CN108148812B

CN108148812B - Method for rapidly preparing PD-1-T cells by efficient gene editing and application thereof

Info

Publication number: CN108148812B
Application number: CN201710677724.3A
Authority: CN
Inventors: 赵永祥; 周素芳; 李桂银
Original assignee: Guangxi Medical University
Current assignee: Guangxi Medical University
Priority date: 2017-08-09
Filing date: 2017-08-09
Publication date: 2021-08-31
Anticipated expiration: 2037-08-09
Also published as: CN108148812A

Abstract

The invention discloses a method for obtaining PD-1-T cells, which comprises the following steps: (1) obtaining human peripheral blood T lymphocytes; (2) plasmid transfection; (3) preparing a fusion cell; (4) expansion of PD-1-T tumor-specific T cells. The PD-1-T cell obtained by the method eliminates negative inhibitory regulatory receptors, has good anti-tumor effect, effectively prolongs the survival time of tumor-bearing mice compared with the cells of the traditional adoptive therapy, and is suitable for popularization in the medical field.

Description

Method for rapidly preparing PD-1-T cells by efficient gene editing and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to a method for quickly preparing PD-1-T cells by efficient gene editing and application thereof.

Background

T cells in the immune system of the body are the major components of lymphocytes, which are used primarily to fight foreign invaders and have diverse biological functions. It is the main "fighter" of the body that acts to combat disease invasion, infection and neoplasia. At present, the methods for treating tumors are generally surgery, radiotherapy and chemotherapy, and there are some problems in these methods, wherein immune escape phenomenon occurs in adoptive therapy, and the anti-tumor effect is affected.

PD-1 (programmed death 1) is a negative inhibitory regulatory receptor, the expression of PD-1 can be obviously increased after T cells are activated, and the activation and cell proliferation capacity of the T cells can be obviously inhibited through the combined action of the PD-L1 ligand, so that the capacity of the T cells for secreting various cytokines is influenced. Blocking or knocking out such molecules can help tumor immune activation, and blocking PD-1 by using an antibody or treating by using a PD-1 inhibitor has been reported to be a promising tumor immune adjuvant therapy method. In experiments, it has been found that the use of blocking agents or PD-1 inhibitors also has some disadvantages such as immunogenicity and high cost of the antibody. It is therefore of great importance to find an efficient and cost-effective process.

CRISPR/Cas9 (Cluster Reg μ larlyInterspaced short Palindormicripeeats) gene technology is a new biotechnology appearing in two years, and the method for forming sgRNA through artificial design to guide Cas9 protein to cut DNA at a target site is a hot spot of current biomedical field research. At present, fusion cells are mostly adopted for stimulating in a method for inducing specific T lymphocytes, so that PD-1 receptor genes on the T cells are knocked out by using a CRISPR/Cas9 gene editing technology in the research, and the anti-tumor effect of the specific T cells induced by the fusion cells is further verified in vitro and in vivo.

However, the existing CRISPR/Cas9 gene technology is applied in many fields and also shows good effect, but the technology is not related to the published technology in the aspect of tumor treatment, particularly the PD-1 gene on a knockout T cell, and in addition, the existing CRISPR/Cas9 knockout PD-1 gene technology does not find the high-penetrating peptide small molecule TAT which is jointly applied, so that the problems of low plasmid transfection efficiency and low PD-1 gene editing efficiency exist. Therefore, the specific PD-1-T cell induced by the fusion cell has the anti-tumor effect on the basis of knocking out the PD-1 gene by using the CRISPR/Cas9 gene technology, and a novel strategy is provided for the immunotherapy of tumors by jointly applying the high-penetration peptide small molecule TAT.

Disclosure of Invention

The invention aims to provide a method for rapidly preparing PD-1-T cells by efficient gene editing and application thereof, aiming at the existing problems, the method can effectively knock out the PD-1 gene, remarkably improve the plasmid transfection efficiency and the PD-1 gene editing efficiency so as to obtain specific PD-1-T cells, generate an anti-tumor effect and be applied to tumor treatment.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention relates to a method for rapidly preparing PD-1-T cells by efficient gene editing, which comprises the following steps:

1) obtaining human peripheral blood T lymphocytes: taking 100ml of peripheral blood, diluting the blood with PBS (equal amount), slowly adding lymphocyte separation liquid, centrifuging, discarding serum, sucking mononuclear cells in a white membrane layer into a new centrifugal tube, centrifuging, discarding supernatant, and washing with PBS (15-30 ml) for three times; and (3) suspending the suspension in an incubator by using incomplete 1640, sucking the suspended cells after the cells adhere to the wall for 1.5-2.5 h, and passing through a nylon cotton column to obtain the T lymphocytes.

2) Plasmid transfection: adjusting the concentration of the T lymphocyte obtained in the step 1) to be 1 × 107/ml, adjusting the concentration of the plasmid to be the same, respectively sucking 90-120 μ l of cell suspension, 2-5 μ l of the plasmid and 2-10 μ l of the high-penetration micromolecule guide peptide TAT aqueous solution to be uniformly mixed, sucking the mixture into a sterile electric rotating cup, quickly transferring the cells to a 12-hole plate after electric rotation, adding 2ml of preheated complete culture medium, placing the mixture in a 37 ℃ and 5% CO2 incubator to continue to culture, and thus completing plasmid transfection and obtaining PD-1T cells.

3) Preparation of fusion cells: adding GM-CSF1000M/ml and IL-4500M/ml to the adherent cells in the step (1) to induce DC cells, then adding target cells HepG2, and inducing DC/HepG2 fusion by using PEG with molecular weight of 1450.

4) Expansion of PD-1-T tumor-specific T cells: the proliferation of PD-1-T cells transfected with the plasmid in step (2) was induced using the DC/HepG2 fusion cells obtained in step (3).

Further, in the step (1), the peripheral blood is diluted by an equal amount of PBS, added into a lymphocyte separation solution, centrifuged and divided into four layers, namely serum, a white membrane layer, a separation solution and red blood cells from top to bottom, and the centrifugation is performed at 1500-2000 rpm for 10-15 min.

Further, the plasmid is a pX330-PD-1-gRNA plasmid, the construction method of the pX330-PD-1-gRNA plasmid is that gRNA is designed according to a CDS region of a PD-1 gene, the pX330 plasmid is cut by BBSI enzyme, the gRNA is connected with the linearized pX330 plasmid through T4 ligase, then escherichia coli is transformed, a monoclonal is selected for sequencing, the pX330-PD-1-gRNA plasmid recombined into the sgRNA is screened out, amplification of the monoclonal bacteria is carried out, and the plasmid is extracted.

Further, the electric cup is set to have an electric condition of 210V, 20ms, 310V, 20ms or 410V, 20 ms.

Further, the complete medium was DMEM medium supplemented with 10% fetal bovine serum and 100M/ml antibiotics.

The invention also provides the application of the method for quickly preparing the PD-1-T cells by high-efficiency gene editing in tumor resistance.

Furthermore, the tumor is a solid tumor, and the solid tumor is one or more of liver cancer, lung cancer, intestinal cancer, prostate cancer, pancreatic cancer and lung adenocarcinoma.

Furthermore, the electric revolving cup in the step 2) comprises a cup and a cup cover, a cleaning dryer capable of cleaning and sterilizing the inner cavity of the cup is further arranged above the cup cover, the cup cover comprises a cover body and a bulge arranged on the upper part of the cover body, a groove matched with the bulge is arranged at the bottom of the cleaning dryer, the cleaning dryer is tightly connected with the bulge of the cover body through the groove at the bottom, the cup comprises a cup body and a positive electrode and a negative electrode which are arranged in the cup body, electric conductors are respectively arranged on two sides of the outer rings of the positive electrode and the negative electrode, connecting leads are further arranged on the electric conductors on the two sides, the positive electrode and the negative electrode are electrically connected with an external electric revolving instrument through the electric conductors and the connecting leads, the cleaning dryer comprises a box-type structure formed by enclosing a left sealing plate, a right sealing plate, an upper cover plate and a lower supporting plate, a hinged seat is arranged in any sealing plate of the box-type structure, articulated seat on be connected with the articulated shaft, the one end that the articulated shaft stretches out is connected with flexible pipe, the other end of flexible pipe still is connected with the heating rod, be connected with wire and plug on the purger switch install the brush on the outer circumference of heating rod be equipped with on the flexible pipe and can make the heating rod heating or the rotatory purger switch of brush, install shrouding and upper cover plate and shrouding and the bottom plate of articulated seat between all install the loose-leaf.

The peripheral blood of the invention is collected from healthy volunteers, and the lymphocyte separation solution is purchased from tertiary ocean biologicals company in Tianjin city.

In summary, due to the adoption of the technical scheme, compared with the prior art, the invention has the prominent substantive features and remarkable progress that:

(1) the CRISPR/Cas9 gene technology is applied to knock out PD-1 gene in T cells, and the specificity is good. The sgRNAs are successfully constructed by using a CRISPR/Cas9 gene technology, the px330-PD-1-gRNA plasmid is transfected to a T lymphocyte from human peripheral blood by using an electroporation technology, and the PD-1 gene on the T cell is knocked out, so that the obtained PD-1-T cell blocks a negative inhibitory regulatory receptor, and has better tumor inhibition activity.

(2) The T cell subjected to gene knockout is amplified by using the fusion cell, so that the anti-tumor effect is stronger. The invention utilizes a PEG chemical method to carry out the fusion of DC/HepG2, and induces specific PD-1-T lymphocyte by using the fusion cell, and according to the experiment of the invention, the induced specific PD-1-T lymphocyte delays the growth of tumor by inhibiting the proliferation of tumor cell and promoting the apoptosis of tumor cell in the human hepatoma SCID mouse, thereby prolonging the survival time of the tumor mouse, indicating that the induced cell has stronger anti-tumor effect and being suitable for medical popularization.

(3) By applying TAT, the gene editing efficiency is improved: the high-penetrability small-molecule guide peptide TAT is used in the plasmid transfection process, and the high-penetrability small-molecule guide peptide TAT is mixed with the plasmid and then transfects the T cell, so that the transfection efficiency of the plasmid and the editing efficiency of the PD-1 gene are obviously improved, the effect of the CRISPR/Cas9 gene knockout technology is exerted to the greatest extent, compared with the PD-1 gene knockout technology in the prior art, the high-penetrability small-molecule guide peptide TAT has higher efficiency, and the expression level effect of the PD-1 gene knockout T cell is obviously shown in the experiment of the invention.

(4) The electric revolving cup is reasonable in structure, the cleaning dryer capable of cleaning, disinfecting and drying the inner cavity of the cup is arranged above the cup cover, the cleaning dryer is matched and tightly connected with the groove at the bottom of the cleaning dryer through the bulge on the upper part of the cover body, the brush can be extended into the cup body for brushing through the extension of the telescopic rod and the upper cover plate and the lower supporting plate when the cleaning dryer is used, the cup body can be heated and dried through the heating rod, the cup body is convenient to clean, the durability of the cup is high, and the experimental expenses are greatly saved.

Drawings

FIG. 1 shows the case of knocking out PD-1 gene according to the present invention.

FIG. 2 is a graph showing the proliferation index of T lymphocyte groups stimulated by various groups of cells according to the present invention.

FIG. 3 shows the killing of HepG2 by specific T lymphocytes induced in vitro according to the invention.

FIG. 4 shows the killing of A549 by specific T lymphocytes induced in vitro according to the invention.

FIG. 5 shows the killing efficiency of HepG2 and A549 by each group of cells under the killing concentration of 30: 1.

FIG. 6 shows the apoptosis of tumor cells after the DC/HepG 2-induced PD-1-T cells are treated in vivo in mice.

FIG. 7 shows the tumor cell apoptosis promotion by each cell after adoptive therapy of mice in the present invention experiment.

FIG. 8 is a graph showing the size of tumor volume after treatment in the experiment of the present invention.

FIG. 9 shows the survival rates of the mice in the present invention experiment.

Fig. 10 is a schematic diagram of a front structure of the electric rotary cup.

Fig. 11 is a schematic diagram of the internal sectional structure of the cup 1.

Fig. 12 is a schematic diagram of an internal cross-sectional structure of the cap 2.

Fig. 13 is a schematic diagram of the internal sectional structure of the washer 3.

Fig. 14 is a schematic diagram of the cup 1 being cleaned by the washer 3.

Fig. 15 is a schematic diagram of the washer 3 for heating and drying the cup 1.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents, and the like used in the following examples. It is commercially available unless otherwise specified.

Materials and reagents used in the examples:

peripheral blood was collected from healthy volunteers and the lymphocyte isolates were purchased from tertiary ocean biologicals of Tianjin.

Human hepatoma HepG2 cell (ATCC, cat No. HB-8065), the material of which is available to the public from the Applicant only for the purpose of repeating the experiments of the invention and not for other uses.

Secondly, a method for rapidly preparing PD-1-T cells by efficient gene editing:

(1) obtaining human peripheral blood T lymphocytes: taking 100ml of peripheral blood, diluting the blood with PBS (equal amount), slowly adding lymphocyte separation solution, centrifuging, discarding serum, sucking mononuclear cells in the tunica albuginea layer into a new centrifuge tube, centrifuging, discarding supernatant, and washing with PBS (20 ml) for three times; and (3) suspending the suspension in an incubator by using incomplete 1640, sucking the suspended cells after the cells are attached to the wall for 2 hours, and passing through a nylon cotton column to obtain the T lymphocytes. The peripheral blood is diluted by equal amount of PBS, added with lymphocyte separation liquid, and centrifuged to be divided into four layers, namely serum, a tunica albuginea layer, the separation liquid and red blood cells from top to bottom, wherein the centrifugation condition is centrifugation for 10min at 1500 rpm.

(2) Plasmid transfection: adjusting the concentration of the T lymphocyte obtained in the step (1) to 1 × 107/ml, adjusting the concentration of the plasmid to the same concentration, respectively sucking 100 μ l of cell suspension, 4 μ l of the plasmid and 5 μ l of the high-penetration micromolecule guide peptide TAT aqueous solution to be uniformly mixed, sucking the mixture into a sterile electric rotating cup, quickly transferring the cells to a 12-hole plate after the electric rotating, adding 2ml of preheated complete culture medium, placing the mixture in a 37 ℃ and 5% CO2 incubator for continuous culture, and finishing the plasmid transfection to obtain the PD-1T cell. The plasmid is a pX330-PD-1-gRNA plasmid, the construction method of the pX330-PD-1-gRNA plasmid comprises the steps of designing gRNA according to a CDS region of a PD-1 gene, carrying out enzyme digestion on the pX330 plasmid by BBSI to connect the gRNA with the linearized pX330 plasmid through T4 ligase, then transforming escherichia coli, selecting a monoclonal for sequencing, screening the pX330-PD-1-gRNA plasmid recombined into the sgRNA, carrying out amplification on the monoclonal bacteria, and extracting the plasmid. The complete medium was DMEM medium supplemented with 10% fetal bovine serum and 100M/ml antibiotics.

The electric revolving cup in the step (2) comprises a cup 1 and a cup cover 2, a cleaning dryer 3 capable of cleaning and sterilizing the inner cavity of the cup 1 is further arranged above the cup cover 2, the cup cover 2 comprises a cover body 9 and a bulge 10 arranged on the upper portion of the cover body 9, a groove 22 matched with the bulge 10 is arranged at the bottom of the cleaning dryer 3, the cleaning dryer 3 is tightly connected with the bulge 10 of the cover body 9 through the groove 22 at the bottom, the cup 1 comprises a cup body 5 and a positive electrode 7 and a negative electrode 7 arranged in the cup body 5, electric conductors 6 are respectively arranged on two sides of the outer ring of the positive electrode 7 and the negative electrode 7, connecting wires 4 are further arranged on the electric conductors 6 on the two sides, the positive electrode 7 and the negative electrode 7 are electrically connected with an external electric revolving apparatus through the electric conductors 6, the cleaning dryer 3 comprises a box-type structure formed by enclosing of a left sealing plate 11, a right sealing plate 11, an upper cover plate 12 and a lower supporting plate 21, install articulated seat 19 in arbitrary shrouding 11 of box structure articulated seat 19 on be connected with articulated shaft 20, the one end that articulated shaft 20 stretched out is connected with flexible pipe 16, the other end of flexible pipe 16 still is connected with heating rod 14, be connected with wire and plug 17 on the purger switch 15 install brush 13 on the outer circumference of heating rod 14 be equipped with on the flexible pipe 16 and can make the heating rod 14 heat or the rotatory purger switch 15 of brush 13, shrouding 11 and upper cover plate 12 and shrouding 11 and the bottom plate 21 of installing articulated seat 19 between all install loose-leaf 18.

(3) Preparation of fusion cells: adding GM-CSF1000M/ml and IL-4500M/ml to the adherent cells in the step (1) to induce DC cells, then adding target cells HepG2, and inducing DC/HepG2 fusion by using PEG with molecular weight of 1450. The electric rotating condition of the electric rotating cup is set to be 210V, 20ms, 310V, 20ms or 410V, 20 ms.

(4) Expansion of PD-1-T tumor-specific T cells: the proliferation of PD-1-T cells transfected with the plasmid in step (2) was induced using the DC/HepG2 fusion cells obtained in step (3).

The tumor is solid tumor, and the solid tumor is one or more of liver cancer, lung cancer, intestinal cancer, prostatic cancer, pancreatic cancer and lung adenocarcinoma. The tumor may be in particular human liver cancer growing on nude mice.

Thirdly, the identification of the PD-1-T cells obtained in the tumor correlation:

1. detection of gene knockout PD-1 gene T cell expression level

According to the condition of T7E1 enzyme digestion identification knockout, after extracting a T cell genome transfected with px330-PD-1-gRNA for PCR amplification, two current bands appear after T7E1 enzyme digestion, the sizes of the two current bands are respectively 370bp and 530bp, and the two current bands accord with the size of an original plasmid fragment (as shown in figure 1).

2. Stimulation of proliferation of induced PD-1-T lymphocytes by DC/HepG2

And (3) detecting the fluorescence attenuation degree of the CFSE by using a flow cytometer, and detecting the proliferation condition of each group of specific T lymphocytes induced by the fusion cells. As shown in FIG. 2, the SPSS statistic results show that the proliferation of PD-1-T lymphocyte induced by DC/HepG2 stimulation is significantly higher than that of PD-1+ T lymphocyte induced by DC/HepG2 stimulation and that of single T cell, and the difference is statistically significant, p < 0.05. The results show that the PD-1-T lymphocyte induced by DC/HepG2 in vitro stimulation has stronger cell proliferation capacity.

3. Detection of DC/HepG 2-induced PD-1-T lymphocytes capable of specifically killing target cells

T cells cultured for 5 to 7 days, PD-1+ T, PD-1-T lymphocytes, and HepG2 cells and A549 cells grown in logarithmic phase were stimulated by T cells and DC/HepG2 fusion cells as effector cells and target cells. The results after calculation by the killing formula showed that the killing efficiency of DC/HepG 2-induced PD-1-T lymphocytes against the target cell HepG2 was higher than that of DC/HepG 2-induced PD-1+ T lymphocytes and other groups at the ratio of 20:1 and 30:1 (see FIG. 3). Under the three target ratios of killing effect, the killing efficiency of DC/HepG 2-induced PD-1-T lymphocytes to A549 is higher than that of DC/HepG 2-induced PD-1+ T lymphocytes and other cells (as shown in FIG. 4). At a killing ratio of 30:1, both DC/HepG 2-induced killing efficiencies of PD-1-T lymphocytes were higher than those of other target cells (see FIG. 5).

4. Detection experiment that PD-1-T cell induced by DC/HepG2 can promote tumor cell apoptosis in tumor tissue

Tumor tissues of tumor-bearing mice are prepared into paraffin sections, and the apoptosis condition of tumor cells is detected by a fluorescence T μm NEL method. And observing the stained tumor tissue section under a fluorescence microscope, wherein the cells emitting green fluorescence are positive cells, namely apoptotic or apoptotic tumor cells, and the blue fluorescence is cell nuclei. The results showed that the number of tumor cell apoptosis of the PD-1-T cell experimental group induced by DC/HepG2 was significantly higher in the human hepatoma-bearing mice than in the other controls (see FIG. 6). Statistical analysis of the number of apoptosis of tumor cells in each group was also performed using the statistical plots for the differences between the PD-1-T lymphocytes induced by DC/HepG2 and the other groups, which were statistically significant (FIG. 7). P < 0.05,. P < 0.01,. P < 0.001.

5. Experiment on Effect of DC/HepG 2-induced PD-1-T cells on mouse survival time

Constructing a mouse liver cancer model, starting adoptive therapy on the induced T lymphocytes after one week of tumor bearing, carrying out treatment once a week for 3 times, and observing and recording the volume of the tumor every 6 days. By observing the differences in tumor size among groups at day 18 after treatment, tumor growth was significantly inhibited by the tumor size of mice treated with DC/HepG 2-induced PD-1-T cells, which was significantly better than other controls and statistically significant in the differences (see FIG. 7). The survival rate of the observed mice was recorded, and the results showed that the DC/HepG 2-induced PD-1-T lymphocytes could significantly prolong the survival time of tumor-bearing mice (see FIG. 8).

The invention is not limited to the specific embodiments described above, but extends to any novel feature or novel combination disclosed herein, or to any novel method or process step or novel combination disclosed.

Claims

1. A method for rapidly preparing PD-1-T cells by efficient gene editing is characterized by comprising the following steps:

1) obtaining human peripheral blood T lymphocytes: taking 100ml of peripheral blood, diluting the blood with PBS (equal amount), slowly adding lymphocyte separation liquid, centrifuging, discarding serum, sucking mononuclear cells in a white membrane layer into a new centrifugal tube, centrifuging, discarding supernatant, and washing with PBS (15-30 ml) for three times; suspending the suspension in an incubator by using incomplete 1640, sucking the suspended cells after the cells adhere to the wall for 1.5-2.5 h, and passing through a nylon cotton column to obtain T lymphocytes;

2) plasmid transfection: adjusting the concentration of the T lymphocyte obtained in the step 1) to be 1 × 10⁷Adding plasmid, respectively sucking 90-120 mu l of cell suspension, 2-5 mu l of plasmid and 2-10 mu l of high-penetration micromolecule guide peptide TAT aqueous solution, uniformly mixing, sucking into a sterile electric rotating cup, quickly transferring cells to a 12-hole plate after electric rotation, adding 2ml of preheated complete culture medium, placing at 37 ℃, continuously culturing in a 5% CO2 incubator, and finishing plasmid transfection to obtain PD-1T cells;

3) preparation of fusion cells: adding GM-CSF1000M/ml and IL-4500M/ml into the adherent cells in the step 1) to induce DC cells, then adding target cells HepG2, and inducing DC/HepG2 fusion by using PEG with molecular weight of 1450;

4) expansion of PD-1-tumor-specific T cells: inducing proliferation of the PD-1-T cells transfected with the plasmid in step (2) using the DC/HepG2 fusion cells obtained in step 3);

in the step 1), diluting the peripheral blood with PBS (equal amount), adding lymphocyte separation liquid, centrifuging, and then dividing the peripheral blood into four layers, wherein the four layers are serum, a white membrane layer, the separation liquid and red blood cells from top to bottom in sequence, and the centrifugation is performed at 1500-2000 rpm for 10-15 min;

in the step 2), the plasmid is a pX330-PD-1-gRNA plasmid, and the pX330-PD-1-gRNA plasmid is constructed by designing a gRNA according to the CDS region of the PD-1 gene, digesting the pX330 plasmid with BBSI enzyme, connecting the gRNA and the linearized pX330 plasmid by T4 ligase, transforming escherichia coli, selecting a monoclonal for sequencing, screening the pX330-PD-1-gRNA plasmid recombined into the sgRNA, amplifying the monoclonal bacteria, and extracting the plasmid;

the complete medium was DMEM medium supplemented with 10% fetal bovine serum and 100M/ml antibiotics.

2. The method for rapid preparation of PD-1-T cells through efficient gene editing as claimed in claim 1, wherein the electroporation conditions of the electroporation cuvette are set to 210V, 20ms, 310V, 20ms or 410V, 20 ms.

3. The method for rapidly preparing PD-1T cells through efficient gene editing according to claim 1, characterized in that the electric revolving cup of the step 2 comprises a cup (1) and a cup cover (2), a cleaning and drying device (3) capable of cleaning, disinfecting and drying the inner cavity of the cup (1) is further arranged above the cup cover (2), the cup cover (2) comprises a cover body (9) and a bulge (10) arranged on the upper portion of the cover body (9), a groove (22) matched with the bulge (10) is arranged at the bottom of the cleaning and drying device (3), the cleaning and drying device (3) is tightly connected with the bulge (10) of the cover body (9) through the groove (22) at the bottom, the cup (1) comprises a cup body (5) and a positive electrode (7) and a negative electrode (7) arranged in the cup body (5), and electric conductors (6) are further respectively arranged on two sides of the outer rings of the positive electrode (7) and the negative electrode (7), the electric conductor (6) is also provided with a connecting lead (4), the positive electrode (7) and the negative electrode (7) are electrically connected with an external electric rotating instrument through the electric conductor (6) and the connecting lead (4), the cleaning dryer (3) comprises a box structure enclosed by a left sealing plate (11), a right sealing plate (11), an upper cover plate (12) and a lower supporting plate (21), a hinged seat (19) is installed in any sealing plate (11) of the box structure, a hinged shaft (20) is connected on the hinged seat (19), one extending end of the hinged shaft (20) is connected with a telescopic pipe (16), the other end of the telescopic pipe (16) is also connected with a heating rod (14), a brush (13) is installed on the outer circumference of the heating rod (14), a cleaner switch (15) capable of heating the heating rod (14) or rotating the brush (13) is arranged on the telescopic pipe (16), the cleaner switch (15) is connected with a lead and a plug (17), and hinges (18) are respectively arranged between the sealing plate (11) provided with the hinged seat (19) and the upper cover plate (12) and between the sealing plate (11) and the lower supporting plate (21).