CN102586262B - Defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), antimicrobial peptide encoded by defensin gene and preparation method for defensin gene - Google Patents
Defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), antimicrobial peptide encoded by defensin gene and preparation method for defensin gene Download PDFInfo
- Publication number
- CN102586262B CN102586262B CN201210073276.3A CN201210073276A CN102586262B CN 102586262 B CN102586262 B CN 102586262B CN 201210073276 A CN201210073276 A CN 201210073276A CN 102586262 B CN102586262 B CN 102586262B
- Authority
- CN
- China
- Prior art keywords
- gene
- defensin
- bemisia tabaci
- antimicrobial peptide
- antibacterial peptide
- 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.)
- Expired - Fee Related
Links
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 74
- 241000254127 Bemisia tabaci Species 0.000 title claims abstract description 65
- 108010002069 Defensins Proteins 0.000 title claims abstract description 51
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 102000044503 Antimicrobial Peptides Human genes 0.000 title abstract description 10
- 108700042778 Antimicrobial Peptides Proteins 0.000 title abstract description 10
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 48
- 102000000541 Defensins Human genes 0.000 claims abstract description 30
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 claims abstract description 18
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 claims abstract description 18
- 238000000034 method Methods 0.000 claims abstract description 14
- 239000002773 nucleotide Substances 0.000 claims abstract description 4
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 4
- 241000894006 Bacteria Species 0.000 claims description 15
- 230000000845 anti-microbial effect Effects 0.000 claims description 15
- 230000000968 intestinal effect Effects 0.000 claims description 6
- 238000005215 recombination Methods 0.000 claims description 4
- 239000013604 expression vector Substances 0.000 abstract description 12
- 102000004169 proteins and genes Human genes 0.000 abstract description 8
- 241000751139 Beauveria bassiana Species 0.000 abstract description 5
- 241000123650 Botrytis cinerea Species 0.000 abstract description 5
- 241000223250 Metarhizium anisopliae Species 0.000 abstract description 5
- 150000001413 amino acids Chemical class 0.000 abstract description 4
- 238000000926 separation method Methods 0.000 abstract description 4
- 230000012010 growth Effects 0.000 abstract description 3
- 241000233866 Fungi Species 0.000 abstract description 2
- 108091005804 Peptidases Proteins 0.000 abstract description 2
- 238000000108 ultra-filtration Methods 0.000 abstract description 2
- 239000004365 Protease Substances 0.000 abstract 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 abstract 1
- 230000000843 anti-fungal effect Effects 0.000 abstract 1
- 230000000967 entomopathogenic effect Effects 0.000 abstract 1
- 239000000047 product Substances 0.000 description 15
- 239000007788 liquid Substances 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 10
- 239000002299 complementary DNA Substances 0.000 description 9
- 230000014509 gene expression Effects 0.000 description 9
- 108090000765 processed proteins & peptides Proteins 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 6
- 235000018102 proteins Nutrition 0.000 description 6
- 230000003321 amplification Effects 0.000 description 5
- 230000000844 anti-bacterial effect Effects 0.000 description 5
- 230000029087 digestion Effects 0.000 description 5
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 4
- 241000238631 Hexapoda Species 0.000 description 4
- 230000001857 anti-mycotic effect Effects 0.000 description 4
- ZAIPMKNFIOOWCQ-UEKVPHQBSA-N cephalexin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@@H]3N(C2=O)C(=C(CS3)C)C(O)=O)=CC=CC=C1 ZAIPMKNFIOOWCQ-UEKVPHQBSA-N 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 244000053095 fungal pathogen Species 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 102000004196 processed proteins & peptides Human genes 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 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 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 244000000010 microbial pathogen Species 0.000 description 3
- 229920001184 polypeptide Polymers 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 108010074860 Factor Xa Proteins 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 241001354013 Salmonella enterica subsp. enterica serovar Enteritidis Species 0.000 description 2
- 241000191967 Staphylococcus aureus Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 239000002543 antimycotic Substances 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000012407 engineering method Methods 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 108091008146 restriction endonucleases Proteins 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 1
- 108010076804 DNA Restriction Enzymes Proteins 0.000 description 1
- 230000004544 DNA amplification Effects 0.000 description 1
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 1
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 1
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 1
- 101710198774 Envelope protein US9 Proteins 0.000 description 1
- 241000255896 Galleria mellonella Species 0.000 description 1
- 241000702463 Geminiviridae Species 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 1
- 241000235058 Komagataella pastoris Species 0.000 description 1
- 241000255908 Manduca sexta Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 102000007079 Peptide Fragments Human genes 0.000 description 1
- 108010033276 Peptide Fragments Proteins 0.000 description 1
- 241000235648 Pichia Species 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 206010044565 Tremor Diseases 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 1
- 150000007854 aminals Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 238000012197 amplification kit Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 101150036080 at gene Proteins 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 238000013016 damping Methods 0.000 description 1
- 230000012969 defense response to bacterium Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000010612 desalination reaction Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229960004756 ethanol Drugs 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 230000005484 gravity Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000005389 magnetism Effects 0.000 description 1
- 239000011259 mixed solution Substances 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
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- -1 vibration 15s Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Images
Landscapes
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The invention relates to antimicrobial peptide and application thereof, and aims to provide a defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), the antimicrobial peptide encoded by the defensin gene and a preparation method for the defensin gene. A nucleotide sequence of the gene of the antimicrobial peptide is shown as SEQ ID NO.1; and an amino acid of the antimicrobial peptide is shown as SEQ ID NO.2. A protease Xa factor is introduced into an expression vector, so that recombinant fusion protein can be effectively digested, and defensin polypeptide is obtained through rapid separation in an ultrafiltration mode according to the magnitude of protein molecules; the whole process is quick and simple; and the obtained antimicrobial peptide has antifungal activity, and can remarkably inhibit the growth of entomopathogenic fungi such as Beauveria bassiana, Metarhizium anisopliae, Botrytis cinerea and the like.
Description
Technical field
The present invention relates to a kind of Bemisia tabaci antibacterial peptide defensin gene and coded antibacterial peptide and application thereof.Relate in particular to method for preparing purified, restructuring Bemisia tabaci antibacterial peptide and the antibacterial application thereof of a kind of Bemisia tabaci antibacterial peptide defensin that recombinates.
Background technology
In recent years, Bemisia tabaci Bemisia tabaci (Gennadius) has become the disastrous insect of a kind of global invasion, all over the world, breaks out and causes disaster, and causes heavy loss example.This worm is a large amount of feeding plant juice not only, causes plant withered, and propagate various plants virus, and especially geminivirus infection, often causes the viroses of plant to be very popular, and makes crop Severe Reduction or total crop failure.Because Bemisia tabaci host range is wide, reproductivity is strong, and hazard approach is many, and the ability that sterilant is developed immunity to drugs is strong especially, therefore, when it intrudes into a new area, very easily breaks out and causes disaster.
Due to the abuse of traditional antibiotic and the continuous generation of Resistant strain, had a strong impact on the clinical therapeutic efficacy of infectious diseases.And that antibacterial peptide has sterilization is strong, be difficult for causing the advantages such as resistance, become the study hotspot that antibacterials are new.Yield of antibacterial peptides is low at present, and the separating difficulty of natural antibacterial peptide is large, thereby tends to utilize gene engineering method to obtain antibacterial peptide.
Alexin (defensin) is the cationoid antimicrobial polypeptide being extensively formed in the bodies of aminal and plant of discovered in recent years, 29~54 amino-acid residues, consists of, and contains 6~8 conservative halfcystines (normally 6), and molecular weight is less than 5KDa.N end has 3 β-bends and 1 γ corner, and there is 1 amphipathic α spiral centre, and C end has 1 antiparallel β-pleated sheet structure, by 3~4 disulfide linkage, connects α spiral and β-bend, forms specific CS α beta structure.Alexin can be synthesized and be stored in cytoplasmic particle in specific cell, or synthetic by pathogenic micro-organism induction, multiple-microorganism is all had to stronger defence capability, Main Function is in the cytolemma of pathogenic micro-organism, make pathogenic micro-organism be difficult for it to produce resistance, alexin is class candidate new antibacterials that have much at present magnetism.
Up to the present, the research report that still there is no Bemisia tabaci defensin antibacterial peptide both at home and abroad.
Summary of the invention
The technical problem to be solved in the present invention is, for current Bemisia tabaci antibacterial peptide research situation and deficiency thereof, provide a kind of Bemisia tabaci antibacterial peptide defensin gene and its coded antimicrobial polypeptide of recombinating, and described gene has the application in the recombinant protein of anti-microbial activity at gene in preparation.
Solution of the present invention is, a kind of Bemisia tabaci defensin antibacterial peptide gene is provided, and the nucleotide sequence of this gene is as shown in SEQ ID NO.1.
The present invention also provides described Bemisia tabaci defensin antibacterial peptide gene to have the application in the recombinant protein of anti-microbial activity in preparation.
The present invention also provides described Bemisia tabaci defensin antibacterial peptide gene preparation to have the method for the recombinant protein of anti-microbial activity, is by gene recombination technology, described gene to be expressed in intestinal bacteria, obtains the recombinant protein with anti-microbial activity.
The present invention also provides the antibacterial peptide of described Bemisia tabaci defensin antibacterial peptide gene coding, and the aminoacid sequence of this antibacterial peptide is as shown in SEQ ID NO.2.
The present invention also provides described antibacterial peptide in the application for the preparation of preventing and treating in the medicine of Bemisia tabaci, and the applicable Bemisia tabaci kind of this medicine is: hidden kind of Middle East-Asia Minor 1 (MEAM1), hidden kind of Mediterranean (Med), hidden kind of ZHJ1, hidden kind of ZHJ2 or hidden kind of ZHJ3.
With respect to prior art, beneficial effect of the present invention is:
1, in the Bemisia tabaci defensin antibacterial peptide gene prokaryotic expression process in the present invention, in expression vector, introduce proteolytic enzyme Xa factor, effectively enzyme is cut recombination fusion protein, and utilizes ultrafiltration mode sharp separation to obtain defensin polypeptide according to protein molecular size.Whole process is simple fast.
2, the restructuring Bemisia tabaci defensin antibacterial peptide in the present invention has anti-mycotic activity, can obviously suppress the insect pathogenic fungus growths such as beauveria bassiana (Beuaveria bassiana), Metarhizium anisopliae (Metarhizium anisopliae) and botrytis cinerea (Botrytis cinerea).
Accompanying drawing explanation
Fig. 1: different hidden kind of Bemisia tabaci antibacterial peptide defensin gene PCR amplified production electrophorograms.In Fig. 1, M is DNA maker, and 1-5 is respectively Bemisia tabaci MEAM1, Med, ZHJ1, ZHJ2 and ZHJ3 antibacterial peptide defensin gene PCR amplified production, and bp represents base pair.
Fig. 2: Bemisia tabaci antibacterial peptide gene and coli expression carrier plasmid pET-32a and defensin mature peptide gene PCR product cleavage map.In Fig. 2, M is DNA maker, 1 be plasmid pET-32a through BamH I and Xho I double digestion after product, 2 and 3 is that defensin mature peptide gene PCR product is through BamH I and Xho I double digestion after product.
Fig. 3: the structure schematic diagram of Bemisia tabaci antibacterial peptide defensin mature peptide segment composition expression vector pET32a-Def.
The SDS-PAGE gel electrophoresis figure of the pET32a-Def-BL21 of Fig. 4: IPTG induction and the restructuring Bemisia tabaci antimicrobial peptide protein of purifying.M is albumen maker, and 1-6 represents different IP TG induced concentration 0mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1mM, and 7 represent recombinant protein Trx-Def after purifying, and kDa represents molecular weight of albumen.
Fig. 5: restructuring Bemisia tabaci antibacterial peptide defensin Tricine-SDS-PAGE gel electrophoresis figure.M is albumen maker, and 1 is defensin protein product.
Specific embodiments
The present invention sets about from Bemisia tabaci antibacterial peptide defensin gene, to its carry out can character, the research of genetic expression and location and the aspect such as recombinant expressed, obtained the restructuring alexin defensin of obligate anti-mycotic activity.
Bemisia tabaci antibacterial peptide defensin gene of the present invention, its nucleotides sequence is as shown in SEQ ID NO.1.Sequence signature: length: 624 base pairs; Type: nucleic acid; Chain: two strands; Topological framework: linearity; Molecule type: cDNA.
The antibacterial peptide of Bemisia tabaci defensin antibacterial peptide gene coding of the present invention, its amino acid is as shown in SEQ ID NO.2.Sequence signature: length: 76 amino acid; Type: amino acid; Chain: strand; Topological framework: linearity; Molecule type: protein.
The cloning process of Bemisia tabaci antibacterial peptide gene cDNA of the present invention is to take the total RNA reverse transcription of Bemisia tabaci to obtain cDNA as template, construction cDNA library, build antibacterial peptide database, transcribe the local Blast screening of group with Bemisia tabaci and obtain defensin Partial cDNA Sequence, then through the amplification of RACE method, obtain end sequence, finally by sequence assembly, obtain Bemisia tabaci antibacterial peptide defensin gene cDNA full length sequence.
The present invention is that to take Bemisia tabaci antibacterial peptide gene full-length gene double-stranded DNA be template for the antibacterial peptide mature peptide gene order of expressing Bemisia tabaci antibacterial peptide recombinant protein, through PCR method amplification, obtain, it derives from the corresponding gene fragment in Bemisia tabaci antibacterial peptide full-length gene region.
Expression vector establishment method of the present invention is according to a conventional method, by by the synthetic Bemisia tabaci antibacterial peptide gene of PCR method through enzyme cut with separation and purification after, be connected to same enzyme and cut having between corresponding restriction enzyme site (being BamH I and Xho I) after purifying, be built into the required expression vector that contains Bemisia tabaci antibacterial peptide gene.
The recombinant expression vector that the Bemisia tabaci antibacterial peptide gene that the above-mentioned bacillus coli gene expression vector that contains Bemisia tabaci antibacterial peptide gene of the present invention is preferentially synthesized by the present invention and coli expression carrier pET-32a build, called after pET32a-Def.
The intestinal bacteria recombinant bacterial strain that the present invention can express Bemisia tabaci antibacterial peptide gene preferentially transforms e. coli bl21 (DE3) by the expression vector pET32a-Def that contains Bemisia tabaci antibacterial peptide and the bacterial strain that obtains, called after pET32a-Def-BL21.
The concrete preparation method of above-mentioned Bemisia tabaci antibacterial peptide gene realizes by following technical solution: choose intestinal bacteria recombinant bacterial strain pET32a-Def-BL21 and be seeded in the LB liquid nutrient medium that 10ml contains penbritin, 37 ℃ of overnight incubation, get 50 μ l bacterium liquid and join 50ml AMP is housed
+in the aseptic triangular flask of 150ml of LB liquid nutrient medium, 37 ℃, shaking in case of 150rpm cultivated 6~8h to logarithmic phase, add 100mM IPTG to final concentration be 1mM, 27 ℃, 250rpm shaking culture.After growing into plateau, collects recombinant bacterium thalline, through the separation and purification Bemisia tabaci antibacterial peptide product that obtains recombinating.
Above-mentioned enzyme is cut rear non-fusion antibacterial peptide sterling, utilize to add Factor Xa sequence, uses Factor Xa enzyme to cut above-mentioned purification of Recombinant Bemisia tabaci antimicrobial peptide protein, and purifying again, obtains about 5kDa left and right Bemisia tabaci antibacterial peptide defensin peptide fragment.
The present invention utilizes the above-mentioned antibacterial peptide that obtains to carry out anti-microbial activity detection, and result shows: it is active that the Bemisia tabaci defensin antibacterial peptide after enzyme is cut purifying has anti-insect pathogenic fungus.
Bemisia tabaci antibacterial peptide gene defensin of the present invention applies in preparation has the recombinant protein of antibacterial peptide activity.
Wherein, the method for described application is by gene recombination technology, described gene to be expressed in intestinal bacteria, obtains the recombinant protein with anti-microbial activity.
For example: the Bemisia tabaci antibacterial peptide gene defensin gene clone obtaining, to pET-32a (Novagen company) expression vector, is transformed into e. coli bl21 cell.Carry out abduction delivering, obtain the activated recombinant protein of tool (being the antibacterial peptide of Bemisia tabaci antibacterial peptide gene defensin genes encoding).
The result of carrying out bacteriostatic experiment after the recombinant protein purification of described anti-microbial activity shows: the polypeptide of the Bemisia tabaci antibacterial peptide gene defensin genetic expression of restructuring has antimycotic and activity yeast.
Further, utilize method of the present invention to produce and also can be used for Bemisia tabaci biological control by existing gene engineering method.
Below in conjunction with the concrete experimental data in laboratory and in conjunction with specific embodiments, further set forth the present invention.These embodiment are only not used in and limit the scope of the invention for the present invention.The experimental program of unreceipted actual conditions in lower routine embodiment, conventionally according to normal condition, for example Sambrook equimolecular is cloned: laboratory manual (New York:Cold Spring Haebor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, can also make some distortion and improvement.
Case study on implementation 1: Bemisia tabaci defensin antibacterial peptide cDNA clone
1.1, TRIzol method is extracted total RNA
(1) by a certain amount of (200 left and right Bemisia tabaci) freezing 1min, put into 1.5ml centrifuge tube, add 1ml TRIzol reagent, fully grind homogenate, the standing 5min of room temperature.
(2) in centrifuge tube, add 0.2ml chloroform, vibration 15s, mixed solution proceeds in TIANGEN centrifuge tube, standing 2min.
(3) 4 ℃, the centrifugal 15min of 12000g, gets supernatant liquor, is proceeded in the centrifuge tube of a new 1.5ml.
(4) in centrifuge tube, add 0.5ml Virahol, liquid in pipe is mixed gently, the standing 10min of room temperature.
(5) 4 ℃, the centrifugal 10min of 12000g, discards supernatant liquor.
(6) in centrifuge tube, add 1ml 75% ethanol, washing precipitation gently, 4 ℃, the centrifugal 5min of 7500g, discards supernatant liquor.(now add dehydrated alcohol, can in-80 ℃ of medium-term and long-term preservations of Ultralow Temperature Freezer)
(7) dry centrifuge tube, add appropriate DEPC H
2o dissolves (65 ℃ of dissolutions), the value of spectrophotometer measurement OD260/OD280, and ratio between 1.8~2.0 time, carries out next step experiment.
1.2, cDNA the first chain is synthetic
(1) toward the RNA that adds respectively in the aseptic centrifuge tube of 0.5ml 1 μ l to extract, 1 μ l SMART IV/5 ' PCR forward primer, 1 μ l CDS III/3 ' PCR reverse primer, adds 2 μ l DEPC H
2o makes cumulative volume reach 5 μ l.
(2) reagent mixing in centrifuge tube is also of short duration centrifugal, hatches 2min for 72 ℃.
(3) rapidly by centrifuge tube ice bath 2min, of short duration centrifugal.
(4) in centrifuge tube, add respectively 2 μ l 5 * Frist-strand Buffer, 1 μ l 20mM dithiothreitol (DTT) (DTT), 1 μ l 10mM dNTP, 1 μ l Reverse Transcriptase ThermoScript II, piping and druming mixes repeatedly, of short duration centrifugal.
Hatch 1h for (5) 42 ℃.
(6) centrifuge tube is placed in to 2~3min on ice, termination reaction.Get a part for further experiment, all the other are in-80 ℃ of freezing preservations of Ultralow Temperature Freezer.
1.3, dsDNA is synthetic
(1) in 0.5 centrifuge tube, add respectively 1 μ l the first chain synthesis reaction product, 5 μ l 10 * LA Buffer (Mg
2+free), 5 μ l Mg
2+, 8 μ l dNTP, 1 μ l SMART IV/5 ' PCR forward primer, 1 μ lCDS III/3 ' PCR reverse primer, 0.5 μ l LA Tag archaeal dna polymerase, 29.5 μ l ddH
2o, fully mixes, of short duration centrifugal.
(2) in PCR instrument by following amplification program (95 ℃, 20s; 25~28 * (95 ℃, 5s; 68 ℃, 6min)) amplification.
(3) get 5 μ l PCR products for gel detection (the contained molecular weight ranges of test strip and band are bright dark), get the good 100 times of templates for PCR after doing of 1 μ l product dilution of detected result, all the other are in-80 ℃ of freezing preservations of Ultralow Temperature Freezer.
1.4, pcr amplification Bemisia tabaci antibacterial peptide defensin gene 5 ' and 3 ' end
The test kit SMARTer of Clontech company is pressed in operation
tMrACE cDNA Amplification Kit specification sheets carries out.According to the requirement of test kit, according to known Defensin gene est sequence, design respectively two upstream and downstream primers (in Table 1), to carry out nest-type PRC.PCR reaction conditions is for the first time: 98 ℃, and 3min; 30 * (98 ℃, 10s; 60 ℃, 20s; 68 ℃, 1min); 68 ℃, 6min.PCR be take for the first time PCR product and is increased as template for the second time, and reaction conditions is: 95 ℃, and 3min; 35 * (95 ℃, 25s; 63 ℃, 20s; 72 ℃, 1min); 72 ℃, 6min.Amplified production is connected on pMD-19 carrier and obtains recombinant plasmid, and with the universal primer M13 of pMD-19 ± carry out sequencing, measurement result and known array fragment compare and sequence assembly.Sequence assembly result is carried out BlastX on NCBI, and it is similar with maduca sexta Manduca sexta antibacterial peptide defensin gene height to greater wax moth Galleria mellonella that result shows to obtain sequence, and similarity is respectively 75% and 74%.
Table 1.PCR amplification Bemisia tabaci antibacterial peptide defensin gene 5 ' and the required primer of 3 ' end
Primer title | Primer sequence (5 '-3 ') |
def-for 1 | GTTGGTGCGAAACCCGTAAATAAGAT |
def-for 2 | GGAGTGAACTACACCTCTGACTGCTTGAA |
def- |
TGAACTACACCTCTGACTGCTTGAAAGA |
def- |
TAAAGGCGGACATTGTGGAAGTGC |
Embodiment 2: Bemisia tabaci antibacterial peptide recombinant expression vector structure, expression and bacteria resistance function are measured
2.1 expression vector establishment
(1) according to the cloning site of the sequence of Bemisia tabaci defensin antibacterial peptide and expression vector pET-32a (Novagen company), design primer, and 5 '-end of gene has designed identification cleavage site (IEGR) encoding sequence of Xa factor:
Def-F:CG
gGATCCaTTGAGGGTCGCGACGTCCTCATCGGAAGTT (underscore is BamH I site, and runic is Xa factor encoding sequence);
Def-R:CCG
cTCGAGtTATTTACGGGTTTCGCACCAAC (underscore is XhoI site).
(2) gene amplification, clone and recombinant plasmid screening
Take Bemisia tabaci dsDNA as template, with above-mentioned primer, carry out PCR reaction, amplification condition is: 94 ℃, and 2min denaturation; 94 ℃, 15s, 63 ℃, 30s, 72 ℃, 45s, 35 circulations; 72 ℃ are extended 6min.
Get 2 μ lPCR products, after 1% agarose gel electrophoresis detects, the clean PCR product that reclaims of Clean UP test kit.Using the DNA of PET-32a plasmid vector and above-mentioned recovery respectively as DNA profiling, prepare following reaction system: Xho I restriction enzyme 1 μ l, BamH I restriction enzyme, DNA profiling 10 μ l, ddH
2o 6 μ l, Buffer 2 μ l.Mix gently, of short duration centrifugal, hatch 1h for 37 ℃.
(3) by the double digestion reaction product of second step, do agarose gel electrophoresis, cut sizeable DNA band and do DNA gel recovery.
(4) in PCR pipe, add respectively the PCR product after 2.5 μ l double digestions, the pET-32a plasmid vector after 0.5 μ l double digestion, the T4Ligase DNA of 0.5 μ l, the T4Ligase DNA Buffer of 1 μ l, the ddH of 5 μ l
2o, hatches under 16 ℃ of conditions, more than connecting 1h.
(5) connection product is transformed in competence, coats on the LB culture medium flat plate of penbritin 37 ℃ of overnight incubation.Choose spot and cultivate bacterium liquid, with T7ter and S-Tag universal primer, carry out r-Tag reaction system PCR and detect, for every part of positive, get 100 μ l and reserve seed for planting and send survey.
(6) comparison sequencing result, chooses the bacterium liquid of reserving seed for planting that result is correct and does next step experiment.
2.2 restructuring Bemisia tabaci defensin peptide expression and purifying
Choosing order-checking positive bacteria liquid is cultivating containing 37 ℃ of overnight shakings in the LB liquid nutrient medium of 50 μ g/mL penbritins, next day, inoculum size with 1% is connected to containing in the liquid nutrient medium of 150 μ g/mL penbritins, treat that thalli growth is to logarithmic phase, OD600 approximately 1.0 o'clock, add 100mM IPTG to make its final concentration be about 1mM, after 27 ℃/250rpm induction 5h, survey OD600 sampling, the centrifugal 5min of 5000rpm, PBS damping fluid (pH7.0) resuspension thalline also adds appropriate sample-loading buffer, and for 12%SDS-PAGE electrophoretic analysis protein expression situation, fusion rotein Trx-Def is with the form successful expression of solubility, finally utilize Clontech His-tag gravity purification kit purifying to obtain Bemisia tabaci defensin antibacterial peptide recombinant protein.The Bemisia tabaci defensin recombinant protein of purifying is through 15% SDS-PAGE gel electrophoresis analysis, and the recombinant protein that Trx-Def merges shows about 25kDa size, in the same size with expection.
2.3 recombinant protein Xa factor proteolytic cleavages
Protein concentration after A280 mensuration purifying, fusion rotein concentration in 50 μ L systems (0.2 μ g/ μ L), by following system cutting, Xa factor dosage is 2U, in 23 ℃ of reaction 24h.Enzyme is cut product and is first utilized the super filter tube (Millipore that molecular weight cut-off is 10kDa, Amicon Ultra-4) remove Trx label and Xa factor etc. and be greater than 10kDa protein molecular, then the super filter tube that is 3kDa with molecular weight cut-off carries out desalination and concentrated to it, adopts 16%Tricine-SDS-PAGE to analyze.
Cutting system is as follows:
2.5 anti-microbial activities detect
Substratum is YDP substratum, and strains tested has gram-positive microorganism, Gram-negative bacteria, insect pathogenic fungus and yeast.Wherein gram-positive microorganism is streptococcus aureus (Staphylococcus aureus); Gram-negative bacteria is Salmonella enteritidis (Salmonella enteritidis) and intestinal bacteria (Escherichia coli); Insect pathogenic fungus is beauveria bassiana (Beuaveria bassiana), Metarhizium anisopliae (Metarhizium anisopliae); Plant pathogenic fungi botrytis cinerea (Botrytis cinerea) yeast is pichia spp (pichia pastoris).By liquid growth inhibition test, undertaken this antibacterial tests, concrete steps are: at substratum, (bacteria culture medium is LB substratum to strains tested, fungi and yeast culture base are YDP substratum) in grow into OD600 and be 0.8 after, with YDP substratum, being diluted to OD600 is 0.001 left and right, in 80 μ L yeast culture things, adds the above-mentioned enzyme of 20 μ L different concns to cut protein product.At 37 ℃, cultivate 24 hours, measure mixed culture OD600 value, determine anti-microbial activity, each test repeats 3 times.Control group is respectively: fusion rotein Trx, Xa factor cutting 0h cutting liquid, penbritin.By growth inhibition test, result shows that defensin has the ability of antimycotic and yeast significantly.
The anti-microbial activity result of Bemisia tabaci defensin antibacterial peptide is as shown in table 2:
Table 2
Claims (3)
1. a Bemisia tabaci defensin antibacterial peptide gene, is characterized in that, the nucleotide sequence of this gene is as shown in SEQ ID NO.1.
2. described in claim 1, Bemisia tabaci defensin antibacterial peptide gene has the application in the recombinant protein of anti-microbial activity in preparation.
3. utilize Bemisia tabaci defensin antibacterial peptide gene preparation described in claim 1 to there is the method for the recombinant protein of anti-microbial activity, it is characterized in that, be by gene recombination technology, described gene to be expressed in intestinal bacteria, obtain the recombinant protein with anti-microbial activity.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201210073276.3A CN102586262B (en) | 2012-03-19 | 2012-03-19 | Defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), antimicrobial peptide encoded by defensin gene and preparation method for defensin gene |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201210073276.3A CN102586262B (en) | 2012-03-19 | 2012-03-19 | Defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), antimicrobial peptide encoded by defensin gene and preparation method for defensin gene |
Publications (2)
Publication Number | Publication Date |
---|---|
CN102586262A CN102586262A (en) | 2012-07-18 |
CN102586262B true CN102586262B (en) | 2014-04-16 |
Family
ID=46475491
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201210073276.3A Expired - Fee Related CN102586262B (en) | 2012-03-19 | 2012-03-19 | Defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), antimicrobial peptide encoded by defensin gene and preparation method for defensin gene |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN102586262B (en) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104480115A (en) * | 2014-12-07 | 2015-04-01 | 浙江大学 | Bemisia tabaci defensin gene segment and dsRNA thereof based on gene silencing technology |
CN105274007B (en) * | 2015-07-27 | 2018-11-27 | 华南农业大学 | One plant of Metarhizium anisopliae var. Anisopliae MaTS02 and its application in terms of preventing and treating Bemisia tabaci |
CN106854657B (en) * | 2016-12-20 | 2018-05-15 | 广州格拉姆生物科技有限公司 | A kind of energy assistant degradation protein simultaneously secretes the prebiotic recombinant Saccharomyces cerevisiae of antibacterial peptide |
CN106884007A (en) * | 2017-03-05 | 2017-06-23 | 中国农业科学院植物保护研究所 | Hidden kind of laccase LAC1 of Bemisia tabaci MED and its gene and application |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101220377A (en) * | 2008-01-23 | 2008-07-16 | 山东省农业科学院高新技术研究中心 | Method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius |
CN101555521A (en) * | 2009-05-08 | 2009-10-14 | 中国农业科学院植物保护研究所 | Pair of Q-type bemisa babaci specificity SCAR primers, fast PCR detection method and kit |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2010193755A (en) * | 2009-02-24 | 2010-09-09 | Sumitomo Chemical Co Ltd | Method for discriminating bemisia tabaci biotype |
-
2012
- 2012-03-19 CN CN201210073276.3A patent/CN102586262B/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101220377A (en) * | 2008-01-23 | 2008-07-16 | 山东省农业科学院高新技术研究中心 | Method for horizontal transfection of exogenesis endosymbiosis bacterium to bemisia tabaci gennadius |
CN101555521A (en) * | 2009-05-08 | 2009-10-14 | 中国农业科学院植物保护研究所 | Pair of Q-type bemisa babaci specificity SCAR primers, fast PCR detection method and kit |
Non-Patent Citations (1)
Title |
---|
JP特开2010-193755A 2010.09.09 |
Also Published As
Publication number | Publication date |
---|---|
CN102586262A (en) | 2012-07-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110643612B (en) | Trachinotus ovatus antimicrobial peptide NK-lysin gene and application thereof | |
CN102586262B (en) | Defensin gene of antimicrobial peptide of bemisia tabaci (Gennadius), antimicrobial peptide encoded by defensin gene and preparation method for defensin gene | |
CN104830825B (en) | A kind of endolysin and its application from salmonella bacteriophage | |
CN110551732A (en) | Trachinotus ovatus antimicrobial peptide LEAP-2 gene and application thereof | |
CN103789317A (en) | Scylla paramamosain antibacterial peptide hyastatin gene clone, encoding protein recombination and application | |
CN105219779B (en) | Red claw crayfish coagulogen and preparation method and application | |
CN101906165B (en) | Expression product in series of two fish antibacterial peptide genes and expression method thereof | |
CN101392249A (en) | Antibiotic peptide gene, preparation method thereof and construction of expression plasmid of the same in pichia vector | |
CN101550183B (en) | Antibacterial peptide and construction and application thereof | |
CN105255935A (en) | Expression method of antimicrobial peptide CC34 in bacillus subtilis | |
CN104962566A (en) | Recombinant bacteriocin and preparation method and application thereof | |
CN110616227A (en) | Gene, recombinant expression vector, engineering strain and application of anti-freeze protein from tenebrio molitor | |
CN107446941A (en) | Cecropin A antibacterial peptide based on self-aggregation short-peptide induction and preparation method thereof | |
CN106496333A (en) | One kind expresses hybrid peptide and preparation method and application using bacillus subtilis | |
CN103103196A (en) | Modified goat defensin gene and preparation method and application thereof | |
CN102816770A (en) | Cotesia plutellae antimicrobial peptide defensin gene, antimicrobial peptide and application | |
CN102337288A (en) | Gene engineering preparation method of tilapia hepcidin | |
CN101886081A (en) | Procambarus clarkia chitin peptide gene and encoded chitin peptide and application thereof | |
CN107475222A (en) | The heat-resisting human lysozyme of genetic engineering transformation | |
CN107266539A (en) | A kind of bacillus licheniformis W10 antibacterial proteins and application | |
CN102061303B (en) | Fusion expression product of antimicrobial peptide genes of two marine animals and preparation method of fusion expression product | |
CN115925847B (en) | Application of MmPI in preparation of trypsin inhibitor | |
CN102094024A (en) | Plutella xylostella cecropin gene, encoded protein, corresponding expression system and application | |
CN104531712A (en) | Preparation and application of Bemisia tabaci peptidoglycan recognition protein with bactericidal activity | |
CN101565703B (en) | Eriocheir sinensis Crustin-2 gene and application of recombinant protein thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
C10 | Entry into substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
C14 | Grant of patent or utility model | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20140416 |
|
CF01 | Termination of patent right due to non-payment of annual fee |