Immortalized goat placenta trophoblast cell line and construction method thereof

Abstract

The invention discloses an immortalized goat placenta trophoblast cell line and a construction method thereof, and relates to the technical field of cell engineering. The construction method comprises the following steps: carrying out isolated culture by using a tissue block culture method to obtain primary goat placenta trophoblast cells; eukaryotic expression plasmids pCI-neo-hTERT are introduced into the primary goat placenta trophoblast cells, positive clones are obtained through screening, subculture is carried out to 80 generations, and the immortalized goat placenta trophoblast cell line is obtained. The genetic structure of the immortalized goat placenta trophoblast cell line constructed by using the construction method can be kept stable in an infinite passage process, and when the immortalized goat placenta trophoblast cell line is transferred to 80 generations, the number of chromosomes is still 60, and the chromosomes are not lost or doubled. Transwell and RT-PCR detection prove that the immortalized goat placenta trophoblast cell line has an invasion characteristic, and TERT protein is stably expressed in the immortalized goat placenta trophoblast cell line.

Description

Technical Field

[0001] The invention relates to the technical field of cell engineering, and in particular to an immortalized goat placental trophoblast cell line and a construction method thereof. Background Art

[0002] Trophoblast cells are an important component of the placental barrier and play a very important role in the process of embryo implantation, implantation, placentogenesis and pregnancy maintenance in mammals. The proliferation and functional regulation of placental trophoblast cells are closely related to the occurrence and development of various pregnancy-related diseases. Many pathogenic factors can cause trophoblastic cell dysfunction and lead to embryo implantation failure or miscarriage. Studies have shown that microbial infection of pregnant females and vertical transmission to the fetus through the placental barrier is one of the main factors causing reproductive disorders in females. Since the biological functions of placental trophoblast cells in vivo are disturbed by many factors (such as hormones, feeding environment, animal stress or individual differences, etc.), it is particularly difficult to accurately explain their physiological functions, and the research progress is relatively slow. In vitro cultured placental trophoblast cells can artificially control a single variable and can be studied in many aspects under ideal conditions. Therefore, obtaining a goat trophoblast cell that can be stably passaged for in vitro study of the interaction mechanism between pathogens and placental trophoblast cells has far-reaching significance for revealing the pathogenesis of reproductive disorders in females.

[0003] The cultivation of placental trophoblast cells is difficult, and it is difficult to obtain trophoblast cells that meet the requirements of the test and that have stable properties. At present, the separation methods of placental trophoblast cells include enzyme digestion, tissue block culture and density gradient centrifugation. After separation and purification, the purity of placental trophoblast cells is up to more than 90%. The primary placental trophoblast cells cultured in vitro can control a single variable, and can exclude the interference of other factors to the experiment. The disadvantage is that primary cells often begin to age and die after 10 generations of cultivation, and the repeated steps of obtaining primary cells are cumbersome, consuming a large amount of manpower, material resources and financial resources, and immortalized cells can overcome these shortcomings, and have advantages that primary cells do not have, such as good uniformity of cells, strong stability and convenient cultivation, etc. Therefore, compared with primary placental trophoblast cells, obtaining immortalized placental trophoblast cells has important practical significance.

[0004] Cell culture technology is now basically mature, but the primary cell culture methods and growth patterns of different animals and different types of cell sources are not the same. Placental trophoblast cell lines have been established in many species, but there have been no reports on goats. Summary of the invention

[0005] The purpose of the present invention is to provide an immortalized goat placental trophoblast cell line and a construction method thereof to solve the problems existing in the above-mentioned prior art. The immortalized goat placental trophoblast cell line constructed by the construction method has invasive characteristics, the genetic structure can remain stable during unlimited passage, and the TERT protein can be stably expressed therein.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides a method for constructing an immortalized goat placental trophoblast cell line, comprising the following steps:

[0008] Primary goat placental trophoblast cells were isolated and cultured by tissue explant culture method.

[0009] The eukaryotic expression plasmid pCI-neo-hTERT is introduced into the primary goat placental trophoblast cells, positive clones are screened, and subcultured to the 80th generation to obtain the immortalized goat placental trophoblast cell line.

[0010] Furthermore, the step of isolating and culturing the primary goat placental trophoblast cells by tissue block culture method comprises:

[0011] The placental tissue is cut into pieces and placed in a DMEM / F12 complete medium containing double antibodies, cultured until a monolayer of cells is formed, and then digested with trypsin to obtain the primary goat placental trophoblast cells.

[0012] Furthermore, the dual antibodies are penicillin and streptomycin.

[0013] Furthermore, the concentrations of penicillin and streptomycin in the DMEM / F12 complete culture medium are both 100 U / mL.

[0014] Furthermore, the eukaryotic expression plasmid pCI-neo-hTERT is introduced into the primary goat placental trophoblast cells by liposome transfection.

[0015] Furthermore, the liposome transfection method uses RFcet transfection reagent.

[0016] Furthermore, the transfection time of the liposome transfection method is 24 hours.

[0017] Furthermore, neomycin was used for screening to obtain the positive clones.

[0018] Furthermore, the concentration of neomycin is 500 μg / mL.

[0019] The present invention also provides an immortalized goat placental trophoblast cell line constructed according to the above construction method.

[0020] The present invention discloses the following technical effects:

[0021] The present invention develops a method for constructing an immortalized goat placental trophoblast cell line. The immortalized goat placental trophoblast cell line constructed by the construction method can maintain a stable genetic structure during unlimited passage. When the chromosome number is passed to the 80th generation, it is still 60 without loss or doubling. Transwell and RT-PCR tests confirm that the immortalized goat placental trophoblast cell line has invasive properties and TERT protein is stably expressed in the immortalized goat placental trophoblast cell line.

[0022] The construction method provided by the present invention can obtain stably passaged goat placental trophoblastic cells, which can be used to study the interaction mechanism between pathogens and placental trophoblastic cells in vitro, which has far-reaching significance for revealing the pathogenesis of reproductive disorders in maternal animals. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0024] Figure 1 This is a microscopic observation of cells that have migrated out from around the placental tissue mass;

[0025] Figure 2 This is a microscopic observation of goat primary placental trophoblast cells;

[0026] Figure 3 This is a microscopic observation of goat placental trophoblast cells transfected for 12 hours;

[0027] Figure 4 Giemsa staining images of primary trophoblast cells (A) and transfected trophoblast cells (B);

[0028] Figure 5 This is the result of immunofluorescence identification;

[0029] Figure 6 This is a growth curve of trophoblast cells at the 50th passage after transfection;

[0030] Figure 7 This is the karyotype analysis of trophoblast cells at the 80th passage after transfection;

[0031] Figure 8 Figure 2 shows the Transwell assay results of primary trophoblast cells (A) and transfected trophoblast cells (B);

[0032] Fig. 9 This is an electrophoresis diagram of RT-PCR detection of the expression of exogenous hTERT mRNA in transfected trophoblast cells; where M: 1000 marker; 1: hTERT-GTCs (30); 2: hTERT-GTCs (50); 3: hTERT-GTCs (80); 4: Primary GTCs;

[0033] Fig.10 This is the electrophoresis diagram of RT-PCR detection of E-Cadherin mRNA expression in transfected trophoblast cells; where M: 1000 marker; 1: hTERT-GTCs (30); 2: hTERT-GTCs (50); 3: hTERT-GTCs (80); 4: Primary GTCs;

[0034] Fig.11 This is an electrophoresis diagram of RT-PCR detection of MHC-I mRNA expression in transfected trophoblast cells; where M: 1000 marker; 1: hTERT-GTCs (30); 2: hTERT-GTCs (50); 3: hTERT-GTCs (80); 4: Primary GTCs;

[0035] Fig.12 This is a Western blot detection result of TERT protein expression in transfected trophoblast cells. DETAILED DESCRIPTION

[0036] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0037] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0038] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0039] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0040] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0041] Example 1

[0042] 1. Isolation and purification of primary goat placental trophoblast cells

[0043] 1. Sample source: A healthy female Boer goat about to give birth to her first litter of one year old was selected, and the placenta sample after delivery was collected from a farmer's home in the vicinity of Changchun City, Jilin Province. The sample was quarantined before sampling and met the experimental requirements.

[0044] 2. Place the placenta of the goat after delivery in a PBS solution containing double antibodies (100U / mL penicillin and 100U / mL streptomycin) for washing, separate the excess uterine caruncle and connective tissue, put it in 75% alcohol for 15-30s and immediately put it in a new PBS buffer for washing. Use the tissue block culture method to separate and culture the placental trophoblast cells, cut the processed placental tissue into 1-2cm pieces and place it in a cell culture dish, turn it upside down in a 37℃, 5% CO2 incubator for 4h, observe that there is no shedding, then add DMEM / F12 complete culture medium containing double antibodies (100U / mL penicillin and 100U / mL streptomycin) to the bottle to ensure that the placental tissue block is completely immersed in the culture solution. Change the cell medium at 24h and 48h, and then change the medium every 2 days until the cells form a monolayer of cells. After washing with PBS, add 1 mL of trypsin. When the cells become round and partially float, add complete culture medium to terminate digestion. Use a pipette to blow the digested cells until they all float and then transfer them into a centrifuge tube at 800 r / min for 5 min. Discard the supernatant, add an appropriate amount of complete culture medium to resuspend the cells, and place them in a new culture flask to preliminarily obtain goat primary placental trophoblast cells.

[0045] Observe the cultured placental tissue blocks under a microscope. On the 5th day or so, cells migrated out from the periphery of the tissue blocks. The cells had various morphologies, such as long spindles or irregular polygons, and were unevenly distributed. Figure 1 After passage, most cells had clear outlines, typical cobblestone shapes, epithelial cell morphology, uniform cell density, and monolayer growth, as shown in Figure 2. Figure 2 shown.

[0046] 2. Transfection, positive cell screening and expansion of continuous culture

[0047] 1. The eukaryotic expression plasmid pCI-neo-hTERT containing the human telomerase reverse transcriptase (hTERT) gene (kindly donated by Professor Wang Aihua of Northwest Agriculture and Forestry University) was introduced into the primary cultured goat placental trophoblast cells by liposome transfection method. The specific steps are as follows:

[0048] 1) Before transfection, the third generation primary cultured goat placental trophoblast cells were inoculated in a 6-well plate, and transfection was performed when the cell confluence was 80-90%. A screening medium containing different concentrations of neomycin (G418) (100, 200, 300, 400, 500, 600, 700, 800, 900 μg / mL) was added, and each gradient was repeated 3 times. The medium was replaced every 3 days, and the cell growth was observed. The minimum G418 concentration that killed all cells within two weeks was selected as the first concentration for screening the cow placental trophoblast cell line. After screening, the first concentration was 500 μg / mL.

[0049] 2) According to RFect PM Primary Cell Plasmid DNA Transfection Reagent Manual Instructions Introduce pCI-neo-hTERT plasmid into primary goat placental trophoblast cells. Dilute 10 μL RFcet with 200 μL Trans buffer per well. PM Transfection reagent, mix thoroughly, prepare RFcet PM -Trans buffer mixture and incubate for 5 min.

[0050] 3) Add 200 μL Trans buffer and 3.2 μL 1 μg / μL pCI-neo-hTERT plasmid to a new EP tube, mix thoroughly to prepare a DNA-Trans buffer mixture, and incubate for 5 min.

[0051] 4) Add the incubated DNA-Trans buffer mixture to RFcet PM -Trans buffer mixture, mix well, and incubate at room temperature for 20 min to obtain a mixture.

[0052] 5) Add the mixture obtained in step 4) dropwise into the cell culture wells, shake the culture plate while adding, and gently mix, and set up 2 wells of non-transfected blank controls. Incubate the cells in a 37°C, 5% CO2 incubator for 12 hours, and analyze the transfected cells.

[0053] 2. 24 hours after the cells were transfected, positive clones were screened with screening medium containing 500 μg / mL G418. The culture was continued for two weeks, and the medium was changed every 2 days. When the negative control cells (untransfected plasmid) died completely and positive clone cell clusters appeared, the G418 concentration was halved and the culture was expanded in a 37°C, 5% CO2 incubator.

[0054] 3. The positive clone cells screened were continuously cultured in a 37°C, 5% CO2 incubator. The single colony was digested with trypsin and then continued to be cultured. The cells were subcultured once every 48 hours. After sorting, the cells showed a cobblestone-like monolayer adherent growth. After subculture to the 50th generation, an immortalized goat placental trophoblast cell line was obtained. The cell morphology after transfection for 12 hours is shown in Figure 3 .

[0055] Screening medium formula: DMEM / F12 medium containing 500 μg / mL G418 and 2% FBS.

[0056] 3. Identification of cell morphological characteristics

[0057] 1. After transfection, the morphological changes of cells were observed under a phase contrast microscope and compared with primary placental trophoblast cells. Primary placental trophoblast cells and trophoblast cells cultured to the 50th generation after transfection were inoculated into 24-well plates. When the cell confluence reached 50-60%, they were fixed with 4% paraformaldehyde, stained with Giemsa, observed under a microscope, and photographed. The results are shown in Figure 4 The results showed that the transfected goat placental trophoblast cells were similar in morphology to the primary cells, with obvious central nuclei and clearly visible nucleoli. Most cells were polygonal or oval in shape, arranged in a typical cobblestone-like pattern, and there was contact inhibition between cells.

[0058] 2. Immunofluorescence detection of CK7 expression in cells

[0059] The trophoblast cells cultured to the 50th generation after transfection were inoculated into a 24-well plate, and staining was performed when the cell confluence reached 50-60%. The operation steps are as follows:

[0060] (1) Discard the cell culture medium, carefully wash with PBS three times, and then add 200 μL of 4% paraformaldehyde to fix the cells at 4°C for 30 min.

[0061] (2) Discard the paraformaldehyde, carefully wash with PBS three times, and block the cell membrane with membrane blocking solution (0.5% Triton X-100 mixed with PBS in a ratio of 1:1, and add 10% goat serum) for 2 h.

[0062] (3) Wash with PBS three times, dilute CK7 antibody and Vimentin at a ratio of 1:200 using primary antibody diluent, add to the culture wells, and incubate at 4°C overnight.

[0063] (5) Wash three times with PBS, add biotin-labeled donkey anti-rabbit IgG, incubate at room temperature for 2 h, and rinse three times with PBS, each time for 5 min.

[0064] (6) Remove the PBS solution, add 100 μL of freshly prepared DAPI working solution, and immediately observe and photograph under an inverted microscope.

[0065] The results of immunofluorescence identification are shown in Figure 5 CK-7 is a specific marker protein for placental trophoblast cells. Immunofluorescence identification revealed that all cells expressed CK-7 after transfection. The cytoplasm of positive cells was stained red and the nucleus was stained blue, but the fibroblast marker protein Vimentin was not expressed, confirming that there was no fibroblast contamination in the cells.

[0066] IV. CCK-8 analysis of trophoblast cell growth curve

[0067] The trophoblast cells at the 50th generation were seeded in a 96-well plate at a rate of 2000 cells / well. 10 μL CCK-8 was added to each well every 24 hours and placed in a 37°C incubator for incubation in the dark for 2 hours. The OD value of each well was measured at a wavelength of 450 nm. The data were read 3 times each time and the average value was taken. The measurement was continued for 7 days, and a growth curve was drawn with time as the horizontal axis and OD value as the vertical axis. The results are shown in Figure 6 As shown, trophoblast cells enter the exponential growth phase on the 2nd day after inoculation and reach the plateau phase on the 5th to 7th day. At this time, the cells should be passaged in time to avoid cell aging or even death.

[0068] 5. Karyotype analysis

[0069] 1. Colchicine treatment of cells: Observe the cell status under a microscope. When the confluence of the cultured cells is about 70-80%, add 0.2μg / mL colchicine to the culture medium and culture in a 37℃, 5% CO2 incubator for 3 hours. Digest and collect the cells into a 15mL centrifuge tube, centrifuge at 800rpm for 5min, and remove the supernatant to keep the cell pellet.

[0070] 2. Hypotonicity: Add 8 mL of 0.075 mol / L KCl hypotonic solution preheated in a 37-degree water bath, use a pipette to mix thoroughly, and incubate in a 37-degree water bath for 15 minutes. Centrifuge at 800 rpm for 5 minutes and discard the supernatant, leaving about 0.5 mL of residual liquid to avoid cell loss.

[0071] 3. Pre-fixation: Gently scrape the bottom of the centrifuge tube, use slight shaking to resuspend the swollen cell pellet, drip freshly prepared fixative (methanol: glacial acetic acid = 3:1) along the tube wall, shake the tube gently while dripping to mix it thoroughly, add about 1mL of fixative, pre-fix for 5 minutes. Centrifuge at 800rpm for 10 minutes, aspirate and discard the supernatant, leaving about 0.5mL of residual liquid.

[0072] 4. First fixation: Slightly shake to resuspend the cells at the bottom, add 5 mL of fixative, gently blow the cells several times, and incubate at room temperature in the dark for 30 minutes. Centrifuge at 800 rpm for 10 minutes, discard the supernatant, and leave about 0.5 mL of residual liquid.

[0073] 5. Second fixation: Repeat step 4.

[0074] 6. Preparation: Wipe the slide with alcohol and dry it for later use. Avoid touching with your fingers to prevent fingerprints from being left on the non-frosted surface. Take a small amount of cell suspension, suspend it at a height of 30-40cm, and gradually drip the suspension onto the slide, and try to avoid the area where it has been dripped. After dripping, immediately fix it several times under the flame of an alcohol lamp. The fixative evaporates and obvious cell particles can be seen on the slide.

[0075] 7. G band staining: Place the baked slides in Giemsa stain solution for 10 minutes. After observing the fission phase under a microscope and seeing that it is purple-red in color, rinse the excess Giemsa stain solution on the slides with running water and take photos of the fission phase for analysis.

[0076] The results are as follows Figure 7 As shown, the number of chromosomes of the immortalized trophoblast cells was still 60 when they were passed to the 80th generation, and there was no loss or doubling, indicating that the genetic structure of the immortalized trophoblast cells remained stable during the unlimited passaging process.

[0077] VI. Determination of trophoblast cell invasion characteristics

[0078] Collect 2×10 4The second generation primary trophoblast cells and the 50th generation trophoblast cells after transfection were resuspended in 200 μL serum-free DMEM / F12 medium and inoculated into Transwell cell chambers (pore size 8 μm), and 500 μL DMEM / F12 complete medium was added to the lower chamber; the cells were cultured in a 37°C, 5% CO2 incubator for 48 h; the cells on the surface of the upper chamber membrane were wiped off with a cotton swab, and the cells that passed through the upper chamber membrane were fixed with 4% paraformaldehyde, stained with crystal violet, observed under a microscope, and photographed. The results are shown in Figure 8 .

[0079] Transwell results showed that both primary trophoblast cells and transfected trophoblast cells could pass through the Transwell chamber and possessed invasive properties.

[0080] 7. RT-PCR detection of exogenous hTERT, MHC-I and E-Cadherin mRNA expression in transfected trophoblast cells

[0081] (1) RNA extraction

[0082] Refer to the steps of Genstar's StarSpin column ultra-speed cell RNA extraction kit, the method is as follows:

[0083] Collect 2×10 6 Place transfected trophoblast cells in a centrifuge tube, add 500μL Lysis Buffer, and immediately shake vigorously until there are no cell clumps. Let stand at room temperature for 1min. Add the mixture to the adsorption column, centrifuge at 12000rpm for 30s, and discard the filtrate. Add 500μL Washing Buffer to the adsorption column, centrifuge at 12000rpm for 30s, discard the filtrate, repeat the wash once, put the adsorption column back into the centrifuge tube, centrifuge at 12000rpm for 2min, and remove the residual rinse solution. Take out the adsorption column, put it in a clean 1.5mL RNase Free centrifuge tube, add 25-50μL Nuclease-free Water in the middle of the adsorption membrane, let it stand at room temperature for 1min, and centrifuge at 10000rpm for 1min. Determine the concentration after aliquoting and store at -80℃ for later use.

[0084] (2) Primer design

[0085] The primer sequences used for RT-PCR of hTERT, MHC-I, E-Cadherin and GAPDH are shown in Table 1 . The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0086] Table 1 Primer sequences

[0087]

[0088] (3) RT-PCR amplification

[0089] cDNA synthesis: refer to the steps of Genstar's StarScript Pro one-tube DNA-free reverse transcription kit, add the following reagents in sequence into an RNase-Free centrifuge tube on ice to prepare the reaction system (see Table 2).

[0090] Table 2 Reverse transcription reaction system

[0091]

[0092] The above liquids were mixed evenly on a vortexer, and the reaction was carried out on a PCR instrument according to the reaction conditions shown in Table 3. After the program was completed, the reverse transcription product was stored at -20°C.

[0093] Table 3 Reverse transcription reaction conditions

[0094]

[0095] Prepare the PCR amplification reaction system as shown in Table 4, add it to the PCR tube, and pre-denature it at 94℃ for 1min on the PCR instrument; then follow the program of 98℃ for 10s, 60℃ for 15s, and 68℃ for 30s for 30 cycles. Take 8μL of PCR product for agarose gel electrophoresis analysis. The voltage is 120V, and after electrophoresis for 30min, use the gel imaging system to observe the results and take pictures.

[0096] Table 4 PCR amplification reaction system

[0097]

[0098] RT-PCR detection revealed that a 384 bp hTERT-specific band was detected in the transfected trophoblast cells. Fig. 9 At the same time, RT-PCR detection revealed that the transfected trophoblast also expressed the epithelial cell-specific adhesion molecule E-cadherin, indicating that the transfected trophoblast cells were of epithelial origin. Fig.10 As shown. RT-PCR detection revealed that the transfected trophoblast cells expressed non-classical MHC-I antigens associated with cell invasion. Fig.11 As shown, combined with the Transwell experiment, it was shown that the transfected trophoblast cells were invasive.

[0099] 8. Western blot detection of TERT protein expression in transfected trophoblast cells

[0100] Collect 5×10 6The 3rd generation primary trophoblast cells, 50th generation and 80th generation trophoblast cells after transfection were washed twice with pre-cooled PBS; the pre-cooled total protein extract was added to lyse the cells on ice for 30 minutes, centrifuged at 4℃12000×g for 15 minutes, and the supernatant was the extracted total cell protein; the protein concentration was determined by BCA method, 1 / 5 volume of 5× protein loading buffer was added to the protein sample, boiled for 10 minutes, cooled to room temperature, and stored at -80℃ for later use. 20μg of cell protein sample was taken for SDS-PAGE electrophoresis, and the protein was transferred to PVDF membrane after the electrophoresis; 5% skim milk powder was used to block the PVDF membrane at room temperature for 2 hours; the PVDF membrane was placed in the TERT primary antibody working solution, shaken on a shaker at 4℃ overnight, and washed with TBST 4 times, 5 minutes each time; the PVDF membrane was placed in the HRP-labeled rabbit anti-mouse IgG working solution, incubated on a shaker at room temperature for 1.5 hours; the membrane was washed with TBST 4 times, 5 minutes each time. After washing, add ECL luminescent liquid droplets onto the PVDF membrane in a dark room and let it act for 1 minute; then take photos and develop them on a machine to analyze the images.

[0101] The results showed that Western blot detection ( Fig.12 ) showed that TERT protein (120kD) was stably expressed in transfected trophoblast cells but not in primary trophoblast cells.

[0102] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing an immortalized goat placental trophoblast cell line, characterized in that: The following steps are involved: Primary goat placental trophoblast cells were isolated and cultured by tissue explant culture method. The eukaryotic expression plasmid pCI-neo-hTERT is introduced into the primary goat placental trophoblast cells, positive clones are screened, and subcultured to the 80th generation to obtain the immortalized goat placental trophoblast cell line.

2. The construction method according to claim 1, characterized in that: The steps of isolating and culturing the primary goat placental trophoblast cells by tissue block culture method include: The placental tissue is cut into pieces and placed in a DMEM / F12 complete medium containing double antibodies, cultured until a monolayer of cells is formed, and then digested with trypsin to obtain the primary goat placental trophoblast cells.

3. The construction method according to claim 2, characterized in that: The dual resistance is penicillin and streptomycin.

4. The construction method according to claim 3, characterized in that: The concentrations of the penicillin and the streptomycin in the DMEM / F12 complete medium are both 100 U / mL.

5. The construction method according to claim 1, characterized in that: The eukaryotic expression plasmid pCI-neo-hTERT is introduced into the primary goat placental trophoblast cells by liposome transfection.

6. The construction method according to claim 5, characterized in that: The liposome transfection method uses RFcet transfection reagent.

7. The construction method according to claim 6, characterized in that: The transfection time of the liposome transfection method is 24h.

8. The construction method according to claim 1, characterized in that: The positive clones were obtained by neomycin screening.

9. The construction method according to claim 8, characterized in that: The concentration of neomycin is 500 μg / mL.

10. An immortalized goat placental trophoblast cell line constructed according to the construction method according to any one of claims 1 to 9.

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