Coding sequence of a recombinant protein, recombinant protein, preparation method, recombinant strain and application

Abstract

The present invention relates to the technical field of genetic engineering, and specifically discloses a coding sequence of a recombinant protein, the recombinant protein, a preparation method, a recombinant strain and applications thereof. The coding sequence of the recombinant protein includes the coding sequences shown in SEQ01 or SEQ03. The amino acid sequence of the recombinant protein is shown in SEQ ID NO.02 or SEQ ID NO.04. The present invention solves the problems in the conventional technology that the expression level of vascular factors after spinal cord injury is relatively low, and the number of regenerative blood vessels repaired by conventional drug treatments is limited. The present invention has the effect of promoting blood vessel formation after spinal cord injury.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and particularly relates to a coding sequence of a recombinant protein, the recombinant protein, a preparation method, a recombinant strain and an application. Background Art

[0002] Spinal cord injury (SCI) is a severe nerve trauma with a high disability rate. After injury, the local microenvironment is damaged, leading to the rupture of the vascular network, which in turn causes ischemia, hypoxia, and blood-spinal cord barrier dysfunction. These pathological changes enable inflammatory cells to infiltrate into the injury site, exacerbating the inflammatory response. Angiogenesis can restore blood supply to the damaged area, improve the local microenvironment, and support neuron survival and axon regeneration. Therefore, in the early stage (3 - 7 days) after injury, timely vascular intervention can optimize the local vascular structure and blood supply, reducing cell loss and neurological deficits.

[0003] After spinal cord injury, the expression level of vascular factors is low, and the number of pro-regenerative blood vessels repaired by conventional drug therapy is limited. Fibroblast growth factors (FGFs) play important roles in various biological processes, including cell proliferation, metabolism, and differentiation. FGFs are divided into paracrine, endocrine, and intracellular subfamilies. Previous studies have shown that FGFs alone or in combination therapy exhibit significant therapeutic effects in SCI animal models. Different from other FGFs subfamilies, intracellular FGFs (iFGFs, FGF11 - FGF14) act intracellularly without activating FGFs receptors. iFGFs are expressed in various cells, including neurons, cardiomyocytes, osteoclasts, and fibroblasts. However, most studies have focused on their role as regulators of voltage-gated ion channels in the nervous system. However, recent studies have begun to reveal their potential role in angiogenesis. Different from mammals, zebrafish have a strong nerve regeneration ability, and even in adults, they can recover normal swimming activities within 6 - 8 weeks after spinal cord injury. However, the function of Fgfs derived from zebrafish has rarely been studied. Summary of the Invention

[0004] In order to overcome the above deficiencies that the expression level of vascular factors is low after spinal cord injury and the number of pro-regenerative blood vessels repaired by conventional drug therapy is limited, the present invention provides a recombinant protein that can significantly increase the expression level of vascular factors after spinal cord injury, and the number of blood vessels is significantly increased, promoting the good effect of blood vessel formation after spinal cord injury.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] A coding sequence of a recombinant protein, comprising: the coding sequence shown in SEQ ID NO.01 or SEQ ID NO.03.

[0007] The present invention also discloses a recombinant protein, which is prepared using the coding sequences shown in SEQ ID NO.01 or SEQ ID NO.03, and the amino acid sequence of the recombinant protein is shown in SEQ ID NO.02 or SEQ ID NO.04.

[0008] The present invention also discloses a method for preparing a recombinant protein, comprising the following steps:

[0009] a. The zebrafish Fgf11a nucleotide sequence or the mouse FGF11b nucleotide sequence is respectively obtained by PCR amplification or gene synthesis to obtain the coding sequences shown in SEQ ID NO.01 or SEQ ID NO.03;

[0010] b. Recombinantly introduce into a prokaryotic expression vector using homologous arms to respectively obtain the corresponding recombinant plasmid vectors.

[0011] Optionally, the primers used in the PCR amplification operation include the primers shown in SEQ ID NO.05 or SEQ ID NO.06.

[0012] Optionally, the primers used in the homologous arm recombination operation include the primers shown in SEQ ID NO.07, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10.

[0013] Optionally, the method for preparing a recombinant protein of the present invention further comprises step c, and the operation method of step c includes: after transforming the engineering bacteria, picking monoclonal colonies and inoculating them into an autoinduction complex medium for culturing, and centrifuging to collect the bacterial cells.

[0014] Adding a bacterial lysate to the bacterial cells, then ultrasonically lysing to obtain a precipitate; after washing the precipitate, adding a urea solution, and shaking and dissolving overnight followed by centrifuging to collect the supernatant; loading the supernatant onto a column, washing away the impurity proteins, eluting and collecting the purified protein.

[0015] Optionally, the method for preparing a recombinant protein of the present invention further comprises step d, and the operation method of step d includes: placing the purified protein in a dialysis bag and soaking it in a gradient dialysis buffer solution for dialysis.

[0016] The present invention also discloses a recombinant strain containing the coding sequences shown in SEQ ID NO.01 or SEQ ID NO.03.

[0017] The present invention also discloses the application of a recombinant protein in promoting angiogenesis after spinal cord injury.

[0018] The beneficial effects of the present invention at least include: the recombinant protein can significantly increase the expression level of vascular factors after spinal cord injury, and the number of blood vessels increases significantly, promoting the formation of blood vessels after spinal cord injury with good effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 It is a detection diagram of prokaryotic expression of the recombinant protein of the present invention;

[0020] Figure 2 It is a schematic diagram of the effect of the recombinant protein of the present invention on the formation of blood vessels in HUVEC cells;

[0021] Figure 3 It is a schematic diagram of the expression of proteins at the spinal cord injury site 4 days after injection of the recombinant protein of the present invention;

[0022] Figure 4 It is a VEGFA protein level diagram at the spinal cord injury site 4 days after injection of the recombinant protein of the present invention;

[0023] Figure 5 It is an immunofluorescence detection diagram of CD31 at the spinal cord injury site 4 days after injection of the recombinant protein of the present invention;

[0024] Among them: M, marker; 1, culture medium; 2, bacterial cells; 3, supernatant after bacterial cell disruption; 4, precipitate after bacterial cell disruption; 5, protein after purification by nickel column. Figure 1 Among them: M, marker; 1, culture medium; 2, bacterial cells; 3, supernatant after bacterial cell disruption; 4, precipitate after bacterial cell disruption; 5, protein after purification by nickel column. DETAILED DESCRIPTION OF THE INVENTION

[0025] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The raw materials and equipment used in the following specific embodiments of the present invention are all known products and can be obtained by purchasing commercially available products. The engineering bacteria in the present invention adopt Transetta(DE3) Chemically Competent Cell of Beijing TransGen Biotech Co., Ltd. Transetta(DE3) is made from imported strains by special processes and can be used for chemical transformation of DNA. The cells are resistant to chloramphenicol (Camr). When detected with pUC19 plasmid DNA, the transformation efficiency can reach 107 cfu / μg DNA. Control Plasmid I (Amp+) is used to detect whether the cells have an expression function, and the size of the expressed protein is 25 kDa. In the present invention, SDS-PAGE is the abbreviation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which is mainly used for separating and analyzing components in a protein mixture. In the present invention, Tris-HCl, that is, tris(hydroxymethyl)aminomethane hydrochloride, is a commonly used buffer and can adopt the products of Solarbio Science & Technology Co., Ltd. The BCA kit of the present invention can purchase the BCA kit of Beyotime Biotechnology Co., Ltd. The PBS buffer solution can purchase the products of Solarbio Science & Technology Co., Ltd. In the present invention, western blot is immunoblotting. Immunoblotting, also known as Western blotting, is a method for detecting a certain protein in a complex sample based on the specific binding of antigen and antibody. The OCT embedding medium can adopt the products of Epredia Experimental Instruments Manufacturing (Shanghai) Co., Ltd.

[0026] In the present invention, PFA (paraformaldehyde) can purchase the products of Solarbio Science & Technology Co., Ltd. BSA (bovine serum albumin) can adopt the products of Thermo Fisher Scientific. The laser confocal C2-2 instrument can adopt C2si of nikon. The Loading solution in the present invention is the loading buffer. In the present invention, CD31 (also known as PECAM-1, PlateletEndothelial Cell Adhesion Molecule-1) is a transmembrane glycoprotein mainly expressed on the surface of endothelial cells and is a specific marker of vascular endothelial cells, which is widely used for detecting angiogenesis (such as tumor angiogenesis, tissue repair, etc.). In this detection, the primary antibody: anti-CD31 antibody (the species needs to match the sample source, such as mouse anti-human CD31 monoclonal antibody); the secondary antibody: fluorescently labeled secondary antibody (such as Alexa Fluor 488 / 594-labeled anti-mouse IgG); the blocking solution: 1-5% BSA; the mounting medium: anti-fluorescence quencher containing DAPI (such as Southern Biotech) PBS buffer solution (pH 7.4).

[0027] The present invention discloses a coding sequence of a recombinant protein, comprising: the coding sequences shown in SEQ ID NO.01 or SEQ ID NO.03. SEQ ID NO.01 or SEQ ID NO.03 of the present invention can be found in the sequence listing. The nucleotide sequence of SEQ ID NO.01 corresponds to the nucleotide sequence XM_021477520.1 of zebrafish Fgf11a (ZFgf11a) (this nucleotide sequence is publicly available on NCBI and was collected by the inventor himself, see the disclosure table of the source of genetic resources for details), and the nucleotide sequence shown in SEQ ID NO.03 corresponds to the nucleotide sequence NM_001291104.2 of mouse FGF11b (MFGF11b) (this nucleotide sequence is publicly available on NCBI and was obtained by purchasing on the market, see the disclosure table of the source of genetic resources for details).

[0028] A recombinant protein is prepared using the coding sequences shown in SEQ ID NO.01 or SEQ ID NO.03, and the amino acid sequence of the recombinant protein is as shown in SEQ ID NO.02 or SEQ ID NO.04. SEQ ID NO.02 and SEQ ID NO.04 of the present invention are referred to the sequence listing.

[0029] The present invention also discloses a recombinant strain containing the coding sequences shown in SEQ ID NO.01 or SEQ ID NO.03.

[0030] The primers used for PCR amplification in the present invention are shown in Table 1 below:

[0031] Table 1 Primers used for PCR amplification in the present invention

[0032] 。

[0033] The primers used for homologous arm recombination in the present invention are shown in Table 2 below:

[0034] Table 2 Primers used for homologous arm recombination in the present invention

[0035] 。

[0036] The present invention discloses a method for preparing a recombinant protein, comprising the following steps:

[0037] a. According to the nucleotide sequences XM_021477520.1 of zebrafish Fgf11a (ZFgf11a) and NM_001291104.2 of mouse FGF11b (MFGF11b) published in the NCBI database, the coding sequences of ZFgf11a and MFGF11b are respectively obtained by PCR amplification or gene synthesis.

[0038] The primers used in the PCR amplification operation include the primers shown as ZFgf11a-F1 and ZFgf11a-R1.

[0039] b. After purification, it is recombinated into the prokaryotic expression vector pET-30a(+)-GFP using homologous arms to obtain the recombinant plasmids pET-30a(+)-GFP-ZFgf11a and pET-30a(+)-GFP-MFGF11b. The primers used in the homologous arm recombination operation include the primers shown as ZFgf11a-F2, ZFgf11a-R2, MFGF11b-F2 or MFGF11b-R2.

[0040] c. Transform the recombinant protein expression vector into engineering bacteria, and screen to obtain recombinant protein expression strains; after transforming the engineering bacteria, pick monoclonal colonies and inoculate them into an auto-inducing complex medium, and culture under predetermined conditions; centrifuge to collect the bacterial cells; add bacterial lysis solution to the bacterial cells, then ultrasonically lyse to obtain a precipitate; wash the precipitate and then add urea solution, and dissolve it by shaking overnight and centrifuge to collect the supernatant; load the supernatant onto a column, wash the column with urea solution to wash away the impurity proteins, elute and collect the purified protein.

[0041] d. Place the purified protein in a dialysis bag and soak it in a gradient dialysis buffer solution for dialysis.

[0042] Example 1: A method for preparing a recombinant protein, comprising the following steps:

[0043] a. According to the nucleotide sequence XM_021477520.1 of zebrafish Fgf11a (ZFgf11a) published in the NCBI database, use PCR amplification to obtain the ZFgf11a coding sequence.

[0044] The primers used in the PCR amplification operation include the primer shown as ZFgf11a-F1.

[0045] b. After purification, it is recombinated into the prokaryotic expression vector pET-30a(+)-GFP using homologous arms to obtain the recombinant plasmid pET-30a(+)-GFP-ZFgf11a. The primers used in the homologous arm recombination operation include the primers shown as ZFgf11a-F2 and ZFgf11a-R2.

[0046] c. Transform the recombinant protein expression vector into engineering bacteria, and screen to obtain recombinant protein expression strains; after transforming the engineering bacteria, pick monoclonal colonies and inoculate them into an auto-induction complex medium, and culture at 37 °C and 200 rpm for 18 h; centrifuge to collect the bacterial cells; add bacterial lysis solution to the bacterial cells, then ultrasonically lyse to obtain a precipitate; wash the precipitate and add urea solution, and centrifuge after shaking and dissolving overnight to collect the supernatant; load the supernatant onto the column, wash the column with urea solution to wash away the impurity proteins, and elute and collect the purified protein.

[0047] d. Place the purified protein in a dialysis bag and soak it in a gradient dialysis buffer solution for dialysis.

[0048] Among them, the steps are specifically as follows: after transforming the recombinant plasmid into the commercial Transetta(DE3) competent cells (TransGen Biotech), pick monoclonal colonies and inoculate them into 250 mL of auto-induction complex medium containing 0.1% kanamycin, and culture at 37 °C and 200 rpm for 18 - 20 h. Centrifuge at 4,000 g at 4 °C for 10 minutes to collect the bacterial cells. Add 30 mL of bacterial lysis solution (containing 1% protease inhibitor mixture) to the bacterial cells, and then ultrasonically lyse (30% power, ultrasonic for 3 s, stop for 3 s) for 30 min. Centrifuge at 10,000 g at 4 °C for 15 minutes to obtain a precipitate. Wash the precipitate with PBS and add 30 mL of high-concentration urea solution (20 mM Tris-HCl, 5 mM imidazole, 0.5 M sodium chloride, and 8 M urea), and shake and dissolve overnight at 4 °C. Centrifuge at 10,000 g for 20 min to collect the supernatant. Load the supernatant onto the column at a flow rate of 10 column volumes per hour. Wash the column with 15 column volumes of high-concentration urea solution to wash away the impurity proteins. Elute with high-concentration imidazole and urea solution, and collect the purified protein solution. Place the purified protein in a dialysis bag with a molecular weight cut-off of 50,000 and soak it in a gradient dialysis buffer solution, and dialyze each dialysis solution for 6 h. The composition of dialysis solution I is 25 mM Tris, 350 mM NaCl, 2 mM reduced glutathione, 196 μL oxidized glutathione, 250 mM imidazole, 4 M urea, 10% glycerol; the composition of dialysis solution II is 25 mM Tris, 200 mM NaCl, 2 mM reduced glutathione, 100 mM imidazole, 2 M urea, 10% glycerol; the composition of dialysis solution III is 25 mM Tris, 100 mM NaCl, 2 mM reduced glutathione, 0 mM imidazole, 100 mM urea, 10% glycerol; the composition of dialysis solution IV is 25 mM Tris, 50 mM NaCl, 2 mM reduced glutathione, 0 mM imidazole, 0 mM urea, 10% glycerol.

[0049] Example 2: A method for preparing a recombinant protein, comprising the following steps:

[0050] a. According to the published and verified mouse FGF11b (MFGF11b) nucleotide sequence NM_001291104.2 in the NCBI database, the MFGF11b coding sequence was obtained by gene synthesis.

[0051] b. After purification, it was recombinantly inserted into the prokaryotic expression vector pET-30a(+)-GFP using homologous arms to obtain the recombinant plasmid pET-30a(+)-GFP-MFGF11b. The primers used in the homologous arm recombination operation included primers such as MFGF11b-F2 or MFGF11b-R2.

[0052] c. The recombinant protein expression vector was transformed into engineering bacteria, and the recombinant protein expression strain was screened; after transforming the engineering bacteria, a single clone was picked and inoculated into an auto-inducing complex medium, and cultured at 37 °C and 200 rpm for 18 h; the cells were collected by centrifugation; bacterial lysate was added to the cells, and then sonicated to obtain a precipitate; after washing the precipitate, a urea solution was added, and it was dissolved by shaking overnight and centrifuged, and the supernatant was collected; the supernatant was loaded onto a column, and the column was rinsed with a urea solution to wash away the miscellaneous proteins, and the purified protein was eluted and collected.

[0053] d. The purified protein was placed in a dialysis bag and dialyzed in a gradient dialysis buffer solution. The remaining specific operation steps were the same as those in Example 1.

[0054] During the expression and purification of the protein in Example 1 and Example 2, 200 μL of the culture medium, cells, supernatant after cell disruption, precipitate after cell disruption, and purified protein were collected respectively, 50 μL of 5× loading buffer was added, boiled at 100 °C for 10 min, and after cooling, the protein was detected by SDS-PAGE electrophoresis. The test results are as Figure 1 shown. After protein purification, the bands of miscellaneous proteins were significantly reduced.

[0055] The present invention discloses the application of a recombinant protein in promoting angiogenesis after spinal cord injury.

[0056] For the identification of the blood vessel-promoting function of the recombinant protein:

[0057] Thaw and resuscitate HUVEC cells and culture them. Plate the cells with good growth status. After 24 h of plating, 2 μg of zebrafish Fgf11a-GFP and mouse FGF11b-GFP recombinant proteins were respectively mixed with the prepared liposomes to prepare recombinant protein-liposome complexes, which were added to the cell culture medium and cultured at 37 °C under 5% CO2 for 24 h. Add commercialized Matrigel (Corning) to a 48-well plate and place it at 37 °C for 30 min. After it solidified, digest and collect the HUVEC cells transiently expressing zebrafish Fgf11a-GFP and mouse FGF11b-GFP recombinant proteins, inoculate them into the Matrigel at a cell density of 50,000 cells per well, and culture them at 37 °C under 5% CO2 for 8 h, then observe the angiogenesis situation under an optical microscope and take pictures. Use ImageJ software for statistical analysis. After transfection of HUVEC cells with Fgf11a-GFP and mouse FGF11b-GFP recombinant proteins, the number of blood vessels formed increased significantly. The specific experimental results are as Figure 2 shown.

[0058] Effect of recombinant protein on angiogenesis after spinal cord injury:

[0059] Raise 8-week-old female C57BL / B6 mice. Set up a GFP control group, a zebrafish ZFgf11a recombinant protein injection group, and a mouse MFGF11b recombinant protein injection group, with 18 mice in each group. The injection dose of the recombinant protein was 20 μg / mouse. Before injecting the recombinant protein, anesthetize the mice by intraperitoneal injection of 1% sodium pentobarbital. Open the vertebral laminae of the 8th - 10th segments, and use a striker to strike the spinal cord at the 10th thoracic vertebra to form a contusive spinal cord injury model. According to the injection dose, mix the recombinant protein with liposomes in equal volume before injection, and then use a microinjector to inject GFP, zebrafish ZFgf11a recombinant protein, and mouse MFGF11b recombinant protein in situ into the injury center at an injection speed of 1 μL. After injection, suture the injury area, and help the mice urinate smoothly by squeezing and massaging every day. Four days after injection, anesthetize the mice, then dissect to obtain the mouse spinal cord, and observe the protein content in the spinal cord using green fluorescence in a live imaging instrument. In the present invention, the method of GFP (green fluorescent protein) is the same as the method of expressing the recombinant protein, and the vector used is the vector of the recombinant protein.

[0060] Mix the spinal cord with a protein extraction reagent, homogenize and extract total tissue proteins. Use a BCA kit to measure the protein concentration. According to the protein concentration, add an appropriate amount of 5×loading buffer solution and 1×PBS buffer solution to prepare WB protein samples. Boil at 100 °C for 10 min, and then use western blot to detect the expression of the angiogenesis factor VEGFA at the spinal cord injury site.

[0061] Meanwhile, the spinal cord was placed in 4% PFA solution and fixed at 4°C for 24 h, and then dehydrated with 20% sucrose solution at 4°C for 24 h; dehydrated with 30% sucrose solution at 4°C for 24 h. After embedding with OCT embedding agent, the frozen tissue blocks were placed in a microtome to cut tissue sections with a thickness of 16 μm. The sections were blocked with 5% BSA at 37°C for 30 min, the primary antibody was diluted with 1% BSA, and incubated overnight at 4°C. Rewarm at 37°C for 1 h. The primary antibody was diluted with 1% BSA at a ratio of 1:500 and incubated at 37°C for 1 h. Wash with 1×PBST buffer three times, 5 min each time. Mount with a mounting medium containing DAPI, and collect images by laser confocal C2-2. Use Image J software to count the random areas of at least 3 images, and the data results were processed by GraphPad Prism 8.0.1 software to generate statistical charts. Four days after protein injection, green fluorescence could still be observed at the spinal cord injury site, indicating that the recombinant protein still existed at the injury site. The specific results are as Figure 3 shown.

[0062] Four days after injection of mouse and zebrafish FGF11 recombinant protein, the protein level of vascular endothelial growth factor VEGFA increased at the spinal cord injury site (see Figure 4 ), and the immunofluorescence detection result of the neovascularization marker protein CD31 showed that the neovascular density increased significantly (see Figure 5 ).

[0063] The specific embodiments described in the present invention are only illustrative of the spirit of the present invention. Those skilled in the art to which the present invention pertains can make various modifications or supplements to the described specific embodiments or use similar ways to replace them, but will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

[0064] Although the present invention has been described in detail and some specific embodiments have been cited, it is obvious that various changes or modifications can be made by those skilled in the art without departing from the spirit and scope of the present invention.

Claims

1. Use of a recombinant protein in the preparation of a drug for promoting angiogenesis after spinal cord injury, characterized in that, The amino acid sequence of the recombinant protein is shown in SEQ ID NO.02 or SEQ ID NO.04.

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