WO2017214944A1 - 促进tigit基因高表达的慢病毒载体及其应用 - Google Patents

促进tigit基因高表达的慢病毒载体及其应用 Download PDF

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WO2017214944A1
WO2017214944A1 PCT/CN2016/086068 CN2016086068W WO2017214944A1 WO 2017214944 A1 WO2017214944 A1 WO 2017214944A1 CN 2016086068 W CN2016086068 W CN 2016086068W WO 2017214944 A1 WO2017214944 A1 WO 2017214944A1
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sequence
tigit
tigit gene
gene
cdna sequence
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毛侃琅
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毛侃琅
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    • 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
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Definitions

  • the present invention belongs to the field of genetic engineering technology, and particularly relates to a lentiviral vector which promotes high expression of a TIGIT gene and an application thereof.
  • T cell immunoglobulin domain and immunoreceptor tyrosine inhibition motif protein T cell immunoglobulin and ITIM domain
  • TIGIT is a newly discovered membrane protein molecule composed of immunoglobulin variable region (IgV), transmembrane region and immunoreceptor tyrosine-based inhibitor motif (ITIM), widely expressed in Treg cells, memory T cells, activating T cells, and NK cell surfaces.
  • IgV immunoglobulin variable region
  • ITIM immunoreceptor tyrosine-based inhibitor motif
  • TIGIT is highly constitutively expressed on Treg, and its expression is further up-regulated after activation, which is low on memory T cells, while naive CD4+ CD25-T is expressed only after activation; 2) TIGIT-Fc fusion protein It can promote DC secretion of IL-10 and reduce pro-inflammatory factor 11 ⁇ 12; 3) The ability of TIGIT-Fc fusion protein-treated mature DC to stimulate T cell proliferation is weakened, which is related to DC secretion of IL-10; 4) In vivo use of TIGIT Proteins can significantly attenuate delayed hypersensitivity reactions with effects comparable to those of CTLA-4 protein. There is increasing evidence that TIGIT is an important immunomodulator and functions similarly to ITIM, which inhibits the cytotoxicity of NK cells and the activation and proliferation of CD4+ T cells and the production of cytokines.
  • TIGIT is a substance that inhibits the action of T cells, and functions to inhibit the excessive activation of T cells under normal conditions; however, this property also makes it easy for tumor cells to utilize to achieve immune escape. Therefore, TIGIT is a potential target for tumor therapy and requires in-depth research. However, the lack of lentiviral expression vectors that specifically promote the high expression of TIG IT gene in the prior art makes the research impossible.
  • the TIGIT 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 TIGIT gene, thereby completing the present invention.
  • the present invention provides a lentiviral expression vector which specifically promotes high expression of the TIGIT gene, including the basic sequence of the pLVX-IRE S-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and the TI GIT a gene cDNA sequence; the multiple cloning site includes an EcoR I cleavage site and a Spe I cleavage site, and the TIGIT gene cDNA sequence includes an EcoR I cleavage site, a TIGIT gene coding sequence, and a Spe I cleavage site The TIGIT gene cDNA sequence is positively inserted into the multiple cloning site sequence.
  • the lentiviral expression vector constructed by inserting the TIGIT gene cDNA sequence of the present invention into the pLVX-IRES-Puro expression vector has high transfection efficiency, low dosage, and can be stably, efficiently and stably improved.
  • the advantages of TIGIT gene expression can be used as a powerful tool in the research and development of drugs for the treatment of diseases such as Alzheimer's disease.
  • the TIGIT 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'-GGAATTCATGCGCTGGTGTCTCCTCCTG-3', ie SEQ ID NO: 1
  • the sequence of the downstream primer is: 5'-GACTAGTCTAACCAGTCTCTGTGAAGAAG -3', ie SEQ ID NO: 2.
  • the TIGIT gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the TIGIT 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 TIGIT gene, comprising the following steps:
  • A) TIGIT gene primer design According to the TIGIT gene coding sequence, using O3 ⁇ 4o 7 analysis, select 5'-GGAATTCATGCGCTGGTGTCTCCTCCTG -3, ie SEQ ID NO: 1 as the upstream bow, select 5 '- GACTAGTCTAACCAGTCTCTGTGAAGAAG -3' , ie, 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 are free of primer dimer, and the annealing temperature difference is small;
  • B) obtaining the TIGIT gene cDNA sequence PCR amplification using the upstream primer and the downstream primer to obtain a large number of TIGIT gene coding sequences, and then adding the A tail reaction to the sequence, using T4 DNA
  • the ligase is ligated to the pGM-T vector to obtain a ligation product, and the ligation product is transformed into competent E. coli ToplO, uniformly coated on an ampicillin-containing LB medium plate, and a positive monoclonal colony culture preservation liquid is picked.
  • the preliminary identification of PCR was carried out, and the preliminary identification results indicated that the TIGIT gene cDNA sequence was inserted into the successful bacterial solution for sequencing.
  • the correct Escherichia coli was identified by liquid LB medium culture and sequencing, and the pGM-T vector carrying the TIGIT gene cDNA sequence was extracted. , using restriction endonuclease EcoR I enzyme and Spe l enzyme double digestion, electrophoresis, gelation recovery 500
  • this fragment is the TIGIT gene cDNA sequence ⁇ IJ;
  • the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of TIGIT gene. After successful identification, it is packaged into a virus and introduced into Jurkat cells. After screening cells with puromycin, real-time quantitative PCR and Westernization are used. The Blot technique verifies the change of TIGIT gene expression from mRNA and protein levels, respectively. The experimental results show that the TIGIT gene cDNA sequence provided by the present invention is successfully inserted into the pLVX-I RES-Puro expression vector, and can promote TIGIT specifically, continuously, efficiently and stably. High expression of genes.
  • the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of a TIGIT gene for the preparation of a medicament for treating a disease associated with abnormal expression of a TIGIT gene.
  • the lentiviral expression vector which specifically promotes the high expression of the TIGIT gene provided by the invention has the advantages of high transfection efficiency, low dosage, and can promote the high expression of the TIGIT gene in a specific, sustained, efficient and stable manner, and can be used as The force tool is applied to drug research and development related to TIGIT; the present invention also provides specific promotion of TIGI
  • a method for constructing a lentiviral expression vector with high expression of T gene which has a good operation effect and reduces the cost of sequence synthesis
  • 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.
  • TIGIT gene coding sequence GenBank NM-173799.3
  • TIGIT gene coding sequence GenBank NM-173799.3
  • upstream primers and downstream primers requiring as little primer-free dimer as possible and the annealing temperature difference is small
  • upstream primers and The protective base and the restriction sites E ⁇ RI 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 S Co., Ltd.
  • Example 2 Construction of a lentiviral vector that specifically promotes high expression of the TIGIT gene
  • the coding sequence of the TIGIT gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A tail reaction, and then ligated to the pGM-T vector by T4 DNA ligase.
  • the ligation product (TIGIT-T vector) was obtained, and the ligation product was transformed into competent E. coli To plO, which was applied to an ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h, and the homologous set was negative.
  • Control group 1 consistentt cells were uniformly coated on ampicillin-free plates
  • negative control group 2 competent 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 TIGIT 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 (the competent cells were uniformly coated on the plate without ampicillin), 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 in good growth state were inoculated into six wells, 1,000,000 cells per well, and the extracted recombinant plasmid pLVX-TIGIT 2 g 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 0.45 ⁇ m sieve for infection of Jurka t cells, and the Lenti-X GoStix kit was used to detect the virus titer of 5,000,000 to 50,000,000 IFU.
  • Jurkat cells were inoculated in a 6-well plate, 1000000 cells per well, and the cell density was about 50% after 12 hours.
  • the virus solution was taken separately, and the virus was diluted 10 times with DMEM complete medium, and then polybmne was added. 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 TIGIT gene.
  • Jurkat cells, pLVX empty vector control Jurkat cell group, and pLVX-TIGIT high expressing cells were each inoculated into a 6-well plate. When the cell density reached 80 ⁇ 3 ⁇ 4-90 ⁇ 3 ⁇ 4, 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 the reverse transcription, add 9 ( ⁇ L of RNase Free dH 20 diluted cDNA, and store at -20 ° C for later detection. Take the cDNA of each group of cells.
  • l L is a template, with GAPDH as an internal reference, real-time quantitative PCR (QPCR) to detect the relative expression of TIGIT, set the reaction conditions: 95 ° C 30s, 1 cycle, 54 ° C 30s 40 cycles, 95 ° C 5s, 60 ° C lmin , 95 ° C 15s , using SYBR Primescript RT-PCR Kit to detect the relative expression of TIGIT gene in each group of cells. After the pLVX-TIGIT cells were continuously cultured for 20 passages, the above experiment was repeated. The summarized results are shown in Figure 2.
  • TIGIT gene is 70-fold higher than that of Jurkat cells, whether it is just after screening or after 20 generations of pLVX-TIGIT cells, and the expression of TIGIT gene in pLVX empty vector cells. There is almost no change compared with Jurkat cells, indicating that the TIGIT gene cDNA sequence provided by the present invention is successfully inserted into the pLVX-IRES-Puro expression vector, and can specifically, stably, efficiently and stably promote the high expression of TIGIT gene.
  • the lentiviral expression vector which specifically promotes the high expression of TIGIT gene provided by the invention has the advantages of high transfection efficiency, low dosage, specific, sustained, efficient and stable promotion of high expression of TIGIT gene, and can be used as a powerful tool for application with TIGIT.
  • Related drug research and development; the present invention also provides specific promotion of TIGI
  • the construction method of the lentiviral expression vector with high expression of T gene has good operation effect, reduces the cost of sequence synthesis, and has low cost.

Abstract

提供一种促进TIGIT基因高表达的慢病毒表达载体，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TIGIT基因cDNA序列；所述多克隆位点包括EcoR I酶切位点和Spe I酶切位点，所述TIGIT基因cDNA序列包括EcoR I酶切位点、TIGIT基因编码序列和Spe I酶切位点，所述TIGIT基因cDNA序列正向插入所述多克隆位点序列中。

Description

发明名称：促进 TIGIT基因高表达的慢病毒载体及其应用 技术领域
[0001] 本发明属于基因工程技术领域， 尤其涉及一种促进 TIGIT基因高表达的慢病毒 载体及其应用。
背景技术
[0002] T细胞免疫球蛋白域和免疫受体酪氨酸抑制基序的蛋白 （T cell immunoglobulin and ITIM domain,
TIGIT) 是一种新发现的膜蛋白分子， 由免疫球蛋白可变区 （IgV) 、 跨膜区以 及受体酪氨酸抑制基序 (ITIM， immunoreceptor tyrosine-based inhibitor motif) 组 成， 广泛表达于 Treg细胞、 记忆性 T细胞、 活化性 T细胞以及 NK细胞表面。 研究发现： 1 ) TIGIT在 Treg上组成性高表达， 活化后表达进一步上调， 它在记 忆性 T细胞上低表达， 而 naive CD4+ CD25- T则仅在活化后表达； 2) TIGIT-Fc 融合蛋白可以促进 DC分泌 IL- 10 , 而减少促炎因子 11^12 ; 3) TIGIT-Fc融合蛋白 处理过的成熟 DC刺激 T细胞增殖的能力减弱， 与 DC分泌 IL- 10有关； 4) 体内使 用 TIGIT蛋白可以显著减弱迟发性超敏反应， 其效应与 CTLA-4蛋白的效果相当 。 越来越多的证据表明： TIGIT是一种重要的免疫调节剂， 与 ITIM的功能相似 ， 它可以抑制 NK细胞的细胞毒作用和 CD4+ T细胞的活化增殖以及细胞因子的产 生。
技术问题
[0003] TIGIT是一种抑制 T细胞发挥作用的物质， 其在正常状态下发挥抑制 T细胞过度 活化的作用； 但该特性也使其容易被肿瘤细胞利用实现免疫逃逸。 因此 TIGIT是 一个潜在的肿瘤治疗的靶点， 需要进行深入研究， 但现有技术缺乏特异促进 TIG IT基因高表达的慢病毒表达载体使得相关研究无法很好地开展。
问题的解决方案
技术解决方案
[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 TIGIT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 TIGIT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 TIGIT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 GIT基因 cDNA序列； 所述多克隆位点包括 EcoR I酶切位点和 Spe I酶切位点， 所 述 TIGIT基因 cDNA序列包括 EcoR I酶切位点、 TIGIT基因编码序列和 Spe I酶切位 点， 所述 TIGIT基因 cDNA序列正向插入所述多克隆位点序列中。
[0006] 釆用上述技术方案， 本发明提供的 TIGIT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 TIGIT基因表达的优点， 可作为有力工具应用于制备治疗 TIGIT基 因表达对阿尔兹海默症等疾病药物的研究和开发中。
[0007] 作为本发明的进一步改进， 所述 TIGIT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'- GGAATTCATGCGCTGGTGTCTCCTCCTG -3' , 即 SEQ ID NO: 1 , 所述下游引 物的序列为： 5'- GACTAGTCTAACCAGTCTCTGTGAAGAAG -3'， 即 SEQ ID NO: 2。 采用上述 PCR引物序列， 通过 PCR可以扩增出 TIGIT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 TIGIT基因 , 减少了序列合成 费用， 成本较低。
[0008] 相应的， 本发明还提供特异促进 TIGIT基因高表达的慢病毒表达载体的构建方 法， 包括如下步骤：
[0009] A) TIGIT基因引物设计： 根据 TIGIT基因编码序列， 使用 O¾o 7分析后选取 5'- GGAATTCATGCGCTGGTGTCTCCTCCTG -3， , 即 SEQ ID NO: 1作为上游弓 |物 , 选取 5 '- GACTAGTCTAACCAGTCTCTGTGAAGAAG -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；
[0010] B) TIGIT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 TIGIT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 TIGIT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 TIGIT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
bp左右的片段， 此片段即为 TIGIT基因 cDNA序歹 IJ；
[0011] C) 特异促进 TIGIT基因高表达的慢病毒载体的构建和鉴定： 提取质粒 pLVX-I RES-Puro, 用限制性内切酶 EcoR I酶和 Spe l酶双酶切， 电泳、 切胶回收载体， 再 用 T4 DNA ligase将所述 TIGIT基因 cDNA序列连接至 IjpLVX-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 TIGIT基因 cDNA序列插入成功的菌液进行测序鉴 定；
[0012] D) 特异促进 TIGIT基因高表达的慢病毒载体的抽提： 将测序结果证实 TIGIT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 TI GIT基因高表达的慢病毒表达载体。
[0013] 本发明利用基因工程技术构建特异促进 TIGIT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 实时荧光定量 PCR和 Western Blot技术分别从 mRNA和蛋白水平验证 TIGIT基因表 达的变化， 实验结果证明本发明提供的 TIGIT基因 cDNA序列成功插入至 pLVX-I RES-Puro表达载体中， 能特异、 持续、 高效、 稳定地促进 TIGIT基因高表达。
[0014] 本发明还提供特异促进 TIGIT基因高表达的慢病毒表达载体在制备治疗 TIGIT基 因表达异常相关疾病的药物中的用途。
发明的有益效果
有益效果
[0015] 本发明提供的特异促进 TIGIT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 TIGIT基因高表达的优点， 可作为有 力工具应用于与 TIGIT相关的药物研究和开发中； 本发明还提供了特异促进 TIGI
T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
附图说明
[0016] 图 1为 pLVX-IRES-Puro表达载体的质粒图谱。
[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。
实施该发明的最佳实施例
本发明的最佳实施方式
[0018] 下面结合附图与具体实施例对本发明做进一步的说明。
[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
Kit购自 QIAGEN公司， pGM-T载体购自天根公司， Endo-Free Plasmid Mini Kit II 购自 Omega bio-tek公司。
[0020] 实施例一 TIGIT基因引物的设计。
[0021] 根据 TIGIT基因编码序列 （GenBank NM— 173799.3) ， 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 E∞R 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 S 务有限公司合成。
[0022] 表 1 TIGIT基因的 PCR弓 |物序列
[] [表 1]
Figure imgf000007_0001
[0023] 实施例二 特异促进 TIGIT基因高表达的慢病毒载体的构建
[0024] 将合成的引物稀释后， 用 Premix PrimeSTAR HS酶对 TIGIT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 （TIGIT-T载体） , 将该连接产物转化到感受态大肠杆菌 To plO中， 均勾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
tig/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。
[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 TIGIT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 TIGI T基因， 接着将重组载体送至上海生工公司测序。
[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 TIGIT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 , 同时设置阴性对照组 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。
[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 TIGIT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 TIGIT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-TIGIT质粒。
[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 lOO 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-TIGIT, 测其纯度和浓度， 结果如表 2所示。
[0029] 表 2重组质粒的纯度和浓度
[] [表 2]
Figure imgf000008_0001
[0030] 实施例三 慢病毒包装
[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-TIGIT 2 g转染到 293FT细胞 , 48h后收集含病毒的上清培养基， 用 0.45μπι的筛子过滤病毒液， 用于感染 Jurka t细胞， Lenti-X GoStix试剂盒检测病毒的滴度为 5000000〜50000000 IFU。
[0032] 实施例四 慢病毒转导 Jurkat细胞
[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%， 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 （polybmne) 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。 [0034] 实施例五 荧光定量 PCR检测 TIGIT基因表达量。
[0035] 根据 GAPDH和 TIGIT基因 mRNA序列， 利用引物设计软件 Oligo 7.0设计引物。
[] [表 3]
Figure imgf000009_0001
[0036] 分别接种 Jurkat细胞、 pLVX空载体对照 Jurkat细胞组、 pLVX-TIGIT高表达细胞 至 6孔板。 细胞密度达到 80<¾-90<¾时， 用 RNeasy Mini Kit提取各组细胞的总 RNA ， 禾1 J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, ∞。 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 L为模板， 以 GAPDH为内参， 实吋荧光定量 PCR (QPCR) 检测 TIGIT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 TIGIT基因相对表达 量。 将 pLVX-TIGIT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-TIGIT细胞， 其 TIGIT基因的表达量较 Jurkat细胞都有 70倍以上的升高， 而 pLVX空载体细胞的 TIGIT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 TIGIT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 TIGIT基因高表达。
[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。 工业实用性
本发明提供的特异促进 TIGIT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 TIGIT基因高表达的优点， 可作为有 力工具应用于与 TIGIT相关的药物研究和开发中； 本发明还提供了特异促进 TIGI
T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。

Claims

[权利要求 1] 一种特异促进 TIGIT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 TIGIT基因 cDNA序列； 所述多克隆位点包括 E co I酶切位点和 Spe I酶切位点， 所述 TIGIT基因 cDNA序列包括 EcoR I酶切位点、 TIGIT基因编码序列和 Spe
I酶切位点 , 所述 TIGIT基因 cDNA序列正向插入所述多克隆位点序列 中。
[权利要求 2] 根据权利要求 1所述的特异促进 TIGIT基因高表达的慢病毒表达载体
, 其特征在于： 所述 TIGIT基因编码序列通过 PCR扩增获得， PCR弓 I 物包括上游引物和下游引物， 所述上游引物的序列为： 5'- GGAATTC ATGCGCTGGTGTCTCCTCCTG - 3 '， 即 SEQ ID NO： 1， 所述下游引物的序列为： 5'-
GACTAGTCTAACCAGTCTCTGTGAAGAAG -3' , IPSEQ ID NO: 2
[权利要求 3] 根据权利要求 2所述的特异促进 TIGIT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：
A) TIGIT基因引物设计： 根据 TIGIT基因编码序列， 使用 Oligo 7分 析后选取 5，- GGAATTCATGCGCTGGTGTCTCCTCCTG -3' , 即 SEQ ID NO: 1作为上游引物， 选取 5'-
GACTAGTCTAACCAGTCTCTGTGAAGAAG -3' , IPSEQ 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；
B) TIGIT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 TIGIT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 PC R初步鉴定， 将初步鉴定结果说明 TIGIT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 TIGIT基因 cDNA序列的 pGM-T载体， 用限制性内切酶 E coR I酶和 Spe l酶双酶切， 电泳、 切胶回收 500 bp左右的片段， 此片段 即为 TIGIT基因 cDN A序列；
C) 特异促进 TIGIT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 TIGIT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 TIGIT基因 cDNA序列插入成功的菌液进行测序鉴 定；
D) 特异促进 TIGIT基因高表达的慢病毒载体的抽提： 将测序结果证 实 TIGIT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 TIGIT基因高表达的慢病毒表达载体。
[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 TIGIT基因高表达的慢病 毒表达载体在制备治疗 TIGIT基因表达异常相关疾病的药物中的用途
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