CN104650242A - Fusion polypeptide as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a fusion polypeptide as well as a preparation method and an application thereof and belongs to the technical field of medicines. The fusion polypeptide is characterized in that the C end of a sequence of dieresis polypeptide HKI is connected with a protein transduction domain TAT polypeptide sequence; the fusion expression is carried out on HKI and the protein transduction domain to build TAT-HKI fusion peptide; HKI quickly passes through an inner membrane of an esophagus system and enters the sites in the body by means of the efficient transmembrane transport action of TAT, thereby ensuring that the dieresis polypeptide can stably exert the function in the transport process in the body, so that cotton bollworms excessively excrete and die. By virtue of the fusion polypeptide, the fusion polypeptide can enter the body of a cotton bollworm by virtue of the behavior of naturally taking food, can be efficiently and stably expressed in the body, shows highly strong poison activity, can overcome the defect of poor poison activity of HKI through the insect digestive tracts, and can be used for preparing insecticides for preventing and treating the cotton bollworms.

Description

A kind of fusion polypeptide and its preparation method and application

Technical field

The invention belongs to medical art, be specifically related to a kind of fusion polypeptide, also relate to the preparation method and application of this fusion polypeptide simultaneously.

Background technology

Bollworm, the one of Noctuidae Helicoverpa insect, be the major pest of cotton cotton buds and bolls phase, be extensively distributed in all over the world, Chinese cotton region and vegetables flake all have generation, main moth food flower bud, flower, bell and tender leaf.Bollworm natural enemy is a lot, has parasite-parasitic wasp, tachinid etc., and predator crow finch and some bacteriums, fungi, virus etc. can play restraining effect to the ovum of bollworm and larva.

The water metabolism activity of diuresis kassinin kinin helicokinin I (HKI) to bollworm stimulates the strongest (Seinsche et al., 2000), after the HKI injection of denier enters the bollworm young, it can be made excessively to drain, and then cause death, what HKI carried out applying as insect killing substance has a high potential.Because it derives from organism, to higher organism and environmental safety, therefore carry out application as insect killing substance and there is inborn environmental advantage.How HKI is used as a kind of insecticide active substance, the trophic behaviour of insect whether can be utilized to be taken in body and play a role; How to make activated short peptide molecules penetrate digestive tube and enter site in body, ensure small peptide stablizing in transport process, this is problem demanding prompt solution simultaneously.

Summary of the invention

In order to overcome the defect of prior art, an object of the present invention is to provide a kind of fusion polypeptide bollworm to strong cytotoxicity.

Two of object of the present invention is the preparation method providing a kind of fusion polypeptide.

Meanwhile, the present invention is also the application providing a kind of fusion polypeptide.

In order to overcome the defect of prior art, the technical solution used in the present invention is as follows:

A kind of fusion polypeptide, its aminoacid sequence is as shown in SEQ ID NO:1.Be specially: methionine(Met)-tyrosine-glycine-arginine-lysine-Lys-Arg-Arg-Gln-Arg-Arg-arginine-glycine-Gly-Gly-Gly-Gly-Tyr-phenylalanine-Ser-Pro-tryptophane-glycine.

The N of described fusion polypeptide holds acetylize.

The preparation method of above-mentioned fusion polypeptide, comprises following operation steps:

1) with 2-(1H-benzo trisazo-L-1-yl)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester is condensing agent, with polystyrene-divinylbenzene resin for initial resin, adopt FMOC solid-phase synthesis to be coupled one by one each protected amino acid, synthesis obtains the peptide chain resin of full guard;

2) the fusion polypeptide N end prepared carries out acetylize.

3) cut with the peptide chain resin of cutting reagent to the surveyor's chain full guard after acetylize process, peptide chain got off from cracking resin and takes out side chain protected group, obtaining polypeptide crude product;

4) by step 3) the polypeptide crude product prepared is through ice ether sedimentation, and centrifugal collecting precipitation, separation and purification, namely lyophilize obtains described fusion polypeptide.

Step 2) described acetylation reagent is the mixed solution of diacetyl oxide and N-Methylimidazole.

Step 3) in cutting reagent be hydrogen fluoride, p-cresol, to the mixture of thin base phenol according to the proportions of volume ratio 90:5:5.

Step 3) in the consumption of cutting reagent be 10 ~ 15mL/g.

The application of above-mentioned fusion polypeptide in preparation control bollworm medicine.

TAT (transactivator of transcription, TAT) effectively transcribes for virus and copies necessary peptide sequence.TAT can be inner by biomacromolecule non-specific ground transducer cell covalently bound with it, and this process had not both relied on acceptor and translocator, also has nothing to do with temperature and energy, and almost do not have toxicity to host cell.This have the specific polypeptide sequence carrying foreign protein permeates cell membranes ability and be called protein transduction domain (Protein transduction Domains, PTDs), also cell-penetrating peptide (cell penetratingpeptides, CPP) is claimed.

Diuresis polypeptide Helicokinin is the small molecule insect neuropeptide belonging to insect kassinin kinin family, and whole family member has similar one of carbon tip pentapeptide sequence Phe-Tyr-Pro-Trp-Gly-NH ₂(FYPWGa), this core 5 peptide is required for its stimulated muscle activity and diuresis activity, research shows that micro-insect kassinin kinin and analogue thereof can cause larger impact (Smagghe et al.Antifeedant activity and high mortality in the peaaphid Acyrthosiphon pisum (Hemiptera:Aphidae) induced by biostable insect kininanalogs to insect physiological metabolism,, 2010; Nachman et al.Nachman et al.Biostable and PEG polymer-conjugatedinsect pyrokinin analogs demonstrate antifeedant activity and induce high mortality in the peaaphid Acyrthosiphon pisum (Hemiptera:Aphidae, 2012)

Fusion polypeptide of the present invention, protein transduction domain TAT peptide sequence is connected at the C end of the sequence of diuresis polypeptide HKI, HKI is carried out amalgamation and expression with protein transduction domain, build TAT-HKI fusogenic peptide, by the efficient transmembrane transport effect of TAT, make HKI enter site in body through esophagus system inner membrance rapidly, guarantee stable in diuresis peptide sequence transport process in vivo playing a role, fusion polypeptide is entered after in Helicoverpa armigera, bollworm is excessively drained, causes death.The behavior that fusion polypeptide of the present invention takes food naturally by bollworm enters in Helicoverpa armigera, and the expression of efficient stable in vivo, shows very strong poisoning vigor, overcome HKI through insect digestive tract virulence poor defect.

The preparation method of fusion polypeptide of the present invention adopts FMOC legal system for fusion polypeptide, and amino acid grafting success ratio is high, easy and simple to handle, is easy to realize, and is suitable for industrial application.

Fusion polypeptide of the present invention high expression in Helicoverpa armigera, shows the characteristic of cytotoxicity, makes it can be used for preparing the medicine of control bollworm.

Embodiment

Below in conjunction with specific embodiment, the present invention is described in further detail, but does not form any limitation of the invention.

Embodiment 1

The present embodiment fusion polypeptide, its aminoacid sequence, as shown in SEQ ID NO:1, is specially methionine(Met)-tyrosine-glycine-arginine-lysine-Lys-Arg-Arg-Arg-Gly-Gly-Gly-Gly-Gly-tyrosine-phenylalanine-Ser-Pro-tryptophane-glycine.

The present embodiment fusion polypeptide N holds acetylize.

The preparation method of the present embodiment fusion polypeptide, concrete operation step is:

1) with 2-(1H-benzo trisazo-L-1-yl)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester is condensing agent, with polystyrene-divinylbenzene resin for initial resin, adopt FMOC solid-phase synthesis to be coupled one by one each protected amino acid, synthesis obtains the peptide chain resin of full guard;

2) with the mixed solution of diacetyl oxide and N-Methylimidazole to step 1) the peptide chain resin of full guard prepared carries out acetylize process;

3) cut with the peptide chain resin of 13 ㎎/L cutting reagents to the surveyor's chain full guard after acetylize process, peptide chain got off from cracking resin and takes out side chain protected group, obtaining polypeptide crude product; Described cutting reagent be hydrogen fluoride, p-cresol, to the mixture of thin base phenol according to the proportions of volume ratio 90:5:5;

4) by step 3) the polypeptide crude product prepared is through ice ether sedimentation, and centrifugal collecting precipitation, separation and purification, the mixed solution adding diacetyl oxide and N-Methylimidazole carries out acetylize process, and namely lyophilize obtains described fusion polypeptide.

Performance Detection: the toxicity test of fusion polypeptide prepared by embodiment

Measuring method, concrete operation step is:

1) first prepare fresh bollworm artificial diet: agar 8g is boiled thawing, temperature adds aseptic wheat bran 70g successively, dry yeast 10g, Semen Maydis oil 3ml, sorbyl alcohol 1g, xitix 15g after 90 DEG C, and solvent is water, and limit edged stirs;

2) test is divided into 3 groups: fusion polypeptide TAT-HKI test group: fusion polypeptide TAT-HKI embodiment prepared is dissolved in sterilized water, when artificial diet heating temperatures to 45 DEG C, add TAT-HKI fusion polypeptide to stir, as fusion polypeptide TAT-HKI test group; Diuresis kassinin kinin HKI test group: be dissolved in by HKI in sterilized water, when artificial diet heating temperatures to 45 DEG C, adds HKI polypeptide and stirs, as HKI test group; Blank group: do not add any additive in artificial diet;

3) by step 2) 3 groups of feeds preparing are cut into 1cm respectively ³square, puts into 2.5 × 10cm flat based tubes (tampon sealing), and accesses normal bollworm and just hatch larva at the beginning of 2 ages, and every treatment group connects worm 200, and often pipe 1, is placed in incubator; The rearing conditions of examination worm after process: temperature 27 ± 1 DEG C, relative humidity 65 ~ 70%, light application ratio (L:D) 16:8h, changed a feed every 4 days, observed the existing state of bollworm every 4 days, and statistics mortality ratio, judges the virulence of fusion polypeptide; Wherein statistic data adopts dsp software to carry out variance analysis.

Bollworm existing state judging criterion: touch polypide with little writing brush, coordinated movement can not think dead.

Detected result is as shown in table 1 below:

The virulence determination of table 1 not homopolypeptide compares

Above-mentioned detection comparative result shows, only take food HKI very low to cotton bollworm larvae kill capacity, not remarkable with blank group difference, and TAT-HKI fusion polypeptide, by the efficient transmembrane transport effect of TAT, TAT-HKI creates very strong poisoning ability to taking food larva, cotton bollworm larvae growth is obviously suppressed, body weight increases less than normal than other groups, and along with taking food the prolongation of time, dead individuality increases gradually, mortality ratio finally reaches 73.6 ± 5.7%, and the test group corrected mortality taking food HKI is only 6.9% (see table 1), take food the test group larval mortality of TAT-HKI fusogenic peptide with difference between other groups extremely significantly (p ﹤ 0.01).This detected result shows that the fusion polypeptide that the present invention builds enters in Helicoverpa armigera by naturally taking food, and high efficiency stable expression in vivo, show very strong poisoning vigor, can be used for cotton bollworm control, for the preparation of the medicine of control bollworm.

Claims (4)

1. a fusion polypeptide, is characterized in that, its aminoacid sequence is as shown in SEQ ID NO:1.

2. fusion polypeptide as claimed in claim 1, is characterized in that, the N of described fusion polypeptide holds acetylize.

3. a preparation method for fusion polypeptide as claimed in claim 1, is characterized in that, comprises following operation steps:

1) with 2-(1H-benzo trisazo-L-1-yl)-1,1,3,3-tetramethyl-urea Tetrafluoroboric acid ester is condensing agent, with polystyrene-divinylbenzene resin for initial resin, adopt FMOC solid-phase synthesis to be coupled one by one each protected amino acid, synthesis obtains the peptide chain resin of full guard;

2) by step 1) the fusion polypeptide N for preparing end carries out acetylize;

3) cut with the peptide chain resin of cutting reagent to the surveyor's chain full guard after acetylize process, peptide chain got off from cracking resin and takes out side chain protected group, obtaining polypeptide crude product;

4) by step 3) the polypeptide crude product prepared is through ice ether sedimentation, and centrifugal collecting precipitation, separation and purification, namely lyophilize obtains described fusion polypeptide.

4. the application of fusion polypeptide as claimed in claim 1 in preparation control bollworm medicine.