WO2017214943A1 - Lentiviral expression vector for promoting tim-3 gene expression and application thereof - Google Patents
Lentiviral expression vector for promoting tim-3 gene expression and application thereof Download PDFInfo
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Definitions
- the present invention belongs to the field of genetic engineering technology, and in particular relates to a lentiviral expression vector which promotes expression of a TIM-3 gene and an application thereof.
- Tumors can be used to strengthen the "army” by various methods: 1) Tumor cells have low expression or lack of expression of specific or related antigens, and down-regulate MHC-I molecules by gene defect or transcription down-regulation, and antigen-presenting cells cannot give T
- B7 molecule such as B7-l, B7-2 or up-regulation of B7-H1, B7-H2, etc., such that T cell activation is deficient in the second signal or induces T cell inactivation, Apoptosis
- Tumor cells can induce autologous apoptosis or induce T cell apoptosis by low expression of Fas and defective Fas signaling molecules or high expression of FasL
- 4) release of tumor-derived factors and induce regulatory T cells (Treg) , Regulatory DCs (DCreg), tumor-associated macrophages (TAM), long-term stimulation of a large number of tumor antigens and immune molecules, leading to T cell depletion.
- DCreg regulatory DCs
- TAM tumor-associated macrophages
- Negative co-stimulatory molecules PD-1 and Tim3 are important marker molecules for early and late viral responsive CD8+ T cell depletion, respectively. Therefore, the study of TIM-3 is expected to enhance the ability of T cells to kill tumor cells and rescue depleted T cells. However, the lack of lentiviral expression vectors that specifically promote the high expression of TIM-3 gene in the prior art makes the related studies not well. exhibition.
- the TIM-3 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 TIM-3 gene, thereby completing the present invention.
- the present invention provides a lentiviral expression vector which specifically promotes high expression of a TIM-3 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 TI M-3 gene cDNA sequence; the multiple cloning site includes Spe I cleavage site and Not
- the TIM-3 gene cDNA sequence includes a Spe l cleavage site, a TIM-3 gene coding sequence and a Not I cleavage site, and the TIM-3 gene cDNA sequence is inserted into the poly Cloning of the site sequence.
- the lentiviral expression vector constructed by inserting the cDNA sequence of the TIM-3 gene into the pLVX-IRES-Puro expression vector has the advantages of high transfection efficiency, low dosage, sustainable, high efficiency and stability.
- the advantages of increasing the expression of TIM-3 gene can be used as a powerful tool for the preparation and treatment of TIM-3 gene expression in the treatment of diseases such as Alzheimer's disease.
- the TIM-3 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'-GACTAGTATGTTTTCACATCTTCCCTTTG -3', ie SEQ ID NO: 1
- the sequence of the downstream primer is: 5, - GGCGGCCGCTGTCGCTTTGCAATGCCATAG -3', ie SEQ ID NO: 2.
- the TIM-3 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the TIM-3 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 the TIM-3 gene, comprising the following steps:
- A) TIM-3 gene primer design According to the TIM-3 gene coding sequence, using Oligo 7 analysis, select 5'-GACTAGTATGTTTTCACATCTTCCCTTTG-3', ie SEQ ID NO: 1 as the upstream bow, select 5 '- GGCGGCCGCTGTCGCTTTGCAATGCCATAG -3, ie, SEQ ID NO: 2 as a downstream primer, then synthesizing the upstream primer and the downstream primer; the upstream primer and the downstream primer are free of primer dimer, and the annealing temperature difference is small;
- the successful bacterial liquid was sequenced and identified; the correct Escherichia coli was identified by liquid LB medium, and the pGM-T vector carrying the TIM-3 gene cDNA sequence was extracted, and the restriction endonuclease Spe I enzyme and Not I enzyme double digestion, electrophoresis, gelatinization to recover a fragment of about 1000 bp, this fragment is the TIM-3 gene cDNA sequence;
- the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of TIM-3 gene. After successful identification, it is packaged into a virus and introduced into Jurkat cells. After screening cells with puromycin, the real-time fluorescence is quantified. The PCR and Western Blot techniques verified the change of TIM-3 gene expression from mRNA and protein levels, respectively. The experimental results confirmed that the TIM-3 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-I RES-Puro expression vector, which can be specific and sustained. Highly and stably promote high expression of TIM-3 gene.
- the present invention also provides a use of a lentiviral expression vector which specifically promotes high expression of a TIM-3 gene for the preparation of a medicament for treating a disease associated with abnormal expression of a TIM-3 gene.
- the lentiviral expression vector which specifically promotes the high expression of the TIM-3 gene provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of the TIM-3 gene in a specific, sustained, efficient and stable manner.
- the present invention also provides specific promotion of TIM-
- the construction method of the lentiviral expression vector with high expression of 3 gene has good operation effect, reduces the cost of sequence synthesis, and has low cost.
- 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.
- Jurkat cells were purchased from the Cell Resource Center of the 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, and Lenti-X GoStix kit. 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.
- TIM-3 gene coding sequence GenBank NMJ 2782.4
- Example 2 Construction of a lentiviral vector that specifically promotes high expression of the TIM-3 gene
- the coding sequence of the TIM-3 gene was sequenced with Premix PrimeSTAR HS enzyme. Amplification, electrophoresis recovery and then adding A tail reaction, using T4
- the DNA ligase was ligated to the pGM-T vector to obtain the ligation product (TIM-3-T vector), and the ligation product was transformed into competent E. coli DH5CC and uniformly coated on an ampicillin-containing LB medium plate at 37 Cultured at °C for 12 h, the same control group was set to negative control group 1 (the competent cells were uniformly coated on the ampicillin-free plate), and the negative control group 2 (the competent cells were uniformly coated with 100 g/ml ampicillin).
- positive control group 1 the connection product of the double enzyme-cut empty vector was uniformly coated on the plate containing 100 g/ml ampicillin
- positive control group 2 the empty carrier was uniformly coated in 100%
- the bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the TIM-3 gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly treated with Spe I enzyme and The Not I enzyme was double-digested, electrophoresed and recovered, and the product was recovered by T4 DNA ligase, and then transformed into competent E. coli DH50C, uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C.
- the same negative control group 1 was set (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 carrier was uniformly coated in 100 g/mL ampicillin) Penicillin on the plate).
- 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-TIM-3 2 ⁇ ⁇ was transfected into 293FT 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 infecting Jur kat cells, and the Lenti-X GoStix kit was used to detect the virus titer of 5000000 to 50000000 IFU.
- Jurkat cells were inoculated into 6-well plates at 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 polybrene was added. ) The final concentration is 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 TIM-3 gene.
- the primer design software Oligo 7.0 was used to design the bow.
- 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.
- ⁇ was used as a template, and GAPDH was used as an internal reference.
- Real-time quantitative PCR was used to detect the relative expression of ⁇ -3, and the reaction conditions were set: 95. C 30s, 1 cycle, 54°C 30s 40 cycles, 95. C 5s, 60.
- TIM-3 gene is more than 60-fold higher than that of Jurkat cells, whether it has just been screened or has been cultured for 20 generations of pLVX-TIM-3 cells, whereas pLVX empty vector cells
- the expression level of TIM-3 gene was almost unchanged compared with Jurkat cells, indicating that the TIM-3 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IRES-Puro expression vector, and the TIM- can be specifically, continuously, efficiently and stably promoted. 3 gene high expression.
- the lentiviral expression vector which specifically promotes the high expression of the TIM-3 gene provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of the TIM-3 gene in a specific, sustained, efficient and stable manner.
- the present invention also provides specific promotion of TIM-
- the construction method of the lentiviral expression vector with high expression of 3 gene has good operation effect, reduces the cost of sequence synthesis, and has low cost.
Abstract
Provided is a lentiviral expression vector for promoting higher expression of TIM-3 genes, comprising a fundamental sequence, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence, and a TIM-3 gene cDNA sequence of a pLVX-IRES-puro expression vector. A multiple cloning site comprises an Spe I enzyme cutting site and a Not I enzyme cutting site; the TIM-3 gene cDNA sequence comprises the Spe I enzyme cutting site, a TIM-3 coding sequence, and the Not I enzyme cutting site; the TIM-3 gene cDNA sequence is inserted into the multiple cloning site sequence in a forward direction.
Description
说明书 发明名称：促进 TIM-3基因表达的慢病毒表达载体及其应用 技术领域 Description: The lentiviral expression vector for promoting TIM-3 gene expression and its application
[0001] 本发明属于基因工程技术领域， 尤其涉及一种促进 TIM-3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 in particular relates to a lentiviral expression vector which promotes expression of a TIM-3 gene and an application thereof.
背景技术 Background technique
[0002] 肿瘤与机体的博弈好比一场持久战， 在战争中肿瘤并非孤军奋战， 而是组建" 肿瘤微环境"这样一支庞大的"军队"。 肿瘤通过各种方法来壮大"军队"： 1) 肿瘤 细胞低表达或缺失表达特异性或者相关的抗原， 通过基因缺陷或转录下调等方 法下调 MHC-I分子， 进而抗原提呈细胞不能给 T [0002] The game between the tumor and the body is like a protracted war. In the war, the tumor is not alone, but a huge "army" such as the "tumor microenvironment". Tumors can be used to strengthen the "army" by various methods: 1) Tumor cells have low expression or lack of expression of specific or related antigens, and down-regulate MHC-I molecules by gene defect or transcription down-regulation, and antigen-presenting cells cannot give T
细胞有效的提呈和激活； 2) B7分子的异常表达， 如下调 B7-l、 B7-2或上调 B7-H1 , B7-H2等使 T细胞激活缺失第二信号或诱导 T细胞失能、 凋亡； 3) 肿 瘤细胞可通过低表达 Fas和缺陷 Fas信号传导分子或高表达 FasL， 避免自身凋 亡或诱导 T细胞凋亡； 4) 释放肿瘤来源的因子， 诱导调节性 T细胞 （Treg) ， 调节性 DCs (DCreg) ， 肿瘤相关巨噬细胞 (TAM)， 长期大量的肿瘤抗原和免疫 分子的刺激， 导致 T细胞耗竭。 Effective presentation and activation of cells; 2) Abnormal expression of B7 molecule, such as B7-l, B7-2 or up-regulation of B7-H1, B7-H2, etc., such that T cell activation is deficient in the second signal or induces T cell inactivation, Apoptosis; 3) Tumor cells can induce autologous apoptosis or induce T cell apoptosis by low expression of Fas and defective Fas signaling molecules or high expression of FasL; 4) release of tumor-derived factors and induce regulatory T cells (Treg) , Regulatory DCs (DCreg), tumor-associated macrophages (TAM), long-term stimulation of a large number of tumor antigens and immune molecules, leading to T cell depletion.
技术问题 technical problem
[0003] 负性协同刺激分子 PD-1和 Tim3分别是早期和后期病毒应答性 CD8+T细胞耗 竭的重要的标志性分子。 因此对 TIM-3的研究预期可以提升 T细胞杀伤肿瘤细胞 的能力， 挽救耗竭的 T细胞， 但现有技术缺乏特异促进 TIM-3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] Negative co-stimulatory molecules PD-1 and Tim3 are important marker molecules for early and late viral responsive CD8+ T cell depletion, respectively. Therefore, the study of TIM-3 is expected to enhance the ability of T cells to kill tumor cells and rescue depleted T cells. However, the lack of lentiviral expression vectors that specifically promote the high expression of TIM-3 gene in the prior art makes the related studies not well. exhibition.
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 Spe I酶切位点和 Not [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 the Spe I cleavage site and Not are included.
I酶切位点的 TIM-3基因 cDNA序列*** pLVX-IRES-Puro表达载体的多克隆位点中 可成功构建特异促进 TIM-3基因高表达的慢病毒表达载体， 从而完成本发明。
[0005] 本发明提供一种特异促进 TIM-3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 TI M-3基因 cDNA序列； 所述多克隆位点包括 Spe I酶切位点和 Not The TIM-3 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 TIM-3 gene, thereby completing the present invention. The present invention provides a lentiviral expression vector which specifically promotes high expression of a TIM-3 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 TI M-3 gene cDNA sequence; the multiple cloning site includes Spe I cleavage site and Not
I酶切位点， 所述 TIM-3基因 cDNA序列包括 Spe l酶切位点、 TIM-3基因编码序列 和 Not I酶切位点， 所述 TIM-3基因 cDNA序列正向***所述多克隆位点序列中。 I cleavage site, the TIM-3 gene cDNA sequence includes a Spe l cleavage site, a TIM-3 gene coding sequence and a Not I cleavage site, and the TIM-3 gene cDNA sequence is inserted into the poly Cloning of the site sequence.
[0006] 采用上述技术方案， 本发明提供的 TIM-3基因 cDNA序列*** 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 TIM-3基因表达的优点， 可作为有力工具应用于制备治疗 TIM-3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 cDNA sequence of the TIM-3 gene into the pLVX-IRES-Puro expression vector has the advantages of high transfection efficiency, low dosage, sustainable, high efficiency and stability. The advantages of increasing the expression of TIM-3 gene can be used as a powerful tool for the preparation and treatment of TIM-3 gene expression in the treatment of diseases such as Alzheimer's disease.
[0007] 作为本发明的进一步改进， 所述 TIM-3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'- GACTAGTATGTTTTCACATCTTCCCTTTG -3' , 即 SEQ ID NO: 1， 所述下游引 物的序列为： 5，- GGCGGCCGCTGTCGCTTTGCAATGCCATAG -3'， 即 SEQ ID NO: 2。 采用上述 PCR引物序列， 通过 PCR可以扩增出 TIM-3基因编码序列， 并 可成功***至 pLVX-IRES-Puro表达载体中持续表达 TIM-3基因， 减少了序列合成 费用， 成本较低。 [0007] As a further improvement of the present invention, the TIM-3 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'-GACTAGTATGTTTTCACATCTTCCCTTTG -3', ie SEQ ID NO: 1, the sequence of the downstream primer is: 5, - GGCGGCCGCTGTCGCTTTGCAATGCCATAG -3', ie SEQ ID NO: 2. Using the above PCR primer sequence, the TIM-3 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the TIM-3 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 TIM-3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 the TIM-3 gene, comprising the following steps:
[0009] A) TIM-3基因引物设计： 根据 TIM-3基因编码序列， 使用 Oligo 7分析后选取 5'- GACTAGTATGTTTTCACATCTTCCCTTTG -3'， 即 SEQ ID NO: 1作为上游弓 |物 ， 选取 5 '- GGCGGCCGCTGTCGCTTTGCAATGCCATAG -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) TIM-3 gene primer design: According to the TIM-3 gene coding sequence, using Oligo 7 analysis, select 5'-GACTAGTATGTTTTCACATCTTCCCTTTG-3', ie SEQ ID NO: 1 as the upstream bow, select 5 '- GGCGGCCGCTGTCGCTTTGCAATGCCATAG -3, ie, SEQ ID NO: 2 as a downstream primer, then synthesizing the upstream primer and the downstream primer; the upstream primer and the downstream primer are free of primer dimer, and the annealing temperature difference is small;
[0010] B) TIM-3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 TIM-3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 DH50C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 TIM-3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 TIM-3基因 cDNA序列的 pGM-T载体， 用限制性内切酶 Spe I酶和 Not I酶双酶切， 电泳、 切胶回收 1000 bp左右的片段， 此片段即为 TIM-3基因 cDNA序 列； [0010] B) obtaining the TIM-3 gene cDNA sequence: using the upstream primer and the downstream primer for PCR amplification, obtaining a large number of TIM-3 gene coding sequences, and then adding the A tail reaction to the sequence, The T4 DNA ligase was ligated to the pGM-T vector to obtain a ligation product, and the ligation product was transformed into competent E. coli DH50C, uniformly coated on an ampicillin-containing LB medium plate, and a positive monoclonal colony culture preservation strain was picked. The liquid was initially identified by PCR, and the preliminary identification results indicated that the TIM-3 gene cDNA sequence was inserted. The successful bacterial liquid was sequenced and identified; the correct Escherichia coli was identified by liquid LB medium, and the pGM-T vector carrying the TIM-3 gene cDNA sequence was extracted, and the restriction endonuclease Spe I enzyme and Not I enzyme double digestion, electrophoresis, gelatinization to recover a fragment of about 1000 bp, this fragment is the TIM-3 gene cDNA sequence;
[0011] C) 特异促进 TIM-3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 Spe I酶和 Not [0011] C) Construction and identification of a lentiviral vector that specifically promotes high expression of the TIM-3 gene: extraction of plasmid pLVX-IR ES-Puro, restriction endonuclease Spe I enzyme and Not
I酶双酶切， 电泳、 切胶回收载体， 再用 T4 DNA ligase将所述 TIM-3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 DH50C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 TIM-3基因 cD NA序列***成功的菌液进行测序鉴定； I enzyme double digestion, electrophoresis, gel extraction of the vector, and then the TIM-3 gene cDNA sequence was ligated into the pLVX-IRES-Puro expression vector using T4 DNA ligase to obtain a ligation product, and the ligation product was transformed into a competent state. Escherichia coli DH50C was uniformly applied to the ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid was picked and preliminarily identified by PCR. The preliminary identification results indicated that the TIM-3 gene cD NA sequence was successfully inserted into the strain. The liquid is subjected to sequencing and identification;
[0012] D) 特异促进 TIM-3基因高表达的慢病毒载体的抽提： 将测序结果证实 TIM-3基 因 cDNA序列***成功的菌液扩增培养， 对重组质粒进行抽提， 得到特异促进 TI M-3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 TIM-3 gene: The sequencing result confirms that the TIM-3 gene cDNA sequence is inserted into a successful bacterial cell expansion culture, and the recombinant plasmid is extracted to obtain a specific promotion. A lentiviral expression vector with high expression of the TI M-3 gene.
[0013] 本发明利用基因工程技术构建特异促进 TIM-3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 Jurkat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 TIM-3基因表 达的变化， 实验结果证明本发明提供的 TIM-3基因 cDNA序列成功***至 pLVX-I RES-Puro表达载体中， 能特异、 持续、 高效、 稳定地促进 TIM-3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 TIM-3 gene. After successful identification, it is packaged into a virus and introduced into Jurkat cells. After screening cells with puromycin, the real-time fluorescence is quantified. The PCR and Western Blot techniques verified the change of TIM-3 gene expression from mRNA and protein levels, respectively. The experimental results confirmed that the TIM-3 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-I RES-Puro expression vector, which can be specific and sustained. Highly and stably promote high expression of TIM-3 gene.
[0014] 本发明还提供特异促进 TIM-3基因高表达的慢病毒表达载体在制备治疗 TIM-3 基因表达异常相关疾病的药物中的用途。 The present invention also provides a use of a lentiviral expression vector which specifically promotes high expression of a TIM-3 gene for the preparation of a medicament for treating a disease associated with abnormal expression of a TIM-3 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 TIM-3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 TIM-3基因高表达的优点， 可作为有 力工具应用于与 TIM-3相关的药物研究和幵发中； 本发明还提供了特异促进 TIM- The lentiviral expression vector which specifically promotes the high expression of the TIM-3 gene provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of the TIM-3 gene in a specific, sustained, efficient and stable manner. As a powerful tool for drug research and development related to TIM-3; the present invention also provides specific promotion of TIM-
3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 ， 成本较低。
对附图的简要说明 The construction method of the lentiviral expression vector with high expression of 3 gene has good operation effect, reduces the cost of sequence synthesis, and has low cost. 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] Jurkat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] Jurkat cells were purchased from the Cell Resource Center of the 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, and Lenti-X GoStix kit. 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 TIM-3基因引物的设计。 Example 1 Design of TIM-3 gene primers.
[0021] 根据 TIM-3基因编码序列 （GenBank NMJ 2782.4) ， 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 Spe l和 Spe 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 TIM-3 gene coding sequence (GenBank NMJ 2782.4), 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), and then upstream primers The protective base and the restriction sites Spe l and Spe 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 TIM-3基因的 PCR弓 |物序列 Table 1 PCR bow of TIM-3 gene |
[] [表 1] [] [Table 1]
[0023] 实施例二特异促进 TIM-3基因高表达的慢病毒载体的构建 Example 2 Construction of a lentiviral vector that specifically promotes high expression of the TIM-3 gene
[0024] 将合成的弓 I物稀释后， 用 Premix PrimeSTAR HS酶对 TIM-3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 [0024] After diluting the synthetic antibody, the coding sequence of the TIM-3 gene was sequenced with Premix PrimeSTAR HS enzyme. Amplification, electrophoresis recovery and then adding A tail reaction, using T4
DNA连接酶连接到 pGM-T载体上得到连接产物 （TIM-3-T载体） ， 将该连接产物 转化到感受态大肠杆菌 DH5CC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。 The DNA ligase was ligated to the pGM-T vector to obtain the ligation product (TIM-3-T vector), and the ligation product was transformed into competent E. coli DH5CC and uniformly coated on an ampicillin-containing LB medium plate at 37 Cultured at °C for 12 h, the same control group was set to negative control group 1 (the competent cells were uniformly coated on the ampicillin-free plate), and the negative control group 2 (the competent cells were uniformly coated with 100 g/ml ampicillin). On the plate of penicillin), positive control group 1 (the connection product of the double enzyme-cut empty vector was uniformly coated on the plate containing 100 g/ml ampicillin), positive control group 2 (the empty carrier was uniformly coated in 100%) g/mL ampicillin on the plate). 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 ΤΙΜ-3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 ΤΙΜ- 3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 the primers of the ΤΙΜ-3 gene for preliminary identification. The results showed that the cultures of 8 single colonies were successful. The ΤΙΜ-3 gene was amplified, and the recombinant vector was sent to Shanghai Biotech Co., Ltd. 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 TIM-3基 因序列的重组 T载体， 将其和 pLVX-IRES-Puro载体分别先用 Spe I酶和 Not 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 DH50C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 TIM-3 gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly treated with Spe I enzyme and The Not I enzyme was double-digested, electrophoresed and recovered, and the product was recovered by T4 DNA ligase, and then transformed into competent E. coli DH50C, uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C. h, the same negative control group 1 was set (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 carrier was uniformly coated in 100 g/mL ampicillin) Penicillin on the plate). 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 ΤΙΜ-3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 ΤΙΜ-3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-TIM-3质粒。 [0027] After picking up 6 single colonies from the experimental group, 0.5 culture solutions were taken and PCR-amplified with the primer of the ΤΙΜ-3 gene for preliminary identification. The results showed that all the 6 culture broths could successfully amplify the ΤΙΜ-3 gene, and then the recombinant vector broth was sent to Shanghai Biotech Co., Ltd. for sequencing, and the sequencing results were exactly as expected, and the pLVX-TIM-3 plasmid was obtained.
[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-TIM-3， 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 get on The recombinant plasmid pLVX-TIM-3 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
[] [表 2]
[] [Table 2]
[0030] 实施例三 慢病毒包装 [0030] Example 3 Lentiviral Packaging
[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-TIM-3 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 Jur kat细胞， Lenti-X GoStix试剂盒检测病毒的滴度为 5000000〜50000000IFU。 [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-TIM-3 2μ § was transfected into 293FT 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 infecting Jur kat cells, and the Lenti-X GoStix kit was used to detect the virus titer of 5000000 to 50000000 IFU.
[0032] 实施例四 慢病毒转导 Jurkat细胞 Example 4 Lentiviral transduction Jurkat cells
[0033] 接种 Jurkat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] Jurkat cells were inoculated into 6-well plates at 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 polybrene was added. ) The final concentration is 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 TIM-3基因表达量。 Example 5 Fluorescence quantitative PCR was used to detect the expression level of TIM-3 gene.
[0035] 根据 GAPDH和 TIM-3基因 mRNA序列， 利用引物设计软件 Oligo 7.0设计弓 |物。 [0035] According to the GAPDH and TIM-3 gene mRNA sequences, the primer design software Oligo 7.0 was used to design the bow.
[] [表 3] [] [table 3]
分别接种 Jurkat细胞、 pLVX空载体对照 Jurkat细胞组、 pLVX-TIM-3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 Inoculated with Jurkat cells, pLVX empty vector control Jurkat cell group, and pLVX-TIM-3 high expressing cells To 6-well plate. 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 ΤΙΜ-3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 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 ΤΙΜ-3, and the reaction conditions were set: 95. C 30s, 1 cycle, 54°C 30s 40 cycles, 95. C 5s, 60. C
lmin, 95°C 15s， 利用 SYBR Primescript RT-PCR Kit检测各组细胞 TIM-3基因相 对表达量。 将 pLVX-TIM-3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-TIM-3 细胞， 其 TIM-3基因的表达量较 Jurkat细胞都有 60倍以上的升高， 而 pLVX空载体 细胞的 TIM-3基因表达量与 Jurkat细胞相比基本没有变化， 说明本发明提供的 TIM -3基因 cDNA序列成功***至 pLVX-IRES-Puro表达载体中， 能特异、 持续、 高效 、 稳定地促进 TIM-3基因高表达。 Lmin, 95 ° C for 15 s, the relative expression of TIM-3 gene in each group was detected by SYBR Primescript RT-PCR Kit. After the pLVX-TIM-3 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 of TIM-3 gene is more than 60-fold higher than that of Jurkat cells, whether it has just been screened or has been cultured for 20 generations of pLVX-TIM-3 cells, whereas pLVX empty vector cells The expression level of TIM-3 gene was almost unchanged compared with Jurkat cells, indicating that the TIM-3 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IRES-Puro expression vector, and the TIM- can be specifically, continuously, efficiently and stably promoted. 3 gene high expression.
[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 TIM-3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 TIM-3基因高表达的优点， 可作为有 力工具应用于与 TIM-3相关的药物研究和幵发中； 本发明还提供了特异促进 TIM- The lentiviral expression vector which specifically promotes the high expression of the TIM-3 gene provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of the TIM-3 gene in a specific, sustained, efficient and stable manner. As a powerful tool for drug research and development related to TIM-3; the present invention also provides specific promotion of TIM-
3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 ， 成本较低。
The construction method of the lentiviral expression vector with high expression of 3 gene has good operation effect, reduces the cost of sequence synthesis, and has low cost.
Claims
[权利要求 1] 一种特异促进 TIM-3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 TIM-3基因 cDNA序歹 ij ; 所述多克隆位点包括 S pe I酶切位点和 Not I酶切位点， 所述 TIM-3基因 cDNA序列包括 Spe I酶 切位点、 TIM-3基因编码序列和 Not l酶切位点， 所述 TIM-3基因 cDNA 序列正向***所述多克隆位点序列中。 [Claim 1] A lentiviral expression vector that specifically promotes high expression of the TIM-3 gene, characterized by: including the basic sequence of the pLVX-IRES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, and the promoter sequence and TIM-3 gene cDNA sequence; the multiple cloning site includes a Spe I restriction site and a Not I restriction site, and the TIM-3 gene cDNA sequence includes a Spe I restriction site, TIM -3 gene coding sequence and Not 1 restriction site, and the TIM-3 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 TIM-3基因高表达的慢病毒表达载体 [Claim 2] The lentiviral expression vector specifically promoting the high expression of TIM-3 gene according to claim 1
， 其特征在于： 所述 TIM-3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'- GACTAGTATGTTTTCACATCTTCCCTTTG -3，， 即 SEQ ID NO: 1， 所述下游引物的序列为： 5'-, which is characterized in that: the TIM-3 gene coding sequence is obtained by PCR amplification, the PCR primer includes an upstream primer and a downstream primer, the sequence of the upstream primer is: 5'-GACTAGTATGTTTTCACATCTTCCCTTTG -3, that is, SEQ ID NO: 1 , the sequence of the downstream primer is: 5'-
GGCGGCCGCTGTCGCTTTGCAATGCCATAG -3，， 即 SEQ ID NO: 2。 GGCGGCCGCTGTCGCTTTGCAATGCCATAG-3, i.e. SEQ ID NO: 2.
[权利要求 3] 根据权利要求 2所述的特异促进 TIM-3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 TIM-3 gene according to claim 2, characterized in that: comprising the following steps:
A) TIM-3基因引物设计： 根据 TIM-3基因编码序列， 使用 01igo 7分 析后选取 5'- GACTAGTATGTTTTCACATCTTCCCTTTG -3'， 即 SEQ ID NO: 1作为上游引物， 选取 5'- A) TIM-3 gene primer design: According to the TIM-3 gene coding sequence, use 01igo 7 to analyze and select 5'- GACTAGTATGTTTTCACATCTTCCCTTTG -3', that is, SEQ ID NO: 1 as the upstream primer, select 5'-
GGCGGCCGCTGTCGCTTTGCAATGCCATAG -3，， 即 SEQ IDGGCGGCCGCTGTCGCTTTGCAATGCCATAG -3, i.e. 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；NO: 2 as the downstream primer, and then synthesize the upstream primer and the downstream primer;
B) TIM-3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 TIM-3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 DH50C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 PC R初步鉴定， 将初步鉴定结果说明 TIM-3基因 cDNA序列***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 TIM-3基因 cDNA序列的 pGM-T载体， 用限制性内切酶 S pe l酶和 Not I酶双酶切， 电泳、 切胶回收 1000 bp左右的片段， 此片段 即为 TIM-3基因 cDNA序列； B) Obtaining the TIM-3 gene cDNA sequence: Use the upstream primer and the downstream primer to perform PCR amplification to obtain a large number of TIM-3 gene coding sequences, and then perform an A-tailing reaction on the sequence, and then connect it with T4 DNA The enzyme is connected to the pGM-T vector to obtain the ligation product. The ligation product is transformed into competent E. coli DH50C, evenly spread on an LB medium plate containing ampicillin, and positive single clone colonies are picked, cultured, and preserved. Preliminary identification by PCR, the preliminary identification results indicate that the TIM-3 gene cDNA sequence was successfully inserted into the bacterial liquid for sequencing and identification; use liquid LB medium to culture and sequence the correct E. coli, And extract the pGM-T vector containing the TIM-3 gene cDNA sequence, double-digest it with the restriction endonuclease Spel enzyme and Not I enzyme, electrophoresis, and gel cutting to recover a fragment of about 1000 bp, which is TIM-3 gene cDNA sequence;
C) 特异促进 TIM-3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 Spe I酶和 Not I酶双酶切， 电泳 、 切胶回收载体， 再用 T4 DNA ligase将所述 TIM-3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 DH50C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 TIM-3基因 cDNA序列***成功的菌液进行测序鉴定 C) Construction and identification of lentiviral vectors that specifically promote high expression of TIM-3 gene: Extract plasmid pLVX-IRES-Puro, double-digest with restriction endonucleases Spe I and Not I, electrophoresis, and gel cutting to recover the vector , and then use T4 DNA ligase to connect the TIM-3 gene cDNA sequence into the pLVX-IRES-Puro expression vector to obtain a ligation product, transform the ligation product into competent E. coli DH50C, and evenly spread it into a solution containing ampicillin On the LB medium plate, select positive single clone colonies, culture and preserve the bacterial liquid and perform preliminary PCR identification. The preliminary identification results indicate that the TIM-3 gene cDNA sequence is successfully inserted into the bacterial liquid for sequencing and identification.
D) 特异促进 TIM-3基因高表达的慢病毒载体的抽提： 将测序结果证 实 TIM-3基因 cDNA序列***成功的菌液扩增培养， 对重组质粒进行 抽提， 得到特异促进 TIM-3基因高表达的慢病毒表达载体。 D) Extraction of lentiviral vector that specifically promotes high expression of TIM-3 gene: Sequencing results confirmed that the TIM-3 gene cDNA sequence was successfully inserted into the bacterial liquid amplification culture, and the recombinant plasmid was extracted to obtain a vector that specifically promotes TIM-3 Lentiviral expression vector for high gene expression.
[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 TIM-3基因高表达的慢病 毒表达载体在制备治疗 TIM-3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 TIM-3 gene according to any one of claims 1 to 3 in the preparation of drugs for the treatment of diseases related to abnormal TIM-3 gene expression
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| WO2003063792A2 (en) * | 2002-01-30 | 2003-08-07 | The Brigham And Women's Hospital, Inc. | Compositions and methods related to tim-3, a th1-specific cell surface molecule |
| CN103079644A (en) * | 2010-06-11 | 2013-05-01 | 协和发酵麒麟株式会社 | Anti-TIM-3 antibody |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003063792A2 (en) * | 2002-01-30 | 2003-08-07 | The Brigham And Women's Hospital, Inc. | Compositions and methods related to tim-3, a th1-specific cell surface molecule |
| CN103079644A (en) * | 2010-06-11 | 2013-05-01 | 协和发酵麒麟株式会社 | Anti-TIM-3 antibody |
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| Title |
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| ZHANG, SHENGTAO ET AL.: "Expression of Human T- cell Immunoglobulin Mucin 3(TIM-3) on Eukaryotic Cells and Establishment of Stable Transfectant Cell Line", CHINESE JOURNAL OF CELLULAR AND MOLECULAR IMMUNOLOGY, vol. 21, no. 6, 31 December 2005 (2005-12-31), pages 707 - 709 and 713 * |
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