WO2023213010A1 - 一种通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法 - Google Patents

一种通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法 Download PDF

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WO2023213010A1
WO2023213010A1 PCT/CN2022/105424 CN2022105424W WO2023213010A1 WO 2023213010 A1 WO2023213010 A1 WO 2023213010A1 CN 2022105424 W CN2022105424 W CN 2022105424W WO 2023213010 A1 WO2023213010 A1 WO 2023213010A1
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silk
fibl
masp
silkworms
bombyx mori
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French (fr)
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贡成良
胡小龙
王崇龙
朱敏
郭仕程
邱群婻
童新宇
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苏州大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the present invention relates to the field of genetic engineering, and specifically relates to a method for producing chimeric silk containing gold web-weaving spider ampulla gland silk using silkworms through Autographa californica nuclear polyhedrovirus.
  • Spider silk has excellent mechanical properties and low immunogenicity, and has broad application prospects in the fields of textiles, biomedical materials and defense industry. Different from ordinary silk-spinning insects, spiders have multiple silk-producing glands, which can produce primary ampulla gland silk, secondary ampulla gland silk, flagellated silk, gelatinous gland silk, botryoidal gland silk, tubular gland silk and aggregated silk. Glandular mucin. Different spider silks or spider silk proteins have different biological functions and their properties are also significantly different. Spider silk protein has three functional regions. The two ends of the amino acid sequence form the N-terminal domain and the C-terminal domain respectively, which are composed of non-repeating amino acid sequences. The middle region of the spider silk protein amino acid sequence is composed of simple amino acids that are highly repeated.
  • Main ampulla gland silk also known as index silk or drag silk
  • MaSp is the silk with the best mechanical strength among spider silks.
  • the main characteristic motifs of the repeated region of MaSp-1 are (GA)n (n:6-14) or (A)n (n:6-14) and GGX, which can form a ⁇ -sheet crystal structure and a 3 10- helix structure respectively. , the former is related to the tensile strength of the silk, and the latter is related to the ductility.
  • the characteristic motifs of MaSp-2 are GPX, QQ, and GSG, which can form a ⁇ -turn helical structure and are related to the elasticity of the silk.
  • spiders In the wild, spiders produce less than 1 mg of silk per day. The amount of silk produced is very small, making it difficult to meet people's demand for spider silk. In addition, spiders are non-social animals and have the habit of killing each other. So far, people have not been able to obtain spider silk through large-scale group breeding. In recent years, with the advancement of biotechnology, people have tried to use genetic engineering technology to express spider silk proteins in bacteria, yeast, mammalian cultured cells, insect cells, and even transgenic animals and plants, and use purified recombinant proteins to create artificial spider-like spiders. Silk or other biological materials, related research has made great progress. The prior art discloses a single polyprotein molecule that improves mechanical and mechanical properties and its application method.
  • One of the four tandem protein sequences type1 to type4 of the spider ampulla silk protein I molecule is used as a single polyprotein molecule.
  • Polyprotein molecules construct an exogenous gene vector corresponding to this single polyprotein sequence, use molecular biology technology to integrate the single polyprotein sequence into the silkworm genome, and finally obtain a new type of composite silk with excellent performance that can stabilize heredity.
  • Bombyx silkworm varieties using new silkworm varieties to produce composite silk.
  • the existing technology uses the natural spinning ability of silkworms to obtain chimeric silk containing spider silk protein components, which improves the mechanical properties of silk fibers to a certain extent. However, it is limited by the introduction of foreign genes into silkworm eggs and the artificial incubation of silkworm eggs.
  • the object of the present invention is to provide a method for producing chimeric silk containing golden silk web-weaving spider large ampulla gland silk through Bombyx mori, which not only obtains silk with improved mechanical properties, especially avoids excessive decrease in silk length, but also is applicable to the present invention. All silkworm species.
  • the present invention uses the recombinant Autographa californica nuclear polyhedrovirus to mediate the expression of golden web-weaving spider gland silk in the posterior silk gland of silkworms to obtain chimeric silk containing spider silk protein. There are no reports on relevant technical solutions.
  • the technical solution adopted by the present invention is: a method for producing chimeric silk from silkworms through Autographa californica nuclear polyhedrosis virus, which includes the following steps: (1) FibL-MaSp-c-polyA The FibL fragment was cloned into the multiple cloning site of pFAST-Bac-Dual to construct plasmid pFAST-FibL-MaSp-c; the sequence of the FibL-MaSp-c-polyA FibL fragment is SEQ ID NO: 1;
  • the cocoons are cocooned at 25°C and cocoons are harvested after 7 days.
  • the larvae of silkworms are 5th instar larvae, which are raised until mature silkworms are seen, and then treated with ecdysone, and then weeded; further preferably, the larvae are treated with antibiotics once during the process of raising the silkworm larvae until mature silkworms are seen.
  • the method of the present invention for producing chimeric silk from silkworms using the Autographa californica nuclear polyhedrosis virus is further described as follows: (1) The 5' end controlled by the promoter of the silk fibroin light chain gene is synthesized with a coding signal peptide sequence, The golden web spider gland silk protein expression cassette FibL-MaSp-c-polyA FibL with a tailing signal at the 3' end has a sequence such as SEQ ID NO: 1; (2) FibL-MaSp-c-polyA FibL fragment Clone into the multiple cloning site of pFAST-Bac Tm -Dual to construct the plasmid pFAST-FibL-MaSp-c; (3) pFAST-FibL-MaSp-c is transformed into E.
  • E. coli containing AcBacmid DH10Ac and then spread on E. coli containing 10 ⁇ g/ml , 50 ⁇ g/ml, 7 ⁇ g/ml, 40 ⁇ g/ml and 100 ⁇ g/ml of tetracycline, kanamycin, gentamicin, IPTG and X-gal on LB agar medium plates, cultured at 37°C.
  • Recombinant virus AcNPV-FibL- MaSp-c is inoculated with 5th instar silkworm larvae, and dried fresh mulberry leaves soaked or sprayed with antibiotic solution (500mg/L) are raised at 24°C ⁇ 25°C for 1 day, and then fresh mulberry leaves are used to raise the silkworms until mature silkworms are seen.
  • the dosage ratio of antibiotic solution and mulberry leaves is 5 ⁇ 7L:100Kg; (6) Feed mature silkworms once with fresh mulberry leaves soaked or sprayed with ecdysone solution; or directly use ecdysone drug Spray the liquid once onto the mature silkworms; (7) Move the mature silkworms to the cluster, create cocoons in an environment of 25°C, and harvest the cocoons after 7 days; (8) After the cocoons are dried, the gold-containing web-weaving spiders are obtained through reeling. Chimeric silk of glandular silk.
  • step (5) the amount of recombinant virus AcNPV-FibL-MaSp-c inoculated into 5th instar silkworm larvae is 10 4 to 10 7 copies/silkworm;
  • the antibiotic solution is ciprofloxacin or norfloxacin or Florfenicol liquid;
  • the concentration of ecdysone liquid is 22.5mg/L, and the dosage ratio of antibiotic liquid and mulberry leaves is 5 ⁇ 7L:100Kg; if you choose to spray mature silkworms, the dosage of ecdysone liquid is: The degree of moistening the body surface of silkworms.
  • FibL-MaSp-c-polyA FibL expression cassette is to adopt a fully chemical synthesis method based on the SEQ ID NO: 1 sequence.
  • FibL-MaSp-c-polyA FibL expression cassette can also be prepared by PCR cloning strategy.
  • the FibL-MaSp-c-polyA FibL fragment was cloned into the multi-cloning site construction plasmid pFAST-FibL-MaSp-c of pFAST-Bac Tm -Dual, and the FibL-MaSp-c-polyA FibL fragment was cloned into pFAST-Bac Tm -
  • the multiple cloning site of Dual can be ligated by enzyme digestion or seamless cloning. Pick white colonies and extract the recombinant AcBacmid-FibL-MaSp-c DNA.
  • the cultured cells can be further inoculated to obtain high
  • the optimized protocol is to purify virus particles from the diseased cell culture supernatant through ultracentrifugation to further increase the virus titer.
  • the preferred silkworm variety is a silkworm variety practical for silk cocoon breeding, such as "Jingsong ⁇ Haoyue".
  • the original silkworm species such as "75 New”
  • the existing silkworm egg injection method cannot be applied to good varieties such as silkworm varieties practical for silk cocoon breeding, and will lead to death by acid immersion.
  • the present invention adopts a new method to successfully solve this problem and is applicable to all silkworm varieties, especially silk cocoon breeding. Practical silkworm species.
  • the development period of the inoculated 5th instar silkworm larvae is 1-3 days after the 5th instar moults. When inoculating the virus, you can use a No.
  • the optimized plan is to inject the recombinant virus AcNPV-FibL-MaSp-c at 10 6 copies/silkworm.
  • the antibiotic selected is ciprofloxacin, norfloxacin or florfenicol; the method of using ecdysone is preferably, when When the humidity is high, fresh mulberry leaves soaked or sprayed with ecdysone liquid can be dried and fed to silkworms; when the climate is drier, silkworms can be directly sprayed with ecdysone liquid.
  • the present invention has the following advantages compared with the prior art: 1. It is difficult to obtain a large amount of spider silk by directly raising spiders.
  • the technology of the present invention can utilize the high-level protein synthesis ability of the silk gland of the silkworm and the biological learning ability of the silkworm to spin cocoons to obtain a large amount of silk containing the large ampulla gland silk of the golden web-weaving spider. Combined 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.
  • 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 large ampulla gland silk of the golden web-weaving spider on a large scale, and the obtained The chimeric silk brings together the advantages of silk and spider index 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 The amino acid sequence of the protein is highly repetitive, the expression level is often very low, and the molecular weight of the expression product is lower than that of natural protein. Therefore, the cost of purifying the recombinant spider silk protein is very high and it is difficult to mass-produce; the present invention uses the recombinant Autographa californica nuclear polyhedrosis virus to mediate the expression of the golden web-weaving spider great ampulla gland silk protein in the posterior silk gland of the silkworm. And the recombinant ampulla gland silk protein enters the cocoon layer through spinning to form chimeric silk. The silk protein material thus prepared does not require complicated purification steps and is convenient for mass production.
  • Figure 1 shows the identification of recombinant AcBacmid-FibH-MaSp-c in Example 1.
  • Extract recombinant AcBacmid-FibL-MaSp-c DNA and use primer lightF (SEQ ID NO:2) and LighR: (SEQ ID NO:3) were identified by PCR.
  • PCR products were separated by 1% agarose gel electrophoresis.
  • M DNA standard molecular weight
  • lane CK wild Bacmid
  • lanes 1 and 2 recombinant AcBacmid-FibL-MaSp-c.
  • Figure 2 shows the relative expression level of MaSp-c in the silk gland of silkworm infected with Ac NPV-FibL-MaSp-c detected by RT-qPCR in Example 1.
  • 10 6 copies of the recombinant virus AcNPV-FibL-MaSp-c were used to inoculate the 5th instar silkworms of "Jingsong ⁇ Haoyue".
  • the posterior silk glands on the 3rd, 4th and 5th day after inoculation were harvested, total RNA was extracted and reverse transcribed into cDNA.
  • the relative expression level of MaSp-c was detected by quantitative PCR using primers qc-F (SEQ ID NO:4) and qc-R (SEQ ID NO:5).
  • primer eIF4-1 SEQ ID NO: 6
  • primer eIF4-2 SEQ ID NO: 7 were used to detect the expression of the internal reference gene eukaryotic cell initiation factor 4A.
  • Figure 3 shows the Western blot detection of MaSp-c expressed in the posterior silk glands of silkworms (Jingsong ⁇ Haoyue) 3 days after being infected with the Ac NPV-FibL-MaSp-c recombinant virus in Example 1.
  • CK control silk gland (uninfected); lane 1, injected with 10 4 copies of Ac NPV-FibL-MaSp-c virus on the 3rd day of the 5th instar; lane 2, injected with 10 4 copies of Ac NPV-FibL-MaSp-c on the 4th day of the 5th instar.
  • lane 5 10 6 copies of Ac NPV-FibL-MaSp-c virus injected on day 4 of 5 instar; lane 6, 10 5 copies of Ac NPV-FibL-MaSp-c virus injected on day 2 of 5 instar; lane 5 7, 10 5 copies of Ac NPV-FibL-MaSp-c virus were injected on the 3rd day of the 5th instar; lane 8, 10 6 copies of the Ac NPV-FibL-MaSp-c virus were injected on the 4th day of the 5th instar.
  • the primary antibody was anti-MaSp-c antibody, and the secondary antibody was HRP-labeled goat anti-rabbit IgG.
  • Figure 4 shows the detection of MaSp-c in cocoon silk by Western blot hybridization in Example 1.
  • Lane M standard molecular weight DNA
  • lane Ck silkworm silk uninfected with the virus AcNPV-FibL-MaSp-c
  • lane 1 silkworm silkworm infected with AcNPV-FibL-MaSp-g.
  • the primary antibody was anti-MaSp-c
  • the secondary antibody was HRP-labeled goat anti-rabbit IgG.
  • Figure 5 shows the immunohistochemical detection of MaSp-c secretion expression in silk glands infected with AcNPV-FibL-MaSp-c in Example 2.
  • A Silk glands of control silkworms not infected with AcNPV-FibL-MaSp-c virus;
  • B and C Silk glands of 5th instar silkworms infected with AcNPV-FibL-MaSp-c virus for 48 and 72 hours.
  • the primary antibody was anti-MaSp-c antibody
  • the secondary antibody was HRP-labeled goat anti-rabbit IgG.
  • Figure 6 shows the Western blot detection of MaSp-c expressed in the posterior silk glands of silkworms (75 new) 3 days after they were infected with the AcNPV-FibL-MaSp-c recombinant virus in Example 2.
  • Figure 7 shows the detection of silk protein by SDS-PAGE in Example 2.
  • Lane M standard molecular weight DNA
  • lanes 1-4 silk fibroin from 5th instar silkworms infected with AcNPV-FibL-MaSp-c virus
  • CK control silk fibroin.
  • Figure 8 shows the Western Blot detection of MaSp-c in silk.
  • Lane M standard molecular weight DNA
  • lanes 1-4 silk fibroin from 5th instar silkworms infected with AcNPV-FibL-MaSp-c virus
  • CK control silk fibroin.
  • Autographa californica nuclear polyhedrosis virus is the model species of baculovirus.
  • the main host of baculovirus is insects.
  • Baculovirus plays an important role in controlling insect populations. With the deepening of research, baculovirus has been widely used in the development of biopesticides, expression of foreign proteins and gene delivery to vertebrate cells.
  • the specific preparation operations of the present invention are conventional methods in the field, and can also be performed according to the commercial instructions, such as sequence synthesis, cloning, transformation, transfection, infection, silkworm rearing, cocooning, cocooning, cocoon harvesting, silk reeling, etc.
  • Example 1 Method for producing chimeric silk containing gold web-weaving spider ampulla gland silk protein through "Jingsong ⁇ Haoyue” variety silkworms: 1.
  • Chemical synthesis Expression cassette FibL-MaSp-c-polyA FibL sequence add Xho I and Sph I sites on both sides of the sequence, and then entrust a commercial company to perform chemical synthesis.
  • the synthesized sequence is shown in SEQ ID NO: 1.
  • Plasmid pFAST-FibL-MaSp-c Clone the FibL-MaSp-c-polyA FibL fragment into the Xho I and Sph I sites of pFAST-Bac Tm -Dual (product of Invitrogen) to construct plasmid pFAST-FibL-MaSp -c.
  • RT-qPCR detection of the relative expression level of MaSp -c in the silk glands of silkworms infected with AcNPV-FibL-MaSp-c Inoculate "Jingsong ⁇ Haoyue" with 10 6 copies of the recombinant virus AcNPV-FibL-MaSp-c from the age of 5
  • For silkworms take the posterior silk glands on days 3, 4, and 5 after inoculation, extract total RNA, and reverse-transcribe it into cDNA using primers qc-F (SEQ ID NO: 4) and qc-R (SEQ ID NO: 5) Detect the relative expression level of MaSp-c by quantitative PCR.
  • primer eIF4-1 SEQ ID NO: 6
  • primer eIF4-2 SEQ ID NO: 7
  • Figure 2 AcNPV-FibL-MaSp-c entered the posterior tissue of the silk gland and transcribed MaSp-c. Its transcription level increased significantly with virus infection.
  • ecdysone to silkworms: Prepare 22.5mg/L ecdysone solution and spray it evenly on the mulberry leaves at 6L/100Kg. Wet the front and back of the mulberry leaves. After drying, feed the above-mentioned mature silkworms once; Then move to the cluster, set the cocoons in an environment of 25°C, and harvest the cocoons after 7 days.
  • the strength elongation curve of the above-mentioned chimeric silk was routinely tested to evaluate its mechanical properties, and the silk of silkworms not infected with the virus AcNPV-FibL-MaSp-c was used as a control.
  • the results are shown in Table 1.
  • the strength and elongation of the chimeric silk of the present invention are improved, but the length of single cocoon silk decreases.
  • the length of single cocoon silk of the present invention remains the same as uninfected domestic silkworm silk. A growth of more than 60% is considered significant progress.
  • Example 2 Method for producing chimeric silk containing golden silk web-weaving spider large ampulla gland silk protein through "75 new" variety of silkworms
  • the recombinant virus AcNPV-FibL-MaSp-c was inoculated into silkworms: the "75 New" variety of silkworms was raised to the fifth instar, and each silkworm was inoculated with 10 6 copies of the virus.
  • the silkworms inoculated with the virus are fed the antibiotic florfenicol: prepare a 500mg/L florfenicol solution and spray it evenly on the mulberry leaves at a rate of 6L/100Kg, and keep the front and back of the mulberry leaves moist.
  • Fresh mulberry leaves sprayed with florfenicol solution were dried and fed to silkworms for 1 day, and then fresh mulberry leaves were used to raise silkworms at about 24°C until mature silkworms were seen.
  • Spray ecdysone on silkworms Prepare 22.5mg/L ecdysone solution and spray it on the body surface of silkworms in step 5, using the humidity as the degree.
  • the clustering, cocooning, and cocoon harvesting are the same as steps 9 in Embodiment 1.
  • the existing technology can obtain chimeric silk containing spider silk protein components through piggyBac-mediated transgenic technology of silkworms, and improve the mechanical properties of the silk fiber to a certain extent, but the content of spider silk protein in the chimeric silk is very limited;
  • Through TALEN-mediated homologous end recombination the multiple-doubled repeating units of the spider main ampulla gland silk protein gene have been used to replace the silk protein heavy chain gene of silkworm.
  • the chimeric silk produced by genetically modified silkworms was obtained by this method.
  • the level of spider silk protein in the silk was significantly increased, and although the extensibility of this chimeric silk increased, its strength decreased.
  • the existing technology introduces the spider silk protein gene into the silkworm genome through the silkworm transgenic method, which requires more complex procedures to screen and identify the transgenic silkworms, and further obtains the pure line of the transgenic silkworms through hybrid screening, which takes 1 to 2 years; through the present invention
  • the technology can obtain recombinant viruses in a shorter time, and by inoculating fifth-instar silkworms with the virus, chimeric silk containing spider silk proteins can be obtained in about a week, achieving significant technological progress.

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Abstract

本发明公开了通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法，构建含FibL-MaSp-c-polyA FibL片段的质粒pFAST-FibL-MaSp-c，转化含有AcBacmid DH10Ac大肠杆菌筛选获得重组AcBacmid-FibL-MaSp-c，转染Sf-9细胞获得重组病毒AcNPV-FibL-MaSp-c，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见熟蚕，用浸渍或喷洒过蜕皮激素药液的新鲜桑叶饲喂见熟蚕；或直接用蜕皮激素药液体喷见熟蚕，将熟蚕移至簇具，营茧后采茧，蚕茧烘干后，经过缫丝获嵌合蚕丝，满足制备各种生物材料对丝蛋白的多样性的需求。

Description

一种通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法 技术领域
本发明涉及基因工程领域，具体涉及一种通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产含金丝织网蜘蛛大壶状腺丝的嵌合蚕丝的方法。
 背景技术
蜘蛛丝机械性能优异、免疫原性小，在纺织、生物医用材料和国防工业领域具有广阔的应用前景。与一般的吐丝昆虫不同，蜘蛛有多个产丝腺体，能分别产生主壶腹腺丝、次壶腹腺丝、鞭毛状丝、胶性腺丝、葡萄状腺丝、管状腺丝和聚合状腺黏液蛋白。不同的蛛丝或蛛丝蛋白具有不同的生物学功能，其性能也有明显的差异。蛛丝蛋白具有3个功能区域，氨基酸序列的两端分别形成N端结构域和C端结构域，由不重复的氨基酸序列构成；蛛丝蛋白氨基酸序列的中间区域为简单氨基酸高度重复。蛛丝蛋白特性以及蛛丝的纤维性能主要取决于重复序列的结构特性。主壶腹腺丝（MaSp）又称索引丝或拖牵丝，是蛛丝中机械强度最好的丝。主壶腹腺丝主要有MaSp-1和-2 种。MaSp-1的重复区域的主要特征基序为（GA）n（n:6-14）或（A）n（n:6-14）和GGX，可分别形成β折叠晶体结构和3 10螺旋结构，前者与丝的抗张强度有关，后者与延展性有关。MaSp-2的特征基序为GPX、QQ、GSG，可形成β-turn螺旋结构，与丝的弹性有关。
在野生状态下，蜘蛛每天产丝量不足1mg，产丝量非常少，难以满足人们对蜘蛛丝的需求。另外，蜘蛛为非群居性动物，且有相互残杀的生活习性，至今为止人们还没有能通过大规模群体饲养获取蜘蛛丝。近年来，随着生物技术的进步，人们已尝试利用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，并用纯化的重组蛋白制造人工仿蜘蛛丝或其他生物材料，相关研究已取得了很大进展。现有技术公开了一种提高力学机械性能的单一多聚蛋白分子及其应用方法，采用蜘蛛大壶状腺丝蛋白I分子4个串联蛋白序列type1～type4中的一种序列作为单一的多聚蛋白分子，构建这种单一多聚蛋白序列对应的外源基因载体，利用分子生物学技术将单一多聚蛋白序列整合到家蚕基因组内，最终获得能够稳定遗传的复合丝性能优异的新型家蚕品种，利用新型家蚕品种生产复合丝。现有技术通过家蚕天然的纺丝能力获得含有蜘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，由于受到将外源基因导入蚕卵和蚕卵人工孵化技术的限制，通过显微注射将基因导入蚕卵进行家蚕遗传修饰基本上局限在无实用生产价值的多化性家蚕品种。作为常识，生产上的实用品种均为二化性，所产卵为越年卵，往往须通过较长时间的低温刺激（冷藏）或即时浸酸（盐酸）处理或冷藏与浸酸组合使用才可以解除滞育，促使胚胎发育。卵显微注射的最佳时期为产下后几小时，而即时浸酸处理通常在卵产下后24小时前后。显微注射后的蚕卵会因浸酸处理而死亡，因此现有技术多选择不需要浸酸处理多化性品种，因此人们仍希望通过一些新的策略和技术利用常规品种家蚕生产含有蛛丝蛋白的嵌合蚕丝。
 技术问题
本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的嵌合蚕丝的方法，不仅获得机械性能提高的蚕丝，尤其是避免丝长的过多下降，并且本发明适用所有家蚕品种。本发明通过重组苜蓿银纹夜蛾核型多角体病毒介导在家蚕后部丝腺表达金丝织网蜘蛛腺丝，获取含有蛛丝蛋白的嵌合蚕丝，相关技术方案没有见报道。
 技术解决方案
为达到上述目的，本发明采用的技术方案是：一种通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法，包括下列步骤：（1）将FibL-MaSp-c-polyA FibL片段克隆进pFAST-Bac-Dual的多克隆位点构建质粒pFAST-FibL-MaSp-c；所述FibL-MaSp-c-polyA FibL片段的序列为SEQ ID NO: 1；
（2）将质粒pFAST-FibL-MaSp-c转化含有AcBacmid DH10Ac大肠杆菌，后涂布于LB琼脂培养基平板上培养，再挑取白色菌落，提取重组AcBacmid-FibL-MaSp-c DNA，优选的，LB琼脂培养基含有四环素、卡那霉素、庆大霉素、IPTG 和X-gal；培养的温度为37℃；（3）将重组AcBacmid-FibL-MaSp-c DNA转染草地夜蛾培养细胞，然后培养至细胞发病，然后取细胞培养上清再次接种培养细胞，然后培养至细胞发病，再收集细胞培养上清，离心纯化得到重组苜蓿银纹夜蛾杆状病毒粒子AcNPV-FibL-MaSp-c，优选的，培养细胞为Sf9培养细胞，培养的温度为26～27℃；（4）将步骤（3）收集的细胞培养上清或者重组苜蓿银纹夜蛾杆状病毒粒子AcNPV-FibL-MaSp-c接种家蚕幼虫，然后饲养至上蔟；然后营茧、采茧、缫丝，得到嵌合蚕丝，25℃环境下营茧，7天后采茧，优选的，家蚕幼虫为5龄家蚕幼虫，饲养至见熟蚕，然后用蜕皮激素处理，再上蔟；进一步优选的，家蚕幼虫饲养至见熟蚕过程中，采用抗生素处理一次。
本发明通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法进一步描述如下：(1)合成家蚕丝素蛋白轻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腺丝蛋白表达盒FibL-MaSp-c-polyA FibL，其序列如SEQ ID NO: 1；(2) 将FibL-MaSp-c-polyA FibL片段克隆进pFAST-Bac Tm-Dual的多克隆位点构建质粒pFAST-FibL-MaSp-c；(3) pFAST-FibL-MaSp-c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，再挑取白色菌落，提取重组AcBacmid-FibL-MaSp-c DNA；(4) 将重组AcBacmid-FibL-MaSp-c DNA转染草地夜蛾Sf9培养细胞，于26～27℃培养至细胞发病，然后取细胞培养上清再次接种培养细胞，细胞发病后，收集细胞培养上清，离心纯化得到重组苜蓿银纹夜蛾杆状病毒粒子AcNPV-FibL-MaSp-c；(5) 重组病毒AcNPV-FibL-MaSp-c接种5龄家蚕幼虫，用浸渍或喷洒过抗生素药液（500mg/L）的凉干后的新鲜桑叶于24℃～25℃饲养1天，后用新鲜桑叶饲养至见熟蚕；优选的，抗生素药液、桑叶的用量比例为5～7L∶100Kg；(6) 用浸渍或喷洒过蜕皮激素药液的新鲜桑叶饲喂见熟蚕1次；或直接用蜕皮激素药液体喷见熟蚕1次；(7) 将熟蚕移至簇具，在25℃环境下营茧，7天后采茧；(8) 蚕茧烘干后，经过缫丝获含金丝织网蜘蛛腺丝的嵌合蚕丝。
上述技术方案中，步骤(5)中，5龄家蚕幼虫接种重组病毒AcNPV-FibL-MaSp-c的量为10 4～10 7拷贝/蚕；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22.5mg/L，抗生素药液、桑叶的用量比例为5～7L∶100Kg；如选择喷见熟蚕的方法，蜕皮激素药液用量以润湿家蚕体表为度。
本发明方法中，构建FibL-MaSp-c-polyA FibL表达盒的优选方案是根据SEQ ID NO：1序列采用全化学合成的方法。FibL-MaSp-c-polyA FibL表达盒也可以通过PCR克隆的策略制备。将FibL-MaSp-c-polyA FibL片段克隆进pFAST-Bac Tm-Dual的多克隆位点构建质粒pFAST-FibL-MaSp-c中，FibL-MaSp-c-polyA FibL片段克隆进pFAST-Bac Tm-Dual（Invitrogen公司产品）的多克隆位点可以采用酶切连接的方法，也可以通过无缝克隆的方法；挑取白色菌落，提取重组AcBacmid-FibL-MaSp-c DNA中，可以用lightF （SEQ ID NO:2）和LighR：（SEQ ID NO:3）引物对AcBacmid-FibL-MaSp-c进行PCR鉴定；制备重组病毒时，对纯化的AcNPV-FibL-MaSp-c DNA，通过PCR扩增并结合PCR产物的序列测定进行确认，通过TCID 50或定量PCR对上清中的病毒滴度进行检测，如获得的培养细胞上清中的病毒滴度较低，可以进一步接种培养细胞，以便获得高病毒滴度的细胞培养上清，优化的方案是，通过超速离心从发病细胞培养上清中纯化病毒粒子，以进一步提高病毒的滴度。
本发明中，家蚕品种优选丝茧育实用家蚕品种，例“菁松×皓月”，也可以选用家蚕原种，如“75新”。现有蚕卵注射的方法无法适用比如丝茧育实用家蚕品种等好的品种，会导致浸酸死亡，本发明采用新的方法，成功解决了此问题，适用所有家蚕品种，尤其是丝茧育实用家蚕品种。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，优化的方案是按10 6拷贝/蚕注射重组病毒AcNPV-FibL-MaSp-c。
本发明中，为了减少接种病毒时因接种伤口污染细菌而引起细菌性败血病的发生，选用抗生素为环丙沙星或氟哌酸或氟苯尼考；使用蜕皮激素的方法优选为，当湿度较大时，浸渍或喷洒过蜕皮激素药液的新鲜桑叶凉干后饲喂蚕；当气候较干燥时，可直接用蜕皮激素药液体喷家蚕。
 有益效果
由于上述技术方案运用，本发明与现有技术相比具有下列优点：1．通过直接饲养蜘蛛难以大量获得蜘蛛丝，本发明技术可以利用家蚕丝腺高水平合成蛋白的能力和家蚕的吐丝纺茧的生物学习性大量获得含有金丝织网蜘蛛大壶状腺丝的嵌合蚕丝。
2．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，要进一步获得蜘蛛丝纤维，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能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的嵌合蚕丝，且所获得的嵌合丝可聚集蚕丝和蜘蛛索引丝的优势。
3.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核型多角体病毒介导在家蚕后部丝腺表达金丝织网蜘蛛大壶状腺丝蛋白，并使重组大壶状腺丝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。
 附图说明
图1为实施例一中重组AcBacmid-FibH-MaSp-c的鉴定。提取重组AcBacmid-FibL-MaSp-c DNA，用引物lightF （SEQ ID NO:2）和LighR：（SEQ ID NO:3）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CK，野性Bacmid；泳道1和泳道2，重组AcBacmid-FibL-MaSp-c。
图2 为实施例一中RT-qPCR检测 AcNPV-FibL-MaSp-c感染家蚕丝腺中MaSp-c的相对表达水平。以10 6拷贝重组病毒AcNPV-FibL-MaSp-c接种“菁松×皓月” 5龄起蚕，取接种后第3、4、5天的后部丝腺，提取总RNA，反转录成cDNA后，用引物qc-F（SEQ ID NO:4）和qc-R（SEQ ID NO:5）通过定量PCR检测MaSp-c相对表达水平。并同时用引物eIF4-1 （SEQ ID NO: 6）和引物 eIF4-2（SEQ ID NO: 7）检测内参基因真核细胞起始因子4A的表达。
图3为实施例一中Western blot检测家蚕（菁松×皓月）感染 AcNPV-FibL-MaSp-c重组病毒3天后的后部丝腺中表达的MaSp-c。CK，对照家蚕丝腺（未感染）；泳道1，5龄第3天注射10 4拷贝的 AcNPV-FibL-MaSp-c病毒；泳道2，5龄第4天注射10 4拷贝的 AcNPV-FibL-MaSp-c病毒；泳道3，5龄第2天注射10 6拷贝的 AcNPV-FibL-MaSp-c病毒；泳道4，5龄第3天注射10 6拷贝的 AcNPV-FibL-MaSp-c病毒；泳道5，5龄第4天注射10 6拷贝的 AcNPV-FibL-MaSp-c病毒；泳道6，5龄第2天注射10 5拷贝的 AcNPV-FibL-MaSp-c病毒；泳道7，5龄第3天注射10 5拷贝的 AcNPV-FibL-MaSp-c病毒；泳道8，5龄第4天注射10 6拷贝的 AcNPV-FibL-MaSp-c病毒。一抗为抗MaSp-c抗体，二抗为HRP标记的羊抗兔IgG。
图4 为实施例一中的Western blot杂交检测茧丝中的MaSp-c。泳道M，标准分子量DNA; 泳道Ck，未感染病毒AcNPV-FibL-MaSp-c蚕的丝；泳道1，感染AcNPV-FibL-MaSp-g蚕的丝。一抗为抗MaSp-c，二抗为HRP标记的羊抗兔IgG。
图5为实施例二中免疫组化检测AcNPV-FibL-MaSp-c感染丝腺中MaSp- c的分泌表达。A，未感染AcNPV-FibL-MaSp-c病毒对照蚕的丝腺；B和C，5龄蚕感染病毒AcNPV-FibL-MaSp-c  48、72小时的丝腺。一抗为抗MaSp- c抗体，二抗为HRP标记的羊抗兔IgG。
图6为实施例二中Western blot检测家蚕（75新）感染AcNPV-FibL-MaSp-c重组病毒3天后的后部丝腺中表达的MaSp-c。
图7为实施例二中SDS-PAGE检测蚕丝蛋白。泳道M，标准分子量DNA； 泳道1-4，5龄感染AcNPV-FibL-MaSp-c病毒的蚕的丝素；CK，对照蚕丝素。
图 8为实施例二中 Western blot检测蚕丝中的MaSp-c。泳道M，标准分子量DNA； 泳道1-4，5龄感染AcNPV-FibL-MaSp-c病毒的蚕的丝素；CK，对照蚕丝素。
 本发明的实施方式
苜蓿银纹夜蛾核型多角体病毒（AcNPV）为杆状病毒的模式种，杆状病毒的主要宿主为昆虫，杆状病毒在控制昆虫种群数量方面发挥重要作用。随着研究的深入，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的研究。杆状病毒种类繁多，不同的杆状病毒的宿主域、感染性、致病性各不相同。本发明具体制备操作为本领域常规方法，也可根据商品说明书操作，比如合成序列、克隆、转化、转染、感染、养蚕、上蔟、营茧、采茧、缫丝等。
下面结合附图及实施例对本发明作进一步描述：实施例一：通过“菁松×皓月”品种家蚕生产含金丝织网蜘蛛大壶状腺丝蛋白的嵌合蚕丝的方法：1、化学合成表达盒FibL-MaSp-c-polyA FibL序列，在序列的二侧分别添加 XhoI和 SphI位点，尔后委托商业公司进行化学合成，合成的序列见SEQ ID NO: 1。
2、质粒pFAST-FibL-MaSp-c构建：将FibL-MaSp-c-polyA FibL片段克隆进pFAST-Bac Tm-Dual（Invitrogen公司产品）的 XhoI和 SphI位点构建质粒pFAST-FibL-MaSp-c。
3、重组Bacmid AcBacmid-FibL-MaSp-c的筛选：将pFAST-FibL-MaSp-c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-FibL-MaSp-c DNA，用引物lightF （SEQ ID NO:/2）和LighR（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-FibL-MaSp-c DNA中可扩增出与理论分子量相符的约2kb的特异性条带，而从野生性AcBacmid DNA中扩增不出，表明重组Bacmid构建成功。
4、重组病毒AcNPV-FibL-MaSp-c构建：将重组AcBacmid-FibL-MaSp-c 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27℃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。
5、AcNPV-FibL-MaSp-c病毒粒子的纯化与病毒拷贝数的测定：步骤4中的细胞培养上清，在4 °C条件下，8000转/分钟离心10分钟，反复2次；取上清，以30000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℃保存备用；取病毒的贮备液，测定其TCID50，计算贮备液中病毒的拷贝数。
6、RT-qPCR检测 AcNPV-FibL-MaSp-c感染家蚕丝腺中MaSp-c的相对表达水平：以10 6拷贝重组病毒AcNPV-FibL-MaSp-c接种“菁松×皓月” 5龄起蚕，取接种后第3、4、5天的后部丝腺，提取总RNA，反转录成cDNA后，用引物qc-F（SEQ ID NO:4）和qc-R（SEQ ID NO: 5）通过定量PCR检测MaSp-c相对表达水平。并同时用引物eIF4-1 （SEQ ID NO: 6）和引物 eIF4-2（SEQ ID NO: 7）检测内参基因真核细胞起始因子4A的表达。结果如图2所示， AcNPV-FibL-MaSp-c进入丝腺后部组织，并转录MaSp-c，其转录水平随着病毒的感染而明显提高。
7、Western blot检测家蚕（菁松×皓月）感染重组病毒 AcNPV-FibL-MaSp-c 3天后的后部丝腺中表达的MaSp-c：5龄第3、4天分别注射10 4拷贝，5龄第2、3、4天分别注射10 6拷贝，5龄第2、3天分别注射10 5拷贝以及5龄第4天注射10 6拷贝的病毒，3天后取后部丝腺组织进行Western blot 检测，一抗用抗MaSp-c抗体，二抗用HRP标记的羊抗兔IgG。结果如图3所示，接种重组病毒组均可检测到代表MaSp-c表达的特异性条带。
8、重组病毒AcNPV-FibL-MaSp-c（贮备液）接种家蚕：“菁松×皓月”品种家蚕常规饲育至5龄起蚕（秋蚕），按每条蚕接种10 6拷贝的病毒。接种病毒的蚕添食抗生素环丙沙星：配制500mg/L的环丙沙星溶液，按6L/100Kg均匀喷洒于桑叶叶面，桑叶正反面湿润。喷洒环丙沙星溶液的新鲜桑叶凉干后喂养家蚕1天，尔后用新鲜桑叶在24℃左右饲养至见熟蚕。
9、家蚕添食蜕皮激素：配制22.5mg/L的蜕皮激素药液，按6L/100Kg均匀喷洒于桑叶叶面，桑叶正反面湿润，凉干后，饲喂上述见熟蚕1次；然后移至簇具，在25℃环境下营茧，7天后采茧。
10、鲜茧烘干后贮藏，缫丝前，贮藏干茧常规脱胶后，经过缫丝获得嵌合蚕丝；取嵌合蚕丝用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4所示，可观察到MaSp-c信号，说明茧丝中含有MaSp-c。
常规测试上述嵌合蚕丝的强力伸长率曲线，评估其机械性能，以没有感染病毒AcNPV-FibL-MaSp-c蚕的丝为对照。结果如表1所示，本发明嵌合蚕丝的强力、伸长率均有提高，但单粒茧丝长下降，不过与现有方法相比，本发明单粒茧丝长保持未感染家蚕丝长的60%以上属于显著的进步。
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实施例二：通过“75新”品种家蚕生产含金丝织网蜘蛛大壶状腺丝蛋白的嵌合蚕丝的方法
1. 重组病毒AcNPV-FibL-MaSp-c的制备：同实施实例一的步骤1-5；
2. 重组病毒AcNPV-FibL-MaSp-c感染的“75新”品种家蚕后部丝腺中MaSp-c的分泌表达检测：5龄起蚕接种10 6拷贝的病毒，48、72小时后，取后丝腺制作切片，通过免疫组化检测AcNPV-FibL-MaSp-c感染丝腺中MaSp- c的分泌表达，检测时一抗用抗MaSp- c抗体，二抗用HRP标记的羊抗兔IgG。结果如图6所示，感染丝腺内腔中可观察到代表MaSp-c的棕色信号，说明MaSp-c表达并分泌至腺腔。
3．Western blot检测重组病毒AcNPV-FibL-MaSp-c感染75新家蚕3天后在后部丝腺中表达的MaSp-c：5龄起蚕接种10 6拷贝的病毒，48、72小时后，取后丝腺通过Western blot检测MaSp- c的表达，结果如图7所示，在病毒感染的丝腺样本中可观察到代表MaSp- c的特异性信号条带，说明MaSp-c已成功表达。
4．重组病毒AcNPV-FibL-MaSp-c接种家蚕：“75新”品种家蚕饲育至5龄起蚕，按每条蚕接种10 6拷贝的病毒。接种病毒的蚕添食抗生素氟苯尼考：配制500mg/L的氟苯尼考溶液，按6L/100Kg均匀喷洒于桑叶叶面，桑叶正反面湿润。喷洒氟苯尼考溶液的新鲜桑叶凉干后喂养家蚕1天，尔后用新鲜桑叶在24℃左右饲养至见熟蚕。
5．家蚕体喷蜕皮激素：配制22.5mg/L的蜕皮激素药液，体喷于步骤5的蚕的体表，以湿润为度。上簇、营茧、采茧同实施例一中的步骤9。
6. 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嵌合蚕丝。SDS-PAGE、Western blot常规检测嵌合蚕丝蛋白，结果如图8所示，在病毒感染的蚕的茧丝样本中可观察到代表MaSp-c条带；Western blot检测结果如图9所示，病毒感染的蚕的茧丝样本中可观察到代表MaSp-c信号条带，说明蚕茧丝中含有MaSp-c。感染家蚕的单粒茧丝长最少为未感染家蚕单粒茧丝长的58%。
现有技术通过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。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。关键的，现有用蚕生产蜘蛛丝嵌合蚕丝的方法，极大降低了丝长，实验研究发现，重组得到的丝长最高为原蚕丝长的41%。现有技术通过家蚕转基因的方法将蜘蛛丝蛋白基因导入家蚕基因组，须经过较复杂的程序进行转基因家蚕的筛选鉴定，进一步通过杂交筛选获得转基因纯系，历时1～2年时间；通过本发明的技术可以在较短的时间获取重组病毒，并通过该病毒接种5龄家蚕能在一周左右获得含有蜘蛛丝蛋白的嵌合蚕丝，取得显著的技术进步。
 序列表自由内容
SEQ ID NO: 1：
CTCGAGGTACGGTTCGTAAAGTTCACCTGCGGCTATATTCCGACTCGCCAAGTTACGTCAGTCGTATTGTAATGAGCGATTTAGTGGGCAACTTCATTCTGTTAATTTTGTGTCACGGTGCGCGCGCATCGTAAAACTTCACTCTCATAGATTTTTCATAACGCGCCTAAAGAAGTATAACTTCAATAATTTAAATTTAAAAAAAAACATGCATAGAATAATTATATGAATTATTTAAAATGTCATTTACCGACATTGACATAACAGACGACGTTAACACTACAAAACATTTTAATTCCACATTGTTACATATTCAACAGTTAAATTTGCGTTAATTCTCGATGCGAACAAATATAAGAACAATCGGATCAATTAGATCGCTTTGTTTCGAACAACACTTAGTTTAACTAGAGGCGTACACCTCAAGAAATCATCTTCATTAGAAACTAAACCTTAAAATCGCAATAATAAAGCATAGTCAATTTTAACTGAAATGCAAAGTCTTTTGAACGTTAGATGCTGTCAGCGTTCGTTGGTACAGTTGTTTGATATTTATTTTAATTGTCTTTTTATATATAAATAGTGGAACATTAATCACGGAATCCTGTATAGTATATACCGATTGGTCACATAACAGACCACTAAAATGAAACCTATCTTCCTCGTTCTGCTGGTGGCTACATCTGCCTATGCCGCCCCATGGTCTTCGACGGAGTTGGCCGACGCTTTTATCAACGCTTTCCTCAATGAAGCCGGAAGAACTGGCGCTTTCACCGCCGACCAACTCGACGATATGTCTACCATTGGTGACACCCTGAAAACAGCTATGGATAAGATGGCCAGATCCAACAAATCATCTCAATCGAAGCTCCAGGCTCTGAATATGGCTTTCGCTTCATCAATGGCTGAAATCGCTGCCGTGGAACAAGGTGGATTGAGCGTTGCTGAAAAAACAAACGCTATTGCCGATTCCCTCAATTCGGCTTTCTACCAAACAACTGGAGCCGTTAACGTCCAGTTCGTCAATGAAATAAGAAGTCTCATCTCAATGTTCGCTCAGGCCAGCGCTAACGAAGCTAGCTACGGCGGTGGATACGGCGGTGGACAAGGCGGTCAATCTGCTGGTGCTGCCGCTGCCGCTGGTGCTGGACAAGGTGGTTACGGTGGACTGGGCGGTCAAGGTGCTGGTAGTGCCGCTGCCGCTGCCGCTTCAGGAGCAGGTCAAGGTGGTTATGGTGGAGTGGGAAACCAGGGTGCTGGAAGAGGCGCCGGAGCCGCTGCCGCTGCCGCTGGCGGTGCTGGTCAAGGTGGTTACAATGGTGGACAAGGACCTTCTGCCGCTGCCGCTGCCGCTGCCAGCGGAGCTGGCCAGGGCGGTTACGGAGGCCCTGGTTCCCAAGGTGCTGGACAAGGAGCTGGAGCTGCCGCTGCCGCTGCCGGTGGAGCTGGACAAGGCGGTTACGGAGGCTTGGGTGGACAGGGAGCTGGAAGAGGCGGTGCTGCCGCTGCCGCTGCCGCTGCCGGTGTGGCTGGACAAGGTGGTCTGGGTTCGCAGGGTGCTGGAAGAGGTGGACTCGGCGGTCAGGGTGCAGGCGCTGCCGCTGCCGCTGGAGGCGCCGGACAGGGTGGATACGGTGGTCTGGGACAAGGTGCTGGTCAAGGAGCTGGAGTCGCCGCTGCCGCTGCCGCTGGAGGCGCTGGCCAAGGTGGATACGGCGGTTTCGGTTCCCAGGGAGCAGGAAGAGGTGGTCAAGGTGGACAAGGTTCGGCCGCTGCCGCTGGCGGTGCTGGGCAAAGAGGTTACGGAGGCCAGGGTGCTGGTCAGGGTGGATTGGGCGGTGGAGAACAGGGAGCTGGCGAAGAAGGTTCTGGTGCCAGCGCTGGCGCTGGTGCCGCTGCCGGAAGAGGCGCTGGCGGTGGAGGCAAGGGTGGACTGGGCGGTCAAGGTGGTAGTGCTGCCGCTGCCGCTGCCGGTGGAGCTGGGCAAGGCGGTTTGGGAGGCTCAAGAGGTGCTGGACAAGGTGCTGGAGCTGCCGCTGCCGCTGCCGGTGGAGCTGGTCAGGGCGGTTATGGAGGCCTGGGCTCACAAGGAGCTGGTAGAGGTGGACAAGGCGCTGGTGCTGCCGCTGCCGCTGCCGGCGGTGCTGGCCAAGGTGGTTACGGTGGACTGGGCGGTCAGGGCGTTGGTAGAGGTGGTCTGGGTGGTCAAGGTGCAGGTGCTGCCGCTGCCGTCGGTGCTGGACAGGGCGGTTACGGAGGCGTGGGATCTGGTGCTTCGGCTGCCAGTGCTGCCAGATCTAGATTGTCGAGTCCTCAAGCTTCATCTAGAGTGAGCTCCGCTGTTTCGAACCTCGTCGCCAGTGGTCCAACAAATTCAGCTGCCCTGTCGAGTACTATTTCAAACGTGGTTTCTCAAATAGGAGCTTCTAATCCTGGACTGAGCGGCTGCGACGTTTTGATACAGGCTCTGTTGGAAGTCGTGTCAGCCTTGATCCAAATTCTCGGTTCATCTAGCATCGGACAGGTCAATTACGGCTCAGCGGGACAGGCTACGCAAATAGTGGGACAGTCAGTCTACCAGGCTTTAGGATAAATAAGAACTGTAAATAATGTATATATATAATTATATAAAAGATATATATAACCATATACAAACATATATATCATTATAAGACAATCTACCTATATAAAAACAGACTAAAATTAATAATTATGTATACTTTAATTGTGTTTAGGACATTTTATGCAAATTGTGTTTGCGTTAGGATTTTTTTTGGAAGTTTTTTAGATTATTTATGAATATATAAATAAATATACGTTAATATAATATATATTATATAAATCAACGACACGGCTTTTCATTTTGGTGATGATCAATCTTATTGTTCTTCTAATTGATTTTTTTGTACAATAAAGATGTATCCAGTTTTCCAGATAAAGAATTTAGTTTGTTATTTCTGGCCCCATTAAAATAAGTACGGTATTCGACAATAGCATGC
SEQ ID NO: 2（lightF）
ATGAAACCTATCTTCCTCGTTCTG
SEQ ID NO: 3 （lightR）
TCCTAAAGCCTGGTAGACTGACTG
SEQ ID NO: 4(qc-F)
AGAGTGAGCTCCGCTGTTTC
SEQ ID NO: 5(qc-R)
TCAAGGCTGACACGACTTCC
SEQ ID NO: 6 eIF4-1
GAATGGACCCTGGGACACTT
SEQ ID NO: 7 eIF4-2
CTGACTGGGCTTGAGCGATA。

Claims (10)

  1. 一种通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法，其特征在于，包括下列步骤：
    （1）将FibL-MaSp-c-polyA FibL片段克隆进pFAST-Bac-Dual的多克隆位点构建质粒pFAST-FibL-MaSp-c；所述FibL-MaSp-c-polyA FibL片段的序列为SEQ ID NO: 1；
    （2）将质粒pFAST-FibL-MaSp-c转化含有AcBacmid DH10Ac大肠杆菌，后涂布于LB琼脂培养基平板上培养，再挑取白色菌落，提取重组AcBacmid-FibL-MaSp-c DNA；
    （3）将重组AcBacmid-FibL-MaSp-c DNA转染草地夜蛾培养细胞，然后培养至细胞发病，然后取细胞培养上清再次接种培养细胞，然后培养至细胞发病，再收集细胞培养上清，离心纯化得到重组苜蓿银纹夜蛾杆状病毒粒子AcNPV-FibL-MaSp-c；
    （4）将步骤（3）收集的细胞培养上清或者重组苜蓿银纹夜蛾杆状病毒粒子AcNPV-FibL-MaSp-c接种家蚕幼虫，然后饲养至上蔟；然后营茧、采茧、缫丝，得到嵌合蚕丝。
  2. 根据权利要求1所述通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法，其特征在于，LB琼脂培养基含有四环素、卡那霉素、庆大霉素、IPTG 和X-gal。
  3. 根据权利要求1所述通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法，其特征在于，步骤（2）中，培养的温度为37℃。
  4. 根据权利要求1所述通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法，其特征在于，步骤（3）中，培养细胞为Sf9培养细胞，培养的温度为26～27℃。
  5. 根据权利要求1所述通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法，其特征在于，家蚕幼虫为5龄家蚕幼虫。
  6. 根据权利要求1所述通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法，其特征在于，饲养至见熟蚕，然后用蜕皮激素处理，再上蔟。
  7. 根据权利要求6所述通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法，其特征在于，家蚕幼虫饲养至见熟蚕过程中，采用抗生素处理一次。
  8. 根据权利要求1所述通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法，其特征在于，25℃环境下营茧，7天后采茧。
  9. 根据权利要求1所述通过苜蓿银纹夜蛾核型多角体病毒用家蚕生产嵌合蚕丝的方法生产的嵌合蚕丝。
  10. 苜蓿银纹夜蛾核型多角体病毒与FibL-MaSp-c-polyA FibL片段在家蚕生产嵌合蚕丝中的应用。
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