CN116239667B - EhEGF recombinant protein with cell proliferation promoting activity, and preparation method and application thereof - Google Patents
EhEGF recombinant protein with cell proliferation promoting activity, and preparation method and application thereof Download PDFInfo
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- CN116239667B CN116239667B CN202210867008.2A CN202210867008A CN116239667B CN 116239667 B CN116239667 B CN 116239667B CN 202210867008 A CN202210867008 A CN 202210867008A CN 116239667 B CN116239667 B CN 116239667B
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- ehegf
- recombinant protein
- cell proliferation
- silkworm
- expression vector
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
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- A—HUMAN NECESSITIES
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- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
- A01K67/0333—Genetically modified invertebrates, e.g. transgenic, polyploid
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/09—Recombinant DNA-technology
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- A—HUMAN NECESSITIES
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- A01K2217/052—Animals comprising random inserted nucleic acids (transgenic) inducing gain of function
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- A—HUMAN NECESSITIES
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- A01K2267/02—Animal zootechnically ameliorated
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses ehEGF recombinant protein with cell proliferation promoting activity, a preparation method and application thereof, wherein ehEGF recombinant protein is obtained by taking human EGF mature peptide as a basis, fusing PTD sequence at the N-terminal of the human EGF mature peptide and fusing PlGF-2 motif at the C-terminal; by utilizing a Ser1 expression system, ehEGF is designed and synthesized according to the preference of the codon usage of a silkworm genome to code ehEGF, a transgenic expression vector PH EHEGFSER1 is constructed, and then ehEGF gene silkworm strain is established, so that the transgenic silkworm which can efficiently express ehEGF in the silkworm is obtained, and ehEGF synthesized by silkworm silk gland has better cell proliferation promoting activity than commercialized EGF, and can be used for cell proliferation promoting activity.
Description
Technical Field
The invention relates to the field of biological materials, in particular to ehEGF recombinant protein with cell proliferation promoting activity, and also relates to a preparation method and application of ehEGF recombinant protein.
Background
As the demand of human beings for functional proteins for various purposes such as medical use, eating use, beauty treatment, health care and the like has been increasing in the 21 st century, the rapidly growing market demands cannot be satisfied by virtue of extracting and producing proteins of natural origin. Various efficient prokaryotic and eukaryotic expression systems are established and perfected, and escherichia coli, yeast, insect cells, mammalian cells, insects, mammals and the like are utilized as host bioreactors, so that the method is an effective and sustainable method for realizing low-cost and large-scale production of recombinant exogenous proteins with biological activity, and becomes a hot spot for research in the world today. The use of Chinese hamster ovary cells as a bioreactor for producing foreign proteins is the most standard expression mode at present, but the operation cost and the requirements on environment are very severe, and the large-scale popularization and application of the foreign proteins are severely limited. In order to establish a low-cost, large-scale, safe and sustainable biological plant for efficiently producing foreign proteins, researchers have been trying to produce recombinant foreign proteins using transgenic organisms such as mammals, birds, insects and plant organs as bioreactors since 2000.
Silkworm is known as silk secretion and is one of the earliest economic animals (insects) fully domesticated and utilized by humans. The silk gland of silkworm is an organ for synthesizing and secreting fibroin, and is the biological foundation of the whole silk industry. After thousands of years of artificial domestication, the silk gland of the silkworm has super-strong protein synthesis and secretion capacity, and about 0.5g of silk protein can be synthesized and secreted by about 5g of silkworm, which is the most known insect at present. The fibroin mainly comprises silk fibroin (fibroin) and sericin (sericin) coated on the outer layer of the fibroin: silk fibroin is the main body of silk, accounting for about 75%, is synthesized by silk glands at the rear part of silkworms, and comprises three main components of fib-H chain, fib-L chain and P25, and is insoluble in water; the rest is Sericin, accounting for about 25 percent, is synthesized by the middle silk gland of silkworm, and comprises three main components of Sericin1 (Sericin 1), sericin2 (Sericin 2) and Sericin3 (Sericin 3), wherein the highest content of Sericin1 protein is used, and the Sericin is soluble in water. With the development of modern molecular biology and transgenic technology, the characteristic of efficient synthesis and secretion of silk protein of silkworm silk gland, and the extremely important post-translational modification processing capacity of protein with glycosylation, methylation and the like for keeping the activity of foreign protein, the method has the characteristics of low feeding cost, industrial production, safety to human beings and animals and the like, and becomes an ideal bioreactor model, and is concerned by researchers in various countries and is competitive for development and utilization.
In 2000, nonomuria et al used pBac transposon-mediated microinjection of silkworm eggs and obtained stably inherited transgenic silkworms; in 2003 Xia Qingyou et al completed the silkworm genome project, and important coding genes involved in silk protein synthesis in silkworm silk gland such as silk fibroin heavy chain (FibH chain) gene, silk fibroin light chain (FibL chain) gene, sericin1 (Sericin 1) gene, sericin2 (Sericin 2) gene, sericin3 (Sericin 3) gene, P25 gene and the like were identified and cloned, and at the same time, a high-efficiency transgenic silkworm silk gum 1 expression system was obtained by optimizing expression system in the early stage. These basic research results make it possible to produce recombinant foreign proteins in large scale in silk glands using transgenic silkworm tissue-specific expression systems. In recent years, attempts have been made to express a plurality of foreign proteins in silk glands at home and abroad using the pBac transposon-mediated transgenic technology and silkworm tissue specific promoter elements, including: EGFP (Zhao et al 2010), cat interferon (Kurihara et al 2007), spider silk traction protein (Zhu et al 2010), human type III collagen partial peptide (Tomita et al 2003), enhanced red fluorescent protein (Tomita et al 2003), silk light chain fused hydroxyproline collagen partial peptide (Adachi et al 2006), fibroblast growth factor (Hino et et al 2006), enhanced green fluorescent protein (Shimizu et al 2007), partial collagen peptide (YANAGISAWA ET et al 2007), and P25 fused red fluorescent protein (Royer et al 2005) were expressed in the posterior silk gland; human serum albumin (Ogawa et al 2007), enhanced green fluorescent protein (Tomita et al 2007), murine monoclonal antibody (Iizuka et al 2009), human collagen alpha chain gene (Adachi et al 2010), and soluble GM-Csf receptor alpha (Urano et al 2010) are expressed in the middle silk gland. The research results at home and abroad are integrated, and show that the silkworm silk gland is used as a bioreactor to produce the exogenous protein with high added value, so that the method has broad market prospect, and meanwhile, the method can break through the barrier that the silkworm can only be used as the traditional industry, thereby providing basic technical system guarantee for the development of novel silkworm industry.
EGF is a low molecular weight single chain polypeptide that exerts its mitogenic effects on epithelial cells, fibroblast-like cells and endothelial cells via tyrosine kinase membrane receptors, thereby stimulating cell proliferation and differentiation (Berlanga-Acosta J et al.2017). EGF is free of glycosyl modification and therefore very stable, and acid and high temperature resistant. EGF has been used in wound healing studies due to its extremely strong ability to promote cell division, and EGF has been found to have excellent therapeutic effects in chronic wound diseases such as diabetic foot ulcers in recent years (Bui T Q et al 2019), and the use of gene recombination technology to produce EGF has great market development prospects.
Disclosure of Invention
Accordingly, it is an object of the present invention to provide ehEGF recombinant proteins having cell proliferation promoting activity; the second purpose of the invention is to provide ehEGF recombinant protein coding genes which are suitable for high-efficiency expression in silk glands of domestic animals; the third object of the invention is to provide an expression vector containing ehEGF recombinant protein coding gene which is suitable for high-efficiency expression of silk gland of domestic animals; the fourth object of the present invention is to provide the use of ehEGF recombinant protein coding gene or expression vector in the production of silk with cell proliferation promoting activity in silkworm; the fifth object of the present invention is to provide a method for producing silk having cell proliferation promoting activity in silkworms using the ehEGF recombinant protein-encoding gene; the sixth object of the present invention is to provide a method for producing a recombinant protein having a cell proliferation promoting activity in silkworms using the ehEGF recombinant protein-encoding gene; the seventh object of the present invention is to provide the application of ehEGF recombinant protein in preparing cell proliferation promoting material.
In order to achieve the aim of the invention, firstly, the humanized epidermal cell growth factor (ehEGF) is designed through artificial transformation, then the coding gene of ehEGF is designed and synthesized according to the preference of the codon usage of the silkworm genome, the coding gene of ehEGF is constructed into a Ser1 expression system hSRSE to obtain a constructed transgenic expression vector PH EHEGFSER1, and the transgenic expression vector is used for establishing a ehEGF gene silkworm strain through microinjection of silkworm embryos, and the specific technical scheme provided by the invention is as follows:
1. a ehEGF recombinant protein with cell proliferation promoting activity, wherein the ehEGF recombinant protein is based on a human EGF mature peptide, and a PTD sequence is fused at the N-terminal end of the human EGF mature peptide, and a PlGF-2 motif is fused at the C-terminal end.
Preferably, the amino acid sequence of ehEGF recombinant protein is shown as SEQ ID NO. 1.
2. The ehEGF recombinant protein coding gene is suitable for high-efficiency expression of the silk gland of the domestic animal, and the nucleotide of the ehEGF recombinant protein coding gene is shown as SEQ ID NO. 2.
3. The expression vector contains ehEGF recombinant protein coding genes suitable for high-efficiency expression of silk glands of domestic animals.
Preferably, the expression vector of the present invention sequentially contains enhancer hr3, secretory sericin 1 gene promoter, ehEGF recombinant protein coding gene and sericin 1 gene terminator.
Preferably, the expression vector is ligated into the BamHI and NotI cleavage sites of psl1180[ hr3PSer1spRedSer PA ] from the sequence shown in SEQ ID NO.2, and then ligated into the same digested pBac [3xp3DsRedaf ] vector after cleavage with AscI.
4. The ehEGF recombinant protein coding gene and the application of the expression vector in the production of silkworms with cell proliferation promoting activity.
5. The method for producing silk with cell proliferation promoting activity in silkworms by utilizing ehEGF recombinant protein coding genes comprises the steps of injecting an expression vector containing ehEGF recombinant protein coding genes into silkworm eggs with diapause removed, sealing the silkworm eggs by using nontoxic glue, sterilizing by formaldehyde steam, hatching, carrying out selfing or backcross seed production after adult, screening transgenic positive moth rings, and obtaining the cocoon shells of positive transgenic silkworms, namely the silk with cell proliferation promoting activity.
6. The ehEGF recombinant protein coding gene and the application of the expression vector in the production of ehEGF recombinant protein with cell proliferation promoting activity in silkworms.
Preferably, the preparation method of the recombinant protein specifically comprises the following steps: injecting an expression vector containing ehEGF recombinant protein coding genes into silkworm eggs with diapause removed, sealing with nontoxic glue, sterilizing with formaldehyde vapor, incubating, performing selfing or backcross seed production after adult, screening transgenic positive moth rings, screening to obtain positive transgenic silkworm eggs for spawning and incubating, feeding to upper cluster spinning cocoons, taking cocoon shells for crushing, dissolving with PBS buffer solution, and performing solid-liquid separation to obtain ehEGF recombinant proteins.
7. The ehEGF recombinant protein is applied to the preparation of materials for promoting cell proliferation.
The invention has the beneficial effects that: the invention aims to provide ehEGF recombinant protein with cell proliferation promoting activity, and the recombinant ehEGF protein can be detected to be expressed efficiently in the cocoon shells of ehEGF transgenic silkworms through a Ser1 expression system hSRSE. Bandscan calculating that the highest content of recombinant protein accounts for 6.24+/-0.59% of cocoon fiber soluble protein; the ehEGF recombinant protein in ehEGF silkworm silks is subjected to crude extraction by using PBS, so that a crude fibroin extract with the concentration of ehEGF of 10-30 mug/mL is obtained, and a foundation is laid for the next large-scale separation and purification. And the cell proliferation activity detection proves that ehEGF synthesized by silkworm silk gland has better cell proliferation promoting activity than the commercialized EGF. Therefore, the method can be used for preparing raw materials for promoting cell proliferation.
Drawings
In order to make the objects, technical solutions and advantageous effects of the present invention more clear, the present invention provides the following drawings for description:
FIG. 1 shows construction of a transgenic ehEGF gene silkworm strain and expression detection of ehEGF recombinant protein (A: a sequence diagram of artificial transformation of EGF; B: a transgenic ehEGF gene vector diagram; C: a transgenic ehEGF gene silkworm fluorescent screening diagram, wherein the first row is a transgenic ehEGF silkworm egg white light diagram, a red fluorescent diagram and a green fluorescent diagram from left to right respectively, the second row is a transgenic ehEGF silkworm pupa white light diagram, a red fluorescent diagram and a green fluorescent diagram from left to right respectively, the third row is a transgenic ehEGF silkworm moth white light diagram, a red fluorescent diagram and a green fluorescent diagram from left to right respectively, and D: SDS-Page detects ehEGF recombinant proteins in silkworm cocoons of different individuals of the transgenic silkworm, the red arrow shows an SDS-Page staining band of ehEGF protein, and the black arrow shows a western blot color band of ehEGF protein.
FIG. 2 shows the concentration calculation of ehEGF recombinant proteins in crude silk extract;
FIG. 3 shows the detection of the proliferation activity of ehEGF recombinant proteins in crude silk extract (A: CCK-8 for detecting the proliferation of cells treated by each sample group at 24 hours, B: CCK-8 for detecting the proliferation of cells treated by each sample group at 48 hours, C: edU staining for detecting the proliferation of cells treated by each sample group at 24 hours, red fluorescence as EdU staining effect, blue fluorescence as nucleus, scale size: 100 μm).
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to limit the invention, so that those skilled in the art may better understand the invention and practice it.
The experimental material of the invention is stored in the laboratory for the test silkworm variety Dazheng (P50). Larvae were fed with artificial feed in a 25 ℃ artificial climatic chamber. Human immortalized epidermal cell HaCaT cell lines were maintained by laboratory and cultured in DMEM medium containing 10% (v/v) fetal bovine serum (FBS, gibco) at 37℃under 5% CO 2. Plasmid vectors pSLfa1180fa, pBac [3xp3EGFPaf ], pBac [3xp3DsRedaf ] were maintained by the laboratory.
Preparation of main reagents and solutions: the conventional culture medium, reagent buffer and the like used in the molecular cloning process are prepared by referring to the laboratory conventional reagent preparation method section (S1-S11) in TaKaRa commodity catalog, third edition of the molecular cloning Experimental guidelines. DNA polymerase Ex-Taq, LA-Taq Kit, conventional restriction enzymes, alkaline phosphatase, sequencing cloning vector pMD19-T simple vector Kit, DNA Ligation Kit Ver.2.0, and fluorescent quantitative PCR Kit SYBR premix Ex TaqTM were all purchased from TaKaRa company. Transformation with E.coli competent cells Trans1-T1, conventional plasmid DNA extraction kit Easypure PLASMID MINIPREP KIT was purchased from full gold company. Agarose gel DNA recovery kit Gel Extraction Mini Kit (50) was purchased from the biological sciences company. The ultrapure plasmid extraction kit QIA PREP SPIN MINIPREP KIT (50) for transgene injection was purchased from QIAGEN. Total RNA Kit II (50) Kit was purchased from Omega Bio-Tec company. Human EGF polyclonal antibody anti-EGF antibody, human EGF protein standard (EGFstd) was purchased from Abcam company.
All data in the present invention are averages of at least 3 values. Data were analyzed by Student's t-test, p <0.05, p <0.01, and p <0.001 were statistically significant, highly significant, and extremely significant, respectively.
Example 1 design modification and Synthesis of EGF Gene
To increase the signaling and ECM binding efficiency of EGF, an engineered ehEGF (FIG. 1A) was created using the amino acid sequence of the human EGF mature peptide (Homo SAPIENS EPIDERMAL growth factor, genBank: AAG 33031) as a template, and fusing HIV-1TAT Protein Transduction Domain (PTD) at the EGFN-terminus to increase its signaling capacity, while fusing PlGF-2 motif at the C-terminus to increase its binding efficiency to extracellular matrix, the amino acid sequence of which is shown in SEQ ID NO. 1. Then, the coding sequence is optimally designed according to the codon usage preference of silkworm, bamHI and NotI digestion sites are respectively connected at the 5 '-end and the 3' -end, and the gene sequence is synthesized by Genscript company, and the nucleotide sequence is shown as SEQ ID NO. 2.
Example 2 construction of transgenic expression vectors
Construction of commercially synthesized ehEGF sequences into a psl1180[ Hr3PSer1spRedSer PA ] (see chinese patent publication No. CN108486123 a) intermediate vector containing a bombyx mori nuclear polyhedrosis virus enhancer Hr3, a bombyx mori sericin1 gene promoter PSer 1-and a 3'-UTR termination sequence Ser1PA, regulation of Red expression by the promoter PSer 1-and the 3' -UTR termination sequence Ser1PA, formation of psl1180[ Hr3PSer1sp-ehEGF-Ser1PA ], construction of ehEGF expression cassette into AscI site of pBac [3xp3DsRed ] vector, formation of transgenic expression vector pBac [3xp3DsRed; hr3Ser1p-rhEGF-Ser1PA ], PHSEHEGFSER (B in FIG. 1), the sequence is shown as SEQ ID NO.3, wherein positions 9-955 are enhancer Hr3, positions 968-1639 are Ser1 promoter, positions 1646-1903 are ehEGF gene, and positions 1912-2290 are SerPA.
EXAMPLE 3 construction of transgenic ehEGF Gene silkworm line
The QIAGEN PLASIMD MINI KIT plasmid extraction kit is used for extracting the transgenic expression vector PHSEHEGFSER1 and the auxiliary vector pHA3PIG plasmid, the plasmid concentration is diluted to 300-500 ng/. Mu.L, and the diluted plasmid concentration is respectively mixed with the auxiliary vector pHA3PIG plasmid according to the molar ratio of 1:1. Injecting the mixed plasmid into the diapause-relieved large-sized early embryo (2-5 h after spawning), sealing the injection hole by using nontoxic glue, sterilizing by using 35% formaldehyde steam for 5 minutes, incubating in an environment with a relative humidity of 85%, rearing the hatched larva (G0 generation) by using artificial feed, carrying out selfing or backcross breeding after adult, detecting the obtained G1 generation silkworm egg (day 6-7) under a macroscopic stereoscopic fluorescent microscope (Olypus MVX, japan), detecting by using excitation light with a wavelength of 510-550 nm in red fluorescent observation, screening out a transgenic positive moth ring with a wavelength of 460-490 nm in green fluorescent observation, and naming sgHSA (C in fig. 1). The fluorescence screening statistics of the transgenic silkworms are shown in Table 1, wherein 8 positive moth rings are screened out of 12G 1 generation moth rings in total, and the positive rate is 67%.
Table 1, table ehEGF Gene transferred silkworm microinjection and fluorescence screening statistical Table
Example 4 detection of expression of ehEGF recombinant protein in cocoons secreted by silkworms transgenic ehEGF Gene
And (3) incubating the screened positive silkworm eggs in an environment with the temperature of 25 ℃ and the relative humidity of 85%, rearing the hatched larvae (G1 generation) to the upper cluster of spinning cocoons by adopting artificial feed or mulberry leaves, screening positive silkworm chrysalis (C in figure 1) with red fluorescent marks on eyes by utilizing a fluorescent microscope, and carrying out exogenous protein expression detection on the silkworm cocoons. The method for extracting and detecting recombinant protein in the total protein of cocoon shells comprises the following steps: crushing cocoon shells into powder in liquid nitrogen, dissolving the powder in a buffer solution of 20mM Tris-Cl, pH7.9 and 8M Urea according to the concentration of 10-50mg/mL cocoon shells, treating the powder in a water bath kettle at 80 ℃ for 30-45min, and then centrifugally collecting the supernatant, wherein the centrifugation conditions are as follows: 18000X rpm,4℃for 15min. The extracted total cocoon shell proteins were subjected to 12% SDS-Page electrophoresis and stained with Coomassie Brilliant blue. The extracted total protein is separated by electrophoresis of 12% SDS-Page gel, and the protein in the SDS-Page gel is transferred onto PVDF membrane by electrotransfer method. PVDF membranes were placed in TBST buffer containing 5% nonfat milk powder and blocked overnight at 4 ℃. Before immunohybridization, PVDF membrane was washed 3 times with TBST at room temperature for 10min each. Preparing an EGF-resistant primary antibody (Abcam) hybridization solution by using TBST containing 5% skimmed milk powder according to 10000-fold dilution ratio, immersing a PVDF film into the hybridization solution, incubating for 2h at room temperature under shaking, and washing the film by TBST for 5 times, each time for 5min. Preparing a goat anti-rabbit secondary antibody (purchased from Biyun Tian Co.) hybridization solution marked by HRP by using TBST according to 20000 times of dilution ratio, immersing a PVDF film cleaned by TBST into the secondary antibody hybridization solution, incubating for 2h at room temperature in an oscillating way, and washing the film by TBST for 5 times each for 5min. And placing the cleaned PVDF film on a clean preservative film, uniformly dripping ECL color development liquid (Amersham Biosciences) on the PDVF film surface, incubating for 5min at room temperature in a dark place, and exposing and imaging by using a Chemiscope Series (Clinx science instruments) instrument. The results showed that, between 10-15kDa protein molecular weight markers, there was a band of difference from the control silkworm in the cocoon protein sample of the transgenic silkworm group, which was presumed to be ehEGF recombinant proteins, and the intensity of this band of difference protein was evident between individuals, presumably caused by different transgene insertion sites (D in FIG. 1). By carrying out gray scale calculation on protein bands, the average content of ehEGF recombinant proteins in silk between different individuals is estimated to be about 6.24+/-0.59%. Further, western Blot is used to verify that the differential protein band is ehEGF recombinant protein (D in figure 1), which shows that the constructed transgenic silkworm successfully expresses ehEGF recombinant protein in silk gland and is secreted into silk. And finally, reserving the transgenic silkworm individual with the highest protein expression quantity for seed production.
Example 5 detection of cell proliferation-promoting Activity of ehEGF recombinant proteins in Silk
To extract the active ehEGF recombinant protein in silk, the ehEGF cocoons secreted and synthesized by the ehEGF gene transgenic silkworm strain are cooled by liquid nitrogen and crushed into cocoon powder. Then, ehEGF cocoon powder was added to PBS buffer (pH=7.0) according to a bath ratio of 1-5% (w/v), stirred well at room temperature for 0.5-2 hours, centrifuged at 9000-12000rpm at 4-16℃for 15-45min, and the supernatant was collected to obtain ehEGF recombinant protein crude extract. Gray scale comparison is carried out on the silk crude extract and EGF standard substances by utilizing SDS-Page and western blot technology, and the concentration of ehEGF recombinant proteins in the silk crude extract is estimated to be 10-30 mug/mL (figure 2).
Subsequently, human immortalized epidermal cell HaCaT cell lines were cultured in DMEM medium containing 10% (v/v) fetal bovine serum (FBS, gibco) at 37℃under 5% CO 2, haCaT cells grown to 96% confluency were collected and plated in 96-well plates adherent at a cell concentration of 500/100. Mu.L/well, and starved for 24 hours in DMEM medium containing 0.5% (v/v) fetal bovine serum (FBS, gibco). According to the working concentration of ehEGF with the final concentration of 50-100ng/mL, adding a certain volume of ehEGF protein crude extract into a 96-well plate, taking an EGF standard substance with the same concentration as a positive control, taking a normal silk crude extract with the same volume as a negative control, and respectively carrying out proliferation detection on cells at 24 and 72 hours of incubation. The method comprises the following steps: first, the growth of cells after silk incubation was observed under a microscope. Subsequently, CCK-8 reagent (available from Biyun Tian Co.) was added to the 96-well plate at a dose of 10. Mu.L/well, and after reaction at 37℃for 4 hours, the absorbance of cells in the wells was measured at a wavelength of 450 nm. Each test group was set up with 3 parallel trials and repeated 3 more times. The results show that ehEGF crude extract group has significant cell proliferation promoting activity on HaCaT cells at 24 hours and 48 hours time points, and the cell proliferation promoting effect is significantly better than that of the EGF commercial standard (a-B in fig. 3) of the same dose, compared with the blank control group and the WT silk crude extract group. Subsequently, DNA replication of cells after 24h of co-culture of each sample with the test cells was examined using EdU staining, red fluorescence (EdU) indicating the cells in the proliferation state, and nuclei were stained using DAPI (blue fluorescence). The results show that: the ehEGF crude extract treated group had significantly enhanced red fluorescence signal at 24 hours of HaCaT cells compared to the blank and WT silk crude extract treated groups, and the intensity of the red fluorescence signal was significantly higher than that of the EGF commercial standard treated group at the same dose (C in fig. 3). The above results indicate that the extracted ehEGF recombinant protein crude extract has cell proliferation promoting activity, and the EGF is artificially engineered to form ehEGF by fusing HIV-1TAT Protein Transduction Domain (PTD) at the N-terminal and PlGF-2 motif at the C-terminal of natural EGF, so that the signal transduction and extracellular matrix binding efficiency of EGF are improved, and the EGF exhibits better cell proliferation promoting activity than commercial EGF.
In conclusion, the efficient secretory expression of ehEGF protein in the transgenic silkworm silk gland can be realized through the silkworm hSRSE expression system, and ehEGF recombinant protein with cell proliferation promoting activity is synthesized. The ehEGF recombinant protein secreted by the synthesis of the silkworm silk gland bioreactor can be separated and purified to form ehEGF preparation, and can be combined with silk to prepare functional silk biological materials for promoting wound healing and tissue repair, and the preparation can be widely applied to the fields of medical cosmetology, tissue engineering and the like in the future.
The above-described embodiments are merely preferred embodiments for fully explaining the present invention, and the scope of the present invention is not limited thereto. Equivalent substitutions and modifications will occur to those skilled in the art based on the present invention, and are intended to be within the scope of the present invention. The protection scope of the invention is subject to the claims.
Claims (9)
1. EhEGF recombinant protein having cell proliferation promoting activity, characterized in that: the ehEGF recombinant protein is obtained by taking human EGF mature peptide as a base, fusing a PTD sequence at the N-terminal of the human EGF mature peptide and fusing a PlGF-2 motif at the C-terminal; the amino acid sequence of ehEGF recombinant protein is shown as SEQ ID NO. 1.
2. EhEGF recombinant protein coding gene suitable for high-efficiency expression of silk gland of domestic animals, and is characterized in that: the nucleotide of the ehEGF recombinant protein coding gene is shown as SEQ ID NO. 2.
3. An expression vector comprising the ehEGF recombinant protein coding gene which is suitable for high-efficiency expression in silk gland of domestic animal according to claim 2.
4. The expression vector of claim 3, wherein: the expression vector sequentially contains an enhancer hr3, a secretory sericin 1 gene promoter, ehEGF recombinant protein coding genes and a terminator of the sericin 1 gene.
5. The expression vector of claim 3 or 4, wherein: the expression vector is connected with BamHI and NotI cleavage sites of psl1180[ hr3PSer1spRedSer PA ] by a sequence shown in SEQ ID NO.2, and then connected with a pBac [3xp3DsRedaf ] vector subjected to the same cleavage after being digested with AscI.
6. Use of ehEGF recombinant protein coding gene according to claim 2 or expression vector according to any one of claims 3-5 in the production of silk with pro-cell proliferation activity in silkworms.
7. A method for producing silk having cell proliferation promoting activity in silkworms using the ehEGF recombinant protein-encoding gene of claim 2, characterized by: injecting an expression vector containing ehEGF recombinant protein coding genes into silkworm eggs with diapause removed, sealing with nontoxic glue, sterilizing with formaldehyde steam, incubating, performing selfing or backcross seed production after adults, screening transgenic positive moth rings, and obtaining the cocoon shells of positive transgenic silkworms, namely silkworms with cell proliferation promoting activity.
8. Use of ehEGF recombinant protein coding gene according to claim 2 or expression vector according to any one of claims 3-5 in production of ehEGF recombinant protein with pro-cell proliferation activity in silkworms.
9. Use of the recombinant protein ehEGF according to claim 1 for the preparation of a material for promoting cell proliferation, characterized in that: the nucleotide of the ehEGF recombinant protein coding gene is shown as SEQ ID NO. 2.
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