Construction method of cell adhesion molecule overexpression plasmid
By obtaining the CDS sequence of the CADM3 gene from the Ensembl database, designing specific primers for high-fidelity PCR amplification, and combining seamless cloning technology and recombinant lentiviral packaging method, the problem of low construction efficiency of cell adhesion molecule overexpression plasmids in the prior art is solved, and efficient and stable expression of the CADM3 gene is achieved.
Patent Information
- Application Number
- CN202510033267.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-09
- Publication Date
- 2025-04-11
AI Technical Summary
The prior art is difficult to effectively construct cell adhesion molecule overexpression plasmids, especially in the efficient expression of CADM3 genes and the construction of stable plasmids.
By obtaining the CDS sequence of the CADM3 gene from the Ensembl database, designing specific primers for high-fidelity PCR amplification, combining seamless cloning technology to connect the target gene with the linearized vector pLX304, and using recombinant lentiviral packaging and infection methods, the efficient overexpression of the CADM3 gene was achieved.
The efficient amplification and stable expression of the CADM3 gene were achieved, which improved the efficiency and accuracy of plasmid construction, and ensured the genetic stability of the recombinant plasmid and the purity of the high titer virus.
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Figure CN119799794A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of cell adhesion molecule overexpression plasmid construction, and in particular to a method for constructing a cell adhesion molecule overexpression plasmid. Background Art
[0002] Cell adhesion molecule 3 (CADM3) is a protein with key biological functions. It is located on human chromosome 1q23.2. Its gene structure is complex, containing 12 exons and 3 different transcripts. It also has up to 201 homologous genes, which shows its importance and diversity in organisms. CADM3 has a unique protein structure, consisting of three extracellular immunoglobulin-like (Ig) domains, a transmembrane domain, and a short cytoplasmic region. This structure enables CADM3 to form stable adhesion connections between cells and the extracellular matrix (ECM), thereby playing a vital role in a variety of cellular functions such as cell movement, proliferation, survival, and differentiation. At the same time, CADM3 is specifically expressed in neural tissue and has a Ca-independent 2+ The cell-cell adhesion activity of homodimers and heterodimers plays an important role in the formation of synapses, axon bundles and myelinated axons;
[0003] In many human diseases, abnormal expression or dysfunction of CADM3 is closely related to the occurrence and development of the disease. In particular, in brain gliomas, the expression of CADM3 is often absent or significantly reduced, which is consistent with its role as a tumor suppressor gene. Studies have shown that the inactivation of CADM3 in brain gliomas may be due to epigenetic modifications such as histone deacetylation, which further emphasizes the key role of CADM3 in tumor suppression.
[0004] During the development of the nervous system, Schwann cell precursors (SCPs) migrate along peripheral nerves and generate glial cells. The interaction between these glial cells and myelinated axons is mediated by the cell adhesion molecules of the Cadm family. CADM3, as a member of the Cadm family, also plays an important role in this process.
[0005] In summary, CADM3, as an important cell adhesion molecule, plays a key role in a variety of biological processes and human diseases. In-depth research and understanding of it will not only help to reveal the pathogenesis of related diseases, but may also provide new targets and strategies for the treatment of diseases. Summary of the invention
[0006] The purpose of the present invention is to solve the shortcomings of the prior art and to propose a method for constructing a cell adhesion molecule overexpression plasmid.
[0007] In order to achieve the above object, the present invention adopts the following technical solutions:
[0008] A method for constructing a cell adhesion molecule overexpression plasmid comprises the following steps:
[0009] S1: Acquisition, amplification and identification of CADM3 gene;
[0010] S2: Packaging of recombinant lentivirus, including selecting 293T cells with good growth status for inoculation, and using Lipo293 when the cell density reaches 70% to 80%. TM The vector plasmid and auxiliary plasmid carrying the target gene are transfected into 293T cells using a transfection reagent. The cell culture supernatant is collected 72 hours after transfection, filtered, and the infection-assisting reagent polybrene is added. The recombinant lentivirus stock solution is obtained by ultracentrifugation or concentration.
[0011] S3: Lentivirus infection of IMR-32 cell line and resistance screening, including infecting IMR-32 cells with virus stock solution of appropriate concentration, replacing the culture medium with complete culture medium containing hygromycin B for screening 48 hours after infection, picking out drug-resistant cells for expansion culture after two generations of screening, and inducing them with different concentrations of doxycycline. After the induction, RNA was extracted for reverse transcription and fluorescence quantitative PCR reaction to detect the expression level of CADM3 gene.
[0012] Preferably: in step S1, it specifically includes:
[0013] Step S11:
[0014] The CDS sequence of the CADM3 transcript was obtained from the Ensembl database, and specific primers containing 15 bp overlapping sequences of the linearized vector pLX304 were designed and synthesized after optimizing the length, GC content, and Tm value parameters of the primers;
[0015] Step S12:
[0016] use High-Fidelity DNA Polymerase (NEB#M0491) was used in a 50 μl reaction system to perform nested PCR to specifically amplify the CADM3-202 CDS 1197 bp target sequence. The upstream primer was 5'-GGGATCAACAAGTTTTGTACATGGGGGCCCCAGCC-3', and the downstream primer was 5'-CTTACCAACCACTTTCAAGATGAAATATTCCTTCT-3'. According to the primer concentration, template DNA concentration and other parameters, an appropriate amount of PCR reaction system was prepared to ensure that the reaction system contained sufficient dNTPs and Mg. 2+Necessary ingredients such as
[0017] Pre-denaturation at 95°C for 3 min to dissociate the double-stranded template DNA, followed by 33 cycles;
[0018] The amplified product was identified by 1% agarose gel electrophoresis and the target DNA fragment was recovered;
[0019] Step S13:
[0020] The linearized vector pLX304 was purified and mixed with the target gene fragment in proportion, and connected using seamless cloning ligase to form a recombinant plasmid. The recombinant plasmid was identified by enzyme digestion and sequenced to ensure that it had inserted the correct CADM3 gene sequence.
[0021] Furthermore: The design of primers in S11 also needs to ensure the specificity of the primer sequence to avoid nonspecific amplification.
[0022] Further: The PCR amplification system in S12 contains sufficient dNTPs, Mg 2+ The necessary components were added, and the amplification conditions included pre-denaturation at 95°C for 3 min, followed by 33 cycles of amplification, each cycle including denaturation at 95°C for 30 s, annealing at 58°C for 20 s, extension at 72°C for 1 min 20 s, and finally extension at 72°C for 5 min.
[0023] As a preferred solution of the present invention: in S13, the ligation product is transformed into DH5-α competent cells, and screening is performed by coating on a solid plate containing corresponding antibiotics, selecting a single colony for expansion culture, and extracting the recombinant plasmid for enzyme digestion identification and sequencing verification.
[0024] As a further solution of the present invention: the auxiliary plasmids in S2 include psPAX2 vector and pMD2G vector, and the concentration and quality of all plasmids must meet the experimental requirements.
[0025] As a further solution of the present invention: during the infection process in S3, it is necessary to ensure that the cells are in a suitable growth environment, and the concentration of hygromycin B should be adjusted according to the sensitivity of the cells and the screening efficiency.
[0026] On the basis of the above scheme: the fluorescent quantitative PCR reaction in S3 uses specific primers to amplify the CADM3 gene fragment, and selects a suitable fluorescent labeling method to detect the PCR amplification product.
[0027] On the basis of the above scheme: S3 collects the fluorescence signal to detect the PCR process in real time, and calculates the expression level of the CADM3 gene according to the fluorescence signal intensity.
[0028] The beneficial effects of the present invention are:
[0029] 1. A method for constructing a cell adhesion molecule overexpression plasmid, by obtaining the CDS sequence of the CADM3 gene from the Ensembl database, designing specific primers, and combining high-fidelity DNA polymerase for PCR amplification, ensuring the accurate acquisition and efficient amplification of the CADM3 gene fragment, which not only improves the accuracy of the experiment, but also greatly shortens the experimental cycle.
[0030] 2. A method for constructing a cell adhesion molecule overexpression plasmid, which uses seamless cloning technology to connect the target gene fragment with the linearized vector pLX304, avoiding the cumbersome restriction enzyme cutting and connection steps in traditional cloning methods, significantly improving the efficiency of plasmid construction, and seamless cloning also reduces potential mutations caused by the introduction of enzyme cutting sites, ensuring the genetic stability of the recombinant plasmid.
[0031] 3. A method for constructing a cell adhesion molecule overexpression plasmid, using 293T cells to package recombinant lentivirus, and obtaining high-titer recombinant lentivirus by optimizing transfection conditions and screening methods. After ultracentrifugation or concentration treatment, the purity and concentration of the virus were further improved, providing strong support for subsequent cell infection experiments.
[0032] 4. A method for constructing a cell adhesion molecule overexpression plasmid. By selecting the appropriate virus infection time and titer, and screening the infected cells for hygromycin B resistance, an IMR-32 cell line that stably expresses the CADM3 gene was successfully obtained.
[0033] 5. A method for constructing a cell adhesion molecule overexpression plasmid, using fluorescent quantitative PCR (qPCR) technology to perform real-time detection of the expression level of the CADM3 gene, and accurately determining the expression level of the CADM3 gene by collecting fluorescent signals and calculating the gene expression amount. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 It is a schematic diagram of the PLX304 band of the expression plasmid construction process of the construction method of the cell adhesion molecule overexpression plasmid proposed by the present invention (the band is located at 8000 bp);
[0035] Figure 2 It is a schematic diagram of the CADM3 amplification band of the vector in the construction process of the expression plasmid of the construction method of the cell adhesion molecule overexpression plasmid proposed by the present invention (the band is located at 1200 bp);
[0036] Figure 3 It is a schematic diagram of the result of enzyme digestion identification of the CADM3 overexpression plasmid in the expression plasmid construction process of the method for constructing a cell adhesion molecule overexpression plasmid proposed by the present invention;
[0037] Figure 4 It is a schematic diagram of plasmid transfection verification of a method for constructing a cell adhesion molecule overexpression plasmid proposed in the present invention (Figure b is for verifying the mRNA level of CADM3 after transfection). DETAILED DESCRIPTION
[0038] The technical solution of this patent is further described in detail below in conjunction with specific implementation methods.
[0039] The embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and cannot be understood as limiting the present invention.
[0040] Embodiment 1:
[0041] A method for constructing a cell adhesion molecule overexpression plasmid comprises the following steps:
[0042] S1: Acquisition, amplification and identification of CADM3 gene:
[0043] S11: Primer design
[0044] The CDS sequence of the CADM3 transcript (Transcript ID: ENST00000368125.9) was obtained from the Ensembl database;
[0045] Primer design should contain 15 bp overlapping sequence of the linearized vector pLX304;
[0046] The primer sequence should be specific to the CADM3 gene;
[0047] The length, GC content, Tm value and other parameters of the primers need to be optimized;
[0048] According to the designed primer sequence, use professional primer synthesis service to synthesize;
[0049] S12: PCR amplification
[0050] use High-Fidelity DNA Polymerase (NEB#M0491) was used in a 50 μl reaction system to perform nested PCR to specifically amplify the CADM3-202 CDS 1197 bp target sequence. The upstream primer was 5'-GGGATCAACAAGTTTTGTACATGGGGGCCCCAGCC-3', and the downstream primer was 5'-CTTACCAACCACTTTCAAGATGAAATATTCCTTCT-3'. According to the primer concentration, template DNA concentration and other parameters, an appropriate amount of PCR reaction system was prepared to ensure that the reaction system contained sufficient dNTPs and Mg. 2+ Necessary ingredients such as
[0051] Pre-denaturation at 95°C for 3 min to dissociate the double-stranded template DNA, followed by 33 cycles, each cycle including: 95°C for 30 s (denaturation), 58°C for 20 s (annealing, primers bind to the single-stranded template DNA), 72°C for 1 min 20 s (extension, synthesis of new DNA chains), and finally 72°C for 5 min to fully extend the remaining primers, and the amplified products were stored at 4°C;
[0052] The amplified products were identified by 1% agarose gel electrophoresis to observe whether there were specific amplified bands, and the target DNA fragments were recovered using a gel recovery kit for subsequent seamless cloning experiments;
[0053] S13: Seamless cloning plasmid construction
[0054] The linearized vector pLX304 was purified to ensure that there was no impurity and nuclease contamination;
[0055] The target gene fragment was mixed with the linearized vector pLX304 in a ratio of 3:1, and a seamless cloning ligase was used for ligation reaction to form a recombinant plasmid;
[0056] The ligation product was transformed into DH5-α competent cells, spread on a solid plate containing the corresponding antibiotics, screened, and a single colony was selected for expansion culture;
[0057] Collect bacterial liquid, use plasmid extraction kit to extract recombinant plasmid, and verify by enzyme digestion and sequencing to ensure that the correct CADM3 gene sequence is inserted into the recombinant plasmid and the seamless cloning connection is correct;
[0058] S2: Packaging of recombinant lentivirus:
[0059] Select 293T cells with good growth status for inoculation, inoculate the cells in a culture dish of appropriate size, usually a 10 cm or 15 cm culture dish, and culture them in Gibico high-glucose DMEM medium containing 10% FBS;
[0060] Observe cell growth and monitor cell density daily. When the cell density reaches 70% to 80%, prepare for transfection.
[0061] Prepare the vector plasmid carrying the target gene (CADM3 overexpression plasmid), prepare the auxiliary plasmids: psPAX2 vector (packaging plasmid) and pMD2G vector (envelope plasmid), and ensure that the concentration and quality of all plasmids meet the experimental requirements;
[0062] Using LiPo293 TM Transfection reagent for transfection. Prepare an appropriate amount of transfection reagent and plasmid mixture according to the instructions of the transfection reagent.
[0063] The vector plasmid carrying the target gene and the auxiliary plasmid were mixed in a certain ratio, and the plasmid mixture was mixed with Lipo293 TM The transfection reagents were mixed to form a transfection complex, and the transfection complex was evenly added to the 293T cell culture dish and gently shaken;
[0064] Place the transfected cells in an incubator and continue culturing. Usually, replace the culture medium with fresh medium within 6 to 24 hours after transfection.
[0065] About 72 hours after transfection, the cell culture supernatant was collected and filtered using a 0.45 μm filter to remove cell debris and impurities. The infection-promoting reagent polybrene was added to the filtered virus culture supernatant at a ratio of 1:1000;
[0066] The virus culture supernatant after adding polybrene is subjected to ultracentrifugation or concentration to obtain a recombinant lentiviral stock solution, which is then packaged and stored in a -80°C refrigerator for later use;
[0067] S3: Lentivirus infection of IMR-32 cell line and resistance screening:
[0068] Remove the IMR-32 cells from the culture flask and digest them with an appropriate digestive enzyme (such as 0.25% trypsin) until the cells begin to round up and fall off the bottom of the culture flask. Gently pipette the cell suspension to disperse it into single cells.
[0069] Inoculate the digested cell suspension into a 6-well plate, add an appropriate amount of cell suspension to each well to ensure that the cells have enough space to grow during the culture process, and gently shake the 6-well plate to evenly distribute the cells;
[0070] When the cell fusion rate reaches about 60%, virus infection is performed. According to the type of virus and infection efficiency, the appropriate virus titer and infection time are determined. During the infection process, ensure that the cells are in a suitable growth environment (such as temperature, humidity, CO2 concentration, etc.);
[0071] 48 hours after infection, replace the medium with 10% FBS complete medium containing hygromycin B for screening culture. The concentration of hygromycin B should be adjusted according to the sensitivity of the cells and the screening efficiency. During the screening process, regularly observe the growth status and resistance performance of the cells;
[0072] After two generations of screening, cells showing drug resistance are selected for further culture. Using aseptic operation techniques, the resistant cells are transferred to new culture bottles and expanded. During the expansion process, the culture medium is regularly replaced to ensure the healthy growth of the cells.
[0073] The cells were induced with different concentrations of doxycycline (0.25ug / ml, 0.5ug / ml, 1ug / ml, 2ug / ml) for 48 hours to ensure that the cells had enough time to respond to doxycycline;
[0074] After induction, collect cells and extract RNA using an appropriate RNA extraction kit or method to ensure that the quality and quantity of the extracted RNA meet the requirements of subsequent experiments;
[0075] Using the extracted reverse RNA as a template, reverse transcriptase and appropriate primers are used to perform reverse transcription to generate cDNA. The conditions of the transcription reaction should be adjusted according to the characteristics of the enzyme and primers used;
[0076] Using the cDNA generated by reverse transcription as a template, a fluorescent quantitative PCR (qPCR) reaction was performed, specific primers were designed to amplify the CADM3 gene fragment, and an appropriate fluorescent labeling method (such as SYBR Green I dye method or TaqMan probe method) was selected to detect the PCR amplification product. During the PCR amplification process, the PCR process was detected in real time by collecting the fluorescent signal, and the expression level of the CADM3 gene was calculated based on the fluorescence signal intensity.
[0077] The above is a preferred specific implementation manner of the present invention, and the protection scope of the present invention is not limited thereto. Any modification, equivalent substitution and improvement made by any technician familiar with the field within the technical scope disclosed by the present invention in combination with the prior art or public common sense, within the spirit and principle of the present invention, shall be covered by the protection scope of the present invention.
Claims
1. A method for constructing a cell adhesion molecule overexpression plasmid, characterized in that: The following steps are involved: S1: Acquisition, amplification and identification of CADM3 gene; S2: Packaging of recombinant lentivirus, including selecting 293T cells with good growth status for inoculation, and using Lipo293 when the cell density reaches 70% to 80%. TM The vector plasmid and auxiliary plasmid carrying the target gene are transfected into 293T cells using a transfection reagent. The cell culture supernatant is collected 72 hours after transfection, filtered, and the infection-assisting reagent polybrene is added. The recombinant lentivirus stock solution is obtained by ultracentrifugation or concentration. S3: Lentivirus infection of IMR-32 cell line and resistance screening, including infecting IMR-32 cells with virus stock solution of appropriate concentration, replacing the culture medium with complete culture medium containing hygromycin B for screening 48 hours after infection, picking out drug-resistant cells for expansion culture after two generations of screening, and inducing them with different concentrations of doxycycline. After the induction, RNA was extracted for reverse transcription and fluorescence quantitative PCR reaction to detect the expression level of CADM3 gene.
2. The method for constructing a cell adhesion molecule overexpression plasmid according to claim 1, characterized in that: In step S1, it specifically includes: Step S11: The CDS sequence of the CADM3 transcript was obtained from the Ensembl database, and specific primers containing 15 bp overlapping sequences of the linearized vector pLX304 were designed and synthesized after optimizing the length, GC content, and Tm value parameters of the primers; Step S12: use High-Fidelity DNA Polymerase (NEB#M0491) was used in a 50 μl reaction system to perform nested PCR to specifically amplify the CADM3-202 CDS 1197 bp target sequence. The upstream primer was 5'-GGGATCAACAAGTTTTGTACATGGGGGCCCCAGCC-3', and the downstream primer was 5'-CTTACCAACCACTTTCAAGATGAAATATTCCTTCT-3'. According to the primer concentration, template DNA concentration and other parameters, an appropriate amount of PCR reaction system was prepared to ensure that the reaction system contained sufficient dNTPs and Mg. 2+ And other necessary ingredients; Pre-denaturation at 95°C for 3 min to dissociate the double-stranded template DNA, followed by 33 cycles; The amplified product was identified by 1% agarose gel electrophoresis and the target DNA fragment was recovered; Step S13: The linearized vector pLX304 was purified and mixed with the target gene fragment in proportion, and connected using seamless cloning ligase to form a recombinant plasmid. The recombinant plasmid was identified by enzyme digestion and sequenced to ensure that it had inserted the correct CADM3 gene sequence.
3. The method for constructing a cell adhesion molecule overexpression plasmid according to claim 2, characterized in that: The design of primers in S11 also needs to ensure the specificity of the primer sequences to avoid nonspecific amplification.
4. The method for constructing a cell adhesion molecule overexpression plasmid according to claim 3, characterized in that: The PCR amplification system in S12 contains sufficient dNTPs, Mg 2+ The necessary components, and the amplification conditions included pre-denaturation at 95°C for 3 min, followed by 33 cycles of amplification, each cycle including denaturation at 95°C for 30 s, annealing at 58°C for 20 s, extension at 72°C for 1 min 20 s, and finally extension at 72°C for 5 min.
5. The method for constructing a cell adhesion molecule overexpression plasmid according to claim 4, characterized in that: In S13, the ligation product is transformed into DH5-α competent cells, and screening is performed by coating on a solid plate containing corresponding antibiotics, a single colony is selected for expansion culture, and the recombinant plasmid is extracted for enzyme digestion identification and sequencing verification.
6. The method for constructing a cell adhesion molecule overexpression plasmid according to claim 5, characterized in that: The auxiliary plasmids in S2 include psPAX2 vector and pMD2G vector, and the concentration and quality of all plasmids must meet the experimental requirements.
7. The method for constructing a cell adhesion molecule overexpression plasmid according to claim 6, characterized in that: During the infection process in S3, it is necessary to ensure that the cells are in a suitable growth environment, and the concentration of hygromycin B should be adjusted according to the sensitivity of the cells and the screening efficiency.
8. The method for constructing a cell adhesion molecule overexpression plasmid according to claim 7, characterized in that: In S3, the fluorescence quantitative PCR reaction uses specific primers to amplify the CADM3 gene fragment, and selects an appropriate fluorescence labeling method to detect the PCR amplification product.
9. The method for constructing a cell adhesion molecule overexpression plasmid according to claim 8, characterized in that: In S3, the fluorescence signal is collected to detect the PCR process in real time, and the expression level of the CADM3 gene is calculated according to the fluorescence signal intensity.