WO2017214833A1 - Lentiviral vector for specifically promoting high expression of erbb2 gene, and applications thereof - Google Patents
Lentiviral vector for specifically promoting high expression of erbb2 gene, and applications thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
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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
- C12N15/09—Recombinant DNA-technology
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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
- C12N15/09—Recombinant DNA-technology
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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
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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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
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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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
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
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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
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
- C12N15/86—Viral vectors
- C12N15/867—Retroviral vectors
Definitions
- the present invention belongs to the field of genetic engineering technology, and particularly relates to a lentiviral vector which specifically promotes high expression of the ERBB2 gene, and a construction method and application thereof.
- ERBB2 is a 185 kDa cell membrane receptor encoded by the proto-oncogene ERBB2 and is a member of the epidermal growth factor receptor (EGFR) family.
- the family includes four members, ERBB1 (also known as EGFR, HER1), ERBB2 (also known as HER2), ERBB3 (HER3), and ERBB4 (HER4).
- ERBB2 is involved in the regulation of growth and development of normal tissues, promotes cell division and secretion of proteolytic enzymes, and enhances cell motility, but under pathological conditions, ERBB2 expression increases, thereby promoting tumor invasion and metastasis.
- ERBB2 has intracellular tyrosine kinase-like activity and plays a role in embryonic development and differentiation. It is widely expressed in epithelial cells of human tissues and is closely related to the survival and progression of various tumors. Therefore, the research on ERBB2 can greatly promote the development of tumor prevention and control, but the existing technology still lacks the lentiviral expression vector which specifically promotes the high expression of ERBB2 gene, which hinders the development of related fields.
- the ERBB2 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 ERBB2 gene, thereby completing the present invention.
- the present invention provides a lentiviral expression vector which specifically promotes high expression of the ERBB2 gene, including the basic sequence of the pLVX-IR ES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and the E RBB2 a cDNA sequence of the gene; the multiple cloning site includes a Xho I cleavage site and a Spe I cleavage site.
- the ERBB2 gene cDNA sequence includes an Xho I restriction site, an ERBB2 gene coding sequence, and a Spe I restriction site, and the ERBB2 gene cDNA sequence is inserted into the multiple cloning site sequence.
- the lentiviral expression vector constructed by inserting the ERBB2 gene cDNA sequence into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase ERBB2.
- the advantages of gene expression can be used as a powerful tool in the preparation and treatment of ERBB2 gene expression for diseases such as Alzheimer's disease.
- the ERBB2 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'-GCTCGAGATGAAGCTGCGGCTCCCTGC-3, ie, SEQ ID NO: 1
- the sequence of the downstream primer is: 5, - GACTAGTTCACACTGGCACGTCCAGACC -3', ie SEQ ID NO: 2.
- the ERBB2 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the ERBB2 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 ERBB2 gene, comprising the following steps:
- A) ERBB2 gene primer design According to the ERBB2 gene coding sequence, use Oligo
- B) obtaining the ERBB2 gene cDNA sequence PCR amplification using the upstream primer and the downstream primer to obtain a large number of ERBB2 gene coding sequences, and then adding the A tail reaction to the sequence, using T4 DNA ligase
- the ligation product is obtained by ligation to the pGM-T vector, and the ligation product is transformed into competent E. coli ToplO, uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid is picked and subjected to PCR.
- Preliminary identification the preliminary identification results indicate that the ERBB2 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification; the correct E.
- coli was identified by liquid LB medium culture sequencing, and the pGM-T vector carrying the ERBB2 gene cDNA sequence was extracted and used. Restriction enzyme Xho I enzyme and Spe I enzyme double digestion, electrophoresis, gelatinization recovery of about 3500 bp fragment, this fragment is ERBB2 base Due to the cDNA sequence;
- the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of ERBB2 gene, and after being successfully constructed, it is packaged into a virus and introduced into Jurkat cells, and after puromycin is used to screen cells, real-time quantitative PCR is used.
- the Western Blot technique verifies the change of ERBB2 gene expression from mRNA and protein levels, respectively.
- the experimental results show that the ERBB2 gene cDNA sequence provided by the present invention is successfully inserted into the pLV X-IRES-Puro expression vector, and can be specifically, continuously, efficiently and stably promoted. High expression of ERBB2 gene
- the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of the ERBB2 gene for the preparation of a medicament for treating a disease associated with abnormal expression of the ERBB2 gene.
- the lentiviral expression vector which specifically promotes the high expression of ERBB2 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 ERBB2 gene, and can be used as a powerful tool.
- the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the E RBB2 gene, which has a good operation effect, reduces the cost of sequence synthesis, and has a low cost.
- DRAWINGS 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.
- ERBB2 gene coding sequence GenBank NM_001005862.2
- the designed PCR primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd.
- Example 2 Construction of a lentiviral vector that specifically promotes high expression of the ERBB2 gene
- the coding sequence of the ERBB2 gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A-tail reaction, and then T4 was used.
- DNA ligase is ligated to the pGM-T vector to obtain a ligation product (ERBB2-T vector), which is produced by the ligation
- ERBB2-T vector a ligation product
- the material was transformed into competent E. coli ToplO, uniformly coated on ampicillin-containing LB medium plate, cultured at 37 ° C for 12 h, and the negative control group 1 was set up (the competent cells were uniformly coated in the absence of ampicillin).
- the negative control group 2 the competent cells were uniformly coated on the plate containing 100 g/ml ampicillin
- the positive control group 1 the connection product of the double enzyme-cut empty vector was uniformly coated in the 100 g/ml ampicillin on the plate
- positive control group 2 the empty carrier was evenly spread on a plate containing 100 g/mL ampicillin.
- the experimental group grew a single colony, 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.
- the bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the ERBB2 gene sequence was extracted, and the pLVX-IRES-Puro vector was separately used with Xho I enzyme and Spe I, respectively.
- the enzyme was double-digested, electrophoresed and recovered, and the product was recovered by T4 DNA ligase, and then transformed into competent E. coli ToplO, uniformly coated on ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h.
- the negative control group 1 was set up (the competent cells were uniformly coated on the ampicillin-free plate), and the negative control group 2 (the competent cells were uniformly coated on the plate containing 100 g/ml ampicillin), Positive control group 1 (the ligation product of the double enzyme-cut empty vector was uniformly coated on a plate containing 100 g/ml ampicillin), positive control group 2 (the empty vector was uniformly coated on 100 g/mL ampicillin) On the tablet).
- the experimental group grew a single colony, and the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, and the positive control group 2 did not grow colonies.
- 293FT cells were cultured, and cells with good growth state were inoculated into six wells, 1,000,000 cells per well, and the extracted recombinant plasmid pLVX-ERBB2 2 ⁇ ⁇ was transfected into 293FT cells using a lentiviral packaging auxiliary kit. After 48 h, the virus-containing supernatant medium was collected, and the virus solution was filtered through a sieve of 0.45 ⁇ m for infecting Jur kat cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 5,000,000 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 ERBB2 gene.
- the primer design software Oligo 7.0 was used to design the bow.
- Jurkat cells, pLVX empty vector control Jurkat cell group, and pLVX-ERBB2 high-expressing cells were inoculated into 6-well plates, respectively.
- Cell density reaches ⁇ , with RNeasy Mini
- Kit extracts total RNA from each group of cells, using PrimeScrip RT reagent Kit reverse transcribes mRNA into cDNA, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ⁇ . After the reverse transcription, add 9 (L RNase Free dH20 diluted cDNA, stored at -20 ° C, for later detection. Take the cDNA of each group of cells
- ERBB2 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 the ERBB2 gene.
- the lentiviral expression vector which specifically promotes the high expression of ERBB2 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 ERBB2 gene, and can be used as a powerful tool.
- the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the E RBB2 gene, which has a good operation effect, reduces the cost of sequence synthesis, and has a low cost.
Abstract
A lentiviral expression vector for specifically promoting high expression of an ERBB2 gene comprises a fundamental sequence, a resistance gene sequence, a multiple-cloning-sites sequence, a promoter sequence, and an ERBB2 gene cDNA sequence of a pLVX-IRES-puro expression vector. The multiple cloning sites comprise an Xho I enzyme cutting site and an Spe I enzyme cutting site, the ERBB2 gene cDNA sequence comprises an Xho I enzyme cutting site, an ERBB2 gene coding sequence and an Spe I enzyme cutting site, and the ERBB2 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 ERBB2 gene, and can serve as a powerful tool applied to research and development of drugs related to ERBB2.
Description
发明名称:特异促进 ERBB2基因高表达的慢病毒载体及其应用 技术领域 Title: Lentiviral Vector for Promoting High Expression of ERBB2 Gene and Its Application
[0001] 本发明属于基因工程技术领域， 尤其涉及特异促进 ERBB2基因高表达的慢病毒 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 lentiviral vector which specifically promotes high expression of the ERBB2 gene, and a construction method and application thereof.
背景技术 Background technique
[0002] ERBB2是原癌基因 ERBB2编码的 185kDa的细胞膜受体， 为表皮生长因子受体（ EGFR)家族成员之一。 该家族包括 ERBB1 (又称 EGFR， HER1) 、 ERBB2 (又 称 HER2) 、 ERBB3 (HER3) 和 ERBB4 (HER4) 四个成员。 [0002] ERBB2 is a 185 kDa cell membrane receptor encoded by the proto-oncogene ERBB2 and is a member of the epidermal growth factor receptor (EGFR) family. The family includes four members, ERBB1 (also known as EGFR, HER1), ERBB2 (also known as HER2), ERBB3 (HER3), and ERBB4 (HER4).
技术问题 technical problem
[0003] ERBB2参与了正常组织的生长与发育调节， 可以促进细胞分裂和蛋白水解酶的 分泌， 并增强细胞的运动能力， 但在病理状态下， ERBB2表达增多， 进而促进 肿瘤的侵袭和转移。 ERBB2具有细胞内酪氨酸激酶样活性， 在胚胎发育形成及 分化中有一定的作用， 在人类组织的上皮细胞中有广泛表达， 并且与多种肿瘤 的生存和演进有密切的关系。 因此对 ERBB2的研究可以大大促进肿瘤防治领域 的发展， 但现有技术仍缺乏特异促进 ERBB2基因高表达的慢病毒表达载体吗， 阻碍了相关领域的发展。 [0003] ERBB2 is involved in the regulation of growth and development of normal tissues, promotes cell division and secretion of proteolytic enzymes, and enhances cell motility, but under pathological conditions, ERBB2 expression increases, thereby promoting tumor invasion and metastasis. ERBB2 has intracellular tyrosine kinase-like activity and plays a role in embryonic development and differentiation. It is widely expressed in epithelial cells of human tissues and is closely related to the survival and progression of various tumors. Therefore, the research on ERBB2 can greatly promote the development of tumor prevention and control, but the existing technology still lacks the lentiviral expression vector which specifically promotes the high expression of ERBB2 gene, which hinders the development of related fields.
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 Xho 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 Xho I cleavage sites and Spe are included.
I酶切位点的 ERBB2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 ERBB2基因高表达的慢病毒表达载体， 从而完成本发明 The ERBB2 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 ERBB2 gene, thereby completing the present invention.
[0005] 本发明提供一种特异促进 ERBB2基因高表达的慢病毒表达载体， 包括 pLVX-IR ES-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 E RBB2基因 cDNA序列； 所述多克隆位点包括 Xho I酶切位点和 Spe I酶切位点， 所
述 ERBB2基因 cDNA序列包括 Xho I酶切位点、 ERBB2基因编码序列和 Spe I酶切 位点， 所述 ERBB2基因 cDNA序列正向插入所述多克隆位点序列中。 The present invention provides a lentiviral expression vector which specifically promotes high expression of the ERBB2 gene, including the basic sequence of the pLVX-IR ES-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and the E RBB2 a cDNA sequence of the gene; the multiple cloning site includes a Xho I cleavage site and a Spe I cleavage site. The ERBB2 gene cDNA sequence includes an Xho I restriction site, an ERBB2 gene coding sequence, and a Spe I restriction site, and the ERBB2 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 ERBB2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 ERBB2基因表达的优点， 可作为有力工具应用于制备治疗 ERBB2 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 ERBB2 gene cDNA sequence into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably increase ERBB2. The advantages of gene expression can be used as a powerful tool in the preparation and treatment of ERBB2 gene expression for diseases such as Alzheimer's disease.
[0007] 作为本发明的进一步改进， 所述 ERBB2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'- GCTCGAGATGAAGCTGCGGCTCCCTGC -3，， 即 SEQ ID NO: 1， 所述下游引 物的序列为： 5，- GACTAGTTCACACTGGCACGTCCAGACC -3'， 即 SEQ ID NO ： 2。 采用上述 PCR引物序列， 通过 PCR可以扩增出 ERBB2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 ERBB2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 ERBB2 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'-GCTCGAGATGAAGCTGCGGCTCCCTGC-3, ie, SEQ ID NO: 1, the sequence of the downstream primer is: 5, - GACTAGTTCACACTGGCACGTCCAGACC -3', ie SEQ ID NO: 2. Using the above PCR primer sequence, the ERBB2 gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the ERBB2 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 ERBB2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 ERBB2 gene, comprising the following steps:
[0009] A) ERBB2基因引物设计： 根据 ERBB2基因编码序列， 使用 Oligo [0009] A) ERBB2 gene primer design: According to the ERBB2 gene coding sequence, use Oligo
7分析后选取 5， - GCTCGAGATGAAGCTGCGGCTCCCTGC -3： 1作为上游引物， 选取 5 '- GACTAGTTCACACTGGCACGTCCAGACC -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； 7 after analysis, select 5, - GCTCGAGATGAAGCTGCGGCTCCCTGC -3: 1 as the upstream primer, select 5 '- GACTAGTTCACACTGGCACGTCCAGACC -3, ie SEQ ID NO: 2 as the downstream primer, then synthesize the upstream primer and the downstream primer; The primer and the downstream primer have no primer dimer, and the annealing temperature difference is small;
[0010] B) ERBB2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 ERBB2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 ERBB2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 ERBB2基因 cDNA序列的 pGM-T载体， 用限制性内切酶 Xho I 酶和 Spe I酶双酶切， 电泳、 切胶回收 3500 bp左右的片段， 此片段即为 ERBB2基
因 cDNA序列； [0010] B) obtaining the ERBB2 gene cDNA sequence: PCR amplification using the upstream primer and the downstream primer to obtain a large number of ERBB2 gene coding sequences, and then adding the A tail reaction to the sequence, using T4 DNA ligase The ligation product is obtained by ligation to the pGM-T vector, and the ligation product is transformed into competent E. coli ToplO, uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid is picked and subjected to PCR. Preliminary identification, the preliminary identification results indicate that the ERBB2 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification; the correct E. coli was identified by liquid LB medium culture sequencing, and the pGM-T vector carrying the ERBB2 gene cDNA sequence was extracted and used. Restriction enzyme Xho I enzyme and Spe I enzyme double digestion, electrophoresis, gelatinization recovery of about 3500 bp fragment, this fragment is ERBB2 base Due to the cDNA sequence;
[0011] C) 特异促进 ERBB2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 Xho l酶和 Spe l酶双酶切， 电泳、 切胶回收载体， 再 用 T4 DNA ligase将所述 ERBB2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 ERBB2基因 cDNA序列插入成功的菌液进行测序 鉴定； [0011] C) Construction and identification of a lentiviral vector that specifically promotes high expression of the ERBB2 gene: extraction of the plasmid pLVX-I RES-Puro, digestion with restriction endonuclease Xho l enzyme and Spe l enzyme, electrophoresis, gelation The vector was recovered, and the ERBB2 gene cDNA sequence was ligated into the pLVX-IRES-Puro expression vector by T4 DNA ligase to obtain a ligation product, which was transformed into competent E. coli ToplO and uniformly coated with ampicillin. On the LB medium plate, the positive monoclonal colonies were cultured and preserved, and the PCR was initially identified. The preliminary identification results indicated that the ERBB2 gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification;
[0012] D) 特异促进 ERBB2基因高表达的慢病毒载体的抽提： 将测序结果证实 ERBB2 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 ERBB2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 ERBB2 gene: The sequencing result confirms that the ERBB2 gene cDNA sequence is inserted into a successful bacterial cell expansion culture, and the recombinant plasmid is extracted to obtain a specific expression of the ERBB2 gene. Lentiviral expression vector.
[0013] 本发明利用基因工程技术构建特异促进 ERBB2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 ERBB2基因 表达的变化， 实验结果证明本发明提供的 ERBB2基因 cDNA序列成功插入至 pLV X-IRES-Puro表达载体中， 能特异、 持续、 高效、 稳定地促进 ERBB2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 ERBB2 gene, and after being successfully constructed, it is packaged into a virus and introduced into Jurkat cells, and after puromycin is used to screen cells, real-time quantitative PCR is used. The Western Blot technique verifies the change of ERBB2 gene expression from mRNA and protein levels, respectively. The experimental results show that the ERBB2 gene cDNA sequence provided by the present invention is successfully inserted into the pLV X-IRES-Puro expression vector, and can be specifically, continuously, efficiently and stably promoted. High expression of ERBB2 gene
[0014] 本发明还提供特异促进 ERBB2基因高表达的慢病毒表达载体在制备治疗 ERBB2 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 ERBB2 gene for the preparation of a medicament for treating a disease associated with abnormal expression of the ERBB2 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 ERBB2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 ERBB2基因高表达的优点， 可作为 有力工具应用于与 ERBB2相关的药物研究和幵发中； 本发明还提供了特异促进 E RBB2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 ERBB2 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 ERBB2 gene, and can be used as a powerful tool. In the drug research and development related to ERBB2; the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the E RBB2 gene, which has a good operation effect, reduces the cost of sequence synthesis, and has a low cost.
对附图的简要说明 Brief description of the drawing
附图说明
[0016] 图 1为 pLVX-IRES-Puro表达载体的质粒图谱。 DRAWINGS 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 ERBB2基因引物的设计。 Example 1 Design of ERBB2 gene primers.
[0021] 根据 ERBB2基因编码序列 （GenBank NM_001005862.2) ， 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 Xho l 和 Xho 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 ERBB2 gene coding sequence (GenBank NM_001005862.2), it was analyzed using 01igo7 to find upstream primers and downstream primers (requiring as little primer-free dimer as possible and the annealing temperature difference is small), then upstream primers and The 5' end of the downstream primer was added with the protecting base and the restriction sites Xho l and Xho I 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 ERBB2基因的 PCR弓 |物序列 Table 1 PCR bow of the ERBB2 gene |
[] [表 1] [] [Table 1]
[0023] 实施例二特异促进 ERBB2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 ERBB2 gene
[0024] 将合成的弓 I物稀释后， 用 Premix PrimeSTAR HS酶对 ERBB2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 ERBB2 gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A-tail reaction, and then T4 was used.
DNA连接酶连接到 pGM-T载体上得到连接产物 （ERBB2-T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 (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。 DNA ligase is ligated to the pGM-T vector to obtain a ligation product (ERBB2-T vector), which is produced by the ligation The material was transformed into competent E. coli ToplO, uniformly coated on ampicillin-containing LB medium plate, cultured at 37 ° C for 12 h, and the negative control group 1 was set up (the competent cells were uniformly coated in the absence of ampicillin). On the plate of penicillin), the negative control group 2 (the competent cells were uniformly coated on the plate containing 100 g/ml ampicillin), and the positive control group 1 (the connection product of the double enzyme-cut empty vector was uniformly coated in the 100 g/ml ampicillin on the plate), positive control group 2 (the empty carrier was evenly spread on a plate containing 100 g/mL ampicillin). The experimental group grew a single colony, 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.
[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 ERBB2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 ERB Β2基因， 接着将重组载体送至上海生工公司测序。 [0025] After picking up 8 single colonies from the experimental group, 0.5 culture solutions were taken and PCR-amplified with primers of ERBB2 gene for preliminary identification. The results showed that the cultures of 8 single colonies could be successfully amplified. The ERB Β2 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 ERBB2基 因序列的重组 T载体， 将其和 pLVX-IRES-Puro载体分别先用 Xho I酶和 Spe I酶进 行双酶切， 电泳回收， 并用 T4 DNA连接酶连接回收产物用， 再次转化到感受态 大肠杆菌 ToplO中， 均匀涂布到含氨苄青霉素 LB培养基平板上， 于 37°C培养 12 h ， 同吋设置阴性对照组 1 (将感受态细胞均匀涂布在不含氨苄青霉素的平板上） 、 阴性对照组 2 (将感受态细胞均匀涂布在含 100 g/ml氨苄青霉素的平板上） 、 阳性对照组 1 (将双酶切空载体的连接产物均匀涂布在含 100 g/ml氨苄青霉素的 平板上） 、 阳性对照组 2 (将空载体均匀涂布在含 100 g/mL氨苄青霉素的平板上 ) 。 实验组长出了单菌落， 阴性对照组 1长出了菌落； 阴性对照组 2、 阳性对照 组 1、 阳性对照组 2没长出菌落。 [0026] The bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the ERBB2 gene sequence was extracted, and the pLVX-IRES-Puro vector was separately used with Xho I enzyme and Spe I, respectively. The enzyme was double-digested, electrophoresed and recovered, and the product was recovered by T4 DNA ligase, and then transformed into competent E. coli ToplO, uniformly coated on ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h. The negative control group 1 was set up (the competent cells were uniformly coated on the ampicillin-free plate), and the negative control group 2 (the competent cells were uniformly coated on the plate containing 100 g/ml ampicillin), Positive control group 1 (the ligation product of the double enzyme-cut empty vector was uniformly coated on a plate containing 100 g/ml ampicillin), positive control group 2 (the empty vector was uniformly coated on 100 g/mL ampicillin) On the tablet). The experimental group grew a single colony, and the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, and the positive control group 2 did not grow colonies.
[0027] 从实验组中挑取 6个单菌落培养保存后， 各取 0.5 培养液， 用 ERBB2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 ERBB2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-ERBB2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 ERBB2 gene for preliminary identification. The results showed that all 6 cultures could successfully amplify the ERBB2 gene, and then the recombinant vector bacteria were sent to Shanghai Biotech Co., Ltd. for sequencing, and the sequencing results were exactly as expected, and the pLVX-ERBB2 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-ERBB2， 测其纯度和浓度， 结果如表 2所示。 [0028] The previously collected recombinant plasmid bacterial solution was taken out, inoculated into 15 ml of LB medium (containing 100 g/ml ampicillin), cultured at 37 ° C, 300 rpm for 16 h, using Endo-Free Plasmid Mini Kit II The recombinant plasmid pLVX-ERBB2 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
[] [表 2]
Table 2 Purification and Concentration of Recombinant Plasmid [] [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-ERBB2 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 5000000〜5000000 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 pLVX-ERBB2 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 Jur kat cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 5,000,000 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 ERBB2基因表达量。 Example 5 Fluorescence quantitative PCR was used to detect the expression level of ERBB2 gene.
[0035] 根据 GAPDH和 ERBB2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 ERBB2 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-ERBB2高表达细 胞至 6孔板。 细胞密度达到 δΟ^^Ο^吋， 用 RNeasy Mini Jurkat cells, pLVX empty vector control Jurkat cell group, and pLVX-ERBB2 high-expressing cells were inoculated into 6-well plates, respectively. Cell density reaches δΟ^^Ο^吋, with RNeasy Mini
Kit提取各组细胞的总 RNA， 利用 PrimeScrip RT reagent
Kit将 mRNA逆转录为 cDNA， 逆转录条件： 37°C， 15min; 85°C， 5s; 4°C， ∞。 反转录结束后， 加入 9( L的 RNase Free dH20稀释 cDNA， -20°C保存， 以便后面 检测使用。 取各组细胞的 cDNA Kit extracts total RNA from each group of cells, using PrimeScrip RT reagent Kit reverse transcribes mRNA into cDNA, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ∞. After the reverse transcription, add 9 (L RNase Free dH20 diluted cDNA, stored 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 ERBB2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ERBB2基因相对表 达量。 将 pLVX-ERBB2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-ERBB2细 胞， 其 ERBB2基因的表达量较 Jurkat细胞都有 80倍以上的升高， 而 pLVX空载体 细胞的 ERBB2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 ER BB2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 ERBB2基因高表达。 Ιμΐ was used as a template, and GAPDH was used as an internal reference. Real-time quantitative PCR (QPCR) was used to detect the relative expression of ERBB2, and the reaction conditions were set: 95. C 30s, 1 cycle, 54. C 30s 40 cycles, 95. C 5s, 60 ° C lmin , 95 ° C 15 s, SYBR Primescript RT-PCR Kit was used to detect the relative expression of ERBB2 gene in each group. After the pLVX-ERBB2 cells were continuously cultured for 20 passages, the above experiment was repeated. The summarized results are shown in Figure 2. It can be seen that the expression level of ERBB2 gene is 80-fold higher than that of Jurkat cells, whether it is just after screening or after 20 generations of pLVX-ERBB2 cells, and the expression of ERBB2 gene in pLVX empty vector cells. There is almost no change compared with Jurkat cells, indicating that the ER BB2 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 the ERBB2 gene.
[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 ERBB2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 ERBB2基因高表达的优点， 可作为 有力工具应用于与 ERBB2相关的药物研究和幵发中； 本发明还提供了特异促进 E RBB2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 ERBB2 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 ERBB2 gene, and can be used as a powerful tool. In the drug research and development related to ERBB2; the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the E RBB2 gene, which has a good operation effect, reduces the cost of sequence synthesis, and has a low cost.
Claims
[权利要求 1] 一种特异促进 ERBB2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 ERBB2基因 cDNA序歹 ij ; 所述多克隆位点包 括 Xho I酶切位点和 Spe [Claim 1] A lentiviral expression vector that specifically promotes high expression of the ERBB2 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 ERBB2 gene cDNA sequence; the multiple cloning sites include Xho I restriction site and Spe
I酶切位点， 所述 ERBB2基因 cDNA序列包括 Xho l酶切位点、 ERBB2 基因编码序列和 Spe I酶切位点， 所述 ERBB2基因 cDNA序列正向插入 所述多克隆位点序列中。 I enzyme cleavage site, the ERBB2 gene cDNA sequence includes an Xhol enzyme cleavage site, an ERBB2 gene coding sequence and a Spe I enzyme cleavage site, and the ERBB2 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 ERBB2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 ERBB2 gene according to claim 1
， 其特征在于： 所述 ERBB2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'- GCTCGAGATGAAGCTGCGGCTCCCTGC -3，， 即 SEQ ID NO: 1， 所述下游引物的序列为： 5'-, which is characterized in that: the ERBB2 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'-GCTCGAGATGAAGCTGCGGCTCCCTGC-3, that is, SEQ ID NO: 1, so The sequence of the downstream primer is: 5'-
GACTAGTTCACACTGGCACGTCCAGACC -3' , 即 SEQ ID NO: 2。 GACTAGTTCACACTGGCACGTCCAGACC -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 ERBB2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 ERBB2 gene according to claim 2, characterized in that: comprising the following steps:
A) ERBB2基因引物设计： 根据 ERBB2基因编码序列， 使用 Oligo 7 分析后选取 5， - GCTCGAGATGAAGCTGCGGCTCCCTGC A) ERBB2 gene primer design: Based on the ERBB2 gene coding sequence, use Oligo 7 to analyze and select 5, - GCTCGAGATGAAGCTGCGGCTCCCTGC
-3，， 即 SEQ ID NO: 1作为上游引物， 选取 5，- -3, that is, SEQ ID NO: 1 as the upstream primer, select 5, -
GACTAGTTCACACTGGCACGTCCAGACC -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； GACTAGTTCACACTGGCACGTCCAGACC-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) ERBB2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 ERBB2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 ERBB2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 ERBB2基因 cDNA序列的 pGM-T载体， 用限制性内切 酶 Xho l酶和 Spe l酶双酶切， 电泳、 切胶回收 3500 bp左右的片段， 此 片段即为 ERBB2基因 cDNA序列； B) Obtaining the cDNA sequence of the ERBB2 gene: Use the upstream primer and the downstream primer to perform PCR amplification to obtain a large number of ERBB2 gene coding sequences, then perform an A-tailing reaction on the sequence, and then connect it to pGM using T4 DNA ligase Obtain the ligation product on the -T vector, transform the ligation product into competent E. coli ToplO, spread it evenly on the ampicillin-containing LB medium plate, pick the positive single clone colony, culture and preserve the bacterial liquid, and perform preliminary PCR identification , the preliminary identification results showed that the ERBB2 gene cDNA sequence was successfully inserted into the bacterial liquid for sequencing and identification; the liquid LB medium was used to culture and sequence the correct E. coli , and extract the pGM-T vector containing the ERBB2 gene cDNA sequence, double-digest it with restriction endonucleases Xhol enzyme and Spel enzyme, perform electrophoresis and gel cutting to recover a fragment of about 3500 bp, which is the ERBB2 gene cDNA sequence;
C) 特异促进 ERBB2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 Xho I酶和 Spe I酶双酶切， 电泳 C) Construction and identification of lentiviral vectors that specifically promote high expression of the ERBB2 gene: Extract the plasmid pLVX-IRES-Puro, double-digest it with restriction endonucleases Xho I and Spe I, and perform electrophoresis.
、 切胶回收载体， 再用 T4 DNA ligase将所述 ERBB2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 ERBB2基因 cDNA序列插入成功的菌液进行测序鉴定 , cut the gel to recover the vector, and then use T4 DNA ligase to connect the ERBB2 gene cDNA sequence into the pLVX-IRES-Puro expression vector to obtain the connection product, transform the connection product into competent E. coli ToplO, and evenly spread it into On the LB medium plate containing ampicillin, select positive single clone colonies, culture and preserve the bacterial liquid, and conduct preliminary PCR identification. The preliminary identification results indicate that the bacterial liquid with successful insertion of the ERBB2 gene cDNA sequence is sequenced and identified.
D) 特异促进 ERBB2基因高表达的慢病毒载体的抽提： 将测序结果证 实 ERBB2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 ERBB2基因高表达的慢病毒表达载体。 D) Extraction of lentiviral vectors that specifically promote the high expression of the ERBB2 gene: The sequencing results confirm that the ERBB2 gene cDNA sequence has been successfully inserted into the bacterial liquid amplification culture, and the recombinant plasmid is extracted to obtain a lentivirus that specifically promotes the high expression of the ERBB2 gene. Expression vector.
[权利要求 4] 根据权利要求 1至 3中任一项所述的特异促进 ERBB2基因高表达的慢 病毒表达载体在制备治疗 ERBB2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 the ERBB2 gene according to any one of claims 1 to 3 in the preparation of drugs for the treatment of diseases related to abnormal ERBB2 gene expression.
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