Novel recombinant protein CAV1-HSA-QTY with tumor cell migration inhibition performance and application of novel recombinant protein CAV1-HSA-QTY

Abstract

The invention discloses a novel recombinant protein CAV1-HSA-QTY with tumor cell migration inhibition performance and application of the novel recombinant protein CAV1-HSA-QTY, and belongs to the technical field of biological medicine. The CAV1 has the advantages that the existing CAV1 is modified and designed by means of synthetic biology, the activity of a protein kinase inhibitor of the CAV1 is reserved to play a role in inhibiting tumor cell migration, and meanwhile, a part of amino acid sequence of highly hydrophilic human serum albumin is fused to improve the water solubility of the CAV1; according to the present invention, the defects of poor water solubility and large toxicity of the small molecule compound type protein kinase inhibitor are effectively overcome, such that the target protein with the tumor cell migration inhibition performance is obtained on a large scale through the synthetic biological means, and the production cost of the natural protein type tumor cell migration inhibitor can be reduced;

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a novel recombinant protein CAV1-HSA-QTY having the ability to inhibit tumor cell migration and an application thereof. Background Art

[0002] Human caveolin-1 is an integral membrane protein, referred to as CAV1, which is mainly located in caveolae on the cell surface. CAV1 plays a certain role in maintaining the integrity of caveolae, signal transduction, and microvesicle transport. Microvesicles are special intracellular depressions located on the surface of lipid rafts on the cell membrane. CAV1 is an integral membrane protein located on the cell membrane and is one of the main components of microvesicles. Research results show that the framework region of CAV1 can exert protein kinase inhibitory activity and can inhibit the activity of signal factors including EGFR, Raf, MEK-1, ERK-2, G protein, C-src tyrosine kinase, etc. These signal factors are widely involved in cell apoptosis, cell proliferation, adhesion and movement. In addition, the results also show that CAV1 is highly expressed in many cells and plays an important regulatory role in the transport of substances, tumor occurrence and invasion, and endothelial permeability. CAV1 may also be a tumor suppressor gene, and its mutations have been detected in many human tumors.

[0003] Currently, the commonly used protein kinase inhibitors are mainly small molecule compounds, such as imatinib, nilotinib, gefitinib, osimertinib, etc. These inhibitors can inhibit the migration and invasion of tumor cells. However, these small molecule protein kinase inhibitors have common disadvantages such as poor water solubility and high toxicity. Summary of the invention

[0004] The purpose of the present invention is to provide a novel recombinant protein CAV1-HSA-QTY with the ability to inhibit tumor cell migration and its application. By means of synthetic biology, the existing CAV1 is redesigned and modified, its protein kinase inhibitor activity is retained to inhibit tumor cell migration, and a highly hydrophilic amino acid sequence (partial amino acid sequence of human serum albumin) is fused to improve its water solubility, which can effectively overcome the shortcomings of small molecule compound protein kinase inhibitors, such as poor water solubility and high toxicity. The present invention obtains the target protein with the ability to inhibit tumor cell migration on a large scale by means of synthetic biology, and can also reduce the production cost of natural protein tumor cell migration inhibitors.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions:

[0006] A novel recombinant protein CAV1-HSA-QTY with the ability to inhibit tumor cell migration is characterized by comprising the entire amino acid sequence of human caveolin-1 and the amino acid sequence of positions 101-300 of human serum albumin, wherein the amino acid sequence of positions 101-300 of human serum albumin comprises valine (V) at position 101, leucine (L) at position 104, leucine (L) at position 127, leucine (L) at position 136, leucine (L) at position 139, valine (V) at position 140, valine (V) at position 144, valine (V) at position 146, The hydrophobic amino acids at positions 140-151 (V), phenylalanine (F), phenylalanine (F), leucine (L), leucine (L), and isoleucine (I) were mutated to hydrophilic amino acids threonine (T), glutamine (Q), glutamine (Q), glutamine (Q), threonine (T), threonine (T), threonine (T), tyrosine (Y), tyrosine (Y), glutamine (Q), glutamine (Q), and threonine (T), respectively, for a total of 13 hydrophobic amino acids;

[0007] The amino acid sequence of the novel recombinant protein is shown in SEQ ID NO.1.

[0008] A recombinant expression vector comprising the novel recombinant protein having the ability to inhibit tumor cell migration as claimed in claim 1.

[0009] Preferably, the types of the vector include plasmid vectors, phage vectors or animal and plant virus vectors.

[0010] A recombinant bacterium expressing the novel recombinant protein having the ability to inhibit tumor cell migration as claimed in claim 1.

[0011] Preferably, the types of the recombinant bacteria include one or more selected from Escherichia coli, Bacillus subtilis, Pichia pastoris and / or Saccharomyces cerevisiae.

[0012] The application of the novel recombinant protein having the ability to inhibit tumor cell migration in the preparation of a pharmaceutical preparation for inhibiting tumor cell migration.

[0013] Preferably, the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier and / or adjuvant.

[0014] Preferably, the drug is for internal use.

[0015] Preferably, the dosage form of the internal medicine includes injection, tablet, pill and the like.

[0016] The application of the novel recombinant protein having the ability to inhibit tumor cell migration in the preparation of an in vitro tumor cell migration inhibitor.

[0017] In summary, the present invention has the following beneficial effects:

[0018] 1. The present invention fuses natural caveolin 1 (CAV1) with the 101-300 amino acid sequence of human serum albumin, and the 101-300 amino acid sequence of human serum albumin is systematically mutated in hydrophilicity. The specific amino acid sequence of the novel recombinant protein is shown in SEQ ID NO.1. The novel recombinant protein has higher performance in inhibiting tumor cell migration and higher hydrophilicity.

[0019] 2. In the early experiments of the present invention, a series of tests on the performance of inhibiting tumor cell migration were carried out on the designed new recombinant protein, including experiments on inhibiting tumor cell migration on 4T1 and A549 cells. The experimental results showed that compared with natural human CAV1, the new recombinant protein has a more efficient ability to inhibit tumor cell migration and has good research application and clinical development value. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0021] Figure 1 This is the electrophoresis band diagram of the novel recombinant protein prepared by the present invention;

[0022] Figure 2 The advanced structure prediction results of the novel recombinant protein CAV1-HSA-QTY designed for the present invention;

[0023] Figure 3 The hydrophilicity prediction results of the novel recombinant protein CAV1-HSA-QTY designed for the present invention;

[0024] Figure 4 Cytotoxicity experiment of the novel recombinant protein CAV1-HSA-QTY designed for the present invention;

[0025] Figure 5 The results of the migration inhibition experiment of 4T1 cells after the novel recombinant protein CAV1-HSA-QTY designed for the present invention was co-cultured with 4T1 cells (mouse breast cancer cells) for 48 hours;

[0026] Figure 6The results of the migration inhibition experiment of A549 cells after the novel recombinant protein CAV1-HSA-QTY designed in the present invention was co-cultured with A549 cells (human non-small cell lung cancer cells) for 48 hours. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0029] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0030] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0031] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0032] In this specification, certain embodiments may be disclosed in a format of being in a certain range. It should be understood that such description of "being in a certain range" is merely for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0033] Materials and Reagents:

[0034] Human embryonic kidney cells 293T were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences, catalog number SCSP-502, cell name: human embryonic kidney cells. 4T1 and A549 cells were also purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences.

[0035] Escherichia coli BL21 (DE3) competent cells were purchased from Beijing Solebow Technology Co., Ltd.

[0036] The pET-22b(+) plasmid is a commercially available E. coli expression vector purchased from Beijing Qingke Biotechnology Co., Ltd. The vector tags are N-pelB and C-His, and the vector resistance is Ampicillin.

[0037] Restriction endonucleases NdeI and XhoI were purchased from NEB (Beijing) Co., Ltd. His-tag protein purification resin (nickel column) was purchased from Shanghai Lianmai Bioengineering Co., Ltd., catalog number LM-616. IPTG, ampicillin, and DMSO (dimethyl sulfoxide) were all purchased from Beijing Solebow Technology Co., Ltd. DMEM medium was purchased from GIBCO. CCK8 reagent was purchased from Chongqing Baoguang Technology Co., Ltd. and used according to the reagent instructions.

[0038] Culture medium:

[0039] Each liter of LB medium contains: 5 g yeast extract, 10 g tryptone, 10 g sodium chloride, and the pH is adjusted to 7.0.

[0040] Preparation method: Dissolve 5g yeast extract, 10g tryptone, and 10g sodium chloride in 950mL double distilled water, adjust the pH to 7.0 with sodium hydroxide solution, and dilute to 1L with double distilled water. If preparing solid culture medium, add agar at 1.5g / 100mL. Sterilize at 121℃ with high pressure steam for 30min.

[0041] The experimental reagents not specifically described in the present invention are all conventional reagents in the art, and can be prepared according to conventional methods in the art or purchased from relevant reagent suppliers; the experimental methods not specifically described are all conventional methods in the art, and reference can be made to relevant experimental manuals, such as molecular cloning experimental manuals or instructions of relevant reagent manufacturers.

[0042] Example 1

[0043] 1. CAV1-HSA-QTY gene design and protein expression

[0044] The gene was designed, and its nucleotide sequence was shown in SEQ ID NO.2, with a total length of 1209 bp. The gene encodes a protein, and its amino acid sequence was shown in SEQ ID NO.1, with a total length of 403 amino acids, and was named as the recombinant protein CAV1-HSA-QTY. When synthesizing the gene, NdeI restriction site (CATATG) and XhoI restriction site (CTCGAG) were added to the 5' end and 3' end of the gene, respectively. The gene sequence with restriction sites is shown in SEQ ID NO.3. The synthesized gene was sequenced and verified, and the gene with the correct sequence was used for subsequent vector construction. The specific sequences involved are shown in Table 1.

[0045] Table 1

[0046]

[0047]

[0048] 2. Vector Construction

[0049] In this embodiment, pET-22b(+) plasmid is preferably used as an expression vector. The pET-22b(+) plasmid and the target gene (SEQ ID NO.1) are subjected to double restriction digestion reaction with restriction endonucleases NdeI and XhoI, respectively, and then the target gene is connected to the pET-22b(+) vector by ligation reaction. It is understood that this embodiment can also be implemented by using phage vectors or animal and plant virus vectors.

[0050] 3. Recombinant Bacterial Transformation

[0051] (1) Take out the competent Escherichia coli BL21 (DE3) cells (Beijing Solebow) from the -80°C freezer and place them on ice for 5 min.

[0052] (2) After the glycerol containing the BL21(DE3) competent cells melts, add the competent cells to the ligation product, pipette and release the pipette three times to mix, and place on ice for 30 min.

[0053] (3) Quickly wipe the water off the tube wall with absorbent paper, then heat shock at 42°C for 90 seconds and immediately place on ice for 2 minutes.

[0054] (4) Add 800 μL of LB liquid culture medium under sterile conditions and culture at 37°C, 150 rpm for 45 min.

[0055] (5) Collect the cells by centrifugation at 8000 rpm for 5 min, discard part of the supernatant, and use the remaining 100 μL of supernatant to resuspend the E. coli. Then, spread it evenly on LB solid culture medium containing 100 μg / mL ampicillin, place it in a 37°C incubator, and invert and culture for about 12 h.

[0056] (6) A single clone was picked and inoculated into liquid LB medium containing 100 μg / mL ampicillin. After culturing at 37°C for about 13 h, positive clones were identified.

[0057] In this embodiment, Escherichia coli is preferably used as the recombinant bacteria. It is understandable that this embodiment can also be implemented using Bacillus subtilis, Pseudomonas aeruginosa or Saccharomyces cerevisiae strains.

[0058] 4. Protein Expression and Purification

[0059] (1) Inoculation: Prepare liquid LB medium and sterilize it. Place the sterilized liquid LB medium in a clean bench and cool it to room temperature. Add ampicillin to the LB medium in the clean bench and mix it to a final concentration of 100 μg / mL. Then, inoculate 200 μL / L of Escherichia coli (positive clone) containing the target gene plasmid into the LB medium. Place the LB medium in a shaker and culture it at a speed of 170 rpm and a temperature of 37°C for 8 h.

[0060] (2) Induction: After 8 h of shaking culture, 2 mL of bacterial culture was taken out and its OD was measured by spectrophotometer. 600 When the OD of the bacterial solution is 600 When the value reached 0.6-0.8, IPTG was added to the bacterial solution at a final concentration of 200 μL / mL, and then cultured in a shaking incubator at 37°C and 170 rpm for 8 h.

[0061] (3) Purification: After adding IPTG and culturing on a shaking platform for 8 h, the bacterial solution was removed and centrifuged at 8000 rpm for 5 min at 4°C. After centrifugation, the supernatant was removed and the precipitate was retained (the precipitate is Escherichia coli).

[0062] (4) Ultrasonic disruption: E. coli was ultrasonically disrupted and then centrifuged. The precipitate obtained by centrifugation contained the target protein. The precipitate obtained by centrifugation was first washed with washing solution I (50 mmol / L Tris-HCl, 1 mol / L urea, 10 mL / L Triton X-100), then washed with washing solution II (50 mmol / LTris-HCl, 2 mol / L urea, 5 mL / L Triton X-100), and then dissolved with inclusion body dissolution solution (50 mmol / LTris-HCl, 8 mol / L urea, 100 mmol / L NaCl).

[0063] (5) Finally, the inclusion body solution was subjected to gradient renaturation as follows: the inclusion body solution was placed in a dialysis bag (Beijing Solebold Technology Co., Ltd., catalog number: YA1071), and the dialysis bag was placed in 6M, 4M, 2M, 1M, and 0.5M urea solutions in turn for gradient renaturation. The renaturation was performed for 3 hours at each urea concentration, and the renaturation was performed at a low temperature of 4°C. The renaturation solution was purified by His-tag protein purification resin (nickel column, Shanghai Lianmai) to obtain the recombinant protein CAV1-HSA-QTY (the gene sequence of the target protein was added with a histidine tag during the design). After purification, polyacrylamide gel electrophoresis (SDS-PAGE) was used to identify whether the purified target protein was successfully obtained. The results are as follows. Figure 1 As shown, a target protein of about 45.834 kDa was obtained.

[0064] The higher-order structure of the recombinant protein CAV1-HSA-QTY prepared in this example was predicted, and the results are shown in Figure 2 The higher-order structure of the novel recombinant protein CAV1-HSA-QTY contains a large number of "α-helical" secondary structures.

[0065] Furthermore, the hydrophilicity and hydrophobicity of the novel recombinant protein CAV1-HSA-QTY prepared in this example were predicted. Figure 3 . Figure 3 The horizontal axis represents the amino acid sequence of the protein; the vertical axis represents hydrophilicity, and the larger the value, the more hydrophobic it is. If it is a negative value, it represents hydrophilicity. The total hydrophilicity value of the new recombinant protein CAV1-HSA-QTY is smaller than the total hydrophilicity value of the natural human CAV1, indicating that the new recombinant protein with kinase inhibition function prepared in the present invention is more hydrophilic.

[0066] Example 2

[0067] Advanced structural prediction of the novel recombinant protein CAV1-HSA-QTY

[0068] Enter the entire amino acid sequence of the novel recombinant protein CAV1-HSA-QTY into the Alphafold3 online prediction website (https: / / alphafoldserver.com / ), submit and start prediction.

[0069] Open the prediction result file using Pymol software, such as Figure 2 As shown, the obtained high-level structure prediction results are analyzed.

[0070] Example 3

[0071] Analysis of the overall hydrophilicity value of the new recombinant protein CAV1-HSA-QTY

[0072] Enter the entire amino acid sequence of the new recombinant protein CAV1-HSA-QTY into the online website https: / / web.expasy.org / protscale / , click Submit and Prediction, and you can obtain the hydrophilicity analysis results of different amino acid sites of the new recombinant protein.

[0073] like Figure 3 As shown in the obtained hydrophilicity result picture, the horizontal axis represents the amino acid site, the vertical axis represents the hydrophilicity, and a positive vertical axis value indicates hydrophobicity, and a negative vertical axis value indicates hydrophilicity. The larger the vertical axis value, the more hydrophobic it is, and the smaller the vertical axis value, the more hydrophilic it is.

[0074] Example 4

[0075] Evaluation of cytotoxicity of the novel recombinant protein CAV1-HSA-QTY

[0076] The toxicity of the recombinant protein CAV1-HSA-QTY prepared in Example 1 to human embryonic kidney cells 293T (cell bank of Type Culture Collection Committee of Chinese Academy of Sciences) was verified by CCK8 method to evaluate its biosafety.

[0077] Method steps:

[0078] (1) In a 96-well plate, 1 × 10 4 Human embryonic kidney 293T cells were cultured in DMEM medium, and 5 experimental groups and 1 control group were set up, with 3 replicate wells in each group.

[0079] (2) After culturing the cells at 37°C for 24 h, the novel recombinant protein CAV1-HSA-QTY was added to the wells of the experimental group (experimental wells), so that the final concentrations of the novel recombinant protein CAV1-HSA-QTY in the five experimental groups were 10 μg / mL, 50 μg / mL, 100 μg / mL, 200 μg / mL, and 400 μg / mL, respectively. No novel recombinant protein CAV1-HSA-QTY was added to the cells in the wells of the control group (control wells).

[0080] (3) Continue to culture at 37°C for 24 hours, and use the CCK8 method to detect the number of live cells. The steps are as follows: add 10 μL of CCK8 reagent to each well and incubate at 37°C for 3 hours. Then use an ELISA reader to measure the absorbance of each well at 450 nm.

[0081] Results Figure 4 , Figure 4It showed that when the concentration of the new recombinant protein CAV1-HSA-QTY reached 400 μg / mL, the survival rate of 293T cells remained above 85%, indicating that the new recombinant protein CAV1-HSA-QTY had no obvious cytotoxicity and no obvious inhibitory or killing effect on normal cells, and could be further applied to in vivo and in vitro research, diagnosis and treatment.

[0082] Example 5

[0083] Tumor cell migration inhibition assay

[0084] Preparation of solution: Accurately weigh the appropriate mass of the new recombinant protein CAV1-HSA-QTY, natural human CAV1 and the small molecule compound kinase inhibitor dacomitinib in turn, dissolve them in DMEM complete medium at room temperature, and the obtained solution is ready for use. Note that the new recombinant protein, natural human CAV1 and the small molecule compound kinase inhibitor dacomitinib all need to be sterilized by UV.

[0085] Co-culture with tumor cells: 4T1 cells were cultured with the prepared CAV1-HSA-QTY, natural human CAV1 and dacomitinib complete culture medium solutions in sequence. After 48 hours of co-culture, the migration rate of 4T1 cells in each experimental group was observed and calculated.

[0086] The experimental steps for A549 cell migration inhibition were the same as those for 4T1 cells.

[0087] The results are as follows Figure 5 and Figure 6 As shown, the new recombinant protein CAV1-HSA-QTY can significantly inhibit the migration of two tumor cells. The inhibitory ability is not significantly different from that of the marketed small molecule compound inhibitor dacomitinib, and is significantly better than the tumor cell migration inhibitory ability of natural human CVA1.

[0088] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A novel recombinant protein CAV1-HSA-QTY with the ability to inhibit tumor cell migration, characterized in that: The invention comprises the entire amino acid sequence of human caveolin-1 and the amino acid sequence of positions 101-300 of human serum albumin, wherein the hydrophobic amino acids of valine at position 101, leucine at position 104, leucine at position 127, leucine at position 136, leucine at position 139, valine at position 140, valine at position 144, valine at position 146, phenylalanine at position 151, phenylalanine at position 158, leucine at position 159, leucine at position 163, and isoleucine at position 166 of the amino acid sequence of positions 101-300 of human serum albumin are mutated to hydrophilic threonine, glutamine, glutamine, glutamine, glutamine, threonine, threonine, threonine, tyrosine, tyrosine, glutamine, glutamine, and threonine, respectively, in sequence, for a total of 13 hydrophobic amino acids mutated; The amino acid sequence of the novel recombinant protein is shown in SEQ ID NO.

1.

2. A recombinant expression vector, characterized in that: The novel recombinant protein having the ability to inhibit tumor cell migration as claimed in claim 1 is included.

3. The recombinant expression vector according to claim 2, characterized in that The types of the vector include plasmid vectors, phage vectors or animal and plant virus vectors.

4. A recombinant bacterium, characterized in that Expressing the novel recombinant protein having the ability to inhibit tumor cell migration as described in claim 1.

5. The recombinant bacterium according to claim 4, characterized in that The types of the recombinant bacteria include one or more selected from Escherichia coli, Bacillus subtilis, Pichia pastoris and / or Saccharomyces cerevisiae.

6. Use of the novel recombinant protein having the ability to inhibit tumor cell migration as claimed in claim 1 in the preparation of a pharmaceutical preparation for inhibiting tumor cell migration.

7. The use according to claim 6, characterized in that The pharmaceutical preparation further comprises a pharmaceutically acceptable carrier and / or adjuvant.

8. The use according to claim 6, characterized in that The medicine is for internal use.

9. The use according to claim 6, characterized in that The dosage forms of the internal medicine include injections, tablets, pills, etc.

10. Use of the novel recombinant protein having the ability to inhibit tumor cell migration according to claim 1 in the preparation of an in vitro tumor cell migration inhibitor.

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