CN104531712B - The preparation and application of Bemisia tabaci peptidoglycan recognition protein with bactericidal activity - Google Patents
The preparation and application of Bemisia tabaci peptidoglycan recognition protein with bactericidal activity Download PDFInfo
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Abstract
The present invention relates to molecular biology and gene engineering technology field, it is desirable to provide a kind of preparation and application of the Bemisia tabaci peptidoglycan recognition protein with bactericidal activity.The nucleotide sequence of the Bemisia tabaci peptidoglycan recognition protein encoding gene is as shown in SEQ ID NO.1.The nucleotide sequence of Bemisia tabaci peptidoglycan recognition protein BtPGRP genes and its coding provided by the present invention, can apply in the new albumen of expression in escherichia coli, coded sequence.After the recombinant protein purification of the antibacterial activity carry out bacteriostatic experiment the result shows that：The recombinant protein of the Bemisia tabaci peptidoglycan identification protein gene BtPGRP prokaryotic expressions of recombination has the ability in conjunction with Gram-negative bacteria and gram-positive bacteria, while can kill Gram-negative bacteria and gram-positive bacteria.Further, it is produced by existing gene engineering method using the method for the present invention, is had potential application in exploitation broad-spectrum antiseptic class drug and novel immune reinforcing agent, feed addictive etc..
Description
Technical field
The invention belongs to molecular biology and gene engineering technology field.The present invention relates to a kind of identifications of Bemisia tabaci peptide glycan
The application of albumen (BtPGRP) gene and its coded albumen more particularly to a kind of Bemisia tabaci peptidoglycan recognition protein (BtPGRP)
In-vitro recombination expression technology, active recombinant protein isolating and purifying and ground to the identification of microorganism and bactericidal bacteriostasis
Study carefully.
Background technology
Innate immune reaction is the important defence line together of body defenses infectious diseases, this system of defense is for all more
Cell biological is survived and species continuity is all very important.Different from mammal, insect lacks and obtains without immunoglobulin
Property it is immune, therefore insect a major challenge of surviving is exactly to identify external pathogen.It has been developed during long-term evolution a set of only
Special pathogen recognition mode, can identify pathogen outer wall lipopolysaccharides (LPS), lipoteichoic acids, lipoprotein, peptide glycan
(PGN), beta-1,3-dextran and defense reaction is made to it.This certain shared highly conserved points in pathogenic microorganism surface
Minor structure is known as pathogen-associated molecular pattern (Pathogen-associated molecular pattern, PAMP).It is immune to know
A variety of pattern recognition receptors (pattern that can identify one or more PAMP molecules are not developed during evolution
Recognition receptor, PRP), such as scavenger receptor (SCRs), C- types agglutinin (CTLs), Gram-negative bacteria knot
Hop protein GNBPs (Gram-negative binding proteins) and peptidoglycan recognition protein PGRPs
(peptidoglycan recognition proteins) etc..Peptidoglycan recognition protein PGRPs is that a kind of important pattern is known
Other receptor (PRR) plays an important role in the invasion of identification pathogen and activation immunomodulatory pathways.Peptidoglycan recognition protein
PGRP has found that the albumen can be in conjunction with peptide glycan, and activation polyphenol oxidase cascade is anti-for the first time in silkworm Bombyx mori
It answers.There are 1 structural domains homologous with T7 lysozymes by PGRP, and the N- acetyl cell wall-l-Alanine amidase relied on Zn
Activity.It is found that 13 PGRP gene codes 17 altogether in Drosophila melanogaster Drosophila melanogaster genomes at present
Find that 7 PGRP genes, homologous comparison find this in a PGRP albumen, anopheles costalis's Anopheles gambiae genomes
A little genes and mammal PGRPs genes are highly conserved.It has now been found that PGRP can be divided into insect congenital immunity it is following
Several classes：A) pro-phenoloxidase cascade reaction is induced after identifying pathogen, generates melanin to wrap up microorganism；B) gram is identified
Positive bacteria, activation Toll signal paths (PGRP-SA)；C) it identifies Gram-negative bacteria, activates Imd signal paths, induce self
Phagocytosis；D) lactamase activity makes PGN inactivate.In addition, PGRP also has antibacterial activity in mammal (people and rat).Most
Nearly research finds that PGRP-SB1, the bear PGRP-S of drosophila in insect have antibacterial action to bacillus.The sterilization machine of PGRP albumen
There are two types of systems：One is for PGRP and the irreversible combination of peptide glycan, to interfere the synthesis of bacteria cell wall；Another kind is
Some PGRP have lactamase activity.Can degrade peptide glycan, so that thalline is ruptured to destroy cell wall, this bactericidal effect class
It is similar to penicillin.
The structure and function of peptidoglycan recognition protein has obtained a degree of research in insect and mammal, but
It is still to be reported both at home and abroad without the research of Bemisia tabaci peptidoglycan recognition protein BtPGRP at present.Bemisia tabaci Bemisia tabaci
(Gennadius) a kind of disastrous insect of global invasive is had become, in population outbreak all over the world, causes heavy damage
It loses.The worm not only a large amount of feeding plant juice cause plant withered, and propagate various plants virus, are important viral biography
Medium is broadcast, the viroses of plant is often resulted in and is very popular, makes crop Severe Reduction or total crop failure.PGRP is in passing malicious insect vector Bemisia tabaci
Research, be conducive to preferably to disclose its in immune response and propagate the effect in plant virus and its working mechanism, simultaneously
Also foundation is provided for exploitation extensive pedigree antibiotic and screening immunopotentiator.
Invention content
The technical problem to be solved by the present invention is to for current Bemisia tabaci immune response research situation and its deficiency, provide
A kind of preparation and application of Bemisia tabaci peptidoglycan recognition protein (BtPGRP) albumen with bactericidal activity.
To solve technical problem, solution of the invention is：
The present invention provides a kind of Bemisia tabaci peptidoglycan recognition protein encoding genes, and the nucleotide sequence of the gene is such as
Shown in SEQID NO.1.
Invention further provides the albumen encoded by Bemisia tabaci peptidoglycan recognition protein encoding gene, the ammonia of the albumen
Base acid sequence is as shown in SEQ ID NO.2.
Invention further provides aforementioned Bemisia tabaci peptidoglycan recognition protein encoding genes to prepare with antibacterial activity
Recombinant protein in method, be so that the gene is expressed in Escherichia coli by gene recombination technology, had
The recombinant protein of antibacterial activity.
Invention further provides foregoing proteins in preparing antimicrobial DP finish, food preservative or feed addictive
Using.
The beneficial effects of the present invention are：
The nucleotide sequence of Bemisia tabaci peptidoglycan recognition protein BtPGRP genes and its coding provided by the present invention, makes it
It can apply in the new albumen of expression in escherichia coli, coded sequence.Pressed down after the recombinant protein purification of the antibacterial activity
Bacterium experiment the result shows that：The recombinant protein of the Bemisia tabaci peptidoglycan identification protein gene BtPGRP prokaryotic expressions of recombination has knot
The ability of Gram-negative bacteria and gram-positive bacteria is closed, while Gram-negative bacteria and gram-positive bacteria can be killed.Into
One step, it is produced by existing gene engineering method using the method for the present invention, in exploitation broad-spectrum antiseptic class drug and Novel free
Epidemic disease reinforcing agent, feed addictive etc. have potential application.
Description of the drawings
Fig. 1 is the recombination Bemisia tabaci peptidoglycan recognition protein of the pET28a-BtPGRP-BL21 and purifying of IPTG inductions
The PAGE gel electrophoretogram of BtPGRP albumen.1：PET28a-BtPGRP-BL21 bacterial strains are not induced；Lane 2：1mM IPTG
The pET28a-BtPGRP-BL21 bacterial strains of induction；3：Purification of recombinant proteins；M：Albumen Marker.
Fig. 2 is recognition reaction figure of the Bemisia tabaci peptidoglycan recognition protein BtPGRP gene recombinant proteins to Escherichia coli.
Fig. 3 is recognition reaction of the Bemisia tabaci peptidoglycan recognition protein BtPGRP gene recombinant proteins to staphylococcus aureus
Figure.
Fig. 4 is growth inhibition of the Bemisia tabaci peptidoglycan recognition protein BtPGRP gene recombinant proteins to E. coli
Action diagram.
Fig. 5 is Bemisia tabaci peptidoglycan recognition protein BtPGRP gene recombinant proteins to staphylococcus aureus S.aureus's
Growth inhibition effect figure.
Specific embodiment
In the present invention, the cloning process of the Bemisia tabaci peptidoglycan identification protein gene cDNA is anti-with Bemisia tabaci total serum IgE
It is template that transcription, which obtains cDNA, and construction cDNA library screens to obtain BtPGRP Partial cDNA Sequences according to Bemisia tabaci transcript profile, so
It expands to obtain end sequence by RACE methods, it is most complete through sequence assembly acquisition Bemisia tabaci peptidoglycan identification protein gene cDNA afterwards
Long sequence.
The present invention is with Bemisia tabaci for expressing the ripe peptide gene sequence of Bemisia tabaci peptidoglycan recognition protein recombinant protein
Peptidoglycan recognition protein full-length gene double-stranded DNA is template, is obtained through PCR method amplification, it derives from Bemisia tabaci peptide glycan
Identify the genetic fragment corresponding to full length protein gene region.
The above-mentioned bacillus coli gene expression vector containing Bemisia tabaci peptidoglycan identification protein gene of the present invention is preferentially by this
The Bemisia tabaci peptidoglycan identification protein gene and the recombinant expression of coli expression carrier pET-28a structures for inventing synthesis carry
Body is named as pET28a-BtPGRP.
The present invention can express the E. coli recombinant stain of Bemisia tabaci peptidoglycan identification protein gene preferentially by containing tobacco powder
Bacterial strain obtained from the expression vector pET28a-BtPGRP conversion e. coli bl21s (DE3) of lice peptidoglycan recognition protein, name
For pET28a-BtPGRP-BL21.
The specific preparation method of recombinant protein of above-mentioned Bemisia tabaci peptidoglycan recognition protein is to come by following technical solution in fact
It is existing：It chooses E. coli recombinant stain pET28a-BtPGRP-BL21 and is seeded in the LB Liquid Cultures that 10ml contains kanamycins
Base, 37 DEG C of overnight incubations take 50 μ l bacterium solutions to be added to equipped with 50ml Kana+The sterile triangular flasks of 150ml of LB liquid medium
In, 37 DEG C, shaking for 150rpm cultivates 6~8h to logarithmic phase in case, is added 100mM IPTG to final concentration of 1mM, 27 DEG C,
250rpm shaken cultivations.Thalline is collected after recombinant bacterium grows into plateau, it is poly- through isolating and purifying to obtain recombination Bemisia tabaci peptide
Sugar identification albumen.
Bemisia tabaci peptidoglycan recognition protein BtPGRP of the present invention is applied in preparing active recombinant protein.Its
In, the method for the application is so that the gene is expressed in Escherichia coli by gene recombination technology, is obtained with anti-
The active recombinant protein of bacterium.Such as：The Bemisia tabaci peptidoglycan identification protein gene of acquisition is cloned into pET-28a, and (Novagen is public
Department) expression vector, it is transformed into e. coli bl21 cell.Induced expression is carried out, active recombinant protein (i.e. tobacco powder is obtained
The activated protein of lice peptidoglycan recognition protein BtPGRP gene codes).
The application of the recombinant protein of Bemisia tabaci peptidoglycan recognition protein, amino acid sequence in the sequence table SEQ ID NO.2
The recombinant protein BtPGRP of row is used to prepare broad-spectrum antiseptic class preparation.
In the sequence table SEQ ID NO.2 recombinant protein BtPGRP of amino acid sequence be used to prepare immunopotentiator or
Feed addictive.
Bemisia tabaci peptidoglycan recognition protein BtPGRP genes of the present invention, nucleotide sequence such as SEQ ID NO.1 institutes
Show, the sequence signature：Length：1027 base-pairs；Type：Nucleic acid；Chain：Double-strand；Topological structure：Linearly；Molecule type：
cDNA。
The albumen of Bemisia tabaci peptidoglycan recognition protein BtPGRP genes coding of the present invention, amino acid is such as
Shown in SEQID NO.2.The sequence signature：Length：235 amino acid；Type：Amino acid；Chain：It is single-stranded；Topological structure：Linearly；
Molecule type：Protein.Characteristic：The molecular weight of albumen is 25.86kDa, and theoretical isoelectric point is 6.21.The wherein 1- of coded sequence
29 are signal peptide sequence, and ripe peptide molecular weight is 22.67kDa, and isoelectric point 5.81, there are one conservative PGRP structures for tool
Domain.
Recombinant protein sequence provided by the invention is Bemisia tabaci peptidoglycan recognition protein BtPGRP mature peptides region.This sequence
Source is that SEQ ID NO.2 remove signal peptide precursor.
Egg is identified using Bemisia tabaci peptidoglycan recognition protein function and plate count the detection peptide glycan of Immunofluorescence test
White antibacterial activity.The result shows that the present invention, which prepares peptidoglycan recognition protein, has kill gram-positive bacteria and Gram-negative bacteria
Activity.Therefore the present invention provides the peptidoglycan recognition proteins to prepare for treating gram-positive bacteria or Gram-negative
Application in the food additives or drug of bacterium infection.
With reference to the specific experimental data in laboratory and in conjunction with specific embodiments, the present invention is further explained.These realities
Example is applied to be only used for the present invention rather than limit the scope of the invention.The experiment side of actual conditions is not specified in lower example embodiment
Case, usually according to normal condition, such as Sambrook equimoleculars clone：Laboratory manual (New York:Cold Spring
Haebor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.It should be understood that
, for those of ordinary skill in the art, without departing from the inventive concept of the premise, several changes can also be made
Shape and improvement.
Case study on implementation 1：Bemisia tabaci peptidoglycan recognition protein BtPGRP full length gene cDNA clones
1.1, TRIzol methods extract total serum IgE
(1) a certain amount of (about 200 Bemisia tabacis) is freezed into 1min, be put into 1.5ml centrifuge tubes, 1ml TRIzol examinations are added
Agent is fully ground homogenate, is stored at room temperature 5min.
(2) 0.2ml chloroforms are added into centrifuge tube, vibrate 15s, mixed liquor is transferred in TIANGEN centrifuge tubes, is stood
2min。
(3) 4 DEG C, 12000g centrifuges 15min, takes supernatant, is transferred in the centrifuge tube of a new 1.5ml.
(4) 0.5ml isopropanols are added into centrifuge tube, by the gently mixing of liquid in pipe, are stored at room temperature 10min.
(5) 4 DEG C, 12000g centrifuges 10min, discards supernatant.
(6) 75% ethyl alcohol of 1ml is added into centrifuge tube, gently washing precipitation, 4 DEG C, 7500g centrifuges 5min, discards supernatant
Liquid.(absolute ethyl alcohol being added at this time, can for a long time be preserved in -80 DEG C of ultra low temperature freezers)
(7) centrifuge tube is dried, suitable DEPC H are added2O dissolves (65 DEG C of dissolutions), spectrophotometer measurement OD260/
The value of OD280, ratio carry out next step experiment when between 1.8~2.0.
1.2, the first chains of cDNA synthesize
(1) RNA of 1 μ l extractions is separately added into the sterile centrifuge tubes of 0.5ml, 1 μ l SMART, IV/5 ' PCR forward directions are drawn
Object, 1 μ l CDS, III/3 ' PCR reverse primers add 2 μ l DEPC H2O makes total volume reach 5 μ l.
(2) reagent in mixing centrifuge tube and of short duration centrifugation, 72 DEG C of incubation 2min.
(3) rapidly by centrifuge tube ice bath 2min, of short duration centrifugation.
(4) 2 μ 5 × Frist-strand of l Buffer, 1 μ l 20mM dithiothreitol (DTT)s are separately added into centrifuge tube
(DTT), 1 μ l 10mM dNTP, 1 μ l Reverse Transcriptase reverse transcriptase blow and beat mixing, of short duration centrifugation repeatedly.
(5) 42 DEG C of incubation 1h.
(6) centrifuge tube is placed in 2~3min on ice, terminates reaction.A part is taken to be used for further experiment, remaining is in -80
DEG C ultra low temperature freezer freezen protective.
1.3, dsDNA is synthesized
(1) 1 μ l the first chain synthesis reaction products, 5 μ l 10 × LA Buffer (Mg are separately added into 0.5 centrifuge tube2+
Free), 5 μ l Mg2+, 8 μ l IV/5 ' PCR forward primers of dNTP, 1 μ l SMART, 1 μ lCDS, III/3 ' PCR reverse primers, 0.5 μ l
LA Tag archaeal dna polymerases, 29.5 μ l ddH2O is mixed well, of short duration centrifugation.
(2) in PCR instrument by following amplification program (95 DEG C, 20s；25~28 × (95 DEG C, 5s；68 DEG C, 6min)) amplification.
(3) it takes 5 μ l PCR products for gel detection (molecular weight ranges and band contained by test strip are bright dark), takes detection
Template of 100 times of the successful 1 μ l product dilutions for being later PCR, remaining is in -80 DEG C of ultra low temperature freezer freezen protectives.
1.4,5 ' and 3 ' end of PCR amplification Bemisia tabaci peptidoglycan recognition protein BtPGRP gene cDNAs
Clontech companies kit SMARTer is pressed in operationTMRACE cDNA Amplification Kit specifications into
Row.According to the requirement of kit, according to known peptidoglycan recognition protein BtPGRP gene est sequences separately design two it is upper,
Downstream primer, to carry out nest-type PRC.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 |
First time PCR reaction condition is：98 DEG C, 3min；30 × (98 DEG C, 10s；60 DEG C, 20s；68 DEG C, 1min)；68 DEG C,
6min.Second of PCR is expanded using first time PCR product as template, and reaction condition 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 carriers and obtains recombinant plasmid, and
With the universal primer M13 of pMD-19 ± carry out sequencing, measurement result is compared with known array segment and sequence assembly.
Sequence assembly result carries out BlastX on NCBI, the results showed that and obtaining sequence and known PGRP sequences has certain similitude,
Similarity about 40%.
Embodiment 2：Bemisia tabaci peptidoglycan recognition protein BtPGRP expression vector establishments, expression and functional activity measure
2.1, expression vector establishment
(1) according to the sequence of Bemisia tabaci peptidoglycan recognition protein BtPGRP and expression vector pET-28a (Novagen companies)
Cloning site, design primer,：PGRP-BamH：CGGGATCC(underscore is ATTGAGGGTCGCCGAGATTCGTGGTACG
BamH I sites)；PGRP-Hind III：CCCAAGCTT(underscore is Hind III to CTATTCGACGAGGAGGGCGAG
Point).
(2) gene magnification, clone and recombinant plasmid screen
Using Bemisia tabaci dsDNA as template, PCR reactions are carried out with above-mentioned primer, amplification condition is：94 DEG C, 2min pre-degenerations；
94 DEG C, 15s, 63 DEG C, 30s, 72 DEG C, 45s, 35 cycles；72 DEG C of extension 6min.
2 μ lPCR products are taken, after 1% agarose gel electrophoresis detection, Clean UP kits cleaning recycling PCR product.
Respectively using PET-28a plasmid vectors and the DNA of above-mentioned recycling as DNA profiling, following reaction system is prepared：Hind III limitations
Property 1 μ l of restriction endonuclease, I restriction enzymes of BamH, 10 μ l of DNA profiling, ddH262 μ l of μ l, Buffer of O.Gently mixing is of short duration
Centrifugation, 37 DEG C of incubation 1h.
(3) agarose gel electrophoresis is done with the double digestion reaction product of second step, cuts sizeable DNA bands and does
DNA gel recycles.
(4) PCR product being separately added into PCR pipe after 2.5 μ l double digestions, the pET-32a plasmids after 0.5 μ l double digestions
Carrier, T4Ligase the DNA Buffer, the ddH of 5 μ l of the T4Ligase DNA, 1 μ l of 0.5 μ l2O is incubated under the conditions of 16 DEG C,
Connect 1h or more.
(5) connection product is transformed into competence, is coated on the LB culture medium flat plates of kanamycins, 37 DEG C were cultivated
Night.Spot culture bacterium solution is chosen, r-Taq reaction systems PCR detection is carried out with T7 and T7ter universal primers, for every part of positive
It takes 100 μ l to reserve seed for planting and send survey.
(6) compare sequencing result, choose result correctly reserve seed for planting bacterium solution do in next step test.
2.2, the expression of recombination Bemisia tabaci peptidoglycan recognition protein BtPGRP
The positive bacterium solution 37 DEG C of overnight shaking cultures in the LB liquid medium containing 50 μ g/mL kanamycins of sequencing are chosen,
Next day is connected to 1% inoculum concentration in the fluid nutrient medium containing 150 μ g/mL kanamycins, waits for thalli growth to logarithmic phase,
When about 1.0 OD600,100mM IPTG, which are added, makes its final concentration be about 1mM, after 27 DEG C/250rpm induces 5h, surveys OD600 samplings,
5000rpm centrifuges 5min, and simultaneously appropriate sample-loading buffer is added in PBS buffer solution (pH7.0) resuspension thalline, and is used for 12%SDS-
PAGE electrophoretic analysis protein expression situations,
2.3, the purifying of recombination Bemisia tabaci peptidoglycan recognition protein BtPGRP and renaturation
Recombinant protein successful expression in the form of soluble, it is pure using Clontech His-TALON gravity purification kits
Change obtains Bemisia tabaci peptidoglycan recognition protein BtPGRP recombinant proteins.The Bemisia tabaci peptidoglycan recognition protein BtPGRP recombinations of purifying
Albumen shows about 25kDa sizes through 15% PAGE gel electrophoretic analysis, in the same size with expection.Specific steps reference
Kit specification.Purifying protein is detected with 12%SDS-PAGE.
The method dialysed using gradient, is made the albumen of purifying carry out dialysis folding, used respectively：
1) urea containing 6M, 1% arginine, 2% glycine, the TBS buffer solutions (pH8.0) of 1mM EDTA；
2) urea containing 4M, 1% arginine, 2% glycine, 1mM EDTA, 0.2mM oxidizeds form of glutathione (GSSG) and
The TBS buffer solutions (pH8.0) of 2mM reduced glutathiones (GSSH)；
3) urea containing 2M, 1% arginine, 2% glycine, 1mM EDTA, 0.2mM oxidizeds form of glutathione (GSSG) and
The TBS buffer solutions (pH8.0) of 2mM reduced glutathiones (GSSH)；
4) urea containing 1M, 1% arginine, 2% glycine, 1mM EDTA, 0.2mM oxidizeds form of glutathione (GSSG) and
The TBS buffer solutions (pH7.4) of 2mM reduced glutathiones (GSSH)；
5) urea containing 0.5M, 1% arginine, 2% glycine, 1mM EDTA, 0.2mM oxidizeds form of glutathione (GSSG) and
The TBS buffer solutions (pH7.4) of 2mM reduced glutathiones (GSSH)；
6) contain TBS buffer solutions (pH7.0) dialysed overnight of 5% glycerine.
Each buffer solution is slowly dialysed 2-3 hour at 4 degree.
Embodiment 3：Bemisia tabaci peptidoglycan recognition protein BtPGRP prokaryotic recombinant proteins identify activity analysis to bacterium
1) Escherichia coli, staphylococcus aureus and the Candida albicans of growth logarithmic phase, 3500rpm centrifugations is taken to go
Clearly, PBS is washed three times, 5 minutes every time.
2) the destination protein suspension bacteria liquid purified with 40 μ l, is stored at room temperature 10min, and last issue is removed in centrifugation.
3) fixer cold acetone fixes bacterium solution, 2min.
4) PBS is washed three times, 5 minutes every time.5%BSA closes 1h.
5) PBS is washed once, and BtPGRP antibody is with 1:PBS is incubated 1h in the buffer solution of 500 (V/V) ratio 0.5%BSA.
6) PBS is washed three times, 5 minutes every time, fluorescence immunoassay antibody 1:400PBS buffer solutions are incubated 1h.
PBS washes five times, 5 minutes every time, is detected under fluorescence microscope.Blank control group is the bacterium of unused recombinant protein processing
Liquid (referring to Fig. 2 and Fig. 3)
Embodiment 4：Bemisia tabaci peptidoglycan recognition protein BtPGRP prokaryotic recombinant protein antibacterial activities detect
To the Cell suppression test of E. coli, staphylococcus aureus S.aureus
The E. coli of growth logarithmic phase, staphylococcus aureus S.aureus, 3500rpm centrifugation is taken to go
Clearly, PBS is washed three times, 5 minutes every time.PBS dilutions 104After times, 1h or 2h is incubated with the BtPGRP (20 μ g/ml) of purifying.Then
It will be applied to LB culture medium flat plates after incubation.After 37 DEG C are incubated overnight, clump count is observed, and count.Each processing is in triplicate
(referring to Fig. 4).As shown, compared with the control, after BtPGRP is incubated 1h with E. coli, 50.4% bacterium is killed
Extremely, after 2h, 72.7% bacterium is killed.After BtPGRP is incubated 1h with staphylococcus aureus S.aureus, 32.6% bacterium quilt
It kills, after 2h, 59.8% bacterium is killed.The above result shows that experiment shows to recombinate Bemisia tabaci peptidoglycan recognition protein
BtPGRP can kill Gram-negative bacteria and gram-positive bacteria.
Claims (1)
1. the Bemisia tabaci peptidoglycan recognition protein with bactericidal activity is preparing anti-Escherichia coli and/or staphylococcus aureus
Application in drug, food preservative or feed addictive；The Bemisia tabaci peptidoglycan recognition protein with bactericidal activity is logical
Following methods are crossed to prepare：Make Bemisia tabaci peptidoglycan recognition protein encoding gene in Escherichia coli by gene recombination technology
It is expressed, obtains the recombinant protein with antibacterial activity；The nucleotide of the Bemisia tabaci peptidoglycan recognition protein encoding gene
Sequence is as shown in SEQ ID NO.1；The amino acid sequence of shown recombinant protein is as shown in SEQ ID NO.2.
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| 斯氏按蚊肽聚糖识别蛋白基因PGRP-LC1的克隆及功能分析;陈杨等;《昆虫学报》;20100228;第53卷(第2期);第132页第1栏第2-3段 * |
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