WO2017214828A1 - 特异促进 Prkcz 基因高表达的慢病毒表达载体及其应用 - Google Patents

特异促进 Prkcz 基因高表达的慢病毒表达载体及其应用 Download PDF

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WO2017214828A1
WO2017214828A1 PCT/CN2016/085633 CN2016085633W WO2017214828A1 WO 2017214828 A1 WO2017214828 A1 WO 2017214828A1 CN 2016085633 W CN2016085633 W CN 2016085633W WO 2017214828 A1 WO2017214828 A1 WO 2017214828A1
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prkcz
sequence
gene
prkcz gene
expression vector
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PCT/CN2016/085633
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石庆学
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石庆学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • 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
    • C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
    • 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

  • lentiviral expression vector for specifically promoting high expression of Prkcz gene and application thereof
  • the present invention belongs to the field of genetic engineering technology, and particularly relates to a lentiviral expression vector which specifically promotes high expression of a Prkcz gene, and a construction method and application thereof.
  • Diabetes is a group of chronic non-infectious, metabolic diseases characterized by elevated blood glucose levels that are a serious hazard to human health. Whether in developed or developing countries, diabetes has become an epidemic in modern society and has become a worldwide public health problem that poses a serious threat to human health. According to the World Health Organization
  • type 2 diabetes T2DM
  • type 2 diabetes T2DM
  • the prevalence of type 2 diabetes has increased dramatically in countries around the world, and the surge in type 2 diabetes is the leading cause of the dramatic increase in the total number of diabetic patients worldwide.
  • the prevalence of diabetes in adults is close to 10%, and type 2 diabetes accounts for about 90% of all diabetic patients.
  • PRKCZ protein kinase cpsilon zeta
  • T2DM T2DM-related genes.
  • the occurrence of T2DM is related to many factors such as genetics, environment and diet, except for genetic variation.
  • Prkcz 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 Prkcz gene, thereby completing the present invention.
  • the present invention provides a lentiviral expression vector which specifically promotes high expression of the Prkcz gene, including the basic sequence of the pLVX-IRE S-puro expression vector, the resistance gene sequence, the multiple cloning site sequence, the promoter sequence and Pr kcz Gene cDNA sequence; the multiple cloning site includes EcoR I cleavage site and Spe
  • a restriction enzyme site, the Prkcz gene cDNA sequence comprising an EcoR I restriction site, a Prkcz gene coding sequence and a Spe I restriction site, and the Prkcz gene cDNA sequence is inserted into the multiple cloning site sequence.
  • the lckviral expression vector constructed by inserting the Prkcz 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 increase Prkcz.
  • the advantages of gene expression can be used as a powerful tool in the preparation and treatment of drugs for the treatment of Prkcz gene expression in diseases such as Alzheimer's disease.
  • the Prkcz 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'-
  • GGAATTCATGGATTCTGTCATGCCTTCC -3 ie SEQ ID NO: 1
  • the sequence of the downstream primer is: 5,- GACTAGTTCACACCGACTCCTCGGTGG -3', ie SEQ ID NO
  • the Prkcz gene coding sequence can be amplified by PCR, and can be successfully inserted into the pLVX-IRES-Puro expression vector to continuously express the Prkcz gene, which reduces the cost of sequence synthesis.
  • the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the Prkcz gene, comprising the following steps:
  • Prkcz gene primer design According to the Prkcz gene coding sequence, using Oligo 7 analysis, select 5'-GGAATTCATGGATTCTGTCATGCCTTCC-3', ie SEQ ID NO: 1 as the upstream bow, select 5 '- GACTAGTTCACACCGACTCCTCGGTGG -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;
  • B) obtaining the Prkcz gene cDNA sequence PCR amplification using the upstream primer and the downstream primer Increasingly, a large number of Prkcz gene coding sequences were obtained, and then the sequence was subjected to an A-tail reaction, and then ligated to the pGM-T vector by T4 DNA ligase to obtain a ligation product, and the ligation product was transformed into competent Escherichia coli DH50C, uniform Applying to the ampicillin-containing LB medium plate, picking up the positive monoclonal colony culture preservation solution and performing preliminary PCR identification. The preliminary identification results indicate that the Prkcz gene cDNA sequence was inserted into the successful bacterial solution for sequencing and identification; The correct E.
  • coli was identified by sequencing, and the pGM-T vector carrying the Prkcz gene cDNA sequence was extracted and digested with restriction endonuclease EcoR I enzyme and Spe I enzyme. The electrophoresis and gel-cutting recovery were about 2500 bp. Fragment, this fragment is the Prkcz gene cDNA sequence;
  • the ligation product was obtained, and the ligation product was transformed into competent Escherichia coli DH50C, uniformly coated on an ampicillin-containing LB medium plate, and the positive monoclonal colony culture preservation liquid was picked and subjected to preliminary identification by PCR, and preliminary identification was carried out.
  • the present invention utilizes genetic engineering technology to construct a lentiviral expression vector which specifically promotes high expression of the Prkcz gene. After successful identification, it is packaged into a virus and transduced into INS-1 cells, and the cells are screened by puromycin. The PCR and Western Blot techniques verified the change of Prkcz gene expression from mRNA and protein levels, respectively. The experimental results showed that the Prkcz gene cDNA sequence provided by the present invention was successfully inserted into the pLVX-IR ES-Puro expression vector, which was specific, sustained, efficient and stable. Promote high expression of the Prkcz gene.
  • the present invention also provides the use of a lentiviral expression vector which specifically promotes high expression of the Prkcz gene for the preparation of a medicament for treating a disease associated with abnormal expression of a Prkcz gene.
  • the lentiviral expression vector which specifically promotes high expression of the Prkcz gene provided by the invention has high transfection efficiency. It has the advantages of low dosage, specific, sustained, efficient and stable promotion of high expression of Prkcz gene, and can be used as a powerful tool in drug research and development related to Prkcz; the present invention also provides specific promotion of high expression of Prkcz gene.
  • the construction method of lentiviral expression vector has good operation effect and reduces the cost of sequence synthesis
  • 1 is a plasmid map of the pLVX-IRES-Puro expression vector.
  • FIG. 2 is a schematic diagram showing the results of fluorescent quantitative PCR detection after puromycin screening of cells.
  • INS-1 cells were purchased from the Cell Resource Center of 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, Lenti-X GoStix reagent. Boxes are 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.
  • Prkcz gene coding sequence GenBank NM_001033581.1
  • the designed PCR primers were synthesized by Shanghai Shenggong Bioengineering Technology Service Co., Ltd. [Table 1]
  • the coding sequence of the Prkcz 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 (Prkcz-T vector) was obtained on the vector, and the ligation product was transformed into competent E. coli DH 5 ⁇ , uniformly coated on an ampicillin-containing LB medium plate, and cultured at 37 ° C for 12 h.
  • Negative control group 1 consistentt cells were uniformly coated on ampicillin-free plates
  • negative control group 2 peripheral cells were uniformly coated in 100
  • positive control group 1 the connection product of the double enzyme-cut empty vector was evenly coated in 100
  • positive control group 2 (the empty carrier was evenly spread on a plate containing 100 g/mL ampicillin).
  • the experimental group grew a single colony, the negative control group 1 grew colonies; the negative control group 2, the positive control group 1, the positive control group 2 did not grow colonies.
  • the bacteria with the correct sequencing result were cultured in liquid LB medium for 14 h, and then the recombinant T vector containing the Prkcz 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 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 grown in good condition were inoculated into six wells, 1,000,000 cells per well, and the extracted recombinant plasmid pLVX-Prkcz 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 infection of INS-1 cells, and the Lenti-X GoStix kit was used to detect a virus titer of 5,000,000 to 50,000,000 IFU.
  • Inoculated INS-1 cells in a 6-well plate 1000000 cells per well, and 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 then polyglycolamine was added. (polybrene) The final concentration was 8 g/mL.
  • the medium in the 6-well plate was added to the virus-containing DMEM complete medium (containing 10% fetal bovine serum). After 24 hours, the virus-containing DMEM complete medium was discarded, and the fresh DMEM complete medium was replaced. After 24 hours, 0.5 was used.
  • the cells were screened at a g/ml concentration of puromycin. After 10 days of screening, the medium was changed once every 3 days, and the concentration of puromycin was continuously increased to 1.00 g/ml.
  • Example 5 Fluorescence quantitative PCR was used to detect the expression level of Prkcz gene.
  • the primer design software Oligo 7.0 was used to design the bow.
  • INS-1 cells pLVX empty vector control INS-1 cell group
  • pLVX-Prkcz 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 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.
  • Prkcz gene is about 150-fold higher than that of INS-1 cells, whether it is just after screening or after 20 generations of pLVX-Prkcz cells, and the Prkcz gene of pLVX empty vector cells.
  • the expression level of the Prkcz gene was successfully inserted into the pLVX-IRES-Puro expression vector, which promoted the high expression of the Prkcz gene specifically, continuously, efficiently and stably.
  • the lentiviral expression vector which specifically promotes the high expression of Prkcz 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 Prkcz gene, and can be used as a powerful tool for application with Prkcz.
  • the present invention also provides a method for constructing a lentiviral expression vector which specifically promotes high expression of the Prkcz gene, which has a good operation effect, reduces the cost of sequence synthesis, and has a low cost.

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Abstract

一种特异促进Prkcz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Prkcz基因cDNA序列；所述多克隆位点包括EcoR I酶切位点和Spe I酶切位点，所述Prkcz基因cDNA序列包括EcoR I酶切位点、Prkcz基因编码序列和Spe I酶切位点，所述Prkcz基因cDNA序列正向插入所述多克隆位点序列中。上述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Prkcz基因的优点，可作为有力工具应用于与Prkcz相关的药物研究与开发。

Description

发明名称:特异促进 Prkcz基因高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， 尤其涉及一种特异促进 Prkcz基因高表达的慢 病毒表达载体及其构建方法与应用。
背景技术
[0002] 糖尿病是一组以血葡萄糖水平增高为特征的、 严重危害人类健康的慢性非传染 性、 代谢性疾病。 无论是在发达国家还是发展中国家， 糖尿病成为现代社会的 流行病， 已成为严重威胁人类健康的世界性公共卫生问题。 按照世界卫生组织
(WHO) 及国际糖尿病联盟 （IDF) 专家组的建议， 糖尿病可分为 1型、 2型、 其他特殊类型及妊娠糖尿病 4种， 其中 2型糖尿病 （T2DM) 是糖尿病人群的主体 。 近年来， 世界各国 2型糖尿病的患病率均有急剧增加的趋势， 2型糖尿病患者 激增是造成全世界糖尿病患者总数剧增的主要原因。 据近年大量流行病学调査 结果显示， 糖尿病在成人中的患病率已接近 10%， 其中 2型糖尿病占全体糖尿病 患者的 90%左右。 WHO统计与预测的结果如下： 1994年糖尿病患者人数为 1.20 亿， 1997年为 1.35亿， 2000年为 1.75亿， 2010年为 2.39亿， 2025年预计将突破 3亿 。 而且近三、 五十年内 2型糖尿病急剧增加的趋势仍将继续。
技术问题
[0003] 近年来有关蛋白激酶 C亚型 ( protein kinasecepsilon zeta， PRKCZ或 ΡΚΟζ)与 糖尿病关系的研究表明， PRKCZ在 2型糖尿病的致病中参与胰岛素的信号传递
， 并且与胰岛的分泌功能密切相关， 是目前发现的重要的 T2DM
相关基因。 T2DM的发生与遗传、 环境、 饮食等多因素相关， 除了遗传变异外
， 表观遗传学研究也是目前研究的重点和热点意义， 但目前现有技术中缺乏特 异促进 Prkcz基因高表达的慢病毒表达载体， 对相关研究的进展造成了一定的阻 碍。
问题的解决方案
技术解决方案 [0004] 为解决现有技术中存在的问题， 发明人在载体的选择、 重组构建方法等方面进 行了大量的探索研究， 发现将包含 EcoR I酶切位点和 Spe
I酶切位点的 Prkcz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 Prkcz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 Prkcz基因高表达的慢病毒表达载体， 包括 pLVX-IRE S-puro表达载体的基本序列、 抗性基因序列、 多克隆位点序列、 启动子序列和 Pr kcz基因 cDNA序列； 所述多克隆位点包括 EcoR I酶切位点和 Spe
I酶切位点， 所述 Prkcz基因 cDNA序列包括 EcoR I酶切位点、 Prkcz基因编码序列 和 Spe I酶切位点， 所述 Prkcz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 Prkcz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 Prkcz基因表达的优点， 可作为有力工具应用于制备治疗 Prkcz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 Prkcz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'-
GGAATTCATGGATTCTGTCATGCCTTCC -3，， 即 SEQ ID NO: 1， 所述下游引 物的序列为： 5，- GACTAGTTCACACCGACTCCTCGGTGG -3'， 即 SEQ ID NO
： 2。 采用上述 PCR引物序列， 通过 PCR可以扩增出 Prkcz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 Prkcz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 Prkcz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) Prkcz基因引物设计： 根据 Prkcz基因编码序列， 使用 Oligo 7分析后选取 5'- GGAATTCATGGATTCTGTCATGCCTTCC -3'， 即 SEQ ID NO: 1作为上游弓 |物 ， 选取 5 '- GACTAGTTCACACCGACTCCTCGGTGG -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) Prkcz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 Prkcz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 Prkcz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 Prkcz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 2500 bp左右的片段， 此片段即为 Prkcz基因 cDNA序 列；
[0011] C) 特异促进 Prkcz基因高表达的慢病毒载体的构建和鉴定： 提取质粒 pLVX-IR ES-Puro, 用限制性内切酶 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 Prkcz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 Prkcz基因 cDNA序列插入成功的菌液进行测序鉴定
[0012] D) 特异促进 Prkcz基因高表达的慢病毒载体的抽提： 将测序结果证实 Prkcz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 Pr kcz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 Prkcz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 INS-1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 Prkcz基因表 达的变化， 实验结果证明本发明提供的 Prkcz基因 cDNA序列成功插入至 pLVX-IR ES-Puro表达载体中， 能特异、 持续、 高效、 稳定地促进 Prkcz基因高表达。
[0014] 本发明还提供特异促进 Prkcz基因高表达的慢病毒表达载体在制备治疗 Prkcz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 Prkcz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 Prkcz基因高表达的优点， 可作为有 力工具应用于与 Prkcz相关的药物研究和幵发中； 本发明还提供了特异促进 Prkcz 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] INS-1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 Prkcz基因引物的设计
[0021] 根据 Prkcz基因编码序列 （GenBank NM_001033581.1) ， 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 EcoR I和 Spe l， 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。 [表 1]
Figure imgf000007_0001
[0022] 实施例二特异促进 Prkcz基因高表达的慢病毒载体的构建
[0023] 将合成的弓 I物稀释后， 用 Premix PrimeSTAR HS酶对 Prkcz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 （Prkcz-T载体） ， 将该连接产物转化到感受态大肠杆菌 DH 5α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。
[0024] 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 Prkcz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 Prkcz 基因， 接着将重组载体送至上海生工公司测序。
[0025] 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 Prkcz基 因序列的重组 T载体， 将其和 pLVX-IRES-Puro载体分别先用 EcoR I酶和 Spe I酶进 行双酶切， 电泳回收， 并用 T4 DNA连接酶连接回收产物用， 再次转化到感受态 大肠杆菌 DH50C中， 均匀涂布到含氨苄青霉素 LB培养基平板上， 于 37°C培养 12 h ， 同吋设置阴性对照组 1 (将感受态细胞均匀涂布在不含氨苄青霉素的平板上） 、 阴性对照组 2 (将感受态细胞均匀涂布在含 100 g/ml氨苄青霉素的平板上） 、 阳性对照组 1 (将双酶切空载体的连接产物均匀涂布在含 100 g/ml氨苄青霉素的 平板上） 、 阳性对照组 2 (将空载体均匀涂布在含 100 g/mL氨苄青霉素的平板上 ) 。 实验组长出了单菌落， 阴性对照组 1长出了菌落； 阴性对照组 2、 阳性对照 组 1、 阳性对照组 2没长出菌落。
[0026] 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 Prkcz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 Prkcz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-Prkcz质粒。
[0027] 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-Prkcz, 测其纯度和浓度， 结果如表 2所示。
[0028] 表 2重组质粒的纯度和浓度
[] [表 2]
Figure imgf000008_0001
[0029] 实施例三 慢病毒包装
[0030] 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-Prkcz 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 INS- 1细胞， Lenti-X GoStix试剂盒检测病毒的滴度为 5000000〜50000000 IFU。
[0031] 实施例四 慢病毒转导 INS-1细胞
[0032] 接种 INS-1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] 实施例五 荧光定量 PCR检测 Prkcz基因表达量。
[0034] 根据 GAPDH和 Prkcz基因 mRNA序列， 利用引物设计软件 Oligo 7.0设计弓 |物。
[] [表 3]
Figure imgf000009_0001
[0035] 分别接种 INS-1细胞、 pLVX空载体对照 INS-1细胞组、 pLVX-Prkcz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 dH 20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 Prkcz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 Prkcz基因相对 表达量。 将 pLVX-Prkcz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-Prkcz细胞 ， 其 Prkcz基因的表达量较 INS-1细胞都有 150倍左右的升高， 而 pLVX空载体细胞 的 Prkcz基因表达量与 INS-1细胞相比基本没有变化， 说明本发明提供的 Prkcz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 Prkcz基因高表达。
[0036] 以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明， 不能认 定本发明的具体实施只局限于这些说明。 对于本发明所属技术领域的普通技术 人员来说， 在不脱离本发明构思的前提下， 还可以做出若干简单推演或替换， 都应当视为属于本发明的保护范围。 工业实用性
本发明提供的特异促进 Prkcz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 Prkcz基因高表达的优点， 可作为有 力工具应用于与 Prkcz相关的药物研究和幵发中； 本发明还提供了特异促进 Prkcz 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 Prkcz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 Prkcz基因 cDNA序歹 ij ; 所述多克隆位点包括 Ec oR I酶切位点和 Spe I酶切位点， 所述 Prkcz基因 cDNA序列包括 EcoR I 酶切位点、 Prkcz基因编码序列和 Spe
I酶切位点， 所述 Prkcz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 Prkcz基因高表达的慢病毒表达载体， 其特征在于： 所述 Prkcz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'- GGAATTCATGGATTCTGTCATGCCTTCC -3，， 即 SEQ ID NO: 1， 所述下游引物的序列为： 5'-
GACTAGTTCACACCGACTCCTCGGTGG -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 Prkcz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) Prkcz基因引物设计： 根据 Prkcz基因编码序列， 使用 Oligo 7分析 后选取 5， - GGAATTCATGGATTCTGTCATGCCTTCC -3，， 即 SEQ ID NO: 1作为上游引物， 选取 5'-
GACTAGTTCACACCGACTCCTCGGTGG -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) Prkcz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 Prkcz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 Prkcz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 Prkcz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 2500 bp左右的片段， 此片段即 为 Prkcz基因 cDNA序列；
C) 特异促进 Prkcz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 Prkcz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 Prkcz基因 cDNA序列插入成功的菌液进行测序鉴定；
D) 特异促进 Prkcz基因高表达的慢病毒载体的抽提： 将测序结果证实 Prkcz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 Prkcz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 Prkcz基因高表达的慢病 毒表达载体在制备治疗 Prkcz基因表达异常相关疾病的药物中的用途
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