Construction and application of lentivirus recombinant tagged vector for stable overexpression of sox18 in mammary gland cells

Abstract

The application discloses a construction method and application of a SOX18 lentivirus recombinant tag carrier stably overexpressed in sheep hair papilla cells, and the method comprises the following steps: amplifying a full-length CDS product of a sheep SOX18 gene, double-enzyme cutting a carrier, connecting the full-length CDS amplification product of the SOX18 gene with a linearized carrier, and expanding the recombinant carrier, mixing the SOX18 lentivirus recombinant tag carrier with a lentivirus packaging auxiliary plasmid and an auxiliary plasmid, and transfecting 293T cells to realize packaging of the SOX18 lentivirus recombinant tag carrier, and transfecting the SOX18 lentivirus recombinant tag carrier into sheep hair papilla cells, and it is found that overexpression of SOX18 can inhibit apoptosis of the sheep hair papilla cells. The application significantly improves the transfection efficiency of the hair papilla cells, greatly increases the probability of integration of the SOX18 gene into the genome of the hair papilla cells, constructs a high-level in-vitro expression model of SOX18, and proposes an application of the SOX18 gene in inhibiting apoptosis of the hair papilla cells.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to the construction and application of a recombinant tag vector of SOX18 lentivirus stably overexpressed in wool papillary cells. Background Technology

[0002] Lambskin with distinctive patterns is highly sought after by consumers. However, due to the extensive efforts by breeders in recent years to select and hybridize lambs for meat-related traits, the pattern characteristic of lambskin has been severely threatened. Therefore, there is an urgent need to study the protection of this trait and the molecular mechanisms underlying its formation. The formation of lambskin patterns is closely related to hair follicle development, and dermal papilla cells are the core cells in this process. Therefore, studying the growth of dermal papilla cells is beneficial for elucidating the developmental mechanisms of wool follicles, thereby clarifying the molecular mechanisms of lambskin pattern formation and providing a theoretical basis for the protection and utilization of lambskin germplasm resources.

[0003] The decline in the quality of lambskin pattern affects the protection and utilization of lambskin germplasm resources. Therefore, conducting research on the function of key genes in wool nipple cells is beneficial to understanding the formation mechanism of lambskin pattern traits.

[0004] Transcription factor SOX18 is a member of the SOX family, which consists of a class of genes related to the SRY (sex determination region of ychomosome) and possessing highly conserved HMG-box DNA-binding domains. These genes encode many SRY-related hMG-box transcription factors, thereby participating in the regulation of vital processes such as embryonic development and cell fate determination. SOX18 mutations can lead to human hypotrichosis-lymphedema-telangiectasia (HLT) syndrome, which is also the root cause of severe cardiovascular and hair follicle defects in ragged (Ra) mice. In RA mice, heterozygotes exhibit uneven hair growth, while homozygotes show complete hairlessness and low survival rates. Currently, there are no reports on the role of SOX18 in wool follicles and dermal papillae. Furthermore, wool papilla cells are difficult to transfect, resulting in low transfection efficiency, which hinders effective research on the function of key genes in wool papilla cells. Summary of the Invention

[0005] This invention addresses the technical problem at hand and overcomes the shortcomings of existing technologies by providing a method for constructing and applying a recombinant tag vector for SOX18 lentiviral stably overexpressed in wool papilla cells.

[0006] One of the objectives of this invention is to provide an application of the sheep SOX18 gene in the apoptosis of wool papillary cells, wherein the expression of the sheep SOX18 gene inhibits the apoptosis of wool papillary cells, and the sequence of the sheep SOX18 gene is shown in SEQ ID NO.1.

[0007] Preferably, the sheep SOX18 gene regulates apoptosis in wool papilla cells by a SOX18 lentiviral recombinant tag vector stably overexpressed in wool papilla cells. The SOX18 lentiviral recombinant tag vector contains the sheep SOX18 gene and is a pLV4ltr-PGK-ZsGreen(2A)Puro-CMV vector.

[0008] The second objective of this invention is to provide a method for constructing a SOX18 lentiviral recombinant tag vector that is stably overexpressed in wool papilla cells, comprising the following steps:

[0009] Step 1: Obtain the full-length CDS amplification product of the sheep SOX18 gene;

[0010] Step 2: Double digest the lentiviral vector pLV4ltr-PGK-ZsGreen(2A)Puro-CMV to obtain a linear lentiviral vector fragment;

[0011] Step 3: Ligate the SOX18 gene amplification fragment obtained in Step 1 and the linear lentiviral vector fragment obtained in Step 2.

[0012] Step 4: Transform the ligation product obtained in Step 3 into TOP10 competent cells, and perform sequencing and comparative analysis on positive clones. The plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18 is the successfully constructed SOX18 gene overexpression vector plasmid.

[0013] Step 5: Extract the constructed SOX18 gene overexpression vector plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18, and co-transfect the extracted SOX18 gene overexpression vector plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18 with lentiviral packaging helper plasmids psPAX2 (pHelper1) and pMD2G (pHelper2) into cells for lentiviral packaging, collection, and titer detection to obtain the SOX18 lentiviral recombinant tag vector.

[0014] This invention amplifies the full-length CDS product of the sheep SOX18 gene, double-digests the pLV4ltr-PGK-ZsGreen(2A)Puro-CMV vector, ligates the full-length CDS amplified product of the SOX18 gene with the linearized pLV4ltr-PGK-ZsGreen(2A)Puro-CMV vector, and proliferates the recombinant vector. The constructed SOX18 lentiviral recombinant tag vector is then mixed with lentiviral packaging helper plasmids psPAX2 (pHelper 1) and pMD2G (pHelper 2) and transfected into 293T cells to achieve packaging of the SOX18 lentiviral recombinant tag vector. Subsequently, transfecting the SOX18 lentiviral recombinant tag vector into wool papilla cells reveals that overexpression of SOX18 can inhibit apoptosis in wool papilla cells.

[0015] The further optimized technical solution of this invention is as follows:

[0016] In step 1, the full-length CDS amplification product of the sheep SOX18 gene is obtained. The specific steps are as follows:

[0017] Step 1.1: Design 22 primers G52005-1_1~1_22 based on the SOX18 CDS sequence. Their sequences are shown in SEQ ID NO.2~23. The 22 primers G52005-1_1~1_22 form a primer combination.

[0018] Step 1.2: Perform the first round of PCR amplification. The reaction system is as follows: primer combination 0.5 μl, polymerase pv2 0.5 μl, 5×PV2 buffer 10 μl, 10 mM dNTP 1 μl, ddH2O 38 μl;

[0019] The PCR reaction conditions were as follows: 95℃ for 3 min, 1 cycle; 95℃ for 25 sec, 60℃ for 20 sec, 72℃ for 40 sec, 25 cycles; 72℃ for 1 min, 1 cycle; 10℃ forever.

[0020] Step 1.3: Perform the second round of PCR amplification. The reaction system is as follows: 0.3 μl of the first round PCR product, 0.5 μl of primer G52005-1_1, 0.5 μl of primer G52005-1_22, 0.5 μl of polymerase pv2, 10 μl of 5×PV2 buffer, 1 μl of 10 mM dNTP, and 37.2 μl of ddH2O.

[0021] The PCR reaction conditions were as follows: 95℃ for 3 min, 1 cycle; 95℃ for 25 sec, 60℃ for 20 sec, 72℃ for 40 sec, 25 cycles; 72℃ for 1 min, 1 cycle; 10℃ forever.

[0022] In step 2, the lentiviral vector pLV4ltr-PGK-ZsGreen(2A)Puro-CMV is double-digested with enzymes to obtain a linear lentiviral vector fragment. The specific steps are as follows: the pLV4ltr-PGK-ZsGreen(2A)Puro-CMV vector is digested with XhoI and BamHI at 37℃ for 2 hours. The digestion system is: 10×Buffer 5μl, pLV4ltr-PGK-ZsGreen(2A)Puro-CMV 1ug, XhoI and BamHI 1μl each, and ultrapure water to make up to 50μl.

[0023] In step 3, the SOX18 gene amplification fragment and the linear lentiviral vector fragment are ligated. The specific steps are as follows: the full-length CDS product of the SOX18 gene and the linear lentiviral vector fragment are recovered using a DNA gel recovery kit; the full-length CDS product of the SOX18 gene obtained by double digestion and the linear lentiviral vector fragment are ligated using recombinase (Smart assemble) and in a 52°C water bath for 40 min.

[0024] The ligation system consisted of: 10 μl of recombinase (Smart assemble), 6 μl of the full-length CDS product of the SOX18 gene, and 4 μl of a linear lentiviral vector fragment.

[0025] In step 4, the ligation product is transformed into TOP10 competent cells. Positive clones are sequenced and analyzed. The plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18, which is the successfully constructed SOX18 gene overexpression vector, is identified. The specific steps are as follows: 10 μl of the ligation product is transformed into TOP10 competent cells and incubated on ice for 10 min; after the ice incubation, the cells are heat-shocked at 42°C for 90 sec and then incubated on ice for 3 min; 800 μl of antibiotic-free LB medium is added and the cells are incubated at 250 rpm for 1 h; the cultured competent cells are centrifuged to remove part of the supernatant, and the remaining supernatant is mixed and spread onto a culture dish and incubated upside down overnight; positive clones are picked for propagation, and the propagated bacterial solution is sequenced and analyzed. The plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18, which is the successfully constructed SOX18 gene overexpression vector, is identified.

[0026] In step 5, the extracted SOX18 gene overexpression vector plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18, along with lentiviral packaging helper plasmids psPAX2 (pHelper1) and pMD2G (pHelper2), was co-transfected into 293T cells for lentiviral packaging, collection, and titer detection. The specific steps are as follows:

[0027] Step 5.1: Mix 10 μg of the SOX18 lentiviral recombinant tag vector stably overexpressed in wool papilla cells or the SOX18 lentiviral recombinant tag vector plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18 stably overexpressed in wool papilla cells constructed in step 4 with 5 μg of lentiviral packaging helper plasmid psPAX2 (pHelper 1) and 5 μg of helper plasmid pMD2G (pHelper 2). Then add the appropriate volume of Opti-MEM and mix well. Adjust the total volume to 0.5 ml and incubate at room temperature for 5 min.

[0028] Step 5.2: Mix 50 μl of transfection reagent with 450 μl of Opti-MEM and incubate at room temperature for 5 min;

[0029] Step 5.3: Mix the DNA diluted in step 5.1 with the transfection reagent diluted in step 5.2, gently invert to mix, do not shake, and incubate at room temperature for 10 minutes after mixing.

[0030] Step 5.4: Add the transfection mixture obtained in step 5.3 dropwise to 293T cell culture medium, shake well, and incubate at 37 ℃ in a 5% CO2 cell culture incubator;

[0031] Step 5.5: After culturing for 6-8 hours, discard the culture medium containing the transfection mixture, replace 10 ml of 10% FBS complete culture medium in each tray of cells, and continue culturing in a 37 ℃, 5% CO2 incubator.

[0032] Step 5.6: 24 hours after transfection, the number of cells containing the tagged fluorescent (ZsGreen) cells is observed under a microscope to determine the transfection efficiency. Once the transfection is confirmed to be successful (fluorescent cell ratio ≥70%), the first virus harvesting operation is performed. The virus-containing culture medium is collected in sterile centrifuge tubes and stored at 4°C. Then, 10 ml of fresh 10% FBS medium is replaced and cultured for another 24 hours.

[0033] Step 5.7: 48 hours after transfection, perform a second virus harvesting operation to harvest the virus-containing culture in sterile 50 ml centrifuge tubes and store at 4°C.

[0034] Step 5.8: Filter the harvested culture medium containing virus particles through a 0.22 μm filter membrane and collect it in a sterile ultracentrifuge tube. Balance and seal the tube. Centrifuge at 80,000 g for 4 hours. Discard the supernatant after centrifugation and resuspend the precipitate using a virus store buffer.

[0035] Step 5.9: Collect the resuspension, filter it again through a 0.22μm filter membrane for sterilization, and dispense it into sterile virus tubes to obtain lentivirus containing the SOX18 gene.

[0036] The third objective of this invention is to provide an application of the lentiviral recombinant tag vector pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18, which contains the SOX18 gene overexpression, in regulating wool papillary cells.

[0037] The above application includes the following steps:

[0038] (1) Transfect wool papillary cells with a recombinant tag vector containing an overexpression lentivirus of the SOX18 gene;

[0039] (2) The effect of SOX18 on apoptosis of wool papilla cells was detected by flow cytometry.

[0040] This invention optimizes the codons and designs PCR amplification primers based on the CDS sequence of the SOX18 gene published in Genebank, and performs PCR amplification of the full-length CDS product of the sheep SOX18 gene. The primer sequences are SEQ ID No. 2 to SEQ ID No. 23. Then, the pLV4ltr-PGK-ZsGreen(2A)Puro-CMV vector sequence is double-digested with enzymes. After purifying the digested product, the pLV4ltr-PGK-ZsGreen(2A)Puro-CMV double-digested product is ligated with the SOX18 gene CDS amplified fragment. The ligation product is transformed into TOP10 competent cells. Positive clones are sequenced and analyzed. The clones with completely correct sequence alignment are the successfully constructed SOX18 gene overexpression lentiviral vector pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18. Then, the constructed pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18 vector plasmid was extracted and co-transfected into 293T cells with the lentiviral packaging helper plasmids psPAX2 (pHelper1) and pMD2G (pHelper2) for lentiviral packaging, collection, and titer detection. Further, the constructed SOX18 gene overexpression lentiviral vector pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18 was transfected into wool papilla cells to confirm the successful construction of the SOX18 gene overexpression lentiviral vector and to detect the effect of SOX18 on wool papilla cell apoptosis.

[0041] This invention significantly improved the transfection efficiency of hair papilla cells by constructing a recombinant tag vector that overexpresses SOX18 using lentiviral vectors, greatly increased the probability of SOX18 gene integration into the hair papilla cell genome, constructed a high-level in vitro expression model of SOX18, and discovered the role of SOX18 in inhibiting apoptosis of hair papilla cells. Attached Figure Description

[0042] Figure 1 This is a gel electrophoresis image of the full-length CDS amplification product of the SOX18 gene in this invention. In the image, Lane 1 represents the PCR product; Lane 1 represents the DNA marker.

[0043] Figure 2 This is a gel electrophoresis image verifying the double enzyme digestion of the SOX18 gene overexpression vector in this invention. In the image, Lane 1 is Plasmid; Lane 2 is Plasmid digested with XhoI-BamHI; and Lane 3 is the DNA Marker.

[0044] Figure 3 This is a fluorescence image of wool papillary cells transfected with the SOX18 gene lentiviral recombinant tag vector in this invention.

[0045] Figure 4 This is a schematic diagram showing the results of Flag tag protein expression detection after transfection with the SOX18 lentiviral recombinant tag vector in this invention.

[0046] Figure 5 This is a schematic diagram illustrating the effect of SOX18 on apoptosis of wool papilla cells in this invention. Detailed Implementation

[0047] The technical solution of the present invention will be further described in detail below with reference to the embodiments: This embodiment is implemented under the premise of the technical solution of the present invention, and provides detailed implementation methods and specific operation processes, but the protection scope of the present invention is not limited to the following embodiments.

[0048] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0049] Example 1: Construction of SOX18 gene expression vector plasmid

[0050] (1) Obtain the sheep SOX18 gene CDS sequence (XM_027976914.2) published on Genebank, which is 1176 bp in length. Based on the SOX18 CDS sequence, codon optimization was performed and 22 primers G52005-1_1 to 1_22 were designed, with sequences SEQ ID NO.2 to SEQ ID NO.23.

[0051] Take 0.1 μl each of primers G52005-1_1 to G52005-1_22, mix them thoroughly to obtain the primer combination consisting of primers G52005-1_1 to G52005-1_22. Take 0.5 μl of this primer combination for the first round of PCR reaction.

[0052] (2) The first round of PCR reaction system was: primer combination 0.5 μl, polymerase pv2 (purchased from Nanjing Qingke) 0.5 μl, 5×PV2 buffer (purchased from Nanjing Qingke) 10 μl, 10 mM dNTP 1 μl, ddH2O 38 μl;

[0053] The PCR reaction conditions were as follows: 95℃ for 3 min, 1 cycle; 95℃ for 25 sec, 60℃ for 20 sec, 72℃ for 40 sec, 25 cycles; 72℃ for 1 min, 1 cycle; 10℃ forever.

[0054] (3) The second round of PCR reaction system is as follows: 0.3 μl of the first round PCR product, 0.5 μl of primer G52005-1_1, 0.5 μl of primer G52005-1_22, 0.5 μl of polymerase pv2, 10 μl of 5×PV2 buffer, 1 μl of 10 mM dNTP, and 7.2 μl of ddH2O;

[0055] The PCR reaction conditions were as follows: 95℃ for 3 min, 1 cycle; 95℃ for 25 sec, 60℃ for 20 sec, 72℃ for 40 sec, 25 cycles; 72℃ for 1 min, 1 cycle; 10℃ forever.

[0056] The PCR products containing the target gene band obtained from the second round of PCR were subjected to gel electrophoresis, and the gene fragment was excised and recovered to obtain the full-length CDS fragment of the SOX18 gene. The gel electrophoresis of the full-length CDS fragment of the SOX18 gene is shown in [link to gel electrophoresis diagram]. Figure 1 .

[0057] (4) The pLV4ltr-PGK-ZsGreen(2A)Puro-CMV vector (purchased from Nanjing Qingke) was digested with XhoI and BamHI at 37℃ for 2 h. The digestion system was: 10×Buffer 5 μl, pLV4ltr-PGK-ZsGreen(2A)Puro-CMV 1 μg, XhoI and BamHI 1 μl each, and ultrapure water to make up to 50 μl. The results of double digestion of the sheep SOX18 gene overexpression vector were obtained by gel electrophoresis. Figure 2 .

[0058] (5) The full-length CDS product of the SOX18 gene and the linear lentiviral vector fragment were recovered using a DNA gel recovery kit (purchased from Axygen, Corning, NY, USA); the full-length CDS product of the SOX18 gene and the linear lentiviral vector fragment obtained by double digestion were ligated with recombinase (Smart assemble) (purchased from Nanjing Qingke), and the ligation was carried out in a water bath at 52℃ for 40 min; the ligation system was: 10 μl of recombinase (Smart assemble), 6 μl of full-length CDS product of SOX18 gene, and 4 μl of linear lentiviral vector fragment.

[0059] (6) Transform the ligation product into TOP10 competent cells (purchased from Nanjing Qingke). Add 10 μl of the ligation product to the TOP10 competent cells, gently tap the tube wall to mix, and place on ice for 10 min; place the centrifuge tube in a 42℃ water bath for 90 sec without shaking the centrifuge tube; quickly transfer the centrifuge tube to an ice bath and place for 3 min; add 800 μl of antibiotic-free LB medium to each centrifuge tube, and then transfer the centrifuge tube to a 37℃ shaker, 250 rpm, and incubate for 1 h to allow the bacteria to recover; centrifuge the cultured competent cells to remove part of the supernatant, and take 200 μl of the cultured cells to spread evenly on an LB plate containing antibiotics, invert the plate in a 37℃ incubator, and incubate for 16 h.

[0060] (7) Pick cloned colonies, extract plasmids and identify them, and pick out positive clones. Pick 6 individual colonies from the LB plate that has been cultured overnight, add them to liquid LB medium containing ampicillin and puromycin (purchased from Beijing Solarbio), and incubate in a shaker at 37°C and 250 rpm for 12 h. Sequencing analysis is performed on a portion of the corresponding bacterial culture.

[0061] (8) The sequencing results were compared with the target sequence. If the sequence alignment results were consistent, it indicates that the SOX18 gene expression vector plasmid was successfully constructed. Based on the sequencing analysis results, the plasmid was extracted from the corresponding bacterial culture to obtain the recombinant plasmid.

[0062] Example 2 Virus Packaging, Collection, and Titer Detection

[0063] (1) 24 hours before transfection, 293T cells in logarithmic growth phase (purchased from ATCC) were digested with trypsin (purchased from Beijing Solarbio) and re-seeded in 10cm cell culture dishes. They were cultured in a 37 ℃, 5% CO2 incubator. When the cell density reached 70% to 80%, they were ready for transfection. The cell culture medium was changed 2 hours before transfection.

[0064] The transfection method is as follows: Add the prepared DNA solutions (10 μg recombinant vector plasmid, 5 μg psPAX2 (pHelper 1) vector (purchased from Nanjing Qingke), 5 μg pMD2G (pHelper 2) vector (purchased from Nanjing Qingke)) to a sterile centrifuge tube, mix thoroughly with the corresponding volume of Opti-MEM (purchased from Gibco, Grand Island, NY, USA) serum-depleted medium, adjust the total volume to 0.5 ml, and incubate at room temperature for 5 min. Mix 50 μl of transfection reagent FZ0001 (purchased from Nanjing Qingke) with 450 μl of Opti-MEM serum-depleted medium, and incubate at room temperature for 5 min. Mix the diluted DNA with the diluted transfection reagent, gently invert to mix, do not shake. After mixing, incubate at room temperature for 10 min. Add the above transfection mixture dropwise to 293T cell culture medium, shake well, and incubate at 37 ℃ in a 5% CO2 cell culture incubator.

[0065] (2) After culturing for 6-8 hours, discard the culture medium containing the transfection mixture, replace 10 ml of 10% FBS complete culture medium in each tray of cells, and continue culturing in a 37 ℃, 5% CO2 incubator.

[0066] (3) 24 hours after transfection, the transfection efficiency was determined by observing the number of tagged fluorescent (ZsGreen) cells under a microscope. After confirming successful transfection (i.e., the proportion of fluorescent cells ≥70%), the first virus harvesting operation was performed. The virus-containing culture was collected in sterile 50 ml centrifuge tubes and stored at 4°C. 10 ml of fresh 10% FBS medium was replaced, and the culture was continued for 24 hours. 48 hours after transfection (i.e., after continuing culture for 24 hours), the second virus harvesting operation was performed. The virus-containing culture was harvested in sterile 50 ml centrifuge tubes at 4°C. The two virus harvesting operations ensured the viral load. The virus-containing culture media collected in the two operations were mixed and the virus particles were harvested.

[0067] (4) The culture medium containing virus particles was filtered through a 0.22 μm filter membrane and collected in a sterile ultracentrifuge tube. The tube was balanced and sealed. It was then ultracentrifuged at 80,000 g for 4 h. The supernatant was discarded and the precipitate was resuspended with Virus Store Buffer (purchased from Nanjing Qingke). The resuspension was collected, filtered through a 0.22 μm filter membrane again for sterilization, and dispensed into sterile virus tubes to obtain lentivirus containing the SOX18 gene.

[0068] (5) Virus titer detection: 24 hours before the experiment, 293T cells were seeded in 96-well plates, approximately 1*102 4Cells / well, 50 μl / well, cultured at 37℃, 5% CO2; before the experiment, observe the cells under a microscope to ensure they are plump, evenly distributed, and free of contamination before proceeding with subsequent experiments; perform serial dilutions of the virus, prepare a new 96-well plate, and label them 10, 1, 10 from top to bottom. -1 10 -2 Add 90 μl of DMEM medium containing 2% FBS to each well; add 10 μl of the original virus solution to the first well (labeled 10), and mix by pipetting; add 10 μl of the diluent from the first well to the second well (labeled 1), mix by pipetting, and continue the same operation until the last well (labeled 10) is diluted. -2 ); Dilute the virus and add samples sequentially, starting from the labeled 10. -2 90 μl of virus was slowly added to a 96-well culture plate containing test cells and placed into a well labeled 1 (dilution gradient). After addition, the virus name, dilution gradient, and test date were labeled. The 96-well culture plate was incubated at 37°C and 5% CO2 for 72 hours, with additional culture medium added once as needed to detect color changes. After 72 hours, the number of cells containing the target virus marker (ZsGreen) was counted under a microscope to determine the virus titer. The virus titer was calculated based on the dilution factor, resulting in a titer of 1*10^8 for the SOX18 lentiviral vector.

[0069] Example 3: Effect of SOX18 gene-overexpressing lentiviral recombinant tag vector on apoptosis of wool papilla cells

[0070] Wool papilla cells were used as experimental material. Cells were seeded in 12-well cell culture plates. Once the cells reached approximately 50% confluence, transfection with the SOX18 gene lentiviral recombinant tag vector was performed. First, the original culture medium was aspirated and replaced with fresh medium. Then, 50 μl of 1*10^8 titer SOX18 gene lentiviral recombinant tag vector and empty vector were transfected, respectively, and the plates were incubated at 37℃ in a 5% CO2 incubator. Fluorescence was observed using a fluorescence inverted microscope 24 h after lentiviral vector transfection (see...). Figure 3 This indicates that the SOX18 gene lentiviral recombinant tag vector has been successfully transfected into wool papillary cells.

[0071] Wool papilla cells were collected, and total protein was extracted. The expression of Flag tag protein in wool papilla cells transfected with the SOX18 gene lentiviral recombinant tag vector was detected using Western blot. Results are as follows: Figure 4As shown, after transfection of wool papillary cells with the SOX18 gene lentiviral recombinant tag vector, the molecular weight of the Flag tag protein was slightly larger than that of the SOX18 protein, indicating that the model of stable high expression of the SOX18 gene lentiviral recombinant tag vector in wool papillary cells was successfully constructed.

[0072] The effect of SOX18 gene lentiviral recombinant tag vector transfection on apoptosis in wool papilla cells was detected by flow cytometry. Results are as follows: Figure 5 As shown, the apoptosis rate of wool papilla cells was significantly reduced after transfection with the SOX18 gene lentiviral recombinant tag vector, indicating that SOX18 can inhibit apoptosis of wool papilla cells.

[0073] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any transformations or substitutions that can be conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for constructing a recombinant tag vector of SOX18 lentiviral stably overexpressed in wool papilla cells, characterized in that, Includes the following steps: Step 1: Obtain the full-length CDS amplification product of the sheep SOX18 gene; the specific steps are as follows: Step 1.1: Design 22 primers G52005-1_1~1_22 based on the SOX18 CDS sequence. Their sequences are shown in SEQ ID NO.2~23. The 22 primers G52005-1_1~1_22 form a primer combination. Step 1.2: Perform the first round of PCR amplification. The reaction system is as follows: primer combination 0.5 μl, polymerase pv2 0.5 μl, 5×PV2 buffer 10 μl, 10 mM dNTP 1 μl, ddH2O 38 μl; The PCR reaction conditions were as follows: 95℃ for 3 min, 1 cycle; 95℃ for 25 sec, 60℃ for 20 sec, 72℃ for 40 sec, 25 cycles; 72℃ for 1 min, 1 cycle; 10℃ forever. Step 1.3: Perform the second round of PCR amplification. The reaction system is as follows: 0.3 μl of the first round PCR product, 0.5 μl of primer G52005-1_1, 0.5 μl of primer G52005-1_22, 0.5 μl of polymerase pv2, 10 μl of 5×PV2 buffer, 1 μl of 10 mM dNTP, and 37.2 μl of ddH2O. The PCR reaction conditions were as follows: 95℃ for 3 min, 1 cycle; 95℃ for 25 sec, 60℃ for 20 sec, 72℃ for 40 sec, 25 cycles; 72℃ for 1 min, 1 cycle; 10℃ forever. Step 2: Double digest the lentiviral vector pLV4ltr-PGK-ZsGreen(2A)Puro-CMV to obtain a linear lentiviral vector fragment; Step 3: Ligate the SOX18 gene amplification fragment obtained in Step 1 and the linear lentiviral vector fragment obtained in Step 2. Step 4: Transform the ligation product obtained in Step 3 into TOP10 competent cells to construct the SOX18 gene overexpression vector plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18; Step 5: Extract the constructed SOX18 gene overexpression vector plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18. Co-transfect the extracted SOX18 gene overexpression vector plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18 with lentiviral packaging helper plasmids psPAX2 (pHelper1) and pMD2G (pHelper2) into cells for lentiviral packaging and collection to obtain the SOX18 lentiviral recombinant tag vector. The SOX18 lentiviral recombinant tag vector contains the sheep SOX18 gene. The sheep SOX18 gene regulates apoptosis of wool papilla cells by stably overexpressing the SOX18 lentiviral recombinant tag vector in wool papilla cells. The expression of the sheep SOX18 gene inhibits apoptosis of wool papilla cells. The sheep SOX18 gene sequence is shown in SEQ ID NO.

1.

2. The construction method according to claim 1, characterized in that, In step 2, the lentiviral vector pLV4ltr-PGK-ZsGreen(2A)Puro-CMV is double-digested with enzymes to obtain a linear lentiviral vector fragment. The specific steps are as follows: the pLV4ltr-PGK-ZsGreen(2A)Puro-CMV vector is digested with XhoI and BamHI at 37℃ for 2 hours. The digestion system is: 10×Buffer 5μl, pLV4ltr-PGK-ZsGreen(2A)Puro-CMV 1ug, XhoI and BamHI 1μl each, and ultrapure water to make up to 50μl.

3. The construction method according to claim 1, characterized in that, In step 3, the SOX18 gene amplification fragment and the linear lentiviral vector fragment are ligated. The specific steps are as follows: the full-length CDS product of the SOX18 gene and the linear lentiviral vector fragment are recovered using a DNA gel recovery kit; the full-length CDS product of the SOX18 gene obtained by double digestion and the linear lentiviral vector fragment are ligated using recombinase, and the ligation is carried out in a water bath at 52°C for 40 min. The ligation system consisted of 10 μl of recombinase, 6 μl of the full-length CDS product of the SOX18 gene, and 4 μl of a linear lentiviral vector fragment.

4. The construction method according to claim 1, characterized in that, In step 4, the ligation product is transformed into TOP10 competent cells. Positive clones are sequenced and analyzed. The plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18, which is the successfully constructed SOX18 gene overexpression vector, is identified. The specific steps are as follows: 10 μl of the ligation product is transformed into TOP10 competent cells and incubated on ice for 10 min; after the ice incubation, the cells are heat-shocked at 42°C for 90 sec and then incubated on ice for 3 min; 800 μl of antibiotic-free LB medium is added and the cells are incubated at 250 rpm for 1 h; the cultured competent cells are centrifuged to remove part of the supernatant, and the remaining supernatant is mixed and spread onto a culture dish and incubated upside down overnight; positive clones are picked for propagation, and the propagated bacterial solution is sequenced and analyzed. The plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18, which is the successfully constructed SOX18 gene overexpression vector, is identified.

5. The construction method according to claim 1, characterized in that, In step 5, the extracted SOX18 gene overexpression vector plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18, along with lentiviral packaging helper plasmids psPAX2 (pHelper1) and pMD2G (pHelper2), was co-transfected into 293T cells for lentiviral packaging, collection, and titer detection. The specific steps are as follows: Step 5.1: Mix 10 μg of the SOX18 lentiviral recombinant tag vector stably overexpressed in wool papilla cells or the SOX18 lentiviral recombinant tag vector plasmid pLV4ltr-PGK-ZsGreen(2A)Puro-CMV-SOX18 stably overexpressed in wool papilla cells constructed in step 4 with 5 μg of lentiviral packaging helper plasmid psPAX2 (pHelper 1) and 5 μg of helper plasmid pMD2G (pHelper 2). Then add the appropriate volume of Opti-MEM and mix well. Adjust the total volume to 0.5 ml and incubate at room temperature for 5 min. Step 5.2: Mix 50 μl of transfection reagent with 450 μl of Opti-MEM and incubate at room temperature for 5 min; Step 5.3: Mix the DNA diluted in step 5.1 with the transfection reagent diluted in step 5.2, gently invert to mix, and incubate at room temperature for 10 minutes. Step 5.4: Add the transfection mixture obtained in step 5.3 dropwise to 293T cell culture medium, shake well, and culture in a 37°C, 5% CO2 cell culture incubator; Step 5.5: After culturing for 6-8 hours, discard the culture medium containing the transfection mixture, replace 10 ml of 10% FBS complete culture medium in each tray of cells, and continue culturing in a 37 ℃, 5% CO2 incubator. Step 5.6: 24 hours after transfection, the number of tagged fluorescent cells is observed under a microscope to determine the transfection efficiency. After confirming successful transfection, the first virus harvesting operation is performed. The virus-containing culture medium is collected in sterile centrifuge tubes and stored at 4°C. Then, 10 ml of fresh 10% FBS medium is replaced and cultured for another 24 hours. Step 5.7: 48 hours after transfection, perform a second virus harvesting operation to harvest the virus-containing culture in sterile 50 ml centrifuge tubes and store at 4°C. Step 5.8: Filter the harvested culture medium containing virus particles through a 0.22 μm filter membrane and collect it in a sterile ultracentrifuge tube. Balance and seal the tube. Centrifuge at 80,000 g for 4 hours. Discard the supernatant after centrifugation and resuspend the precipitate in Virus Store Buffer. Step 5.9: Collect the resuspension, filter it again through a 0.22μm filter membrane for sterilization, and dispense it into sterile virus tubes to obtain lentivirus containing the SOX18 gene.

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