WO2023213009A1 - 一种含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法 - Google Patents

一种含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法 Download PDF

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WO2023213009A1
WO2023213009A1 PCT/CN2022/105407 CN2022105407W WO2023213009A1 WO 2023213009 A1 WO2023213009 A1 WO 2023213009A1 CN 2022105407 W CN2022105407 W CN 2022105407W WO 2023213009 A1 WO2023213009 A1 WO 2023213009A1
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silk
web
ampulla
spider
silk protein
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French (fr)
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贡成良
王崇龙
胡小龙
李继杰
朱敏
张星
童新宇
邱群婻
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苏州大学
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43513—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae
    • C07K14/43518—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from arachnidae from spiders
    • 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
    • A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
    • A01K67/0337—Genetically modified Arthropods
    • A01K67/0339—Genetically modified insects, e.g. Drosophila melanogaster, medfly
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43586—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from silkworms
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    • 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
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A01K2207/00—Modified animals
    • A01K2207/15—Humanized animals
    • 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
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/072—Animals genetically altered by homologous recombination maintaining or altering function, i.e. knock in
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/70—Invertebrates
    • A01K2227/706—Insects, e.g. Drosophila melanogaster, medfly
    • 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
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/01—Animal expressing industrially exogenous proteins
    • C—CHEMISTRY; METALLURGY
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
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    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14021—Viruses as such, e.g. new isolates, mutants or their genomic sequences
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    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vectore

Definitions

  • the invention relates to the field of genetic engineering, and specifically relates to a method of producing gold-containing silk web-weaving spider large ampulla gland silk protein composite silk through silkworms.
  • Silkworms are the only insects that can be raised indoors to provide large amounts of silk fiber.
  • Silk protein is mainly composed of sericin and silk fibroin
  • silk fiber is mainly composed of water-insoluble silk fibroin heavy chain (350kDa), silk fibroin light chain (25.8kDa) and P25 protein (25.7kDa) according to 6: Assembled at a molar ratio of 6:1, the mechanical properties of silk fibroin are mainly determined by the high molecular weight of the silk fibroin heavy chain and the high degree of repetition of the amino acid sequence.
  • the spider botryoidal silk protein gene is black widow spider botryoidal silk or garden spider botryoidal silk.
  • the gene sequence composed of silk 1-fold base repeating units with 1-8 times continuous repeats has applications such as improving the performance of silkworm silk.
  • the pBac-ACSP plasmid a vector for silkworm synthesis and secretion of botryoidal silk protein, was constructed, and the plasmid and auxiliary plasmid were introduced.
  • transposons were used to introduce the fluorescent protein gene and botryoidal silk protein gene into the silkworm genome, and stable inheritance and expression were achieved to breed transgenic silkworms that secrete spider botryoidal silk protein.
  • the existing technology uses a method of introducing plasmids and auxiliary plasmids into fertilized eggs of silkworms, which is time-consuming and unsuitable for silkworm eggs of practical varieties with duality. Only the silkworms that hatch from these eggs can obtain high-performance and high-quality silk.
  • the object of the present invention is to provide a method for producing composite silk containing gold silk web-weaving spider ampulla gland silk protein through silkworms.
  • the technical solution adopted by the present invention is: a preparation method of gold-containing web-weaving spider large ampulla gland silk protein composite silk, which is characterized by including the following steps: inoculating the recombinant virus into 5th instar silkworm larvae; After feeding the mulberry leaves, collect the silk to obtain composite silk containing the golden silk web-weaving spider large ampulla silk protein; the recombinant virus contains a sequence expressing the golden silk web-weaving spider large ampulla silk protein, and the recombinant virus is inoculated into 5-year-old silkworms The number of larvae is 10 4 to 10 8 copies/silkworm.
  • recombinant DNA is transfected into cultured cells, and then cultured until the cells develop disease, the cell culture supernatant is taken to inoculate the cultured cells again, and then cultured until the cells develop disease, the cell culture supernatant is collected to obtain the recombinant virus; the recombinant DNA contains expression Sequence of the major ampulla gland silk protein of the golden web-weaver spider.
  • the recombinant plasmid is transformed into DH10Ac Escherichia coli, and then spread on an LB agar medium plate, and then cultured, and then picks white colonies and extracts recombinant DNA;
  • the DH10Ac Escherichia coli contains AcBacmid;
  • the recombinant plasmid contains expression gold Sequence of silk web spider major ampulla gland silk protein, preferably, the LB agar medium contains tetracycline, kanamycin, gentamicin, IPTG and X-gal, tetracycline, kanamycin, gentamicin
  • concentrations of mycin, IPTG and X-gal were 10 ⁇ g/ml, 50 ⁇ g/ml, 7 ⁇ g/ml, 40 ⁇ g/ml and 100 ⁇ g/ml respectively.
  • the DNA fragment containing the sequence of the golden silk-weaving spider's large ampulla silk protein is cloned into a plasmid to obtain a recombinant plasmid; the DNA fragment containing the DNA fragment expressing the golden silk-weaving spider's large ampulla silk protein sequence is obtained
  • the sequence is SEQ ID NO: 1; the plasmid is pFAST-Bac-Dual.
  • the preferred method for synthesizing the FibH-MaSp-g-polyA Fib expression cassette shown in SEQ ID NO: 1 is to use a fully chemical synthesis method; the promoter sequence and the addition of a coding signal peptide sequence can also be obtained by PCR using the silkworm genome as a template.
  • the MaSp-g sequence was obtained by RT-PCR using the RNA of the great ampulla gland of the golden silk web spider as a template, and then spliced to obtain the FibH-MaSp-g-polyA Fib expression cassette; it can also be amplified by PCR
  • the FibH-MaSp-g-polyA Fib expression cassette was prepared by a method combined with chemical synthesis. Cloning can be done by enzyme ligation or seamless cloning.
  • antibiotics and ecdysone are added when feeding mulberry leaves.
  • mulberry leaves containing antibiotics are fed for one day, and then conventional mulberry leaves are fed until the silkworms are mature, and then fed
  • the mulberry leaves containing ecdysone are sprayed once or the ecdysone is sprayed once, and then the silk is collected; the silk collection is a conventional technique, including reeling, cocooning, cocoon picking, and silk reeling.
  • the preferred silkworm varieties to be inoculated in the present invention are silkworm varieties practical for silk cocoon breeding, such as Jingsong ⁇ Haoyue, Zhong 2016 ⁇ Ri 2016.
  • the original silkworm species, such as J14-Hua, can also be used.
  • the method of the present invention has universal applicability and successfully solves the current problems.
  • the preferred development period of the inoculated fifth-instar silkworm larvae is 1-3 days after the fifth-instar molt. When inoculating the virus, you can use a No. 4 insect needle to take the collected cell culture supernatant or the centrifuged purified virus to inoculate the silkworm larvae.
  • the amount of the recombinant virus inoculated into the 5th instar silkworm larvae is 10 5 to 10 7 copies/silkworm.
  • the present invention first synthesizes the large ampulla gland silk protein gene expression cassette FibH of the golden web-weaving spider with a coding signal peptide sequence at the 5' end and a tailing signal at the 3' end controlled by the promoter of the silk fibroin heavy chain gene of Bombyx mori.
  • -MaSp-g-polyA FibH whose sequence is as SEQ ID NO: 1; then clone it into the multiple cloning site of pFAST-Bac Tm -Dual to construct plasmid pFAST-FibH-MaSp-g; then transform the plasmid to contain AcBacmid DH10Ac Escherichia coli, and then spread it on an LB agar medium plate containing tetracycline, kanamycin, gentamicin, IPTG and g DNA; then transfect the recombinant AcBacmid-FibH-MaSp-g DNA into Spodoptera frugiperda Sf9 cultured cells, and culture them routinely until the cells develop disease.
  • the cocoons were harvested after 7 days. After the cocoons were dried, they were reeled to obtain composite silk containing the golden silk web-weaving spider's ampulla gland silk protein.
  • the development period of the inoculated 5th instar silkworm larvae is 1 to 3 days after the 5th instar molt; the antibiotic solution is ciprofloxacin or norfloxacin or florfenicol; the concentration of the ecdysone solution is 20 to 25mg/ L.
  • Silk protein has been widely used in new materials. Spider silk protein and silk protein each have unique characteristics. People hope to obtain a mixture of spider silk protein and silk protein through biological methods to meet the diverse requirements for silk protein in preparing various materials. sexual needs. Chimeric silk containing spider silk protein can be obtained through piggyBac-mediated transgenic technology of silkworms, and the mechanical properties of the silk fiber are improved to a certain extent. However, the content of spider silk protein in the chimeric silk is very limited; through TALEN-mediated Guided homologous terminal recombination has achieved the replacement of the silk protein heavy chain gene of the silkworm with multiple doubled repeating units of the spider main ampulla gland silk protein gene.
  • the spider silk protein in the chimeric silk produced by the genetically modified silkworm obtained by this method Silk protein levels increased significantly, and although the extensibility of this chimeric silk increased, its strength decreased.
  • current genetic modification of silkworms is basically limited to polymorphic silkworms with no practical production value.
  • Baculoviruses are pathogens of many insects, and recombinant baculoviruses have been widely used to develop bioinsecticides, express foreign proteins, and deliver genes to vertebrate cells. There are many types of baculoviruses, and their host domains, infectivity, and pathogenicity are different.
  • the present invention uses recombinant Autographa californica nuclear polyhedrovirus to mediate the expression of golden silk web spider gland silk protein in the posterior silk gland of silkworm. , from the acquisition of composite silk containing spider silk protein, the relevant technical solutions have not been reported. It is difficult to obtain spider silk in large quantities by directly raising spiders.
  • the technology of the present invention can be used to obtain a large amount of composite silk containing the silk protein of the large ampulla gland of golden web-weaving spiders.
  • the technology of the present invention can be used to obtain the advantages of high production performance of practical silkworm varieties. Chimeric silk with excellent properties of silk and spider silk.
  • Figure 1 shows the identification of recombinant AcBacmid-FibH-MaSp-g in Example 1.
  • Figure 2 shows the PCR identification of the recombinant virus Ac NPV-FibH-MaSp-g in Example 1.
  • Figure 3 shows the PCR detection of Ac NPV-FibH-MaSp-g copy number in the blood and silk glands of silkworms infected in Example 2.
  • Figure 4 shows the MaSp-g expression of Ac NPV-FibH-MaSp-g in the posterior silk gland detected by Western blot in Example 2.
  • Figure 5 shows the Western blot detection of MaSp-g in cocoon silk in Example 2.
  • Figure 6 shows the RT-PCR detection of MaSp-g transcription in silk glands infected with AcNPV-FHP-MaSp-g in Example 3.
  • Figure 7 shows the qRT-PCR detection of MaSp-g transcription at different phases in silk glands infected with AcNPV-FHP-MaSp-g in Example 3.
  • Figure 8 shows Western blot detection of silk gland tissue of Bombyx mori after injection of different titers of viruses in Example 3.
  • Figure 9 RT-PCR in Example 4 detects the expression of MaSp-g gene in silk glands.
  • FIG. 10 Western blot in Example 4 detects the expression of MaSp-g gene in silk glands.
  • the specific preparation operations as well as the cultivation and test characterization of silkworms involved in the present invention are all conventional techniques.
  • the present invention will be further described below in conjunction with the accompanying drawings and examples.
  • the silkworms are spring silkworms.
  • Example 1 Entrust a commercial company to perform chemical synthesis.
  • the synthesized sequence is as SEQ ID NO: 1, which contains a sequence expressing the golden silk web spider's large ampulla gland silk protein. It is named FibH-MaSp-g-polyA FibH .
  • the sequence Add Not I and Pst I sites on both sides respectively.
  • FibH-MaSp-g-polyA FibH was cloned into the NotI/PstI site of pFAST-Bac Tm- Dual (product of Invitrogen) to construct plasmid pFAST-FibH-MaSp-g.
  • Plasmid pFAST-FibH-MaSp-g was transformed into E. coli containing AcBacmid DH10Ac, and then spread on tetracycline and kanax containing 10 ⁇ g/ml, 50 ⁇ g/ml, 7 ⁇ g/ml, 40 ⁇ g/ml and 100 ⁇ g/ml respectively.
  • the wild AcBacmid can amplify a product of about 300 bp using primers M13-F (SEQ ID NO: 2) and M13-R (SEQ ID NO: 4), indicating that the recombinant Bacmid was successfully constructed.
  • Figure 1 recombinant AcBacmid-FibH-MaSp-g DNA was extracted, and primers M13-F and HC-left-R were used for PCR identification; wild-type AcBacmid was identified by PCR using primers M13-F and M13-R, and the PCR product was identified with 1 % agarose gel electrophoresis separation, M, DNA standard molecular weight; lane 1, wild Bacmid; lane 2, recombinant AcBacmid-FibH-MaSp-g.
  • a specific band of approximately 8 kb can be amplified from the total DNA of virus-infected cells, indicating that AcNPV-FibH-MaSp-g was successfully constructed.
  • Figure 2 the total DNA of the diseased cells after transfection with AcBacmid-FibH-MaSp-g was extracted, PCR identification was performed using primers EcoRI-FG-F and XhoI-FG-R, and the amplified product was subjected to agarose gel electrophoresis, lane M , standard molecular weight DNA; lanes 1 and 2, recombinant virus Ac NPV-FibH-MaSp-g.
  • the virus stock solution was stored at -20°C for use in the following examples. Take the stock solution of the virus, extract the viral DNA, and determine the copy number of the virus by quantitative PCR using P4-F (SEQ ID NO:7) and P4-R (SEQ ID NO:8).
  • Example 2 The silkworms of the "China 2016 ⁇ Japan 2016" variety are raised to the fifth age, and each silkworm is inoculated with 10 6 copies of the virus (the stock solution of the AcNPV-FibH-MaSp-g recombinant virus), and then the silkworms are fed and sprayed with ciprofloxacin solution of fresh mulberry leaves for 1 day, and then raise the silkworms with fresh mulberry leaves at 24°C until they are mature.
  • the virus the stock solution of the AcNPV-FibH-MaSp-g recombinant virus
  • Detection of MaSp-g in silk Take the above composite silk, add the protein solution dissolved in conventional lithium bromide solution to the dialysis membrane, and perform Western blot detection after 72 hours of dialysis. The results are shown in Figure 5, and Masp-g can be observed. Signal, indicating that cocoon silk contains MaSp-g, lane M, standard molecular weight DNA; lane con, silkworm silkworm not infected with the virus AcNPV-FHP-MaSp-g; lanes 1-3, silkworm infected with AcNPV-FHP-MaSp-g Silk. The primary antibody was anti-MaSp-g.
  • Example 3 1. Inoculate silkworms with the recombinant virus AcNPV-FibH-MaSp-g: The "J14-Hua" variety of silkworms was raised to the fifth instar, and each silkworm was inoculated with 10 4 , 10 5 , and 10 6 copies of the virus.
  • PCR detection of the proliferation of virus AcNPV-FibH-MaSp-g in silk glands Take silk gland tissues at different stages of virus infection, extract RNA, reverse-transcribe into cDNA, and use primer MaSP-gF (SEQ ID NO: 9) and primer MaSP-gR (SEQ ID NO: 10) for PCR amplification.
  • the electrophoresis results of the PCR product are shown in Figure 6. Each detection group can amplify specific bands, indicating that AcNPV-FibH-MaSp-g enters the silkworm.
  • lane M standard molecular weight DNA
  • lanes 1 and 2 pFAST-FibH-MaSp-g positive control
  • lane 3 silk gland inoculated with 10 4 copies of the virus infected for 72 hours
  • lane 4 silk glands infected with 10 5 copies of the virus for 72 hours
  • Detection of recombinant protein MaSp-g in silk gland tissue by Western blotting Take the silk glands of silkworms infected with the virus for 72 hours and use the antibody of MaSp-g for Western blotting detection. The detection results are shown in Figure 8.
  • the signal band representing MaSp-g can be detected in the virus-infected samples, indicating that the MaSp-g gene has been translated into protein, lane con, no virus injection AcNPV-FibH-MaSp-g control; lanes 1 and 2, injection 10 4. 10 5 copies of the virus infected the posterior silk gland for 72 hours.
  • step 3 After drying the fresh mulberry leaves sprayed with ecdysone solution, feed the mature silkworms in step 2 once, then move the mature silkworms to the cluster, and create cocoons at 25°C. After 7 days, harvest the cocoons and freshly After drying and storage, before reeling, the stored dry cocoons are degummed and reeled to obtain composite silk containing gold silk web-weaving spider large ampulla gland silk protein.
  • Example 4 On the 2nd day after the silkworms of the variety "Jingsong ⁇ Haoyue" were reared to the 5th instar, the AcNPV-FibH-MaSp-g virus-infected cultured cells supernatant collected in Example 1 was puncture-inoculated with No. 4 insect needles, and then inoculated with The fresh mulberry leaves sprayed with norfloxacin solution were dried and then fed with silkworms for 1 day, and then the fresh mulberry leaves were used to raise the silkworms at about 24°C until the silkworms matured.
  • 1RT-PCR detection of the transcription of MaSp-g in the silk glands of silkworms inoculated with the virus Take the posterior silk glands of silkworms 72 hours after being inoculated with the virus, extract total RNA, reverse-transcribe into cDNA, and use primer MaSP-g-F (SEQ ID NO: 9) and primer MaSP-g-R (SEQ ID NO: 10) for PCR amplification.
  • the electrophoresis results of the PCR products are shown in Figure 9.
  • Detection of recombinant protein MaSp-g in silk gland tissue by Western blotting Take the posterior silk gland of silkworm infected with the virus for 72 hours, and use the antibody of MaSp-g for Western blotting detection. The detection results are shown in Figure 10.
  • the signal band representing MaSp-g can be detected in the virus-infected samples, indicating that the MaSp-g gene has been translated into protein, lane con, the posterior silk gland of AcNPV-FHP-MaSp-g silkworm that has not been inoculated with the virus; lane H1 -H3, posterior silk gland of silkworm inoculated with virus AcNPV-FHP-MaSp-g for 72 hours.
  • the average breaking strength of conventional silk is 666.04MPa, and the average elongation is 11.81%; the composite silk of the present invention has an average tensile strength of 8.18 N, an average breaking strength of 1042.08 MPa, and an average elongation of 6.9%. Average of 50 samples. It can be seen that the length of the composite silk prepared by the method of the present invention reaches 70% of the length of uninfected silk.
  • the existing spider protein modified silk is only about 40% of the length of unmodified silk, or even lower. The present invention not only improves the It improves the mechanical strength of silk and significantly increases the length of existing composite silk.
  • the existing technology can express spider silk proteins through E. coli, yeast, animal cells or transgenic animals and plants. To further obtain spider silk fibers, it is necessary to purify the recombinant protein through tedious steps and then further achieve it through artificial spinning. This process is not only time-consuming but It is expensive, and the current technical level is difficult to produce on a large scale. The mechanical properties of the prepared silk fibers are still much lower than those of natural spider silk. Utilizing the technology of the present invention, the ability of the silk gland tissue of the silkworm to synthesize proteins efficiently and the natural ability of the silkworm to spin silk and form cocoons can be directly used to obtain chimeric silk containing the golden silk web-weaving spider ampulla gland silk protein on a large scale.
  • the obtained chimeric silk can combine the advantages of silk and spider silk.
  • Silk protein materials have been widely used in various fields. After the repeating units of the spider silk protein gene have been doubled many times, genetic engineering technology has been used to achieve expression in E. coli, yeast, animal cells or transgenic animals and plants. However, due to the highly repetitive amino acid sequence of spider silk protein, the expression level is often very low. And the molecular weight of the expression product is lower than natural.
  • the cost of purifying recombinant spider silk protein is very high and it is difficult to mass produce; the present invention uses recombinant Autographa californica baculovirus to mediate the expression of golden silk web ( Trichonephila clavipes) spider large ampulla gland silk in the posterior silk gland of silkworm. Protein, and can make the recombinant protein enter the cocoon layer through spinning to form chimeric silk.
  • the silk protein material thus prepared does not require complicated purification steps and is easy to mass produce.

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Abstract

本发明公开了含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法，将重组病毒接种5龄家蚕幼虫，桑叶添食后收集蚕丝，得到含金丝织网蜘蛛大壶状腺丝蛋白复合丝；所述重组病毒含有表达金丝织网蜘蛛大壶状腺丝蛋白的序列；重组病毒AcNPV-FibH-MaSp-g接种5龄家蚕幼虫，用浸渍或喷洒过抗生素药液的凉干后的新鲜桑叶饲养，后用新鲜桑叶饲养至熟蚕；用浸渍或喷洒过蜕皮激素药液的新鲜桑叶饲喂熟蚕；或直接用蜕皮激素药液体喷熟蚕，将熟蚕移至簇具，营茧，采茧，经过缫丝获含金丝织网蜘蛛大壶状腺丝蛋白的复合丝。利用本发明可获取含有金丝织网蜘蛛大壶状腺丝蛋白的蚕丝，以满足制备各种生物材料对丝蛋白的多样性的需求。

Description

一种含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法 技术领域
本发明涉及基因工程领域，具体涉及一种通过家蚕生产含金丝织网蜘蛛大壶状腺丝蛋白复合丝。
 背景技术
近年来，人们已可以通过基因工程技术在细菌、酵母、哺乳类动物培养细胞、昆虫细胞甚至通过转基因动物和植物表达蛛丝蛋白，但这些表达的蛋白不能自主装配成丝纤维，须通过人工纺丝的技术进一步加工成丝纤维，但目前通过这种技术还难以大量获取机械性优异的蛛丝纤维。另外由于主壶腹腺丝蛋白的分子量巨大，且高度重复，当用异源系统表达时往往重组蛛丝蛋白分子量低于自然状态，且表达水平低下，而人工纺丝所获得的丝的机械性与丝蛋白的分子量有正相关性。几千年来，饲养家蚕是丝绸工业的基础，家蚕是唯一能室内规模饲养大量提供丝纤维的昆虫。蚕丝蛋白主要由丝胶蛋白和丝素蛋白组成，而丝纤维主要由不溶于水的丝素蛋白重链（350kDa）、丝素蛋白轻链（25.8kDa）以及P25蛋白(25.7kDa)按6:6:1的摩尔比装配而成，蚕丝蛋白的机械性能主要由丝素蛋白重链的高分子量和氨基酸序列的高度重复决定。很长时间以来，人们一直希望利用家蚕高效合成丝蛋白以及天然的纺丝能力生产蛛丝。目前，将合成的络新妇蛛（ Nephila clavipes）、大腹园蛛( Araneus ventricosus)主壶腹腺丝蛋白基因的重复模序经过多次重复后，通过piggyBac介导的转基因已实现了非完整蛛丝蛋白基因在家蚕中的表达，并通过家蚕天然的纺丝能力获得含有蛛丝蛋白成份的嵌合蚕丝，在一定程度上改善了丝纤维的机械性能，但该丝纤维中蛛丝蛋白的含量非常有限；现有技术公开了一种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，蜘蛛葡萄状腺丝蛋白基因为黑寡妇蜘蛛葡萄状腺丝或者园蛛葡萄状腺丝1倍碱基重复单元以1‑8倍连续重复构成的基因序列，具有改良家蚕丝性能等应用；先构建家蚕合成分泌葡萄状腺丝蛋白的载体pBac‑ACSP质粒，将质粒与辅助质粒导入到家蚕受精卵内，用转座子使荧光蛋白基因和葡萄状腺丝蛋白基因导入到家蚕基因组内，并稳定遗传和表达，育成分泌蜘蛛葡萄状腺丝蛋白的转基因家蚕，其发现了一种蜘蛛葡萄状腺丝蛋白基因的用途，并开发了一种新型家蚕蜘蛛仿生丝的改良家蚕丝性能的生产方法。现有技术采用将质粒与辅助质粒导入到家蚕受精卵内的方法耗时长，且不适用二化性实用品种的蚕卵，而饲养这些蚕卵孵化的蚕才可以得到高性能优质蚕丝。
技术问题
本发明的发明目的是提供一种通过家蚕生产含金丝织网蜘蛛大壶状腺丝蛋白的复合蚕丝的方法。
 技术解决方案
为达到上述目的，本发明采用的技术方案是：一种含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法，其特征在于，包括以下步骤：将重组病毒接种5龄家蚕幼虫，桑叶添食后收集蚕丝，得到含金丝织网蜘蛛大壶状腺丝蛋白复合丝；所述重组病毒含有表达金丝织网蜘蛛大壶状腺丝蛋白的序列，重组病毒接种5龄家蚕幼虫的量为10 4～10 8拷贝/蚕。
本发明中，将重组DNA转染培养细胞，然后培养至细胞发病，取细胞培养上清再次接种培养细胞，然后培养至细胞发病，收集细胞培养上清，得到重组病毒；所述重组DNA含有表达金丝织网蜘蛛大壶状腺丝蛋白的序列。进一步的，将重组质粒转化DH10Ac大肠杆菌，后涂布于LB琼脂培养基平板上，然后培养，再挑取白色菌落，提取重组DNA；所述DH10Ac大肠杆菌含有AcBacmid；所述重组质粒含有表达金丝织网蜘蛛大壶状腺丝蛋白的序列，优选的，所述LB琼脂培养基含有四环素、卡那霉素、庆大霉素、IPTG和X-gal，四环素、卡那霉素、庆大霉素、IPTG 和X-gal的浓度分别为10 µg/ml、50 µg/ml、7 µg/ml、40µg/ml和100 µg/ml。
本发明中，将含有表达金丝织网蜘蛛大壶状腺丝蛋白序列的DNA片段克隆入质粒，得到重组质粒；所述含有表达金丝织网蜘蛛大壶状腺丝蛋白序列的DNA片段的序列为SEQ ID NO: 1；所述质粒为pFAST-Bac-Dual。合成SEQ ID NO: 1所示FibH-MaSp-g-polyA Fib表达盒的优选方案是采用全化学合成的方法；也可以家蚕基因组为模板通过PCR获得带有编码信号肽序列的启动子序列和加尾信号序列，以金丝织网蜘蛛大壶状腺的RNA为模板通过RT-PCR的方法获得MaSp-g序列，然后通过拼接获得FibH-MaSp-g-polyA Fib表达盒；也可以通过PCR扩增与化学合成相结合的方法制备FibH-MaSp-g-polyA Fib表达盒。克隆可以采用酶切连接的方法，也可以通过无缝克隆的方法。
本发明，桑叶添食时添加抗生素、蜕皮激素，具体的，将重组病毒接种5龄家蚕幼虫后，添食含有抗生素的桑叶一天，然后添食常规桑叶，至熟蚕，然后添食含有蜕皮激素的桑叶一次或者喷洒蜕皮激素一次，再进行收集蚕丝；其中收集蚕丝为常规技术，包括上蔟、营茧、采茧、缫丝。
本发明接种的家蚕品种优选丝茧育实用家蚕品种，如菁松×皓月、中2016×日2016，也可以选用家蚕原种，如J14-花，本发明方法具有普适性，成功解决了现有方法不适用二化性蚕种的技术缺陷。优选的接种的5龄家蚕幼虫的发育时期为5龄蜕皮后1-3天。接种病毒时可以用4号昆虫针醮取收集的细胞培养上清或离心纯化的病毒穿刺接种家蚕幼虫，优选的，重组病毒接种5龄家蚕幼虫的量为10 5～10 7拷贝/蚕。本发明首先合成家蚕丝素蛋白重链基因启动子控制的5’端带有编码信号肽序列、3’端带有加尾信号的金丝织网蜘蛛的大壶状腺丝蛋白基因表达盒FibH-MaSp-g-polyA FibH，其序列如SEQ ID NO: 1；再将其克隆进pFAST-Bac Tm-Dual的多克隆位点构建质粒pFAST-FibH-MaSp-g；然后将质粒转化含有AcBacmid DH10Ac大肠杆菌，后涂布于含有四环素、卡那霉素、庆大霉素、IPTG 和X-gal的LB 琼脂培养基平板上，常规培养，再挑取白色菌落，提取重组AcBacmid-FibH-MaSp-g DNA；然后将重组AcBacmid-FibH-MaSp-g DNA转染草地夜蛾Sf9培养细胞，常规培养至细胞发病，然后取细胞培养上清再次接种草地夜蛾Sf9培养细胞，常规培养至细胞发病后，收集细胞培养上清，离心纯化得到重组病毒粒子AcNPV-FibH-MaSp-g；然后将重组病毒AcNPV-FibH-MaSp-g接种5龄家蚕幼虫，用浸渍或喷洒过抗生素药液的凉干后的新鲜桑叶饲养1天，后用新鲜桑叶饲养至熟蚕；再用浸渍或喷洒过蜕皮激素药液的新鲜桑叶饲喂蚕1次或直接用蜕皮激素药液体喷家蚕1次；然后将熟蚕移至簇具，在25℃环境下营茧，7天后采茧；蚕茧烘干后，经过缫丝获含金丝织网蜘蛛大壶状腺丝蛋白的复合丝。接种的5龄家蚕幼虫的发育时期为5龄蜕皮后1～3天；抗生素药液为环丙沙星或氟哌酸或氟苯尼考药液；蜕皮激素药液的浓度为20～25mg/L。
有益效果
丝蛋白已广泛用于新型材料，蜘蛛丝蛋白和蚕丝蛋白各具独特的特点，人们希望通过生物学的方法获取蜘蛛丝蛋白和蚕丝蛋白的混合体，以满足制备各种材料对丝蛋白的多样性的需求。通过piggyBac介导的家蚕转基因技术可获得含有蛛丝蛋白成份的嵌合蚕丝，且在一定程度上改善了丝纤维的机械性能，但该嵌合蚕丝中蛛丝蛋白的含量非常有限；通过TALEN介导的同源末端重组已实现了用蜘蛛主壶腹腺丝蛋白基因经多次加倍的重复单元替代家蚕丝蛋白重链基因，这种方法获得的遗传修饰家蚕所生产的嵌合蚕丝中的蛛丝蛋白水平提高明显，尽管这种嵌合蚕丝的延伸性增加，但强度下降。此外由于受到通过显微注射将基因导入蚕卵的技术限制，目前对家蚕的遗传修饰基本上局限于无实用生产价值的多化性家蚕。杆状病毒是众多昆虫的病原，重组杆状病毒已广泛用于研发生物杀虫剂、表达外源蛋白和基因传递至脊椎动物细胞。杆状病毒种类繁多，且宿主域、感染性、致病率各不相同，本发明通过重组苜蓿银纹夜蛾核型多角病毒介导在家蚕后部丝腺表达金丝织网蜘蛛腺丝蛋白，从获取含有蛛丝蛋白的复合蚕丝，相关技术方案没有见报道。通过直接饲养蜘蛛难大量获得蜘蛛丝，利用本发明技术可以大量获得含有金丝织网蜘蛛大壶状腺丝蛋白的复合蚕丝，利用本发明的技术可以利用实用家蚕品种高生产性能的优势获取具有蚕丝和蛛丝优秀性能的嵌合蚕丝。
 附图说明
图1为实施例一中 重组AcBacmid-FibH-MaSp-g的鉴定。
图2为实施例一中重组病毒 AcNPV-FibH-MaSp-g的PCR鉴定。
图3 为实施例二中PCR检测 AcNPV-FibH-MaSp-g感染家蚕血液、丝腺中的拷贝数。
图4为实施例二中Western blot检测后部丝腺中 AcNPV-FibH-MaSp-g表达的MaSp-g。
图5 为实施例二中的Western blot 检测茧丝中的MaSp-g。
图 6 为实施例三中的RT-PCR检测感染AcNPV-FHP-MaSp-g蚕丝腺中的MaSp-g的转录。
图7为实施例三中的qRT-PCR检测感染AcNPV-FHP-MaSp-g蚕丝腺中不同时相的MaSp-g的转录。
图8为实施例三中Western blot检测注射不同滴度病毒后的家蚕丝腺组织。
图9 实施例四中的RT-PCR检测丝腺中MaSp-g基因的表达。
图10实施例四中的Western blot检测丝腺中MaSp-g基因的表达。
 本发明的实施方式
本发明涉及的具体制备操作以及家蚕培养、测试表征都为常规技术，下面结合附图及实施例对本发明作进一步描述，家蚕为春蚕。
实施例一：委托商业公司进行化学合成，合成的序列如SEQ ID NO: 1，为含有表达金丝织网蜘蛛大壶状腺丝蛋白的序列，命名为FibH-MaSp-g-polyA FibH，序列的二侧分别添加 NotI和 PstI位点。
将FibH-MaSp-g-polyA FibH克隆进pFAST-Bac Tm-Dual（Invitrogen公司产品）的NotI/PstI位点构建质粒pFAST-FibH-MaSp-g。将质粒pFAST-FibH-MaSp-g转化含有AcBacmid DH10Ac大肠杆菌，后涂布于分别含有10 µg/ml、50 µg/ml、7 µg/ml、40µg/ml和100 µg/ml的四环素、卡那霉素、庆大霉素、IPTG 和X-gal的LB 琼脂培养基平板上，于37℃培养12小时后，挑取白色菌落，接种在含有10 µg/ml、50 µg/ml和7 µg/ml的四环素、卡那霉素、庆大霉素LB培养基中，振荡培养8小时，提取重组AcBacmid-FibH-MaSp-g DNA，用引物M13-F （SEQ ID NO:2）和HC-left-R：（SEQ ID NO:3）进行PCR鉴定，扩增产物的琼脂糖凝胶电泳结果如图1所示，从AcBacmid-FibH-MaSp-g DNA中可扩增5kb的特异性条带，而野生性AcBacmid用引物M13-F（SEQ ID NO:2）和M13-R（SEQ ID NO: 4）可扩增出300bp左右的产物，表明重组Bacmid构建成功。图1中，提取重组AcBacmid-FibH-MaSp-g DNA，用引物M13-F和HC-left-R进行PCR鉴定；野生型AcBacmid用M13-F和M13-R引物进行PCR鉴定，PCR产物用1%的琼脂糖凝胶电泳分离，M，DNA标准分子量；泳道1，野性Bacmid；泳道2，重组AcBacmid-FibH-MaSp-g。
将重组AcBacmid-FibH-MaSp-g DNA 2μg与脂质体Lipofectamine 2000（Invitrogen公司）混合，转染草地夜蛾Sf9培养细胞，于26℃培养4天，然后取细胞培养上清再次接种培养细胞，细胞发病后，收集细胞和细胞培养上清。提取细胞总DNA，用引物EcoRI-FG-F（SEQ ID NO:5）和XhoI-FG-R（SEQ ID NO:6）进行PCR鉴定，扩增产物的琼脂糖凝胶电泳结果如图2所示。从感染病毒的细胞总DNA可扩增约8kb的特异性条带，表明AcNPV-FibH-MaSp-g构建成功。图2中，提取AcBacmid-FibH-MaSp-g转染后发病细胞的总DNA，用引物EcoRI-FG-F和XhoI-FG-R进行PCR鉴定，扩增产物进行琼脂糖凝胶电泳，泳道M，标准分子量DNA；泳道1和2，重组病毒 AcNPV-FibH-MaSp-g。
取上述细胞培养上清，在4 °C条件下，8,000转/分钟离心10分钟，反复2次；取上清，以30,000转/分钟离心30分钟，取沉淀，用磷酸缓冲液溶解沉淀获重组病毒的贮备液，-20°C保存备用，用于以下实施例。取病毒的贮备液，提取病毒DNA，用P4-F（SEQ ID NO:7）和P4-R （SEQ ID NO:8）通过定量PCR测定病毒的拷贝数。
配制500mg/L的环丙沙星溶液，按10L/100Kg均匀喷洒于桑叶叶面，晾干备用。配制500mg/L的氟苯尼考溶液，按10L/100Kg均匀喷洒于桑叶叶面，晾干备用。配制500mg/L的氟哌酸溶液，按10L/100Kg均匀喷洒于桑叶叶面，晾干备用。配制22.5mg/L的蜕皮激素药液，按8L/100Kg均匀喷洒于桑叶叶面，晾干备用。
实施例二：  “中2016×日2016”品种家蚕饲育至5龄起蚕，按每条蚕接种10 6拷贝的病毒（AcNPV-FibH-MaSp-g重组病毒的贮备液），然后添食喷洒环丙沙星溶液的新鲜桑叶1天，尔后用新鲜桑叶在24℃饲养至熟蚕。
AcNPV-FibH-MaSp-g接种家蚕后，按感染后24、48、72和96小时取蚕的血液200μl和100mg后部丝腺，提取DNA，用P4-F（SEQ ID NO:7）和P4-R （SEQ ID NO:8）通过定量PCR检测病毒的拷贝数。检测结果如图3所示，接种病毒24-72小时，血液、丝腺中病毒的拷贝数增加不明显，至接种病毒96小时，病毒的拷贝数急速增加，血液中病毒的拷贝数高于丝腺组织。
取病毒感染不同时相的后部丝腺组织，经过SDS-PAGE分离后，用MaSp-g抗体进行Western blot检测，同时，用微管蛋白（Tubulin）抗体检测内参基因表达的蛋白如图4所示，在感染24-120小时的丝腺中均能检测到MaSp-g的特异性条带，说明已经表达金丝织网蜘蛛大壶状腺丝蛋白，M，标准分子量DNA；泳道1，大肠杆菌表达的重组MaSp-g；泳道2,未感染病毒对照；泳道3-7，分别为感染病毒24、48、72、96和120小时的丝腺。一抗为抗MaSp-g抗体和抗微管蛋白抗体。
取蜕皮激素药液喷洒的桑叶，凉干后，饲喂上述熟蚕1次，再将熟蚕移至簇具，在25℃环境下营茧，7天后采茧，鲜茧烘干后贮藏，缫丝前，贮藏干茧脱胶后，经过缫丝获得含金丝织网蜘蛛大壶状腺丝蛋白的复合丝。
蚕丝中MaSp-g的检测：取上述复合丝，用常规溴化锂溶液溶解后的蛋白溶液加入到透析膜中，透析72小时后进行Western blot检测，结果如图5所示，可观察到Masp-g信号，说明茧丝中含有MaSp-g，泳道M，标准分子量DNA; 泳道con，未感染病毒AcNPV-FHP-MaSp-g蚕的丝；泳道1-3，感染AcNPV-FHP-MaSp-g蚕的丝。一抗为抗MaSp-g。
实施例三：1. 重组病毒AcNPV-FibH-MaSp-g接种家蚕：“J14-花”品种家蚕饲育至5龄起蚕，按每条蚕接种10 4、10 5、10 6拷贝的病毒。
2. 用喷洒氟苯尼考溶液的新鲜桑叶凉干后添食接种家蚕1天，尔后用新鲜桑叶在24℃左右饲养至熟蚕。
PCR检测病毒AcNPV-FibH-MaSp-g在丝腺中的增殖：取病毒感染不同时相的丝腺组织，提取RNA，反转录成cDNA后，用引物MaSP-g-F（SEQ ID NO: 9）和引物MaSP-g-R （SEQ ID NO:10）进行PCR扩增，PCR产物的电泳结果如图6所示，各个检测组均可扩增出特异条带，说明AcNPV-FibH -MaSp-g进入家蚕后部丝腺，并转录MaSp-g；泳道M，标准分子量DNA; 泳道1和2，pFAST-FibH-MaSp-g阳性对照；泳道3，接种10 4拷贝的病毒感染72小时的丝腺；泳道4，接种10 5拷贝的病毒感染72小时的丝腺；泳道5、6和7，为接种10 6拷贝的病毒分别感染24、72、48小时的丝腺。取接种10 6拷贝病毒24、48和72小时蚕的后部丝腺，提取RNA，反转录成cDNA后，用引物MaSP-g-F（SEQ ID NO:9）和引物MaSP-g-R （SEQ ID NO:10）进行qRT-PCR检测,并同时用引物eIF4-1 （SEQ ID NO: 11）和引物 eIF4-2（SEQ ID NO: 12）检测内参基因真核细胞起始因子4A的表达，计算相对表达水平，结果如图7所示，随着病毒感染进程，MaSp-g的转录水平增加。
Western blotting检测丝腺组织中的重组蛋白MaSp-g：取病毒感染72小时的家蚕丝腺，用MaSp-g的抗体进行Western blotting检测，检测结果如图8。在感染病毒的样本中可检测到代表MaSp-g的信号条带，说明MaSp-g基因已翻译成蛋白，泳道con，未注射病毒AcNPV-FibH-MaSp-g对照；泳道1和2，注射10 4、10 5拷贝的病毒感染72小时的后部丝腺。
3. 用喷洒蜕皮激素药液的新鲜桑叶凉干后，饲喂步骤2的熟蚕1次，再将熟蚕移至簇具，在25℃环境下营茧，7天后采茧，鲜茧烘干后贮藏，缫丝前，贮藏干茧脱胶后，经过缫丝获得含金丝织网蜘蛛大壶状腺丝蛋白的复合丝。
实施例四： “菁松×皓月”品种家蚕饲育至5龄第2天，用4号昆虫针醮取实施例一收集的AcNPV-FibH-MaSp-g病毒感染培养细胞上清穿刺接种，再用喷洒氟哌酸溶液的新鲜桑叶凉干后添食家蚕1天，尔后用新鲜桑叶在24℃左右饲养至熟蚕。
①RT-PCR检测接种病毒家蚕丝腺中的MaSp-g的转录：取接种病毒72小时蚕的后部丝腺，提取总RNA，反转录成cDNA后，用引物MaSP-g-F（SEQ ID NO:9）和引物MaSP-g-R （SEQ ID NO:10）进行PCR扩增，PCR产物的电泳结果如图9所示，在检测的样本中均可观察到代表MaSp-g表达的特异性条带，说明AcNPV-FibH-MaSp-g进入家蚕后部丝腺并表达MaSp-g，泳道M，标准分子量DNA；泳道FHP，pFAST-FibH-MaSp-g阳性对照；泳道H1-H3:接种病毒AcNPV-FHP-MaSp-g 72小时的家蚕后部丝腺。
②Western blotting检测丝腺组织中的重组蛋白MaSp-g：取病毒感染72小时的家蚕后部丝腺，用MaSp-g的抗体进行Western blotting检测，检测结果如图10。在感染病毒的样本中可检测到代表MaSp-g的信号条带，说明MaSp-g基因已翻译成蛋白，泳道con，未接种病毒AcNPV-FHP-MaSp-g蚕的后部丝腺；泳道H1-H3，接种病毒AcNPV-FHP-MaSp-g 72小时的家蚕后部丝腺。
用喷洒蜕皮激素药液新鲜桑叶凉干后，饲喂上述熟蚕1次，再将熟蚕移至簇具，在25℃环境下营茧，7天后采茧，鲜茧烘干后贮藏，缫丝前，贮藏干茧脱胶后，经过缫丝获得含金丝织网蜘蛛大壶状腺丝蛋白的复合丝。
③嵌合蚕丝的特征检测:蚕茧单粒茧缫丝，测定丝长，对照蚕（未感染病毒）茧丝长为964米，丝平均半径为6.5μm，横截面为132.75μm 2；感染AcNPV-FibH-MaSp-g蚕的茧丝长677米，丝平均半径为5μm，横截面为78.5μm 2。常规蚕丝（未感染病毒）平均断裂强力为666.04MPa，平均伸长率为11.81%；本发明复合丝分别为：平均拉力8.18 N，平均断裂强力1042.08 MPa，平均伸长率6.9%。50个样本平均。可以看出，本发明方法制备的复合丝丝长达到未感染蚕丝长的70%，现有蜘蛛蛋白改性蚕丝仅为未改性蚕丝丝长40%左右，甚至更低，本发明不仅提升了蚕丝的力学强度，而且显著提升现有复合丝的丝长。
现有技术通过大肠杆菌、酵母、动物细胞或转基因动植物能够表达出蜘蛛丝蛋白，要进一步获得蛛丝纤维，需要通过繁琐步骤纯化重组蛋白，再进一步通过人工纺丝实现，该过程不仅耗时费钱，而且目前的技术水平难以规模化生产，所制备的丝纤维的机械性仍大大低于天然蛛丝。利用本发明的技术，可以直接利用家蚕丝腺组织高效合成蛋白的能力和家蚕吐丝结茧的天然本领，大规模获得含有金丝织网蜘蛛大壶状腺丝蛋白的嵌合蚕丝，且所获得的嵌合丝可聚集蚕丝和蛛丝优势。丝蛋白材料已广泛用于各个领域。蛛丝蛋白基因的重复单元经多次加倍后，利用基因工程技术已在大肠杆菌、酵母、动物细胞或转基因动植物中实现表达，但由于蛛丝蛋白氨基酸序列高度重复，往往表达水平极低，且表达产物的分子量低于天然。因此纯化重组蛛丝蛋白的成本很大，难以量产；本发明通过重组苜蓿银纹夜蛾杆状病毒介导在家蚕后部丝腺表达金丝织网( Trichonephila clavipes)蜘蛛大壶状腺丝蛋白，并可以使重组蛋白通过吐丝进入茧层形成嵌合蚕丝，由此制备丝蛋白材料无需复杂的纯化步骤，便于量产。
SEQ ID NO: 1
GCGGCCGCTCAAAGCCTCATCCCAATTTGGAGTCACTCAAGACATCCTTGATTAAGGCAGCTGCCGATATTGACATGGACCTCGTTCGTGCTGCGATAGACGACTGGCCGCGCAGATTGAAGGCCTGTATTCAAAATCACGGAGGTCATTTTGAATAAACTTTAGTGTCATAAGAATCTATGTTTTGTTAAGTTCATTTTGGTATATGAATGGTTACATAATGAATAAACTTGTTTCAATTATTTTACATTAAACATGTGACAGAATTTATGACCTGACTAGGTAGGTACAAACAGCCTTTTTGATATTAGAAAACTAAGTAAAATAGCCTACGGTCACATCTCTTTCCGTGGGTGTCGTTAAAGGGCGACTTAGAGAACCACCAAGAACGTAGCAGAATCCTCAGAGTGTCATACCAGCATACAGCCATCGCTAACTGCTATTTACTGGTAATAGGGCACATTGTAATCTCACTTAACCATACTGTCGGGCCACCATCTAGCCTATTTCTGCCACGAATCAATCGTGAGTGATGGACATAGAGAAACTATTAGTTGAGAAGAAAACAAGAGCACTAAAGGTTTGATATTGACAAAAATCTACTTCGCCGTCACTCCATAGGTTTATTGTCTCTCATTAGTCCAGAACAGCAGTTACAGACGTAAGCTTTTACGCACAAACTACAGGGTTGCTCTTTATTGTATCGAAAATATGGGACCTGAATAAGGGCGATTTTGACGCGTCCTGCCCGCCCATTCCCGATCCTACGGACAGAATGGCAAGCAGTCGACGTCGCCCCAAACACGTCATTTCGGATCCTCACGATCCACTAACGGTGCTTTAGGTACCTCAAGCACCGGTCATCGTTCTCGTCGGACCCGTCGCTTGCGACGAAGGGCTCGACGAGCAAATTAACCCTCAGACACAGCCCACTGAGTTTCTCGCCGGATCTTCTCAGCGGGTCGCGTTTCCGATCCGGTGGTAGATTCTGCGAAGCACGGCTCTTGCTAGGATTCGTGTTAGCAACGTCGTCAGGTTTGAGCCCCGTGAGCTCACTTACTAGTTAAGGTTACGCTGAAATAGCCTCTCAAGGCTCTCAGCTAGGTAGGAAACAAAAAAAAAAGTCCTGCCCTTAACACCGTTGCGATGGCTTGTCTTTGCAGAAAGATGTTTTGTACGGAAAGTTTGAATAAGTGCTTAATTGCAAGTAACGTAACAATGTTTTAGGGTTCGGTCCTCAATAAATTCGACCAATAAACCATATATGTCGTGCTAATTACTGGACACATTGTATAACAGTTCCACTGTATTGACAATAATAAAACCTCTTCATTGACTTGAGAATGTCTGGACAGATTTGGCTTTGTATTTTTGATTTACAAATGTTTTTTTGGTGATTTACCCATCCAAGGCATTCTCCAGGATGGTTGTGGCATCACGCCGATTGGCAAACAAAAACTAAAATGAAACTAAAAAGAAACAGTTTCCGCTGTCCCGTTCCTCTAGTGGGAGAAAGCATGAAGTAAGTTCTTTAAATATTACAAAAAAATTGAACGATATTATAAAATTCTTTAAAATATTAAAAGTAAGAACAATAAGATCAATTAAATCATAATTAATCACATTGTTCATGATCACAATTTAATTTACTTCATACGTTGTATTGTTATGTTAAATAAAAAGATTAATTTCTATGTAATTGTATCTGTACAATACAATGTGTAGATGTTTATTCTATCGAAAGTAAATACGTCAAAACTCGAAAATTTTCAGTATAAAAAGGTTCAACTTTTTCAAATCAGCATCAGTTCGGTTCCAACTCTCAAGATGAGAGTCAAAACCTTCGTGATCTTGTGCTGTGCTCTCCAATACGTGGCCTACACAAACGCTCCATGGAGCGACACCGCTACAGCCGATGCTTTCATTCAAAATTTCCTCGGTGCCGTCTCCGGATCTGGTGCTTTCACCCCTGACCAGCTGGACGATATGGCTACTGTGGGAGACACCATTATGTCCGCCATCGATAAGATGGCTAGAAACAATAAGTCATCTAAGAGTAAGCTCCAGTCACTGAAAATGGCCTTCGCTTCATCAATCGCTGGTATTGCTGCCGTTGAACAAGGTGGACAGTCGATGGACATCAAGACCAACGCCATTGCTAATGCCTTGGATTCGGCTTTCTACATGACAACTGGAAGTACAAACCAACAGTTCGTCAATGAAATGAGAAGTCTCATATCAATGATCTCTGCTGCCAGCGCCAACGAAGCTAGCTACGGCGGTGGAGCTTCCGCTGCCGCTGCCACAGCTGGCGGTTACGGTCAAGGAGCTTCCGGTTACGATCCTGGACTGTCCCCAGCTTCGGCTGCCGCTCCTAGTGGCTACGGTCCATCAAAGAGAGAACCTTCAGGTATTGGTGCCGCTGCCGCTGCCCCATCTGAATACGGTTCGAGTCAACAGGGCCCGAGTGGTACAAAAGCTGCCACTATCGCTGCCGCTAAGAGAGGCCCCACTAGCTACGGTCCTAGACAACAACGCCCTGGTGGTTCTGGAGCTCCTGCCGCTACCGCTGGTAGAGGACCGGGTGGATACGGACCCGAACAACAAGGACCTAGAGGCTCAGGAGCCGCTGCCGACGAAGCTGGACCAGGACAACAGGAACCGGGTGCTGATGCTGCCGCTGCCTTCGGTAGTGGATCAGGCGAACAGGGTCCAGGAAGATTCGACGCTGCCGCTGCCACTGCTAAATCGAGAGGCAATGGTCCTGGACAACAGGGCTCTGGTGTCGCTTCAGCTGCTGCTGCTGGTAGTGAACCCAGAGGATACGGCCCTGGTCAACAAGCTCACAGAGGACACGGCGCTGCCGCTGCCGCTACTGGAAGCGGCGGTTACGAACCAGGACAACAAGGACCTGGTGGTCCTTCCGCCGCTGCCGCTGGTTTGGGACCAGGTGGATACGGTCCGAGAAAACAAGGACAAAGAAGACCCGCCGCTACCGCCGCTGCCGCTGAAACAGGCGGTTACGGTCCTAGAATACAGGGAACAGGAGCCGCTGCCGCTGCCGCTACCGGAAGAGGACCCGGAGGCTACGGTCCTGGACAACAGGTTCCAGGTGGATCTGGAGCTGTCAAGGCCGCTGATGGACCTGAAAGTTTCGGACCTGGTCAGCCTGGCGGTCCTGGAGCCGCTGCCACAGCTGGCGCCAGAAGAGGACCGGGAGGCTACGGACCTGGACAACAAGAACCTGGAAGACCATCTGTGGCTGCCGCTAGTGCTGGCTCAGGTGGATACGGTCCTAGACAACAGGGACCAGGCGGTTACGCTCCGGGACAACAGGGTCCTGGAGTTCCTGGTGCTACTGGAGCCGCTGCCGCTGGCAGAGGTTCAGGATACGCTAATGGCAAAAAGGTCCCGGGAGGCCCTGGCGCCGCTGCCGCTGCCGCTACTGGGTCTACACCTGGAGCTTACGGCCCTGGTCAACAGGGACCAGGTGGAGACGATCCGAAACAACAGGCTCCCGCCTCATCTAGCGCTACAGAAGCCGCTGCCGGACCTAGAGGATACGGCCCAGGTAAACAAGGTCCTGGTGCTGCCGTCGCTGTTGCTGCCGGTTCTGGACCCGGCGGTTACGGCCCTCGTCAGCAGGGTCCTGGAGGCCCAGCTATAGGCCCAGGTGTTTACGGACCGGGCCAACAGGGTAAAAGAGTCTACGGTCCCGGTCAGCAAGGACCTGGTGGATTCGGTGCTGCCGCTGCCACTGCTGCCGGCCCTGGTGACTACGGTCCTGATAAGAGAGGACCGGGCGGTCCTGGAGTTGCTGCCGCTGGAAGAGGCAGCGGTAGACCAGGATCCGCCGCTGACGCTACAGCCGGATCTGGTCCCGGAGGCTACGGTCCAGGACAACAAGGACCAGGAGCCGCTGCCACTGCTGCCTCTGGATCTGGACCGGGTGTTTACAGACCCAGACAATCTGGTGGACCAGGTGCTGCCGTCGGAGCTGCTACTAGAAGAGGATACGGCTACGGACCAGGACAACAGGGTCCTGAGGGACCAGGAGCTGTTGCTGCCGCTGCCGCTGGATCTGAACCTGGCGGTTACGGACCAGGCCAACAGGGCAAGGAAGGTTACGTCAGTGGTGAACAGGAGCCAGGAGATTCTGGATCGGCCGCTGCCGCTTTCGGTCCTGGAGTGTCTGGACCCAAACAACAGGGCCCTGGTGAAAAGGCCGCTGCCGCTAGTGGATCAGGCACAAGAGGTTATGGTCCAGGCCAACAAGGTCCGGGAGGCCCTGGTGCCGCTGCCGCTACTGAAGCTGGTAGAGGATCAGGTGGATACGGCCCAGGTCAACAGGGTCCGGAAGGATCTGGCGTTGCCGCTGCCGCTGCCGCTCGTCCCGGCGGTTACGGTCTCGGACAAGAAGGCCCAGGTTCGGCCGCTGCCACAGCTGCCGGAAGAGGAATAGAAGGTCACGGACCTGGCCAACAAGGACCTGGAGGCCCAGGTGCTGCCGCTGCCGCTGCCACCGGTAGAGGACAAGGTGGATACAAACCCGGTCAGAAGGGACCTGGCGGTTACGGAACAAGACAACAAGGACCTGAAGAACCTGGTTCTGATGCTGCCGCTACTAATGGCACCGGTCTCGGACAGGAAGGACCTGGAGGCCCTGTTACTGCCGCTGTCGCCGCTGGCTCTGGTCAACAGAAGTTGAGTGCCGCTGCCGCTGCCACCGCTGGAAGAGGATTGGGTGGATATGGACCAGGACAACAAGGTCCGGCTGCCACTGCTACCACAGCTGGCCGCGGTCTGGGCGGTACTGGAGCTGCCGCTGAAGCCGCTGCCGGACGTGGTCCCGGAGGCTATGGACCTGGACAACAGGAAGCTGGCGTGTCGGGTGAAGCTGCCGAAGCTGCCGGCCCTGGTCCTCCACCGCAAGGACCTGGCACTGCTGCCATCGCTGCCGCTGGTAGTGTGCCAGGTGGATACGTTCCTGGACAGAGAGGTACCGGCGGTCCAGCCGCTGCCGCTGCCACTGGTCTCGGAGGCTACAAACCCGGTCAACAGGGACCTGGTGGATACGCTCCAGGCCAAAAGGGTCTGGAAGCTACCGCTGCCGGTAGAGGAAGCGGCTACGGTCCCGCTAAACAGGTGCCGGGCGGTCCTGGAGCTGCCGCTGCCGCTGCCGAACCTGGACCCCCTGGCGAATACGGTACAGAAAAAAGAGGACCGAAAGGAGACGGACCAAAACAGCAAGCTGCCGCTGGATCCTCGGCCGCTGCCGCTGCCGGCAGTTCAGCTGCCGCTGCCGCTACAGGTCCTCAAGGTTATGGTCCTGGACAACAAGGTCCTGGAGCTACTGCCTCGGCCGCTGCCGGAAGTAGACCCGTCAGATACGGACCTGGTCAAAAGGGACCTGGTGCAGGACCCGGAGGCTACGAACCTGGTCAGCAAGGTCCTGGTGGACCTGGAAGCGCTGCCGCTGGCCCAGGCGGTTACGGTCCGGCTCAACAAGGACCTGGTGTGCCATCCGCCGCTGCCGGCAGAAGAGGTTTGGGATACGGCCCCGGTAAACATGGACCTAGCGCTGCCGCTGCCGCTGCCGCTGGAAGCGGCCCTGGTGGTTACGGTCCGGGACAACAGGGTAAAGGTGGATATGGTCCCGGTAAACAAGAACCTGGTAACTTCGGGGCCGCTGCCGCTGCCTCGGGACCAGGCGGTTACGGACCGGGCAAAGAAGGTCCCGGAAGTGCTGATGCTGCCGCTGCCAGAAGAGGACCTGGAGGCTACGGCCCAAAACAAAAAGGTGCTGCCGCTATGGCCGCTGCCGCTGCCGGTTCAATCCCTGAAGGCTACGGTCCCGTCCAACAAGGACCTGGCGTGTCAGGAGCTGCCGCTGCCACTACCTCTGAACCGGTGGGTTACGGAGCTGGCCAAGAAGGTCACGGAGCAGTCGCTGCCGCTACAGCTGGCAGAGGTCCAGGTGGATACAGACCGGGCCTGTACGGTCCCGGCGGTTCTGGTAGCGCCGCTGAAGCCGCTGGACCTGGAGGCTATGGTTCAAAACAACAGGGTACAATTTCTACTGCCGCTGCCGCTGCCGGATCAGAACCTGGTGGATACGGACCTGGTCAGCAAGGACCGGGCGGTTCTGGAGTTGCTGCCGCTACCGAAGAAAGAAGAGAACCCGGAGGCTACAAGCCTGGTCAGCAAGGCCCTGGTGGACCATCTGTGGCCGCTGCCTCTGCTGGCCTCGGCGGTTACGGTCCAGGACAGCAAGGTCCGGGAGGCCCAAATGGACCTGGTCAACAGGGTCCTGGTGGATCAGGTGTTGCTGCCGCTACTGAAGAAAGAAGAGAACCAGGCGGTTACAAGCCGGGTCAACAAGGTCCTGGTGGTCCTTCTGTGGCCGCTGCCTCCGCTGGACTGGGTGGATACGGCCCTGGACAACAAGGACCCGGCGGTCCTTCTGTTGCTGCCGCTAGTGCTGAATTGGGAGGCTACGGCCCCAGACAGCAAGGCCCTGGTGGATACGCTCCTGGTCAGCAGGGTCCGGGCGGTTACGCTCCAGGTAGACAAGGTCCAGGAGTTCCTTGTGCTGCTACAGCCGCTGGCGCTGGTTCTGGTTATGGTCCTGGCCAACAGGTCCCCGGAGGCCCAGGAACAACTGCCGCTGCCGCTGCCGGAAGCACTTCTGTCGAATACGGACCTGGCCAACAGGGTAGAAAAGGTGACGGACCTAAGCAACAGGCTCCAGCCGGATCTAGCGATGCTGCCGCTGCCGCTGGCCCGAGAGGCTATGGCCCTGGACAACAGGGACCTGTTGCCGCTGCCTTGGCTGCCGCTGGCTCTGGTCCAGTGGGTTATGGACCTGGTCAAAGAGGACCTGGTGCCGCTGTGGCTGCTTCTGCTGGTAGCGGACCTCTCGGCTACGGTCCAAGACAACAGGGTCAAGTGGGACACGGCAGAGCCGCTACTGCTGAAGCCGGTAGAGGACCGGGCGTTTACGAGCCTGGAGAACAAGGTCCAGGTGGACCTGGTTCAGCCGCTGCCGCTGCCGGTCCTAGAGGATACAGACCACGTCAGCAAGGTCCTGGAGTTCACGGAGCTGCTACCGCTAGAAGAGGCTCTGGATACGGACCAGGCCAACAAGGACCTGAAGCTCCAGGTGCTGCCGCTGCCACAGCTGCCGGTTCTGGTCCCGGCGGTTACGGACCTGGTAAACAGGGTAAAGGTGGTTACGTCCCAGGACAACAGGAGCCTGGCGACTTTGGAGCTGCCGCTGCCGCTAGTGGTTCAGGTGGATACGGACCTGGAAGCGCCGCTGCCGCTGCCGCTGGTAGAGGACCCGGCGGTTACGGTCCTAAACAACAGGGCGCTGGTGCTATGGCTTCAACCGCCGCTGGATCTATCCCTGGTGGTTACGGACCTGGACAGCAAGGTCCTGGTCAGCAAGGACCAGGTGACTTCGGTGCCGCTGCCGCTGAAGCTGCTTCCGGACCAGGTGGATATGGTCCTGGACAGGAAGTTCCTGTTCCTGTGGCTGTTGCCGCTGCCGGTAGAGGACCAGGCGGTTACAGATCAGGACAACAAGGACCGGGAGGCTTCGGATCTACTGCTGCCGCTGCCGGTCCCGGTGGATATGGTCCTGGTCAACAAGGTCCCGGAACAGTTGCTGTGGCTGCCGCTGAATCTGGTCCTGGCGGTTACGGTACTGGTCAACAAGGCCCTGGTGGTCCTAGCGCCGCTGCCGCTTCCGCTGGTCCGGGTGGATATGGCCCTGGTCAGCAAGGACCTGGAGTGCCTGGAGCTGTTGCTACCGCCGCTGCCGTGAGAGGTTCTGGATACGGCGCTGGTCAACAAGTTCCAGGCGGTCCTGGTGCTGCCGCTGCCACCGTCACCGGTAGAAGACCTGGAGGCTATGGCCCAGGCCAACAAGGTCCTGGAAGATTGGATGCTGCCAGCGCTGCCGCTGGCCCTGGTTCCTACGGTCCTGAACAACAGGGACCAGTTGCTAGTGCCGCTGGAAGAGGCCCCGGTAGATACGGTACTGAACAACAGGGACCTGGCAGATACGGTACCGGTCAACAGGGCCCCGGTAGACCTGTCACAGCCGCTGTGGATTCTGGCAGCGAACAACAGGGTCTGTCGGCCGCTGCCGCTGCCGCTGCCGGACGTGGCAACGGTGGATACTTGCCTGGTCAACAAGGACCCGCTGTGGCTGCCGCTGCCGCTGGTCGTGGACTGGGCGGTTACGGCCCGGGTCAACAGGAACCTGGTGGTCCGGGAGCCGCTTTGGCCAATGCTGGCCCTGAAGGTTATGGTCCTGGTCAACAGGGTACTGACGCCGCTGCCGCTACCGCTATTGTTTCAGGACCAGGCGCCGCTACATCCACTGGAAGATCGCCGGAATGCTACGGATCTGAGCAGCAAGGACCCGCTGGTCCTGGAGCTGCCACTGCCGCTGCCGCTGGCAGGGGTCCTGGTGGATACAGATCAGGTGAGCAAGGTCCAGAGGGACCTGGTGCCGCTGCCGCTACTGTGGCTGGTATTGGACCTGGCGGTTACGGTAGCAGACAGGAAGGACCCGGAGGCCCTGTTGCCGCTGCCGATGCTTCCGGCCCAGGTGGATATAGACCAGGACAGCCGGGCGGTCCTGTGGCTACCGCTGCCACAGCTGGCCAGGGTCCGAGAGGTTACGTGCCCGGACAACAGGGCCCTGTGGGAGCTGCCGCTGCCACTTCCAGATCGGGACCTGGTGGTTATGGTCCGGGCAAACAAGGACCTGGAGCTGCCTCCGCTGCCTCGGGACCTGGTGGATACGGTCCAGAACAACAAGGACCTGGTGCTGCCCTCGCTGCCGCTGCCGGATCAGGTCCTGGCGGTTATGGTCCAGGACCTCAGGCTAGTGCTGCCAGATCTAGACTGGCTTTCCCAGACAGTAGATCAAGAGTCTCCTCGGCTGCCTCGAACTTGGTGGCTAGTGGTCCGACAAATTCTGCTGCCCTCAGCAACGCTATTTCCAATACTGTGTCGGAAATAGGAGCTTCATACCCAGGACTGTCTGGCTGTGATGTTCTGGTCCAAGCTTTGATGGAAATTGTTAGCGCCCTCGTCGCTATACTGAGTTCATCTAGCATCGGACAGGTTAACTACGTGGCCGTTTCTCAAAGCGCTCAGGTGGTTTCCCAATCGCTGTTGCAGGCTTTGTACTAATTTTTAATATAAAATAACCCTTGTTTCTTACTTCGTCCTGGATACATCTATGTTTTTTTTTTCGTTAATAAATGAGAGCATTTAAGTTATTGTTTTTAATTACTTTTTTTTAGAAAACAGATTTCGGATTTTTTGTATGCATTTTATTTGAATGTACTAATATAATCAATTAATCAATGAATTCATTTATTTAAGGGATAACAATAATCCATGAATTCACATGCACATTTAAAACAAAACTAAATTACAATAGGTTCATATAAAAACAACAAGTATGCCTTCTCAACTAAGAATACTATACTGCAG
SEQ ID NO: 2 （M13-F）
CGCCAGGGTTTTCCCAGTCACGAC
SEQ ID NO: 3 （HC-left-R）
TGCAGAGCGCAGCACAAGATCAC
SEQ ID NO: 4 （M13-R）
ACACAGGAAACAGCTATGAC
SEQ ID NO: 5 EcoRI-FG-F
GGAATTCCATGACAGCCGATGCTTTCATTCAAAATTTCCTCGGTGC
SEQ ID NO: 6 XhoI-FG-R
CCCTCGAGGGGTACAAAGCCTGCAACAGCGATTGGGAAACCACCTGA
SEQ ID NO: 7 P4-F
TATATTCGCGGCGTTGTGAC
SEQ ID NO: 8 P4-R
AAGTTGGGCATACGGGAAGA
SEQ ID NO: 9 MaSP-g-F
TCTTGTGCTGTGCTCTCCAA
SEQ ID NO: 10 MaSP-g-R
TCAGGGGTGAAAGCACCAGA
SEQ ID NO: 11 eIF4-1
GAATGGACCCTGGGACACTT
SEQ ID NO: 12 eIF4-2
CTGACTGGGCTTGAGCGATA

Claims (10)

  1. 一种含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法，其特征在于，包括以下步骤：将重组病毒接种5龄家蚕幼虫，添食桑叶后收集蚕丝，得到含金丝织网蜘蛛大壶状腺丝蛋白复合丝；所述重组病毒含有表达金丝织网蜘蛛大壶状腺丝蛋白的序列。
  2. 根据权利要求1所述含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法，其特征在于，添食桑叶时添加抗生素、蜕皮激素。
  3. 根据权利要求1所述含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法，其特征在于，将重组DNA转染培养细胞，然后培养至细胞发病，取细胞培养上清再次接种培养细胞，然后培养至细胞发病，收集细胞培养上清，得到重组病毒；所述重组DNA含有表达金丝织网蜘蛛大壶状腺丝蛋白的序列。
  4. 根据权利要求3所述含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法，其特征在于，将重组质粒转化DH10Ac大肠杆菌，后涂布于LB琼脂培养基平板上，然后培养，再挑取白色菌落，提取重组DNA；所述DH10Ac大肠杆菌含有AcBacmid；所述重组质粒含有表达金丝织网蜘蛛大壶状腺丝蛋白的序列。
  5. 根据权利要求4所述含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法，其特征在于，所述LB琼脂培养基含有四环素、卡那霉素、庆大霉素、IPTG和X-gal。
  6. 根据权利要求4所述含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法，其特征在于，将含有表达金丝织网蜘蛛大壶状腺丝蛋白序列的DNA片段克隆入质粒，得到重组质粒。
  7. 根据权利要求6所述含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法，其特征在于，所述含有表达金丝织网蜘蛛大壶状腺丝蛋白序列的DNA片段的序列为SEQ ID NO: 1；所述质粒为pFAST-Bac-Dual。
  8. 根据权利要求1所述含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法，其特征在于，重组病毒接种5龄家蚕幼虫的量为10 4～10 8拷贝/蚕。
  9. 如SEQ ID NO: 1所述的DNA片段在制备含金丝织网蜘蛛大壶状腺丝蛋白复合丝中的应用。
  10. 根据权利要求1所述含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法制备的复合丝。
PCT/CN2022/105407 2022-05-05 2022-07-13 一种含金丝织网蜘蛛大壶状腺丝蛋白复合丝的制备方法 WO2023213009A1 (zh)

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CN114957485B (zh) * 2022-05-05 2023-11-10 苏州大学 一种含多种蜘蛛腺丝蛋白的高强度蚕丝及其制备方法

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