide for purification of recombinant proteins | |
| CN113151227B (en) | Protease gene and heterologous expression thereof | |
| CN117801123B (en) | Wo Suoli peptide soluble intermediate, intermediate preparation method and Wo Suoli peptide preparation method | |
| CN117466992B (en) | Fibronectin mutant and preparation and application thereof | |
| CN110564634A (en) | Engineering bacterium for extracellularly secreting and expressing inonotus obliquus dipeptidase | |
| CN112689674B (en) | Dextran affinity tag and application thereof | |
| CN110511951B (en) | Application of PLB protein in construction of fusion protein expression vector with chaperone-like protein effect | |
| CN114807101B (en) | Fusion protein containing bovine enterokinase light chain protein, expression vector and recombinant engineering bacteria thereof |
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: 20220823 Address after: 6-1106, Xingguang Commercial Center, Xinwu District, Wuxi City, Jiangsu Province, 214000 Patentee after: Jiangsu Yuan Pharmaceutical Research Co.,Ltd. Address before: 430000 no.1-22, 10 / F, building A4, financial port backstage service center phase I, No.77, Guanggu Avenue, Donghu New Technology Development Zone, Wuhan City, Hubei Province Patentee before: YINZHI CAI BIOTECHNOLOGY (WUHAN) Co.,Ltd. |
|
| TR01 | Transfer of patent right |