EpCAM gene high expression vector based on lentiviral vector and cell strain thereof
By constructing a lentiviral vector-based EpCAM gene high-expression vector and screening transgenic cell lines, the deficiencies in the study of the molecular mechanism of EpCAM in colorectal cancer were resolved, efficient EpCAM expression was achieved, and the research and treatment of colorectal cancer were promoted.
Patent Information
- Application Number
- CN202510982771.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-10-10
AI Technical Summary
The existing technology has not conducted in-depth research on the molecular mechanism of EpCAM in colorectal cancer, and there is a lack of vectors and cell lines that highly express the EpCAM gene, which limits the understanding of the mechanism of EpCAM and the occurrence and development of colorectal cancer and the development of treatment methods.
A lentiviral vector-based EpCAM gene high-expression vector was constructed, including the pCHD-CMV-MCS-EF1-RFP-T2A-puro backbone structure and the EpCAM gene expression structure. The vector was recombined with Escherichia coli and transfected into HT-29 and HT-115 colorectal cancer cells to screen and obtain transgenic cell lines with high EpCAM expression.
It provides efficient EpCAM gene expression vectors and cell lines, supports the research on the molecular mechanism of EpCAM in colorectal cancer, and promotes the development of prevention, diagnosis and treatment methods for colorectal cancer.
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Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to a lentivirus vector-based EpCAM gene high-expression vector and cell strain thereof, and belongs to the field of genetic engineering and biotechnology. BACKGROUND
[0002] Colorectal cancer is one of the common digestive system malignant tumors after gastric cancer and esophageal cancer, and its treatment effect is poor. In the investigation of all malignant tumors in China, the morbidity and mortality rank the third and the fifth. In China, the population aging and the change of dietary structure make the morbidity and mortality significantly increase compared with the previous ten years, and there is a rising trend in the future. The most common treatment method for early colorectal cancer is surgical resection of cancer, combined with some adjuvant therapy, but for advanced colorectal cancer, the situation is opposite, surgery is only used as a palliative treatment method for patients, or is only used to eliminate the symptoms that have greater impact on patients. With the development of medicine, the position of targeted therapy and immunotherapy in the treatment of colorectal cancer is increasingly prominent.
[0003] Epithelial specific adhesion molecule (EpCAM) is a 40kD type I transmembrane glycoprotein, which is a specific antigen of colorectal cancer as well as a tumor stem cell antigen. As a cell adhesion molecule, EpCAM is involved in cell adhesion and connection, and plays an important role in maintaining the integrity and function of tissues. In colorectal cancer, abnormal expression of EpCAM may lead to decreased intercellular adhesion, promoting tumor cell invasion and metastasis. In addition, EpCAM may also participate in the occurrence and development of colorectal cancer by affecting cell proliferation, apoptosis and other biological processes. In summary, there is a close relationship between EpCAM and colorectal cancer. The high expression of EpCAM in colorectal cancer is positively correlated with the malignant degree of tumor, and its abnormal expression may promote the invasion and metastasis of tumor cells.
[0004] Although some studies have revealed the relationship between EpCAM and colorectal cancer, there are still many aspects that need further in-depth study. For example, the specific mechanism of action of EpCAM in colorectal cancer: a deeper understanding of the specific mechanism of action of EpCAM in the occurrence and development of colorectal cancer, especially the differences in its role in different subtypes and different pathological stages of colorectal cancer. Research on how EpCAM affects the biological processes of colorectal cancer cells such as proliferation, apoptosis, invasion, and metastasis, as well as the interactions between these processes. Explore how EpCAM promotes the distant metastasis of colorectal cancer cells by regulating intercellular adhesion, cell migration, and other processes. Explore how EpCAM affects the interaction between colorectal cancer and microenvironment components such as immune cells and interstitial cells, and in turn affects tumor development. In summary, the molecular mechanism of EpCAM and colorectal cancer is a complex and important research field that needs to be studied and explored from multiple angles. These studies will help us better understand the pathogenesis of colorectal cancer and provide new ideas and methods for the prevention, diagnosis, and treatment of colorectal cancer. Therefore, in basic research, constructing a vector for high expression of EpCAM gene and then constructing a colorectal cancer cell line with overexpression of EpCAM gene is very important for the study of the molecular mechanism of EpCAM and the occurrence and development of colorectal cancer. SUMMARY
[0005] In view of the need for research on the molecular mechanism of EpCAM and the occurrence and development of colorectal cancer, the present application provides an EpCAM gene high-expression vector based on a lentiviral vector and a cell line thereof.
[0006] The first object of the present application is to provide an EpCAM gene high-expression vector based on a lentiviral vector, which comprises:
[0007] a pCHD-CMV-MCS-EF1-RFP-T2A-puro backbone structure and an EpCAM gene expression structure;
[0008] The pCHD-CMV-MCS-EF1-RFP-T2A-puro backbone structure comprises the promoter CMV Promoter of the target gene, the reporter gene promoter EF1a Promoter, the marker gene green fluorescent protein RFP for eukaryotic cell screening, and the puromycin puro, and the vector map is as shown in Figure 1 The nucleotide sequence of the EpCAM gene expression structure is shown in SEQ ID NO. 2.
[0009] The nucleotide sequence of the EpCAM gene expression structure is shown in SEQ ID NO. 2.
[0010] In one embodiment of the present application, the nucleotide sequence of the promoter CMV Promoter of the target gene is shown as SEQ ID NO. 3; the nucleotide sequence of the reporter gene promoter EF1a Promoter is shown as SEQ ID NO. 4; the nucleotide sequence of the marker gene red fluorescent protein RFP for eukaryotic cell screening is shown as SEQ ID NO. 5; and the nucleotide sequence of the puromycin puro is shown as SEQ ID NO. 6.
[0011] A second object of the present application is to provide a construction method of the recombinant expression vector, which comprises the following steps:
[0012] S1. Xba1 and Nhe1 enzyme cutting sites are added at both ends of the synthesized EpCAM gene sequence, and the sequence is connected to the pCHD-CMV-MCS-EF1-RFP-T2A-puro empty vector through the two enzyme cutting sites to construct a pCDH-OE-EpCAM recombinant expression vector. Figure 2 )。
[0013] S2. The E. coli containing the recombinant expression vector is streaked on an ampicillin-resistant LB plate and cultured at 37°C overnight. Several single colonies are picked for sequencing.
[0014] S3. The single colony with correct sequencing result is shaken and cultured, and the plasmid is extracted by using an endotoxin-free plasmid extraction kit. After detecting the purity and concentration, the plasmid is stored at -20°C.
[0015] A third object of the present application is to provide a recombinant microbial cell of the recombinant expression vector.
[0016] In one embodiment of the present application, the recombinant microbial cell is a recombinant E. coli.
[0017] In one embodiment of the present application, the recombinant microbial cell is a recombinant E. coli.
[0018] A fourth object of the present application is to provide a transgenic cell of the lentivirus recombinant expression vector.
[0019] In one embodiment of the present application, the transgenic cell is an HT-29-OE-EpCAM cell.
[0020] In one embodiment of the present application, the transgenic cell is obtained by transfecting HT-29 cells with the pCDH-OE-EpCAM recombinant expression vector and screening single colonies.
[0021] In one embodiment of the present invention, the transgenic cells are HT-115-OE-EpCAM cells.
[0022] In one embodiment of the present invention, the transgenic cells are obtained by transfecting HT-115 cells with the pCDH-OE-EpCAM recombinant expression vector and then screening for single clones.
[0023] A fifth object of the present invention is to provide a method for constructing the above-mentioned transgenic cell, the method comprising the following steps:
[0024] S1. The pCDH-OE-EpCAM recombinant expression vector was transfected into HT-29 and HT-115 cells using the transfection reagent Lipo2000.
[0025] S2. Subculture the transfected cells in a 10 mm culture dish at an area ratio of 1:10 to 1:45, depending on the transfection efficiency.
[0026] S3. Based on the results of the puromycin cytotoxicity assay on two colorectal cancer cell lines, add appropriate amounts of puromycin and screen until monoclonal cell lines are formed.
[0027] S4. Use cloning rings to collect monoclonal cells for passage, and identify each monoclonal cell line by flow cytometry to obtain the transgenic cell line required for this study.
[0028] The present invention also claims protection for the use of the pCDH-OE-EpCAM recombinant expression vector and its recombinant microbial cells, HT-29-OE-EpCAM cells and HT-115-OE-EpCAM cells in the fields of biology, medicine and medical treatment. Beneficial effects
[0029] The present invention constructs a vector capable of causing eukaryotic cells to highly express the EpCAM gene, recombinant microbial cells, and colorectal cancer cell lines that highly express the EpCAM gene, laying a very important foundation for studying the molecular mechanism of the EpCAM gene and the occurrence and development of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS
[0030] Figure 1 Map of the pCHD-CMV-MCS-EF1-RFP-T2A-puro vector.
[0031] Figure 2 Map of the pCDH-OE-EpCAM recombinant expression vector.
[0032] Figure 3 The transfection efficiency of HT-29 was observed under a fluorescence microscope.
[0033] Figure 4 Observe the transfection efficiency of HT-115 under fluorescence microscope.
[0034] Figure 5 Observe the monoclonal cells of HT-29 under fluorescence microscope.
[0035] Figure 6 Observe the monoclonal cells of HT-115 under fluorescence microscope.
[0036] Figure 7 The expression of EpCAM gene in HT29 colorectal cancer cell strain was 24.39% detected by flow cytometry.
[0037] Figure 8 The expression of EpCAM gene in HT-29-OE-EpCAM-2 transgenic colorectal cancer cell strain was 73.63% detected by flow cytometry.
[0038] Figure 9 The expression of EpCAM gene in HT115 colorectal cancer cell strain was 0.00% detected by flow cytometry.
[0039] Figure 10 The expression of EpCAM gene in HT-115-OE-EpCAM-19 transgenic colorectal cancer cell strain was 24.06% detected by flow cytometry.
[0040] Figure 11 HT-115-OE-EpCAM-19-1 transgenic colorectal cancer cell strain was obtained by flow cytometry sorting. DETAILED DESCRIPTION
[0041] Material description in the example:
[0042] 1. Strain information: E. coli DH5a competent, commercial strain.
[0043] 2. Vector information: Plasmid pCHD-CMV-MCS-EF1-RFP-T2A-puro empty vector is a commercial vector.
[0044] 3. DMEM high-sugar medium, McCoy'5A medium, fetal bovine serum (FBS), opti-MEM medium, PBS buffer, Lipo2000, puromycin, CD326-PE are all commercial products.
[0045] 4. LB medium formula: 10 g / L peptone, 5 g / L yeast powder, 10 g / L Nacl; solid medium adds 20 g / L agar powder.
[0046] Construction of pCDH-OE-EpCAM recombinant expression vector
[0047] The human EpCAM gene sequence was found in NCBI, and Xbal and Nhel enzyme sites were added at both ends of the full-length DNA sequence of the synthesized gene. The sequence was connected to the pCHD-CMV-MCS-EF1-RFP-T2A-puro empty vector through the two enzyme sites to construct the pCDH-OE-EpCAM recombinant expression vector. The vector was transformed into E. coli DH5a competent cells, and after transformation, it was plated on ampicillin-resistant LB solid medium plates and incubated at 37°C overnight. Several single colonies were picked and sent to a company for sequencing. The single colonies with correct sequencing results were cultured in LB medium, and the plasmid was extracted using an endotoxin-free plasmid extraction kit. After detecting the purity and concentration, it was stored at -20°C.
[0048] Example 2: Culture of colorectal cancer cell lines HT-29 and HT-115
[0049] The culture medium for the HT-29 colorectal cancer cell line was 90% McCoy'5A medium + 10% FBS, and the culture medium for the HT-115 colorectal cancer cell line was 90% DMEM high glucose + 10% FBS. The cells to be thawed were taken from the liquid nitrogen tank and immediately placed in a 37°C water bath, shaken rapidly, and the frozen cells were thawed as quickly as possible within 1 minute. The frozen tube was quickly moved to a clean bench after alcohol disinfection. The cell solution was transferred to a 1.5 ml tube using a pipette, centrifuged at 1500 rpm / min for 5 min, the supernatant was discarded, 1 ml of culture medium was added to suspend the cells, and the cell count was counted. The cells were inoculated in T25 culture bottles at a cell number of 1x106 per bottle, and cultured in a 37°C, 5% CO2 incubator. The cells were cultured to about 90% confluence, and the trypsin digestion time was about 2 min.
[0050] Example 3: Detection of the cytotoxicity of puromycin on colorectal cancer cell lines
[0051] Two colorectal cancer cell lines were inoculated in two 24-well plates at a density of 1-2x10 4 The concentration of puromycin was set at 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 ng / ul, and 3 replicates were set for each concentration. On the first day, each concentration of puromycin was added, and the medium was changed every 2 days. The lowest concentration at which all cells died within 7-10 days was used as the screening concentration for transgenic cells of the colorectal cancer cell line. After detection, the puromycin screening concentrations for HT-29 and HT-115 were 4 ng / ul and 6 ng / ul, respectively.
[0052] Example 4 Transfection of colorectal cancer cell lines
[0053] Two colorectal cancer cell lines were cultured at a concentration of 1-2×10 5 The cells were seeded into 24-well plates at a density of 1000 cells / well. When the cells grew to 80% confluence, the serum-free medium was replaced and transfection was performed 2 hours later. Transfection steps: The transfection reagent Lipo2000 and the target plasmid were added to the Opti-MEM medium (the ratio of total plasmid volume to Lipo2000 was 1:2). After 5 minutes, the two liquids were mixed and incubated in the dark for 20 minutes. The mixture was then added dropwise to the cell culture wells. After 6 hours, the normal culture medium containing serum was replaced. After 18-24 hours, the HT-29 cells were observed under a fluorescence microscope ( Figure 3 ) and HT-115 ( Figure 4 ) transfection efficiency.
[0054] Example 5 Construction of transgenic colorectal cancer cell lines
[0055] The transfected colorectal cancer cells were passaged at an area ratio of 1:10 to 1:45 according to the transfection efficiency and were transferred to 10mm culture dishes. According to the results of the puromycin cytotoxicity test of the two colorectal cancer cell lines, the corresponding puromycin content was added and the screening medium was replaced every 3 days. After 10-14 days of screening, the formation of monoclonal cell lines could be seen. Observe under a fluorescence microscope and select several HT-29 ( Figure 5 ) and HT-115 ( Figure 6 ) are formed into monoclonal cells, which are collected using cloning rings and passaged. When a certain cell number is reached, each monoclonal cell line is identified by flow cytometry to ultimately obtain the desired transgenic cell line.
[0056] Example 6 Flow cytometry analysis and screening of transgenic cell lines
[0057] The non-transgenic HT-29 and HT-115 colorectal cancer cell lines were used as control groups, and the expression of EpCAM gene in the two colorectal cancer cell lines and their corresponding transgenic cell lines was analyzed by flow cytometry. 6 After 100 cells were grown, the cells were washed twice with PBS to remove the culture medium and serum. After staining with CD326-PE flow cytometry antibody for 30 minutes, the cells were washed twice with PBS to remove the influence of free antibody. The cells were resuspended in PBS and then analyzed by flow cytometry. The expression of EpCAM gene in HT29 colorectal cancer cell line was 24.39% ( Figure 7 ), the expression of EpCAM gene in HT-29-OE-EpCAM-2 transgenic colorectal cancer cell line was detected to be 73.63% ( Figure 8); the expression of EpCAM gene in HT-115 colorectal cancer cell strain was 0.00% (FACS) Figure 9 ), and the expression of EpCAM gene in HT-115-OE-EpCAM-19 transgenic colorectal cancer cell strain was 24.06% (FACS) Figure 10 ). Since the expression of EpCAM gene in HT-115-OE-EpCAM-19 transgenic colorectal cancer cell strain was low, the cell strain formed by the single clone was sorted by flow cytometry to obtain HT-115-OE-EpCAM-19-1 transgenic colorectal cancer cell strain. Figure 11 ). The cell strains were subcultured and cryopreserved to finally establish HT-29-OE-EpCAM-2 cell strain and HT-115-OE-EpCAM-19-1 cell strain.
[0058] Although the present application has been disclosed with reference to the preferred embodiments, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, and therefore the protection scope of the present application should be defined by the claims.
Claims
1. A eukaryotic expression vector, characterized in that: The EpCAM gene expression structure was connected to the lentiviral vector pCHD-CMV-MCS-EF1-RFP-T2A-puro backbone structure to obtain the recombinant lentiviral expression vector pCDH-OE-EpCAM; the nucleotide sequence of the EpCAM gene structure is shown in SEQ ID NO. 2, and the nucleotide sequence of the lentiviral expression vector pCHD-CMV-MCS-EF1-RFP-T2A-puro is shown in SEQ ID NO.
1.
2. The recombinant lentiviral expression vector according to claim 1, wherein The EpCAM gene expression construct was ligated between the multiple cloning sites Xba1 and Nhe1 of the vector pCHD-CMV-MCS-EF1-RFP-T2A-puro and expressed at a high level under the drive of the CMV promoter.
3. The recombinant lentiviral expression vector according to claim 1, wherein The vector contains marker genes RFP gene structure and puro gene structure for transgenic cell screening, which are expressed at a medium level under the drive of EF1α promoter.
4. A recombinant microbial cell, characterized in that Contains the recombinant lentiviral expression vector according to any one of claims 1 to 3.
5. The recombinant microbial cell according to claim 4, wherein The recombinant microbial cell uses Escherichia coli DH5α as a host.
6. The recombinant microbial cell according to claim 5, wherein The preparation method of the recombinant microbial cell is to connect the EpCAM gene expression structure between the multiple cloning sites Xba1 and Nhe1 of the vector pCHD-CMV-MCS-EF1-RFP-T2A-puro to obtain the recombinant plasmid pCDH-OE-EpCAM, and transform the recombinant plasmid into Escherichia coli DH5α.
7. A eukaryotic transgenic cell, characterized in that: Contains the recombinant lentiviral expression vector according to any one of claims 1 to 3.
8. The eukaryotic transgenic cell according to claim 7, wherein The eukaryotic transgenic cells are the objects of gene editing of colorectal cancer cell lines HT-29 and HT-115.
9. The eukaryotic transgenic cell according to claim 8, wherein The pCDH-OE-EpCAM recombinant expression vector was transfected into colorectal cancer cell lines HT-29 and HT-115 using Lipo2000 transfection reagent, and then single clones were screened to obtain transgenic cells.
10. Use of the recombinant lentiviral expression vector according to any one of claims 1 to 3, or the recombinant microbial cell according to any one of claims 4 to 6, or the eukaryotic transgenic cell according to any one of claims 7 to 9 in the fields of biology, medicine, and healthcare.