Expression method of non-proteolytic thrombin peptide TP508 in bombyx mori silk gland, and product and application thereof

Abstract

The invention discloses an expression method of a non-proteolytic thrombin peptide TP508 in bombyx mori silk gland and a product and application thereof, a fusion expression vector of the non-proteolytic thrombin peptide TP508 and a red fluorescent protein DsRED is constructed, the fusion protein is expressed in silkworm cocoon silk, the bred transgenic strain silk successfully expresses the fusion protein, and the non-proteolytic thrombin peptide TP508 is obtained. The thrombin peptide TP508 fusion protein expressed by the transgenic silk not only can remarkably promote cell proliferation, cell migration and vascularization, but also keeps the characteristic of red fluorescence, and is expected to be applied to the fields of biomedicine and tissue cell engineering.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to an expression method of non-proteolytic thrombin peptide TP508 in silkworm silk glands, and a product and application thereof. Background Art

[0002] TP508 is a synthetic peptide composed of 23 amino acids, representing the sequence of amino acids 508 to 530 of human prothrombin. It was originally found to compete with thrombin for binding to high-affinity thrombin receptors on fibroblasts and produce mitogenic signals that depend on receptor occupancy. However, TP508 itself does not have thrombin activity. Studies have shown that TP508 can activate endothelial NO synthase (eNOS) and stimulate the production of NO in human endothelial cells. TP508 activates endothelial cells and stem cells to regenerate blood vessels and tissues. TP508 activates cells to promote cell proliferation, blood vessel regeneration and tissue regeneration, promotes tissue repair and accelerates wound healing. It can also reduce radiation-induced gastrointestinal damage by activating stem cells and maintaining crypt integrity. As a synthetic peptide with multiple biological functions, TP508 shows great potential in tissue repair, inflammation regulation, angiogenesis and reducing radiation damage. It accelerates tissue regeneration and repair by activating a series of cellular events and signaling pathways.

[0003] Silk is the earliest and most widely used natural animal protein fiber by humans. With the release of the silkworm genome map and the establishment of the silkworm molecular breeding technology system, the molecular and technical foundation for the genetic improvement of silk and material innovation research has been laid. In recent years, the performance of silk and the genetic manipulation of silkworms have become a hot topic in the study of silk protein. In 2003, Japanese scholars established a silkworm transgenic technology system with piggyBac transposon as the core, and transgenic overexpression technology has become a method that can efficiently transform the silk gland and silk performance of silkworms. Unlike gene editing technology, transgenic overexpression technology only recombines and expresses functional exogenous target proteins in specific tissues of silkworms. It is relatively mild and is not easy to have a significant impact on the constitution of silkworms themselves and the characteristics of silk. With the help of these genetic operating systems, researchers have successfully transformed the performance of silk, endowed silk with new biological functions, and expanded the application field of silk. Therefore, using the silk gland transgenic incremental expression system to express TP508 fusion protein is of great significance for obtaining multifunctional silk. Summary of the invention

[0004] In view of this, one of the objects of the present invention is to provide a method for expressing non-proteolytic thrombin peptide TP508 in the silk gland of silkworm, a second object of the present invention is to provide a silkworm strain overexpressing non-proteolytic thrombin peptide TP508 fusion protein obtained by the method, a third object of the present invention is to provide a thrombin peptide TP508 fusion protein prepared by the silkworm strain, and a fourth object of the present invention is to provide the application of the non-proteolytic thrombin peptide TP508 fusion protein in promoting cell proliferation, migration and angiogenesis.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: 1. A method for expressing the non-proteolytic thrombin peptide TP508 in the silk gland of silkworms, by overexpressing a fusion gene of the non-proteolytic thrombin peptide TP508 and the red fluorescent protein DsRed in silkworms to obtain transgenic positive individuals, and then extracting the non-proteolytic thrombin peptide TP508 fusion protein from silk.

[0006] In some embodiments of the present invention, the amino acid sequence encoded by the fusion gene of the non-proteolytic thrombin peptide TP508 and the red fluorescent protein DsRed is shown in SEQ ID NO.2.

[0007] In some embodiments of the present invention, the nucleotide sequence of the fusion gene of the non-proteolytic thrombin peptide TP508 and the red fluorescent protein DsRed is shown in SEQ ID NO.1.

[0008] In some embodiments of the present invention, the overexpression method is to construct a transgenic vector containing a fusion gene of the non-proteolytic thrombin peptide TP508 and the red fluorescent protein DsRed, and then inject the transgenic vector into silkworms, raise the G0 generation, mate and lay eggs, and fluorescently screen the G1 generation silkworm eggs to obtain transgenic positive individuals.

[0009] In some embodiments of the present invention, the transgenic vector is a Piggybac vector.

[0010] In some embodiments of the present invention, the transgenic vector further comprises a promoter, an Hr3CQ enhancer and a Ser1PA terminator of the Fib-h gene.

[0011] 2. A silkworm strain overexpressing the non-proteolytic thrombin peptide TP508 fusion protein obtained by the method.

[0012] 3. The non-proteolytic thrombin peptide TP508 fusion protein prepared by the silkworm strain.

[0013] 4. Application of the non-proteolytic thrombin peptide TP508 fusion protein in promoting cell proliferation, migration and angiogenesis.

[0014] The beneficial effects of the present invention are as follows: the present invention breeds a silkworm strain that overexpresses TP508 fusion protein in silkworm cocoons by overexpressing TP508 and DsRED fusion genes through transgenic overexpression; for the first time, the simultaneous expression of fluorescent protein and functional peptide is achieved, and the bred strain has the following characteristics: 1) For the first time, the fluorescent properties of silk were retained without affecting the TP508 functional peptide; 2) Overexpression of non-proteolytic thrombin peptide TP508 fusion protein promotes cell proliferation and migration; 3) Overexpression of the non-proteolytic thrombin peptide TP508 fusion protein has pro-angiogenic and fluorescent properties and is expected to be applied in the fields of biomedicine and tissue cell engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the present invention provides the following drawings for illustration: Figure 1 The diagram for obtaining and testing transgenic lines (A. Schematic diagram of the TP508 fusion gene transgenic overexpression vector; B. Screening of positive individuals; 3xp3 is the eye-specific promoter; DsRed is the red fluorescent protein; SV40 is the terminator; hr3CQ is the enhancer, Fib-H is the gene promoter; Ser1PA is the terminator).

[0016] Figure 2 Phenotypic observation of transgenic positive individuals (A. The silkworm individuals appear pink; B. The cocoons also appear pink; C. After dissecting the positive silkworm individuals, it was found that the entire silk gland emitted bright red fluorescence).

[0017] Figure 3 Purification of TP508 fusion protein (A. The process of purifying the target protein from silkworm cocoons. The silkworm cocoons are pink, the sericin solution after degumming is also red, and the protein solution collected after purification is still pink; B. SDS-PAGE detection of TP508 fusion protein in silkworm cocoons; C. TP508 fusion protein was successfully purified from silkworm cocoons).

[0018] Figure 4 The effect of TP508 fusion protein on the proliferation of NIH / 3T3 cells was detected (A. CCK8 detection found that 50μg / ml TP508 had a significant proliferation effect on mouse NIH / 3T3 cells; B. Live-Dead staining results also showed that the number of mouse NIH / 3T3 cells increased significantly after adding 50μg / ml TP508).

[0019] Figure 5 To detect the effect of TP508 fusion protein on HUVEC cell tube formation (A. TP508 stimulation of HUVEC cells can promote cell tube formation; B. Statistical analysis of branch points, junctions, meshes and total length of blood vessels in the vascular network, significant difference analysis: *p < 0.05, ** p < 0.01, ***p < 0.001).

[0020] Figure 6 Detection of the effect of TP508 fusion protein on HUVEC cell migration (A. TP508 stimulation of HUVEC cells can promote cell migration; B. The relative migration distance of cells was statistically analyzed after 12 hours of TP508 stimulation).

[0021] Figure 7 This is the detection of TP508 fusion protein on LPS-induced macrophage inflammatory response (TP508 stimulates the upregulation of NO and three pro-inflammatory factors TNF-α, IL-6, and IL-1β in macrophages). DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0023] Example 1 We synthesized the full-length TP508 and DsRED fusion gene (SEQ ID No. 1~2) by gene synthesis. After successful sequencing, we used a seamless cloning kit to connect it and successfully cloned it into the transition vector containing the promoter (Fib-h) of the Fib-h gene specifically expressed in the posterior silk gland of the silkworm, the Hr3CQ enhancer and the Ser1PA terminator, and obtained pSL1180 [Hr3CQ Fib-hTP508 Ser1PA]. The expression frame after the transition vector was cut with AscI single enzyme was connected with the dephosphorylated silkworm transgenic basic expression vector piggyBac [3xp3 DsRed SV40] to obtain the final transgenic expression vector piggyBac [3xp3 DsRed SV40; Hr3 Fib-h TP508 Ser1PA], named Over-TP508 ( Figure 1 Middle A).

[0024] The constructed transgenic vector was extracted and ultrapure plasmid was injected into silkworms. After the G0 generation was raised, the moths mated and laid eggs, and the G1 generation silkworm eggs were further screened by fluorescence to successfully obtain Over-TP508 transgenic positive individuals ( Figure 1B), hereinafter named Over-TP508 strain. The transgenic silkworms were reared and found that compared with the control, the silk glands and cocoons of the positive silkworms were pink ( Figure 2 ).

[0025] Example 2 Silkworm cocoon SDS-PAGE showed that we successfully overexpressed TP508 fusion protein ( Figure 3 , B), the cocoons were crushed into powder using a grinder and degummed with 8M urea. The degummed solution was treated and purified using a nickel column. ( Figure 3 , A), and then the purified protein was stained with Coomassie Brilliant Blue and analyzed by Western Blot. The results showed that the best elution effect was obtained under 250 mM imidazole ( Figure 3 , C), the purified protein also has fluorescent properties ( Figure 3 , A), indicating that the purification method can successfully isolate TP508 fusion protein (TP508-RED) from silkworm cocoons with high purity and specificity.

[0026] The CCK-8 kit was further used to resuscitate, culture, passage, plate, starve and add protein to mouse embryonic fibroblasts NIH / 3T3, with different concentrations of TP508-RED protein as the experimental group, BSA as the control group, and untreated cells as the blank control group. Protein was added and finally observed under a microplate reader. Figure 4 , A shows that different concentrations of TP508-RED can promote the adhesion and proliferation of NIH / 3T3 cells, and the effect is significant at a concentration of 50μg / mL, with the best effect. We used 50μg / mL of TP508-RED for Live-Dead staining and observed the cells under green, red and white light under a fluorescence microscope. Figure 4 As can be seen from Figure B, the Live-Dead stain is not cytotoxic, that is, no dead cells were found under red light. It can also be seen in the figure that the number of live cells treated with TP508-RED protein is significantly higher than that in the BSA group and the control group, so this further confirms that TP508-RED has the effect of promoting cell proliferation.

[0027] Example 3 In order to verify that TP508-RED protein has the function of promoting cell tube formation, different concentrations of TP508-RED protein were used as the experimental group, BSA was used as the control group, and untreated cells were used as the blank control group. Human umbilical vein endothelial cells (HUVEC) were revived, cultured, passaged, starved, and plated in six-well plates. Protein was added, and then the cells in different treatment groups in the six-well plates were digested, resuspended, and plated in 96-well plates pre-coated with matrix gel. Finally, the cell tube formation was observed under a microscope. Figure 5, A shows that different concentrations of TP508-RED can promote HUVEC cell tube formation, and at a concentration of 50 μg / mL, the number of branch points, junctions, meshes and total length of blood vessels in the vascular network formed by cells are more significant ( Figure 5 , B).

[0028] At the same time, we used different concentrations of TP508-RED protein as the experimental group, BSA as the control group, and untreated cells as the blank control group. Human umbilical vein endothelial cells HUVEC were revived, cultured, passaged, starved, and plated in six-well plates pre-treated with different concentrations of TP508-RED. Finally, the migration of HUVEC cells was observed under a microscope. Figure 6 , A and Figure 6 As can be seen from B, compared with the BSA group and the control group, different concentrations of TP508-RED treatment groups can promote HUVEC cell migration.

[0029] In addition, studies have shown that TP508 increases NO production by activating nitric oxide synthase (eNOS), a mechanism that is essential for maintaining endothelial function and promoting angiogenesis. The NO detection kit was used to detect the NO content in the culture medium with TP508-RED, BSA bovine serum albumin (negative control), and no protein solution. The results showed that the NO production increased ( Figure 7 ). Other studies have shown that TP508 can induce cells to express a variety of cytokines, which play an important role in early immune responses, promoting the recruitment and activation of inflammatory cells, thereby initiating tissue repair and angiogenesis. Therefore, we also tested the inflammatory response of TP508-RED protein to LPS-induced macrophages, and the expression of three pro-inflammatory factors TNF-α, IL-6, and IL-1β were all upregulated ( Figure 7 ). These results indicate that TP508-RED can promote angiogenesis by promoting NO and inducing upregulation of inflammatory factors.

[0030] The above-described embodiments are only preferred embodiments for fully illustrating the present invention, and the protection scope of the present invention is not limited thereto. Equivalent substitutions or changes made by those skilled in the art based on the present invention are within the protection scope of the present invention. The protection scope of the present invention shall be subject to the claims.

Claims

1. A method for expressing the non-proteolytic thrombin peptide TP508 in the silk gland of silkworm, characterized in that: The non-proteolytic thrombin peptide TP508 and the red fluorescent protein DsRed fusion gene were overexpressed in silkworms to obtain transgenic positive individuals, and then the non-proteolytic thrombin peptide TP508 fusion protein was extracted from silk.

2. The method according to claim 1, characterized in that The amino acid sequence encoded by the fusion gene of the non-proteolytic thrombin peptide TP508 and the red fluorescent protein DsRed is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that The nucleotide sequence of the fusion gene of the non-proteolytic thrombin peptide TP508 and the red fluorescent protein DsRed is shown in SEQ ID NO.

1.

4. The method according to claim 5, characterized in that The overexpression method is to construct a transgenic vector containing a fusion gene of the non-proteolytic thrombin peptide TP508 and the red fluorescent protein DsRed, then inject the transgenic vector into silkworms, raise the G0 generation, mate and lay eggs, and fluorescently screen the G1 generation silkworm eggs to obtain transgenic positive individuals.

5. The method according to claim 4, characterized in that The transgenic vector is a Piggybac vector.

6. The method according to claim 5, characterized in that The transgenic vector also contains the promoter, Hr3CQ enhancer and Ser1PA terminator of the Fib-h gene.

7. A silkworm strain overexpressing a non-proteolytic thrombin peptide TP508 fusion protein obtained by the method according to any one of claims 1 to 6.

8. A non-proteolytic thrombin peptide TP508 fusion protein prepared by the silkworm strain of claim 7.

9. Use of the non-proteolytic thrombin peptide TP508 fusion protein according to claim 8 in promoting cell proliferation, migration and angiogenesis.

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