CN108264563A - Covalent trimeric polypeptides of glucan-modified Hrps and preparation method thereof - Google Patents

Abstract

The invention discloses a kind of covalent trimeric polypeptides of glucan-modified Hrps and preparation method thereof, it forms covalent trimeric polypeptide by three kinds of Hrps polypeptides and glucan by covalent modification, and general structure is：(3Hrps)_n‑(C₆H₁₀O₅)_n；Wherein, three kinds of Hrps polypeptides are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:Arbitrary three kinds in amino acid sequence shown in 4.By glucan-modified several Hrps polypeptides in combination segments, in a manner of covalently compound, glucan and several Hrps functional polypeptides combination segment are prepared into fully and partially incomplete covalent trimeric polypeptide, i.e., (3Hrps)_n‑(C₆H₁₀O₅)_nCovalent trimeric polypeptide, the emulsion stability, homogeneity and structural stability higher, timeliness for making the Hrps glucan new products of acquisition are longer, and function is more, and application range is wider.

Description

Covalent trimeric polypeptides of glucan-modified Hrps and preparation method thereof

Technical field

The invention belongs to biotechnologies, relate generally to the skill of glucan-modified several Hrps functional polypeptides combination segments Art method and by the technical method, it is raw by different degrees of covalent compound of glucan and several Hrps functional polypeptides segments Into fully and partially not exclusively covalent trimeric polypeptide and covalent trimerization peptide prod in multi-field application.

Background technology

The zoic cell surface of institute is all coated with, and there is also various in the cell by many different types of sugar chains The glycosylation of type.Oligonucleotide chain is covalently attached in a variety of forms with the amino acid in protein and peptide chain, forms the sugar of glycoprotein Peptide connection chain.Glycoprotein is the base substance for participating in many bioprocess, these processes include cell growth, cell and cell Adhesion, immune response, fertilization, the degradation of sludged blood, virus multiplication, parasitic infection and inflammatory reaction etc..With protein group The continuous development of technology, the research of glycosylated protein group increasingly will widely be paid attention to.

Plant is in long-term evolution, in existence various with around and growing environment condition (including biology and abiotic) In interaction, a kind of common adaptability is formd, here it is the common defence system and growing system formed in evolution, This is a kind of defense mechanism and growth mechanism that plant shares, and is realized by a plurality of signal path and metabolic pathway.

Research shows that stream signal substance of the Hrp protides as plant, with the receptor being widely present in plant cell wall Hrp binding proteins (Harpin-binding proteins) combine and a plurality of signal path in activated plant, these signals lead to The activation on road will realize the cytophylaxis intrinsic to plant and grow the startup and regulation and control of a plurality of metabolic pathway and mechanism, this is just It is Hrp protides as plant growth regulator and the mechanism of action of biological pesticide.At present, the Hrp albumen of referred to as super quick albumen Class product has been widely studied, development and application, is had broad prospects.

With the development of technique for gene engineering, the protides system such as more and more polypeptides, enzyme, signaling molecule, cell factor Product are studied exploitation, these albumen based articles have many advantages, such as that specificity is strong, timeliness is high, are widely used, safety is good, but simultaneously Several significant shortcomings are had also discovered, for example (1) is soluble and uniformity is poor；(2) antigenicity and immunogenicity；(3) Easily quickly removed from the circulatory system；(4) it is unstable, easily degraded by the enzyme of inside and outside；(5) it is the outer used time, easily outer The microorganism fast degradation of boundary's environment and light degradation.

For Hrps albuminoids, since floristics is various, between different plant species, between kind biological characteristics sex differernce compared with Greatly, internal and external environment is also different and the differences of Interaction among genes, separate sources and under the conditions of the Hrps albuminoids that obtain and its system The property of agent in use condition and effect there are apparent selective difference, the Hrps albumen of single kind in effect Significantly show deficiency；Hrps albuminoids as a kind of upstream functional signal protein applied to plant, it is soluble and Stability is poor, meanwhile, use when Gen Shi (foliage-spray and) under field conditions (factors), it is easy to fast by the microorganism of external environment Prompt drop solution and light degradation, so as to influence their timeliness and effect.

For these reasons, it needs to carry out structure of modification and chemical modification to Hrps albuminoids.

Structure of modification and chemical modification method refer to carry out local flow improvement and modification to protein on a molecular scale, i.e., in body The outer side-chain radical by protein passes through manual method and some chemical groups, the particularly molecular radical with biocompatibility It is covalently attached with macromolecular, forms fully and partially incomplete covalent complex, so as to which part changes the property of protein Matter and structure, and more superior performance when showing than being respectively individually present, the technology and methods are in medical and health and food The application of the protein product of product industry above causes more and more extensive attention.

It requires emphasis, it is noted that the Hrps albuminoid polypeptides of separate sources are combined, structure of modification and chemistry are repaiied Decorations form covalent complex, are not reported at present both at home and abroad.

There are many molecular radical and the macromolecular for being commonly used for protein modification, mainly have polyethylene glycols (including PEG and MPEG etc.), polysaccharide and homologous protein and artificial synthesized polypeptide, polyaminoacid etc..To the structure of modification of protein Have developed rapidly in recent years with chemical modification technology, for different enzymes, polypeptide or protein chemical modification form it is a variety of Technology and methods still, there is its scope of application and limitation, and there are no pervasive general technology and method is available so far And utilization, going deep into and developing with protein modification technical research, it will further contribute to improve to various different albumen The modified specificity of matter is good, modification rate is high, the production of easy to operate, product stability and both effectiveness good new technology and method It is raw.

Invention content

It is an object of the present invention to provide a kind of covalent trimeric polypeptides of glucan-modified Hrps and preparation method thereof, make Hrps polypeptides Emulsion stability, homogeneity and structural stability higher, timeliness are longer, and function is more, and application range is wider.

The technical scheme is that：The covalent trimeric polypeptides of glucan-modified Hrps, it is gathered by three kinds of Hrps polypeptides and Portugal Sugar forms covalent trimeric polypeptide by covalent modification, and general structure is：(3Hrps)_n-(C₆H₁₀O₅)_n；Wherein, three kinds of Hrps polypeptides For SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:Arbitrary three in amino acid sequence shown in 4 Kind.

Further, SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 quality proportioning is 3:2:3:2。

Further, the quality proportioning of three kinds of Hrps polypeptides and glucan is 10:1 to 1:10.

Further, the quality proportioning of three kinds of Hrps polypeptides and glucan is 1:2.

Further, glucan is avenabeta glucosan, molecular mass 5300-257200.

Further, glucan is avenabeta glucosan, molecular mass 32000-38000.

The preparation method of the covalent trimeric polypeptides of glucan-modified Hrps, preparation process are as follows：

(1) from SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:Four kinds of Hrps shown in 4 are more Three kinds of Hrps polypeptides are arbitrarily selected in peptide；

(2) quality of glucan, three kinds of Hrps polypeptides and glucan is added in into three kinds of Hrps polypeptides that step (1) selects It matches and is：10:1 to 1:10；Phosphate buffer is added in, it is 2-10 to make system pH；0-5 hours are stood, is 15-80 in temperature DEG C, mixing speed is reacts 3-25 hours under the conditions of 30-300r/min；

(3) after the completion of reacting, cryogenic vacuum is continuous drying, collects product up to covalent trimeric polypeptide, (3Hrps)_n- (C₆H₁₀O₅)_n。

Further, in step (2), a concentration of 10-200mmol/L of phosphate buffer, phosphate buffer addition is 10-100 times of three kinds of Hrps polypeptides and glucan gross mass.

Preferably, preparation method is as follows：

(1) from SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:Four kinds of Hrps shown in 4 are more Three kinds of Hrps polypeptides are arbitrarily selected in peptide；Wherein, SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4 The quality proportioning of four kinds of Hrps polypeptides is 3:2:3:2.

(2) glucan is added in into three kinds of Hrps polypeptides that step (1) selects, glucan is avenabeta glucosan, molecule Quality is 32000-38000；The quality proportioning of three kinds of Hrps polypeptides and glucan is：1:2；Add in the phosphorus of a concentration of 50mmol/L Acid buffer, phosphate buffer addition are 40 times of three kinds of Hrps polypeptides and glucan gross mass, and it is 7 to make system pH, quiet It puts 1 hour；It is reacted 12 hours under the conditions of temperature is 46 DEG C, mixing speed is 60r/min；

(3) after the completion of reacting, cryogenic vacuum is continuous drying, collects product up to covalent trimeric polypeptide, (3Hrps)_n- (C₆H₁₀O₅)_n。

Four Hrps functional polypeptide segments are respectively:

SEQ NO ID:Hrps polypeptide fragments shown in 1 are derived to be compiled by the HrpsNEccs genes that 356 amino acid form The highly conserved region segment of Harpin family proteins contained by code albumen, is made of 200 amino acid, positioned at the C- ends of albumen (amino acid 1 57-356).

SEQ NO ID:Hrps polypeptide fragments shown in 2 are derived to be compiled by the HrpsZpst genes that 370 amino acid form The highly conserved region segment of Harpin family proteins contained by code albumen, is made of 312 amino acid, positioned at the N- ends of albumen And C- ends (amino acid 1-270 and 329-370).

SEQ NO ID:Hrps polypeptide fragments shown in 3 are derived to be compiled by the HrpsNEcb genes that 370 amino acid form The highly conserved region segment of Harpin family proteins contained by code albumen, is made of 200 amino acid, positioned at the C- ends of albumen (amino acid 1 71-370).

SEQ NO ID:Hrps polypeptide fragments shown in 4 are derived to be compiled by the HrpsNEch genes that 339 amino acid form The highly conserved region segment of Harpin family proteins contained by code albumen, is made of 326 amino acid, positioned at the stage casing of albumen (amino acid 9-334).

These functional polypeptide segments are all the gene and its expression product achievement in research that patent inventor independently completes, by Patent inventor registers in GenBank, their number of registrations are respectively：AY999002.1, AY999004, DQ355519.1 and AY999001；According to the size of these functional polypeptide segments, in conservative region position and sequence difference and Hrp protein gene Carry out the feature of derived bacterium, selected these segments complementaryly, be configured with four combinations, each combination is different more containing 3 Peptide fragment, the four of the modified polypeptide fragment combinations that will be carried out are I respectively:No. 1, No. 2, No. 3, II:No. 1, No. 2, No. 4, III:No. 1, No. 3, No. 4 and IV:No. 2, No. 3, No. 4, their expression general formula is (3Hrps)_n, the expression formula of four fragment combinations Respectively (3Hrps)_n(I)、(3Hrps)_n(II)、(3Hrps)_n(III) and (3Hrps)_n(IV)。

Above 4 Hrps functional polypeptide segments are further described：

1st, the Harpin albumen of the source gene code in several Hrps functional polypeptides sources, their size and amino acid sequence Row composition all differs greatly.No. 1 HrpNEccs albumen is made of 356 amino acid；No. 2 HrpZpst albumen are by 370 amino acid Composition；No. 3 HrpNEcb albumen are made of 370 amino acid；And No. 4 HrpNEch albumen only have 339 amino acid.

2nd, the highly conserved region (DOMAIN) of Harpin albumen all family proteins containing Harpin of four gene codes. The highly conserved region of Harpin family proteins is the main functional areas of Harpin albuminoids, and remarkable biological effect is main It is determined by the sequence in the contained highly conserved region of Harpin family proteins and the polypeptide higher structure of these conservative regions It is fixed.

3, the amino acid sequence between four Harpin albumen and its contained highly conserved region of Harpin family proteins No matter formed from sequence size, position and amino acid, the difference between them is all very big.The Harpin family proteins of No. 1 albumen The highly conserved region of matter is made of 200 amino acid, positioned at the C- ends (amino acid 1 57-356) of albumen；The Harpin of No. 2 albumen The highly conserved region of family protein is made of 312 amino acid, positioned at the N- ends of albumen and C- ends (amino acid 1-270 and 329-370)；The highly conserved region of Harpin family proteins of No. 3 albumen is made of 200 amino acid, positioned at the C- of albumen It holds (amino acid 1 71-370)；The highly conserved region of Harpin family proteins of No. 4 albumen is made of 326 amino acid, is located at The stage casing (amino acid 9-334) of albumen.The polypeptide fragment amino acid sequence in this 4 highly conserved regions of Harpin family proteins And the polypeptide higher structure of conservative region determines the remarkable biological effect that they have.

Oligonucleotide chain is covalently attached in a variety of forms with the amino acid in protein and peptide chain, forms the glycopeptide chain of glycoprotein, Hrps functional protein polypeptide fragments involved by us, they are only one, two, three structure and the non-enzymatic egg without quaternary structure White matter, without cystine and cysteine, rich in the amino to form glycopeptide chain can be covalently attached with other 3 types with oligonucleotide chain Acid.There is serine 40 in HrpNEccs gene coded proteins, wherein in the highly conserved region segment of Harpin family proteins There is serine 19；There is threonine 13, wherein have threonine 10 in the highly conserved region segment of Harpin family proteins； There is lysine 18, wherein have lysine 16 in the highly conserved region segment of Harpin family proteins；There is proline 9, Wherein there is proline 6 in the highly conserved region segment of Harpin family proteins；There are glycine 75, wherein Harpin families There is glycine 26 in the segment of high conserved protein domain；There is arginine 5, wherein Harpin family proteins are highly conserved There is arginine 6 in region segments；There is aspartic acid 15, wherein have in the highly conserved region segment of Harpin family proteins Aspartic acid 13；There is 9, glutamic acid, wherein have 7, glutamic acid in the highly conserved region segment of Harpin family proteins. There is serine 38 in HrpZpst gene coded proteins, wherein have silk in the highly conserved region segment of Harpin family proteins Propylhomoserin 32；There is threonine 23, wherein have threonine 18 in the highly conserved region segment of Harpin family proteins；Depend on There is lysine 11 in the highly conserved region segment of propylhomoserin 11, wherein Harpin family proteins；There is proline 12, wherein There is proline 8 in the highly conserved region segment of Harpin family proteins；There are glycine 57, wherein Harpin family proteins There is glycine 46 in the highly conserved region segment of matter；There are arginine 4, the wherein highly conserved region of Harpin family proteins There is arginase 12 in segment；There is aspartic acid 27, wherein have asparagus fern in the highly conserved region segment of Harpin family proteins Propylhomoserin 25；There is 9, glutamic acid, wherein have 9, glutamic acid in the highly conserved region segment of Harpin family proteins. There is serine 34 in HrpNEcb gene coded proteins, wherein have silk in the highly conserved region segment of Harpin family proteins Propylhomoserin 16；There is threonine 13, wherein have threonine 9 in the highly conserved region segment of Harpin family proteins；Depend on There is lysine 15 in the highly conserved region segment of propylhomoserin 17, wherein Harpin family proteins；There is proline 8, wherein There is proline 7 in the highly conserved region segment of Harpin family proteins；There are glycine 87, wherein Harpin family proteins There is glycine 29 in the highly conserved region segment of matter；There are arginine 4, the wherein highly conserved region of Harpin family proteins There is arginine 3 in segment；There is aspartic acid 17, wherein have asparagus fern in the highly conserved region segment of Harpin family proteins Propylhomoserin 14；There is 8, glutamic acid, wherein have 6, glutamic acid in the highly conserved region segment of Harpin family proteins；And There is serine 40 in HrpNEch gene coded proteins, wherein have silk in the highly conserved region segment of Harpin family proteins Propylhomoserin 40；There is threonine 14, wherein have threonine 13 in the highly conserved region segment of Harpin family proteins；Depend on There is lysine 20 in the highly conserved region segment of propylhomoserin 21, wherein Harpin family proteins；There is proline 5, wherein There is proline 5 in the highly conserved region segment of Harpin family proteins；There are glycine 55, wherein Harpin family proteins There is glycine 55 in the highly conserved region segment of matter；There are arginine 3, the wherein highly conserved region of Harpin family proteins There is arginine 3 in segment；There is aspartic acid 22, wherein have asparagus fern in the highly conserved region segment of Harpin family proteins Propylhomoserin 22；There is 4, glutamic acid, wherein have 4, glutamic acid in the highly conserved region segment of Harpin family proteins.These knots Structure determines the mode that they can in a variety of forms be covalently attached with oligonucleotide chain, forms the glycopeptide connection chain of glycoprotein, realizes Avenabeta glucosan prepares covalent trimeric polypeptide through modifying several Hrps functional polypeptides segments.

Oligonucleotide chain is covalently attached in a variety of forms with the amino acid in protein and peptide chain, forms the glycopeptide connection of glycoprotein Chain, abbreviation glycopeptide chain.According to glycopeptide chain type, protein glycosylation can be divided into four classes, i.e., relied with serine, threonine, hydroxyl The hydroxyl of propylhomoserin and hydroxyproline be tie point, formation-O-glycosides bond type；With the amide groups of asparagine, N₂End amino acid Alpha-amido and lysine or arginic omega-amino be tie point, formation-N- glucosides bond types；With aspartic acid or paddy ammonia The free carboxy of acid is tie point, forms ester glucosides bond type and the cardohydrata-peptide linkage using cysteine as tie point.

β-(1 → 3,1 → 4) glucan in oat, abbreviation avenabeta glucosan is a kind of non-starch polysaccharide.It is by list Body β-D- glucopyranoses are got up a kind of high molecular polymer to be formed by β-(1 → 3) and β-(1 → 4) glucosides key connection. There is 1 β-(1 → 3) glucosides key connection between every 2-3 β-(1 → 4) glycosidic bond in more than 85% avenabeta glucosan molecule, 15% is made of long-chain β -1, (1 → 4) glycosidic bond interval β-(1 → 3) glycosidic bond, and length may have 4,5 or 8 Portugals Grape saccharide residue.With β-straight-chain of (1 → 3) key and branchiess polysaccharide body structure, wherein β-(1 → 4) and β-(1 → 3) sugar The ratio of glycosidic bond is about 2.4:1.In oat endosperm and gluten cell wall ingredient, beta glucan accounts for more than 85%.Oat β- Glucan is a kind of smaller short chain glucan of relative molecular mass, and the variation range of relative molecular mass is 5300- 257200。

The present invention has the following advantages that compared with prior art：

In the present invention, by glucan-modified several Hrps polypeptides in combination segments, in a manner of covalently compound, by glucan Fully and partially incomplete covalent trimeric polypeptide is prepared into several Hrps functional polypeptides combination segment, i.e., (3Hrps)_n- (C₆H₁₀O₅)_nCovalent trimeric polypeptide makes emulsion stability, homogeneity and the structural stability of Hrps- glucan new products of acquisition more Height, timeliness are longer, and function is more, and application range is wider.

Specific embodiment

Technology implementation process and condition include：

(1) quality (with mM measured) proportioning of the Hrps functional polypeptides 1-4 segments in combination：Typically with identical to not Conjunction is grouped with mM mass；(2) selection of glucan：Relative molecular mass ranging from 5300-257200；(3) polypeptide fragment four The quality proportioning of 3 Hrps functional polypeptides segments and glucan in a combination：10:1 to 1:Between 10；(4) reaction solution is dense Degree：3 Hrps polypeptide fragments and glucan mixing, 10-200mM phosphate buffers are diluted to 10 to 100 times of mixture quality； (5) pH and stewing process are reacted：10-200mM phosphate buffer pH 2-10, stewing process 0-5 hours；(6) reaction temperature and when Between：Reaction temperature is 15 DEG C -80 DEG C, and in reaction time 3-25 hour, during reaction, reaction mixture needs to stir, and mixing speed is 30-300 per minute turns；(7) optimum condition of the Hrps functional polypeptides combination covalent trimeric polypeptide of segment-glucan is prepared to react molten Liquid, which staticly settles the time and induces balsamine blade, occurs anaphylactoid time and the oat plumular axis speed of growth as final Screening is according to (biological effect)；(8) after the completion of reacting, cryogenic vacuum is continuous drying, and the product finally collected is：(3Hrps is more Peptide)_n-(C₆H₁₀O₅)_nCovalent trimeric polypeptide, simplifying general formula is：(3Hrps)_n-(C₆H₁₀O₅)_n；(9) analyte detection is generated：Using dodecane Base sodium sulfonate polyacrylamide gel electrophoresis (SDS-PAGE), the results showed that, (3Hrps)_n-(C₆H₁₀O₅)_nCovalent trimeric polypeptide shape Into macromolecular, there is the new bands of a spectrum of macromolecule, the electrophoretic band of 3 Hrps polypeptide fragments in separation gel and concentration glue interface As the progress for preparing reaction gradually weakens, it was demonstrated that covalent cross-linking occurs between Hrps polypeptides in combination segment and glucan, generates Covalent complex.Contrast test shows Hrps polypeptides in combination segments after glucan-modified, (3Hrps)_n-(C₆H₁₀O₅)_nAltogether The emulsion stability of valency trimeric polypeptide significantly improves；Significant change occurs for zeta current potentials, and isoelectric point declines more than 1 pH unit.

(3Hrps) of four combinations of Hrps Peptide of Protein_n-(C₆H₁₀O₅)_nThe preparation of covalent trimeric polypeptide, uses Same procedure carries out side by side simultaneously, the result agreed.

Embodiment 1

Hrps functional polypeptides combine the preparation of the covalent trimeric polypeptide of segment-glucan:

(1) quality (with mM measured) proportioning of the Hrps functional polypeptides 1-4 segments in combination：Typically with identical to not Conjunction is grouped with mM mass；

(2) selection of glucan：The usual range of avenabeta glucosan molecular mass is 5300-257200；

(3) the Hrps functional polypeptides segment in the combination of polypeptide fragment four and the quality proportioning of glucan, usually 10:1 To 1:Between 10；

(4) reaction density, Hrps polypeptides in combination segment and glucan mixing, 10-200mM phosphate buffers are diluted to mix 10 to 100 times of amount of substance；

(5) pH and stewing process time are reacted, usually 10-200mM phosphate buffers pH2-10 and 0-5 hours；

(6) reaction temperature and time：Reaction temperature is usually 15 DEG C -80 DEG C, and the reaction time is usually 3-25 hours, reaction When, reaction mixture needs to stir, and mixing speed per minute is usually that 30-300 turns；

(7) optimum condition for preparing the Hrps functional polypeptides combination covalent trimeric polypeptide of segment-glucan is stood with reaction solution Anaphylactoid time and the oat plumular axis speed of growth occur for sedimentation time and induction balsamine blade to finally screen foundation (biological effect)；

(8) after the completion of reacting, cryogenic vacuum is continuous drying, and the product finally collected is：(3Hrps)_n-(C₆H₁₀O₅)_nCovalently Trimeric polypeptide；

(9) product detection：Shown using sodium dodecyl sulfate-polyacrylamide gel electrophoresis method (SDS-PAGE), (3Hrps)_n-(C₆H₁₀O₅)_nCovalent trimeric polypeptide forms macromolecular, occurs macromolecule in separation gel and concentration glue interface and newly composes Band, the electrophoretic bands of 3 Hrps polypeptide fragments gradually weaken with the progress for preparing reaction, it was demonstrated that Hrps polypeptides in combination segment with Covalent cross-linking occurs between glucan, produces covalent complex.Contrast test shows Hrps polypeptides in combination segments through glucan After modification, (3Hrps)_n-(C₆H₁₀O₅)_nThe emulsion stability of covalent trimeric polypeptide significantly improves；Zeta current potential significant changes, wait electricity Point declines more than 1 pH unit.

(3Hrps) of four combinations of Hrps Peptide of Protein_n-(C₆H₁₀O₅)_nThe preparation of covalent trimeric polypeptide, uses Same method carries out side by side simultaneously, the result agreed.

Preparing Hrps polypeptides function combination segment-glucan, covalently polymeric optimum condition is (raw to finally screen foundation Object effect) be response when, through repeatedly being tested preparation condition and being screened, it is (1) to obtain preferred control condition Quality (with mM measured) proportioning of the Hrps functional polypeptide 1-4 segments in combination：Typically with identical to different mM quality groups Into combination, preferably 1,2,3, No. 4 segment is identical mM mass, more preferably No. 1 2mM, No. 2 3mM, No. 3 2mM and No. 4 3mM, most preferably No. 1 3mM, No. 2 2mM, No. 3 3mM and No. 4 2mM composition combinations；(2) selection of avenabeta glucosan：Molecular weight Usual range be 5300-257200, it is preferred molecular weight 60000-70000, more preferably 50000-60000, further excellent It is selected as 40000-50000, most preferably 32000-38000；(3) quality proportioning of Hrps polypeptides function fragment and glucan：It is logical It is often 10:1 to 1:10, preferably 8:1, more preferably 1:8, further preferably 4:1, it is further preferably 1:4, most preferably 1:2； (4) reaction density：Hrps polypeptides in combination segment and glucan mixing, usual 50mM phosphate buffers are diluted to mixture quality It it is 10-100 times, preferably 20 times, more preferably 80 times, further preferably 30 times are further preferably 60 times, most preferably 40 Times；(5) pH and stewing process time are reacted：Usual 10-200mM phosphate buffers pH is 2-10 and the time is 0-5 hours, phosphoric acid Buffer solution, preferably 10mM, more preferably 150mM, further preferably 30mM are further preferably 80mM, most preferably 50mM；Instead Answer pH, preferably pH is 4, more preferably pH8, further preferably pH 6, most preferably pH 7；It is preferred that the stewing process time is 0 Hour, more preferably 4 hours, further preferably 2 hours, most preferably 1 hour；(6) reaction temperature and time：Reaction temperature Usually 35 DEG C -75 DEG C, preferably 40 DEG C, more preferably 60 DEG C, further preferably 50 DEG C, most preferably 46 DEG C, are important to , it is noted that the temperature for improving heat treatment can increase the grafting degree of glycation product, still, for Hrps functional polypeptides Speech, not be increased to temperature 80 DEG C, in order to avoid influence the biological effect of Hrps functional polypeptides；Reaction time is usually 3-25 small When, preferably 7 hours, more preferably 20 hours, further preferably 10 hours are further preferably 14 hours, and most preferably 12 is small When；During reaction, reaction mixture needs to stir, and mixing speed is usually that 30-300 per minute turns, preferably 30 turns, more preferably 250 turns, further preferably 50 turns are further preferably 100, most preferably 60 turns；(7) prepare Hrps functional polypeptides combination segment- The optimum condition of the covalent trimeric polypeptide of glucan finally screens foundation：Reaction solution staticly settle the time must not be less than 2 hours, lure The hair balsamine blade generation anaphylactoid time must not exceed 4 hours and the oat plumular axis speed of growth must not be less than 0.03cm/h (biological effect)；(8) after the completion of reacting, cryogenic vacuum is continuous drying, and the product finally collected is：(3Hrps is more Peptide)_n-(C₆H₁₀O₅)_nCovalent trimeric polypeptide, simplifying general formula is：(3Hrps)_n-(C₆H₁₀O₅)_n。

(3Hrps) of four combinations of Hrps Peptide of Protein_n-(C₆H₁₀O₅)_nPreferred control prepared by covalent trimeric polypeptide Condition test using same method is carried out, obtains above-mentioned consistent result side by side simultaneously.Complete end product (3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) preparation of covalent trimeric polypeptide and product detection.

Further using similary technology of preparing and condition, preferably control condition, with glucan-modified more Hrps albuminoids (Harpin_Eccs、Harpin_Ecc、Harpin_Ea、Harpin_Ech、Harpin_pst、Harpin_psg、Harpin_pssDeng and pass through base The fusion protein Harpin obtained by recombination form_Eccs+Harpin_Ecc、Harpin_Eccs+Harpin_Ea、Harpin_Eccs+ Harpin_Ech、Harpin_Ecc+Harpin_Eca、Harpin_Ecc+Harpin_Ech、Harpin_Ea+Harpin_EchDeng), prepare polymorphic type Heteroconjugate dimer, heteroconjugate tripolymer, the heteroconjugate tetramer and heteroconjugate polymer.

Embodiment 2

(3Hrps) of four combinations prepared by glucan-modified Hrps Peptide of Protein_n-(C₆H₁₀O₅)_n(I-IV) covalently Trimeric polypeptide dosage form designs:

(3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) covalently design dosage form of the trimeric polypeptide in preparing, storing and applying has：It is former Medicine, including solid, that is, original powder and liquid, that is, crude oil；Storage and application product forms include granule, wettable powder, aqua, Sustained release agent and with the mix preparation of other fertilizer or fungicide, insecticide or plant growth regulator modulation etc..In addition, (3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) relevant auxiliary materials and auxiliary agent of covalent trimeric polypeptide, a large amount of nutrition of solubility including food-grade Ingredient and a small amount of nutritional ingredient.Since the covalent condensate of this kind of functional polypeptide (covalent trimeric polypeptide) and its preparation are nontoxic, nuisanceless, Without special protection during use.

Embodiment 3

(3Hrps) of four combinations prepared by glucan-modified Hrps Peptide of Protein_n-(C₆H₁₀O₅)_n(I-IV) covalently The application method of trimeric polypeptide and requirement:

During condensate covalent using this kind of functional polypeptide：(1) proper method of processing plant includes seed soaking, seed dressing, seed Coating, root dipping, foliar spray, root are applied, are injected, flowers and fruits spraying etc.；(2) suitable condition of processing plant includes selecting suitable use Meteorological Elements in China when medicine time and medication；Granule, wettable powder or aqua are using free from chloride river water, well water, underground Water or tap water are made into the use concentration of requirement, are never mixed with acid, alkali, and drug should be stored at shady and cool drying；(3) processing is planted The specific requirement of object is adapted to medication including the entire season of growth, one season crop can dispenser 3-6 times, per minor tick 10-20 days, apply With time and number depending on being determined using purpose, during use prepared liquid suggestion be finished in 24 hours.

Embodiment 4

(3Hrps) of four combinations prepared by glucan-modified Hrps functional polypeptides segment_n-(C₆H₁₀O₅)_n(I-IV) covalently The Detection of Stability of trimeric polypeptide:

We press (3Hrps) prepared by 1 method of embodiment_n-(C₆H₁₀O₅)_n(I-IV) covalent trimeric polypeptide and corresponding Hrps The mixing segment of polypeptides in combination, stability under several conditions compares, as a result as follows：

At room temperature, the aqueous solution of content 3% is configured to, the mixing segment aqueous solution of Hrps polypeptides in combination stands 20 points There is apparent layering in Zhong Hou, after 1 hour, forms flocculent deposit；(3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) covalent trimeric polypeptide water After solution left standstill 6 hours, solution is still uniform, is not layered.

It is exposed to be configured to the aqueous solution of content 3% under field conditions (factors), it is prepared respectively in culture dish injection a thin layer Aqueous solution, the mixing segment aqueous solution of Hrps polypeptides in combination is after 3 days, and Hrp albumen bands are very weak when sampling electrophoresis, 5 days Afterwards, Hrps albumen bands have disappeared, and show that Hrps polypeptides disintegrate substantially；(3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) covalent trimerization After peptide aqueous solution 7 days, their bands of a spectrum are also high-visible during electrophoresis.

Under the conditions of 100 DEG C, it is configured to the aqueous solution of content 3%, the mixing segment aqueous solution heating of Hrps polypeptides in combination After 25 minutes, injection no longer causes tobacco leaf allergic reaction, the basic loss of activity of Hrps polypeptides；(3Hrps)_n-(C₆H₁₀O₅)_n (I-IV) after covalently trimeric polypeptide aqueous solution heats 30 minutes, injection causes tobacco leaf allergic reaction, and covalent trimeric polypeptide still has work Property, there is weaker activity to keep after 50 minutes.

It is exposed under 30000Lux high light conditions, is configured to the aqueous solution of content 3%, inject one in culture dish respectively The aqueous solution that thin layer is prepared, after sixty minutes, injection no longer causes Tobacco Leaf to Hrps polypeptides in combination mixing segment aqueous solution intense light irradiation Piece allergic reaction, Hrps polypeptide loss of activity；(3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) covalent trimeric polypeptide intense light irradiation after 120 minutes, Injection causes tobacco leaf allergic reaction, still active, and tobacco leaf allergic reaction is no longer caused after 150 minutes, just loses and lives Property.

(3Hrps) of four combinations of Hrps Peptide of Protein_n-(C₆H₁₀O₅)_n(I-IV) stability of covalent trimeric polypeptide Experiment, same method carry out side by side simultaneously, obtain similar result.

Embodiment 5

(3Hrps) of four combinations prepared by glucan-modified Hrps functional polypeptides segment_n-(C₆H₁₀O₅)_n(I-IV) covalently More functions of trimeric polypeptide

For soaking seed, dressing seed, make seed germination early, germination percentage is high, and budding is neat, and sprout is healthy and strong, and diseases prevention is degeneration-resistant, root system hair It reaches；It is applied for seedling foliage-spray, root, robust plant, growth potential is good, and diseases prevention is degeneration-resistant, well developed root system；It is sprayed for seedling blade face Apply, root is applied, and improves the photosynthetic efficiency of crop, with luxuriant foliage and spreading branches in leafy profusion, branch tiller is more, and plant moulding is good, improve it is pregnant spends pregnant fruit rate, enhancing The defence capability that crop endangers pest and disease damage and multiclass poor environment (low temperature, high fever, arid, waterlogging etc.), is greatly reduced danger Evil, and have powerful repair ability；For preceding foliage-spray, root to be spent to apply, the photosynthetic efficiency of crop is improved, spray flower spike is more, carries Before bloom fruiting, fruit branch fruit ear is more, prevents fruit drop, enhancing crop to pest and disease damage and multiclass poor environment (low temperature, high fever, Arid, waterlogging etc.) harm defence capability, harm is greatly reduced, and have powerful repair ability；For blade face of yielding positive results Spray, root is applied, and improves the photosynthetic efficiency of crop, improve fruit-setting rate, setting percentage and maturity, fruit size is uniform, significantly improves Yield and quality promotes agricultural product marketing quality, mitigates disease hazard after harvest, extends agricultural product shelf freshness date, and volume increase increases It receives.

Embodiment 6

(3Hrps) of four combinations prepared by glucan-modified Hrps functional polypeptides segment_n-(C₆H₁₀O₅)_n(I-IV) covalently The wider array of use scope of trimeric polypeptide

It carries out (3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) covalent trimeric polypeptide and single HrpEcb albumen and corresponding Hrps polypeptides Three kinds of protein formulation biological activities of mixing segment-anaphylactoid comparative test of combination, carried out 36 kinds of plant leaf blades Quick reaction test, after 24 hours, the anaphylactoid floristics number of characteristic feature occurs for observation statistics, and allergic reaction is plant The external manifestation of induced resistance, thus whether allergic reaction or anaphylactoid power are occurred by plant, it is possible to compare The biological activity of anti-albumen and the ability of inducing plant resistance are accordingly lured, (3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) covalent trimerization Peptide can induce 35 kinds of test plants and generate allergic reactions, they be tobacco, capsicum, eggplant, tomato, potato, strawberry, cucumber, Water spinach, cockscomb, glass Malus spectabilis, chrysanthemum in September, heartsease, Radix primulae maximowiczii, petunia, grape, Chinese rose, Chinese scholartree, pea, peach, Red sage, sponge gourd, kidney bean, cauliflower, spinach, rape, Chinese yam, cowpea, broad bean, corn, rice, soybean, cyclamen, mulberry Tree, pumpkin, Ailanthus altissima, allergic reaction spot is larger, and tissue necrosis degree (allergic reaction intensity) is higher；Single HrpEcb albumen can lure Lead 16 kinds of test plants and generate allergic reactions, they be tobacco, capsicum, eggplant, tomato, potato, strawberry, cucumber, water spinach, Cockscomb, glass Malus spectabilis, chrysanthemum in September, heartsease, Radix primulae maximowiczii, petunia, grape, Chinese rose, allergic reaction spot or tissue necrosis journey It is general or weaker to spend (allergic reaction intensity)；The mixing segment of corresponding Hrps polypeptides in combination can induce 26 kinds of test plants and generate Allergic reaction, they are tobacco, capsicum, eggplant, tomato, potato, strawberry, cucumber, water spinach, cockscomb, glass Malus spectabilis, nine Month chrysanthemum, heartsease, Radix primulae maximowiczii, petunia, grape, Chinese rose, Chinese scholartree, pea, peach, red sage, sponge gourd, kidney bean, cauliflower, Spinach, rape, Chinese yam, allergic reaction spot or tissue necrosis degree (allergic reaction intensity) are general or weaker.

The result shows that (3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) covalent trimeric polypeptide, single HrpEcb albumen and corresponding Hrps The mixing segment of polypeptides in combination all has the ability and biological activity of induction test plant resistance, in the plant of induced hypersensitivity reaction In species range and the ability and biological activity of induction of resistance, (3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) covalent trimeric polypeptide It is optimal, most strong, secondly the mixing segment of corresponding Hrps polypeptides in combination, is thirdly single HrpEcb albumen, it is clear that (3Hrps)_n-(C₆H₁₀O₅)_n(I-IV) covalently trimeric polypeptide has wider array of use scope and stronger lures anti-ability.

Embodiment 7

(3Hrps) of four combinations prepared by glucan-modified Hrps functional polypeptides segment_n-(C₆H₁₀O₅)_n(I-IV) covalently The more powerful synergy power of trimeric polypeptide

It, can be with applied in fertilizer product, being a kind of unique and powerful synergy power as the intermediate complement of addition Significantly strengthen and promote Fertilizer application effect and efficiency, more than 30% Fertilizer application amount can be saved, and realize volume increase, increase It receives, degeneration-resistant, safe effect.

It, can be with applied in Pesticidal products, being a kind of unique and powerful synergy power as the intermediate complement of addition Significantly strengthen and promote Pesticide use effect and efficiency, it is particularly very notable to the control effect of bacterium and virus disease, it can To save 80% Pesticide use amount, and realize volume increase, increase income, is degeneration-resistant, safe effect.

As the intermediate complement of addition, applied in plant growth regulator product, being a kind of unique and powerful increasing Power is imitated, can significantly strengthen and promote plant growth regulator using effect and efficiency, 80% plant growth can be saved Conditioning agent usage amount, and realize volume increase, increase income, is degeneration-resistant, safe effect.

Embodiment 8

(3Hrps) of four combinations prepared by glucan-modified Hrps functional polypeptides segment_n-(C₆H₁₀O₅)_n(I-IV) covalently Trimeric polypeptide is a variety of for studying the using effect on object

(1) 5 towns village of 2 cities and counties of Sichuan Province's Deyang City has carried out the covalent trimerization peptide test in more than 20 batch crop fields and has shown Model implements more than 360 mu of area total amount, mainly for study object for potato, cucumber, corn, capsicum, tomato, suncured tabacco, red oil dish, Cauliflower, rice, mulberry tree, nectarine etc., amount of increase in production are 8% to 36%, show significantly to lure anti-work to pest and disease damage and adverse circumstance With improving product quality and commodity, and extend the storage life of product.

(2) 7 villages in 5 districts in Sichuan Province Chengdu have carried out the covalent trimerization in three more than 10 batch crop fields of the season of growth More than 280 mu of area total amount is implemented in peptide test and demonstration, mainly for study object for potato, cucumber, capsicum, eggplant, celery, Chinese cabbage, It is tealeaves (white tea), strawberry, fruit mulberry, grape, loquat, Chinese yam and Zijin flower, cyclamen, iris, heartsease, poinsettia, short It leads a cow, African balsamine, chrysanthemum, fuchsia, toad's-mouth, glass Malus spectabilis etc. more than 20 plant flowers, vegetables and fruits volume increase Amplitude is 12% to 32%, shows significantly to lure anti-effect to pest and disease damage and adverse circumstance, improves product quality and commodity, and Extend the storage life of product；Flowers show significantly to lure anti-effect to pest and disease damage and adverse circumstance, and plastotype effect is stronger, improves Product commodity, and the florescence is extended, it increases and spends number and color more bright-coloured；Tealeaves improves quality, and can pick 1 more It is secondary, remarkable in economical benefits.

(3) the covalent trimeric polypeptide effect test demonstration in 2 districts of Sichuan Province's Luzhou City, chief crop is raspberry, lotus And sorghum, more than 60 mu of the gross area, more than 30% output increased of raspberry and lotus, Output of Sorghum improve 24%, show pair Pest and disease damage and adverse circumstance significantly lure anti-effect, improve product quality and commodity.

(4) the covalent trimeric polypeptide effect test demonstration in the cloud and mist planting base in village of 2, Yunnan Province, plants more than 120 mu tobaccos altogether, After tobacco leaf harvest, tobacco leaf degree generally proposes one level higher, yield increase by 24%, and vega tobacco grower improves more than 380 yuan of income per acre (increasing income up to 30%).

(5) flower and plant base in 1 county of Yunnan Province, in a variety of flower culture plantations of more than 60 mus, demonstration uses covalent trimeric polypeptide Product, the results showed that, there is notable repellent to pest and disease damage and adverse circumstance and lure anti-effect, to colored plant shape, plastotype effect is stronger, carries High product commodity, and the florescence is extended, it increases and spends number and color more bright-coloured.

(6) the vegetable cultivation base in 2 villages and small towns of Yunnan Province's Yuxi, in more than 50 mu corns, capsicum, eggplant, celery In plantation, demonstration has used covalent trimerization peptide product, the results showed that, during harvest, except there is notable repellent to pest and disease damage and adverse circumstance With lure outside anti-effect, output increased 15% to 38%, and improve the commodity of product.

(7) the Pu'er tea base in county of 1, Yunnan Province is demonstrated in more than 60 mu Pu'er tea planting sites and is produced using covalent trimeric polypeptide Product are advanced by harvest time, more than 30% output increased, and curing process faster, significantly promotes mouthfeel and tea smell.

(8) the agriculture section proving ground of Shanxi Province Yuci uses and verifies covalently in cucumber, romaine lettuce, the demonstration of celery planting site The effect of trimerization peptide product, through strictly surveying production, cucumber production promoting 25% increases production more than 900 kilograms of cucumber, celery volume increase per acre 10.8%, romaine lettuce volume increase 7.6% achieves good economic benefit.

(9) cotton planting in 1 county in Shanxi Province Yuncheng, 8 mu of First Year, 60 mu of second year, in cotton planting demonstrating makes It the effect of with covalent trimerization peptide product is verified, strictly surveys and produces in person through relevant department expert, Yield Increase In Cotton amplitude is up to 31.46% More than.

(10) cucumber of 2 counties and cities in Shanxi Province Yuncheng, pakchoi, celery use the experiment and demonstration of covalent trimerization peptide product, It is checked and accepted through strictly surveying production, more than 50% cucumber production promoting, pakchoi, more than 20% celery volume increase are brought significantly economical to peasant household Benefit.

(11) wheat, corn, watermelon, muskmelon, tomato, apricot, apple, pears, the jujube in 1 villages and small towns in 1 county of Shanxi Province Deng the experiment and demonstration using covalent trimerization peptide product, the experiment of 3 years, more than 280 mu of the gross area have been carried out, the results showed that, except to disease Insect pest and adverse circumstance have notable repellent and lure outside anti-effect, and wheat yield improves 8.7%-13%, the output increased of corn More than 22.6%, particularly wheat have spent cold spell in later spring, and corn is anti-to have crossed serious spring drought, and tomato improves 28%, watermelon production Amount improves 28%, and muskmelon improves 32%, and apricot improves 30%, apple, pears, jujube output increased 25%, improve the quotient of product Moral character and extend Storage period.

(12) the artificial growth herbage in the protection of the steppe vegetation in Qinghai Province's sources of three rivers and pastoral area and pastoral area, in 30000 mu of grass Large-scale demonstration uses covalent trimerization peptide product on field, the results showed that, differently using covalent trimerization peptide product, herbage Output increased 27% to 33%.

(13) various vegetables in 2, county of 1, Qinghai Province villages and small towns and cereal crops are shown using the experiment of covalent trimerization peptide product Model, the crops such as highland barley, wheat, shallot, tomato experiments have shown that, except there is notable repellent to pest and disease damage and adverse circumstance and lure anti- Effect is outer, highland barley output increased 11.47%, and wheat improves 15%, and shallot improves 19%, and tomato improves 13%, and carries significantly The high commodity of product.

(14) the extraordinary medicinal material planting base of Hexi Region of Gansu Province is related with the covalent trimeric polypeptide of potato planting base Experiment and demonstration, more than 300 mu of cultivated area, the results showed that, the yield and quality of extraordinary medicinal material active ingredient is significantly promoted, and is added Work quality is especially prominent, and more than 21% potato production promoting substantially increases economic benefit.

(15) cotton on 2 farms of Xinjiang Production and Construction Army Corps uses the experiment and demonstration of covalent trimerization peptide product, two farms Implement 120 mu of experiment and demonstration, the results showed that, the apparent repellent bollworm of cotton plants, and resisting verticillium, output increased 19%-20%, remarkable in economical benefits.

(16) jujube in 1 county in Xinjiang South Sinkiang uses the experiment and demonstration of covalent trimerization peptide product, implements 180 mu of area, The result shows that after using covalent trimerization peptide product, Cracking fruit reduces 80%, and commodity rate increases substantially, output increased 10%- 25%, remarkable in economical benefits.

(17) city of Jixi of Heilongjiang in relation to covalent polymer products test and demonstrate, Mishan City implement 26.5 mu of area, 40 mu of area is implemented in land reclamation and cultivation, the results showed that, in the case of very favourable weather (for the crops) in 2014, the yield in common crop field is also very Height, in such a situa-tion, corn yield increasing 11.8%, soybean yield-increasing 10.4%, increasing production of rice 7.7% really belonged to exception.

(18) the covalent polymer products of the use in Heilungkiang Qiqihar implement soybean acreage in relation to testing and demonstrating More than 110 mus, the results showed that, averagely increase production 16.7%, by the trial zone of test requirements document management, amount of increase in production is achieved up to 27% Good result of the test.

(19) technology and its it associated products prepared can be widely applied to the protein and peptide agricultural fertilizer of agriculture field, agriculture Medicine, growth regulator production, applied to the protein and peptide externally applied drug of field of medicaments, medicine for oral administration, intramuscular injection medicine and quiet Arteries and veins injects the production and processing of the related polypeptide product of medicine production and grocery trade.

Comparison between Hrps albumen and between functional polypeptide segment (DOMAIN)

SEQUENCE LISTING

<110>Sichuan basis crop Science and Technology Ltd.

<120>Covalent trimeric polypeptides of glucan-modified Hrps and preparation method thereof

<160> 4

<170> PatentIn Version 2.1

<210> 1

<211> 356

<212> PRT

<213>Carrot soft rot Erwinia Chinese cabbage subspecies（Erwiniacarotovorum subsp. Carotovorum strain CSSY002 ）

<220>

<221> DOMAIN

<222> (157)-(357)

<400> 1

Met Leu Asn Ser Leu Gly Gly Gly Ala Ser Leu Gln Ile Thr Ile Lys

1 5 10 15

Ala Gly Gly Asn Gly Gly Leu Phe Pro Ser Gln Ser Ser Gln Asn Gly

20 25 30

Gly Ser Pro Ser Gln Ser Ala Phe Gly Gly Gln Arg Ser Asn Ile Ala

35 40 45

Glu Gln Leu Ser Asp Ile Met Thr Thr Met Met Phe Met Gly Ser Met

50 55 60

Met Gly Gly Gly Met Gly Gly Gly Leu Gly Gly Leu Gly Ser Ser Leu

65 70 75 80

Gly Gly Leu Gly Gly Gly Leu Leu Gly Gly Gly Leu Gly Gly Gly Leu

85 90 95

Gly Ser Ser Leu Gly Ser Gly Leu Gly Ser Ala Leu Gly Gly Gly Leu

100 105 110

Gly Gly Val Leu Gly Ala Gly Met Asn Ala Met Asn Pro Ser Ala Met

115 120 125

Met Gly Ser Leu Leu Phe Ser Ala Leu Glu Asp Leu Leu Gly Gly Gly

130 135 140

Met Ser Gln Gln Gln Gly Gly Leu Phe Gly Asn Lys Gln Pro Ser Ser

145 150 155 160

Pro Glu Ile Ser Ala Tyr Thr Gln Gly Val Asn Asp Ala Leu Ser Ala

165 170 175

Ile Leu Gly Asn Gly Leu Ser Gln Thr Lys Gly Gln Thr Ser Pro Leu

180 185 190

Gln Leu Gly Asn Asn Gly Leu Gln Gly Leu Ser Gly Ala Gly Ala Phe

195 200 205

Asn Gln Leu Gly Ser Thr Leu Gly Met Ser Val Gly Gln Lys Ala Gly

210 215 220

Leu Gln Glu Leu Asn Asn Ile Ser Thr His Asn Asp Ser Pro Thr Arg

225 230 235 240

Tyr Phe Val Asp Lys Glu Asp Arg Ala Met Ala Lys Glu Ile Gly Gln

245 250 255

Phe Met Asp Gln Tyr Pro Glu Val Phe Gly Lys Ala Glu Tyr Gln Lys

260 265 270

Asp Asn Trp Gln Thr Ala Lys Gln Glu Asp Lys Ser Trp Ala Lys Ala

275 280 285

Leu Ser Lys Pro Asp Asp Asp Gly Met Thr Lys Gly Ser Met Asp Lys

290 295 300

Phe Met Lys Ala Val Gly Met Ile Lys Ser Ala Ile Ala Gly Asp Thr

305 310 315 320

Gly Asn Thr Asn Leu Ser Ala Arg Gly Asn Gly Gly Ala Ser Leu Gly

325 330 335

Ile Asp Ala Ala Met Ile Gly Asp Arg Ile Val Asn Met Gly Leu Lys

340 345 350

Lys Leu Ser Ser

355

<210> 2

<211> 370

<212> PRT

<213>Tomato leaf plaque（Pseudomonas syringae pv. tomato strain CSCS008）

<220>

<221> DOMAIN

<222>(1)-(270), (329)-(370)

<400> 2

Met Gln Ala Leu Asn Ser Ile Ser Ser Leu Gln Thr Ser Ala Ser Leu

1 5 10 15

Phe Pro Val Ser Leu Asn Ser Asp Val Ser Ala Asn Thr Ser Thr Ser

20 25 30

Ser Lys Glu Leu Lys Ala Val Ile Asp Gln Leu Val Gln Ala Leu Thr

35 40 45

Gln Ser Gly Gln Leu Asp Glu Thr Ser Pro Leu Gly Lys Met Leu Ala

50 55 60

Lys Ala Met Ala Ala Asp Gly Lys Ser Ala Asn Ser Ile Asp Asp Ile

65 70 75 80

Thr Ala Ser Leu Asp Lys Leu Ile His Glu Lys Leu Gly Asp Asn Phe

85 90 95

Gly Ala Ser Ala Gly Ile Gly Ala Gly Gly Gly Gly Gly Gly Ile Gly

100 105 110

Gly Ala Gly Ser Gly Ser Gly Val Gly Gly Gly Leu Ser Ser Asp Ala

115 120 125

Gly Ala Gly Gln Ser Asp Leu Met Ser Gln Val Leu Asn Gly Leu Gly

130 135 140

Lys Ala Val Leu Asp Asp Leu Leu Thr Pro Ser Gly Glu Gly Gly Thr

145 150 155 160

Thr Phe Ser Ser Asp Asp Met Pro Thr Leu Glu Lys Val Ala Gln Phe

165 170 175

Met Asp Asp Asn Lys Ala Gln Phe Pro Thr Arg Asp Gly Gly Ser Trp

180 185 190

Met Asn Glu Leu Lys Glu Asp Asn Gly Leu Asp Ala Gln Glu Thr Ala

195 200 205

Gln Phe Arg Ser Ala Leu Asp Val Ile Gly Gln Gln Leu Gly Gln Gln

210 215 220

Gln Gly Asp Ala Ser Gly Val Thr Ser Gly Gly Gly Leu Gly Ser Pro

225 230 235 240

Val Ser Asp Ser Ser Leu Gly Asn Pro Ala Ile Asp Ala Asn Thr Gly

245 250 255

Pro Ala Ala Asn Gly Asn Ala Ser Val Asp Val Gly Gln Leu Ile Gly

260 265 270

Gln Leu Ile Asp Arg Gly Leu Gln Ser Val Ser Ser Gly Gly Gly Leu

275 280 285

Gly Thr Pro Val Asp Asn Ser Thr Gln Pro Thr Gly Gly Thr Pro Ala

290 295 300

Ala Asn Pro Thr Gly Asn Val Ser Asn Gln Asp Leu Gly Gln Leu Leu

305 310 315 320

Ser Gly Leu Leu Gln Arg Gly Leu Glu Ala Thr Leu Gln Asp Ala Gly

325 330 335

Asn Thr Gly Ala Asp Leu Gln Ser Ser Ala Ala Gln Val Ala Ala Gln

340 345 350

Leu Ile Asn Ala Leu Leu Gln Gly Thr Asn Asn Gln Thr Asn Gln Ala

355 360 365

Val Ala

370

<210> 3

<211> 370

<212> PRT

<213>Carrot soft rot Erwinia beet subspecies（Pectobacteriumbetavasculorum strain EcbCSL101）

<220>

<221> DOMAIN

<222> (171)-(370)

<400> 3

Met Leu Asn Ser Leu Gly Gly Gly Thr Ser Leu Gln Ile Thr Ile Lys

1 5 10 15

Ala Gly Gly Asn Gly Asp Leu Phe Gln Ser Gln Ser Ser Gln Asn Gly

20 25 30

Gly Ala Pro Ser Gln Leu Gly Leu Gly Gly Gln Arg Ser Asn Ile Ala

35 40 45

Glu Gln Leu Ser Asp Ile Met Thr Thr Met Met Phe Met Gly Ser Met

50 55 60

Met Gly Gly Gly Leu Gly Gly Leu Gly Gly Met Gly Gly Gly Leu Gly

65 70 75 80

Gly Ala Leu Gly Gly Leu Gly Ser Ser Leu Gly Gly Leu Gly Gly Gly

85 90 95

Leu Leu Gly Gln Gly Leu Gly Gly Gly Leu Ala Gly Gly Leu Gly Ser

100 105 110

Ser Leu Gly Ser Gly Leu Gly Gly Ala Leu Gly Gly Gly Leu Gly Gly

115 120 125

Ala Leu Gly Ala Gly Met Asn Ala Met Asn Pro Ser Ala Met Met Gly

130 135 140

Ser Leu Leu Phe Ser Ala Leu Glu Asp Leu Leu Gly Gly Gly Met Ser

145 150 155 160

Gln Gln Gln Gly Gly Leu Phe Gly Asn Lys Gln Pro Ala Ser Pro Glu

165 170 175

Ile Ser Ala Tyr Thr Gln Gly Val Asn Asp Thr Leu Ser Ala Ile Leu

180 185 190

Gly Asn Gly Leu Ser Gln Ala Lys Gly Gln His Ser Pro Leu Gln Leu

195 200 205

Gly Asn Asn Gly Leu Gln Gly Leu Ser Gly Ala Gly Ala Phe Asn Gln

210 215 220

Leu Gly Ser Thr Leu Gly Met Gly Val Gly Gln Lys Ala Gly Leu Gln

225 230 235 240

Glu Leu Asn Asn Ile Ser Thr His Asn Gly Ser Pro Thr Arg Tyr Phe

245 250 255

Val Asp Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly Gln Phe Met

260 265 270

Asp Gln Tyr Pro Glu Val Phe Gly Lys Pro Glu Tyr Gln Lys Asp Asn

275 280 285

Trp Gln Thr Ala Lys Gln Asp Asp Lys Ser Trp Ala Lys Ala Leu Ser

290 295 300

Lys Pro Asp Asp Asp Gly Met Thr Lys Gly Ser Met Asp Lys Phe Met

305 310 315 320

Lys Ala Val Gly Met Ile Lys Ser Ala Val Ala Gly Asp Thr Gly Asn

325 330 335

Thr Asn Leu Asn Ala Arg Gly Asn Gly Gly Ala Ser Leu Gly Ile Asp

340 345 350

Ala Ala Met Ile Gly Asp Arg Ile Val Asn Met Gly Leu Gln Lys Leu

355 360 365

Ser Ser

370

<210> 4

<211> 339

<212> PRT

<213>Chrysanthemum soft rot Erwinia（Erwinia chrysanthemi strain CSCL006）

<220>

<221> DOMAIN

<222> (9)-(334)

<400> 4

Met Gln Ile Thr Ile Lys Ala His Ile Gly Gly Asp Leu Gly Val Ser

1 5 10 15

Gly Leu Gly Leu Gly Ala Gln Gly Leu Lys Gly Leu Asn Ser Ala Ala

20 25 30

Ser Ser Leu Gly Ser Ser Val Asp Lys Leu Ser Ser Thr Ile Asp Lys

35 40 45

Leu Thr Ser Ala Leu Thr Ser Met Met Phe Gly Gly Ala Leu Ala Gln

50 55 60

Gly Leu Gly Ala Ser Ser Lys Gly Leu Gly Met Ser Asn Gln Leu Gly

65 70 75 80

Gln Ser Phe Gly Asn Gly Ala Gln Gly Ala Ser Asn Leu Leu Ser Val

85 90 95

Pro Lys Ser Gly Gly Asp Ala Leu Ser Lys Met Phe Asp Lys Ala Leu

100 105 110

Asp Asp Leu Leu Gly His Asp Thr Val Thr Lys Leu Thr Asn Gln Ser

115 120 125

Asn Gln Leu Ala Asn Ser Met Leu Asn Ala Ser Gln Met Thr Gln Gly

130 135 140

Asn Met Asn Ala Phe Gly Ser Gly Val Asn Asn Ala Leu Ser Ser Ile

145 150 155 160

Leu Gly Asn Gly Leu Gly Gln Ser Met Ser Gly Phe Ser Gln Pro Ser

165 170 175

Leu Gly Ala Gly Gly Leu Gln Gly Leu Ser Gly Ala Gly Ala Phe Asn

180 185 190

Gln Leu Gly Asn Ala Ile Gly Met Gly Val Gly Gln Asn Ala Ala Leu

195 200 205

Ser Ala Leu Ser Asn Val Ser Thr His Val Asp Gly Asn Asn Arg His

210 215 220

Phe Val Asp Lys Glu Asp Arg Gly Met Ala Lys Glu Ile Gly Gln Phe

225 230 235 240

Met Asp Gln Tyr Pro Glu Ile Phe Gly Lys Pro Glu Tyr Gln Lys Asp

245 250 255

Gly Trp Ser Ser Pro Lys Thr Asp Asp Lys Ser Trp Ala Lys Ala Leu

260 265 270

Ser Lys Pro Asp Asp Asp Gly Met Thr Gly Ala Ser Met Asp Lys Phe

275 280 285

Arg Gln Ala Met Gly Met Ile Lys Ser Ala Val Ala Gly Asp Thr Gly

290 295 300

Asn Thr Asn Leu Asn Leu Arg Gly Ala Gly Gly Ala Ser Leu Gly Ile

305 310 315 320

Asp Ala Ala Val Val Gly Asp Lys Ile Ala Asn Met Ser Leu Val Ala

325 330 335

Ala Asn Ala

Claims (9)

1. the covalent trimeric polypeptides of glucan-modified Hrps, which is characterized in that it is passed through covalent by three kinds of Hrps polypeptides and glucan Modification forms covalent trimeric polypeptide, and general structure is：(3Hrps)_n-(C₆H₁₀O₅)_n；Wherein, three kinds of Hrps polypeptides are SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:Arbitrary three kinds in amino acid sequence shown in 4.

2. the covalent trimeric polypeptides of glucan-modified Hrps according to claim 1, which is characterized in that SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 quality proportioning is 3:2:3:2.

3. the covalent trimeric polypeptides of glucan-modified Hrps according to claim 1, which is characterized in that three kinds of Hrps polypeptides with The quality proportioning of glucan is 10:1 to 1:10.

4. the covalent trimeric polypeptides of glucan-modified Hrps according to claim 1, which is characterized in that three kinds of Hrps polypeptides with The quality proportioning of glucan is 1:2.

5. the covalent trimeric polypeptides of glucan-modified Hrps according to claim 1, which is characterized in that glucan for oat β- Glucan, molecular mass 5300-257200.

6. the covalent trimeric polypeptides of glucan-modified Hrps according to claim 1, which is characterized in that glucan for oat β- Glucan, molecular mass 32000-38000.

7. according to the preparation method of the glucan-modified covalent trimeric polypeptides of Hrps of claim 1-6 any one of them, feature It is, preparation process is as follows：

(1) from SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:In four kinds of Hrps polypeptides shown in 4 Arbitrarily three kinds of Hrps polypeptides of selection；

(2) quality proportioning of glucan, three kinds of Hrps polypeptides and glucan is added in into three kinds of Hrps polypeptides that step (1) selects For：10:1 to 1:10；Phosphate buffer is added in, it is 2-10 to make system pH；0-5 hours are stood, is 15-80 DEG C, stirs in temperature Speed is mixed to be reacted 3-25 hours under the conditions of 30-300r/min；

(3) after the completion of reacting, cryogenic vacuum is continuous drying, collects product up to covalent trimeric polypeptide, (3Hrps)_n-(C₆H₁₀O₅)_n。

8. the preparation method of the covalent trimeric polypeptides of glucan-modified Hrps according to claim 7, which is characterized in that step (2) in, a concentration of 10-200mmol/L of phosphate buffer, phosphate buffer addition is that three kinds of Hrps polypeptides and glucan are total 10-100 times of quality.

9. the preparation method of the covalent trimeric polypeptides of glucan-modified Hrps according to claim 7, which is characterized in that prepare Method is as follows：

(1) from SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:In four kinds of Hrps polypeptides shown in 4 Arbitrarily three kinds of Hrps polypeptides of selection；Wherein, SEQ ID NO:1、SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4 four kinds The quality proportioning of Hrps polypeptides is 3:2:3:2；

(2) glucan is added in into three kinds of Hrps polypeptides that step (1) selects, glucan is avenabeta glucosan, molecular mass For 32000-38000；The quality proportioning of three kinds of Hrps polypeptides and glucan is：1:2；The phosphoric acid for adding in a concentration of 50mmol/L delays Fliud flushing, phosphate buffer addition are 40 times of three kinds of Hrps polypeptides and glucan gross mass, and it is 7 to make system pH, and it is small to stand 1 When；It is reacted 12 hours under the conditions of temperature is 46 DEG C, mixing speed is 60r/min；

(3) after the completion of reacting, cryogenic vacuum is continuous drying, collects product up to covalent trimeric polypeptide, (3Hrps)_n-(C₆H₁₀O₅)_n。