Recombinant cell for expressing DspB protein and application thereof

Abstract

The invention relates to a recombinant cell for expressing DspB protein and application thereof, and belongs to the technical field of genetic engineering. The invention provides a recombinant cell for expressing a DspB protein. The recombinant cell comprises a DspB protein gene and host bacteria, the host cell is a CHO cell; the nucleotide sequence of the DspB protein gene is as shown in SEQ ID NO. 1. The method for constructing the recombinant cell is simple, the constructed recombinant cell can stably express a large amount of DspB protein, the enzymatic activity of the expressed DspB protein is higher than that of the DspB protein expressed by prokaryotic cells, and the bacterial biofilm removal effect is better.

Description

Technical Field

[0001] The present invention relates to the technical field of gene engineering, and in particular to a recombinant cell expressing DspB protein and application thereof. Background Art

[0002] Many microorganisms in nature tend to form multicellular communities. The surface of the colony is coated with a layer of self-synthesized polysaccharides, lipids, proteins and other substances to form bacterial biofilms to help them survive attacks by antimicrobial agents and the host immune system. Bacterial biofilms present on the surface of implanted biomedical devices often cause serious infections and inflammations. For example, biofilms of Staphylococcus epidermidis and Escherichia coli often cause intravascular catheter-related infections, and biofilms of Staphylococcus aureus are often associated with infections caused by metal implants. Bacterial biofilms can protect bacteria from being recognized and eliminated by the host immune system, while antibiotics cannot reach pathogens due to the obstruction of bacterial biofilms and cannot achieve the expected bactericidal effect.

[0003] Bacteria in biofilms are coated in a self-synthesized extracellular polysaccharide matrix, the main component of which is poly-β-(1,6)-N-acetylglucosamine connected by β-(1,6) glycosidic bonds. After the biofilm formed by bacteria is destroyed by proteases, deoxyribonucleases and glycosidases, the bacteria are easily exposed to antimicrobial agents and killed. Glycoside hydrolase GH20 protein can hydrolyze the β-(1,4) glycosidic bonds between N-acetylglucosamine residues, and dispersin B (DspB), as one of the glycoside hydrolase family GH20 proteins, has the function of catalyzing the degradation of the β-(1,6) glycosidic bonds of poly-β-(1,6)-N-acetylglucosamine. DspB protein can degrade the biofilm formed by bacteria, making the bacteria form free cells, which is convenient for the body's immune system to clear and the penetration and diffusion of antibiotics. By utilizing the specificity of DspB binding to substrates and combining it with medical devices, it is expected to provide an effective treatment for bacterial biofilm infections of implanted medical devices. Currently, the protein DspB has been expressed and purified in Escherichia coli. It has degradation activity for poly-β-(1,6)-N-acetylglucosamine and can destroy bacterial biofilms. However, the recombinant protein expressed by it has low activity and low efficiency in removing bacterial biofilms.

[0004] Based on this, the present invention is proposed. Summary of the invention

[0005] The object of the present invention is to provide a recombinant cell expressing DspB protein and its application, so as to solve the problems in the prior art that the DspB protein expressed and purified in Escherichia coli has low activity and low efficiency in removing bacterial biofilm.

[0006] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0007] The present invention provides a recombinant cell expressing DspB protein, wherein the recombinant cell comprises a DspB protein gene and a host bacterium; the host cell is a CHO cell;

[0008] The nucleotide sequence of the DspB protein gene is shown in SEQ ID NO.1.

[0009] The present invention provides a method for constructing the recombinant cell, comprising the following steps:

[0010] (1) inserting the DspB protein gene into the pB transposon vector to obtain a recombinant vector;

[0011] (2) using the recombinant vector obtained in step (1) to transfect CHO cells to obtain transfected cells;

[0012] (3) culturing the transfected cells using a culture medium, and when the cell confluence of the transfected cells reaches 85-95%, obtaining recombinant cells expressing the DspB protein;

[0013] The DspB protein gene in step (1) is the DspB protein gene.

[0014] Preferably, the pB transposon vector in step (1) is pPB-HSA.

[0015] Preferably, the transfection method in step (2) is calcium phosphate precipitation method, electrofection method, liposome transfection method or PEI transfection method.

[0016] Preferably, the culture medium in step (3) is a DMEM-F12 complete culture medium containing blasticidin; the final concentration of blasticidin in the DMEM-F12 complete culture medium is 8 to 17 μg / mL.

[0017] The present invention provides the use of the recombinant cell or the recombinant cell obtained by the construction method in preparing a product for removing bacterial biofilm.

[0018] The present invention provides a method for preparing DspB protein, comprising the following steps:

[0019] (1) inoculating the recombinant cells expressing the DspB protein into a serum-free suspension medium for culture, and when the cell survival rate is 65-75%, centrifuging and collecting the supernatant to obtain a supernatant;

[0020] (2) filtering, purifying and eluting the supernatant obtained in step (1) to obtain DspB protein;

[0021] The recombinant cell expressing the DspB protein in step (1) is the recombinant cell or the recombinant cell obtained by the construction method.

[0022] Preferably, the serum-free suspension medium in step (1) is CHO serum-free suspension medium; the culture speed is 115-125 rpm, the culture temperature is 36-38°C, and the culture CO 2 The concentration is 4-6%; the initial concentration of the recombinant cells expressing DspB protein in the serum-free suspension medium is 4-6×10 5 Pieces / mL.

[0023] Preferably, the filtration in step (2) is microporous membrane filtration, the pore size of the microporous membrane is 0.2-0.45 μm; the purification is nickel column purification; the elution solution is a phosphate buffer containing imidazole, and the final concentration of the imidazole is 180-220 mmol / L.

[0024] The invention provides application of the DspB protein obtained by the preparation method in preparing a product for removing bacterial biofilm.

[0025] The present invention has the following technical effects and advantages:

[0026] The present invention optimizes the DspB protein gene so that it can be expressed in eukaryotic animal cells and can increase the expression amount of the DspB protein. The present invention provides a recombinant cell expressing the DspB protein, the construction method of the recombinant cell is simple, and the DspB protein can be stably expressed in large quantities, the enzyme activity of the expressed DspB protein is higher than that of the DspB protein expressed by prokaryotic cells, and the effect of removing bacterial biofilm is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the structural diagram of the recombinant vector pPB-DspB;

[0028] Figure 2 To stabilize the cell line, Western Blot was used to detect the expression of DspB protein;

[0029] Figure 3 The results of Western Blot analysis of DspB protein expression in monoclonal cell lines;

[0030] Figure 4 The SDS-PAGE results of the eluates with different concentrations of imidazole;

[0031] Figure 5 is the enzymatic activity of DspB protein expressed by different recombinant cells;

[0032] Figure 6 Bacterial biofilm removal rate of DspB protein expressed by different recombinant cells. DETAILED DESCRIPTION

[0033] The present invention provides a recombinant cell expressing DspB protein, wherein the recombinant cell comprises a DspB protein gene and a host bacterium; the host cell is a CHO cell;

[0034] The nucleotide sequence of the DspB protein gene is shown in SEQ ID NO.1;

[0035]

[0036] The present invention provides a method for constructing the recombinant cell, comprising the following steps:

[0037] (1) inserting the DspB protein gene into the pB transposon vector to obtain a recombinant vector;

[0038] (2) using the recombinant vector obtained in step (1) to transfect CHO cells to obtain transfected cells;

[0039] (3) culturing the transfected cells using a culture medium, and when the cell confluence of the transfected cells reaches 85% to 95%, preferably 90%, obtaining recombinant cells expressing the DspB protein;

[0040] The DspB protein gene in step (1) is the DspB protein gene.

[0041] In the present invention, the pB transposon vector in step (1) is pPB-HSA, and the method for preparing the recombinant vector is to replace the HAS gene of the pPB-HSA vector with the DspB gene using seamless cloning technology.

[0042] In the present invention, the transfection method in step (2) is calcium phosphate precipitation method, electrofection method, liposome transfection method or PEI transfection method.

[0043] In the present invention, the culture medium in step (3) is a DMEM-F12 complete culture medium containing blasticidin; the final concentration of blasticidin in the DMEM-F12 complete culture medium is 8 to 17 μg / mL, preferably, the culture medium is cultured for 10 days with a DMEM-F12 complete culture medium containing blasticidin at a final concentration of 15 μg / mL, and then cultured for 18 days with a DMEM-F12 complete culture medium containing blasticidin at a final concentration of 10 μg / mL, and the culture medium is replaced every 2 days during the culture period.

[0044] The present invention provides the use of the recombinant cell or the recombinant cell obtained by the construction method in preparing a product for removing bacterial biofilm.

[0045] The present invention provides a method for preparing DspB protein, comprising the following steps:

[0046] (1) inoculating the recombinant cells expressing the DspB protein into a serum-free suspension medium for cultivation, and when the cell survival rate is 65-75%, preferably 70%, centrifuging and collecting the supernatant to obtain a supernatant;

[0047] (2) filtering, purifying and eluting the supernatant obtained in step (1) to obtain DspB protein;

[0048] The recombinant cell expressing the DspB protein in step (1) is the recombinant cell or the recombinant cell obtained by the construction method.

[0049] In the present invention, the serum-free suspension medium in step (1) is a CHO serum-free suspension medium; the rotation speed of the culture is 115 to 125 rpm, preferably 120 rpm, the temperature of the culture is 36 to 38°C, preferably 37°C, and the CO 2 The concentration is 4-6%, preferably 5%; the initial concentration of the recombinant cells expressing DspB protein in the serum-free suspension medium is 4-6×10 5 / mL, preferably 5×10 5 Pieces / mL.

[0050] In the present invention, the filtration in step (2) is microporous membrane filtration, the pore size of the microporous membrane is 0.2-0.45 μm, preferably 0.22 μm; the purification is nickel (Ni) column purification; the elution solution is a phosphate buffer containing imidazole, and the final concentration of the imidazole is 180-220 mmol / L, preferably 200 mmol / L.

[0051] The invention provides application of the DspB protein obtained by the preparation method in preparing a product for removing bacterial biofilm.

[0052] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0053] The pPB-HSA described in the embodiment is prepared according to the construction method of the pPB-HSA vector described in "A highly efficient transposon vector system for recombinant protein expression in CHO cells" published by Tian-Yun W; the CHO cells are CHO-S cells purchased from the cell bank of the Chinese Academy of Sciences; 2000 transfection reagent was purchased from Hefei Lanjieke Technology Co., Ltd. with the catalog number BL623B; the N-acetyl-β-D-glucosidase (NAG) test kit was purchased from Shanghai Jining Biotechnology Co., Ltd. with the catalog number JN24601; CHO serum-free suspension culture medium was purchased from Henan Punuoyi Biological Products Research Institute Co., Ltd.

[0054] Example 1: Construction of recombinant cells expressing DspB protein

[0055] 1. Optimization and synthesis of DspB gene and construction of recombinant vector containing DspB gene

[0056] The amino acid sequence of DspB protein was obtained from the GenBank database, and its number was AAP31025.1. Then the DspB gene sequence was obtained. The DspB gene sequence was optimized by General Biosystems (Anhui) Co., Ltd. for expression in mammalian cells. The optimized DspB gene sequence is shown in SEQ ID NO.1.

[0057] The optimized DspB gene was cloned downstream of the CMV promoter of the pPB-HSA vector using seamless cloning technology to replace the HAS gene of the pPB-HSA vector and construct a recombinant vector pPB-DspB containing the DspB gene. Its structure is as follows: Figure 1 shown.

[0058] 2. Transfection and culture of recombinant cells

[0059] (1) Transfection of recombinant cells

[0060] CHO-S cells in good growth state were selected and inoculated into 12-well culture plates containing DMEM F12 medium for culture. When the cell confluence reached 80%, cells to be transfected were obtained and transfected. A control group was set up without transfection.

[0061] The transfection method is as follows:

[0062] ① Add 125 μL of DMEM-F12 serum-free medium to each well, and then add 5 μL of 2000 μL of transfection reagent, mix gently and leave at room temperature for 5 min to obtain the transfection reagent dilution solution;

[0063] ② Add 2 μg of the recombinant vector pPB-DspB to 125 μL of DMEM-F12 serum-free medium in each well, mix gently, and prepare the recombinant vector pPB-DspB dilution solution;

[0064] ③Take 130 μL of the diluted transfection reagent solution in ① and 125 μL of the recombinant vector pPB-DspB dilution solution in ②, mix gently, and incubate at room temperature for 15 minutes to obtain a mixed solution;

[0065] ④ Change the medium of the cells to be transfected, add 1 mL of fresh DMEM F12 complete medium, and add the mixed solution obtained in ③ to the 12-well plate containing the cells to be transfected;

[0066] ⑤ Incubate the transfected cells at 37°C and 5% CO 2 The cells were cultured in a constant temperature incubator, and fresh DMEM F12 complete medium was replaced 5 hours after transfection. Transfected cells were obtained 48 hours after transfection.

[0067] (2) Culture of transfected cells

[0068] The transfected cells were cultured using DMEM-F12 complete medium containing blasticidin at a final concentration of 15 μg / mL, and the medium was replaced every 2 days. The culture was carried out for 10 days. After the cells in the control group died, the cells were cultured for another 18 days using DMEM-F12 complete medium containing blasticidin at a final concentration of 10 μg / mL, and the medium was replaced every 2 days. When the confluence of the transfected cells reached 90%, recombinant cells stably expressing the DspB protein were obtained.

[0069] Example 2: Verification of the expression of recombinant cell DspB protein

[0070] (1) Screening of monoclonal cell lines

[0071] The recombinant cells stably expressing DspB protein obtained in Example 1 were subjected to gradient dilution to obtain a cell suspension, and the cells were plated in a 96-well plate. After the cells adhered to the wall, they were observed under an inverted microscope, and the wells containing only one cell were marked. After 5 days, the wells with cells that formed a monoclonal cell were marked again. When the monoclonal cell line was full in the 96-well plate, the cells were digested with trypsin, and the cells in the wells with the monoclonal cell line were transferred to a new culture flask for expanded culture.

[0072] (2) Cell suspension culture

[0073] The cells in the monoclonal cell culture flask were digested with trypsin to collect the monoclonal cell lines. The stable cell lines and monoclonal cell lines obtained in Example 1 were added to CHO serum-free suspension medium and resuspended to obtain cell concentrations of 5×10 5 The stable cell line suspension was prepared with a cell concentration of 5×10 5 Take 150 mL of the stable cell line suspension and the monoclonal cell line suspension, respectively, and pour them into a 500 mL shake flask. Incubate at 37°C and 5% CO 2 The cells were suspended and cultured in a cell culture incubator at 120 rpm. Cell growth was monitored using a cell counter during the culture process. The culture was terminated when the cell viability dropped to 70%. The culture supernatant was collected separately, centrifuged at 1000 rpm for 5 min, and then centrifuged again at 13000 rpm for 10 min, and the supernatant was collected separately.

[0074] (3) Detection of DspB protein expression by Western Blot

[0075] Add 5×SDS sample buffer to the supernatant of the collected stable cell lines and monoclonal cell lines, respectively, and boil at 100℃ for 10 min to fully denature the protein. Prepare 10% SDS-PAGE gel and take 10μL of protein samples for electrophoresis. After the electrophoresis, use a fast transfer instrument to transfer the membrane at a constant current of 400mA for 30min, block with 5% skim milk powder at room temperature for 2h, and incubate His antibody at 4℃ overnight; wash the membrane with TBST 3 times, 5min each time, and incubate the secondary antibody at room temperature for 1.5h; prepare ECL ultrasensitive luminescent liquid and observe the expression of DspB protein on a gel imager. The expression results of DspB protein detected by Western Blot in stable cell lines are shown in the figure. Figure 2 As shown in the figure, DspB 1-2 represent two stable cell lines, and the expression results of DspB protein detected by Western Blot method of monoclonal cell lines are shown in Figure 3 As shown, Clone 1 to 4 represent 4 monoclonal cell lines.

[0076] according to Figures 2-3 It can be seen that the expression of DspB protein can be detected in both stable cell lines and monoclonal cell lines, indicating that CHO cells successfully expressed DspB protein.

[0077] Example 3: Purification of protein DspB

[0078] The supernatant of the monoclonal cell line collected in step (2) of Example 2 was filtered through a microporous filter membrane with a pore size of 0.22 μm, purified using a nickel (Ni) column on an AKTApure 25L protein purification chromatography system, and the supernatant was gradient eluted using a phosphate buffer containing different concentrations of imidazole. The eluate was collected, and then the collected eluate with different concentrations of imidazole was subjected to Coomassie Brilliant Blue SDS-PAGE. The results are as follows: Figure 4 As shown in the figure, lanes 1 to 11 are respectively the original solution, the flow-through solution, the 20mM imidazole eluent, the 50mM imidazole eluent, the 50mM imidazole eluent, the 100mM imidazole eluent, the 100mM imidazole eluent, the 200mM imidazole eluent, the 200mM imidazole eluent, the 200mM imidazole eluent, and the 500mM imidazole eluent.

[0079] according to Figure 4 It can be seen that high-purity DspB protein can be obtained by eluting with phosphate buffer containing 200 mmol / L imidazole.

[0080] Example 4: Preparation of DspB protein

[0081] (1) The recombinant cells expressing DspB protein obtained in Example 1 were added to CHO serum-free suspension medium and resuspended to obtain a cell concentration of 5×10 5150 mL of cell suspension was placed in a 500 mL shake flask and incubated at 37 °C and 5% CO 2 The cells were suspended and cultured in a cell culture incubator at 120 rpm. During the culture process, a cell counter was used to monitor the cell growth. The culture was terminated when the cell viability dropped to 70%. The cell culture supernatant was collected, centrifuged at 1000 rpm for 5 min, and then centrifuged again at 13000 rpm for 10 min, and the supernatant was collected to obtain the supernatant.

[0082] (2) The supernatant was filtered through a microporous filter membrane with a pore size of 0.22 μm, purified using a nickel (Ni) column on an AKTApure 25L protein purification chromatography system, eluted with a phosphate buffer containing 200 mmol / L imidazole, and the imidazole in the eluate was removed to obtain a DspB protein solution.

[0083] Example 5: Activity detection of DspB protein

[0084] The DspB protein solution obtained in Example 4 was quantified using a BCA protein assay kit, and the concentration of the DspB protein in the solution was measured to be 0.745 mg / mL. The DspB protein solution was diluted to obtain DspB protein solutions with concentrations of 0.4 mg / mL and 0.6 mg / mL, and the DspB protein solutions with concentrations of 0.4 mg / mL and 0.6 mg / mL were used as experimental groups, and commercially available prokaryotically expressed DspB proteins were added to phosphate buffer to prepare DspB protein solutions with concentrations of 0.4 mg / mL and 0.6 mg / mL, and the DspB protein solutions prepared with commercially available prokaryotically expressed DspB proteins were used as control groups, and the DspB protein solutions prepared with commercially available prokaryotically expressed DspB proteins were used as control groups, and the DspB protease activity of the experimental group and the control group was detected using an N-acetyl-β-D-glucosidase (NAG) test kit, respectively. The results are as follows: Figure 5 shown.

[0085] according to Figure 5 It can be seen that the enzyme activity of the DspB protein expressed by CHO cells at concentrations of 0.4 mg / mL and 0.6 mg / mL is 16.35 nmol / min / mL and 32.38 nmol / min / mL, respectively, which is significantly higher than the DspB protease activity expressed by prokaryotic cells at the same concentration (2.36 nmol / min / mL and 5.45 nmol / min / mL, respectively). This shows that the DspB protease activity expressed by the recombinant cell CHO prepared in the present application scheme is higher than the DspB protease activity expressed by prokaryotic cells.

[0086] Example 6: Determination of the efficacy of DspB protein in removing bacterial biofilm

[0087] The concentration of 10 6A solution of Staphylococcus aureus of 100 mg / mL was inoculated into TSB broth medium in a 96-well plate, mixed and incubated in a constant temperature incubator at 37°C for 72 hours, and collected at the bottom of the well plate to form a bacterial biofilm. The collected bacterial biofilm was washed with sterile saline for 3 times, and DspB solution was added respectively, incubated at 37°C for 24 hours, washed with saline, fixed with methanol for 5 minutes, dried at room temperature, and 0.1% crystal violet was added for staining for 20 minutes, washed with saline, dried, and the biofilm was dissolved with 95% ethanol, and the absorbance value at A580nm wavelength was measured to calculate the biofilm removal rate. The DspB protein solution expressed by the recombinant cells of Example 1 with a concentration of 0.06 mg / mL and 0.1 mg / mL was used as the experimental group, and the DspB protein solution expressed by the commercially available prokaryotes with a concentration of 0.06 mg / mL and 0.1 mg / mL was used as the control group. The bacterial biofilm removal rates of the experimental group and the control group were measured, and the results are as follows. Figure 6 shown.

[0088] according to Figure 6 It can be seen that the bacterial biofilm removal rates of the DspB protein solution expressed by CHO cells at concentrations of 0.06 mg / ml and 0.1 mg / ml are 56.78% and 68.25%, respectively, which are higher than the bacterial biofilm removal rate of the DspB protein solution expressed by prokaryotes, indicating that the biofilm removal rate of the DspB protein expressed by recombinant cells prepared in the present application scheme is higher than the biofilm removal rate of the DspB protein expressed by prokaryotic cells.

[0089] As can be seen from the above embodiments, the present invention provides a recombinant cell expressing DspB protein and its application. The method for constructing the recombinant cell of the present invention is simple, and the constructed recombinant cell can stably and massively express DspB protein, the enzyme activity of the expressed DspB protein is higher than that of the DspB protein expressed by prokaryotic cells, and the effect of removing bacterial biofilm is better.

[0090] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A recombinant cell expressing DspB protein, characterized in that: The recombinant cell comprises a DspB protein gene and a host bacterium; the host cell is a CHO cell; The nucleotide sequence of the DspB protein gene is shown in SEQ ID NO.

1.

2. The method for constructing a recombinant cell according to claim 1, characterized in that: The steps include: (1) inserting the DspB protein gene into the pB transposon vector to obtain a recombinant vector; (2) using the recombinant vector obtained in step (1) to transfect CHO cells to obtain transfected cells; (3) culturing the transfected cells using a culture medium, and when the cell confluence of the transfected cells reaches 85-95%, obtaining recombinant cells expressing the DspB protein; The DspB protein gene in step (1) is the DspB protein gene according to claim 1.

3. The construction method according to claim 2, characterized in that: The pB transposon vector in step (1) is pPB-HSA.

4. The construction method according to claim 2, characterized in that: The transfection method in step (2) is calcium phosphate precipitation method, electrofection method, liposome transfection method or PEI transfection method.

5. The construction method according to claim 2, characterized in that: The culture medium in step (3) is a DMEM-F12 complete culture medium containing blasticidin; the final concentration of blasticidin in the DMEM-F12 complete culture medium is 8 to 17 μg / mL.

6. Use of the recombinant cell according to claim 1 or the recombinant cell obtained by the construction method according to any one of claims 2 to 5 in preparing a product for removing bacterial biofilm.

7. A method for preparing DspB protein, characterized in that: The steps include: (1) inoculating the recombinant cells expressing the DspB protein into a serum-free suspension medium for cultivation, and when the cell survival rate reaches 65-75%, centrifuging and collecting the supernatant to obtain a supernatant; (2) filtering, purifying and eluting the supernatant obtained in step (1) to obtain DspB protein; The recombinant cell expressing the DspB protein in step (1) is the recombinant cell according to claim 1 or the recombinant cell obtained by the construction method according to any one of claims 2 to 5.

8. The preparation method according to claim 7, characterized in that: The serum-free suspension medium in step (1) is CHO serum-free suspension medium; the rotation speed of the culture is 115-125 rpm, the temperature of the culture is 36-38° C., and the CO2 concentration of the culture is 4-6%; the initial concentration of the recombinant cells expressing the DspB protein in the serum-free suspension medium is 4-6×10 5 Pieces / mL.

9. The preparation method according to claim 7, characterized in that: The filtration in step (2) is microporous membrane filtration, the pore size of the microporous membrane is 0.2-0.45 μm; the purification is nickel column purification; the elution solution is a phosphate buffer containing imidazole, and the final concentration of the imidazole is 180-220 mmol / L.

10. Use of the DspB protein obtained by the preparation method according to any one of claims 7 to 9 in the preparation of a product for removing bacterial biofilm.

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