WO2022148253A1 - 一种人klf7基因启动子及其构建方法与应用 - Google Patents

一种人klf7基因启动子及其构建方法与应用 Download PDF

Info

Publication number
WO2022148253A1
WO2022148253A1 PCT/CN2021/141244 CN2021141244W WO2022148253A1 WO 2022148253 A1 WO2022148253 A1 WO 2022148253A1 CN 2021141244 W CN2021141244 W CN 2021141244W WO 2022148253 A1 WO2022148253 A1 WO 2022148253A1
Authority
WO
WIPO (PCT)
Prior art keywords
klf7
human
gene promoter
seq
promoter
Prior art date
Application number
PCT/CN2021/141244
Other languages
English (en)
French (fr)
Inventor
张志威
陈月婵
Original Assignee
石河子大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 石河子大学 filed Critical 石河子大学
Publication of WO2022148253A1 publication Critical patent/WO2022148253A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • 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
    • 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

Definitions

  • the invention relates to the technical field of genetic engineering, in particular to a human KLF7 gene promoter and a construction method and application thereof.
  • KLFs Kruppel-like factors
  • Human KLF7 (human KLF7, hKLF7) gene is located in the antisense strand of chromosome 2 2q33.3 region, quantitative genetics report shows that human 2q33.3 and early-onset arthritis (early-onset osteoarthritis, FOA) and early-onset obesity (age of obesity rebound).
  • KLF7 single nucleotide polymorphism single nucleotide polymorphism, SNP
  • SNP single nucleotide polymorphism
  • SNP locus located between human KLF7 and cAMP responsive element binding protein 1 (CREB1) was shown by the combination of SNP microarrays and DNA pooling (SNP Microarrays and Pooling, SNP-MAP). ) is associated with human mild intellectual disability (MMI), and this locus may be in linkage disequilibrium with KLF7 and/or CREB1.
  • MMI human mild intellectual disability
  • KLF7 gene copy number variation CNV
  • Transcription factor KLF7 is a nuclear protein with nuclear localization sequence (NLS), which is mainly located in the nucleus.
  • NLS nuclear localization sequence
  • the NCBI Gene database search showed that hKLF7 can transcribe at least 5 different transcripts, encoding 4 different proteins and 1 long non-coding RNA.
  • the human transcript encoding the longest KLF7 protein encodes 302 amino acids (amino acid, aa), the N-terminal is a transcriptional regulatory domain, and the C-terminal is a DNA-binding domain composed of three highly conserved C2H2 zinc finger structures.
  • the protein expression level of KLF7 is regulated by the N-terminal sequence, and the deletion of the N-terminal 1-76aa sequence significantly increases the protein expression level of KLF7.
  • various drugs regulate the expression of KLF7 while exerting their effects.
  • morphine can increase the level of KLF7 transcription and translation in human lymphocytes, and this promotion is naloxone-reversible, suggesting that KLF7 plays an important role in morphine-mediated physiological responses.
  • the polyphenol-catechin [(-)-catechin] in green tea can significantly promote the expression and secretion of adiponectin and the uptake of glucose in 3T3-L1 adipocytes while inhibiting the expression of KLF7, and regulate the expression of KLF7. Levels help improve endocrine function and insulin sensitivity of adipocytes.
  • KLF7 expression is an independent predictor of poor prognosis in pediatric acute lymphoblastic leukemia.
  • animal-level studies have shown similar activity of HSPCs in embryonic livers of KLF7 -/- and KLF7 +/+ mice, and serial transplantation experiments have shown long-term multilineage engraftment ability of KLF7 -/- cells engraftment) and self-renewal capacity were not significantly different compared to KLF7 +/+ cells.
  • KLF7 inhibits the growth of myeloid progenitors and causes loss of short- and long-term repopulation activity
  • overexpression of KLF7 inhibits multi-lineage growth from hematopoietic stem cells to common lymphoid progenitors, but does not affect T cell growth Conversely enhances early thymocyte survival.
  • RNA expression analysis showed that KLF7 inhibited the growth of myeloid progenitors not by regulating the expression of Cdkn1a (p21Cip1/Waf1), and deletion of Cdkn1a did not rescue the replantation defect.
  • KLF7 is a regulatory gene for the occurrence of various human diseases, and revealing the expression and regulation mechanism of KLF7 is of great significance for the treatment of various human diseases.
  • promoters for the expression of various human KLF7 transcripts Confirming the human KLF7 gene promoter has application value for revealing the regulatory mechanism of the expression of various human KLF7 transcriptional spliceosomes and developing drugs targeting KLF7 expression. .
  • the purpose of the present invention is to provide a human KLF7 gene promoter and its construction method and application, so as to solve the problems existing in the above-mentioned prior art, which can be used to detect a variety of human KLF7 gene transcript spliceosome expression regulation mechanisms.
  • the present invention provides the following scheme:
  • the present invention provides a human KLF7 gene promoter, and the nucleotide sequence of the human KLF7 gene promoter is selected from any sequence shown in SEQ ID NO: 1-SEQ ID NO: 18.
  • human KLF7 gene promoter has promoter activity in eukaryotic cells.
  • the present invention also provides a primer set, the primer set comprises upstream primers as shown in SEQ ID NO: 21-SEQ ID NO: 31, and downstream primers as SEQ ID NO: 19, SEQ ID NO: 20 and SEQ ID NO: 32 shown;
  • the human KLF7 gene promoter is obtained by PCR amplification.
  • the present invention also provides a recombinant vector comprising the human KLF7 gene promoter.
  • the present invention also provides a recombinant bacteria, comprising the recombinant vector.
  • the present invention also provides a kit including the primer set.
  • the eukaryotic cells are derived from human or chicken cells.
  • the present invention also provides an application of the KLF7 gene promoter, which is used in the preparation of medicines targeting KLF7 gene expression.
  • the human KLF7 gene promoter disclosed in the present invention takes DNA extracted from human semen as a template, and uses primers to carry out PCR amplification to obtain 16 kinds of human KLF7 promoter sequences, which are named as A1-A7 and B1-B9 respectively. Recombination technology is used. It was constructed into pGL4.10 vector, and the corresponding recombinant plasmids A1-A7 and B1-B9 were constructed. Then use restriction enzymes XhoI or Hind III to digest B1 separately to obtain two other human KLF7 promoters, named C and D, and use T4 DNA ligase to circularize the corresponding DNA fragments recovered to obtain recombinant plasmids C and D.
  • Figure 1 is the agarose gel electrophoresis image of PCR amplification of human KLF7 gene promoter sequences (A1-A7 and B1-B9); M is the DL5000 DNA marker, 1-16 are the KLF7 promoter sequences A1-A7 and B1- In the PCR amplification results of B9, the brightest band in each electropherogram is the target band, and the rest are PCR non-specific bands, which will be discarded during subsequent gel recovery and will not be used for subsequent vector construction;
  • Fig. 4 is the digestion identification map of human KLF7 gene promoter luciferase reporter gene vector; wherein, swimming lane 1 is the empty vector pGL4.10 that utilizes Xho I single digestion; swimming lane 2-8 is the recombination utilizing Xho I single digestion Plasmids A1-A7 and lane 9 are DL5000 DNA markers; lanes 10-17 are the recombinant plasmids B9, B6, B5, B8, B4, B7, B3 and B2 digested with Xho I in turn; lanes 18 and 19 are Kpn1 respectively Recombinant plasmid D double digested with Hind III and recombinant plasmid C double digested by Kpn1 and Xho I; swimming lane 20 is the recombinant plasmid B1 single digested by Bgl 1;
  • Figure 6 is a graph showing the activity analysis of the human KLF7 gene promoter firefly luciferase reporter gene; among them, pGL4.10 without the promoter sequence is a negative control, pGL3-promoter is a positive control, and "**" indicates a significant difference between pGL4.10 and pGL4.10. Difference (P ⁇ 0.01);
  • Figure 7 is a graph showing the relative activity of the human KLF7 gene promoter luciferase reporter gene; among them, pGL4.10 without promoter sequence is a negative control group, pGL3-promoter is a positive control group, and "**" indicates that it has the same characteristics as pGL4.10. Significant difference (P ⁇ 0.01).
  • proteinase K 20mg/mL proteinase K (proteinase K): add 200mg proteinase K to 9.5mL of double distilled water, shake gently until proteinase K is completely dissolved, make up to 10ml, and then store in aliquots at -20°C.
  • SDS sodium dodecyl sulfate
  • step 4 Add 500 ⁇ L of phenol:chloroform:isoamyl alcohol (25:24:1) to the collected aqueous phase in step 3, invert and mix for 10 min, then centrifuge at 15000 r/min for 10 min, and place the aqueous phase of the absorbent layer in a 1.5 mL EP tube;
  • step 5 Add 1 mL of absolute ethanol to the aqueous phase obtained in step 4), wash the white precipitate by inversion, centrifuge at 12000 r/min for 5 min, and discard the liquid.
  • step 6) Add 1 mL of 70% ethanol to the white precipitate obtained in step 5) to wash, centrifuge at 12000 r/min for 5 min, and remove the supernatant.
  • the PCR product was subjected to agarose gel electrophoresis (as shown in Figure 1), and the target band was recovered using the AXYGEN gel recovery and purification kit.
  • the digestion conditions were: 37°C for 1 h.
  • the Xho I digestion results are shown in the first lane of Figure 4.
  • the digestion products were identified by 1% agarose gel electrophoresis and purified and recovered by AXYGEN gel recovery and purification kit to obtain recombinant plasmids that can be used for A1-A7 and B2-B9 Constructed linearized pGL4.10 vector.
  • the pGL4.10 vector used for the recombination of the promoter B1 sequence was first digested with the endonuclease SAC I, and then the gel was recovered and purified, and then the gel recovered product was digested with Nhe I, and then the gel was recovered and purified to obtain a recombinant plasmid that could be used for B1 construction.
  • the linearized pGL4.10 vector was first digested with the endonuclease SAC I, and then the gel was recovered and purified, and then the gel recovered product was digested with Nhe I, and then the gel was recovered and purified to obtain a recombinant plasmid that could be used for B1 construction.
  • the linearized pGL4.10 vector was first digested with the endonuclease SAC I, and then the gel was recovered and purified, and then the gel recovered product was digested with Nhe I, and then the gel was recovered and purified to obtain a recombinant
  • the reaction system was reacted at 37 °C for 30 min to complete the plasmid recombination, and the luciferase reporter gene bodies containing 16 KLF7 promoters, A1-A7 and B1-B9, were constructed as shown in Figure 2.
  • These 16 recombinant plasmids were purchased from PROMEGA
  • the company's pGL4.10 vector is the backbone (as shown in Figure 3), and it was successfully constructed after introducing the above-mentioned 16 human KLF7 promoters A1-A7 and B1-B9 through homologous recombination.
  • the insert sequence is shown in SEQ ID NO in the Sequence Listing. : 1 to SEQ ID NO: 16.
  • the recombinant product was transformed into Escherichia coli JM109 competent cells, screened for AMP resistance and cultured. After picking a single clone, the single clone was inoculated in LB liquid medium supplemented with 1% AMP for culture. After 12 hours of culture, the plasmid was extracted with AXYGEN. The plasmid was extracted from the kit, and the recombinant plasmids A1-A7 and B2-B9 were identified by enzyme digestion with Xho I, and the recombinant plasmid B1 was identified by single enzyme digestion with Bgl I. The results are shown in Figure 4.
  • the plasmids were sequenced and verified, and the obtained 16 positive recombinant plasmid markers were named as recombinant plasmids A1-A7 and B1-B9 according to the difference of the inserted sequences.
  • T4 DNA ligation system is shown in Table 6:
  • the reaction conditions were: 16°C overnight.
  • the plasmids C and D were identified by enzyme digestion with Kpn1 and Xho I or Kpn1 and Hind III, respectively.
  • the electrophoresis results are shown in Figure 4.
  • the 4200bp band and the predicted size fragments can be cut out, indicating that the vector construction was successful.
  • the 18 human KLF7 gene promoter luciferase reporter gene plasmids that showed significant activity were not spurious phenomena caused by differences in transfection efficiency and cell state.
  • the constructed 18 human KLF7 gene promoters had significant relative activities (P ⁇ 0.01, two-tailed unpaired t test). It shows that all the constructed human KLF7 promoter luciferase reporter gene plasmids have promoter activity.
  • promoter fragments numbered A7, B9 and D respectively, have weak promoter activity (as shown in Figure 4), combined with the physical map displayed by sequence structure analysis (as shown in Figure 2), A7, B9 and D are located upstream of the transcription start site (TSS) of the three transcripts NM_001270942.1, transcripts NM_003709.4 and NM_001270943.1 of KLF7, respectively, they do not have sequence crossover, and all have obvious promoter activity, Therefore, the above three promoter fragments may respectively contain the core promoter sequences of the above three different transcripts of human KLF7.
  • TSS transcription start site

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biotechnology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Plant Pathology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

提供了一种人KLF7基因启动子及其构建方法与应用，属于基因工程技术领域。所述人KLF7基因启动子核苷酸序列选自如SEQ ID NO：1-SEQ ID NO：18所示任一条序列；以人体精液中提取DNA作为模板，利用引物进行PCR扩增和限制性酶切的方法获取了18种人KLF7启动子，通过试验验证，发现18种启动子在鸡前脂肪细胞中都具有启动子活性，这为揭示多种人KLF7转录剪接体表达的调控机制和开发靶向KLF7表达的药物具有重要意义，并且为研究人类多种与KLF7基因调控的疾病治疗奠定基础。

Description

一种人KLF7基因启动子及其构建方法与应用
本申请要求于2021年01月08日提交中国专利局、申请号为“202110021958.9”、发明名称为“一种人KLF7基因启动子及其构建方法与应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及基因工程技术领域，特别是涉及一种人KLF7基因启动子及其构建方法与应用。
背景技术
Krüppel样因子(Kruppel-like factors，KLFs)是一类存在于动物体内的转录因子，它们参与细胞增殖、分化和凋亡等多个生命过程的调控。目前，人体内共发现了17种KLF因子，它们的蛋白质结构特征是羧基端(carboxyl terminus，C端)具有三个典型的C2H2锌指结构作为DNA结合结构域，氨基端(amino terminus，N端)序列作为转录调控结构域在家族成员间保守性差。1998年，Matsumoto等通过简并PCR技术从人血管内皮细胞中克隆得到一个之前从未报道过的KLF因子，根据其广泛表达于成人多种组织的特性，将其命名为普遍存在的Krüppel样因子(Ubiquitous KLF，UKLF)。根据系统命名法，UKLF又被命名为KLF7。
人KLF7(human KLF7，hKLF7)基因位于2号染色体2q33.3区域的反义链，数量遗传学报道显示人2q33.3与早发性关节炎(early-onset osteoarthritis，FOA)和早发性肥胖(肥胖反弹的年龄)相关。KLF7单核苷酸多态性(single nucleotide polymorphism，SNP)研究表明，hKLF7是人类肥胖和2型糖尿病(type 2 diabetes)发生的相关基因。日本人群体研究表明，hKLF7第二内含子的一个SNP(rs2302870，chr2:207953406A/C)与2型糖尿病的发生相关。丹麦人群体研究显示rs2302870位点与2型糖尿病和肥胖没有显著相关，但是位于hKLF7 5’UTR的另一个SNP(rs7568369，chr2:208031315 C/A)与肥胖相关。
采用SNP基因芯片与DNA池相结合(SNP Microarrays andPooling，SNP-MAP)的研究显示位于人KLF7和cAMP反应元件结合蛋白1(cAMP  responsive element binding protein 1，CREB1)之间的一个SNP位点(rs991684)与人轻度智力障碍(Mild mental impairment，MMI)相关，该位点可能与KLF7和(或)CREB1处于连锁不平衡状态。一个瘢痕疙瘩家系5个个体(4例瘢痕疙瘩患者，1例健康人)的全基因组重测序结果显示，KLF7基因拷贝数变异(copy number variation,CNV)可能与瘢痕疙瘩的形成有关。
转录因子KLF7是一个核蛋白，具有核定位序列(Nuclear localization sequence，NLS)，主要定位于细胞核中。NCBI Gene数据库检索显示，hKLF7至少能够转录5种不同转录本，编码4种不同的蛋白质和1种长链非编码RNA。
目前被广泛研究的是编码最长链蛋白质的KLF7转录本，该转录本翻译的蛋白在脊椎动物间高度保守，各个结构域在高等动物(哺乳动物和鸟类)间的序列相似性大于85％。人编码最长KLF7蛋白质的转录本，编码302个氨基酸(amino acid，aa)，N端为转录调控域，C端是由3个高度保守的C2H2锌指结构组成的DNA结合结构域。KLF7蛋白质表达水平受到N端序列调控，缺失N端1-76aa的序列明显提高KLF7的蛋白质表达水平。另外，多种药物在发挥作用的同时调控KLF7的表达。例如：吗啡可以提高人淋巴细胞中的KLF7转录和翻译水平，并且这种促进作用是纳洛酮可逆的(naloxone-reversible)，提示了KLF7在吗啡介导的生理反应中具有重要作用。绿茶中的多酚-儿茶素[(-)-catechin]在抑制KLF7表达的同时，能明显促进3T3-L1脂肪细胞脂联素的表达和分泌水平以及对葡萄糖的摄取能力，调控KLF7的表达水平有助改善脂肪细胞的内分泌功能和胰岛素敏感性。
KLF7表达量增加是小儿急性淋巴细胞白血病(pediatric acute lymphoblastic leukemia)预后不良的独立预测因子。虽然动物水平研究显示，KLF7 -/-小鼠和KLF7 +/+小鼠胚肝中HSPCs的活性相似，并且连续移植实验显示，KLF7 -/-细胞的长期多谱系植入能力(long-term multilineage engraftment)和自我更新能力与KLF7 +/+细胞相比没有明显差别。但是，体外研究显示过表达KLF7抑制髓系祖细胞生长，并且造成短期和长期再植活性的丢失；过表达KLF7抑制从造血干细胞到普通淋巴系祖细胞的多 谱系生长，但不影响T细胞生长相反增强早期胸腺细胞的存活。这些结果提示，虽然KLF7不是正常的造血干细胞和祖细胞发挥功能必需的，但是增加KLF7表达能抑制骨髓细胞的增殖并促进早期胸腺细胞存活。此外，RNA表达分析显示，KLF7抑制髓系祖细胞生长不通过调控Cdkn1a(p21Cip1/Waf1)表达实现，缺失Cdkn1a不能拯救再植缺陷。
综上所述，KLF7是多种人类疾病发生的调控基因，揭示KLF7的表达调控机制对于治疗人类多种疾病具有重要意义。但是目前还没针对多种人KLF7多种转录本表达的启动子报道，确认人KLF7基因启动子，对于揭示多种人KLF7转录剪接体表达的调控机制和开发靶向KLF7表达的药物具有应用价值。
发明内容
本发明的目的是提供一种人KLF7基因启动子及其构建方法与应用，以解决上述现有技术存在的问题，可用于检测多种人KLF7基因转录本剪接体表达调控机制。
为实现上述目的，本发明提供了如下方案：
本发明提供一种人KLF7基因启动子，所述人KLF7基因启动子核苷酸序列选自如SEQ ID NO：1-SEQ ID NO：18所示任一条序列。
进一步地，所述人KLF7基因启动子在真核细胞中具有启动子活性。
本发明还提供一种引物组，所述引物组包含上游引物如SEQ ID NO：21-SEQ ID NO：31所示，下游引物如SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：32所示；
利用任一条所述上游引物和任一条所述下游引物组合，经PCR扩增获得所述的人KLF7基因启动子。
本发明还提供一种重组载体，包含所述的人KLF7基因启动子。
本发明还提供一种重组菌，包含所述的重组载体。
本发明还提供一种所述的人KLF7基因启动子的构建方法，包括利用所述的引物组PCR扩增获取人KLF7基因启动子核苷酸序列的步骤。
本发明还提供一种试剂盒，包括所述的引物组。
本发明还提供一种所述的KLF7基因启动子的应用，应用于启动萤火虫荧光素酶在真核细胞中表达。
优选的是，所述真核细胞来源于人或者鸡细胞。
进一步地，所述鸡细胞为鸡前脂肪细胞。
本发明还提供一种所述的KLF7基因启动子的应用，应用于制备靶向KLF7基因表达的药物中。
本发明公开了以下技术效果：
本发明公开的人KLF7基因启动子，是以人体精液中提取DNA作为模板，利用引物进行PCR扩增获取了16种人KLF7启动子序列，分别命名为A1-A7和B1-B9，利用重组技术将其构建到pGL4.10载体，构建相应的重组质粒A1-A7和B1-B9。再利用限制性酶XhoI或Hind III分别单酶切B1获得另外2种人KLF7启动子，命名为C和D，利用T4DNA连接酶环化回收的相应DNA片段，获得重组质粒C和D。通过试验验证，发现18种启动子在鸡前脂肪细胞中都具有启动子活性，说明每种启动子都能直接调控人KLF7转录表达，并且这18种启动子序列分别位于三种人KLF7转录剪接体的转录起始位点上游不同位置，这对于揭示多种人KLF7转录剪接体表达的调控机制和开发靶向KLF7表达的药物具有重要意义，并且为研究KLF7基因参与调控的人类多种疾病治疗奠定基础。
附图说明
图1为PCR扩增人KLF7基因启动子序列(A1-A7和B1-B9)的琼脂糖凝胶电泳图；M为DL5000 DNA marker，1-16分别为KLF7启动子序列A1-A7和B1-B9的PCR扩增结果，每一电泳图中最亮的条带为目的带，其余为PCR非特异带，在后续胶回收时会弃掉，不用于后续载体构建；
图2为18种人KLF7基因启动子荧光素酶报告基因载体构建示意图和相关序列定位的物理图谱；其中，LUC指的萤火虫荧光素酶基因；图中注明了人KLF7基因的染色体定位和不同转录剪接体的转录起始位点；
图3为用于构建KLF7基因启动子荧光素酶报告基因的空载体pGL4.10质粒结构图；
图4为人KLF7基因启动子荧光素酶报告基因载体的酶切鉴定图；其中，泳道1为利用Xho I单酶切的空载体pGL4.10；泳道2-8为利用Xho I单酶切的重组质粒A1-A7、泳道9为DL5000 DNA marker；泳道10-17 依次为利用Xho I单酶切的重组质粒B9、B6、B5、B8、B4、B7、B3和B2；泳道18和19分别为Kpn1和Hind III双酶切的重组质粒D和Kpn1和Xho I双酶切的重组质粒C；泳道20为Bgl 1单酶切的重组质粒B1；
图5为人KLF7基因启动子荧光素酶报告基因载体B1的单酶切电泳图；其中，泳道1为Xho1单酶切人KLF7基因启动子荧光素酶报告基因载体B1；泳道2为Hind III单酶切人KLF7基因启动子荧光素酶报告基因载体B1；泳道M为DL5000 DNA marker；
图6为人KLF7基因启动子萤火虫荧光素酶报告基因活性分析图；其中，不含有启动子序列的pGL4.10为阴性对照，pGL3-promoter为阳性对照，“**”表示与pGL4.10具有显著差异(P<0.01)；
图7为人KLF7基因启动子荧光素酶报告基因相对活性图；其中，不含有启动子序列的pGL4.10为阴性对照组，pGL3-promoter为阳性对照组，“**”表示与pGL4.10具有显著差异(P<0.01)。
具体实施方式
现详细说明本发明的多种示例性实施方式，该详细说明不应认为是对本发明的限制，而应理解为是对本发明的某些方面、特性和实施方案的更详细的描述。
实施例1人KLF7基因启动子的制备
1、人精子基因组DNA提取
1.1试剂配置
1)酚：氯仿：异戊醇＝25：24：1(按照体积比配制)
2)氯仿：异戊醇＝24：1(按照体积比配制)
3)20mg/mL蛋白酶K(proteinase K)：将200mg的蛋白酶K加入到9.5mL双蒸水中，轻轻摇动，直至蛋白酶K完全溶解，定容至10ml，然后分装储存于-20℃。
4)10％SDS(十二烷基硫酸钠)：称取100g SDS慢慢转移到0.9L双蒸水的烧杯中，加热至68℃并用磁力搅拌器搅拌至完全溶解。若需要，用1mol/LNaOH调节至pH值7.2，用双蒸水定容至1L，室温保存，无需灭菌。
5)STE配制
10mmol/L Tris-HCl(pH8.0)
0.1mol/L NaCl
1mmol/L EDTA(pH8.0)
121℃高压灭菌15min，储存于4℃。
6)1mol/L二硫苏糖醇配制标准(DTT)：二硫苏糖醇5g，加32.4mL去离子水，分成小份储存于-20℃。
1.2精子DNA提取
1)取冻存精液50μL放入1.5mL离心管中，用1mL PBS洗涤，10000r/min离心10min，弃上清，重复洗涤一次。
2)弃上清，加0.5mL STE，50μL 10％SDS，20μL蛋白酶K(20mg/mL)，加入40μL DTT(1mol/L)，55℃消化过夜，摇床上进行。
3)从水浴锅中取出，冷却至室温加入500μLTris饱和酚，颠倒混10min，再于15000r/min离心10min，用宽口移液管(口径0.3cm)吸水层水相于1.5mL EP管；勿吸入蛋白层；再用Tris饱和酚抽提2次，收集水相。
4)步骤3)收集的水相中加入500μL酚：氯仿：异戊醇(25：24：1)，颠倒混匀10min，再于15000r/min离心10min，吸水层水相于1.5mL EP管；
5)步骤4)得到的水相中加入1mL无水乙醇，颠倒洗涤白色沉淀，12000r/min离心5min，弃液体。
6)步骤5)得到的白色沉淀中加1mL 70％乙醇洗涤，12000r/min离心5min，去上清。
7)自然干燥30min，用pH8.0 TE溶解DNA，-20℃保存备用。
2、人KLF7基因启动子区克隆
以提取的人基因组DNA为模板，利用表1所示的引物组合进行PCR扩增人KLF7基因启动子区，获得编号为A1-A7和B1-B9的16种质粒(如图2所示)中所包含的人KLF7启动子基因序列。
表1用于获得KLF7启动子片段A1-A7和B1-B9的引物组合
Figure PCTCN2021141244-appb-000001
Figure PCTCN2021141244-appb-000002
PCR反应体系如表2：
表2
Figure PCTCN2021141244-appb-000003
PCR反应条件如表3：
表3
Figure PCTCN2021141244-appb-000004
Figure PCTCN2021141244-appb-000005
将PCR产物进行琼脂糖凝胶电泳(如图1所示)，并用AXYGEN凝胶回收纯化试剂盒回收目的条带备用。
3、人KLF7启动子荧光素酶报告基因载体构建
以pGL4.10质粒为酶切底物，利用限制性内切酶进行酶切，酶切体系以Xho I为例如表4：
表4
Figure PCTCN2021141244-appb-000006
酶切条件为：37℃酶切1h。
Xho I酶切结果如图4第1泳道所示，酶切产物经1％琼脂糖凝胶电泳鉴定并用AXYGEN凝胶回收纯化试剂盒纯化回收，获得可以用于A1-A7和B2-B9重组质粒构建的线性化pGL4.10载体。
用于启动子B1序列重组的pGL4.10载体先利用内切酶SAC I酶切后进行胶回收纯化，之后胶回收产物再利用Nhe I酶切后胶回收纯化，获得可以用于B1重组质粒构建的线性化pGL4.10载体。
质粒重组采用Vazyme的One Step Cloning Kit试剂盒，按照说明书在冰上配置好体系，体系如表5：
表5
Figure PCTCN2021141244-appb-000007
Figure PCTCN2021141244-appb-000008
反应体系在37℃反应30min完成质粒重组，构建如图2所示的含有A1-A7和B1-B9这16种KLF7启动子的荧光素酶报告基因体，这16种重组质粒均以购自PROMEGA公司的pGL4.10载体为骨架(如图3所示)，通过同源重组导入上述A1-A7和B1-B9这16种人KLF7启动子后构建成功，其插入序列详见序列表SEQ ID NO：1至SEQ ID NO：16。重组产物转化到大肠杆菌JM109感受态细胞中，AMP抗性筛选培养，挑取单克隆后，将单克隆接种于加了1％AMP的LB液体培养基中培养，培养12h后，采用AXYGEN提质粒试剂盒提取质粒，利用Xho I进行酶切鉴定重组质粒A1-A7和B2-B9，采用Bgl I单酶切鉴定重组质粒B1，结果如图4所示，重组质粒A1和A7均切出了4200bp(空载体)和相应大小的插入序列片段；由于B2-B9序列中本身具有一个内源性Xho I酶切位点加上载体构建时引入的2个Xho I酶切位点，所以切出了3条带，分别为4200bp片段和一条比目的片段小200bp的DNA片段，和一条200bp的片段；由于B8目的片段为400左右，所以切出了4200条带和一条较宽的200bp左右条带；B1由于序列内含有3个内源性Bgl 1酶切位点以及2个载体上自带的Bgl 1酶切位点，所以获得了符合预期大小的4条带；以上结果表明上述16种KLF7启动子质粒酶切鉴定正确。
酶切验证正确后，将质粒进行测序验证，将得到的16个阳性重组质粒标记按照插入序列的不同分别命名为重组质粒A1-A7、B1-B9。
将获得的重组质粒B1分别采用Xho I和Hind III单独酶切后，如图5所示回收大于5000bp的较长片段，采用T4 DNA连接酶连接，可以获得含有人KLF7启动子序列荧光素酶报告基因质粒C和D(如图2所示)。质粒C和D在空载体pGL4.10的多克隆位点间的插入序列详见序列表SEQ ID NO：17和SEQ ID NO：18。T4 DNA连接的体系和反应条件如下。
T4 DNA连接体系如表6：
表6
Figure PCTCN2021141244-appb-000009
Figure PCTCN2021141244-appb-000010
反应条件为：16℃过夜。
利用Kpn1和Xho I或Kpn1和Hind III分别进行酶切鉴定质粒C和D，电泳结果如图4显示，能切出4200bp条带和预测大小的片段，表明载体构建成功。
实施例2人KLF7基因启动子活性分析
将生长状态良好的鸡前脂肪细胞接种于24孔细胞培养板中，接种密度为2.5×10 4个/孔，24h之后，将Fugene HD(promega)转染试剂按照说明书将pGL4.10空质粒、pGL3-promoter和18种人KLF7基因启动子荧光素酶报告基因质粒(即实施例1中构建的重组质粒A1-A7、B1-B9、C和D)分别转染到细胞中，其中，pGL4.10空质粒(empty vector，EV)为阴性对照组，转染pGL3-promoter质粒作为阳性对照组。每孔细胞转染500ng的萤火虫荧光素酶报告基因质粒，并同时转染45ng的pRL-TK质粒为实验内参，每组重复至少三次，转染48h之后收细胞，按照Promega公司的
Figure PCTCN2021141244-appb-000011
Luciferase Assay System说明书进行荧光活性测定。
结果如图6所示，与阴性对照相比，所构建的18种人KLF7基因启动子荧光素酶报告基因质粒均能够表现出显著的荧光素酶活性(P<0.01，双尾不配对t检验)。
如图7所示，与阴性对照相比，表现出显著活性的18种人KLF7基因启动子荧光素酶报告基因质粒，并不是由于转染效率和细胞状态差异造成的虚假现象。与阴性对照相比，构建的18种人KLF7基因启动子具有显著的相对活性(P<0.01，双尾不配对t检验)。表明构建的所有人KLF7启动子荧光素酶报告基因质粒均具有启动子活性。其中，3个启动子片段，编号分别为A7、B9和D具有较弱的启动子活性(如图4所示)，结合序列结构分析显示的物理图谱(如图2所示)，A7、B9和D分别位于KLF7的3种转录本NM_001270942.1、转录本NM_003709.4和NM_001270943.1的转录起始位点(TSS)的上游，它们不存在序列交叉， 并且均具有明显的启动子活性，因此上述三个启动子片段可能分别含有了上述人KLF7的三种不同转录本的核心启动子序列。
以上所述的实施例仅是对本发明的优选方式进行描述，并非对本发明的范围进行限定，在不脱离本发明设计精神的前提下，本领域普通技术人员对本发明的技术方案做出的各种变形和改进，均应落入本发明权利要求书确定的保护范围内。

Claims (11)

  1. 一种人KLF7基因启动子，其特征在于，所述人KLF7基因启动子核苷酸序列选自如SEQ ID NO：1-SEQ ID NO：18所示任一条序列。
  2. 一种引物组，其特征在于，所述引物组包含上游引物如SEQ ID NO：21-SEQ ID NO：31所示，下游引物如SEQ ID NO：19、SEQ ID NO：20和SEQ ID NO：32所示；
    利用任一条所述上游引物和任一条所述下游引物组合，经PCR扩增获得权利要求1所述的人KLF7基因启动子。
  3. 根据权利要求2所述的引物组，其特征在于，所述引物组如下表所示：
    Figure PCTCN2021141244-appb-100001
  4. 一种重组载体，其特征在于，包含权利要求1所述的人KLF7基因 启动子。
  5. 一种重组菌，其特征在于，包含权利要求4所述的重组载体。
  6. 一种如权利要求1所述的人KLF7基因启动子的构建方法，其特征在于，包括利用权利要求2或3所述的引物组进行PCR扩增获取人KLF7基因启动子核苷酸序列的步骤。
  7. 一种试剂盒，其特征在于，包括权利要求2或3所述引物组。
  8. 一种如权利要求1所述的KLF7基因启动子的应用，其特征在于，应用于启动萤火虫荧光素酶在真核细胞中表达。
  9. 如权利要求8所述的KLF7基因启动子的应用，其特征在于，所述真核细胞来源于人细胞或者鸡细胞。
  10. 如权利要求9所述的KLF7基因启动子的应用，其特征在于，所述鸡细胞为鸡前脂肪细胞。
  11. 一种如权利要求1所述的KLF7基因启动子的应用，其特征在于，应用于制备靶向KLF7基因表达的药物中。
PCT/CN2021/141244 2021-01-08 2021-12-24 一种人klf7基因启动子及其构建方法与应用 WO2022148253A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110021958.9A CN112662673B (zh) 2021-01-08 2021-01-08 一种人klf7基因启动子及其构建方法与应用
CN202110021958.9 2021-01-08

Publications (1)

Publication Number Publication Date
WO2022148253A1 true WO2022148253A1 (zh) 2022-07-14

Family

ID=75413668

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/141244 WO2022148253A1 (zh) 2021-01-08 2021-12-24 一种人klf7基因启动子及其构建方法与应用

Country Status (2)

Country Link
CN (1) CN112662673B (zh)
WO (1) WO2022148253A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112662673B (zh) * 2021-01-08 2022-06-28 石河子大学 一种人klf7基因启动子及其构建方法与应用
CN113881678B (zh) * 2021-11-01 2024-04-12 石河子大学 一种c/ebpz基因启动子及其应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004091660A1 (ja) * 2003-04-17 2004-10-28 Kowa Co., Ltd. Lklf/klf2遺伝子発現促進剤
JP2006081414A (ja) * 2004-09-14 2006-03-30 Shionogi & Co Ltd 新規糖尿病関連因子
KR20070106822A (ko) * 2006-05-01 2007-11-06 (주)아모레퍼시픽 (-)카테친을 함유하는 아디포넥틴 발현 증강제
CN105969768A (zh) * 2016-05-10 2016-09-28 石河子大学 一种klf7基因启动子及其活性和应用
WO2019178283A1 (en) * 2018-03-13 2019-09-19 Baylor Research Institute Methods and compositions for treating and prognosing colorectal cancer
CN112662673A (zh) * 2021-01-08 2021-04-16 石河子大学 一种人klf7基因启动子及其构建方法与应用

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101899500B (zh) * 2010-02-09 2012-10-17 西北农林科技大学 一种黄牛klf7基因的单核苷酸多态性检测方法
SG11201405545XA (en) * 2012-03-27 2014-11-27 Curevac Gmbh Artificial nucleic acid molecules comprising a 5'top utr
EP2873727B1 (en) * 2012-07-12 2019-11-06 Kyoto Prefectural Public University Corporation Brown fat cells and method for preparing same
CN104511028B (zh) * 2013-10-08 2018-03-02 上海市肿瘤研究所 Klf9基因在抑制肝癌中的应用
US11845960B2 (en) * 2016-09-12 2023-12-19 President And Fellows Of Harvard College Transcription factors controlling differentiation of stem cells
US20180305719A1 (en) * 2017-04-19 2018-10-25 The Board Of Trustees Of The University Of Illinois Vectors For Integration Of DNA Into Genomes And Methods For Altering Gene Expression And Interrogating Gene Function
CN111500590B (zh) * 2020-05-11 2022-09-16 石河子大学 一种调控鸡脂肪细胞形成的转录因子gata2的应用
CN113881678B (zh) * 2021-11-01 2024-04-12 石河子大学 一种c/ebpz基因启动子及其应用

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004091660A1 (ja) * 2003-04-17 2004-10-28 Kowa Co., Ltd. Lklf/klf2遺伝子発現促進剤
JP2006081414A (ja) * 2004-09-14 2006-03-30 Shionogi & Co Ltd 新規糖尿病関連因子
KR20070106822A (ko) * 2006-05-01 2007-11-06 (주)아모레퍼시픽 (-)카테친을 함유하는 아디포넥틴 발현 증강제
CN105969768A (zh) * 2016-05-10 2016-09-28 石河子大学 一种klf7基因启动子及其活性和应用
WO2019178283A1 (en) * 2018-03-13 2019-09-19 Baylor Research Institute Methods and compositions for treating and prognosing colorectal cancer
CN112662673A (zh) * 2021-01-08 2021-04-16 石河子大学 一种人klf7基因启动子及其构建方法与应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE NUCLEOTIDE 4 October 2017 (2017-10-04), ANONYMOUS : "Homo sapiens Kruppel like factor 7 (KLF7), RefSeqGene on chromosome 2", XP055949492, retrieved from NCBI Database accession no. NG_052989 *
LIN TAO, CHEN YUECHAN, ZHANG YANLING, LI YAOYAO, GAO LINGYU, ZHANG ZHIWEI: "Transcriptional control of chicken promoter in preadipocytes", ACTA BIOCHIMICA BIOPHYSICA SINICA, BLACKWELL PUBLISHING, INC., MALDEN, MA, US, vol. 53, no. 2, 1 December 2020 (2020-12-01), US , pages 149 - 159, XP055949490, ISSN: 1672-9145, DOI: 10.1093/abbs/gmaa149 *

Also Published As

Publication number Publication date
CN112662673B (zh) 2022-06-28
CN112662673A (zh) 2021-04-16

Similar Documents

Publication Publication Date Title
WO2022148253A1 (zh) 一种人klf7基因启动子及其构建方法与应用
Govoroun et al. Isolation of chicken homolog of the FOXL2 gene and comparison of its expression patterns with those of aromatase during ovarian development
Wang et al. Structural diversity and functional implications of the eukaryotic TDP gene family
AU2023201019A1 (en) C/EBP alpha short activating RNA compositions and methods of use
CN110628766B (zh) 与羊骨骼肌发育相关的lncRNA编码基因及其应用
Guja et al. Hitting the brakes: termination of mitochondrial transcription
LaPointe et al. Upstream sequences confer atrial-specific expression on the human atrial natriuretic factor gene.
US7767881B2 (en) Utilization of histamine receptor h3 gene participating in body weight or food intake control
Beckers et al. Active genes in junk DNA? Characterization of DUX genes embedded within 3.3 kb repeated elements
Mordes et al. Identification of photoreceptor genes affected by PRPF31 mutations associated with autosomal dominant retinitis pigmentosa
WO2013144663A2 (en) Method of determination of neutral dna sequences in the genome, system for targeting sequences obtained thereby and methods for use thereof
CN110204605B (zh) 转录因子C/EBPα作为ACOX1启动子区的转录因子的应用
AU720798B2 (en) Regulators of UCP2 gene expression
CN113881678B (zh) 一种c/ebpz基因启动子及其应用
Smith et al. Genomic organization of the mammalian SLC14a2 urea transporter genes
Wang et al. Expression of the cardiac maintenance and survival factor FGF-16 gene is regulated by Csx/Nkx2. 5 and is an early target of doxorubicin cardiotoxicity
Pérez-Ibave et al. Expression of growth hormone gene in the baboon eye
CN113201559B (zh) 一种人源胰腺炎相关cel-hyb1基因变异敲入小鼠的构建方法与应用
CN106831975B (zh) 热休克转录因子1在调控15kDa硒蛋白表达中的应用
AU726567B2 (en) Regulators of UCP3 gene expression
AU2004223739B2 (en) Cyclic AMP response element activator proteins and uses related thereto
CN1209843A (zh) 新的鉴定刺激stf－1在胰岛细胞内表达的化合物的方法
JPH10500311A (ja) 核タンパク質と相互作用する因子
Nam et al. Isolation and transient expression of a cDNA encoding l-gulono-γ-lactone oxidase, a key enzyme for l-ascorbic acid biosynthesis, from the tiger shark Scyliorhinus torazame
Mogayzel Jr et al. CFTR intron 1 increases luciferase expression driven by CFTR 5′-flanking DNA in a yeast artificial chromosome

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21917293

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21917293

Country of ref document: EP

Kind code of ref document: A1