WO2019037051A1 - Mcf-7 cell strain for overexpression of hdac6 gene, and construction method therefor - Google Patents

Mcf-7 cell strain for overexpression of hdac6 gene, and construction method therefor Download PDF

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WO2019037051A1
WO2019037051A1 PCT/CN2017/098908 CN2017098908W WO2019037051A1 WO 2019037051 A1 WO2019037051 A1 WO 2019037051A1 CN 2017098908 W CN2017098908 W CN 2017098908W WO 2019037051 A1 WO2019037051 A1 WO 2019037051A1
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hdac6
mcf
gene
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毛吉炎
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深圳市博奥康生物科技有限公司
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  • the present invention relates to the field of cell biotechnology, a MCF-7 cell line overexpressing the HDAC6 gene and a method of constructing the same.
  • HDAC6 also known as B cell activating factor, interacts with its receptor transmembrane activator and chiral cyclophilin ligand (transmembrane activator or calcium-modulating cyclophylin).
  • BAFF-R binds, promotes B cell survival, growth, antigen presentation, and class switching and recombination of immunoglobulins at different stages, playing an important role in B cell survival, proliferation and activation.
  • HDAC6 is mainly derived from bone marrow family cells such as monocytes, giant cells, dendritic cells and the like. Activated T cells also produce HDAC6, and express both BAFF-R and TACI receptors, regulate T cell function, promote T cell activation and survival, and play an important role in T cell-mediated autoimmune diseases.
  • a large amount of potential transformation value requires a lot of research, but the lack of a cell line that overexpresses the HDAC6 gene in the prior art has hindered the progress of related research.
  • the object of the present invention is to overcome the deficiencies of the prior art and to provide a MCF-7 cell line overexpressing the HDAC6 gene to break through the deficiencies in the existing HDAC6 gene research by increasing the HDAC 6 gene in MCF-7 cells.
  • the amount of expression that promotes the role and mechanism of HDAC6 gene is to provide a method for constructing the above MCF-7 cell line overexpressing the HDAC6 gene.
  • the present invention provides a MCF-7 cell line overexpressing the HDAC6 gene, which contains an overexpressed HDAC6 gene. Another object of the present invention is achieved by the following technical solutions:
  • a MCF-7 cell line overexpressing HDAC6 was constructed by constructing a plasmid containing the HDAC6 gene, transfecting the MCF-7 cells with lentivirus, and then screening the stably transfected cell line with puromycin.
  • the construction method of the present invention includes the following steps:
  • a fragment of the target gene HDAC6 was obtained by PCR, and the primers used in the PCR were:
  • Upstream Primer 5'- GTCTAGAATGAGTGGAGCGAACCGCG -3'
  • Downstream primer 5'- GGGATCCTTAGTGTGGGTGGGGCATATC -3';
  • the PCR product is purified and ligated into the pCDH-CMV-MCS-EF1-Puro plasmid as an exogenous DNA fragment, transformed, screened, and recombinantly cloned by PCR to construct a HDAC6 plasmid;
  • the MCF-7 cells were transfected with the target gene lentivirus LV-HDAC6, and the stably transfected cell lines were screened with a concentration of 2 g/ml of puromycin to obtain a MCF-7 cell line overexpressing HDAC6.
  • the present invention breaks the research direction of the prior art on the HDAC6 gene, and overcomes the current lack of related biological reagents for studying the action and mechanism of the HDAC6 gene, by establishing a MCF-7 cell line stably overexpressing HDAC6, This cell line can directly develop the related research of HDAC6 gene, which has the advantages of intuitive, real and stable passage, and is of great significance for the research and development of tumor therapy technology.
  • MCF-7 cell line stably overexpressing HDAC6
  • FIG. 1 Schematic diagram of the expression of HDAC6 gene by fluorescence quantitative PCR after puromycin screening of cells.
  • a fragment of the target gene HDAC6 was obtained by PCR, and the bow used in PCR was:
  • Upstream Primer 5'- GTCTAGAATGAGTGGAGCGAACCGCG -3'
  • Downstream primer 5'- GGGATCCTTAGTGTGGGTGGGGCATATC -3'
  • the PCR product was then purified using a full-gold PCR Cleanup kit, and the purified PCR product was successfully ligated to the pCDH-CMV-MCS-EF1-Puro plasmid as an exogenous DNA fragment: Xba I and Bam HI double-digested PCR fragments And pCDH-CMV-MCS-EFl-Puro plasmid, respectively, were recovered and ligated with T4 DNA Ligase, and 5 ⁇ was used for transformation after transformation; Subsequently, E. coli transformation and recombinant strain screening, PCR screening of recombinant clones were performed. The selected plasmid DNA was extracted, and the plasmid DNA was digested and sequenced to obtain a Nur l plasmid containing the gene of interest.
  • DNA-EndoFectin lentivirus complex was ligated into the cell plate and the vortex plate was gently vortexed to disperse the complex.
  • the cells were cultured overnight (8-14 ⁇ ) at 37 ° C under C02 conditions, and the old medium was replaced with fresh DMEM medium supplemented with 10% heat-inactivated fetal bovine serum. 0.2% volume of TiterBoost titer was added to the medium. The enhancer was continued and cultured at 37 ° C under C02 conditions. The medium containing the DNA-End oFectin lentivirus mixture was replaced within 16 hours after transfection. After 48 hours of transfection, the medium was collected and centrifuged at 500 g for 10 minutes to remove cell debris, and a lentivirus-containing medium was obtained. After centrifugation, to 0.45
  • the medium was filtered with a low protein binding of PNY filter membrane to obtain the target gene lentivirus LV-HDAC6.
  • the minimum lethal concentration of puromycin in the cell was screened by normal MCF-7 cells to be 3 g/mL, and the drug screening of the stably transformed strain was performed at the minimum lethal concentration.
  • MCF-7 cells were counted and plated in 6-well plates, and 2 mL of 1640 medium was added to each well (add 10%).
  • the infected cells were trypsinized and transferred to a 6-well plate (each l-well plate corresponds to one lentivirus, leaving one well for negative control cells) to contain 3 g/ml puromycin 1640 medium (add 10% FBS) for drug screening culture; continuous dosing culture for 7 days (pyromycin concentration 3 g / m l), after the cells are over, get MCF-7 cells stably overexpressing HDAC6, collect The sample is subjected to subsequent testing.
  • Example 2 Fluorescence quantitative PCR was used to detect the expression level of HDAC6 gene.
  • MCF-7 cells overexpressing MCF-7 cells, overexpressing the HDAC6 gene, were plated into 6-well plates, respectively. The cell density reached ⁇ , and the total RNA of each group was extracted with RNeasy Mini Kit. The mRNA was reverse transcribed into cDNA using PrimeScrip RT reagent Kit. Reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, co. After the reverse transcription, add 9 (L RNase Free dH 2 0 diluted cDNA, and store at -20 °C for later detection.
  • the present invention breaks through the research direction of the prior art regarding the HDAC6 gene, and overcomes the current lack of related biological reagents for studying the action and mechanism of the HDAC6 gene by establishing a MCF-7 cell line stably overexpressing HDAC6.
  • This cell line can directly develop the related research of HDAC6 gene, which has the advantages of intuitive, real and stable passage, and is of great significance for the research and development of tumor therapy technology.

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Abstract

Provided are an MCF-7/HDAC6 cell strain for overexpression of HDAC6, and a construction method therefor. The cell strain is MCF-7 and contains a stable overexpression HDAC6 gene.

Description

一种过表达 HDAC6基因的 MCF-7细胞株及其构建方法  MCF-7 cell strain overexpressing HDAC6 gene and construction method thereof
技术领域 Technical field
[0001] 本发明涉及细胞生物技术领域， 一种过表达 HDAC6基因的 MCF-7细胞株及其 构建方法。  [0001] The present invention relates to the field of cell biotechnology, a MCF-7 cell line overexpressing the HDAC6 gene and a method of constructing the same.
背景技术  Background technique
[0002] HDAC6, 又称 B细胞活化因子， 与其受体跨膜激活剂及巧调亲环素配体相互作 用分子 (transmembrane activator or calcium-modulating cyclophylin  [0002] HDAC6, also known as B cell activating factor, interacts with its receptor transmembrane activator and chiral cyclophilin ligand (transmembrane activator or calcium-modulating cyclophylin).
ligand-interactor, TACl) 、B细胞成熟抗原 (cell maturation  Ligand-interactor, TACl), B cell maturation antigen (cell maturation)
antigen, BCMA) 和 B细胞活化因子受体 (B cell-activating factor  Antigen, BCMA) and B cell-activating factor
receptor, BAFF-R) 结合， 促进 B细胞的存活、生长、抗原呈递以及不同阶段免疫 球蛋白的类别转换和重组， 在 B细胞存活、增殖和活化中起重要作用。  Receptor, BAFF-R) binds, promotes B cell survival, growth, antigen presentation, and class switching and recombination of immunoglobulins at different stages, playing an important role in B cell survival, proliferation and activation.
技术问题  technical problem
[0003] HDAC6主要来源于骨髓家族细胞如单核细胞、巨隨细胞、树突状细胞等。 活化 的 T细胞也产生 HDAC6, 且表达 BAFF-R和 TACI两种受体， 调节 T细胞的功能， 促进 T细胞的活化和存活， 在 T细胞介导的自身免疫病中发挥重要作用， 具有较 大的潜在转化价值， 需做大量研究， 但现有技术中缺乏过表达 HDAC6基因的细 胞株， 对相关研究的进展造成了一定的阻碍。  [0003] HDAC6 is mainly derived from bone marrow family cells such as monocytes, giant cells, dendritic cells and the like. Activated T cells also produce HDAC6, and express both BAFF-R and TACI receptors, regulate T cell function, promote T cell activation and survival, and play an important role in T cell-mediated autoimmune diseases. A large amount of potential transformation value requires a lot of research, but the lack of a cell line that overexpresses the HDAC6 gene in the prior art has hindered the progress of related research.
问题的解决方案  Problem solution
技术解决方案  Technical solution
[0004] 本发明的目的在于克服现有技术的不足， 提供一种过表达 HDAC6基因的 MCF- 7细胞株， 以突破现有 HDAC6基因研究中的不足， 通过提高 MCF-7细胞中 HDAC 6基因的表达量， 促进 HDAC6基因作用与机制的研究。 本发明的另一目的在于提 供上述过表达 HDAC6基因的 MCF-7细胞株的构建方法。  [0004] The object of the present invention is to overcome the deficiencies of the prior art and to provide a MCF-7 cell line overexpressing the HDAC6 gene to break through the deficiencies in the existing HDAC6 gene research by increasing the HDAC 6 gene in MCF-7 cells. The amount of expression that promotes the role and mechanism of HDAC6 gene. Another object of the present invention is to provide a method for constructing the above MCF-7 cell line overexpressing the HDAC6 gene.
[0005] 本发明的目的通过以下技术方案予以实现：  [0005] The object of the present invention is achieved by the following technical solutions:
[0006] 本发明提供的一种过表达 HDAC6基因的 MCF-7细胞株， 所述细胞含有过表达 的 HDAC6基因。 [0007] 本发明的另一目的通过以下技术方案予以实现： The present invention provides a MCF-7 cell line overexpressing the HDAC6 gene, which contains an overexpressed HDAC6 gene. Another object of the present invention is achieved by the following technical solutions:
[0008] 本发明提供的上述过表达 HDAC6的 MCF-7细胞株的构建方法如下：  The method for constructing the above-mentioned MCF-7 cell line overexpressing HDAC6 provided by the present invention is as follows:
[0009] 通过构建含目的基因 HDAC6质粒， 经慢病毒包装后转染 MCF-7细胞， 然后用 嘌呤霉素筛选稳定转染细胞株而构建得到过表达 HDAC6的 MCF-7细胞株。  [0009] A MCF-7 cell line overexpressing HDAC6 was constructed by constructing a plasmid containing the HDAC6 gene, transfecting the MCF-7 cells with lentivirus, and then screening the stably transfected cell line with puromycin.
[0010] 进一步地， 本发明构建方法包括以下步骤： [0010] Further, the construction method of the present invention includes the following steps:
[0011] (1) 构建含目的基因 HDAC6质粒 [0011] (1) Construction of the HDAC6 plasmid containing the gene of interest
[0012] 通过 PCR获得目的基因 HDAC6的片段， PCR采用的引物为：  [0012] A fragment of the target gene HDAC6 was obtained by PCR, and the primers used in the PCR were:
[0013] 上游引物： 5'- GTCTAGAATGAGTGGAGCGAACCGCG -3'  [0013] Upstream Primer: 5'- GTCTAGAATGAGTGGAGCGAACCGCG -3'
[0014] 下游引物： 5'- GGGATCCTTAGTGTGGGTGGGGCATATC -3'；  [0014] Downstream primer: 5'- GGGATCCTTAGTGTGGGTGGGGCATATC -3';
[0015] PCR产物经纯化后作为外源 DNA片段与 pCDH-CMV-MCS-EFl-Puro质粒连接， 经转化、 筛选， 通过 PCR重组克隆， 构建得到 HDAC6质粒；  [0015] The PCR product is purified and ligated into the pCDH-CMV-MCS-EF1-Puro plasmid as an exogenous DNA fragment, transformed, screened, and recombinantly cloned by PCR to construct a HDAC6 plasmid;
[0016] (2) 慢病毒包装 [0016] (2) Lentiviral packaging
[0017] 获得目的基因慢病毒 LV-HDAC6; Obtaining the target gene lentivirus LV-HDAC6;
[0018] (3) 转染和筛选 (3) Transfection and screening
[0019] 以目的基因慢病毒 LV-HDAC6转染 MCF-7细胞， 再用嘌呤霉素浓度 2 g/ml筛选 稳定转染细胞株， 得到过表达 HDAC6的 MCF-7细胞株。  [0019] The MCF-7 cells were transfected with the target gene lentivirus LV-HDAC6, and the stably transfected cell lines were screened with a concentration of 2 g/ml of puromycin to obtain a MCF-7 cell line overexpressing HDAC6.
发明的有益效果  Advantageous effects of the invention
有益效果  Beneficial effect
[0020] 本发明突破了现有技术关于 HDAC6基因的研究方向， 克服了目前缺乏用于研 究 HDAC6基因作用与机制的相关生物试剂的问题， 通过建立稳定过表达 HDAC6 的 MCF-7细胞株， 以此细胞株可直接幵展 HDAC6基因的相关研究， 具有直观、 真实、 可稳定传代等优点， 对于肿瘤治疗技术的研究和发展具有重要意义。 对附图的简要说明  [0020] The present invention breaks the research direction of the prior art on the HDAC6 gene, and overcomes the current lack of related biological reagents for studying the action and mechanism of the HDAC6 gene, by establishing a MCF-7 cell line stably overexpressing HDAC6, This cell line can directly develop the related research of HDAC6 gene, which has the advantages of intuitive, real and stable passage, and is of great significance for the research and development of tumor therapy technology. Brief description of the drawing
附图说明  DRAWINGS
[0021] 图 1嘌呤霉素筛选细胞后荧光定量 PCR检测 HDAC6基因表达结果示意图。  [0021] Fig. 1 Schematic diagram of the expression of HDAC6 gene by fluorescence quantitative PCR after puromycin screening of cells.
实施该发明的最佳实施例  BEST MODE FOR CARRYING OUT THE INVENTION
本发明的最佳实施方式 [0022] 实施例一构建过表达 HDAC6的 MCF-7细胞株 BEST MODE FOR CARRYING OUT THE INVENTION Example 1 Construction of MCF-7 cell line overexpressing HDAC6
[0023] (1) 构建质粒 (1) constructing a plasmid
[0024] 通过 PCR获得目的基因 HDAC6的片段， PCR采用的弓 |物为：  [0024] A fragment of the target gene HDAC6 was obtained by PCR, and the bow used in PCR was:
[0025] 上游引物： 5'- GTCTAGAATGAGTGGAGCGAACCGCG -3' [0025] Upstream Primer: 5'- GTCTAGAATGAGTGGAGCGAACCGCG -3'
[0026] 下游引物： 5'- GGGATCCTTAGTGTGGGTGGGGCATATC -3' [0026] Downstream primer: 5'- GGGATCCTTAGTGTGGGTGGGGCATATC -3'
[0027] 然后用全式金的 PCR Cleanup试剂盒纯化 PCR产物， 纯化 PCR产物成功后作为外 源 DNA片段与 pCDH-CMV-MCS-EFl-Puro质粒连接： Xba I和 Bam HI双酶切 PCR 片段和 pCDH-CMV-MCS-EFl-Puro质粒后， 分别回收并用 T4 DNA Ligase进行连 接， 连接后取 5 μΐ用于转化； 随后进行大肠杆菌的转化及重组菌的筛选、 PCR法 筛选基因重组克隆， 提取筛选后的质粒 DNA、 对质粒 DNA进行酶切及测序， 得 到含目的基因 Nurr l质粒。 [0027] The PCR product was then purified using a full-gold PCR Cleanup kit, and the purified PCR product was successfully ligated to the pCDH-CMV-MCS-EF1-Puro plasmid as an exogenous DNA fragment: Xba I and Bam HI double-digested PCR fragments And pCDH-CMV-MCS-EFl-Puro plasmid, respectively, were recovered and ligated with T4 DNA Ligase, and 5 μΐ was used for transformation after transformation; Subsequently, E. coli transformation and recombinant strain screening, PCR screening of recombinant clones were performed. The selected plasmid DNA was extracted, and the plasmid DNA was digested and sequenced to obtain a Nur l plasmid containing the gene of interest.
[0028] (2) 病毒包装 [0028] (2) Virus packaging
[0029] 在进行转染前， 提前两天将 13-15万的 293T细胞移入 10 cm培养皿中进行培养， 板内加入 10ml含 10%热灭活胎牛血清的 DMEM培养基， 使细胞在进行转染吋达 到 70〜80%融合率。 在 5%C02条件下， 37°C培养细胞。 通过包装细胞使慢病毒 质粒增殖。  [0029] Before transfection, 13-15 million 293T cells were transferred to a 10 cm culture dish for culture two days in advance, and 10 ml of DMEM medium containing 10% heat-inactivated fetal bovine serum was added to the plate to make the cells in Transfection 进行 achieved a fusion rate of 70 to 80%. The cells were cultured at 37 ° C under 5% CO 2 conditions. The lentiviral plasmid is propagated by packaging the cells.
[0030] 往无菌 EP管加入上述含目的基因 HDAC6的 ORF/shRNA表达质粒 2.5 g、 LentiPa cHIV混合试剂 （GeneCopoeia) 5.0 μΐ以 Opti-MEMI稀释至 200 μ1。 在另一无菌 ΕΡ 管内， 以 Opti-MEMI稀释 15 μΐ EndoFectin转染试剂 （GeneCopoeia) 。 边轻柔进 行涡旋， 边往 DNA和 LentiPacHIV试剂的混合溶液中滴加以上稀 EndoFectin转染 试剂。 室温培养该溶液 10-25 min， 使 DNA-EndoFectin混合物产生。  [0030] To the sterile EP tube, the above ORF/shRNA expression plasmid containing the desired gene HDAC6 2.5 g, LentiPa cHIV mixed reagent (GeneCopoeia) 5.0 μΐ was diluted to 200 μl with Opti-MEMI. In another sterile tube, 15 μΐ EndoFectin Transfection Reagent (GeneCopoeia) was diluted with Opti-MEMI. While gently swirling, add the diluted EndoFectin transfection reagent to the mixed solution of DNA and LentiPacHIV reagent. The solution was incubated at room temperature for 10-25 min to produce a DNA-EndoFectin mixture.
[0031] 将 DNA-EndoFectin慢病毒复合物接加入细胞板， 轻柔涡旋平板使复合物分散。  [0031] The DNA-EndoFectin lentivirus complex was ligated into the cell plate and the vortex plate was gently vortexed to disperse the complex.
细胞在 C02条件下， 37°C过夜培养 （8-14小吋） ， 以加入 10 %热灭活胎牛血清的 新鲜 DMEM培养基更换旧培养基， 培养基中加入 0.2%体积的 TiterBoost滴度增强 剂并继续在 C02条件下， 以 37°C继续培养。 转染后的 16小吋内， 替换含 DNA-End oFectin慢病毒混合物的培养基。 转染 48小吋后收集培养基， 以 500 g离心 10分钟 去除细胞碎片， 可获得含慢病毒的培养基。 离心后， 以 0.45  The cells were cultured overnight (8-14 吋) at 37 ° C under C02 conditions, and the old medium was replaced with fresh DMEM medium supplemented with 10% heat-inactivated fetal bovine serum. 0.2% volume of TiterBoost titer was added to the medium. The enhancer was continued and cultured at 37 ° C under C02 conditions. The medium containing the DNA-End oFectin lentivirus mixture was replaced within 16 hours after transfection. After 48 hours of transfection, the medium was collected and centrifuged at 500 g for 10 minutes to remove cell debris, and a lentivirus-containing medium was obtained. After centrifugation, to 0.45
μηι的低蛋白结合 PES过滤膜过滤该培养基， 得到目的基因慢病毒 LV-HDAC6。 [0032] (3) 转染和筛选 The medium was filtered with a low protein binding of PNY filter membrane to obtain the target gene lentivirus LV-HDAC6. (3) Transfection and screening
[0033] 采用正常的 MCF-7细胞筛选出该细胞嘌呤霉素的最小致死浓度为 3 g/mL， 按最 小致死浓度进行稳转株的药物筛选。  [0033] The minimum lethal concentration of puromycin in the cell was screened by normal MCF-7 cells to be 3 g/mL, and the drug screening of the stably transformed strain was performed at the minimum lethal concentration.
[0034] 将 MCF-7细胞计数后铺到 6孔板中培养， 各孔加入 2 mL1640培养基 （添加 10%[0034] MCF-7 cells were counted and plated in 6-well plates, and 2 mL of 1640 medium was added to each well (add 10%).
FBS) ， 在 5% FBS) at 5%
C02条件下， 37°C培养 24h; 铺板 24h后每孔加入目的基因慢病毒 LV-HDAC6 5(V1 ， 培养 48h。  Under the condition of C02, culture at 37 °C for 24 h; after 24 h of plating, add the target gene lentivirus LV-HDAC6 5 (V1, culture for 48 h.
[0035] 用胰酶消化感染过的细胞并转移至 6孔板 （每块 6孔板对应一种慢病毒， 留出一 孔用于阴性对照细胞） ， 以含 3 g/ml嘌呤霉素的 1640培养基 （添加 10%FBS) 进 行药筛培养； 连续加药培养 7天 （嘌呤霉素浓度 3 g/ml) ， 待细胞长满后， 得到 稳定过表达 HDAC6的 MCF-7细胞， 收取样品进行后续检测。 [0035] The infected cells were trypsinized and transferred to a 6-well plate (each l-well plate corresponds to one lentivirus, leaving one well for negative control cells) to contain 3 g/ml puromycin 1640 medium (add 10% FBS) for drug screening culture; continuous dosing culture for 7 days (pyromycin concentration 3 g / m l), after the cells are over, get MCF-7 cells stably overexpressing HDAC6, collect The sample is subjected to subsequent testing.
[0036] 实施例二荧光定量 PCR检测 HDAC6基因表达量。  [0036] Example 2 Fluorescence quantitative PCR was used to detect the expression level of HDAC6 gene.
[0037] 分别接种 MCF-7细胞、 过表达 HDAC6基因的 MCF-7细胞至 6孔板。 细胞密度达 到 δΟ^^Ο^吋， 用 RNeasy Mini Kit提取各组细胞的总 RNA， 利用 PrimeScrip RT reagent Kit将mRNA逆转录为cDNA， 逆转录条件： 37°C， 15min; 85°C， 5s; 4°C ， co。 反转录结束后， 加入 9( L的 RNase Free dH 20稀释 cDNA， -20°C保存， 以 便后面检测使用。 取各组细胞的 cDNA ^l为模板， 以 GAPDH为内参， 实吋荧光 定量 PCR检测 HDAC6相对表达量， 设置反应条件： 95°C 30s， 1循环， 54°C 30s 40循环， 95。C 5s， 60°C lmin, 95。C [0037] MCF-7 cells overexpressing MCF-7 cells, overexpressing the HDAC6 gene, were plated into 6-well plates, respectively. The cell density reached δΟ^^Ο^吋, and the total RNA of each group was extracted with RNeasy Mini Kit. The mRNA was reverse transcribed into cDNA using PrimeScrip RT reagent Kit. Reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, co. After the reverse transcription, add 9 (L RNase Free dH 2 0 diluted cDNA, and store at -20 °C for later detection. Take cDNA from each group as template, GAPDH as internal reference, real fluorescence quantitative PCR to detect the relative expression of HDAC6, set the reaction conditions: 95 ° C 30s, 1 cycle, 54 ° C 30s 40 cycles, 95 ° C 5s, 60 ° C lmin, 95 ° C
15s , 结果如图 1所示。 可以看到， 过表达 HDAC6基因的 MCF-7的 HDAC6基因表 达量较正常 MCF-7细胞有 50倍以上的升高， 说明本发明提供的 HDAC6基因 cDNA 序列成功插入至 pCDH-CMV-MCS-EFl-Puro表达载体中， 能特异、 持续、 高效、 稳定地促进 HDAC6基因高表达。  15s, the result is shown in Figure 1. It can be seen that the expression level of HDAC6 gene of MCF-7 overexpressing HDAC6 gene is more than 50-fold higher than that of normal MCF-7 cells, indicating that the HDAC6 gene cDNA sequence provided by the present invention is successfully inserted into pCDH-CMV-MCS-EFl. -Puro expression vector can promote high expression of HDAC6 gene specifically, continuously, efficiently and stably.
工业实用性  Industrial applicability
[0038] 本发明突破了现有技术关于 HDAC6基因的研究方向， 克服了目前缺乏用于研 究 HDAC6基因作用与机制的相关生物试剂的问题， 通过建立稳定过表达 HDAC6 的 MCF-7细胞株， 以此细胞株可直接幵展 HDAC6基因的相关研究， 具有直观、 真实、 可稳定传代等优点， 对于肿瘤治疗技术的研究和发展具有重要意义。  The present invention breaks through the research direction of the prior art regarding the HDAC6 gene, and overcomes the current lack of related biological reagents for studying the action and mechanism of the HDAC6 gene by establishing a MCF-7 cell line stably overexpressing HDAC6. This cell line can directly develop the related research of HDAC6 gene, which has the advantages of intuitive, real and stable passage, and is of great significance for the research and development of tumor therapy technology.

Claims

权利要求书 [权利要求 1] 一种过表达 HDAC6的 MCF-7/HDAC6细胞株， 其特征在于： 所述细胞 含有过表达的 HDAC6基因， 所述细胞是 MCF-7。 [权利要求 2] 权利要求 1所述过表达 HDAC6的 MCF-7/HDAC6细胞株的构建方法， 其特征在于： 通过构建含目的基因 HDAC6质粒， 经慢病毒包装后转 染 MCF-7细胞， 然后用嘌呤霉素筛选稳定转染细胞株而构建得到过表 达 HDAC6的 MCF-7细胞株。 [权利要求 3] 根据权利要求 2所述的过表达 HDAC6的 MCF-7/HDAC6细胞株的构建 方法， 其特征在于包括以下步骤： Claims [Claim 1] A MCF-7/HDAC6 cell line overexpressing HDAC6, characterized in that the cell contains an overexpressed HDAC6 gene, and the cell is MCF-7. [Claim 2] The method for constructing a MCF-7/HDAC6 cell line overexpressing HDAC6 according to claim 1, wherein: by constructing a plasmid containing the HDAC6 gene of interest, transfecting the MCF-7 cells by lentivirus packaging, and then A MCF-7 cell line overexpressing HDAC6 was constructed by screening a stably transfected cell line with puromycin. [Claim 3] The method for constructing a MCF-7/HDAC6 cell line overexpressing HDAC6 according to claim 2, comprising the steps of:
(1) 构建含目的基因 HDAC6质粒  (1) Construction of the HDAC6 plasmid containing the gene of interest
通过 PCR获得目的基因 HDAC6的片段， PCR采用的引物为： 上游引物： 5'- GTCTAGAATGAGTGGAGCGAACCGCG -3' 下游引物： 5'- GGGATCCTTAGTGTGGGTGGGGCATATC -3'； PCR产物经纯化后作为外源 DNA片段与 pCDH-CMV-MCS-EF 1 -Puro质 粒连接， 经转化、 筛选， 通过 PCR重组克隆， 构建得到 HDAC6质粒  The target gene HDAC6 fragment was obtained by PCR. The primers used for PCR were: upstream primer: 5'- GTCTAGAATGAGTGGAGCGAACCGCG -3' downstream primer: 5'- GGGATCCTTAGTGTGGGTGGGGCATATC -3'; PCR product was purified as exogenous DNA fragment and pCDH-CMV -MCS-EF 1 -Puro plasmid ligation, transformation, screening, recombinant ligation by PCR, construction of HDAC6 plasmid
(2) 慢病毒包装 (2) Lentiviral packaging
获得目的基因慢病毒 LV-HDAC6;  Obtaining the target gene lentivirus LV-HDAC6;
(3) 转染和筛选  (3) Transfection and screening
以目的基因慢病毒 LV-HDAC6转染 MCF-7细胞， 再用嘌呤霉素浓度 3 g/ml筛选稳定转染细胞株， 得到过表达 HDAC6的 MCF-7细胞株。  The MCF-7 cells were transfected with the target gene lentivirus LV-HDAC6, and the stably transfected cell lines were screened with a concentration of 3 g/ml of puromycin. The MCF-7 cell line overexpressing HDAC6 was obtained.
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