WO2017214910A1

WO2017214910A1 - Lentiviral expression vector for specifically promoting high expression of tβ4 gene, and applications thereof

Info

Publication number: WO2017214910A1
Application number: PCT/CN2016/085926
Authority: WO
Inventors: 石庆学
Original assignee: 石庆学
Priority date: 2016-06-15
Filing date: 2016-06-15
Publication date: 2017-12-21

Abstract

A lentiviral expression vector for specifically promoting high expression of a Tβ4 gene comprises a fundamental sequence, a resistance gene sequence, a multiple-cloning-sites sequence, a promoter sequence, and a Tβ4 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 Tβ4 gene cDNA sequence comprises an EcoR I enzyme cutting site, a Tβ4 gene coding sequence and an Spe I enzyme cutting site, and the Tβ4 gene cDNA sequence is forwardly inserted into the multiple-cloning-sites sequence. The lentiviral expression vector has the advantages of high transfection efficiency and small usage and being capable of specifically, continuously, efficiently and stably expressing the Tβ4 gene, and can serve as a powerful tool applied to research and development of drugs related to Tβ4.

Description

特异促进 Τβ4基因高表达的慢病毒表达载体及其应用技术领域 Lentiviral expression vector for specifically promoting high expression of Τβ4 gene and application thereof

[0001] 本发明属于基因工程技术领域，尤其涉及一种特异促进 Τβ4基因高表达的慢病毒表达载体及其构建方法与应用。 [0001] The present invention belongs to the field of genetic engineering technology, and particularly relates to a chronic virus expression vector which specifically promotes high expression of Aβ4 gene, and a construction method and application thereof.

背景技术 Background technique

[0002] 胸腺素 β4 (Τβ4) 是由 129个碱基组成的小分子多肽，多以单链形式存和发挥作用，整个分子中富含谷氨酸和赖氧酸， Ν端为乙醜丝氨酸，谷氧酸和赖氨酸的量占到胸腺素氧基酸总量的 70%_80<¾，是真核生物细胞中典型的肌动蛋白整合分子，存在于大多数的哺乳动物和某些脊椎动物的多种组织和有核细胞中。通过免疫定位的方法发现， Τβ4在肾脏、膀胱、子宫、卵巢中都有表达。 Τβ4在生物体中具有重要的作用，有多种生物学活性，它可以调节肌动蛋白 G与 F之间的分布比例，进而诱导细胞骨架结构的形成;参与机体的免疫调节、哺乳动物毛囊的发育、与神经的发育也密切相关，对创伤的修复也有促进功效，具体表现为能够促进创伤部位的细胞发生迁移、诱导胶原的沉积等修复过程，调节器官发育及肿瘤的发生与转移等功能。 [0002] Thymosin β4 (Τβ4) is a small molecule polypeptide consisting of 129 bases, which is mostly in a single-stranded form. The whole molecule is rich in glutamic acid and lysine, and the terminal is ugly-serine. The amount of glutamic acid and lysine accounts for 70%_80<3⁄4 of the total amount of thymosinic acid, which is a typical actin-integrating molecule in eukaryotic cells, present in most mammals and certain A variety of tissues and nucleated cells of vertebrates. By immunolocalization, it was found that Τβ4 is expressed in the kidney, bladder, uterus, and ovary. Τβ4 plays an important role in organisms and has a variety of biological activities. It can regulate the distribution ratio between actin G and F, and then induce the formation of cytoskeletal structure; participate in the body's immune regulation, mammalian hair follicles. Development, and nerve development are also closely related, and also promote the repair of trauma, specifically to promote the migration of cells in the wound site, induce collagen deposition and other repair processes, regulate organ development and tumor occurrence and metastasis.

技术问题 technical problem

[0003] Τβ4在肿瘤的发生、转移和血管生成方面具有重要作用，因此 Τβ4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 Τβ4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] Τβ4 plays an important role in tumorigenesis, metastasis, and angiogenesis. Therefore, Τβ4 is a potential target for tumor therapy and requires in-depth study. However, the prior art lacks lentiviral expression that specifically promotes high expression of Τβ4 gene. The carrier makes the relevant research 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 [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 EcoR I cleavage site and Spe are included.

I酶切位点的 Τβ4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 Τβ4基因高表达的慢病毒表达载体，从而完成本发明。 The Τβ4 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 the high expression of the Τβ4 gene, thereby completing the present invention.

[0005] 本发明提供一种特异促进 Τβ4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 Τβ4 基因 cDNA序列；所述多克隆位点包括 EcoR I酶切位点和 Spe The present invention provides a lentiviral expression vector which specifically promotes high expression of the Aβ4 gene, including pLVX-IRES- The basic sequence of the puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence, and the Τβ4 gene cDNA sequence; the multiple cloning site includes the EcoR I cleavage site and Spe

I酶切位点，所述 Τβ4基因 cDNA序列包括 EcoR I cleavage site, the Τβ4 gene cDNA sequence including EcoR

I酶切位点、 Τβ4基因编码序列和 Spe I酶切位点，所述 Τβ4基因 cDNA序列正向插入所述多克隆位点序列中。 The I cleavage site, the Τβ4 gene coding sequence and the Spe I cleavage site, and the Τβ4 gene cDNA sequence is inserted into the multiple cloning site sequence.

[0006] 采用上述技术方案，本发明提供的 Τβ4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 Τβ4基因表达的优点，可作为有力工具应用于制备治疗 Τβ4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 Τβ4 gene into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase Τβ4. The advantages of gene expression can be used as a powerful tool in the preparation and treatment of Τβ4 gene expression for diseases such as Alzheimer's disease.

[0007] 作为本发明的进一步改进，所述 Τβ4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 Τβ4 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'-

GGAATTCATGTCTGACAAACCCGATATG -3' , 即 SEQ ID NO: 1，所述下游引物的序列为： 5，- GACTAGTTTACGATTCGCCTGCTTGCTTC -3'，即 SEQ ID NOGGAATTCATGTCTGACAAACCCGATATG -3' , ie SEQ ID NO: 1, the sequence of the downstream primer is: 5,- GACTAGTTTACGATTCGCCTGCTTGCTTC -3', ie SEQ ID NO

： 2。采用上述 PCR引物序列，通过 PCR可以扩增出 Τβ4基因编码序列，并可成功 ***至 pLVX-IRES-Puro表达载体中持续表达 Τβ4基因，减少了序列合成费用，成本较低。 : 2. Using the above PCR primer sequence, the coding sequence of Τβ4 gene can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the Τβ4 gene, which reduces the cost of sequence synthesis and has a low cost.

[0008] 相应的，本发明还提供特异促进 Τβ4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 Aβ4 gene, and comprises the following steps:

[0009] Α) Τβ4基因引物设计：根据 Τβ4基因编码序列，使用 01igo 7分析后选取 5'- GGAATTCATGTCTGACAAACCCGATATG -3'，即 SEQ ID NO: 1作为上游弓 |物，选取 5 '- GACTAGTTTACGATTCGCCTGCTTGCTTC -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] Τβ4 gene primer design: According to the Τβ4 gene coding sequence, using 01igo 7 analysis, select 5'-GGAATTCATGTCTGACAAACCCGATATG -3', ie SEQ ID NO: 1 as the upstream bow, select 5 '- GACTAGTTTACGATTCGCCTGCTTGCTTC -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) Τβ4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 Τβ4基因编码序列，然后将该序列进行加 Α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 Top 10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 Τβ4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 Τβ4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 200 bp左右的片段，此片段即为 Τβ4基因 cDNA序列；[0010] B) obtaining a cDNA sequence of the Τβ4 gene: PCR amplification is performed using the upstream primer and the downstream primer to obtain a large number of Τβ4 gene coding sequences, and then the sequence is subjected to a tail-flick reaction, and T4 DNA ligase is used. The ligation product is obtained by ligation to the pGM-T vector, and the ligation product is transformed into competent E. coli Top 10, 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, the preliminary identification results indicate that the cDNA sequence of Τβ4 gene was successfully inserted. The bacterial liquid was sequenced and identified; the correct Escherichia coli was identified by liquid LB medium, and the pGM-T vector carrying the cDNA sequence of Τβ4 gene was extracted, and the restriction enzymes EcoR I enzyme and Spe I enzyme double enzyme were used. Cutting, electrophoresis, and gel-cutting recover a fragment of about 200 bp, which is the cDNA sequence of Τβ4 gene;

[0011] C) [0011] C)

特异促进 Τβ4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 Τβ4基因 cDNA序列连接 ajpLVX-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 Τβ4基因 cDNA序列***成功的菌液进行测序鉴定； Construction and identification of a lentiviral vector that specifically promotes high expression of Τβ4 gene: The plasmid pLVX-IRES-Puro was extracted, digested with restriction endonuclease EcoR I enzyme and Spe I enzyme, electrophoresis, gel-removed recovery vector, and T4 DNA ligase The Τβ4 gene cDNA sequence was ligated into the ajpLVX-IRES-Puro expression vector to obtain a ligation product, which was transformed into competent E. coli ToplO and uniformly coated onto an ampicillin-containing LB medium plate. The positive monoclonal colonies were cultured and preserved, and the PCR was initially identified. The preliminary identification results indicated that the Τβ4 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification;

[0012] D) 特异促进 Τβ4基因高表达的慢病毒载体的抽提：将测序结果证实 Τβ4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 Τβ 4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 Τβ4 gene: The sequencing result confirms that the Τβ4 gene cDNA sequence is inserted into a successful bacterial cell expansion culture, and the recombinant plasmid is extracted to obtain a specific Τβ 4 gene. Expressed lentiviral expression vector.

[0013] 本发明利用基因工程技术构建特异促进 Τβ4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 Hs27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 Τβ4基因表达的变化，实验结果证明本发明提供的 Τβ4基因 cDNA序列成功***至 pLVX-IRES-Pu ro表达载体中，能特异、持续、高效、稳定地促进 Τβ4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 Τβ4 gene. After successful identification, it is packaged into a virus and introduced into Hs27 cells, and after puromycin is used to screen cells, real-time quantitative PCR is used. The Western Blot technique verified the change of Τβ4 gene expression from mRNA and protein levels, respectively. The experimental results confirmed that the Τβ4 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IRES-Pu ro expression vector, and can be specifically, continuously, efficiently and stably promoted. The Τβ4 gene is highly expressed.

[0014] 本发明还提供特异促进 Τβ4基因高表达的慢病毒表达载体在制备治疗 Τβ4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 the Aβ4 gene for the preparation of a medicament for treating a disease associated with abnormal expression of Aβ4 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 Τβ4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 Τβ4基因高表达的优点，可作为有力工具应用于与 Τβ4相关的药物研究和幵发中；本发明还提供了特异促进 Τβ4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 lentiviral expression vector which specifically promotes the high expression of Τβ4 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 Τβ4 gene, and can be used as a powerful tool. In the drug research and development related to Τβ4, the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of Τβ4 gene, which has good operation effect, reduces sequence synthesis cost, and has low cost.

对附图的简要说明附图说明 Brief description of the drawing 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] Hs27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] Hs27 cells were purchased from the Cell Resource Center of Shanghai Institute of Biological Sciences, and 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 Τβ4基因引物的设计。 Example 1 Design of the Τβ4 gene primer.

[0021] 根据 Τβ4基因编码序列（GenBank NM_021109.3) ，使用 01igo7对其进行分析 [0021] According to the Τβ4 gene coding sequence (GenBank NM_021109.3), it was analyzed using 01igo7

，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。 , looking for upstream primers and downstream primers (requires no primer dimer as much as possible and the annealing temperature difference is small), then add the protective base and the restriction sites EcoR I and EcoR I at the 5' end of the upstream primer and the downstream primer, respectively. 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 Τβ4基因的 PCR弓 |物序列 Table 1 PCR bow of the Τβ4 gene |

[] [表 1] [] [Table 1]

[0023] 实施例二特异促进 Τβ4基因高表达的慢病毒载体的构建 Example 2 Construction of a lentiviral vector specifically promoting the high expression of Aβ4 gene

[0024] 将合成的弓 I物稀释后，用 Premix PrimeSTAR HS酶对 Τβ4基因的编码序列进行扩增，电泳回收后然后将其进行加 Α尾反应后，用 T4 DNA连接酶连接到 pGM-T载体上得到连接产物（Τβ4-Τ载体），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，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[0024] After diluting the synthetic antibody, the coding sequence of the Τβ4 gene was amplified with Premix PrimeSTAR HS enzyme, and after electrophoresis recovery, the tailing reaction was carried out, and then connected to pGM-T by T4 DNA ligase. Load The ligation product (Τβ4-Τ vector) was obtained on the body, and the ligation product was transformed into competent E. coli ToplO, uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h, and the homologous set was negative. Control group 1 (consistent cells were uniformly coated on ampicillin-free plates), negative control group 2 (competent cells were uniformly coated on plates containing 100 g/ml ampicillin), positive control group 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 ligation product of the double enzyme-cut empty vector was uniformly coated on a plate containing 100 g/ml ampicillin), and the positive control group 2 (the empty carrier was uniformly coated on a plate containing 100 g/mL ampicillin). In the experimental group, a single colony grew, and 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 Τβ4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 Τβ4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 Τβ4 gene for preliminary identification. The results showed that the cultures of 8 single colonies could be successfully amplified. The β4 gene was exported, 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 Τβ4基因序列的重组 Τ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 sputum vector containing the Τβ4 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 an 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 Τβ4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 Τβ4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-Tp4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 Τβ4 gene for preliminary identification. The results showed that all 6 culture broths could successfully amplify the Τβ4 gene, and then the recombinant vector broth was sent to Shanghai Biotech Co., Ltd. for sequencing. The sequencing results were exactly as expected, and the pLVX-Tp4 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 ρίνΧ-Τβ4，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 [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 ρίνΧ-Τβ4 was extracted and its purity and concentration were measured. The results are shown in Table 2. Table 2 Purification and Concentration of Recombinant Plasmid

[] [表 2]

[] [Table 2]

[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 ρίνΧ-Τβ4 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 Hs27 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 with good growth state were inoculated into six wells, 1,000,000 cells per well, and the extracted recombinant plasmid ρίνΧ-Τβ4 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 infecting Hs27 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 Hs27细胞 Example 4 Lentiviral transduction Hs27 cells

[0033] 接种 Hs27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] Hs27 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 Τβ4基因表达量。 Example 5 Fluorescence quantitative PCR was used to detect the expression level of Τβ4 gene.

[0035] 根据 GAPDH和 Τβ4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 Τβ4 gene mRNA sequences, the primer design software Oligo 7.0 was used to design the bow.

[] [表 3] [] [table 3]

分别接种 Hs27细胞、 pLVX空载体对照 Hs27细胞组、 pLVX-Tp4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 Hs27 cells, pLVX empty vector control Hs27 cell group, and pLVX-Tp4 high expressing cells were inoculated into 6-well plates, 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. Wo ¹ J with 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 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.

Ιμί为模板，以 GAPDH为内参，实吋荧光定量 PCR (QPCR) 检测 Τβ4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 Ιμί 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 Τβ4, 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 Τβ4基因相对表达量。将 ρίνΧ-Τβ4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 ρίνΧ-Τβ4细胞，其 Τβ4基因的表达量较 Hs27细胞都有 260倍以上的升高，而 pLVX空载体细胞的 Τβ 4基因表达量与 Hs27细胞相比基本没有变化，说明本发明提供的 Τβ4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 T β4基因高表达。 Lmin, 95 ° C for 15 s, using SYBR Primescript RT-PCR Kit to detect the relative expression of Τβ4 gene in each group. After continuously culturing ρίνΧ-Τβ4 cells for 20 passages, the above experiment was repeated. The summarized results are shown in Figure 2. It can be seen that whether ρίνΧ-Τβ4 cells, which have just been screened or have been cultured for 20 generations, have a 260-fold increase in the expression of Τβ4 gene compared with Hs27 cells, whereas Τβ 4 gene expression in pLVX empty vector cells. The amount of the Τβ4 gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IRES-Puro expression vector, and the T β4 gene expression was promoted 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 Τβ4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 Τβ4基因高表达的优点，可作为有力工具应用于与 Τβ4相关的药物研究和幵发中；本发明还提供了特异促进 Τβ4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 lentiviral expression vector which specifically promotes the high expression of Τβ4 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 Τβ4 gene, and can be used as a powerful tool. In the drug research and development related to Τβ4, the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of Τβ4 gene, which has good operation effect, reduces sequence synthesis cost, and has low cost.

Claims

权利要求书 claims

[权利要求 1] 一种特异促进 Τβ4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 Τβ4基因 cDNA序歹 ij ; 所述多克隆位点包括 EcoR I 酶切位点和 Spe I酶切位点，所述 Τβ4基因 cDNA序列包括 EcoR I酶切位点、 Τβ4基因编码序列和 Spe l酶切位点，所述 Τβ4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 Tβ4 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 The cDNA sequence of Tβ4 gene is ij; the multiple cloning site includes EcoR I restriction site and Spe I restriction site, and the Tβ4 gene cDNA sequence includes EcoR I restriction site, Tβ4 gene coding sequence and Spe I enzyme Cutting site, the Tβ4 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 Τβ4基因高表达的慢病毒表达载体，其特征在于：所述 Τβ4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'- GGAATTCATGTCTGACAAACCCGATATG -3，，即 SEQ ID NO: 1，所述下游引物的序列为： 5'- [Claim 2] The lentiviral expression vector specifically promoting the high expression of Tβ4 gene according to claim 1, characterized in that: the Tβ4 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'- GGAATTCATGTCTGACAAACCCGATATG -3, that is, SEQ ID NO: 1, and the sequence of the above-mentioned downstream primer is: 5'-

GACTAGTTTACGATTCGCCTGCTTGCTTC -3'，即 SEQ ID NO: 2。 GACTAGTTTACGATTCGCCTGCTTGCTTC-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 Τβ4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 Tβ4 gene according to claim 2, characterized in that: it includes the following steps:

Α) Τβ4基因引物设计：根据 Τβ4基因编码序列，使用 01igo 7分析后选取 5， - GGAATTCATGTCTGACAAACCCGATATG -3，，即 SEQ ID NO: 1作为上游引物，选取 5'- Α) Tβ4 gene primer design: According to the Tβ4 gene coding sequence, 5'-GGAATTCATGTCTGACAAACCCGATATG-3 was selected after analysis using 01igo 7, that is, SEQ ID NO: 1 as the upstream primer, and 5'- was selected.

GACTAGTTTACGATTCGCCTGCTTGCTTC -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； GACTAGTTTACGATTCGCCTGCTTGCTTC-3', that is, SEQ ID NO: 2, is used as the downstream primer, and then the upstream primer and the downstream primer are synthesized;

B) Τβ4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 Τβ4基因编码序列，然后将该序列进行加 Α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 Τβ4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 Τβ4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 200 B) Obtaining the Tβ4 gene cDNA sequence: Use the upstream primer and the downstream primer to perform PCR amplification to obtain a large number of Tβ4 gene coding sequences, and then perform an A-tail addition 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 indicate that the Tβ4 gene cDNA sequence was successfully inserted into the bacterial liquid for sequencing and identification; the E. coli that was correctly identified by sequencing was cultured in liquid LB medium, and the pGM-T vector carrying the Tβ4 gene cDNA sequence was extracted and used with restriction endonuclease Dicer EcoR I Enzyme and Spe l enzyme double digestion, electrophoresis, gel recovery 200

bp左右的片段，此片段即为 Τβ4基因 cDNA序列; 特异促进 Τβ4基因高表达的慢病毒载体的构建和鉴定：提取质粒 pLV X-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 A fragment of about bp, this fragment is the Tβ4 gene cDNA sequence; Construction and identification of lentiviral vectors that specifically promote high expression of Tβ4 gene: Extract plasmid pLV X-IRES-Puro, use restriction endonuclease EcoR I enzyme and Spe I Double enzyme digestion, electrophoresis, and gel cutting to recover the vector, and then use T4 DNA

ligase将所述 Τβ4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 Τβ4基因 cDNA序列***成功的菌液进行测序鉴定；特异促进 Τβ4基因高表达的慢病毒载体的抽提：将测序结果证实 Τβ4 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 Τβ4基因高表达的慢病毒表达载体。 Ligase connects the Tβ4 gene cDNA sequence to the pLVX-IRES-Puro expression vector to obtain a ligation product. The ligation product is transformed into competent E. coli Top10 and evenly spread on an LB medium plate containing ampicillin. Take positive single clone colonies, culture and preserve the bacterial liquid and perform preliminary PCR identification. The preliminary identification results indicate that the Tβ4 gene cDNA sequence is successfully inserted into the bacterial liquid for sequencing identification; Extraction of lentiviral vectors that specifically promote the high expression of Tβ4 gene: Sequencing results It was confirmed that the Tβ4 gene cDNA sequence was successfully inserted into the bacterial liquid amplification culture, and the recombinant plasmid was extracted to obtain a lentiviral expression vector that specifically promoted the high expression of the Tβ4 gene.

[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 Τβ4基因高表达的慢病毒表达载体在制备治疗 Τβ4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 Tβ4 gene according to any one of claims 1 to 3 in the preparation of drugs for treating diseases related to abnormal expression of Tβ4 gene.