CN108085310A - A kind of diamondback moth identification albumen PxIDGF products and its preparation method and application - Google Patents
A kind of diamondback moth identification albumen PxIDGF products and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of diamondback moth identification albumen PxIDGF products and its preparation method and application, the amino acid sequence such as SEQ ID NO of the protein product:Shown in 1.Total serum IgE is extracted from diamondback moth four-age larva body, reverse transcription synthesizes the first chain cDNA, diamondback moth identification albumen PxIDGF genes are expanded from cDNA using primer is designed, identify albumen PxIDGF gene clonings into pCzn1 carriers diamondback moth, then recombinant plasmid is transferred to Escherichia coli Arctic Express expression bacterial strains, induced fusion protein expression, by way of refolding strategy, the molten target protein of weight obtains destination protein by Ni columns affinity purification.Diamondback moth prepared by the present invention identifies that albumen PxIDGF products purity is high, activity is high, has combination and the ability of aggegation Escherichia coli and staphylococcus aureus.The present invention can be that further investigation diamondback moth identifies that technical foundation is laid in the application of albumen PxIDGF.
Description
Technical field
Identify field the invention belongs to the insect innate immunity, more particularly to a kind of diamondback moth identification albumen PxIDGF products and
Its preparation method and application.
Background technology
One of the countries with the most serious ... is endangered as information system of crop pest distribution in China in the world, the great agricultural pests in China there are 30 kinds
As many as, harm is serious, and loss is surprising.Diamondback moth (Plutella xylostella) is one of which.As a kind of harm ten
The worldwide agricultural pests of Zi Hua sections plant, to many insecticides such as pyrethroid, organophosphor and carbamates
Generate the notable resistance to the action of a drug.It is it is first to DDT generate resistance crop pest and it is first to thuringiensis bacillus toxin generate
The insect (Baxter etc., 2010) of resistance.Therefore there is an urgent need to study new diamondback moth controlling way.And develop biological control skill
Art is the inexorable trend of persistent high efficiency control diamondback moth harm.The virulence factor generated currently with insect pathogenic bacteria or pathogen
It is one of forward position of biological control of insect pests research to carry out pest control, and successful key is the immune defense for understanding host insect
Mechanism.Therefore theoretical base can be provided to the immune response mechanism of various pathogens for new pest control method by illustrating diamondback moth
Plinth.
Due to lacking acquired immunity, insect relies primarily on the innate immunity of its own to resist entering for various pathogens
It invades.The innate immunity of insect includes cellular immunity and humoral immunity.Insect such as loses the protection of the innate immunity, and existence will be subject to
It influences.Some researches show that the innate immunity of the insect (caused by gene mutation) or artificial inhibition insect of congenital immunity defect, elder brothers
Worm will weaken the resistance of pathogen.In humoral immunity, from pathogen invasion and attack to finally by host remove generally will pass through such as
Lower process：It is identification alien material first；Then trigger activation serine protease and release serpin
Extracellular cascade reaction；Then priming signal transduction pathway and activating genes of interest are transcribed.This process need 4 it is important because
Son identifies molecule, the Signal Regulation factor, signal transduction system and effector molecule.
Cause of disease identification is the key that start the insect humoral immunity first step, is mainly completed by different identification molecules.Know
Other molecule, also known as pattern recognition protein are the key factors of Insect immunity response, can identify the cell wall constituent of pathogen
(i.e. cause of disease molecule associative mode pathogen associated molecular patterns, PAMPs), such as gram-negative
Property bacterium lipopolysaccharides, the peptide glycan of gram-positive bacterium, β -1 of fungi, 3- glucans and virus double-stranded RNA.It is different
Identification molecule have different 26S Proteasome Structure and Functions, with reference to different PAMPs.Model insects Diptera drosophila is identified at present
The identification molecule gone out mainly has peptidoglycan recognition protein (PGRPs), thioester albumen, gramnegative bacterium to combine egg more than 8 kinds
(GNBPs), beta-1,3-dextran identify albumen (β GRPs), scavenger receptor, c-type agglutinin, galactose agglutinin and are immunized in vain
Globulin etc..There is extremely important influence for the existence of insect just because of the innate immunity, have become injurious insect control
An important target.The research of molecule is identified in the insect innate immunity at present has become the emphasis and hot spot of immunological investigation.
To inquire into the immune defence mechanism that diamondback moth invades disease fungus, we infect diamondback moth larvae to causal organism
Transcript profile be sequenced, and gene involved in immunity is screened.It identifies some and may participate in diamondback moth and be immunized
The immunogene that causal organism infects, including：Identify molecule, the Signal Regulation factor, the antimicrobial effect factor and some other possibility
Participate in immune gene etc..Wherein identify that molecule includes PxIDGF, CTL (C-type lectins) and PGRP
(peptidoglycan recognition protein) etc..IDGF, i.e. imaginal disk growth factor, are under the jurisdiction of glycosyl hydrolase
18th family is secreted by the expression of insect fat-body in the hemolymph of insect, and major function is to adjust insect growth and growth.
It may be by speculating due to having 1 with reference to the Lectin domain of chitin and 1 lactic acid structural domain with reference to chitin
It works in the cause of disease identification of invertebrate.There is not experimental data to prove at present on this function.
In order to which whether diamondback moth identification albumen PxIDGF has clearly during diamondback moth humoral immunity pathogenic microorganism
There is immune identification action, it is very necessary to obtain the active albumen.There is not the system of diamondback moth identification albumen PxIDGF at present
Invention in terms of product and preparation method.
The content of the invention
The present invention provides a kind of diamondback moth identification albumen PxIDGF products and its preparation method and application.
The present invention is to be achieved by the following technical programs：
The present invention provides a kind of diamondback moths to identify albumen PxIDGF products, is by gene gram from diamondback moth larvae
A kind of albumen that grand, protein expression, purifying obtain, theoretical molecular weight 47.904KD, isoelectric point 7.67；The protein product
Overall amino acid sequence be MNHKVHHHHHHMNQVVSTKKVICYYDSKSYVRESNARLLPPDLEPALPYCTHLVYG YAGVQ
PDTYKMISTNQNLDIDSAHANYRTITSFKTKYPQLKMLLAVGGDADLEDPQKYNALLESQQARTAFVNSGVVLAEQH
GFDGIDLAWQFPKVKPKKIRSGWGNFWHGVKKTFKTTPVDEKESEHREGFTALVRELKAALSLKPHLELGVTILPNV
NSTIYCDVPAIINFVDYVNLLAFDYFTPERNEKEADYTAPIYAPQNRHPEQNVDAAVKYWRNAGAPPTKIVVGIATY
ARTWKLDSDSEVAGVPPIHTDGAGEPGPYTKTEGLLSYPEVCMKLIAPPAGLRANIRKVTDPSKRFGTYAFRLPDSD
GNGGIWVSYEDADTAGQKADYVKKNNLGGISIVDLSMDDFRELCTGNKYPILRAAKYRL。
Invention also provides the preparation methods of diamondback moth identification albumen PxIDGF products：The present invention selects diamondback moth
(Plutella xylostella) is used as material, and total serum IgE is extracted from diamondback moth four-age larva body, and reverse transcription synthesizes the first chain
CDNA is expanded diamondback moth identification albumen PxIDGF genes from cDNA using the primer of design, passes through conventional molecular cloning side
Diamondback moth identification albumen PxIDGF genes are connected in pCzn1 carriers by method, and plasmid then is transferred to Escherichia coli Arctic-
Express expresses bacterial strain, and overnight, next day presses 1 for 37 DEG C of shakings:100 dilution proportion bacterium solutions shake bacterium to OD600It is worth for 0.6-0.8, adds
Entering final concentration 0.5mMIPTG, 15 DEG C of shakings are stayed overnight, induced fusion protein expression, by way of refolding strategy, the molten target egg of weight
In vain, destination protein PxIDGF is obtained by Ni columns affinity purification.
The preparation method comprises the following steps：
(1) pCzn1-IDGF plasmid constructions；
(2) pCzn 1-IDGF carriers are converted to Escherichia coli Arctic-Express；
(3) expression of pCzn 1-IDGF carrier fusions；
(4) refolding strategy of inclusion body protein；
(5) the Ni column affinity purifications of fusion protein.
Step (1) the pCzn1-IDGF plasmid constructions method is as follows：Total serum IgE is extracted from diamondback moth four-age larva body,
Reverse transcription synthesizes the first chain cDNA, designed for a pair of of upstream and downstream primer of amplification diamondback moth identification albumen PxIDGF genes, if
Restriction enzyme site is introduced in the primer of meter, expands diamondback moth identification albumen PxIDGF genes, profit from cDNA using the primer of design
The linearisation target gene diamondback moth of acquisition is identified into albumen PxIDGF and destination carrier pCzn1 with the method that common molecular is cloned
Connection, connection product conversion TOP10 clone strains, the sequencing of picking positive clone molecule, screening, which obtains, is sequenced correct positive colony,
Extract plasmid；
Wherein, the upstream primer sequence is 5 '-GGAATTCCATATGAACCAGGTCGTCTCC-3 ', underlined sequences
For Nde I restriction enzyme site sequences, nucleotide sequence such as SEQ ID NO:Shown in 2.
Downstream primer sequence is 5 '-GCTCTAGATTACAGACGGTACTTGG-3 ', underlined sequences are Xba I digestions position
Point sequence, nucleotide sequence such as SEQ ID NO:Shown in 3.
Step (2) the pCzn 1-IDGF carriers, which are converted to the method for Escherichia coli Arctic-Express, is：It will sequencing
Correct positive colony extraction plasmid, 1 μ L plasmids are added in 100 μ L competent bacterias, put 20min on ice；42 DEG C of heat shock 90s
Put 5min in ice rapidly；Add in 600 μ L LB culture solutions；37 DEG C, 220rpm shaking 1h are all coated on after centrifugation containing 50 μ g/ml
The LB tablets of Amp, 37 DEG C of inversion overnight incubations.
The expression of step (3) the pCzn 1-IDGF carrier fusions is：Monoclonal on picking conversion tablet
It is inoculated in the test tube of the 3ml LB culture solutions containing 50 μ g/ml AMP, 37 DEG C, 220rpm shakings are overnight；Next day presses 1：100 inoculations
In the 30ml LB culture solutions of 50 μ g/ml AMP, 37 DEG C of 220rpm are shaken to thalline OD600For 0.6-0.8；IPTG is added in end
Concentration is 0.5mM, and 15 DEG C, 220rpm shakings are stayed overnight, induced fusion protein expression；Under the conditions of 4000g, 10min is centrifuged, is abandoned
Clearly, precipitation is collected.
The denaturation and renaturation method of step (4) described inclusion body protein is：By the table Jing Guo pCzn 1-IDGF carrier fusions
Precipitation after reaching is resuspended in 20ml lysis buffers, ultrasonication；By the cell pyrolysis liquid of ultrasonication at 4 DEG C, 10000g
Under the conditions of centrifuge 20min, collect precipitation；Precipitation inclusion body is washed using inclusion body cleaning solution 3 times；Again one is pressed with dissolving buffer solution
Certainty ratio dissolves inclusion body, and 4 DEG C stand overnight；Then 15min is centrifuged under the conditions of room temperature 15000rpm；20mM is added dropwise in supernatant
In Tris-HCL 5mM EDTA Buffer PH7.8 buffer solutions, progressively gradient dilution is slowly stirred at double, and protein solution is filled
Enter bag filter dialysed overnight in PBS pH7.4 solution, collect protein solution in bag filter；
Wherein, the lysis buffer is 20mM Tris-HCl, contains 1mM PMSF and bacterialprotease inhibitor mixed
Object, pH 8.0；
The ultrasonication condition is：Power 400W, work 4s, interval 8s, common 20min；
The inclusion body cleaning solution be 20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1%Triton X-100,
pH8.0；
The buffer solution that dissolves is 20mM Tris, 5mM DTT, 8M urea, pH8.0.
The Ni column affinity purification methods of step (5) described fusion protein are：Using low pressure chromatography system, inclusion body will be passed through
The protein solution that the denaturation and renaturation method of albumen obtains is with 0.5ml/min flow velocitys loading to Ni-IDA combination buffer pre-equilibrations
Ni-IDA-Sepharose CL-6B affinity columns；It is rinsed with Ni-IDA combination buffers with 0.5ml/min flow velocitys, until outflow
Liquid OD280Value reaches baseline, then is rinsed with Ni-IDA lavation buffer solutions with 1ml/min flow velocitys, until efflux OD280Value reaches base
Line, to remove foreigh protein removing；Destination protein is eluted with 1ml/min flow velocitys with Ni-IDA elution buffers, collects albumen efflux；Egg
White efflux is added in bag filter, is carried out dialysed overnight using the PBS of pH7.4, is collected protein solution in bag filter, this albumen is molten
Liquid is cold dry rear up to diamondback moth identification albumen PxIDGF products；
Wherein, the Ni-IDA lavation buffer solutions be 20mM Tris-HCl, 20mM imidazoles, 0.15M NaCl, pH8.0；
The Ni-IDA elution buffers be 20mM Tris-HCl, 250mM imidazoles, 0.15M NaCl, pH8.0.
The identification albumen PxIDGF products of preparation of the present invention have the ability with reference to microorganism, aggegation microorganism.
The present invention has the beneficial effect that：
The present invention has many advantages, such as that raw material sources enrich, is easy to be quick, and expression system has increase protein translation efficiency, increases
The features such as adding protein expression level, can induce foreign protein high level expression and increase the activity of expressing protein under cryogenic
Probability.Diamondback moth prepared by present invention identification albumen PxIDGF products purity is high, activity is high, have with reference to microorganism ability and
Be aggregated the ability of microorganism, eliminate from diamondback moth the tedious steps of extraction identification albumen, greatly save experimental cost and
Time.Preparation method of the present invention large-scale industrialized can produce, with short production cycle, and production cost is relatively low, and from external environment
Influence.The present invention can be that further investigation diamondback moth identifies that technical foundation is laid in the application of albumen PxIDGF.Importantly, this
Invention first passage microorganism combines and agglutination test confirms that diamondback moth identification albumen PxIDGF is identified with microorganism cause of disease
Ability, and the binding ability has the microbes composition and division in a proportion do not reported in the prior art under the support of test data
Example and agglutinability.
Description of the drawings
Fig. 1 is the result of restructuring plasmid enzyme restriction identification；
In figure：M for DNA molecular amount Marker (from top to bottom be respectively 100bp, 250bp, 500bp, 750bp, 1000bp,
1500bp, 2000bp, 3000bp and 5000bp)；1 is plasmid before digestion；2 be plasmid after digestion；
Fig. 2 is expressing fusion protein qualification result；
In figure：M is molecular weight of albumen Marker；1 is not induce bacterium solution；2 induce bacterium solution for IPTG；3 lure for 15 DEG C of IPTG
Lead supernatant after thalline ultrasonication；4 be to be precipitated after 15 DEG C of IPTG induce thalline ultrasonications；
Fig. 3 is the destination protein SDS-PAGE results of purifying；
In figure：M is molecular weight of albumen Marker；1 is non-purification of samples；2 be Ni-IDA lavation buffer solution washing samples；3
Pure protein is eluted for Ni-IDA elution buffers；
Fig. 4 identifies that albumen PxIDGF is combined measurement result with microorganism for diamondback moth；
In figure：1 is supernatant after incubation；2-5 is washed four times to be precipitated after incubation with TBS respectively；6 use to be precipitated after incubation
Supernatant after 7%SDS elutions；7 be that precipitation precipitates after being eluted with 7%SDS after being incubated；8 identify albumen for the diamondback moth of purifying
PxIDGF；
Fig. 5 identifies that albumen PxIDGF is aggregated microorganism result for diamondback moth；
Control is 1mM CaCl2；+ BSA is 1mM CaCl2With the TBS of 75ng BSA；+ IDGF is 1mM CaCl2
With the TBS of 75ng PxIDGF；+ IDGF+EDTA is 1mM CaCl2, 75ng PxIDGF and 1mM EDTA TBS.
Specific embodiment
It is further described the present invention in the following with reference to the drawings and specific embodiments, but embodiments of the present invention are unlimited
In this.
Embodiment 1
1st, pCzn1-IDGF plasmid constructions
Total serum IgE is extracted from diamondback moth four-age larva body using Trizol reagents (Takara), uses cDNA synthetic agents
Box (Takara) reverse transcription synthesizes the first chain cDNA, in a pair designed for amplification diamondback moth identification albumen PxIDGF genes,
Anti-sense primer, 5 '-GGAATTC of upstream primer sequenceCATATG(underlined sequences are Nde I enzymes to AACCAGGTCGTCTCC-3 '
Enzyme site sequence), 5 '-GC of downstream primer sequenceTCTAGA(underlined sequences are Xba I enzymes to TTACAGACGGTACTTGG-3 '
Enzyme site sequence).The primer of design middle can introduce restriction enzyme site.Diamondback moth identification egg is expanded from cDNA using the primer of design
White PxIDGF genes, PCR system：2.0 μ L of template DNA, 1.0 μ L of sense primer, 1.0 μ L, 10X buffer of anti-sense primer, 5.0 μ
1.0 μ L of L, dNTP Mixture 4.0ul, Pfu archaeal dna polymerase, use ddH2O is mended to 50 μ L.PCR programs：94℃5min；94℃
30s, 65 DEG C of 30s, 72 DEG C of 45s 30 are cycled totally；Last 72 DEG C of 5min.Using the method for common molecular clone by the linear of acquisition
Change target gene diamondback moth identification albumen PxIDGF to be connected with destination carrier pCzn1, connection product conversion TOP10 clone strains,
Picking positive clone molecule is sequenced, and screening, which obtains, is sequenced correct positive colony.And it is identified using digestion.Digestion system：Plasmid
0.25 1.0 μ L of μ L, 10 × Buffer of 3ul, restriction endonuclease Nde I 0.25 μ L, restriction endonuclease Xba I, use ddH2O is mended to 10 μ L.Enzyme
It is as shown in Figure 1 to cut qualification result.
2nd, pCzn 1-IDGF carriers are converted to Escherichia coli Arctic-Express
Correct positive colony extraction plasmid will be sequenced, 1 μ L plasmids are added in 100 μ L competent bacterias, are put on ice
20min；42 DEG C of heat shock 90sec, put rapidly 5min in ice；Add in 600 μ L LB culture solutions；37 DEG C, 220rpm shaking 1h, centrifugation
The LB tablets containing 50 μ g/ml Amp, 37 DEG C of inversion overnight incubations are all coated on afterwards.
3rd, IPTG induces the expression of pCzn 1-IDGF carrier fusions
(1) monoclonal on picking conversion tablet is inoculated in the test tube of the 3ml LB culture solutions containing 50 μ g/ml AMP, and 37
DEG C 220rpm shaking is overnight；
(2) next day presses 1：100 are inoculated in the 30ml LB culture solutions of 50 μ g/ml AMP, and 37 DEG C of 220rpm are shaken to thalline
OD600 is 0.6-0.8；
(3) 1ml cultures are taken out, 10000g room temperatures centrifugation 2min abandons supernatant, is resuspended with 100 1 × sample-loading buffers of μ l
Bacterial sediment；
(4) IPTG to final concentration of 0.5mM is added in into remaining culture, overnight, induction is melted for 15 DEG C of 220rpm shakings
Hop protein is expressed；
(5) 1ml cultures are taken out, 10000g room temperatures centrifugation 2min abandons supernatant, is resuspended with 100 1 × sample-loading buffers of μ l
Bacterial sediment.Remaining culture 4000g centrifuges 10min, abandons supernatant, and bacterial sediment is resuspended with PBS；Re-suspension liquid carries out ultrasonic wave
After broken, supernatant is taken to add in sample-loading buffer with precipitated liquid respectively and is resuspended.
(6) 12%SDS-PAGE detection and analysis are carried out, coomassie brilliant blue staining shows band, and the results are shown in Figure 2.
4th, the refolding strategy of inclusion body protein
Precipitation after expression by pCzn 1-IDGF carrier fusions is resuspended in 20ml lysis buffers (20mM
Tris-HCl contains 1mM PMSF and bacterioprotein Protease Inhibitor Cocktail, pH 8.0), ultrasonication (power 400W, work
4sec, interval 8sec, common 20min)；By 4 DEG C of 10000g centrifugation 20min of cell pyrolysis liquid of ultrasonication, precipitation is collected；It uses
Inclusion body cleaning solution (20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1%Triton X-100, pH8.0) washing precipitation
Inclusion body 3 times；Again with dissolving buffer solution (20mM Tris, 5mM DTT, 8M urea pH8.0), inclusion body is dissolved by a certain percentage,
4 DEG C stand overnight；Then 15min is centrifuged under the conditions of room temperature 15000rpm；20mM Tris-HCL 5mM are added dropwise in supernatant
In EDTABuffer PH7.8 buffer solutions, progressively gradient dilution is slowly stirred at double, and protein solution is packed into bag filter in pH7.4
PBS solution in dialysed overnight, collect bag filter in protein solution.
5th, the Ni column affinity purifications of fusion protein
Using low pressure chromatography system, by the protein solution obtained by the denaturation and renaturation method of inclusion body protein with 0.5ml/
Min flow velocitys loading to Ni-IDA combination buffer pre-equilibrations Ni-IDA-Sepharose CL-6B affinity columns；Use Ni-
IDA combination buffers are rinsed with 0.5ml/min flow velocitys, until efflux OD280Value reaches baseline, then with Ni-IDA lavation buffer solutions
(20mM Tris-HCl, 20mM imidazoles, 0.15M NaCl, pH8.0) is rinsed with 1ml/min flow velocitys, until efflux OD280Value reaches
Baseline, to remove foreigh protein removing；With Ni-IDA elution buffers (20mM Tris-HCl, 250mM imidazoles, 0.15M NaCl,
PH8.0 destination protein) is eluted with 1ml/min flow velocitys, collects albumen efflux；Albumen efflux is added in bag filter, uses PBS
(PH7.4) carry out dialysed overnight, collect protein solution in bag filter, this protein solution it is cold it is dry after be the diamondback moth identification
Albumen PxIDGF products.
The diamondback moth identification albumen PxIDGF product samples obtained using 1 method of embodiment are analyzed：
1st, 12%SDS-PAGE is analyzed
100 μ L samples is taken to add in 20 μ L 5 × SDS Loading Buffer and boil 5min, 12000rpm centrifugation 5min take
20 μ L loadings.12%SDS-PAGE is carried out, 2h is dyed with coomassie brilliant blue staining liquid, was decolourized with Coomassie brilliant blue destainer
Night.It is as shown in Figure 3 to carry out 12%SDS-PAGE analysis results.
2nd, western blot analysis
Sample carries out 12%SDS-PAGE, and is transferred on nitrocellulose filter.Then, with Block buffer (5%BSA)
When close membrane room 1.5 is small at room temperature, and with PxIDGF polyclonal antibodies (1 in Block buffer：2500 dilutions) together at 4 DEG C
Be incubated overnight and with IgG at room temperature altogether be incubated 2 it is small when.Band is observed with DAB dye reagents (Boster Biotech).
3rd, microorganism binding assay
The bacterium (Escherichia coli and staphylococcus aureus) in mid-log phase is taken, 5min is centrifuged with 6000rpm, it is heavy to take
It forms sediment with Tris- buffered salines (TBS；50mM Tris-HCl, 150mM NaCl, pH7.5) it washs three times, and be suspended in TBS,
Concentration is made into as 2 × 108The bacterial suspension of cells/ml.Bacterial suspension (200 μ l) and 200 μ lPxIDGF (150 μ g/ml) is mixed
It closes.Mixture is incubated at room temperature 1 it is small when, it is mild to rotate.By bacterial precipitation, washed four times with TBS, and washed with 7%SDS
It is de-.Washing and elution solution are analyzed using Western blotting.The diamondback moth identification albumen PxIDGF products of preparation have
With reference to the ability of Escherichia coli and staphylococcus aureus, as a result as shown in Figure 4.Diamondback moth identification albumen PxIDGF as seen from the figure
Allocation proportion in being precipitated after being incubated with Escherichia coli reaches 90%, the distribution in being precipitated after being incubated with staphylococcus aureus
Ratio reaches 95%.
4th, microorganism agglutination test
By 2 × 106A Escherichia coli or aureus cell are added to 1mM CaCl2(control), has
1mM CaCl 2With the TBS of 75ng BSA, there is 1mM CaCl2With TBS the and 1mM CaCl of 75ng PxIDGF2、75ng
The TBS of PxIDGF and 1mM EDTA.By it is all processing be incubated at room temperature 2 it is small when.With Eclipse Ti inverted microscopes
(Nikon) aggregates of bacteria can be found by checking, as a result as shown in Figure 5.The result is shown in can be observed to put down under 20 times of object lens visuals field
Equal 5 zoogloeas (taking 50 visual field average values).
Sequence table
<110>Agricultural University Of Anhui
<120>A kind of diamondback moth identification albumen PxIDGF products and its preparation method and application
<130> 2017
<141> 2017-12-15
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 428
<212> PRT
<213> Plutella xylostella
<400> 1
Met Asn His Lys Val His His His His His His Met Asn Gln Val Val
1 5 10 15
Ser Thr Lys Lys Val Ile Cys Tyr Tyr Asp Ser Lys Ser Tyr Val Arg
20 25 30
Glu Ser Asn Ala Arg Leu Leu Pro Pro Asp Leu Glu Pro Ala Leu Pro
35 40 45
Tyr Cys Thr His Leu Val Tyr Gly Tyr Ala Gly Val Gln Pro Asp Thr
50 55 60
Tyr Lys Met Ile Ser Thr Asn Gln Asn Leu Asp Ile Asp Ser Ala His
65 70 75 80
Ala Asn Tyr Arg Thr Ile Thr Ser Phe Lys Thr Lys Tyr Pro Gln Leu
85 90 95
Lys Met Leu Leu Ala Val Gly Gly Asp Ala Asp Leu Glu Asp Pro Gln
100 105 110
Lys Tyr Asn Ala Leu Leu Glu Ser Gln Gln Ala Arg Thr Ala Phe Val
115 120 125
Asn Ser Gly Val Val Leu Ala Glu Gln His Gly Phe Asp Gly Ile Asp
130 135 140
Leu Ala Trp Gln Phe Pro Lys Val Lys Pro Lys Lys Ile Arg Ser Gly
145 150 155 160
Trp Gly Asn Phe Trp His Gly Val Lys Lys Thr Phe Lys Thr Thr Pro
165 170 175
Val Asp Glu Lys Glu Ser Glu His Arg Glu Gly Phe Thr Ala Leu Val
180 185 190
Arg Glu Leu Lys Ala Ala Leu Ser Leu Lys Pro His Leu Glu Leu Gly
195 200 205
Val Thr Ile Leu Pro Asn Val Asn Ser Thr Ile Tyr Cys Asp Val Pro
210 215 220
Ala Ile Ile Asn Phe Val Asp Tyr Val Asn Leu Leu Ala Phe Asp Tyr
225 230 235 240
Phe Thr Pro Glu Arg Asn Glu Lys Glu Ala Asp Tyr Thr Ala Pro Ile
245 250 255
Tyr Ala Pro Gln Asn Arg His Pro Glu Gln Asn Val Asp Ala Ala Val
260 265 270
Lys Tyr Trp Arg Asn Ala Gly Ala Pro Pro Thr Lys Ile Val Val Gly
275 280 285
Ile Ala Thr Tyr Ala Arg Thr Trp Lys Leu Asp Ser Asp Ser Glu Val
290 295 300
Ala Gly Val Pro Pro Ile His Thr Asp Gly Ala Gly Glu Pro Gly Pro
305 310 315 320
Tyr Thr Lys Thr Glu Gly Leu Leu Ser Tyr Pro Glu Val Cys Met Lys
325 330 335
Leu Ile Ala Pro Pro Ala Gly Leu Arg Ala Asn Ile Arg Lys Val Thr
340 345 350
Asp Pro Ser Lys Arg Phe Gly Thr Tyr Ala Phe Arg Leu Pro Asp Ser
355 360 365
Asp Gly Asn Gly Gly Ile Trp Val Ser Tyr Glu Asp Ala Asp Thr Ala
370 375 380
Gly Gln Lys Ala Asp Tyr Val Lys Lys Asn Asn Leu Gly Gly Ile Ser
385 390 395 400
Ile Val Asp Leu Ser Met Asp Asp Phe Arg Glu Leu Cys Thr Gly Asn
405 410 415
Lys Tyr Pro Ile Leu Arg Ala Ala Lys Tyr Arg Leu
420 425
Claims (11)
1. a kind of diamondback moth identifies albumen PxIDGF products, which is characterized in that the identification protein product is from diamondback moth larvae
In a kind of protein product for being obtained by gene cloning, protein expression, purifying, theoretical molecular weight 47.904KD, isoelectric point is
7.67；The amino acid sequence of the protein product such as SEQ ID NO:Shown in 1.
2. the preparation method of diamondback moth identification albumen PxIDGF products described in a kind of claim 1, which is characterized in that including following
Step：
(1) pCzn1-IDGF plasmid constructions；
(2) pCzn 1-IDGF carriers are converted to Escherichia coli Arctic-Express；
(3) IPTG induces the expression of pCzn 1-IDGF carrier fusions；
(4) refolding strategy of inclusion body protein；
(5) the Ni column affinity purifications of fusion protein.
A kind of 3. preparation method of diamondback moth identification albumen PxIDGF products according to claim 2, which is characterized in that institute
It is as follows to state step (1) pCzn1-IDGF plasmid construction methods：Total serum IgE, reverse transcription synthesis are extracted from diamondback moth four-age larva body
First chain cDNA, designed for a pair of of upstream and downstream primer of amplification diamondback moth identification albumen PxIDGF genes, in the primer of design
Restriction enzyme site is introduced, diamondback moth identification albumen PxIDGF genes is expanded from cDNA using the primer of design, utilizes common molecular
The linearisation target gene diamondback moth identification albumen PxIDGF of acquisition is connected by the method for clone with destination carrier pCzn1, is connected
Product converts TOP10 clone strains, and the sequencing of picking positive clone molecule, screening, which obtains, is sequenced correct positive colony, extracts plasmid；
The upstream primer sequence is 5 '-GGAATTCCATATGAACCAGGTCGTCTCC-3 ', nucleotide sequence such as SEQ ID
NO:Shown in 2,
Downstream primer sequence is 5 '-GCTCTAGATTACAGACGGTACTTGG-3 ', nucleotide sequence such as SEQ ID NO:3 institutes
Show.
A kind of 4. preparation method of diamondback moth identification albumen PxIDGF products according to claim 2, which is characterized in that institute
It states step (2) pCzn 1-IDGF carriers and converts to the method for Escherichia coli Arctic-Express and be：It will the correct positive of sequencing
Clone's extraction plasmid, 1 μ L plasmids are added in 100 μ L competent bacterias, put 20min on ice；42 DEG C of heat shock 90s are put in ice rapidly
5min；Add in 600 μ L LB culture solutions；37 DEG C, 220rpm shaking 1h are all coated on the LB containing 50 μ g/ml Amp and put down after centrifugation
Plate, 37 DEG C of inversion overnight incubations.
A kind of 5. preparation method of diamondback moth identification albumen PxIDGF products according to claim 2, which is characterized in that institute
The expression for stating step (3) pCzn 1-IDGF carrier fusions is：Monoclonal on picking conversion tablet is inoculated in containing 50
In the test tube of the 3ml LB culture solutions of μ g/ml AMP, 37 DEG C, 220rpm shakings are overnight；Next day presses 1：100 are inoculated in 50 μ g/ml
In the 30ml LB culture solutions of AMP, 37 DEG C of 220rpm are shaken to thalline OD600For 0.6-0.8；IPTG is added in final concentration of
0.5mM, 15 DEG C, 220rpm shakings are stayed overnight, induced fusion protein expression；Under the conditions of 4000g, 10min is centrifuged, abandons supernatant, received
Collection precipitation.
A kind of 6. preparation method of diamondback moth identification albumen PxIDGF products according to claim 2, which is characterized in that institute
The denaturation and renaturation method for stating step (4) inclusion body protein is：By the precipitation after the expression by pCzn 1-IDGF carrier fusions
It is resuspended in 20ml lysis buffers, ultrasonication；By 4 DEG C of the cell pyrolysis liquid of ultrasonication, 10000g centrifugation 20min are received
Collection precipitation；Precipitation inclusion body is washed using inclusion body cleaning solution 3 times；Again inclusion body is dissolved by a certain percentage with dissolving buffer solution, 4
It DEG C stands overnight；Then 15min is centrifuged under the conditions of room temperature 15000rpm；20mM Tris-HCL 5mM EDTA are added dropwise in supernatant
In Buffer PH7.8 buffer solutions, progressively gradient dilution is slowly stirred at double, and protein solution is packed into bag filter in PBS pH7.4
Dialysed overnight in solution collects protein solution in bag filter；
Wherein, the lysis buffer is 20mM Tris-HCl, containing 1mM PMSF and bacterioprotein Protease Inhibitor Cocktail,
pH 8.0；
The ultrasonication condition is：Power 400W, work 4s, interval 8s, common 20min；
The inclusion body cleaning solution be 20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1%Triton X-100,
pH8.0；
The buffer solution that dissolves is 20mM Tris, 5mM DTT, 8M urea, pH8.0.
A kind of 7. preparation method of diamondback moth identification albumen PxIDGF products according to claim 2, which is characterized in that institute
The Ni column affinity purification methods for stating step (5) fusion protein are：Using low pressure chromatography system, will be answered by the change of inclusion body protein
Ni-IDA- of the protein solution that property method obtains with 0.5ml/min flow velocitys loading to Ni-IDA combination buffer pre-equilibrations
Sepharose CL-6B affinity columns；It is rinsed with Ni-IDA combination buffers with 0.5ml/min flow velocitys, until efflux OD280
Value reaches baseline, then is rinsed with Ni-IDA lavation buffer solutions with 1ml/min flow velocitys, until efflux OD280Value reaches baseline, to remove
Foreigh protein removing；Destination protein is eluted with 1ml/min flow velocitys with Ni-IDA elution buffers, collects albumen efflux；Albumen flows out
Liquid is added in bag filter, and dialysed overnight is carried out using the PBS of pH7.4, collects protein solution in bag filter, this protein solution is cold dry
Afterwards albumen PxIDGF products are identified up to diamondback moth；
Wherein, the Ni-IDA lavation buffer solutions be 20mM Tris-HCl, 20mM imidazoles, 0.15M NaCl, pH8.0；
The Ni-IDA elution buffers be 20mM Tris-HCl, 250mM imidazoles, 0.15M NaCl, pH8.0.
8. application of the diamondback moth identification albumen PxIDGF products as described in claim 1 in microbial infection is resisted, special
Sign is that the identification albumen PxIDGF products have the ability with reference to microorganism, aggegation microorganism.
9. application of the diamondback moth identification albumen PxIDGF products as claimed in claim 8 in microbial infection is resisted, special
Sign is that the microorganism is Escherichia coli or staphylococcus aureus.
10. application of the diamondback moth identification albumen PxIDGF products as claimed in claim 9 in microbial infection is resisted, special
Sign is, in microorganism Binding experiment, 30 μ g protein products and Escherichia coli (4 × 107Cells/ml) at ambient temperature
Mild rotation be incubated 1 it is small when cause albumen precipitate after incubation in allocation proportion reach 90%, 30 μ g protein products with it is golden yellow
Color staphylococcus (4 × 107Cells/ml in so that albumen precipitates after incubation when) mild rotation incubation 1 is small at ambient temperature
Allocation proportion reach 95%.
11. application of the diamondback moth identification albumen PxIDGF products as claimed in claim 9 in microbial infection is resisted, special
Sign is, in microorganism aggegation experiment, 75ng protein products and Escherichia coli or staphylococcus aureus (2 × 106) in room
Be incubated under the conditions of temperature 2 it is small when after under 20 times of object lens visuals field can average observation to 5 zoogloeas.
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Citations (3)
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|---|---|---|---|---|
| US6060590A (en) * | 1998-03-31 | 2000-05-09 | The Regents Of The University Of California | Chitinase related proteins and methods of use |
| US20020120008A1 (en) * | 2000-06-29 | 2002-08-29 | Seymour Benzer | Life extension of drosophila by a drug treatment |
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2017
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|---|---|---|---|---|
| US6060590A (en) * | 1998-03-31 | 2000-05-09 | The Regents Of The University Of California | Chitinase related proteins and methods of use |
| US20020120008A1 (en) * | 2000-06-29 | 2002-08-29 | Seymour Benzer | Life extension of drosophila by a drug treatment |
| CN110923240A (en) * | 2019-12-21 | 2020-03-27 | 山西省农业科学院植物保护研究所 | Function of insect adult disc growth factor and application of dsRNA (double-stranded ribonucleic acid) thereof |
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