CN108264543A

CN108264543A - A kind of ribosomal toxin and its encoding gene and application

Info

Publication number: CN108264543A
Application number: CN201810085980.8A
Authority: CN
Inventors: 李洪波; 夏玉先
Original assignee: Huaihua University
Current assignee: Huaihua University
Priority date: 2018-01-29
Filing date: 2018-01-29
Publication date: 2018-07-10
Anticipated expiration: 2038-01-29
Also published as: CN108264543B

Abstract

The present invention relates to a kind of ribosomal toxin and its encoding gene and application, gene provided by the invention is as follows（1）‑（3）Any one of DNA molecular：（1）As the DNA molecular shown in sequence in sequence table 1；（2）Under strict conditions with（1）The DNA molecular of the albumen of DNA sequence dna hybridization and coding with poisoning insect active of restriction；（3）With（1）The DNA sequence dna of restriction is at least with 95%, at least with 96%, at least with the 97%, DNA molecular at least with 98% or at least with the albumen of 99% homology and coding with poisoning insect active.Present invention optimizes the DNA sequence dna of HtA, and provide it is a kind of can be with high efficient expression and the method for rapidly purifying HtA recombinant proteins, for pest-resistant field, particularly cultivating zoophobous kind has important value, has great importance to the yield for improving crops.

Description

A kind of ribosomal toxin and its encoding gene and application

Technical field

The present invention relates to biotechnologies, and in particular to a kind of ribosomal toxin from thompson crinosity bacterium （HtA）The method and application of recombination and its Prepare restructuring albumen.

Background technology

Thompson crinosity bacterium（Hirsutella thompsonii）It is a kind of fungi acaricide, early in the seventies in last century Just it is used to the pests such as prevention mite.In recent years the study found that thompson crinosity bacterium generate sugared body toxin（HtA）With its desinsection Exist between activity and be closely connected, zymotic fluid is there is also insecticidal activity, and the ribosomal toxin purified from zymotic fluid is similary Insect pest can be killed.Although HtA has the important potential value of control agricultural pests, the content of native ribosome toxin It is low and difficult extraction, and often can only obtain very small amount of sterling, it dies from 1 be only capable of purifying not in right zymotic fluid at present To 1 milligram of albumen, and utilize the heterologous gene expression system also middle recombination egg that milligram grade can be obtained from 1 liter of culture In vain, this just further studies the bioactivity of the toxin to us and insecticidal properties bring very big difficulty.Therefore, it establishes high What the heterologous gene expression system of effect and its preparation method of albumen will be helpful to solve currently to be encountered can not obtain a large amount of eggs White bottleneck.

And current Escherichia coli and yeast expression system express the albumen that also there are clearly disadvantageous parts：The prior art By ribosomal toxin construction of expression vector, then it is transformed intoE. coliBL21 (DE3) is expressed, but what is obtained is all no work Property inclusion body, by being dissolved under the conditions of suitable in vitro, being denaturalized, renaturation and purifying, be only capable of from every liter of culture medium To micro soluble protein, output is extremely low；The method that the expression vector of structure is expressed in yeast, but its expression quantity It is low and isolate and purify very difficult.Therefore, it is expressed by DNA sequence dna and optimization HtA that HtA is transformed and purification process is come efficiently raw Production HtA recombinant proteins can will thoroughly solve the determination of activity of HtA, desinsection spectrum analysis and the application in pest-resistant the problems such as.This Invention will provide a kind of complete and efficiently solves the problems, such as this feasible method for the first time.

Invention content

For the defects in the prior art, present invention aims at provide a kind of ribosomal toxin HtA recombinations and its Prepare method and the application of albumen.

The present invention provides gene, is the gene of encoding ribosomal toxin HtA albuminoids, is as follows（1）-（3）In it is any one The DNA molecular of kind：

（1）As the DNA molecular shown in sequence in sequence table 1；

（2）Under strict conditions with（1）DNA points of the albumen of DNA sequence dna hybridization and coding with poisoning insect active of restriction Son；

（3）With（1）The DNA sequence dna of restriction is at least with 95%, at least with 96%, at least with 97%, at least with 98% or extremely DNA molecular less with the albumen of 99% homology and coding with poisoning insect active.

The stringent condition can be as follows：50 DEG C, in 7% lauryl sodium sulfate（SDS）、0.5 M Na₃PO₄With 1 mM Hybridize in the mixed solution of EDTA, rinsed in 50 DEG C, 2 × SSC, 0.1% SDS；Can also be：50 DEG C, in 7% SDS, 0.5 M Na₃PO₄Hybridize in the mixed solution of 1 mM EDTA, rinsed in 50 DEG C, 1 × SSC, 0.1% SDS；Can also be：50 DEG C, 7% SDS, 0.5 M Na₃PO₄Hybridize in the mixed solution of 1 mM EDTA, floated in 50 DEG C, 0.5 × SSC, 0.1% SDS It washes；Can also be：50 DEG C, in 7% SDS, 0.5 M Na₃PO₄Hybridize in the mixed solution of 1 mM EDTA, at 50 DEG C, 0.1 × It is rinsed in SSC, 0.1% SDS；Can also be：50 DEG C, in 7% SDS, 0.5 M Na₃PO₄With it is miscellaneous in the mixed solution of 1 mM EDTA It hands over, is rinsed in 65 DEG C, 0.1 × SSC, 0.1% SDS；Or：In 6 × SSC, the solution of 0.5% SDS, 65^oIt is miscellaneous under C It hands over, then with 2 × SSC, 0.1% SDS and 1 × SSC, it is primary that 0.1% SDS respectively washes film.

Wherein, the sequence 1 in sequence table is made of 411 deoxynucleotides, this sequence includesHtAThe maturation protein of gene The major sites such as overall length reading frame, terminator codon and gene cloning site, amino acid of the coding with sequence 2 in sequence table are residual The recombinant protein of basic sequence.

Obtained albumen is encoded by the sequence 1 in above-mentioned sequence table to belong to the scope of protection of the present invention.

The present invention provides albumen, is as follows（1）Or（2）：

（1）The protein being made of the amino acid sequence shown in sequence in sequence table 2；

（2）By substitution of the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues and/or missing And/or add and have the protein as derived from sequence 2 of poisoning insect active.

Wherein, the sequence 2 in sequence table is made of 130 amino acid residues.

It the substitution of one or several amino acid residues and/or lacks and ors add and refers to not more than ten amino acid It the substitution of residue and/or lacks and ors add.

Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing said gene also belong to the protection of the present invention Range.

The recombinant vector is specially that said gene is inserted into expression vector, and the recombination for obtaining expressing above-mentioned albumen carries Body.The recombinant vector is particularly preferred as said gene being inserted into the Xho of expression vector pPICZ α AAnd XbaRestriction enzyme site Between, obtain expressing the recombinant vector of above-mentioned albumen.

Second object of the present invention is to provide a kind of method for preparing albumen, includes the following steps：

S1：Gene described in claim 1 and expression vector pPICZ α with Xho I and I double digestions of Xba and are purified into recycling respectively Then target DNA fragments are connected at 16 °C using ligase, obtain recombinant vector pPICZ α A-HtA；

S2：The recombinant vector pPICZ α A-HtA I single endonuclease digestions of Sac are linearized, and are transformed into lithium chloride conversion method complete red In yeast host bacterium, screen to obtain positive colony using Zeocin；

S3：The positive colony is forwarded to the YPD tablets containing 1000 ~ 1500 μ g/mL Zeocin, screening obtains height The transformant of Zeocin resistances, by the resistance transformant BMGY medium cultures to OD₆₀₀It is 10 ~ 15, is collected by centrifugation thin Born of the same parents are precipitated, and cell is resuspended with BMMY culture mediums, add in methanol and its mass fraction is made to be 1.0% ~ 1.5%, induce 24 hours, collect The supernatant of fermentation；Before induced expression, preferably by the transformant of the high Zeocin resistances a small amount of induced expressions of BMMY, sieve Choosing obtains the transformant of high-level secretory expression target protein, then with BMGY medium culture induced expressions.

Preferably, after the induction of step S3 24 hours and collect fermentation supernatant before, further include and further lure Lead the following steps of expression：

S4：Continue Fiber differentiation 72 hours at 28 °C, adding methanol within every 24 hours makes its mass fraction be maintained at 1.0% ~ 1.5%.

Preferably, after step s4, the following steps of purifying protein are further included：

S5：The medium centrifugal through 72 hours methanol inductions obtained by S4 steps is detached and takes supernatant, supernatant is utilized After the Tris-HCl dialysed overnights of 20 mM of the pH 8.0 ~ 8.5 of 50 ~ 100 times of volumes, adjust pH to 8.0 ~ 8.5, to be more than or Rotating speed equal to 15000 g centrifuges 10 ~ 20 minutes, and the supernatant of acquisition is loaded to and is buffered through 8.0 ~ 8.5 Tris-HCl of pH CM cation-exchange chromatography posts after liquid balance, the pH's 8.0 ~ 8.5 for chromatographing column volume with 5 ~ 10 times contains 20 mM Tris-HCl The CM cation-exchange chromatography posts are rinsed with the buffer solution of 50 ~ 100 mM NaCl；

S6：The CM cation-exchange chromatography posts are eluted with containing the buffer solution of 20 mM Tris-HCl and 400 ~ 800 mM NaCl, Obtained eluent is dialysed using the bag filter that molecular weight is 10 kDa in 10 mM Tris-Cl buffer solutions, then ultrafiltration Concentration.

Preferably, after step S6, the following steps for preserving albumen are further included：

S7：The product that the ultrafiltration concentration is obtained is quick-frozen under -80 °C, is then freeze-dried.

The albumen that the method for preparing albumen according to any of the above-described kind is prepared also belongs to protection scope of the present invention.

Pass through the step S3 stabilizations screened and the yeast transformant of energy high-level secretory expression ribosomal toxin HtA Belong to the scope of protection of the invention.

Above-mentioned albumen, said gene or above-mentioned recombinant vector, expression cassette, transgenic cell line or recombinant bacterium are in pest-resistant field In application be also the scope of protection of the invention.

Third object of the present invention is to provide a kind of method for cultivating genetically modified plants, is by the coding base of above-mentioned albumen Because importeding into purpose plant, genetically modified plants are obtained, the insect resistace of the genetically modified plants is higher than the purpose plant.

Above-mentioned genetically modified plants are interpreted as not only comprising the first generation transgenosis for obtaining the genetic transformation purpose plant Plant, also including its filial generation.For genetically modified plants, the gene can be bred in the species, it is also possible to traditional breeding method The gene transfer is entered to other kinds of same species, particularly including in commercial variety.By the channel genes purpose plant, Can make to synthesize in the protein purpose plant, into but the pest-resistant performance of purpose plant improved.

The present invention also provides a kind of methods for cultivating transgenic virus, are that the encoding gene of above-mentioned albumen is transferred to purpose In virus, transgenic virus is obtained, the insect resistace of the transgenic virus is higher than purpose virus, and the purpose virus is preferably For one kind in baculoviral, nucleopolyhedrosis element and cytoplasmic polyhedrosis virus.

Technical solution provided by the invention has the following advantages：First, the expression secretion according to the technical program The biologically active recombination ribosomal toxin HtA that expression and purifying obtain can effectively prevent host strain to expression product Degradation, mitigate host cell metabolism load and expression product to the toxic effect of host；Second is that utilize yeast vector pPICZ α The gene secretion expression of secretion signal α-factor signal peptide guiding destination protein on A-HtA, destination protein can be largely secreted into In culture solution, and accurate space structure can be formed, so as to keep the natural activity of ribosomal toxin HtA；Third, pass through screening Stablized and can high-level secretory expression obtain the yeast transformant of ribosomal toxin HtA；Eucaryon host is used fourth, finding out Pichia pastoris expresses the method for insect toxins HtA and the rapidly and efficiently method of purifying ribosomal toxin HtA, can reduce cost And realize mass production；Fifth, the recombinant protein of purifying has significant insecticidal activity to insect pest.

The additional aspect and advantage of the present invention will be set forth in part in the description, and will partly become from the following description It obtains significantly or is recognized by the practice of the present invention.

Description of the drawings

Fig. 1 is that the carrier for expression of eukaryon pPICZ α A-HtA in the embodiment of the present invention build schematic diagram；

Fig. 2 is the SDS-PAGE testing result figures of the yeast transformant that high-level secretory expression recombinates HtA in the embodiment of the present invention；

Fig. 3 is the SDS-PAGE detection knots of the different time points target protein expression under methanol induction in the embodiment of the present invention Fruit is schemed；

Fig. 4 be the embodiment of the present invention in elution gained purify HtA albumen SDS-PAGE testing result figures；

Fig. 5 is the SDS-PAGE testing result figures after purifying HtA protein concentrations in the embodiment of the present invention；

Fig. 6 is the SDS-PAGE of the yeast transformant induced product detection knots that natural HtA genescreens arrive in comparative example of the present invention Fruit；

Fig. 7 is the cellular level biological activity result schematic diagram of the recombination HtA albumen after optimizing in the embodiment of the present invention；

Fig. 8 is the flat biology Activity Results schematic diagram of insect injection water that HtA albumen is recombinated after optimizing in the embodiment of the present invention.

Specific embodiment

Below in conjunction with the attached drawing in the embodiment of the present invention, the technical solution in the embodiment of the present invention is carried out clear, complete Site preparation describes, it is clear that described embodiment is only part of the embodiment of the present invention, instead of all the embodiments.It is based on Embodiment in the present invention, those of ordinary skill in the art are obtained every other without making creative work Embodiment shall fall within the protection scope of the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

% in following embodiments is mass percentage unless otherwise specified.Quantitative test in following embodiment, Three repeated experiments are respectively provided with, data are the average value or mean+SD of three repeated experiments.

The present invention selects Pichi strain and conformability expression plasmid pPICZ α A to be purchased from U.S. Invritrogen public affairs Department.

Used medium formula is as follows：

1）Yeast growth medium（BMGY）：

It is completely dissolved 10 g yeast extracts, 20 g peptones, constant volume to 800 mL.121 DEG C of steam high pressure sterilization 15-20 Min is cooled to room temperature, adds in 100 mL, 1 M potassium phosphate solutions, 100 mI YNB, 2 500 × biotins of mL, 20 mL 50% Sterile glycerol；

2）Yeast inducing culture（BMMY）

It is completely dissolved 10 g yeast extracts, 20 g peptones, constant volume to 800 mL.121 DEG C of steam high pressure sterilization 15-20 Min is cooled to room temperature, adds in 100 mL, 1 M potassium phosphate solutions, 100 mL YNB, 2 mL500 × biotin, 10 mL methanol；

3）YPD culture mediums

It is completely dissolved 10 g yeast extracts, 20 g peptones, constant volume to 900 mL, 121 DEG C of steam high pressure sterilization 15-20 Min is cooled to 70 DEG C or so and adds 100 mL20% sterile dextrose solution.Adding in the agar of 1.5-1.8% wherein can make Obtain YPD solid mediums；

4）YPG culture mediums

It is completely dissolved 10 g yeast extracts, 20 g peptones, 20 g glycerine, constant volume to 1000 mL, 121 DEG C of steam high pressures Sterilize 15-20 min.

Embodiment one

Present embodiments provide a kind of artificial synthesized HtA genes of optimization, particular sequence as shown in the sequence 1 in sequence table, Protein sequence corresponding to the gene is as shown in the sequence 2 in sequence table.Sequence before the optimization of the present embodiment is basis The DNA sequence dna that ncbi database provides is the n DNA for synthesizing HtA, then according to toxin gene and Pichia pastoris password sublist Up to feature, optimize the DNA after simultaneously synthesis optimizing.Natural DNA sequence (the GenBank accession number of DNA sequence dna and HtA after optimization M27706 it) is compared through NCBI, without apparent similitude.

DNA after the n DNA of HtA before optimization and optimization is respectively connected to Pichia pastoris secreted expression carrier On pPICZ α A, recombinant vector is obtained, then respectively will using the lithium chloride conversion method that Invitrogen companies operation manual provides Recombinant vector is transformed into Pichia pastoris host strain X-33, respectively with containing 100 μ g/mL Zeocin antibiotic after conversion YPD tablets are screened, and transformant is verified using PCR, and the Pichia pastoris transformant scribing line after PCR is verified connects respectively The YPD tablets of the kind extremely Zeocin antibiotic containing various concentration, the height of the HtA n DNAs before screening is obtained containing optimization respectively The Pichia pastoris transformant of resistance and the resistance Pichia pastoris transformant containing the artificial synthesized DNA after optimization, Ran Houfen Not with a small amount of induced expressions of BMMY, then carry out SDS-PAGE analyses.HtA n DNAs structure before analysis result display optimization Obtained Pichia pastoris transformant will not express target protein, and be built with the artificial synthesized DNA after optimization complete red Yeast transformant can express and secrete target protein.

Embodiment two

The present embodiment provides a kind of methods for preparing albumen, specifically comprise the following steps：

S1：Construction of expression vector and conversion：Artificial synthesized DNA after the optimization of embodiment one is connected to Pichia pastoris secretion Type expression vector pPICZ α A, obtain recombinant vector pPICZ α A-HtA, as shown in FIG. 1, FIG. 1 is the embodiment of the present invention for vector construction In carrier for expression of eukaryon pPICZ α A- HtA structure schematic diagram.Main vector construction step is preferably as follows：

（1）With Xho I and I double digestions of Xba containing the plasmid of HtA genes synthesized after optimization, purpose segment is obtained, reaction system is such as Under（Restriction endonuclease and buffer solution used are purchased from Dalian TAKARA companies）：

The 15 μ L of plasmid of HtA genes containing synthesis

10 × M buffer solutions, 5 μ L

XhoⅠ 5 U

XbaⅠ 5 U

Sterile water is to 50 μ L

（2）With I double digestion pPICZ α A of Xho I and Xba, carrier segment is obtained, reaction system is as follows（Restriction endonuclease and buffer solution used It is purchased from Dalian TAKARA companies）：

15 μ L of plasmid pPICZ α A

10 × M buffer solutions, 5 μ L

XhoⅠ 5U

XbaⅠ 5U

Sterile water is to 50 μ L

（3）By step（1）With（2）Obtained purpose segment and carrier segment DNA gel withdraws kit recycling, the kit Purchased from Dalian TAKARA companies, concrete operations are carried out by kit specification.

（4）By step（3）Recycle obtained purpose segment and carrier T4DNA ligases（Purchased from Dalian TAKARA companies） Reaction is attached, target gene is properly inserted in the excretion vector reading frame containing secretion signal α-factor, reactant System is as follows：

1 μ L of carrier pPICZ α A segments

Target fragment falls 3 μ L

10 × buffer solution, 1 μ L

0.5 μ L of T4 ligases

Sterile water is to 10 μ L

S2：The conversion of recombinant plasmid：The recombinant vector pPICZ α A-HtA I single endonuclease digestions of Sac are linearized, according to The lithium chloride conversion method that Invitrogen companies operation manual provides, recombinant vector is transformed into Pichia pastoris host strain, this That embodiment is selected is X-33.It is screened, is utilized with the YPD tablets containing 100 μ g/mL Zeocin antibiotic after conversion PCR verifies transformant.

S3：The screening of high-level secretory expression yeast transformant and the expression of albumen：Pichia pastoris after PCR is verified turns Beggar's streak inoculation screens to the YPD tablets of the Zeocin antibiotic containing various concentration and obtains 1000-1500 μ g/mL high The transformant of Zeocin resistances, by the resistance transformant a small amount of induced expressions of BMMY, screening obtains high-level secretory expression The transformant of target protein chooses the yeast transformant single bacterium colony of high-level secretory expression, is seeded to equipped with 50 mL YPG liquid In 250 mL triangular flasks of culture medium, cultivated under 28 DEG C, 300 rpm condition of culture to thalline OD₆₀₀=6.0, take above-mentioned bacterium Liquid is seeded to , Mei in 2 L shaking flasks of the culture mediums of BMGY containing 500mL and is inoculated with 5 mL, and in 28 DEG C, 250 rpm are cultivated to OD₆₀₀= 12；2500 g centrifuge 5 min at room temperature, collect thalline, and thalline is resuspended with the BMMY of 1/7 former volume of culture, adds into culture medium Add 100% methanol to final concentration（Mass fraction）It is 1.0% ~ 1.5%, induces 24 hours, continues Fiber differentiation 72 hours at 28 °C, Adding methanol within every 24 hours makes its mass fraction be maintained at 1.0% ~ 1.5%, collects the supernatant of fermentation.

It should be noted that by the transformant of the high Zeocin resistances a small amount of induced expressions of BMMY, analyzed through SDS-PAGE, Screening obtains one plant of stabilization and height from ten plant height resistances (resistance level is greater than or equal to 1000 μ g/mL Zeocin) transformant The yeast transformant of level secretion expression ribosomal toxin HtA, and it is less than 500 μ g/mL Zeocin from 50 plants of resistance levels The transformant of YPD tablets does not screen the transformant of high-level secretory expression target protein, the high-level transformant screening in part SDS-PAGE the results are shown in Figure 2, wherein 1-8 transformants for high Zeocin resistances ribosomal toxin HtA yeast conversion Son, yeast transformant of 9 and 10 transformants for the ribosomal toxin HtA of low Zeocin resistances, it is evident that 1-8 transformants are in target There is apparent protein expression in position.

Different time target protein expression under methanol induction is detected with SDS-PAGE to analyze, electrophoresis result such as Fig. 3 institutes Show, Fig. 3 is the SDS-PAGE detection knots of the different time points target protein expression under methanol induction in the embodiment of the present invention Fruit is schemed.Through induction 1 ~ 4 day, there is apparent destination protein to express, and by continuing to add methanol, induction obtains in 15 kDa or so Destination protein content higher, the total amount result that specific purposes albumen accounts for whole bacterial protein is as shown in table 1 below.

The destination protein that the different induction times of table 1 obtain accounts for the degree result of Supernatant protein

Induction time	1 day	2 days	3 days	4 days
					Degree（%）	8	17	22	20

Embodiment three

The present embodiment provides a kind of methods for purifying HtA albumen, after step s 3, specifically further include following steps：

S4：By S3 cultivate 3 days obtained supernatants in 4 DEG C centrifugation, by gained supernatant using NaOH adjust pH 8.0 ~ 8.5 with Rotating speed more than or equal to 15000 g centrifuges 10 ~ 20 minutes, and the supernatant of acquisition is loaded to 20 mM through pH 8.0 ~ 8.5 CM cation-exchange chromatography posts after Tris-HCl buffer solutions balance, the pH's 8.0 ~ 8.5 for chromatographing column volume with 5 ~ 10 times contains The buffer solution of 20 mM Tris-HCl and 50 ~ 100 mM NaCl rinse the CM cation-exchange chromatography posts；

S5：The CM sun is eluted with buffer solutions of the pH 8.0 ~ 8.5 containing 20 mM Tris-HCl and 400 ~ 800 mM NaCl Ion exchange column, by obtained eluent using the bag filter that molecular weight is 10 kDa in 10 mM Tris-Cl buffer solutions Middle dialysis, is then concentrated by ultrafiltration.

S6：The recombination HtA protein freeze-dried powders to get high-purity are freeze-dried after concentrated product is quick-frozen in -80 °C of refrigerators.

It should be noted that the purifying protein that step S5 is eluted carries out SDS-PAGE analyses, as a result such as Fig. 4 institutes Show, Fig. 4 is in the embodiment of the present invention it can be seen that having afforded the target protein of purifying.Eluent is utilized into molecular weight 10 The bag filter of kDa utilizes the 15 mL super filter tubes of 10 kDa of molecular cut off will after dialysing in 10 mM Tris-HCl buffer solutions 20 times of sample concentration.The purifying protein of 20 micrograms is taken, SDS-PAGE testing results in target position as shown in figure 5, only have albumen one Band illustrates to have obtained the HtA recombinant proteins of high-purity.

Comparative example

Artificial synthesized ribosomal toxin HtA n DNAs are connected to pPICZ α A carriers in the same way, with identical enzyme into Row linearizes and converts Pichia pastoris X33 bacterial strains in the same way, is used after conversion and contains 100 μ g/mL Zeocin antibiotic YPD tablets screened, transformant is verified using PCR.The transformant of gained is applied to 1000 ~ 1500 μ again The YPD tablets of g/mL Zeocin screen 50 transformants, are seeded to 250 mL equipped with 50 mL YPG fluid nutrient mediums respectively In triangular flask, cultivated under 28 DEG C, 300 rpm condition of culture to thalline OD₆₀₀=6.0, above-mentioned bacterium solution is taken, is seeded to containing 500mL , Mei are inoculated with 5 mL in 2 mL shaking flasks of BMGY culture mediums, and in 28 DEG C, 250 rpm are cultivated to OD₆₀₀=12；2500 g at room temperature 5 min are centrifuged, collect thalline, thalline is resuspended with the BMMY of 1/7 former volume of culture, it is dense to end that 100% methanol is added into culture medium Degree（Mass fraction）It is 1.0 ~ 1.5%, induces 24 hours, continues Fiber differentiation 72 hours at 28 °C, add methanol and make within every 24 hours Its mass fraction is maintained at 1.0 ~ 1.5%, collects the supernatant of fermentation.SDS-PAGE results, which are shown, to fail to see target protein Band of expression, SDS-PAGE testing results are as shown in Figure 6.

The expression quantity of technical solution destination protein HtA provided in this embodiment is greatly improved, and the supernatant of fermentation is collected from S3 Liquid, by step S4 and S5 purify, from the induction broth of 1 L can with nearly 30 mg of HtA albumen of the purity more than 95%, And in comparative example, without the expression of target protein.

The present embodiment is obtained into HtA albumen and carries out analysis of biological activity, specific analytical method and result are as follows.

Experimental method one：Insect cell SF9 is cultivated in insect cell medium to 90% life in 10cm culture dishes Long density, pancreatin digest and adjust cell concentration to 5 × 10⁵Cell/mL is added in into 96 porocyte culture plates per hole After overnight incubation, A, B or C group of different final concentrations are separately added into each culture hole for the insect SF9 cell suspensions of 100 μ l HtA albumen (0,1,2,4,16,32 ng/ml), each 3 parallel holes of concentration, 48 are cultivated by above-mentioned cell under standard conditions Hour, cell is measured under microscope grows into condition.

Experimental result one：Fig. 7 is that the cell-proliferation activity of the recombination HtA albumen after optimizing in the embodiment of the present invention measures knot Fruit schematic diagram.As shown in fig. 7, the recombination HtA albumen of 1 ng/ml be with regard to that can inhibit the growth of insect cell SF9, what mtt assay measured Light absorption value is decreased obviously, the cell mortality when concentration reaches 16 ng/ml, the light absorption value measured and 32 ng/ml mono- Sample is all close to 0.Cell-proliferation activity measurement result shows that HtA recombinant proteins prepared by the method applied in the present invention have Very high biological activity.

Experimental method two：HtA recombinant proteins after purification are diluted to 1 mg/ml with PBS, are injected by 5 μ g/g weight Large wax borer larva（0.3±0.05g）, every group 30, each 3 groups of injection；Simultaneously using inject the large wax borer larva of same volume PBS as The poisoning symptom and death rate of large wax borer larva are observed in control.

Experimental result two：Experimental result 2：As a result, it has been found that inject the large wax borer larva table of HtA recombinant proteins after purification Revealed the i.e. insect of poisoning symptom of ribosomal toxin polyphenol seizing and turning over enzyme system be destroyed from face cause polypide present brown Even black, the color for injecting elder brother's body of the insect of HtA and PBS are as shown in Figure 8.The insect for injecting HtA is dead in 24 hours Rate reaches 80%, and the death rate reaches 100% within 48 hours, and inject same volume PBS control group larva none be poisoned Symptom.Animal injection experimental result equally embodies the insecticidal activity that the recombinant protein has.After injecting HtA recombinant proteins, greatly Cured snout moth's larva larval mortality concrete outcome is as shown in table 2：

2 biology of table is lived

In the description of this specification, reference term " one embodiment ", " some embodiments ", " example ", " specific example ", Or the description of " some examples " etc. means to combine the embodiment or example particular features, structures, materials, or characteristics described packet Contained at least one embodiment of the present invention or example.In the present specification, schematic expression of the above terms are necessarily It is directed to identical embodiment or example.Moreover, particular features, structures, materials, or characteristics described can be at any one Or it is combined in an appropriate manner in multiple embodiments or example.In addition, without conflicting with each other, those skilled in the art Member the different embodiments or examples described in this specification and the feature of different embodiments or examples can be combined and Combination.

Although the embodiments of the present invention has been shown and described above, it is to be understood that above-described embodiment is example Property, it is impossible to limitation of the present invention is interpreted as, those of ordinary skill in the art within the scope of the invention can be to above-mentioned Embodiment is changed, changes, replacing and modification, and it does not separate the essence of the corresponding technical solution various embodiments of the present invention skill The range of art scheme should all cover in the claim of the present invention and the range of specification.

SEQUENCE LISTING

<110>Huaihua College

<120>A kind of ribosomal toxin and its encoding gene and application

<130> 1

<160> 2

<170> PatentIn version 3.3

<210> 1

<211> 411

<212> DNA

<213> Hirsutella thompsonii

<400> 1

ctcgagaaaa gagccccaat cgtcacctgc agaccaaagt tggacggtag agagaagcca 60

ttcaaggtcg acgtcgccac tgcccaggct caagccagaa aggccggttt gaccaccggt 120

aagtccggtg acccacacag atacttcgcc ggagaccaca tcagatgggg tgtcaacaac 180

tgcgacaagg ccgacgccat cttgtgggag tacccaatct actgggtcgg taagaacgcc 240

gagtgggcca aggacgtcaa gacctcccag cagaagggtg gaccaacccc aatcagagtt 300

gtctacgcca actccagagg tgccgtccaa tactgcggag tcatgaccca ctccaaggtc 360

gacaagaaca accagggaaa ggagttcttc gagaagtgcg actaatctag a 411

<210> 2

<211> 130

<212> PRT

<213> Hirsutella thompsonii

<400> 2

Ala Pro Ile Val Thr Cys Lys Pro Lys Leu Asp Gly Lys Glu Lys Pro

1 5 10 15

Phe Lys Val Asp Val Ala Thr Ala Gln Ala Gln Ala Lys Lys Ala Gly

20 25 30

Leu Thr Thr Gly Lys Ser Gly Asp Pro His Lys Tyr Phe Ala Gly Asp

35 40 45

His Ile Lys Trp Gly Val Asn Asn Cys Asp Lys Ala Asp Ala Ile Leu

50 55 60

Trp Glu Tyr Pro Ile Tyr Trp Val Gly Lys Asn Ala Glu Trp Ala Lys

65 70 75 80

Asp Val Lys Thr Ser Gln Gln Lys Gly Gly Pro Thr Pro Ile Lys Val

85 90 95

Val Tyr Ala Asn Ser Arg Gly Ala Val Gln Tyr Cys Gly Val Met Thr

100 105 110

His Ser Lys Val Asp Lys Asn Asn Gln Gly Lys Glu Phe Phe Glu Lys

115 120 125

Cys Asp

130

Claims

1. a kind of gene is as follows（1）-（3）Any one of DNA molecular：

（1）As the DNA molecular shown in sequence in sequence table 1；

2. the albumen that DNA molecular according to claim 1 encodes.

3. albumen according to claim 2, it is characterised in that：It is as follows（1）Or（2）：

4. recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing gene described in claim 1；

The recombinant vector be specially will gene described in claim 1 be inserted into expression vector in, obtain expression claim 2 or The recombinant vector of 3 albumen, the recombinant vector are particularly preferred as said gene being inserted into the Xho of expression vector pPICZ α A Between I and Xba, I restriction enzyme sites, obtain expressing the recombinant vector of above-mentioned albumen.

5. a kind of method that albumen is prepared using gene described in claim 1, is included the following steps：

S1：Gene described in claim 1 and expression vector pPICZ α with Xho I and I double digestions of Xba and are purified into recycling respectively, Then it is connected using ligase at 16 °C, obtains recombinant vector pPICZ α A-HtA；

S3：The positive colony is forwarded to the YPD tablets containing 1000 ~ 1500 μ g/mL Zeocin, screening obtains height The transformant of Zeocin resistances, by the resistance transformant BMGY medium cultures to OD₆₀₀It is 10 ~ 15, is collected by centrifugation thin Born of the same parents are precipitated, and cell is resuspended with BMMY culture mediums, add in methanol and its mass fraction is made to be 1.0% ~ 1.5%, induce collect within 72 hours The supernatant of fermentation.

6. the method according to claim 5 for preparing albumen, it is characterised in that：After the induction 24 hours of step S3 and Before the supernatant for collecting fermentation, following steps are further included：

7. the method according to claim 5 or 6 for preparing albumen, it is characterised in that：After step s 3, it further includes as follows Step：

S5：The supernatant is adjusted into pH to 8.0 ~ 8.5 using NaOH, to be greater than or equal to the rotating speed of 15000 g centrifugation 10 ~ 20 Minute, the supernatant of acquisition is loaded to the CM cation exchanges after the 20 mM Tris-HCl buffer solutions balance of pH 8 ~ 8.5 Chromatographic column, with the delaying containing 20 mM Tris-HCl and 50 ~ 100 mM NaCl of the pH 8.0 ~ 8.5 of 5 ~ 10 times of chromatography column volumes Fliud flushing rinses the CM cation-exchange chromatography posts；

S6：The CM cation exchange layers are eluted with containing the buffer solution of 20 mM Tris-HCl and 400 ~ 800 mM NaCl Column is analysed, obtained eluent is dialysed using the bag filter that molecular weight is 10 kDa in 10 mM Tris-Cl buffer solutions, then It is concentrated by ultrafiltration.

8. the method according to claim 7 for preparing albumen, it is characterised in that：After step S6, following step is further included Suddenly：

9. the albumen being prepared according to the method that claim 5-8 any one of them prepares albumen.

10. recombinant vector, expression described in gene described in the albumen of claim 2,3 or 9, claim 1 or claim 4 The application of box, transgenic cell line or recombinant bacterium in pest-resistant field.