WO2004076662A1 - Sequences de regions associees de support et leur utilisation - Google Patents

Sequences de regions associees de support et leur utilisation Download PDF

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WO2004076662A1
WO2004076662A1 PCT/CN2004/000146 CN2004000146W WO2004076662A1 WO 2004076662 A1 WO2004076662 A1 WO 2004076662A1 CN 2004000146 W CN2004000146 W CN 2004000146W WO 2004076662 A1 WO2004076662 A1 WO 2004076662A1
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sar
nuclear matrix
sequence
expression
cells
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PCT/CN2004/000146
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French (fr)
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Mujun Zhao
Liang Da
Suiquan Wang
Zaiping Li
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Shanghai Institutes For Biological Sciences, Chinese Academy Of Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the invention relates to the fields of molecular cell biology and regulation of eukaryotic gene expression. Specifically, it relates to a new human nuclear matrix protein binding sequence (SAR), and applications of the SAR sequence, including the construction of gene expression vectors, and the enhancement of the expression level and stability of foreign genes in eukaryotic cells.
  • SAR nuclear matrix protein binding sequence
  • SAR nuclear matrix binding regions
  • SAR nuclear scaffold associated regions
  • SAR is an important cis-acting element. SAR plays an important role in the formation and change of advanced structures of chromatin, and plays an important role in regulating DNA replication and gene transcription from the level of chromatin.
  • a nuclear matrix protein binding sequence is a nucleotide sequence that has the characteristic of specifically binding to the nuclear matrix of a cell.
  • Bode J. et al. Discovered in the early 1990s that SAR can enhance the expression of huIFN- ⁇ gene in mouse L cells (Bode J, et al., Biochemistry, 1991, 30: 1264-1270) .
  • SAR element of the rRNA gene cluster of castor silkworm can enhance the expression of foreign genes (Zhao Mujun et al., Science in China (Series C), 1998, 28 (1): 42 ⁇ 49).
  • SAR spans a large number of chromosomes and requires a genomic sequence as a probe, it is difficult to find and clone SAR. Not many SAR sequences have been reported so far.
  • an isolated nuclear matrix protein binding sequence is provided, which contains the nucleotide sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.
  • the nuclear matrix protein binding sequence has a nucleotide sequence shown in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8.
  • a vector which contains the nuclear matrix protein binding sequence of the present invention.
  • the nuclear matrix protein binding sequence is located upstream of the promoter.
  • a host cell which is the vector of the present invention.
  • the host cell is a eukaryotic cell.
  • a method for increasing the expression intensity of a foreign gene includes:
  • step (b) transferring the construct of step (a) into a host cell
  • step (c) culturing the host cell of step (b), thereby expressing the encoded product of the foreign gene.
  • the nuclear matrix protein binding sequence of the present invention which are used to construct an expression vector that enhances the expression amount and / or stability of foreign genes in eukaryotic cells.
  • the expression vector is an animal or plant transgenic vector.
  • Figure 1 shows that the results of Southern blot experiments proved that 17 SAR-positive clones could hybridize to the total SAR probes of 7721 liver cancer cells and L02 liver cells.
  • FIG. 2 shows that Southwestern experiments demonstrated the specific binding of SAR to nuclear matrix proteins of S-band C-7721 cells.
  • c ⁇ i DNA is a non-specific competitive substrate with concentrations of 0, 10, and 50 g / ml, respectively.
  • Figure 3 shows a pLu plasmid map with SAR inserted between the BamHI-Sall restriction sites of the plasmid.
  • the present invention provides eight DNA fragments (SAR No. 1-8) located in the 8p23. 1 region of the human chromosome, and their sequences are shown in SEQ ID NOs: 1-8.
  • All eight SARs can bind to nuclear matrix proteins, and all eight DNA fragments can hybridize to the total SAR of SMMC-7721 cells (human liver cancer cell line) and L02 cells (human immortalized normal liver cells) (see Figure 1 ).
  • the experiments of the present invention also demonstrated that these SARs can specifically bind to nuclear matrix proteins (as shown in Figure 2).
  • the eukaryotic expression vector constructed by using the SAR (SAR No.1-S) according to the present invention can improve the stable expression of the foreign gene product (as shown in Figure 4).
  • the inventor selected human BAC clone RP11-177H2 from
  • the clone contains a 162 kb human chromosome 8p23. 1 DNA fragment. Then, by separating the nuclear matrix proteins to precipitate the DNA fragments bound to nuclear matrix proteins, a total SAR library of cells was prepared. Using the BAC clone RP11-177H2 DNA as a probe, the SAR of 8p23. 1 region was screened from the total SAR library. DNA-protein in vitro binding experiments confirmed that the SAR has the activity of specifically binding to nuclear backbone proteins.
  • isolated refers to the separation of a substance from its original environment (if it is a natural substance, the original environment is the natural environment).
  • polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotides or polypeptides are separated or purified if they are separated from other substances existing in the natural state. of.
  • the SAR of the present invention is in the form of DNA.
  • the form of DNA includes genomic DNA or synthetic DNA.
  • the invention also relates to a variant of said SAR.
  • These variants can be naturally occurring or non-naturally occurring variants.
  • These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is a replacement form of a polynucleotide that may be a substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the occurrence of SAR and nuclear matrix proteins. Combined functionality.
  • the invention also relates to a nucleic acid fragment that hybridizes to the sequence described above.
  • nucleic acid fragment contains at least 15 nucleotides in length, preferably at least 30 nucleotides, more preferably at least 50 nucleotides, and most preferably at least 100 nucleotides. Nucleic acid fragments can be used in nucleic acid amplification techniques such as PCR to determine and / or isolate the nucleotide sequence of SAR.
  • the polynucleotide in the present invention is preferably provided in an isolated form, and is more preferably purified to homogeneity.
  • the full-length human SAR nucleotide sequence or a fragment thereof of the present invention can usually be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method.
  • primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, and a commercially available genomic library or DNA prepared according to a conventional method known to those skilled in the art can be used as a template for amplification.
  • a commercially available genomic library or DNA prepared according to a conventional method known to those skilled in the art can be used as a template for amplification.
  • About the sequence When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then stitch the amplified fragments together in the correct order.
  • the recombination method can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it into a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic methods can also be used to synthesize related sequences, especially when the fragment length is short.
  • long fragments can be obtained by synthesizing multiple small fragments first and then performing ligation.
  • the SAR of the present invention can be preferably obtained by PCR amplification.
  • the present invention also relates to a vector comprising the SAR of the present invention, and a host cell produced by a genetic manipulation technique using the vector of the present invention, and a method for enhancing the expression amount and / or stability of a foreign gene in a eukaryotic cell using the SAR of the present invention. method.
  • the SAR element of the present invention can be applied to construct a eukaryotic gene expression vector and improve the stable expression of foreign genes.
  • the SAR element is cloned into a eukaryotic expression vector pLu plasmid (see FIG. 3) to construct a pLu-SAR expression vector.
  • This vector contains the SV40 promoter and luciferase reporter gene, which is introduced into eukaryotic cells, and the level of expression of foreign genes can be determined by detecting the activity of luciferase.
  • the pLu-SAR clone was stably transformed into CH0 cells (Chinese hamster ovarian cancer cells), and it was found that the SAR of the present invention can increase the expression of the luciferase gene of the insect more than seven times.
  • CH0 cells Choinese hamster ovarian cancer cells
  • the SAR sequence of the present invention is a SAR regulatory element of human origin, which is used to construct a high expression vector, which can be used in humans and other eukaryotic cells, and is used to enhance the expression of foreign genes in eukaryotic cells. It is suitable for genetic engineering to prepare genes and their products, and is suitable for transgenic research of human body.
  • the SAR element of the present invention is located in the human chromosome 8p23. 1 region, and is isolated from tumor cells (SMMC-7721 liver cancer cell line).
  • the human chromosome 8p23. 1 region is a highly sensitive region for the occurrence of liver cancer.
  • the SAR of the present invention is an active regulatory element in liver cancer cells, so it can be applied to tumor cells, especially gene therapy of liver cancer, and has important significance.
  • the eukaryotic expression vector pLu-SAR constructed by SAR is used to introduce foreign genes into eukaryotic cells and integrate them on the chromosome.
  • SAR can enhance the promoter activity on the chromosome and enhance gene expression. Because the foreign gene is integrated on the chromosome and is distributed to the progeny daughter cells with cell division, it is not easy to lose. Therefore, the ability of SAR to enhance gene expression has the advantage of being stable and efficient. It is especially suitable for stably expressing the target gene in eukaryotic cells. For gene or protein therapy to improve the expression and stability of the therapeutic gene.
  • the SAR of the present invention is effective when inserted either upstream or downstream of the promoter or downstream of the 3 'end of the expressed gene.
  • SAR / MAR can be combined on the nuclear matrix, it is conducive to improving the level of protein expression and its stability, and is suitable for eukaryotic cells to produce genetically engineered products and improve the quality. Therefore, it has broad application prospects in genetic engineering production and gene therapy.
  • the present invention is further described below with reference to specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are generally based on conventional conditions, for example, Sambrook et al., Molecular Cloning: Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to manufacturing conditions Conditions recommended by the manufacturer.
  • Example 1 Screening and cloning of human chromosome 8p23. 1 region SAR
  • nuclear matrix proteins were isolated from commercially available hepatocellular carcinoma cell line Sband C-7721 and normal liver cell line L02, that is, 5 X 10 7 cells were collected and suspended in 5 ml TM-2 solution (10 mM Tris-HCl [pH 7. 4], 2 mM M g Cl 2, 0. 5 mM PMSF) in. Leave on ice for 5 minutes, add 0.25 ml 10% Triton ⁇ -100, mix well, and then place on ice for 5 minutes. Nuclei were pelleted by centrifugation (800 rpm ⁇ 10 min, 4 ° C), and then washed twice with TM-2 solution.
  • TM-2 solution 10 mM Tris-HCl [pH 7. 4], 2 mM M g Cl 2, 0. 5 mM PMSF
  • the BAC clone RP11-177H2 (available from Children's Hospital Oakland Research Institute, CHRI) was selected as a probe. This clone contained a 162 kb human chromosome 8p23. 1 DNA fragment.
  • the BAC clone was completely digested with s 6I restriction enzyme (Takara), and the ends of the digested fragments were connected with PCR adapters:
  • Primer 1 ACTGAGCTCGAGTATCCATGAACA (SEQ ID NO: 9)
  • Primer 2 TATGTTCATGG (SEQ ID NO: 10)
  • the PCR adapter was used as a primer and the small DNA fragment mixture was used as a template for PCR amplification.
  • the amplified product was then subjected to an in vitro binding assay with the nuclear matrix of S-C7721 cells.
  • the nuclear matrix protein suspension obtained in Example 1a was digested with proteinase K, the protein was removed by extraction with phenol and chloroform, and the DNA was finally precipitated by ethanol to obtain the total SAR of SMMC-7721 cells and L02 cells.
  • the total SAR of the two cells was labeled with [ ⁇ - 32 P] dATP (random primer labeling kit, Takara).
  • a single primer (SEQ ID NO: 9) was used to amplify inserts on approximately 80 clones in the candidate SAR library by PCR, and these PCR products were spotted on nylon membranes, respectively. All the clones in the candidate SAR library were further screened by Southern experiments (rapid hybridization kit, Amersham Life Science) using the probes labeled earlier, and 17 positive clones were obtained (Figure 1).
  • Example la The nuclear matrix protein obtained in Example la was subjected to 8% SDS-polyacrylamide gel electrophoresis, and
  • Bio-Rad's protein transfer device transfers proteins to nitrocellulose membranes.
  • Method using PCR Digoxin (Roche Applied Science) was labeled on the SAR fragments and subjected to Southwestern hybridization experiments.
  • the positive control established was the 3 'SAR of the apo gene
  • the negative control was the cDNA sequence of the LPTS gene.
  • E. coli DNA was used as a non-competitive substrate in the hybridization solution at concentrations of 0, 10, and 50 ug / ml, respectively.
  • SAR fragment to be tested has strong binding effect with nuclear matrix proteins, so that the fragment is identified as a nuclear matrix-specific binding element, that is, a SAR element. All eight SARs (SEQ ID NO: 1-8) have been verified by the above experiments and have the characteristics of binding to nuclear matrix proteins.
  • SAR element enhances expression of foreign genes in eukaryotic cells
  • This embodiment takes SAR NO. 8 as an example (SEQ ID NO: 8), which proves that the SAR element of the present invention can enhance the expression of foreign genes in eukaryotic cells.
  • the nucleotide sequence of the SAR N0. 8 element obtained in Example 2 was used, or a primer with BamHI and Sai l restriction sites and corresponding to 20 nucleotide sequences at the head and tail of SEQ ID N0: 8 sequence, respectively Using the BAC clone RP11-177H2 (available from CHORI) as a template, the SAR No. 8 sequence was obtained by PCR amplification. Then the SA ⁇ fragment was digested with enzyme and inserted into the BamHI-Sail site of pLu plasmid to construct a pLu-SAR eukaryotic expression vector.
  • pLu plasmid is a commonly used plasmid in the laboratory.
  • the structure diagram is shown in Figure 3.
  • the pLu plasmid contains the penicillin resistance gene (Amp R ) and the neomycin resistance gene Neo as a selection marker. It contains the SV40 promoter and the luciferase reporter gene (Luc) as an exogenous gene. Enzyme activity to determine the expression of foreign genes.
  • the pLu plasmid expresses Luc with low activity and can be used as a control.
  • the SAR element was inserted upstream of the SV40 promoter of pLu plasmid (between BamH I and Sal I sites) to construct the pLu-SAR plasmid. If the SAR element can enhance gene expression, the activity of luciferase will be increased after detection. b. Detection of SAR element enhancing expression activity of foreign genes in eukaryotic cells
  • the eukaryotic expression vector pLu-SAR and pLu as control were transfected into CH0 cells.
  • CH0 cells were cultured on 6-well plates, the number of cells was about 2.5 X 10 5 / well, pLu-SAR plasmid was added 2 ug / well, liposome transfection reagent (product of Invitrogen) 5 ⁇ 1 / well, transfection method
  • liposome transfection reagent product of Invitrogen
  • transfection method Follow the operation manual provided by the company. 48 hours after transfection, screening was performed with 0.7 mg / mL G418 to obtain anti-G418 resistant clones, and then luciferase activity was detected. The luciferase activity was measured using a luciferase detection system (Promega). For the method, see the Promega product manual, LB9507 Fluorometer. The counter is a product of EG & G Berthold.
  • SAR element enhances expression of foreign genes in different cells
  • the results are shown in Table 1.
  • the SAR of the present invention has the function of enhancing gene expression not only in CH0 cells, but also in liver cancer cells SMMC-7721 and liver cells L02. This indicates that SAR, as a regulatory element that enhances gene expression, is non-cellular or tissue-specific, and has activity inserted upstream of the promoter in the forward or reverse direction, but the activity varies.
  • Table 1 SAR No. 8 enhances the expression of foreign genes in CH0, SMMC-7721 and L02 cells SAR No. 8 MAR forward MAR reverse
  • SAR is a new element that regulates gene expression at the chromosomal level.
  • the action site of the SAR element of the present invention can be placed 5 'upstream or 3' downstream of the gene, and the position can be inserted forward or reverse upstream of the promoter, and there is no strict distance requirement. Therefore, SAR is widely used in regulating gene expression.

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Description

核基质蛋白结合序列及其应用 技术领域
本发明涉及分子细胞生物学和真核基因表达调控领域。具体地涉及新的人核 基质蛋白结合序列 (SAR)， 以及所述 SAR序列的应用， 包括用于基因表达载体的 构建、 以及增强外源基因在真核细胞中的表达量和稳定性。 背景技术
在真核生物细胞核内， DNA发生多级折叠包装，经历 10nm核小体串珠结构、 30nm螺线管结构， 进一步折叠形成染色质环状结构。 染色质环通过特殊的位点 附着在细胞核基质上， 这些特殊的 DNA区域被称为核基质结合区或核骨架结合 区 (scaffold associated regions, SAR)。 SAR是一种重要的顺式作用元件， SAR在染 色质高级结构的形成与变化中起重要作用， 并且从染色质水平上对 DNA复制， 基因的转录起着重要的调控作用。 简而言之， 核基质蛋白结合序列 (SAR)是具有 与细胞核基质特异性结合的特征的核苷酸序列。
国外最早由 Mirkovitch J等报导了组蛋白基因簇 SAR 的检测及其特性 (Mirkovitch J, et al., Cell, 1984, 39:223-232), 发现 SAR是富含 A、 T的 DNA序列 (A+T〉70%)，长约 400〜1000 bp,能特异地与核骨架蛋白结合。 而后， Cockerill和 Garrard报导了免疫球蛋白 κ基因的 MAR(Garrard W. T.， et al.， Cell, 1986, 44:273- 282)， 并发现该 MAR / SAR含有拓扑异构酶 II的识别位点等。
随着研究的逐步深入， 九十年代初 Bode J.等人发现， SAR能够增强 huIFN- β基因在鼠 L细胞中的表达 (Bode J， et al., Biochemistry, 1991, 30:1264-1270)。
对 SAR功能的研究也越来越引起注意。 例如发现蓖麻蚕 rRNA基因簇的 SAR 元件对外源基因表达具有增强作用（赵慕钧等，中国科学（C 辑）， 1998， 28 (1) : 42〜49)。 但由于 SAR在染色体上的跨度较大， 需要有基因组序列作为探针， 因此 SAR的发现和克隆有一定难度。 目前报道的 SAR序列并不多。
因此， 本领域迫切需要开发新的 SAR调控序列， 尤其是随着人类基因组测序 的初步完成， 更迫切需要开发人源的 SAR调控序列。 发明内容
本发明的目的就是提供新的来源于人的 SAR调控序列及其用途。 确 认 本 在本发明的第一方面， 提供了一种分离的核基质蛋白结合序列， 它含有 SEQ ID NO: l、 2、 3、 4、 5、 6、 7或 8所示的核苷酸序列。
较佳地， 所述的核基质蛋白结合序列具有 SEQ ID NO: 1、 2、 3、 4、 5、 6、 7 或 8所示的核苷酸序列。
在本发明的第二方面， 提供了一种载体， 它含有本发明的核基质蛋白结合序 列。 较佳地， 所述的核基质蛋白结合序列位于启动子的上游。
在本发明的第三方面， 提供了一种宿主细胞， 它本发明所述的载体。 较佳地， 所述的宿主细胞是真核细胞。
在本发明的第四方面， 提供了一种提高外源基因的表达强度的方法， 该方法 包含：
(a)构建从 5'至 3'依次还有以下元件的构建物： 权利要求 1所述的核基质蛋白结 合序列、 启动子序列、 外源基因和终止序列；
(b)将步骤 (a)的构建物转入宿主细胞，
(c)培养步骤 (b)的宿主细胞， 从而表达外源基因的编码产物。
在本发明的第五方面， 提供了一种本发明核基质蛋白结合序列的用途， 它们 被用于构建增强外源基因在真核细胞中的表达量和 /或稳定性的表达载体。较佳地， 所述的表达载体是动植物的转基因载体。
本发明的其它方面由于本文的技术的公开， 对本领域的技术人员而言是显而 易见的。 附图说明
图 1显示 Southern印迹实验结果证明 17个 SAR阳性克隆能与 7721肝癌细 胞和 L02肝细胞的总 SAR探针杂交。
图 2显示 Southwestern实验证明 SAR与 S匪 C- 7721细胞核基质蛋白的特异 性结合。 其中， （a) , apo基因 3' SAR为阳性对照。 （b) . LPTS cDNA为阴性对 照。 （c) . SAR No. 8与核基质蛋白特异性结合。 c^i DNA为非特异性竞争底 物， 浓度分别为 0、 10、 50 g/ml。
图 3显示 pLu质粒图谱， SAR插入到该质粒的 BamHI-Sall酶切位点之间。 图 4显示了 SAR元件增强虫萤光素酶相对活力的检测结果。 其中， 将 pLu、 pLu-SAR质粒稳定转染 CH0细胞， 测定转染细胞株的 Luciferase 活性。 以 pLu 作为对照（Luc if erase活性 = 1)。 具体实施方式
本发明人通过广泛而深入的研究， 通过独特的技术路线和实验方案， 检测并 克隆人染色体 8p23. 1区域上的 SAR。将克隆到的新的 SAR序列在体外组建成真核 基因表达载体， 并携带外源基因导入真核细胞， 获得增强外源基因稳定高效表达 的 SAR元件， 在此基础上完成了本发明。 本发明提供了 8个定位于人染色体 8p23. 1区域的 DNA片段（SAR No. 1-8) , 其序列 SEQ ID NO : 1-8所示。 这 8个 SAR都能与核基质蛋白结合， 并且此 8个 DNA片段都能与 SMMC- 7721细胞（人肝癌细胞株）和 L02细胞（人永生化正常肝细胞) 的总 SAR杂交（见图 1)。 本发明的实验还证明了这些 SAR能与细胞核基质蛋白特 异结合（如图 2所示）。 用本发明所述 SAR(SARNo.l- S)构建的真核表达载体， 可 提高外源基因稳定表达其产物 (如图 4所示)。
在本发明的一个实施例中， 本发明人选择人 BAC 克隆 RP11-177H2 可从
CHORI , Children Hospital Oakland Research Institute购得）作为研究材料， 该克隆含有 162 kb的人染色体 8p23. 1区域的 DNA片段。 然后通过分离细胞核基 质蛋白来沉淀结合在核基质蛋白上的 DNA片段， 制备细胞总的 SAR库。 以 BAC 克隆 RP11- 177H2的 DNA为探针， 从总的 SAR库中筛选获得 8p23. 1区域的 SAR。 DNA-蛋白质体外结合实验证实， 上述 SAR具有核骨架蛋白特异性结合的活性。 如本文所用， "分离的"是指物质从其原始环境中分离出来 (如果是天然的物 质， 原始环境即是天然环境) ,。 如活体细胞内的天然状态下的多聚核苷酸和多肽是 没有分离纯化的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质 中分开， 则为分离或纯化的。
本发明的 SAR是 DNA形式。 DNA形式包括基因组 DNA或人工合成的 DNA。
本发明还涉及所述 SAR 的变异体。 这些变异体可以是天然发生的变异体或非 天然发生的变异体。 这些核苷酸变异体包括取代变异体、 缺失变异体和插入变异 体。 如本领域所知的， 等位变异体是一个多核苷酸的替换形式， 它可能是一个或 多个核苷酸的取代、 缺失或插入， 但不会从实质上改变 SAR 与核基质蛋白发生结 合的功能。 本发明还涉及与上述的序列杂交的核酸片段。 如本文所用， "核酸片段" 的 长度至少含 15个核苷酸， 较好是至少 30个核苷酸， 更好是至少 50个核苷酸， 最 好是至少 100个核苷酸以上。 核酸片段可用于核酸的扩增技术（如 PCR)以确定和 / 或分离 SAR的核苷酸序列。
本发明中多核苷酸优选以分离的形式提供， 更佳地被纯化至均质。
本发明的人 SAR核苷酸全长序列或其片段通常可以用 PCR扩增法、 重组法或人 工合成的方法获得。 对于 PCR扩增法， 可根据本发明所公开的有关核苷酸序列来设 计引物， 并用市售的基因组库或按本领域技术人员已知的常规方法所制备的 DNA作 为模板， 扩增而得有关序列。 当序列较长时， 常常需要进行两次或多次 PCR扩增， 然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列， 就可以用重组法来大批量地获得有关序列。 这通常 是将其克隆入载体， 再转入细胞， 然后通过常规方法从增殖后的宿主细胞中分离 得到有关序列。
此外， 还可用人工合成的方法来合成有关序列， 尤其是片段长度较短时。 通 常， 通过先合成多个小片段， 然后再进行连接可获得序列很长的片段。
根据本发明所公开的具体序列（SEQ ID NO : 1-8)， 可优选应用 PCR技术扩增 获得本发明的 SAR。
本发明也涉及包含本发明的 SAR的载体， 以及用本发明的载体经基因操作技 术产生的宿主细胞， 以及用本发明 SAR增强外源基因在真核细胞中的表达量和 /或 稳定性的方法。
本发明的 SAR元件可应用于构建真核基因表达载体，并提高外源基因的稳定 表达。 在一优选例中， 将该 SAR元件克隆到真核表达载体 pLu质粒（见图 3)中， 构建成 pLu- SAR表达载体。 该载体含有 SV40启动子和虫萤光素酶报告基因， 将 其导入真核细胞， 通过检测虫萤光素酶的活力， 来确定外源基因的表达高低。 将 pLu-SAR克隆稳定转化 CH0细胞（中国仓鼠卵巢癌细胞）， 发现本发明的 SAR能增 强虫萤光素酶基因表达七倍以上。 本发明主要优点在于：
(1)本发明的 SAR序列， 是人源的 SAR调控元件， 用于构建高表达载体， 可在 人体及其它真核细胞中应用， 用于增强外源基因在真核细胞中的表达量， 适合于 基因工程制备基因及其产物， 并适合应用于人体的转基因治疗研究。 (2)本发明的 SAR元件定位于人染色体 8p23. 1 区域， 并且从肿瘤细胞中 (SMMC-7721肝癌细胞株）分离获得。人染色体 8p23. 1区域是肝癌发生的高敏感区 域。 本发明的 SAR是在肝癌细胞中有活性的调控元件， 因此可应用于肿瘤细胞， 特别是肝癌的基因治疗， 有重要意义。
(3)利用 SAR构建的真核表达载体 pLu-SAR，将外源基因导入真核细胞并整合 到染色体上， SAR能在染色体上增强启动子活力， 使基因表达增强。 由于外源基 因整合在染色体上， 随细胞分裂而分配到后代子细胞中， 不易丢失， 因此 SAR增 强基因表达的能力具有稳定高效的优点，特别适用于在真核细胞中稳定表达目的 基因， 用于基因或蛋白治疗， 以提高治疗基因的表达量和稳定性。
(4) 本发明的 SAR在正向或反向插入启动子的上游或表达基因 3'末端的下游 时， 都有效。
由于 SAR/MAR能结合在核基质上，有利于提高表达蛋白的水平及其稳定性， 适用于真核细胞生产基因工程产品， 提高质量。 因此在基因工程生产和基因治疗 中有广阔的应用前景。 下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规条件，例如 Sambrook等人， 分子克隆：实验室手册（New York : Cold Spring Harbor Laboratory Press, 1989)中所述的条件， 或按照制造厂商所建 议的条件。 实施例 1 : 人染色体 8p23. 1区域 SAR的筛选和克隆
a. 细胞核基质的制备
首先从市售的肝癌细胞株 S匪 C-7721 和正常肝细胞株 L02分离出核基质蛋 白， 即分别收集 5 X 107细胞， 悬于 5 ml TM-2溶液 （10 mM Tris-HCl [pH 7. 4] , 2 mM MgCl2， 0. 5 mM PMSF)中。 冰上放置 5分钟， 加入 0. 25 ml 10% Triton χ- 100， 混匀， 再放置冰上 5分钟。 通过离心（800 rpmX IO min, 4°C )将细胞核沉 淀下来， 然后用 TM- 2溶液洗两次。 得到的细胞核用溶液 I (10 mM NaCl, 10 mM Tris-HCl [pH 7. 4]， 3 mM MgCl2， 0. 25 M sucrose, 0. 5 mM PMSF)洗两次， 再悬 于 0· 6 ml溶液 I， 加入 350 U DNase I (Takara公司）， 于 23°C反应 2小时。 离 心， 得到的沉淀用溶液 II (2M NaCl, 10 mM EDTA, 10 mM Tris-HCl [pH 7. 4] , 0. 5 mM PMSF)悬浮， 再离心后沉淀即为细胞核基质， 悬于 0. 05 ml溶液 I。 b.人染色体 8p23. 1区域候选 SAR库的建立
选择 BAC 克隆 RP11- 177H2 (可从 CH0RI， Children Hospital Oakland Research Institute购得）作为探针， 该克隆含有 162 kb的人染色体 8p23. 1区 域的 DNA片段。将 BAC克隆用 s 6I限制性内切酶（Takara公司）完全酶解，在酶 解片段的两端装连上 PCR接头：
引物 1 : ACTGAGCTCGAGTATCCATGAACA (SEQ ID NO : 9)
引物 2 : TATGTTCATGG (SEQ ID NO : 10)
以 PCR接头为引物， DNA小片段混合物为模板， 进行 PCR扩增。 然后将扩增 产物与 S應 C- 7721细胞的核基质做体外结合试验。
向步骤 a分离制备的基质蛋白加入 0. 04 mg鲑精 DNA， 室温温浴并振荡 30 分钟之后， 加入 20 ng PCR扩增得到的 DNA混合物， 继续温浴振荡 2小时。 离心 得到的沉淀用蛋白酶 K消化过夜， 用酚、氯仿抽提去除蛋白， 最后乙醇沉淀 DNA。 筛选得到的 DNA混合物装入 pMD 18- T 载体 （Takara公司）， 转化 E. coli细胞， 得到的阳性克隆， 即为人染色体 8p23. 1区域候选的 SAR克隆库。 c Southern点杂交筛选 SAR
将实施例 la中得到的核基质蛋白悬液用蛋白酶 K消化， 经酚、 氯仿抽提去 除蛋白， 最后乙醇沉淀 DNA， 得到 SMMC- 7721细胞和 L02细胞的总 SAR。 用 [ α - 32P] dATP分别标记（随机引物标记试剂盒， Takara公司）两种细胞的总 SAR。
用单引物（SEQ ID NO : 9)， 通过 PCR扩增候选 SAR库中约 80个克隆上的插 入片段， 将这些 PCR 产物分别点在尼龙膜上。 用前面标记得到的探针通过 Southern实验（快速杂交试剂盒， Amersham Life Science公司）， 对候选 SAR库 中所有克隆做进一步筛选， 得到的 17个阳性克隆（图 1)。
对 17个阳性克隆全部进行测序，结果获得定位于 8个 SAR，其序列分别列于 SEQ ID NO :卜 8。 实施例 2: SAR与细胞核基质蛋白的特异性结合
将实施例 la中得到的核基质蛋白进行 8% SDS-聚丙烯酰胺凝胶电泳， 釆用
Bio - Rad公司的蛋白转膜装置， 将蛋白转移到硝酸纤维素膜上。 采用 PCR的方法 将地高辛（Roche Appl i ed Science公司）标记到 SAR片段上， 进行 Southwestern 杂交实验。 杂交反应中， 设立的阳性对照为 apo基因 3' SAR, 阴性对照为 LPTS 基因的 cDNA序列。 杂交液中用 E. col i DNA作为非竞争性底物， 浓度分别为 0、 10和 50 ug/ml o
实验结果如图 2所示， 待测的 SAR片段与核基质蛋白有强的结合作用， 从而 鉴定该片段为细胞核基质特异的结合元件， 即 SAR元件。 8个 SAR(SEQ ID N0 : 1-8) 都经上述实验验证， 具有与核基质蛋白结合的特征。 实施例 3 : SAR元件增强外源基因在真核细胞中的表达
本实施例以 SAR N0. 8为例（SEQ ID NO : 8)， 证明本发明 SAR元件可增强外 源基因在真核细胞中的表达。
a. 真核表达载体的构建
用实施例 2中获得 SAR N0. 8元件的核苷酸序列， 或者用分别带有 BamHI和 Sai l酶切位点且对应于 SEQ ID N0 : 8序列头尾各 20个核苷酸序列的引物， 以 BAC 克隆 RP11- 177H2 (可从 CH0RI购得）为模板， 通过 PCR扩增获得 SAR No. 8序列。 然后将 SA^片段经酶切后插入 pLu质粒的 BamHI- Sai l位点， 构建成 pLu- SAR真 核表达载体。 pLu质粒是一种实验室常用的质粒， 结构图见图 3。 pLu质粒含有青 霉素抗性基因（AmpR)和新霉素抗性基因 Neo 为筛选标记， 含有 SV40启动子和 作为外源基因的虫萤光素酶报告基因（Luc)，通过测定虫萤光素酶的活力来确定 外源基因的表达高低。 pLu质粒表达 Luc的活力很低， 因此可以作为对照。
将 SAR元件插入到 pLu质粒 SV40启动子的上游（BamH I和 Sal I位点之间）， 构建成 pLu-SAR质粒。 如果该 SAR元件能够增强基因的表达， 经检测虫萤光素酶 的活力就提高。 b. SAR元件增强外源基因在真核细胞中表达活性的检测
将构建的真核表达载体 pLu-SAR, 以及作为对照的 pLu分别转染 CH0细胞。 CH0细胞培养在 6孔板上， 细胞数约为 2. 5 X 105/孔， 加入 pLu- SAR质粒 2 ug / 孔， 脂质体转染试剂（Invitrogen公司产品） 5μ1 /孔， 转染方法按公司提供的操 作手册进行。 在转染 48小时后用 0. 7mg/mL G418进行筛选， 得到抗 G418抗性克 隆后， ， 然后检测虫萤光素酶活性。 虫萤光素酶的活性用虫萤光素酶检测系统 (Promega公司产品）进行测定， 方法见 Promega公司产品说明书， LB9507荧光计 数仪为 EG&G Berthold公司产品。
测定结果如图 4所示， CHO/pLu- SAR细胞相对荧光强度比 CHO/ pLu细胞大 7倍。 这说明 SAR No. 8应用于构建真核细胞表达载体可显著增强外源基因表达 的能力。
用相同的方法测试 SAR No : l-7， 结果表明它们也可不同程度地增强外源基因 表达的能力。 实施例 4: SAR元件增强外源基因在不同细胞中的表达
在本实施例中， 按实施例 3相同的方法测试 SEQ ID NO : 8所示 SAR的增强 活性， 不同点仅在于宿主细胞和插入位置不同（表 1)。
结果如表 1所示， 本发明的 SAR不仅在 CH0细胞中有增强基因表达的功能， 在肝癌细胞 SMMC- 7721和肝细胞 L02中也有同样的功能。 这表明， SAR作为一种 增强基因表达的调控元件， 具有非细胞或组织特异性， 并且以正向或反向插入启 动子的上游都有活性， 只是活性大小有所不同。 表 1 SAR No. 8增强外源基因在 CH0、 SMMC- 7721和 L02细胞中的表达 SAR No. 8 MAR正向 MAR反向
细胞株 CHO SMMC7721 L02 CHO SMMC7721 L02 增倍数 6.83 8.21 5.79 3.65 4.38 2.33
SAR是一种新的在染色体水平调控基因表达的元件。 本发明 SAR元件的作用 位点，可以置于基因 5'上游或 3'下游， 位置可以正向或反向插入启动子的上游， 并且没有严格的距离要求。 因此， SAR在调控基因表达中， 应用十分广泛。 在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被 单独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本 领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所 附权利要求书所限定的范围。

Claims

权 利 要 求
1.一种分离的核基质蛋白结合序列，其特征在于，它含有 SEQ ID NO: 1、 2、 3、 4、 5、 6、 7或 8所示的核苷酸序列。
2.如权利要求 1所述的核基质蛋白结合序列， 其特征在于， 具有 SEQ ID NO: 1、
2、 3、 4、 5、 6、 7或 8所示的核苷酸序列。
3.—种载体， 其特征在于， 它含有权利要求 1所述的核基质蛋白结合序列。
4.如权利要求 3所述的载体， 其特征在于， 所述的核基质蛋白结合序列位于启 动子的上游。
5.—种宿主细胞， 其特征在于， 它含有权利要求 3所述的载体。
6.如权利要求 5所述的宿主细胞， 其特征在于， 所述的宿主细胞是真核细胞。
7. 一种提高外源基因的表达强度的方法， 其特征在于， 该方法包含：
(a)构建从 5'至 3'依次还有以下元件的构建物： 权利要求 1所述的核基质蛋白结 合序列、 启动子序列、 外源基因和终止序列；
(b)将步骤 (a)的构建物转入宿主细胞，
(c)培养步骤 (b)的宿主细胞， 从而表达外源基因的编码产物。
8.如权利要求 7所述的方法， 其特征在于， 所述的宿主细胞是真核细胞。
9.一种权利要求 1所述的核基质蛋白结合序列的用途，其特征在于，用于构建 增强外源基因在真核细胞中的表达量和 /或稳定性的表达载体。
10.如权利要求 9所述的用途， 其特征在于， 所述的表达载体是动植物的转基 因载体。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996019573A1 (en) * 1994-12-19 1996-06-27 Cangene Corporation Recombinant dna molecules and expression vectors for erythropoietin
WO1997027207A1 (en) * 1996-01-26 1997-07-31 North Carolina State University A plant nuclear scaffold attachment region which increases gene expression
WO1998016650A1 (en) * 1996-10-17 1998-04-23 E.I. Du Pont De Nemours And Company Enhanced transgene expression in a population of monocot cells employing scaffold attachment regions
WO1998044139A1 (en) * 1997-03-28 1998-10-08 Mycogen Plant Science, Inc. Improved sar plant transformation process
WO2000006757A1 (en) * 1998-07-31 2000-02-10 Mycogen Plant Science, Inc. Improved plant transformation process by scaffold attachment regions (sar)

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996019573A1 (en) * 1994-12-19 1996-06-27 Cangene Corporation Recombinant dna molecules and expression vectors for erythropoietin
WO1997027207A1 (en) * 1996-01-26 1997-07-31 North Carolina State University A plant nuclear scaffold attachment region which increases gene expression
WO1998016650A1 (en) * 1996-10-17 1998-04-23 E.I. Du Pont De Nemours And Company Enhanced transgene expression in a population of monocot cells employing scaffold attachment regions
WO1998044139A1 (en) * 1997-03-28 1998-10-08 Mycogen Plant Science, Inc. Improved sar plant transformation process
WO2000006757A1 (en) * 1998-07-31 2000-02-10 Mycogen Plant Science, Inc. Improved plant transformation process by scaffold attachment regions (sar)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [online] 1 April 2002 (2002-04-01), BIRREN B. ET AL.: "Homo sapiens chromosome 8, clone CTD-2135J3, complete sequence", Database accession no. (AC105001) *
DATABASE GENBANK [online] 1 December 2001 (2001-12-01), BIRREN B. ET AL.: "Homo sapiens chromosome 8, clone RP11-177H2, complete sequence", Database accession no. (AC011008) *
DATABASE GENBANK [online] 31 March 2002 (2002-03-31), BIRREN B. ET AL.: "Homo sapiens chromosome 8, clone CTD-3103L12, complete sequence", Database accession no. (AC105107) *

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