CN103276016B - A kind of dedicated silkworm breed breeding method secreting medical silk - Google Patents
A kind of dedicated silkworm breed breeding method secreting medical silk Download PDFInfo
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- CN103276016B CN103276016B CN201310207988.4A CN201310207988A CN103276016B CN 103276016 B CN103276016 B CN 103276016B CN 201310207988 A CN201310207988 A CN 201310207988A CN 103276016 B CN103276016 B CN 103276016B
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Abstract
The present invention relates to the breeding technique of cultivated silkworm breed variety, aim to provide a kind of dedicated silkworm breed breeding method secreting medical silk.The method is that calcium functional gene territory is operated by genetically engineered, the carrier with reporter gene based on the PiggyBACK factor, build restructuring transgene carrier, the silkworm with reporter gene is obtained with Visuble self-sucking extrinsic gene induction of insect egg, and biomineralization function is detected in the fibroin of transgenic bombyx mori, be bred as and can secrete the medical new silkworm variety with biomineralization function.By Juvenile Hormone in the present invention after implantable bioartificial body has biomineralization function, gives the biological characteristics that biologic-organ is intrinsic.By Juvenile Hormone can cut open acquisition from domestic silkworm silk body of gland Directly solution, and not only biological safety is high, and can significantly improve the physical strength of finished product in pole, overcomes this limited resource that common silk is applied in biological medicine material.Preparation technology is simple, significantly reduces cost.<!--1-->
Description
Technical field
The present invention relates to the selection of medical silk cultivated silkworm breed variety, by the design of recombinant fibroin molecule, the structure of expression vector and silkworm egg microinjection transgenic technology the gene order with biomineralization function be incorporated into domestic silkworm gene group and reach at posterior division of silkgland body surface, selecting the breeding novel method secreting the new silkworm variety with biomineralization function silk.
Background technology
Silk is the important source material of light textile and chemical industry, is the protein fibre that consumption is maximum in the world.China is traditional state of silkworm sparetime university, for a long time, because the object product of Sericultural production is raw silk, the intrinsic thoughtcast that people define " having planted mulberry; support good silkworm; produced silk ", the breeding goal of Silkworm varieties is silk cocoon high-quality, strong raising and the breakthrough of the economic characters aspect such as easily to support of high yield, silkworm.Through the effort of silkworm breeding man, current China produces the upper Silkworm varieties promoted and is in the leading level in the world, practical Silkworm varieties to have reached capacity state at the trim point of silk amount silk quality and physique, and application traditional breeding technology will have larger breakthrough quite difficult.Traditional breeding method can not meet the needs of multi-faceted, the high-level development of silkworm industry completely.Along with the research of molecular biology particularly genetic engineering technique breaks through, utilize DNA recombinant technology to improve cultivated silkworm breed variety and Innovation Germplasm resource has seemed more and more urgent.Exploring and developing new breeding technique is obtain effective object proterties thus realize one of effective way of silkworm Germplasm enhancement.By the molecular breeding that genetically engineered is carried out, than traditional cross-breeding, not only there is breeding objective clear and definite and shorten the advantage of breeding cycle, and foreign gene imports in silkworm body by gene transfer technique by it, make it stable integration on chromosome, cultivate novel transgenic bombyx mori kind, thus reach the target of unconventional property breeding character.
Fibroin is by fibroin light chain protein, fibroin heavy chain protein, P25 albumen, and the fibrous protein that a few albumen such as 5 kinds of sericins combines by a certain percentage, that one has good permeability, nontoxic, non-stimulated, with the biomaterial of human tissue organ close friend, one of study hotspot is in recent years exactly pass through physics, the method of chemistry carries out character reforming processing to silk, so that can as artificial skin, tendon, ligament, the artificial material of the tissue such as bone and tooth, relevant research also achieves gratifying progress, multiple product is had to enter clinical experimental stage.But the method for the physics and chemistry adopted is carried out character to silk and is transformed not only treatment process more complicated, and can change the biological characteristics of silk, affects the efficiency about function.Therefore utilize gene recombination technology and silkworm transgenic technology, creating can the direct production new silkworm variety that the is applicable to new medical material silk key of will deal with problems.This technological invention is exactly the design by recombinant fibroin gene, by in conjunction with the gene function territory of calcium ion and fibroin gene recombination, cultivation can produce the new silkworm variety of calcium binding function silk, the silk that this silkworm tells is as joint prosthesis and tendon materials, By Juvenile Hormone after implantable bioartificial body has biomineralization function, have more the biological characteristics that biologic-organ is intrinsic, therefore can be medical science and novel biomaterial is provided.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes deficiency of the prior art, provides a kind of dedicated silkworm breed breeding method secreting medical silk, and silkworm can be made to secrete the silk with biomineralization function.
For solving the problems of the technologies described above, solution of the present invention is:
A kind of dedicated silkworm breed breeding method secreting medical silk is provided, that calcium functional gene territory is operated by genetically engineered, the carrier with reporter gene based on the PiggyBACK factor, build restructuring transgene carrier, the silkworm with reporter gene is obtained with Visuble self-sucking extrinsic gene induction of insect egg, and biomineralization function is detected in the fibroin of transgenic bombyx mori, be bred as and can secrete the medical new silkworm variety with biomineralization function;
Described calcium functional gene territory is the design by recombinant protein molecule, i.e. the crystallizable fragment (AGSGAG) of silk fibroin protein primary structure
6combine with the aminoacid sequence YDYDDDSDDDDEWD in the conchiolin MSI60 of black lip (Pinctadafucata), design can promote the recombinant fibroin of biomineralization function, and its primary sequence is ((AGSGAG)
6aSEYDYDDDSDDDDEWD) n; By crystallizing field and the combination of calcium in conjunction with amino acid fragment, restructuring silk is designed to lamellar structure, namely (AGSGAG) 6 formation β-sheet are folding, and calcium binding amino acid sequence is exposed to β-sheet folds outside crystallizing field, promote the formation of hydroxylapatite crystal with calcium binding.
In the present invention, specifically comprise the steps:
(1) selection of function fragment and combination
Adopt the crystallizable fragment (AGSGAG) of silk fibroin protein primary structure
6combine with the aminoacid sequence YDYDDDSDDDDEWD in black lip (Pinctadafucata) conchiolin MSI60, design can promote the recombinant fibroin of biomineralization function, and its primary sequence is ((AGSGAG)
6aSEYDYDDDSDDDDEWD)
n; By crystallizing field and the combination of calcium in conjunction with amino acid fragment, restructuring silk is designed to lamellar structure, that is, (AGSGAG)
6form β-sheet to fold, and calcium binding amino acid sequence is exposed to β-sheet and folds outside crystallizing field, promotes the formation of hydroxylapatite crystal with calcium binding;
(2) repeated fragment (AGSGAG)
6the sign of the biomineralization function of ASEYDYDDDSDDDDEWD
After determining two different fragments combinations, whether there is biomineralization function, first will to the polypeptide (AGSGAG) of synthesis
6aSEYDYDDDSDDDDEWD carries out biomineralization function sign;
First calcium-ion-binding ability test is carried out: by the polypeptide CaCl of above-mentioned synthesis
2solution soaking 0.5h, then carries out stain test with Feng Kusa dye liquor; Coloration result clearly can observe polypeptide laking, illustrates that this polypeptide is provided with stronger calcium-ion-binding ability;
Then biomineralization and sign is carried out: add 1.5SBF solution (simulatedbodyfluid simulated body fluid) at the polypeptide solution of above-mentioned synthesis, and concussion makes to dissolve completely, mineralising is induced at 37.2 DEG C, 1,3,5, sample respectively during 7d, by sample after lyophilize, dry again after cleaning, detect for X-ray diffraction (XRD) and infrared absorption spectrum (FT-IR); Polypeptide after induction mineralising, composes the characteristic peak of existing hydroxylapatite crystal in XRD figure, confirm the generation of hydroxylapatite crystal, this polypeptide has biomineralization function;
(3) the constructing of cloning vector and expression vector
To above-mentioned (AGSGAG)
6the Nucleotide of ASEYDYDDDSDDDDEWD is optimized, and is cloning vector with pUC57, obtains ((AGSGAG)
6aSEYDYDDDSDDDDEWD)
2cloning vector and expression vector;
(4) target protein gene order is imported silkworm
The method of signal peptide, target protein gene order fusion DNA vaccine is coupled together, obtains LSP+SM2 fragment; Again this fragment is connected with promotor fusion DNA vaccine, obtains a complete expression cassette; With " Visuble self-sucking extrinsic gene induction of insect egg " (Chinese invention patent 200410073439.3), above-mentioned plasmid is mixed in concentration 1:1 ratio with providing the helper plasmid of transposase gene, import to P50 Silkworm varieties lay eggs after in fertilization silkworm seed within 8h; To import the silkworm seed of expression plasmid by 25 DEG C, 90% humidity cultivation hatching, raise the silkworm G0 generation of hatching with mulberry leaf, the selfing of experiment silkworm moth is laid eggs G1 generation;
(5) detection of transgenic bombyx mori
G1 silkworm seed is pressed silkworm egg hatching breeding to two age, detect with fluor stereomicroscope, the individuality that every silkworm body can be observed obvious green eye is transgenic positive silkworm, shows that desired polypeptides sequence has proceeded to domestic silkworm gene group; Transgenic positive of selecting and remain silkworm rearing is to matured silkworm.
Because technique scheme is used, beneficial effect of the present invention is:
(1) compare with common cultivated silkworm breed variety, the silk secreted by silkworm that the present invention cultivates is directly used in the material such as joint prosthesis and tendon, and the By Juvenile Hormone after implantable bioartificial body has biomineralization function, gives the biological characteristics that biologic-organ is intrinsic.
(2) the physical chemistry treating processes be used for before biomaterial with common silk compares, the By Juvenile Hormone of biomineralization function that what the present invention produced have can cut open acquisition from domestic silkworm silk body of gland Directly solution, not only biological safety is high, and the physical strength of finished product can be significantly improved in pole, overcome this limited resource that common silk is applied in biological medicine material.
(3) transgenic bombyx mori that the present invention obtains can be bred by conventional eggs of silkworm raising technology, and silkworm larva standard feed technology is raised, simple for preparation technology during medical biotechnology material, significantly reduces cost.
Embodiment
Method and the technical operating procedure of medical silk dedicated silkworm breed breeding of the present invention is described in detail below in conjunction with embodiment.
1, the selection of function fragment and combination
Adopt the crystallizable fragment (AGSGAG) of silk fibroin protein primary structure
6combine with the aminoacid sequence YDYDDDSDDDDEWD in black lip (Pinctadafucata) conchiolin MSI60, design can promote the recombinant fibroin of biomineralization function, and its primary sequence is ((AGSGAG)
6aSEYDYDDDSDDDDEWD)
n.By crystallizing field and the combination of calcium in conjunction with amino acid fragment, restructuring silk is designed to lamellar structure, that is, (AGSGAG)
6form β-sheet to fold, and calcium binding amino acid sequence is exposed to β-sheet and folds outside crystallizing field, promotes the formation of hydroxylapatite crystal with calcium binding.
2, the sign of the biomineralization function of repeated fragment (AGSGAG) 6ASEYDYDDDSDDDDEWD
After determining two different fragments combinations, whether there is biomineralization function, first will to the polypeptide (AGSGAG) of synthesis
6aSEYDYDDDSDDDDEWD carries out biomineralization function sign.
First calcium-ion-binding ability test is carried out: by the polypeptide aCl of above-mentioned synthesis
2solution soaking 0.5h, then carries out stain test with Feng Kusa dye liquor, and coloration result clearly can observe polypeptide laking, illustrates that this polypeptide is provided with stronger calcium-ion-binding ability.
Then biomineralization and sign is carried out: add 1.5SBF solution at the polypeptide solution of above-mentioned synthesis, and concussion makes to dissolve completely, mineralising is induced at 37.2 DEG C, 1,3,5, sample respectively during 7d, by sample after lyophilize, dry again after cleaning, detect for X-ray diffraction (XRD) and infrared absorption spectrum (FT-IR).Polypeptide after induction mineralising, occur obvious peak in 2 θ=32.77 of XRD figure spectrum, this peak is the characteristic peak of hydroxylapatite crystal, confirms the generation of hydroxylapatite crystal.1036cm in infrared absorption spectrum
-1locate v in corresponding HAp molecule
3pO
3-the stretching vibration of key, 596cm
-1locate v in corresponding HAp molecule
4pO
-3the flexural vibration of key are all charateristic avsorption bands of hydroxylapatite crystal.Use after 1.5SBF solution mineralising 7d, polypeptide powder is at 1036cm
-1and 596cm
-1all there is obvious absorption peaks in place, the appearance of these characteristic peaks illustrates, under 37.2 DEG C of conditions, through mineralising in 1.5SBF solution, polypeptid induction generates hydroxylapatite crystal, demonstrates this polypeptide and has biomineralization function.
1.5SBF solution (simulatedbodyfluid simulated body fluid) refers to 1.5 times of standard SBF.SBF has disclosed standard recipe, belongs to changeless prior art content, and 1.5SBF is exactly 1.5 times of preparations according to standard.
3, the constructing of cloning vector and expression vector
To above-mentioned (AGSGAG)
6the Nucleotide of ASEYDYDDDSDDDDEWD is optimized, and is cloning vector with pUC57, obtains ((AGSGAG)
6aSEYDYDDDSDDDDEWD)
2cloning vector and expression vector.
4, target protein gene order is imported silkworm
By signal peptide, the method for target protein gene order fusion DNA vaccine couples together, and obtains LSP+SM2 fragment.Again this fragment is connected with promotor fusion DNA vaccine, obtains a complete expression cassette.With " Visuble self-sucking extrinsic gene induction of insect egg " (Chinese invention patent 200410073439.3), above-mentioned plasmid is mixed in concentration 1:1 ratio with providing the helper plasmid of transposase gene, import to P50 Silkworm varieties lay eggs after in fertilization silkworm seed within 8h.To import the silkworm seed of expression plasmid by 25 DEG C, 90% humidity cultivation hatching, raise the silkworm (G0 generation) of hatching with mulberry leaf, the selfing of experiment silkworm moth is laid eggs (G1 generation).
5, the detection of transgenic bombyx mori
G1 silkworm seed is pressed silkworm egg hatching breeding to two age, detect with fluor stereomicroscope, the individuality that every silkworm body can be observed obvious green eye is transgenic positive silkworm, and desired polypeptides sequence has proceeded to domestic silkworm gene group.Transgenic positive of selecting and remain silkworm rearing is to matured silkworm.
6, the detection of desired polypeptides sequence
Dissect transgenic positive matured silkworm, take out the silk fibroin in sericterium body, carry out biomineralization and sign by the method for above-mentioned 2nd, prove that the fibroin that transgenic positive silkworm secretes has biomineralization function, successfully obtain the dedicated silkworm kind of secretion medical silk.
The all distortion derived from content disclosed by the invention or associate it is to be noted that above enumerating is only some specific embodiments of the present invention, the invention is not restricted to above example, also can have many distortion, if all should think protection scope of the present invention.
Claims (1)
1. secrete the dedicated silkworm breed breeding method of medical silk for one kind, it is characterized in that, that calcium functional gene territory is operated by genetically engineered, the carrier with reporter gene based on the PiggyBAC factor, build restructuring transgene carrier, obtain the silkworm with reporter gene with Visuble self-sucking extrinsic gene induction of insect egg, and biomineralization function detected in the fibroin of transgenic bombyx mori, be bred as and can secrete the medical new silkworm variety with biomineralization function; Described calcium functional gene territory is the design by recombinant protein molecule, i.e. the crystallizable fragment (AGSGAG) of silk fibroin protein primary structure
6combine with the aminoacid sequence YDYDDDSDDDDEWD in the conchiolin MSI60 of black lip, design can promote the recombinant fibroin of biomineralization function, and its primary sequence is ((AGSGAG)
6aSEYDYDDDSDDDDEWD) n; By crystallizing field and the combination of calcium in conjunction with amino acid fragment, restructuring silk is designed to lamellar structure, namely (AGSGAG) 6 formation β-sheet are folding, and calcium binding amino acid sequence is exposed to β-sheet folds outside crystallizing field, promote the formation of hydroxylapatite crystal with calcium binding;
Described method specifically comprises the steps:
(1) selection of function fragment and combination
Adopt the crystallizable fragment (AGSGAG) of silk fibroin protein primary structure
6combine with the aminoacid sequence YDYDDDSDDDDEWD in black lip conchiolin MSI60, design can promote the recombinant fibroin of biomineralization function, and its primary sequence is ((AGSGAG)
6aSEYDYDDDSDDDDEWD)
n; By crystallizing field and the combination of calcium in conjunction with amino acid fragment, restructuring silk is designed to lamellar structure, that is, (AGSGAG)
6form β-sheet to fold, and calcium binding amino acid sequence is exposed to β-sheet and folds outside crystallizing field, promotes the formation of hydroxylapatite crystal with calcium binding;
(2) repeated fragment (AGSGAG)
6the sign of the biomineralization function of ASEYDYDDDSDDDDEWD
After determining two different fragments combinations, whether there is biomineralization function, first will to the polypeptide (AGSGAG) of synthesis
6aSEYDYDDDSDDDDEWD carries out biomineralization function sign;
First calcium-ion-binding ability test is carried out: by the polypeptide CaCl of above-mentioned synthesis
2solution soaking 0.5h, then carries out stain test with Feng Kusa dye liquor; Coloration result clearly can observe polypeptide laking, illustrates that this polypeptide is provided with stronger calcium-ion-binding ability;
Then biomineralization and sign is carried out: add 1.5SBF solution at the polypeptide solution of above-mentioned synthesis, and concussion makes to dissolve completely, mineralising is induced at 37.2 DEG C, 1,3,5, sample respectively during 7d, by sample after lyophilize, dry again after cleaning, detect for X-ray diffraction and infrared absorption spectrum; Polypeptide after induction mineralising, can compose the characteristic peak of existing hydroxylapatite crystal in XRD figure; This confirms the generation of hydroxylapatite crystal, and this polypeptide has biomineralization function;
(3) the constructing of cloning vector and expression vector
To above-mentioned (AGSGAG)
6the Nucleotide of ASEYDYDDDSDDDDEWD is optimized, and is cloning vector with pUC57, obtains ((AGSGAG)
6aSEYDYDDDSDDDDEWD)
2cloning vector and expression vector;
(4) target protein gene order is imported silkworm
The method of signal peptide, target protein gene order fusion DNA vaccine is coupled together, then this fragment is connected with promotor fusion DNA vaccine, obtain a complete expression cassette; With Visuble self-sucking extrinsic gene induction of insect egg, above-mentioned plasmid is mixed in concentration 1:1 ratio with providing the helper plasmid of transposase gene, import to P50 Silkworm varieties lay eggs after in fertilization silkworm seed within 8h; To import the silkworm seed of expression plasmid by 25 DEG C, 90% humidity cultivation hatching, raise the silkworm G0 generation of hatching with mulberry leaf, the selfing of experiment silkworm moth is laid eggs G1 generation;
(5) detection of transgenic bombyx mori
G1 silkworm seed is pressed silkworm egg hatching breeding to two age, detect with fluor stereomicroscope, the individuality that every silkworm body can be observed obvious green eye is transgenic positive silkworm, shows that desired polypeptides sequence has proceeded to domestic silkworm gene group; Transgenic positive of selecting and remain silkworm rearing is to matured silkworm.
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| CN103688910B (en) * | 2014-01-09 | 2015-04-15 | 浙江省农业科学院 | Method for inducing silkworm meiosis type parthenogernesis by liquid refrigerants |
| CN106480199B (en) * | 2016-10-31 | 2019-11-05 | 广西壮族自治区蚕业技术推广总站 | The homozygous method for genetic of single copy transgenic bombyx mori strain |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101177692A (en) * | 2007-12-05 | 2008-05-14 | 浙江大学 | Method for developing transgenic domestic silkworm by using A3 promotor defect piggyBac transposon |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101177692A (en) * | 2007-12-05 | 2008-05-14 | 浙江大学 | Method for developing transgenic domestic silkworm by using A3 promotor defect piggyBac transposon |
Non-Patent Citations (2)
| Title |
|---|
| Conformational Study of Silklike Peptides Modified by the Addition of the Calcium-Binding Sequence from the Shell Nacreous Matrix Protein MSI60 Using 13C CP/MAS NMR Spectroscopy;Tetsuo Asakura, Megumi Hamada, Sung-Won Ha, David P.;《Biomacromolecules》;20060630;第7卷(第6期);721-729 * |
| Synthesis and characterization of silklike materials containing the calcium-binding sequence from calbindin D9k or the shell nacreous matrix protein MSI60;Yang M, Muto T, Knight D, Collins AM, Asakura T.;《Biomacromolecules》;20071208;第9卷(第1期);第416页摘要，正文第1、3-5段，第417页第1-2段 * |
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