CN116023450A - Recombinant antibacterial peptide PmHis9, recombinant expression vector, engineering bacteria and application thereof - Google Patents
Recombinant antibacterial peptide PmHis9, recombinant expression vector, engineering bacteria and application thereof Download PDFInfo
- Publication number
- CN116023450A CN116023450A CN202310032193.8A CN202310032193A CN116023450A CN 116023450 A CN116023450 A CN 116023450A CN 202310032193 A CN202310032193 A CN 202310032193A CN 116023450 A CN116023450 A CN 116023450A
- Authority
- CN
- China
- Prior art keywords
- pmhis9
- recombinant
- antibacterial peptide
- seq
- plectasin
- 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
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 66
- 241000894006 Bacteria Species 0.000 title claims abstract description 24
- 239000013604 expression vector Substances 0.000 title claims abstract description 22
- 238000003259 recombinant expression Methods 0.000 title claims abstract description 20
- 241000191967 Staphylococcus aureus Species 0.000 claims abstract description 14
- 241000499912 Trichoderma reesei Species 0.000 claims abstract description 14
- 239000002773 nucleotide Substances 0.000 claims abstract description 11
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 11
- 150000001413 amino acids Chemical class 0.000 claims abstract description 10
- 241000222122 Candida albicans Species 0.000 claims abstract description 9
- 229940095731 candida albicans Drugs 0.000 claims abstract description 9
- 206010012735 Diarrhoea Diseases 0.000 claims abstract description 8
- 241000186779 Listeria monocytogenes Species 0.000 claims abstract description 8
- 241000589517 Pseudomonas aeruginosa Species 0.000 claims abstract description 8
- 241000607760 Shigella sonnei Species 0.000 claims abstract description 8
- 206010048038 Wound infection Diseases 0.000 claims abstract description 8
- 229940115939 shigella sonnei Drugs 0.000 claims abstract description 8
- 241000588748 Klebsiella Species 0.000 claims abstract description 7
- 241000607142 Salmonella Species 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 239000012634 fragment Substances 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 8
- 230000004151 fermentation Effects 0.000 claims description 8
- 210000001938 protoplast Anatomy 0.000 claims description 6
- 238000000246 agarose gel electrophoresis Methods 0.000 claims description 5
- 238000012216 screening Methods 0.000 claims description 4
- 238000012163 sequencing technique Methods 0.000 claims description 4
- 238000012795 verification Methods 0.000 claims description 4
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 claims description 2
- 238000012220 PCR site-directed mutagenesis Methods 0.000 claims description 2
- 239000002202 Polyethylene glycol Substances 0.000 claims description 2
- 239000003674 animal food additive Substances 0.000 claims description 2
- 239000003814 drug Substances 0.000 claims description 2
- 230000001404 mediated effect Effects 0.000 claims description 2
- 229920001223 polyethylene glycol Polymers 0.000 claims description 2
- 238000011426 transformation method Methods 0.000 claims description 2
- 108010078656 plectasin Proteins 0.000 abstract description 32
- 230000000844 anti-bacterial effect Effects 0.000 abstract description 15
- 108700042778 Antimicrobial Peptides Proteins 0.000 abstract description 12
- 102000044503 Antimicrobial Peptides Human genes 0.000 abstract description 12
- 230000005764 inhibitory process Effects 0.000 abstract description 11
- 230000000694 effects Effects 0.000 abstract description 7
- 238000002741 site-directed mutagenesis Methods 0.000 abstract description 5
- 239000003899 bactericide agent Substances 0.000 abstract description 4
- 239000000022 bacteriostatic agent Substances 0.000 abstract description 3
- 102000038379 digestive enzymes Human genes 0.000 abstract description 2
- 108091007734 digestive enzymes Proteins 0.000 abstract description 2
- 230000002349 favourable effect Effects 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 238000006243 chemical reaction Methods 0.000 description 10
- 230000001580 bacterial effect Effects 0.000 description 8
- 108090000623 proteins and genes Proteins 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 241000223259 Trichoderma Species 0.000 description 6
- 102000004142 Trypsin Human genes 0.000 description 6
- 108090000631 Trypsin Proteins 0.000 description 6
- 238000000137 annealing Methods 0.000 description 6
- 239000012588 trypsin Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 102000057297 Pepsin A Human genes 0.000 description 5
- 108090000284 Pepsin A Proteins 0.000 description 5
- 238000004925 denaturation Methods 0.000 description 5
- 230000036425 denaturation Effects 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 229940111202 pepsin Drugs 0.000 description 5
- 239000013598 vector Substances 0.000 description 5
- 238000010276 construction Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108010006654 Bleomycin Proteins 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- 230000004544 DNA amplification Effects 0.000 description 2
- 241000192125 Firmicutes Species 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 239000008118 PEG 6000 Substances 0.000 description 2
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 description 2
- 241000383853 Pseudoplectania nigrella Species 0.000 description 2
- 108010084455 Zeocin Proteins 0.000 description 2
- 235000001014 amino acid Nutrition 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 229960001561 bleomycin Drugs 0.000 description 2
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 2
- 210000002421 cell wall Anatomy 0.000 description 2
- MTHSVFCYNBDYFN-UHFFFAOYSA-N diethylene glycol Chemical compound OCCOCCO MTHSVFCYNBDYFN-UHFFFAOYSA-N 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- CWCMIVBLVUHDHK-ZSNHEYEWSA-N phleomycin D1 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC[C@@H](N=1)C=1SC=C(N=1)C(=O)NCCCCNC(N)=N)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C CWCMIVBLVUHDHK-ZSNHEYEWSA-N 0.000 description 2
- 238000012257 pre-denaturation Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- NOIIUHRQUVNIDD-UHFFFAOYSA-N 3-[[oxo(pyridin-4-yl)methyl]hydrazo]-N-(phenylmethyl)propanamide Chemical compound C=1C=CC=CC=1CNC(=O)CCNNC(=O)C1=CC=NC=C1 NOIIUHRQUVNIDD-UHFFFAOYSA-N 0.000 description 1
- 208000035143 Bacterial infection Diseases 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- -1 anionic amino acid Chemical class 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000029087 digestion Effects 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
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000004744 fabric Substances 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 230000002949 hemolytic effect Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000000411 inducer Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- ULXTYUPMJXVUHQ-OVTFQNCVSA-N lipid II Chemical compound OC(=O)[C@@H](C)NC(=O)[C@@H](C)NC(=O)[C@H](CCCCN)NC(=O)CC[C@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](C)O[C@@H]1[C@@H](NC(C)=O)[C@@H](OP(O)(=O)OP(O)(=O)OC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(\C)CC\C=C(/C)CC\C=C(/C)CCC=C(C)C)O[C@H](CO)[C@H]1O[C@H]1[C@H](NC(C)=O)[C@@H](O)[C@H](O)[C@@H](CO)O1 ULXTYUPMJXVUHQ-OVTFQNCVSA-N 0.000 description 1
- ULXTYUPMJXVUHQ-UHFFFAOYSA-N lipid ii Chemical compound OC(=O)C(C)NC(=O)C(C)NC(=O)C(CCCCN)NC(=O)CCC(C(O)=O)NC(=O)C(C)NC(=O)C(C)OC1C(NC(C)=O)C(OP(O)(=O)OP(O)(=O)OCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)CCC=C(C)C)OC(CO)C1OC1C(NC(C)=O)C(O)C(O)C(CO)O1 ULXTYUPMJXVUHQ-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 229940031000 streptococcus pneumoniae Drugs 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to a recombinant antibacterial peptide PmHis9, a recombinant expression vector, engineering bacteria and application thereof, belonging to the fields of genetic engineering and biotechnology, wherein the amino acid sequence of the recombinant antibacterial peptide PmHis9 is shown as SEQ ID NO. 1. The nucleotide sequence of the coded amino acid shown in SEQ ID NO.1 is shown as SEQ ID NO.2, the invention provides a preparation method of heterologous expression of recombinant antimicrobial peptide PmHis9 in Trichoderma reesei, the recombinant antimicrobial peptide PmHis9 is produced by site-directed mutagenesis of mycelial mycin plectasin, has good heat stability, pH stability and digestive enzyme stability, has stronger inhibition effect on Klebsiella, salmonella, pseudomonas aeruginosa, shigella sonnei, listeria monocytogenes, staphylococcus aureus and candida albicans than the mycelial mycin plectasin, and is favorable for application of the recombinant antimicrobial peptide PmHis9 as a bacteriostatic agent or bactericide in treating wound infection and treating diarrhea.
Description
Technical Field
The invention belongs to the field of genetic engineering and biotechnology, and in particular relates to a recombinant antibacterial peptide PmHis9, a recombinant expression vector, engineering bacteria and application thereof.
Background
The plectasin is a cationic antibacterial peptide derived from pseudoplectania nigrella (Pseudoplectania nigrella), has 3 disulfide bonds and a unique CS alpha beta structure, has a strong inhibition effect on gram-positive bacteria, has good stability and no hemolytic activity, and has no obvious inhibition effect on gram-negative bacteria. Mycelial mycin plectasin is similar to vancomycin in that it binds to the cell wall precursor material Lipid-ii of gram-positive bacteria such as staphylococcus aureus, thereby inhibiting the formation of cell walls; however, the binding site is mainly a phosphate group of Lipid-II, so that bacteria are less likely to develop drug resistance. At present, the NoveXin company is applying the mutant NZ2114 of mycelial mycin plectasin to the treatment of gram-positive bacterial infection and has obtained clinical development permission, and the result shows that the novel mycelial mycin is effective to streptococcus pneumoniae and can effectively reduce the bacterial concentration of cerebrospinal fluid, thus indicating that the mycelial mycin has great application prospect and reconstruction value.
The mycelial mycin plectasin has a motif consisting of four amino acids "DEDD", and has been reported to be capable of associating with Ca 2+ The isodivalent cations combine to achieve some of their antimicrobial properties. At present, the modification of mycelial mycin is mainly focused on the aspects of increasing the overall positive charge of the antibacterial peptide, changing the hydrophobicity and the like, and the modification of adding anionic amino acid to the region is not performed.
At present, the application of mycelial mycin plectasin to bacteriostats or bactericides has several main problems. Firstly, the antibacterial activity of mycelial mycin plectasin in the prior art has a larger gap compared with that of the traditional antibiotics, and compared with the traditional antibiotics, the mycelial mycin plectasin has a narrower antibacterial spectrum, is difficult to cope with diseases such as wound infection, diarrhea and the like caused by various pathogenic bacteria, and is unstable to trypsin; secondly, the safety problem in the production process is that host engineering strains for producing plectasin such as pichia pastoris and the like need to be added with inducers such as methanol and the like before fermentation production.
Disclosure of Invention
The invention aims to provide a recombinant antibacterial peptide PmHis9, a recombinant expression vector, engineering bacteria and application thereof. In order to improve the antibacterial activity of the plectasin, the invention designs a mutant PmHis9 of the plectasin by adopting a site-directed mutagenesis method, the mutant PmHis9 mutates methionine after DEDD motif in an original sequence into glutamic acid, and the inhibition activity of the PmHis9 on microorganisms such as staphylococcus aureus and the like is further improved compared with the plectasin, so that the antibacterial spectrum is further widened.
The invention is realized based on the following technical scheme:
a recombinant antibacterial peptide PmHis9, wherein the amino acid sequence of the recombinant antibacterial peptide PmHis9 is shown as SEQ ID NO. 1. The SEQ ID NO.1 is a mutant obtained by site-directed mutagenesis of the amino acid sequence fragment of SEQ ID NO. 3. The nucleic acid of the prosequence plectasin codes for methionine at position 13 in SEQ ID NO.3 and is mutated to glutamic acid coding for SEQ ID NO. 1.
The nucleotide sequence of the amino acid sequence of the coded SEQ ID NO.1 is shown as SEQ ID NO.2, and the SEQ ID NO.2 is translated into the coded nucleotide sequence on the basis of the antibacterial peptide PmHis9 of the original amino acid sequence SEQ ID NO. 1.
SEQ ID NO.1:GFGCNGPWDEDDEQCHNHCKSIKGYKGGYCAKGGFV CKCY；
SEQ ID NO.2:gaattcggatttggatgtaacggtccgtgggatgaagatgatgaacaatgtcataaccat tgtaagtctattaagggatacaagggaggttactgtgctaagggtggttttgtttgtaagtgttactaataagcggccgc；
SEQ ID NO.3:GFGCNGPWDEDDMQCHNHCKSIKGYKGGYCAKGGF VCKCY；
A recombinant expression vector comprising the gene of the recombinant antimicrobial peptide PmHis9.
A recombinant engineering bacterium, which comprises the recombinant expression vector and a strain Trichoderma reesei Tu6.
The invention comprises a preparation method of recombinant antibacterial peptide PmHis9, which specifically comprises the following steps: firstly, constructing a recombinant antibacterial peptide trichoderma reesei expression vector plectasin-pCBHG; constructing a recombinant expression vector PmHis9-pCBHG of the recombinant antibacterial peptide PmHis9 by taking plectasin-pCBHG as a template through PCR site-directed mutagenesis, detecting the PCR fragment by agarose gel electrophoresis, and performing sequencing verification on the PCR fragment to convert the PCR fragment into escherichia coli DH5 alpha; transferring the recombinant expression vector into Trichoderma reesei Tu6 by adopting a polyethylene glycol-mediated protoplast transformation method, screening positive transformants and verifying, and obtaining the recombinant antibacterial peptide PmHis9 through fermentation expression.
The invention also provides application of the recombinant antibacterial peptide PmHis9 in preparing feeds and feed additives.
The invention also provides application of the recombinant antibacterial peptide PmHis9 in preparing medicaments for treating wound infection and diarrhea, wherein the wound infection and diarrhea are caused by bacteria including klebsiella, salmonella, pseudomonas aeruginosa, shigella sonnei, listeria monocytogenes, staphylococcus aureus and candida albicans.
Compared with the prior art, the invention has the beneficial effects that:
the recombinant antibacterial peptide PmHis9 has good thermal stability and pH stability, and compared with mycelial mycin plectasin, the stability of trypsin and pepsin is improved. Has stronger inhibition effect on klebsiella, salmonella, pseudomonas aeruginosa, shigella sonnei, listeria monocytogenes, staphylococcus aureus and candida albicans, and is favorable for being used as a bacteriostatic agent or bactericide in treating wound infection and treating diarrhea.
The trichoderma reesei used in the invention is safe and harmless, can be directly added into feed according to national standards, has the advantages of strong promoter, mammal-like protein modification capability, high-efficiency synthesis and secretion mechanism and the like, and has unique advantages in the aspect of large-scale fermentation.
Drawings
FIG. 1 is a schematic diagram of gene amplification nucleic acid electrophoresis of recombinant expression vectors;
FIG. 2 is a schematic diagram of construction of recombinant expression vector PmHis 9-pCBHG;
FIG. 3 is a schematic illustration of the thermal stability of the recombinant antimicrobial peptide PmHis 9;
FIG. 4 is a schematic illustration of pepsin stability for recombinant antimicrobial peptide PmHis 9;
FIG. 5 is a schematic illustration of trypsin stability of recombinant antimicrobial peptide PmHis 9;
FIG. 6 is a schematic diagram showing the pH stability of the recombinant antimicrobial peptide PmHis9.
Detailed Description
The technical scheme of the present invention is further explained below by means of examples in combination with the accompanying drawings, but the scope of the present invention is not limited in any way by the examples.
EXAMPLE 1 construction of the Trichoderma reesei recombinant expression vector plectasin-PCBGG of plectasin
The amino acid sequence of plectasin published in NCBI UQI49964.1 is taken as an original amino acid sequence, and is translated into a nucleotide sequence by using SnapGene 3.2.1 software for artificial synthesis. Constructing a Trichoderma reesei recombinant expression vector plec-PCBHG of plectasin by taking the synthesized nucleotide sequence as a template.
Linearized primer pair PCBHG-f using trichoderma reesei Tu6 vector backbone: 5'-GCTCCGTGGCGAAAGCCT-3' and PCBHG-r:5'-AGCACGAGCTGTGGCCAAG-3' it is linearized by a PCR reaction using the enzyme
Super-Fidelity DNA Polymerase, PCR reaction system (50. Mu.L) was: 5 XPhanta Buffer 25. Mu.L; dNTP 1. Mu.L; />
0.5μL；pf 2μL；pr 2μL is; 1 mu L of carrier PCBHG; ddH 2 O18.5. Mu.L. The Trichoderma reesei Tu6 vector skeleton PCR adopts gradient annealing temperature, and the reaction conditions are as follows: pre-denaturation at 94℃for 3 min; denaturation at 94℃for 30sec, annealing at 62℃for 30sec, extension at 72℃for 7min, 5 cycles; denaturation at 94℃for 30sec, annealing at 60℃for 30sec, extension at 72℃for 7min, 5 cycles; denaturation at 94℃for 30sec, annealing at 58℃for 30sec, elongation at 72℃for 7min, 5 cycles; denaturation at 94℃for 30sec, annealing at 56℃for 30sec, elongation at 72℃for 7min, 25 cycles; total extension at 72℃for 10 min.
The PCR product verified by agarose gel electrophoresis was digested, and 5. Mu. L rCutSmart Buffer was added using 1. Mu.L of DpnI enzyme. The reaction conditions were 37℃for 2h. And (3) recovering and purifying by using a Cycle Pure Kit PCR purification kit to obtain the linearized fragment of the PCHG vector. Carrying out in vitro homologous recombination connection on the artificially synthesized plectasin nucleic acid fragment and a linearization vector, wherein the used homologous recombination ligase is Exnase II, and a 5 mu L connection system is as follows: exnase II 0.5. Mu.L, CE II Buffer 2. Mu.L, linear vector backbone 0.5. Mu.L, ddH 2 O1. Mu.L, plectasin gene 1. Mu.L.
The reaction was carried out at 37℃for 30min to form a ring, and then the above-mentioned product was transformed into E.coli DH 5. Alpha. And cultured in LB solid medium supplemented with bleomycin (zeocin) at 37℃for 16h. After a single colony grows, selecting the single colony for colony PCR and sequencing, and performing comparison verification to complete construction of the plectasin-PCBHG recombinant expression vector of plectasin.
EXAMPLE 2 site-directed mutagenesis of the recombinant antibacterial peptide PmHis9 Gene
A site-directed mutagenesis PCR reaction was performed using plectasin-PCBHG of example 1 as a template to prepare a PmH is9 nucleotide fragment. The PCR amplification enzyme used was
The high-fidelity amplifying enzyme and the PCR reaction system (50 mu L) are as follows: />
Buffer 25μL；dNTP 1μL；/>
0.5. Mu.L; pf 2 μl; pr 2. Mu.L, 1. Mu.L of synthetic template; ddH 2 O18.5. Mu.L. The PCR amplification reaction conditions of the PmHis9 gene are as follows: pre-denaturation at 94℃for 3 min; denaturation at 94℃for 30sec, annealing at 57℃for 30sec, extension at 72℃for 7min, 35 cycles; total extension at 72℃for 10 min; 4 ℃. Wherein the sequence of the forward primer PmH is9-f of the PmHis9 gene is 5'-ATGATGAACAATGTCATAACCATTGTAAG-3', and the sequence of the reverse primer PmHis 9-r is 5'-GACATTGTTCATCATCTTCATCCC-3'.
The band of interest for gene amplification of the antibacterial peptide PmHis9 was detected by agarose gel electrophoresis, as shown in FIG. 1. The PCR product verified by agarose gel electrophoresis was digested, and 5. Mu. LrCutSmart Buffer was added using 1. Mu.LDpnI enzyme. The reaction conditions were 37℃for 2h. The linearized fragment of PmHis9-PCBHG was recovered and purified using a Cycle Pure Kit PCR purification kit, as shown in FIG. 2. The linearized recombinant expression vector PmHis9-PCBHG is connected into a circular plasmid by using Exnase II, and a 5 mu L connection system is as follows: exnase II 0.5. Mu.L, CE II Buffer 2. Mu.L, ddH 2 O1. Mu.L, plectasin gene 1.5. Mu.L. The reaction was carried out at 37℃for 30min to form a ring, and then the above-mentioned product was transformed into E.coli DH 5. Alpha. And cultured in LB solid medium supplemented with bleomycin (zeocin) at 37℃for 16h. After a single colony grows, the single colony is selected for colony PCR and sequencing, and the construction of the Trichoderma reesei recombinant expression vector PmHis9-PCBHG of PmHis9 is completed after comparison and verification, as shown in figure 2.
Example 3 transformation of Trichoderma recombinant expression vector of antibacterial peptide PmHis9
After subculturing Trichoderma reesei host strain Tu6 on PDA+U solid medium plate at 30deg.C for 5-6 days, spores were washed out with 0.1% Triton solution, inoculated into YEG medium, and cultured in a constant temperature shaker at 30deg.C at 180rpm for 20h.
Filtering the bacterial liquid which grows mature after enzymolysis by using sterile filter cloth and a funnel, flushing the bacterial liquid by using sterile water, taking a proper amount of hypha, and filtering and sterilizing by using an enzymolysis liquid by adopting a filter with the aperture of 0.45 mu L; incubate at 30℃for 2h on a thermostatted shaker at 80 rpm. The bacterial enzymolysis solution was filtered and centrifuged, and the protoplast was resuspended in 1M sorbitol solution, centrifuged at 3500rpm for 4min, and then washed repeatedly with sterile water 2 times, and the protoplast was resuspended in 1mL of 1M sorbitol solution, thereby completing the preparation of protoplast.
The recombinant plasmid constructed in example 2 was extracted using the E.Z.N.A.plasmid Mini Kit I Kit. 150. Mu.L of protoplast, 10. Mu.L of recombinant PmHis9 plasmid, 50. Mu.L of 25% PEG6000 were taken, and mixed well and then ice-bathed for 25min. The above system was transferred to a 10mL centrifuge tube containing 2mL 25% PEG6000 and left at room temperature for 20min. Adding the solution into the trichoderma upper layer culture medium, mixing, pouring onto a plate paved with the trichoderma lower layer culture medium, and culturing at 30 ℃ for 5-6 days.
EXAMPLE 4 fermentative expression of the antibacterial peptide PmHis9 in Trichoderma
The transformant growing in the trichoderma transformation culture medium is inoculated on a new plate for screening, and after hypha and spore grow out, genome extraction is carried out, and the target gene is verified. The screened positive transformant is inoculated into a trichoderma fermentation culture medium, firstly grows for 2 days in a constant temperature shaking table at 30 ℃ and 180rpm, reduces the temperature to 25 ℃ after the obvious growth of the thalli, continues fermentation and induction expression for 4-5 days, and then is centrifuged to obtain a fermentation product. Detecting, screening and separating the fermentation product to obtain the antibacterial peptide PmHis9, and determining that the amino acid sequence is the same as SEQ ID NO.1 and the coding nucleotide sequence is the same as SEQ ID NO.2.
Example 5 determination of antibacterial Activity of the antibacterial peptide PmHis9
Determination of Minimum Inhibitory Concentration (MIC)
The indicator bacteria used in this example were Klebsiella, salmonella, E.coli, pseudomonas aeruginosa, shigella sonnei, listeria monocytogenes, staphylococcus aureus, and Candida albicans.
MIC was determined by microdilution: preparing bacterial suspension, and respectively diluting four bacteria cultured to the logarithmic phase of growth to 10 5 CFU/mL, the purified antibacterial peptide PmHis9 solution and the antibacterial peptide plectasin solution are mixed with bacterial suspension in equal quantity in the first column of a 96-well plate, and the initial concentration of the antibacterial peptide sample is 150 mug/mL at the moment, and then concentration gradient dilution is sequentially carried out. All samples of the 96-well plate are added with an equivalent diluted bacterial suspension, and each bacterium is incubated for 18 hours at 37 ℃ and is whiteCandida albicans was incubated at 30 ℃ for 24h. The haze level of each well was visually observed after the culture, wherein the minimum concentration capable of clarifying the well was the MIC of the antibacterial peptide PmHis9 for each bacteria.
The results are shown in Table 1, and the antibacterial activity of the recombinant antibacterial peptide PmHis9 is improved relative to plectasin. The original sequence has no inhibition effect on klebsiella and salmonella under the test concentration, and the MIC of the recombinant antibacterial peptide PmHis9 on the two bacteria is 150 mug/mL. The MIC of the two to the escherichia coli is 150 mug/mL; the MIC of the recombinant antibacterial peptide PmHis9 on pseudomonas aeruginosa, shigella sonnei, listeria monocytogenes, staphylococcus aureus and candida albicans is 75 mug/mL, 37.5 mug/mL, 18.8 mug/mL and 18.8 mug/mL respectively, which shows that the antibacterial activity of the recombinant antibacterial peptide PmHis9 on the pseudomonas aeruginosa, the Shigella sonnei, the listeria monocytogenes, the staphylococcus aureus and the candida albicans is doubled compared with the antibacterial activity of the recombinant antibacterial peptide PmHis on the primary sequence plectasin.
TABLE 1 MIC of mycelial mycin plectasin and recombinant antibacterial peptide PmHis9 for different bacteria (μg/mL)
Determination of zone of inhibition
And (3) coating 150 mu L of the bacterial liquid on a culture medium suitable for growth of each bacterium, respectively adding 100 mu L of recombinant antibacterial peptide PmHis9 and mycelial mycin plectasin with the concentration of 300 mu g/mL into an oxford cup, incubating each bacterium for 18 hours at 37 ℃, and incubating candida albicans for 24 hours at 30 ℃. The measurement results are shown in Table 2, and the antibacterial activity of the recombinant antibacterial peptide PmHis9 on each bacterium is improved.
TABLE 2 antibacterial circle size (mm) of mycelial mycin plectasin and recombinant antibacterial peptide PmHis9 against different bacteria
The data show that the recombinant antimicrobial peptide PmHis9 has improved antimicrobial activity on gram-negative klebsiella, salmonella, escherichia coli, pseudomonas aeruginosa, shigella sonnei, gram-positive listeria monocytogenes, staphylococcus aureus and candida albicans in fungi, and is beneficial to the application of the recombinant antimicrobial peptide PmHis9 as a bacteriostatic agent or bactericide in treating wound infection and treating diarrhea.
EXAMPLE 6 determination of the thermal stability of the recombinant antibacterial peptide PmHis9
The indicator bacteria used in this example are staphylococcus aureus, the thermal stability results are shown in fig. 3, wherein the abscissa is the treatment time of boiling water bath, the inhibition activities of the recombinant antibacterial peptide PmHis9 and mycelial mycin plectasin on each bacteria are reduced to different degrees after high-temperature treatment, and the PmHis9 can still keep 97% inhibition rate after 30min treatment and is slightly lower than the original sequence. The data show that the recombinant antibacterial peptide PmHis9 has relatively good heat stability and can withstand high temperature for a long time.
EXAMPLE 7 determination of the digestive enzyme stability of the recombinant antibacterial peptide PmHis9
The indicator bacteria used in this example were staphylococcus aureus, and the results of the digestion enzyme stability measurement of the recombinant antimicrobial peptide PmHis9 are shown in fig. 4 and 5, wherein fig. 4 is pepsin and fig. 5 is trypsin. The recombinant antibacterial peptide PmHis9 and the original sequence plectasin have better stability in pepsin, and the inhibition rate of the recombinant antibacterial peptide PmHis9 is higher than that of the original sequence after the pepsin is treated for 180 min. Because of the reduced negative charge, the recombinant antimicrobial peptide PmHis9 has better trypsin stability compared with the original sequence. After being treated by trypsin for 60min, the inhibition rate of PmHis9 to staphylococcus aureus is obviously higher than that of the original sequence.
Example 8 determination of the pH stability of the recombinant antibacterial peptide PmHis9
The indicator used in this example was staphylococcus aureus and the pH stability was measured as shown in fig. 6, where the abscissa indicates pH. The recombinant antibacterial peptide PmHis9 and the original sequence plectasin can maintain more than 85% of antibacterial activity after being treated for 120min in alkaline and acidic environments, which shows that the recombinant antibacterial peptide PmHis9 and the original sequence plectasin have good pH stability and tolerance to acid and alkali.
Claims (7)
1. The recombinant antibacterial peptide PmHis9 is characterized in that the amino acid sequence of the recombinant antibacterial peptide PmHis9 is shown as SEQ ID NO. 1.
2. The nucleotide sequence of the amino acid shown in SEQ ID NO.1 is characterized in that the nucleotide sequence is shown in SEQ ID NO.2.
3. A recombinant expression vector comprising the nucleotide sequence of SEQ ID No.2 of claim 2.
4. A recombinant engineering bacterium is characterized by comprising the nucleotide sequence SEQ ID NO.2 as set forth in claim 2, wherein the host strain is Trichoderma reesei Tu6.
5. The preparation method of the recombinant antibacterial peptide PmHis9 is characterized by comprising the steps of firstly constructing a recombinant antibacterial peptide Trichoderma reesei expression vector plectasin-pCBHG; constructing a recombinant expression vector PmHis9-pCBHG of the recombinant antibacterial peptide PmHis9 by taking plectasin-pCBHG as a template through PCR site-directed mutagenesis, detecting the PCR fragment by agarose gel electrophoresis, and performing sequencing verification on the PCR fragment to convert the PCR fragment into escherichia coli DH5 alpha; transferring the recombinant expression vector into Trichoderma reesei Tu6 by adopting a polyethylene glycol-mediated protoplast transformation method, screening positive transformants and verifying, and obtaining the recombinant antibacterial peptide PmHis9 through fermentation expression.
6. The use of the recombinant antibacterial peptide PmHis9 according to claim 1 for the preparation of feed and feed additives.
7. Use of the recombinant antibacterial peptide PmHis9 according to claim 1, for the preparation of a medicament for the treatment of wound infections and diarrhoea, characterised in that said wound infections and diarrhoea are caused by bacteria comprising klebsiella, salmonella, pseudomonas aeruginosa, shigella sonnei, listeria monocytogenes, staphylococcus aureus, candida albicans.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310032193.8A CN116023450B (en) | 2023-01-10 | 2023-01-10 | Recombinant antibacterial peptide PmHis, recombinant expression vector, engineering bacteria and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310032193.8A CN116023450B (en) | 2023-01-10 | 2023-01-10 | Recombinant antibacterial peptide PmHis, recombinant expression vector, engineering bacteria and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116023450A true CN116023450A (en) | 2023-04-28 |
| CN116023450B CN116023450B (en) | 2024-05-03 |
Family
ID=86072028
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310032193.8A Active CN116023450B (en) | 2023-01-10 | 2023-01-10 | Recombinant antibacterial peptide PmHis, recombinant expression vector, engineering bacteria and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116023450B (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113831395A (en) * | 2021-11-26 | 2021-12-24 | 中国海洋大学 | Recombinant antibacterial peptide Trsub, preparation method and application thereof |
| CN115124604A (en) * | 2022-06-20 | 2022-09-30 | 中国海洋大学 | Recombinant antibacterial peptide E-EJ97, recombinant expression vector, engineering bacterium and application thereof |
-
2023
- 2023-01-10 CN CN202310032193.8A patent/CN116023450B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113831395A (en) * | 2021-11-26 | 2021-12-24 | 中国海洋大学 | Recombinant antibacterial peptide Trsub, preparation method and application thereof |
| CN115124604A (en) * | 2022-06-20 | 2022-09-30 | 中国海洋大学 | Recombinant antibacterial peptide E-EJ97, recombinant expression vector, engineering bacterium and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116023450B (en) | 2024-05-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20230329283A1 (en) | Recombinant antibacterial peptide trsub, preparation method and application for same | |
| KR101679549B1 (en) | Novel Lactobacillus plantarum bacteriophage Lac-PLP-1 and its use for preventing proliferation of Lactobacillus plantarum | |
| WO2024031921A1 (en) | Pichia pastoris with enhanced lactoferrin expression, method for constructing same, and use thereof | |
| CN103937830B (en) | A kind of recombinant bacterium of efficient secretory expression Nattokinase | |
| CN111420037B (en) | Application of phage lyase Lysep3 in preparation of broad-spectrum antibacterial drugs | |
| CN115124604B (en) | Recombinant antibacterial peptide E-EJ97, recombinant expression vector, engineering bacteria and application thereof | |
| CN116023450B (en) | Recombinant antibacterial peptide PmHis, recombinant expression vector, engineering bacteria and application thereof | |
| CN111117942B (en) | Genetic engineering bacterium for producing lincomycin and construction method and application thereof | |
| CN105732790B (en) | A kind of source of fish antibacterial peptide hepcidin mutants and its application | |
| CN111518851B (en) | Immobilized enzyme continuous preparation 14/15 N]Process for preparing L-citrulline | |
| CN113684223A (en) | Multi-copy recombinant expression vector capable of efficiently expressing LL-37 polypeptide and application thereof | |
| CN116333067B (en) | Antibacterial peptide mutant Sub168-QC/R, recombinant strain and application thereof | |
| CN115960187A (en) | Recombinant antibacterial peptide PmHis3, recombinant expression vector, engineering bacterium and application thereof | |
| CN108948210B (en) | Hybrid antibacterial peptide PA-MO and preparation method and application thereof | |
| KR101460861B1 (en) | Expression of bovine lactoferrin using transformed Chlorella | |
| CN116947985B (en) | Recombinant antibacterial peptide Sub168-1/3R, recombinant strain, vector, preparation method and feed additive | |
| CN115992117B (en) | Recombinant human lysozyme and preparation method and application thereof | |
| CN116333067A (en) | Antibacterial peptide mutant Sub168-QC/R, recombinant strain and application thereof | |
| WO2024103822A1 (en) | Mature polypeptide for synthesizing oligosaccharides | |
| CN117924435A (en) | Pichia pastoris for secretory expression of immunomodulatory peptide Y6, and construction method and application thereof | |
| CN114853863B (en) | Recombinant mussel-like mucin purification method | |
| CN114044816B (en) | Recombinant crassostrea gigas Jiao Kongsu protein rCgGSDME-N, preparation method and application thereof | |
| CN113637659B (en) | Acidocella acetylhexosidase Amuc_2109 protein prepared by genetic engineering and preparation method and application thereof | |
| CN106520770B (en) | The non-coding tiny RNA s224 of Lactococcus lactis cream subspecies YF11 | |
| CN117069818A (en) | Oyster galactose lectin recombinant protein rCgGal-CUB and application 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 |