Sarachnoid silk protein transgene production method based on lentivirus transfection and sperm mediation and application

Abstract

The invention discloses a spider silk protein transgene production method and application based on lentiviral transfection and sperm mediation in the field of biotechnology and genetic engineering. The spider silk protein transgene production method comprises the following steps: firstly, constructing a vector pLC-[3xP3-DsRed]-MaSp2-Amyloids for synthesizing and secreting spider silk protein genes by bombyx mori; the plasmid is introduced into a silkworm fertilized egg by using a lentivirus transfection and sperm-mediated gene transfer method, the red fluorescent protein gene and the black widow spider dragline silk protein 2 and amyloid polypeptide composite gene are introduced into a silkworm genome by using the characteristics of the lentivirus, and stable inheritance and expression are obtained; therefore, a transgenic silkworm capable of specifically synthesizing and secreting spider silk protein in bombyx mori silk gland cells is created, then the spider silk protein is synthesized and secreted by the silkworm, and the spider silk protein-containing composite silk with bombyx mori silk as the background is obtained. The method lays a foundation for mass production of black spider silk protein and also lays a foundation for improvement of mechanical properties of silk fibers.

Description

Technical Field

[0001] This invention relates to the fields of biotechnology and genetic engineering, specifically to a method and application of transgenic production of spider silk protein based on lentivirus transfection combined with sperm-mediated synthesis. Background Technology

[0002] Natural spider silk possesses excellent mechanical properties, being the strongest known natural fiber. Its mechanical strength is 3-5 times that of ordinary steel, comparable to the highest strength carbon fibers and high-strength synthetic fibers (such as Kevlar fiber). Its overall toughness is 5 times that of Kevlar, a material commonly used in bulletproof vests. Due to its unique physical and chemical properties, such as high strength, high toughness, and good biocompatibility, spider silk protein shows great promise for applications in the pharmaceutical industry. However, the low yield of spider silk and the fact that cannibalism among spiders prevents the large-scale acquisition of spider silk through breeding limit its widespread application.

[0003] With the rapid development of biotechnology and a deeper understanding of the coding sequence and spinning mechanism of spider silk, various heterologous expression systems have been used to express spider silk proteins. Examples include *E. coli*, yeast (*Pichia pastoris*), and insect cells (*sf9*, *BmN*). However, due to the large molecular weight and high repetition of spider silk proteins, the expressed spider silk proteins from various heterologous expression systems used to date have relatively small molecular weights. Many recombinant proteins lack key protein motifs, and many are not pure spider silk protein molecules but fusion proteins fused with molecules such as fluorescent proteins. These factors ultimately affect the mechanical properties of the exogenous proteins. Furthermore, the success rate of exogenous gene introduction is very low, and the operation is highly complex.

[0004] With the development of genetic engineering technology, expressing recombinant spider silk proteins using exogenous hosts (such as silkworms) through transgenic methods has become a research hotspot. However, due to the limitations and complexity of the technology itself, the yield and performance of spider silk proteins are difficult to improve. For example, the spider silk genes of the earliest transgenic silkworms obtained through the Bac-to-Bac baculovirus expression system cannot be stably inherited; the recognition site of the piggyBac transposon-mediated system is fixed at TTAA, which can only randomly insert the spider silk gene into the silkworm genome, thus resulting in low efficiency, and it cannot simultaneously knock out the silkworm heavy chain protein gene (Fibrin heavy chain, Fib-H), leading to very low expression levels of spider silk proteins; while transcription activator-like effector nucleases (TALENs) are complex to operate, the assembly process is very cumbersome, the cost is high, and they have certain cytotoxicity.

[0005] Furthermore, the development of a mature methodology for introducing exogenous genes has been a pressing issue in silkworm transgenic research. Domestic and international experts have made various attempts at silkworm transgenic technology, including gene guns for fertilized eggs, electroporation, sperm-mediated methods, and testicular injection. Substantial breakthroughs in silkworm transgenic technology only came after Tamura et al. successfully obtained transgenic silkworms using the piggyBac transposon vector combined with microinjection technology in 2000. More importantly, silkworm egg microinjection technology requires expensive equipment and skilled operators. The trauma caused by piercing the hard eggshell during injection significantly reduces the survival rate, affecting its application in transgenic technology systems. Moreover, microinjection technology can only inject a limited number of silkworm eggs at a time, making the operation cumbersome and time-consuming, unsuitable for large-scale production involving the introduction of exogenous genes. Summary of the Invention

[0006] The purpose of this invention is to address existing technical problems by proposing a method and application for the production of spider silk protein transgenics based on lentivirus transfection combined with sperm-mediated synthesis. This method allows for the mass introduction of the target spider silk protein gene, enabling the development of a production process that can synthesize and secrete complete spider silk protein, and obtaining hybrid silk fibers based on silkworm silk for the development and utilization of spider silk. It can also be used to improve the mechanical properties of silk fibers.

[0007] The present invention achieves the above objectives through the following technical solutions: As a first aspect of the present invention, the present invention provides a method for producing spider silk protein transgenic products based on lentivirus transfection combined with sperm-mediated transgenesis, comprising the following steps: (1) The pLC-[3xP3-DsRed]-MaSp2-Amyloids plasmid was constructed using molecular biology methods and used as a gene vector for expressing spider silk proteins in the silk glands of silkworms; (2) The pLC-[3xP3-DsRed]-MaSp2-Amyloids plasmid was transfected into cells using a lentiviral vector transfection-mediated gene delivery method. The transfected cells were cultured, filtered, and centrifuged to concentrate the recombinant virus. (3) The recombinant virus was introduced into the fertilized eggs of silkworms via sperm-mediated infection; (4) After being infected with recombinant virus, silkworm fertilized eggs were hatched and mated for multiple generations to produce transgenic silkworms with homozygous red-eye gene and spider silk protein gene and silk gland cells capable of synthesizing and secreting spider silk protein. (5) Spider silk protein is synthesized and secreted by the silk gland cells of the transgenic silkworm and enters the cocoon along with the silkworm spinning and cocooning behavior of the transgenic silkworm. The silk is collected to obtain the composite silk containing spider silk protein based on silkworm silk.

[0008] As a further optimization of the present invention, the pLC-[3xP3-DsRed]-MaSp2-Amyloids plasmid includes two functional expression boxes. One functional expression box is a red fluorescent protein gene expression box initiated by the 3×P3 promoter, namely 3×P3Promoter–DsRed-SV40. The other functional expression box contains the C-terminus of the silkworm heavy chain protein and the sequence of the spider silk protein gene MaSp2. Furthermore, the sequence of the spider silk protein MaSp2 gene is modified with amyloids containing 4-128 repeating sequences.

[0009] As a further optimization of the present invention, the amyloid peptides include any one of GDVIEV (PDB:3SGS), KLVFFAE (PDB:3OW9), and FGAILSS (PDB:5E61).

[0010] As a further optimization of the present invention, step (2) specifically includes: (2-1) Prepare plasmid mixture: mix pLC-[3xP3-DsRed]-MaSp2 plasmid and Cas9 helper plasmid containing sgRNA1 and sgRNA2 vectors with psPAX2 plasmid and pMD2.G plasmid in a ratio of 4:3:1, add to 100 μL serum-free culture medium, and mix to obtain plasmid mixture; Among them, the psPAX2 plasmid (packaging plasmid) carries the gag, pol, rev, and tat genes and is a plasmid that can express the lentiviral coat. Its expression product can more easily cross the cell membrane through the adhesion mechanism. The pMD2.G plasmid (enveloping plasmid) contains the VSV-G envelope protein gene and is often used as a vector for gene cloning and expression. It is an auxiliary plasmid for lentiviral packaging with high copy number, good stability, and easy operation. It can enhance the envelope stability and infection efficiency of lentiviral particles. In the second-generation viral packaging system, the target plasmid, psPAX2 plasmid, and pMD2.G plasmid are transfected into cells (such as 293T cells) together to induce the cells to produce lentivirus, providing a powerful tool for gene delivery and cell engineering research. (2-2) Incubate the plasmid mixture with the transfection reagent, then add it to the culture medium containing cells for transfection. After successful transfection, collect the culture supernatant, take the cell culture supernatant and re-inoculate the cultured cells, then culture the cells again, collect the cell culture supernatant, filter the collected supernatant to remove cell residues and impurities, and obtain crude virus solution. Centrifuge and concentrate the crude virus solution using an ultrafiltration tube to obtain recombinant virus. Further, add the transfection reagent to serum-free culture medium and mix well to obtain a transfection reagent mixture. The transfection reagent can be any one of PolyJet, Lipofectamine 2000, Lipofectamine 3000, Lipofectamine LTX and Lipofectamine MessengerMAX, with PolyJet being preferred.

[0011] As a further optimization of the present invention, the amount of fertilized egg infected by the recombinant virus is 10. 4 -10 8 Copy / fertilized egg, more preferably 10 7 Copy / fertilized egg.

[0012] As a further optimization of the present invention, in step (3), the method of introducing the recombinant virus into the fertilized egg through sperm-mediated means is any one of the following: Method 1: Inject the recombinant virus into the mating sac of a virgin moth using a glass needle made from capillary glass, and then allow it to mate with a male moth to lay eggs and hatch. Method 2: Separate the male and female moths 30 minutes after mating, and then inject recombinant virus into the female moth's mating sac to induce egg laying and hatching; Method 3: Separate the male and female moths 30 minutes after mating, dissect the female moth, extract the sperm from the copulatory sac, mix the sperm with the recombinant virus, and inject it into the copulatory sac of the virgin moth to induce egg laying and hatching.

[0013] As a further optimization of the present invention, step (4) specifically includes: (4-1) After the silkworm fertilized eggs infected with recombinant virus are hatched and raised to adulthood, they are mated with non-transgenic silkworms to produce the G1 generation. During the greening period of the G1 generation silkworm eggs, transgenic silkworms expressing the red fluorescent DsRed marker gene in one eye are screened out. After being raised to adulthood, they are mated with non-transgenic silkworms to produce the G2 generation. (4-2) In the G2 generation, silkworms that express the red fluorescent DsRed marker gene under a fluorescence stereomicroscope were screened out, and the G3 generation was produced by crossbreeding silkworms from the same moth area; (4-3) The G3 generation silkworms were raised by single moths, and the silkworm moths expressing the red fluorescent DsRed marker gene in the same moth area were crossbred to produce the G4 generation; (4-4) Starting from the G4 generation and for three consecutive generations, the same methods of raising moths in moth-producing areas with a pure red-eye phenotype, raising single moths, and mating with silkworm moths in the same moth-producing area were used to select and mate them, so as to breed transgenic silkworms with homozygous red-eye gene and spider silk protein gene and whose silk gland cells can synthesize and secrete spider silk protein.

[0014] As a second aspect of the present invention, a spider silk protein composite filament prepared by any of the methods described above is also provided.

[0015] As a further optimization of the present invention, the spider silk composite filament has a strength of 312~1534MPa, an elastic modulus of 4935~11045MPa, and a toughness of 58~369MJ·m. -3 .

[0016] The beneficial effects of this invention are as follows: This invention first constructs a vector pLC-[3xP3-DsRed]-MaSp2-Amyloids for the gene that synthesizes and secretes spider silk protein in silkworms. Then, using lentiviral transfection combined with sperm-mediated gene delivery, the plasmid is introduced into silkworm fertilized eggs. Utilizing the characteristics of lentiviruses, the red fluorescent protein gene and the complex gene of black widow spider puller silk protein 2 and amyloid polypeptide are introduced into the silkworm genome and stably inherited and expressed. This creates a transgenic silkworm capable of specifically synthesizing and secreting spider puller silk protein in silk gland cells. The silkworm is then used to synthesize and secrete spider silk protein, laying the foundation for the large-scale production of black spider silk protein and also for improving the mechanical properties of silk fibers.

[0017] (2) In this invention, amyloid polypeptides are embedded into the spider silk protein gene. The amyloid polypeptide fragments are easy to form β-nano crystals during the silk formation process, which helps to improve the mechanical properties of spider silk protein and obtain silk fibers with ultra-high strength and ultra-toughness.

[0018] (3) This invention utilizes recombinant lentivirus transfection and sperm combination to integrate the target gene into the silkworm genome. This integration is stable and has high transfection efficiency, increasing the amount of exogenous DNA entering the silkworm eggs and improving the efficiency of sperm-mediated gene transfer. In addition, a silkworm moth can produce hundreds of eggs, and a single copulatory sac injection can be completed instantly. The transgenic silkworms are obtained in large quantities, allowing the target gene to be continuously expressed in multiple generations in silkworms. The combination of lentivirus transfection and sperm vector methods allows for the batch introduction of the target gene, screening out stable transgenic silkworms, and is suitable for large-scale production of transgenic silkworms. (4) This invention uses CRISPR-Cas9 gene editing technology to mediate the production of full-length spider silk protein (MaSp2). The gene sequence of the heavy chain (Fib-H) repeat region of silk fibroin is replaced by the gene sequence of full-length spider silk protein (MaSP2), retaining the CT and part of the NT of Fib-H. The light chain (Fib-L) and glycoprotein P25 remain unchanged, and the ratio of MaSP2, light chain and glycoprotein does not change after replacement. The expressed spider silk protein also binds to the light chain through the C-terminal region of the heavy chain protein via disulfide bonds and other means. During the silkworm spinning process, this type of protein, along with p25 protein, sericin, etc., exists in the silkworm cocoon in the form of silk fibers. Macroscopically, there is no significant difference in diameter and appearance between wild-type and transgenic silk fibers. Microscopically, its secondary structure also includes β-sheets that provide strength and α-helix structures that provide elasticity. Attached Figure Description

[0019] Figure 1 The expression of transgenic silk protein obtained in Example 1 of this invention. Detailed Implementation

[0020] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0021] I. Materials All target plasmids involved were synthesized by the commercial company Nanjing GenScript.

[0022] The accession number for the black widow spider's traction silk protein 2 gene MaSp2 is EF595245; Amyloid peptides include GDVIEV (PDB: 3SGS), KLVFFAE (PDB: 3OW9), and FGAILSS (PDB: 5E61). The viral packaging helper plasmids involved were all purchased from Shanghai Zeye Biotechnology Co., Ltd., including psPAX2 plasmid (catalog number: ZY1444) and pMD2.G plasmid (catalog number: ZY1443). Viral infection enhancement reagent: polybrene, Beijing Lanbokangsi Technology Co., Ltd., catalog number C0351-1ml; Transfection reagent: PolyJet, purchased from Changzhou Cisco Biotechnology Co., Ltd., product number SL100688; Serum-free culture medium was purchased from Shanghai Yanhui Biotechnology Co., Ltd., product number 88-581-CM; Complete culture medium was purchased from GeoBlue (Guangdong) Life Science and Technology Center, catalog number JNO173-01; 293T cells were purchased from Shanghai Nuobai Biotechnology Co., Ltd., catalog number C01-BD.

[0023] Unless otherwise specified, all reagents and materials used are commercially available products.

[0024] method Unless otherwise specified, the methods used below are conventional methods known to those skilled in the art.

[0025] 1. Construction of recombinant viruses (1) Cell preparation: One day before transfection, 293T cells were plated at an appropriate density (using a 6 cm culture dish as an example). (2) Adjusting cell density: On the second day, when the density of 293T cells reaches about 70%-80%, plasmid transfection is started. Before transfection, the culture medium in the culture dish is replaced with 2.8 ml of fresh complete culture medium. (3) Preparation of plasmid mixture: Mix the three plasmids in a total amount of 2.5 µg at a ratio of 4:3:1, i.e. (target plasmid and Cas9 helper plasmid containing sgRNA1 and sgRNA2 vectors): psPAX2: pMD2G, add to 100 µL serum-free culture medium and mix gently. (4) Preparation of PolyJet mixture: Add 7.5µL of PolyJet transfection reagent to another 100µL serum-free culture medium and mix gently; (5) Complex mixing and incubation: Add the PolyJet mixture to the plasmid mixture at a ratio of 1:1, mix well, and then incubate the resulting complex at room temperature for 15 minutes. (6) Adding cells for transfection: Add the above complex evenly to a 6 cm culture dish and gently shake to ensure even distribution; (7) Change the culture medium and collect the virus supernatant: After 12 hours, replace the culture medium with 4 ml of fresh complete culture medium to optimize the cell growth environment. Collect the culture supernatant 48 hours after transfection; (8) Filtration to remove impurities: The collected supernatant was filtered using a 0.45 µm filter membrane to remove cell residues and other impurities, thereby obtaining crude virus solution. This step ensures that unwanted cell debris is removed, leaving only pure liquid containing the virus. (9) Virus concentration: To further increase the virus concentration, centrifugation was performed using a 100 kDa ultrafiltration tube. The centrifuge was set to 3000 rpm and run for 20 minutes to obtain concentrated virus liquid, which is the recombinant virus.

[0026] 2. Screening, succession, and pure line acquisition of gene-edited silkworms The amount of 10 μL of concentrated viral fluid (recombinant virus infected silkworm eggs (fertilized eggs)) is 1 × 10⁻⁶. 7 Simultaneously, 5µg / ml of Polybrene was added and introduced into the fertilized egg using a sperm-mediated method; There are three methods of sperm-mediated induction: Sperm-mediated method 1: After injecting the recombinant virus into the copulatory sac of the virgin moth using a glass needle made from a capillary glass tube, the female moth mates with the male moth. The female moth separates from the male moth 24 hours after mating, allowing the female moth to lay eggs. Sperm-mediated method two: Separate the male and female moths 30 minutes after mating, and then inject recombinant virus into the female moth's copulatory sac to induce egg laying; Sperm-mediated method three: Separate the male and female moths 30 minutes after mating, dissect the female moth, extract the sperm from the copulatory sac, mix the sperm with the recombinant virus, and inject it into the copulatory sac of the virgin moth to induce egg laying; The obtained silkworm eggs were incubated at 25℃ and 85% humidity. After about 14 days, the eggs gradually hatched. The hatching rate was calculated. The silkworm larvae were cultured to the third to fifth instar. The larvae were gently removed and their heads were fixed. The fluorescence microscope was adjusted to excitation light of ~554. At a distance of nm, the eyes of the larvae were observed, and silkworms with red fluorescence in their eyes were selected. The positive rate was calculated, and red-eyed silkworm moths (G0 generation) were collected and crossed with wild-type (WT). The laid silkworm eggs were collected, hatched, and raised to the third to fifth instar. Red-eyed silkworms (G1 generation) were selected by fluorescence microscopy. During the egg-to-green stage of the G1 generation in the transgenic experiment, 10 transgenic silkworm moths expressing the DsRed marker gene were obtained by fluorescence microscopy. These moths were raised to adulthood and crossed with non-transgenic silkworms for generation, which is called G2. From the G2 generation onwards, transgenic silkworms were raised by single moths. During the egg stage, transgenic silkworms expressing the DsRed marker gene were selected by fluorescence stereomicroscopy, raised to adulthood, and mated with moths in the same moth area to make the full-length spider silk gene homozygous, and then the G3 and G4 generations were bred. Starting with the G5 generation, we selected moths from the same moth-producing area to raise, and then bred them by mating them with moths from the same area. This resulted in a new transgenic silkworm variety that is homozygous for red eyes and whose posterior silk gland cells can synthesize and secrete full-length spider silk genes.

[0027] III. Experiments and Conclusions 1. To investigate the effects of different target plasmids on transfection efficiency and the mechanical properties of transgenic silk protein, different target plasmids were selected during the construction of recombinant viruses to prepare concentrated virus liquids 1-18. Using virus liquids 1-18 as materials, and following the screening, subculture, and pure line acquisition methods of gene-edited silkworms described above, new transgenic silkworm varieties 1-18 were obtained.

[0028] Different types of target plasmids are shown in Table 1.

[0029] Table 1. Different types of target plasmids ; Note: The recombinant virus construction in Example 8 differs from that in Example 1 in that: Prepare plasmid mixtures of the two separately: Three plasmids, totaling 2.5 µg, were mixed in a 4:3:1 ratio, i.e., pLC-[3xP3-DsRed]-MaSp2-128×FGAILSS:psPAX2:pMD2G. The mixture was added to 100 µL of serum-free medium and gently mixed to obtain plasmid mixture ①. The virus was constructed according to the recombinant virus construction method to obtain pLC-[3xP3-DsRed]-MaSp2-128×FGAILSS virus concentrate ①. Three plasmids, totaling 2.5 µg, were mixed in a 4:3:1 ratio (Cas9 helper plasmid containing sgRNA1 and sgRNA2 vectors): psPAX2: pMD2G. This mixture was added to 100 µL of serum-free medium and gently mixed to obtain plasmid mixture ②. Following the method for constructing recombinant viruses, the virus was constructed to obtain Cas9 virus concentrate ② containing sgRNA1 and sgRNA2 vectors.

[0030] When screening, subculturing, and obtaining pure lines of gene-edited silkworms, virus concentrate ① and virus concentrate ② are injected separately, as follows: First, inject 5 microliters of concentrated virus solution ② into the mating sac of the virgin moth using a glass needle made from a capillary glass tube. Then, allow the female moth to mate with the male moth. 24 hours after mating, separate the female moth from the male moth and allow the female moth to lay eggs.

[0031] Collect the eggs and incubate the virus-infected silkworm eggs at 25℃ and 85% humidity. After about 14 days, the silkworm eggs gradually hatch. Raise the silkworm larvae to the third to fifth instar, gently remove the larvae and fix their heads, adjust the fluorescence microscope to the excitation light, observe the larvae's body, screen out the silkworms with green fluorescence, and collect the silkworm moths.

[0032] Using collected silkworm moths as material, 5 microliters of concentrated virus solution ① were injected into the mating sac of virgin moths using a glass needle made from capillary glass. Then, the female moth was allowed to mate with the male moth. After 24 hours of mating, the female moth separated from the male moth and allowed the female moth to lay eggs.

[0033] The remaining steps are the same as in Example 1.

[0034] Using the transgenic silkworm silk protein 1 obtained in Example 1 as material, the expression of transgenic silkworm silk protein 1 was analyzed by SDS-PAGE electrophoresis and Western blot technique with full-length spider silk gene antibody. The results showed that specific protein bands consistent with the expected molecular weight were obtained. Figure 1 ).

[0035] The transfection results of Examples 1-18, including the number of silkworm eggs, the number of hatched silkworms (rate%), and the number of G0 silkworms with positive results (rate%), are shown in Table 2.

[0036] Table 2. Statistical analysis of transfection results in the examples ; Table 2 shows that the successful hatching rate of silkworm eggs after introducing the spider silk protein gene MaSp2 into silkworm eggs via lentiviral transfection was 61%–85%, close to that of normal silkworm eggs. This indicates that lentiviral transfection has almost no impact on the hatching rate of silkworm eggs. Furthermore, the positive rate of G0 silkworms was 64%–97%, demonstrating that lentiviral transfection can effectively introduce the target gene into silkworm eggs and achieve successful expression. In addition, embedding different types and lengths of amyloid peptides into the target plasmid helps improve the transfection effect, such as increasing the positive rate and hatching rate.

[0037] Furthermore, the number of hatchings and the positive rate in Example 1 were significantly higher than those in Examples 2 and 3, indicating that the method of introducing the target gene using sperm vectors, namely, "sperm-mediated method 1: injecting the recombinant virus into the mating sac of a virgin moth using a glass needle made of capillary glass, and then allowing it to mate with a male moth to lay eggs and hatch", is more effective than methods 2 and 3.

[0038] Example 8 differs from Example 1 in that the target plasmid and the Cas9 helper plasmid are introduced separately, resulting in a higher positive rate.

[0039] The mechanical properties of the above-mentioned transgenic silkworm silk protein were tested, including strength, Young's modulus and toughness, with ordinary silkworm silk as a control. The results are shown in Table 3.

[0040] Table 3. Test results of mechanical properties of transgenic silkworm silk protein ; As shown in Table 3, compared with ordinary silkworm silk, the mechanical properties of transgenic silkworm silk protein are significantly improved, with a strength of 312~1534 MPa, an elastic modulus of 4935~11045 MPa, and a toughness of 58~369 MJ·m. -3The results of Examples 1-15 compared with those of Example 16 show that embedding amyloid peptides into the target plasmid can improve the mechanical properties of transgenic silkworm silk protein. Moreover, for the same type of amyloid peptide, the mechanical properties of transgenic silkworm silk protein are better as the number of repeating sequences of the amyloid peptide increases.

[0041] 2. To investigate the effects of different virus inoculation methods on transfection efficiency and the mechanical properties of transgenic silkworm silk protein, the following comparative examples were set up: Comparative Example 1 The difference from Example 1 is that the concentrated virus solution pLC-[3xP3-DsRed]-MaSp2-4×FGAILSS obtained in Example 1 (the amount of recombinant virus infected silkworm eggs is 1×10) 7 The drug was injected into fertilized eggs within 6 hours after silkworm laying using a microinjection method, with a total injection volume of 10 microliters.

[0042] Comparative Example 2 The difference from Example 1 is in the construction step (3) of the recombinant virus: pLC-[3xP3-DsRed]-MaSp2-4×FGAILSS plasmid containing spider silk gene is mixed with Cas9 helper plasmid containing sgRNA1 and sgRNA2 vectors at a ratio of 1:1. The total concentration of the two plasmids is 0.4 μg / μl. The plasmid is dissolved in 0.5mM phosphate buffer at pH=7.

[0043] The remaining steps are the same as in Example 1.

[0044] Comparative Example 3 Similar to Comparative Example 2, the method of introducing foreign genes was changed to: Sperm-mediated method 2: A total volume of 10 microliters was injected into the copulatory sac of the virgin moth using a glass needle made of capillary glass, and then the female moth was allowed to mate with the male moth. 24 hours after mating, the female moth and the male moth separated, and the female moth laid eggs.

[0045] Comparative Example 4 Similar to Comparative Example 2, the method of introducing exogenous genes was changed to: Sperm-mediated method 3: The male and female moths were separated 30 minutes after mating, the female moth was dissected, the sperm in the copulatory sac was taken out, mixed with 5 microliters of concentrated virus solution and injected into the copulatory sac of the virgin moth, so that it could lay eggs and hatch.

[0046] Comparative Example 5 The experimental steps were the same as in Example 1, except that the target plasmid for preparing the concentrated virus solution was changed to pLC-[3xP3-DsRed]-MaSp2.

[0047] Comparative Example 6 The experimental steps were the same as in Example 1, except that the target plasmid for preparing the concentrated virus solution was changed to pLC-[3xP3-DsRed]-4×FGAILSS.

[0048] Comparative Example 7 The experimental steps were the same as in Example 1, except that the target plasmid for preparing the concentrated virus solution was changed to pLC-[3xP3-DsRed]-4×GDVIEV.

[0049] Comparative Example 8 The experimental steps were the same as in Example 1, except that the target plasmid for preparing the concentrated virus solution was changed to pLC-[3xP3-DsRed]-4×KLVFFAE.

[0050] Comparative Example 9 The experimental steps were the same as in Example 2, except that the target plasmid for preparing the concentrated virus solution was changed to pLC-[3xP3-DsRed]-S-128×FGAILSS, where S represents the following amino acid sequence:

[0051] Comparative Example 10 The experimental steps were the same as in Example 2, except that the target plasmid for preparing the concentrated virus solution was changed to pLC-[3xP3-DsRed]-MaSp2.

[0052] Comparative Example 11 The experimental steps were the same as in Example 3, except that the target plasmid for preparing the concentrated virus solution was changed to pLC-[3xP3-DsRed]-MaSp2.

[0053] The statistical comparison results of transfection, including the number of silkworm eggs, the number of hatched silkworms (rate%), and the number of G0 silkworms with positive results (rate%), are shown in Table 4.

[0054] Table 4. Transfection results of the comparative example ; As can be seen from Table 4, A comparison of the results of Comparative Example 1 and Example 1 shows that microinjection significantly reduced the hatching rate of silkworm eggs. Because silkworm eggs have a hard shell, the internal pressure caused by the injection during shell breaking often leads to the outflow of egg contents and embryonic death. Furthermore, due to the limitations of microinjection technology, the positive rate of G0 silkworms is very low, only 5.6%. Lentiviral transfection can greatly improve the success rate of transgenic technology.

[0055] Comparative Examples 2-4 were not transfected with lentivirus, and their hatching rate and positive rate were lower than those of Example 1. It can be seen that the lentivirus transfection method is more effective.

[0056] The above-mentioned new transgenic silkworm varieties were raised using conventional silkworm rearing methods, and transgenic silkworm silk protein was obtained. The mechanical properties of the transgenic silk protein, including strength, Young's modulus and toughness, were tested, and the results are shown in Table 5.

[0057] Table 5. Test results of mechanical properties of transgenic silkworm silk protein ; As can be seen from Tables 3 and 5, compared with Comparative Example 1, due to the different expression vectors constructed, the virus-packaged plasmid vector can better express spider silk protein, and the mechanical properties of the transgenic silk are better than those of the microinjection method.

[0058] Compared to the examples, the spider silk protein MaSP2 expressed in Comparative Example 2 was only a partial fragment, resulting in a significant reduction in the molecular weight of the silk fibers and consequently a decrease in mechanical properties. All examples expressed the full-length spider silk protein MaSP2, therefore the mechanical properties of the silk fibers were significantly better than those in Comparative Example 2. Furthermore, due to the introduction of the repetitive FGAILSS sequence, the mechanical properties of the silk fibers from the examples were significantly better than those of Comparative Examples 5, 10, and 11.

[0059] The above research results demonstrate that the spider silk protein MaSP2×Amyloids gene has been inserted into the transgenic silkworm and can synthesize and secrete spider silk protein in the posterior silk gland cells. This protein can enter the cocoon during the silk spinning and cocooning behavior. This trait has been stably inherited and expressed, and the mechanical properties of the transgenic silkworm silk have been significantly improved.

[0060] 4. Secondary Structure Analysis The specific steps are as follows: The silkworm cocoons obtained in the examples and comparative examples were weighed and chopped, then added to deionized water at a mass-to-volume ratio of 1:100. The mixture was degummed at 125℃ for 70 min to obtain transgenic silkworm fibroin protein, which was then dried. The protein was prepared into a circular dichroism (CD) sample with a final concentration of 0.2 mg / mL. 200 μL of the sample was placed in a 300 μL quartz glass tube with a light path of 0.1 cm, and analyzed by CD at 20℃. The parameters were set as follows: wavelength step 0.5 nm, response time 1 s, bandwidth 1 nm, and scanning range 190–260 nm. To detect protein folding, the molar residual ellipticity (MRE) value at 222 nm was used to calculate the protein folding ratio. The MRE value at 222 nm recorded by the instrument in real time was set as CDO, CDN in the native state, and CDD when completely denatured. The formula for calculating protein folding rate is: Protein folding rate (%) = (CDO - CDD) / (CDN - CDD).

[0061] All the above sample spectra are averages of three consecutive scans. Blank and control samples were also tested under the same conditions and were subtracted from all spectra. Origin 8.0 software was used to generate the curves, and CDPro and CDSSTR software were used to estimate the content of each secondary structure of spider silk protein under different conditions, with the mode selected as set 4 with 43 soluble proteins. The control samples were natural spider silk MaSP2 and natural silkworm silk.

[0062] The results are shown in Table 6.

[0063] Table 6. Results of Secondary Structure Analysis ; ; As shown in Table 6, the secondary structure analysis of transgenic silkworm silk proteins by circular dichroism (CD) chromatography revealed that, although the number of repeating units varied, the CD CD chromatograms of different transgenic silkworm silk proteins were basically consistent, exhibiting typical α-helix structural features (positive peak at 192 nm, negative peaks at 208 nm and 222 nm) and β-sheet structural features (1605–1639 cm⁻¹). -1 1690~1695 cm -1(Representing β-sheet). Combined with the mechanical property test results, it can be seen that the higher the proportion of each of the β-sheet, which provides strength, and the α-helix structure, which provides elasticity, the better the mechanical properties of the transgenic silkworm silk protein, and it can have both high strength and high toughness. A high proportion of β-sheet alone cannot produce transgenic silkworm silk protein with high toughness.

[0064] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A method for producing transgenic spider silk protein based on lentiviral transfection combined with sperm-mediated synthesis, characterized in that: Includes the following steps: (1) The pLC-[3xP3-DsRed]-MaSp2-Amyloids plasmid was constructed using molecular biology methods and used as a gene vector for expressing spider silk proteins in the silk glands of silkworms; (2) The pLC-[3xP3-DsRed]-MaSp2-Amyloids plasmid was transfected into cells using a lentiviral vector transfection-mediated gene delivery method. The transfected cells were cultured, filtered, and centrifuged to concentrate the recombinant virus. (3) The recombinant virus was introduced into the fertilized eggs of silkworms via sperm-mediated infection; (4) After being infected with recombinant virus, silkworm fertilized eggs were hatched and mated for multiple generations to produce transgenic silkworms with homozygous red-eye gene and spider silk protein gene and silk gland cells capable of synthesizing and secreting spider silk protein. (5) Spider silk protein is synthesized and secreted by the silk gland cells of the transgenic silkworm and enters the cocoon along with the silkworm spinning and cocooning behavior of the transgenic silkworm. The silk is collected to obtain the composite silk containing spider silk protein based on silkworm silk.

2. The method for producing spider silk protein transgenic products based on lentivirus transfection combined with sperm-mediated transgenesis according to claim 1, characterized in that, In the pLC-[3xP3-DsRed]-MaSp2-Amyloids plasmid, the sequence of the spider silk protein MaSp2 gene is modified with amyloids containing 4-128 repeating sequences.

3. The method for producing spider silk protein transgenic products based on lentivirus transfection combined with sperm-mediated transgenesis according to claim 2, characterized in that, The amyloid peptides include any one of GDVIEV, KLVFFAE, and FGAILSS.

4. The method for producing spider silk protein transgenic products based on lentivirus transfection combined with sperm-mediated transgenesis according to claim 1, characterized in that, Step (2) specifically involves: (2-1) Prepare plasmid mixture: mix pLC-[3xP3-DsRed]-MaSp2 plasmid and Cas9 helper plasmid containing sgRNA1 and sgRNA2 vectors with psPAX2 plasmid and pMD2.G plasmid in a ratio of 4:3:1, add to 100 μL serum-free culture medium, and mix to obtain plasmid mixture; (2-2) Incubate the plasmid mixture with the transfection reagent, then add it to the culture medium containing cells for transfection. After successful transfection, collect the culture supernatant, take the cell culture supernatant and inoculate the cultured cells again, then culture the cells again, collect the cell culture supernatant, filter the collected supernatant to remove cell residues and impurities, and obtain crude virus solution. Use an ultrafiltration tube to centrifuge and concentrate the crude virus solution to obtain recombinant virus.

5. The method for producing spider silk protein transgenic material based on lentivirus transfection combined with sperm-mediated transgenesis according to claim 1, characterized in that, The amount of recombinant virus that infects fertilized eggs is 10. 4 -10 8 Copy / fertilized egg.

6. The method for producing spider silk protein transgenic products based on lentivirus transfection combined with sperm-mediated transgenesis according to claim 1, characterized in that, In step (3), the method of introducing the recombinant virus into the fertilized egg through sperm-mediated delivery is any one of the following: Method 1: Inject the recombinant virus into the mating sac of a virgin moth using a glass needle made from capillary glass, and then allow it to mate with a male moth to lay eggs and hatch. Method 2: Separate the male and female moths 30 minutes after mating, and then inject recombinant virus into the female moth's mating sac to induce egg laying and hatching; Method 3: Separate the male and female moths 30 minutes after mating, dissect the female moth, extract the sperm from the copulatory sac, mix the sperm with the recombinant virus, and inject it into the copulatory sac of the virgin moth to induce egg laying and hatching.

7. The method for producing spider silk protein transgenic products based on lentivirus transfection combined with sperm-mediated transgenesis according to claim 1, characterized in that, Step (4) specifically involves: (4-1) After the silkworm fertilized eggs infected with recombinant virus are hatched and raised to adulthood, they are mated with non-transgenic silkworms to produce the G1 generation. During the greening period of the G1 generation silkworm eggs, transgenic silkworms expressing the red fluorescent DsRed marker gene in one eye are screened out. After being raised to adulthood, they are mated with non-transgenic silkworms to produce the G2 generation. (4-2) In the G2 generation, silkworms that express the red fluorescent DsRed marker gene under a fluorescence stereomicroscope were screened out, and the G3 generation was produced by crossbreeding silkworms from the same moth area; (4-3) The G3 generation silkworms were raised by single moths, and the silkworm moths expressing the red fluorescent DsRed marker gene in the same moth area were crossbred to produce the G4 generation; (4-4) Starting from the G4 generation and for three consecutive generations, the same methods of raising moths in moth-producing areas with a pure red-eye phenotype, raising single moths, and mating with silkworm moths in the same moth-producing area were used to select and mate them, so as to breed transgenic silkworms with homozygous red-eye gene and spider silk protein gene and whose silk gland cells can synthesize and secrete spider silk protein.

8. A spider silk protein composite filament prepared by the method according to any one of claims 1-6.

9. A spider silk protein composite filament according to claim 7, characterized in that, The spider silk composite filament has a strength of 312~1534 MPa, an elastic modulus of 4935~11045 MPa, and a toughness of 58~369 MJ·m. -3 .

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