Recombinant vector for expressing human neurotrophic factor NT-3 by bombyx mori silk gland as well as preparation and application of recombinant vector

Abstract

The invention relates to the field of gene engineering, in particular to a recombinant vector of a bombyx mori silk gland expression human neurotrophic factor NT-3 as well as preparation and application of the recombinant vector. An optimized gene is constructed in a bombyx mori silk gland expression vector pBac [DsRed] plasmid by modifying a human-derived neurotrophic factor NT-3 gene, such as a sequence shown in SEQ ID NO 2, so that a pBac [DsRed, NT-3] recombinant plasmid is obtained, the recombinant plasmid and an auxiliary plasmid are mixed and microinjected into fresh silkworm eggs which are just laid by bombyx mori within 2 hours, a G0 generation is obtained, then a G1 generation is copulated and bred, and the G0 generation and the G1 generation are subjected to separation and purification. And screening out silkworms expressing the human neurotrophic factor NT-3 from the G1 generation. The optimized human neurotrophic factor NT-3 gene vector expressed in bombyx mori is constructed, the human neurotrophic factor NT-3 protein hydrogel is obtained, and the human neurotrophic factor NT-3 has potential application value in cell proliferation, cell migration and repair.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and relates to a recombinant vector for expressing human neurotrophic factor NT-3 in silkworm silk gland, and the preparation and application thereof. Background Art

[0002] Silkworms secrete silk protein through their silk glands to form silk fibers. This natural material is widely used in textiles, medicine, and bioengineering. Silkworms can produce a large amount of protein and have a short growth cycle, which is suitable for large-scale breeding. Silk has excellent biocompatibility, biodegradability, and mechanical properties, making it suitable for drug delivery and tissue engineering. The silk gland environment helps the correct folding and post-modification of target proteins, thereby improving the biological activity of proteins. The research on silkworm silk gland bioreactors aims to use the ability of silk glands to efficiently synthesize silk protein to express other exogenous proteins, expand the function of silk protein, and explore the application of silk protein in new fields. The silk gland bioreactor of silkworms can efficiently produce exogenous proteins in a controlled environment. By regulating environmental conditions (such as temperature, humidity, nutrients, etc.), the synthesis of silk protein and exogenous proteins can be optimized.

[0003] Through transgenic technology, the silk glands of silkworms can be transformed, and exogenous target genes can be introduced into the silk gland cells of silkworms, so that they can synthesize the required proteins in the silk glands. The target proteins are then harvested through the silkworm spinning process, while the yield and performance of silk protein are enhanced, thereby promoting the development of the silk industry. The extracted silk protein can be used in many fields such as medical treatment (such as sutures, stents), biomaterials (such as coatings, drug release carriers), etc., and has broad market prospects. Compared with synthetic materials, silkworm silk protein is of natural origin, has strong biodegradability, and is more in line with the concept of sustainable development.

[0004] Neurotrophic factors are a class of proteins that are essential for the survival, development and function of nerve cells. These factors play a key role in the function of the nervous system by promoting the growth, differentiation and repair of neurons. Among them, the neurotrophic factor NT-3 has been widely studied, and NT-3 plays an important role in neural development, survival and differentiation. NT-3 has the effect of promoting growth and repair of various neurons, especially in sensory neurons and motor neurons. During the embryonic development stage, NT-3 is essential for the development of the nervous system, helping neurons to connect correctly and form synapses. After nerve damage, NT-3 helps promote the regeneration and repair of damaged nerves. NT-3 can participate in the regulation of synaptic plasticity and is closely related to cognitive functions such as learning and memory. Comprehensive existing studies have shown that NT-3 may have potential application value in the treatment of neurodegenerative diseases, nerve damage and nerve regeneration. However, the current acquisition of NT-3 protein is very limited. The present invention can use the silkworm silk gland bioreactor to produce highly active NT-3 protein on a large scale, providing a basis for its wide application in neurobiology. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a recombinant vector for constructing silkworm silk gland to express human neurotrophic factor NT-3; to prepare a protein containing human neurotrophic factor NT-3 through silkworm silk gland expression, and to prepare a silk hydrogel using the protein; the silk hydrogel material containing human neurotrophic factor NT-3 protein can promote the proliferation and repair activity of nerve cells, and has a wide range of medical and non-medical uses.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a recombinant vector for expressing human neurotrophic factor NT-3 in silkworm silk glands. The recombinant vector is an optimized human neurotrophic factor NT-3 gene, and its sequence is shown in SEQ ID NO: 2. The recombinant vector also includes silkworm enhancer Hr3, promoter Ser1P for specifically expressing Ser1 gene in the middle silk gland of silkworm, and terminator Ser1PA for specifically expressing Ser1 gene in the middle silk gland of silkworm;

[0008] The recombinant vector for expressing human neurotrophic factor NT-3 in silkworm silk gland, the nucleotide sequence of the promoter Ser1P is shown in SEQ ID NO: 5, the nucleotide sequence of the enhancer Hr3 is shown in SEQ ID NO: 6, and the nucleotide sequence of the terminator Ser1PA is shown in SEQ ID NO: 4;

[0009] The amino acid sequence of the human neurotrophic factor NT-3 protein with a His tag expressed by the recombinant vector is shown in SEQ ID NO:3.

[0010] Furthermore, the preparation method of the recombinant vector expressing human NT-3 in the silkworm silk gland is as follows:

[0011] S1: The synthesized optimized human NT-3 gene was double-digested with NotI and BamHI, and the basic vector pSL1180 plasmid was cut with these two enzymes at the same time. After ligation, the positive clone plasmid was extracted to obtain the pSL1180[NT-3] vector;

[0012] S2: The constructed pSL1180[NT-3] and pBac[DsRed] plasmids were digested with AscI, respectively, the digestion products were recovered and ligated, and the positive plasmid extracted after ligation was the recombinant vector pBac[DsRed, NT-3] vector of the human neurotrophic factor NT-3;

[0013] Furthermore, the silk gland of the silkworm expresses the silk protein of the recombinant vector of human neurotrophic factor NT-3;

[0014] Further, a hydrogel containing human neurotrophic factor NT-3 protein;

[0015] Furthermore, the application of hydrogel containing human neurotrophic factor NT-3 protein in the preparation of products promoting the proliferation and repair of nerve cells.

[0016] The beneficial effects of the present invention are:

[0017] Based on the codon preference of endogenous genes expressed in the silk gland in the silkworm genome sequence database, the present invention optimizes the sequence of the human neurotrophic factor NT-3 gene, thereby enhancing the expression efficiency of the modified NT-3 gene in the silk gland of the silkworm, and obtaining an NT-3 protein with a structure and activity close to that of the human source. This method can be used for large-scale production of human NT-3 protein, promote the application of NT-3 protein in the fields of neuroscience and medicine, and has broad prospects for medical application.

[0018] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0020] Figure 1The structure diagram of the transgenic vector pBac[DsRed, NT-3] for the successful construction of silkworm silk gland expressing human neurotrophic factor NT-3;

[0021] Figure 2 This is a fluorescence screening image of silkworm eggs expressing human neurotrophic factor NT-3 in transgenic silkworms;

[0022] Figure 3 This is the immunohistochemical image of the silk gland section of the fifth instar three-day-old silkworm (WT is the silk gland of the wild-type silkworm; NT-3 is the silk gland of the transgenic silkworm; immunohistochemistry was detected using NT-3 antibody);

[0023] Figure 4 The analysis diagram of the expression detection of human NT-3 protein in silk cocoons (A is SDS-PAGE electrophoresis diagram; B is Western blot diagram; Marker is protein size; WT is the control sample; NT-3 is the transgenic silkworm sample; BSA is the protein standard sample; anti-NT-3 is the NT-3 antibody detection);

[0024] Figure 5 The toxicity test diagram of the prepared human NT-3 silk hydrogel on cell culture (Control is the PBS treatment group; WT is the normal silk cocoon hydrogel group; NT-3 is the transgenic silk cocoon hydrogel group; the cytotoxicity is detected by LIVE / DEAD cell activity staining);

[0025] Figure 6 This is a diagram showing that human NT-3 silk hydrogel promotes the proliferation of mouse hippocampal neuron cells HT22 (A is CCK-8 detection of cell proliferation; B is EdU cell staining detection; Control is the PBS treatment group; WT is the normal silkworm cocoon hydrogel group; NT-3 is the transgenic silkworm cocoon hydrogel group; NT-3Std is the standard NT-3 protein sample group);

[0026] Figure 7 This is a diagram of the human NT-3 silk gel hydrogel promoting the migration of mouse hippocampal neuron cells HT22 cells (A is the observation of the cell scratch experiment at 0h and 24h; B is the statistical migration rate of the cell scratch experiment data; Control is the PBS treatment group; WT is the normal silk cocoon hydrogel group; NT-3 is the transgenic silk cocoon hydrogel group; NT-3Std is the standard NT-3 protein sample group);

[0027] Figure 8 This is a test to detect the effect of human NT-3 silk worm sericin hydrogel on the differentiation of mouse hippocampal neuron HT22 cells (Control is the PBS treatment group; WT is the normal silkworm cocoon hydrogel group; NT-3 is the transgenic silkworm cocoon hydrogel group). DETAILED DESCRIPTION

[0028] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0029] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0030] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0031] Example 1 Construction of a transgenic vector expressing human NT-3 protein in the middle silk gland of silkworm

[0032] The human neurotrophic factor NT-3 gene sequence (accession number: NM_002527) was downloaded from NCBI to obtain the sequence shown in SEQ ID NO: 1. The NT-3 gene coding sequence was optimized and designed according to the preference of silkworm codon usage to obtain the sequence shown in SEQ ID NO: 2, and the optimized NT-3 gene sequence was synthesized by the company. The amino acid sequences of the human NT-3 protein and His tag protein encoded by the optimized NT-3 gene are shown in SEQ ID NO: 3. The synthesized NT-3 gene was double-digested with NotI and BamHI, and the basic vector pSL1180 plasmid was cut with these two enzymes at the same time. After connection, the positive clone plasmid was extracted and named pSL1180 [NT-3] vector. The constructed pSL1180[NT-3] and pBac[DsRed] plasmids (see Wang, F., Xu, H., Yuan, L. et al. An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms. Transgenic Res 22, 925–938 (2013). https: / / doi.org / 10.1007 / s11248-013-9695-6) were digested with AscI, and the digestion products were recovered and ligated. After ligation, the positive target plasmid was extracted and named pBac[DsRed, NT-3] vector, which is the optimized plasmid that can be used to specifically express human NT-3 protein in the middle silk gland of silkworm. Figure 1 This is the structure diagram of the pBac[DsRed,NT-3] expression vector. Figure 1 Hr3 is an enhancer sequence (as shown in SEQ ID NO: 4), Ser1P is a promoter sequence for specifically expressing Ser1 gene in the middle silk gland of Bombyx mori (as shown in SEQ ID NO: 5), and Ser1PA is a terminator sequence of Ser1 gene (as shown in SEQ ID NO: 6).

[0033] Example 2 Obtaining a transgenic strain expressing human NT-3 protein in the middle silk gland of silkworm

[0034] First, pBac[DsRed,NT-3] plasmid and auxiliary plasmid pHA3PIG (see Wang, F., Xu, H., Yuan, L. et al. An optimized sericin-1 expression system for mass-producing recombinant proteins in the middle silk glands of transgenic silkworms. Transgenic Res 22, 925–938 (2013). https: / / doi.org / 10.1007 / s11248-013-9695-6) were mixed at a mass ratio of 1:1, and then the mixed plasmids were injected into fresh silkworm eggs within 2 hours after being laid by the silkworm using a microinjector. The G0 generation larvae hatched from the injected silkworm eggs were carefully raised to silkworm moths, and mated and laid eggs to obtain the G1 generation. When the G1 generation silkworm eggs developed to the 6th-7th day, the injected silkworm egg embryos were observed under a fluorescence microscope to screen the transgenic individuals with red fluorescence in the eyes of the silkworm ( Figure 2 To detect whether NT-3 protein is expressed in the middle silk gland of silkworm, the middle silk gland of silkworms at the fifth instar and third day of age was frozen and sectioned for anti-NT-3 immunohistochemical observation. The immunofluorescence results showed that NT-3 was specifically expressed in the middle silk gland of silkworms ( Figure 3 ). These results indicate that the NT-3 transgenic silkworm strain was successfully constructed and has the ability to secrete NT-3 protein. To confirm that the cocoons obtained from the transgenic silkworms synthesized NT-3 protein, 8M urea solution was used to extract the sericin of WT and NT-3 silk and perform SDS-PAGE ( Figure 4 A) and Western blot ( Figure 4 B) analysis, the results showed that NT-3 cocoons had specific protein bands, and the bands could be specifically recognized by anti-NT-3 antibodies. The above results indicate that we have successfully obtained a transgenic strain expressing human NT-3 protein in the middle silk gland of the silkworm, which can be continuously subcultured and preserved for large-scale production of human NT-3 protein.

[0035] Example 3 Silk hydrogel material is non-toxic to cell culture

[0036] Normal WT silk hydrogel and transgenic NT-3 silk hydrogel were added to the culture plate of mouse hippocampal neuron HT22 cells to treat the cells. After 48 hours, the Live-Dead double staining kit was used to detect cytotoxicity. The cell staining was observed under a fluorescence microscope. The live cells were stained green by calcein and the dead cells were stained red by propidium iodide. The cells cultured normally without any treatment were used as controls. The results showed that there were no dead cells in the WT and NT-3 silk hydrogel treatment groups, and the number of live cells was not significantly different from that in the control group ( Figure 5 ), indicating that silk hydrogel materials and NT-3 protein had no toxic effects on HT22 cells.

[0037] Example 4 Silk hydrogel containing human NT-3 protein promotes cell proliferation

[0038] PBS (Control), normal WT silk hydrogel, transgenic NT-3 silk hydrogel and NT-3 protein standard Std were added to the culture plate for culturing mouse hippocampal neuron HT22 cells to treat the cells. After 24 hours, the OD value of the cells at a wavelength of 450nm was measured using a CCK-8 kit. The results showed that the OD value of the transgenic NT-3 silk hydrogel treatment group was significantly higher than that of the PBS and WT groups, and was not much different from that of the standard Std group. This indicates that the NT-3 protein expressed in the silk gland of the silkworm has the biological activity of promoting the proliferation of HT22 ( Figure 6 A). The nuclear DNA replication was detected by EdU-488 kit. DAPI staining showed that the nuclear DNA was blue fluorescence, while EdU-488 green fluorescence indicated cells with active DNA replication. The number of cells with new DNA replication in the transgenic NT-3 silk hydrogel treatment group and the standard Std treatment group was significantly higher than that in the PBS and WT treatment groups ( Figure 6 B), indicating that the NT-3 protein expressed in the silk gland of Bombyx mori can effectively promote cell proliferation.

[0039] Example 5 Silk hydrogel containing human NT-3 protein promotes cell migration and repair

[0040] Mouse hippocampal neuron HT22 cells were cultured in a 6-well cell culture plate. After the cells grew all over the cell culture plate, a straight line was drawn in the central area of ​​each culture well. The culture medium was replaced with a low-concentration serum culture medium containing 0.25% FBS. PBS (Control), normal WT silk hydrogel, transgenic NT-3 silk hydrogel, and NT-3 protein standard Std were added to treat the cells. After 24 hours, the changes in the crossed-out blank area were observed under a microscope. The results showed that the surrounding cells migrated to the central blank area. The blank areas of the transgenic NT-3 silk hydrogel and NT-3 protein standard Std treatment groups were significantly narrower than those of the PBS and WT treatment groups ( Figure 7A and 7B), indicating that NT-3 has the effect of promoting cell migration and repair.

[0041] Example 6 Silk hydrogel containing human NT-3 protein promotes cell differentiation

[0042] PBS (Control), normal WT silk hydrogel, and transgenic NT-3 silk hydrogel were added to the culture plate for culturing mouse hippocampal neuron HT22 cells. After 72 hours of culture, the culture medium was discarded, washed twice with pH7.4 PBS, and Trizol reagent was directly added to extract RNA and reverse transcribe it. Then, RT-qPCR was used to detect the cell differentiation activity of silk hydrogel containing human NT-3 protein. RT-qPCR results showed that silk hydrogel containing NT-3 protein could significantly upregulate the expression of neuronal marker genes (NSE, β3-Tubulin, MAP), myelin synthesis marker genes (MPZ, MBP), and astrocyte marker protein (GFAP) compared with PBS and wild-type silk hydrogel ( Figure 8 ).

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. A recombinant vector expressing human neurotrophic factor NT-3 in silkworm silk gland, characterized in that: The recombinant vector is an optimized human neurotrophic factor NT-3 gene, whose sequence is shown in SEQ ID NO:

2. The recombinant vector also includes the silkworm enhancer Hr3, the promoter Ser1P of the silkworm middle silk gland-specific expression of the Ser1 gene, and the terminator Ser1PA of the silkworm middle silk gland-specific expression of the Ser1 gene.

2. The recombinant vector expressing human neurotrophic factor NT-3 in silkworm silk gland according to claim 1, characterized in that: The nucleotide sequence of the promoter Ser1P is shown in SEQ ID NO:5, the nucleotide sequence of the enhancer Hr3 is shown in SEQ ID NO:6, and the nucleotide sequence of the terminator Ser1PA is shown in SEQ ID NO:

4.

3. The recombinant vector expressing human neurotrophic factor NT-3 in silkworm silk gland according to claim 2, characterized in that: The amino acid sequence of the human neurotrophic factor NT-3 protein with a His tag expressed by the recombinant vector is shown in SEQ ID NO:

3.

4. The recombinant vector expressing human neurotrophic factor NT-3 in the silkworm silk gland according to any one of claims 1 to 3, characterized in that: The preparation method of the recombinant vector expressing human NT-3 in the silkworm silk gland is as follows: S1: The synthesized optimized human NT-3 gene was double-digested with NotI and BamHI, and the basic vector pSL1180 plasmid was cut with these two enzymes at the same time. After ligation, the positive clone plasmid was extracted to obtain the pSL1180[NT-3] vector; S2: The constructed pSL1180[NT-3] and pBac[DsRed] plasmids were digested with AscI, respectively, and the digestion products were recovered and ligated. After ligation, the positive plasmid extracted was the recombinant vector pBac[DsRed, NT-3] vector of the human neurotrophic factor NT-3.

5. Silk protein comprising the recombinant vector expressing human neurotrophic factor NT-3 in the silk gland of silkworm as claimed in any one of claims 1 to 3.

6. A method comprising the human neurotrophic factor NT-3 protein hydrogel according to claim 5.

7. The human neurotrophic factor NT-3 protein hydrogel according to claim 6, characterized in that: Application of human neurotrophic factor NT-3 protein hydrogel in the preparation of products promoting nerve cell proliferation and repair.

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