Monoclonal antibody of porcine pseudorabies virus EP0 protein, preparation method and application
阅读说明：本技术 一种猪伪狂犬病毒ep0蛋白的单克隆抗体、制备方法和应用 (Monoclonal antibody of porcine pseudorabies virus EP0 protein, preparation method and application ) 是由 王迪 赵鸿远 陈冬杰 于 2021-10-26 设计创作，主要内容包括：本发明公开了一种猪伪狂犬病毒EP0蛋白的单克隆抗体、制备方法和应用,属于基因工程技术领域,其单克隆抗体包括重链可变区和轻链可变区,所述重链可变区的氨基酸序列如SEQ ID No.5所示,所述轻链可变区的氨基酸序列如SEQ ID No.11所示。本发明筛选获得了3株能够稳定分泌与EP0蛋白发生特异性反应的单克隆抗体的杂交瘤细胞株2C5、4C6和3B5。经单抗亚型鉴定,发现2C5为IgG2b/κ型,3B5和4C6均为IgG1/κ型。本发明所制备的针对RPV EP0蛋白的单克隆抗体能够为进一步分析EP0蛋白的功能提供研究基础。(The invention discloses a monoclonal antibody of porcine pseudorabies virus EP0 protein, a preparation method and application, belonging to the technical field of genetic engineering, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.5, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 11. The invention screens and obtains 3 hybridoma cell strains 2C5, 4C6 and 3B5 which can stably secrete monoclonal antibodies which can specifically react with EP0 protein. Through monoclonal antibody subtype identification, 2C5 is IgG 2B/kappa type, and 3B5 and 4C6 are both IgG 1/kappa type. The monoclonal antibody aiming at the RPV EP0 protein prepared by the invention can provide a research basis for further analyzing the function of the EP0 protein.)
1. The monoclonal antibody of the porcine pseudorabies virus EP0 protein is characterized by comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.5, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 11.
2. The monoclonal antibody of claim 1, further comprising a leader sequence and a constant region; the amino acid sequence of the heavy chain leader sequence is shown as SEQ ID No.4, and the amino acid sequence of the heavy chain constant region is shown as SEQ ID No. 6; the amino acid sequence of the light chain leader sequence is shown as SEQ ID No.10, and the amino acid sequence of the light chain constant region is shown as SEQ ID No. 12.
3. The gene encoding the monoclonal antibody of claim 1, wherein the nucleotide sequence encoding the heavy chain variable region of said monoclonal antibody is represented by SEQ ID No.2 and the nucleotide sequence encoding the light chain variable region of said monoclonal antibody is represented by SEQ ID No. 8.
4. An expression vector comprising the gene of claim 3.
5. A host cell comprising the expression vector of claim 4.
6. A method for producing the monoclonal antibody according to claim 1 or 2, comprising the step of culturing the host cell according to claim 5 to express a monoclonal antibody against porcine pseudorabies virus EP0 protein.
7. Use of the monoclonal antibody according to claim 1 or 2 for the preparation of a reagent for the detection or the auxiliary diagnosis of porcine pseudorabies virus.
8. Use of the monoclonal antibody according to claim 1 or 2 for the preparation of a reagent for detecting or aiding in the diagnosis of porcine pseudorabies virus EP0 protein.
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a monoclonal antibody of porcine pseudorabies virus EP0 protein, a preparation method and application.
Background
Porcine Pseudorabies virus (PRV) belongs to the family herpesviridae, the subfamily alphaherpesviridae, the genus porcine herpesvirus. PRV can infect various domestic animals and wild animals, mainly causes symptoms such as fever, extreme itching (except pigs), encephalomyelitis and the like, has extremely serious harm, and is one of epidemic diseases which must be reported by OIE regulations. Sow reproductive disorders such as miscarriage, stillbirth, weak births and even mummy births in pregnant sows can be caused after PRV infection in the sows. After the piglets are infected with PRV, the piglets can be caused to have nervous symptoms, the piglets within 2 weeks of age can die, and the fatality rate reaches 100 percent. The adult pigs have the characteristic of latent infection after tolerance, which is also an important reason for difficult control. The PRV virion is oval or round, has a diameter of 110-150nm, and mainly consists of four parts, namely an interplipidic membrane, a mesosphere, a nucleocapsid and a viral genome. Its genome is linear double-stranded DNA, is about 150kb in length, and can be divided into unique long fragment (UL), unique short fragment (US), Terminal Repeat Sequence (TRS) and Internal Repeat Sequence (IRS) at both sides of short segment. The full-length genome can code 73 genes, and can be divided into four types of genes, namely early, early/late and late according to the transcription time sequence. EP0 is the most prominent early gene encoded by the PRV genome. To date, the function of the EP0 protein has not been fully studied, but the homology of EP0 for PRV with ICP0 for HSV-1 should be similar in structure and function, so it is speculated that EP0 protein may play an important role in the latent infection and activation of PRV. It has been shown that EP0 is a transcription activator of viral genome transcription and replication, and in vitro extracted recombinant EP0 protein promotes initiation of transcription of the promoter of the artificially synthesized TATA box in cell nuclear extracts. In addition, the EP0 protein has antiviral effects against interferon modulation, which facilitates the establishment of latent infection by PRV in the host.
In order to deeply research the role of the EP0 protein in virus infection and realize accurate diagnosis of PRV virus infection, the development of the EP0 protein specific monoclonal antibody has important significance for deeply researching the biological function of the EP0 protein and is also helpful for the development of PRV diagnosis and detection reagents.
Disclosure of Invention
The invention aims to provide a monoclonal antibody of porcine pseudorabies virus EP0 protein, a preparation method and application, so as to solve the problems in the prior art, the invention uses a prokaryotic expression system to express and purify the EP0 protein, immunizes BALB/c mice, prepares a monoclonal antibody specifically aiming at PRV EP0, analyzes the characteristics of the monoclonal antibody, and provides a material basis for better researching the function of the EP0 protein.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a monoclonal antibody of porcine pseudorabies virus EP0 protein, which comprises a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.5, and the amino acid sequence of the light chain variable region is shown as SEQ ID No. 11.
Further, a leader sequence and a constant region are also included; the amino acid sequence of the heavy chain leader sequence is shown as SEQ ID No.4, and the amino acid sequence of the heavy chain constant region is shown as SEQ ID No. 6; the amino acid sequence of the light chain leader sequence is shown as SEQ ID No.10, and the amino acid sequence of the light chain constant region is shown as SEQ ID No. 12.
The invention also provides a gene for coding the monoclonal antibody, wherein the nucleotide sequence for coding the heavy chain variable region of the monoclonal antibody is shown as SEQ ID No.2, and the nucleotide sequence for coding the light chain variable region of the monoclonal antibody is shown as SEQ ID No. 8.
The invention also provides an expression vector containing the gene.
The present invention also provides a host cell comprising the above expression vector.
The invention also provides a preparation method of the monoclonal antibody, which comprises the steps of culturing the host cell and expressing the monoclonal antibody of the porcine pseudorabies virus EP0 protein.
The invention also provides application of the monoclonal antibody in preparation of a reagent for detecting or assisting in diagnosis of porcine pseudorabies virus.
The invention also provides application of the monoclonal antibody in preparing a reagent for detecting or assisting in diagnosing the porcine pseudorabies virus EP0 protein.
The invention discloses the following technical effects:
the GC content of the full-length EP0 gene is 69%, the GC content is high, the amplification difficulty of the EP0 gene is high, the method adopts a mode of adding DMSO into an amplification system, the amplification efficiency is improved, and the full-length EP0 gene is successfully amplified.
The invention utilizes a prokaryotic expression system to express PRV EP0 protein, uses a nickel column for purification, immunizes a BALB/C mouse with the purified recombinant EP0 protein, takes spleen cells of the BALB/C mouse to fuse with myeloma cells SP2/0 after the serum titer of the BALB/C mouse meets the requirement, obtains 3 hybridoma cell strains 2C5, 4C6 and 3B5 which can stably secrete monoclonal antibodies which have specific reaction with EP0 protein through ELISA screening, and successfully prepares the monoclonal antibodies of the EP0 protein. IFA and Western blot prove that the 3-strain monoclonal antibodies can be specifically reacted with PK-15 cells infected with PRV, and the specificity is proved to be good. The monoclonal antibody subtype identification result shows that 2C5 is IgG 2B/kappa type, and 3B5 and 4C6 are both IgG 1/kappa type. In conclusion, the monoclonal antibody aiming at the RPV EP0 protein prepared by the invention can provide a research basis for further analyzing the function of the EP0 protein.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art to obtain other drawings without creative efforts.
FIG. 1 shows PCR amplification of the EP0 gene; wherein, M: DNA molecular mass standard; 1: EP0 gene amplification product;
FIG. 2 shows SDS-PAGE and Western Blot identification of EP0 protein; wherein, A: SDS-PAGE identification of EP0 protein; b: western Blot identification of EP0 protein; m: prestained protein molecular mass standard; 1: EP0 protein expression precipitation at 16 ℃; 2: EP0 protein expression supernatant at 16 ℃; 3: EP0 protein expression precipitation at 25 ℃; 4: EP0 protein expression supernatant at 25 ℃; 5: EP0 protein expression precipitation at 37 ℃; 6: EP0 protein expression supernatant at 37 ℃; 7: pET28a empty control;
FIG. 3 shows the purification of EP0 protein; wherein, M: prestained protein molecular mass standard; 1: before purification of EP 0; 2: after purification of EP 0; 3: pET28a empty control;
FIG. 4 shows the WB assay of the reaction of MAb 4C6, 2C5 and 3B5 with recombinant EP0 protein; wherein, 1: recombinant EP0 protein; 2: pET28a empty control;
FIG. 5 shows the IFA results for MAbs 4C6, 2C5, and 3B5 with PRV.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that each intervening value, between the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
Example 1
1. Materials and methods
1.1 strains, vectors and reagents
The PRV HB1201 strain is isolated from Hebei in 2012 and stored in the health academy of Shanxi province; the PET28a vector, PK-15 cell and SP2/0 cell were all preserved by the health academy of Shanxi province. The virus genome DNA extraction kit is purchased from Tiangen Biotechnology limited; EcoRI and HindIII restriction enzymes were purchased from NEB; pEASY-blunt vector, Trans 5 alpha competence, BL21 competence, BL21(DE3) competence, gel recovery kit, plasmid extraction kit, anti-His tag monoclonal antibody, HRP-labeled anti-mouse secondary antibody, FITC-labeled anti-mouse secondary antibody andwestern Blot kits were purchased from Beijing Quanji Biotechnology Ltd; KOD Fx Neo was purchased from ToyoBo, Inc.; the monoclonal antibody subclass identification kit is purchased from Boolong immuno-technology GmbH.
1.2 EP0 Gene amplification
The full-length gene of EP0 is amplified by taking the gene sequence of PRV HB1201 strain EP0 (No. KU057086.1) in GenBank as a template.
The upstream primer is EP0-F (SEQ ID No. 13): CG (CG)GAATTCGACTGCCCCATCTGCCTGGACGTC, the downstream primer is EP0-R (SEQ ID No. 14): CCCAAGCTTTCAGTCGTCGTCCTGGGTGA, wherein the underlined sections represent EcoRI and HindIII sites, respectively.
EP0 gene amplification system: KOD Fx Neo 1. mu.l; 2 XPCR Buffer: 25. mu.l; 10. mu.l of 2mM dNTPs; EP 0-F0.5. mu.l; EP 0-R0.5. mu.l; 2. mu.l of PRV DNA; DMSO 0.5 μ l; ddH2O:10.5μl。
Amplification program, 2min at 98 ℃; 10s at 98 ℃, 30s at 60 ℃ and 30s at 68 ℃ (35 cycles); 7min at 68 ℃; infinity at 4 ℃.
The amplified EP0 gene is recovered by glue, then connected with a pEasy-blunt vector, transformed into a Trans 5 alpha competent cell, and subjected to PCR identification and sequencing analysis to obtain correct recombinant pEASY-blunt-EP0 and extract a plasmid thereof.
1.3 construction of prokaryotic expression vector of EP0 Gene
The recombinant plasmid pEASY-blunt-EP0 was digested with EcoRI and HindIIII, ligated to the similarly digested pET28a vector to construct a recombinant pET28a-EP0 plasmid, which was transformed into Trans 5. alpha. competent cells. And carrying out PCR identification on the positive colonies, carrying out sequencing analysis, selecting positive clones with correct sequencing, carrying out propagation, and extracting plasmids of the positive clones.
1.4 prokaryotic expression identification and purification of recombinant EP0 protein
The pET28a-EP0 recombinant plasmid is transformed into BL21(DE3) competent cells, and a single colony is picked up for propagation and expression. Bacteria solution OD600When the concentration reaches 0.6-0.8, isopropyl thiogalactoside (IPTG) with the final concentration of 0.5mM is added, the overnight induction expression is carried out at 16 ℃, 25 ℃ and 37 ℃ respectively, and meanwhile pET28a no-load expression bacteria are set as negative control. After induction, mycoprotein precipitation and supernatant are collected respectively, and the expression condition is verified by SDS-PAGE and Western Blot. After determining the optimal expression conditions of the recombinant EP0 protein, it was expressed in large quantities and subjected to Ni column purification.
1.5 preparation of monoclonal antibodies (mAbs) against the EP0 protein
And (3) taking the purified recombinant EP0 protein and Freund's complete or incomplete adjuvant to immunize the mice for multiple times, and taking tail blood of the mice after the second immunization for 7d and measuring the titer of the serum antibody by an indirect ELISA method. When the titer of the serum antibody reaches more than 1:10000, the immunity effect of the surface recombinant protein is good, after three times of immunity and boosting immunity, the spleen of a mouse can be taken for cell fusion, and the positive screening is carried out on hybridoma cell holes. The specific immunization, titer determination, fusion and positive clone screening operation steps are carried out by adopting the conventional method in the field.
1.6 MAb WB identification
The recombinant EP0 protein after expression and purification is separated by SDS-PAGE, transferred to a nitrocellulose membrane (NC membrane) and blocked with 40g/L skim milk-PBS for 2h at room temperature. Selected MAbs were used as primary antibody, SP2/0 cell culture was used as negative control, and incubation was performed at 37 ℃ for 1 h. PBST was washed 3 times for 5min each. HRP-labeled goat anti-mouse IgG was used as a secondary antibody (diluted 1: 10000), and the secondary antibody was washed after reacting at 37 ℃ for 1 hour. And observing the result after color development is carried out by using an ECL substrate color developing solution.
1.7 MAb subtype identification
The obtained MAb subtype is identified by using a mouse monoclonal antibody subtype identification kit, and the specific steps are as follows: the purified recombinant EP0 protein was coated on an enzyme-labeled plate (100. mu.l/well) overnight at 4 ℃. Adding culture supernatant of the screened positive hybridoma cells into a coated enzyme label plate (100 mu l/hole), incubating for 30min at 37 ℃, and washing for 5 times by PBST; adding eight HRP markers of the kit into an enzyme label plate (100 mu l/hole), incubating for 30min at 37 ℃, washing PBST for 5 times, then adding TMB developing solution (100 mu l/hole), developing for 20min in a dark place at room temperature, and developing for 2M H2SO4And (5) after the reaction is terminated, reading by using a microplate reader to judge the result.
1.8 MAb Indirect immunofluorescence assay
PK-15 cells in a good state were plated on a 24-well cell culture plate, and when the cells grew to 90% or more, PRV HB1201 strain at 1MOI was infected, and an uninfected group was set as a negative control. After PRV infection for 6h, the culture medium is discarded, anhydrous glacial ethanol is added to fix the cells, the cells are fixed for 15min at room temperature and washed by PBS for 3 times, culture supernatant of the positive hybridoma cells is added, the cells are incubated for 1h at 37 ℃ and washed by PBS for 3 times, FITC-labeled goat anti-mouse IgG is added, the cells are incubated for 1h at 37 ℃ in a dark place and observed under an inverted fluorescence microscope.
2. Results
2.1 amplification of the EP0 Gene
By taking RPV HB1201 strain as a template, the EP0 gene (shown in figure 1) is successfully amplified, the size of a target gene fragment is 1101bp, and the size is consistent with a theoretical value. It was further ligated into vectors such as pEASY-blunt and pET28a, and the sequence and insertion position were confirmed to be correct by sequencing.
2.2 expression and characterization of EP0 protein
BL21(DE3) competent cells were transformed with the correctly constructed pET28a-EP0 recombinant plasmid, and then subjected to SDS-PAGE and Western Blot identification after inducible expression at different temperatures (16 ℃, 25 ℃, 37 ℃). As shown in FIG. 2, the results of SDS-PAGE (FIG. 2A) revealed that an expression band of EP0 was observed in both the pellet and the supernatant at different expression temperatures, and the expression band was about 65ku, which was much more expressed in the pellet at 16 ℃, the pellet at 25 ℃, the supernatant at 25 ℃ and the pellet at 37 ℃. The Western Blot (FIG. 2B) results also show that the reaction bands are evident in the precipitation at 16 deg.C, the precipitation at 25 deg.C, the supernatant at 25 deg.C and the precipitation at 37 deg.C. For purification, the experiment selects 25 ℃ induction expression conditions for mass expression, and the expression supernatant is purified by a nickel column.
2.3 purification of EP0 protein
The induction temperature of 25 ℃ is selected to carry out mass expression on EP0, the supernatant is purified by a nickel column, and the purified supernatant is identified by SDS-PAGE, the result is shown in figure 3, the concentration of the purified EP0 protein reaches more than 85 percent, and the purified EP0 protein is subpackaged and stored at the temperature of minus 20 ℃ and is used for mouse immunization.
2.4 screening and subtype identification of monoclonal antibodies
After successful ELISA detection by cell fusion and three subcloning, three hybridoma positive cell lines were selected, as shown in table 1, and named 2C5, 3B5, and 4C6, respectively. The antibody subtype secreted by the three obtained positive hybridomas is respectively determined by using a monoclonal antibody subtype identification kit, and the result shows that 2C5 is of an IgG 2B/kappa type, and 3B5 and 4C6 are both of IgG 1/kappa types.
TABLE 1 ELISA and subtype identification of monoclonal antibodies
2.5 WB identification of monoclonal antibodies
In order to further verify the properties of the monoclonal antibodies screened, the obtained three-strain antibodies were tested for their reactivity with recombinant EP0 protein using WB. As shown in FIG. 4, MAb 4C6, 2C5 and 3B5 were reactive with recombinant EP0 protein and were not reactive with the empty vector control pET-28a, indicating that the three monoclonal antibodies obtained were able to specifically recognize the EP0 protein.
2.6 IFA identification of monoclonal antibodies
IFA analysis was performed on PRV-infected PK-15 cells using the obtained three MAbs as primary antibodies. As shown in FIG. 5, both MAb 2C5 and 4C6 reacted with PRV-infected PK-15 cells, while 3B5 did not react with PRV-infected PK-15 cells.
Through titer determination, the titers of MAb 2C5 and 4C6 can both reach 1: 32000 above, the potency of MAb 3B5 can reach 1: 8000.
2.7 monoclonal antibody variable region genes and sequences
Monoclonal antibody 2C5 was sequenced and the results were as follows:
(1) heavy chain:
1) mouse IgG1, DNA sequence (D46642/G943401H):
leader sequence (SEQ ID No. 1): ATGGAAAGGCACTGGATCTTTCTACTCCTGTTGTCAGTAACTGCAGGTGTCCACTCC, respectively;
variable region sequence (SEQ ID No. 2): CAGGTCCAGCTGCAGCAGTCTGGGGCTGAACTGGCAAGACCTGGGGCCTCAGTGAAGATGTCCTGCAAGGCTTCTGGCTACACCTTTACCAGCAACACTATGCACTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGCATACATTAATCCTAGCAGTGGTTATGTATATTACAATCAGAAGTTCAAGGACAAGGCCACATTGACTGCAGACAAATCCTCCAGCACAGCCTACATGCAACTGAGCAGCCTGACATCTGAGGACTCTGCACTCTATTACTGTGCAAGAGGAGGTTACCACGGTGGTAACTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCGCCGTCTCCTCA, respectively;
constant region (SEQ ID No. 3): GCCAAAACAACACCCCCATCAGTCTATCCACTG, respectively;
2) amino acid sequence:
leader sequence (SEQ ID No. 4): MERHWIFLLLLSVTAGVHS, respectively;
variable region sequence (SEQ ID No. 5): QVQLQQSGAELARPGASVKMSCKASGYTFTSNTMHWVKQRPGQGLEWIAYINPSSGYVYYNQKFKDKATLTADKSSSTAYMQLSSLTSEDSALYYCARGGYHGGNYAMDYWGQGTSVAVSS, respectively;
constant region (SEQ ID No. 6): AKTTPPSVYPL, respectively;
(2) light chain:
1) mouse κ, DNA sequence (D46652/G943401K):
leader sequence (SEQ ID No. 7): ATGGATTCACAGGCCCAGGTTCTTATATTGCTGCTGCTATGGGTATCTGGTACCTGTGGG, respectively;
variable region sequence (SEQ ID No. 8): GACATTGTGATGTCACAGTCTCCATCCTCCCTGGCTGTGTCAGCAGGAGAGAAGGTCACTATGAGCTGCAAATCCAGTCAGAGTCTGCTCAACAGTAGAACCCGAAAGAACTACTTGGCTTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATCTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGCAGGCTGAAGACCTGGCAGTTTATTACTGCAAGCAATCTTATAATCTGTACGCGTTCGGAGGGGGGACCAAGCTGGAAATAAAA, respectively;
constant region (SEQ ID No. 9): CGGGCTGATGCTGCACCAACTGTATCCATCTTCCAATCGTCGACC, respectively;
2) amino acid sequence:
leader sequence (SEQ ID No. 10): MDSQAQVLILLLLWVSGTCG, respectively;
variable region sequence (SEQ ID No. 11): DIVMSQSPSSLAVSAGEKVTMSCKSSQSLLNSRTRKNYLAWYQQKPGQSPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLAVYYCKQSYNLYAFGGGTKLEIK, respectively;
constant region (SEQ ID No. 12): RADAAPTVSIFQSST are provided.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements of the technical solutions of the present invention can be made by those skilled in the art without departing from the spirit of the present invention, and the technical solutions of the present invention are within the scope of the present invention defined by the claims.
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