CN108486153B

CN108486153B - Application and method of FGF2 and TGF-beta 1 fusion gene in promotion of silk cell proliferation activity and anti-inflammatory function

Info

Publication number: CN108486153B
Application number: CN201810214294.6A
Authority: CN
Inventors: 夏庆友; 王峰; 王元成; 许胜�; 田弛; 赵萍
Original assignee: Southwest University
Current assignee: Southwest University
Priority date: 2018-03-15
Filing date: 2018-03-15
Publication date: 2020-12-11
Anticipated expiration: 2038-03-15
Also published as: CN108486153A

Abstract

The invention relates to an application of FGF2 and TGF-beta 1 fusion gene in promoting silk cell proliferation activity and anti-inflammatory function and a method thereof, which can express FGF2 and TGF-beta 1 in silkworm cocoon silk at the same time, and cultivate silk material capable of secreting two functional proteins, endow silk with cell proliferation promoting activity and immunoinflammatory regulation, have no cytotoxicity, and can be applied in the field of medicine as wound dressing raw material.

Description

Application and method of FGF2 and TGF-beta 1 fusion gene in promotion of silk cell proliferation activity and anti-inflammatory function

Technical Field

The invention belongs to the technical field of biology, and relates to an application of FGF2 and TGF-beta 1 fusion genes in promoting silk cell proliferation activity and anti-inflammatory function; also relates to a method for preparing silk with cell proliferation promoting activity and anti-inflammatory function.

Background

Wound healing after trauma is a long, complex process. The wound healing stage is mainly divided into an inflammatory stage, a proliferation stage and a structure remodeling stage, and each stage needs a plurality of growth factors to participate in regulation, such as epidermal growth factor, platelet-derived growth factor, fibroblast growth factor, transfer cell growth factor, interleukin and the like. For example, research reports indicate that Basic fibroblast growth factor (Basic fibroblast growth factor, FGF2, 16-18.5 kD) can activate ERK pathway in cells, enhance expression of transcription factor ElK1, thereby accelerating cell proliferation, and meanwhile, FGF2 also has the effect of reducing scar formation and has an important effect of promoting rapid wound healing; on the other hand, Transforming cell growth factor (TGF-beta 1, 12.5kD) participates in various stages of inflammation, proliferation, structural remodeling and the like in the wound healing process, not only can promote the proliferation of cells, but also can regulate immune response in the wound healing process and prevent wound inflammation. Therefore, the TGF-beta 1 is added into the biological material for helping the regeneration of the cartilage, so that the immunological rejection of the biological material in vivo can be effectively slowed down, and the healing of the cartilage is further accelerated.

The silk is protein fiber, has good biocompatibility, biodegradability and morphological plasticity, is a good biological material, and has wide application prospect in the field of medical tissue engineering. After the silkworm transgenic operation system is established, researchers develop a transgenic expression system capable of specifically expressing exogenous proteins in the rear silk gland and the middle silk gland of the silkworm in succession, and develop genetic improvement research of silk. For example, a novel silk with stronger mechanical property is obtained by specifically expressing spider silk-silk fusion protein on a silk fibroin layer for improvement; the novel colorful silk is obtained by specifically expressing fluorescent protein improvements such as red fluorescent protein, green fluorescent protein and the like on a silk fibroin layer; and the novel functional silk capable of promoting cell proliferation and accelerating wound healing is obtained by specifically expressing human fibroblast growth factor FGF1 in silk. In our earlier studies, a polygene expression system based on 2A lytic peptide (2A) is established, the system can express multiple target genes in the silk gland and cocoon silk of the silkworm simultaneously, and the system can simply and efficiently recombine and express multiple target genes so as to improve the performance of the silk. However, there is no report of simultaneous expression of FGF2 and TGF-. beta.1 in silkworm cocoon filaments by using a silkworm silk gland 2A polygene expression system.

Disclosure of Invention

In view of the above, an object of the present invention is to provide an application of FGF2 and TGF- β 1 fusion gene in promoting silk cell proliferation activity and anti-inflammatory function; the invention also aims to provide a method for preparing silk with cell proliferation promoting activity and anti-inflammatory function; the invention also aims to provide the silk prepared by the method for preparing the silk with the cell proliferation activity promoting and anti-inflammatory functions; the fourth purpose of the invention is to provide the application of the silk in preparing the wound dressing for promoting cell proliferation and resisting inflammation.

In order to achieve the above purpose, the invention provides the following technical scheme:

the application of the fusion gene of FGF2 and TGF-beta 1 in promoting the proliferation activity and anti-inflammatory function of silk cells is disclosed, wherein the nucleotide sequence of the fusion gene of FGF2 and TGF-beta 1 is shown in SEQ ID NO. 1.

Preferably, the amino acid sequence of the FGF2 and TGF-beta 1 fusion gene is shown as SEQ ID NO. 2.

2. The method for preparing the silk with the cell proliferation activity promoting and anti-inflammatory functions comprises the following steps: connecting FGF2 and TGF-beta 1 fusion gene into silkworm transgenic expression vector to obtain injection plasmid, then injecting silkworm eggs, culturing and screening transgenic silkworms; the nucleotide sequence of the FGF2 and TGF-beta 1 fusion gene is shown in SEQ ID NO. 1.

Preferably, the recombinant vector is prepared by the following method: the FGF2 and TGF-beta 1 fusion gene is connected into a transition vector pSL1180[ Hr3Ser1DsRedSer1PA ] which is cut by BamHI and NotI, then AscI is used for single enzyme cutting, a target fragment is recovered and then connected to a gene expression vector Piggybab [3xp3DsRed SV40], and an injection plasmid is obtained and named as Piggybab [3xp3DsRed SV 40; hr3Ser1FGF 2-2A-TGF-. beta.1 Ser1PA ].

Preferably, the method for injecting the silkworm eggs comprises the following steps: preparing a mixed solution of the plasmid to be injected and the Help plasmid containing Piggybab transposase in a mass ratio of 1:1, and injecting the mixed solution of the plasmid into silkworm eggs by using a micromanipulator and a microinjector under a microscope.

Preferably, the transgenic silkworm is obtained by placing the injected silkworm eggs at 25 ℃ and 90% relative humidity for incubation for 11 days to obtain G0 silkworms to be hatched, feeding fresh mulberry leaves to the cocooning frame, carrying out moth breeding and selfing to obtain G1 silkworms to be hatched, and screening silkworms expressing DsRed marker genes in eyes and nerves of silkworm embryos after the G1-generation embryos develop for 6 days.

3. The silk prepared by the method for preparing the silk with the cell proliferation activity promoting and anti-inflammatory functions.

4. The silk is applied to the preparation of biological materials for promoting cell proliferation and resisting inflammation.

The invention has the beneficial effects that: the invention discloses an application of FGF2 and TGF-beta 1 fusion gene in promoting silk cell proliferation activity and anti-inflammatory function, which expresses FGF2 and TGF-beta 1 simultaneously in silk cocoon by using a silk silkworm silk gland 2A polygene expression system, and cultivates a silk material capable of secreting two functional proteins simultaneously, so that the silk material is endowed with cell proliferation promoting activity and immunoinflammatory regulation, and can be applied in the field of medicine as a wound dressing raw material.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the cultivation and identification of silkworm with FGF2 and TGF _ beta 1 fusion gene; (A: a structure diagram of a transgenic expression vector, Hr3 is an enhancer of silkworm nuclear polyhedrosis virus, Ser1-P is a promoter of silkworm endogenous sericin 1 gene, Ser1PA is a 3-UTR sequence of silkworm endogenous sericin 1 gene, 3xp3DsRedSV40 is a screening gene of the transgenic vector, B: a transgenic screening result, C and D: quantitative PCR detection of expression conditions of FGF2 and TGF-beta 1 in middle silk glands of normal silkworms and transgenic silkworms, and E: expression detection of recombinant proteins FGF2 and TGF-beta 1 in cocoon shells).

FIG. 2 is an SEM observation of transgenic silk FT (A: 100-fold magnification; B: 500-fold magnification).

FIG. 3 shows the infrared spectrum analysis of normal cocoon shell and FT cocoon shell.

FIG. 4 is the analysis of mechanical properties of silk (A: stress-strain curve of normal cocoon shell and FT cocoon shell; B: breaking strength of normal cocoon shell and FT cocoon shell; C: elastic modulus of normal cocoon shell and FT cocoon shell; D: Young's modulus of normal cocoon shell and FT cocoon shell).

FIG. 5 shows the release characteristics of recombinant FGF2 and TGF-. beta.1 proteins in transgenic silk FT (A: release of recombinant FGF2 in transgenic silk FT; B: release of recombinant TGF-. beta.1 in transgenic silk FT).

FIG. 6 shows that transgenic silk promotes the proliferation of NIH3T3 cells (A: EdU staining result after co-culture of transgenic silk FT and NIH3T3 cells; B: Live-Dead staining result after co-culture of transgenic silk FT and NIH3T3 cells; C: CCK-8 measures growth rate after co-culture of transgenic silk FT and NIH3T3 cells).

FIG. 7 shows the cytotoxicity and inflammatory assay of transgenic silk FT (A: Live-Dead staining after co-culture of transgenic silk FT and NIH3T3 cells; B: CCK-8 measures the growth rate after co-culture of transgenic silk FT and NIH3T3 cells; C: expression of iNOS in cells after co-culture of transgenic silk FT and Raw264.7 cells; D: expression of TNF-alpha in the medium after co-culture of transgenic silk FT and Raw264.7 cells; E: expression of NO in the medium after co-culture of transgenic silk FT and Raw264.7 cells).

FIG. 8 shows the anti-inflammatory activity of transgenic silk FT (A and B: quantitative determination of iNOS and IL-1. beta.; C and D: TNF-. alpha.and NO content in cell culture medium; E and F: expression of iNOS in cells and its quantitative analysis results).

Detailed Description

Preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

The experimental materials used in the examples of the present invention are as follows:

cell line: mouse embryonic cell NIH3T3, mouse macrophage Raw264.7. Both NIH3T3 cells and raw264.7 cells were cultured using DMEM medium (Gibico) containing 10% fetal bovine serum (Gibico).

A target gene: human basic fibroblast growth factor (FGF2) and human metastatic cell growth factor (TGF _ β 1).

Silkworm strain: non-diapauzing variety D9L.

Example 1 vector construction

According to amino acid sequences of a human bFGF gene (NCBI: NM-002006.4) and a human TGF-beta 1 gene (GenBank: E03028.1), after the bFGF and the TGF-beta 1 are connected by GSG-P2A, a base sequence of a fusion gene is optimized by silkworm specific codons and is shown as SEQ ID NO.1, and an amino acid sequence is shown as SEQ ID NO. 2; the fusion gene FGF 2-2A-TGF-. beta.1 was synthesized by the Kinsley company. The fragment cut with BamHI and NotI was subcloned into the transition vector pSL1180[ Hr3Ser1DsRed Ser1PA ]. Ser1 is the promoter of sericin 1 gene specifically expressed by middle silk gland of silkworm, Hr3 is enhancer in NPV virus of silkworm, Ser1PA is the termination sequence of sericin 1 gene of silkworm. Then, the target fragment is recovered by using AscI single enzyme digestion and is connected to a final transgenic expression vector Piggybab [3xp3DsRed SV40] to obtain Piggybab [3xp3DsRed SV 40; hr3Ser1FGF2-2A-TGF _ beta 1Ser1PA ], and the vector structure is shown as A in figure 1.

Example 2 preparation of transgenic silkworms

Preparation of transgenic injection plasmid: and (3) after the constructed plasmid is converted again, selecting a single bacterial plaque and shaking the bacteria for 12h, taking 10mL of bacterial liquid, extracting the plasmid by using a Qiagen ultrapure kit, measuring the purity of the plasmid by using a DNA concentration meter, and keeping the 260/280 ratio at 1.8-1.9 and the concentration at 400-500 ng/microliter for later use.

Silkworm seed preparation for preparing transgenic silkworms: high-quality mulberry leaves are fed to silkworms to be cocooning, the silkworms are copulated for 6 to 8 hours in a self-copulation mode after being pupated and the silkworms are refrigerated for later use at 4 degrees.

Culturing and screening transgenic silkworms: and preparing a mixed solution of the plasmid to be injected and the Help plasmid containing Piggybab transposase according to the mass ratio of 1: 1. The female moths were removed and the plasmid mixture was injected within 2h after their eggs under a SZX16 microscope (Olympus) using a TransferMan NK2 micromanipulator and a Femto Jet 5247 microsyringe (Eppendorf). Placing the injected silkworm eggs at 25 ℃ and 90% relative humidity for incubation for 11 days to obtain G0 silkworms to be formicary, carefully feeding fresh mulberry leaves for cocooning, and selfing after moth formation to obtain G1 silkworms. After the G1 generation embryos developed for 6 days, the eyes and nerves of the silkworm embryos were screened for DsRed marker genes using an Olympus SZX12 fluorescence stereomicroscope (Olympus), and the results are shown in Table 1.

TABLE 1 statistics of transgenic silkworm injections

The result shows that 400 silkworm diversified varieties D9L non-diapause silkworm eggs are injected in a microinjection mode, after the incubation, 95 silkworm eggs are normally hatched, the hatching rate is 24%, after the silkworm eggs are carefully raised to five-year old, 35G 1 moth rings are obtained after selfing and oviposition in the adult stage through the pupal stage, 18 positive moth rings (shown as B in figure 1) with red fluorescence emitted by eyes are screened, and the positive rate is 51%. Through preliminary identification, the strain (FT) with the highest foreign protein expression amount is taken as a research object to carry out subsequent research.

Example 3 detection of transgenic silkworms

1. Quantitative PCR

Dissecting the five-instar middle silk glands of the WT normal silkworms and the transgenic silkworms, and extracting the total RNA by using the total RNA extraction kit. 2 mu g of total RNA was taken and reverse transcription was performed using a reverse transcription kit. Detection of mRNA of endogenous sericin 1 gene (Ser1), FGF2 and TGF-. beta.1 gene in a sample by SYBR Premix ExTaq kit (Takara) in ABI Fast 7000 quantitative PCR apparatus (Applied Biosystems). Sw22934 was used as an internal reference gene. RT-PCR primers are listed in Table 1.

TABLE 1 quantitative primers used in the experiments

The quantitative PCR results are shown in fig. 1C and D, and show that a large amount of FGF2mRNA and TGF _ β 1mRNA, respectively, was contained in the middle silk gland of the transgenic silkworm FT relative to the WT silkworm, relative to the expression levels of the endogenous Ser1 gene, 8.25% and 7.74%.

2. Protein detection

Cell eggExtraction of white samples: the medium was discarded, and 1mL of PBS (135mM NaCl, 2.7mM KCl, 1.5mM KH) was used₂PO₄And 8mM K₂HPO₄pH7.4), adding 150 μ l of newly prepared RIPA lysate containing protease inhibitor PSPF, placing on ice for 30min, collecting cells, centrifuging at 13400rpm for 5min, and collecting supernatant as cell protein sample.

Extracting a middle silk gland protein sample of the silkworm: dissecting the middle silk gland of silkworm, cutting, soaking in PBS for 4 deg.C, and extracting overnight. Centrifuging the extract at 13400rpm for 5min, and taking the supernatant as a middle silk gland protein sample of the silkworm;

extracting a cocoon shell protein sample: the cocoon shell is pulverized by a pulverizer, 20mg of cocoon powder is extracted by 1mL of protein extract (8M urea, 25mM Tris-HCl, pH7.0) at 80 ℃ for 30min, and the cocoon is fully vibrated once every 10 min. Centrifuging the extract at 13400rpm for 5min, and taking supernatant as a cocoon shell protein sample;

SDS-PAGE and Western Blotting: and (3) determining the protein concentration (Beyotime) of the cell sample, the silkworm middle silk gland sample and the cocoon shell sample by using an enhanced BCA protein determination kit. Protein samples of equal mass were subjected to SDS-PAGE and detected by Coomassie blue staining. After the electrophoresis was completed, the protein sample was transferred to PVDF membrane by a membrane transfer apparatus, and after 5% nonfat dry milk blocking, 5 times of PBST membrane washing, and secondary antibody (anti-mouse or rabbit IgG antibody) washing, ECL Western Blotting Detection System (Amersham Biosciences) was used to visualize the bands on the membrane. The exposure mode adopts automatic exposure.

SDS-PAGE and Western blotting are shown in E in figure 1, and recombinantly expressed bFGF and TGF-beta 1 protein are also detected in transgenic cocoon FT, and the contents are 1.79 mug/g and 0.25 mug/g respectively. It was shown that FGF2 and TGF _ β 1 fusion genes were successfully transcribed and translated into two independent proteins, which provides the basis for their biological activity.

3. Observation by electron microscope

A cocoon shell intermediate layer cocoon sheet sample is taken for gold spraying treatment, and observation and photographing are carried out by utilizing a scanning electron microscope (Supra 55sapphire, Zeiss). The photographing process is carried out at room temperature, and the voltage is 3.0 kV; three independent samples, 10 beats of results. The results are shown in FIG. 2A. The results show that there is no obvious difference between the transgenic silk FT and the normal silkworm WT in the structure and the gap of cocoon shell, the size of silk (the diameters of WT and FT silk are respectively 18.5 +/-0.4 μm and 19.3 +/-0.4 μm), and the like.

4. Infrared spectrometry

Taking a cocoon shell middle layer cocoon sheet as a detection sample, and then measuring by using an infrared spectrum measuring instrument (Thermo fisher scientific); each sample was measured 30 times and the results were averaged for analysis and are shown in figure 3. The result shows that the infrared spectrum of the transgenic silk FT and the infrared spectrum of the normal silkworm WT have high similarity in the wavelength range of 800-4000, and the result shows that no obvious difference exists in secondary structures such as alpha helix, beta folding and the like in the silk.

5. Measurement of mechanical Properties

And (3) extracting a silk sample from the cocoon shell, fixing the silk sample on a paper tape with a gap of about 2cm, and measuring the mechanical property by using a universal tester. The tensile rate was 1mm/min, the test temperature was 25 degrees, the humidity was 60%, and each sample was measured 40 times. The results of the assay showed that the transgenic silk FT had a slightly lower maximum breaking strength and elastic modulus than the normal silk, but there was no significant difference between the two (as shown in fig. 2, C). The maximum breaking strength of WT and FT silk is 158.4 + -7.32 Mpa, 139.0 + -9.78 Mpa respectively; the maximum breaking elastic modulus of WT and FT silk is 14.89 + -1.0% and 13.45 + -1.02%, respectively; young's moduli of WT and FT silk were 37.98. + -. 2.75MPa and 38.77. + -. 2.67MPa, respectively (as shown in FIG. 4). It is shown that the transgenic silk FT has no obvious difference from natural silk, and has the excellent properties of the natural silk.

6. Characterization of transgenic silkworm silk FT

The release characteristics of FGF2 and TGF _ beta 1 protein which are recombinantly expressed in the transgenic silk FT are detected by using an ELISA technology, and the result is shown in figure 5, and the result shows that FGF2 and TGF _ beta 1 in the transgenic silk FT can be effectively and slowly released from silk. Within the first 30h, only a small amount of FGF2 and TGF _ beta 1 are released from silk, and the release rate is slow; after 30h, FGF2 and TGF-. beta.1 were gradually released from silk at a relatively constant rate, and finally, more than 11.9. mu.g of FGF2 and 4.2. mu.g of TGF-. beta.1 recombinant protein could be released from 30mg of transgenic FT silk.

7. Silk NIH3T3 cell proliferation assay

The cocoon shells are cut into cocoon sheets with the diameter of 0.5cm after being layered, and are disinfected by ultraviolet direct irradiation for 6 hours. NIH3T3 cells were plated in 96-well plates with 500 cells per well in 100. mu.l of the system in DMEM medium with 0.25% serum overnight. After soaking the cocoon pieces with the medium, placing the cocoon pieces in a 96-well plate for continuous culture for 1 day, 2 days and 3 days, carefully taking out the cocoon pieces, and replacing the old medium with fresh 100 mul of the medium. Cell wells containing FGF1 or FGF2 standard proteins were set as positive controls. Staining of proliferating cells using the Click-iT EdU kit (Invitrogen); using Live-Dead staining kit (Molecular Probes)^TM) Staining live cells and dead cells respectively; the number of cells was analyzed using CCK-8 kit (Beyotime). Total RNA of NIH3T3 cells after 1 day of treatment of each sample was extracted using a total RNA extraction kit (Sigma) for the later RT-PCR experiment, and the results are shown in FIG. 6.

The EdU staining result shows that compared with cells of Null and WT groups, cells with red signals in a transgenic cocoon sheet FT group are obviously increased, the number of the cells with red fluorescent signals in an FGF2 standard group is similar, and the cells undergoing cell division and proliferation in the cells after transgenic cocoon sheet FT co-culture are more. Live-Dead staining kit was able to stain Live cells green and Dead cells red. After staining NIH3T3 cells after FT co-culture of transgenic cocoon sheets, the number of green living cells is found to be significantly more than that in normal WT cocoon sheet groups, and is basically the same as that after the positive control group FGF2 standard is cultured. Also, there was no significant difference in the number of dead cells exhibiting red signals between groups. In addition, the proliferation of each group of cells was quantitatively determined by using the CCK-8 kit. The results show that the number of cells in the transgenic cocoon sheet FT group is significantly higher than that of the Null and WT groups after 1 day, 2 days and 3 days after co-culture, and even slightly higher than that of the positive control FGF2 group. The results of the EdU staining, Live-Dead staining and CCK-8 kit are combined to show that the transgenic cocoon sheet FT can remarkably enhance the proliferation and growth of NIH3T3 cells.

8. Anti-inflammatory and cytotoxic experiment of silk

To examine the biosafety of transgenic silk FT, the cocoon sheets were co-cultured with NIH3T3 cells and raw264.7 cells, and it was observed whether the transgenic silk was sufficiently cytotoxic and inflammatory response-inducing. The specific method comprises the following steps: raw264.7 cells were plated in 24-well plates with 500 cells per well in 500ul serum DMEM medium and incubated overnight. Adding 25ng/ml lipopolysaccharide (Sigma) of Escherichia coli 0111: B4, soaking cocoon sheets in a culture medium, putting the soaked cocoon sheets into a 24-well plate, 8 cocoon sheets/well, taking a standard TGF-B1 as a sample control, culturing continuously for 1 day and 2 days, carefully taking out the cocoon sheets, and directly taking a cell culture solution for later use. After cocoon sheets of transgenic silk FT are co-cultured with NIH3T3 cells for several days, the Live-Dead staining kit staining result shows that the number of NIH3T3 cells is small after the cocoon sheets of the transgenic silk FT are co-cultured for one day, the number of Dead cells is small, the number of NIH3T3 cells is remarkably increased after the culture time reaches 3 days and 5 days, particularly, the NIH3T3 cells grow over the whole culture plate at the 5 th day, and a large number of cells do not appear (as shown in A in figure 7). In addition, the number of cells after the FT silk co-culture was measured by using the CCK-8 cell quantification kit, and it was found that the number was not significantly different from that of the normally cultured NIH3T3, and the number of cells rapidly increased with the time of the culture (as shown in B in FIG. 7), indicating that the transgenic silk FT was not cytotoxic. After cocoon sheets of transgenic silk FT are co-cultured with Raw264.7 cells for several days, the expression condition of proinflammatory signal molecule nitric oxide synthase iNOS in the Raw264.7 cells is detected, and the result shows that after the transgenic silk FT and the cells are co-cultured for 1 day and 2 days, compared with LPS-induced cells, a large amount of inflammatory signals are generated, and the FT silk cultured cells do not induce macrophages to generate serious inflammatory signals (as shown in C in figure 7) like the normal cultured cells. This is also confirmed by measuring the levels of the pro-inflammatory molecules tumor cell necrosis factor TNF-alpha and NO in the cell culture medium. Compared with the macrophage group cultured normally, the transgenic silk FT did not detect a large amount of TNF-alpha and NO production in the culture after 1 day and 2 days of co-culture, while the LPS-induced group cells induced a large amount of TNF-alpha and NO (as shown in D and E in FIG. 7), indicating that the transgenic silk FT did not induce the cell inflammatory production.

In summary, transgenic silk FT is not cytotoxic and does not induce severe inflammatory cellular responses, with a biologically good biosafety.

9. Experiment on cell inflammation

After normal WT cocoon sheets, transgenic cocoon sheets FT and a standard TGF _ beta 1 are treated for 1 day, expression conditions of proinflammatory signal molecules iNOS and IL-1 beta in a cellular inflammatory molecular pathway are detected, and the result is shown in FIG. 8. RT-PCR results show that LPS induced group remarkably enables Raw264.7 cells to generate a large amount of inflammatory responses, and when a standard TGF _ beta 1 is added, the expression levels of iNOS mRNA and IL-1 beta mRNA are remarkably low, which indicates that the standard TGF _ beta 1 has anti-inflammatory activity. WT cocoon sheets failed to effectively reduce iNOS mRNA produced by LPS induction, while transgenic FT cocoon sheets significantly reduced iNOS mRNA produced by LPS induction by about 0.6 times; for IL-1 beta, both WT and transgenic FT cocoon sheets can effectively reduce IL-1 beta produced by LPS induction, but the transgenic FT cocoon sheets have stronger activity compared with WT cocoon sheets. Furthermore, we also examined the levels of the proinflammatory molecules TNF- α and NO in cell culture. The results show that after LPS-induced Raw64.7 cells are cultured with silk or a standard substance for 6h and 12h, the content of TNF-alpha in the culture medium of the transgenic cocoon piece FT group is significantly lower than that in the culture medium of the normal cocoon piece WT group and the LPS-induced group, namely 0.64 times and 0.78 times of that of the WT group respectively, and the content of NO is also significantly reduced, namely only 0.49 times and 0.66 times of that of the WT group. In addition, I also detected the expression of iNOS in Raw64.7 cells. The result shows that after the transgenic cocoon sheets FT and the induced Raw264.7 cells are co-cultured for 6 hours, the expression level of iNOS in the cells between WT and the transgenic cocoon sheets FT is not obviously different, and after 12 hours, the expression level of iNOS in the cells cultured with the transgenic cocoon sheets FT is obviously lower than that of a normal WT group and is only 0.59 times of that of the normal WT group, which indicates that the transgenic cocoon sheets FT can also obviously reduce the content of iNOS generated by Raw264.7 cells induced by LPS. The results show that the transgenic cocoon sheet FT can obviously reduce the expression of iNOS, IL-1 beta, TNF-alpha and NO generated by Raw264.7 cells induced by LPS as the standard TGF _ beta 1, and the transgenic silk FT has anti-inflammatory bioactivity.

Finally, it is noted that the above-mentioned preferred embodiments illustrate rather than limit the invention, and that, although the invention has been described in detail with reference to the above-mentioned preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

Sequence listing

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

gcgagcactt tggagtattg g 21

<210> 7

<211> 23

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atctgaagac ggtttctggt ggt 23

<210> 8

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

aactgcctga agtggttgtg c 21

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ttcgtactgg ctcttctcgt 20

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

caaagttgat agcaattccc t 21

<210> 11

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

gctccccaca cataccttga c 21

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

actggacgga aactggaagg a 21

<210> 13

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tgaaatgcca ccttttgaca gt 22

<210> 14

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

atgagtgata ctgcctgcct ga 22

<210> 15

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

acccagagac aagcctaccc 20

<210> 16

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

tgctacagtt ccgagcgtca 20

<210> 17

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gctatccaga aaacccctca a 21

<210> 18

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

catgtctcga tcccagtaga cggt 24

Claims

1. The method for preparing the silk with the cell proliferation activity promoting and anti-inflammatory functions is characterized by comprising the following steps: connecting FGF2 and TGF-beta 1 fusion gene into silkworm transgenic expression vector to obtain injection plasmid, then injecting silkworm eggs, culturing and screening transgenic silkworms; the nucleotide sequence of the FGF2 and TGF-beta 1 fusion gene is shown in SEQ ID NO. 1.

2. The method for preparing silk with cell proliferation promoting activity and anti-inflammatory function according to claim 1, wherein: the preparation method of the transgenic expression vector comprises the following steps: the FGF2 and TGF-beta 1 fusion gene is connected into a BamHI and NotI cut transition vector pSL1180, then the single cut of AscI is carried out, a target fragment is recovered and then the target fragment is connected to a gene expression vector Piggybac, and an injection plasmid is obtained.

3. The method for preparing silk with cell proliferation promoting activity and anti-inflammatory function according to claim 1, wherein the method for injecting silkworm eggs comprises the following steps: preparing a mixed solution of the plasmid to be injected and the Help plasmid containing Piggybab transposase according to the mass ratio of 1:1, and injecting the mixed solution of the plasmid into silkworm eggs by using a micromanipulator and a microinjector under a microscope.

4. The method for preparing silk with cell proliferation promoting activity and anti-inflammatory function according to claim 1, wherein: the transgenic silkworm is obtained by placing injected silkworm eggs at 25 ℃ and 90% relative humidity for 11 days to accelerate green growth to obtain G0 generation ant silkworms, feeding fresh mulberry leaves to a cocooning frame, carrying out moth transformation and selfing to obtain G1 generation silkworms, and screening silkworms expressing DsRed marker genes in eyes and nerves of silkworm embryos after the G1 generation embryos develop for 6 days.

5. Silk obtained by the method for producing silk having cell proliferation promoting activity and anti-inflammatory function according to any one of claims 1 to 4.

6. Use of silk according to claim 5 for the preparation of a wound dressing for promoting cell proliferation and anti-inflammatory.