Collagen binding domain fusion ccl14 recombinant protein and application thereof

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to a collagen binding domain fusion CCL14 recombinant protein and application thereof. The amino acid sequence of the recombinant protein is shown as SEQ ID NO. 1, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 2. The application innovatively applies the chemokine CCL14 to tumor immunotherapy. Compared with the simple CCL14 protein, after the recombinant protein treatment, the tumor weight of the mouse is lighter, and the tumor volume is smaller. Therefore, the collagen binding domain fusion CCL14 recombinant protein is a feasible, innovative and scientific scheme for tumor immunotherapy.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a collagen-binding domain fusion CCL14 recombinant protein and application thereof. BACKGROUND

[0002] Tumor is the primary threat to human health at present. In 2021, there were 1929 million new cancer cases in the world, including 1006 million cases in men and 923 million cases in women. In 2021, there were 996 million cancer deaths in the world, including 553 million cases in men and 443 million cases in women. Due to insufficient understanding of the mechanism of tumor, the survival rate of tumor patients is not optimistic, and therefore the treatment of tumor is one of the key problems in the scientific community at present.

[0003] Compared with traditional treatment methods, tumor immunotherapy is an effective treatment method for tumor in recent years. For example, the treatment of tumor by targeting immune checkpoint PD-1 (programmed cell death protein-1) as a treatment strategy has made remarkable progress. However, due to different tumor phenotypes of different patients, tumor immunotherapy cannot work for all patients, and therefore it is very important to find new immunotherapy targets.

[0004] Chemokines are an important class of cytokines that can cause changes in the tumor microenvironment, and are also the largest subfamily of cytokines. Because of the ability to direct chemotaxis of cells, chemokines have become key factors for regulating the tumor microenvironment. The tumor microenvironment is the main place for the interaction between tumor cells and the host immune system, and a variety of immune cells migrate to the tumor microenvironment through the interaction between chemokines and chemokine receptors. The role of these immune cells and chemokines in the tumor microenvironment has an important influence on the occurrence, development and treatment results of tumor. Tumor immunotherapy targeting chemokines has made great progress and has a bright development prospect.

[0005] CCL14 is a CC-type chemokine, and its high expression in various tumor databases is associated with good prognosis of tumor patients, which suggests that CCL14 may have an anticancer function. Although CCL14 is considered to have an anticancer function, research on the anticancer effect of CCL14 through the immune system is not in-depth, and only a few papers have preliminarily mentioned it. How to apply CCL14 to tumor immunotherapy has not yet been effectively realized. SUMMARY

[0006] To solve the above technical problems, the application provides a collagen-binding domain fusion CCL14 recombinant protein and application thereof.

[0007] In a first aspect, the present application provides a collagen-binding domain fusion CCL14 recombinant protein, the amino acid sequence of which is shown in SEQ ID NO. 1.

[0008] In a second aspect, the present application provides a gene encoding the recombinant protein, the nucleotide sequence of which is shown in SEQ ID NO. 2.

[0009] In a third aspect, the present application provides a recombinant expression vector containing the gene.

[0010] In a fourth aspect, the present application provides a construction method of the recombinant expression vector, which comprises the following steps:

[0011] The gene sequence shown in SEQ ID NO. 2 is inserted into the multiple cloning site EcoR I and Xho I of pcDNA3.1 plasmid by seamless cloning method.

[0012] In a fifth aspect, the present application provides a preparation method of the recombinant protein, characterized in that it comprises the following steps:

[0013] S1, transfecting the recombinant expression vector into 293F cells and culturing;

[0014] S2, centrifuging the cells cultured for seven days to collect the supernatant; incubating the supernatant with nickel ion beads in a His-tag protein purification kit at 4℃ on a shaker for 3h; after incubation, adding the supernatant into a chromatography column, and performing washing and elution; eluted protein is dialyzed using a dialysis bag with a pore size of 5KDa, and the obtained liquid is the collagen-binding domain fusion CCL14 recombinant protein.

[0015] In a sixth aspect, the present application provides the use of the recombinant protein, the gene or the recombinant expression vector in the preparation of a medicament for tumor immunotherapy.

[0016] Further, the tumor includes melanoma.

[0017] The present application has the following beneficial effects:

[0018] The present application innovatively fuses the collagen-binding domain with the chemokine CCL14, uses the recombinant protein to treat tumors, and achieves more significant effects than the treatment of pure CCL14 protein. Therefore, the present application provides a feasible, innovative and scientific scheme for tumor immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 Schematic diagram of pCDNA3.1-CCL14-his-CON recombinant plasmid and pCDNA3.1-CCL14-CBD-his.

[0020] Figure 2Western blot identification results of CCL14-his-CON and CCL14-CBD-his before purification.

[0021] Figure 3 Western blot identification results of CCL14-his-CON and CCL14-CBD-his after purification.

[0022] Figure 4 For the therapeutic effect of CCL14-his-CON and CCL14-CBD-his on tumors, Figure A is the tumor image analysis result, Figure B is the quantitative statistical diagram of tumor volume, and Figure C is the statistical diagram of tumor weight.

[0023] Figure 5 For the immunofluorescence staining of tumor sections, the upper three graphs are 80 μm, and the lower three graphs are 20 μm. DETAILED DESCRIPTION

[0024] The present application will be described in detail below in conjunction with the accompanying drawings and specific examples, but should not be understood as limiting the present application. If not specifically stated, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specifically stated, can be obtained from commercial channels.

[0025] Example 1: Obtaining collagen binding domain fusion CCL14 recombinant protein

[0026] 1. Experimental method

[0027] 1.1 Vector design, construction and identification

[0028] 1.1.1 Vector construction

[0029] The collagen binding domain fusion CCL14 gene (CCL14-CBD) shown in SEQ ID NO. 2 and the control gene (CCL14-CON) shown in SEQ ID NO. 3 were obtained by conventional molecular cloning means or artificial synthesis, and were subjected to enzyme digestion with EcoR1 and Xho1, electrophoresis and gel recovery; then the target fragments recovered by enzyme digestion and the enzyme-digested pCDNA3.1 vector were connected using T4 ligase, i.e. the overexpression plasmid pCDNA3.1-CCL14-his-CON and the pCDNA3.1-CCL14-CBD-his containing the collagen binding domain were obtained.

[0030] 1.1.2 Vector identification

[0031] The overexpression plasmid pCDNA3.1-CCL14-his-CON and pCDNA3.1-CCL14-CBD-his containing the collagen binding domain were transfected in 293T cells. Protein was collected 48h post transfection and Western blot was performed, both His-tag antibody and CCL14 antibody detected a band around 10KDa.

[0032] 1.2 Expression of recombinant protein

[0033] 1.2.1 Culture of 293F cells in suspension

[0034] We used 293F cells in suspension to express the recombinant protein we needed. 293F cells were cultured using FreeStyle 293 Expression Medium from Gibco, and passaged every 2-3 days. When passaging, we centrifuged at 800rpm for 5min, and discarded the supernatant. The culture conditions were 37℃, 125rpm, 8% CO2 in a cell culture incubator. TM

[0035] 1.2.2 Plasmid transfection

[0036] The concentration of the plasmid should be 1 x 10 6 , and the transfection system was 4.5μL of Lipofectamine EZ Transfection Reagent, 1.5μg of plasmid, and 60μL of serum-free medium per milliliter of cells. After mixing the transfection system and incubating for 20min, we added it to the culture flask of 293F cells. The culture flask was placed in the incubator for continuous culture for 7 days.

[0037] 1.3 Purification and identification of recombinant protein

[0038] 1.3.1 Purification of recombinant protein

[0039] We used the His-tag protein purification kit from Biyun Tian to purify the protein. After centrifugation at 1200rpm for 5min, we collected the supernatant from the cells cultured for 7 days after transfection. We incubated the culture supernatant with nickel ion beads in the His-tag protein purification kit on a 4℃ shaker for 3h, and as a reference, we used 500μL of nickel ion beads per 50mL of culture supernatant. After incubation, we added the supernatant to the chromatography column, and used the traditional NI-NTA buffer for washing and elution. As a reference, the concentration of imidazole in the eluate was 250mM.

[0040] 1.3.2 Dialysis of recombinant protein

[0041] The protein eluted with imidazole cannot be directly used for mouse tumor treatment, so we used a dialysis bag with a pore size of 5KDa from Youbo to dialyze it in excess PBS for 24h. After dialysis, we collected the liquid in the dialysis bag, which was the recombinant protein we needed. ​

[0042] 1.3.3 Recombinant protein identification

[0043] After obtaining the recombinant protein, BCA quantitative method was used for quantification, and after quantification, protein gel electrophoresis and Coomassie blue staining method were used for identification. The gel after electrophoresis was stained with 30 mL of Coomassie blue staining solution for 3 h, and then eluted with Coomassie blue elution solution overnight, and imaged.

[0044] 2 Experimental results

[0045] 2.1 According to the experimental purpose, the overexpression plasmid pCDNA3.1-CCL14-his-CON of CCL14 and the pCDNA3.1-CCL14-CBD-his containing collagen binding domain were designed and obtained. The schematic diagram is Figure 1 .

[0046] 2.2 pCDNA3.1-CCL14-his-CON and pCDNA3.1-CCL14-CBD-his containing collagen binding domain were successfully expressed in 293F cells Figure 2 , using antibody His tag).

[0047] 2.3 CCL14-his-CON (amino acid sequence as shown in SEQ ID NO. 4) and CCL14-CBD-his (amino acid sequence as shown in SEQ ID NO. 1) containing collagen binding domain were successfully expressed in 293F cells, and were purified and dialyzed. The protein after dialysis was subjected to western blot and Coomassie staining Figure 3 ).

[0048] Example 2: Therapeutic effect of collagen binding domain fusion CCL14 recombinant protein on tumors

[0049] 1 Experimental method

[0050] 1.1 C57BL / c mice subcutaneously inoculated with tumor cells

[0051] 6-8 week old male C57BL / c mice were subcutaneously inoculated with mouse melanoma cells B16 / F10. The tumor cells with a confluence rate of 80%-90% were digested into centrifuge tubes with trypsin, the supernatant was discarded after centrifugation, and the centrifuge tubes were placed on ice. Washed twice with sterile PBS. Take part of the cell suspension and count it on a hemocytometer under a light microscope. According to the calculated concentration, adjust the cell suspension concentration to 2.5×10 6 6 / mL with PBS. Inject 200 μL of mixed cell suspension with a 1 mL disposable syringe under the epidermis between the back and hips of the mouse, and the number of tumor cells is 5×10 5A total of 21 mice were used. After 7 days, the mice were shaved, and the subcutaneous tumors were numbered according to their relative size, and then the 21 mice were randomly divided into 3 groups with 3 mice in each group.

[0052] 1.2 Tail vein injection of recombinant proteins

[0053] At 7 and 14 days after tumor inoculation, 300 μL PBS, 25 μg of control recombinant protein CCL14-his-CON, and an equimolar amount of collagen-binding recombinant protein CBD-CCL14-his were injected into the tail vein of the mice. The subcutaneous tumor volume was measured every 2 days using a vernier caliper. At 18 days, the two mice with the largest and smallest tumor volume in each group were excluded, and the tumors of the remaining 5 mice were collected and weighed.

[0054] 1.3 Detection of tumor infiltration of recombinant proteins

[0055] At 10 days after subcutaneous tumor inoculation of B16 / F10 in C57BL / c mice, 300 μL PBS, 25 μg of control recombinant protein CCL14-his-CON, and an equimolar amount of collagen-binding recombinant protein CBD-CCL14-his were injected into the tail vein of the mice. The tumors were collected after 48 h, fixed with paraformaldehyde for 6 h, and dehydrated with a 15% sucrose PBS solution at 4°C overnight. The next day, OCT embedding agent was used for embedding, and after freezing at -80°C, the sections were cut into 12 μm slices and subjected to immunofluorescence staining. Blocking was performed using a 3% BSA solution for 30 min, staining was performed using a His-tag antibody from proteintech (dilution ratio 1:1000, 1% BSA, and 0.3% Triton), secondary antibody staining (1:500, 1% BSA), Hoechst staining of the cell nucleus (1:4000, 1% BSA) for 7-10 min, mounting, and observation under a confocal microscope.

[0056] 2 Experimental results

[0057] 2.1 The recombinant protein CBD-CCL14-his played a tumor inhibitory role in a mouse subcutaneous tumor model. Compared with the control group, the tumor volume and weight of the tumor-bearing mice treated with the CBD-CCL14-his recombinant protein were significantly reduced. There was no significant difference in the tumor volume and weight of the mice injected with the control protein CCL14-his-CON and the PBS group ( Figure 4 ).

[0058] 2.2 Immunofluorescence showed that compared with the tumors of mice injected with CCL14-his-CON protein, the his tag (shown in the box) could be detected in the tumors of mice injected with CBD-CCL14-his protein, indicating that after injection, CBD-CCL14-his could better target the tumor microenvironment ( Figure 5).

[0059] In conclusion, the immunotherapy of tumor using collagen-binding domain fusion CCL14 recombinant protein is a feasible, innovative and scientific solution.

[0060] While the preferred embodiments of the application have been described, additional modifications and changes can occur to those skilled in the art once they learn of the basic creative principles disclosed herein. Accordingly, it is intended that the appended claims shall cover all such modifications and changes as fall within the true spirit and scope of the application.

[0061] It will be apparent to those skilled in the art that various modifications and variations can be made to the present application without departing from the spirit or scope of the application. Thus, it is intended that the present application cover modifications and variations of this application provided they come within the scope of the appended claims and their equivalents.

Claims

1. A recombinant protein with a collagen-binding domain fused to CCL14, characterized in that, The amino acid sequence is shown in SEQ ID NO.

1.

2. The gene encoding the recombinant protein according to claim 1, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

3. A recombinant expression vector containing the gene of claim 2.

4. The method for constructing the recombinant expression vector according to claim 3, characterized in that, Includes the following steps: The gene sequence shown in SEQ ID NO.2 was inserted into the multiple cloning site between EcoRI and XhoI of the pcDNA3.1 plasmid using a seamless cloning method.

5. The method for preparing the recombinant protein according to claim 1, characterized in that, Includes the following steps: S1. Transfect the recombinant expression vector according to claim 3 into 293F cells and culture them; S2. Centrifuge the cells cultured for seven days and collect the supernatant. Incubate the supernatant with nickel ion beads from the His-tagged protein purification kit in a shaker at 4°C for 3 h. After incubation, add the supernatant to the chromatography column for washing and elution. Dialyze the eluted protein through a 5 kDa dialysis bag. The resulting liquid is the collagen-binding domain fused CCL14 recombinant protein.

6. The use of the recombinant protein of claim 1, the gene of claim 2, or the recombinant expression vector of claim 3 in the preparation of a medicament for tumor immunotherapy, characterized in that, The tumor is melanoma.

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