Application of RESP18 gene in preparation of cleft lip and palate early screening product and treatment drug

Abstract

The invention discloses application of an RESP18 gene in preparation of a cleft lip and palate early screening product and a treatment medicine, expression of the RESP18 gene of human palate embryo mesenchymal cells is up-regulated, and proliferation, migration and osteogenic differentiation movement ability of the human palate embryo mesenchymal cells can be improved. The application has the advantages that the RESP18 gene which is a very effective protective gene is provided for early screening and treatment of cleft lip and palate, an effective direction is provided for developing corresponding gene treatment measures, and the application has great significance in preparation of cleft lip and palate early screening products and treatment drugs.

Description

Technical Field

[0001] The present invention relates to the technical field of preparing disease screening products and therapeutic drugs, and in particular to early screening and therapeutic drugs for cleft lip and palate. Background Art

[0002] Cleft lip and palate is one of the most common congenital malformations. Because it occurs on the face, it has a great impact on the patient's appearance and psychology, and can also cause problems such as feeding difficulties and susceptibility to infection. At present, most of the research on cleft lip and palate at home and abroad focuses on improving surgical methods and surgical time. Most patients can repair the defect through surgery, but the slight maxillofacial deformity, pronunciation problems and psychological problems that remain after surgery cannot be effectively solved.

[0003] Intervention during embryonic development may be a fundamental solution. In fact, the concept of early non-invasive screening has been fully promoted in the diagnosis and treatment of cancer. For example, AFP, CEA and CA199 are widely used in clinical practice as markers for early diagnosis of hepatocellular carcinoma, colorectal cancer and pancreatic cancer, bringing huge benefits to patients. In the non-tumor field, free nucleic acid examination of maternal peripheral blood in early pregnancy has also been initially applied in DiGeorge syndrome. However, there are currently few studies on diagnostic markers for cleft lip and palate, and early non-invasive screening for cleft lip and palate has not received sufficient attention. There are very few studies on screening and intervention for cleft lip and palate during embryonic development. Although studies have shown that exosome-derived piRNA has the potential to be used as a molecular marker for the early diagnosis of cleft lip and palate, its low specificity, complex extraction and unclear mechanism have hindered its use in the diagnosis of cleft lip and palate.

[0004] RESP18 (regulated endocrine specific protein 18) is a protein with a molecular weight of 18kDa. It is expressed in various tissue cells of the human body and can be detected in the blood circulation. The role of RESP18 in blood pressure regulation, gastrointestinal secretion, and protection of dopaminergic neurons has been reported. RESP18 also plays an important role in the reproductive system. RESP18 can be specifically expressed in mature spermatids and spermatocytes as a marker of the completion of meiosis. The homozygous deletion of RESP18 will affect the survival of fetal mice. Most importantly, the expression of RESP18 in mice coincides with the development of lip and palate tissues. At present, there are few studies on RESP18, and the mechanism of action of RESP18 in cleft lip and palate has not yet been elucidated. Summary of the invention

[0005] The purpose of the present invention is to provide the use of RESP18 gene in the preparation of early screening products and therapeutic drugs for cleft lip and palate, so as to solve the current research gaps in cleft lip and palate markers and the problems that hinder the development of early screening and therapeutic drugs.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] Application of RESP18 gene in the preparation of early screening products for cleft lip and palate.

[0008] The present invention also provides the use of a reagent for detecting the expression level of the RESP18 gene in the preparation of an early screening product for cleft lip and palate.

[0009] The present invention also provides application of RESP18 gene in preparing medicine for treating cleft lip and palate.

[0010] The present invention also provides the use of RESP18 in preparing a medicine for treating cleft lip and palate.

[0011] The present invention also provides an application of a vector for overexpressing the RESP18 gene in preparing a drug for treating cleft lip and palate.

[0012] The present invention also provides a method for constructing the vector for overexpressing the RESP18 gene, and the construction process is: inserting RESP18 cDNA into the NheI / AscI site of the lentiviral expression vector to obtain a RESP18 overexpression lentiviral vector; incubating the RESP18 overexpression lentiviral vector, the lentiviral packaging auxiliary vector and the host cells to obtain the RESP18 overexpression lentivirus.

[0013] Furthermore, the construction process is as follows: synthesizing RESP18 cDNA, digesting the lentiviral expression vector at the NheI / AscI site, inserting the RESP18 cDNA into the linearized lentiviral expression vector after digestion, and obtaining the RESP18 overexpression lentiviral vector; mixing and incubating the RESP18 overexpression lentiviral vector, the lentiviral packaging auxiliary vector pSPAX2, and the lentiviral packaging auxiliary vector pMD2.G with Opti-MEM to obtain the three diluted vectors; mixing and incubating the Lipofectamine 2000 reagent with Opti-MEM to obtain the diluted Lipofectamine 2000; quickly mixing the three diluted vectors with the diluted Lipofectamine 2000 to form a transfection complex of the three vectors and the Lipofectamine 2000 dilution, incubating the transfection complex with the host cells, and then culturing the cells transfected with the RESP18 overexpression lentiviral vector, purifying and concentrating the cell supernatant, and obtaining the RESP18 overexpression lentiviral concentrate.

[0014] Furthermore, the A260 / A280 of the plasmid DNA extracted from the RESP18 overexpression lentiviral vector, the lentiviral packaging auxiliary vector pSPAX2, and the lentiviral packaging auxiliary vector pMD2.G is between 1.8 and 2.0.

[0015] Furthermore, the host cell is a human embryonic kidney cell 293T cell.

[0016] Furthermore, 24 h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were digested and the cell density was adjusted to 1.2×10 7 cells / 20mL, re-inoculated in a 15cm cell culture dish, cultured at 37°C and 5% CO2, and used for transfection after 24h when the cell density reaches 70% to 80%; 2h before transfection, replace the cell culture medium with serum-free medium, mix 10μg of pLVX-RESP18 overexpression lentiviral vector, 10μg of lentiviral packaging auxiliary vector pSPAX2, and 10μg of lentiviral packaging auxiliary vector pMD2.G with the corresponding volume of Opti-MEM, adjust the total volume to 2.5mL, incubate at room temperature for 5 minutes, mix 100μL of Lipofectamine 2000 reagent with 2.4mL Opti-MEM, and incubate at room temperature for 5 minutes; gently invert and mix the diluted three vectors and the diluted Lipofectamine 2000, do not shake, and mix within 5 minutes; after mixing, incubate at room temperature for 2 minutes to form a transfection complex of the three vectors and the Lipofectamine 2000 dilution; Transfer 2000 of the mixture to the culture medium of 293T cells and mix well. After culturing at 37°C, 5% CO2 for 8 hours, pour off the culture medium containing the transfection complex and wash the residue; add 25 mL of cell culture medium containing 10% serum to each bottle of cells, and continue to culture in a 37°C, 5% CO2 incubator for 48 hours; collect the supernatant of 293T cells, centrifuge at 4°C, 4000g for 10 min to remove cell debris; filter the supernatant with a 0.45μm filter into an ultracentrifuge tube, centrifuge at 4000×g to the required virus concentration volume, collect the virus concentrate of RESP18 overexpression lentivirus, and store it at -80°C for a long time.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] For the first time, a RESP18 overexpression lentivirus was constructed, which can efficiently and conveniently infect human palate embryo mesenchymal cells and significantly upregulate the expression of the RESP18 gene in the latter, and can improve the proliferation, migration and osteogenic differentiation movement ability of human palate embryo mesenchymal cells, thereby providing a very effective protective gene such as the RESP18 gene for the early screening and treatment of cleft lip and palate, and providing an effective direction for the development of corresponding gene therapy measures, which is of great significance for the preparation of early screening products and therapeutic drugs for cleft lip and palate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings described herein are used to provide a further understanding of the present invention, constitute a part of the present application, and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 This is the result of detecting stable double-site mutations in the RESP18 gene in blood samples of 5 patients with familial cleft lip and palate;

[0021] Figure 2 It is the statistical graph of RESP18 expression level of each embryonic palate tissue sample of cleft lip and palate sequencing data GSE238062 in Table 1;

[0022] Figure 3 It is a statistical graph showing that the expression level of RESP18 in each embryonic palate tissue sample of the cleft lip and palate sequencing data GSE93301 decreases over time in Table 1;

[0023] Figure 4 This is a graph showing the experimental results of Western blot experiment to determine the expression of RESP18 in the induced group after HEPM cells were induced by atRA in the present invention;

[0024] Figure 5 is the DNA map of PDS407_pL-CMV-RFP-ccdB-puro lentiviral expression vector;

[0025] Figure 6 is the DNA map of the lentiviral packaging helper vector psPAX 2;

[0026] Figure 7 It is the DNA map of the lentiviral packaging helper vector pMD2.G;

[0027] Figure 8 These are experimental pictures of cells in the OE-RESP18 and OE-CON groups after infection;

[0028] Fig. 9 is the result graph of RESP18 expression level in cells of OE-RESP18 group and OE-CON group;

[0029] Fig.10This is an experimental diagram showing the effect of RESP18 overexpression on the proliferation capacity of human palatal embryo mesenchymal cells;

[0030] Fig.11 This is an experimental diagram showing the effect of RESP18 overexpression on the migration and motility of human palate embryo mesenchymal cells;

[0031] Fig.12 This is an experimental diagram showing the effect of RESP18 overexpression on the osteogenic differentiation ability of human palate embryo mesenchymal cells. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The exemplary embodiments and descriptions of the present invention are used to explain the present invention, but are not intended to limit the present invention.

[0033] 1. Experimental study on the specific expression of RESP18 in cleft lip and palate tissues

[0034] Blood samples from 5 patients with familial cleft lip and palate were obtained, and then the total DNA contained in the blood samples was extracted for Sanger sequencing analysis. The Sanger sequencing products were checked using an ABI 3730 genetic analyzer, and data were analyzed using Chromas V1.0.0.1. Figure 1 As shown, there are stable double-site mutations in the RESP18 gene in the pedigree of cleft lip and palate patients.

[0035] Two mouse cleft lip and palate sequencing data (GSE238062, GSE93301) were downloaded from the Gene Expression Omnibus public database. The data were imported into R studio software, and the columns of gene names and sample expression values ​​were extracted. The average expression value of each row was calculated. The row with the highest expression average value was retained for different probes of the same gene. The expression values ​​were log2(N+1) logarithmically transformed, and the "normalizeBetweenArrays package" was used for batch correction. The RESP18 expression values ​​in each sample were extracted, as shown in Table 1. In the table, atRA1_count, atRA2_count, atRA3_count, and atRA4_count are embryonic palate tissue samples constructed by treating embryonic stem cells with high concentrations of all-trans retinoic acid (atRA). GraphPad Prism software was used for data analysis and visualization. The results are shown in Table 1. Figure 2As shown in the figure, compared with the average expression level of RESP18 in embryonic palate tissues of CON1_count, CON2_count, CON3_count, and CON4_count, the average expression level of RESP18 in embryonic palate tissues of atRA1_count, atRA2_count, atRA3_count, and atRA4_count was lower, but the difference was not statistically significant, which may be related to the small sample size. After atRA treatment of embryonic stem cells for 36 hours, as shown in Figure 3 As shown, the expression level of RESP18 decreased with time.

[0036] Table 1 RESP18 expression values ​​in various samples

[0037]

[0038]

[0039] Retinoic acid is an active derivative of vitamin A. High concentrations of all-trans retinoic acid (atRA) can effectively induce cleft lip and palate deformities. It is easy to operate and has a high success rate. It is often used to construct cleft lip and palate models. Human palate embryo mesenchymal cells (HEPM cells) are a biochemical cell line isolated from mesenchymal cells of human embryos and are commonly used in in vitro studies of cleft lip and palate. The present invention cultured HEPM cells in an incubator at 37°C and 5% CO2, added an appropriate amount of atRA during the exponential growth phase of the cells, and maintained the atRA concentration of the solution at 6.6 μg / mL. The culture was terminated after 2 days, the total protein was extracted, and the expression of RESP18 was determined using a Western blot experiment. Figure 4 As shown, the expression of RESP18 in the atRA-induced group was lower than that in the blank control group.

[0040] 2. Construction of RESP18 overexpression lentivirus and negative control lentivirus

[0041] The RESP18 gene sequence was obtained from the National Center for Biotechnology Information (NCBI) website, as shown in Table 2. According to the RESP18 gene sequence, RESP18 cDNA was synthesized in vitro. The DNA map of the PDS407_pL-CMV-RFP-ccdB-puro lentiviral expression vector is shown in Figure 5, the PDS407_pL-CMV-RFP-ccdB-puro lentiviral expression vector was digested at the NheI / AscI site, and the RESP18 cDNA was inserted into the linearized PDS407_pL-CMV-RFP-ccdB-puro lentiviral expression vector. PCR identification experiments were performed on the target gene sequence in the PDS407_pL-CMV-RFP-ccdB-puro lentiviral expression vector, and the results are shown in Table 3. The clones identified positive by PCR were sequenced and aligned, and the clones with correct alignment were the successfully constructed lentiviral expression vectors containing the RESP18 gene sequence, named pLVX-RESP18 overexpression lentiviral vector.

[0042] Prepare the pLVX-RESP18 overexpression lentiviral vector, lentiviral packaging helper vector psPAX 2, and lentiviral packaging helper vector pMD2.G. The DNA map of the lentiviral packaging helper vector psPAX 2 is shown in Figure 6 The DNA map of the lentiviral packaging auxiliary vector pMD2.G is shown in Figure 7 The above vector plasmid DNA was extracted and dissolved in sterilized TE, and its concentration and purity were determined by ultraviolet absorption method to ensure that the A260 / A280 of the extracted plasmid DNA was between 1.8 and 2.0. If it reached this standard, it was qualified and could be used for subsequent transfection of cells.

[0043] 24 h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were digested with trypsin and the cell density was adjusted to 1.2×10 7Cells / 20mL, re-inoculated in a 15cm cell culture dish, cultured in a 37℃, 5% CO2 incubator, and used for transfection after 24h when the cell density reaches 70%-80%. Replace the cell culture medium with serum-free medium 2h before transfection. Add 10μg of the prepared pLVX-RESP18 overexpression lentiviral vector, 10μg of the lentiviral packaging auxiliary vector pSPAX2, and 10μg of the lentiviral packaging auxiliary vector pMD2.G to a sterile centrifuge tube, mix evenly with the corresponding volume of Opti-MEM, adjust the total volume to 2.5mL, and incubate at room temperature for 5 minutes. Gently shake the Lipofectamine 2000 reagent, take 100μL and mix with 2.4mL Opti-MEM in another tube, and incubate at room temperature for 5 minutes. Mix the diluted three vectors mentioned above with the diluted Lipofectamine 2000, gently invert and mix, do not shake. It must be mixed within 5 minutes. After mixing, incubate at room temperature for 2 minutes to form a transfection complex of the three vectors and Lipofectamine 2000 dilution. Transfer the three vectors and Lipofectamine 2000 mixture to the culture medium of 293T cells, mix well, and culture in a cell culture incubator at 37°C and 5% CO2. After continuing to culture for 8 hours, pour out the culture medium containing the transfection complex, wash the remaining transfection complex with PBS solution, and then pour it out. Add 25mL of cell culture medium containing 10% serum to each bottle of cells and continue to culture in a 37°C and 5% CO2 incubator for 48 hours. The supernatant of 293T cells was collected 48 hours after transfection, and centrifuged at 4000g for 10 min at 4°C to remove cell debris; the supernatant was filtered through a 0.45μm filter in a 40mL ultracentrifuge tube, and centrifuged at 4000×g to the required virus concentration volume, and the pLVX-RESP18 overexpression lentivirus in the filter cup was transferred to the sample collection cup, and the virus concentrate of RESP18 overexpression lentivirus was collected, and the virus biological titer was determined and stored at -80°C for a long time. The lentivirus constructed by the same method with PDS407_pL-CMV-RFP-ccdB-puro empty vector plasmid instead of pLVX-RESP18 plasmid was the negative control virus.

[0044] Table 2 RESP18 gene sequence

[0045]

[0046]

[0047] Table 3 PCR identification results of target gene sequence in PDS407_pL-CMV-RFP-ccdB-puro lentiviral expression vector

[0048]

[0049] 3. Lentivirus infection of human palatal embryo mesenchymal cells (HEPM cells)

[0050] Human palate mesenchymal cells (HEPM cells) in the logarithmic growth phase were digested with trypsin to produce cells with a density of approximately 5×10 4 cells / mL of cell suspension were inoculated into 6-well plates and cultured until the cell confluence reached about 50%. The multiplicity of infection (MOI) of the RESP18 overexpression lentivirus was 80, and the MOI of the negative control lentivirus was 80. According to the MOI value, appropriate amounts of lentivirus were added, and the medium was changed after 12 hours of culture. After 48 hours of infection, the expression of the reporter gene RFP (red fluorescent protein) on the lentivirus was observed, and the fluorescence rate was found to be greater than 80%, indicating that the infection was satisfactory. HEPM cells infected with RESP18 overexpression lentivirus and negative control lentivirus were called OE-RESP18 (Over-expression) and OE-CON (Over-expression Control, negative control), respectively. The conditions after infection of OE-RESP18 and OE-CON are shown in Figure 8 .

[0051] 4. Western blot detection of RESP18 overexpression

[0052] Proteins of OE-RESP18 and OE-CON cells with good growth status were extracted respectively; the extracted proteins were quantified according to the instructions of the BCA kit; and the differences in protein expression of each group were recorded using Image J analysis. Fig. 9 As shown, it can be seen that the relative expression level of RESP18 in the OE-RESP18 group was significantly higher than that in the OE-CON group, proving that RESP18 was overexpressed in the OE-RESP18 group.

[0053] 5. Effect of RESP18 overexpression on the proliferation of human palatal embryo mesenchymal cells

[0054] Prepare a 96-well plate, digest the HEPM cells OE-RESP18 and OE-CON in the logarithmic growth phase, centrifuge and resuspend them, take 10 μL of the cell suspension and mix it with 10 μL of trypan blue dye, take 10 μL of the mixture to count and record the cell concentration. Depending on the cell growth status, 2-5×10 3Cells, keep the cell amount of each group consistent, and add culture medium to 100μL. Set up 12 duplicate wells for each group of cells and culture them in a 37℃, 5% CO2 incubator. Select three duplicate wells at 0h, 34h, 68h, and 102h to add 10μL CCK8 solution. After incubating in the incubator for 1-4h, use a Bio-Tek microplate reader to detect the absorbance at OD450, and calculate the proliferation effect according to the formula. Fig.10 As shown, the proliferation rate of cells in the OE-RESP18 group was significantly higher than that in the OE-CON group, indicating that RESP18 overexpression significantly enhanced the proliferation ability of HEPM cells.

[0055] 6. Effect of RESP18 overexpression on the migration and motility of human palatal embryo mesenchymal cells

[0056] Take HEPM cells OE-RESP18 and OE-CON in the logarithmic growth phase, digest and centrifuge, resuspend the cells in serum-free culture medium, take 10 μL for trypan blue staining and count. Wet the upper and lower layers of the Transwell chamber with serum-free culture medium for 5 minutes in advance, add the cells to the upper layer of the chamber, and adjust the number of cells in each chamber to 3-8×10 5 The upper layer of the chamber contains 200 μL of serum-free culture medium, and the lower layer contains 600 μL of complete culture medium. Culture in a 37°C, 5% CO2 incubator for 24-72 hours. Terminate the culture before the end of a cell growth cycle and remove the culture medium. Wash the upper and lower layers of the chamber with PBS, fix with a fixative (4% paraformaldehyde or a mixture of methanol and glacial acetic acid in a ratio of 1:3), and wash with PBS. Add crystal violet staining solution to the upper and lower layers of the chamber to immerse the cells for staining. Wipe off the cells on the upper layer of the chamber that have not passed through the chamber holes, and remove excess staining solution. After the chamber is air-dried or immersed in water, use a microscope to take pictures of each group and perform statistical analysis later. The results are as follows: Fig.11 As shown, the migration speed of cells in the RESP18 overexpression group was accelerated.

[0057] 7. Effect of RESP18 overexpression on osteogenic differentiation of human palatal embryo mesenchymal cells

[0058] Take HEPM cells in the logarithmic growth phase, digest and centrifuge them, resuspend the cells in serum-free culture medium, take 10 μL, stain with trypan blue, and count them. Add 3×10 cells per well according to the cell growth status. 3 Cells were treated with RESP18 overexpression lentivirus / control lentivirus. The medium was changed after 48 hours, and then every 2-3 days. The culture was terminated after 2-3 weeks. The cells were gently washed with PBS and fixed with 4% paraformaldehyde. Alizarin red stain was used in dark and room temperature conditions. Calcium deposition was observed under a microscope and photographed. The results are shown in Fig.12 As shown, the osteogenic differentiation ability of cells in the OE-RESP18 group was enhanced.

[0059] The role of early screening and treatment in the field of cleft lip and palate shows strong application prospects. Therefore, analyzing the relationship between the expression level of RESP18 in blood samples during pregnancy and cleft lip and palate, constructing RESP18 overexpression lentiviral vectors and corresponding lentiviruses, and observing the effects produced after infecting palatal mesenchymal stem cells are very important for studying the role and mechanism of RESP18 in the occurrence and development of cleft lip and palate. The present invention can provide a basis for the development of means and methods for efficiently and stably increasing the expression level of RESP18 in palatal mesenchymal stem cells.

[0060] The technical solutions provided by the embodiments of the present invention are introduced in detail above. Specific examples are used herein to illustrate the principles and implementation methods of the embodiments of the present invention. The description of the above embodiments is only applicable to help understand the principles of the embodiments of the present invention. At the same time, for those skilled in the art, according to the embodiments of the present invention, there will be changes in the specific implementation methods and application scopes. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. Application of RESP18 gene in the preparation of early screening products for cleft lip and palate.

2. Application of reagents for detecting RESP18 gene expression levels in the preparation of early screening products for cleft lip and palate.

3. Application of RESP18 gene in the preparation of drugs for the treatment of cleft lip and palate.

4. Application of RESP18 in the preparation of drugs for the treatment of cleft lip and palate.

5. Use of a vector that overexpresses the RESP18 gene in the preparation of a drug for treating cleft lip and palate.

6. A method for constructing a vector for overexpressing RESP18 gene as claimed in claim 5, characterized in that: The construction process is as follows: insert RESP18 cDNA into the NheI / AscI site of the lentiviral expression vector to obtain a RESP18 overexpression lentiviral vector; incubate the RESP18 overexpression lentiviral vector, the lentiviral packaging auxiliary vector and the host cells to obtain a RESP18 overexpression lentivirus.

7. A method for constructing a vector for overexpressing RESP18 gene according to claim 6, characterized in that: The construction process is as follows: synthesizing RESP18 cDNA, digesting the NheI / AscI site of the lentiviral expression vector, inserting the RESP18 cDNA into the linearized lentiviral expression vector after digestion, and obtaining the RESP18 overexpression lentiviral vector; mixing and incubating the RESP18 overexpression lentiviral vector, the lentiviral packaging auxiliary vector pSPAX2, and the lentiviral packaging auxiliary vector pMD2.G with Opti-MEM to obtain three diluted vectors; mixing and incubating the Lipofectamine 2000 reagent with Opti-MEM to obtain diluted Lipofectamine 2000; quickly mixing the three diluted vectors with the diluted Lipofectamine 2000 to form a transfection complex of the three vectors and the Lipofectamine 2000 dilution, incubating the transfection complex with host cells, and then culturing the cells transfected with the RESP18 overexpression lentiviral vector, purifying and concentrating the cell supernatant, and obtaining the RESP18 overexpression lentiviral concentrated solution.

8. The method for constructing a vector for overexpressing the RESP18 gene according to claim 6, characterized in that: The A260 / A280 of the plasmid DNA extracted from the RESP18 overexpression lentiviral vector, the lentiviral packaging auxiliary vector pSPAX2, and the lentiviral packaging auxiliary vector pMD2.G is between 1.8 and 2.

0.

9. The method for constructing a vector for overexpressing the RESP18 gene according to claim 6, characterized in that: The host cell is human embryonic kidney cell 293T cell.

10. The method for constructing a vector for overexpressing the RESP18 gene according to claim 6, characterized in that: 24 h before transfection, human embryonic kidney 293T cells in logarithmic growth phase were digested and the cell density was adjusted to 1.2×10 7 cells / 20mL, re-inoculated in a 15cm cell culture dish, cultured at 37°C and 5% CO2, and used for transfection after 24h when the cell density reaches 70% to 80%; 2h before transfection, replace the cell culture medium with serum-free medium, mix 10μg of pLVX-RESP18 overexpression lentiviral vector, 10μg of lentiviral packaging auxiliary vector pSPAX2, and 10μg of lentiviral packaging auxiliary vector pMD2.G with the corresponding volume of Opti-MEM, adjust the total volume to 2.5mL, incubate at room temperature for 5 minutes, mix 100μL of Lipofectamine 2000 reagent with 2.4mL Opti-MEM, and incubate at room temperature for 5 minutes; gently invert and mix the diluted three vectors and the diluted Lipofectamine 2000, do not shake, and mix within 5 minutes; after mixing, incubate at room temperature for 2 minutes to form a transfection complex of the three vectors and the Lipofectamine 2000 dilution; Transfer 2000 ml of the mixture to the culture medium of 293T cells and mix well. After culturing at 37°C and 5% CO2 for 8 hours, pour out the culture medium containing the transfection complex and wash the residue. Add 25 mL of cell culture medium containing 10% serum to each bottle of cells and continue culturing in a 37°C and 5% CO2 incubator for 48 hours. Collect the supernatant of 293T cells and centrifuge at 4°C and 4000g for 10 minutes to remove cell debris. The supernatant was filtered through a 0.45 μm filter into an ultracentrifuge tube and centrifuged at 4000×g to the required virus concentration volume. The virus concentrate of RESP18 overexpression lentivirus was collected and stored at -80°C for a long term.

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