WO2023213008A1

WO2023213008A1 - High-strength silk comprising various spider silk proteins and preparation method therefor

Info

Publication number: WO2023213008A1
Application number: PCT/CN2022/105404
Authority: WO
Inventors: 贡成良; 胡小龙; 王崇龙; 李楠; 张星; 朱敏; 童新宇; 邱群婻
Original assignee: 苏州大学
Priority date: 2022-05-05
Filing date: 2022-07-13
Publication date: 2023-11-09
Also published as: CN114957485B; CN114957485A

Abstract

The present invention provides high-strength silk comprising various spider silk proteins and a preparation method therefor. The obtained silk comprises gold silk web spider major ampullate silk proteins MaSp‑g and MaSp‑c. By means of cloning the gene sequences of the spider silk proteins into plasmids, followed by transformation and recombination, and then transfecting cultured cells, recombinant virus particles are obtained. The recombinant virus particles are inoculated into 5-instar Bombyx mori for culturing to obtain chimeric silk comprising various spider silk proteins. The breaking strength of the silk reaches 1116.55 MPa, and the length of the silk reaches 65% of the length of pure silk.

Description

High-strength silk containing multiple spider gland silk proteins and preparation method thereof

Technical field

The invention relates to the field of genetic engineering, and specifically to a modified silk containing a variety of golden silk web-weaving spider ampulla gland silk proteins and a preparation method thereof.

Background technique

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 demand for the diversity of silk proteins in the preparation of various materials. However, the breaking stress of chimeric silk is 371.3Mpa, which is 17.4% lower than the 449.5Mpa of wild silk. The breaking strains of chimeric silk and wild silk are 32.2% and 22.5% respectively, that is, chimeric silk The elongation increases but the strength decreases. In addition, due to the technical limitations of introducing foreign genes into silkworms, genes can usually only be introduced into eggs produced by polymorphic varieties of silkworms through microinjection. As a common sense, all practical varieties for production are bipolar, so through microinjection Most of the transgenic silkworms obtained through genetic transformation are limited to polymorphic silkworms with no practical production value.

Technical solutions

The object of the present invention is to provide a modified silk containing a variety of golden web-weaving spider ampulla gland silk proteins and a preparation method thereof, with a breaking strength of 1116.55MPa. In order to achieve the above object, the technical solution adopted by the present invention is: a high-strength silk containing a variety of spider gland silk proteins, and the multiple spider gland silk proteins are two or more different spider gland silk proteins, preferably , the spider gland silk protein is spider ampulla silk protein; the spider is a golden silk web-weaving spider.

The invention discloses a method for producing the above-mentioned high-strength silkworm silk containing a variety of spider gland silk proteins. Multiple sequences expressing spider gland silk proteins are cloned into plasmids, then transformed and recombined, and then transfected into cultured cells to obtain recombinant virus particles. ; Inoculate the recombinant virus particles into silkworm larvae and feed them to mature silkworms to obtain the silkworms that produce the high-strength silk containing a variety of spider gland silk proteins. Bombyx mori larvae are not genetically modified. The invention discloses a silk fibroin solution prepared by utilizing the above-mentioned high-strength silk containing a variety of spider gland silk proteins. The application of the high-strength silk containing a variety of spider gland silk proteins in the preparation of silk products is also disclosed. The preparation method of the high-strength silk containing multiple spider gland silk proteins according to the present invention is to clone multiple sequences expressing spider gland silk proteins into plasmids, then transform and recombine them, and then transfect the cultured cells to obtain recombinant virus particles; The recombinant virus particles are inoculated into silkworm larvae, fed to mature silkworms, and then cocoons, cocoons, and silk reeling are obtained to obtain high-strength silk containing a variety of spider gland silk proteins; specifically, the sequences of multiple expression spider gland silk proteins are: SEQ ID NO: 1 and SEQ ID NO: 2.

In the present invention, Escherichia coli containing AcBacmid DH10Ac is used for transformation, white colonies are picked after culture, and recombinant DNA is extracted; the transformed recombinant DNA is transfected into Spodoptera frugiperda Sf9 culture cells to obtain recombinant virus particles.

In the present invention, mulberry leaves containing antibiotics are used to feed the inoculated silkworms for one day, and then untreated mulberry leaves are used to feed the silkworms until they are mature, and then treated with ecdysone once, and then the silkworms are weeded.

The varieties of silkworms of the present invention include varieties of silkworms practical for silk cocoon breeding and original varieties of silkworms. Compared with the existing method of preparing spider protein composite silk, the present invention is suitable for dimorphic silkworm species for the first time and solves the problem that the existing technology is difficult to apply to dimorphic varieties. As a common sense, the practical varieties in production are all bisexual, and the eggs they lay are over-the-year eggs, which often require long-term low-temperature stimulation (refrigeration) or immediate pickling (hydrochloric acid) or a combination of cold storage and pickling. It can relieve diapause and promote embryonic development. The best time for egg microinjection is a few hours after laying, while immediate pickling treatment is usually around 24 hours after egg laying. Silkworm eggs after microinjection will die due to pickling treatment, so the existing technology can only select polymorphic varieties that do not require pickling treatment; and when the existing technology uses spider silk protein to modify silk, the silk length decreases significantly. They are less than 42% of the length of pure silk.

The present invention synthesizes the large ampulla gland of the golden web-weaving spider (Trichonephila clavipes), which is controlled by the promoter of the silk fibroin light chain (FibL) gene and has a coding signal peptide sequence at the 5' end and a tailing signal at the 3' end. Silk protein (major ampullate spidroin, MaSp) c gene expression cassette FibL-MaSp-c-polyA _FibL , its sequence is as SEQ ID NO: 1; the 5' end band controlled by the promoter of the synthetic silk fibroin heavy chain (FibH) gene There is a major ampullate spidroin (MaSp) g gene expression cassette FibH-MaSp-g-polyA _FibH of Trichonephila clavipes encoding a signal peptide sequence and a tailing signal at the 3' end. , its sequence is as SEQ ID NO: 2; FibL-MaSp-c-polyA _FibL and FibH-MaSp-g-polyA _FibH fragments were cloned into the XhoI/SphI site and NotI/PstI site of pFAST-Bac ^Tm -Dual respectively. Construct plasmid pFAST-FibH/L-MaSp-g/c.

Further, the above plasmid pFAST-FibH/L-MaSp-g/c was transformed to contain AcBacmid DH10Ac E. coli, coated with tetracycline, kanamycin, gentamicin, IPTG, and X- Gal's LB agar medium plate, culture at 37°C, then pick white colonies, extract recombinant AcBacmid-FibH/L-MaSp-g/c DNA; transform the recombinant AcBacmid-FibH/L-MaSp-g/c DNA into Infect Spodoptera frugiperda Sf9 cells, culture them at 26-27°C until the cells develop disease, and then take the cell culture supernatant and inoculate the cultured cells again. After the cells develop disease, collect the cell culture supernatant and centrifuge to purify the recombinant Spodoptera alfalfa rods. Virion AcNPV-FibH/L-MaSp-g/c.

Finally, the above-mentioned recombinant virus AcNPV-FibH/L-MaSp-g/c was inoculated into 5th instar silkworm larvae, and dried fresh mulberry leaves soaked or sprayed with antibiotic solution were raised for 1 day at about 24°C, and then fresh mulberry leaves were used. Feed the silkworms on the leaves until mature silkworms appear; then feed the silkworms once with fresh mulberry leaves soaked or sprayed with ecdysone liquid or directly spray the silkworms with ecdysone liquid once; move the mature silkworms to clusters and place them in a 25°C environment The cocoon is cocooned and harvested after 7 days; after the cocoon is dried, the silk containing the golden web-weaving spider ampulla silk proteins g and c is obtained through reeling, which is a high-strength silk containing two spider gland silk proteins.

The preferred solution for constructing FibL-MaSp-c-polyA _FibL and FibH-MaSp-g-polyA _FibH expression cassettes according to the present invention is to adopt a fully chemical synthesis method based on the sequences of SEQ ID NO: 1 and SEQ ID NO: 2; it can also be used in silkworms The genome was used as a template to obtain the promoters of Bombyx mori silk fibroin light chain (FibL) and heavy chain (FibH) genes and the corresponding 3'-end tailed signal regions polyA _FibL and polyA _FibH through PCR method; using the silk gland tissue of Bombyx mori Total RNA and total RNA from the main ampulla gland of the golden silk-weaving spider were used as templates. The signal peptide coding sequences of the silk fibroin light chain and heavy chain genes and the golden silk-weaving spider silk major ampulla gland silk protein were obtained by RT-PCR. g and c gene coding sequences, and then synthesize the expression cassette by bridging method, or the expression cassette can be prepared by combining PCR amplification and chemical synthesis. FibL-MaSp-c-polyA _FibL and FibH-MaSp-g-polyA _FibH fragments were cloned into the XhoI/SphI site and NotI/PstI site of pFAST-Bac ^Tm -Dual (product of Invitrogen) and can be connected by restriction enzyme digestion method, or through seamless cloning. The optimized protocol is to purify virions from diseased cell culture supernatants by ultracentrifugation. The silkworm species inoculated with the recombinant virus AcNPV-FibH/L-MaSp-g/c is preferably a silkworm species practical for silk cocoon breeding, such as Zhong 2016×Day 2016, or the original silkworm species can be used; the preferred development period of the inoculated 5th instar silkworm larvae It is 1-3 days after the 5th instar molts. 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 puncture and inoculate the silkworm larvae. The optimized plan is to inject the recombinant virus AcNPV-FibH/L-MaSp-g/ at 10 ⁶ copies/silkworm. c.

In order to reduce the occurrence of bacterial septicemia caused by bacteria contaminating the wound when inoculating the virus, the preferred antibiotic in the present invention is ciprofloxacin, norfloxacin or florfenicol. The method of using ecdysone depends on the climate conditions. 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, the ecdysone liquid can be sprayed directly. silkworm.

Baculoviruses are pathogenic to 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 with different host domains, infectivity, and pathogenicity. There are two types of major ampulla silk protein genes in the golden web-weaving spider ( Trichonephila clavipes) , namely MaSp-g and MaSp-c. There are significant differences in sequence and molecular weight between the two. MaSp-g consists of 2466 amino acid residues. The molecular weight is huge. MaSp-c has a small molecular weight and is composed of 658 amino acid residues. Autographa californica nuclear polyhedrosis virus is a model species of baculovirus. The present invention uses recombinant Autographa californica nuclear polyhedrosis virus to simultaneously express the golden silk web-weaving spider large pot-shaped in the posterior silk gland of silkworm. Gland silk proteins g and c utilize the natural spinning habits of silkworms to obtain chimeric silk containing spider silk proteins g and c. No relevant technical solutions have been reported.

beneficial effects

Due to the application of the above technical solutions, the present invention has the following advantages compared with the prior art: 1. Spider silk proteins can be expressed 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 and expensive, Moreover, the current technical level is difficult to produce on a large scale, and 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 to synthesize proteins efficiently and the natural ability of the silkworm to spin silk and form cocoons can be directly exploited to obtain chimeric silk containing the golden web-weaving spider ampulla gland silk proteins g and c on a large scale. , and the obtained chimeric silk can gather the advantages of silk and spider silk.

2. Introducing the spider silk protein gene into the silkworm genome through the method of transgenic silkworms requires more complex procedures to screen and identify the transgenic silkworms, and further obtain transgenic pure lines through hybrid screening, which takes 1-2 years, and most of them are expression-specific. spider silk protein genes; the recombinant virus can be obtained in a short time through the technology of the present invention, and chimeric silk containing two spider silk proteins can be obtained in about a week by inoculating 5-year-old silkworms with the virus, and its breaking strength reaches 1116.55 MPa, the silk length reaches 65% of the length of pure silk, which not only solves the problem that the existing technology can only be used for diverse varieties with poor practicality, but also overcomes the problem that the existing technology's composite silk length is less than 42% of the length of pure silk. Defects.

Description of the drawings

Figure 1 shows the PCR identification of the recombinant virus AcNPV-FH/LP-MaSp-g/c in Example 1.

Figure 2 shows the PCR detection of viral gene expression in the silk gland of silkworm infected with AcNPV-FH/LP-MaSp-g/c in Example 1.

Figure 3 shows the Western blot detection of MaSp-g and MaSp-c expressed in AcNPV-FH/LP-MaSp-g/c in the posterior silk gland (Middle 2016 × Day 2016) in Example 1.

Figure 4 shows the characteristics of cocoons produced by silkworms infected with AcNPV-FH/LP-MaSp-g/c in Example 1.

Figure 5 shows the infrared spectral characteristics of cocoon silk produced by AcNPV-FH/LP-MaSp-g/c infected silkworms in Example 1.

Figure 6 shows the Western blot detection of MaSp-g/c in silk glands in Example 2.

Figure 7 shows the immunohistochemical detection of MaSp-g/c secretion in AcNPV-FHP/FLP-MaSp-g/c-infected silk glands in Example 2.

Figure 8 shows the Western blot detection of MaSp-g/c in silk in Example 2.

Figure 9 shows the mechanical properties of cocoon silkworms infected with AcNPV-FHP/FLP-MaSp-g/c in Example 2.

Figure 10 shows the infrared spectral characteristics of silk in Example 2 and the secondary structure analysis of silk protein based on the infrared spectral characteristics.

Embodiments of the invention

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. In addition, due to technical limitations of introducing genes into silkworm eggs through microinjection, current genetic modification of silkworms is basically limited to polymorphic silkworms with no practical production value. The technology of the present invention can take advantage of the high production performance of practical silkworm varieties to obtain chimeric silk with excellent properties 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. Therefore, 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 simultaneously express the golden silk web ( Trichonephila clavipes) in the posterior silk gland of silkworm and the large ampulla gland of spider Silk proteins g and c can be used to make the recombinant protein enter the cocoon layer through spinning to form chimeric silk. The preparation of silk protein materials does not require complicated purification steps and is convenient for mass production.

The present invention will be further described below in conjunction with the accompanying drawings and examples. The specific operations involved in the present invention as well as the method and characterization of silkworm raising are all conventional techniques. A commercial company was entrusted to perform chemical synthesis. The synthesized sequence is such as SEQ ID NO: 1, with Xho I and Sph I sites on both sides of the sequence. A commercial company was entrusted to perform chemical synthesis. The synthesized sequence was such as SEQ ID NO: 2. There were Not I and Pst I sites on both sides of the sequence.

In Example 1, 2016 × 2016 varieties of silkworms: (1) Construction of recombinant plasmid pFast-FH/LP-MaSp-g/c: clone SEQ ID NO: 1 and SEQ ID NO: 2 sequences into pFAST-Bac ^Tm respectively -Dual (product of Invitrogen) Prepare recombinant plasmid pFAST-FH/LP-MaSp-g/c between Xho I and Sph I sites, NotI and PstI sites.

(2) Recombinant Bacmid Screening of AcBacmid-FH/LP-MaSp-g/c: pFast-FH/LP-MaSp-g/c was transformed into Escherichia coli containing AcBacmid DH10Ac, and then spread on cells containing 10 µg/ml, 50 µg/ml, 7 µg/ml, 40µg/ml and 100 On the LB agar medium plate containing tetracycline, kanamycin, gentamicin, IPTG and 50 µg/ml and 7 µg/ml of tetracycline, kanamycin, and gentamicin in LB medium, culture with shaking for 8 hours, and extract recombinant AcBacmid-pFast-FH/LP-MaSp-g/c DNA.

(3) Recombinant virus AcNPV- Construction and identification of FH/LP-MaSp-g/c: Mix 2 μg of recombinant AcBacmid- FH/LP-MaSp-g/c DNA with lipofectamine 2000 (Invitrogen Company), transfected into Spodoptera frugiperda Sf9 cultured cells, cultured at 27°C for 4 days, and then the cell culture supernatant was taken to inoculate the cultured cells again. After the cells became ill, the cells and cell culture supernatant were collected to improve the viral resistance. Titer. Extract the total DNA of the cells and use the primer pair lightF (SEQ ID NO: 3) and lightR (SEQ ID NO:4), and the primer pair heavyF (SEQ ID NO: 5) and heavy R (SEQ ID NO: 6) for PCR detection. MaSp-c and MaSp-g genes, the agarose gel electrophoresis results of the amplified products are shown in Figure 1, which can be obtained from AcNPV-FH/LP-MaSp-g/c Gene fragments representing MaSp-c (A in Figure 1) and MaSp-g (B in Figure 1) were amplified from the DNA respectively, indicating that the DNA of the recombinant AcNPV-FibH-MaSp-g/c contains MaSp-c and MaSp -g gene. Purification of AcNPV-FH/LP-MaSp-g/c virus particles and determination of virus copy number: centrifuge the above cell culture supernatant at 8,000 rpm for 10 minutes at 4 °C, repeat twice; take the supernatant , centrifuge at 30,000 rpm for 30 minutes, take the precipitate, dissolve the precipitate in phosphate buffer to obtain a stock solution of the recombinant virus, and store it at -20°C for later use. 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).

(4) Inoculate silkworms with the recombinant virus AcNPV-FH/LP-MaSp-g/c (stock solution): The "Zhong 2016×Day 2016" variety of silkworms is raised to the fifth instar, and each silkworm is inoculated with 10 ⁶ copies of the virus. Compare 10 ³ , 10 ⁴ , and 10 ⁵ .

(5) Add the antibiotic ciprofloxacin to the virus-inoculated silkworms: Prepare 500mg/L ciprofloxacin solution, spray 6L solution/100kg mulberry leaves evenly on the mulberry leaves, spray fresh ciprofloxacin solution After the mulberry leaves are dried, the above-mentioned inoculated silkworms are fed for one day, and then fresh mulberry leaves are used to raise the silkworms at about 24°C until mature silkworms are seen. PCR detection of the proliferation of virus AcNPV-FH/LP-MaSp-g/c in silkworms: After inoculating silkworms with AcNPV-FH/LP-MaSp-g/c, the blood of silkworms was taken at 24, 48, 72 and 96 hours after infection. 200 μl and 100 mg of posterior silk gland, DNA was extracted, and viral copy number was detected by quantitative PCR using P4-F (SEQ ID NO:7) and P4-R (SEQ ID NO:8). The detection results are shown in Figure 2. With viral infection, the copy number of the virus in the posterior silk gland tissue shows an increasing trend. Western blot detection of recombinant protein MaSp-g/c in silk gland tissue: 96 hours after AcNPV-FH/LP-MaSp-g/c infected 5th instar silkworm, the posterior silk glands of different silkworms were taken and separated by SDS-PAGE , Western blot detection was performed with MaSp-g/c antibody. As shown in Figure 3, representative MaSp-c can be detected in virus-infected silk gland tissue (55 kDa) and MaSp-g (>170 kDa) signal band, indicating that MaSp-c and MaSp-g genes have been translated into proteins.

(6) Add ecdysone to silkworms: Prepare 22.5mg/L ecdysone solution, spray fresh mulberry leaves according to 5L solution/100kg mulberry leaves, dry them, and feed the above-mentioned mature silkworms once; move the mature silkworms The cocoons are grown in clusters at 25°C and harvested after 7 days. Investigation on the appearance and quality of silkworm cocoons: Silkworms from the 5th instar were inoculated with 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ copies/ silkworm virus AcNPV-FH/LP-MaSp-g/c, and the cocoons in each treatment area were dried. , observing the cocoon shape, no obvious changes in cocoon shape were found due to inoculation with the virus. The dry cocoon weight, cocoon shell weight and silk length of the silkworms inoculated with 10 ⁶ copies of the virus were investigated, compared with the control without virus inoculation, see Figure 4.

(7) Preparation of chimeric silk containing gold web-weaving spider major ampulla gland silk protein MaSp-g/c: cocoons are dried and stored. Before reeling, after the dry cocoons were stored and degummed, chimeric silk containing the golden web-weaving spider ampulla gland silk protein MaSp-g/c was obtained through reeling. The performance test results of MaSp-g/c chimeric silk are shown in Table 1. The control is the silkworm silkworm that has not been injected with virus; MaSp-g/c is the silkworm silkworm inoculated with 10 ⁶ virus. The breaking stress, Young modulus, and breaking energy of silk increased significantly, the average cross-sectional area decreased significantly, and the breaking strain slightly decreased.

.

Secondary structure detection of chimeric silk protein of MaSp-g/c: cocoons produced by silkworms infected with AcNPV-FH/LP-MaSp-g/c were degummed and reeled, and analyzed by infrared spectroscopy. According to the characteristics of the infrared spectrum, the proportion of secondary structure in silk protein is calculated, see Figure 5. Compared with the silk of control silkworms that were not injected with the virus, the content of β-sheets in the secondary structure of the silk proteins of silkworms infected with the virus AcNPV-FHP/FLP-MaSp-g/c increased by 15.8%, while the proportion of α-helices was basically the same. constant.

Figure 1 shows the PCR identification of the above recombinant virus AcNPV-FH/LP-MaSp-g/c. Extract the total DNA of the diseased cells after transfection with AcBacmid-FH/LP-MaSp-g/c, and use the primer pair lightF (SEQ ID NO: 3) and lightR (SEQ ID NO: 4) PCR to detect the MaSp-c gene. The primer pair heavyF (SEQ ID NO: 5), primer heavyR (SEQ ID NO: 6) PCR detection of MaSp-g. The amplified products were subjected to agarose gel electrophoresis. Figure A, MaSp-c gene detection results. Lane M, standard molecular weight DNA; lane con, recombinant plasmid pFast-FH/LP-MaSp-g/c; lanes 1, 2 and 3, recombinant virus AcNPV-FH/LP-MaSp-g/c. Figure B, MaSp-g gene detection results. Lane M, standard molecular weight DNA; lane con, wild virus; lanes 1, 2 and 3, recombinant virus AcNPV-FH/LP-MaSp-g/c. Figure 2 shows the above-mentioned PCR detection of viral gene expression in the silk gland of silkworm infected with AcNPV-FH/LP-MaSp-g/c. After inoculating 5-instar silkworms of "Zhong 2016 × Day 2016" with 10 ⁶ copies of AcNPV-FH/LP-MaSp-g/c, the posterior silk glands of the silkworms were taken at 24, 48, 72 and 96 hours after infection, and DNA was extracted. Primers P4-F and P4-R were used to detect the gentamicin gene fragment in the recombinant virus by qPCR. With virus infection, the virus copy number in the posterior silk gland increased. Figure 3 shows the above-mentioned Western blot detection of MaSp-g and MaSp-c expression in AcNPV-FH/LP-MaSp-g/c in the posterior silk gland (middle 2016 × day 2016). Lane M, standard molecular weight DNA; lane 2, uninfected virus control; lanes 3-5, silk glands of different silkworms infected with the virus for 96 hours. The primary antibody is anti-MaSp-g/c antibody. Figure 4 shows the characteristics of cocoons produced by silkworms infected with AcNPV-FH/LP-MaSp-g/c. A, the appearance of silkworm cocoons, 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ , the titers of the inoculated virus AcNPV-FH/LP-MaSp-g/c are 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ copies/strip respectively Silkworm; B, dry cocoon weight; C, cocoon shell weight; D, cocoon silk length. con, control; MaSp-g/c, inoculated with virus AcNPV-FH/LP-MaSp-g/c. Figure 5 shows the infrared spectral characteristics of cocoon silk produced by silkworms infected with AcNPV-FH/LP-MaSp-g/c. A, Infrared spectrum of silk. con, silk from silkworms not infected with the virus; 13-1, silk from silkworms infected with 10 ⁶ copies of the virus. B, Secondary structure analysis of silk proteins based on infrared spectral characteristics. Negative control, silk from silkworms not infected with the virus; 10^6, silk from silkworms infected with 10 ⁶ copies of the virus.

Example 2 7532 variety of silkworm: (1) The recombinant virus AcNPV-FH/LP-MaSp-g/c is the cell culture supernatant from step (3) of Example 1.

(2) Inoculate silkworms with the recombinant virus AcNPV-FibH-MaSp-g: The " 7532 " variety of silkworms is raised to the fifth instar, and each silkworm is inoculated with 10 ⁶ copies of the virus. Compare 10 ³ , 10 ⁴ , and 10 ⁵ .

(3) Add the antibiotic florfenicol to the virus-inoculated silkworms: Prepare a 500mg/L florfenicol solution, spray 6L solution/100kg mulberry leaves evenly on the mulberry leaves, and spray fresh florfenicol solution After the mulberry leaves are dried, the silkworms are fed for 1 day, and then fresh mulberry leaves are used to raise the silkworms at about 24°C until mature silkworms are seen. Detection of recombinant protein MaSp-g/c in silk gland tissue by Western blotting: Take 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ copies of virus AcNPV-FHP/FLP-MaSp-g/c to infect the rear part of silkworms for 72 hours. Silk glands were detected by Western blotting using MaSp-g/c antibodies, and tubulin was used as an internal control. The test results are shown in Figure 6. Signal bands representing MaSp-g (>170 kDa) and MaSp-c (55kDa) can be detected in the posterior silk gland infected with the virus, indicating that the MaSp-g and MaSp-c genes have been translated into proteins.

Tissue immunofluorescence detection of secretion of recombinant MaSp-g/c in silk gland cells: 48, 72, and 96 hours after infection of fifth-instar silkworms with 10 ⁶ copies of the recombinant virus AcNPV-FHP/FLP-MaSp-g/c, silk gland tissues were taken Paraffin sections were made, and MaSp-g/c antibody was used to detect the secretion of recombinant spider silk protein expressed by silk gland cells through tissue immunology. The results are shown in Figure 7. A brown signal representing MaSp-g/c can be observed in the silk gland lumen infected with AcNPV-FHP/FLP-MaSp-g/c, and with virus infection, the signal intensity in the gland lumen increases, indicating that recombinant MaSp-g /c is secreted into the gland lumen and accumulates.

(4) Spray ecdysone on silkworms: Prepare 22.5mg/L ecdysone solution and spray the silkworms in step 3 so that the body surface of the silkworms is moist; move the mature silkworms to the cluster and create cocoons at 25°C. , pick the cocoons after 7 days, dry the cocoons and store them. Before reeling, after dry cocoons were stored and degummed, chimeric silk containing gold web-weaving spider ampulla gland silk proteins MaSp-g and MaSp-c was obtained through reeling. The length of the silk reached the control silkworm (uninfected with virus, i.e. pure silkworm). Silk) 63% of the silk length. Western blotting to detect MaSp-g/c in silk: Take the above chimeric silk and dissolve the silk with regular lithium bromide solution. The protein solution dissolved in lithium bromide was added to the dialysis membrane, and after dialysis for 72 hours, Western blot detection was performed using MaSp-g/c antibody. As shown in Figure 8, signal bands representing MaSp-g (>170 kDa) and MaSp-c (55kDa) can be detected in silk samples of fifth-instar silkworms infected with the virus AcNPV-FHP/FLP-MaSp-g/c. , indicating that the prepared silk contains MaSp-g and MaSp-c. Performance testing of chimeric silkworm silkworms infected with AcNPV-FHP/FLP-MaSp-g/c: Determine the stress-strain curve of silkworms inoculated with 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ copies of the virus group (Figure 9), Further calculation of the mechanical properties of the silk showed that the breaking stress and Young modulus of the silk in the virus-inoculated group were significantly higher than those in the uninoculated control group, and were dependent on the copy number of the inoculated virus; the cross-sectional area of the silk The average areas were significantly lower than the control group without virus, and the breaking strain was slightly reduced compared with the control, but the breaking energy was lower than the control group, and it was dependent on the copy number of the inoculated virus (Table 2). The infrared spectrum detection results are shown in Figure 10. According to the infrared spectrum detection results, the proportion of secondary levels in the silk protein was calculated. The proportion of β-sheets in the silk protein of the virus-inoculated group was higher than that of the control group, and as the number of inoculated virus copies increased, , the proportion of β-sheets in silk proteins increases in a dose-dependent manner.

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Figure 6 shows the above Western blot detection of MaSp-g/c in silk glands. 10 ³ , 10 ⁴ , 10 ⁵ , and 10 ⁶ copies of the virus AcNPV-FHP/FLP-MaSp-g/c were used to infect fifth-instar silkworms. After 72 hours, silk gland tissue was taken and Western blot detection was performed with MaSp-g/c antibody. Lane M, standard molecular weight protein; lane con, silk glands of silkworms not infected with the virus AcNPV-FHP/FLP-MaSp-g/c; lanes 10 ³ , 10 ⁴ , 105 and 10 ⁶ , infected with 10 ³ , 10 ⁴ , 10 ⁵ , 10 ⁶ copies of the virus AcNPV-FHP/FLP-MaSp-g/c silk glands. The primary antibody was anti-MaSp-g/c. The internal reference is tubulin, and the detection antibody is anti-tubulin antibody. Figure 7 shows the above immunohistochemical detection of MaSp-g/c secretion in AcNPV-FHP/FLP-MaSp-g/c-infected silk glands. A, silk glands of control silkworms; B, C and D, silk glands of fifth-instar silkworms infected with virus AcNPV-FHP/FLP-MaSp-g/c for 48, 72 and 96 hours. The primary antibody is anti-MaSp-g/c antibody. Figure 8 shows the above Western blot detection of MaSp-g/c in silk. Lane M, standard molecular weight protein; lane con, uninfected virus AcNPV-FHP/FLP-MaSp-g/c control; lanes 1, 2 and 3, 5th instar silkworms inoculated with 106 copies of virus AcNPV-FHP/FLP-MaSp-g /c Silkworm silk. The primary antibody was anti-MaSp-g/c. Figure 9 shows the mechanical properties of cocoons of silkworms infected with AcNPV-FHP/FLP-MaSp-g/c. A, Mechanical properties of silkworm cocoons from 5th instar silkworms inoculated with different titers of virus AcNPV-FHP/FLP-MaSp-g/c. Con, the stress-strain curve of the silk group without virus inoculation; 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ , the stress-strain curve of the virus group inoculated with 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ copies respectively. Figure 10 shows the infrared spectral characteristics of the above-mentioned silk and the secondary structure analysis of silk protein based on the infrared spectral characteristics; Con, the infrared spectrum of the silk of the uninoculated virus group; 1, 2, 3 and 4 were inoculated with 10 ³ , 10 ⁴ , 10 ⁵ and 4 respectively. Silk infrared spectrum of 10 ⁶ copies of virus group; Negative control, silkworm silk uninfected with virus; 10^3, 10^4, 10^5, 10^6, inoculated with 10 ³ , 10 ⁴ , 10 ⁵ and 10^6 respectively 10 ⁶ copies of the virus group in silkworm silk.

With the advancement of DNA sequencing technology, people have elucidated the sequences of multiple spider silk protein genes. For more than ten years, people have tried to express spider silk proteins in large quantities through genetic engineering technology, and further mechanically prepared spider silk through spinning engineering technology, and have made a lot of progress. At present, it is possible to express a variety of spider silk protein full-length genes or repeated fragments of repeated regions in expression systems such as bacteria, yeast, mammalian cells, and insects. Spider silk proteins can even be expressed through transgenic animals and plants. In nature, spiders spin silk proteins into silk fibers through the autonomous assembly of silk proteins and the spider's innate spinning habits. Since the recombinant spider silk protein expressed through genetic engineering cannot assemble independently, it must be further processed into fibers through artificial spinning. However, it is currently not possible to obtain fibers with mechanical properties comparable to natural spider silk in large quantities through artificial spinning. Spider silk proteins have a unique composition, especially the main ampulla gland silk protein, which has a huge molecular weight. Studies have shown that when the molecular weight of the foreign protein is higher than 60kDa, the expression level shows a significant decrease. When E. coli is used to express the spider drag silk protein gene fragment exceeding 3kb, not only the gene expression efficiency is reduced, but the expression is terminated prematurely. In addition, spider silk proteins usually have 4 universal amino acid modules: (1) GPGXX, (2) GGx, (3)An/ (GA)n and (4) spacer. Spider silk proteins are mostly composed of these modules that are highly repeated on a regular basis. Therefore, when expressed in a heterologous system, the molecular weight of the recombinant spider silk protein is often lower than that of the natural state, and the expression level is extremely low. Since the molecular weight of the recombinant spider silk protein is lower than that of the natural state, and people have not been able to completely imitate the spinning process of spiders, the mechanical properties of the silk obtained by artificial spinning are lower than those of natural spider silk. Bombyx mori are the only insects that can be reared on an indoor scale to provide large amounts of silk fiber. Silk protein is mainly composed of sericin and silk fibroin, while 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. In recent years, silk fiber or silk protein has been widely used in the development of medical biomaterials. Introducing the spider silk protein gene into the silkworm genome through the method of transgenic silkworms requires more complex procedures to screen and identify the transgenic silkworms, and further obtain transgenic pure lines through hybrid screening, which takes 1-2 years and mostly expresses a species of spider. Silk protein gene; the recombinant virus can be obtained in a short time through the technology of the present invention, and by inoculating the 5th instar silkworm with the virus, chimeric silk containing two kinds of spider silk proteins can be obtained in about a week, with a breaking strength of 1116.55MPa. The silk length reaches 65% of the existing technology, which not only solves the problem that the existing technology can only be used for diverse varieties with poor practicality, but also overcomes the problem that the existing spider protein composite silk silk is less than 42% of the length of pure silk. Defects.

Sequence Listing Free Content

SEQ ID NO: 1: ctcgaggtacggttcgtaaagttcacctgcggctatattccgactcgccaagttacgtcagtcgtattgtaatgagcgatttagtgggcaacttcattctgttaattttgtgtcacggtgcgcgcgcatcgtaaaacttcactctcatagatttttcataacgcgcctaaagaagtataacttca ataatttaaatttaaaaaaaaacatgcatagaataattatgaattatttaaaaatgtcatttaccgacattgacataacagacgacgttaacactacaaaacattttaattccacattgttacatattcaacagttaaatttgcgttaattctcgatgcgaacaaataagaacaatcggatcaattagatcgctttgtttcgaacaacacttagtttaactagaggcgtaca cctcaagaaatcatcttcattagaaactaaaccttaaaatcgcaataataaagcatagtcaattttaactgaaatgcaaagtcttttgaacgttagatgctgtcagcgttcgttggtacagttgtttgatatttattttaattttaattgtctttttatatataaatagtggaacattaatcacggaatcctgtatagtatataccgattggtcacata acagaccactaaaatgaaacctatcttcctcgttctgctggtggctacatctgcctatgccgccccatggtcttcgacggagttggccgacgcttttatcaacgctttcctcaatgaagccggaagaactggcgctttcaccgccgaccaactcgacgatatgtctaccattggtgacaccctgaaaacagctatggataagatggcc agatccaacaaatcatctcaatcgaagctccaggctctgaatatggctttcgcttcatcaatggctgaaatcgctgccgtggaacaaggtggattgagcgttgctgaaaaaacaaacgctattgccgattccctcaattcggctttctaccaaacaactggagccgttaacgtccagttcgtcaatgaaataagaagtctcatctca atgttcgctcaggccagcgctaacgaagctagctacggcggtggatacggcggtggacaaggcggtcaatctgctggtgctgccgctgccgctggtgctggacaaggtggttacggtggactgggcggtcaaggtgctggtagtgccgctgccgctgccgcttcaggagcaggtcaaggtggttatggtggagtgggaa accagggtgctggaagaggcgccggagccgctgccgctgccgctggcggtgctggtcaaggtggttacaatggtggacaaggaccttctgccgctgccgctgccgctgccagcggagctggccagggcggttacggaggccctggttcccaaggtgctggacaaggagctggagctgccgctgccgctgccggtggagctggac aaggcggttacggaggcttgggtggacagggagctggaagaggcggtgctgccgctgccgctgccgctgccgctgccgctgccgctgccggtgtggctggacaaggtggtctgggttcgcagggtgctggaagaggtggactcggcggtcagggtgcaggcgctgccgctgccgctggaggcgccggacagggtggatacggtggtctgggaca aggtgctggtcaaggagctggagtcgccgctgccgctgccgctggaggcgctggccaaggtggatacggcggtttcggttcccagggagcaggaagaggtggtcaaggtggacaaggttcggccgctgccgctggcggtgctgggcaaagaggttacggaggccagggtgctggtcagggtggattgggcggtgg agaacagggagctggcgaagaaggttctggtgccagcgctggcgctggtgccgctgccggaagaggcgctggcggtggaggcaagggtggactgggcggtcaaggtggtagtgctgccgctgccgctgccggtggagctgggcaaggcggtttgggaggctcaagaggtgctggacaaggtgctggagctgccgctgccgct gccggtggagctggtcagggcggttatggaggcctgggctcacaaggagctggtagaggtggacaaggcgctggtgctgccgctgccgctgccggcggtgctggccaaggtggttacggtggactgggcggtcagggcgttggtagaggtggtctgggtggtcaaggtgcaggtgctgccgctgccgtcggtg ctggacagggcggttacggaggcgtgggatctggtgcttcggctgccagtgctgccagatctagattgtcgagtcctcaagcttcatctagagtgagctccgctgtttcgaacctcgtcgccagtggtccaacaaattcagctgccctgtcgagtactatttcaaacgtggtttctcaaataggagcttctaatcc tggactgagcggctgcgacgttttgatacaggctctgttggaagtcgtgtcagccttgatccaaattctcggttcatctagcatcggacaggtcaattacggctcagcgggacaggctacgcaaatagtgggacagtcagtctaccaggctttaggataaataagaactgtaaataatgtatatatataattatataaaagatatatataac catatacaaacatatatatcattataagacaatctacctatataaaaacagactaaaattaataattatgtatactttaattgtgtttaggacattttatgcaaattgtgtttgcgttaggatttttttttggaagtttttttagattatttatgaatatataaataaatatacgttaatataatatatatattatataaatcaacgacacggcttttcattttggtga tgatcaatcttattgttcttctaattgatttttttgtacaataaagatgtatccagttttccagataaagaatttagtttgttattctggccccattaaaataagtacggtattcgacaatagcatgc.

SEQ ID NO: 2: GCGCCGCGCTCAAGCCTCCCCCCCCAATTGGGGGGGGGGGAgAgAgAgAgcgcccccgACACACCTCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCCGCGCCGCCGCCCCCCCCAATTCAATCCCCCCCCCCATCCCAAT. GCTGTAAAAAAAAAAAAATCACACGGGTCATTTTGAAATTTTTTGTCAAGAATTTTTTTTTTTTTTTTTTTTTTTTAATGAAAAACTTTTTTTTTTTAAACAACAACAACAACAACAACAAACAAACAAACAAAAAAAAAAAATTT GAATTTTTTTTTACTGACTGTAGTAGTAGTACAACACAGCCTTTTTTTTGAAACTAAGTAAAAAAAAAAAATAGCTCACACACACACACACACTTCCGGCACCCAAGAACAACACGT AGCAGAATCCTCAGTGTGTCACAGCAGCAGCCCCAactgctgctggggggggggggggggggggggTCTCACCACCACCCCCCCCCCCCCCCCAATCGAATCGAATCGAATCGAATCGAATCGAATCGAATCAAT AGTGATGACACACAAAACTACTTAGAGAGAGAAAACAAGCAGCAGCAAGGGAAGAAAAAAAAAAAAAAAAAAAAAAAATCTCCCCTCCCTCTTCTCCAgCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCAGCACCCCAACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAAGCCCCCCCCCCCCCCCCCC CGTAGCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCACAGGTT's TCGCCCCAACACACACACACATTCGGGACCCCCCCCCCCCCCCCCCTACGTTTCTCCAGCACCCACCGGTCGTCGGGGCGCGCGCGCGACGAAACCAACCCCCTC AGACACAGCCCCCCCCCCCCCCATGAGAGCGCGGGGGGGAGAGCGGGGCGCGCGTCCCCCGGGGTGTGCGCAgCAgCTCTTGTCGTCGTCGTCGCCCCCCCGGCCCCCCCCCGCCCCCCCCG TGAGCTCTCTTCTACTAGTTTTTTTTACGCTGAAAAATAGCTCTCTCTCTCTCTCTCTCTAAAAAAAAAAAAAAAAAGCTGCCCCCCCCGCGAgCGAgCAGCAGAAGAgAgAgAgAgAgAgAgAgAgAgAAGCCCCTGCCCCTGCCCCTCTCTCT TGTACGGAAGAAGAAGAATGAAGTGCTTGCAACGTAACGTAACGTTTTTTCGTCTCAATCGACCAAAAAAAACCATAATGTGACACACACACACACACACACACACACACACACACACACAT AttgacaataAAAAAAAAAAAAAAAAAAAAAAAAAAAATTGACTGAGAATGGGGGGGACAGGGGGGGGGGGGTTTTTTTTTTTTTAAAAAAAAAAAAATTTTTTTTCCCCAgCAGCCAGGGCACCACACCACACACACCCCGATT GGCAAAAAAAAAAAAAAAAAAAATGAAAAAAAAAAAAAGAAAAACAGTTTTTTCCCCCCCCCTCTCTGGGGGGGGGAAGAAGAAGTTTTTTTTAAAAAAAAAAAAAAAAAAAAATATATA TAAAAGTAAGAACAATAATCAATCAATCAATCATAATCATCATCATCAATTTCATTCATCATCATCATCGTTGTTTTTTAAAAAAAGAATTAATAATACAATACAATACAATACAATAATAATAATAAT GTTTTTATTCTCGAAAAAAAAATACGTCAAAAACGAAAAAAATTTTTTTTTAAAAAGTTCAAAATCAGCAGCAGTCCCTCTCTCTCTCAAGTCAACTCTCTGTGTGTGTGTGTGTCTCAAGTCAAGCAAGCAAGCAAGCAAGCAATCAACAAGTAAGTAAAGTAAAAAAAAAAAAAAAAAGTAAAAGTAAGTCAAT TGCTCTCCAATACGGCCTACACAACAACGCACCCCCGACCGCGCGCCCCGAAAAAAAAAAAAAAAAAAATTCTCTCCCCCGGGGGGGGGGGCCCCCCCCTGACCCTGACCCTGACCCTGACCCCCCCCCCCCCCCCCCCCCCCGGGACCCACACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC CTACTGTGGGGGGAGAGAGACCATTATGTCCCCCCCATCGAAGGGCTAGGCTAGAAATCAAGTCCTCCTCCTCCTCCTGAAAAAAAAAAAAAAAAAAAAAAATGCTCAATCCTGCCCCCGCGTGTGCCCCCCCTGCCCCCCCCCCCCCCCCCTCAT GGACAGTCGATGATGAAGACCAACCAACCAAATGCTGCTTGGGGGGGGGGGGGGACTACTGGGGGGGGGGGGGGGGGGGGGAACCAATCAAAAAAGAAGAATCTCTCTGCCCCCAGC GCCAACGAAGCTACTACGGGGGGGGGGGGGGGCTCCGCCCCCCCCAGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCCCCCCCTCGCGCTCTCTCTCTCTCTCTCCTCTCTCTCTCTCCTCGCCTCCTCCCCTCCT AcggtcatCAAAGAGACTTCAGGTGTGCCCCCCCCCCCCCCCCCCCCCCCGGGGGCACCCCCCCCCGCCGCCCCCCCTCTCTCCTCTGCCTAGCCCCCCCC ActagCTACGTCTCTAGACAACAACGCCCCCCCTGGGGGGGGGGCTGCCGCGCGGGGGGGGGGGGGGGGGGGGGGGAacaacaacaacaCCTCTCTCTCTCCCCCGCCCCCGCCCCGCCCCCCCCG AcgaagCTGACACACACACACACCCCGGGGTGCTGCCTGCTGCCTGCGGGGGCGCGCGCGGGGTCCCGCGCCCCTGCCTGCTGCGCGCAGCATCAGCAAT cctggacaacaacagGctCTGGTGTGTCGCTCGCTGCTGCTGGGGGGAacCCCAGGGCCCTCTCACACACACACACACACACACACGCCGCCTGCTGCTGGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC GTTACGACACACAGACAACAAGGACTGGGGGTGTGTCTCCCCCCCGCGCGCGGGGGGGGGGGGGGGGGGGGGTCCGACAAGCCCCCCCCCCCCCCCCCCCCCCCCCGCCGCGCGCTGA is AACACGCGCGTTCGTCTAGGGGGGGCCCCCCCCGCCGCGCGGGAgggggggggggggggacgggggggggggggggggggggggcccccccccccccccccccccccccgCGCGCGCGCGCGCGCGCGGAGAGGAGAGAGGAGAGGACGCGCCTGCCTGCCCCTGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC CTGATGGACTGAAGAAGGGGACTGGGGTCAGGGGGGGGGGGGGCCCCCCCCCCGCGCCGCCGCCGCCCCCCCGGGGGGGGGGGGGGGGGGGGGGGGGGACTGACCCCACCCACCACCACCACCACCACCACCACCACCCACCACCCACCCACCCCACCCCACCCCCACCCCT is GCTGCCGCTAGTGGGGCGGGGGGGGGGGGGTCTACACACCACCGCGCGCGCCGGGGGGGGGGGGGGGGGGGGGGGCCCCGCGCGCGCGCGCGCGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCGCGCGCGCGCGCGCGCGCCGCCGCCCCCCCCCCCCT CaggataCGCTAATAATGCAAAAAGTCCCCCCGGGGCCCCGCGCCCCCCTGCGCGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGACCCCGACGACGACGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCT AggCTCCCCTCTCTCGCGCGCGCAGCCCCCCCCCGGACTAGGGCCCCCCCCCCCCCCCCCCAAGTCTGCTGCCTGCGCTGCTGCCCCCCGCGGGGGGGGGGCGGCGGCGGCGCGGCGCGGCGCGCGCGCGCGCGCGCGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCGCCCCCCCCCCCCCCCCCC CGTCAGCGGGTCTGGGCCCCCCCCTAGCCCCCCCCCCAGGGGGGGGGCCCAACGGGGGGGTCTCCCCCCCCCCCCCCCAgCAgCTGGGGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC TGCCCCCCTGGTGTACTACGTCCTGAgAgAggggggggggcccccccccccgcgcgcgcgcgcgCCCCCCCCCCCCGCCCCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCTGCGCGCGCCCT CTACGGTCAgGacaacaacaaggaccCAgCCCCCCCCCCCCCTGCTGGGGGGGGGGGGGGGGGGGGGGGACCCCCCCAATGGGGGGCCCCTCGGGGGGGGGCTGGCTGGCTCTGGCTCTCCCCCCCCCCCCCCTGCTGCTGCTGCTGCTGCTGCTGCCTGCCTGCCTGCCCTGCCT GataCGGCTACGGAGACACACACACAgGGGGGGGGACCAGGAGGGCTGCCCCGCGCTGGGGGCGGGGGCCCCCCCCCCCAggggggggggggggggttchgtcgtcagtcgtcgtcgtcgtcgtcAgGCCCTGACGGAgGaccccccccgggggggggggggggggggggggggggggggggggggggaccgggGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC GAACAGGAGAGAGAGAGAGAGAGGGGGGGGCGCCGCCCCGCGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCCCCTGCCCCCCCCCCCCCCAgCAGCAGGCAGGCAGGCAGGCAGGCAGGCAGCAGGCAGCAGCAGCAGCAGCCCCCCCCCCT CaggCAACAACAACAGGGGGGGGCCCCCCCTGCCCCCTGCGCGCGCGCGCTGAGGGTCAgGGGGGGCCCCCCCCCCCCCCCCCGGGGCCCCCCCCCCCTGCCTGCCCCCGCTC GTCCCGGGCGTTTCGTCGGGacaagAggCCCCCCCCGGCCCCCCCCCCCCCCGGGGGAGGGGGGGGGCCCAacgCCCCCCCCCCCCCCCCCCCCTGCCTGCCCGCTGC CGCTGCCCCCCCCGGTAGAGAGGGGGGGGGTGAAAAAAACCCCCCCGGTCAGGGGGGGGGGGGGGGGGGGGGAacaacaacaCTGAACTGTGTGCTGCCTAATGCCAATGCAATGCATAAT CGGACAGGAGAGAGAGAGGGGCCCCCTGTGCCCGCGCGCTGGGGGGGGGGGGGGTCACAGAGCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGGGGGGACCACACACACAACAC AggtcgcgCTGCCTGCCCACACAGCCTGCGCGCGGGGGGGGGGGGGGGGCCCTGCCCCCCCCCCCCCCGGGGGGGGGGGGGGGGGGGCTGCTGCGGGCGGCGGCGGGGGGCGGCGGCGGCGGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCT GGTGAAGCTGCCCGCTGCCCCCCCCCCCCCCCGCGCGCACTGCTGCCCCCCGCCGCCCGCCGCCTGCCCCCCCCCCCCGGGGGGGGGGGCGCGCGCGGCGCGGCGGCGGCGCGCGCCCCCCCAGC is CGCTGCGCGCCTGCCCTGGTCTCGGGGGGGGCTACCCCCCGGTCAACACACGGGGGGGGGGCTCCCCCCCAAAAAAGGGGGGGGGCCCCCGCGGGGGGGGGGGGGGGTCCCCGCGCT AAACAGGTGCCCGCGCGGTCTGGGGGCTGCCGCCGCGCGCGCCCCCCCCCCCCCCGGGGGGGGGGGGGGAAGAGAGACGACCGACCCAGCCCCCCCTGCCCTGCTGGGGATC is CTCGGCCGCCTGCCCCCCCCCGGCGCAGCAGCTGCGCGCGCGCGCGTCTCTCTCTCTCTGGGGGGGGGGGGGTCTGGGCTCTCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCT AcggacctgtcaaAgggggaccgcgcgccccccggggggAcGACTGTCAGGTCTGGGGGGGGGCGCCCCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCGGGGCGGGCGCGCCCCCCCCCCCACTACTACTACTAC CTGGTGTGCCCCCCCCCCCGCCCCGCGGCAGGGGGGGGGGGCCCCCCCCCCCCCCCCCACACCTGCCTGCCCCGCCGCCCGCGCGCGCCCCCCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCCCCTGCCCCCCCCCCCCCCCCCCCCCCCCCCCCC GTAAGGGTGGGTGATATGTCCCCCCGGTAACAACAACCTGGGGGGGGGGGGGGCCGCGCCTGCTCTCTCGGGGGCGGGGGGGGGGGGGGGGCCCCTGCCTGCTGCTGCTGCCA is GaagggAccccgggggCTACGCCCCCAAAAAAAAAAAAGGCTGCCTGCCCCCCCGCGCGCCTGCCCCCCCCCTGGGGGTCCCCCCCCCCCCCACCACCACCTGCGTCGTCGCGTCTCCCCCCCCCCCCCCCCCCCCCTGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC GCTGCCCTACTACTGACCGGGGGGGGGGGGGGGGGGCCAAGGTCAGGGGGCGCGCGCGCGCGCGCTGGCGGGGGGGGGGGGGGCGGCCCCCCCCCCCCCCCCCCCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCT CTGGTAGCGCGCGCGCCCCCCGCTGGGGGGGGGGGCTAAACAACACAACAATCTCTGCCCCCCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGACAGACAGACCAGACCAGACCAGACCAGACCAGACCAGACAGACACTGCCCCTCAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAACAAC CGGTTCTGGGAGCTGCCCCGCGCGCGCGAAGAGAGACCCCCCCCCCGGGCTAGCTGTCAGCAGCCCCCCCCCCCCCCTGCTGCGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTCCA is GGACAGCAAGGTCGGGGGGCCCCCAAAAAAAAATGACTGTCAACGGGTCTGGGGGGGTGTGCTGCTGCTGCTGCGCGCTGAAGAACCCAACCCCCCCCGGGGGGGGGGTCAGGTCCTGGG TGGTCTCTCTGTGCCCTGCTGCTCCTCTGGGGGGGGGGGGCCCCCCCCCCCCCCCCCCCCCCGGGGTCCTCTGCCTGCCTGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTCCCCCC GCCCTGGGGGATACTCTCTGGTCAGGGGGGGGGGGGGGGGGGGCGCTCCCCCCAGTCCAGGGGGGGGGCTGCTGCCCCGCCGCGCGCGCGCTGTCCTGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCCCCCCCCCCCCCCCCCCCCCCT GTCCCCCCGGGGCCAGAACAACAACTGCCCGCCCCGCGCGCGGCAGGGTCGAACGGGGCCCAACCCAACGGGGGGGACGGGCCCCCCCCCCCCCGGGGGAgCGCGCGAT is GCTGCCGCTGCCGCCCCCCGCGCTAGGCCTGCCTGACAcaggggaccccccccccccccccccccgcgCTGGGGGGGGGGGGGGGGGGGGGGGGGCCCGC is TGTGGCTGCTTCTCTCTGGGTGTAGGACGACTCTCTCTACCCAACACACACAGGGGGGGGGGGGGCCCCCTGCCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGGGAGAGAg AcaagGTCCAGGGGGACCTGTGTCCCCCGCCGCGCGCCCCCTCTAGAGAGAGACCCAGCAGTCAGGGGGGGGGGGGGGGCTCTCTAgCTGGGGGGGGGGGGACGACCCAg GCCAACAACCCCCCTCTCCCCAGTGCCTGCCTGCACAGCCCTGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGTCCCCCCCCCCCCCCAgCAgCCCCCCCCTGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGCGC ActTTTTTTGGAGAGCTGCGCTGCCCTGGGGGGTCAGGGGGGGGGGGGGGGCGCGCCCCCTGCGCCGCGCGCCCCCCCCCGGGGGGGGGGCGCGCGCGCGCTGTGTGGGGCT is TCACCGCGCTGATGATCCCCCTCTGGGGGGGGGGACTGACAGGTCTCTCAGGCAggaccCAgGTGTCGCCCCGCGCGCGCCTGCCCCGGGGGGGGGGGGTCCTG GACACGAAGAGAGAGAGAGAGAGAGTCCCTCTGTGTGCTGCCCCCCCCGCGCGTAGCCGCGCGCGCGACACAACCCGGGGGGGGGGGGGGGCCCCCCCCGGGGGGGGGGATGGGGG TCCTGGTCAACAACAACAGTCCCCCCGGAACACAGCTGCTGCTGCTGCGCGCTGAATGTGGGGGGGGGGGGGTCAACAACCCCCTGCCCCCCGCCGCGCGCCTCTGGGGGGGGGGT GGATATGCCTGGTGTGTGTGCAGACCTGAGGGCCTGGGGGGCTGCCCCGCCGCCCTGTGTGGGGGGGCGGGGGGGGGGGCCCCCCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCTGCCTGCTGCCTGCCTGCCTGCCTGCCCTGCCCCCCTGCCCTGCCT GTCACCGTAGGACCTGGGGCTAGCCCCCCCAACCAAGGGGGGGGGGCCCGCCGCGCCTGCCTGCCTGGGGGGGGGGGGGGGGGAacgggggggggggcttgcgCagcagcagcagcagcagcagcaggggggggggggggggggggggggggacacccccccccccccccccccccccccgCCCCTGCCCCCCCCCCTCTCT GCTGGAGGCCCCCCCCCGGTAGTACTGAacAacaggggcgcgcagGGGGGGGGGGGGGCCCCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGGGGGGGGGGGACGGACGACGGGGGGGGGGGGGGGGGCGGGGGGGGGGCGCGCCCCCCCCCGGCACGACGACGACGACGCGCGCCCCCCCGCCCCCCCCCC TCGGCGCGCTGCCTGCCGCCTGCCCCCGGCGCAACGGGGGTGCCTGGTCAACCCCCCCTGCCTGCCCCCCCGCGCCGCGGGGGGGGGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCACGCACGTGCCCTGCCCACGCCCCCCCCCCCCCCCCCCCCCCCCCCC AACTGGGTGGTGGTCCCCCCCGCGCCCCAATGCTGCTGCTGAgGGGGGGGGGGGGGTCACACGTGCCCCCCGCCCGCGCGCGCGCGCCGCCGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCAT AAGAGATCGCGGGAATGGGGGGGGAGAGCAGCCCCCCCCGGGGGGGGGGGGGCCCCCCTGCCTGCGCGCGCGGGGGGGGGGGGGGGGGGGGGGGTCCAGGGGGGGGGGGGGGGGGACGACGACTGAGGCCTGCCTGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGCCCTGGGGCCCCTGTGCCCCCTGCCCCCCTGCCCCCCTGCCCCCCTGCCCCC CCGCTGCGCGCGCGCTACTGGGGGGGGTACTGGGGGGGGGGGGGGTACGTAGCAgGAGACCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCGCGCGGGCGGCGCGCGCGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCC TCCTGTGGCTACCCTGCCCACAGCCCCCCCAGGGTCGGGGCCCCCCCCCGGGCCCCTGGGGGCCCCCCCCCCCCCCCCCCCCCGGGGGGGGGGGGGGGGCAAC is AAGGACCTGGGAGCCCCTCTCTGCTCTCGGGGGGGGGGGGGGGGGGGTCCAACAACACCTGCTGCCTGCCTGCCCCCCCCCCGGGGGGGGGGGGGGGGGGGTCCCCCCTCCCCCCCTCCTCTCACTCT CTAGTGCTGCCAGCTAGGGGGGCTTTTTTCCCCAGAGAGAGAGGGGGCTCTCTCTCTCTCGGGGGGGGGTCCCTCTCTCTCTCTCCAATTCCAATTCCAATTCCAATTCAATTCAATTCCAATTCAATTCCAATTCAATTCCAATTCCAATTCCAATTCCAATTCCAATTCCAATTCCAATTCCAATTCCAATTCTCCTCAT TCGGAAAATAGGAGAGAGAGCATCCCCCCACACGACTGTGGGGTGTGTGTGTGTGTCCAGTTTGGGAAAAATGCGCCTCTCTCTCTCTCACGGGGCCCGT TTCTCAAGCGCGCGGGTGTTTTCCCCAATCCTGTGCAGCAGTTTTTTTTTTTTTTTAACCCCCCTTTTTTTTTTTTTTTTTTTTTTTT TaagtttttttttttttttttaattTTTTTTTTTTTTTTTTTTTTTTTCGGGGGGGGGGGGTTTTTTTTTTTGAATCAATCAATCAATTTTTTTTTTTTTTTTTTTTTTTTTTTT TAAAAAAAAACTAAATTACAATATATCATAAAAAAACAAAACAAGTAACTCTCTCTACTACTACTGCAG.

SEQ ID NO: 3:ATGAAACCTATCTTCCTCGT.

SEQ ID NO: 4: TTATCCTAAAGCCTGGTAGA.

SEQ ID NO: 5:ATGAGAGTCAAAACCTTCGTG.

SEQ ID NO: 6: GCTTTGAGAAACGGCCACGTA.

SEQ ID NO: 7: TATATTCGCGGCGTTGTGAC.

SEQ ID NO: 8: AAGTTGGGCATACGGGAAGA.

Claims

A high-strength silk containing multiple spider gland silk proteins, characterized in that the multiple spider gland silk proteins are two or more different spider gland silk proteins.
The high-strength silk containing multiple spider gland silk proteins according to claim 1, characterized in that the spider gland silk protein is spider ampulla silk protein.
The high-strength silk containing multiple spider gland silk proteins according to claim 1, characterized in that the spider is a golden silk web-weaving spider.
A method of producing high-strength silkworm silk containing a variety of spider gland silk proteins according to claim 1, characterized in that multiple sequences expressing spider gland silk proteins are cloned into plasmids, transformed and recombined, and then cultured cells are transfected, Recombinant virus particles are obtained; the recombinant virus particles are inoculated into silkworm larvae and fed to mature silkworms to obtain silkworms that produce the high-strength silk containing a variety of spider gland silk proteins.
The method for preparing high-strength silk containing multiple spider gland silk proteins according to claim 1, characterized in that multiple sequences expressing spider gland silk proteins are cloned into plasmids, transformed and recombined, and then cultured cells are transfected to obtain the recombinant Virus particles; the recombinant virus particles are inoculated into silkworm larvae, fed to mature silkworms, and then reeled, cocooned, and reeled to obtain high-strength silk containing a variety of spider gland silk proteins.
The method for preparing high-strength silk containing multiple spider gland silk proteins according to claim 5, characterized in that the sequences expressing multiple spider gland silk proteins are SEQ ID NO: 1 and SEQ ID NO: 2.
The method for preparing high-strength silk containing a variety of spider gland silk proteins according to claim 5, which is characterized in that: Escherichia coli containing AcBacmid DH10Ac is used for transformation, white colonies are picked after cultivation, and recombinant DNA is extracted; and the recombinant DNA obtained by transformation is The Spodoptera frugiperda Sf9 cultured cells were transfected to obtain recombinant virus particles.
The method for preparing high-strength silk containing multiple spider gland silk proteins according to claim 5, characterized in that the inoculated silkworms are fed with mulberry leaves containing antibiotics for one day, and then fed with untreated mulberry leaves until the silkworms are mature, and then Treat once with ecdysone and then weed.
The method for preparing high-strength silk containing multiple spider gland silk proteins according to claim 5, characterized in that the varieties of silkworms include varieties of silkworms practical for silk cocoon breeding and original species of silkworms.
The use of high-strength silk containing a variety of spider gland silk proteins in the preparation of silk products according to claim 1.