Engineered cell, construction method thereof and application of engineered cell in detection or screening of TLR4 receptor stimulant

Abstract

The invention belongs to the technical field of biology, and relates to detection of receptor protein agonists, in particular to an engineered cell, a construction method thereof and application of the engineered cell in detection or screening of TLR4 receptor agonists. The engineering cell is obtained by transfecting a lentivirus containing a TLR4 gene with an EYFP tag, a lentivirus containing a TLR4 gene with an ECFP tag and a lentivirus containing an MD2-CD14 gene to an HEK293T cell. The engineered cell provided by the invention highly expresses TLR4 with an EYFP tag, TLR4 with an ECFP tag, MD2, CD14 and other proteins, and can realize cellular level screening of an agonist capable of inducing TLR4 dimerization by utilizing FRET, so that a screening platform is established for the TLR4 receptor agonist.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the detection of receptor protein agonists, and specifically relates to an engineered cell and a construction method thereof and application thereof in the detection or screening of TLR4 receptor agonists. Background Art

[0002] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention, and should not necessarily be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art.

[0003] Toll-like receptor 4 (TLR4) receptor agonists can regulate the body's immune function and play a variety of roles, such as promoting immune response, anti-infection, and promoting immune memory. Among them, the role of TLR4 receptor agonists in activating antigen presenting cells (APCs) makes them promising for development as immune adjuvants, because the activation of APCs can promote antigen presentation, thereby enhancing the body's specific immune response to antigens. Immune adjuvants are key components of vaccines and can enhance the intensity, speed, and durability of vaccine immune responses. According to their mechanism of action, adjuvants are generally divided into immunostimulatory molecules, delivery systems, or a combination of the two. Immunostimulatory molecules mainly work by activating innate immune receptors (such as TLRs), which, after receptor activation, produce signals that initiate downstream adaptive immune responses. In order to trigger immune responses to specific pathogens, vaccines require new, efficient, and safe adjuvants, among which TLR4 receptor agonists are very promising adjuvants. However, there is currently a lack of efficient screening methods for TLR4 receptor agonists, which greatly limits the development of such adjuvants. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide an engineered cell and a construction method thereof and use thereof in detecting or screening TLR4 receptor agonists. The engineered cell provided by the present invention highly expresses TLR4 with an EYFP label, TLR4 with an ECFP label, and proteins such as MD2 and CD14, and can utilize fluorescence resonance energy transfer (FRET) to achieve cellular-level screening of agonists that can induce TLR4 dimerization, thereby building a screening platform for TLR4 receptor agonists.

[0005] In order to achieve the above object, the technical solution of the present invention is: In the first aspect, an engineered cell is obtained by transfecting HEK293T cells with a lentivirus containing a TLR4 gene with an EYFP tag, a lentivirus containing a TLR4 gene with an ECFP tag, and a lentivirus containing an MD2-CD14 gene, and the HEK293T cells can highly express TLR4 with an EYFP tag, TLR4 with an ECFP tag, MD2, and CD14. HEK293T cells are human embryonic kidney cells, which rarely express endogenous receptors required for extracellular ligands, are relatively easy to transfect and have a high transfection efficiency, and are a very commonly used tool cell line for expressing exogenous genes in research.

[0006] The engineered cells provided by the present invention can highly express TLR4 with an EYFP label, TLR4 with an ECFP label, MD2 and CD14, and can interact with and activate TLR4 with the help of CD14 (leukocyte differentiation antigen) and MD2 (myeloid differentiation protein 2) in the presence of a TLR4 receptor agonist, thereby causing receptor dimerization of TLR4; since the TLR4 expressed by the engineered cells provided by the present invention respectively carries EYFP and ECFP, after receptor dimerization of TLR4 occurs, the distance between EYFP and ECFP is shortened, so that the distance between the fluorescent groups of the two fluorescent proteins is less than 10 nm, thereby the energy between the groups is transferred from the donor to the acceptor in a non-radiative manner through dipole-dipole coupling, thereby generating fluorescence resonance energy transfer (FRET), and further realizing the screening of TLR4 receptor agonists through FRET.

[0007] In order to obtain the above-mentioned engineered cells, the present invention, on the other hand, provides a method for constructing the above-mentioned engineered cells, comprising the following steps: The lentivirus containing the TLR4 gene with an EYFP tag was transfected into HEK293T cells to obtain transformed cells; The transformed cells were transfected with a lentivirus containing the TLR4 gene with an ECFP tag and a lentivirus containing the MD2-CD14 gene.

[0008] First, in order to make HEK293T cells highly express TLR4 with an EYFP tag, TLR4 with an ECFP tag, MD2 and CD14, the general method is to transfect lentivirus carrying relevant genes. For the engineered cells constructed by the present invention, since TLR4 needs to have an EYFP tag and an ECFP tag respectively, the molecular weight of its gene is relatively large, it is difficult to successfully transfect and obtain relevant engineered cells by using one lentivirus to carry three related genes at the same time. Therefore, the present invention uses three lentiviruses to carry the TLR4 gene with an EYFP tag, the TLR4 gene with an ECFP tag, and the MD2-CD14 gene respectively.

[0009] In the process of transfecting three lentiviruses, the transfection step can be to transfect three lentiviruses simultaneously, or to transfect three lentiviruses separately and sequentially. However, the present invention has found that transfecting three lentiviruses simultaneously will cause defects in cells, thereby causing cells to be misidentified or contaminated, thereby affecting the accuracy and reliability of the results of screening TLR4 receptor agonists; it is difficult to transfect and obtain the target engineered cells by transfection if the three lentiviruses are transfected separately and sequentially. Through further research of the present invention, it was unexpectedly found that when the above-mentioned steps provided by the present invention are used for transfection (i.e., first transfecting a lentivirus containing a TLR4 gene with an EYFP label, and then simultaneously transfecting a lentivirus containing a TLR4 gene with an ECFP label and a lentivirus containing an MD2-CD14 gene), the target engineered cells can be obtained.

[0010] In some embodiments, the transfected HEK293T cells are in the logarithmic growth phase. Because in this growth phase, the cell division and proliferation rate are the fastest, it is suitable for a series of experiments and studies. The cell growth rate in the logarithmic growth phase is constant, the cell density increases rapidly, and the cell morphology and biochemical properties are relatively stable, which is very important for studying the functions and properties of cells.

[0011] In some embodiments, the screening process includes a culture medium containing Blasticidin (BSD), Geneticin (G418) and Puromycin (Puro).

[0012] Specifically, the concentration of blasticidin in the culture medium is 45-55 µg / mL.

[0013] Specifically, the concentration of geneticin in the culture medium was 360-440 µg / mL.

[0014] Specifically, the concentration of puromycin in the culture medium is 0.9~1.1 µg / mL.

[0015] In a third aspect, a use of the above-mentioned engineered cells in detecting or screening TLR4 receptor agonists.

[0016] Specifically, the detection or screening method is: incubating the sample to be detected or screened with the engineered cells, and then performing fluorescence detection.

[0017] In some embodiments, LPS is used as a positive control. Lipopolysaccharide (LPS) is a typical TLR4 ligand that can be recognized by TLR4. LPS binds to lipopolysaccharide binding protein (LBP) and forms a complex, which can interact with and activate TLR4 with the help of CD14 (LPS receptor, leukocyte differentiation antigen) and MD2 (myeloid differentiation protein 2), thereby causing TLR4 receptor dimerization. Therefore, LPS can be used as a positive control for detecting or screening TLR4 receptor agonists.

[0018] In some embodiments, flow cytometry is used for fluorescence detection, wherein the excitation wavelength of the fluorescence detection is 404-407 nm, and the emission wavelength of the fluorescence detection is 524-526 nm.

[0019] In some embodiments, the incubation time is 8 to 10 h.

[0020] The beneficial effects of the present invention are: The present invention constructs an engineered cell and utilizes the FRET principle to screen agonists that can induce TLR4 dimerization at the cellular level, thereby building a screening platform for TLR4 receptor agonists and further providing a rich material basis for the research and development of new immune adjuvants. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0022] Figure 1 This is a result diagram of HEK293T cells transfected with lentivirus under a fluorescence microscope in an embodiment of the present invention.

[0023] Figure 2 The results of screening the working concentration of BSD in the examples of the present invention are shown in Figure 1; the BSD concentration in A is 50 µg / mL, the BSD concentration in B is 40 µg / mL, the BSD concentration in C is 30 µg / mL, the BSD concentration in D is 20 µg / mL, and the BSD concentration in E is 10 µg / mL.

[0024] Figure 3The results of screening the working concentration of G418 in the embodiments of the present invention are shown in Figure 1; the concentration of G418 in A is 1000 µg / mL, the concentration of G418 in B is 900 µg / mL, the concentration of G418 in C is 800 µg / mL, the concentration of G418 in D is 700 µg / mL, the concentration of G418 in E is 600 µg / mL, the concentration of G418 in F is 500 µg / mL, the concentration of G418 in G is 400 µg / mL, and the concentration of G418 in H is 300 µg / mL.

[0025] Figure 4 The results of screening the working concentration of Puro in the examples of the present invention are shown in Figure 1. The Puro concentration in A is 0 µg / mL, the Puro concentration in B is 1 µg / mL, the Puro concentration in C is 2 µg / mL, the Puro concentration in D is 3 µg / mL, the Puro concentration in E is 4 µg / mL, and the Puro concentration in F is 5 µg / mL.

[0026] Figure 5 This is a diagram showing the protein expression results after transfection verified by Western Blot in an embodiment of the present invention.

[0027] Figure 6 This is a graph showing the results of flow cytometry to explore the LPS incubation time in an embodiment of the present invention.

[0028] Figure 7 This is a result diagram of the proportion of positive cells for receptor fluorescence EYFP detected by flow cytometry in an embodiment of the present invention. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0030] Example Instruments, reagents and materials 1. Instrument ChemiDoc XRS chemiluminescence imaging system (Bio-Rad, USA); CytoFLEX S flow cytometer (Beckman, USA); inverted fluorescence microscope (Olympus, China); Milli-Q pure water system (Millipore, USA); CO2 constant temperature cell culture incubator (Thermo Fisher Scientific, USA); TDZ5-W desktop low-speed centrifuge (Hunan Xiangyi Instrument Co., Ltd.); TW12 constant temperature water bath (Julabo, Germany); WB electrophoresis tank (Bio-Rad, USA); WB electrotransfer tank (Bio-Rad, USA).

[0031] 2. Reagents and Materials DMEM medium (VivaCell); sterile PBS buffer (Beijing Solebao Company); fetal bovine serum (Wuhan Sevier Company); cell culture flask (Hangzhou Bellanber Company); 6-well plate (Shanghai Wohong Biological Company); loading buffer (Shanghai Yazyme Company); RIPA lysis buffer (Shanghai Yazyme Company); rapid blocking solution (Shanghai Yazyme Company); electrophoresis buffer (Shanghai Yazyme Company); transfer buffer (Shanghai Yazyme Company); protease inhibitors (Shanghai Yazyme Company); phosphatase inhibitors (Shanghai Yazyme Company); ECFP antibody (Jiangsu Qinke Biological); EYFP antibody (Jiangsu Qinke Biological); MD2 antibody (Jiangsu Qinke Biological); GAPDH antibody (Jiangsu Qinke Biological); HRP-labeled goat anti-rabbit secondary antibody (Jiangsu Qinke Biological); BCA protein concentration kit (Beijing Biyuntian Company).

[0032] Overexpression lentivirus: LV-TLR4-ECFP-OE (Shanghai GeneCare, referred to as lentivirus ECFP-TLR4), LV-TLR4-EYFP-OE (Shanghai GeneCare, referred to as lentivirus EYFP-TLR4), LV-MD2-T2A-CD14-OE (Shanghai GeneCare, referred to as lentivirus MD2-CD14); vector elements are CMV enhancer-MCS-sv40-puromycin, Ubi-MCS-SV40-Neomycin, Ubi-MCS-SV40-blasticidin; transfection enhancement solution P solution (25×, Shanghai GeneCare); HEK293T cells were maintained by the laboratory of the inventor.

[0033] 1. Transfection of lentiviral EYFP-TLR4, lentiviral ECFP-TLR4, and lentiviral MD2-CD14 into HEK293T cells HEK293T cells in the logarithmic growth phase were plated in 6-well plates, and 1×10 5 / well, with a plate volume of 2 mL, and cultured in a cell culture incubator at 37 ℃ and 5% CO2 for 24 h. When the cell confluence is 30%, the original culture medium in the 6-well plate is aspirated and fresh complete culture medium (DMEM culture medium + 10% fetal bovine serum) is added, 800 µL per well. According to the virus transfection kit, the culture medium (480 µL), transfection enhancement solution P solution (120 µL) and lentivirus EYFP-TLR4 (30 µL) are prepared in the EP tube, and the operation is carried out on an ice box. Gently blow with a gun to obtain the first virus solution. Take out the prepared first virus solution from the EP tube and add it to the 6-well plate, 200 µL per well, so that the final volume of each well is 1 mL, and the 6-well plate is evenly placed in a 37 ℃, 5% CO2 incubator for culture. Change the medium after 16 hours, and pass the cells in a six-well plate after 72 hours, 1×10 5 / well, with a plate volume of 2 mL, and cultured in a cell culture incubator at 37 ℃ and 5% CO2 for 24 h. When the cell confluence was 30%, the lentivirus ECFP-TLR4 and lentivirus MD2-CD14 were transfected. According to the virus transfection kit, the culture medium (800 µL), transfection enhancement solution P solution (200 µL), lentivirus ECFP-TLR4 (15 µL) and lentivirus MD2-CD14 (18.75 µL) were prepared in the EP tube. The operation was performed on an ice box and the second virus solution was obtained by gently blowing with a gun. The prepared second virus solution was taken out from the EP tube and added to the 6-well plate, 200 µL per well, so that the final volume of each well was 1 mL. The 6-well plate was evenly placed in a 37 ℃, 5% CO2 incubator for culture. The medium was changed after 16 h, and photos were taken under a fluorescence microscope 48 h after transfection. Figure 1 As shown. Cells were passaged after 72 h. After transfection, corresponding antibiotics were added to the corresponding culture medium for cell selection.

[0034] 2. Screening of working concentrations of BSD, G418 and Puro HEK293T cells in the logarithmic growth phase were plated in 24-well plates, and 3 × 10 4 / well, shake well, and culture overnight in a cell culture incubator at 37°C and 5% CO2. Dilute 10 mg / mL BSD stock solution to 50, 40, 30, 20, and 10 µg / mL, dilute 20 mg / mL G418 stock solution to 1000, 900, 800, 700, 600, 500, 400, and 300 µg / mL, and dilute 1 mg / mL Puro stock solution to 1, 2, 3, 4, and 5 µg / mL. 24-well plates were administered, and control wells were given DMEM culture medium. Cultured in an incubator, the cells were observed every day. On the 7th day, the cells in the BSD group obviously fell and died at a concentration of 50 µg / mL, as shown in Figure 2. Figure 2As shown in A, B, C, D and E in Figure 1, the cells in the G418 group obviously fell and died at a concentration of 400 µg / mL. Figure 3 As shown in A, B, C, D, E, F, G and H. On the second day, the cells in the Puro group obviously fell and died at a concentration of 1 µg / mL, as shown in Figure 4 As shown in A, B, C, D, E and F in Figure 1. Therefore, the working concentrations of BSD, G418 and Puro are 50 µg / mL, 400 µg / mL and 1 µg / mL respectively. The medium containing these three antibiotics was prepared and used as the medium for cell replacement and subculture, but normal medium was used for the first subculture after transfection.

[0035] 3. Western Blot Verification The transfected cells were screened with antibiotics for more than half a month, and the total proteins of blank control and HEK293T cells overexpressing the target protein after transfection were extracted using RIPA cell lysis buffer containing protease inhibitors, and protein quantification was performed using Beijing Biotech Protein Quantification Kit (BCA method).

[0036] Wherein, the blank control (abbreviated as NC) is HEK293T cells that are not transfected with lentivirus. The specific method for extracting total protein from the cells is as follows: Take out the cells from the 6-well plate and pour off the culture medium. Rinse twice with PBS. Add 80 μL RIPA lysis buffer to each well on ice and scrape the cells with a cell scraper. Collect the cells into a 1.5 mL centrifuge tube and lyse on ice for 20 min. Centrifuge at 12000 r for 15 min, and collect the cell supernatant as the total protein solution. According to the protein quantification results, take 30 μg of protein for Western Blot. The protein sample was loaded on SDS-PAGE gel, separated by electrophoresis, and then transferred to PVDF membrane by wet method. After blocking with rapid blocking solution for 30 min, ECFP antibody (1:2000 dilution), EYFP antibody (1:2000 dilution), MD2 antibody (1:1000 dilution) and GAPDH antibody (1:10000 dilution) were added respectively, and incubated overnight at 4 ° C shaker. HRP-labeled goat anti-rabbit secondary antibody (1:3000 dilution) was added and incubated at room temperature for 1 h. After ECL color development, it was exposed in a chemiluminescence instrument and the results were recorded. The results showed that compared with the blank control (i.e. Figure 5 Compared with the NC group shown in , the ECFP, EYFP, and MD2 protein levels in HEK293T cells increased significantly (see Figure 5 ), the virus was successfully transferred and can be used for subsequent research.

[0037] 4. Flow cytometry to detect the proportion of positive cells for receptor fluorescence EYFP HEK293T cells in the logarithmic growth phase were plated in 6-well plates the day before, and 1×10 6 The cells were plated in 2 mL per well and cultured in a 37°C, 5% CO2 incubator for 24 h. Subsequently, the cells were treated with LPS (1 µg / mL). The cells were collected at 1 h, 3 h, 6 h, 9 h, and 12 h after administration, and the proportion of positive cells for the receptor fluorescence EYFP was detected by flow cytometry at an excitation wavelength of 405 nm and an emission wavelength of 525 nm. The results are shown in Figure 6 As shown, it was found that the proportion of positive cells for the receptor fluorescence EYFP was the highest after 9 h of incubation, so 9 h was selected as the detection time point.

[0038] HEK293T cells in the logarithmic growth phase were plated in 6-well plates the day before, and 1×10 6 / well, with a plate volume of 2 mL, and cultured in a cell culture incubator at 37 ℃ and 5% CO2 for 24 hours. Subsequently, the cells were treated with LPS, SIP (cuttlefish polysaccharide), HA (hyaluronic acid), lentinan, dendrobium polysaccharide, ginseng polysaccharide, atractylodes polysaccharide, angelica polysaccharide, astragalus polysaccharide, polygonatum polysaccharide, maitake polysaccharide, schisandra polysaccharide, and Acanthopanax polysaccharide (all at a concentration of 1 µg / mL). The cells were collected 9 hours after administration, and the proportion of positive cells for the receptor fluorescence EYFP was detected by flow cytometer at an excitation wavelength of 405 nm and an emission wavelength of 525 nm. The results are shown in Figure 7 As shown, it was found that Astragalus polysaccharide, Polygonatum sibiricum polysaccharide, Grifola frondosa polysaccharide, Schisandra chinensis polysaccharide, and Acanthopanax senticosus polysaccharide underwent obvious fluorescence shift, indicating that they may have interacted with TLR4 protein.

[0039] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. An engineered cell, characterized in that: HEK293T cells that can highly express TLR4 with an EYFP tag, TLR4 with an ECFP tag, MD2 and CD14 are obtained by transfecting HEK293T cells with a lentivirus containing the TLR4 gene with an EYFP tag, a lentivirus containing the TLR4 gene with an ECFP tag and a lentivirus containing the MD2-CD14 gene.

2. A method for constructing the engineered cell according to claim 1, characterized in that: The steps include: The lentivirus containing the TLR4 gene with an EYFP tag was transfected into HEK293T cells to obtain transformed cells; The transformed cells were transfected with a lentivirus containing the TLR4 gene with an ECFP tag and a lentivirus containing the MD2-CD14 gene.

3. The construction method according to claim 2, characterized in that: Transfected HEK293T cells were in the logarithmic growth phase.

4. The construction method according to claim 2, characterized in that: The invention comprises a screening process, wherein the culture medium used in the screening process contains blasticidin, geneticin and puromycin.

5. The construction method according to claim 4, characterized in that the culture medium The concentration of blasticidin in the medium is 45~55 µg / mL; or, the concentration of geneticin in the culture medium was 360–440 µg / mL; Alternatively, the concentration of puromycin in the culture medium is 0.9~1.1 µg / mL.

6. Use of the engineered cell according to claim 1 in detecting or screening a TLR4 receptor agonist.

7. The use according to claim 6, characterized in that: The detection or screening method is: incubating the sample to be detected or screened with the engineered cells, and then performing fluorescence detection.

8. The use according to claim 7, characterized in that: LPS was used as a positive control.

9. The use according to claim 7, characterized in that: Fluorescence detection was performed using flow cytometry.

10. The use according to claim 7, characterized in that: The incubation time is 8~10 h.

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