CN114250244A - Colon cancer vaccine using recombinant attenuated listeria monocytogenes as carrier and preparation method thereof - Google Patents
Colon cancer vaccine using recombinant attenuated listeria monocytogenes as carrier and preparation method thereof Download PDFInfo
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- CN114250244A CN114250244A CN202111529548.1A CN202111529548A CN114250244A CN 114250244 A CN114250244 A CN 114250244A CN 202111529548 A CN202111529548 A CN 202111529548A CN 114250244 A CN114250244 A CN 114250244A
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- listeria monocytogenes
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Abstract
The invention provides a colon cancer vaccine taking recombinant attenuated and single-increased listeria monocytogenes as a vector and a preparation method thereof, relating to the field of genetic engineering, wherein a background strain Lemo-C07 of the attenuated and single-increased listeria monocytogenes is taken as a background vector, and the hly gene 498bp of Lemo-C07 and the hly gene downstream 500bp are amplified by a PCR technology and respectively taken as a fragment A and a fragment B; connecting three fragments (an antigen fragment AH1 is designed on a primer) by using an SOE-PCR technology to obtain a target fragment of A-B, wherein the target fragment is shown as a sequence SEQ ID NO. 1; kpn I and Pst I are used as enzyme cutting sites, and are connected to a listeria shuttle plasmid pKSV7 through enzyme cutting and enzyme digestion to obtain a recombinant plasmid pSL1885, and the recombinant attenuated single-increment listeria vector vaccine is obtained through plasmid electrotransformation, homologous recombination and plasmid loss. Integrating tumor specific antigen genes into the attenuated Listeria genome to ensure that the antigen genes can be stably expressed and are not easy to lose; compared with a wild strain, the attenuated strain Lemo-C07 has the toxicity reduced by nearly 10000 times; does not contain resistant plasmids, has no antibiotic resistance risk and has higher safety.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to a colon cancer vaccine taking recombinant attenuated Listeria monocytogenes as a vector and a preparation method thereof.
Background
Colon cancer is a common malignancy of the digestive tract that occurs in the colon, well at the junction of the rectum and sigmoid colon. The colon cancer is mainly adenocarcinoma, mucus adenocarcinoma and undifferentiated carcinoma. The general morphology is polypoid, ulcer type, etc. The colon cancer can circulate along the intestinal wall, spread up and down along the longitudinal diameter of the intestinal canal or infiltrate into the deep layer of the intestinal wall, and can be planted in the abdominal cavity or spread and transferred along the suture line and the incision surface besides the lymphatic vessel, blood flow transfer and local invasion. The onset of colon cancer is mainly associated with a high fat and low cellulose diet. Chronic inflammation of the colon causes a higher incidence of intestinal cancer than the general population.
Traditional methods of treating cancer are surgical resection, radiation therapy, chemotherapy, and therapeutic vaccines. Compared with immunotherapy, immunotherapy has the advantages of small damage to organisms and low toxicity, but the existing tumor-related vaccines are mainly traditional inactivated vaccines and subunit vaccines, and have the defects of long immune cycle, poor immune effect and the like.
The vaccine based on bacteria as an antigen delivery vector has the advantages of low production cost, genetic stability, safety, high-efficiency anti-tumor effect and the like. How to provide a tumor immunotherapy vaccine can effectively solve the limitations of traditional vaccines and other treatment methods, can better improve the immunotherapy effect, shorten the immune cycle, and improve the survival rate of cancer patients, is a problem which needs to be solved at present.
Disclosure of Invention
Solves the technical problem
Aiming at the defects of the prior art, the invention provides a colon cancer vaccine taking recombinant attenuated listeria monocytogenes as a vector and a preparation method thereof, and solves at least one problem provided by the background art.
Technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme:
in a first aspect: the invention provides a recombinant plasmid, which comprises a gene segment with a gene sequence as SEQ ID NO. 1.
In a second aspect: the present invention provides a recombinant attenuated listeria monocytogenes comprising the recombinant plasmid of claim 1.
In a third aspect: the invention provides a colon cancer vaccine strain taking recombinant attenuated single-increment listeria monocytogenes as a carrier, which is named as LADS-AlfaH1 and is preserved in common microorganisms of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 21383.
In a fourth aspect: the invention provides a colon cancer vaccine using recombinant attenuated listeria monocytogenes as a vector, which contains the colon cancer vaccine strain using the recombinant attenuated listeria monocytogenes as the vector shown in claim 3.
In a fifth aspect: the invention provides a preparation method of a colon cancer vaccine taking recombinant attenuated Listeria monocytogenes as a vector, which comprises the following steps:
constructing a homologous recombinant plasmid pSL1885, wherein the recombinant plasmid comprises a gene segment with a gene sequence shown as SEQ ID NO. 1;
preparing attenuated listeria monocytogenes competent cells;
electrically transferring the recombinant plasmid pSL1885 into the attenuated Listeria monocytogenes competent cells, and culturing to obtain a monoclonal antibody;
homologous recombination and screening to obtain the recombinant attenuated single-increment listeria carrier vaccine for expressing the LLO-AH1 fusion protein.
Preferably, the step of constructing the homologous recombinant plasmid pSL1885 is as follows:
using Lemo-C07 whole genome DNA as a template and a primer containing an AH1 gene sequence, and amplifying a 498bp fragment A and a 500bp fragment B respectively containing enzyme cutting sites Kpn I and Pst I by utilizing a PCR technology; the target fragment of AB is obtained by connecting the two fragments by SOE-PCR technology, and the target fragment is cloned to a shuttle plasmid pKSV7 by enzyme digestion and enzyme ligation by taking Kpn I and Pst I as enzyme digestion sites, so as to obtain a homologous recombination plasmid pSL 1885.
Preferably, the step of obtaining the monoclonal antibody is:
and (2) electrically transferring the recombinant plasmid pSL1885 into the prepared competent cells by an electrotransfer, quickly adding a preheated BHI liquid culture medium after electric shock, fully and uniformly mixing, transferring into an EP tube, culturing at constant temperature, centrifuging, coating the resuspended thallus into a BHI solid culture medium containing chloramphenicol resistance, and culturing in a constant-temperature culture box to obtain the monoclonal antibody.
Preferably, the steps of obtaining the recombinant attenuated listeria monocytogenes vector vaccine expressing the LLO-AH1 fusion protein through homologous recombination and screening are as follows:
inoculating the obtained monoclonal colony into a BHI liquid culture medium, and placing the culture medium at 37 ℃ for overnight culture by shaking;
carrying out PCR verification on the bacterial liquid by using a primer; inoculating the corresponding cloned antibody into a BHI liquid culture medium, carrying out subculture at 42 ℃ for homologous recombination, and streaking the bacterial liquid until the subculture reaches a proper generation number to obtain the monoclonal antibody;
sequencing the PCR products, and if the sequencing results are successfully compared, considering that the homologous recombination and integration are successful; inoculating the screened monoclonal into a BHI liquid culture medium without resistance, carrying out passage at 30 ℃ to discard plasmids after homologous recombination, streaking and picking the monoclonal to carry out resistance screening to obtain a non-resistant clone, and obtaining the recombinant attenuated single-increment Listeria vector vaccine expressing the LLO-AH1 fusion protein after gene sequencing verification is correct. .
Advantageous effects
The invention provides a colon cancer vaccine using recombinant attenuated Listeria monocytogenes as a vector and a preparation method thereof. Compared with the prior art, the method has the following beneficial effects:
1. according to the recombinant attenuated and singly-increased listeria carrier vaccine provided by the invention, the tumor specific antigen gene is integrated into the attenuated listeria genome, so that the antigen gene can be stably expressed and is not easy to lose; compared with a wild strain, the attenuated strain Lemo-C07 has the toxicity reduced by nearly 10000 times (which is in accordance with the biological safety level of the vaccine); the recombinant vaccine does not contain resistant plasmids, does not have antibiotic resistance risk and has higher safety.
2. The recombinant attenuated and single-increased listeria monocytogenes vector vaccine provided by the invention carries and expresses the tumor specific antigen AH1, can activate the immunity of organism specific anti-tumor cells and stimulate the tumor killing effect, does not damage normal cells, and has specificity. The fusion expression of tumor specific antigen AH1 and Listeria monocytogenes self-virulence gene LLO can obviously activate the anti-tumor immune mechanism of an organism, and has a high-efficiency tumor killing mechanism.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a map of recombinant plasmid pSL1885 of the present invention, comprising the C-terminal 498bp (hly stop codon removed) of the hly gene of attenuated Listeria monocytogenes Lemo-C07 and the homologous sequence of 500bp of the hly downstream gene, as well as the AH1 gene sequence;
FIG. 2 is a schematic diagram of Westernblotting detection of expression of related antigenic proteins Lemo-C07 and LADS-AlfaH1 according to an embodiment of the present invention;
FIG. 3 shows the construction strategy and immunotherapy procedure of BALB/c mouse colon cancer model according to the present invention;
FIG. 4 is a schematic diagram showing the evaluation of the effect of the inventive vaccine treatment on the inhibition of tumor size on a BALB/c mouse colon cancer model;
FIG. 5 is a schematic diagram of the evaluation of the immunity of anti-tumor cells treated with the vaccine of the present invention on a BALB/c mouse colon cancer model in accordance with an embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described, and it is obvious that the described embodiments are a part of the embodiments of the present invention, but not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in fig. 1 to 5, the present invention aims to provide a colon cancer vaccine using recombinant attenuated listeria monocytogenes as a vector and a preparation method thereof, wherein a background strain Lemo-C07 of attenuated listeria monocytogenes is used as a background vector, and PCR technology is used to amplify the hly gene 498bp of Lemo-C07 and the downstream 500bp of the hly gene respectively as a fragment a and a fragment B; connecting three fragments (an antigen fragment AH1 is designed on a primer) by using an SOE-PCR technology to obtain a target fragment of A-B, wherein the target fragment is shown as a sequence SEQ ID NO. 1; kpn I and Pst I are used as enzyme cutting sites, and are connected to a listeria monocytogenes shuttle plasmid pKSV7 through enzyme cutting and enzyme digestion to obtain a recombinant plasmid pSL1885, and a recombinant attenuated single-increment listeria monocytogenes vector vaccine expressing colon cancer-associated antigen AH1 is finally obtained through plasmid electrotransformation, homologous recombination and plasmid loss, and is named as LADS-AlfaH1, the preservation number is CGMCC 21383, and the preservation date is 12, 16 days 2020; the preservation organization is the common microorganism center of China Committee for culture Collection of microorganisms (address: No. 3 of West Lu No.1 of Beijing, Kyoho, national academy of sciences, microbiological research institute; zip code 100101), and is named Listeria monocytogenes.
Wherein the background carrier used is attenuated Listeria monocytogenes Lemo-C07, the attenuated Listeria monocytogenes Lemo-C07 is preserved in China general microbiological culture collection management center (address: Beijing City & Inward area Beichen Xilu No.1, institute of microbiology, China academy of sciences; postal code 100101), and the public can obtain the strain according to the regulation, and the preservation number is 18647;
based on the above explanation, the embodiment of the invention provides a recombinant plasmid pSL1885, wherein the recombinant plasmid pSL1885 comprises a gene segment with a gene sequence shown as SEQ ID NO. 1.
Furthermore, the embodiment of the invention also provides a recombinant attenuated listeria monocytogenes, which contains the recombinant plasmid pSL 1885.
Further, the embodiment of the invention also provides a colon cancer vaccine strain taking recombinant attenuated listeria monocytogenes as a carrier, wherein the vaccine strain is named as LADS-AlfaH1 and is preserved in common microorganisms of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 21383.
Further, the embodiment of the invention provides a preparation method of a colon cancer vaccine using recombinant attenuated listeria monocytogenes as a vector, which comprises the following steps:
constructing a homologous recombinant plasmid pSL1885, wherein the recombinant plasmid comprises a gene segment with a gene sequence shown as SEQ ID NO. 1;
preparing attenuated listeria monocytogenes competent cells;
electrically transferring the recombinant plasmid pSL1885 into the attenuated Listeria monocytogenes competent cells, and culturing to obtain a monoclonal antibody;
homologous recombination and screening to obtain the recombinant attenuated single-increment listeria carrier vaccine for expressing the LLO-AH1 fusion protein.
The following specific description is made in conjunction with detailed examples:
the main reagents required for vaccine production are:
LB medium, agarose H, agar, BHI medium, PCR purification kit, gel recovery kit, plasmid extraction kit, total cell RNA extraction kit, BCA protein concentration determination kit, PCR-related reagents, DNA ligase (Ligation high.2), restriction enzymes, NEB, ampicillin and kanamycin, Sangon, TOYOBO, DMEM, FBS, PBS, protein marker, flow fluorescent antibody, transfection Factor Buffer Set and Stain Buffer FBS, were purchased from Shanghai Bioengineering, Inc., UK Oxoid, PCR purification kit, gel recovery kit, Goji-Chi Biotech, plasmid extraction kit, BCA protein concentration determination kit, Byun-Chi, PCR-related reagents from Nanjing Nozan, DNA ligase (Ligation high.2), Toyobo, restriction enzymes from NEB, ampicillin and kanamycin from Sangon, and kits from TOYOBO, DMEM, FBS, PBS, protein marker from Sammer, flow fluorescent antibody, transfection Factor Buffer Set and Stain Buffer FBS from BD corporation.
The main instruments required for vaccine production are:
shaking table (HZ-9211K), vortex oscillator (ZEALWAY GI541), multi-functional enzyme labeling instrument (BioTek synergy TM H1), gradient PCR instrument (Eppendorf), gel imaging system (UVP), metal bath (Thermo), biological safety cabinet (BSC-II), electric shock conversion instrument (BTX ECM 630) protein electrophoresis instrument (BIORAD), cell carbon dioxide incubator (Thermo).
1. Constructing a homologous recombinant plasmid pSL1885, wherein the recombinant plasmid comprises a gene segment with a gene sequence shown as SEQ ID NO. 1;
fragments A (498bp, amplification primers P2 and P3) and B (500 bp, amplification primers P4 and P5) respectively containing restriction enzyme sites Kpn I and Pst I are amplified by PCR using Lemo-C07 whole genome DNA as a template and a primer containing an AH1 gene sequence. On the basis, the two fragments are connected together by an overlapping PCR technology (SOE-PCR) to obtain the target fragment of the 'AB' (the sequence is shown in SEQ ID NO. 1). Taking Kpn I and Pst I as enzyme cutting sites, carrying out enzyme cutting, connecting and cloning to Listeria shuttle plasmid pKSV7 to obtain homologous recombination plasmid pSL1885, and storing at-20 ℃ after sequencing verification is correct. The relevant primers are shown in the following table:
note: p1 is a primer which is used for verifying the recombinant plasmid and is 208bp upstream from the A fragment on the listeria genome; p2 is an upstream primer for amplifying the A fragment; p3 is a downstream primer for amplifying the A fragment; p4 is an upstream primer for amplifying the B fragment; p5 shows the restriction site underlined in the downstream primer for amplifying the B fragment, and the protective base of the restriction site is bold.
The method comprises the following specific operations: the A fragment was amplified with primers P2 and P3, the B fragment was amplified with primers P4 and P5, the three fragments were ligated together by SOE-PCR to obtain the target fragment "AB", the target fragment and the vector (pKSV7) were digested simultaneously with Kpn I and Pst I, and the digestion products were purified according to the kit instructions. mu.L of the target fragment, 4. mu.L of the vector and 10. mu.L of DNA ligase were mixed and then placed in a 16 ℃ metal bath for enzyme ligation to obtain the recombinant plasmid pSL1885 (the construction map is shown in FIG. 1).
2. Preparing attenuated listeria monocytogenes competent cells;
and (3) dipping the strain ring with the Lemo-C07 frozen strain liquid of the attenuated Listeria monocytogenes, streaking on a BHI solid plate culture, and culturing overnight at 37 ℃ to obtain a single colony. Single colonies were picked and inoculated into 5mLBHI liquid medium and incubated overnight at 37 ℃. Inoculating overnight culture liquid into 1-100 mL BHI culture medium (containing 0.5M sucrose) at a ratio of 1:100, and shake culturing at 37 deg.C until OD600 nm value is about 0.18-0.25; adding penicillin G (filter sterilized) with the final concentration of 20 mu G/mL, and continuing to culture for 2 h; 3500rpm, 10min, 4 ℃, collecting thalli, discarding supernatant, adding a proper amount of precooled buffer (containing 1mM HEPES and 0.5M sucrose), and washing twice; centrifuging, removing supernatant, adding washing buffer solution into the precipitate, resuspending thallus, packaging, and placing in-80 deg.C refrigerator for use.
3. Electrically transferring the recombinant plasmid pSL1885 into the attenuated Listeria monocytogenes competent cells, and culturing to obtain a monoclonal antibody;
transferring 1 μ g of recombinant plasmid pSL1885 into the competent cells prepared above by electrotransfer (under electrotransfer setting conditions of 2500V, 200 Ω and 25 μ F), rapidly adding 1mL of preheated (37 ℃) BHI liquid culture medium (containing 0.5M sucrose) after electric shock, transferring to a new EP tube after full mixing, placing in a 30 ℃ constant temperature incubator for static culture for 2-3h, centrifuging (6000rpm and 2min), coating the resuspended thallus in the BHI solid culture medium containing chloramphenicol resistance, placing in a 37 ℃ constant temperature incubator for culture for 24-48h, and culturing the monoclonal antibody.
4. Homologous recombination and screening to obtain recombinant attenuated single-increment listeria carrier vaccine for expressing LLO-AH1 fusion protein
The monoclonal colonies obtained above were inoculated into BHI broth (chloramphenicol resistance) and cultured overnight with shaking at 37 ℃. PCR product sequencing verification is carried out by using primers P2 and P5, and the successful transformation of the homologous recombination plasmid is judged by comparing sequencing results. Inoculating the corresponding single clone into a BHI liquid culture medium (chloramphenicol resistance), subculturing at 42 ℃ for homologous recombination, streaking a bacterial liquid at a proper passage to obtain the single clone, performing colony PCR by using primers P1 and P5, sequencing a PCR product, and determining that the homologous recombination and integration are successful if the results are compared successfully. Inoculating the selected monoclonal into a non-resistant BHI liquid culture medium, carrying out passage at 30 ℃ to discard plasmids after homologous recombination, carrying out resistance screening on the monoclonal by appropriate passage streaking to obtain a non-resistant clone (the plasmid is lost), and finally obtaining the recombinant attenuated Listeria monocytogenes (namely LADS-AlfaH1 disclosed by the embodiment of the invention) expressing the LLO-AH1 fusion protein after gene sequencing verification is correct, wherein the recombinant listeria monocytogenes is added with 60% of glycerol and frozen in a refrigerator at-80 ℃.
The specific verification steps are as follows:
evaluation of immunotherapeutic effect of recombinant listeria vaccine LADS-AlfaH1 on mouse colon cancer model:
verification of LADS-ALfaH1 expression recombinant tumor antigen AH1 protein
Single colonies were picked in 5mLBHI broth and placed on a shaker at 37 ℃ for overnight incubation. Transferring 1mL of overnight culture liquid into 100mLBHI liquid culture medium, shaking and culturing for 8-9h at 37 ℃ in a shaking table, centrifuging, and filtering supernatant; precipitating the filtered supernatant with trichloroacetic acid overnight at 12000rpm for 20min, centrifuging at 4 ℃ to remove the supernatant, resuspending the precipitate with 1mL of 1M NaOH, and centrifuging to obtain the supernatant, namely the secreted protein; and washing the precipitate with 50mM PBS after centrifugation, then resuspending the precipitate with 1mL Listeria lysate, ultrasonically crushing the precipitate, and centrifuging the supernatant to obtain the cytoplasmic protein. And (3) measuring the concentration of the secretory protein and the cytoplasmic protein by using a BCA protein concentration measuring kit, quantifying, adding a protein Loading Buffer (4 multiplied by Loading Buffer), boiling for 6-7min, completing the preparation of a protein sample, and verifying the protein expression condition by Western blotting. As shown in FIG. 2, LADS-AlfaH1 was fusion-expressed with LLO-AH1 protein, and AH1 protein was expressed only in LADS-AlfaH1, as compared to control Lemo-C07.
2. Construction of mouse colon cancer model and strategy for immunotherapy by using LADS-AlfaH1
The left flank of BALB/c female mouse with 6-8 weeks of age is selected to be injected with about 2X 105 CT26 tumor cells subcutaneously, and the tumor diameter reaches about 2-3mm after about 6-7 days, so that the next tumor immunotherapy experiment can be carried out. The vaccine of the invention was administered via tail vein injection of LADS-AlfaH1 (approximately 108CFU per mouse) on days 6 and 13, respectively, and changes in tumor size were observed during immunization, while mouse tumor cells and spleen cells were taken on day 20 for subsequent analysis. Specific immunotherapy strategies are shown in figure 3.
Evaluation of the Effect of LADS-AlfaH1 in the treatment of mouse Colon cancer model
Abdominal tumor sizes were measured with a vernier caliper on days 6, 9, 13, 16 and 20, respectively, and tumor volumes were measured according to the formula (L × W2)/2 (L and W are length and width, respectively). As shown in FIG. 4, the tumor growth of mice injected with the inventive vaccine LADS-AlfaH1 (injection amount was about 108CFU) was suppressed and the tumor of 1 mouse in this treatment group had been completely eliminated by day 2022 throughout the treatment period; the growth of the tumors of the mice in the control group injected with both PBS and Lemo-C07 was not inhibited at all, and the tumor size continued to increase. The experiment shows that the vaccine LADS-ALfaH1 has obvious treatment effect on a mouse colon cancer model.
Detection of T lymphocyte distribution in a model of LADS-AlfaH1 treatment of mouse colon cancer
The spleen of the mouse was taken on day 2022, ground thoroughly in a mortar, added with PBS and filtered through a filter to obtain mouse splenocytes. Changes in T lymphocytes were analyzed by flow cytometry using antibodies such as anti-Fluorescein Isothiocyanate (FITC) -labeled anti-CD 3, Phycoerythrin (PE) -labeled anti-CD 4, anti-phycocyanin (APC) -labeled anti-CD 8, Fluorescein Isothiocyanate (FITC) -labeled anti-CD 4, phycocyanin (APC) -labeled anti-CD 25, and Phycoerythrin (PE) -labeled FoxP 3. As shown in fig. 5, compared with the control PBS, the distribution of CD4+ and CD8+ T lymphocytes in the spleen of the mice treated by the vaccine LADS-AlfaH1 of the present invention is significantly increased, while the proportion of regulatory T lymphocytes (Tregs) that inhibit T cell activation and proliferation in vivo through an active regulation mode is decreased, which indicates that LADS-AlfaH1 can inhibit tumor growth in the body of the mice and eliminate tumors by activating effector T lymphocytes and regulatory T lymphocytes, thereby achieving the effect of killing tumor cells.
After the listeria monocytogenes infects host cells, the specific cellular immune response of organisms can be obviously activated. The virulence factor LLO contains residues (PEST domain) rich in proline, glutamic acid, serine and threonine, and clusters containing positively charged residues are arranged on two sides of the virulence factor LLO, and the clusters can induce the exogenous tumor specific antigen expressed by fusion with the LLO to be degraded in proteasomes and presented by MHC class I molecules to activate the immunity of anti-tumor cells, thereby achieving the effect of killing the tumor cells.
It should be noted that, in this document, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.
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Claims (8)
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