CN104531712A - Preparation and application of Bemisia tabaci peptidoglycan recognition protein with bactericidal activity - Google Patents
Preparation and application of Bemisia tabaci peptidoglycan recognition protein with bactericidal activity Download PDFInfo
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Abstract
Relating to the technical field of molecular biology and genetic engineering, the invention aims to provide preparation and application of a Bemisia tabaci peptidoglycan recognition protein with bactericidal activity. The nucleotide sequence of a Bemisia tabaci peptidoglycan recognition protein coding gene is shown as SEQ ID NO.1. The Bemisia tabaci peptidoglycan recognition protein BtPGRP gene and the coding nucleotide sequence provided by the invention enable application in escherichia coli to express new protein and coding sequence. Bacteriostatic experiment of the purified recombinant protein with bactericidal activity shows that: recombinant Bemisia tabaci peptidoglycan recognition protein gene BtPGRP prokaryotically expressed recombinant protein has the ability of combining gram-negative bacteria and gram-positive bacteria, and can kill gram-negative bacteria and gram-positive bacteria at the same time. Further, application of the method to production through existing genetic engineering approaches has potential application value in development of broad-spectrum antibacterial drugs, novel immunopotentiators, feed additives and other aspects.
Description
Technical field
The invention belongs to molecular biology and gene engineering technology field.The present invention relates to the application of a kind of Bemisia tabaci peptidoglycan recognition protein (BtPGRP) gene and coded albumen thereof, particularly relate to a kind of in-vitro recombination expression technology of Bemisia tabaci peptidoglycan recognition protein (BtPGRP), the separation and purification of active recombinant protein and to the identification of microorganism and bactericidal Effect study.
Background technology
Innate immune reaction is the important defence line together of body defenses infectious diseases, and this system of defense is all very important for all multicellular organism existence and species continuity.Be different from Mammals, insect, without immunoglobulin (Ig), lacks acquired immunity, and therefore insect a major challenge of surviving identifies the exotic disease original exactly.The pathogen recognition mode of a set of uniqueness has been developed in long-term evolution process, pathogen outer wall lipopolysaccharides (LPS), lipoteichoic acids, lipoprotein, peptidoglycan (PGN), β-1,3-dextran defensive raction is made to it can be identified.The molecular structure of some total high conservative of this pathogenic micro-organism surface is called pathogen-associated molecular pattern (Pathogen-associated molecular pattern, PAMP).Immune discrimination develops multiple pattern recognition receptors (the pattern recognition receptor that can identify one or more PAMP molecules during evolution, PRP), as scavenger receptor (SCRs), C-type lectin (CTLs), Gram-negative bacteria associated proteins GNBPs (Gram-negative binding proteins) and peptidoglycan recognition protein PGRPs (peptidoglycan recognition proteins) etc.Peptidoglycan recognition protein PGRPs is the important pattern recognition receptors (PRR) of a class, plays an important role in the invasion of identification pathogenic agent and activate immunity regulatory pathway.Peptidoglycan recognition protein PGRP is Late Cambrian in silkworm Bombyx mori, and this albumen can binding peptide glycan, activates polyphenol oxidase enzyme cascade.PGRP exist 1 with the structural domain of T7 N,O-Diacetylmuramidase homology, and there is N-acetyl cell wall-ALANINE lactamase activity that Zn relies on.Found altogether at present to find 7 PGRP genes in 13 PGRP genes encodings, 17 PGRP albumen, anopheles costalis Anopheles gambiae genome in drosophila melanogaster Drosophila melanogaster genome, homology comparison finds these genes and Mammals PGRPs gene high conservative.Current research finds that PGRP can be divided into following a few class in insect congenital immunity: bring out pro-phenoloxidase cascade reaction after a) identifying pathogenic agent, produces black and usually wraps up microorganism; B) identify gram-positive microorganism, activate Toll signal path (PGRP-SA); C) identify Gram-negative bacteria, activate Imd signal path, induction autophagy; D) lactamase activity, makes PGN inactivation.In addition, in Mammals (people and rat), PGRP also has antibacterial activity.Nearest research finds that PGRP-SB1, the bear PGRP-S of fruit bat in insect have anti-microbial effect to genus bacillus.The sterilization Machine of PGRP albumen is shaped with two kinds: one is for PGRP and the irreversible combination of peptidoglycan, thus the synthesis of interference bacteria cell wall; Another kind is that some PGRP have lactamase activity.Can to degrade peptidoglycan, thus to destroy cell walls thalline is broken, this germicidal action is similar to penicillin.
The structure and function of peptidoglycan recognition protein in insect and Mammals research to a certain extent, but, still do not have the research of Bemisia tabaci peptidoglycan recognition protein BtPGRP to report both at home and abroad at present.Bemisia tabaci Bemisia tabaci (Gennadius) has become the disastrous insect of a kind of global invasive, at population outbreak all over the world, causes heavy loss.This worm is a large amount of feeding plant juice not only, causes plant to wither, and propagate various plants virus, is important virus disseminating medium, often causes the viroses of plant to be very popular, make crop Severe Reduction or total crop failure.PGRP is passing the research in malicious insect vector Bemisia tabaci, is conducive to better disclosing it in immune response and the effect of propagating in plant virus and working mechanism thereof, simultaneously also for exploitation extensive pedigree antibiotic and screening immunostimulant provide foundation.
Summary of the invention
The technical problem to be solved in the present invention is, for current Bemisia tabaci immune response research situation and deficiency thereof, provides a kind of preparations and applicatio with Bemisia tabaci peptidoglycan recognition protein (BtPGRP) albumen of fungicidal activity.
For technical solution problem, solution of the present invention is:
The invention provides a kind of Bemisia tabaci peptidoglycan recognition protein encoding gene, the nucleotide sequence of this gene is as shown in SEQID NO.1.
Invention further provides by the albumen of Bemisia tabaci peptidoglycan recognition protein encoding gene encodes, the aminoacid sequence of this albumen is as shown in SEQ ID NO.2.
Invention further provides aforementioned Bemisia tabaci peptidoglycan recognition protein encoding gene and prepare the method had in the recombinant protein of anti-microbial activity, be by gene recombination technology, described gene is expressed in intestinal bacteria, obtain the recombinant protein with anti-microbial activity.
Invention further provides foregoing proteins and prepare the application in antimicrobial DP finish, food preservative or fodder additives.
Beneficial effect of the present invention is:
The nucleotide sequence of Bemisia tabaci peptidoglycan recognition protein BtPGRP gene provided by the present invention and coding thereof, can be applied in the new albumen of expression in escherichia coli, encoding sequence.The result of carrying out bacteriostatic experiment after the recombinant protein purification of described anti-microbial activity shows: the recombinant protein of the Bemisia tabaci peptidoglycan identification protein gene BtPGRP prokaryotic expression of restructuring has the ability in conjunction with Gram-negative bacteria and gram-positive microorganism, can kill Gram-negative bacteria and gram-positive microorganism simultaneously.Further, utilize method of the present invention to be produced by existing gene engineering method, in exploitation broad-spectrum antimicrobial class medicine and Novel immune toughener, fodder additives etc., there is potential using value.
Accompanying drawing explanation
Fig. 1 is the SDS-PAGE gel electrophoresis figure of the pET28a-BtPGRP-BL21 of IPTG induction and the restructuring Bemisia tabaci peptidoglycan recognition protein BtPGRP albumen of purifying.1: do not induce pET28a-BtPGRP-BL21 bacterial strain; The pET28a-BtPGRP-BL21 bacterial strain that Lane 2:1mM IPTG induces; 3: purification of recombinant proteins; M: albumen Marker.
Fig. 2 is that Bemisia tabaci peptidoglycan recognition protein BtPGRP gene recombinant protein is to colibacillary recognition reaction figure.
Fig. 3 is that Bemisia tabaci peptidoglycan recognition protein BtPGRP gene recombinant protein is to the recognition reaction figure of streptococcus aureus.
Fig. 4 is that Bemisia tabaci peptidoglycan recognition protein BtPGRP gene recombinant protein is to the growth-inhibiting action diagram of E. coli.
Fig. 5 is that Bemisia tabaci peptidoglycan recognition protein BtPGRP gene recombinant protein is to the growth-inhibiting action diagram of streptococcus aureus S.aureus.
Specific embodiments
In the present invention, the cloning process of described Bemisia tabaci peptidoglycan identification protein gene cDNA obtains cDNA for template with the reverse transcription of Bemisia tabaci total serum IgE, construction cDNA library, BtPGRP Partial cDNA Sequence is obtained according to the screening of Bemisia tabaci transcript profile, then obtain end sequence through the amplification of RACE method, obtain Bemisia tabaci peptidoglycan identification protein gene cDNA full length sequence finally by sequence assembly.
The present invention is for template with Bemisia tabaci peptidoglycan recognition protein full-length gene double-stranded DNA for expressing the mature peptide gene order of Bemisia tabaci peptidoglycan recognition protein recombinant protein, obtain through PCR method amplification, it derives from the gene fragment corresponding to Bemisia tabaci peptidoglycan recognition protein full-length gene region.
The Bemisia tabaci peptidoglycan identification protein gene that the above-mentioned bacillus coli gene expression vector containing Bemisia tabaci peptidoglycan identification protein gene of the present invention is preferentially synthesized by the present invention and the recombinant expression vector that coli expression carrier pET-28a builds, called after pET28a-BtPGRP.
The bacterial strain that the E. coli recombinant stain that the present invention can express Bemisia tabaci peptidoglycan identification protein gene is preferentially obtained by expression vector pET28a-BtPGRP transformation of E. coli BL21 (DE3) containing Bemisia tabaci peptidoglycan recognition protein, called after pET28a-BtPGRP-BL21.
The concrete preparation method of recombinant protein of above-mentioned Bemisia tabaci peptidoglycan recognition protein realizes by following technical solution: choose E. coli recombinant stain pET28a-BtPGRP-BL21 and be seeded in the LB liquid nutrient medium that 10ml contains kantlex, 37 DEG C of overnight incubation, get 50 μ l bacterium liquid and join 50ml Kana is housed
+in the aseptic triangular flask of 150ml of LB liquid nutrient medium, 37 DEG C, shaking in case of 150rpm cultivates 6 ~ 8h to logarithmic phase, and adding 100mM IPTG to final concentration is 1mM, 27 DEG C, 250rpm shaking culture.After recombinant bacterium grows into plateau, collect thalline, obtain restructuring Bemisia tabaci peptidoglycan recognition protein through separation and purification.
Bemisia tabaci peptidoglycan recognition protein BtPGRP of the present invention applies preparing in the activated recombinant protein of tool.Wherein, the method for described application by gene recombination technology, described gene is expressed in intestinal bacteria, obtains the recombinant protein with anti-microbial activity.Such as: the Bemisia tabaci peptidoglycan identification protein gene of acquisition is cloned into pET-28a (Novagen company) expression vector, is transformed into e. coli bl21 cell.Carry out abduction delivering, obtain the activated recombinant protein of tool (i.e. the activated protein of Bemisia tabaci peptidoglycan recognition protein BtPGRP genes encoding).
The application of the recombinant protein of Bemisia tabaci peptidoglycan recognition protein, in described sequence table SEQ ID NO.2, the recombinant protein BtPGRP of aminoacid sequence is for the preparation of broad-spectrum antimicrobial class preparation.
In described sequence table SEQ ID NO.2, the recombinant protein BtPGRP of aminoacid sequence is for the preparation of immunostimulant or fodder additives.
Bemisia tabaci peptidoglycan recognition protein BtPGRP gene of the present invention, its nucleotide sequence as shown in SEQ ID NO.1, this sequence signature: length: 1027 base pairs; Type: nucleic acid; Chain: double-strand; Topological framework: linear; Molecule type: cDNA.
The albumen of Bemisia tabaci peptidoglycan recognition protein BtPGRP gene coding of the present invention, its amino acid is as shown in SEQID NO.2.This sequence signature: length: 235 amino acid; Type: amino acid; Chain: strand; Topological framework: linear; Molecule type: protein.Characteristic: this molecular weight of albumen is 25.86kDa, theoretical iso-electric point is 6.21.Wherein the 1-29 position of encoding sequence is signal peptide sequence, and mature peptide molecular weight is 22.67kDa, and iso-electric point is 5.81, has a conservative PGRP structural domain.
Recombinant protein sequence provided by the invention is Bemisia tabaci peptidoglycan recognition protein BtPGRP mature peptide region.This sequence source is SEQ ID NO.2 removal signal peptide precursor.
The Bemisia tabaci peptidoglycan recognition protein function of Immunofluorescence test and plate count is utilized to detect peptidoglycan recognition protein anti-microbial activity.Result shows that the present invention prepares peptidoglycan recognition protein and has that to kill gram-positive microorganism and Gram-negative bacteria active.Therefore the invention provides described peptidoglycan recognition protein for the preparation of the application in treatment gram-positive microorganism or the foodstuff additive of gram positive bacterial infection or medicine.
The experimental data concrete below in conjunction with laboratory and in conjunction with specific embodiments, sets forth the present invention further.These embodiments are only not used in for the present invention and limit the scope of the invention.The experimental program of unreceipted actual conditions in lower routine embodiment, usual conveniently condition, such as Sambrook equimolecular clone: laboratory manual (New York:Cold SpringHaebor Laboratory Press, 1989) condition described in, or according to the condition that manufacturer advises.It should be pointed out that for the person of ordinary skill of the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.
Case study on implementation 1: Bemisia tabaci peptidoglycan recognition protein BtPGRP full length gene cDNA clones
1.1, TRIzol method extracts total serum IgE
(1) by a certain amount of (about 200 Bemisia tabaci) freezing 1min, put into 1.5ml centrifuge tube, add 1ml TRIzol reagent, fully grind homogenate, room temperature leaves standstill 5min.
(2) in centrifuge tube, add 0.2ml chloroform, vibration 15s, mixed solution proceeds in TIANGEN centrifuge tube, leaves standstill 2min.
(3) 4 DEG C, 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, mixed gently by liquid in pipe, room temperature leaves standstill 10min.
(5) 4 DEG C, the centrifugal 10min of 12000g, discards supernatant liquor.
(6) in centrifuge tube, add 1ml 75% ethanol, washing precipitation gently, 4 DEG C, the centrifugal 5min of 7500g, discards supernatant liquor.(now add dehydrated alcohol, can in-80 DEG C of medium-term and long-term preservations of Ultralow Temperature Freezer)
(7) dry centrifuge tube, add appropriate DEPC H
2o dissolves (65 DEG C of dissolutions), the value of spectrophotometer measurement OD260/OD280, when ratio is between 1.8 ~ 2.0, carries out next step experiment.
1.2, cDNA first chain synthesis
(1) in the aseptic centrifuge tube of 0.5ml, add the RNA that 1 μ l extracts respectively, 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 in mixing centrifuge tube is also of short duration centrifugal, hatches 2min for 72 DEG C.
(3) rapidly by centrifuge tube ice bath 2min, of short duration centrifugal.
(4) in centrifuge tube, add 2 μ l 5 × Frist-strand Buffer respectively, 1 μ l 20mM dithiothreitol (DTT) (DTT), 1 μ l 10mM dNTP, 1 μ l Reverse Transcriptase ThermoScript II, repeatedly blow and beat mixing, of short duration centrifugal.
Hatch 1h for (5) 42 DEG C.
(6) centrifuge tube is placed in 2 ~ 3min on ice, termination reaction.Get a part for further experiment, all the other are in-80 DEG C of Ultralow Temperature Freezer freezen protective.
1.3, dsDNA synthesis
(1) in 0.5 centrifuge tube, 1 μ l first chain synthesis reaction product is added respectively, 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 DEG C, 20s; 25 ~ 28 × (95 DEG C, 5s; 68 DEG C, 6min)) amplification.
(3) 5 μ l PCR primer are got for gel detection (molecular weight ranges contained by test strip and band bright dark), get 1 good μ l product dilution 100 of detected result doubly for the template of PCR after doing, all the other are in-80 DEG C of Ultralow Temperature Freezer freezen protective.
1.4, pcr amplification Bemisia tabaci peptidoglycan recognition protein BtPGRP gene cDNA 5 ' and 3 ' end
Clontech company test kit SMARTer is pressed in operation
tMrACE cDNA Amplification Kit specification sheets carries out.According to the requirement of test kit, design two upstream and downstream primers respectively, to carry out nest-type PRC according to known peptidoglycan recognition protein BtPGRP gene est sequence.The primer is respectively:
Primer | Primer sequence (5 '-3 ') | Purposes |
PGRP-F 1 | CACCACTCCCGACTTCCACCTTGC | 3’RACE |
PGRP-F 2 | CTCGTGGGCTACTCGGAGCAGGAC | 3’RACE |
PGRP-R 1 | AAGGTGGAAGTCGGGAGTGGTGGAT | 5’RACE |
PGRP-R 2 | CGCCGAGAAATGCTATGTTGATGC | 5’RACE |
PCR reaction conditions is for the first time: 98 DEG C, 3min; 30 × (98 DEG C, 10s; 60 DEG C, 20s; 68 DEG C, 1min); 68 DEG C, 6min.Second time PCR increases for template with first time PCR primer, and reaction conditions is: 95 DEG C, 3min; 35 × (95 DEG C, 25s; 63 DEG C, 20s; 72 DEG C, 1min); 72 DEG C, 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 carries out BlastX on NCBI, and result shows to obtain sequence and known PGRP sequence has certain similarity, similarity about 40%.
Embodiment 2: Bemisia tabaci peptidoglycan recognition protein BtPGRP expression vector establishment, expression and functionally active measure
2.1, expression vector establishment
(1) according to the sequence of Bemisia tabaci peptidoglycan recognition protein BtPGRP and the cloning site of expression vector pET-28a (Novagen company), design primer: PGRP-BamH:CG
gGATCCaTTGAGGGTCGCCGAGATTCGTGGTACG (underscore is BamH I site); PGRP-Hind III:CCC
aAGCTTcTATTCGACGAGGAGGGCGAG (underscore is Hind III site).
(2) gene amplification, clone and recombinant plasmid screen
With Bemisia tabaci dsDNA for template, carry out PCR reaction with above-mentioned primer, amplification condition is: 94 DEG C, 2min denaturation; 94 DEG C, 15s, 63 DEG C, 30s, 72 DEG C, 45s, 35 circulations; 72 DEG C extend 6min.
Get 2 μ lPCR products, after the agarose gel electrophoresis detection of 1%, Clean UP test kit is clean reclaims PCR primer.Respectively using the DNA of PET-28a plasmid vector and above-mentioned recovery as DNA profiling, prepare following reaction system: Hind III 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 DEG C.
(3) do agarose gel electrophoresis by the double digestion reaction product of second step, cut sizeable DNA band and do DNA gel recovery.
(4) in PCR pipe, the PCR primer after 2.5 μ l double digestions is added respectively, the pET-32a plasmid vector after 0.5 μ l double digestion, the T4Ligase DNA Buffer of the T4Ligase DNA of 0.5 μ l, 1 μ l, the ddH of 5 μ l
2o, hatches under 16 DEG C of conditions, connects more than 1h.
(5) product conversion will be connected in competence, coat on the LB culture medium flat plate of kantlex, 37 DEG C of overnight incubation.Choose spot and cultivate bacterium liquid, carry out r-Taq reaction system PCR with T7 and T7ter universal primer and detect, 100 μ l are got for positive every part and reserves seed for planting and send survey.
(6) comparison sequencing result, chooses the correct bacterium liquid of reserving seed for planting of result and does next step experiment.
2.2, the expression of restructuring Bemisia tabaci peptidoglycan recognition protein BtPGRP
Choose order-checking positive bacteria liquid 37 DEG C of overnight shakings in the LB liquid nutrient medium containing 50 μ g/mL kantlex to cultivate, next day, inoculum size with 1% is connected in the liquid nutrient medium containing 150 μ g/mL kantlex, treat that thalli growth is to logarithmic phase, when OD600 about 1.0, adding 100mM IPTG makes its final concentration be about 1mM, after 27 DEG C/250rpm induces 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
2.3, to recombinate the purifying of Bemisia tabaci peptidoglycan recognition protein BtPGRP and renaturation
Recombinant protein, with the form successful expression of solubility, utilizes Clontech His-TALON gravity Purification Kit to obtain Bemisia tabaci peptidoglycan recognition protein BtPGRP recombinant protein.The Bemisia tabaci peptidoglycan recognition protein BtPGRP recombinant protein of purifying, through the SDS-PAGE gel electrophoresis analysis of 15%, shows about 25kDa size, in the same size with expection.Concrete steps reference reagent box specification sheets.Purifying protein 12%SDS-PAGE detects.
Adopt the method for gradient dialysis, make the albumen of purifying carry out dialysis and fold, use respectively:
1) containing 6M urea, 1% arginine, 2% glycine, the TBS damping fluid (pH8.0) of 1mM EDTA;
2) containing 4M urea, 1% arginine, 2% glycine, the TBS damping fluid (pH8.0) of 1mM EDTA, 0.2mM Sleep-promoting factor B (GSSG) and 2mM reduced glutathion (GSSH);
3) containing 2M urea, 1% arginine, 2% glycine, the TBS damping fluid (pH8.0) of 1mM EDTA, 0.2mM Sleep-promoting factor B (GSSG) and 2mM reduced glutathion (GSSH);
4) containing 1M urea, 1% arginine, 2% glycine, the TBS damping fluid (pH7.4) of 1mM EDTA, 0.2mM Sleep-promoting factor B (GSSG) and 2mM reduced glutathion (GSSH);
5) containing 0.5M urea, 1% arginine, 2% glycine, the TBS damping fluid (pH7.4) of 1mM EDTA, 0.2mM Sleep-promoting factor B (GSSG) and 2mM reduced glutathion (GSSH);
6) containing TBS damping fluid (pH7.0) dialysed overnight of 5% glycerine.
Each damping fluid is slowly dialysed 2-3 hour at 4 degree.
Embodiment 3: Bemisia tabaci peptidoglycan recognition protein BtPGRP prokaryotic recombinant protein is to bacterium identification activation analysis
1) get the intestinal bacteria of growth logarithmic phase, streptococcus aureus and Candida albicans, 3500rpm is centrifugal removes supernatant, and PBS washes three times, each 5 minutes.
2) with the target protein suspension bacteria liquid that 40 μ l are purified, room temperature leaves standstill 10min, centrifugally removes last issue.
3) stationary liquid cold acetone fixes bacterium liquid, 2min.
4) PBS washes three times, each 5 minutes.5%BSA closes 1h.
5) PBS washes once, and BtPGRP antibody hatches 1h with PBS in the damping fluid of 1:500 (V/V) ratio 0.5%BSA.
6) PBS washes three times, each 5 minutes, and fluorescence immunoassay antibody 1:400PBS damping fluid hatches 1h.
PBS washes five times, each 5 minutes, detects under fluorescent microscope.Blank group is not with the bacterium liquid (see Fig. 2 and Fig. 3) of recombinant protein process
Embodiment 4: Bemisia tabaci peptidoglycan recognition protein BtPGRP prokaryotic recombinant protein anti-microbial activity detects
To the Cell suppression test of E. coli, streptococcus aureus S.aureus
Get the E. coli of growth logarithmic phase, streptococcus aureus S.aureus, 3500rpm are centrifugal removes supernatant, PBS washes three times, each 5 minutes.PBS dilution 10
4doubly, hatch 1h or 2h with the BtPGRP (20 μ g/ml) of purifying.Then LB culture medium flat plate will be applied to after hatching.After 37 DEG C of incubated overnight, observe colony number, and count.Each process is (see Fig. 4) in triplicate.As shown in the figure, compared with the control, after BtPGRP and E. coli hatch 1h, 50.4% bacterium is killed, and after 2h, 72.7% bacterium is killed.After BtPGRP and streptococcus aureus S.aureus hatches 1h, 32.6% bacterium is killed, and after 2h, 59.8% bacterium is killed.Above result shows, experiment shows, restructuring Bemisia tabaci peptidoglycan recognition protein BtPGRP can kill Gram-negative bacteria and gram-positive microorganism.
Claims (4)
1. Bemisia tabaci peptidoglycan recognition protein encoding gene, is characterized in that, the nucleotide sequence of this gene is as shown in SEQ IDNO.1.
2. by the albumen of Bemisia tabaci peptidoglycan recognition protein encoding gene encodes, it is characterized in that, the aminoacid sequence of this albumen is as shown in SEQ ID NO.2.
3. Bemisia tabaci peptidoglycan recognition protein encoding gene described in claim 1 is preparing the method had in the recombinant protein of anti-microbial activity, it is characterized in that, be by gene recombination technology, described gene is expressed in intestinal bacteria, obtain the recombinant protein with anti-microbial activity.
4. albumen described in claim 2 is preparing the application in antimicrobial DP finish, food preservative or fodder additives.
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CN112724257B (en) * | 2019-10-14 | 2023-09-19 | 江西缘生生物科技有限公司 | Hybrid antibacterial protein with strong bactericidal effect and application thereof |
CN116178521A (en) * | 2023-03-21 | 2023-05-30 | 中国海洋大学 | Fish-source peptidoglycan recognition protein mutant, preparation method and application thereof |
CN116178521B (en) * | 2023-03-21 | 2024-04-09 | 中国海洋大学 | Fish-source peptidoglycan recognition protein mutant, preparation method and application thereof |
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