WO2020000327A1 - CELL LINE FOR RXRα PROTEIN STABLE HIGH EXPRESSION AND PREPARATION METHOD THEREOF - Google Patents

Abstract

The present invention falls within the technical field of biological preparations, and particularly relates to a RXRα gene stably transfected cell line for RXRα protein stable high expression and a preparation method therefor. The cell line is a cell clone where the RXRα gene is randomly integrated into the chromosomes of an SK-N-SH cell, and the genotype for the cell line is stable and can be stably inherited with cell proliferation, and the progeny cells stably and highly express the RXRα protein, thereby greatly improving the stability of the high expression of the RXRα protein to ensure the functionality of the RXRα gene and the reliability of the tested materials. Provided is a method for preparing the cell line, comprising the following steps: designing and preparing a eukaryotic expression vector, transfecting SK-N-SH cells with the eukaryotic expression vector by means of the nuclear transfection technique, obtaining the cell clones stably transfected with the RXRα gene by means of G418 drug screening, identifying whether the cell clone is a positive cell clone that is stably transfected with the RXRα gene by means of PCR and gene sequencing, identifying the expression level of the RXRα protein in positive cloned cells by using Western blot, and then obtaining the cell line for RXRα protein stable high expression in which genotypes can be stably passaged.

Description

RXRα protein stable and highly expressed cell line and preparation method thereof Technical field

The invention belongs to the technical field of biological preparation, and particularly relates to a RXRα protein stable and highly expressed cell line and a preparation method thereof.

Background technique

There are three main types of spliceosome of RXR (Homo sapiens, retinoid X receptor) genes, which are RXRα (NM_002957.5), RXRβ (NM_001291920.1), and RXRγ (NM_001291921.1).

The intracellular RXRα receptor can be combined with a variety of drugs and is closely related to most human diseases including but not limited to tumors, diabetes, neurodegenerative diseases, and steatohepatitis. Constructing a cell line with high and low expression of RXRα protein will be beneficial to study the function of RXRα receptor, help to explore the pathogenesis of diseases related to RXRα receptor, and develop effective drugs for disease targets.

Cell transfection technology is a process of introducing foreign genetic material into a target cell, making it express in the target cell, and obtaining a new genetic marker. It is a common technique for studying gene expression and gene function. Cell transfection can be divided into two categories, one is transient transfection and the other is stable transfection. The former exogenous genetic material does not integrate into the host chromosome, there are multiple copies of exogenous genes in the cell, resulting in high-level expression, which usually lasts only a few days, until the exogenous gene is lost due to various factors during cell division until. Stable transfection can integrate the foreign target gene into the chromosome of the cell by nuclear transfection technology and become a part of the cell genome, so that the target gene is stably expressed in the cell, and the progeny cells of the stably transfected cell also express the foreign gene. Formation of stably transfected cell lines. The probability of gene integration into the chromosome is small, and a selectable marker gene (antibiotic resistance gene) is needed to screen transfected cells. The target gene and the antibiotic resistance gene were linked to co-transfect the target cells, and then the corresponding antibiotics were used to screen the transfected cells. Only the stably transfected cells will acquire resistance to antibiotics and survive in long-term culture, thereby achieving the screening and expansion of target cells and obtaining stable transfected homologous cell lines.

As mentioned above, the effective construction of a RXRα protein stable and highly expressing cell line is important for the study of the biological effects of RXRα protein in vivo and the development of drugs for related diseases. The present invention intends to solve this problem.

Summary of the invention

In view of the problems existing in the prior art, the primary object of the present invention is to provide a RXRα protein stable and highly expressed cell line, which is characterized in that the cell lines are RXR / TG-SK-N-SH-97 # and RXR / TG, respectively. -SK-N-SH-202 #, the cell line is prepared by the following preparation method.

Another object of the present invention is to provide a method for preparing a RXRα protein stable and highly expressed cell line:

(1) Preparation of eukaryotic expression vector with high expression of RXRα. This vector contains CMV promoter, pcDNA3.1 sequence, EGFP tracer gene and target gene RXRα. The final vector is pcDNA3.1-RXRα-2A-EGFP eukaryotic expression. Carrier

(2) inserting the final gene into the immortalized human SK-N-SH cells of human neuroblastoma by nuclear transfer, and using G418 drug screening to obtain stable cell clones;

(3) The obtained cell clones are amplified by RXRα specific sequence by PCR, and the transgenic positive cell clones are initially selected by agarose gel electrophoresis, and the suspected positive clone cells are submitted for core sequence detection;

(4) Using qPCR and Westernblot detection technology to identify the RXRα gene mRNA and protein expression level of positive clone cells with correct core sequence sequencing, and finally obtain a cell line with stable and high expression of RXRα protein.

A further preferred solution is that the preparation method includes the following preparation steps:

(1) Construction of eukaryotic expression vector:

(1.1) Primer design;

(1.2) EGFP-PA fragment acquisition;

(1.3) pcDNA3.1 (+) was digested with BamHI to obtain the vector backbone;

(1.4) ligating the EGFP-PA fragment with the pcDNA3.1 backbone into pcDNA3.1-EGFP;

(1.5) RXRα fragment acquisition;

(1.6) pcDNA3.1-EGFP was digested with BamHI to obtain the vector backbone;

(1.7) The RXRα-2A amplification product Infusion is connected to the pcDNA3.1-EGFP backbone to obtain pcDNA3.1-RXRα-2A-EGFP;

(1.8) Enzymatic digestion to verify whether the pcDNA3.1-RXRα-2A-EGFP plasmid was successfully constructed;

(2) Cell transfection;

(3) Screening and cloning of positive cells;

(4) Identification of positive cloned cells:

(4.1) Identification of positive clones of eukaryotic expression vectors;

(4.2) Identification of positive clone RXRα mRNA expression level;

(4.3) Identification of high expression level of RXRα protein in positive clone cells.

The technical effects of the present invention relative to the prior art include:

(1) The present invention effectively constructs a RXRα protein stably overexpressing cell line. The cell line, RXRα overexpressing cell line 97 (RXR / TG-SK-N-SH-97 #) and cell line 202 (RXR / TG-SK-N-SH-202 #) The relative expression of RXRα protein compared to wild type (SK-N-SH) cell group, RXR / TG-SK-N-SH-97 # and RXR / TG-SK The expression of RXRα protein of -N-SH-202 # was significantly increased, and the difference was statistically significant (P <0.01).

Among them, the expression of RXRα protein in RXR / TG-SK-N-SH-97 # cell line was 1.87 times (P <0.01) the expression of RXRα protein in wild-type cells, and RXR / TG-SK-N-SH-202 # cells The expression of RXRα protein was 1.90 times higher than that of wild type cells (P <0.01).

(2) The cell line is stable in genotype, can stably and highly express RXRα, and does not require regular drug screening and identification of protein expression levels, thereby greatly improving the reliability and research efficiency of RXRα protein-related functional test materials and reducing RXRα Difficulty in proliferating and culturing high protein-expressing cells.

(3) The preparation method simplifies the procedure of cell construction, has good reproducibility, and is conducive to industrial application.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 Schematic diagram of the amplified product of EGFP-SV40polyA.

Figure 2 Schematic diagram of pcDNA3.1 (+) digested product with BamHI.

FIG. 3 is a schematic diagram of the PCR results of the cloned bacteria liquid after the EGFP + SV40polyA amplification product is connected to the backbone.

Figure 4 pcDNA3.1-EGFP intermediate vector map.

Figure 5 Schematic diagram of pcDNA3.1-EGFP double restriction digestion verification.

Figure 6 Schematic diagram of RXRα-2A amplification products.

Figure 7 Schematic diagram of the single digested product of pcDNA3.1-EGFP by BamHI.

Fig. 8 Schematic diagram of infusion connection reaction system and reaction procedure.

Figure 9 PCR results of the cloned bacteria liquid after the RXRα-2A amplification product was linked to the backbone.

Figure 10 pcDNA3.1-RXRα-2A-EGFP eukaryotic expression vector final vector plasmid map.

Figure 11 Schematic diagram of pcDNA3.1-RXRα-2A-EGFP plasmid digestion and verification.

Figure 12 Schematic diagram of SK-N-SH wild type cells.

Figure 13 Schematic diagram of cell morphology before nuclear transfer.

Figure 14 Schematic diagram of EGFP fluorescent protein expression in cells after transfection.

Figure 15 The process of cell clone formation and the morphology of cell clones.

Figure 16. PCR results of picked cell clones.

Figure 17. Agarose gel electrophoresis of suspected positive cloned cells.

FIG. 18 is a schematic diagram of a qPCR three-step amplification procedure.

FIG. 19 is a schematic diagram of a qPCR amplification curve.

Figure 20 Schematic analysis results of RXRαmRNA relative expression data in suspected positive cell clone cells.

Figure 21 Schematic diagram of RXRα protein expression identification in SK-N-SH wild-type and RXRα gene high-expressing cells.

FIG. 22 is a schematic diagram showing the determination of the relative expression of RXRα protein in SK-N-SH wild-type and RXRα gene high-expressing cells.

Note: SK-N-SH is a wild-type cell; RXR / TG-SK-N-SH-97 # RXRα gene high expression type 97 cell; RXR / TG-SK-N-SH-202 # is a high expression RXRα gene Type 202 cells.

detailed description

The present invention is described in further detail below with reference to the embodiments and the accompanying drawings, but the embodiments of the present invention are not limited thereto.

Example 1

RXRα protein stable and highly expressed cell line and preparation method thereof,

(1) Preparation of pcDNA3.1-RXRα-2A-EGFP eukaryotic expression vector

(1.1) Primer design:

Design primers to amplify the EGFP-SV40polyA fragment from the EGFP-N1 vector plasmid;

Design primers to amplify the CDS sequence of RXRα from the cDNA of SK-N-SH cells (the stop codon TAG needs to be removed during design) to amplify the target gene RXRα and add 2A sequence at the back end and about 15bp required for IN-Fusion connection Vector backbone fragment.

Design primers Primer information is shown in Table 1.

Table 1 Primer information table

Note: Primer RXRα-2A-infusion-F / RXRα-clone-R uses SK-N-SH cell cDNA as template to amplify the target gene RXRα; primer RXRα-2A-infusion-F / RXRα-2A-infusion- The amplification template used by R is the PCR product of the first step. The purpose is to add the 2A sequence and the 15 bp homologous sequence required at the 3 'end to the PCR product of the first step.

(1.2) EGFP-PA fragment acquisition:

The PCR amplification technology was used to amplify the EGFP-SV40polyA fragment from the EGFP-N1 plasmid. The reaction system is shown in Table 2.

Reaction procedure: (98 ℃ / 10s, 55 ℃ / 30s, 72 ℃ / 1min) X35cycles. After the reaction, EGFP-PA agarose gel electrophoresis results are shown in Figure 1.

Table 2 PCR reaction system

(1.3) pcDNA3.1 (+) was digested with BamHI to obtain the vector backbone:

The endonuclease BamHI digestion was used to obtain the backbone fragment pcDNA3.1 (+). The BamHI digestion reaction system is shown in Table 3. The agarose gel electrophoresis identified the linearized pcDNA3.1 (+) fragment size as shown in Figure 2. The gel was recovered to obtain the enzyme. Cut skeleton fragments.

The reaction procedure was: 37 ° C, 3h.

Table 3 BamHI digestion system

(1.4) Link the EGFP-PA fragment with the pcDNA3.1 backbone to form pcDNA3.1-EGFP:

Purify the amplified product of EGFP-PA fragment, Infusion ligation (In-Fusion HD Cloning Kits, Clontech), transformation, plating, picking bacteria, and bacterial liquid PCR. Extract the plasmid.

The connection and conversion steps are as follows:

① Connect the sample on ice: 5 μl In-Fusion + 1.5 μl carrier backbone + 2.5 μl PCR product + 1 μl water, react at 16 ° C for 2h or 4 ° C overnight, and place on ice.

② Take competent cells, dissolve them on ice, and gently mix the competent cells with a pipette;

③ Take a 1.5 ml centrifuge tube, add 5 μl of the vector-linked product, add 50 μl of competent cells, mix gently, and immediately place on ice;

④ Incubate on ice for 30min, heat shock in a 42 ℃ water bath for 90s, and immediately cool on ice for 1 ~ 2min;

⑤ Add 150 μl of anti-LB-free medium, activate at 37 rpm, 200 rpm for 1 h, and simultaneously take the corresponding number of Amp-resistant plates in an oven;

⑥ Take 30 ～ 50μl of the activated bacterial solution in a pre-heated Amp-resistant plate, spread it evenly, place it for 2 ~ 3min in the forward direction, and invert and incubate it in a 37 ° C incubator overnight.

The steps of PCR for picking clones are as follows:

① Observe the colony morphology on the plate. When visible to the naked eye and can be distinguished from other colonies, you can pick clones;

② Determine the number of clones to be picked for each plate. If it is a short fragment of the T vector, pick 6 to 8 clones per plate. For difficult fragments, you need to choose 16 to 48 clones.

③ In a clean bench, take a corresponding amount of 1.5ml sterile centrifuge tubes on a test tube rack. After labeling, add 6μl of resistant medium to each centrifuge tube and set aside.

④ Carefully pick larger colonies from the plate with a pipette and pipette in a centrifuge tube with 6 μl of culture medium (for colonies with smaller colonies, pick them out and mix them and place them in a shaker) PCR identification after shaking for 1h);

⑤ Take 1μl of the above bacterial solution for PCR identification, and leave the remaining 5μl of the bacterial solution in a 37 ° C oven or shaker;

⑥ After the result of PCR identification, select the positive strain and send it for sequencing.

Among them, the PCR result of EGFP + SV40polyA amplification product after connecting to the backbone is shown in Figure 3. The intermediate vector map of pcDNA3.1-EGFP is shown in Figure 4; the plasmid was verified by double digestion with BamHI and EcoRI, and with 50 μl EcoRI single digestion. The double digestion result should be: 6301bp = 948bp + 5353bp, and the result is shown in Figure 5 .

(1.5) RXRα fragment acquisition:

The reaction system for RXRα fragment amplification is shown in Table 4.

Reaction procedure: (98 ° C / 10s, 55 ° C / 30s, 72 ° C / 1.5min) X35 cycles, agarose gel electrophoresis of RXRα-2A fragment is shown in Figure 6.

Table 4 PCR reaction system

Note: The amplification of RXRα-2A fragment is divided into two steps: The primer used for the first step of amplification is RXRα-2A-infusion-F / RXRα-clone-R, and the amplification template used is SK-N-SH cell cDNA. The purpose is RXRα was amplified first; the primers used in the second step were RXRα-2A-infusion-F / RXRα-2A-infusion-R, and the amplification template used was the PCR product of the first step. The PCR product is followed by the 2A sequence and the 15 bp homologous sequence required at the 3 'end.

(1.6) pcDNA3.1-EGFP was digested with BamHI to obtain the vector backbone:

PcDNA3.1-EGFP (the homologous sequence when infusion ligated to EGFP complemented the BamBI digestion site) was digested with the restriction enzyme BamHI to obtain a vector backbone with sticky ends. The reaction system is shown in Table 5.

The reaction procedure was: 37 ° C, 3h. The agarose gel electrophoresis was performed to recover the digested skeletal fragments as shown in Figure 7.

Table 5BamHI digestion system

(1.7) Obtaining pcDNA3.1-RXRα-2A-EGFP:

Purify the RXRα-2A amplification product and pcDNA3.1-EGFP single enzyme-digested backbone fragment. The two fragments are ligated by Infusion, transformed, plated, picked, and used for bacterial liquid PCR, selected positive colonies, expanded shake culture, protected bacteria to improve Plasmid. The results of PCR identification are shown in Figure 9 and the plasmid map of the eukaryotic expression vector final vector is shown in Figure 9.

Operating procedures:

a. Infusion ligation: The above prepared double-digested cohesive ends of the vector and the DNA fragments with 15bp homologous sequences at both ends were ligated using Clontech's Infusion HD cloning kit. The infusion ligation reaction system and reaction procedure are shown in Figure 8. .

b. Conversion coating: The steps for connecting the product conversion coating are as follows

① Take an appropriate amount of ice with an ice box, remove the competent cells from the -80 ℃ refrigerator and place on ice;

② In a clean bench, take a 1.5ml centrifuge tube with burned and cooled forceps, add 3-10 μl of the ligation product, and mix with 35 μl competent cells (TAKARA is generally 100 μl in a tube, each tube can be transferred three times) Mix gently by pipetting, quickly place on ice, and incubate for 30min;

③ Adjust the temperature of the incubator to 42 ° C. After the temperature is reached, incubate the above mixture at 42 ° C for 90s, take it out of the ice and leave it for 1-3 minutes;

④ In a clean bench, add 500 μl of anti-LB-free medium to the above mixture, activate it in a shaker at 37 ° C / 200rpm for 1 h, and at the same time, take a corresponding number of resistant plates from the refrigerator and preheat in a 37 ° C oven;

⑤ After activation for 1 h, remove the mash and centrifuge at 6000 rpm for 5 min to collect the precipitate;

⑥ During centrifugation of mash, stir and wash the coated bar in 75% alcohol in a clean bench for several times and over-disinfect it on an alcohol lamp, and stand on a test tube rack to cool.

After the mash was centrifuged, the upper 400 μl of culture medium was aspirated and discarded from the ultra-clean workbench, and the remaining culture medium and the mash solution were gently pipetted and mixed. After the coating bar is cooled, add the remaining mash to a pre-heated resistant plate, and use the coating bar to gently spread the mash.

The coated plate was placed in a forward direction for 2 minutes until the solid medium fully absorbed the mash, and then placed in an overnight incubator at 37 ° C for incubation.

Note: The operations on the competent and diarrhea must be completed in a clean bench to avoid contamination with bacteria, which will result in a lot of "background" on the clones grown after coating the plate.

c. PCR identification of picking clone liquid and identification of plasmid digestion

The steps for selecting cloning mash PCR are as follows:

① Observe the colony morphology on the plate. When visible to the naked eye and can be distinguished from other colonies, you can pick clones;

② Determine the number of clones to be picked for each plate. If it is a short fragment of the T vector, pick 6 to 8 clones per plate. For difficult fragments, you need to choose 16 to 48 clones.

③ In a clean bench, take a corresponding amount of 1.5ml sterile centrifuge tubes on a test tube rack. After labeling, add 5μl of sterile water or resistant medium to each centrifuge tube, and set aside.

④ Carefully pick larger colonies from the plate with a pipette, and mix by pipetting in a centrifuge tube with 5 μl of sterile water or culture medium;

⑤ Take 1 μl of the above-mentioned mash solution for PCR identification, and incubate the remaining 4 μl of mash solution in a 37 ° C oven.

⑥ After the result of PCR identification, positive strains were selected, and 4 μl of mash was added to 5 ml of resistant medium and shaken overnight.

The steps to identify and extract plasmids are as follows:

① On the next day, take 5ml of mash, in a clean bench, take 1ml of mash, and divide it into two centrifuge tubes: 500μl of mash, add 500μl of 60% glycerol, mix, and temporarily put in 4 ℃ refrigerator save;

② The remaining 4 ml of mash was centrifuged at 8000 rpm for 5 min, the supernatant was discarded, and the plasmid was extracted according to the instructions of the plasmid small extraction kit;

③ Identification of the extracted plasmids by double digestion (at least two groups). Strains that meet the expected results were electrophoresed. Take 500 μl from glycerol bacteria for sequencing, and transfer the rest to -80 ° C for storage.

Among them, the PCR result of the RXRα-2A amplification product after connecting to the backbone is shown in FIG. 9. The constructed final vector map is shown in Figure 10.

(8) Enzymatic digestion to verify whether the pcDNA3.1-RXRα-2A-EGFP plasmid was successfully constructed:

The digestion results are shown in Figure 11. Double enzyme digestion verified SpeI + XhoI = 3339bp + 4455bp (not digested completely in the figure, the size of the top band is equal to the size of the linearized plasmid); single enzyme digestion SpeI = 7794bp. The size was exactly the same, indicating that the plasmid construction was correct, and the SpeI linearized plasmid was recovered.

(2) Cell transfection

SK-N-SH was passaged into a culture plate and cultured stably. When the confluence of cells in the 6-well plate reaches 80-90%, nuclear transfer is performed. The transfection system is:

The nuclear transfer steps are as follows:

a) will

Supplement all added

Solution (stable storage at 4 ° C for 3 months).

b) Take out a tube of complete culture medium from the refrigerator, preheat it at 37 ° C, then add 1.5ml to a 6-well plate and put it in a 37 ° C incubator.

c) The cells in the 6-well plate were digested and centrifuged, then the medium was removed, and the cells were washed once in PBS.

d) Use 100μl Supplement

Solution (removed from the refrigerator in advance and returned to room temperature), resuspend the cells, and mix with a total of 2 μg of plasmid (according to the kit instructions).

e) Use the dropper provided with the kit to carefully add the mixed solution of cells and plasmids to the provided nuclear rotor to avoid air bubbles. After nuclear transfer using the corresponding procedure, add 500 μl of pre-warmed culture medium and transfer the cells to the In a hot 12-well plate, shake and let stand for at least 24 hours.

f) Observe the next day, take photos to record the adherence of the transfected cells, observe the cell conditions and take photos after changing the fresh medium.

(3) Screening and cloning of positive cells

After transfection, the cells continued to be cultured. When they grew to 80% -90%, they were plated into 10cm dishes, and the plates were divided according to the cell culture status and transfection efficiency. (If the cell state is good and the transfection efficiency is high, the cell density can be slightly lower; if the cell state is poor and the transfection efficiency is low, a large number of deaths will occur after dosing, so the cell density should be slightly higher.) It is advisable to observe 7 to 15 cells in the microscopic field.

When most single cells divide into 4 to 8 cells, dosing is started, and drug screening is performed with the optimal screening concentration of the pre-experiment (after pre-experiment, this experiment is set to 300 μg / ml), during which some cells die, Change the fresh medium with dosing every two days. The first day of dosing was recorded as Day0.

When adding medicine to Day6-Day7, cells will start to die in large numbers. At the same time, some formed monoclonal cell clusters are observed. Among Day8-Day12, according to the situation, select the cell state and the clone shape that are better. Use a gun to gently scrape it down and Aspirate to a 96-well plate and change to fresh medium before picking clones.

Note: When pipetting to a 96-well plate, you can bring 100 to 120 μl of culture medium, and gently blow the cell mass into the 96-well plate at the same time. Pay attention to the process gently, and try not to generate air bubbles. Shear force).

The clones were observed every day after selection. When the cells grew to more than 90% in the 96-well plate, they could be transferred to the 48-well plate and then passed to the 12-well plate in turn.

(4) Identification of positive cloned cells

(4.1) Identification of positive clones of eukaryotic expression vectors:

Half of the cells in the 12-well plate were collected and lysed to extract cell DNA, and amplified according to the previously designed primers (Table 8).

When the confluence in the 12-well plate reaches more than 90%, the cells are digested half (the remaining half of the original wells are cultured and numbered), centrifuged at 5000 rpm for 10 minutes, the supernatant is discarded, and the lysate is lysed for identification.

a) In a 1.5ml centrifuge tube, centrifuge at 5000rpm for 10min, try to remove the cell culture medium as much as possible. If there is any residue, it is recommended to open the EP tube cover and dry it in an oven at 70 ℃ for 10min;

b) Lysate formulation: 15 μl DirectPCR reagent + 1 μl proteinaseK + 4 μl H ₂ O = 20 μl; where DirectPCR reagent (Viagen Biotech, Cat # 101-T or # 301-C), proteinase K (from the DNA extraction kit of Tiangen) band);

c) Add the prepared 20μl lysate to the cell pellet, mix it and transfer it to a 200μl PCR tube, and number it. 55 ° C / 2h, 85 ° C / 45min, take 1μl for PCR identification (20μl system does not exceed 1.5μl).

d) Before batch cell clone lysis identification, it is best to explore the lysate primer conditions. Some primers may not be suitable for the lysate environment.

e) 24-well plate cells are also suitable for identification in this method step.

f) The lysis and identification is best completed within 1-2 days. After the positive clones are obtained, the target cells can be cultured, and the negative cells can be discarded. Eliminate the need for a large number of cell culture operations.

The amplification reaction system is shown in Table 6; the amplification identification primers are shown in Table 7.

Reaction procedure: (98 ° C / 10s, 55 ° C / 30s, 72 ° C / 1.5min) X35 cycles.

Table 6 PCR reaction system

Table 7 Identification primer information table

A total of 89 cell clones were obtained in this experiment by G418 drug selection. A total of 41 clones were positively identified by PCR after passaging. Finally, a total of 11 positive clones were identified. The identification PCR results are shown in Figure 16:

(4.2) Identification of positive clone RXRα mRNA expression level:

Six positive cells and one wild-type cell were selected for culture, RNA extraction and reverse transcription. The results of RNA purity and integrity testing are shown in Figure 17 and Table 8.

Resuscitate the cells to a 12-well plate. After the cells are full, digest the cells, wash them with PBS, centrifuge, remove the supernatant, extract RNA from the cells, and perform reverse transcription. The steps are as follows:

a) After trypsin digestion of the cells (digestion of the medium is terminated), the cells are aspirated and placed in a 1.5 ml centrifuge tube (imported centrifuge tube), the cells are taken out, and the subsequent steps are performed between the molecules.

b) Centrifuge at 1000 rpm for 5 min and aspirate the supernatant. Add 500 μl of TRIZOL reagent, mix by pipetting up and down, and let stand for 5 min at room temperature (lysis of nucleoprotein complex);

c) Add 100 μl of chloroform to Trizol-cell homogenate (0.2ml of chloroform per 1ml of Trizol), cover with a lid and shake vigorously for 15s, and let stand at room temperature for 3min;

d) Centrifuge at 4 ° C for 15 min at 12,000 rpm, carefully suck the upper aqueous layer into a new centrifuge tube (note that the protein layer in the middle layer cannot be sucked), add 250 μl isopropanol (0.5 ml isopropanol per 1 ml Trizol), and invert Mix several times and let stand at room temperature for 10min;

e) Centrifuge at 12000 rpm for 10 min at 4 ° C and discard the supernatant;

f) Wash the pellet with 500 μl of 75% ethanol (prepared in DEPC water) (carefully invert several times), centrifuge at 7500 rpm for 5 min at 4 ° C, and discard the supernatant;

g) Dry the precipitate in a clean bench for 5 min. Dissolve the precipitate in 20 μl of enzyme-free water. After measuring the concentration, dilute a portion of the RNA to 250 ng / μl and store at -80 ° C or directly reverse transcription.

The results of RNA electrophoresis identification are shown in Figure 17.

Table 8 RNA concentration and OD value information

Sample serial number	RNA concentration	260/280	260/230
SK-N-SH	480ng / μl	1.96	2.10
38	523ng / μl	1.93	2.03
56	563ng / μl	1.90	2.04
97	494ng / μl	1.83	1.96
137	512ng / μl	2.03	1.99
160	479ng / μl	1.94	1.89
202	533ng / μl	2.01	1.92

After RNA extraction is completed, reverse transcription is performed, and the reaction system is prepared according to Tables 9 and 10:

Table 9 Reaction system

The above system was reacted at 65 ° C for 5 minutes, and then cooled on ice;

Table 10 Reaction system

42 ° C × 60min, 70 ° C × 15min. After the reaction is completed, PCR identification or storage at -20 ° C is required.

After qPCR identification:

Six samples (cell cDNA), each pair of primers (RXRα and GAPDH), the system is shown in Table 11:

Table 11 Reaction system

This qPCR experiment uses ABI 7500 model qPCR instrument. The procedure is as follows. The three-step amplification program is used, as shown in Figure 18:

The analysis of qPCR results is shown in Figure 19, Figure 20 and Table 12.

Table 12. Sorting and analysis of qPCR raw CT data

qPCR analysis results:

In this study, qPCR was used to detect the expression of RXRα in SK-N-SH cell lines identified as positive by PCR. The results showed that the RXRα mRNA of the three positive clones was significantly higher than that of SK-N-SH wild-type cells, respectively: 97 # is wild 17 times of the type, 202 # is 8 times of the wild type, 38 # is 6 times of the wild type, so 97 # and 202 #, two cells with higher expression, were expanded and cultured.

The cell lines obtained were RXR / TG-SK-N-SH-97 # and RXR / TG-SK-N-SH-202 #.

(4.3) Identification of RXRα protein expression level in positive cloned cell lines

The cells of Example 1 were subjected to extraction of total protein from the cells, and protein quantification was performed using the BCA protein quantification method. After SDS-PAGE gel electrophoresis, the protein was transferred to the PVDF membrane by a semi-dry transfer membrane instrument, and the 5% skimmed milk powder was blocked for 2 hours. RXRα and the internal control GAPDH primary antibody diluent were added, and incubated at room temperature for 2 hours. HPR-labeled goat anti-rabbit and goat anti-mouse secondary antibodies were diluted at a ratio of 1: 4000 for 10 times, incubated at room temperature for 1 h, and washed with TBST three times for 10 minutes each time. The ECL chemiluminescence kit developed the color, developed with a cold CCD imaging system, Image J The software 2.0 quantitatively analyzes the gray value of protein bands.

(4.4) Identification of RXRα protein expression levels in positive clones

The results of Western Blot identification of RXRα protein expression are shown in Figure 21 and Figure 22. The RXRα gene high-expressing cell line 97 (RXR / TG-SK-N-SH-97 #) and the cell line 202 (RXR / TG- SK-N-SH-202 #) The relative expression of RXRα protein compared to the wild-type cell group, RXRα protein of RXR / TG-SK-N-SH-97 # and RXR / TG-SK-N-SH-202 # The expression level was significantly increased, and the difference was statistically significant (P <0.01). Among them, the expression of RXRα protein in RXR / TG-SK-N-SH-97 # cell line was 1.87 times (P <0.01) the expression of RXRα protein in wild-type cells, and RXR / TG-SK-N-SH-202 # cells The expression of RXRα protein was 1.90 times higher than that of wild type cells (P <0.01).

Based on the above identification results, through this part of the research, our laboratory successfully constructed the eukaryotic vector RXRα high expression pcDNA3.1-RXRα-2A-EGFP and two RXRα protein high expression cell lines. The RXRα protein high expression cell lines were RXR / TG-SK-N-SH-97 # and RXR / TG-SK-N-SH-202 # provide test materials for subsequent experiments.

The above embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above embodiment. Any other changes, modifications, substitutions, combinations, and modifications made without departing from the spirit and principle of the present invention, Simplified, all should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (10)

1. A method for preparing a RXRα protein stable and high-expressing cell line, characterized in that:

   (1) Preparation of eukaryotic expression vector with high expression of RXRα gene, the vector contains CMV promoter, pcDNA3.1 sequence, EGFP tracer gene and target gene RXRα, and the final vector is pcDNA3.1-RXRα-2A-EGFP. Nuclear expression vector

   (2) Inserting the final gene into the immortalized cells of human neuroblastoma, that is, SK-N-SH cells by nuclear transfer, and using G418 drug screening to obtain stable cell clones;

   (3) Screen the obtained cell clones, use PCR to amplify the RXRα-specific sequence, and initially select transgenic positive cell clones by agarose gel electrophoresis, and send suspected positive cell clones for inspection to detect the core sequence;

   (4) The mRNA and protein expression levels of positively cloned cells with positive core sequence sequencing were identified using qPCR and Western blot detection techniques, and finally a cell line with stable and high expression of RXRα protein was obtained.
2. The method according to claim 1, further comprising the following steps:

   (1) Construction of eukaryotic expression vector:

   (1.1) Primer design;

   (1.2) EGFP-PA fragment acquisition;

   (1.3) pcDNA3.1 (+) was digested with BamHI to obtain the vector backbone;

   (1.4) ligating the EGFP-PA fragment with the pcDNA3.1 backbone into pcDNA3.1-EGFP;

   (1.5) RXRα fragment acquisition;

   (1.6) pcDNA3.1-EGFP was digested with BamHI to obtain the vector backbone;

   (1.7) The RXRα-2A amplification product Infusion is connected to the pcDNA3.1-EGFP backbone to obtain pcDNA3.1-RXRα-2A-EGFP;

   (1.8) Enzymatic digestion to verify whether the pcDNA3.1-RXRα-2A-EGFP plasmid was successfully constructed;

   (2) cell transfection;

   (3) Screening and cloning of positive cells;

   (4) Identification of positive cloned cells:

   (4.1) Identification of positive clones of eukaryotic expression vectors;

   (4.2) Identification of positive clone RXRα mRNA expression level;

   (4.3) Identification of RXRα protein expression level in positive cloned cells.
3. The preparation method according to claim 2, characterized in that:

   The step (1.1) is to design primers to amplify the EGFP-SV40polyA fragment from the EGFP-N1 vector plasmid; to design primers to amplify the CDS sequence of RXRα from the SK-N-SH cell cDNA. The design needs to remove the stop codon TAG to Amplify the target gene RXRα and add a 2A sequence at the back end and a 15 bp vector backbone fragment required for IN-Fusion ligation;

   The step (1.2) is to amplify the EGFP-SV40polyA fragment from the EGFP-N1 plasmid by using PCR amplification technology; the reaction procedure: (98 ° C / 10s, 55 ° C / 30s, 72 ° C / 1min) × 35 cycles;

   The step (1.3) is to obtain the scaffold fragment pcDNA3.1 (+) by digestion with the endonuclease BamHI, and then digest with the BamHI digestion reaction system, and identify and identify the size of the linearized pcDNA3.1 (+) fragment by agarose gel electrophoresis. The gel fragments were recovered by digestion, and the reaction procedure was 37 ° C for 3h.
4. The preparation method according to claim 2, characterized in that the step (1.4) is to purify the amplified product of EGFP-PA fragment, Infusion (In-Fusion HD Cloning Kits, Clontech), transform, plate, pick bacteria The results of the bacterial liquid PCR are shown in Fig. 3, and two positive colonies were selected to expand the shake bacteria, protect the bacteria, and extract the plasmid;

   The connection and conversion steps are as follows:

   a) Connect the sample on ice: 5μl In-Fusion + 1.5μl carrier backbone + 2.5μl PCR product + 1μl water, react at 16 ° C for 2h or 4 ° C overnight, and place on ice;

   b) take competent cells, dissolve them on ice, and gently mix the competent cells with a pipette;

   c) Take a 1.5 ml centrifuge tube, add 5 μl of the vector-linked product, add 50 μl of competent cells, mix gently, and immediately place on ice;

   d) Incubate on ice for 30min, heat shock in a 42 ° C water bath for 90s, and immediately cool on ice for 1 ~ 2min;

   e) Add 150 μl of anti-LB-free medium, activate at 37 ° C with a shaker at 200 rpm for 1 h, and simultaneously take the corresponding number of Amp-resistant plates in an oven;

   f) Take 30-50 μl of the activated bacterial solution in a pre-heated Amp-resistant plate, spread it evenly, place it for 2 to 3 minutes in the forward direction, and incubate it in a 37 ° C incubator overnight;

   The steps of PCR for picking clones are as follows:

   a) Observe the morphology of the colonies on the plate. When visible to the naked eye and can be distinguished from other colonies, you can pick clones;

   b) In a clean bench, take a corresponding amount of 1.5ml sterile centrifuge tubes on a test tube rack. After labeling, add 6μl of resistant medium to each centrifuge tube and set aside.

   c) Carefully pick larger colonies from the plate with a pipette and pipette in a centrifuge tube with 6 μl of culture medium (for colonies with smaller colonies, pick them out and mix them and place them on a shaker) PCR identification after shaking for 1h);

   d) Take 1μl of the above bacterial solution for PCR identification, and leave the remaining 5μl of the bacterial solution in a 37 ° C oven or shaker;

   After the results of PCR identification came out, positive strains were selected and sent to the test sequence.
5. The preparation method according to claim 2, characterized in that:

   The step (1.5) is:

   The reaction system of RXRα fragment amplification is as follows:

   

   Reaction procedure: (98 ° C / 10s, 55 ° C / 30s, 72 ° C / 1.5min) × 35cycles, agarose gel electrophoresis cut gel to recover RXRα-2A fragment;

   The step (1.6) is:

   PcDNA3.1-EGFP (the homologous sequence when infusion is connected to EGFP complements the BamBI digestion site) with restriction enzyme BamHI, the reaction system is shown in the table below;

   

   The reaction procedure is: 37 ° C, 3h, agarose gel electrophoresis is performed to recover the enzyme-digested skeleton fragments;

   The step (1.7) is:

   Purify the RXRα-2A amplification product and pcDNA3.1-EGFP single enzyme-digested backbone fragment. The two fragments are ligated by Infusion, transformed, plated, picked, and used for bacterial liquid PCR, selected positive colonies, expanded shake culture, protected bacteria to improve Plasmid

   The step (1.8) is:

   Double enzyme digestion verified SpeI + XhoI = 3339bp + 4455bp; single enzyme digestion SpeI = 7794bp.
6. The preparation method according to claim 2, characterized in that the step (2) is: pass SK-N-SH into a culture plate and stably culture; perform when the confluence of the cells in the 6-well plate reaches 80-90% Nuclear transfection; the transfection system is:

   

   The nuclear transfer steps are as follows:

   a) will

   

   Supplement all added

   

   Solution, stored stable at 4 ℃ for 3 months;

   b) Take out a tube of complete culture medium from the refrigerator, preheat it at 37 ° C, then take 1.5ml into a 6-well plate and put it in a 37 ° C incubator;

   c) the cells of the 6-well plate were digested and centrifuged, the medium was removed, and the cells were washed once in PBS;

   d) Use 100μl Supplement

   

   Solution resuspend the cells and mix with a total of 2 μg of plasmid;

   e) Using the dropper provided in the kit, carefully add the cell and plasmid mixture to the provided nuclear spinneret.

   Avoid generating air bubbles. After nuclear transfer using the corresponding program, add 500 μl of pre-warmed culture medium, transfer the cells to a pre-warmed 12-well plate, shake and let stand for at least 24 h;

   f) Observe the next day, take photos to record the adherence of the transfected cells, observe the cell conditions and take photos after changing the fresh medium.
7. The preparation method according to claim 2, wherein the step (3) is:

   After transfection, the cells continued to be cultured. When they grew to 80% -90%, they were plated into 10cm dishes. According to the state of cell culture and transfection efficiency, the plates were plated. The density was controlled in the microscopic field. 7 to 15 cells were observed. Better

   When most single cells divide to 4-8 cells, dosing is started, and the optimal screening concentration of pre-experiment is 300 μg / ml for drug sieving, during which some cells die, and the fresh medium for dosing is replaced every two days; The first day of dosing is recorded as Day0;

   When adding medicine to Day6-Day7, cells will start to die in large numbers. At the same time, some formed monoclonal cell clusters are observed. Among Day8-Day12, according to the situation, select the cell state and the clone shape that are better. Use a gun to gently scrape it down and Aspirate to a 96-well plate and change to fresh medium before picking clones;

   The clones were observed every day after selection. When the cells grew to more than 90% in the 96-well plate, they could be transferred to the 48-well plate and then passed to the 12-well plate in turn.
8. The preparation method according to claim 2, characterized in that:

   The step (4.1) is:

   Collect half of the cells in the 12-well plate to lyse and extract the cell DNA, and use the primers designed previously to expand. When the confluency in the 12-well plate reaches more than 90%, digest the cells in half, and culture the remaining half in the original wells. Centrifuge at 5000 rpm for 10 min, discard the supernatant, and identify the lysate after lysis. The lysis step is as follows:

   a) Centrifuge the 1.5ml centrifuge tube at 5000rpm for 10min, dry the culture medium, and if there is any residue, open the EP tube cover and dry it in an oven at 70 ℃ for 10min;

   b) Lysate formulation: 15 μl DirectPCR reagent + 1 μl proteinaseK + 4 μl H ₂ O = 20 μl; where DirectPCR reagent (Viagen Biotech, Cat # 101-T or # 301-C), proteinase K (from the DNA extraction kit of Tiangen) band);

   c) Add the prepared 20μl lysate to the cell pellet, mix and transfer to 200μl PCR tube, and number it. 55 ℃ / 2h, 85 ℃ / 45min, take 1μl for PCR identification;

   d) the identification is completed, and the positive clones are subjected to key cultivation;

   The reaction system for PCR amplification is as follows:

   

   Reaction program: (98 ℃ / 10s, 55 ℃ / 30s, 72 ℃ / 1.5min) × 35cycles;

   The step (4.2) is:

   Select 6 positive cells and 1 wild type cell for culture, RNA extraction and reverse transcription;

   Resuscitate the cells to a 12-well plate. After the cells are full, digest the cells, wash them with PBS, centrifuge, remove the supernatant, extract RNA from the cells, and perform reverse transcription. The steps are as follows:

   a) After trypsin digests the cells, aspirate the cells and place them in a 1.5ml centrifuge tube, take out the cells, and perform subsequent steps to operate between the molecules;

   b) Centrifuge at 1000 rpm for 5 min and aspirate the supernatant. Add 500 μl of TRIZOL reagent, mix by pipetting up and down, and let stand at room temperature for 5 min;

   c) Add 100 μl of chloroform to Trizol-cell homogenate, cover with a lid and shake vigorously for 15s, and let stand at room temperature for 3min;

   d) Centrifuge at 4 ° C for 15 min at 12,000 rpm, carefully suck the upper aqueous layer into a new centrifuge tube, add 250 μl of isopropanol, 0.5 ml of isopropanol per 1 ml of Trizol, mix by inversion several times, and leave at room temperature for 10 min;

   e) Centrifuge at 12000 rpm for 10 min at 4 ° C and discard the supernatant;

   f) Wash the pellet with 500 μl of 75% ethanol, centrifuge at 7500 rpm for 5 min at 4 ° C, and discard the supernatant;

   g) Dry the precipitate in a clean bench for 5 min. Dissolve the precipitate in 20 μl of enzyme-free water. After measuring the concentration, dilute a portion of the RNA to 250 ng / μl and store at -80 ° C or directly reverse transcription.
9. The preparation method according to claim 2, characterized in that:

   The step (4.3) is:

   Total cell protein was extracted and protein quantification was performed by BCA protein quantification method. After SDS-PAGE gel electrophoresis, the protein was transferred to a PVDF membrane using a semi-dry membrane transfer device, and 5% skimmed milk powder was blocked for 2 hours. RXRα and GAPDH primary antibody were added for dilution. Solution, incubate at room temperature for 2h, wash the membrane with TBST buffer 3 times for 10min each time, dilute HPR-labeled goat anti-rabbit and goat anti-mouse secondary antibodies at 1: 4000 ratio, incubate for 1h at room temperature, wash the membrane 3 times with 10min each time, ECL chemistry The light-emitting kit was used for color development, and the cold CCD imaging system was developed. The Image 2.0 software was used to quantitatively analyze the gray value of the protein band.
10. A RXRα protein stable and highly expressed cell line, characterized in that the cell lines are RXR / TG-SK-N-SH-97 #, RXR / TG-SK-N-SH-202 #, according to claim 1- It is obtained by the preparation method according to any one of 9.

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