CN108085310B

CN108085310B - Plutella xylostella recognition protein PxIDGF product and preparation method and application thereof

Info

Publication number: CN108085310B
Application number: CN201711347198.0A
Authority: CN
Inventors: 党向利; 李双双; 王丽芳; 李胜男; 江俊起; 李桂亭; 缪勇
Original assignee: Anhui Agricultural University AHAU
Current assignee: Anhui Agricultural University AHAU
Priority date: 2017-12-15
Filing date: 2017-12-15
Publication date: 2021-03-30
Anticipated expiration: 2037-12-15
Also published as: CN108085310A

Abstract

The invention discloses a diamondback moth recognition protein PxIDGF product, a preparation method and application thereof, wherein the amino acid sequence of the protein product is shown as SEQ ID NO. 1. Extracting total RNA from young plutella xylostella at four ages, carrying out reverse transcription to synthesize first-chain cDNA, amplifying a plutella xylostella recognition protein PxIDGF gene from the cDNA by using a designed primer, cloning the plutella xylostella recognition protein PxIDGF gene into a pCzn1 vector, transferring the recombinant plasmid into an Escherichia coli Arctic-Express expression strain, inducing expression of fusion protein, redissolving target protein in a renaturation mode, and carrying out affinity purification on a Ni column to obtain the target protein. The plutella xylostella recognition protein PxIDGF product prepared by the invention has high purity and high activity, and has the capacity of combining and agglutinating escherichia coli and staphylococcus aureus. The invention can lay a technical foundation for the application of the diamondback moth recognition protein PxIDGF.

Description

Plutella xylostella recognition protein PxIDGF product and preparation method and application thereof

Technical Field

The invention belongs to the field of natural insect immune recognition, and particularly relates to a diamondback moth recognition protein PxIDGF product, and a preparation method and application thereof.

Background

As one of the most serious countries in the world, the number of the major agricultural pests in China is as much as 30, the harm is serious, and the loss is remarkable. Plutella xylostella (Plutella xylostella) is one of them. As a worldwide agricultural pest which damages crucifers, a remarkable resistance has been developed to many insecticides such as pyrethroids, organophosphates and carbamates. It is the first crop pest to develop resistance to DDT and is also the first insect to develop resistance to bacillus thuringiensis toxins (Baxter et al, 2010). Therefore, a new way for preventing and controlling the plutella xylostella is urgently needed to be researched. The development of biological control technology is a necessary trend for controlling the damage of the plutella xylostella in a lasting and efficient manner. At present, the control of pests by using insect pathogenic bacteria or virulence factors generated by the pathogenic bacteria is one of the frontiers of biological pest control research, and the key point of success lies in understanding the immune defense mechanism of host insects. Therefore, the elucidation of the immune response mechanism of plutella xylostella to various pathogens can provide a theoretical basis for a new pest control method.

Due to the lack of adaptive immunity, insects rely primarily on their own innate immunity to combat the invasion of various pathogens. Natural immunity of insects includes cellular immunity and humoral immunity. If the insects lose the protection of natural immunity, the survival of the insects is affected. It has been shown that by innate immunodeficiency insects (caused by genetic mutations) or by artificial suppression of the natural immunity of the insect, the resistance of the insect to the pathogenic agent is reduced. In humoral immunity, the following processes generally take place from the invasion of the pathogen to its eventual elimination by the host: firstly, identifying foreign matters; subsequently initiating an extracellular cascade of activation of the serine protease and release of the serine protease inhibitor; then, signal transduction pathways are initiated to activate transcription of the target gene. This process requires 4 important factors, namely recognition molecules, signal-regulating factors, signal transduction systems and effector molecules.

Pathogenic recognition is the key first step in the initiation of humoral immunity in insects, and is mainly accomplished by different recognition molecules. Recognition molecules, also known as pattern recognition proteins, are key factors in the immune response of insects and can recognize cell wall components of pathogens (i.e., pathogen associated patterns of molecular patterns, PAMPs), such as lipopolysaccharides from gram-negative bacteria, peptidoglycans from gram-positive bacteria, beta-1, 3-glucans from fungi, and double-stranded RNA from viruses. Different recognition molecules have different structures and functions, binding to different PAMPs. The pattern insect Drosophila diptera has now identified over 8 recognition molecules, mainly peptidoglycan recognition proteins (PGRPs), sulfatoproteins, gram negative bacterial binding proteins (GNBPs), beta-1, 3-glucan recognition proteins (beta GRPs), scavenger receptors, C-type lectins, galectins and immunoglobulins, among others. It is because innate immunity has an extremely important effect on the survival of insects that it has become an important target for pest control. At present, the research of recognition molecules in insect natural immunity has become the focus and hot spot of immunological research.

In order to discuss the immune defense mechanism of plutella xylostella against pathogenic fungus invasion, sequencing transcriptome of plutella xylostella larvae infected by pathogenic organisms and screening immune related genes are carried out. Identifying some immune genes which are possibly involved in the immune protozoan infection of diamondback moth comprises: recognition molecules, signal regulating factors, antibacterial effect factors and other genes possibly involved in immunity. The recognition molecules include PxIDGF, CTL (C-type molecules), PGRP (peptidoglycan recognition protein), and the like. IDGF, an organ bud growth factor, belongs to glycosyl hydrolase family 18, is secreted by insect fat body expression into the blood lymph of insects, and its primary function is to regulate insect development and growth. It is postulated that it may play a role in invertebrate pathogen recognition due to having 1 lectin domain that binds chitin and 1 lactate domain that binds chitin. No experimental data currently exists for this function.

In order to determine whether the diamondback moth recognition protein PxIDGF has an immunological recognition effect in the process of immunizing pathogenic microorganisms with the diamondback moth body fluid, the protein with activity is very necessary to obtain. At present, the invention in the aspects of the product and the preparation method of the diamondback moth recognition protein PxIDGF is not available.

Disclosure of Invention

The invention provides a diamondback moth recognition protein PxIDGF product, a preparation method and application thereof.

The invention is realized by the following technical scheme:

the invention provides a diamondback moth recognition protein PxIDGF product, which is a protein obtained from diamondback moth larvae through gene cloning, protein expression and purification, and has the theoretical molecular weight of 47.904KD and the isoelectric point of 7.67; the complete amino acid sequence of the protein preparation is MNHKVHHHHHHMNQVVSTKKVICYYDSKSYVRESNARLLPPDLEPALPYCTHLVYGYAGVQPDTYKMISTNQNLDIDSAHANYRTITSFKTKYPQLKMLLAVGGDADLEDPQKYNALLESQQARTAFVNSGVVLAEQHGFDGIDLAWQFPKVKPKKIRSGWGNFWHGVKKTFKTTPVDEKESEHREGFTALVRELKAALSLKPHLELGVTILPNVNSTIYCDVPAIINFVDYVNLLAFDYFTPERNEKEADYTAPIYAPQNRHPEQNVDAAVKYWRNAGAPPTKIVVGIATYARTWKLDSDSEVAGVPPIHTDGAGEPGPYTKTEGLLSYPEVCMKLIAPPAGLRANIRKVTDPSKRFGTYAFRLPDSDGNGGIWVSYEDADTAGQKADYVKKNNLGGISIVDLSMDDFRELCTGNKYPILRAAKYRL.

The invention also provides a preparation method of the diamondback moth recognition protein PxIDGF product, which comprises the following steps: the present invention selects diamondback moth (Plutella xylostella) as material, extracts total RNA from diamondback moth four-age larva, makes reverse transcription to synthesize first chain cDNA, utilizes designed primer to amplify diamondback moth identification protein PxIDGF gene from cDNA, utilizes conventional molecular cloning method to connect diamondback moth identification protein PxIDGF gene into pCzn1 carrier, then transfers the plasmid into Escherichia coli Arctic-Express expression strain, shakes overnight at 37 deg.C, next day dilutes bacterial liquid according to 1:100 ratio and shakes bacterial to OD₆₀₀The value is 0.6-0.8, the final concentration is 0.5mMIPTG, the mixture is shaken overnight at 15 ℃, fusion protein expression is induced, target protein is re-dissolved in a renaturation mode, and the target protein PxIDGF is obtained through Ni column affinity purification.

The preparation method comprises the following steps:

(1) constructing pCzn1-IDGF plasmid;

(2) the pCzn1-IDGF carrier is transformed into escherichia coli Arctic-Express;

(3) expressing pCzn1-IDGF carrier fusion protein;

(4) renaturation of inclusion body protein;

(5) and (3) carrying out Ni column affinity purification on the fusion protein.

The pCzn1-IDGF plasmid construction method in the step (1) is as follows: extracting total RNA from a plutella xylostella four-instar larva body, carrying out reverse transcription to synthesize first-strand cDNA, designing a pair of upstream and downstream primers for amplifying a plutella xylostella identification protein PxIDGF gene, introducing an enzyme cutting site into the designed primers, amplifying the plutella xylostella identification protein PxIDGF gene from the cDNA by using the designed primers, connecting the obtained linearized target gene plutella xylostella identification protein PxIDGF with a target vector pCzn1 by using a conventional molecular cloning method, transforming a connecting product into a TOP10 cloning strain, picking positive clones for sequencing, screening to obtain positive clones with correct sequencing, and extracting plasmids;

wherein the upstream primer sequence is 5' -GGAATTCCATATGAACCAGGTCGTCTCC-3', the underlined sequence is Nde I restriction site sequence, and the nucleotide sequence is shown in SEQ ID NO: 2.

The sequence of the downstream primer is 5' -GCTCTAGATTACAGACGGTACTTGG-3', the underlined sequence is the Xba I restriction site sequence, and the nucleotide sequence is shown in SEQ ID NO. 3.

The method for transforming the pCzn1-IDGF carrier into the escherichia coli Arctic-Express in the step (2) comprises the following steps: extracting plasmid from positive clone with correct sequencing, adding 1 μ L plasmid into 100 μ L competent bacteria, and placing on ice for 20 min; rapidly placing in ice for 5min under 42 deg.C heat shock for 90 s; adding 600 μ L LB culture solution; after centrifugation, the whole was spread on LB plates containing 50. mu.g/ml Amp after shaking at 220rpm for 1 hour at 37 ℃ and cultured overnight in an inverted state at 37 ℃.

The expression method of the pCzn1-IDGF carrier fusion protein in the step (3) comprises the following steps: picking single clone on the transformation plate and inoculating to a test tube containing 3ml LB culture solution containing 50. mu.g/ml AMP, shaking at 37 ℃, 220rpm overnight; the following day is as follows: 100 was inoculated into 30ml of LB medium containing 50. mu.g/ml AMP, and the mixture was shaken at 37 ℃ and 220rpm until the OD of the cells was reached₆₀₀0.6-0.8; adding IPTG to the final concentration of 0.5mM, shaking at 15 ℃ and 220rpm overnight, and inducing the expression of the fusion protein; centrifuging for 10min under 4000g, discarding the supernatant, and collecting the precipitate.

The method for renaturation of the inclusion body protein in the step (4) comprises the following steps: resuspending the precipitate after the expression of the pCzn1-IDGF carrier fusion protein in 20ml of lysis buffer solution, and carrying out ultrasonic disruption; centrifuging the ultrasonically-broken cell lysate for 20min at 4 ℃ under the condition of 10000g, and collecting the precipitate; washing and precipitating the inclusion body for 3 times by using an inclusion body washing solution; dissolving the inclusion body with a dissolving buffer solution according to a certain proportion, and standing overnight at 4 ℃; then centrifuging for 15min at the room temperature of 15000 rpm; dropwise adding the supernatant into 20mM Tris-HCL 5mM EDTA Buffer PH7.8 Buffer solution, gradually diluting in a gradient manner, slowly stirring, putting the protein solution into a dialysis bag, dialyzing in PBS pH7.4 solution overnight, and collecting the protein solution in the dialysis bag;

wherein the lysis buffer is 20mM Tris-HCl, contains 1mM PMSF and a bacterial protease inhibitor mixture and has a pH value of 8.0;

the ultrasonic crushing conditions are as follows: the power is 400W, the work time is 4s, the intermittence time is 8s, and the total time is 20 min;

the inclusion body washing solution is 20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1% Triton X-100, and the pH value is 8.0;

the dissolution buffer was 20mM Tris, 5mM DTT, 8M Urea, pH 8.0.

The Ni column affinity purification method of the fusion protein in the step (5) comprises the following steps: loading a protein solution obtained by a renaturation method of the inclusion body protein to a Ni-IDA-Sepharose CL-6B affinity chromatographic column pre-balanced by a Ni-IDA binding buffer solution at the flow rate of 0.5ml/min by using a low-pressure chromatographic system; washing with Ni-IDA binding buffer at flow rate of 0.5ml/min to obtain effluent OD₂₈₀The value reached baseline, and the flow rate was then flushed with Ni-IDA wash buffer at 1ml/min to the effluent OD₂₈₀The value reached baseline to remove the contaminating proteins; eluting the target protein by using Ni-IDA elution buffer solution at the flow rate of 1ml/min, and collecting protein effluent; adding the protein effluent into a dialysis bag, dialyzing with PBS (phosphate buffer solution) with pH of 7.4 overnight, collecting the protein solution in the dialysis bag, and cooling and drying the protein solution to obtain a plutella xylostella identification protein PxIDGF product;

wherein the Ni-IDA washing buffer solution is 20mM Tris-HCl, 20mM imidazole, 0.15M NaCl, pH8.0;

the Ni-IDA elution buffer is 20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH8.0.

The recognition protein PxIDGF product prepared by the invention has the capability of binding and agglutinating microorganisms.

The invention has the following beneficial effects:

the invention has the advantages of rich raw material sources, simplicity, convenience, rapidness and the like, and the expression system has the characteristics of increasing the protein translation efficiency, increasing the protein expression level and the like, and can induce the high-level expression of the foreign protein and increase the activity probability of the expressed protein under the low-temperature condition. The diamondback moth identification protein PxIDGF product prepared by the invention has high purity and high activity, has the capability of combining microorganisms and the capability of agglutinating the microorganisms, avoids the complicated step of extracting the identification protein from the diamondback moth, and greatly saves the experiment cost and time. The preparation method of the invention can be used for large-scale industrial production, has short production period and lower production cost, and is not influenced by the external environment. The invention can lay a technical foundation for the application of the diamondback moth recognition protein PxIDGF. More importantly, the invention proves that the plutella xylostella recognition protein PxIDGF has the capability of recognizing microbial pathogens through microbial binding and agglutination tests for the first time, and the binding capability has the microbial binding proportion and the agglutination capability which are not reported in the prior art under the support of test data.

Drawings

FIG. 1 shows the results of restriction enzyme identification of recombinant plasmids;

in the figure: m is DNA molecular weight Marker (100 bp, 250bp, 500bp, 750bp, 1000bp, 1500bp, 2000bp, 3000bp and 5000bp from bottom to top respectively); 1 is plasmid before enzyme digestion; 2 is plasmid after enzyme digestion;

FIG. 2 shows the results of the identification of the expression of the fusion protein;

in the figure: m is a protein molecular weight Marker; 1 is non-induced bacterial liquid; 2 is IPTG induced bacteria liquid; 3, inducing the thalli to carry out ultrasonic crushing at 15 ℃ by IPTG, and then obtaining a supernatant; 4, IPTG induction thalli at 15 ℃ and precipitation after ultrasonic crushing;

FIG. 3 shows SDS-PAGE results of purified target protein;

in the figure: m is a protein molecular weight Marker; 1 is an unpurified sample; 2 is Ni-IDA washing buffer solution to wash the sample; 3, eluting pure protein by using a Ni-IDA elution buffer solution;

FIG. 4 shows the result of the binding assay of Plutella xylostella recognition protein PxIDGF with microorganisms;

in the figure: 1 is the supernatant after incubation; 2-5 the precipitates after incubation were washed four times with TBS respectively; 6 is supernatant after the precipitation is eluted by 7% SDS after incubation; 7, after incubation, the precipitate is eluted by 7% SDS and then precipitated; 8 is purified diamondback moth recognition protein PxIDGF;

FIG. 5 shows the result of the agglutination microorganism of the Plutella xylostella recognition protein PxIDGF;

control is 1mM CaCl₂(ii) a + BSA 1mM CaCl₂And TBS of 75ng BSA; + IDGF is 1mM CaCl₂And TBS of 75ng PxIDGF; + IDGF + EDTA 1mM CaCl₂TBS 75ng PxIDGF and 1mM EDTA.

Detailed Description

The present invention will be described in further detail below with reference to the drawings and specific examples, but the embodiments of the present invention are not limited thereto.

Example 1

1. Construction of pCzn1-IDGF plasmid

Extracting total RNA from young plutella xylostella with Trizol reagent (Takara), reverse transcribing to synthesize first strand cDNA with cDNA synthesizing kit (Takara), and designing a pair of upstream and downstream primers for amplifying plutella xylostella identification protein PxIDGF gene, wherein the upstream primer sequence is 5' -GGAATTCCATATGAACCAGGTCGTCTCC-3 '(underlined sequence is Nde I cleavage site sequence), and the sequence of the downstream primer is 5' -GCTCTAGATTACAGACGGTACTTGG-3' (the underlined sequence is the Xba I cleavage site sequence). Designed primers can be introduced with enzyme cutting sites. Amplifying a diamondback moth recognition protein PxIDGF gene from cDNA by using a designed primer, wherein a PCR system comprises: 2.0. mu.L of template DNA, 1.0. mu.L of upstream primer, 1.0. mu.L of downstream primer, 5.0. mu.L of 10 XBuffer, 4.0ul of dNTP mix, 1.0. mu.L of Pfu DNA polymerase, and the like using ddH₂And O is supplemented to 50 mu L. PCR procedure: 5min at 94 ℃; 30 cycles of 94 ℃ for 30s, 65 ℃ for 30s and 72 ℃ for 45 s; finally 5min at 72 ℃. The obtained linearized target gene plutella xylostella recognition protein PxIDGF is connected with a target vector pCzn1 by using a conventional molecular cloning method, a connecting product is converted into a TOP10 cloning strain, a positive clone is selected for sequencing, and a positive clone with correct sequencing is obtained by screening. And utilizing enzyme digestion to identify. Enzyme digestion system: plasmid 3ul, endonuclease Nde I0.25 uL, endonuclease Xba I0.25 uL, 101.0 μ L of Xbuffer using ddH₂And O is supplemented to 10 mu L. The results of enzyme cleavage identification are shown in FIG. 1.

2. Transformation of pCzn1-IDGF vector into Escherichia coli Arctic-Express

Extracting plasmid from positive clone with correct sequencing, adding 1 μ L plasmid into 100 μ L competent bacteria, and placing on ice for 20 min; thermally shocking at 42 deg.C for 90sec, and rapidly placing in ice for 5 min; adding 600 μ L LB culture solution; after centrifugation, the whole was spread on LB plates containing 50. mu.g/ml Amp after shaking at 220rpm for 1 hour at 37 ℃ and cultured overnight in an inverted state at 37 ℃.

3. IPTG induction of expression of pCzn1-IDGF carrier fusion protein

(1) Picking single clone on the transformation plate to inoculate in a tube containing 50 ug/ml AMP in 3ml LB culture solution, shaking at 37 ℃ and 220rpm overnight;

(2) the following day is as follows: 100 is inoculated into 30ml LB culture solution of 50 mug/ml AMP, and is shaken at 37 ℃ and 220rpm until the thallus OD600 is 0.6-0.8;

(3) taking out 1ml of culture, centrifuging at 10000g of room temperature for 2min, discarding supernatant, and resuspending the thallus precipitate with 100 μ l of 1 Xloading buffer;

(4) adding IPTG to the rest culture until the final concentration is 0.5mM, shaking at 15 ℃ and 220rpm overnight, and inducing the expression of the fusion protein;

(5) 1ml of the culture was removed, centrifuged at 10000g for 2min at room temperature, the supernatant was discarded, and the pellet was resuspended in 100. mu.l of 1 Xloading buffer. Centrifuging the residual culture at 4000g for 10min, discarding the supernatant, and resuspending the thallus precipitate with PBS; after the resuspension liquid is subjected to ultrasonic crushing, supernatant and precipitation liquid are respectively taken and added into a sample loading buffer solution for resuspension.

(6) Analysis by 12% SDS-PAGE showed bands on Coomassie blue staining, which is shown in FIG. 2.

4. Renaturation of inclusion body proteins

Resuspending the pellet after expression of pCzn1-IDGF vector fusion protein in 20ml lysis buffer (20mM Tris-HCl, containing 1mM PMSF and bacterial protease inhibitor cocktail, pH8.0), ultrasonication (power 400W, working 4sec, pause 8sec, total 20 min); centrifuging the ultrasonically-broken cell lysate at 4 ℃ and 10000g for 20min, and collecting the precipitate; the inclusion bodies were washed 3 times with inclusion body wash (20mM Tris, 1mM EDTA, 2M urea, 1M NaCl, 1% Triton X-100, pH 8.0); dissolving the inclusion body with dissolving buffer solution (20mM Tris, 5mM DTT, 8M urea pH8.0) according to a certain proportion, and standing overnight at 4 ℃; then centrifuging for 15min at the room temperature of 15000 rpm; the supernatant was added dropwise to 20mM Tris-HCl 5mM EDTABuffer pH7.8 buffer, gradually diluted in stepwise gradient with slow stirring, and the protein solution was placed in a dialysis bag and dialyzed overnight in PBS at pH7.4, and the protein solution in the dialysis bag was collected.

5. Ni-column affinity purification of fusion proteins

Loading a protein solution obtained by a renaturation method of the inclusion body protein to a Ni-IDA-Sepharose CL-6B affinity chromatographic column pre-balanced by a Ni-IDA binding buffer solution at the flow rate of 0.5ml/min by using a low-pressure chromatographic system; washing with Ni-IDA binding buffer at flow rate of 0.5ml/min to obtain effluent OD₂₈₀The value reached baseline, and the flow rate was further washed with Ni-IDA washing buffer (20mM Tris-HCl, 20mM imidazole, 0.15M NaCl, pH8.0) at 1ml/min until the effluent OD₂₈₀The value reached baseline to remove the contaminating proteins; eluting the target protein with Ni-IDA elution buffer (20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1ml/min, and collecting the protein effluent; and adding the protein effluent into a dialysis bag, dialyzing overnight by using PBS (PH7.4), collecting the protein solution in the dialysis bag, and cooling and drying the protein solution to obtain the plutella xylostella recognition protein PxIDGF product.

The sample of the diamondback moth recognition protein PxIDGF product obtained by the method of example 1 is analyzed:

1. 12% SDS-PAGE analysis

mu.L of the sample was added to 20. mu.L of 5 XSDS Loading Buffer and boiled for 5min, centrifuged at 12000rpm for 5min, and 20. mu.L of the sample was applied. 12% SDS-PAGE was performed, stained with Coomassie Brilliant blue stain for 2h, and destained overnight with Coomassie Brilliant blue stain. The results of 12% SDS-PAGE analysis are shown in FIG. 3.

2. Western blot analysis

Samples were subjected to 12% SDS-PAGE and transferred onto nitrocellulose membranes. Subsequently, the membrane compartment was blocked with blocking buffer (5% BSA) at room temperature for 1.5 hours and incubated with PxIDGF polyclonal antibody (1: 2500 dilution in blocking buffer) overnight at 4 ℃ and co-incubated with IgG at room temperature for 2 hours. The bands were visualized with DAB dye reagent (Boster Biotech).

3. Microbial binding assays

Bacteria (Escherichia coli and Staphylococcus aureus) in middle logarithmic phase were centrifuged at 6000rpm for 5min, and the pellet was washed three times with Tris-buffered saline (TBS; 50mM Tris-HCl, 150mM NaCl, pH7.5) and suspended in TBS at a concentration of 2X 10⁸cells/ml bacterial suspension. The bacterial suspension (200. mu.l) was mixed with 200. mu.l PxIDGF (150. mu.g/ml). The mixture was incubated at room temperature for 1 hour with gentle rotation. The bacteria were pelleted, washed four times with TBS and eluted with 7% SDS. The washing and elution solutions were analyzed using western blot. The prepared plutella xylostella recognition protein PxIDGF product has the capacity of combining escherichia coli and staphylococcus aureus, and the result is shown in a figure 4. The distribution ratio of the diamondback moth recognition protein PxIDGF in the precipitate after the incubation with the escherichia coli reaches 90 percent and the distribution ratio of the diamondback moth recognition protein PxIDGF in the precipitate after the incubation with the staphylococcus aureus reaches 95 percent.

4. Microbiological agglutination test

2 x 10 to⁶Coli or Staphylococcus aureus cells were added with 1mM CaCl₂(control) with 1mM CaCl₂TBS with 75ng BSA, 1mM CaCl₂TBS with 75ng PxIDGF and 1mM CaCl₂TBS 75ng PxIDGF and 1mM EDTA. All treatments were incubated at room temperature for 2 hours. Bacterial aggregates were found by examination with Eclipse Ti inverted microscope (Nikon) and the results are shown in FIG. 5. The results show that an average of 5 colonies of bacteria were observed under a 20-fold field of view of the objective lens (taking the average of 50 fields).

Sequence listing

<110> agriculture university of Anhui

<120> plutella xylostella recognition protein PxIDGF product, and preparation method and application thereof

<130> 2017

<141> 2017-12-15

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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

1. The application of a diamondback moth recognition protein PxIDGF product in resisting microbial infection is characterized in that the recognition protein PxIDGF product has the capacity of binding microorganisms and agglutinating the microorganisms, and the microorganisms are escherichia coli or staphylococcus aureus;

the diamondback moth recognition protein PxIDGF product is a protein product obtained by gene cloning, protein expression and purification from diamondback moth larvae, the theoretical molecular weight is 47.904KD, and the isoelectric point is 7.67; the amino acid sequence of the protein product is shown as SEQ ID NO. 1.

2. The use of a plutella xylostella recognition protein PxIDGF preparation according to claim 1 for combating microbial infections, wherein the preparation method of the plutella xylostella recognition protein PxIDGF preparation comprises the following steps:

(1) constructing pCzn1-IDGF plasmid;

(2) the pCzn1-IDGF carrier is transformed into escherichia coli Arctic-Express;

(3) IPTG induces the expression of pCzn1-IDGF carrier fusion protein;

(4) renaturation of inclusion body protein;

(5) and (3) carrying out Ni column affinity purification on the fusion protein.

3. The use of a plutella xylostella recognition protein PxIDGF preparation according to claim 2 for defending against microbial infection, wherein the pCzn1-IDGF plasmid in the step (1) is constructed as follows: extracting total RNA from a plutella xylostella four-instar larva body, carrying out reverse transcription to synthesize first-strand cDNA, designing a pair of upstream and downstream primers for amplifying a plutella xylostella identification protein PxIDGF gene, introducing an enzyme cutting site into the designed primers, amplifying the plutella xylostella identification protein PxIDGF gene from the cDNA by using the designed primers, connecting the obtained linearized target gene plutella xylostella identification protein PxIDGF with a target vector pCzn1 by using a conventional molecular cloning method, transforming a connecting product into a TOP10 cloning strain, picking positive clones for sequencing, screening to obtain positive clones with correct sequencing, and extracting plasmids;

the sequence of the upstream primer is 5'-GGAATTCCATATGAACCAGGTCGTCTCC-3', the nucleotide sequence is shown as SEQ ID NO. 2,

the sequence of the downstream primer is 5'-GCTCTAGATTACAGACGGTACTTGG-3', and the nucleotide sequence is shown as SEQ ID NO. 3.

4. The use of a plutella xylostella recognition protein PxIDGF preparation according to claim 2, wherein the step (2) of transforming the pCzn1-IDGF vector into E.coli Arctic-Express is as follows: extracting plasmid from positive clone with correct sequencing, adding 1 μ L plasmid into 100 μ L competent bacteria, and placing on ice for 20 min; rapidly placing in ice for 5min under 42 deg.C heat shock for 90 s; adding 600 μ L LB culture solution; after centrifugation, the whole was spread on LB plates containing 50. mu.g/ml Amp after shaking at 220rpm for 1 hour at 37 ℃ and cultured overnight in an inverted state at 37 ℃.

5. The use of a plutella xylostella recognition protein PxIDGF preparation in resisting microbial infection as claimed in claim 2, wherein the expression method of the pCzn1-IDGF carrier fusion protein in the step (3) is as follows: picking single clone on the transformation plate and inoculating to a test tube containing 3ml LB culture solution containing 50. mu.g/ml AMP, shaking at 37 ℃, 220rpm overnight; the following day is as follows: 100 was inoculated into 30ml of LB medium containing 50. mu.g/ml AMP, and the mixture was shaken at 37 ℃ and 220rpm until the OD of the cells was reached₆₀₀0.6-0.8; adding IPTG to the final concentration of 0.5mM, shaking at 15 ℃ and 220rpm overnight, and inducing the expression of the fusion protein; centrifuging for 10min under 4000g, discarding the supernatant, and collecting the precipitate.

6. The use of a plutella xylostella recognition protein PxIDGF preparation according to claim 2, wherein the inclusion body protein renaturation method of the step (4) is as follows: resuspending the precipitate after the expression of the pCzn1-IDGF carrier fusion protein in 20ml of lysis buffer solution, and carrying out ultrasonic disruption; centrifuging the ultrasonically-broken cell lysate for 20min at 4 ℃ and 10000g, and collecting precipitates; washing and precipitating the inclusion body for 3 times by using an inclusion body washing solution; dissolving the inclusion body with a dissolving buffer solution according to a certain proportion, and standing overnight at 4 ℃; then centrifuging for 15min at the room temperature of 15000 rpm; dropwise adding the supernatant into 20mM Tris-HCL 5mM EDTA Buffer PH7.8 Buffer solution, gradually diluting in a gradient manner and slowly stirring, putting the protein solution into a dialysis bag, dialyzing in PBS PH7.4 solution overnight, and collecting the protein solution in the dialysis bag;

the dissolution buffer was 20mM Tris, 5mM DTT, 8M Urea, pH 8.0.

7. The use of a plutella xylostella recognition protein PxIDGF preparation according to claim 2 for defending against microbial infection, wherein the Ni column affinity purification method of the fusion protein in the step (5) is as follows: loading a protein solution obtained by a renaturation method of the inclusion body protein to a Ni-IDA-Sepharose CL-6B affinity chromatographic column pre-balanced by a Ni-IDA binding buffer solution at the flow rate of 0.5ml/min by using a low-pressure chromatographic system; washing with Ni-IDA binding buffer at flow rate of 0.5ml/min to obtain effluent OD₂₈₀The value reached baseline, and the flow rate was then flushed with Ni-IDA wash buffer at 1ml/min to the effluent OD₂₈₀The value reached baseline to remove the contaminating proteins; eluting the target protein by using Ni-IDA elution buffer solution at the flow rate of 1ml/min, and collecting protein effluent; adding the protein effluent into a dialysis bag, dialyzing with PBS (phosphate buffer solution) with pH of 7.4 overnight, collecting the protein solution in the dialysis bag, and cooling and drying the protein solution to obtain a plutella xylostella identification protein PxIDGF product;

the Ni-IDA elution buffer is 20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH8.0.

8. The use of a plutella xylostella recognition protein PxIDGF preparation according to claim 1 for combating microbial infestation, wherein 30 μ g of the protein preparation is combined with 4 x 10 in a microbial binding assay⁷cells/ml of E.coli were incubated at room temperature for 1 hour with gentle rotation so that the distribution ratio of protein in the pellet after incubation reached 90%, 30. mu.g of protein preparation and 4X 10⁷cells/ml of Staphylococcus aureus were incubated at room temperature for 1 hour with gentle rotation so that the distribution of protein in the pellet after incubation reached 95%.

9. The use of a plutella xylostella recognition protein PxIDGF preparation according to claim 1 for combating microbial infestation, wherein 75ng of the protein preparation is mixed with 2 x 10 of the protein preparation in a microbial agglutination test⁶Escherichia coli or 2X 10⁶An average of 5 colonies of S.aureus were observed in a 20-fold field of view of the objective lens after 2 hours incubation at room temperature.