WO2012151718A1 - Pig myostatin gene promoter and its applications - Google Patents
Pig myostatin gene promoter and its applications Download PDFInfo
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- WO2012151718A1 WO2012151718A1 PCT/CN2011/000831 CN2011000831W WO2012151718A1 WO 2012151718 A1 WO2012151718 A1 WO 2012151718A1 CN 2011000831 W CN2011000831 W CN 2011000831W WO 2012151718 A1 WO2012151718 A1 WO 2012151718A1
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Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/108—Swine
Definitions
- the invention relates to the field of biotechnology, in particular to a porcine myostatin gene promoter and application thereof. Background technique
- transgenic technology In the past decade, the rapid development and wide application of transgenic technology, especially the combination of gene targeting and somatic cell cloning technology, has made the genetic modification of transgenic animals a reality.
- the key to this technology is to allow site-specific integration and stable expression of foreign genes on the genome of transgenic animals, and the expression of foreign genes is closely related to the transcriptional activity of their promoters. Therefore, obtaining a promoter with stable expression activity will greatly promote transgenic animal research.
- the types and quantities of promoters currently used in transgenic animals are very limited, mainly from viral promoters, such as cytomegalovirus (CMV), simian virus 40 (SV40), simple blisters.
- a constitutive promoter such as herpes simplex virus HSV.
- Another advantage of using the endogenous promoter of transgenic animals is that the "in situ" site-specific integration of the foreign gene can be achieved by gene targeting, driven by the endogenous promoter "in situ” to express the foreign gene. Control at a certain physiological level. Therefore, research in this area as soon as possible has important application value for the research and production of GM animals.
- the myostatin gene was first cloned in mice by McPherron et al. in 1997. This gene belongs to the TGF- ⁇ family and is a transforming growth factor. Gene knockout proves that the inactivation of this gene causes muscle tissue hyperplasia and weight gain in mice; and the mice can survive normally and have fertility. Subsequently, it was found in cattle, sheep and other animals that the main function of the myostatin gene is to negatively regulate muscle growth and development, and the inactivation of myostatin does not cause abnormalities in the physiological functions of the above animals. Therefore, myostatin is a candidate modified gene for transgenic animals that has proven to be a phenotypic effect and relatively safe.
- the DNA molecule provided by the present invention which is derived from a pig, is a DNA molecule according to any one of the following 1) -4):
- the DNA molecule shown in 4) is the DNA molecule shown in SEQ ID NO: 1 in the Sequence Listing.
- the stringent conditions were hybridization in a 6XSSC, 0.5% SDS solution at 65 °C followed by one wash with 2 XSSC, 0.1% SDS and 1X SSC, 0.1% SDS.
- a recombinant vector, expression cassette, transgenic cell line or recombinant strain containing the DNA molecule is also within the scope of protection of the present invention.
- the recombinant vector is as follows 1) or 2):
- Primer pairs that amplify the DNA molecule are also within the scope of the invention.
- the primer pair is as follows 1) or 2) :
- the animal cell is a mouse cell or a human cell.
- the mouse cell is a mouse myoblast
- the human cell is a human cervical cancer cell.
- the mouse myoblast is a C2C12 cell
- the human cervical cancer cells are Hela cells.
- Figure 1 is a schematic diagram of the tissue structure and promoter candidate regions of porcine myostatin gene.
- Figure 2 shows the amplification of the porcine myostatin gene promoter.
- Figure 3 is a porcine myostatin gene promoter reporter vector digestion verification
- Figure 4 shows the cloning, mutation and activity assay of porcine myostatin gene promoter.
- Figure 5 is a functional verification of the expression of green fluorescent protein driven by the porcine myostatin gene promoter.
- Figure 6 shows the activity assay of porcine myostatin gene promoter reporter vector in human cultured cells. The best way to implement the invention
- pGL3-bas ic both purchased from pGL3-bas ic, pGL3-p:romote:r, Promega (USA) pGL3- promoter expression firefly incinerated pRL-TK reporter vector photozyme, and pRL-TK reporter vector expresses Renilla fluorescence Prime enzyme reporter gene high fidelity polymerase K0D Toyobo (Japan) amplification of porcine myostatin gene promoter plus sub
- ThermoCycler PCR Thermo- Fisher nucleic acid PCR amplification Thermostat, shaker Shanghai Zhicheng Bacterial culture
- Each sample was added with 300 ⁇ l DNA extraction buffer (Tris-Hcl 10 mM, EDTA 10 mM, SDS 2%, NaCl 300 mM, pH 8.0), 8 ⁇ l proteinase K (10 mg/ml), and digested at 55 ° C for 6 hours.
- DNA extraction buffer Tris-Hcl 10 mM, EDTA 10 mM, SDS 2%, NaCl 300 mM, pH 8.0
- 8 ⁇ l proteinase K 10 mg/ml
- the mixture was extracted twice with phenol, centrifuged at 10, OOO rpm for 10 minutes, and the supernatant was removed to remove the protein and the lower phenol.
- the extract was again extracted with chloroform/isoamyl alcohol phenol, and the supernatant was taken by centrifugation at 10, OOO rpm for 10 minutes.
- the precipitate was washed once with 75% cold ethanol, and centrifuged for 5 minutes to remove the ethanol to take a precipitate.
- the precipitate is fully dried at 4 ° C, added with three distilled water ⁇ ⁇ ⁇ ⁇ , electrophoresis check quantitative, -20 ° C frozen storage available
- the pig genomic DNA was obtained by PCR amplification.
- M is the molecular weight standard of lkb DNA, and the length is 0.5 kb, lkb, 1. 5 kb, 2 kb, 3 kb, 4 kb, 5kb, 6kb, 8kb, lOkb, PCR amplification to obtain 1. lkb fragment.
- the PCR product was sent to the Huada gene for sequencing, and the sequencing primer was Glprimer2.
- the PCR product had the nucleotide shown in SEQ ID NO: 1 in the sequence listing, and the gene of the PCR product was named MSTN.
- 1031-1038 is the 5' end transcriptional element TATA box (5' end TATA box), from the 5' end 1056-1063 to the 3' end transcriptional element TATA box (3 ' end TATA box) ), from the 5' end to the 991-996 position for the 3'-end transcriptional element CAAT box.
- Sequence 1 can also be synthesized manually.
- FIG. 1 is a schematic diagram showing the tissue structure and promoter candidate region of porcine myostatin gene. It can be seen that the porcine myostatin gene contains three exons and two introns, and the full length is 3789 bp. The exon lengths were 373 bp, 374 bp and 381 bp, respectively; the intron lengths were 1809 bp and 1980 bp, respectively.
- the sequence immediately upstream of exon 1 contains a TATA cassette (2) and a CAAT box (1), and a MEF (myocyte enhancer factor) binding sequence. 3) Cloning and identification of porcine myostatin gene promoter fragment
- reaction conditions were: 16 ° C, and allowed to stand overnight.
- the ligation product was transformed into Escherichia coli DH5a according to standard methods, and cultured overnight at 37 ° C to obtain a transformant.
- the transformants obtained above were identified by colony PCR using the MSTN-F and MSTN-R primers, and the recombinant which can obtain the lkb amplification product was a positive plasmid;
- the positive plasmid was digested with Mlu I and Bgl II, and pGL3-basi C was used as a control.
- the results are shown in Fig. 3.
- lanes 1, 3 are pGL3-basic
- 2, 4 is a p-port GL3_basic plasmid digested with Mlul and BglU; it can be seen that the pGL3-basi C plasmid has no insert, so the plasmid is electrophoretically displayed after double digestion. It is a single strip with a length of 4. 8 kb, which is consistent with the theoretical analysis.
- Lanes 5, 7, 9 are recombinants, 6, 8, and 10, respectively, 5, 7, and 9 recombinants were digested with Mlul and BglU; it can be seen that lanes 6, 8, 10 were digested and electrophoresed. The results showed that 1. lkb (shown by the black arrow on the right, the brightness was weak) and the 4.8 kb two fragments were positive plasmids, which was consistent with the theoretical analysis, indicating that lanes 5, 7, and 9 were positive plasmids.
- the plasmid which was positive by PCR and restriction enzyme digestion was sent for sequencing. The result was that the plasmid was inserted into the plasmid between the Mlu I and Bgl II cleavage sites of pGL3-baisc, and the plasmid was named. For pGL3-MSTN.
- mutant vector adopts a Dpnl-mediated point mutation method
- the reaction system of the mutant PCR is as follows:
- reaction conditions are:
- Sequence 2 or Sequence 3 can also be synthesized manually.
- the reaction was carried out in a water bath at 37 ° C for 2 hours.
- the ⁇ digested product was separately transformed into E. coli competent cells DH5 a according to standard procedures.
- the linear digested product has a homology arm at both ends, which can be cyclized in E. coli. This is the principle of the classical mutation method. Developed by Stratagene, USA, three transformants were obtained: positive plasmid 1, positive plasmid 2, transformant 3.
- the plasmid of transformant 1 was extracted and sent for sequencing. As a result, the plasmid contained the sequence 2 in the sequence listing, and the plasmid was inserted between the Mlu I and Bgl II cleavage sites of pGL3-basic.
- the plasmid was named PGL3-TATA1;
- the plasmid of transformant 2 was extracted and sent for sequencing.
- the plasmid contained the sequence 3 in the sequence listing, which was obtained by inserting sequence 3 in the sequence listing between the Mlu I and Bgl II cleavage sites of pGL3-basic.
- the plasmid of transformant 3 was extracted and sent for sequencing.
- the plasmid contained the sequence 4 in the sequence listing, which was obtained by inserting the sequence 4 in the sequence listing between the Mlu I and Bgl II cleavage sites of pGL3-basic.
- the plasmid was named pGL3-3-boxes.
- transfection complexes were prepared according to the system listed in the table, and dropped into 24-well plates, supplemented with 400 ⁇ l complete medium, placed. Incubate at 37 ° C under 5% CO 2 . Cells were harvested 48 h after transfection. Luciferase activity assays were performed according to Promega's DLR Assay kit instructions.
- the transfection system is as follows:
- FIG. 4 A schematic diagram of the partial structure of each reporter vector and the relative ratio of enzyme activity are shown in Figure 4.
- the TATA box and the CAAT box are key components of the transcription of the porcine myostatin gene, and the deletion of the three components can lead to the report. Loss of gene activity (not significantly different from the enzyme activity of the negative control pGL3-basi C ).
- Two TATA boxes were deleted, respectively, and they were all able to efficiently drive transcription. It was proved that these two TATA boxes are effective elements for the transcription initiation of porcine myostatin gene (mutant and wild type enzyme activities are almost identical), but the two are not superimposed. effect.
- *, p ⁇ 0.05 the difference is significant, indicating that the DNA molecule shown in SEQ ID NO: 2 and the DNA molecule shown in SEQ ID NO: 3 are also promoters.
- ThermoCycler PCR Thermo-Fi she nucleic acid PCR amplification instrument r
- the pGL3-MSTN plasmid DNA was used as a template, and a specific primer with a predetermined restriction site (promoter upstream primer and promoter downstream primer) was used to amplify the porcine myostatin gene promoter sequence.
- the reaction system was:
- reaction conditions are:
- the lj PCR product was subjected to 1% agarose gel electrophoresis to obtain a 1122 bp PCR product.
- reaction conditions were: 16 ° C, and allowed to stand overnight.
- the ligation product was transformed into E. coli DH5a according to standard methods and cultured overnight at 37 ° C to obtain a transformant.
- the plasmid of the transformant was extracted, and the primer upstream of the promoter and the primer downstream of the promoter were subjected to PCR amplification to obtain a 1122 bp fragment as a positive plasmid.
- the positive plasmid was digested with Nhel to obtain 1122 bp.
- the fragment was further proved to be a positive plasmid, and the plasmid which was positive by PCR and restriction enzyme digestion was sent for sequencing, and the result was that the plasmid 1 was inserted between the sel and Nhel cleavage sites of pEGFP-Cl.
- the plasmid was named pMSTN-EGFP.
- the constructed pMSTN-EGFP reporter vector was transfected (transfection method is described in detail in the kit). Mouse myoblast C2C12 was observed 24 hours later, and pEGFP-Cl was used as a control.
- the transfection system is as follows:
- transfected complexes were prepared separately in the system listed, and were added to a 24-well plate, supplemented with 400 ⁇ l of complete medium, and cultured at 37 ° C and 5% CO 2 . Cells were harvested 48 h after transfection. Luciferase activity assays were performed according to Promega's DLR M Assay kit instructions.
- the transfection system is as follows:
- porcine myostatin gene promoter reporter vector PGL3-MSTN was transfected into human cervical cancer Hela cells, and the transcriptional activity of the porcine myostatin gene promoter in human cells was examined by a reporter gene. The results showed that the cloned promoter fragment was highly active in human cells, demonstrating that the identified porcine myostatin gene promoter is efficiently expressed in mammalian cells.
- the experiments of the present invention prove that the promoter provided by the present invention can drive the expression of the reporter gene firefly luciferase, and can also be used to construct the promoter into the reporter vector and then transfect the pig source and the human cultured cell.
- reporter gene testing the activity and efficiency of the promoter are identified by accurate quantitative methods, and the problems of introducing foreign genes after the promoter, solving the unsteadiness of the expression of foreign genes in transgenic pigs, and the unpredictability of positional effects are provided. A reliable and valuable genetic resource.
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Abstract
A pig myostatin gene promoter and its applications are disclosed in the present invention. The DNA fragment provided in the present invention which is derived from pigs is anyone of the following DNA molecules 1)-4): 1) the DNA molecule of SEQ ID NO:2 shown in the Sequence Listing; 2) the DNA molecule of SEQ ID NO:3 shown in the Sequence Listing; 3) the DNA molecule which hybridizes to the DNA sequence in 1) or 2) under strict conditions and has promoter function; 4) the DNA molecule which has more than 90% homology with the DNA sequence in 1) or 2) and has promoter function. The experiments in the present invention prove that the promoter provided in the present invention can drive the expression of firefly luciferase reporter gene. The promoter can also be built into the reporter vector and then the vector is transfected into cultured swine and human cells, and the activity and efficiency of the promoter are identified by accurate quantitative methods through reporter gene test.
Description
猪肌抑素基因启动子及其应用 技术领域 Porcine myostatin gene promoter and its application
本发明涉及生物技术领域, 尤其涉及一种猪肌抑素基因启动子及其应用。 背景技术 The invention relates to the field of biotechnology, in particular to a porcine myostatin gene promoter and application thereof. Background technique
近十年来, 转基因技术得到迅猛发展和广泛应用, 尤其是基因打靶与体细 胞克隆技术的结合, 使得转基因动物的基因定点修饰成为现实。 该技术的关键 是使外源基因在转基因动物基因组上定点整合并且稳定表达, 而外源基因的表 达与其启动子的转录活性密切相关。 因此, 获得具有稳定表达活性的启动子将 极大促进转基因动物研究。 但目前应用于转基因动物的启动子种类和数量则非 常有限, 主要是来源于病毒的启动子, 如巨细胞病毒(cytomegalovirus , CMV)、 猿猴空泡病毒 40 (simian virus 40, SV40)、单纯疱疫病毒 (herpes simplex virus HSV)等组成型启动子。 它们可以在几乎所有种类真核生物细胞内调控外源基因 表达, 而且没有时空特异性。 用上述启动子调控外源基因表达时, 由于外源基 因在不需要的细胞或不需要的发育阶段大量表达和积累, 往往造成动物形态和 生理功能异常, 以致早衰或死亡。 另外, 由于它们属于异源基因, 容易在转基 因动物体内遭受免疫排斥和表观遗传修饰, 导致外源基因表达的不稳定性。 鉴 于此, 寻找来源于转基因动物自身的内源性启动子就成为解决该问题的突破口。 而且, 利用转基因动物内源性启动子的另外一个优势是通过基因打靶, 可以实 现外源基因的 "原位 " 定点整合, 由内源性启动子 "原位"驱动, 将外源基因 的表达控制在一定的生理水平。 因此, 尽快开展此方面的研究, 对于转基因动 物的研究和生产具有重要的应用价值。 In the past decade, the rapid development and wide application of transgenic technology, especially the combination of gene targeting and somatic cell cloning technology, has made the genetic modification of transgenic animals a reality. The key to this technology is to allow site-specific integration and stable expression of foreign genes on the genome of transgenic animals, and the expression of foreign genes is closely related to the transcriptional activity of their promoters. Therefore, obtaining a promoter with stable expression activity will greatly promote transgenic animal research. However, the types and quantities of promoters currently used in transgenic animals are very limited, mainly from viral promoters, such as cytomegalovirus (CMV), simian virus 40 (SV40), simple blisters. A constitutive promoter such as herpes simplex virus HSV. They regulate foreign gene expression in almost all types of eukaryotic cells and are not spatio-temporal specific. When the above-mentioned promoter is used to regulate the expression of a foreign gene, since the foreign gene is abundantly expressed and accumulated in an undesired cell or an undesired developmental stage, the morphological and physiological functions of the animal are often abnormal, resulting in premature aging or death. In addition, because they belong to heterologous genes, they are susceptible to immunological rejection and epigenetic modification in transgenic animals, leading to instability of foreign gene expression. In view of this, finding an endogenous promoter derived from the transgenic animal itself has become a breakthrough in solving this problem. Moreover, another advantage of using the endogenous promoter of transgenic animals is that the "in situ" site-specific integration of the foreign gene can be achieved by gene targeting, driven by the endogenous promoter "in situ" to express the foreign gene. Control at a certain physiological level. Therefore, research in this area as soon as possible has important application value for the research and production of GM animals.
肌抑素基因最早于 1997年由 McPherron等人在小鼠中克隆。 该基因属于 TGF- β家族, 是一种转化生长因子。 基因敲除证明此基因的失活引起小鼠肌肉组织 增生, 体重增大; 而且小鼠可以正常存活, 也具有生育能力。 随后在牛、 羊等 动物中发现, 肌抑素基因的主要功能是负调控肌肉生长发育, 而且肌抑素的失 活并未导致上述动物的生理功能出现异常。 因此, 肌抑素是迄今为止证明表型 效应明显、 相对安全的转基因动物候选修饰基因。 鉴于肌抑素基因序列和蛋白 质序列在物种进化上的高度保守, 人们有理由推测猪肌抑素基因也具有相似的 功能, 因而它正成为目前转基因猪研究的热点基因, 潜力巨大。 但现阶段研究 多着眼于肌抑素基因的敲除以获得产肉率更高的转基因猪, 关于猪肌抑素基因 启动子的克隆、 鉴定和活性分析的研究还是空白。 The myostatin gene was first cloned in mice by McPherron et al. in 1997. This gene belongs to the TGF-β family and is a transforming growth factor. Gene knockout proves that the inactivation of this gene causes muscle tissue hyperplasia and weight gain in mice; and the mice can survive normally and have fertility. Subsequently, it was found in cattle, sheep and other animals that the main function of the myostatin gene is to negatively regulate muscle growth and development, and the inactivation of myostatin does not cause abnormalities in the physiological functions of the above animals. Therefore, myostatin is a candidate modified gene for transgenic animals that has proven to be a phenotypic effect and relatively safe. In view of the fact that the sequence of myostatin gene and protein sequence are highly conserved in species evolution, it is reasonable to speculate that the porcine myostatin gene also has similar functions, and thus it is becoming a hot gene for transgenic pig research, with great potential. However, the current research focuses on the knockout of the myostatin gene to obtain transgenic pigs with higher meat production rate. The research on the cloning, identification and activity analysis of the pig myostatin gene promoter is still blank.
发明公开 Invention disclosure
本发明的目的是提供一种猪肌抑素基因启动子及其应用。 It is an object of the present invention to provide a porcine myostatin gene promoter and use thereof.
本发明提供的 DNA分子, 来源于猪, 为如下 1 ) -4 )中任一所述的 DNA分子: The DNA molecule provided by the present invention, which is derived from a pig, is a DNA molecule according to any one of the following 1) -4):
1 ) 序列表的序列 2所示的 DNA分子; 1) a DNA molecule as shown in Sequence 2 of the Sequence Listing;
2 ) 序列表的序列 3所示的 DNA分子;
3) 在严格条件下与 1) 或 2) 所述 DNA序列杂交且具有启动子功能的 DNA 分子; 2) a DNA molecule as shown in SEQ ID NO: 3 of the Sequence Listing; 3) a DNA molecule which hybridizes under stringent conditions to the DNA sequence of 1) or 2) and has a promoter function;
4) 与 1) 或 2) 中所述 DNA序列具有 90%以上同源性, 且具有启动子功能的 DNA分子。 4) A DNA molecule having 90% or more homology with the DNA sequence described in 1) or 2) and having a promoter function.
所述 4) 所示的 DNA分子为序列表中序列 1所示的 DNA分子。 The DNA molecule shown in 4) is the DNA molecule shown in SEQ ID NO: 1 in the Sequence Listing.
所述严格条件为在 6XSSC, 0.5% SDS的溶液中, 在 65°C下杂交,然后用 2 XSSC, 0.1% SDS和 1XSSC, 0.1% SDS各洗膜一次。 The stringent conditions were hybridization in a 6XSSC, 0.5% SDS solution at 65 °C followed by one wash with 2 XSSC, 0.1% SDS and 1X SSC, 0.1% SDS.
含有所述 DNA 分子的重组载体、 表达盒、 转基因细胞系或重组菌也是本发 明保护的范围。 A recombinant vector, expression cassette, transgenic cell line or recombinant strain containing the DNA molecule is also within the scope of protection of the present invention.
所述重组载体为如下 1) 或 2) : The recombinant vector is as follows 1) or 2):
1) 为将所述 DNA分子插入 pGL3-basic的 Μ1 禾 P Bgl II位点间得到的重 组载体; 1) a recombinant vector obtained by inserting the DNA molecule between the Μ1 and P Bgl II sites of pGL3-basic;
2) 为将所述 DNA分子插入 pEGFP-Cl 的 el和 Nhel位点间得到的重组载 体。 2) A recombinant vector obtained by inserting the DNA molecule between the el and Nhel sites of pEGFP-Cl.
扩增所述 DNA分子的引物对也是本发明保护的范围。 Primer pairs that amplify the DNA molecule are also within the scope of the invention.
所述引物对为如下 1) 或 2) : The primer pair is as follows 1) or 2) :
1) 所示的引物对中的一条引物的序列为序列 5, 另一条引物的序列为序列 1) The sequence of one of the primer pairs shown is sequence 5, and the sequence of the other primer is sequence
6; 6;
2) 所示的引物对中的一条引物的序列为序列 7, 另一条引物的序列为序列 8。 2) The sequence of one of the primer pairs shown is sequence 7, and the sequence of the other primer is sequence 8.
所述 DNA分子在使目的基因在离体动物细胞中的表达中的应用也是本发明 保护的范围。 The use of the DNA molecule in the expression of the gene of interest in ex vivo animal cells is also within the scope of the invention.
所述动物细胞为鼠源细胞或人源细胞。 The animal cell is a mouse cell or a human cell.
所述鼠源细胞为小鼠成肌细胞; The mouse cell is a mouse myoblast;
所述人源细胞为人宫颈癌细胞。 The human cell is a human cervical cancer cell.
所述小鼠成肌细胞为 C2C12细胞; The mouse myoblast is a C2C12 cell;
所述人宫颈癌细胞为 Hela细胞。 The human cervical cancer cells are Hela cells.
除非特别指出或是单独定义, 本文所使用的科学和技术术语具有本发明所 属领域技术人员共知的、 无歧义的相同含义。 另外, 本文所述的材料、 方法以 及实施案例本意在于说明和阐述而非限制或限定。 Unless otherwise indicated or individually defined, the scientific and technical terms used herein have the same meaning of the meaning of the meaning of the invention. In addition, the materials, methods, and examples described herein are intended to be illustrative and illustrative, and not limiting or limiting.
附图说明 DRAWINGS
图 1为猪肌抑素基因组织结构和启动子候选区域示意图 Figure 1 is a schematic diagram of the tissue structure and promoter candidate regions of porcine myostatin gene.
图 2为猪肌抑素基因启动子扩增 Figure 2 shows the amplification of the porcine myostatin gene promoter.
图 3为猪肌抑素基因启动子报告载体酶切验证 Figure 3 is a porcine myostatin gene promoter reporter vector digestion verification
图 4为猪肌抑素基因启动子克隆、 突变与活性检测 Figure 4 shows the cloning, mutation and activity assay of porcine myostatin gene promoter.
图 5为猪肌抑素基因启动子驱动绿色荧光蛋白表达的功能验证 Figure 5 is a functional verification of the expression of green fluorescent protein driven by the porcine myostatin gene promoter.
图 6为猪肌抑素基因启动子报告载体在人源培养细胞中的活性检测
实施发明的最佳方式 Figure 6 shows the activity assay of porcine myostatin gene promoter reporter vector in human cultured cells. The best way to implement the invention
下述实施例中所使用的实验方法如无特殊说明, 均为常规方法。 The experimental methods used in the following examples are all conventional methods unless otherwise specified.
下述实施例中所用的材料、 试剂等, 如无特殊说明, 均可从商业途径得到。 下面结合具体实施例, 进一步阐述本发明。 应当理解, 这些实施例仅用于 说明本发明而不用于限制本发明要求保护的范围, 下列实施例中未注明具体实 验条件和方法, 通常按照常规条件如 J. 萨姆布鲁克, D. W. 拉塞尔等著, 黄培 堂等译, 科学出版社, 2002, 分子克隆实验指南第三版所建议的条件。 The materials, reagents and the like used in the following examples are commercially available unless otherwise specified. The invention is further illustrated below in conjunction with specific embodiments. It is to be understood that the examples are merely illustrative of the invention and are not intended to limit the scope of the invention, which is not intended to limit the scope of the invention, and the specific conditions and methods, such as J. Sambrook, DW. Er et al., Huang Peitang et al., Science Press, 2002, The conditions recommended in the third edition of the Molecular Cloning Experiment Guide.
实施例 1、 猪肌抑素基因启动子的克隆与验证 Example 1. Cloning and verification of porcine myostatin gene promoter
1、 实验材料 1. Experimental materials
表 1实验材料 Table 1 Experimental materials
材料 来源 说明 湖北白猪耳组织 湖北省农科院畜 液氮速冻, -8CTC冻存 Material Source Description Hubei white pig ear tissue Hubei Provincial Academy of Agricultural Sciences animal liquid nitrogen freezing, -8CTC cryopreservation
牧兽医研究所实验猪场 Pastoral Veterinary Research Institute Experimental Pig Farm
初生仔猪 Newborn piglet
pGL3-bas ic 、 均 购 自 pGL3-bas ic 、 pGL3 - p:romote:r、 Promega (USA) pGL3- promoter表达萤火虫焚 pRL-TK报告载体 光素酶, 而 pRL-TK报告载体 表达海肾荧光素酶报告基因 高保真聚合酶 K0D Toyobo (Japan) 扩增猪肌抑素基因启动 plus 子 pGL3-bas ic, both purchased from pGL3-bas ic, pGL3-p:romote:r, Promega (USA) pGL3- promoter expression firefly incinerated pRL-TK reporter vector photozyme, and pRL-TK reporter vector expresses Renilla fluorescence Prime enzyme reporter gene high fidelity polymerase K0D Toyobo (Japan) amplification of porcine myostatin gene promoter plus sub
Tag DNA 聚合酶、 上海申能博彩 鉴定阳性转化子 dNTP Tag DNA polymerase, Shanghai Shenneng Gaming Identification of positive transformants dNTP
MSTN-F 上游引物 上海英骏合成 TTCAACGCGTGCTCTATTCT MSTN-F upstream primer Shanghai Yingjun synthesis TTCAACGCGTGCTCTATTCT
(5, →3 ' ) CTGCTCCCAGACC (Mlul) (序列 (5, →3 ' ) CTGCTCCCAGACC (Mlul) (sequence
5 ) 5)
MSTN-R 下游引物 上海英骏合成 CATCAGATCTCGCCAAGCAA MSTN-R downstream primer Shanghai Yingjun synthesis CATCAGATCTCGCCAAGCAA
(5, →3 ' ) AATTTTAATGCC (Bgl II) (序 列 6 ) (5, →3 ' ) AATTTTAATGCC (Bgl II) (Sequence 6)
GLprimer2 (5 J → 上海英骏合成 CTTTATGTTTTTGGCGTCTTGLprimer2 (5 J → Shanghai Yingjun Synthetic CTTTATGTTTTTGGCGTCTT
3 ' ) CC 3 ' ) CC
Bgl II、 Mlul限制 Takara (Japan) 酶切 DNA片段 Bgl II, Mlul restriction Takara (Japan) digestion DNA fragment
性内切酶 Endonuclease
DNA割胶回收试剂盒 北京天根 割胶回收 PCR产物 超纯质粒提取试剂 北京天根 制备无内毒素质粒用于 合 细胞转染 DNA Cutting Recycling Kit Beijing Tiangen Cutting Rubber Recovery PCR Product Ultrapure Plasmid Extraction Reagent Beijing Tiangen Preparation of endotoxin-free plasmid for cell transfection
T4 DNA连接酶 Fermentas 载体与插入片段连接
SeaKem琼脂糖 美国 FMC 电泳检测与分析 溴化乙啶(ethidium S igma 核酸染色 T4 DNA ligase Fermentas vector linked to the insert SeaKem agarose US FMC electrophoresis detection and analysis of ethidium bromide (ethidium S igma nucleic acid staining)
bromide) Bromide)
Tris饱和酚、 氯仿、 国药集团(分析 核酸提取 Tris saturated phenol, chloroform, Sinopharm Group (analytical nucleic acid extraction
异戊醇、 无水乙醇、 醋酸 纯) Isoamyl alcohol, absolute ethanol, acetic acid pure)
钠 Sodium
lkb DNA分子量标准 广州东盛 核酸片段大小识别 蛋白酶 K S igma 核酸提取过程中去除蛋 白质 Lkb DNA molecular weight standard Guangzhou Dongsheng nucleic acid fragment size recognition protease K S igma nucleic acid extraction process protein removal
核糖核酸酶 Rnase Amresco 去除 RNA Ribonuclease Rnase Amresco RNA removal
氨节抗生素 Amp Amresco 转化与筛选 Ammonia antibiotic Amp Amresco transformation and screening
2、 仪器设备 2, equipment
表 2仪器设备 Table 2 Instrumentation
名称 来源 说明 Name Source Description
超净工作台 北京半导体设备 细菌转化等无菌操作使 厂 用 Ultra-clean workbench Beijing semiconductor equipment, aseptic operation such as bacterial transformation
高速离心机 贝克曼-库尔特 DNA、 质粒提取 High speed centrifuge Beckman-Coulter DNA, plasmid extraction
(USA) (USA)
水平电泳仪 北京六一 电泳检测 Horizontal Electrophoresis System Beijing Liuyi Electrophoresis Detection
凝胶成像系统 Uvitek, UK 图像捕获 Gel Imaging System Uvitek, UK Image Capture
ThermoCycler PCR仪 Thermo - Fisher 核酸的 PCR扩增 温箱、 摇床 上海智城 细菌培养 ThermoCycler PCR Thermo- Fisher nucleic acid PCR amplification Thermostat, shaker Shanghai Zhicheng Bacterial culture
3、 实验方法 3. Experimental methods
1 ) 猪基因组 DNA提取 1) Pig genome DNA extraction
取仔猪的离体猪耳肌肉组织样 0. 1克, 洗净, 剪碎。 Take the isolated pig ear muscle tissue sample 0. 1 gram, wash, cut.
每个样品加 300μ1 DNA抽提缓冲液(Tris-Hcl 10mM, EDTA 10mM, SDS 2%, NaCl 300 mM, pH8. 0 ) , 8μ1 蛋白酶 K ( lOmg/ml ) , 55 °C消化 6小时。 Each sample was added with 300 μl DNA extraction buffer (Tris-Hcl 10 mM, EDTA 10 mM, SDS 2%, NaCl 300 mM, pH 8.0), 8 μl proteinase K (10 mg/ml), and digested at 55 ° C for 6 hours.
用苯酚抽提两次, 10, OOOrpm 离心 10分钟取上清液, 去掉蛋白质和下层苯 酚。 The mixture was extracted twice with phenol, centrifuged at 10, OOO rpm for 10 minutes, and the supernatant was removed to remove the protein and the lower phenol.
再用氯仿 /异戊醇苯酚抽提一次, 10, OOOrpm 离心 10分钟取上清液。 The extract was again extracted with chloroform/isoamyl alcohol phenol, and the supernatant was taken by centrifugation at 10, OOO rpm for 10 minutes.
在上清液中加 30μ1 3Μ NaAC, 600μ1无水乙醇充分混匀, 10, OOOrpm 离心 10分钟取沉淀。 Add 30 μl of 3 Μ NaAC to the supernatant, mix well with 600 μl of absolute ethanol, and centrifuge at 10, OOO rpm for 10 minutes to remove the precipitate.
沉淀用 75%冷乙醇洗涤一次, 离心 5分种去乙醇取沉淀。 The precipitate was washed once with 75% cold ethanol, and centrifuged for 5 minutes to remove the ethanol to take a precipitate.
沉淀在 4°C充分晾干, 加三蒸水 Ιθθμΐ , 电泳检查定量, -20°C 冻存可用
于 PCR扩增, 得到猪基因组 DNA。 The precipitate is fully dried at 4 ° C, added with three distilled water Ι θ θ μΐ, electrophoresis check quantitative, -20 ° C frozen storage available The pig genomic DNA was obtained by PCR amplification.
2 ) 猪肌抑素基因启动子片段的 PCR扩增 2) PCR amplification of porcine myostatin gene promoter fragment
以上述得到的基因组 DNA为模板, 带有预设酶切位点的特异引物 (MSTN-F 和 MSTN-R) 扩增猪肌抑素基因启动子序列, 反应体系为: Using the genomic DNA obtained above as a template, specific primers (MSTN-F and MSTN-R) with predetermined restriction sites were used to amplify the promoter sequence of the porcine myostatin gene. The reaction system was:
表 3反应体系 成分 初始浓度 用量 终浓度 Table 3 Reaction system Ingredients Initial concentration Amount Final concentration
PCR buffer 10 X 5μ1 I X PCR buffer 10 X 5μ1 I X
dNTP 2mmol/L 5μ1 200μπιο1/1 硫 酸 镁 25 mmol/L 2μ1 lmmol/L dNTP 2mmol/L 5μ1 200μπιο1/1 magnesium sulphate 25 mmol/L 2μl lmmol/L
MgS04 MgS0 4
KOD plus lunit/μΐ ΐμΐ 0. 02 unit/μΐ KOD plus lunit/μΐ ΐμΐ 0. 02 unit/μΐ
MSTN-F 10μπιο1/1 4μ1 800nmo/LMSTN-F 10μπιο1/1 4μ1 800nmo/L
MSTN-R 10μπιο1/1 4μ1 800nmo/L 基因组 DNA 25ng/ l ΐμΐ ο. Sng/μΐ 双蒸水 28μ1 MSTN-R 10μπιο1/1 4μ1 800nmo/L Genomic DNA 25ng/ l ΐμΐ ο. Sng/μΐ Double distilled water 28μ1
总体积 50μ1 反应条件为: Total volume 50μ1 Reaction conditions are:
预变性 95 °C 2分钟; Pre-denaturation at 95 °C for 2 minutes;
变性 95 °C 30秒; Denaturation at 95 °C for 30 seconds;
退火 55 °C 30秒; 30 Annealing 55 °C 30 seconds; 30
延伸 68 °C 1. 5分钟; Extension 68 °C 1. 5 minutes;
反应结束后, 以 1%琼脂糖凝胶电泳检测 PCR产物, 结果如图 2所示, M为 lkb DNA 分子量标准, 长度大小依次为 0. 5kb, lkb, 1. 5kb, 2kb, 3kb, 4kb, 5kb, 6kb, 8kb, lOkb, PCR扩增得到 1. lkb片段。 After the reaction, the PCR product was detected by 1% agarose gel electrophoresis. The results are shown in Fig. 2. M is the molecular weight standard of lkb DNA, and the length is 0.5 kb, lkb, 1. 5 kb, 2 kb, 3 kb, 4 kb, 5kb, 6kb, 8kb, lOkb, PCR amplification to obtain 1. lkb fragment.
将该 PCR产物送至华大基因测序, 测序引物为 Glprimer2, 结果为该 PCR产 物具有序列表中序列 1所示的核苷酸, 将该 PCR产物的基因命名为 MSTN。 其中 从 5 ' 末端第 1031-1038位为 5 ' 端转录必需元件 TATA盒(5 ' 端 TATA盒),从 5 ' 末端第 1056-1063为 3 ' 端转录必需元件 TATA盒(3 ' 端 TATA盒), 从 5 ' 末端 第 991-996位为 3 ' 端转录必需元件 CAAT盒。 The PCR product was sent to the Huada gene for sequencing, and the sequencing primer was Glprimer2. As a result, the PCR product had the nucleotide shown in SEQ ID NO: 1 in the sequence listing, and the gene of the PCR product was named MSTN. From the 5' end, 1031-1038 is the 5' end transcriptional element TATA box (5' end TATA box), from the 5' end 1056-1063 to the 3' end transcriptional element TATA box (3 ' end TATA box) ), from the 5' end to the 991-996 position for the 3'-end transcriptional element CAAT box.
也可人工合成序列 1。 Sequence 1 can also be synthesized manually.
图 1为猪肌抑素基因组织结构和启动子候选区域示意图, 可以看出, 猪肌抑 素基因包含三个外显子和两个内含子, 全长 3789bp。 外显子长度分别为 373bp、 374bp和 381bp; 内含子长度分别为 1809bp和 1980bp。 紧靠外显子 1上游的序 列,含有真核生物转录必需元件 TATA盒(2个)与 CAAT盒(1个),以及 MEF (myocyte enhancer factor, 肌肉细胞增强因子)结合序列等。
3 ) 猪肌抑素基因启动子片段的克隆与鉴定 Figure 1 is a schematic diagram showing the tissue structure and promoter candidate region of porcine myostatin gene. It can be seen that the porcine myostatin gene contains three exons and two introns, and the full length is 3789 bp. The exon lengths were 373 bp, 374 bp and 381 bp, respectively; the intron lengths were 1809 bp and 1980 bp, respectively. The sequence immediately upstream of exon 1 contains a TATA cassette (2) and a CAAT box (1), and a MEF (myocyte enhancer factor) binding sequence. 3) Cloning and identification of porcine myostatin gene promoter fragment
将上述 2 ) 得到的 PCR产物经 Μ1Λ和 Bgl II酶切得到的片段与经过同样酶 切得到的报告载体 pGL3-basiC片段以下述体系做连接: The fragment obtained by digesting the PCR product obtained in the above 2) with Μ1Λ and Bgl II was ligated to the reporter vector pGL3-basi C fragment obtained by the same digestion:
表 4连接体系 成分 初始浓度 用量 终浓度 酶切后 PCR 5μ1 Table 4 Connection system Ingredients Initial concentration Dosage Final concentration After digestion, PCR 5μ1
产物 Product
pGL3~bais o ΐμΐ lOng/μΙ pGL3~bais o ΐμΐ lOng/μΙ
o o o o
c片段 口口 c fragment mouth
10 X缓冲 10 X ΐμΐ I X 10 X buffer 10 X ΐμΐ I X
T4 DNA lOunits/μΙ ΐμΐ 1 unit/μΐ l igase T4 DNA lOunits/μΙ ΐμΐ 1 unit/μΐ l igase
双蒸水 2μ1 Double distilled water 2μ1
总体积 Ιθμΐ Total volume Ιθμΐ
反应条件为: 16°C, 连接过夜。 The reaction conditions were: 16 ° C, and allowed to stand overnight.
按照标准方法将连接产物转化大肠杆菌 DH5 a, 37°C培养过夜, 得到转化子。 以 MSTN-F和 MSTN-R引物对上述获得的转化子进行菌落 PCR鉴定, 能够得到 1. lkb扩增产物的重组子即为阳性质粒; The ligation product was transformed into Escherichia coli DH5a according to standard methods, and cultured overnight at 37 ° C to obtain a transformant. The transformants obtained above were identified by colony PCR using the MSTN-F and MSTN-R primers, and the recombinant which can obtain the lkb amplification product was a positive plasmid;
将阳性质粒用 Mlu I和 Bgl I I酶切, 以 pGL3-basiC为对照, 结果如图 3所 示。其中,泳道 1, 3为 pGL3-basic, 2, 4为经 Mlul和 BglU双酶切的 p口GL3_basic 质粒; 可以看出, pGL3-basiC 质粒无插入片段, 故双酶切后该质粒电泳显示为 单一条带, 长度为 4. 8kb, 与理论分析相符。 泳道 5, 7, 9为重组子, 6, 8, 10分 别为 5、 7、 9的重组子经 Mlul和 BglU双酶切的产物; 可以看出, 泳道 6, 8, 10 双酶切后电泳结果显示为 1. lkb (右侧黑色箭头所示,亮度较弱)和 4. 8kb两条片 段的为阳性质粒, 与理论分析相符, 说明泳道 5, 7, 9为阳性质粒。 The positive plasmid was digested with Mlu I and Bgl II, and pGL3-basi C was used as a control. The results are shown in Fig. 3. Among them, lanes 1, 3 are pGL3-basic, 2, 4 is a p-port GL3_basic plasmid digested with Mlul and BglU; it can be seen that the pGL3-basi C plasmid has no insert, so the plasmid is electrophoretically displayed after double digestion. It is a single strip with a length of 4. 8 kb, which is consistent with the theoretical analysis. Lanes 5, 7, 9 are recombinants, 6, 8, and 10, respectively, 5, 7, and 9 recombinants were digested with Mlul and BglU; it can be seen that lanes 6, 8, 10 were digested and electrophoresed. The results showed that 1. lkb (shown by the black arrow on the right, the brightness was weak) and the 4.8 kb two fragments were positive plasmids, which was consistent with the theoretical analysis, indicating that lanes 5, 7, and 9 were positive plasmids.
经 PCR、 酶切均鉴定为阳性的质粒送去测序, 结果为该质粒为将序列表中的 序列 1插入 pGL3-baisc的 Mlu I和 Bgl I I酶切位点间得到的质粒, 将该质粒 命名为 pGL3-MSTN。 实施例 2、 猪肌抑素基因启动子的功能鉴定 The plasmid which was positive by PCR and restriction enzyme digestion was sent for sequencing. The result was that the plasmid was inserted into the plasmid between the Mlu I and Bgl II cleavage sites of pGL3-baisc, and the plasmid was named. For pGL3-MSTN. Example 2. Functional identification of the porcine myostatin gene promoter
一、 猪肌抑素基因启动子转录活性检测 I. Detection of transcriptional activity of porcine myostatin gene promoter
A、 猪肌抑素基因启动子报告系统突变载体的制备与验证 A. Preparation and verification of mutation vector for porcine myostatin gene promoter reporter system
1、 实验材料 1. Experimental materials
表 5实验材料 Table 5 experimental materials
主要材料 来源 说明 Main material Source Description
高保真聚合酶 K0D Toyobo 扩增环状质粒
plus (Japan) High fidelity polymerase K0D Toyobo amplification circular plasmid Plus (Japan)
5 ' TATA盒删除突变 上海英 CGACACTTGTCTCATCAAGTGGAAAGC 上游引物 骏合成 CACTTGGAATACAGTATAAAAG 5 'TATA box deletion mutation Shanghai Ying CGACACTTGTCTCATCAAGTGGAAAGC upstream primer Jun synthesis CACTTGGAATACAGTATAAAAG
5 ' TATA盒删除突变 上海英 CTTTTATACTGTATTCCAAGTGGCTTTC 下游引物 骏合成 CTTGATGAGACAAGTGTCG 5 'TATA box deletion mutation Shanghai Ying CTTTTATACTGTATTCCAAGTGGCTTTC Downstream primer Jun synthesis CTTGATGAGACAAGTGTCG
3 ' TATA盒删除突变 上海英 GGAATATAAAAAGCCACTTGGAATACA 上游引物 骏合成 GGATTCACTGGTGTGGCAAGTTGTCTCTC 3 'TATA box deletion mutation Shanghai Ying GGAATATAAAAAGCCACTTGGAATACA upstream primer Jun synthesis GGATTCACTGGTGTGGCAAGTTGTCTCTC
3 ' TATA盒删除突变 上海英 GAGAGACAACTTGCCACACCAGTGAAT 下游引物 骏合成 CCTGTATTCCAAGTGGCTTTTTATATTCC 三元件删除突变引 上海英 CATCAGATCTCCACAATGAATCTCGCT 物 骏合成 GTC (BgllT) 3 'TATA box deletion mutation Shanghai Ying GAGAGACAACTTGCCACACCAGTGAAT Downstream primer Jun synthesis CCTGTATTCCAAGTGGCTTTTTATATTCC Three-element deletion mutation introduction Shanghai Ying CATCAGATCTCCACAATGAATCTCGCT Material Jun synthesis GTC (BgllT)
Glprimer2 上海英 CTTTATGTTTTTGGCGTCTTCC Glprimer2 Shanghai Ying CTTTATGTTTTTGGCGTCTTCC
骏合成 Jun synthesis
Dpnl限制性内切酶 ΡΘΓΠΙΘΠ 去除甲基化质粒 DNA Dpnl restriction enzyme ΡΘΓΠΙΘΠ removal of methylated plasmid DNA
tas Tas
SeaKem琼脂糖 美 国 电泳检测与分析 SeaKem Agarose US Electrophoresis Detection and Analysis
FMC FMC
溴化乙啶(ethidium Sigma 核酸染色 Ethidium bromide (ethidium Sigma nucleic acid staining)
bromide) Bromide)
lkb DNA分子量标准 广州东 核酸片段大小识别 Lkb DNA molecular weight standard Guangzhou East nucleic acid fragment size recognition
盛 Sheng
氨苄抗生素 Amp Amresc 转化与筛选 Ampicillin Amp Amresc Transformation and Screening
2、 仪器设备 2, equipment
表 6仪器设备 名称 来源 说明 超净工作台 北京半导体设 细菌转化等无菌操作 备厂 使用 Table 6 Instrument and equipment Name Source Description Ultra-clean workbench Beijing Semiconductor Design Aseptic operation such as bacterial transformation Preparation plant Use
高速离心机 贝克曼 -库尔 DNA、 质粒提取 High speed centrifuge Beckman-Chur DNA, plasmid extraction
特(USA) Special (USA)
水平电泳仪 北京六一 电泳检测 Horizontal Electrophoresis System Beijing Liuyi Electrophoresis Detection
凝胶成像系统 Uvitek, UK 图像捕获
ThermoCycler PCR Thermo - Fishe 核酸的 PCR扩增 Gel Imaging System Uvitek, UK Image Capture ThermoCycler PCR Thermo - PCR amplification of Fishe nucleic acid
r r
变预退延 Pre-deferred
温箱、 摇床 上海智城 细菌培养 Thermostat, shaker, Shanghai Zhicheng, bacterial culture
变火伸性 Fire resistance
3、 试验方法 3. Test method
1) 突变载体构建 本发明采用 Dpnl介导的点突变法, 1) Construction of mutant vector The present invention adopts a Dpnl-mediated point mutation method,
突变 PCR的反应体系如下: The reaction system of the mutant PCR is as follows:
表 7反应体系 Table 7 reaction system
PCR buffer 10X 5μ1 IX PCR buffer 10X 5μ1 IX
dNTP 2mmol/L 5μ1 200μπιο1/1 硫 酸 镁 25 mmol/L 2μ1 lmmol/L dNTP 2mmol/L 5μ1 200μπιο1/1 magnesium sulphate 25 mmol/L 2μl lmmol/L
MgS04 MgS0 4
KOD plus lunit/μΐ ΐμΐ 0.02 unit/μΐ 上游引物 10μπιο1/1 4μ1 800nmo/L 下游引物 10μπιο1/1 4μ1 800nmo/L PGL3-MSTN song/μΐ ΐμΐ lng/μΐ 双蒸水 28μ1 KOD plus lunit/μΐ ΐμΐ 0.02 unit/μΐ upstream primer 10μπιο1/1 4μ1 800nmo/L downstream primer 10μπιο1/1 4μ1 800nmo/L PGL3-MSTN song/μΐ ΐμΐ lng/μΐ double distilled water 28μ1
总体积 50μ1 Total volume 50μ1
反应条件为: The reaction conditions are:
性 95 °C 2分钟; 95 ° C for 2 minutes;
95 °C 30秒; 3Q 95 °C 30 seconds; 3Q
55 °C 30秒; 55 ° C for 30 seconds;
68 °C 1.5分钟; 68 ° C for 1.5 minutes;
具体如下: details as follows:
以 PGL3-MSTN为模板, 以 5' TATA盒删除突变上游引物和 5' TATA盒删除突 变下游引物作为引物, 进行 PCR扩增, 得到 5.9kbPCR产物 1, 经过测序, 具有 序列表中序列 2所示的核苷酸 (5' TATA盒缺失) ; Using PGL3-MSTN as a template, 5' TATA box deletion mutation upstream primer and 5' TATA box deletion mutation downstream primer were used as primers, and PCR amplification was performed to obtain 5.9 kb PCR product 1 which was sequenced and shown in sequence 2 in the sequence listing. Nucleotide (5' TATA box deletion);
以 PGL3-MSTN为模板, 以 3' TATA盒删除突变上游引物和 3' TATA盒删除突 变下游引物作为引物, 进行 PCR扩增, 得到 5.9kbPCR产物 2, 经过测序, 具有 序列表中序列 3所示的核苷酸 (3' TATA盒) ; Using PGL3-MSTN as a template, the 3' TATA box deletion mutation upstream primer and the 3' TATA box deletion mutation downstream primer were used as primers, and PCR amplification was performed to obtain a 5.9 kb PCR product 2, which was sequenced and shown in sequence 3 in the sequence listing. Nucleotide (3' TATA box);
以 pGL3-MSTN为模板, 以三元件删除突变引物和 MSTN-F作为引物, 进行 PCR 扩增, 得到 981bpPCR产物 3, 经过酶切、 连接、 转化、 鉴定、 测序, 具有序列 表中序列 4所示的核苷酸; Using pGL3-MSTN as a template, using the three-component deletion mutant primer and MSTN-F as primers, PCR amplification was performed to obtain 981 bp PCR product 3, which was digested, ligated, transformed, identified, and sequenced, and shown in sequence 4 in the sequence listing. Nucleotide
也可人工合成序列 2或序列 3。 Sequence 2 or Sequence 3 can also be synthesized manually.
用 Dpnl分别消化上述 PCR产物(去除环化的模板 PGL3-MSTN), 体系如下: 表 8反应体系
成分 初始浓度 用量 终浓度 The above PCR product (removal of the cyclized template PGL3-MSTN) was separately digested with Dpnl, and the system was as follows: Table 8 Reaction system Initial concentration of component
Buffer 10 X 3μ1 I X Buffer 10 X 3μ1 I X
l ango l ango
Dpn I lOunits/μΙ ΐμΐ 0. 33 unit/μΐ Dpn I lOunits/μΙ ΐμΐ 0. 33 unit/μΐ
PCR产物 26μ1 PCR product 26μ1
总体积 30μ1 Total volume 30μ1
反应在 37 °C水浴, 消化时间 2小时。 The reaction was carried out in a water bath at 37 ° C for 2 hours.
消化结束后,分别取 Ιθμΐ消化产物按照标准步骤转化大肠杆菌感受态 细胞 DH5 a (线性的消化产物两端有同源臂, 在大肠杆菌中可以进行环化, 这是经典突变方法的原理, 由美国 Stratagene公司开发) , 得到 3种转化 子: 阳性质粒 1、 阳性质粒 2、 转化子 3。 After digestion, the Ιθμΐ digested product was separately transformed into E. coli competent cells DH5 a according to standard procedures. (The linear digested product has a homology arm at both ends, which can be cyclized in E. coli. This is the principle of the classical mutation method. Developed by Stratagene, USA, three transformants were obtained: positive plasmid 1, positive plasmid 2, transformant 3.
提取转化子 1的质粒, 送去测序, 结果为该质粒中含有序列表中的序列 2, 该质粒为将序列表中的序列 2插入 pGL3-basic的 Mlu I和 Bgl I I酶切位点间 得到的质粒, 命名为 PGL3-TATA1 ; The plasmid of transformant 1 was extracted and sent for sequencing. As a result, the plasmid contained the sequence 2 in the sequence listing, and the plasmid was inserted between the Mlu I and Bgl II cleavage sites of pGL3-basic. The plasmid was named PGL3-TATA1;
提取转化子 2的质粒, 送去测序, 结果为该质粒中含有序列表中的序列 3, 该质粒为将序列表中的序列 3插入 pGL3-basic的 Mlu I和 Bgl I I酶切位点间 得到的质粒, 命名为 PGL3-TATA2; The plasmid of transformant 2 was extracted and sent for sequencing. As a result, the plasmid contained the sequence 3 in the sequence listing, which was obtained by inserting sequence 3 in the sequence listing between the Mlu I and Bgl II cleavage sites of pGL3-basic. Plasmid, named PGL3-TATA2;
提取转化子 3的质粒, 送去测序, 结果为该质粒中含有序列表中的序列 4, 该质粒为将序列表中的序列 4插入 pGL3-basic的 Mlu I和 Bgl I I酶切位点间 得到的质粒, 命名为 pGL3-3-boxes。 The plasmid of transformant 3 was extracted and sent for sequencing. As a result, the plasmid contained the sequence 4 in the sequence listing, which was obtained by inserting the sequence 4 in the sequence listing between the Mlu I and Bgl II cleavage sites of pGL3-basic. The plasmid was named pGL3-3-boxes.
B、 猪肌抑素基因启动子转录活性检测 B. Detection of transcriptional activity of porcine myostatin gene promoter
1、 实验材料 1. Experimental materials
表 9实验材料 主要材料 来源 说明 Table 9 Experimental materials Main materials Source Description
Lipof ectamine2000 Invitr 细胞转染 Lipof ectamine2000 Invitr Cell Transfection
ogen Gene
小鼠成肌细胞 C2C12 ATCC 细 肌肉细胞模型 Mouse myoblast C2C12 ATCC fine muscle cell model
胞系编号为 Cell line number is
CRL-1772 CRL-1772
双荧光素酶试剂盒 Promeg 用于报告基因酶活测定 Dual luciferase kit Promeg for reporter gene assay
DLRM Assay a DLR M Assay a
超纯质粒提取试剂 北京天 制备无内毒素质粒用 合 根 于细胞转染 Ultrapure plasmid extraction reagent Beijing Tian Preparation of endotoxin-free plasmids for root transfection
Opti-MEM Gibco 细胞转染辅助试剂
24孔细胞培养板 Cornin 细胞培养 Opti-MEM Gibco Cell Transfection Auxiliary Reagent 24-well cell culture plate Cornin cell culture
g g
2、 仪器设备 2, equipment
表 10实验材料 Table 10 experimental materials
名称 来源 说明 Name Source Description
C02培养箱 Sanyo (Japan) 哺乳动物细胞培养 超净工作台 北京半导体设 细胞实验专用 C02 Incubator Sanyo (Japan) Mammalian Cell Culture Super Clean Bench Beijing Semiconductor Design Cell Experiment
备厂 Preparation plant
GloMAX Promega 报告基因测试 GloMAX Promega Reporter Gene Test
3、 试验方法 3. Test method
1 ) 、 细胞转染 1) , cell transfection
转染前一天, 将 4 X 104 C2C12细胞铺到 24孔板中,隔夜生长后按照表中 所列体系配制转染复合物, 滴于 24孔板中, 补加 400μ1完全培养基, 置于 37 °C, 5%C02的条件下培养。在转染 48h后收集细胞。按照 Promega公司的 DLR Assay试剂盒说明书进行荧光素酶活性测定。 One day before transfection, 4 ×10 4 C2C12 cells were plated into 24-well plates. After overnight growth, transfection complexes were prepared according to the system listed in the table, and dropped into 24-well plates, supplemented with 400 μl complete medium, placed. Incubate at 37 ° C under 5% CO 2 . Cells were harvested 48 h after transfection. Luciferase activity assays were performed according to Promega's DLR Assay kit instructions.
转染体系如下: The transfection system is as follows:
MEM mine2000 MEM mine2000
pGL3~basi lOOng Ιθθμΐ 0. 5μ1 pGL3~basi lOOng Ιθθμΐ 0. 5μ1
PGL3-prom lOOng Ιθθμΐ 0. 5μ1 PGL3-prom lOOng Ιθθμΐ 0. 5μ1
oter Oter
PGL3-MSTN lOOng Ιθθμΐ 0. 5μ1 PGL3-MSTN lOOng Ιθθμΐ 0. 5μ1
pGL3- TATA lOOng Ιθθμΐ 0. 5μ1 pGL3- TATA lOOng Ιθθμΐ 5μ1 pGL3-3~bo lOOng Ιθθμΐ 5μ1 pGL3- TATA lOOng Ιθθμΐ 0. 5μ1 pGL3- TATA lOOng Ιθθμΐ 5μ1 pGL3-3~bo lOOng Ιθθμΐ 5μ1
xes Xes
RL-TK (内 lOOng Ιθθμΐ 5μ1 实验设置三个生物学重复, 结果取平均值。 RL-TK (intraOOOOng Ιθθμΐ 5μ1 experiment set three biological replicates, the results were averaged.
各个报告载体的酶活绝对值与相对比值如下表 12所示: The absolute and relative enzymatic activities of the individual reporter vectors are shown in Table 12 below:
表 12各个报告载体的酶活绝对值与相对比值
Table 12 Absolute and relative values of enzyme activity of each reporter vector
各个报告载体的部分结构示意图和酶活相对比值作图如图 4所示,从上述可 以看出, TATA盒与 CAAT盒是猪肌抑素基因转录的关键元件, 三个元件的缺失可 导致报告基因活性的丧失(与阴性对照 pGL3-basiC的酶活差异不显著)。 分别缺 失两个 TATA盒发现它们均可有效驱动转录, 证明这两个 TATA盒是猪肌抑素基 因转录起始的有效元件(突变型与野生型的酶活几乎一致), 但是二者无叠加效 应。 *, p<0. 05,差异显著, 说明序列 2所示的 DNA分子和序列 3所示的 DNA分 子也为启动子。 A schematic diagram of the partial structure of each reporter vector and the relative ratio of enzyme activity are shown in Figure 4. As can be seen from the above, the TATA box and the CAAT box are key components of the transcription of the porcine myostatin gene, and the deletion of the three components can lead to the report. Loss of gene activity (not significantly different from the enzyme activity of the negative control pGL3-basi C ). Two TATA boxes were deleted, respectively, and they were all able to efficiently drive transcription. It was proved that these two TATA boxes are effective elements for the transcription initiation of porcine myostatin gene (mutant and wild type enzyme activities are almost identical), but the two are not superimposed. effect. *, p < 0.05, the difference is significant, indicating that the DNA molecule shown in SEQ ID NO: 2 and the DNA molecule shown in SEQ ID NO: 3 are also promoters.
二、 猪肌抑素基因启动子驱动绿色荧光蛋白表达的功能验证 2. Functional verification of porcine myostatin gene promoter driving green fluorescent protein expression
A、 连接绿色荧光蛋白载体的制备与检测 A. Preparation and detection of green fluorescent protein carrier
1、 实验材料 1. Experimental materials
表 13实验材料 主要材料 来源 说明 Table 13 Experimental materials Main materials Source Description
pEGFP-Cl Invitr 绿色荧光蛋白报告载 ogen 体 pEGFP-Cl Invitr green fluorescent protein reporter containing ogen
T4 DNA连接酶 Fermen 基因克隆
tas T4 DNA ligase Fermen gene cloning Tas
Asel、 Nhel Fermen 限制性内切酶 Asel, Nhel Fermen restriction enzyme
tas Tas
启动子上游引物 上海英 TTCATTAATGCTCTATTC 骏 TCTGCTCCCAGACC (AseT) Promoter upstream primer Shanghai Ying TTCATTAATGCTCTATTC Jun TCTGCTCCCAGACC (AseT)
(序列 7 ) (sequence 7)
启动子下游引物 上海英 CATCGCTAGCCGCCAAGC 骏 AAAATTTTAATGCC {Nhel) Promoter downstream primer Shanghai Ying CATCGCTAGCCGCCAAGC Jun AAAATTTTAATGCC {Nhel)
(序列 8 ) (sequence 8)
超纯质粒提取试剂 北京天 制备无内毒素质粒 合 根 Ultrapure plasmid extraction reagent Beijing Tian Preparation of endotoxin-free plasmid
2、 仪器设备 2, equipment
表 14仪器设备 Table 14 Instrumentation
名称 来源 说明 超净工作台 北京半导体设 细菌转化等无菌操作 备厂 使用 Name Source Description Ultra-clean workbench Beijing Semiconductor Design Aseptic operation such as bacterial transformation Preparation plant
高速离心机 贝克曼 -库尔 DNA、 质粒提取 High speed centrifuge Beckman-Chur DNA, plasmid extraction
特(USA) Special (USA)
水平电泳仪 北京六一 电泳检测 Horizontal Electrophoresis System Beijing Liuyi Electrophoresis Detection
凝胶成像系统 Uvitek, UK 图像捕获 Gel Imaging System Uvitek, UK Image Capture
ThermoCycler PCR Thermo-Fi she 核酸的 PCR扩增 仪 r ThermoCycler PCR Thermo-Fi she nucleic acid PCR amplification instrument r
温箱、 摇床 上海智城 细菌培养 Thermostat, shaker, Shanghai Zhicheng, bacterial culture
3、 实验方法 3. Experimental methods
1 ) 猪肌抑素基因启动子片段的 PCR扩增 1) PCR amplification of porcine myostatin gene promoter fragment
以 pGL3-MSTN质粒 DNA为模板, 带有预设酶切位点的特异引物 (启动子上 游引物和启动子下游引物) 扩增猪肌抑素基因启动子序列, 反应体系为: The pGL3-MSTN plasmid DNA was used as a template, and a specific primer with a predetermined restriction site (promoter upstream primer and promoter downstream primer) was used to amplify the porcine myostatin gene promoter sequence. The reaction system was:
PCR 10 X 5μ1 I X PCR 10 X 5μ1 I X
buffer Buffer
dNTP 2mmol/L 5μ1 200μπιο1/1 硫 画
25 mmol/L 2μ1 lmmol/LdNTP 2mmol/L 5μ1 200μπιο1/1 sulfur painting 25 mmol/L 2μ1 lmmol/L
MgS04
KOD plus lunit/μΐ ΐμΐ 0.02 unit/μΐ 启动子上 10μπιο1/1 4μ1 800nmo/L 游引物 MgS0 4 KOD plus lunit/μΐ ΐμΐ 0.02 unit/μΐ 10μπιο1/1 4μ1 800nmo/L on the promoter
启动子下 10μπιο1/1 4μ1 800nmo/L 游引物 Under the promoter 10μπιο1/1 4μ1 800nmo/L
质 粒 25ng/ l ΐμΐ ο. Sng/μΐ Grain 25ng/ l ΐμΐ ο. Sng/μΐ
PGL3-MSTN PGL3-MSTN
双蒸水 28μ1 Double steamed water 28μ1
总体积 50μ1 Total volume 50μ1
反应条件为: The reaction conditions are:
预变性 95 °C 2分钟; Pre-denaturation at 95 °C for 2 minutes;
变性 95 °C 30秒; Denaturation at 95 °C for 30 seconds;
退火 55 °C 30秒; 30 Annealing 55 °C 30 seconds; 30
延伸 68 °C 1.5分钟; Extend at 68 °C for 1.5 minutes;
反应结束后,以 1%琼脂糖凝胶电泳检领 lj PCR产物,得到 1122bp的 PCR产物。 After the completion of the reaction, the lj PCR product was subjected to 1% agarose gel electrophoresis to obtain a 1122 bp PCR product.
2) 猪肌抑素基因启动子片段的克隆与鉴定 2) Cloning and identification of porcine myostatin gene promoter fragment
将经 和 Nhel酶切的上述 1)得到的 PCR产物与经过同样酶切得到的报 告载体 pEGFP-Cl片段以下述体系做连接: The PCR product obtained by the above 1) digested with Nhel and the reporter vector pEGFP-Cl fragment obtained by the same digestion were linked by the following system:
表 16连接体系 Table 16 connection system
成分 初始浓度 用量 终浓度 酶切后 PCR 100 ng/μΐ 5μ1 10 ng/μΐ 产物 Ingredients Initial concentration Amount Final concentration After digestion, PCR 100 ng/μΐ 5μ1 10 ng/μΐ Product
pEGFP-Cl 片 100 ng/μΐ ΐμΐ lOng/μΙ 段 pEGFP-Cl sheet 100 ng/μΐ ΐμΐ lOng/μΙ segment
10 X缓冲液 10X ΐμΐ IX 10 X Buffer 10X ΐμΐ IX
T4 DNA lOunits/μΙ ΐμΐ 1 unit/μΐ ligase T4 DNA lOunits/μΙ ΐμΐ 1 unit/μΐ ligase
双蒸水 2μ1 Double distilled water 2μ1
总体积 Ιθμΐ Total volume Ιθμΐ
反应条件为: 16°C, 连接过夜。 The reaction conditions were: 16 ° C, and allowed to stand overnight.
按照标准方法将连接产物转化大肠杆菌 DH5 a, 37°C培养过夜, 得到转 化子。 The ligation product was transformed into E. coli DH5a according to standard methods and cultured overnight at 37 ° C to obtain a transformant.
提取转化子的质粒, 以启动子上游引物和启动子下游引物进行 PCR扩增, 得 到 1122bp的片段为阳性质粒。 将阳性质粒用 和 Nhel酶切, 得到 1122bp
片段, 进一步证明为阳性质粒, 经 PCR、 酶切均鉴定为阳性的质粒送去测序, 结 果为该质粒为将序列表中的序列 1插入 pEGFP-Cl的 ^sel和 Nhel酶切位点间得 到的质粒, 将该质粒命名为 pMSTN-EGFP。 The plasmid of the transformant was extracted, and the primer upstream of the promoter and the primer downstream of the promoter were subjected to PCR amplification to obtain a 1122 bp fragment as a positive plasmid. The positive plasmid was digested with Nhel to obtain 1122 bp. The fragment was further proved to be a positive plasmid, and the plasmid which was positive by PCR and restriction enzyme digestion was sent for sequencing, and the result was that the plasmid 1 was inserted between the sel and Nhel cleavage sites of pEGFP-Cl. The plasmid was named pMSTN-EGFP.
B、 猪肌抑素基因启动子驱动绿色荧光蛋白表达的功能验证 B. Functional verification of porcine myostatin gene promoter driving green fluorescent protein expression
1、 实验材料 1. Experimental materials
表 17实验材料 Table 17 Experimental materials
主要材料 来源 说明 Main material Source Description
Lipof ectamine2000 Invi tr 细胞转染 Lipof ectamine2000 Invi tr Cell Transfection
ogen Gene
小鼠成肌细胞 C2C12 ATCC 细 肌肉细胞模型 Mouse myoblast C2C12 ATCC fine muscle cell model
胞系编号为 Cell line number is
CRL-1772 CRL-1772
超纯质粒提取试剂 北京天 制备无内毒素质粒用 合 根 于细胞转染 Ultrapure plasmid extraction reagent Beijing Tian Preparation of endotoxin-free plasmids for root transfection
Opt i-MEM Gibco 细胞转染辅助试剂 Opt i-MEM Gibco Cell Transfection Auxiliary Reagent
24孔细胞培养板 Cornin 细胞培养 24-well cell culture plate Cornin cell culture
g g
2、 仪器设备 2, equipment
表 18仪器设备 名称 来源 说明 Table 18 Instrument Equipment Name Source Description
C02培养箱 Sanyo (Japan) 哺乳动物细胞培养 超净工作台 北京半导体设 细胞实验专用 C02 Incubator Sanyo (Japan) Mammalian Cell Culture Super Clean Bench Beijing Semiconductor Design Cell Experiment
备厂 Preparation plant
荧光显微镜 Le i ca (German 观察荧光 Fluorescence microscope Le i ca (German observation fluorescence
y) y)
3、 试验方法 3. Test method
1 ) 细胞转染 1) Cell transfection
将上述构建好的 pMSTN-EGFP报告载体转染 (转染方法在试剂盒的说明书中有 详细说明) 小鼠成肌细胞 C2C12 , 24小时后观察荧光, 以 pEGFP-Cl为对照。 The constructed pMSTN-EGFP reporter vector was transfected (transfection method is described in detail in the kit). Mouse myoblast C2C12 was observed 24 hours later, and pEGFP-Cl was used as a control.
结果如图 5所示, 阳性对照质粒 pEGFP-Cl转染细胞后, 可见大量绿色荧光蛋 白表达; pMSTN-EGFP转染细胞后, 也有部分细胞表达绿色荧光蛋白, 但是其总亮度 明显低于 pEGFP-Cl样品。 这一方面说明所鉴定的 MSTN启动子可以驱动报告基因绿 色荧光蛋白的表达, 另一方面也说明所鉴定 MSTN启动子的活性确实低于组成型启 动子 CMV的效率,这与荧光素酶报告基因测试的结果是一致的,二者可以交互验证。 由此可以认为, 通过采用不同的报告基因系统, 均检测到了所克隆片段的启动子活
证明该片段确为猪肌抑素基因的启动子功能序列, The results are shown in Figure 5. After transfection of the positive control plasmid pEGFP-Cl, a large amount of green fluorescent protein was expressed. After pMSTN-EGFP transfected cells, some cells expressed green fluorescent protein, but the total brightness was significantly lower than that of pEGFP- Cl sample. This aspect indicates that the identified MSTN promoter can drive the expression of the reporter gene green fluorescent protein, and on the other hand, the activity of the identified MSTN promoter is indeed lower than that of the constitutive promoter CMV, which is related to the luciferase reporter gene. The results of the test are consistent and the two can be verified interactively. Therefore, it can be considered that the promoter activity of the cloned fragment was detected by using different reporter gene systems. Prove that the fragment is a promoter sequence of the porcine myostatin gene.
转染体系如下: The transfection system is as follows:
表 19转染体系 Table 19 Transfection system
质粒 用量 Opti- Lipof ectami Plasmid dosage Opti- Lipof ectami
MEM ne2000 MEM ne2000
pEGFP-Cl lOOOng Ιθθμΐ 2μ1 pEGFP-Cl lOOOng Ιθθμΐ 2μ1
pMSTN-EGF lOOOng Ιθθμΐ 2μ1 pMSTN-EGF lOOOng Ιθθμΐ 2μ1
P 三、 猪肌抑素基因启动子报告载体在人源细胞的表达 P. Expression of porcine myostatin gene promoter reporter vector in human cells
1、 实验材料 1. Experimental materials
Lipof ectamine2000 Invitr 细胞转染 Lipof ectamine2000 Invitr Cell Transfection
ogen Gene
Hela细胞 ATCC 细 人源细胞 Hela cell ATCC fine human cell
胞系编号为 Cell line number is
CCL-2 CCL-2
超纯质粒提取试剂 北京天 制备无内毒素质粒用于 合 根 细胞转染 Ultrapure plasmid extraction reagent Beijing Tian Preparation of endotoxin-free plasmid for root cell transfection
Opti-MEM Gibco 细胞转染辅助试剂 Opti-MEM Gibco Cell Transfection Auxiliary Reagent
24孔细胞培养板 Cornin 细胞培养 24-well cell culture plate Cornin cell culture
g g
2、 仪器设备 2, equipment
表 21仪器设备 名称 来源 说明 Table 21 Instrument Equipment Name Source Description
C02培养箱 Sanyo (Japan) 哺乳动物细胞培养 超净工作台 北京半导体设 细胞实验专用 C02 Incubator Sanyo (Japan) Mammalian Cell Culture Super Clean Bench Beijing Semiconductor Design Cell Experiment
备厂 Preparation plant
GloMAX Promega 报告基因测试 GloMAX Promega Reporter Gene Test
3、 试验方法 3. Test method
1 ) 细胞转染 1) Cell transfection
转染前一天, 将 4 X 104个 hela细胞铺到 24孔板中,隔夜生长后按照表
中所列体系分别配制转染复合物, 滴于 24孔板中,补加 400μ1完全培养基, 置于 37 °C、 5%C02的条件下培养。 在转染 48h后收集细胞。 按照 Promega公 司的 DLRM Assay试剂盒说明书进行荧光素酶活性测定。 One day before transfection, 4 X 10 4 hela cells were plated into 24-well plates and grown overnight. The transfected complexes were prepared separately in the system listed, and were added to a 24-well plate, supplemented with 400 μl of complete medium, and cultured at 37 ° C and 5% CO 2 . Cells were harvested 48 h after transfection. Luciferase activity assays were performed according to Promega's DLR M Assay kit instructions.
实验设置三个生物学重复, 结果取平均值。 Three biological replicates were set up in the experiment and the results were averaged.
转染体系如下: The transfection system is as follows:
MEM 000 MEM 000
pGL3-basic lOOOng Ιθθμΐ 2μ1 pGL3-basic lOOOng Ιθθμΐ 2μ1
PGL3-promo lOOOng Ιθθμΐ 2μ1 PGL3-promo lOOOng Ιθθμΐ 2μ1
ter Ter
PGL3-MSTN lOOOng Ιθθμΐ 2μ1 PGL3-MSTN lOOOng Ιθθμΐ 2μ1
RL-TK (内 500ng Ιθθμΐ 2μ1 荧光素酶活性检测结果如下表所示: The results of RL-TK (inner 500ng Ιθθμΐ 2μ1 luciferase activity are shown in the following table:
表 23荧光素酶活性 Table 23 Luciferase activity
将结果取平均值作图如图 6所示。 The results are averaged as shown in Figure 6.
可以看出, 将猪肌抑素基因启动子报告载体 PGL3-MSTN转染人宫颈癌 Hela 细胞, 通过报告基因检测了猪肌抑素基因启动子在人源细胞的转录活性。 结果 显示, 所克隆的启动子片段在人源细胞中具有很高的活性, 证明所鉴定的猪肌 抑素基因启动子在哺乳动物细胞中可有效表达。 It can be seen that the porcine myostatin gene promoter reporter vector PGL3-MSTN was transfected into human cervical cancer Hela cells, and the transcriptional activity of the porcine myostatin gene promoter in human cells was examined by a reporter gene. The results showed that the cloned promoter fragment was highly active in human cells, demonstrating that the identified porcine myostatin gene promoter is efficiently expressed in mammalian cells.
工业应用 Industrial application
本发明的实验证明, 本发明提供的启动子, 其可以驱动报告基因萤火虫荧 光素酶的表达, 也可以将启动子构建到报告载体后转染猪源以及人源培养细胞,
通过报告基因测试, 以准确定量的方法鉴定启动子的活性和效率, 为在该启动 子后引入外源基因、 解决转基因猪外源基因表达不稳定性以及位置效应的不可 预知性等问题, 提供了可靠、 有价值的基因资源。
The experiments of the present invention prove that the promoter provided by the present invention can drive the expression of the reporter gene firefly luciferase, and can also be used to construct the promoter into the reporter vector and then transfect the pig source and the human cultured cell. Through reporter gene testing, the activity and efficiency of the promoter are identified by accurate quantitative methods, and the problems of introducing foreign genes after the promoter, solving the unsteadiness of the expression of foreign genes in transgenic pigs, and the unpredictability of positional effects are provided. A reliable and valuable genetic resource.
Claims
1、 DNA分子, 为如下 1) -4) 中任一所述的 DNA分子: 1. A DNA molecule, which is a DNA molecule as described in any one of the following 1) -4):
1) 序列表的序列 2所示的 DNA分子; 1) a DNA molecule as shown in Sequence 2 of the Sequence Listing;
2) 序列表的序列 3所示的 DNA分子; 2) the DNA molecule shown in sequence 3 of the sequence listing;
3) 在严格条件下与 1) 或 2) 所述 DNA序列杂交且具有启动子功能的 DNA 分子; 3) a DNA molecule that hybridizes under stringent conditions to the DNA sequence of 1) or 2) and has a promoter function;
4) 与 1) 或 2) 中所述 DNA序列具有 90%以上同源性, 且具有启动子功能的 DNA分子。 4) A DNA molecule having 90% or more homology with the DNA sequence described in 1) or 2) and having a promoter function.
2、 根据权利要求 2所述的 DNA分子, 其特征在于: 所述 4) 所示的 DNA分 子为序列表中序列 1所示的 DNA分子。 The DNA molecule according to claim 2, wherein the DNA molecule shown in the above 4) is a DNA molecule represented by SEQ ID NO: 1 in the Sequence Listing.
3、 含有权利要求 1或 2所述 DNA分子的重组载体、 表达盒、 转基因细胞系 或重组菌。 3. A recombinant vector, expression cassette, transgenic cell line or recombinant strain comprising the DNA molecule of claim 1 or 2.
4、 根据权利要求 3所述的重组载体, 其特征在于: 所述重组载体为如下 1) 或 2) : 4. The recombinant vector according to claim 3, wherein: the recombinant vector is as follows 1) or 2):
1) 为将权利要求 1或 2中所述 DNA分子插入 pGL3-basic的多克隆位点得 到的重组载体; 1) a recombinant vector obtained by inserting the DNA molecule of claim 1 or 2 into a multiple cloning site of pGL3-basic;
2) 为将权利要求 1或 2中所述 DNA分子插入 pEGFP-Cl的多克隆位点得到 的重组载体。 2) A recombinant vector obtained by inserting the DNA molecule of claim 1 or 2 into the multiple cloning site of pEGFP-Cl.
5、 扩增权利要求 1或 2所述 DNA分子的引物对。 5. A primer pair for amplifying the DNA molecule of claim 1 or 2.
6、 根据权利要求 5所述的引物对, 其特征在于: 所述引物对为如下 1) 或 6. The primer pair according to claim 5, wherein: the primer pair is as follows 1) or
2) : 2) :
1) 所示的引物对中的一条引物的序列为序列 5, 另一条引物的序列为序列 1) The sequence of one of the primer pairs shown is sequence 5, and the sequence of the other primer is sequence
6; 6;
2) 所示的引物对中的一条引物的序列为序列 7, 另一条引物的序列为序列 2) The sequence of one of the primer pairs shown is sequence 7, and the sequence of the other primer is sequence
8。 8.
7、权利要求 1或 2所述 DNA分子在使目的基因在离体动物细胞中的表达中 的应用。 7. Use of a DNA molecule according to claim 1 or 2 for expression of a gene of interest in an ex vivo animal cell.
8、 根据权利要求 7中所述的应用, 其特征在于: 所述动物细胞为鼠源细胞 或人源细胞。 8. Use according to claim 7, characterized in that the animal cells are mouse cells or human cells.
9、 根据权利要求 7或 8中所述的应用, 其特征在于: 9. Use according to claim 7 or 8, characterized in that:
所述鼠源细胞为小鼠成肌细胞; The mouse cell is a mouse myoblast;
所述人源细胞为人宫颈癌细胞。 The human cell is a human cervical cancer cell.
10、 根据权利要求 7-9中任一所述的应用, 其特征在于: 10. Use according to any of claims 7-9, characterized in that:
所述小鼠成肌细胞为 C2C12细胞; The mouse myoblast is a C2C12 cell;
所述人宫颈癌细胞为 Hela细胞。 The human cervical cancer cells are Hela cells.
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN113151502A (en) * | 2021-05-18 | 2021-07-23 | 华中农业大学 | Genetic marker associated with meat production traits of live pigs and application |
CN113151502B (en) * | 2021-05-18 | 2022-06-03 | 华中农业大学 | Genetic marker associated with meat production traits of live pigs and application |
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CN102725408B (en) | 2014-03-12 |
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