WO2017214942A1 - Lentiviral expression vector for improving expression of tctp gene, and applications thereof - Google Patents
Lentiviral expression vector for improving expression of tctp gene, and applications thereof Download PDFInfo
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- WO2017214942A1 WO2017214942A1 PCT/CN2016/086066 CN2016086066W WO2017214942A1 WO 2017214942 A1 WO2017214942 A1 WO 2017214942A1 CN 2016086066 W CN2016086066 W CN 2016086066W WO 2017214942 A1 WO2017214942 A1 WO 2017214942A1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/867—Retroviral vectors
Definitions
- the present invention belongs to the field of genetic engineering technology, and particularly relates to a lentiviral expression vector for enhancing expression of a TCTP gene and an application thereof.
- Diabetes is a group of chronic non-infectious, metabolic diseases characterized by elevated blood glucose levels that are a serious hazard to human health. Whether in developed or developing countries, diabetes has become an epidemic in modern society and has become a worldwide public health problem that poses a serious threat to human health. According to the World Health Organization
- type 2 diabetes T2DM
- type 2 diabetes T2DM
- the prevalence of type 2 diabetes has increased dramatically in countries around the world, and the surge in type 2 diabetes is the leading cause of the dramatic increase in the total number of diabetic patients worldwide.
- the prevalence of diabetes in adults is close to 10%, and type 2 diabetes accounts for about 90% of all diabetic patients.
- TCTP is a protein widely expressed in eukaryotes. Its structure is highly conserved and participates in a series of cellular physiological functions such as cell cycle, apoptosis, protein translation, cell morphology, etc. Death protein. Recent studies have shown that TCTP plays an important role in the survival of pancreatic islet ⁇ cells and needs further study. However, the lack of lentiviral expression vectors that specifically promote the high expression of TCTP gene in the prior art makes the relevant studies not well developed.
- the present invention provides a lentiviral expression vector which specifically promotes high expression of a TCTP gene, including a basic sequence of a pLVX-IRE S-puro expression vector, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence, and a T CTP a gene cDNA sequence; the multiple cloning site comprises an EcoR I cleavage site and a Spe l cleavage site, and the TCTP gene cDNA sequence comprises an EcoR I cleavage site, a TCTP gene coding sequence, and a Spe I cleavage site The TCTP gene cDNA sequence is positively inserted into the multiple cloning site sequence.
- the lentiviral expression vector constructed by inserting the TCTP gene cDNA sequence into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase TCTP.
- the advantages of gene expression can be used as a powerful tool in the preparation and treatment of drugs for the treatment of TCTP gene expression in diseases such as Alzheimer's disease.
- the TCTP gene coding sequence is obtained by PCR amplification
- the PCR primer comprises an upstream primer and a downstream primer
- the sequence of the upstream primer is: 5'-
- GGAATTCATGATTATCTACCGGGACCTC -3 ie SEQ ID NO: 1
- sequence of the downstream primer is: 5,- GACTAGTCTAAAGATGTATTCCCCAATTC -3', ie SEQ ID NO: 2
- the TCTP gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the TCTP gene, which reduces the cost of sequence synthesis and lowers the cost.
- the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of a TCTP gene, comprising the following steps:
- A) TCTP gene primer design According to the TCTP gene coding sequence, using Oligo 7 analysis, select 5'-GGAATTCATGATTATCTACCGGGACCTC-3', ie SEQ ID NO: 1 as the upstream bow, select 5 '- GACTAGTCTAAAGATGTATTCCCCAATTC -3, , that is, SEQ ID NO: 2 as a downstream primer, and then synthesizing the upstream primer and the downstream primer; the upstream primer and the downstream primer have no primer dimer, and the annealing temperature difference is small;
- coli was identified by liquid LB medium, and the pGM-T vector carrying the TCTP gene cDNA sequence was extracted and digested with the restriction enzymes EcoR I and Spe I, electrophoresis and gelatinization. Recover a fragment of about 500 bp, which is the TCTP gene cDNA sequence;
- the ligation product is obtained, and the ligation product is transformed into competent E. coli ToplO, uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid is picked and subjected to preliminary identification by PCR, and preliminary identification is performed.
- the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of TCTP gene, and after successful identification, packaging into a virus and transfecting into INS-1 cells, and screening cells with puromycin, using real-time fluorescence quantitative
- the PCR and Western Blot techniques were used to verify the changes of TCTP gene expression from mRNA and protein levels, respectively.
- the experimental results showed that the TCTP gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IR ES-Puro expression vector, which was specific, sustained, efficient and stable. Promote high expression of TCTP gene.
- the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of a TCTP gene for the preparation of a medicament for treating a disease associated with abnormal expression of a TCTP gene.
- the lentiviral expression vector which specifically promotes the high expression of the TCTP gene provided by the invention has the advantages of high transfection efficiency, low dosage, specific, sustained, high efficiency and stable promotion of high expression of the TCTP gene, and can be used as a powerful tool.
- the present invention also provides specific promotion of TCTP
- 1 is a plasmid map of the pLVX-IRES-Puro expression vector.
- FIG. 2 is a schematic diagram showing the results of fluorescent quantitative PCR detection after puromycin screening of cells.
- INS-1 cells were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences. 293FT cells were purchased from Thermo Fisher, Premix PrimeSTAR HS enzyme, lentiviral expression vector pLVX-IRES-Puro, virus packaging auxiliary kit, Lenti-X GoStix reagent. Boxes are purchased from Takara, RNeasy Mini
- Kit was purchased from QIAGEN, pGM-T was purchased from Tiangen, and Endo-Free Plasmid Mini Kit II was purchased from Omega bio-tek.
- Example 2 Construction of a lentiviral vector that specifically promotes high expression of TCTP gene
- the coding sequence of the TCTP gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A tail reaction, and then ligated to pGM-T with T4 DNA ligase.
- the ligation product (TCTP-T vector) was obtained on the vector, and the ligation product was transformed into competent E. coli To plO, uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h.
- Negative control group 1 consistentt cells were uniformly coated on ampicillin-free plates
- negative control group 2 peripheral cells were uniformly coated in 100
- positive control group 1 the connection product of the double enzyme-cut empty vector was evenly coated in 100
- positive control group 2 (the empty carrier was evenly spread on a plate containing 100 g/mL ampicillin).
- the experimental group grew a single colony, the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, the positive control group 2 did not grow colonies.
- the bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the TCTP gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly used with EcoR I enzyme and Spe I, respectively.
- the enzyme was double-digested, electrophoresed and recovered, and the product was recovered by T4 DNA ligase, and then transformed into competent E. coli ToplO, uniformly coated on ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h.
- the negative control group 1 was set up (the competent cells were uniformly coated on the ampicillin-free plate), and the negative control group 2 (the competent cells were uniformly coated on the plate containing 100 g/ml ampicillin), Positive control group 1 (the ligation product of the double enzyme-cut empty vector was uniformly coated on a plate containing 100 g/ml ampicillin), positive control group 2 (the empty vector was uniformly coated on 100 g/mL ampicillin) On the tablet).
- the experimental group grew a single colony, and the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, and the positive control group 2 did not grow colonies.
- 293FT cells were cultured, and cells grown in good condition were inoculated into six wells, 1,000,000 cells per well, and the extracted recombinant plasmid pLVX-TCTP 2 ⁇ ⁇ was transfected into 293FT cells using a lentiviral packaging auxiliary kit. After 48 h, the virus-containing supernatant medium was collected, and the virus solution was filtered through a sieve of 0.45 ⁇ m for infection of INS-1 cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 50,000,000 IFU.
- Inoculated INS-1 cells in a 6-well plate 1000000 cells per well. After 12 hours, the cell density was about 50 ⁇ 3 ⁇ 4, and the virus solution was taken separately. The virus was diluted 10 times with DMEM complete medium, and then polyglycolamine was added. (polybrene) The final concentration was 8 g/mL. The medium in the 6-well plate was added to the virus-containing DMEM complete medium (containing 10% fetal bovine serum). After 24 hours, the virus-containing DMEM complete medium was discarded, and the fresh DMEM complete medium was replaced. After 24 hours, 0.5 was used. The cells were screened at a g/ml concentration of puromycin. After 10 days of screening, the medium was changed once every 3 days, and the concentration of puromycin was continuously increased to 1.00 g/ml.
- Example 5 Fluorescence quantitative PCR was used to detect the expression level of TCTP gene.
- INS-1 cells, pLVX empty vector control INS-1 cell group, and pLVX-TCTP high expressing cells were inoculated to a 6-well plate, respectively.
- the cell density reached 80 ⁇ 3 ⁇ 4-90 ⁇ 3 ⁇ 4 ⁇ , and the total RNA of each group was extracted with RNeasy Mini Kit, and PrimeScrip RT reagent was used for He 1 J.
- Kit reverse transcribes mRNA into cDNA, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ⁇ . After the end of reverse transcription, add 9 (L of RNase Free dH 2 0 diluted cDNA, and store at -20 ° C for later detection. Take the cDNA of each group of cells.
- the expression level of TCTP gene is 60-fold higher than that of INS-1 cells, whether it is just after screening or after 20 generations of pLVX-TCTP cells, and the TCTP gene of pLVX empty vector cells.
- the expression level of the TCTP gene was successfully inserted into the pLVX-IRES-Puro expression vector, which promoted the high expression of TCTP gene specifically, continuously, efficiently and stably.
- the lentiviral expression vector provided by the invention specifically promoting the high expression of the TCTP gene has high transfection efficiency. It has the advantages of low dosage, specific, sustained, efficient and stable promotion of high expression of TCTP gene, and can be used as a powerful tool in drug research and development related to TCTP.
- the present invention also provides specific promotion of high expression of TCTP gene.
- the construction method of lentiviral expression vector has good operation effect and reduces the cost of sequence synthesis
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Abstract
A lentiviral expression vector for specifically promoting high expression of a TCTP gene comprises a fundamental sequence, a resistance gene sequence, a multiple-cloning-sites sequence, a promoter sequence, and a TCTP gene cDNA sequence of a pLVX-IRES-puro expression vector. The multiple cloning sites comprise an EcoR I enzyme cutting site and an Spe I enzyme cutting site, the TCTP gene cDNA sequence comprises an EcoR I enzyme cutting site, a TCTP gene coding sequence and an Spe I enzyme cutting site, and the TCTP gene cDNA sequence is forwardly inserted into the multiple-cloning-sites sequence. The lentiviral expression vector can serve as a tool applied to the research and development of drugs related to TCTP.
Description
提升 TCTP基因表达的慢病毒表达载体及其应用 技术领域 Lentiviral expression vector for enhancing TCTP gene expression and application thereof
[0001] 本发明属于基因工程技术领域, 尤其涉及一种提升 TCTP基因表达的慢病毒表 达载体及其应用。 [0001] The present invention belongs to the field of genetic engineering technology, and particularly relates to a lentiviral expression vector for enhancing expression of a TCTP gene and an application thereof.
背景技术 Background technique
[0002] 糖尿病是一组以血葡萄糖水平增高为特征的、 严重危害人类健康的慢性非传染 性、 代谢性疾病。 无论是在发达国家还是发展中国家, 糖尿病成为现代社会的 流行病, 已成为严重威胁人类健康的世界性公共卫生问题。 按照世界卫生组织 [0002] Diabetes is a group of chronic non-infectious, metabolic diseases characterized by elevated blood glucose levels that are a serious hazard to human health. Whether in developed or developing countries, diabetes has become an epidemic in modern society and has become a worldwide public health problem that poses a serious threat to human health. According to the World Health Organization
(WHO) 及国际糖尿病联盟 (IDF) 专家组的建议, 糖尿病可分为 1型、 2型、 其他特殊类型及妊娠糖尿病 4种, 其中 2型糖尿病 (T2DM) 是糖尿病人群的主体 。 近年来, 世界各国 2型糖尿病的患病率均有急剧增加的趋势, 2型糖尿病患者 激增是造成全世界糖尿病患者总数剧增的主要原因。 据近年大量流行病学调査 结果显示, 糖尿病在成人中的患病率已接近 10%, 其中 2型糖尿病占全体糖尿病 患者的 90%左右。 WHO统计与预测的结果如下: 1994年糖尿病患者人数为 1.20 亿, 1997年为 1.35亿, 2000年为 1.75亿, 2010年为 2.39亿, 2025年预计将突破 3亿 。 而且近三、 五十年内 2型糖尿病急剧增加的趋势仍将继续。 According to the recommendations of the (WHO) and International Diabetes Federation (IDF) expert groups, diabetes can be divided into type 1, type 2, other special types and gestational diabetes. Among them, type 2 diabetes (T2DM) is the main body of diabetes. In recent years, the prevalence of type 2 diabetes has increased dramatically in countries around the world, and the surge in type 2 diabetes is the leading cause of the dramatic increase in the total number of diabetic patients worldwide. According to a large number of epidemiological surveys in recent years, the prevalence of diabetes in adults is close to 10%, and type 2 diabetes accounts for about 90% of all diabetic patients. The results of WHO statistics and predictions are as follows: In 1994, the number of people with diabetes was 120 million, in 1997 it was 135 million, in 2000 it was 175 million, in 2010 it was 239 million, and in 2025 it is expected to exceed 300 million. And the trend of a sharp increase in type 2 diabetes in the last three or fifty years will continue.
技术问题 technical problem
[0003] TCTP是一种广泛表达于真核生物的蛋白质, 其结构高度保守, 参与了一系列 的细胞生理功能, 如细胞周期, 凋亡, 蛋白翻译, 细胞形态等, 而且是一种抗 凋亡蛋白。 最近有研究显示, TCTP在胰岛 β细胞的生存中扮演着重要的角色, 需要进一步深入研究, 但现有技术缺乏特异促进 TCTP基因高表达的慢病毒表达 载体使得相关研究无法很好地幵展。 [0003] TCTP is a protein widely expressed in eukaryotes. Its structure is highly conserved and participates in a series of cellular physiological functions such as cell cycle, apoptosis, protein translation, cell morphology, etc. Death protein. Recent studies have shown that TCTP plays an important role in the survival of pancreatic islet β cells and needs further study. However, the lack of lentiviral expression vectors that specifically promote the high expression of TCTP gene in the prior art makes the relevant studies not well developed.
问题的解决方案 Problem solution
技术解决方案 Technical solution
[0004] 为解决现有技术中存在的问题, 发明人在载体的选择、 重组构建方法等方面进 行了大量的探索研究, 发现将包含 EcoR I酶切位点和 Spe
I酶切位点的 TCTP基因 cDNA序列插入 pLVX-IRES-Puro表达载体的多克隆位点中 可成功构建特异促进 TCTP基因高表达的慢病毒表达载体, 从而完成本发明。 [0004] In order to solve the problems existing in the prior art, the inventors conducted extensive research on the selection of vectors, the method of recombinant construction, and the like, and found that it will contain EcoR I cleavage sites and Spe. The TCTP gene cDNA sequence of the I cleavage site was inserted into the multiple cloning site of the pLVX-IRES-Puro expression vector to construct a lentiviral expression vector which specifically promotes high expression of the TCTP gene, thereby completing the present invention.
[0005] 本发明提供一种特异促进 TCTP基因高表达的慢病毒表达载体, 包括 pLVX-IRE S-puro表达载体的基本序列、 抗性基因序列、 多克隆位点序列、 启动子序列和 T CTP基因 cDNA序列; 所述多克隆位点包括 EcoR I酶切位点和 Spe l酶切位点, 所 述 TCTP基因 cDNA序列包括 EcoR I酶切位点、 TCTP基因编码序列和 Spe I酶切位 点, 所述 TCTP基因 cDNA序列正向插入所述多克隆位点序列中。 The present invention provides a lentiviral expression vector which specifically promotes high expression of a TCTP gene, including a basic sequence of a pLVX-IRE S-puro expression vector, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence, and a T CTP a gene cDNA sequence; the multiple cloning site comprises an EcoR I cleavage site and a Spe l cleavage site, and the TCTP gene cDNA sequence comprises an EcoR I cleavage site, a TCTP gene coding sequence, and a Spe I cleavage site The TCTP gene cDNA sequence is positively inserted into the multiple cloning site sequence.
[0006] 采用上述技术方案, 本发明提供的 TCTP基因 cDNA序列插入 pLVX-IRES-Puro 表达载体构建得到的慢病毒表达载体具有转染效率高, 用量少, 可持续、 高效 、 稳定地提高 TCTP基因表达的优点, 可作为有力工具应用于制备治疗 TCTP基因 表达对阿尔兹海默症等疾病药物的研究和幵发中。 [0006] According to the above technical solution, the lentiviral expression vector constructed by inserting the TCTP gene cDNA sequence into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase TCTP. The advantages of gene expression can be used as a powerful tool in the preparation and treatment of drugs for the treatment of TCTP gene expression in diseases such as Alzheimer's disease.
[0007] 作为本发明的进一步改进, 所述 TCTP基因编码序列通过 PCR扩增获得, PCR引 物包括上游引物和下游引物, 所述上游引物的序列为: 5'- As a further improvement of the present invention, the TCTP gene coding sequence is obtained by PCR amplification, and the PCR primer comprises an upstream primer and a downstream primer, and the sequence of the upstream primer is: 5'-
GGAATTCATGATTATCTACCGGGACCTC -3,, 即 SEQ ID NO: 1, 所述下游引 物的序列为: 5,- GACTAGTCTAAAGATGTATTCCCCAATTC -3', 即 SEQ IDGGAATTCATGATTATCTACCGGGACCTC -3, ie SEQ ID NO: 1, the sequence of the downstream primer is: 5,- GACTAGTCTAAAGATGTATTCCCCAATTC -3', ie SEQ ID
NO: 2。 采用上述 PCR引物序列, 通过 PCR可以扩增出 TCTP基因编码序列, 并 可成功插入至 pLVX-IRES-Puro表达载体中持续表达 TCTP基因, 减少了序列合成 费用, 成本较低。 NO: 2. Using the above PCR primer sequence, the TCTP gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the TCTP gene, which reduces the cost of sequence synthesis and lowers the cost.
[0008] 相应的, 本发明还提供特异促进 TCTP基因高表达的慢病毒表达载体的构建方 法, 包括如下步骤: Correspondingly, the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of a TCTP gene, comprising the following steps:
[0009] A) TCTP基因引物设计: 根据 TCTP基因编码序列, 使用 Oligo 7分析后选取 5'- GGAATTCATGATTATCTACCGGGACCTC -3', 即 SEQ ID NO: 1作为上游弓 |物 , 选取 5 '- GACTAGTCTAAAGATGTATTCCCCAATTC -3,, 即 SEQ ID NO: 2作 为下游引物, 然后合成所述上游引物和所述下游引物; 所述上游引物和所述下 游引物无引物二聚体, 且退火温度差距较小; [0009] A) TCTP gene primer design: According to the TCTP gene coding sequence, using Oligo 7 analysis, select 5'-GGAATTCATGATTATCTACCGGGACCTC-3', ie SEQ ID NO: 1 as the upstream bow, select 5 '- GACTAGTCTAAAGATGTATTCCCCAATTC -3, , that is, SEQ ID NO: 2 as a downstream primer, and then synthesizing the upstream primer and the downstream primer; the upstream primer and the downstream primer have no primer dimer, and the annealing temperature difference is small;
[0010] B) TCTP基因 cDNA序列的获得: 用所述上游引物和所述下游引物进行 PCR扩 增, 获得大量 TCTP基因编码序列, 然后将该序列进行加 A尾反应后, 用 T4 DNA 连接酶连接到 pGM-T载体上得到连接产物, 将该连接产物转化到感受态大肠杆
菌 ToplO中, 均匀涂布到含氨苄青霉素 LB培养基平板上, 挑取阳性单克隆菌落培 养保存菌液并进行 PCR初步鉴定, 将初步鉴定结果说明 TCTP基因 cDNA序列插入 成功的菌液进行测序鉴定; 用液体 LB培养基培养测序鉴定正确的大肠杆菌, 并 抽提其中带 TCTP基因 cDNA序列的 pGM-T载体, 用限制性内切酶 EcoR I酶和 Spe I酶双酶切, 电泳、 切胶回收 500 bp左右的片段, 此片段即为 TCTP基因 cDNA序 列; [0010] B) obtaining the TCTP gene cDNA sequence: PCR amplification using the upstream primer and the downstream primer to obtain a large number of TCTP gene coding sequences, and then adding the A tail reaction to the sequence, using T4 DNA ligase Attached to the pGM-T vector to obtain a ligation product, which is transformed into a competent colon In the ToplO strain, uniformly coated onto the ampicillin-containing LB medium plate, picking up the positive monoclonal colony culture preservation liquid solution and performing preliminary PCR identification. The preliminary identification results indicate that the TCTP gene cDNA sequence was inserted into the successful bacterial solution for sequencing identification. The correct E. coli was identified by liquid LB medium, and the pGM-T vector carrying the TCTP gene cDNA sequence was extracted and digested with the restriction enzymes EcoR I and Spe I, electrophoresis and gelatinization. Recover a fragment of about 500 bp, which is the TCTP gene cDNA sequence;
[0011] C) 特异促进 TCTP基因高表达的慢病毒载体的构建和鉴定: 提取质粒 pLVX-IR ES-Puro, 用限制性内切酶 EcoR I酶和 Spe I酶双酶切, 电泳、 切胶回收载体, 再 用 T4 DNA ligase将所述 TCTP基因 cDNA序列连接 ajpLVX-IRES-Puro表达载体中 [0011] C) Construction and identification of a lentiviral vector that specifically promotes high expression of the TCTP gene: extraction of the plasmid pLVX-IR ES-Puro, digestion with restriction endonuclease EcoR I enzyme and Spe I enzyme, electrophoresis, gelation The vector was recovered, and the TCTP gene cDNA sequence was ligated into the ajpLVX-IRES-Puro expression vector by T4 DNA ligase.
, 得到连接产物, 将该连接产物转化到感受态大肠杆菌 ToplO中, 均匀涂布到含 氨苄青霉素 LB培养基平板上, 挑取阳性单克隆菌落培养保存菌液并进行 PCR初 步鉴定, 将初步鉴定结果说明 TCTP基因 cDNA序列插入成功的菌液进行测序鉴 定; The ligation product is obtained, and the ligation product is transformed into competent E. coli ToplO, uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid is picked and subjected to preliminary identification by PCR, and preliminary identification is performed. The results indicated that the TCTP gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification;
[0012] D) 特异促进 TCTP基因高表达的慢病毒载体的抽提: 将测序结果证实 TCTP基 因 cDNA序列插入成功的菌液扩增培养, 对重组质粒进行抽提, 得到特异促进 TC TP基因高表达的慢病毒表达载体。 [0012] D) Extraction of a lentiviral vector that specifically promotes high expression of the TCTP gene: The sequencing result confirms that the TCTP gene cDNA sequence is inserted into a successful bacterial cell expansion culture, and the recombinant plasmid is extracted to obtain a specific TC TP gene. Expressed lentiviral expression vector.
[0013] 本发明利用基因工程技术构建特异促进 TCTP基因高表达的慢病毒表达载体, 经鉴定构建成功后, 包装成病毒转导入 INS-1细胞, 嘌呤霉素筛选细胞后, 使用 实吋荧光定量 PCR和 Western Blot技术分别从 mRNA和蛋白水平验证 TCTP基因表 达的变化, 实验结果证明本发明提供的 TCTP基因 cDNA序列成功插入至 pLVX-IR ES-Puro表达载体中, 能特异、 持续、 高效、 稳定地促进 TCTP基因高表达。 [0013] The present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of TCTP gene, and after successful identification, packaging into a virus and transfecting into INS-1 cells, and screening cells with puromycin, using real-time fluorescence quantitative The PCR and Western Blot techniques were used to verify the changes of TCTP gene expression from mRNA and protein levels, respectively. The experimental results showed that the TCTP gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IR ES-Puro expression vector, which was specific, sustained, efficient and stable. Promote high expression of TCTP gene.
[0014] 本发明还提供特异促进 TCTP基因高表达的慢病毒表达载体在制备治疗 TCTP基 因表达异常相关疾病的药物中的用途。 The present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of a TCTP gene for the preparation of a medicament for treating a disease associated with abnormal expression of a TCTP gene.
发明的有益效果 Advantageous effects of the invention
有益效果 Beneficial effect
[0015] 本发明提供的特异促进 TCTP基因高表达的慢病毒表达载体具有转染效率高, 用量少, 能特异、 持续、 高效、 稳定地促进 TCTP基因高表达的优点, 可作为有 力工具应用于与 TCTP相关的药物研究和幵发中; 本发明还提供了特异促进 TCTP
基因高表达的慢病毒表达载体的构建方法, 操作效果好, 减少了序列合成费用The lentiviral expression vector which specifically promotes the high expression of the TCTP gene provided by the invention has the advantages of high transfection efficiency, low dosage, specific, sustained, high efficiency and stable promotion of high expression of the TCTP gene, and can be used as a powerful tool. In drug research and development related to TCTP; the present invention also provides specific promotion of TCTP A method for constructing a lentiviral expression vector with high expression of a gene, which has a good operation effect and reduces the cost of sequence synthesis
, 成本较低。 , the cost is lower.
对附图的简要说明 Brief description of the drawing
附图说明 DRAWINGS
[0016] 图 1为 pLVX-IRES-Puro表达载体的质粒图谱。 1 is a plasmid map of the pLVX-IRES-Puro expression vector.
[0017] 图 2为嘌呤霉素筛选细胞后荧光定量 PCR检测结果示意图。 2 is a schematic diagram showing the results of fluorescent quantitative PCR detection 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 BEST MODE FOR CARRYING OUT THE INVENTION
[0018] 下面结合附图与具体实施例对本发明做进一步的说明。 [0018] The present invention will be further described below in conjunction with the drawings and specific embodiments.
[0019] INS-1细胞购自上海生命科学院细胞资源中心, 293FT细胞购自 Thermo Fisher公 司, Premix PrimeSTAR HS酶、 慢病毒表达载体 pLVX-IRES-Puro、 病毒包装辅助 试剂盒、 Lenti-X GoStix试剂盒均购自 Takara公司, RNeasy Mini [0019] INS-1 cells were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences. 293FT cells were purchased from Thermo Fisher, Premix PrimeSTAR HS enzyme, lentiviral expression vector pLVX-IRES-Puro, virus packaging auxiliary kit, Lenti-X GoStix reagent. Boxes are purchased from Takara, RNeasy Mini
Kit购自 QIAGEN公司, pGM-T载体购自天根公司, Endo-Free Plasmid Mini Kit II 贝勾自 Omega bio-tek公司。 Kit was purchased from QIAGEN, pGM-T was purchased from Tiangen, and Endo-Free Plasmid Mini Kit II was purchased from Omega bio-tek.
[0020] 实施例一 TCTP基因引物的设计。 Example 1 Design of TCTP Gene Primers.
[0021] 根据 TCTP基因编码序列 (GenBank NM_001033581.1) , 使用 01igo7对其进行 分析, 寻找上游引物和下游引物 (要求尽可能无引物二聚体且退火温度差距较 小) , 然后在上游引物和下游引物的 5'端分别加入保护碱基与酶切位点 EcoR I和 EcoR I, 设计得到的引物序列如表 1所示。 设计的 PCR引物由上海生工生物工 程技术服务有限公司合成。 [0021] According to the TCTP gene coding sequence (GenBank NM_001033581.1), it was analyzed using 01igo7 to find upstream primers and downstream primers (requiring as little primer-free dimer as possible and the annealing temperature difference is small), then upstream primers and The protective base and the restriction sites EcoR I and EcoR I were added to the 5' end of the downstream primer, respectively, and the designed primer sequences are shown in Table 1. The designed PCR primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
[0022] 表 1 TCTP基因的 PCR弓 |物序列 Table 1 PCR bow of the TCTP gene |
[] 1] [] 1]
[0023] 实施例二特异促进 TCTP基因高表达的慢病毒载体的构建 [0023] Example 2 Construction of a lentiviral vector that specifically promotes high expression of TCTP gene
[0024] 将合成的弓 I物稀释后, 用 Premix PrimeSTAR HS酶对 TCTP基因的编码序列进行 扩增, 电泳回收后然后将其进行加 A尾反应后, 用 T4 DNA连接酶连接到 pGM-T 载体上得到连接产物 (TCTP-T载体) , 将该连接产物转化到感受态大肠杆菌 To plO中, 均匀涂布到含氨苄青霉素 LB培养基平板上, 于 37°C培养 12 h, 同吋设置 阴性对照组 1 (将感受态细胞均匀涂布在不含氨苄青霉素的平板上) 、 阴性对照 组 2 (将感受态细胞均匀涂布在含 100[0024] After diluting the synthetic antibody, the coding sequence of the TCTP gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A tail reaction, and then ligated to pGM-T with T4 DNA ligase. The ligation product (TCTP-T vector) was obtained on the vector, and the ligation product was transformed into competent E. coli To plO, uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h. Negative control group 1 (consistent cells were uniformly coated on ampicillin-free plates), negative control group 2 (peripheral cells were uniformly coated in 100)
g/ml氨苄青霉素的平板上) 、 阳性对照组 1 (将双酶切空载体的连接产物均匀涂 布在含 100 g/ml ampicillin on the plate), positive control group 1 (the connection product of the double enzyme-cut empty vector was evenly coated in 100
g/ml氨苄青霉素的平板上) 、 阳性对照组 2 (将空载体均匀涂布在含 100 g/mL 氨苄青霉素的平板上) 。 实验组长出了单菌落, 阴性对照组 1长出了菌落; 阴性 对照组 2、 阳性对照组 1、 阳性对照组 2没长出菌落。 g/ml ampicillin on the plate), positive control group 2 (the empty carrier was evenly spread on a plate containing 100 g/mL ampicillin). The experimental group grew a single colony, the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, the positive control group 2 did not grow colonies.
[0025] 从实验组中挑取 8个单菌落培养保存后, 各取 0.5 培养液, 用 TCTP基因的引 物进行 PCR扩增来初步鉴定, 结果表明 8个单菌落的培养液均能成功扩增出 TCTP 基因, 接着将重组载体送至上海生工公司测序。 [0025] After picking up 8 single colonies from the experimental group, 0.5 culture solutions were taken and PCR-amplified with primers of TCTP gene for preliminary identification. The results showed that the cultures of 8 single colonies could be successfully amplified. The TCTP gene was released, and the recombinant vector was then sent to Shanghai Biotech for sequencing.
[0026] 取测序结果正确的菌, 置于液体 LB培养基中培养 14 h, 然后提取包含 TCTP基 因序列的重组 T载体, 将其和 pLVX-IRES-Puro载体分别先用 EcoR I酶和 Spe I酶进 行双酶切, 电泳回收, 并用 T4 DNA连接酶连接回收产物用, 再次转化到感受态 大肠杆菌 ToplO中, 均匀涂布到含氨苄青霉素 LB培养基平板上, 于 37°C培养 12 h , 同吋设置阴性对照组 1 (将感受态细胞均匀涂布在不含氨苄青霉素的平板上) 、 阴性对照组 2 (将感受态细胞均匀涂布在含 100 g/ml氨苄青霉素的平板上) 、 阳性对照组 1 (将双酶切空载体的连接产物均匀涂布在含 100 g/ml氨苄青霉素的 平板上) 、 阳性对照组 2 (将空载体均匀涂布在含 100 g/mL氨苄青霉素的平板上 ) 。 实验组长出了单菌落, 阴性对照组 1长出了菌落; 阴性对照组 2、 阳性对照 组 1、 阳性对照组 2没长出菌落。 [0026] The bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the TCTP gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly used with EcoR I enzyme and Spe I, respectively. The enzyme was double-digested, electrophoresed and recovered, and the product was recovered by T4 DNA ligase, and then transformed into competent E. coli ToplO, uniformly coated on ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h. The negative control group 1 was set up (the competent cells were uniformly coated on the ampicillin-free plate), and the negative control group 2 (the competent cells were uniformly coated on the plate containing 100 g/ml ampicillin), Positive control group 1 (the ligation product of the double enzyme-cut empty vector was uniformly coated on a plate containing 100 g/ml ampicillin), positive control group 2 (the empty vector was uniformly coated on 100 g/mL ampicillin) On the tablet). The experimental group grew a single colony, and the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, and the positive control group 2 did not grow colonies.
[0027] 从实验组中挑取 6个单菌落培养保存后, 各取 0.5 培养液, 用 TCTP基因的引 物进行 PCR扩增来初步鉴定。 结果表明全部 6支培养液均能成功扩增出 TCTP基因 , 接着将这些重组载体菌液送至上海生工公司测序, 测序结果与预期完全相符
, 获得 pLVX-TCTP质粒。 [0027] After picking up 6 single colonies from the experimental group, 0.5 culture solutions were taken and PCR-amplified with primers of TCTP gene for preliminary identification. The results showed that all the six culture broths could successfully amplify the TCTP gene, and then the recombinant vector broth was sent to Shanghai Biotech Co., Ltd. for sequencing. The sequencing results were exactly in line with expectations. , obtained pLVX-TCTP plasmid.
[0028] 取出之前保存的重组质粒菌液, 取 20 接种到 15 ml LB培养基 (含 100 g/ml 氨苄青霉素) 中, 37°C, 300 rpm培养 16 h, 用 Endo-Free Plasmid Mini Kit II进行 抽提重组质粒 pLVX-TCTP, 测其纯度和浓度, 结果如表 2所示。 [0028] The previously collected recombinant plasmid bacterial solution was taken out, inoculated into 15 ml of LB medium (containing 100 g/ml ampicillin), cultured at 37 ° C, 300 rpm for 16 h, using Endo-Free Plasmid Mini Kit II The recombinant plasmid pLVX-TCTP was extracted and its purity and concentration were measured. The results are shown in Table 2.
[0029] 表 2重组质粒的纯度和浓度 [0029] Table 2 recombinant plasmid purity and concentration
[0031] 培养 293FT细胞, 取生长状态良好的细胞接种到六孔中, 每孔 1000000个细胞, 用慢病毒包装辅助试剂盒, 取抽提的重组质粒 pLVX-TCTP 2μ§转染到 293FT细胞 , 48h后收集含病毒的上清培养基, 用 0.45μηι的筛子过滤病毒液, 用于感染 INS- 1细胞, Lenti-X GoStix试剂盒检测病毒的滴度为 5000000〜50000000 IFU。 [0031] 293FT cells were cultured, and cells grown in good condition were inoculated into six wells, 1,000,000 cells per well, and the extracted recombinant plasmid pLVX-TCTP 2μ § was transfected into 293FT cells using a lentiviral packaging auxiliary kit. After 48 h, the virus-containing supernatant medium was collected, and the virus solution was filtered through a sieve of 0.45 μm for infection of INS-1 cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 50,000,000 IFU.
[0032] 实施例四 慢病毒转导 INS- 1细胞 Example 4 Lentiviral transduction of INS-1 cells
[0033] 接种 INS-1细胞于 6孔板中, 每孔 1000000个细胞, 12h后细胞密度约为 50<¾, 分 别取病毒液, 用 DMEM完全培养基 10倍稀释病毒, 再加入聚凝胺 (polybrene) 至终浓度为 8 g/mL。 去 6孔板中的培养基, 加入含病毒的 DMEM完全培养基 ( 含 10%胎牛血清) , 24h后弃去含病毒的 DMEM完全培养基, 更换新鲜的 DMEM 完全培养基, 24h后用 0.5 g/ml浓度的嘌呤霉素筛选细胞。 筛选 10d, 每隔 3d更换 培养基一次, 并不断的增加嘌呤霉素的浓度至 1.00 g/ml。 [0033] Inoculated INS-1 cells in a 6-well plate, 1000000 cells per well. After 12 hours, the cell density was about 50<3⁄4, and the virus solution was taken separately. The virus was diluted 10 times with DMEM complete medium, and then polyglycolamine was added. (polybrene) The final concentration was 8 g/mL. The medium in the 6-well plate was added to the virus-containing DMEM complete medium (containing 10% fetal bovine serum). After 24 hours, the virus-containing DMEM complete medium was discarded, and the fresh DMEM complete medium was replaced. After 24 hours, 0.5 was used. The cells were screened at a g/ml concentration of puromycin. After 10 days of screening, the medium was changed once every 3 days, and the concentration of puromycin was continuously increased to 1.00 g/ml.
[0034] 实施例五 荧光定量 PCR检测 TCTP基因表达量。 Example 5 Fluorescence quantitative PCR was used to detect the expression level of TCTP gene.
[0035] 根据 GAPDH和 TCTP基因 mRNA序列, 利用引物设计软件 Oligo 7.0设计弓 |物。
[表 3] [0035] According to the GAPDH and TCTP gene mRNA sequences, the primer design software Oligo 7.0 was used to design the bow. [table 3]
[0036] 分别接种 INS-1细胞、 pLVX空载体对照 INS-1细胞组、 pLVX-TCTP高表达细胞 至 6孔板。 细胞密度达到 80<¾-90<¾吋, 用 RNeasy Mini Kit提取各组细胞的总 RNA , 禾1 J用 PrimeScrip RT reagent [0036] INS-1 cells, pLVX empty vector control INS-1 cell group, and pLVX-TCTP high expressing cells were inoculated to a 6-well plate, respectively. The cell density reached 80<3⁄4-90<3⁄4吋, and the total RNA of each group was extracted with RNeasy Mini Kit, and PrimeScrip RT reagent was used for He 1 J.
Kit将 mRNA逆转录为 cDNA, 逆转录条件: 37°C, 15min; 85°C, 5s; 4°C, ∞。 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 Kit reverse transcribes mRNA into cDNA, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ∞. After the end of reverse transcription, add 9 (L of RNase Free dH 2 0 diluted cDNA, and store at -20 ° C for later detection. Take the cDNA of each group of cells.
Ιμΐ为模板, 以 GAPDH为内参, 实吋荧光定量 PCR (QPCR) 检测 TCTP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 15s, 利用 SYBR Primescript RT-PCR Kit检测各组细胞 TCTP基因相对 表达量。 将 pLVX-TCTP细胞连续培养 20代后, 重复以上实验。 汇总后的结果如 图 2所示。 可以看到, 不管是刚筛选完, 还是已经培养 20代后的 pLVX-TCTP细胞 , 其 TCTP基因的表达量较 INS-1细胞都有 60倍以上的升高, 而 pLVX空载体细胞 的 TCTP基因表达量与 INS-1细胞相比基本没有变化, 说明本发明提供的 TCTP基 因 cDNA序列成功插入至 pLVX-IRES-Puro表达载体中, 能特异、 持续、 高效、 稳 定地促进 TCTP基因高表达。 Ιμΐ was used as a template, and GAPDH was used as an internal reference. Real-time quantitative PCR (QPCR) was used to detect the relative expression of TCTP, and the reaction conditions were set: 95. C 30s, 1 cycle, 54°C 30s 40 cycles, 95. C 5s, 60. C lmin, 95 ° C for 15 s, the relative expression of TCTP gene in each group was detected by SYBR Primescript RT-PCR Kit. After the pLVX-TCTP cells were continuously cultured for 20 passages, the above experiment was repeated. The summarized results are shown in Figure 2. It can be seen that the expression level of TCTP gene is 60-fold higher than that of INS-1 cells, whether it is just after screening or after 20 generations of pLVX-TCTP cells, and the TCTP gene of pLVX empty vector cells. The expression level of the TCTP gene was successfully inserted into the pLVX-IRES-Puro expression vector, which promoted the high expression of TCTP gene specifically, continuously, efficiently and stably.
[0037] 以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明, 不能认 定本发明的具体实施只局限于这些说明。 对于本发明所属技术领域的普通技术 人员来说, 在不脱离本发明构思的前提下, 还可以做出若干简单推演或替换, 都应当视为属于本发明的保护范围。 [0037] The above is a further detailed description of the present invention in conjunction with the specific preferred embodiments. It is not intended that the specific embodiments of the invention are limited to the description. It will be apparent to those skilled in the art that the present invention may be practiced without departing from the spirit and scope of the invention.
工业实用性 Industrial applicability
[0038] 本发明提供的特异促进 TCTP基因高表达的慢病毒表达载体具有转染效率高,
用量少, 能特异、 持续、 高效、 稳定地促进 TCTP基因高表达的优点, 可作为有 力工具应用于与 TCTP相关的药物研究和幵发中; 本发明还提供了特异促进 TCTP 基因高表达的慢病毒表达载体的构建方法, 操作效果好, 减少了序列合成费用The lentiviral expression vector provided by the invention specifically promoting the high expression of the TCTP gene has high transfection efficiency. It has the advantages of low dosage, specific, sustained, efficient and stable promotion of high expression of TCTP gene, and can be used as a powerful tool in drug research and development related to TCTP. The present invention also provides specific promotion of high expression of TCTP gene. The construction method of lentiviral expression vector has good operation effect and reduces the cost of sequence synthesis
, 成本较低。
, the cost is lower.
Claims
[权利要求 1] 一种特异促进 TCTP基因高表达的慢病毒表达载体, 其特征在于: 包 括 pLVX-IRES-puro表达载体的基本序列、 抗性基因序列、 多克隆位 点序列、 启动子序列和 TCTP基因 cDNA序歹 ij ; 所述多克隆位点包括 E coR I酶切位点和 Spe I酶切位点, 所述 TCTP基因 cDNA序列包括 EcoR I酶切位点、 TCTP基因编码序列和 Spe [Claim 1] A lentiviral expression vector that specifically promotes high expression of the TCTP gene, characterized by: including the basic sequence of the pLVX-IRES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and TCTP gene cDNA sequence: the multiple cloning site includes EcoR I enzyme cut site and Spe I enzyme cut site, the TCTP gene cDNA sequence includes EcoR I enzyme cut site, TCTP gene coding sequence and Spe I enzyme cut site
I酶切位点, 所述 TCTP基因 cDNA序列正向插入所述多克隆位点序列 中。 I enzyme cutting site, the TCTP gene cDNA sequence is inserted into the multiple cloning site sequence in the forward direction.
[权利要求 2] 根据权利要求 1所述的特异促进 TCTP基因高表达的慢病毒表达载体, 其特征在于: 所述 TCTP基因编码序列通过 PCR扩增获得, PCR引物 包括上游引物和下游引物, 所述上游引物的序列为: 5'- GGAATTCATGATTATCTACCGGGACCTC -3,, 即 SEQ ID NO: 1, 所述下游引物的序列为: 5'- [Claim 2] The lentiviral expression vector that specifically promotes high expression of TCTP gene according to claim 1, characterized in that: the TCTP gene coding sequence is obtained by PCR amplification, and the PCR primers include upstream primers and downstream primers, so The sequence of the above-mentioned upstream primer is: 5'- GGAATTCATGATTATCTACCGGGACCTC-3, that is, SEQ ID NO: 1. The sequence of the above-mentioned downstream primer is: 5'-
GACTAGTCTAAAGATGTATTCCCCAATTC -3' , 即 SEQ ID NO: 2 GACTAGTCTAAAGATGTATTCCCCAATTC -3' , which is SEQ ID NO: 2
[权利要求 3] 根据权利要求 2所述的特异促进 TCTP基因高表达的慢病毒表达载体 的构建方法, 其特征在于: 包括如下步骤: [Claim 3] The construction method of a lentiviral expression vector that specifically promotes high expression of TCTP gene according to claim 2, characterized in that: comprising the following steps:
A) TCTP基因引物设计: 根据 TCTP基因编码序列, 使用 01igo 7分析 后选取 5, - GGAATTCATGATTATCTACCGGGACCTC -3,, 即 SEQ ID NO: 1作为上游引物, 选取 5'- A) TCTP gene primer design: According to the TCTP gene coding sequence, use 01igo 7 to analyze and select 5, - GGAATTCATGATTATCTACCGGGACCTC -3, that is, SEQ ID NO: 1 as the upstream primer, select 5'-
GACTAGTCTAAAGATGTATTCCCCAATTC -3' , 即 SEQ ID NO: 2 作为下游弓 I物, 然后合成所述上游弓 I物和所述下游弓 I物; GACTAGTCTAAAGATGTATTCCCCAATTC-3', i.e. SEQ ID NO: 2 as the downstream primer, and then synthesize the upstream primer and the downstream primer;
B) TCTP基因 cDNA序列的获得: 用所述上游引物和所述下游引物进 行 PCR扩增, 获得大量 TCTP基因编码序列, 然后将该序列进行加 A尾 反应后, 用 T4 DNA连接酶连接到 pGM-T载体上得到连接产物, 将该 连接产物转化到感受态大肠杆菌 ToplO中, 均匀涂布到含氨苄青霉素 LB培养基平板上, 挑取阳性单克隆菌落培养保存菌液并进行 PCR初 步鉴定, 将初步鉴定结果说明 TCTP基因 cDNA序列插入成功的菌液进
行测序鉴定; 用液体 LB培养基培养测序鉴定正确的大肠杆菌, 并抽 提其中带 TCTP基因 cDNA序列的 pGM-T载体, 用限制性内切酶 EcoR I 酶和 Spe l酶双酶切, 电泳、 切胶回收 500 B) Obtaining the TCTP gene cDNA sequence: Use the upstream primer and the downstream primer to perform PCR amplification to obtain a large number of TCTP gene coding sequences, and then perform an A-tailing reaction on the sequence, and then connect it to pGM using T4 DNA ligase Obtain the ligation product on the -T vector, transform the ligation product into competent E. coli ToplO, spread it evenly on an LB medium plate containing ampicillin, pick out positive single clone colonies, culture and preserve the bacterial liquid, and perform preliminary PCR identification. The preliminary identification results showed that the TCTP gene cDNA sequence was successfully inserted into the bacterial liquid. Perform sequencing identification; use liquid LB medium to culture the correct E. coli for sequencing identification, and extract the pGM-T vector containing the TCTP gene cDNA sequence, double-digest with restriction endonuclease EcoR I enzyme and Spel enzyme, and perform electrophoresis. , cut rubber recycling 500
bp左右的片段, 此片段即为 TCTP基因 cDNA序列; A fragment of about bp, this fragment is the TCTP gene cDNA sequence;
C) 特异促进 TCTP基因高表达的慢病毒载体的构建和鉴定: 提取质 粒 pLVX-IRES-Puro, 用限制性内切酶 EcoR I酶和 Spe I酶双酶切, 电 泳、 切胶回收载体, 再用 T4 DNA ligase将所述 TCTP基因 cDNA序列 连接到 pLVX-IRES-Puro表达载体中, 得到连接产物, 将该连接产物 转化到感受态大肠杆菌 ToplO中, 均匀涂布到含氨苄青霉素 LB培养基 平板上, 挑取阳性单克隆菌落培养保存菌液并进行 PCR初步鉴定, 将 初步鉴定结果说明 TCTP基因 cDNA序列插入成功的菌液进行测序鉴定 C) Construction and identification of lentiviral vectors that specifically promote the high expression of TCTP genes: Extract plasmid pLVX-IRES-Puro, double-digest with restriction endonucleases EcoR I and Spe I, electrophoresis, and gel cutting to recover the vector, and then Use T4 DNA ligase to connect the TCTP gene cDNA sequence into the pLVX-IRES-Puro expression vector to obtain a ligation product, transform the ligation product into competent E. coli Top10, and spread it evenly onto an LB medium plate containing ampicillin On the top, select positive single clone colonies, culture and preserve the bacterial liquid and perform preliminary PCR identification. The preliminary identification results indicate that the TCTP gene cDNA sequence has been successfully inserted into the bacterial liquid for sequencing and identification.
D) 特异促进 TCTP基因高表达的慢病毒载体的抽提: 将测序结果证 实 TCTP基因 cDNA序列插入成功的菌液扩增培养, 对重组质粒进行抽 提, 得到特异促进 TCTP基因高表达的慢病毒表达载体。 D) Extraction of lentiviral vectors that specifically promote the high expression of the TCTP gene: The sequencing results confirm that the TCTP gene cDNA sequence has been successfully inserted into the bacterial liquid amplification culture, and the recombinant plasmid is extracted to obtain the lentivirus that specifically promotes the high expression of the TCTP gene. Expression vector.
[权利要求 4] 根据权利要求 1至 3中任一项所述的特异促进 TCTP基因高表达的慢病 毒表达载体在制备治疗 TCTP基因表达异常相关疾病的药物中的用途
[Claim 4] Use of the lentiviral expression vector that specifically promotes high expression of TCTP gene according to any one of claims 1 to 3 in the preparation of drugs for the treatment of diseases related to abnormal TCTP gene expression
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