Rhcol-ctgf fusion protein and preparation method and application thereof

Abstract

The preparation method of the HLC-CTGF fusion protein disclosed in the application comprises the following steps: designing a target gene and synthesizing and enzyme cutting sites, then reactivating a bacterial strain, and then sequentially performing expression vector construction of the fusion protein HLC-CTGF, expression of the fusion protein HLC-CTGF, purification of the fusion protein HLC-CTGF, and Western blotting of the fusion protein HLC-CTGF. The rhCol-CTGF fusion protein prepared in the application is non-toxic and has good cell compatibility. The fusion protein can be cross-linked to prepare a hydrogel scaffold, collagen provides necessary nutrition and structural support, and creates a good environment for the growth of osteoblasts; the degradation of the hydrogel is controlled to release CTGF, the time-dependent disadvantage of the expression of CTGF in the body is overcome, and the bone tissue repair is accelerated.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to rhCol-CTGF fusion protein, a method for preparing rhCol-CTGF fusion protein, and applications of rhCol-CTGF fusion protein. Background Technology

[0002] Connective tissue growth factor (CTGF) is a cysteine-rich polypeptide that promotes the synthesis of the extracellular matrix (ECM) and participates in tissue repair processes. In bone tissue, it supports bone regeneration by enhancing the adhesion and migration of bone marrow stromal stem cells; CTGF overexpression also increases type I collagen synthesis, providing a basis for calcium salt deposition and thus promoting bone formation. Furthermore, CTGF regulates angiogenesis during endochondral ossification, supporting osteocyte metabolism by enhancing endothelial cell migration. However, CTGF expression during bone tissue repair is time-dependent. For example, in the in vitro induction of osteogenic differentiation of mesenchymal stem cells, CTGF is only expressed within the first 7 days of osteogenic induction, followed by a sharp decline in expression. This means that after playing its role at a specific stage of bone repair, it cannot provide sufficient sustained support, affecting the integrity and long-term effectiveness of bone repair.

[0003] Collagen is a major component of the organic matter in bones. Its fibrous structure weaves together to provide structural support for bones, giving them elasticity and resilience, helping them withstand pressure and maintain their shape. Secondly, collagen can stimulate the activity of osteoblasts, promoting their proliferation and differentiation. Osteoblasts are the cells responsible for generating new bone tissue, and their activity level directly affects bone growth and repair. Collagen creates a favorable environment for osteoblast growth by providing essential nutrients and structural support. Summary of the Invention

[0004] The purpose of this invention is to provide an rhCol-CTGF fusion protein, which solves the problem that the expression of CTGF in the prior art has a time-dependent effect on the integrity and long-term effect of bone repair.

[0005] A second objective of this invention is to provide a method for preparing the rhCol-CTGF fusion protein.

[0006] A third objective of this invention is to provide the application of the rhCol-CTGF fusion protein in the preparation of hydrogels.

[0007] The first technical solution adopted in this invention is an rhCol-CTGF fusion protein, the rhCol-CTGF amino acid sequence of which is shown in Sequence 1.

[0008] The invention is further characterized by:

[0009] The nucleic acid sequence of rhCol-CTGF is shown in Sequence 2.

[0010] The second technical solution adopted in this invention is a method for preparing rhCol-CTGF fusion protein, which is specifically implemented according to the following steps:

[0011] Step 1: Target gene synthesis and restriction enzyme site design;

[0012] Step 2, activate the bacterial strain;

[0013] Step 3: Construction of the expression vector for the fusion protein rhCol-CTGF;

[0014] Step 4: Expression of the fusion protein rhCol-CTGF;

[0015] Step 5: Purification of the fusion protein rhCol-CTGF;

[0016] Step 6: Western blot of the fusion protein rhCol-CTGF.

[0017] The second technical solution of the present invention is further characterized in that,

[0018] Step 1 specifically involves: designing the rhCol-CTGF amino acid sequence and predicting the spatial structure of the fusion protein based on sequence 1; designing a nucleic acid sequence for rhCol-CTGF containing double restriction enzyme sites using E. coli codon bias; and obtaining the pUC57-rhCol-CTGF strain and plasmid.

[0019] Step 2 is implemented in the following steps:

[0020] Step 2.1: Prepare solid culture medium, sterilize at high temperature and cool, then add ampicillin to prepare LB agar plate medium;

[0021] Step 2.2: After the inoculation loop is heated and cooled under an alcohol lamp, a small amount of bacterial solution is taken onto a plate, streaked with the inoculation loop, and placed in a bacterial incubator for overnight incubation.

[0022] Step 2.3: After culturing, add ampicillin-resistant LB liquid culture to a sterile conical flask, then pick single colonies into a shake flask, culture for 6-8 hours, send the bacterial culture for sequencing and add 60% glycerol to preserve the bacterial strain.

[0023] Step 3 is implemented in the following steps:

[0024] Step 3.1: The obtained plasmid pUC57-rhCol-CTGF is digested with two enzymes to obtain the target fragment, and the expression vector pET28a is linearized by double enzyme digestion.

[0025] Step 3.2: Add 1 μL each of T4 DNA Ligase and T4 DNA Ligase buffer to a centrifuge tube at a volume ratio of rhCol-CTGF:pET28a = 3:1, and ligate overnight at 4°C to obtain the ligation product pET28a-rhCol-CTGF plasmid.

[0026] Step 3.3: Spread the bacterial culture onto kanamycin-resistant LB agar plates, then invert the plates and incubate them overnight in an incubator;

[0027] Step 3.4: Single clones should be cultured in kanamycin-resistant liquid medium and then verified by double enzyme digestion;

[0028] Step 3.5: Sequencing the plasmids whose band positions were verified by double enzyme digestion, then comparing and analyzing the sequencing results, and storing the bacterial strains with correct sequencing results in a -80°C freezer with 60% glycerol.

[0029] Step 4 is implemented in the following steps:

[0030] Step 4.1: Transform the plasmid that was correctly sequenced in Step 3 into E. coli BL21(DE3) competent cells, then plate them on kanamycin-resistant LB plates and incubate overnight in an incubator;

[0031] Step 4.2: Select single colonies, culture them overnight in kanamycin-resistant LB liquid medium, then transfer them to fresh medium for culture, take the bacterial culture, add 60% glycerol and store at -80℃, then add IPTG to the final concentration of 0.5mM, and continue to culture on a shaker for 4-5 hours;

[0032] Step 4.3: Centrifuge to collect bacteria, resuspend the bacterial cells in ddH2O, dilute with 5×SDS-PAGE Loading Buffer, boil in a water bath, and detect protein expression by SDS-PAGE electrophoresis.

[0033] Step 4.4: After electrophoresis, the gel is stained with Coomassie Brilliant Blue and then destained with destaining solution. The protein expression is observed under a gel imaging system and photographs are taken.

[0034] Step 5 is implemented in the following steps:

[0035] Step 5.1, Pretreatment of nickel column: Add nickel packing to the packing tube, open the cap to let the liquid in it flow out, and then add an equal volume of Binding-Buffer to resuspend the nickel packing.

[0036] Step 5.2: Add 3-6 times the volume of protein solution to the nickel column, cover it, and invert it to ensure that the nickel filler and protein solution are thoroughly mixed. Let it bind overnight on a rotary shaker.

[0037] Step 5.3: Open the cap, let stand to allow the protein solution to flow out into the collection tube, add 3 times the volume of Binding-Buffer, place on a rotating shaker, and then let stand to collect the outflow. Repeat once.

[0038] Step 5.4, elution column: Add 2 volumes of Elution Buffer to the nickel packing material, seal the column, place it on a rotary shaker, let it stand and collect the eluent, repeat twice;

[0039] Step 5.5: Cut the dialysis bag with a molecular weight cutoff of 30kD, boil it in a ddH2O bath, cool it, fix one end with a clip, add the purified protein into the dialysis bag, and then fix the other end of the dialysis bag.

[0040] Step 5.6: Place the dialysis bag containing protein into a measuring cup containing ddH2O, dialyze at 4°C, change the ddH2O every 4 hours, and dialyze for another 48 hours.

[0041] Step 5.7: Pour the dialyzed protein solution into a culture dish, freeze overnight, then freeze-dry under vacuum in a freeze dryer and store in a desiccator.

[0042] Step 6 is implemented in the following steps:

[0043] Step 6.1: After preparing the protein sample, spot the sample and perform SDS-PAGE electrophoresis;

[0044] Step 6.2, Transfer: Soak the prepared filter paper and sponge in the freshly prepared transfer solution, then soak the PVDF membrane in methanol to activate it. Then place the sponge, filter paper, protein gel, PVDF membrane, filter paper and sponge in sequence, clamp them and place them in the transfer tank, pour in the transfer solution, and then place the entire tank in an ice-water mixture for constant flow transfer.

[0045] Step 6.3: After the transfer is complete, open the transfer clamp, take out the PVDF membrane, soak it in skim milk powder, and seal it on a shaker at room temperature.

[0046] Step 6.4: Dilute the His antibody and incubate overnight;

[0047] Step 6.5: After rewarming, recover the primary antibody and wash with PBST;

[0048] Step 6.6: Dilute the HRP secondary antibody and incubate on a shaker at room temperature;

[0049] Step 6.7: Recover the secondary antibody and wash with PBST.

[0050] Step 6.8, Development: Mix the color developing solution in a 1:1 volume ratio, apply it evenly to the membrane, and then develop and photograph it.

[0051] The third technical solution adopted in this invention is the application of rhCol-CTGF fusion protein in the preparation of hydrogels. Specifically, rhCol and rhCol-CTGF fusion protein are taken, TG is dissolved in PBS, and then they are taken out and placed in centrifuge tubes. After standing, their gelation time is observed to obtain the final product.

[0052] The beneficial effects of this invention are:

[0053] The rhCol-CTGF fusion protein prepared by this invention is non-toxic and has good cell compatibility. It can be cross-linked to prepare a hydrogel scaffold. Collagen provides necessary nutrition and structural support, creating a favorable environment for osteoblast growth. By controlling the degradation of the hydrogel to release CTGF, the time-dependent expression of CTGF in vivo is overcome, thus accelerating bone tissue repair. Attached Figure Description

[0054] Figure 1 This is a schematic diagram of the spatial structure prediction results in this invention;

[0055] Figure 2 This is a schematic diagram of the double enzyme digestion verification results in this invention;

[0056] Figure 3 This is a diagram showing the results of exploring and verifying the expression conditions of the fusion protein in this invention;

[0057] Figure 4 This is a diagram showing the exploration of expression conditions for the fusion protein in this invention and the results of Western blot verification.

[0058] Figure 5 This is a cytotoxicity detection diagram from the present invention;

[0059] Figure 6 This is a physical image of the hydrogel used in this invention. Detailed Implementation

[0060] The following detailed description is provided in conjunction with specific implementation methods.

[0061] The present invention relates to an rhCol-CTGF fusion protein, the rhCol-CTGF amino acid sequence of which is shown in Sequence 1.

[0062] The nucleic acid sequence of rhCol-CTGF is shown in Sequence 2.

[0063] Example 1

[0064] The preparation method of rhCol-CTGF fusion protein is carried out according to the following steps:

[0065] Step 1: Target gene synthesis and restriction enzyme site design;

[0066] Step 2, activate the bacterial strain;

[0067] Step 3: Construction of the expression vector for the fusion protein rhCol-CTGF;

[0068] Step 4: Expression of the fusion protein rhCol-CTGF;

[0069] Step 5: Purification of the fusion protein rhCol-CTGF;

[0070] Step 6: Western blot of the fusion protein rhCol-CTGF.

[0071] Example 2

[0072] The present invention discloses a method for preparing the rhCol-CTGF fusion protein, wherein step 1 specifically comprises: designing the rhCol-CTGF amino acid sequence and, based on sequence 1, predicting the spatial structure of the fusion protein using online protein structure prediction software (https: / / zhanggroup.org / I-TASSER / ). The spatial structure prediction results are as follows: Figure 1 As shown, the pUC57-rhCol-CTGF strain and plasmid were obtained by designing a nucleic acid sequence containing a (BamHI / XhoI) double restriction site for rhCol-CTGF using Escherichia coli codon preference.

[0073] Step 2 is implemented in the following steps:

[0074] Step 2.1: Prepare 100 mL of LB solid culture medium, sterilize at high temperature and cool, then add 100 μL of 100 mg / mL ampicillin to prepare LB plate culture medium;

[0075] Step 2.2: After the inoculation loop is heated and cooled under an alcohol lamp, a small amount of bacterial solution is taken onto a plate, streaked with the inoculation loop, and placed in a bacterial incubator at 37°C for overnight incubation.

[0076] Step 2.3: The next day, add 5 mL of ampicillin-resistant LB liquid culture medium to a 50 mL sterile Erlenmeyer flask, then pick single colonies into a shake flask, incubate for 6 h, send the bacterial culture for sequencing and add 60% glycerol to preserve the strain.

[0077] Step 3 is implemented in the following steps:

[0078] Step 3.1: The obtained plasmid pUC57-rhCol-CTGF was digested with double enzymes (BamHI / XhoI) to obtain the target fragment, and the expression vector pET28a was linearized by double enzyme digestion.

[0079] Step 3.2: Then, according to the volume ratio of rhCol-CTGF:pET28a = 3:1, add 1 μL each of T4 DNA Ligase and T4 DNA Ligase buffer to a 1.5 mL centrifuge tube, and ligate overnight at 4°C to obtain the ligation product pET28a-rhCol-CTGF plasmid; ligate the target fragment to the expression vector pET28a (with a SUMO tag), and verify the results with double enzyme digestion as shown below. Figure 2 As shown in the figure, 1 is the pET28a-rhCol-CTGF plasmid; 2 is the pET28a-rhCol-CTGF plasmid double digested with BamH1 and Xho1.

[0080] Step 3.3, transformation, the experimental method is the same as in step 5.3, spread the bacterial culture on kanamycin-resistant LB plates, then invert them in a 37°C incubator and incubate overnight;

[0081] Step 3.4: Single clones should be cultured in 5 mL of kanamycin-resistant liquid medium for 12 h, followed by double enzyme digestion for verification;

[0082] Step 3.5, Sequence determination and alignment: Plasmids with correctly positioned bands verified by double enzyme digestion were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were then compared and analyzed using MAGA software. Strains with correct sequencing results were stored in a -80°C freezer with 60% glycerol.

[0083] The results of the exploration and verification of expression conditions for the fusion protein are as follows: Figure 3 As shown in the figure, 1. rhCol-pET28a (SUMO) uninduced; 2. total rhCol-pET28a protein; 3. rhCol-pET28a supernatant; 4. rhCol-pET28a precipitate; 5. pET28a-rhCol-CTGF uninduced; 6. total pET28a-rhCol-CTGF protein; 7. pET28a-rhCol-CTGF supernatant; 8. pET28a-rhCol-CTGF precipitate.

[0084] The expression conditions for the fusion protein were explored and the results of Western blot validation are as follows: Figure 4 As shown.

[0085] Step 4 is implemented in the following steps:

[0086] Step 4.1: Transform the plasmids that were correctly sequenced in step 3 into E. coli BL21(DE3) competent cells, then plate them on kanamycin-resistant LB plates and incubate overnight at 37°C.

[0087] Step 4.2: On the second night, pick single colonies and culture them overnight in 10 mL of kanamycin-resistant LB liquid medium. Then, transfer them to fresh medium and culture for 4 hours. Take 1 mL of the bacterial culture, add 60% glycerol and store it at -80°C. Then add IPTG to the final concentration of 0.5 mM and continue to culture at 37°C on a shaker for 4 hours.

[0088] Step 4.3: Centrifuge to collect bacteria, resuspend the bacterial cells in 100 μL of ddH2O, and dilute to 1× with 25 μL of 5×SDS-PAGE Loading Buffer. Boil in a water bath for 8 min and detect protein expression by SDS-PAGE electrophoresis.

[0089] Step 4.4: After electrophoresis, the gel was stained with Coomassie Brilliant Blue for 5 hours, then destained with destaining solution. The protein expression was observed and photographed using a gel imaging system (Shanghai Peiqing, JS1085P).

[0090] Step 5 is implemented in the following steps:

[0091] Step 5.1, Pretreatment of nickel column: Add nickel packing to the packing tube, open the cap to let the liquid in it flow out, and then add an equal volume of Binding-Buffer to resuspend the nickel packing.

[0092] Step 5.2: Then add 3 times the volume of protein solution to the nickel column, cover it, and invert it to ensure that the nickel filler and protein solution are thoroughly mixed. Incubate overnight on a rotary shaker at 4°C.

[0093] Step 5.3: Open the cap and let stand to allow the protein solution to flow out into the collection tube; add 3 times the volume of Binding-Buffer, incubate on a rotating shaker at 4°C for 10 minutes, then let stand to collect the outflow, and repeat once.

[0094] Step 5.4, elution column: Add 2 volumes of Elution Buffer to the nickel packing, seal, incubate on a rotary shaker at 4°C for 10 min, collect the eluent, and repeat twice;

[0095] Step 5.5: Cut the dialysis bag with a molecular weight cutoff of 30kD into 15cm lengths, boil it in ddH2O for 10min, cool it, fix one end with a clip, add the purified protein into the dialysis bag, and then fix the other end of the dialysis bag.

[0096] Step 5.6: Place the dialysis bag containing protein into a measuring cup containing ddH2O, dialyze at 4°C, change the ddH2O every 4 hours, and dialyze for 48 hours.

[0097] Step 5.7: Pour the dialyzed protein solution into a culture dish and freeze at -80°C overnight. Then, freeze-dry it under vacuum for 4 hours in a freeze dryer (Thermo Scientific, Heto PowerDry PL3000) and store it in a desiccator.

[0098] Step 6 is implemented in the following steps:

[0099] Step 6.1: After preparing the protein sample, spot the sample and perform SDS-PAGE electrophoresis;

[0100] Step 6.2, Transfer: Immerse the prepared filter paper and sponge in the freshly prepared transfer solution, then activate the PVDF membrane by immersing it in methanol for 10 seconds. Then, place the sponge, filter paper, protein gel, PVDF membrane, filter paper, and sponge sequentially (from negative to positive electrode). Next, clamp the membrane and place it in the transfer tank, pour in the transfer solution, and then place the entire tank in an ice-water mixture. Perform constant current transfer at 250 mA for 2 hours.

[0101] Step 6.3: After the transfer is complete, open the transfer clamp, take out the PVDF membrane, soak it in 5% skim milk powder, and seal it on a shaker at room temperature for 2 hours.

[0102] Step 6.4: Dilute His antibody (Abmart, M30111, mouse-derived) at 1:1000 and incubate overnight at 4°C;

[0103] Step 6.5: After warming for 1 hour, recover the primary antibody and wash with PBST 3 times, 10 min each time;

[0104] Step 6.6: Dilute HRP secondary antibody (Beyotime, P0612, goat anti-mouse) at a ratio of 1:1000 and incubate on a shaker at room temperature for 2 hours;

[0105] Step 6.7: Recover the secondary antibody and wash three times with PBST, 10 min each time;

[0106] Step 6.8, Development: Mix the color developing solution in a 1:1 volume ratio, coat it evenly on the membrane, and develop and photograph it using a chemiluminescence imaging system (Tanon 5200).

[0107] Example 3

[0108] The preparation method of rhCol-CTGF fusion protein of the present invention includes culturing for 7 hours in step 2.3, culturing for another 5 hours in a shaker at 37°C in step 2.4, and adding 5 times the volume of protein solution to a nickel column in step 5.2. Other preparation conditions are the same as in Example 2.

[0109] Example 4

[0110] The preparation method of HLC-CTGF fusion protein of the present invention includes culturing for 8 hours in step 2.3, culturing for another 5 hours in a shaker at 37°C in step 2.4, and adding 6 times the volume of protein solution to a nickel column in step 5.2. Other preparation conditions are the same as in Example 2.

[0111] Example 5

[0112] Cytotoxicity assays of the rhCol-CTGF fusion protein of this invention:

[0113] Step 1: MSCs were cultured until the confluence reached approximately 80%. After digestion and centrifugation, the cells were resuspended in 3% FBS medium and counted. The medium was then diluted to a density of 2 × 10⁻⁶ cells / mL. 4 cells / mL;

[0114] Step 2: Add cell suspension at 100 μL / well to a 96-well plate and incubate at 37°C for 8 h to allow cell adhesion. Then remove the culture medium and add 0.5 μg / mL of fusion protein rhCol-CTGF prepared in 3% FBS medium, 200 μg / mL of filtered sterilized recombinant protein (Tβ4)12 solution, and 3% FBS medium alone as a control at 100 μL / well. Each group has six replicates.

[0115] Step 3: On days 1, 3, and 5, remove the culture medium, add 100 μL of PBS containing 10% CCK-8 to each well, incubate at 37°C for 2 hours, and measure the OD450 value using a microplate reader (BioTek, Epoch). Results are as follows... Figure 5 As shown.

[0116] Example 6

[0117] The application of the rhCol-CTGF fusion protein in the preparation of hydrogels in this invention is as follows: 0.04 g of rhCol and the fusion protein rhCol-CTGF were weighed separately, and 0.005 g of TG was weighed and dissolved in 500 μL of PBS. Then, 200 μL of each solution was transferred to a 0.5 mL centrifuge tube and placed at 37°C. The gelation time was observed, and the results are as follows. Figure 6 As shown.

[0118] sequence list

[0119] <110> Xi'an Medical College

[0120] <120>rhCol-CTGF fusion protein, preparation method and application

[0121] <160>2

[0122] <210>1

[0123] <211>516

[0124] <212>PRT

[0125] <213>rhCol-CTGF

[0126] <400>1

[0127] GPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQPGNKG55

[0128] SPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKPGQPG109

[0129] EQGSPGNQGPAGNEGPKGQPGQNGKPGSPGPPGEPGNPGKPGSPGPAGSNGEPGP164

[0130] AGSPGEKGSQGSNGNPGPAGNQGQPGNKGSPGNPGKPGEPGSNGPQGEPGSQGN218

[0131] PGKNGQPGSPGSQGSPGNQGQPGKPGQPGEQGSPGNQGPAGNEGPKGQPGQNGK272

[0132] PGTPGPPGEPGNPGKPGSPGPAGSNGEPGPAGSPGEKGSQGSNGNPGPAGNQGQP327

[0133] GNKGSPGNPGKPGEPGSNGPQGEPGSQGNPGKNGQPGSPGSQGSPGNQGQPGKP381

[0134] GQPGEQGSPGNQGPAGNEGPKGQPGQNGKPELGGGGSGGGGSGKKCIRTPKISKPI437

[0135] KFELSGCTSMKTYRAKFCGVCTDGRCCTPHRTTTLPVEFKCPDGEVMKKNMMFIKTCA495

[0136] CHYNCPGDNDIFESLYYRKMY516

[0137] <210>2

[0138] <211>1548

[0139] <212>DNA

[0140] <213>rhCol-CTGF

[0141] <400>2

[0142] GGTCCGCCGGGCGAACCGGGTAATCCGGGTAAACCGGGTTCTCCGGGCCCGGCG54

[0143] GGTAGCAACGGCGAACCGGGTCCGGCCGGCTCACCGGGCGAAAAAGGCTCTCAG108

[0144] GGTAGTAATGGCAACCCGGGTCCGGCAGGTAATCAGGGTCAGCCGGGCAACAAAG163

[0145] GTAGCCCGGGTAATCCAGGTAAACCGGGCGAACCAGGCTCTAACGGTCCGCAGGG218

[0146] TGAACCAGGCAGCCAGGGCAATCCGGGCAAAAACGGTCAGCCGGGCTCACCGGG272

[0147] CAGCCAGGGTAGTCCGGGTAATCAGGGCCAGCCGGGCAAACCGGGTCAGCCGGG326

[0148] TGAACAGGGCAGCCCGGGCAACCAGGGTCCGGCGGGTAATGAAGGCCCGAAAGG380

[0149] TCAGCCGGGTCAGAACGGTAAACCGGGCAGCCCGGGTCCGCCGGGCGAACCGGG434

[0150] TAATCCGGGTAAACCGGGTTCTCCGGGCCCGGCGGGTAGCAACGGCGAACCGGGT489

[0151] CCGGCCGGCTCACCGGGCGAAAAAGGCTCTCAGGGTAGTAATGGCAACCCGGGTC544

[0152] CGGCAGGTAATCAGGGTCAGCCGGGCAACAAAGGTAGCCCGGGTAATCCAGGTAA599

[0153] ACCGGGCGAACCAGGCTCTAACGGTCCGCAGGGTGAACCAGGCAGCCAGGGCAAT654

[0154] CCGGGCAAAAACGGTCAGCCGGGCTCACCGGGCAGCCAGGGTAGTCCGGGTAATC709

[0155] AGGGCCAGCCGGGCAAACCGGGTCAGCCGGGTGAACAGGGCAGCCCGGGCAACC763

[0156] AGGGTCCGGCGGGTAATGAAGGCCCGAAAGGTCAGCCGGGTCAGAACGGTAAACC818

[0157] GGGTACCCCGGGTCCGCCGGGCGAACCGGGTAATCCGGGTAAACCGGGTTCTCC872

[0158] GGGCCCGGCGGGTAGCAACGGCGAACCGGGTCCGGCCGGCTCACCGGGCGAAAA926

[0159] AGGCTCTCAGGGTAGTAATGGCAACCCGGGTCCGGCAGGTAATCAGGGTCAGCCG981

[0160] GGCAACAAAGGTAGCCCGGGTAATCCAGGTAAACCGGGCGAACCAGGCTCTAACG1036

[0161] GTCCGCAGGGTGAACCAGGCAGCCAGGGCAATCCGGGCAAAAACGGTCAGCCGG1090

[0162] GCTCACCGGGCAGCCAGGGTAGTCCGGGTAATCAGGGCCAGCCGGGCAAACCGG1144

[0163] GTCAGCCGGGTGAACAGGGCAGCCCGGGCAACCAGGGTCCGGCGGGTAATGAAG1198

[0164] GCCCGAAAGGTCAGCCGGGTCAGAACGGTAAACCGGAGCTCGGCGGTGGGGGAA1252

[0165] GCGGCGGTGGGGGAAGCGGCAAAAAATGCATTCGTACCCCGAAAATTAGCAAACC1307

[0166] GATTAAATTTGAACTGAGCGGCTGCACCAGCATGAAAACCTATCGTGCGAAATTTTG1364

[0167] CGGCGTGTGCACCGATGGCCGTTGCTGCACCCCGCATCGTACCACGACCCTGCCG1419

[0168] GTGGAATTTAAATGCCCGGATGGCGAAGTGATGAAAAAAAATATGATGTTTATTAAA1476

[0169] ACCTGCGCGTGCCATTATAATTGCCCGGGCGATAATGATATTTTTGAAAGCCTGTAT1533

[0170] TATCGTAAAATGTAT1548

Claims

1. rhCol-CTGF fusion protein, characterized in that, The amino acid sequence of the rhCol-CTGF fusion protein is shown in Sequence 1.

2. The rhCol-CTGF fusion protein according to claim 1, characterized in that, The nucleic acid sequence of the fusion protein is shown in Sequence 2.

3. The method for preparing the rhCol-CTGF fusion protein according to any one of claims 1-2, characterized in that, The specific steps are as follows: Step 1: Target gene synthesis and restriction enzyme site design; Step 2, activate the bacterial strain; Step 3: Construction of the expression vector for the fusion protein rhCol-CTGF; Step 4: Expression of the fusion protein rhCol-CTGF; Step 5: Purification of the fusion protein rhCol-CTGF; Step 6: Western blot of the fusion protein rhCol-CTGF.

4. The method for preparing the rhCol-CTGF fusion protein according to claim 3, characterized in that, Step 1 specifically involves: designing the rhCol-CTGF amino acid sequence and predicting the spatial structure of the fusion protein based on sequence 1; designing a nucleic acid sequence containing double restriction enzyme sites for rhCol-CTGF using E. coli codon bias; and obtaining the pUC57-rhCol-CTGF strain and plasmid.

5. The method for preparing the rhCol-CTGF fusion protein according to claim 3, characterized in that, Step 2 is implemented in the following steps: Step 2.1: Prepare solid culture medium, sterilize at high temperature and cool, then add ampicillin to prepare LB agar plate medium; Step 2.2: After the inoculation loop is heated and cooled under an alcohol lamp, a small amount of bacterial solution is taken onto a plate, streaked with the inoculation loop, and placed in a bacterial incubator for overnight incubation. Step 2.3: After culturing, add ampicillin-resistant LB liquid culture to a sterile conical flask, then pick single colonies into a shake flask, culture for 6-8 hours, send the bacterial culture for sequencing and add 60% glycerol to preserve the bacterial strain.

6. The method for preparing the rhCol-CTGF fusion protein according to claim 3, characterized in that, Step 3 is implemented in the following steps: Step 3.1: The obtained rhCol-CTGF plasmid pUC57-rhCol-CTGF is digested with two enzymes to obtain the target fragment, and the expression vector pET28a is linearized by double enzyme digestion. Step 3.2: Add 1 μL each of T4 DNA Ligase and T4 DNA Ligase buffer to a centrifuge tube at a volume ratio of rhCol-CTGF:pET28a = 3:1, and ligate overnight at 4°C to obtain the ligation product pET28a-rhCol-CTGF plasmid. Step 3.3, transformation: spread the bacterial culture on kanamycin-resistant LB agar plates and then incubate them upside down in an incubator overnight; Step 3.4: Single clones are cultured in kanamycin-resistant liquid medium and then verified by double enzyme digestion; Step 3.5: Sequencing the plasmids whose band positions were verified by double enzyme digestion, then comparing and analyzing the sequencing results, and storing the bacterial strains with correct sequencing results in a -80°C freezer with 60% glycerol.

7. The method for preparing the rhCol-CTGF fusion protein according to claim 3, characterized in that, Step 4 is implemented in the following steps: Step 4.1: Transform the plasmid that was correctly sequenced in Step 3 into E. coli BL21(DE3) competent cells, then plate them on kanamycin-resistant LB plates and incubate overnight in an incubator; Step 4.2: Select single colonies, culture them overnight in kanamycin-resistant LB liquid medium, then transfer them to fresh medium for culture, take the bacterial culture, add 60% glycerol and store at -80℃, then add IPTG to the final concentration of 0.5mM, and continue to culture on a shaker for 4-5 hours; Step 4.3: Centrifuge to collect bacteria, add ddH2O to resuspend the bacterial cells, add 5×SDS-PAGE Loading Buffer to dilute, boil in a water bath, and detect whether the protein is expressed by SDS-PAGE electrophoresis. Step 4.4: After electrophoresis, the gel is stained with Coomassie Brilliant Blue and then destained with destaining solution. The protein expression is observed under a gel imaging system and photographs are taken.

8. The method for preparing the rhCol-CTGF fusion protein according to claim 3, characterized in that, Step 5 is implemented in the following steps: Step 5.1, Pretreatment of nickel column: Add nickel packing to the packing tube, open the cap to let the liquid in it flow out, and then add an equal volume of Binding-Buffer to resuspend the nickel packing. Step 5.2: Add 3-6 times the volume of protein solution to the nickel column, cover it, and invert it to ensure that the nickel filler and protein solution are thoroughly mixed. Let it bind overnight on a rotary shaker. Step 5.3: Open the cap, let stand to allow the protein solution to flow out into the collection tube, add 3 times the volume of Binding-Buffer, place on a rotating shaker, and then let stand to collect the outflow. Repeat once. Step 5.4, elution column: Add 2 volumes of Elution Buffer to the nickel packing material, seal the column, place it on a rotary shaker, let it stand and collect the eluent, repeat twice; Step 5.5: Cut the dialysis bag with a molecular weight cutoff of 30kD, boil it in a ddH2O bath, cool it, fix one end with a clip, add the purified protein into the dialysis bag, and then fix the other end of the dialysis bag. Step 5.6: Place the dialysis bag containing protein into a measuring cup containing ddH2O, dialyze at 4°C, change the ddH2O every 4 hours, and dialyze for another 48 hours. Step 5.7: Pour the dialyzed protein solution into a culture dish, freeze overnight, then freeze-dry under vacuum in a freeze dryer and store in a desiccator.

9. The method for preparing the rhCol-CTGF fusion protein according to claim 3, characterized in that, Step 6 is implemented in the following steps: Step 6.1: After preparing the protein sample, spot the sample and perform SDS-PAGE electrophoresis; Step 6.2, Transfer: Soak the prepared filter paper and sponge in the freshly prepared transfer solution, then soak the PVDF membrane in methanol to activate it. Then place the sponge, filter paper, protein gel, PVDF membrane, filter paper and sponge in sequence, clamp them and place them in the transfer tank, pour in the transfer solution, and then place the entire tank in an ice-water mixture for constant flow transfer. Step 6.3: After the transfer is complete, open the transfer clamp, take out the PVDF membrane, soak it in skim milk powder, and seal it on a shaker at room temperature. Step 6.4: Dilute the His antibody and incubate overnight; Step 6.5: After rewarming, recover the primary antibody and wash with PBST; Step 6.6: Dilute the HRP secondary antibody and incubate on a shaker at room temperature; Step 6.7: Recover the secondary antibody and wash with PBST. Step 6.8, Development: Mix the color developing solution in a 1:1 volume ratio, apply it evenly to the membrane, and then develop and photograph it.

10. The use of the rhCol-CTGF fusion protein according to any one of claims 1-2 in the preparation of hydrogels.

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