WO2017067423A1 - 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法 - Google Patents

利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法 Download PDF

Info

Publication number
WO2017067423A1
WO2017067423A1 PCT/CN2016/102266 CN2016102266W WO2017067423A1 WO 2017067423 A1 WO2017067423 A1 WO 2017067423A1 CN 2016102266 W CN2016102266 W CN 2016102266W WO 2017067423 A1 WO2017067423 A1 WO 2017067423A1
Authority
WO
WIPO (PCT)
Prior art keywords
silkworm
gene
black widow
silk
widow spider
Prior art date
Application number
PCT/CN2016/102266
Other languages
English (en)
French (fr)
Inventor
钟伯雄
Original Assignee
浙江大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN201510698099.1A external-priority patent/CN105400815B/zh
Priority claimed from CN201510697644.5A external-priority patent/CN105463022B/zh
Priority claimed from CN201510698879.6A external-priority patent/CN105400817B/zh
Application filed by 浙江大学 filed Critical 浙江大学
Publication of WO2017067423A1 publication Critical patent/WO2017067423A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
    • A01K67/04—Silkworms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/866—Baculoviral vectors

Definitions

  • the invention relates to a method for synthesizing secreted foreign proteins, in particular to a method for synthesizing secreted black widow spider traction silk protein by using a silkworm gland bioreactor using transgenic technology.
  • Spider silk is a natural high molecular protein fiber secreted by spider silk gland. It has excellent mechanical properties such as good elasticity, high strength, toughness, high temperature and low temperature resistance, impact resistance, small specific gravity and good biocompatibility. As well as excellent properties such as biodegradability, its unique comprehensive properties are incomparable with other natural and synthetic fibers.
  • the dragline silk which is secreted by the major saccular glands, is radially distributed and forms the basic framework of the spider web.
  • the strength of the traction wire is five times that of steel, so it has the reputation of “bio-steel”.
  • the protein gene of the traction wire is highly conserved, and is mainly composed of two pairs of proteins, the paired traction silk protein I (MaSp1) and the traction silk protein II (MaSp2), having a molecular weight of about 350 kDa.
  • the black widow spider (Latrodectus hesperus) is the most powerful traction wire in spiders.
  • the black widow spider traction silk protein I (MaSp1) gene has a single exon, 9390 bp in length, encoding 3129 amino acids, the gene contains a large number of repeat units, and each repeat unit consists of 4 primary type repeats Type1, Type2.
  • Type 3 and Type 4 are formed by concatenation of head and tail (Type1-Type2-Type3-Type4), and each of the primary type repeats contains typical (GA)n, An, and GGX protein motifs.
  • MaSpI is considered to be the main molecular basis for the strong strength of the traction wire.
  • the black widow spider traction silk protein II (MaSp2) gene has a single exon, 11340 bp in length, encoding 3779 amino acids.
  • the gene contains a large number of repeating units, and each repeat unit consists mainly of two primary type repeats of Type1-Type1.
  • -Type4 is made up in tandem, and each of the primary type repeats contains typical An, GPGXX, and GGX protein motifs.
  • MaSp2 is considered to be the main molecular basis for the toughness of the traction wire.
  • Heterologous expression systems include Escherichia coli and Pichia pastoris. Insect cells (sf9, BmN), hamster kidney cells, transgenic potatoes, transgenic tobacco, transgenic goats, transgenic mice, and transgenic silkworms.
  • the spider silks expressed are mainly Nephila clavipes and Nephila spiders. Clavata), a sequence fragment of four spider silks of Araneus ventricosus and Araneus diadematus.
  • the genes used in various heterologous expression systems used so far are not the black widow spider traction silk gene with the best mechanical properties, and the expressed spider silk protein has a small molecular weight, and many recombinant proteins lack some key proteins.
  • the motif, which has more proteins than pure spider silk protein molecules, is a fusion protein that is fused to molecules such as fluorescent proteins, which ultimately affect the mechanical properties of the expressed product. Therefore, the prior art lacks a method for synthesizing bionic spider silk with excellent secretion properties.
  • the object of the present invention is to provide a method for synthesizing secreted black widow spider traction silk protein using a silkworm gland bioreactor.
  • the black widow spider silk fibroin I gene and protein II gene are respectively introduced into the silkworm genome by transgenic silkworm technology, and are specifically expressed in the silk gland cells, respectively, and the black silk widow spider silk protein I can be separately synthesized and secreted.
  • protein II of the transgenic silkworm is a transgenic silkworm
  • the two transgenic silkworm cultivars are crossed and the hybrid cultivars are bred, and the hybrid cultivars are self-crossed with the moth-positive individuals, and the cultivar homozygous for both the silk fibroin I gene and the protein II gene is selected.
  • transgenic silkworm technology combined with cross breeding technology, the black widow spider traction silk protein I and protein II genes were simultaneously introduced into the silkworm genome and specifically expressed in silk gland cells, and the simultaneous synthesis and secretion of black widow spider traction silk protein I and protein were developed.
  • the silkworm of II one of the bionic silk fibers of the hybrid is composed of the traction silk protein I and the protein II.
  • the invention obtains a super-bionic spider silk which is similar to the natural black widow spider traction wire in synthesizing the black silk widow spider traction silk protein, and has similar performance and even surpasses the natural black widow spider traction wire.
  • the pBac[3xP3-DsRed]-MaSp1 plasmid was constructed by molecular biology method as the black widow spider traction silk protein I gene vector expressed in the silk gland of the silk, and the pBac[3xP3-DsRed]-MaSp1 plasmid was shown in Figure 1-3. It is indicated that the pBac[3xP3-DsRed]-MaSp1 plasmid comprises a black widow spider traction silk protein I gene containing a foreign gene and a red fluorescent DsRed gene expression cassette as a marker gene based on a piggyBac transposon;
  • the pBac[3xP3-DsRed]-MaSp1 plasmid and the helper plasmid pHA3PIG capable of providing piggyBac transposase were introduced into the fertilized egg within 6 hours after the silkworm lays eggs at a concentration ratio of 1:1.
  • the black widow spider traction silk protein I gene was inserted into the silkworm genome using the piggyBac transposon in the pBac[3xP3-DsRed]-MaSp1 plasmid;
  • G1 generation After the eggs are hatched, they are reared to adult worms, and then they are mixed with non-transgenic silkworms to prepare the cultivars. This generation is G1 generation.
  • G1 generation In the greening stage of G1 silkworm eggs, single-eye expression of red fluorescent DsRed markers is screened by fluorescence stereomicroscope. The transgenic silkworm of the gene is raised to the adult and then mixed with the non-transgenic silkworm to form a G2 generation;
  • G3 generation silkworms adopt single moth breeding, and silkworm moths expressing red fluorescent DsRed marker genes in the moth area are mated to each other to form G4 generation;
  • the red-eye gene obtained by the step A6) and the black widow spider traction silk protein I gene are homozygous, and the silkworm gland cells capable of synthesizing the transgenic silkworm cultivar secreting the black widow spider traction silk protein I can synthesize and secrete the black widow spider traction silk protein I, And with the silkworm silkworm crusting behavior into the silkworm cocoon.
  • the pBac[3xP3-DsRed]-MaSp2 plasmid was constructed by molecular biology method as the black widow spider traction silk protein II gene vector expressed in silk gland of the silk, and the pBac[3xP3-DsRed]-MaSp2 plasmid was shown in Fig. 4 to Fig. 5
  • the pBac[3xP3-DsRed]-MaSp2 plasmid comprises two expression cassettes of a black widow spider traction silk protein II gene as a foreign gene and a red fluorescent DsRed gene as a marker gene based on a piggyBac transposon;
  • the pBac[3xP3-DsRed]-MaSp2 plasmid and the helper plasmid pHA3PIG capable of providing the piggyBac transposase were introduced into the fertilized egg within 6 hours after the silkworm was laid at a concentration ratio of 1:1.
  • the black widow spider traction silk protein II gene was inserted into the silkworm genome using the piggyBac transposon in the pBac[3xP3-DsRed]-MaSp2 plasmid;
  • the silkworm eggs are raised to adult worms, and then mixed with non-transgenic silkworms to prepare cultivars.
  • This generation is G1 generation.
  • single-eye expression of red fluorescent DsRed markers was screened by fluorescence stereomicroscope.
  • the transgenic silkworm of the gene is raised to the adult and then mixed with the non-transgenic silkworm to form a G2 generation;
  • the G2 generation silkworm adopts single moth breeding, and the silkworm expressing the red fluorescent DsRed marker gene under the fluorescence microscope is screened, and the G3 generation is made by mating with the moth moth in the same moth area;
  • the G3 generation silkworm adopts a single moth, and the silk moths expressing the red fluorescent DsRed marker gene in the moth area are mated to each other to form the G4 generation;
  • the red-eye gene obtained by the step B6) and the black widow spider traction silk protein II gene are homozygous, and the silkworm gland cells capable of synthesizing the transgenic silkworm cultivar secreting the black widow spider traction silk protein II can synthesize and secrete the black widow spider traction silk protein II. And with the silkworm silkworm crusting behavior into the silkworm cocoon.
  • F1 generation hybrids were homozygous for the F2 generation, and the F2 generation silkworms were treated with single moths, and the silkworms expressing the red fluorescent DsRed marker gene under the fluorescence microscope were screened. Mating with each other to make F3 generation;
  • F3 generation silkworms were treated with single moths, and all the individuals expressing the red fluorescent DsRed marker gene under the fluorescence microscope were screened in the same moth area, and simultaneously detected by Western Blot technique, and the black widow spiders were simultaneously screened.
  • the silkworm moths of the moth area of the traction silk protein I and the protein II are mated to each other to form the F4 generation;
  • the plasmid pBac[3xP3-DsRed]-MaSp1 is based on a piggyBac transposon and carries an Amp resistance gene, including two transposable arms PBL and PBR of a piggyBac transposon, and two transposome arms.
  • Two functional expression cassettes one of which is the red fluorescent protein gene expression cassette initiated by the 3 ⁇ P3 promoter, namely 3 ⁇ P3Promoter–DsRed-SV40, and the other functional expression cassette contains the silk fibroin heavy chain gene.
  • the plasmid pBac[3xP3-DsRed]-MaSp2 is based on a piggyBac transposon and carries an Amp resistance gene, including two transposable arms PBL and PBR of a piggyBac transposon, and two transposable arms.
  • Two functional expression cassettes one of which is the red fluorescent protein gene expression cassette initiated by the 3 ⁇ P3 promoter, namely 3 ⁇ P3Promoter–DsRed-SV40, and the other functional expression cassette contains the silk fibroin heavy chain gene.
  • the black widow spider traction silk protein I gene comprises a 2-fold to 16-fold MaSp1 gene repeating unit Type1-Type2-Type3-Type4, as shown in Figures 1-3.
  • the black widow spider traction silk protein II gene comprises 3 to 24 times the MaSp2 gene repeating unit Type1-Type1-Type4, as shown in Figures 4-5.
  • the invention firstly constructs a vector pBac[3xP3-DsRed]-MaSp1 and pBac[3xP3-DsRed]-MaSp2 for synthesizing the silk fibroin I gene and protein II gene of the black widow spider, and then using the microinjection transgenic silkworm technology.
  • These five plasmids were introduced into the silkworm fertilized egg together with the helper plasmid pHA3PIG plasmid (shown in Figure 6) which can provide the piggyBac transposase, and the red fluorescent protein gene and the black widow were made by the transposable nature of the piggyBac transposon.
  • the spider silk protein I gene, as well as the red fluorescent protein gene and the black widow spider traction silk protein II gene were introduced into the silkworm genome, and stably inherited and expressed, thereby creating a black widow that can specifically synthesize and secrete silk fibroblasts in the body.
  • transgenic silkworm of the spider silk protein I gene and the protein II gene self-crossing, homozygous the black widow spider traction silk protein I gene and protein II gene, and bred a transgenic silkworm capable of secreting black widow spider traction silk protein I and protein II; Hybridization of the above two transgenic silkworms, breeding of the silk fibroin I gene and protein II base by hybrid breeding technology Homozygous hybrids, then using this type of synthesis and secretion of silkworm black widow spider dragline silk.
  • the invention screens the transgenic silkworm by means of the fluorescent marker gene, and the transgenic silkworm can specifically synthesize and secrete the black widow spider traction silk protein I and protein II in the silk fibroblasts, respectively, and the black widow spider traction silk protein I and protein II have functional activity.
  • bionic silk fiber structure obtained by the invention is similar to the natural black widow spider traction wire, and is a super bionic spider silk with close performance or even surpassing the natural black widow spider traction wire.
  • the present invention develops a novel production process capable of separately or simultaneously obtaining black widow spider traction silk protein I and protein II, laying a foundation for separately or simultaneously mass production of black widow spider traction silk protein I and protein II.
  • the performance is close to, even beyond the super black bionic spider silk of the natural black widow spider laying the foundation, also laid the foundation for improving the mechanical properties of silk.
  • Fig. 1 is a diagram showing the structure of pBac[3 ⁇ P3-DsRed]-MaSp1 ⁇ 2 plasmid synthesized by the silk gland cells of the silkworm, which is secreted and secreted by recombinant black widow.
  • Fig. 2 is a diagram showing the structure of pBac[3 ⁇ P3-DsRed]-MaSp1 ⁇ 12 plasmid synthesized by the silk gland cells of the silkworm, which secretes and recombines the recombinant black widow.
  • Fig. 3 is a diagram showing the structure of pBac[3 ⁇ P3-DsRed]-MaSp1 ⁇ 16 plasmid synthesized by the silk gland cells of the silkworm, which is secreted and secreted by the black widow.
  • Fig. 4 is a diagram showing the plasmid structure of pBac[3 ⁇ P3-DsRed]-MaSp2 ⁇ 24 synthesized by the silk gland cells of the silkworm, which is secreted and secreted by the recombinant black widow.
  • Figure 5 is a pBac synthesis and secretion of recombinant black widow traction silk protein II in the silk gland cells of the silkworm, Bombyx mori [3 ⁇ P3-DsRed]-MaSp2 ⁇ 3 plasmid structure map.
  • Figure 6 is a diagram showing the structure of a helper plasmid of the piggyBac transposase involved in the present invention.
  • the pBac[3xP3-DsRed]-MaSp1 plasmid of the present invention in the following examples was prepared in the following manner:
  • the silk fibroin heavy chain protein gene promoter (Fib-HP) was ligated into the pMD18-T simple (Takara) vector, and the basic piggyBac plasmid piggy6212 vector containing the A3 promoter and the green fluorescent protein expression cassette was passed. XhoI/NcoI double digestion, the gel-recovery heavy chain promoter sequence was ligated to the piggyBac vector backbone to obtain the piggy6609 vector.
  • the plasmid was designed according to the sequence characteristics of the full-length black widow spider traction silk protein I gene (MaSp1), and the base was optimized according to the codon preference of the silkworm. The detailed information is as follows.
  • a signal peptide of a silk fibroin heavy chain protein gene, a repeating unit sequence of a 2-fold MaSp1 molecule (the base sequence and the expressed protein sequence are SEQ ID NO. 1 and SEQ ID NO. 2, respectively), and a MaSp1 molecule
  • the C-terminus, the C-terminus of the silk fibroin heavy chain protein gene and the sequence of PolyA, and the sequence of the restriction enzyme site required for subsequent vector construction, were ligated to the pUC57 vector and designated as pUC4423 plasmid, which contains 2 Repeat the sequence of the MaSp1 gene.
  • the pUC4423 plasmid was digested with two homologous enzymes SpeI and NheI, respectively, and sequentially ligated with different multiple repeating unit sequences.
  • the specific steps of the superposition process are as follows:
  • the pUC4423 plasmid was digested with SpeI/Hind ⁇ , and a fragment of 1698 bp containing 2 times of the MaSp1 gene repeat unit was recovered.
  • pUC4423 was digested with NheI/Hind ⁇ , and a fragment of 3457 bp containing 2 times of the MaSp1 gene repeat unit was recovered, and the above 1698 bp was ligated. Fragment and 3457 bp fragment, obtained pUC5155 plasmid (containing 4 times the MaSp1 gene repeat unit);
  • pUC5155 plasmid was digested with SpeI/Hind ⁇ , and a fragment of 2430 bp containing 4 times of the MaSp1 gene repeat unit was recovered.
  • pUC5155 was digested with NheI/Hind ⁇ , and a fragment of 4189 bp containing 4 times of the MaSp1 gene repeat unit was recovered, and the above 2430 bp was ligated. Fragment and 4189 bp fragment, obtained pUC6619 plasmid (containing 8-fold MaSp1 gene repeat unit);
  • the pUC6619 plasmid was digested with SpeI/Hind ⁇ , and a fragment of 3894 bp containing 8 times of the MaSp1 gene repeat unit was recovered.
  • pUC6619 was digested with NheI/Hind ⁇ , and a fragment of 5653 bp containing 8 times of the MaSp1 gene repeat unit was recovered, and the above 3894 bp was ligated. Fragment and 5653 bp fragment, the pUC9547 plasmid was obtained, containing 16-fold MaSp1 gene repeat unit;
  • the above 2430 bp fragment (containing a 4-fold MaSp1 fragment) was ligated with a 5653 bp fragment (containing an 8-fold MaSp1 gene repeat unit) to obtain a pUC8083 plasmid containing 12-fold MaSp1 gene repeat unit;
  • the above plasmid containing the repeating unit sequence of the MaSp1 gene of 2, 8, 12, and 16 is digested with NcoI/MunI to recover a sequence fragment containing the repeating unit fragments of different lengths and the constituent elements, and the above-mentioned A) has been constructed to contain the heavy weight.
  • the chain promoter and the piggyBac backbone vector piggy6609 were digested with the same restriction endonuclease, and the target fragment containing the piggyBac vector backbone and the heavy chain promoter sequence element was recovered and ligated to the above-mentioned different length repeat unit fragments to obtain piggy7540, Piggy9736, piggy11200 and piggy12664 vectors, which contain 2-fold, 8-fold, 12-fold and 16-fold MaSp1 gene repeat unit sequences, respectively.
  • the piggy7540, piggy9736, piggy11200 and piggy12664 vectors were digested with BglII/AflII, respectively, and the target fragment containing the MaSp1 gene repeat unit and the piggyBac vector backbone of different lengths was recovered, and the 3 ⁇ P3 promoter-red eye gene expression cassette and A3 were contained.
  • the promoter-green fluorescent protein expression cassette vector piggy7785 was digested with the same enzyme, and a 1230 bp target fragment containing the 3 ⁇ P3 promoter and the DsRed coding frame of the marker gene was recovered, and the two target fragments were ligated to add the DsRed marker gene.
  • the specific steps are as follows:
  • the piggy7785 vector was digested with AflIII/BglII, and a 1230 bp fragment was recovered.
  • the piggy7540 vector was digested with AflII/BglII, and the 7198 bp fragment was recovered and ligated with the 1230 bp fragment to obtain pBac[3xP3-DsRed]-MaSp1 ⁇ 2 vector (containing 2-fold MaSp1 gene repeat unit) (Fig. 1);
  • the piggy11200 vector was digested with AflII/BglII, and a 10858pb fragment was recovered and ligated with the 1230pb fragment to obtain pBac[3xP3-DsRed]-MaSp1 ⁇ 12 vector (containing 12-fold MaSp1 gene repeat unit) (Fig. 2);
  • the piggy12664 vector was digested with AflII/BglII, and a 12322 bp fragment was recovered and ligated with a 1230 bp fragment to obtain pBac[3xP3-DsRed]-MaSp1 ⁇ 16 vector (containing 16-fold MaSp1 gene repeat unit) (Fig. 3).
  • the pBac[3xP3-DsRed]-MaSp2 plasmid in the examples of the present invention was prepared by the following method:
  • the silk fibroin heavy chain protein gene promoter (Fib-HP) was ligated into the pMD18-T simple (Takara) vector, and the basic piggyBac plasmid piggy6212 vector containing the A3 promoter and the green fluorescent protein expression cassette was passed. XhoI/NcoI double digestion, the gel-recovery heavy chain promoter sequence was ligated to the piggyBac vector backbone to obtain the piggy6609 vector.
  • the plasmid was designed according to the sequence characteristics of the full-length black widow spider traction silk protein II gene (MaSp2), and the base was optimized according to the codon preference of the silkworm. The detailed information is as follows.
  • MaSp2 gene repeat unit sequence The signal peptide of the silk fibroin heavy chain protein gene, the 3 fold MaSp2 gene repeat unit sequence (the base sequence and the expressed protein sequence are SEQ ID NO. 3 and SEQ ID NO. 4, respectively), and the MaSp2 molecule C
  • the pUC4301 plasmid was digested with two homologous enzymes SpeI and NheI, respectively, and the repeat unit sequences of different multiples were sequentially connected.
  • the specific steps of the superposition process are as follows:
  • the pUC4301 plasmid was digested with SpeI/Hind ⁇ , and a fragment of 1629 bp containing 3 times MaSp2 gene was recovered.
  • pUC4301 was digested with NheI/Hind ⁇ , and a fragment of 3326 bp containing 3 times of the MaSp2 gene repeat unit was recovered, and the 1629 bp fragment was ligated and 3326 bp fragment, obtaining pUC4955 plasmid (containing 6-fold MaSp2 gene repeat unit);
  • the pUC4955 plasmid was digested with SpeI/Hind ⁇ as a vector, and a fragment of 2283 bp containing MaSp2 gene was recovered.
  • the pUC4955 was digested with NheI/Hind ⁇ , and a fragment of 3980 bp containing 6-fold MaSp2 gene repeat unit was recovered, and the above 2283 bp was ligated. Fragment and 3980 bp fragment, obtaining pUC6263 plasmid containing 12-fold MaSp2 gene repeat unit;
  • the pUC6263 plasmid was digested with SpeI/Hind ⁇ , and a fragment of 3961 bp containing 12-fold MaSp2 gene repeat unit was recovered.
  • pUC6263 was digested with NheI/Hind ⁇ , and a fragment of 5288 bp containing 12-fold MaSp2 gene repeat unit was recovered and ligated to the above 3591 bp. Fragment and 5288 bp fragment, the pUC8879 plasmid was obtained, containing 24 times the MaSp2 gene repeat unit;
  • the plasmid containing the different multiples of the MaSp2 gene repeat unit sequence obtained above was digested with NcoI/MunI, and the sequence fragment containing the repeat fragment and the constituent elements was recovered, and the heavy chain promoter containing the A) step was constructed and
  • the piggyBac backbone vector Piggy6609 was digested with the same restriction endonuclease, and the target fragment containing the piggyBac vector backbone and the heavy chain promoter sequence element was recovered and ligated to the above-described different length repeat unit fragments to obtain piggy 7701, and piggy12049 vectors.
  • These two plasmids contain three-fold and 24-fold MaSp2 gene repeat unit sequences, respectively.
  • the piggy7471 and piggy12049 vectors were digested with BglII/AflII, respectively, and the target fragment containing the MaSp2 gene repeat unit of different length and the piggyBac vector backbone was separately recovered, and the 3 ⁇ P3 promoter-red eye gene expression cassette and the A3 promoter-green were contained.
  • the fluorescent protein expression cassette vector piggy7785 was digested with the same enzyme, and a 1230 bp target fragment containing the 3 ⁇ P3 promoter and the DsRed coding frame of the marker gene was recovered, and the two target fragments were ligated to add the DsRed marker gene. The specific steps are as follows:
  • the piggy7785 vector was digested with AflIII/BglII, and a 1230 bp fragment was recovered.
  • the piggy7471 vector was digested with AflII/BglII, and the 7129 bp target fragment containing the MaSp2 repeat unit and the piggyBac vector backbone was recovered and ligated with the 1230 bp fragment to obtain the pBac[3xP3-DsRed]-MaSp2 ⁇ 3 vector (containing 3 times the MaSp2 gene repeat).
  • a unit, wherein the repeat unit of the 3X MaSp2 gene is SEQ ID NO. 3, and the expressed protein sequence is SEQ ID NO. 4) (Fig. 5);
  • the piggy12049 vector was digested with AflII/BglII, and the 11707 bp target fragment containing the MaSp2 repeat unit and the piggyBac vector backbone was recovered and ligated with a 1230 bp size to obtain pBac[3xP3-DsRed]-MaSp2 ⁇ 24 vector (containing 24 times MaSp2 gene repeat). Unit) ( Figure 4).
  • Example 1-3 Synthetic secretion of silk fibroin I by black widow spider
  • the pBac[3xP3-DsRed]-MaSp1 ⁇ 2 plasmid (Fig. 1) constructed above and the helper plasmid pHA3PIG plasmid (Fig. 6) capable of providing the piggyBac transposase were mixed at a ratio of 1:1.
  • the microinjected silkworm eggs were raised to adults at 25 ° C and 85% humidity, and crossed with non-transgenic silkworms for G1 generation.
  • the transgenic silkworm 2 moth expressing the DsRed marker gene was observed by fluorescence microscopy (Olympus, SZX12, Japan), and the silkworm was reared to the adult and the non-transgenic silkworm was crossed and passed, which was G2. Since the G2 generation, the transgenic silkworms have adopted single moth breeding.
  • the transgenic silkworms expressing the DsRed marker gene were selected by fluorescence microscopy, and they were raised to adults and mated with the moth area to make the black widow spider pull silk protein I. The gene is homozygous, and the G3 generation and the G4 generation are further cultivated.
  • the genomic DNA of the silk gland cells of the 5th instar of the 5th instar was used as a template, and the insert of the black widow spider traction silk protein I gene in the silkworm genome was analyzed by Inverse PCR, and the amplified fragment was cloned, sequenced and sequenced. Chromosome localization analysis showed that the progeny insertion sites of the two moths were at the 14033724 locus of chromosome 3, which proved that the transposon had been inserted into the silkworm genome.
  • the red-eyed genotype homozygous moth area was selected, and the red-eye gene was homozygously mated with the moth moth, and the rear silk gland cells were able to synthesize a new transgenic silkworm variety that secreted the black widow spider traction silk protein I. MASP1-2.
  • the silkworm protein of MASP1-2 silkworm was extracted as the material.
  • the expression of MASP1-2 protein in transgenic silkworm was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody.
  • the specific protein was obtained according to the expected molecular weight. Bands.
  • the gray scale analysis of SDS-PAGE electrophoresis showed that the black widow spider traction silk protein I accounted for 3.1% of the silk light chain.
  • the pBac[3xP3-DsRed]-MaSp1 ⁇ 12 plasmid (Fig. 2) constructed above was introduced into the silkworm in the same manner as in Example 1, and the expression of DsRed was obtained by fluorescence microscopy in the G1 generation of the transgenic experiment.
  • the transgenic silkworm 10 moth of the marker gene is then incubated until the G3 generation and the G4 generation are obtained.
  • the red-eyed genotype homozygous moth area was selected, and the red-eye gene was homozygously mated with the moth moth, and the rear silk gland cells were able to synthesize a new transgenic silkworm variety that secreted the black widow spider traction silk protein I. MASP1-12.
  • the silkworm silk fibroin of the four families of the above-mentioned insertion sites was extracted as the material.
  • the expression of MASP1-12 protein in transgenic silkworm was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody. The results were obtained with the expected molecular weight. A specific protein band of consistent size.
  • the gray scale analysis of SDS-PAGE electrophoresis showed that the black widow spider silk protein I accounted for up to 2.4% of the silk light chain.
  • the pBac[3xP3-DsRed]-MaSp1 ⁇ 16 plasmid (Fig. 3) constructed above was introduced into the silkworm in the same manner as in Example 1, and the DsRed marker gene was obtained by fluorescence microscopy during the G1 generation.
  • the red-eyed genotype homozygous moth area was selected, and the red-eye gene was homozygously mated with the moth moth, and the rear silk gland cells were able to synthesize a new transgenic silkworm variety that secreted the black widow spider traction silk protein I. MASP1-16.
  • the silkworm silk fibroin of the four families of the above-mentioned insertion sites was extracted as the material.
  • the expression of MASP1-16 protein in transgenic silkworm was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody. The results were obtained with the expected molecular weight. A specific protein band of consistent size.
  • the gray scale analysis of SDS-PAGE electrophoresis showed that the black widow spider traction silk protein I accounted for up to 2.3% of the silk light chain.
  • the black widow spider traction silk protein I can be efficiently synthesized in the silk gland cells of the silk, and the black widow spider traction silk protein I can be secreted into the glandular cavity by the silk gland like silk. Further spit out the silkworm. This trait has been stably expressed and inherited.
  • the method can mass produce black widow spider traction silk protein I, can improve the mechanical properties of silk, can improve the economic benefits of silkworm and increase the income of silkworm farmers.
  • Example 4-8 Synthetic secretion of silk fibroin II by black widow spider
  • the pBac[3xP3-DsRed]-MaSp2 ⁇ 24 plasmid (Fig. 4) constructed above was introduced into the silkworm according to the method of Example 1.
  • the transgenic silkworm expressing the DsRed marker gene was obtained by fluorescence microscopy during the G1 generation. 1 moth, then incubated until G3 generation, G4 generation.
  • the genomic DNA of the silk gland cells of the 5th instar of the 5th instar of the 5th instar was used as a template, and the chromosome insertion site analysis was carried out in the same manner as in Example 1. The result showed that the progeny insertion site of this moth area was inserted. At the 2966967 locus on chromosome 22, it was confirmed that the transposon has been inserted into the silkworm genome.
  • the red-eyed genotype homozygous moth area was selected, and the red-eye gene was homozygous and the rear silk gland cells were able to synthesize a new transgenic silkworm variety that secreted black widow spider traction silk protein II. MASP2-24-1.
  • the silkworm protein of MASP2-24-1 silkworm was extracted as material.
  • the expression of MASP2-24-1 protein in transgenic silkworm was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody. The results were consistent with the expected molecular weight. Specific protein bands. Gray scale analysis of SDS-PAGE electrophoresis showed that the content of silk fibroin II in black widow spiders was up to 2.2% of silk light chain content.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 64.5%, the maximum strain increased by 31.4%, the Young's modulus increased by 49.3%, and the toughness increased by 108.3%.
  • the pBac[3xP3-DsRed]-MaSp2 ⁇ 24 plasmid (Fig. 4) constructed above was introduced into the silkworm according to the method of Example 1.
  • the transgenic silkworm expressing the DsRed marker gene was obtained by fluorescence microscopy during the G1 generation. 2 moths, It is then incubated until the G3 generation and the G4 generation are obtained.
  • the red-eyed genotype homozygous moth area was selected, and the red-eye gene was homozygous and the rear silk gland cells were able to synthesize a new transgenic silkworm variety that secreted black widow spider traction silk protein II. MASP2-24-3.
  • the above silkworm silk fibroin was used as the material to determine the insertion site.
  • the expression of MASP2-24-3 protein in transgenic silkworm was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody. The results were consistent with the expected molecular weight. Specific protein bands.
  • the gray scale analysis of SDS-PAGE electrophoresis showed that the black widow spider traction silk protein II accounted for up to 1.3% of the silk light chain.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 55.5%, the maximum strain increased by 21.2%, the Young's modulus increased by 49.3%, and the toughness increased by 86.4%.
  • the pBac[3xP3-DsRed]-MaSp2 ⁇ 24 plasmid (Fig. 4) constructed above was introduced into the silkworm according to the method of Example 1.
  • the transgenic silkworm expressing the DsRed marker gene was obtained by fluorescence microscopy during the G1 generation. 1 moth, then incubated until G3 generation, G4 generation.
  • the red-eyed genotype homozygous moth area was selected, and the red-eye gene was homozygous and the silk gland cells were able to synthesize a transgenic silkworm new type of black widow spider silk fibroin II, which was named MASP2- 24-4.
  • the above silkworm silk fibroin was extracted as the material, and the expression of MASP2-24-4 protein in transgenic silkworm was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody. The result showed that the specific protein strip was consistent with the expected molecular weight. band. Gray scale analysis of SDS-PAGE electrophoresis showed that black widow spider traction silk protein II accounted for up to 2.2% of silk light chain.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 50.3%, the maximum strain increased by 19.9%, the Young's modulus increased by 48.9%, and the toughness increased by 76.0%.
  • the pBac[3xP3-DsRed]-MaSp2 ⁇ 24 plasmid (Fig. 4) constructed above was introduced into the home according to the method of Example 1.
  • the transgenic silkworm 2 moth expressing the DsRed marker gene was obtained by fluorescence microscopy, and then cultured until the G3 generation and the G4 generation were obtained.
  • the red-eyed genotype homozygous moth area was selected, and the red-eye gene was homozygous and the rear silk gland cells were able to synthesize a new transgenic silkworm variety that secreted black widow spider traction silk protein II. MASP2-24-5.
  • the above silkworm silk fibroin was extracted from the above-mentioned insertion site.
  • the expression of MASP2-24-5 protein in transgenic silkworm was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody. The results were consistent with the expected molecular weight. Specific protein bands. Gray scale analysis of SDS-PAGE electrophoresis showed that black widow spider traction silk protein II accounted for up to 1.9% of silk light chain.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 51.2%, the maximum strain increased by 31.8%, the Young's modulus increased by 36.2%, and the toughness increased by 94.3%.
  • the pBac[3xP3-DsRed]-MaSp2 ⁇ 3 plasmid (Fig. 5) constructed above was introduced into the silkworm by the method of Example 1, and the transgenic silkworm expressing the DsRed marker gene was obtained by fluorescence microscopy. 1 moth is then incubated until G3 generation, G4 generation is obtained.
  • one silkworm was randomly selected from the third day of 5th instar, and the genomic DNA of the posterior silk gland cells was extracted as a template.
  • the chromosome insertion site analysis was carried out in the same manner as in Example 1, and the result showed that the progeny insertion site of this moth area was inserted.
  • the transposon has been inserted into the genome of the transgenic silkworm family.
  • the red-eyed genotype homozygous moth area was selected, and the red-eye gene was homozygous and the rear silk gland cells were able to synthesize a new transgenic silkworm variety that secreted black widow spider traction silk protein II. MASP2-3-1.
  • the above silkworm silk fibroin was used as the material to determine the insertion site.
  • the expression of MASP2-3-1 protein in transgenic silkworm was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody. The results were consistent with the expected molecular weight. Specific protein bands. Gray scale analysis of SDS-PAGE electrophoresis showed that black widow spider traction silk protein II accounted for up to 2.2% of silk light chain.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 18.4%, the maximum strain increased by 9.2%, the Young's modulus increased by 13.2%, and the toughness increased by 31.8%.
  • the black widow spider traction silk protein II can be efficiently synthesized in the silk gland cells, and the black widow spider traction silk protein II can be secreted into the glandular cavity by the silk gland like silk. Spit the silkworm in one step. This trait has been stably expressed and inherited.
  • the method can mass produce black widow spider traction silk protein II, can improve the mechanical properties of silk, can improve the economic benefits of silkworm and increase the income of silkworm farmers.
  • Example 9-14 Simultaneous synthesis and secretion of silk fibroin I and protein II by black widow spider
  • the black silk widow spider traction silk protein I gene silkworm variety MASP1-2-1, and the black widow spider traction silk protein II gene silkworm variety MASP2-3-1 were selected, and the two varieties were crossed and named MASP1-2 ⁇ 2. -3.
  • the silkworm protein of MASP1-2 ⁇ 2-3 silkworm was extracted as the material.
  • the expression of MASP1-2 ⁇ 2-3 protein in transgenic silkworm was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody. Two specific protein bands with expected molecular weight sizes are expected. Gray scale analysis of SDS-PAGE electrophoresis showed that the content of silk fibroin I in black widow spider reached 2.1% of silk light chain content. Black widow spider traction silk protein II content reached 1.1% of silk light chain protein content.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 20.5%, the maximum strain increased by 9.8%, the Young's modulus increased by 14.0%, and the toughness increased by 33.9%.
  • the black silk widow spider traction silk protein I gene silkworm variety MASP1-12-1, and the black widow spider traction silk protein II gene silkworm variety MASP2-24-1 were selected, and the two varieties were crossed and named MASP1-12 ⁇ 2 -twenty four.
  • the silkworm protein of MASP1-12 ⁇ 2-24 silkworm was extracted as material.
  • the expression of MASP1-12 ⁇ 2-24 protein in transgenic silkworm was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody. Two specific protein bands with expected molecular weight sizes are expected. Gray scale analysis of SDS-PAGE electrophoresis showed that the content of silk fibroin I in black widow spider reached 2.2% of silk light chain protein content. Black widow spider traction silk protein II content reached 1.4% of silk light chain protein content.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 62.3%, the maximum strain increased by 30.4%, the Young's modulus increased by 28.1%, and the toughness increased by 98.9%.
  • the black widow spider traction silk protein I gene silkworm variety MASP1-16-8 and the black widow spider traction silk protein II gene silkworm variety MASP2-24-5 were selected, and the two varieties were crossed and named MASP1-16-8. ⁇ 2-24-5.
  • the silkworm protein of MASP1-16-8 ⁇ 2-24-5 silkworm was extracted as material, and the transgenic silkworm MASP1-16-8 ⁇ 2-24-5 was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody.
  • the expression of the protein resulted in two specific protein bands consistent with the expected molecular weight. Gray-scale analysis of SDS-PAGE strips showed that black widow spider silk protein I accounted for 1.9% of silk light chain.
  • the black widow spider traction silk protein II accounts for up to 1.0% of the silk light chain.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 57.3%, the maximum strain increased by 27.4%, the Young's modulus increased by 41.1%, and the toughness increased by 92.9%.
  • the black widow spider traction silk protein I gene silkworm variety MASP1-16-6 and the black widow spider traction silk protein II gene silkworm variety MASP2-24-1 were selected, and the two varieties were crossed to obtain F1 generation.
  • the F1 generation hybrids were self-intercropped with the F2 generation, and the F2 generation silkworms were treated with single moths.
  • the silkworms expressing the red fluorescent DsRed marker gene under the fluorescence microscope were screened and prepared by intercropping with the moth moths. Into the F3 generation.
  • F3 generation silkworms were cultured with single moths, and all the individuals under the fluorescence microscope were selected to express the red fluorescent DsRed marker gene in the moth area.
  • the Western Blot technique was used to screen and simultaneously express the black widow spider traction silk protein I and protein.
  • the silk moths of the moth area of II are mated to each other to form the F4 generation.
  • the F3 generation was used to raise, screen, and seed, until the F8 generation, and the two loin proteins were homozygous; the name was MASP1-16-6 ⁇ 2-24-1-HO.
  • Hybridization of the exogenous gene was carried out by crossing the wild type silkworm variety twice.
  • the specific method is to take the F1 generation of 6 moths of MASP1-16-6 ⁇ 2-24-1-HO and the wild type silkworm, and the F1 generation of the silkworms all display the red fluorescent DsRed marker gene, which is taken for each F1 generation.
  • Two moths were further crossed with wild-type silkworm cultivars to obtain F2 generation.
  • Each silkworm of F2 moth area had 3/4 individuals showing red fluorescent DsRed marker gene, which proved MASP1-16-6 ⁇ 2-24-1-
  • the exogenous gene of the HO variety, the black widow spider has been homozygous for the traction protein I and protein II genes.
  • the silkworm protein of MASP1-16-6 ⁇ 2-24-1-HO silkworm was extracted as material, and the transgenic silkworm MASP1-16-6 ⁇ 2-24 was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody.
  • the expression of the 1-HO protein resulted in two specific protein bands consistent with the expected molecular weight.
  • the gray scale analysis of SDS-PAGE electrophoresis showed that the content of silk fibroin I in black widow spider reached 2.0% of silk light chain protein content.
  • Black widow spider traction silk protein II content reached 1.7% of silk light chain protein content.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 69.4%, the maximum strain increased by 33.9%, the Young's modulus increased by 46.3%, and the toughness increased by 97.1%.
  • the black widow spider traction silk protein I gene silkworm variety MASP1-16-2 and the black widow spider traction silk protein II gene silkworm variety MASP2-24-3 were selected, and the two varieties were crossed to obtain F1 generation.
  • the F1 generation hybrids were self-intercropped with the F2 generation, and the F2 generation silkworms were treated with single moths.
  • the silkworms expressing the red fluorescent DsRed marker gene under the fluorescence microscope were screened and prepared by intercropping with the moth moths. Into the F3 generation.
  • F3 generation silkworms were cultured with single moths, and all the individuals under the fluorescence microscope were selected to express the red fluorescent DsRed marker gene in the moth area.
  • the Western Blot technique was used to screen and simultaneously express the black widow spider traction silk protein I and protein.
  • the silk moths of the moth area of II are mated to each other to form the F4 generation.
  • the F3 generation was used to raise, screen, and seed, until the F8 generation, and the two loin proteins were homozygous; the name was MASP1-16-2 ⁇ 2-24-3-HO.
  • Example 12 The same detection method as in Example 12 was carried out, that is, hybridization with wild-type silkworm cultivar twice to detect the purification of the foreign gene, and the result showed that the foreign gene of the MASP1-16-2 ⁇ 2-24-3-HO variety-black
  • the widow spider traction silk protein I and protein II genes have been homozygous.
  • the silkworm protein of MASP1-16-2 ⁇ 2-24-3-HO silkworm was extracted as material, and the transgenic silkworm MASP1-16-2 ⁇ 2-24 was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody.
  • the expression of the -3-HO protein resulted in two specific protein bands consistent with the expected molecular weight.
  • the gray scale analysis of SDS-PAGE electrophoresis showed that the black widow spider traction silk protein I accounted for 1.8% of the silk light chain content.
  • the black widow spider traction silk protein II accounts for up to 1.3% of the silk light chain.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 65.6%, the maximum strain increased by 31.8%, the Young's modulus increased by 45.6%, and the toughness increased by 95.3%.
  • the black widow spider traction silk protein I gene silkworm variety MASP1-16-14, and the black widow spider traction silk protein II gene silkworm variety MASP2-24-4 were selected, and the two varieties were crossed to obtain F1 generation.
  • the F1 generation hybrids were self-intercropped with the F2 generation, and the F2 generation silkworms were treated with single moths.
  • the silkworms expressing the red fluorescent DsRed marker gene under the fluorescence microscope were screened and prepared by intercropping with the moth moths. Into the F3 generation.
  • F3 generation silkworms were cultured with single moths, and all the individuals under the fluorescence microscope were selected to express the red fluorescent DsRed marker gene in the moth area.
  • the Western Blot technique was used to screen and simultaneously express the black widow spider traction silk protein I and protein.
  • the silk moths of the moth area of II are mated to each other to form the F4 generation.
  • the F3 generation was used to raise, screen, and seed, until the F8 generation, and the two loin proteins were homozygous; the name was MASP1-16-14 ⁇ 2-24-4-HO.
  • Example 12 The same detection method as in Example 12 was carried out, that is, hybridization with wild-type silkworm cultivar twice to detect the purification of the foreign gene, and the result was confirmed that the foreign gene of MASP1-16-14 ⁇ 2-24-4-HO variety-black
  • the widow spider traction silk protein I and protein II genes have been homozygous.
  • the silkworm protein of MASP1-16-14 ⁇ 2-24-4-HO silkworm was extracted as material, and the transgenic silkworm MASP1-16-14 ⁇ 2-24 was analyzed by SDS-PAGE electrophoresis and Western blot analysis of black widow spider silk antibody.
  • the expression of the -4-HO protein resulted in two specific protein bands consistent with the expected molecular weight.
  • the gray scale analysis of SDS-PAGE electrophoresis showed that the black widow spider silk protein I accounted for up to 2.0% of the silk light chain.
  • the black widow spider traction silk protein II accounts for up to 1.7% of the silk light chain.
  • the mechanical properties of the transgenic silk showed that compared with the wild type control Lan10, the maximum stress increased by 69.4%, the maximum strain increased by 33.9%, the Young's modulus increased by 46.3%, and the toughness increased by 97.1%.
  • the black widow spider traction silk protein I and protein II are efficiently synthesized in the silk gland cells by the method of the present invention, and the black widow spider traction silk protein I and protein II can be secreted by the silk gland like silk. Enter the glandular cavity and further spit out the silkworm, and the trait has been stably expressed and inherited.
  • the method can mass produce black widow spider traction silk protein I and protein II, can improve the mechanical properties of silk, can improve the economic benefits of silkworm, and increase the income of silkworm farmers.
  • SEQ ID NO. 1 base sequence encoding 2 fold re-MaSp1
  • SEQ ID NO. 2 Protein sequence encoding 2 fold re-MaSp1
  • SEQ ID NO 3 base sequence encoding 3 fold MaSp2 gene repeat unit
  • SEQ ID NO. 4 3X MaSp2 gene repeating unit protein sequence encoding

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Environmental Sciences (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Plant Pathology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Animal Husbandry (AREA)
  • Biodiversity & Conservation Biology (AREA)
  • Biophysics (AREA)
  • Peptides Or Proteins (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法。通过转基因育成黑寡妇蜘蛛牵引丝蛋白基因纯合且能够合成分泌黑寡妇蜘蛛牵引丝蛋白的家蚕品种；所述的家蚕品种为能合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕、能合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕或者上述两种杂交后的家蚕品种；通过上述家蚕品种的家蚕丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白，并随家蚕吐丝结茧行为进入蚕茧。

Description

利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法 技术领域
本发明涉及一种合成分泌外源蛋白的方法，尤其是涉及了利用转基因技术的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法。
背景技术
蜘蛛种类繁多，至今在全世界已命名的蜘蛛有112个科，3905个属，44000多种。蜘蛛丝是由蜘蛛丝腺分泌的一种天然高分子蛋白纤维，具有优良的机械性能，如弹性好、强度大、韧性强、耐高温和低温、耐冲击、比重小、生物相容性好、以及生物可降解等优良特性，其独特的综合性能是其他天然纤维和合成纤维所无法相比的。典型的蜘蛛丝有7种类型，分别由7种不同丝腺分泌的不同的蛋白质分子组成。其中由大囊状腺(Major ampullate gland)分泌的主牵引丝(dragline silk)，呈放射状分布，构成蜘蛛网基本框架。牵引丝的强度是钢的5倍，因而拥有“生物钢”之美誉。牵引丝的蛋白基因是高度保守的，主要由分子量350kDa左右的、成对的牵引丝蛋白Ⅰ(MaSp1)和牵引丝蛋白Ⅱ(MaSp2)2种蛋白组成。
黑寡妇蜘蛛(Latrodectus hesperus)牵引丝是蜘蛛中性能最优秀的牵引丝。黑寡妇蜘蛛牵引丝蛋白Ⅰ(MaSp1)基因具有一个单一外显子，全长9390bp，编码3129个氨基酸，基因含有大量的重复单元，每1个重复单元由4种初级类型重复序列Type1、Type2、Type3和Type4通过头尾串联而成(Type1-Type2-Type3-Type4)，而每1种初级类型重复序列都包含了典型的(GA)n、An和GGX蛋白基序。MaSpⅠ被认为是牵引丝具有很强强度的主要分子基础。
黑寡妇蜘蛛牵引丝蛋白Ⅱ(MaSp2)基因具有一个单一外显子，全长11340bp，编码3779个氨基酸，基因含有大量的重复单元，每一个重复单元主要由2种初级类型重复序列以Type1-Type1-Type4串联而成，而每一种初级类型重复序列都包含了典型的An、GPGXX和GGX蛋白基序。MaSp2被认为是牵引丝具有很强韧性的主要分子基础。
由于蜘蛛丝自身的产量低，且由于其相互残杀而不能通过大规模的饲养获得批量的蜘蛛丝，所以限制了蜘蛛丝的广泛应用。随着生物技术的快速发展和对蜘蛛丝的编码序列及吐丝机理的深入了解，已有采用各种异源表达系统来表达蜘蛛丝蛋白的研究，异源表达系统包括大肠杆菌、毕赤酵母、昆虫细胞(sf9、BmN)、仓鼠的肾脏细胞、转基因土豆、转基因烟草、转基因山羊、转基因老鼠、以及转基因家蚕，表达的蜘蛛丝主要是金纺蜘蛛(Nephila clavipes)、棒络新妇蛛(Nephila clavata)、大腹圆蛛(Araneus ventricosus)、十字圆蛛(Araneus diadematus)4种蜘蛛丝的序列片段。
但是，至今为止使用的各种异源表达系统采用的基因不是机械性能最优异的黑寡妇蜘蛛牵引丝基因，而且表达出来的蜘蛛丝蛋白的分子量较小，有较多重组蛋白缺少一些关键的蛋白基序，有较多蛋白不是纯的蜘蛛丝蛋白分子，而是与荧光蛋白等分子融合的融合蛋白，这些因素最终影响了表达产物的机械性能。因此，现有技术缺少了一种方法能合成分泌性能优异的仿生蜘蛛丝。
发明内容
为了解决背景技术中存在的问题，本发明的目的在于提出了一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法。
本发明一方面，利用转基因家蚕技术分别将黑寡妇蜘蛛牵引丝蛋白Ⅰ基因和蛋白Ⅱ基因导入家蚕基因组内，并分别在家蚕丝腺细胞中特异表达，开发出能分别合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的转基因家蚕；
本发明另一方面，将两种转基因家蚕品种杂交，育成杂交品种，通过杂交品种同蛾区阳性个体自交留种，选择出牵引丝蛋白Ⅰ基因和蛋白Ⅱ基因2种基因都纯合的品种。利用转基因家蚕技术结合杂交育种技术，将黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ基因同时导入家蚕基因组内，并在家蚕丝腺细胞中特异表达，开发出能同时合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的家蚕，使杂交种的1根仿生丝纤维由牵引丝蛋白Ⅰ和蛋白Ⅱ组成。
本发明获得合成分泌黑寡妇蜘蛛牵引丝蛋白的仿生丝纤维结构与天然黑寡妇蜘蛛牵引丝相似，性能接近、甚至超越天然黑寡妇蜘蛛牵引丝的超级仿生蜘蛛丝。
为了达到上述目的，本发明采用的技术方案的步骤如下：
一、第一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法：
A1)采用分子生物学方法构建pBac[3xP3-DsRed]-MaSp1质粒作为在家蚕丝腺中表达的黑寡妇蜘蛛牵引丝蛋白Ⅰ基因载体，pBac[3xP3-DsRed]-MaSp1质粒如图1～图3所示，pBac[3xP3-DsRed]-MaSp1质粒以piggyBac转座子为基础包含外源基因的黑寡妇蜘蛛牵引丝蛋白Ⅰ基因和作为标记基因的红色荧光DsRed基因表达框；
A2)采用显微注射转基因家蚕方法将pBac[3xP3-DsRed]-MaSp1质粒及能够提供piggyBac转座酶的辅助质粒pHA3PIG按浓度比1：1的比例导入家蚕产卵后6小时以内的受精卵内，利用pBac[3xP3-DsRed]-MaSp1质粒中的piggyBac转座子将黑寡妇蜘蛛牵引丝蛋白Ⅰ基因插入到家蚕基因组内；
A3)蚕卵孵化后饲养至成虫，然后与非转基因家蚕交配制种续代，此代为G1代，在G1代蚕卵的转青期，通过荧光体视显微镜观察筛选出单眼表达红色荧光DsRed标记基因的转基因家蚕，饲养至成虫再与非转基因家蚕交配制种续代成为G2代；
A4)G2代家蚕采用单蛾育，筛选出荧光体视显微镜下表达红色荧光DsRed标记基因的家蚕，采用同蛾区蚕蛾相互交配制成G3代；
A5)G3代家蚕采用单蛾育，同蛾区表达红色荧光DsRed标记基因的蚕蛾相互交配，制成G4代；
A6)从G4代开始并经连续3代采用红眼表型纯一的蛾区饲养、单蛾育和同蛾区蚕蛾交配的相同方法进行选择和交配，育成红眼基因和黑寡妇蜘蛛牵引丝蛋白Ⅰ基因纯合、能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕。
通过所述步骤A6)获得的红眼基因和黑寡妇蜘蛛牵引丝蛋白Ⅰ基因纯合、能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕品种的家蚕丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ，并随家蚕吐丝结茧行为进入蚕茧。
二、第二种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法：
B1)采用分子生物学方法构建pBac[3xP3-DsRed]-MaSp2质粒作为在家蚕丝腺中表达的黑寡妇蜘蛛牵引丝蛋白Ⅱ基因载体，pBac[3xP3-DsRed]-MaSp2质粒如图4～图5所示，pBac[3xP3-DsRed]-MaSp2质粒以piggyBac转座子为基础包含有作为外源基因的黑寡妇蜘蛛牵引丝蛋白Ⅱ基因和作为标记基因的红色荧光DsRed基因的两个表达框；
B2)采用显微注射转基因家蚕方法将pBac[3xP3-DsRed]-MaSp2质粒及能够提供piggyBac转座酶的辅助质粒pHA3PIG按浓度比1：1的比例导入家蚕产卵后6小时以内的受精卵内，利用pBac[3xP3-DsRed]-MaSp2质粒中的piggyBac转座子将黑寡妇蜘蛛牵引丝蛋白Ⅱ基因插入到家蚕基因组内；
B3)蚕卵孵化后饲养至成虫，然后与非转基因家蚕交配制种续代，此代为G1代，在G1代蚕卵的转青期，通过荧光体视显微镜观察筛选出单眼表达红色荧光DsRed标记基因的转基因家蚕，饲养至成虫再与非转基因家蚕交配制种续代成为G2代；
B4)G2代家蚕采用单蛾育，筛选出荧光体视显微镜下表达红色荧光DsRed标记基因的家蚕，采用同蛾区蚕蛾相互交配制成G3代；
B5)G3代家蚕采用单蛾育，同蛾区表达红色荧光DsRed标记基因的蚕蛾相互交配，制成G4代；
B6)从G4代开始并经连续3代采用红眼表型纯一的蛾区饲养、单蛾育和同蛾区蚕蛾交配的相同方法进行选择和交配，育成红眼基因和黑寡妇蜘蛛牵引丝蛋白Ⅱ基因纯合、能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕。
通过所述步骤B6)获得的红眼基因和黑寡妇蜘蛛牵引丝蛋白Ⅱ基因纯合、能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕品种的家蚕丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ，并随家蚕吐丝结茧行为进入蚕茧。
三、第三种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法：
(1)通过转基因育成带有红色荧光DsRed标记基因且能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕和带有红色荧光DsRed标记基因且能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕，这两种家蚕的培育见上文；
(2)将上述两种转基因家蚕品种杂交，育成F1代杂交品种；
(3)将F1代杂交品种同蛾区个体自交留种为F2代，F2代家蚕采用单蛾育，筛选出荧光体视显微镜下表达红色荧光DsRed标记基因的家蚕饲养，采用同蛾区蚕蛾相互交配制成F3代；
(4)F3代家蚕采用单蛾育，筛选出荧光体视显微镜下所有个体全部表达红色荧光DsRed标记基因的蚕蛾在同一蛾区饲养，并同时采用Western Blot技术检测，筛选出同时表达黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的蛾区的蚕蛾相互交配制成F4代；
(5)F4代开始均采用与F3代同样的方法饲养、筛选、制种，一直到F8代，育成两个黑寡妇蜘蛛牵引丝蛋白基因都纯合的家蚕品种。
通过所述步骤(5)获得的两个黑寡妇蜘蛛牵引丝蛋白基因都纯合的家蚕品种的家蚕丝腺细胞同时合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ，黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的基因在家蚕丝腺细胞特异表达，在家蚕丝素蛋白重链信号肽的作用下分泌到丝腺腺腔，并随吐丝行为分泌到蚕茧。
所述的质粒pBac[3xP3-DsRed]-MaSp1是以piggyBac转座子为基础并带有Amp抗性基因，包括piggyBac转座子的两个转座臂PBL和PBR，以及两个转座臂之间的两个功能表达框，一个功能表达框是3×P3启动子启动的红色荧光蛋白基因表达框，即3×P3Promoter–DsRed-SV40，另一个功能表达框是包含家蚕丝素蛋白重链基因启动子、丝素蛋白重链基因信号肽、黑寡妇蜘蛛牵引丝蛋白Ⅰ基因和家蚕丝素蛋白重链基因3’末端的表达框，即Fibroin H chain Promoter-Fibroin H chain signal peptide-MaSp1-Fibroin H chain PolyA。
所述的质粒pBac[3xP3-DsRed]-MaSp2是以piggyBac转座子为基础并带有Amp抗性基因，包括piggyBac转座子的两个转座臂PBL和PBR，以及两个转座臂之间的两个功能表达框，一个功能表达框是3×P3启动子启动的红色荧光蛋白基因表达框，即3×P3Promoter–DsRed-SV40，另一个功能表达框是包含家蚕丝素蛋白重链基因启动子、丝素蛋白重链基因信号肽、黑寡妇蜘蛛牵引丝蛋白Ⅱ基因和家蚕丝素蛋白重链基因3’末端的表达框，即Fibroin H chain Promoter-Fibroin H chain signal peptide-MaSp2-Fibroin H chain PolyA。
所述的黑寡妇蜘蛛牵引丝蛋白Ⅰ基因是包含2倍到16倍MaSp1基因重复单元Type1-Type2-Type3-Type4，如图1-3所示。
所述的黑寡妇蜘蛛牵引丝蛋白Ⅱ基因是包含3倍到24倍MaSp2基因重复单元Type1-Type1-Type4，如图4-5所示。
本发明是先分别构建家蚕合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ基因和蛋白Ⅱ基因的载体pBac[3xP3-DsRed]-MaSp1和pBac[3xP3-DsRed]-MaSp2，再利用显微注射转基因家蚕技术将这5种质粒分别与能够提供piggyBac转座酶的辅助质粒pHA3PIG质粒(如图6所示)一起导入到家蚕受精卵内，依靠piggyBac转座子的转座特性，使红色荧光蛋白基因和黑寡妇蜘蛛牵引丝蛋白Ⅰ基因、以及红色荧光蛋白基因和黑寡妇蜘蛛牵引丝蛋白Ⅱ基因导入到家蚕基因组内，并得到稳定遗传和表达，从而创制成一种能够在家蚕丝腺细胞特异性合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ基因和蛋白Ⅱ基因的转基因家蚕，自交使黑寡妇蜘蛛牵引丝蛋白Ⅰ基因和蛋白Ⅱ基因纯合，育成能分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的转基因家蚕；再将上述2种转基因家蚕杂交，利用杂交育种技术，育成牵引丝蛋白Ⅰ基因和蛋白Ⅱ基因纯合的杂交品种，然后利用该种家蚕合成分泌黑寡妇蜘蛛牵引丝。
本发明具有的有益效果是：
本发明借助荧光标志基因筛选转基因家蚕，这种转基因家蚕能够分别或者同时在家蚕丝腺细胞特异地合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ，黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ具有功能活性。
并且本发明获得的仿生丝纤维结构与天然黑寡妇蜘蛛牵引丝相似，是性能接近、甚至超越天然黑寡妇蜘蛛牵引丝的超级仿生蜘蛛丝。
综合来说，本发明开发了一种新型的能够分别或同时获得黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的生产工艺，为分别或者同时大量生产黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ奠定了基础，为获得结构与天然黑寡妇蜘蛛牵引丝相似，性能接近、甚至超越天然黑寡妇蜘蛛牵引丝的超级仿生蜘蛛丝奠定了基础，也为提高蚕丝的机械性能奠定了基础。
附图说明
图1是家蚕后部丝腺细胞合成分泌重组黑寡妇牵引丝蛋白Ⅰ的pBac[3×P3-DsRed]-MaSp1×2质粒结构图。
图2是家蚕后部丝腺细胞合成分泌重组黑寡妇牵引丝蛋白Ⅰ的pBac[3×P3-DsRed]-MaSp1×12质粒结构图。
图3是家蚕后部丝腺细胞合成分泌黑寡妇重组牵引丝蛋白Ⅰ的pBac[3×P3-DsRed]-MaSp1×16质粒结构图。
图4是家蚕后部丝腺细胞合成分泌重组黑寡妇牵引丝蛋白Ⅱ的pBac[3×P3-DsRed]-MaSp2×24质粒结构图。
图5是家蚕后部丝腺细胞合成分泌重组黑寡妇牵引丝蛋白Ⅱ的pBac[3× P3-DsRed]-MaSp2×3质粒结构图。
图6是本发明涉及的piggyBac转座酶的辅助质粒结构图。
具体实施方式
下面结合附图和实施例对本发明作进一步说明。
本发明的实施例如下：
本发明以下实施例中的pBac[3xP3-DsRed]-MaSp1质粒是采用以下方式制备合成：
A)先将家蚕丝素重链蛋白基因启动子(Fib-H-P)连到pMD18-T simple(Takara)载体，并和基本的含有A3启动子和绿色荧光蛋白表达框的piggyBac质粒piggy6212载体分别经过XhoI/NcoI双酶切，胶回收重链启动子序列部分与piggyBac载体骨架连接，获得piggy6609载体。
B)pBac[3xP3-DsRed]-MaSp1质粒构建方法和步骤如下：
根据已经报导的全长黑寡妇蜘蛛牵引丝蛋白Ⅰ基因(MaSp1)序列特征设计质粒，并根据家蚕的密码子偏好性作了碱基优化，详细信息如下。
人工合成家蚕丝素重链蛋白基因的信号肽、2倍的MaSp1分子的重复单元序列(其碱基序列和表达的蛋白序列分别如SEQ ID NO.1和SEQ ID NO.2)、MaSp1分子的C末端、家蚕丝素重链蛋白基因的C末端及其PolyA的序列，以及后续载体构建所需的酶切位点序列，将这些序列连接到pUC57载体上，命名为pUC4423质粒，该质粒含2倍MaSp1基因重复单元序列。
将pUC4423质粒分别利用2个同尾酶SpeI和NheI酶切，依次与不同倍数的重复单元序列连接起来，其叠加过程的具体操作步骤如下：
pUC4423质粒用SpeI/HindШ双酶切，回收长度为1698bp含2倍MaSp1基因重复单元的片段，pUC4423用NheI/HindШ双酶切，回收长度为3457bp含2倍MaSp1基因重复单元的片段，连接上述1698bp片段和3457bp片段，获得pUC5155质粒(含4倍MaSp1基因重复单元)；
pUC5155质粒用SpeI/HindШ双酶切，回收长度为2430bp含4倍MaSp1基因重复单元的片段，pUC5155用NheI/HindШ双酶切，回收长度为4189bp含4倍MaSp1基因重复单元的片段，连接上述2430bp片段和4189bp片段，获得pUC6619质粒(含8倍MaSp1基因重复单元)；
pUC6619质粒用SpeI/HindШ双酶切，回收长度为3894bp含8倍MaSp1基因重复单元的片段，pUC6619用NheI/HindШ双酶切，回收长度为5653bp含8倍MaSp1基因重复单元的片段，连接上述3894bp片段和5653bp片段，获得pUC9547质粒，含16倍MaSp1基因重复单元；
将上述的2430bp片段(含4倍的MaSp1片段)与5653bp的片段(含8倍的MaSp1基因重复单元)连接，获得pUC8083质粒，含12倍MaSp1基因重复单元；
将上述含有2、8、12、16倍数MaSp1基因重复单元序列的质粒用NcoI/MunI双酶切回收含有不同长度重复单元片段以及组成元件的序列片段，同时将上述A)已构建好的包含重链启动子和piggyBac骨架的载体piggy6609用同样的限制性内切酶酶切，回收包含piggyBac载体骨架及重链启动子序列元件的目的片段，并分别与上述不同长度重复单元片段连接，获得piggy7540、piggy9736、piggy11200和piggy12664载体，这4种载体分别包含有2倍、8倍、12倍和16倍MaSp1基因重复单元序列。
将piggy7540、piggy9736、piggy11200和piggy12664载体分别用BglII/AflII双酶切，回收包含有不同长度的MaSp1基因重复单元和piggyBac载体骨架的目的片段，对含有3×P3启动子-红眼基因表达框和A3启动子-绿色荧光蛋白表达框的载体piggy7785用同样的酶切，回收1230bp含有3×P3启动子和标记基因DsRed编码框的目的片段，分别连接两目的片段以加上DsRed标记基因。具体的操作步骤如下：
piggy7785载体用AflⅢ/BglⅡ双酶切，回收1230bp的片段。
piggy7540载体用AflⅡ/BglⅡ双酶切，回收7198bp的片段，并与1230bp的片段连接，获得pBac[3xP3-DsRed]-MaSp1×2载体(含2倍MaSp1基因重复单元)(图1)；
piggy11200载体用AflⅡ/BglⅡ双酶切，回收10858pb的片段，并与1230pb的片段连接，获得pBac[3xP3-DsRed]-MaSp1×12载体(含12倍MaSp1基因重复单元)(图2)；
piggy12664载体用AflⅡ/BglⅡ双酶切，回收12322bp的片段，并与1230bp的片段连接，获得pBac[3xP3-DsRed]-MaSp1×16载体(含16倍MaSp1基因重复单元)(图3)。
本发明实施例中的pBac[3xP3-DsRed]-MaSp2质粒是采用以下方式制备合成：
A)先将家蚕丝素重链蛋白基因启动子(Fib-H-P)连到pMD18-T simple(Takara)载体，并和基本的含有A3启动子和绿色荧光蛋白表达框的piggyBac质粒piggy6212载体分别经过XhoI/NcoI双酶切，胶回收重链启动子序列部分与piggyBac载体骨架连接，获得piggy6609载体。
B)pBac[3xP3-DsRed]-MaSp2质粒构建方法和步骤如下：
根据已经报导的全长黑寡妇蜘蛛牵引丝蛋白Ⅱ基因(MaSp2)序列特征设计质粒，并根据家蚕的密码子偏好性作了碱基优化，详细信息如下。
人工合成家蚕丝素重链蛋白基因的信号肽、3倍的MaSp2基因重复单元序列(其碱基序列和表达的蛋白序列分别如SEQ ID NO.3和SEQ ID NO.4)、MaSp2分子的C末端、家蚕丝素重链蛋白基因的C末端及其PolyA的序列、以及后续载体构建所需的酶切位点序列，将这些序列连接到pUC57载体上，命名为pUC4301质粒，该质粒含3倍MaSp2基因重复单元序列。
将pUC4301质粒分别利用2个同尾酶SpeI和NheI酶切，依次将不同倍数的重复单元序列连接起来，其叠加过程的具体操作步骤如下：
pUC4301质粒用SpeI/HindШ双酶切，回收长度为1629bp含3倍MaSp2基因的片段，pUC4301用NheI/HindШ双酶切，回收长度为3326bp含3倍MaSp2基因重复单元的片段，连接上述1629bp片段和3326bp片段，获得pUC4955质粒(含6倍MaSp2基因重复单元)；
pUC4955质粒作为载体用SpeI/HindШ双酶切，回收长度为2283bp含6倍MaSp2基因的片段，pUC4955用NheI/HindШ双酶切，回收长度为3980bp含6倍MaSp2基因重复单元的片段，连接上述2283bp片段和3980bp片段，获得pUC6263质粒，含12倍MaSp2基因重复单元；
pUC6263质粒用SpeI/HindШ双酶切，回收长度为3591bp含12倍MaSp2基因重复单元的片段，pUC6263用NheI/HindШ双酶切，回收长度为5288bp含12倍MaSp2基因重复单元的片段，连接上述3591bp片段和5288bp片段，获得pUC8879质粒，含24倍MaSp2基因重复单元；
将上述获得的含有不同倍数的MaSp2基因重复单元序列的质粒用NcoI/MunI双酶切，回收含有重复片段以及组成元件的序列片段，将A)步骤已经构建好的包含重链启动子和 piggyBac骨架的载体Piggy6609用同样的限制性内切酶酶切，回收包含piggyBac载体骨架及重链启动子序列元件的目的片段，分别与上述不同长度重复单元片段连接，获得piggy7471、和piggy12049载体。这2种质粒分别包含有3倍和24倍MaSp2基因重复单元序列。
将piggy7471和piggy12049载体分别用BglII/AflII双酶切，分别回收包含有不同长度MaSp2基因重复单元和piggyBac载体骨架的目的片段，对含有3×P3启动子-红眼基因表达框和A3启动子-绿色荧光蛋白表达框的载体piggy7785用同样的酶切，回收1230bp含有3×P3启动子和标记基因DsRed编码框的目的片段，分别连接两目的片段以加上DsRed标记基因。具体的操作步骤如下：
piggy7785载体用AflⅢ/BglⅡ双酶切，回收1230bp的片段。
piggy7471载体用AflⅡ/BglⅡ双酶切，回收7129bp含有MaSp2重复单元和piggyBac载体骨架的目的片段，并与1230bp的片段连接，获得pBac[3xP3-DsRed]-MaSp2×3载体(含3倍MaSp2基因重复单元，其3倍MaSp2基因的重复单元如SEQ ID NO.3，其表达的蛋白序列如SEQ ID NO.4)(图5)；
piggy12049载体用AflⅡ/BglⅡ双酶切，回收11707bp含有MaSp2重复单元和piggyBac载体骨架的目的片段，并与1230bp大小的连接，获得pBac[3xP3-DsRed]-MaSp2×24载体(含24倍MaSp2基因重复单元)(图4)。
实施例1－3：黑寡妇蜘蛛牵引丝蛋白Ⅰ的合成分泌
实施例1：
将上述构建的pBac[3xP3-DsRed]-MaSp1×2质粒(图1)及能够提供piggyBac转座酶的辅助质粒pHA3PIG质粒(图6)按1:1比率混合，2种质粒的总浓度为0.4μg/μl，质粒溶解在pH＝7、0.5mM的磷酸缓冲液中，然后采用显微注射方法导入家蚕产卵后6小时以内的受精卵内，导入总体积为10nl。将显微注射的蚕卵在25℃、85％湿度条件下饲养至成虫，与非转基因家蚕杂交传代，是为G1代。在转基因实验的G1代卵转青期，通过荧光显微镜(Olympus，SZX12，日本)观察获得表达DsRed标志基因的转基因家蚕2蛾，将蚕饲养至成虫与非转基因家蚕杂交传代，是为G2。自第G2代以后的转基因家蚕均采用单蛾育，在卵期通过荧光体视显微镜观察，挑选表达DsRed标志基因的转基因家蚕，饲养至成虫，同蛾区交配，使黑寡妇蜘蛛牵引丝蛋白Ⅰ基因纯合，进而培育得到G3代、G4代。
在G4代时，取5龄第3天家蚕后部丝腺细胞基因组DNA为模板，采用Inverse PCR分析黑寡妇蜘蛛牵引丝蛋白Ⅰ基因在家蚕基因组中的插入片段，对扩增片段进行克隆、测序和染色体定位分析，结果显示2个蛾区的后代插入位点都是在第3号染色体14033724位点处，证明转座子已经插入到家蚕基因组内。
从G5代开始选择红眼基因型纯合的蛾区饲养，采用同蛾区蚕蛾交配，育成红眼基因纯合、后部丝腺细胞能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕新品种，定名为MASP1-2。
提取MASP1-2家蚕的茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP1-2蛋白的表达情况，结果得到与预期分子量大小相符的特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅰ占蚕丝轻链含量高达3.1％。
对转基因家蚕丝的机械性能测定结果显示(下表1)，与野生型对照品种Lan10相比，最大应力提高19.31％，最大应变提高9.71％，杨氏模量提高13.51％，韧性提高32.82％。
表1实施例1转基因家蚕MASP1-2茧丝机械性能测定结果
Figure PCTCN2016102266-appb-000001
研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅰ基因已插入到转基因家蚕新品种MASP1-2基因组的第3染色体内，并能够在后部丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ，该蛋白能够随吐丝结茧行为进入蚕茧，此性状已稳定遗传和表达。转基因家蚕丝的机械性能得到显著改善。
实施例2：
按照与实施例1相同的方法，将上述构建的pBac[3xP3-DsRed]-MaSp1×12质粒(图2)导入家蚕，在转基因实验的G1代卵转青期，通过荧光显微镜观察获得了表达DsRed标志基因的转基因家蚕10蛾，接着培育直到得到G3代、G4代。
在G4代时，随机取4个蛾区的5龄第3天家蚕各1条，提取后部丝腺细胞基因组DNA为模板，采用与实施例1相同方法进行染色体插入位点分析，结果显示MASP1-12-1这个蛾区的后代插入位点是在第5号染色体17280911位点处，MASP1-12-5插入位点是在第8号染色体11229702位点处，MASP1-12-10插入位点是在第8号染色体11385343位点处，MASP1-12-8插入位点是在第9号染色体13448009位点处，证明转座子已经插入到每一个转基因家蚕家系的基因组内。
从G5代开始选择红眼基因型纯合的蛾区饲养，采用同蛾区蚕蛾交配，育成红眼基因纯合、后部丝腺细胞能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕新品种，定名为MASP1-12。
提取上述测定插入位点的4个家系的家蚕茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP1-12蛋白的表达情况，结果得到与预期分子量大小相符的特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅰ占蚕丝轻链含量最高达2.4％。
对转基因家蚕丝的机械性能测定结果显示(下表2)，与野生型对照品种Lan10相比，最大应力提高58.82％，最大应变提高19.93％，杨氏模量提高38.93％，韧性提高89.94％。
表2实施例2转基因家蚕MASP1-12茧丝机械性能测定结果
Figure PCTCN2016102266-appb-000002
Figure PCTCN2016102266-appb-000003
上述研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅰ基因已插入到4个转基因家蚕家系MASP1-12基因组的不同染色体内，并能够在后部丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ，该蛋白能够随吐丝结茧行为进入蚕茧，此性状已稳定遗传和表达。转基因家蚕丝的机械性能得到显著改善。
实施例3：
按照与实施例1相同的方法，将上述构建的pBac[3xP3-DsRed]-MaSp1×16质粒(图3)导入家蚕，在G1代卵转青期，通过荧光显微镜观察获得了表达DsRed标志基因的转基因家蚕16蛾，接着培育直到得到G3代、G4代。
在G4代时，随机取4个蛾区的5龄第3天家蚕各1条，提取后部丝腺细胞基因组DNA为模板，采用与实施例1相同方法进行染色体插入位点分析，结果显示MASP1-16-2这个蛾区的后代插入位点是在第18号染色体11456596位点处，MASP1-16-6插入位点是在第6号染色体5682251位点处，MASP1-16-8和MASP1-16-14这2个家系的插入位点都是在第10号染色体3608552位点处，证明转座子已经插入到每一个转基因家蚕家系的基因组内。
从G5代开始选择红眼基因型纯合的蛾区饲养，采用同蛾区蚕蛾交配，育成红眼基因纯合、后部丝腺细胞能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕新品种，定名为MASP1-16。
提取上述测定插入位点的4个家系的家蚕茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP1-16蛋白的表达情况，结果得到与预期分子量大小相符的特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅰ占蚕丝轻链含量最高达2.3％。
对转基因家蚕丝的机械性能测定结果显示(下表3)，与野生型对照品种Lan10相比，最大应力提高61.34％，最大应变提高29.17％，杨氏模量提高43.32％，韧性提高96.90％。
表3实施例3转基因家蚕MASP1-16茧丝机械性能测定结果
Figure PCTCN2016102266-appb-000004
Figure PCTCN2016102266-appb-000005
上述研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅰ基因已插入到4个转基因家蚕家系MASP1-16基因组的不同染色体内，并能够在后部丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ，该蛋白能够随吐丝结茧行为进入蚕茧，此性状已稳定遗传和表达。转基因家蚕丝的机械性能得到显著改善。
综合从上述3个实施例可以看出，利用本发明方法，可以在家蚕丝腺细胞高效合成黑寡妇蜘蛛牵引丝蛋白Ⅰ，黑寡妇蜘蛛牵引丝蛋白Ⅰ可以像蚕丝一样由丝腺分泌进入腺腔，并进一步吐出蚕体。该性状已能够稳定表达并遗传。采用本方法能够大量生产黑寡妇蜘蛛牵引丝蛋白Ⅰ，能够提高蚕丝机械性能，能够提高蚕桑经济效益，提高蚕农收入。
实施例4－8：黑寡妇蜘蛛牵引丝蛋白Ⅱ的合成分泌
实施例4：
按照实施例1的方法，将上述构建的pBac[3xP3-DsRed]-MaSp2×24质粒(图4)导入家蚕，在G1代卵转青期，通过荧光显微镜观察获得了表达DsRed标志基因的转基因家蚕1蛾，接着培育直到得到G3代、G4代。
在G4代时，蛾区中取1条5龄第3天家蚕后部丝腺细胞基因组DNA为模板，采用与实施例1相同方法进行染色体插入位点分析，结果显示这个蛾区的后代插入位点分别在第22号染色体2966967位点处，证明转座子已经插入到家蚕基因组内。
从G5代开始选择红眼基因型纯合的蛾区饲养，采用同蛾区蚕蛾交配，育成红眼基因纯合、后部丝腺细胞能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕新品种，定名为MASP2-24-1。
提取MASP2-24-1家蚕的茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP2-24-1蛋白的表达情况，结果得到与预期分子量大小相符的特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅱ含量最高达蚕丝轻链含量的2.2％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高了64.5％，最大应变提高31.4％，杨氏模量提高49.3％，韧性提高108.3％。
研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅱ基因已插入到转基因家蚕新品种MASP2-24-1基因组的第22染色体内，并能够在丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ，该蛋白能够随吐丝结茧行为进入蚕茧，此性状已稳定遗传和表达。转基因家蚕丝的机械性能得到显著改善。
实施例5：
按照实施例1的方法，将上述构建的pBac[3xP3-DsRed]-MaSp2×24质粒(图4)导入家蚕，在G1代卵转青期，通过荧光显微镜观察获得了表达DsRed标志基因的转基因家蚕2蛾， 接着培育直到得到G3代、G4代。
在G4代时，随机取1个蛾区的5龄第3天家蚕1条，提取后部丝腺细胞基因组DNA为模板，采用与实施例1相同方法进行染色体插入位点分析，结果显示这个蛾区的后代插入位点是在第11号染色体2512160位点处，证明转座子已经插入到每一个转基因家蚕家系的基因组内。
从G5代开始选择红眼基因型纯合的蛾区饲养，采用同蛾区蚕蛾交配，育成红眼基因纯合、后部丝腺细胞能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕新品种，定名为MASP2-24-3。
提取上述测定插入位点的家蚕茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP2-24-3蛋白的表达情况，结果得到与预期分子量大小相符的特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅱ占蚕丝轻链含量最高达1.3％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高55.5％，最大应变提高21.2％，杨氏模量提高49.3％，韧性提高86.4％。
上述研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅱ基因已插入到转基因家蚕家系MASP2-24-3基因组的11号染色体内，并能够在丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ，该蛋白能够随吐丝结茧行为进入蚕茧，此性状已稳定遗传和表达。转基因家蚕丝的机械性能得到显著改善。
实施例6：
按照实施例1的方法，将上述构建的pBac[3xP3-DsRed]-MaSp2×24质粒(图4)导入家蚕，在G1代卵转青期，通过荧光显微镜观察获得了表达DsRed标志基因的转基因家蚕1蛾，接着培育直到得到G3代、G4代。
在G4代时，取5龄第3天家蚕1条，提取后部丝腺细胞基因组DNA为模板，采用与实施例1相同方法进行染色体插入位点分析，结果显示这个蛾区的后代插入位点是在第26号染色体7476231位点处，证明转座子已经插入到转基因家蚕家系的基因组内。
从G5代开始选择红眼基因型纯合的蛾区饲养，采用同蛾区蚕蛾交配，育成红眼基因纯合、丝腺细胞能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕新品种，定名为MASP2-24-4。
提取上述家蚕茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP2-24-4蛋白的表达情况，结果得到与预期分子量大小相符的特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅱ占蚕丝轻链含量最高达2.2％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高50.3％，最大应变提高19.9％，杨氏模量提高48.9％，韧性提高76.0％。
上述研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅱ基因已插入到转基因家蚕家系MASP2-24-4基因组的26号染色体内，并能够在丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ，该蛋白能够随吐丝结茧行为进入蚕茧，此性状已稳定遗传和表达。转基因家蚕丝的机械性能得到显著改善。
实施例7：
按照实施例1的方法，将上述构建的pBac[3xP3-DsRed]-MaSp2×24质粒(图4)导入家 蚕，在G1代卵转青期，通过荧光显微镜观察获得了表达DsRed标志基因的转基因家蚕2蛾，接着培育直到得到G3代、G4代。
在G4代时，随机取1个蛾区的5龄第3天家蚕1条，提取后部丝腺细胞基因组DNA为模板，采用与实施例1相同方法进行染色体插入位点分析，结果显示这个蛾区的后代插入位点是在第22号染色体2966967位点处，证明转座子已经插入到转基因家蚕家系的基因组内。
从G5代开始选择红眼基因型纯合的蛾区饲养，采用同蛾区蚕蛾交配，育成红眼基因纯合、后部丝腺细胞能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕新品种，定名为MASP2-24-5。
提取上述测定插入位点的家蚕茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP2-24-5蛋白的表达情况，结果得到与预期分子量大小相符的特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅱ占蚕丝轻链含量最高达1.9％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高51.2％，最大应变提高31.8％，杨氏模量提高36.2％，韧性提高94.3％。
上述研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅱ基因已插入到转基因家蚕家系MASP2-24-5基因组的22号染色体内，并能够在丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ，该蛋白能够随吐丝结茧行为进入蚕茧，此性状已稳定遗传和表达。转基因家蚕丝的机械性能得到显著改善。
实施例8：
按照实施例1的方法，将上述构建的pBac[3xP3-DsRed]-MaSp2×3质粒(图5)导入家蚕，在G1代卵转青期，通过荧光显微镜观察获得了表达DsRed标志基因的转基因家蚕1蛾接着培育直到得到G3代、G4代。
在G4代时，随机取5龄第3天家蚕1条，提取后部丝腺细胞基因组DNA为模板，采用与实施例1相同方法进行染色体插入位点分析，结果显示这个蛾区的后代插入位点是在第25号染色体2869975位点处，证明转座子已经插入到转基因家蚕家系的基因组内。
从G5代开始选择红眼基因型纯合的蛾区饲养，采用同蛾区蚕蛾交配，育成红眼基因纯合、后部丝腺细胞能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕新品种，定名为MASP2-3-1。
提取上述测定插入位点的家蚕茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP2-3-1蛋白的表达情况，结果得到与预期分子量大小相符的特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅱ占蚕丝轻链含量最高达2.2％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高18.4％，最大应变提高9.2％，杨氏模量提高13.2％，韧性提高31.8％。
上述研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅱ基因已插入到转基因家蚕家系MASP2-3-1基因组的25号染色体内，并能够在丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ，该蛋白能够随吐丝结茧行为进入蚕茧，此性状已稳定遗传和表达。转基因家蚕丝的机械性能得到显著改善。
综合从上述5个实施例可以看出，利用本发明方法，可以在家蚕丝腺细胞高效合成黑寡妇蜘蛛牵引丝蛋白Ⅱ，黑寡妇蜘蛛牵引丝蛋白Ⅱ可以像蚕丝一样由丝腺分泌进入腺腔，并进 一步吐出蚕体。该性状已能够稳定表达并遗传。采用本方法能够大量生产黑寡妇蜘蛛牵引丝蛋白Ⅱ，能够提高蚕丝机械性能，能够提高蚕桑经济效益，提高蚕农收入。
实施例9-14：黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的同时合成分泌
实施例9：
选取转黑寡妇蜘蛛牵引丝蛋白Ⅰ基因家蚕品种MASP1-2-1，以及转黑寡妇蜘蛛牵引丝蛋白Ⅱ基因家蚕品种MASP2-3-1，将这2个品种杂交，定名为MASP1-2×2-3。
提取MASP1-2×2-3家蚕的茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP1-2×2-3蛋白的表达情况，结果得到与预期分子量大小相符的2条特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅰ含量达到蚕丝轻链含量的2.1％。黑寡妇蜘蛛牵引丝蛋白Ⅱ含量达到蚕丝轻链蛋白含量的1.1％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高了20.5％，最大应变提高9.8％，杨氏模量提高14.0％，韧性提高33.9％。
研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ基因能够同时在丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ，这2个蛋白能够随吐丝结茧行为进入蚕茧，转基因家蚕丝的机械性能得到显著改善。
实施例10：
选取转黑寡妇蜘蛛牵引丝蛋白Ⅰ基因家蚕品种MASP1-12-1，以及转黑寡妇蜘蛛牵引丝蛋白Ⅱ基因家蚕品种MASP2-24-1，将这2个品种杂交，定名为MASP1-12×2-24。
提取MASP1-12×2-24家蚕的茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP1-12×2-24蛋白的表达情况，结果得到与预期分子量大小相符的2条特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅰ含量达到蚕丝轻链蛋白含量的2.2％。黑寡妇蜘蛛牵引丝蛋白Ⅱ含量达到蚕丝轻链蛋白含量的1.4％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高了62.3％，最大应变提高30.4％，杨氏模量提高28.1％，韧性提高98.9％。
研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ基因能够同时在丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ，这2个蛋白能够随吐丝结茧行为进入蚕茧，转基因家蚕丝的机械性能得到显著改善。
实施例11：
选取转黑寡妇蜘蛛牵引丝蛋白Ⅰ基因家蚕品种MASP1-16-8，以及转黑寡妇蜘蛛牵引丝蛋白Ⅱ基因家蚕品种MASP2-24-5，将这2个品种杂交，定名为MASP1-16-8×2-24-5。
提取MASP1-16-8×2-24-5家蚕的茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP1-16-8×2-24-5蛋白的表达情况，结果得到与预期分子量大小相符的2条特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅰ占蚕丝轻链含量高达1.9％。黑寡妇蜘蛛牵引丝蛋白Ⅱ占蚕丝轻链含量高达1.0％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高了57.3％，最大应变提高27.4％，杨氏模量提高41.1％，韧性提高92.9％。
研究结果证明黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ基因能够同时在丝腺细胞合成分泌黑寡 妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ，这2个蛋白能够随吐丝结茧行为进入蚕茧，转基因家蚕丝的机械性能得到显著改善。
实施例12：
选取转黑寡妇蜘蛛牵引丝蛋白Ⅰ基因家蚕品种MASP1-16-6，以及转黑寡妇蜘蛛牵引丝蛋白Ⅱ基因家蚕品种MASP2-24-1，将这2个品种杂交，得F1代。
将F1代杂交品种同蛾区个体自交留种为F2代，F2代家蚕采用单蛾育，筛选出荧光体视显微镜下表达红色荧光DsRed标记基因的家蚕饲养，采用同蛾区蚕蛾相互交配制成F3代。
F3代家蚕采用单蛾育，筛选出荧光体视显微镜下所有个体全部表达红色荧光DsRed标记基因的蛾区饲养，并同时采用Western Blot技术检测，筛选出同时表达黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的蛾区的蚕蛾相互交配制成F4代。
F4代开始，采用F3代同样方法饲养、筛选、制种，一直到F8代，育成2个牵引丝蛋白基因都纯合的品种；定名为MASP1-16-6×2-24-1-HO。
采用与野生型家蚕品种杂交2次以检测外源基因的纯化性。具体方法是取MASP1-16-6×2-24-1-HO的6个蛾子与野生型家蚕品种杂交得F1代，F1代的家蚕全部表现红色荧光DsRed标志基因，每1个F1代分别取2个蛾子进一步再与野生型家蚕品种杂交得F2代，每一个F2代蛾区的家蚕都有3/4的个体表现红色荧光DsRed标志基因，证明MASP1-16-6×2-24-1-HO品种的外源基因-黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ基因已经纯合。
提取MASP1-16-6×2-24-1-HO家蚕的茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP1-16-6×2-24-1-HO蛋白的表达情况，结果得到与预期分子量大小相符的2条特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅰ含量最高达到蚕丝轻链蛋白含量的2.0％。黑寡妇蜘蛛牵引丝蛋白Ⅱ含量最高达到蚕丝轻链蛋白含量的1.7％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高了69.4％，最大应变提高33.9％，杨氏模量提高46.3％，韧性提高97.1％。
研究结果证明MASP1-16-6×2-24-1-HO品种的黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ基因已经纯合，能够同时在丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ，这2个蛋白能够随吐丝结茧行为进入蚕茧，转基因家蚕丝的机械性能得到显著改善。
实施例13：
选取转黑寡妇蜘蛛牵引丝蛋白Ⅰ基因家蚕品种MASP1-16-2，以及转黑寡妇蜘蛛牵引丝蛋白Ⅱ基因家蚕品种MASP2-24-3，将这2个品种杂交，得F1代。
将F1代杂交品种同蛾区个体自交留种为F2代，F2代家蚕采用单蛾育，筛选出荧光体视显微镜下表达红色荧光DsRed标记基因的家蚕饲养，采用同蛾区蚕蛾相互交配制成F3代。
F3代家蚕采用单蛾育，筛选出荧光体视显微镜下所有个体全部表达红色荧光DsRed标记基因的蛾区饲养，并同时采用Western Blot技术检测，筛选出同时表达黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的蛾区的蚕蛾相互交配制成F4代。
F4代开始，采用F3代同样方法饲养、筛选、制种，一直到F8代，育成2个牵引丝蛋白基因都纯合的品种；定名为MASP1-16-2×2-24-3-HO。
采用与实施例12相同的检测方法，即与野生型家蚕品种杂交2次以检测外源基因的纯化性，结果证明MASP1-16-2×2-24-3-HO品种的外源基因-黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ基因已经纯合。
提取MASP1-16-2×2-24-3-HO家蚕的茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP1-16-2×2-24-3-HO蛋白的表达情况，结果得到与预期分子量大小相符的2条特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅰ占蚕丝轻链含量高达1.8％。黑寡妇蜘蛛牵引丝蛋白Ⅱ占蚕丝轻链含量高达1.3％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高了65.6％，最大应变提高31.8％，杨氏模量提高45.6％，韧性提高95.3％。
研究结果证明MASP1-16-2×2-24-3-HO品种的黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ基因已经纯合，能够同时在丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ，这2个蛋白能够随吐丝结茧行为进入蚕茧，转基因家蚕丝的机械性能得到显著改善。
实施例14：
选取转黑寡妇蜘蛛牵引丝蛋白Ⅰ基因家蚕品种MASP1-16-14，以及转黑寡妇蜘蛛牵引丝蛋白Ⅱ基因家蚕品种MASP2-24-4，将这2个品种杂交，得F1代。
将F1代杂交品种同蛾区个体自交留种为F2代，F2代家蚕采用单蛾育，筛选出荧光体视显微镜下表达红色荧光DsRed标记基因的家蚕饲养，采用同蛾区蚕蛾相互交配制成F3代。
F3代家蚕采用单蛾育，筛选出荧光体视显微镜下所有个体全部表达红色荧光DsRed标记基因的蛾区饲养，并同时采用Western Blot技术检测，筛选出同时表达黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的蛾区的蚕蛾相互交配制成F4代。
F4代开始，采用F3代同样方法饲养、筛选、制种，一直到F8代，育成2个牵引丝蛋白基因都纯合的品种；定名为MASP1-16-14×2-24-4-HO。
采用与实施例12相同的检测方法，即与野生型家蚕品种杂交2次以检测外源基因的纯化性，结果证明MASP1-16-14×2-24-4-HO品种的外源基因-黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ基因已经纯合。
提取MASP1-16-14×2-24-4-HO家蚕的茧丝蛋白为材料，采用SDS-PAGE电泳和黑寡妇蜘蛛牵引丝抗体的Western blot技术分析转基因家蚕MASP1-16-14×2-24-4-HO蛋白的表达情况，结果得到与预期分子量大小相符的2条特异性蛋白条带。SDS-PAGE电泳条带灰度分析显示，黑寡妇蜘蛛牵引丝蛋白Ⅰ占蚕丝轻链含量高达2.0％。黑寡妇蜘蛛牵引丝蛋白Ⅱ占蚕丝轻链含量高达1.7％。
对转基因家蚕丝的机械性能测定结果显示，与野生型对照品种Lan10相比，最大应力提高了69.4％，最大应变提高33.9％，杨氏模量提高46.3％，韧性提高97.1％。
研究结果证明MASP1-16-14×2-24-4-HO品种的黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ基因已经纯合，能够同时在丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ，这2个蛋白能够随吐丝结茧行为进入蚕茧，转基因家蚕丝的机械性能得到显著改善。
综合从上述6个实施例可看出，利用本发明方法在家蚕丝腺细胞高效合成了黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ，黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ可以像蚕丝一样由丝腺分泌进入腺腔，并进一步吐出蚕体，且该性状已能够稳定表达并遗传。采用本方法能够大量生产黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ，能够提高蚕丝机械性能，能够提高蚕桑经济效益，提高蚕农的收入。
本发明中涉及的序列如下：
SEQ ID NO.1：编码2倍re-MaSp1的碱基序列
来源：人工合成
Figure PCTCN2016102266-appb-000006
SEQ ID NO.2：编码2倍re-MaSp1的蛋白序列
来源：根据编码2倍re-MaSp1的碱基序列翻译
Figure PCTCN2016102266-appb-000007
SEQ ID NO 3：编码3倍MaSp2基因重复单元碱基序列
来源：人工合成
Figure PCTCN2016102266-appb-000008
SEQ ID NO.4：编码3倍MaSp2基因重复单元蛋白序列
来源：根据编码3倍MaSp2基因重复单元碱基序列表达
Figure PCTCN2016102266-appb-000009
上述具体实施方式用来解释说明本发明，而不是对本发明进行限制，在本发明的精神和权利要求的保护范围内，对本发明作出的任何修改和改变，都落入本发明的保护范围。

Claims (11)

  1. 一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于该方法的步骤如下：
    (1)通过转基因育成黑寡妇蜘蛛牵引丝蛋白基因纯合且能够合成分泌黑寡妇蜘蛛牵引丝蛋白的家蚕品种；
    (2)通过上述家蚕品种的家蚕丝腺细胞合成分泌黑寡妇蜘蛛牵引丝蛋白，并随家蚕吐丝结茧行为进入蚕茧。
  2. 根据权利要求1所述的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于：所述的黑寡妇蜘蛛牵引丝蛋白基因纯合且能够合成分泌黑寡妇蜘蛛牵引丝蛋白的家蚕品种为带有红色荧光DsRed标记基因且能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕、带有红色荧光DsRed标记基因且能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕或者上述两种转基因家蚕杂交后的家蚕品种。
  3. 根据权利要求1所述的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于：所述带有红色荧光DsRed标记基因且能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕和带有红色荧光DsRed标记基因且能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕杂交后的家蚕品种具体是采用以下方式进行培育杂交获得：
    (1)通过转基因育成带有红色荧光DsRed标记基因且能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕和带有红色荧光DsRed标记基因且能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕；
    (2)将上述两种转基因家蚕品种杂交，育成F1代杂交品种；
    (3)将F1代杂交品种同蛾区个体自交留种为F2代，F2代家蚕采用单蛾育，筛选出荧光体视显微镜下表达红色荧光DsRed标记基因的家蚕饲养，采用同蛾区蚕蛾相互交配制成F3代；
    (4)F3代家蚕采用单蛾育，筛选出荧光体视显微镜下所有个体全部表达红色荧光DsRed标记基因的蚕蛾在同一蛾区饲养，并同时采用Western Blot技术检测，筛选出同时表达黑寡妇蜘蛛牵引丝蛋白Ⅰ和蛋白Ⅱ的蛾区的蚕蛾相互交配制成F4代；
    (5)F4代开始均采用与F3代同样的方法饲养、筛选、制种，一直到F8代，育成两个黑寡妇蜘蛛牵引丝蛋白基因都纯合的家蚕品种。
  4. 根据权利要求2或3所述的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于：所述带有红色荧光DsRed标记基因且能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕采用以下方式转基因培育获得：
    A1)采用分子生物学方法构建pBac[3xP3-DsRed]-MaSp1质粒作为在家蚕丝腺中表达的黑寡妇蜘蛛牵引丝蛋白Ⅰ基因载体，pBac[3xP3-DsRed]-MaSp1质粒以piggyBac转座子为基础包含外源基因的黑寡妇蜘蛛牵引丝蛋白Ⅰ基因和作为标记基因的红色荧光DsRed基因的两个表达框；
    A2)采用显微注射转基因家蚕方法将pBac[3xP3-DsRed]-MaSp1质粒及能够提供piggyBac转座酶的辅助质粒pHA3PIG按浓度比1：1的比例导入家蚕产卵后6小时以内的受精卵内，利用pBac[3xP3-DsRed]-MaSp1质粒中的piggyBac转座子将黑寡妇蜘蛛牵引丝蛋白Ⅰ基因插入到家蚕基因组内；
    A3)蚕卵孵化后饲养至成虫，然后与非转基因家蚕交配制种续代，此代为G1代，在G1代蚕卵的转青期，通过荧光体视显微镜观察筛选出单眼表达红色荧光DsRed标记基因的转基因家蚕，饲养至成虫再与非转基因家蚕交配制种续代成为G2代；
    A4)G2代家蚕采用单蛾育，筛选出荧光体视显微镜下表达红色荧光DsRed标记基因的家蚕，采用同蛾区蚕蛾相互交配制成G3代；
    A5)G3代家蚕采用单蛾育，同蛾区表达红色荧光DsRed标记基因的蚕蛾相互交配，制成G4代；
    A6)从G4代开始并经连续3代采用红眼表型纯一的蛾区饲养、单蛾育和同蛾区蚕蛾交配的相同方法进行选择和交配，育成红眼基因和黑寡妇蜘蛛牵引丝蛋白Ⅰ基因纯合、能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅰ的转基因家蚕。
  5. 根据权利要求2或3所述的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于：
    所述带有红色荧光DsRed标记基因且能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕采用以下方式转基因培育获得：
    B1)采用分子生物学方法构建pBac[3xP3-DsRed]-MaSp2质粒作为在家蚕丝腺中表达的黑寡妇蜘蛛牵引丝蛋白Ⅱ基因载体，pBac[3xP3-DsRed]-MaSp2质粒以piggyBac转座子为基础包含有作为外源基因的黑寡妇蜘蛛牵引丝蛋白Ⅱ基因和作为标记基因的红色荧光DsRed基因的两个表达框；
    B2)采用显微注射转基因家蚕方法将pBac[3xP3-DsRed]-MaSp2质粒及能够提供piggyBac转座酶的辅助质粒pHA3PIG按浓度比1：1的比例导入家蚕产卵后6小时以内的受精卵内，利用pBac[3xP3-DsRed]-MaSp2质粒中的piggyBac转座子将黑寡妇蜘蛛牵引丝蛋白Ⅱ基因插入到家蚕基因组内；
    B3)蚕卵孵化后饲养至成虫，然后与非转基因家蚕交配制种续代，此代为G1代，在G1代蚕卵的转青期，通过荧光体视显微镜观察筛选出单眼表达红色荧光DsRed标记基因的转基因家蚕，饲养至成虫再与非转基因家蚕交配制种续代成为G2代；
    B4)G2代家蚕采用单蛾育，筛选出荧光体视显微镜下表达红色荧光DsRed标记基因的家蚕，采用同蛾区蚕蛾相互交配制成G3代；
    B5)G3代家蚕采用单蛾育，同蛾区表达红色荧光DsRed标记基因的蚕蛾相互交配，制成G4代；
    B6)从G4代开始并经连续3代采用红眼表型纯一的蛾区饲养、单蛾育和同蛾区蚕蛾交配的相同方法进行选择和交配，育成红眼基因和黑寡妇蜘蛛牵引丝蛋白Ⅱ基因纯合、能够合成分泌黑寡妇蜘蛛牵引丝蛋白Ⅱ的转基因家蚕。
  6. 根据权利要求4所述的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于：所述的质粒pBac[3xP3-DsRed]-MaSp1是以piggyBac转座子为基础并带有Amp抗性基因，包括piggyBac转座子的两个转座臂PBL和PBR，以及两个转座臂之间的两个功能表达框，一个功能表达框是3×P3启动子启动的红色荧光蛋白基因表达框，即3×P3Promoter–DsRed-SV40，另一个功能表达框是包含家蚕丝素蛋白重链基因启动子、丝素蛋白重链基因信号肽、黑寡妇蜘蛛牵引丝蛋白Ⅰ基因和家蚕丝素蛋白重链基因3’末端的表达框，即Fibroin H chain Promoter-Fibroin H chain signal peptide-MaSp1-Fibroin H chain PolyA。
  7. 根据权利要求5所述的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于：所述的质粒pBac[3xP3-DsRed]-MaSp2是以piggyBac转座子为基础并带有Amp抗性基因，包括piggyBac转座子的两个转座臂PBL和PBR，以及两个转座臂之间的两个功能表达框，一个功能表达框是3×P3启动子启动的红色荧光蛋白基因表达框，即3×P3Promoter–DsRed-SV40，另一个功能表达框是包含家蚕丝素蛋白重链基因启动子、丝素蛋白重链基因信号肽、黑寡妇蜘蛛牵引丝蛋白Ⅱ基因和家蚕丝素蛋白重链基因3’末端的表达框，即Fibroin H chain Promoter-Fibroin H chain signal peptide-MaSp2-Fibroin H chain PolyA。
  8. 根据权利要求2-4和6中任一所述的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于：所述的黑寡妇蜘蛛牵引丝蛋白Ⅰ基因是包含2倍到16倍MaSp1基因重复单元Type1-Type2-Type3-Type4。
  9. 根据权利要求2、3、5和7中任一所述的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于：所述的黑寡妇蜘蛛牵引丝蛋白Ⅱ基因是包含3倍到24倍MaSp2基因重复单元Type1-Type1-Type4。
  10. 根据权利要求1所述的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于：所述黑寡妇蜘蛛牵引丝蛋白基因在家蚕丝腺细胞特异表达，在家蚕丝素蛋白重链信号肽的作用下分泌到丝腺腺腔，并随吐丝行为分泌到蚕茧。
  11. 根据权利要求1或10所述的一种利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法，其特征在于：所述的家蚕丝腺细胞合成分泌的黑寡妇蜘蛛牵引丝蛋白为黑寡妇蜘蛛牵引丝蛋白Ⅰ或者黑寡妇蜘蛛牵引丝蛋白Ⅱ或者上述两种的混合。
PCT/CN2016/102266 2015-10-23 2016-10-17 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法 WO2017067423A1 (zh)

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
CN201510698099.1A CN105400815B (zh) 2015-10-23 2015-10-23 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白1的方法
CN201510697644.5 2015-10-23
CN201510697644.5A CN105463022B (zh) 2015-10-23 2015-10-23 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白2的方法
CN201510698099.1 2015-10-23
CN201510698879.6A CN105400817B (zh) 2015-10-23 2015-10-23 利用家蚕同时合成分泌黑寡妇蜘蛛牵引丝蛋白1和蛋白2的方法
CN201510698879.6 2015-10-23

Publications (1)

Publication Number Publication Date
WO2017067423A1 true WO2017067423A1 (zh) 2017-04-27

Family

ID=58556713

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2016/102266 WO2017067423A1 (zh) 2015-10-23 2016-10-17 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法

Country Status (1)

Country Link
WO (1) WO2017067423A1 (zh)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012050919A2 (en) * 2010-09-28 2012-04-19 The University Of Notre Dame Chimeric spider silk and uses thereof
CN104846011A (zh) * 2015-03-18 2015-08-19 浙江大学 利用家蚕后部丝腺合成分泌蜂王浆主蛋白1的方法
CN105400815A (zh) * 2015-10-23 2016-03-16 浙江大学 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白1的方法
CN105400817A (zh) * 2015-10-23 2016-03-16 浙江大学 利用家蚕同时合成分泌黑寡妇蜘蛛牵引丝蛋白1和蛋白2的方法
CN105463022A (zh) * 2015-10-23 2016-04-06 浙江大学 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白2的方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012050919A2 (en) * 2010-09-28 2012-04-19 The University Of Notre Dame Chimeric spider silk and uses thereof
CN104846011A (zh) * 2015-03-18 2015-08-19 浙江大学 利用家蚕后部丝腺合成分泌蜂王浆主蛋白1的方法
CN105400815A (zh) * 2015-10-23 2016-03-16 浙江大学 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白1的方法
CN105400817A (zh) * 2015-10-23 2016-03-16 浙江大学 利用家蚕同时合成分泌黑寡妇蜘蛛牵引丝蛋白1和蛋白2的方法
CN105463022A (zh) * 2015-10-23 2016-04-06 浙江大学 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白2的方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YUANSONG ZHANG ET AL.: "Expression of EGFP-spider dragline silk fusion protein in BmN cells and larvae of silkworm showed the solubility is primary limit for dragline proteins yield", MOLECULAR BIOLOGY REPORTS, vol. 35, 31 December 2008 (2008-12-31), pages 329 - 335, XP019611880, ISSN: 0301-4851 *
ZHANG, YUANSONG ET AL.: "zhuang3ji1yin1 jialcan2 shenglchan3 han2 zhulsildan4bai2 de xinlxing2 fu4he2 jian3si1 xianlweil", JOURNAL OF TEXTILE RESEARCH, vol. 33, no. 5, 31 May 2012 (2012-05-31), pages 1 - 5, ISSN: 0253-9721 *

Similar Documents

Publication Publication Date Title
CN105400815B (zh) 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白1的方法
US11089767B2 (en) Transgenic silkworms expressing spider silk
US20210400936A1 (en) Methods, compositions and systems for production of recombinant spider silk polypeptides
CN111518832B (zh) 蜘蛛梨状腺丝蛋白基因序列的应用及其改良家蚕丝性能的方法
CN111518831B (zh) 蜘蛛葡萄状腺丝蛋白基因序列的应用及其改良家蚕丝性能的方法
CN111500591B (zh) 蜘蛛聚状腺丝蛋白基因序列的应用及其改良家蚕丝性能的方法
Li et al. Construction of transgenic silkworm spinning antibacterial silk with fluorescence
US8394942B2 (en) Nucleic acid encoding a silk protein and a method of producing a silk thread using same
CN111850039A (zh) 表达蛋白分布于家蚕丝胶层的家蚕丝心蛋白重链表达系统及其制备方法和应用
CN105400817B (zh) 利用家蚕同时合成分泌黑寡妇蜘蛛牵引丝蛋白1和蛋白2的方法
CN102321650B (zh) 一种利用转基因家蚕生产荧光抗菌丝的方法
JP5098039B2 (ja) 化合物の結合効率が向上した絹糸
JP5997772B2 (ja) カイコフィブロイン重鎖遺伝子の突然変異配列、及び突然変異を誘発する方法と応用
CN105463022B (zh) 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白2的方法
JP2011103816A (ja) トランスジェニックカイコ
WO2017067423A1 (zh) 利用家蚕丝腺生物反应器合成分泌黑寡妇蜘蛛牵引丝蛋白的方法
JP2006521802A (ja) 鱗翅目の後部絹糸腺における有用ポリペプチド発現を指令する核酸およびその応用
CN105907786B (zh) 用于表达t4连接酶的家蚕中部丝腺生物反应器双启动子通用型质粒及其应用与方法
US20210047655A1 (en) Transgenic silkworms expressing hagfish thread keratin
WO2023190453A1 (ja) キメラ絹糸を生産するゲノム改変カイコ
CN115820736B (zh) 家蚕丝胶蛋白Ser4在提高蚕丝性能中的应用及其方法
CN105907784B (zh) 用于表达t4连接酶的家蚕后部丝腺生物反应器双启动子通用型质粒及其应用与方法
JP2004344123A (ja) 外来遺伝子発現を促進するポリヌクレオチド
CN117025672A (zh) 一种基于基因打靶家蚕制备蜘蛛家蚕复合丝纤维的方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 16856854

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 16856854

Country of ref document: EP

Kind code of ref document: A1