WO2017214937A1 - 一种促进 app 基因表达的慢病毒表达载体及其应用 - Google Patents

一种促进 app 基因表达的慢病毒表达载体及其应用 Download PDF

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WO2017214937A1
WO2017214937A1 PCT/CN2016/086061 CN2016086061W WO2017214937A1 WO 2017214937 A1 WO2017214937 A1 WO 2017214937A1 CN 2016086061 W CN2016086061 W CN 2016086061W WO 2017214937 A1 WO2017214937 A1 WO 2017214937A1
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app gene
sequence
app
expression vector
vector
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PCT/CN2016/086061
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毛侃琅
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毛侃琅
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C12N15/867—Retroviral vectors

Definitions

  • the present invention belongs to the field of genetic engineering technology, and particularly relates to a lentiviral expression vector for promoting APP gene expression and an application thereof.
  • AD Alzheimer's disease
  • ⁇ -amyloid precursor protein ( ⁇ ) is a single transmembrane protein having a membrane receptor-like protein structure which is widely present in various tissue cells of the whole body.
  • --amyloid ( ⁇ ) is a 39-43 amino acid fragment that is cleaved by ⁇ and ⁇ secretase in a pathological condition.
  • the relative molecular mass is about 4.2x103, deposited in the extracellular space. Forming age spots.
  • AD has become the fourth largest disease causing human death, seriously affecting the physical and mental health and quality of life of the elderly.
  • Alzheimer's disease may cause many family social problems in susceptible populations.
  • the role of the disease in the pathogenesis of Alzheimer's disease is of great significance for the early prevention and treatment of Alzheimer's disease.
  • the existing research puts most of the energy on the ⁇ , and the research on ⁇ itself is seriously insufficient. Therefore, the prior art also lacks a vector capable of continuously, stably and efficiently expressing the A PP gene in human cells.
  • the APP 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 APP gene, thereby completing the present invention.
  • the present invention provides a lentiviral expression vector which specifically promotes high expression of an APP gene, including a basic sequence of a pLVX-IRES- puro expression vector, a resistance gene sequence, a multiple cloning site sequence, a promoter sequence, and an APP gene cDNA.
  • the multiple cloning site includes a Spe l cleavage site and an Xba l cleavage site
  • the APP gene cDNA sequence includes a Spe I cleavage site, an APP gene coding sequence, and Xba
  • the enzyme gene cleavage site, the APP gene cDNA sequence is inserted into the multiple cloning site sequence.
  • the lentiviral expression vector constructed by inserting the APP gene cDNA sequence of the present invention into the pLVX-IRES-Puro expression vector has high transfection efficiency and low dosage, and can stably, efficiently and stably improve APP.
  • the advantages of gene expression can be used as a powerful tool in the preparation and treatment of drugs for the treatment of APP gene expression in diseases such as Alzheimer's disease.
  • the APP 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'-
  • CCGCTCGAGACATGGCTCTCATCCCAGACTT -3 ie SEQ ID NO: 1
  • sequence of the downstream primer is: 5,- ATCTAGATCAGGCTCCACTTACGGTGCCATCC
  • the APP gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the APP gene, which reduces the cost of sequence synthesis and has a lower cost.
  • the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of an APP gene, comprising the following steps:
  • A) APP gene primer design According to the APP gene coding sequence, use Oligo 7 analysis and select 5'-
  • NO: 2 is used as a downstream primer, and then the upstream primer and the downstream primer are synthesized; the upstream primer and the downstream primer have no primer dimer, and the annealing temperature difference is small;
  • B) obtaining the APP gene cDNA sequence PCR amplification using the upstream primer and the downstream primer to obtain a large number of APP 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 D ⁇ 5 ⁇ , uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation solution is picked and subjected to Preliminary identification by PCR, the preliminary identification results indicate that the cDNA sequence of the ⁇ gene was inserted into The bacterial liquid was identified by sequencing; the correct E.
  • coli was identified by liquid LB medium, and the pGM-T vector carrying the APP gene cDNA sequence was extracted, and the restriction endonuclease Spe I was digested with Xba I enzyme. Double-digestion, electrophoresis, and gel-cutting recover a fragment of about 2500 bp, which is the APP gene cDNA sequence;
  • the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of APP gene, and after being successfully constructed, it is packaged into a virus and introduced into RGC5 nerve cells, and after puromycin is used to screen cells, real-time quantitative PCR is used. And Western Blot technology verified the change of APP gene expression from mRNA and protein levels respectively.
  • the experimental results show that the APP gene cDNA sequence provided by the present invention can be successfully inserted into the pLVX-IRE S-Puro expression vector, which can be specific, sustained, efficient and stable. Promote high expression of APP gene.
  • the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of an APP gene for the preparation of a medicament for treating a disease associated with abnormal expression of an APP gene.
  • the lentiviral expression vector which specifically promotes the high expression of APP 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 APP gene, and can be used as a powerful tool.
  • the invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of APP gene, has good operation effect, reduces sequence synthesis cost, and has low cost.
  • 1 is a plasmid map of the pLVX-IRES-Puro expression vector.
  • FIG. 2 is a schematic diagram showing the results of fluorescent quantitative PCR detection after puromycin screening of cells.
  • RGC5 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 was purchased from QIAGEN, pGM-T was purchased from Tiangen, and Endo-Free Plasmid Mini Kit II was purchased from Omega bio-tek.
  • Example 1 Design of an APP gene primer.
  • upstream primers and downstream primers (requires as little primer-free dimer as possible and have a small annealing temperature difference), then add protective bases and restriction sites Spe I and Xba I at the 5' end of the upstream and downstream primers, respectively.
  • the designed primer sequences are shown in Table 1.
  • 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 APP gene
  • the coding sequence of the APP gene was amplified by Premix PrimeSTAR HS enzyme, and then electrophoresed and then subjected to A tail reaction, and then ligated to pGM-T with T4 DNA ligase.
  • the ligation product (APP-T vector) was obtained on the vector, and the ligation product was transformed into competent Escherichia coli DH5. In oc, evenly spread onto ampicillin-containing LB medium plate, culture at 37 ° C for 12 h, and set up negative control group 1 (consistent cells uniformly coated on ampicillin-free plates), negative Control group 2 (perfect cells were uniformly coated on a 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
  • the positive control group 2 the empty carrier was uniformly coated on a plate containing 100 g/mL ampicillin.
  • 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.
  • the bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the APP gene sequence was extracted, and the pLVX-IRES-Puro vector was firstly treated with Spe I enzyme and Xba I.
  • the enzyme was double-digested, electrophoresed and recovered, and the product was recovered by T4 DNA ligase, and then transformed into competent E. coli DH50C, uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C 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-APP 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 RGC 5 nerve cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 50,000,000 IFU.
  • Example 5 Fluorescence quantitative PCR was used to detect the expression of APP gene.
  • the primer design software Oligo 7.0 was used to design the bow.
  • RGC5 cells, pLVX empty vector control RGC5 cell group, and pLVX-APP high expression cells were inoculated into 6-well plates, respectively.
  • the cell density reached 80 ⁇ 3 ⁇ 4-90 ⁇ 3 ⁇ 4 ⁇ , and the total RNA of each group was extracted with RNeasy Mini Kit, and PrimeScrip RT reagent was used for He 1 J. Kit reverse transcribes mRNA into cDNA, reverse transcription conditions: 37 ° C, 15 min; 85 ° C, 5 s; 4 ° C, ⁇ . After the 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
  • ⁇ was used as a template
  • GAPDH was used as an internal reference
  • QPCR real-time quantitative PCR
  • the lentiviral expression vector which specifically promotes the high expression of APP 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 APP gene, and can be used as a powerful tool.
  • the invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of APP gene, has good operation effect, reduces sequence synthesis cost, and has low cost.

Abstract

一种特异促进APP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APP基因cDNA序列；所述多克隆位点包括Spe I酶切位点和Xba I酶切位点，所述APP基因cDNA序列包括Spe I酶切位点、APP基因编码序列和Xba I酶切位点，所述APP基因cDNA序列正向插入所述多克隆位点序列中。该慢病毒表达载体具有转染效率高、用量少、可特异、持续、高效、稳定地表达APP基因的优点，可作为有力工具应用于与APP相关的药物研究与开发。

Description

一种促进 APP基因表达的慢病毒表达载体及其应用 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 APP基因表达的慢病毒表达 载体及其应用。
背景技术
[0002] 阿尔茨海默病 （Alzheimer's disease , AD) ， 是引起老年性痴呆的最常见原因
。 此疾病常见于老年人， 是一种进行性认知障碍和记忆能力损害为主的中枢神 经系统退行性变性疾病， 临床表现为认知和记忆功能不断恶化， 日常生活能力 进行性减退， 并有各种神经精神症状和行为障碍。 多起病于老年期， 潜隐起病 ， 病程缓慢且不可逆， 临床上以智能损害为主。 β-淀粉样前体蛋白 （ΑΡΡ) 为广 泛存在于全身各组织细胞、 具有膜受体样蛋白结构的单跨膜蛋白。 β-淀粉样蛋白 (β-amyloid, Αβ)为病理状况下 ΑΡΡ经 β和 γ分泌酶协同作用裂解成的含有 39〜43个 氨基酸的片段， 相对分子质量约为 4.2x103， 沉积于细胞外间隙形成老年斑。 技术问题
[0003] 目前， AD已成为引起人类死亡的第四大疾病， 严重影响老年人的身心健康与 生命质量， 在易感人群中阿尔茨海默病可能产生诸多家庭社会问题， 研究 ΑΡΡ在 阿尔兹海默症发病过程中的作用对于阿尔兹海默症的早期防控、 对症治疗有着 很大的意义。 但现有的研究将大多数精力都放在了 Αβ上， 对于 ΑΡΡ本身的研究 投入严重不足， 因此现有技术中也缺乏可在人源细胞中持续、 稳定且高效表达 A PP基因的载体。
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 Spe I酶切位点和 Xba
I酶切位点的 APP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 APP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 APP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 APP 基因 cDNA序列； 所述多克隆位点包括 Spe l酶切位点和 Xba l酶切位点， 所述 APP 基因 cDNA序列包括 Spe I酶切位点、 APP基因编码序列和 Xba
I酶切位点， 所述 APP基因 cDNA序列正向插入所述多克隆位点序列中。
[0006] 采用上述技术方案， 本发明提供的 APP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 APP基因表达的优点， 可作为有力工具应用于制备治疗 APP基因表达 对阿尔兹海默症等疾病药物的研究和幵发中。
[0007] 作为本发明的进一步改进， 所述 APP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'-
CCGCTCGAGACATGGCTCTCATCCCAGACTT -3，， 即 SEQ ID NO: 1， 所述下 游引物的序列为： 5，- ATCTAGATCAGGCTCCACTTACGGTGCCATCC
-3'， 即 SEQ ID NO: 2。 采用上述 PCR引物序列， 通过 PCR可以扩增出 APP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 APP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 APP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) APP基因引物设计： 根据 APP基因编码序列， 使用 Oligo 7分析后选取 5'-
GCACTAGTGTCGCGATGCTGCCCGGTTTG -3'， 即 SEQ ID NO: 1作为上游引 物， 选取 5， - ATCTAGACTAGTTCTGCATCTGCTCAAAGAAC -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) APP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 APP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 D Η5α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 ΑΡΡ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 APP基因 cDNA序列的 pGM-T载体， 用限制性内切酶 Spe I酶切和 Xba I酶 进行双酶切， 电泳、 切胶回收 2500 bp左右的片段， 此片段即为 APP基因 cDNA序 列；
[0011] C) 特异促进 APP基因高表达的慢病毒载体的构建和鉴定： 提取质粒 pLVX-IRE S-Puro, 用限制性内切酶 Spe I酶切和 Xba I酶进行双酶切， 电泳、 切胶回收载体 ， 再用 T4 DNA ligase将所述 APP基因 cDNA序列连接到 pLVX-IRES-Puro表达载体 中， 得到连接产物， 将该连接产物转化到感受态大肠杆菌 DH50C中， 均匀涂布到 含氨苄青霉素 LB培养基平板上， 挑取阳性单克隆菌落培养保存菌液并进行 PCR 初步鉴定， 将初步鉴定结果说明 APP基因 cDNA序列插入成功的菌液进行测序鉴 定；
[0012] D) 特异促进 APP基因高表达的慢病毒载体的抽提： 将测序结果证实 APP基因 c DNA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 APP 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 APP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 RGC5神经细胞， 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 APP基因表 达的变化， 实验结果证明本发明提供的 APP基因 cDNA序列成功插入至 pLVX-IRE S-Puro表达载体中， 能特异、 持续、 高效、 稳定地促进 APP基因高表达。
[0014] 本发明还提供特异促进 APP基因高表达的慢病毒表达载体在制备治疗 APP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 APP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 APP基因高表达的优点， 可作为有力工 具应用于与 APP相关的药物研究和幵发中； 本发明还提供了特异促进 APP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] RGC5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 贝勾自 Omega bio-tek公司。
[0020] 实施例一 APP基因引物的设计。
[0021] 根据 APP基因编码序列 （GenBank NM_000484.3) ， 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 Spe I和 Xba 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。
[0022] 表 1 APP基因的 PCR弓 |物序列
[] [表 1]
Figure imgf000006_0001
[0023] 实施例二特异促进 APP基因高表达的慢病毒载体的构建
[0024] 将合成的弓 I物稀释后， 用 Premix PrimeSTAR HS酶对 APP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 （APP-T载体） ， 将该连接产物转化到感受态大肠杆菌 DH5 oc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。
[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 ΑΡΡ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 ΑΡΡ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 APP基因 序列的重组 T载体， 将其和 pLVX-IRES-Puro载体分别先用 Spe I酶和 Xba I酶进行 双酶切， 电泳回收， 并用 T4 DNA连接酶连接回收产物用， 再次转化到感受态大 肠杆菌 DH50C中， 均匀涂布到含氨苄青霉素 LB培养基平板上， 于 37°C培养 12 h， 同吋设置阴性对照组 1 (将感受态细胞均匀涂布在不含氨苄青霉素的平板上） 、 阴性对照组 2 (将感受态细胞均匀涂布在含 100 g/ml氨苄青霉素的平板上） 、 阳 性对照组 1 (将双酶切空载体的连接产物均匀涂布在含 100 g/ml氨苄青霉素的平 板上） 、 阳性对照组 2 (将空载体均匀涂布在含 100 g/mL氨苄青霉素的平板上） 。 实验组长出了单菌落， 阴性对照组 1长出了菌落； 阴性对照组 2、 阳性对照组 1 、 阳性对照组 2没长出菌落。
[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 ΑΡΡ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 ΑΡΡ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-APP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 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-APP， 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-APP 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 RGC 5神经细胞， 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 RGC5神经细胞
[0033] 接种 RGC5神经细胞于 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 （polybren e) 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 DME M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 APP基因表达量。
[0035] 根据 GAPDH和 APP基因 mRNA序列， 利用引物设计软件 Oligo 7.0设计弓 |物。
[] [表 3]
Figure imgf000008_0002
分别接种 RGC5细胞、 pLVX空载体对照 RGC5细胞组、 pLVX-APP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 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
Ιμΐ为模板， 以 GAPDH为内参， 实吋荧光定量 PCR (QPCR) 检测 APP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 APP基因相对 表达量。 将 pLVX- APP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-APP细胞， 其 APP基因的表达量较 RGC5细胞都有 80倍左右的升高， 而 pLVX空载体细胞的 A PP基因表达量与 RGC5细胞相比基本没有变化， 说明本发明提供的 APP基因 cDN A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 APP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
[0038] 本发明提供的特异促进 APP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 APP基因高表达的优点， 可作为有力工 具应用于与 APP相关的药物研究和幵发中； 本发明还提供了特异促进 APP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

权利要求书
[权利要求 1] 一种特异促进 APP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 APP基因 cDNA序歹 ij ; 所述多克隆位点包括 Spe I 酶切位点和 Xba I酶切位点， 所述 APP基因 cDNA序列包括 Spe I酶切位 点、 APP基因编码序列和 Xba I酶切位点， 所述 APP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 APP基因高表达的慢病毒表达载体， 其特征在于： 所述 APP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'- GCACTAGTGTCGCGATGCTGCCCGGTTTG -3' , 即 SEQ ID NO: 1 ， 所述下游引物的序列为： 5'-
ATCTAGACTAGTTCTGCATCTGCTCAAAGAAC -3'， 即 SEQ ID NO
： 2。
[权利要求 3] 根据权利要求 2所述的特异促进 APP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) APP基因引物设计： 根据 APP基因编码序列， 使用 01igo 7分析后 选取 5， - GCACTAGTGTCGCGATGCTGCCCGGTTTG -3，， 即 SEQ ID NO: 1作为上游引物， 选取 5'-
ATCTAGACTAGTTCTGCATCTGCTCAAAGAAC -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；
B) APP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 APP基因编码序列， 然后将该序列进行加 A尾 反应后， 用 T4 DNA连接酶连接到 pGM-T载体上得到连接产物， 将该 连接产物转化到感受态大肠杆菌 DH50C中， 均匀涂布到含氨苄青霉素 LB培养基平板上， 挑取阳性单克隆菌落培养保存菌液并进行 PCR初 步鉴定， 将初步鉴定结果说明 APP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 APP基因 cDNA序列的 pGM-T载体， 用限制性内切酶 Spe I酶 切和 Xba l酶进行双酶切， 电泳、 切胶回收 2500 bp左右的片段， 此片 段即为 APP基因 cDNA序列；
C) 特异促进 APP基因高表达的慢病毒载体的构建和鉴定： 提取质粒 p LVX-IRES-Puro， 用限制性内切酶 Spe I酶切和 Xba I酶进行双酶切， 电泳、 切胶回收载体， 再用 T4 DNA ligase将所述 APP基因 cDNA序列 连接到 pLVX-IRES-Puro表达载体中， 得到连接产物， 将该连接产物 转化到感受态大肠杆菌 DH50C中， 均匀涂布到含氨苄青霉素 LB培养基 平板上， 挑取阳性单克隆菌落培养保存菌液并进行 PCR初步鉴定， 将 初步鉴定结果说明 APP基因 cDNA序列插入成功的菌液进行测序鉴定
D) 特异促进 APP基因高表达的慢病毒载体的抽提： 将测序结果证实 APP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 APP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 APP基因高表达的慢病毒 表达载体在制备治疗 APP基因表达异常相关疾病的药物中的用途。
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