CN105441454A - SCAP gene mutant and application thereof - Google Patents
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- CN105441454A CN105441454A CN201510980242.6A CN201510980242A CN105441454A CN 105441454 A CN105441454 A CN 105441454A CN 201510980242 A CN201510980242 A CN 201510980242A CN 105441454 A CN105441454 A CN 105441454A
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Abstract
本发明公开了SCAP基因突变体及其应用，具体涉及分离的编码SCAP突变体的核酸，分离的多肽，筛选易患早发心肌梗死的生物样品的方法，筛选易患早发心肌梗死的生物样品的系统，以及用于筛选易患早发心肌梗死的生物样品的试剂盒。其中，该分离的编码SCAP突变体的核酸，与SEQ？ID？NO：1相比，具有c.3035C>T突变。通过检测该突变体在生物样品中是否存在，可以有效地检测生物样品是否易患早发心肌梗死。The invention discloses SCAP gene mutants and applications thereof, in particular to isolated nucleic acids encoding SCAP mutants, isolated polypeptides, methods for screening biological samples susceptible to premature myocardial infarction, and screening biological samples susceptible to premature myocardial infarction system, and a kit for screening biological samples predisposed to premature myocardial infarction. Wherein, the isolated nucleic acid encoding the SCAP mutant is identical to SEQ? ID? NO: Compared with 1, it has c.3035C>T mutation. By detecting whether the mutant exists in the biological sample, it can effectively detect whether the biological sample is susceptible to premature myocardial infarction.
Description
技术领域technical field
本发明涉及SCAP基因突变体及其应用。具体地，本发明涉及分离编码SCAP突变体的核酸，分离的多肽，筛选易患早发心肌梗死的生物样品的方法，筛选遗憾早发心肌梗死的生物样品的系统，用于筛选易患早发心肌梗死的生物样品的试剂盒，构建体以及重组细胞。The present invention relates to SCAP gene mutant and application thereof. In particular, the present invention relates to isolated nucleic acids encoding SCAP mutants, isolated polypeptides, methods for screening biological samples for predisposition to premature myocardial infarction, systems for screening biological samples for unfortunately premature myocardial infarction, for screening for predisposition to premature myocardial infarction Kits, constructs and recombinant cells for myocardial infarction biological samples.
背景技术Background technique
心肌梗死(MyocardialInfarction,MI)是一种由于冠状动脉粥样硬化斑块的纤维帽破裂而引起后续的血栓形成，从而使得血管堵塞并引起相应区域的心肌发生缺血缺氧，最终引起心肌坏死的严重致猝死性疾病。其中，部分人群呈现出发病年龄早(男性<50，女性<60)、家族聚集倾向等典型临床特点，称为早发心肌梗死(PrematureMyocardialInfarction,PMI)。有研究显示，早发心肌梗死的遗传度高达63％。由于这类患者的首发症状即可表现为由急性心肌坏死引起的猝死，之前可无任何冠心病及其危险因素的相关症状，因此相比非早发性心肌梗死，其危险度更高。这使得通过早期检测潜在患病者的遗传信息，在出现致死性症状前提供风险预测、实现对早发心肌梗死的“初级预防”显得尤为重要。而目前该病的发病机制尚未完全清楚，致病基因和致病突变仍不明确，且尚未有足够的用于早期识别早发心梗患者的遗传标志物。Myocardial infarction (MI) is a kind of subsequent thrombosis caused by the rupture of the fibrous cap of coronary atherosclerotic plaque, which makes the blood vessel blockage and causes myocardial ischemia and hypoxia in the corresponding area, and finally causes myocardial necrosis. Severe sudden death disease. Among them, some populations present typical clinical characteristics such as early age of onset (male <50, female <60) and family clustering tendency, which is called premature myocardial infarction (PMI). Studies have shown that the heritability of premature myocardial infarction is as high as 63%. Since the first symptoms of such patients can be sudden death caused by acute myocardial necrosis, and they may not have any symptoms related to coronary heart disease and its risk factors before, their risk is higher than that of non-premature myocardial infarction. This makes it particularly important to provide risk prediction and achieve "primary prevention" of premature myocardial infarction through early detection of genetic information of potential patients before fatal symptoms appear. At present, the pathogenesis of the disease is not yet fully understood, the pathogenic genes and mutations are still unclear, and there are not enough genetic markers for early identification of patients with premature myocardial infarction.
因而，目前对早发心肌梗死的致病基因和致病突变的研究仍有待深入。Therefore, the research on the pathogenic genes and pathogenic mutations of premature myocardial infarction still needs to be further studied.
发明内容Contents of the invention
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提出一种能够有效筛选易患早发心肌梗死的生物样品的方法。The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, an object of the present invention is to propose a method capable of effectively screening biological samples susceptible to premature myocardial infarction.
本发明是基于发明人的下列工作完成的：发明人高通量外显子组测序联合候选基因突变验证的方法确定了SCAP基因为早发心肌梗死的致病基因，并且SCAP基因18号外显子上的c.3035C>T突变为早发心肌梗死的致病突变。The present invention is completed based on the inventor's following work: the inventor's method of high-throughput exome sequencing combined with candidate gene mutation verification has determined that the SCAP gene is the causative gene of premature myocardial infarction, and the No. 18 exon of the SCAP gene The c.3035C>T mutation is the pathogenic mutation of premature myocardial infarction.
根据本发明的第一方面，本发明提出了一种分离的编码SCAP突变体的核酸。根据本发明的实施例，所述核酸与SEQIDNO：1相比，具有c.3035C>T突变，即相对于野生型SCAP基因，本发明的SCAP基因突变体的第3035位碱基从C突变为T。根据本发明的实施例，发明人确定了SCAP基因以及该突变体与早发心肌梗死的发病密切相关，从而通过检测该突变体在生物样品中是否存在，可以有效地检测生物样品是否易患早发心肌梗死。According to a first aspect of the present invention, the present invention proposes an isolated nucleic acid encoding a SCAP mutant. According to an embodiment of the present invention, compared with SEQ ID NO: 1, the nucleic acid has a c.3035C>T mutation, that is, relative to the wild-type SCAP gene, the 3035th base of the SCAP gene mutant of the present invention is mutated from C to T. According to the embodiments of the present invention, the inventors have determined that the SCAP gene and the mutant are closely related to the onset of premature myocardial infarction, so that by detecting whether the mutant exists in the biological sample, it is possible to effectively detect whether the biological sample is susceptible to premature myocardial infarction. Myocardial infarction.
根据本发明的第二方面，本发明提出了一种分离的多肽。根据本发明的实施例，与SEQIDNO：2相比，所述分离的多肽具有p.A1012V突变，即该突变是由于c.3035C>T的无义突变而引起的，具体地，该突变表示：该分离的多肽，由野生型SCAP的第1012位氨基酸A(丙氨酸)突变为V(缬氨酸)。通过检测生物样品中是否表达该多肽，可以有效地检测生物样品是否易患早发心肌梗死。According to a second aspect of the present invention, the present invention provides an isolated polypeptide. According to an embodiment of the present invention, compared with SEQ ID NO: 2, the isolated polypeptide has a p.A1012V mutation, that is, the mutation is caused by a nonsense mutation of c.3035C>T, specifically, the mutation represents: The isolated polypeptide is mutated from amino acid A (alanine) at position 1012 of wild-type SCAP to V (valine). By detecting whether the polypeptide is expressed in the biological sample, it can effectively detect whether the biological sample is susceptible to premature myocardial infarction.
根据本发明的第三方面，本发明提出了一种筛选易患早发心肌梗死的生物样品的方法。根据本发明的实施例，该方法包括以下步骤：从所述生物样品提取核酸样本；确定所述核酸样本的核酸序列；所述核酸样本的核酸序列与SEQIDNO：1相比，具有c.3035C>T突变是所述生物样品易患早发心肌梗死的指示。通过根据本发明实施例的筛选易患早发心肌梗死的生物样品的方法，可以有效地筛选易患早发心肌梗死的生物样品。According to a third aspect of the present invention, the present invention proposes a method for screening biological samples susceptible to premature myocardial infarction. According to an embodiment of the present invention, the method includes the following steps: extracting a nucleic acid sample from the biological sample; determining the nucleic acid sequence of the nucleic acid sample; comparing the nucleic acid sequence of the nucleic acid sample with SEQ ID NO: 1, it has c.3035C> A T mutation is indicative of a predisposition of said biological sample to premature myocardial infarction. By the method for screening biological samples susceptible to premature myocardial infarction according to the embodiments of the present invention, biological samples susceptible to premature myocardial infarction can be effectively screened.
根据本发明的第四方面，本发明提出了一种筛选易患早发心肌梗死的生物样品的系统。根据本发明的实施例，该系统包括：核酸提取装置，所述核酸提取装置用于从所述生物样品提取核酸样本；核酸序列确定装置，所述核酸序列确定装置与所述核酸提取装置相连，用于对所述核酸样本进行分析，以便确定所述核酸样本的核酸序列；判断装置，所述判断装置与所述核酸序列确定装置相连，以便基于所述核酸样本的核酸序列与SEQIDNO：1相比，是否具有c.3035C>T突变，判断所述生物样品是否易患早发心肌梗死。利用该系统，能够有效地实施前述筛选易患早发心肌梗死的生物样品的方法，从而可以有效地筛选易患早发心肌梗死的生物样品。According to a fourth aspect of the present invention, the present invention proposes a system for screening biological samples susceptible to premature myocardial infarction. According to an embodiment of the present invention, the system includes: a nucleic acid extraction device, the nucleic acid extraction device is used to extract a nucleic acid sample from the biological sample; a nucleic acid sequence determination device, the nucleic acid sequence determination device is connected to the nucleic acid extraction device, It is used to analyze the nucleic acid sample so as to determine the nucleic acid sequence of the nucleic acid sample; a judging device, the judging device is connected to the nucleic acid sequence determining device so that the nucleic acid sequence of the nucleic acid sample is consistent with SEQ ID NO: 1 ratio, whether it has the c.3035C>T mutation, to determine whether the biological sample is susceptible to premature myocardial infarction. Using this system, the aforementioned method for screening biological samples susceptible to premature myocardial infarction can be effectively implemented, thereby effectively screening biological samples susceptible to premature myocardial infarction.
根据本发明的第五方面，本发明提出了一种用于筛选易患早发心肌梗死的生物样品的试剂盒。根据本发明的实施例，该试剂盒含有：适于检测SCAP基因突变体的试剂，其中与SEQIDNO：1相比，该SCAP基因突变体具有c.3035C>T突变。利用根据本发明的实施例的试剂盒，能够有效地筛选易患早发心肌梗死的生物样品。According to the fifth aspect of the present invention, the present invention proposes a kit for screening biological samples susceptible to premature myocardial infarction. According to an embodiment of the present invention, the kit contains: a reagent suitable for detecting a SCAP gene mutant, wherein compared with SEQ ID NO: 1, the SCAP gene mutant has a c.3035C>T mutation. Using the kit according to the embodiment of the present invention, biological samples susceptible to premature myocardial infarction can be efficiently screened.
根据本发明的第六方面，本发明还提出了一种构建体。根据本发明的实施例，该构建体包含前面所述的分离的编码SCAP突变体的核酸。由此，利用本发明的构建体转化受体细胞获得的重组细胞，能够有效地用于筛选治疗早发心肌梗死的药物。According to the sixth aspect of the present invention, the present invention also proposes a construct. According to an embodiment of the present invention, the construct comprises the aforementioned isolated nucleic acid encoding a SCAP mutant. Thus, the recombinant cells obtained by transforming recipient cells with the construct of the present invention can be effectively used for screening drugs for treating premature myocardial infarction.
根据本发明的第七方面，本发明还提出了一种重组细胞。根据本发明的实施例，该重组细胞是通过前面所述的构建体转化受体细胞而获得的。根据本发明的一些实施例，利用本发明的重组细胞，能够有效地筛选治疗早发心肌梗死的药物。According to the seventh aspect of the present invention, the present invention also provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell is obtained by transforming the recipient cell with the aforementioned construct. According to some embodiments of the present invention, the recombinant cells of the present invention can be used to effectively screen drugs for treating premature myocardial infarction.
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。Additional aspects and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
附图说明Description of drawings
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：The above and/or additional aspects and advantages of the present invention will become apparent and comprehensible from the description of the embodiments in conjunction with the following drawings, wherein:
图1显示了根据本发明实施例的筛选易患早发心肌梗死的生物样品的系统及其组成部分的示意图，其中，1 shows a schematic diagram of a system for screening biological samples susceptible to premature myocardial infarction and its components according to an embodiment of the present invention, wherein,
图1A为根据本发明实施例的筛选易患早发心肌梗死的生物样品的系统的示意图，1A is a schematic diagram of a system for screening biological samples susceptible to premature myocardial infarction according to an embodiment of the present invention,
图1B为根据本发明实施例的核酸提取装置的示意图，Figure 1B is a schematic diagram of a nucleic acid extraction device according to an embodiment of the present invention,
图1C为根据本发明实施例的核酸序列确定装置的示意图；Fig. 1C is a schematic diagram of a device for determining a nucleic acid sequence according to an embodiment of the present invention;
图2显示了根据本发明一个实施例的早发心肌梗死患者家系的家系图；Fig. 2 has shown the pedigree diagram of the pedigree of patients with premature myocardial infarction according to one embodiment of the present invention;
图3显示了根据本发明的一个实施例，早发心肌梗死患者家系中患者、家系内正常人以及家系外正常人的SCAP基因c.3035C>T突变位点的代表性Sanger测序验证峰图。Figure 3 shows a representative Sanger sequencing verification profile of the c.3035C>T mutation site of the SCAP gene in a family of patients with premature myocardial infarction, normal people in the family, and normal people outside the family, according to an embodiment of the present invention.
具体实施方式detailed description
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，仅用于解释本发明，而不能理解为对本发明的限制。Embodiments of the present invention are described in detail below, examples of which are shown in the drawings, wherein the same or similar reference numerals designate the same or similar elements or elements having the same or similar functions throughout. The embodiments described below by referring to the figures are exemplary only for explaining the present invention and should not be construed as limiting the present invention.
SCAP基因突变体SCAP mutant
根据本发明的第一方面，本发明提出了一种分离的编码SCAP突变体的核酸。根据本发明的实施例，所述核酸与SEQIDNO：1相比，具有c.3035C>T突变。在本文中所使用的表达方式“编码SCAP突变体的核酸”，是指与编码SCAP突变体的基因相对应的核酸物质，即核酸的类型不受特别限制，可以是任何包含与SCAP突变体的编码基因相对应的脱氧核糖核苷酸和/或核糖核苷酸的聚合物，包括但不限于DNA、RNA或cDNA。根据本发明的一个具体示例，前面所述的编码SCAP突变体的核酸为DNA。根据本发明的实施例，发明人确定了SCAP基因的及该突变体与早发心肌梗死的发病密切相关，从而通过检测该突变体在生物样品中是否存在，可以有效地检测生物样品是否易患早发心肌梗死，也可以通过检测该突变体在生物体中是否存在，可以有效地预测生物体是否易患早发心肌梗死。According to a first aspect of the present invention, the present invention proposes an isolated nucleic acid encoding a SCAP mutant. According to an embodiment of the present invention, compared with SEQ ID NO: 1, the nucleic acid has a c.3035C>T mutation. The expression "nucleic acid encoding a SCAP mutant" as used herein refers to a nucleic acid substance corresponding to a gene encoding a SCAP mutant, that is, the type of nucleic acid is not particularly limited, and may be any Polymers of deoxyribonucleotides and/or ribonucleotides corresponding to coding genes, including but not limited to DNA, RNA or cDNA. According to a specific example of the present invention, the aforementioned nucleic acid encoding the SCAP mutant is DNA. According to the embodiments of the present invention, the inventors have determined that the SCAP gene and the mutant are closely related to the onset of premature myocardial infarction, so by detecting whether the mutant exists in the biological sample, it is possible to effectively detect whether the biological sample is susceptible to Premature myocardial infarction can also be effectively predicted whether the organism is susceptible to premature myocardial infarction by detecting whether the mutant exists in the organism.
对于本发明说明书和权利要求书中，提及核酸，本领域技术人员应当理解，实际包括互补双链的任意一条，或者两条。为了方便，在本说明书和权利要求书中，虽然多数情况下只给出了一条链，但实际上也公开了与之互补的另一条链。例如，提及SEQIDNO:1，实际包括其互补序列。本领域技术人员还可以理解，利用一条链可以检测另一条链，反之亦然。As for nucleic acid mentioned in the specification and claims of the present invention, those skilled in the art should understand that it actually includes any one or both of the complementary double strands. For convenience, in this specification and claims, although only one chain is given in most cases, another chain complementary to it is actually disclosed. For example, reference to SEQ ID NO: 1 actually includes its complement. It will also be understood by those skilled in the art that one strand can be used to detect the other, and vice versa.
该编码SCAP突变体的核酸，是本申请的发明人通过高通量外显子组测序联合候选基因突变验证的方法确定的早发心肌梗死的致病基因SCAP上的致病突变。该突变位点在现有技术中并未被提到。The nucleic acid encoding the SCAP mutant is the pathogenic mutation in the premature myocardial infarction pathogenic gene SCAP determined by the inventors of the present application through the method of high-throughput exome sequencing combined with candidate gene mutation verification. This mutation site has not been mentioned in the prior art.
其中，野生型SCAP基因的cDNA具有如下所示的核苷酸序列：Wherein, the cDNA of the wild-type SCAP gene has the nucleotide sequence shown below:
其编码的蛋白质具有如下所示的氨基酸序列：The encoded protein has the amino acid sequence shown below:
发明人发现的该突变体与SEQIDNO：1相比，具有c.3035C>T突变，即相对于野生型SCAP基因，本发明的SCAP基因突变体的第3035位碱基从C突变为T。由此，其所编码的产物与野生型的SCAP相比，具有p.A1012V突变，即该突变是由于c.3035C>T的无义突变而引起的，具体地，该突变表示：该分离的多肽，由野生型SCAP的第1012位氨基酸A(丙氨酸)突变为V(缬氨酸)。Compared with SEQ ID NO: 1, the mutant found by the inventors has a c.3035C>T mutation, that is, relative to the wild-type SCAP gene, the 3035th base of the SCAP gene mutant of the present invention is mutated from C to T. Therefore, compared with the wild-type SCAP, the encoded product has p.A1012V mutation, that is, the mutation is caused by the nonsense mutation of c.3035C>T. Specifically, the mutation indicates that the isolated The polypeptide is mutated from amino acid A (alanine) at position 1012 of wild-type SCAP to V (valine).
需要说明的是，SCAP基因位于3号染色体，由23个外显子组成，共包含1279个氨基酸。它是哺乳动物脂质合成和摄入的“中心调节因子”，在细胞内胆固醇水平降低时，其可发生构象改变，并通过其酶切作用激活SREBP基因、促进其进入细胞核完成一系列的转录调控反应，进而上调包括LDLR、HMGCoA等一系列脂质代谢的关键酶，从而促进胆固醇的形成；相反，当细胞内胆固醇水平增高时，SCAP基因则可感受细胞内过高的胆固醇水平，并停止发挥其酶切作用。因此，SCAP是胆固醇代谢环节中的重要负反馈介质。有研究表明，位于SCAP基因上的点突变可造成其对于细胞内胆固醇水平变化的敏感性下降，导致其不能为升高的胆固醇水平抑制，进而使得细胞内胆固醇水平过度增高：当该过程发生于肝细胞等机体主要的胆固醇合成组织时，则会导致机体出现高胆固醇血症；而当该过程发生于巨噬细胞及平滑肌细胞时，则会导致其行为“泡沫细胞”，而“泡沫细胞”的形成，则是心肌梗死发生发展的重要机制及细胞学标志。进而，本发明的发明人经过一系列的实验发现并验证了SCAP基因为早发心肌梗死的致病基因，并进一步发现SCAP基因18号外显子上的c.3035C>T突变能够引起早发心肌梗死，为早发心肌梗死的致病突变。但是，本发明的SCAP基因突变位点c.3035C>T并未见报道。It should be noted that the SCAP gene is located on chromosome 3, consists of 23 exons, and contains a total of 1279 amino acids. It is the "central regulator" of lipid synthesis and uptake in mammals. When the intracellular cholesterol level decreases, it can undergo conformational changes, activate SREBP gene through its enzymatic cleavage, and promote its entry into the nucleus to complete a series of transcriptions Regulate the reaction, and then up-regulate a series of key enzymes of lipid metabolism including LDLR, HMGCoA, etc., thereby promoting the formation of cholesterol; on the contrary, when the intracellular cholesterol level increases, the SCAP gene can sense the excessively high intracellular cholesterol level and stop play its role in enzymatic cleavage. Therefore, SCAP is an important negative feedback mediator in cholesterol metabolism. Studies have shown that point mutations on the SCAP gene can cause a decrease in its sensitivity to changes in intracellular cholesterol levels, resulting in its inability to be inhibited by elevated cholesterol levels, resulting in an excessive increase in intracellular cholesterol levels: when this process occurs in When the main cholesterol-synthesizing tissues of the body, such as liver cells, will lead to hypercholesterolemia in the body; and when the process occurs in macrophages and smooth muscle cells, it will lead to their behavior as "foam cells", and "foam cells" The formation of is an important mechanism and cytological marker of the occurrence and development of myocardial infarction. Furthermore, the inventors of the present invention discovered and verified that the SCAP gene is the causative gene of premature myocardial infarction through a series of experiments, and further found that the c.3035C>T mutation on exon 18 of the SCAP gene can cause premature myocardial infarction Infarction, a pathogenic mutation for premature myocardial infarction. However, the SCAP gene mutation site c.3035C>T of the present invention has not been reported.
根据本发明的第二方面，本发明提出了一种分离的多肽。根据本发明的实施例，与野生型SCAP相比，该分离的多肽具有p.A1012V突变，即该突变是由于c.3035C>T的无义突变而引起的，具体地，该突变表示：该分离的多肽，由野生型SCAP的第1012位氨基酸A(丙氨酸)突变为V(缬氨酸)。根据本发明的一些具体示例，该多肽是由前述分离的编码SCAP突变体的核酸编码的。通过检测生物样品中是否表达该多肽，可以有效地检测生物样品是否易患早发心肌梗死，也可以通过检测这些多肽在生物体中是否存在，可以有效地预测生物体是否易患早发心肌梗死。According to a second aspect of the present invention, the present invention provides an isolated polypeptide. According to an embodiment of the present invention, compared with the wild-type SCAP, the isolated polypeptide has a p.A1012V mutation, that is, the mutation is caused by the c.3035C>T nonsense mutation, specifically, the mutation means: the The isolated polypeptide is mutated from amino acid A (alanine) at position 1012 of wild-type SCAP to V (valine). According to some specific examples of the present invention, the polypeptide is encoded by the aforementioned isolated nucleic acid encoding a SCAP mutant. By detecting whether the polypeptide is expressed in the biological sample, it can effectively detect whether the biological sample is prone to premature myocardial infarction, and by detecting whether these polypeptides exist in the organism, it can effectively predict whether the organism is prone to premature myocardial infarction .
根据本发明的第三方面，本发明提出了一种筛选易患早发心肌梗死的生物样品的方法。根据本发明的实施例，该方法包括以下步骤：According to a third aspect of the present invention, the present invention proposes a method for screening biological samples susceptible to premature myocardial infarction. According to an embodiment of the present invention, the method includes the following steps:
从所述生物样品提取核酸样本。根据本发明的实施例，生物样品的类型并不受特别限制，只要从该生物样品中能够提取到反映生物样品SCAP是否存在突变的核酸样本即可。根据本发明的实施例，生物样品可以为选自人体血液、皮肤、皮下组织的至少一种，优选外周血。由此，可以方便地进行取样和检测，从而能够进一步提高筛选易患早发心肌梗死的生物样品的效率。根据本发明的实施例，这里所使用的术语“核酸样本”应做广义理解，其可以是任何能够反映生物样品中SCAP是否存在突变的样本，例如可以是从生物样品中直接提取的全基因组DNA，也可以是该全基因组中包含SCAP编码序列的一部分，可以是从生物样品中提取的总RNA，也可以是从生物样品中提取的mRNA。根据本发明的一个实施例，所述核酸样本为全基因组DNA。由此，可以扩大生物样品的来源范围，并且可以同时对生物样品的多种信息进行确定，从而能够提高筛选易患早发心肌梗死的生物样品的效率。另外，根据本发明的实施例，针对采用RNA作为核酸样本，从生物样品提取核酸样本可以进一步包括：从生物样品提取RNA样本，优选RNA样本为mRNA；以及基于所得到的RNA样本，通过反转录反应，获得cDNA样本，所得到的cDNA样本构成核酸样本。由此，可以进一步提高利用RNA作为核酸样本筛选易患早发心肌梗死的生物样品的效率。A nucleic acid sample is extracted from the biological sample. According to the embodiments of the present invention, the type of the biological sample is not particularly limited, as long as a nucleic acid sample reflecting whether there is a mutation in the SCAP of the biological sample can be extracted from the biological sample. According to an embodiment of the present invention, the biological sample may be at least one selected from human blood, skin, and subcutaneous tissue, preferably peripheral blood. Thus, sampling and detection can be conveniently performed, thereby further improving the efficiency of screening biological samples susceptible to premature myocardial infarction. According to an embodiment of the present invention, the term "nucleic acid sample" used here should be understood in a broad sense, and it can be any sample that can reflect whether there is a mutation in SCAP in a biological sample, for example, it can be whole genome DNA directly extracted from a biological sample , can also be a part of the whole genome containing the SCAP coding sequence, can be total RNA extracted from biological samples, and can also be mRNA extracted from biological samples. According to an embodiment of the present invention, the nucleic acid sample is whole genome DNA. In this way, the range of sources of biological samples can be expanded, and various information of biological samples can be determined at the same time, thereby improving the efficiency of screening biological samples susceptible to premature myocardial infarction. In addition, according to an embodiment of the present invention, for using RNA as a nucleic acid sample, extracting a nucleic acid sample from a biological sample may further include: extracting an RNA sample from a biological sample, preferably the RNA sample is mRNA; and based on the obtained RNA sample, by inversion The reaction is recorded to obtain a cDNA sample, and the obtained cDNA sample constitutes a nucleic acid sample. Thus, the efficiency of using RNA as a nucleic acid sample to screen biological samples susceptible to premature myocardial infarction can be further improved.
接下来，在得到核酸样本之后，可以对核酸样本进行分析，从而能够确定所得到核酸样本的核酸序列。根据本发明的实施例，确定所得到核酸样本的核酸序列的方法和设备并不受特别限制。根据本发明的具体实施例，可以通过测序方法，确定核酸样本的核酸序列。根据本发明的实施例，可以用于进行测序的方法和设备并不受特别限制。根据本发明的实施例，可以采用第二代测序技术，也可以采用第三代以及第四代或者更先进的测序技术。根据本发明的具体示例，可以利用选自IlluminaHiSeq4000、SOLiD、454和单分子测序装置的至少一种对核酸序列进行测序。由此，结合最新的测序技术，针对单个位点可以达到较高的测序深度，检测灵敏度和准确性大大提高，因而能够利用这些测序装置的高通量、深度测序的特点，进一步提高对核酸样本进行检测分析的效率。从而，能够提高后续对测序数据进行分析时的精确性和准确度。由此，根据本发明的实施例，确定核酸样本的核酸序列可以进一步包括：首先，针对所得到的核酸样本，构建核酸测序文库；以及对所得到的核酸测序文库进行测序，以便获得由多个测序数据构成的测序结果。根据本发明的一些实施例，可以采用选自IlluminaHiSeq4000、SOLiD、454和单分子测序装置的至少一种对所得到的核酸测序文库进行测序。另外，根据本发明的实施例，可以对核酸样本进行筛选，富集SCAP外显子，该筛选富集可以在构建测序文库之前，构建测序文库过程中，或者构建测序文库之后进行。根据本发明的一个实施例，针对核酸样本，构建核酸测序文库进一步包括：利用SCAP外显子特异性引物，对核酸样本进行PCR扩增；以及针对所得到的扩增产物，构建核酸测序文库。由此，可以通过PCR扩增，富集SCAP外显子(尤其是第18号外显子)，从而能够进一步提高筛选易患早发心肌梗死的生物样品的效率。根据本发明的实施例，SCAP外显子特异性引物的序列不受特别限制，根据本发明的优选实施例，这些SCAP外显子特异性引物(针对SCAP的18号外显子)具有SEQIDNO：37和38所示的核苷酸序列：Next, after the nucleic acid sample is obtained, the nucleic acid sample can be analyzed, so that the nucleic acid sequence of the obtained nucleic acid sample can be determined. According to the embodiments of the present invention, the method and device for determining the nucleic acid sequence of the obtained nucleic acid sample are not particularly limited. According to a specific embodiment of the present invention, the nucleic acid sequence of the nucleic acid sample can be determined by a sequencing method. According to the embodiments of the present invention, the methods and devices that can be used for sequencing are not particularly limited. According to the embodiment of the present invention, the second-generation sequencing technology may be used, and the third-generation and fourth-generation or more advanced sequencing technologies may also be used. According to a specific example of the present invention, the nucleic acid sequence can be sequenced by using at least one selected from Illumina HiSeq4000, SOLiD, 454, and a single-molecule sequencing device. Therefore, combined with the latest sequencing technology, a higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved. Therefore, the high-throughput and deep sequencing characteristics of these sequencing devices can be used to further improve the detection of nucleic acid samples. Efficiency in conducting assays. Therefore, the precision and accuracy of the subsequent analysis of the sequencing data can be improved. Thus, according to an embodiment of the present invention, determining the nucleic acid sequence of the nucleic acid sample may further include: first, constructing a nucleic acid sequencing library for the obtained nucleic acid sample; and sequencing the obtained nucleic acid sequencing library, so as to obtain multiple Sequencing results composed of sequencing data. According to some embodiments of the present invention, at least one selected from Illumina HiSeq4000, SOLiD, 454 and single-molecule sequencing devices can be used to sequence the obtained nucleic acid sequencing library. In addition, according to the embodiments of the present invention, nucleic acid samples can be screened to enrich SCAP exons, and the screening and enrichment can be performed before, during, or after the construction of the sequencing library. According to an embodiment of the present invention, constructing a nucleic acid sequencing library for a nucleic acid sample further includes: performing PCR amplification on the nucleic acid sample using SCAP exon-specific primers; and constructing a nucleic acid sequencing library for the obtained amplification product. Thus, the SCAP exon (especially the No. 18 exon) can be enriched by PCR amplification, thereby further improving the efficiency of screening biological samples susceptible to premature myocardial infarction. According to an embodiment of the present invention, the sequence of the SCAP exon-specific primer is not particularly limited. According to a preferred embodiment of the present invention, these SCAP exon-specific primers (for Exon 18 of SCAP) have SEQ ID NO: 37 and the nucleotide sequence shown in 38:
SCAP-18F：gactccccaggctatgact(SEQIDNO：37)；SCAP-18F: gactccccaggctatgact (SEQ ID NO: 37);
SCAP-18R：acagcagttgaagagaaccag(SEQIDNO：38)。SCAP-18R: acagcagttgaagagaaccag (SEQ ID NO: 38).
发明人惊奇地发现，通过采用这些引物，可以在PCR反应体系中通过显著有效地完成对SCAP外显子的扩增。需要说明的是，这些SEQIDNO：37和SEQIDNO：38所示的核苷酸序列是本发明的发明人在付出了艰苦的劳动后，意外获得的。The inventors surprisingly found that by using these primers, the amplification of SCAP exons can be accomplished significantly and efficiently in the PCR reaction system. It should be noted that the nucleotide sequences shown in SEQ ID NO: 37 and SEQ ID NO: 38 were accidentally obtained by the inventors of the present invention after hard work.
关于针对核酸样本，构建测序文库的方法和流程，本领域技术人员可以根据不同的测序技术进行适当选择，关于流程的细节，可以参见测序仪器的厂商例如Illumina公司所提供的规程，例如参见Illumina公司MultiplexingSamplePreparationGuide(Part#1005361；Feb2010)或Paired-EndSamplePrepGuide(Part#1005063；Feb2010)，通过参照将其并入本文。根据本发明的实施例，从生物样品提取核酸样本的方法和设备，也不受特别限制，可以采用商品化的核酸提取试剂盒进行。Regarding the method and process for constructing a sequencing library for nucleic acid samples, those skilled in the art can make appropriate choices according to different sequencing technologies. For details on the process, refer to the procedures provided by manufacturers of sequencing instruments such as Illumina, for example, see Illumina MultiplexingSamplePreparationGuide (Part#1005361; Feb2010) or Paired-EndSamplePrepGuide (Part#1005063; Feb2010), which are incorporated herein by reference. According to the embodiments of the present invention, the method and equipment for extracting nucleic acid samples from biological samples are not particularly limited, and commercial nucleic acid extraction kits can be used.
需要说明的是，在这里所使用的术语“核酸序列”应作广义理解，其可以是在对核酸样本进行测序得到的测序数据进行组装后，得到的完整的核酸序列信息，也可以是直接采用通过对核酸样本进行测序所得到的测序数据(reads)作为核酸序列，只要这些核酸序列中含有对应SCAP的编码序列即可。It should be noted that the term "nucleic acid sequence" used here should be understood in a broad sense. It can be the complete nucleic acid sequence information obtained after the sequencing data obtained by sequencing nucleic acid samples are assembled, or it can be obtained directly. Sequencing data (reads) obtained by sequencing nucleic acid samples are used as nucleic acid sequences, as long as these nucleic acid sequences contain coding sequences corresponding to SCAP.
最后，在确定核酸样本的核酸序列之后，将所得到的核酸样本的核酸序列与SEQIDNO：1的序列相比对。如果在所得到的核酸序列中具有c.3035C>T突变，则指示生物样品易患早发心肌梗死。由此，通过根据本发明实施例的筛选易患早发心肌梗死的生物样品的方法，可以有效地筛选易患早发心肌梗死的生物样品。根据本发明的实施例，对核酸序列与SEQIDNO：1进行比对的方法和设备并不受特别限制，可以采用任意常规的软件进行操作，根据本发明的具体实例，可以采用SOAP软件进行比对。Finally, after the nucleic acid sequence of the nucleic acid sample is determined, the obtained nucleic acid sequence of the nucleic acid sample is compared with the sequence of SEQ ID NO:1. If there is a c.3035C>T mutation in the resulting nucleic acid sequence, it indicates that the biological sample is susceptible to premature myocardial infarction. Thus, biological samples susceptible to premature myocardial infarction can be effectively screened through the method for screening biological samples prone to premature myocardial infarction according to the embodiments of the present invention. According to the embodiments of the present invention, the method and equipment for comparing the nucleic acid sequence with SEQIDNO: 1 are not particularly limited, and any conventional software can be used for operation. According to specific examples of the present invention, SOAP software can be used for comparison .
根据本发明的实施例，所述早发心肌梗死为常染色体遗传性心肌梗死。According to an embodiment of the present invention, the premature myocardial infarction is autosomal hereditary myocardial infarction.
需要说明的是，根据本发明实施例的“筛选易患早发心肌梗死的生物样品的方法”的用途不受特别限制，例如可以用作非诊断目的的筛选方法。It should be noted that the application of the "method for screening biological samples prone to premature myocardial infarction" according to the embodiment of the present invention is not particularly limited, for example, it can be used as a screening method for non-diagnostic purposes.
筛选易患早发心肌梗死的生物样品的系统和试剂盒Systems and kits for screening biological samples predisposed to premature myocardial infarction
根据本发明的第四方面，本发明提出了一种能够有效实施上述筛选易患早发心肌梗死的生物样品的方法的系统。According to a fourth aspect of the present invention, the present invention proposes a system capable of effectively implementing the above method of screening biological samples susceptible to premature myocardial infarction.
参考图1，根据本发明的实施例，该筛选易患早发心肌梗死的生物样品的系统1000包括核酸提取装置100、核酸序列确定装置200以及判断装置300。Referring to FIG. 1 , according to an embodiment of the present invention, the system 1000 for screening biological samples susceptible to premature myocardial infarction includes a nucleic acid extraction device 100 , a nucleic acid sequence determination device 200 and a judging device 300 .
根据本发明的实施例，核酸提取装置100用于从生物样品提取核酸样本。如前所述，根据本发明的实施例，核酸样本的类型并不受特别限制，对于采用RNA作为核酸样本，则核酸提取装置进一步包括RNA提取单元101和反转录单元102，其中，提取单元101用于从生物样品提取RNA样本，反转录单元102与RNA提取单元101相连，用于对RNA样本进行反转录反应，以便获得cDNA样本，所得到的cDNA样本构成核酸样本。According to an embodiment of the present invention, the nucleic acid extraction device 100 is used to extract nucleic acid samples from biological samples. As mentioned above, according to the embodiment of the present invention, the type of nucleic acid sample is not particularly limited. For using RNA as a nucleic acid sample, the nucleic acid extraction device further includes an RNA extraction unit 101 and a reverse transcription unit 102, wherein the extraction unit 101 is used to extract RNA samples from biological samples, and the reverse transcription unit 102 is connected to the RNA extraction unit 101, and is used to perform reverse transcription reaction on the RNA samples to obtain cDNA samples, and the obtained cDNA samples constitute nucleic acid samples.
根据本发明的实施例，核酸序列确定装置200与核酸提取装置100相连，用于对核酸样本进行分析，以便确定核酸样本的核酸序列。如前所示，可以采用测序的方法确定核酸样本的核酸序列。由此，根据本发明的一个实施例，所述核酸序列确定装置200可以进一步包括：文库构建单元201以及测序单元202。文库构建单元201用于针对核酸样本，构建核酸测序文库；测序单元202与文库构建单元201相连，用于对核酸测序文库进行测序，以便获得由多个测序数据构成的测序结果。如前所述，可以通过PCR扩增，富集SCAP外显子，进一步提高筛选易患早发心肌梗死的生物样品的效率。由此，文库构建单元201可以进一步包括PCR扩增模块(图中未示出)，在该PCR扩增模块中设置有SCAP外显子特异性引物，以便利用SCAP外显子特异性引物，对所述核酸样本进行PCR扩增，根据本发明的具体实施例，SCAP外显子特异性引物(针对SCAP的18号外显子)具有如SEQIDNO：37和38所示的核苷酸序列。根据本发明的实施例，测序单元202可以包括选自ILLUMINAHISEQ4000、SOLiD、454和单分子测序装置的至少一种。由此，结合最新的测序技术，针对单个位点可以达到较高的测序深度，检测灵敏度和准确性大大提高，因而能够利用这些测序装置的高通量、深度测序的特点，进一步提高对核酸样本进行检测分析的效率。从而，提高后续对测序数据进行分析时的精确性和准确度。According to an embodiment of the present invention, the nucleic acid sequence determination device 200 is connected to the nucleic acid extraction device 100 and is used to analyze the nucleic acid sample so as to determine the nucleic acid sequence of the nucleic acid sample. As mentioned above, the nucleic acid sequence of the nucleic acid sample can be determined by sequencing. Therefore, according to an embodiment of the present invention, the nucleic acid sequence determination device 200 may further include: a library construction unit 201 and a sequencing unit 202 . The library construction unit 201 is used for constructing a nucleic acid sequencing library for nucleic acid samples; the sequencing unit 202 is connected to the library construction unit 201 and used for sequencing the nucleic acid sequencing library, so as to obtain a sequencing result composed of multiple sequencing data. As mentioned above, the SCAP exons can be enriched by PCR amplification to further improve the efficiency of screening biological samples susceptible to premature myocardial infarction. Thus, the library construction unit 201 can further include a PCR amplification module (not shown in the figure), in which the SCAP exon-specific primers are set, so that the SCAP exon-specific primers can be used to The nucleic acid sample is subjected to PCR amplification. According to a specific embodiment of the present invention, the SCAP exon-specific primer (for exon 18 of SCAP) has the nucleotide sequences shown in SEQ ID NO: 37 and 38. According to an embodiment of the present invention, the sequencing unit 202 may include at least one selected from ILLUMINAHISEQ4000, SOLiD, 454, and single-molecule sequencing devices. Therefore, combined with the latest sequencing technology, a higher sequencing depth can be achieved for a single site, and the detection sensitivity and accuracy are greatly improved. Therefore, the high-throughput and deep sequencing characteristics of these sequencing devices can be used to further improve the detection of nucleic acid samples. Efficiency in conducting assays. Therefore, the precision and accuracy of the subsequent analysis of the sequencing data are improved.
根据本发明的实施例，判断装置300与核酸序列确定装置200相连，适于将核酸样本的核酸序列进行比对，以便基于核酸样本的核酸序列与SEQIDNO：1的区别判断生物样品是否易患早发心肌梗死。具体地，基于核酸样本的核酸序列与SEQIDNO：1相比，是否具有c.3035C>T突变，判断生物样品是否易患早发心肌梗死。如前所述，根据本发明的一个实施例，核酸样本的核酸序列与SEQIDNO：1相比，具有c.3035C>T突变，是生物样品易患早发心肌梗死的指示。如前所述，根据本发明的实施例，对核酸序列与SEQIDNO：1进行比对的设备并不受特别限制，可以采用任意常规的软件进行操作，根据本发明的具体实例，可以采用SOAP软件进行比对。According to an embodiment of the present invention, the judging device 300 is connected to the nucleic acid sequence determining device 200, and is suitable for comparing the nucleic acid sequences of the nucleic acid samples, so as to judge whether the biological sample is susceptible to premature disease based on the difference between the nucleic acid sequence of the nucleic acid sample and SEQ ID NO: 1. Myocardial infarction. Specifically, based on whether the nucleic acid sequence of the nucleic acid sample has the c.3035C>T mutation compared with SEQ ID NO: 1, it is determined whether the biological sample is susceptible to premature myocardial infarction. As mentioned above, according to an embodiment of the present invention, the nucleic acid sequence of the nucleic acid sample has a c.3035C>T mutation compared with SEQ ID NO: 1, which is an indication that the biological sample is susceptible to premature myocardial infarction. As mentioned above, according to the embodiments of the present invention, the equipment for comparing the nucleic acid sequence with SEQIDNO: 1 is not particularly limited, and any conventional software can be used for operation. According to specific examples of the present invention, SOAP software can be used Compare.
由此，利用该系统，能够有效地实施前述筛选易患早发心肌梗死的生物样品的方法，从而可以有效地筛选易患早发心肌梗死的生物样品。Thus, using this system, the aforementioned method for screening biological samples susceptible to premature myocardial infarction can be effectively implemented, thereby effectively screening biological samples susceptible to premature myocardial infarction.
根据本发明的第五方面，本发明提出了一种用于筛选易患早发心肌梗死的生物样品的试剂盒。根据本发明的实施例，该用于筛选易患早发心肌梗死的生物样品的试剂盒包括：适于检测SCAP基因突变体的试剂，其中与SEQIDNO：1相比，该SCAP基因突变体具有c.3035C>T突变。利用根据本发明的实施例的试剂盒，能够有效地筛选易患早发心肌梗死的生物样品。在本文中，所使用的术语“适于检测SCAP基因突变体的试剂”应做广义理解，即可以是检测SCAP编码基因的试剂，也可以是检测SCAP突变体多肽的试剂，例如可以采用识别特异性位点的抗体。根据本发明的一个实施例，所述试剂为核酸探针或引物，优选地，所述核酸探针或引物具有如SEQIDNO：37-38所示的核苷酸序列。由此，可以高效地筛选易患早发心肌梗死的生物样品。According to the fifth aspect of the present invention, the present invention proposes a kit for screening biological samples susceptible to premature myocardial infarction. According to an embodiment of the present invention, the kit for screening biological samples susceptible to premature myocardial infarction includes: a reagent suitable for detecting a SCAP gene mutant, wherein compared with SEQ ID NO: 1, the SCAP gene mutant has c .3035C>T mutation. Using the kit according to the embodiment of the present invention, biological samples susceptible to premature myocardial infarction can be efficiently screened. In this paper, the term "reagents suitable for detecting SCAP gene mutants" should be understood in a broad sense, that is, it can be a reagent for detecting SCAP coding genes, or a reagent for detecting SCAP mutant polypeptides, for example, it can be used to identify specific Antibodies to sex loci. According to an embodiment of the present invention, the reagent is a nucleic acid probe or primer, preferably, the nucleic acid probe or primer has a nucleotide sequence as shown in SEQ ID NO: 37-38. Thus, biological samples susceptible to premature myocardial infarction can be efficiently screened.
需要说明的是，在本文前面筛选易患早发心肌梗死的生物样品的方法部分中所描述的特征和优点，同样适用于筛选易患早发心肌梗死的生物样品的系统或者试剂盒，在此不再赘述。It should be noted that the features and advantages described in the method for screening biological samples susceptible to premature myocardial infarction are also applicable to the system or kit for screening biological samples susceptible to premature myocardial infarction. Herein No longer.
构建体及重组细胞Constructs and Recombinant Cells
根据本发明的第六方面，本发明还提出了一种构建体。根据本发明的实施例，该构建体包含前面所述的分离的编码SCAP突变体的核酸，即本发明的SCAP基因突变体。由此，利用本发明的构建体转化受体细胞获得的重组细胞，能够有效地用于筛选治疗早发心肌梗死的药物。其中，所述受体细胞的种类不受特别限制，例如可以为大肠杆菌细胞、哺乳动物细胞，优选该受体细胞来源于哺乳动物。According to the sixth aspect of the present invention, the present invention also proposes a construct. According to an embodiment of the present invention, the construct comprises the aforementioned isolated nucleic acid encoding a SCAP mutant, that is, the SCAP gene mutant of the present invention. Thus, the recombinant cells obtained by transforming recipient cells with the construct of the present invention can be effectively used for screening drugs for treating premature myocardial infarction. Wherein, the type of the recipient cells is not particularly limited, for example, Escherichia coli cells, mammalian cells, preferably the recipient cells are derived from mammals.
在本发明中所使用的术语“构建体”是指这样的一种遗传载体，其包含特定核酸序列，并且能够将目的核酸序列转入宿主细胞中，以获得重组细胞。根据本发明的实施例，构建体的形式不受特别限制。根据本发明的实施例，其可以为质粒、噬菌体、人工染色体、粘粒(Cosmid)、病毒的至少一种，优选质粒。质粒作为遗传载体，具有操作简单，可以携带较大片段的性质，便于操作和处理。质粒的形式也不受特别限制，既可以是环形质粒，也可以是线性质粒，即可以是单链的，也可以是双链的。本领域技术人员可以根据需要进行选择。在本发明中所使用的术语“核酸”可以是任何包含脱氧核糖核苷酸或者核糖核苷酸的聚合物，包括但不限于经过修饰的或者未经修饰的DNA、RNA，其长度不受任何特别限制。对于用于构建重组细胞的构建体，优选所述核酸为DNA，因为DNA相对于RNA而言，其更稳定，并且易于操作。The term "construct" used in the present invention refers to a genetic carrier that contains a specific nucleic acid sequence and is capable of transferring the target nucleic acid sequence into a host cell to obtain a recombinant cell. According to the embodiments of the present invention, the form of the construct is not particularly limited. According to an embodiment of the present invention, it may be at least one of a plasmid, a phage, an artificial chromosome, a cosmid (Cosmid), and a virus, preferably a plasmid. As a genetic carrier, plasmids are easy to operate and can carry large fragments, which is convenient for operation and processing. The form of the plasmid is also not particularly limited, and it can be either a circular plasmid or a linear plasmid, that is, it can be single-stranded or double-stranded. Those skilled in the art can make selections as needed. The term "nucleic acid" used in the present invention can be any polymer comprising deoxyribonucleotides or ribonucleotides, including but not limited to modified or unmodified DNA, RNA, its length is not subject to any Special restrictions. For constructs for constructing recombinant cells, the nucleic acid is preferably DNA, because DNA is more stable and easier to handle than RNA.
根据本发明的第七方面，本发明还提出了一种重组细胞。根据本发明的实施例，该重组细胞是通过前面所述的构建体转化受体细胞而获得的。从而，本发明的重组细胞能够表达构建体所携带的SCAP基因突变体。根据本发明的一些实施例，利用本发明的重组细胞，能够有效地筛选治疗早发心肌梗死的药物。根据本发明的实施例，受体细胞的种类不受特别限制，例如可以为大肠杆菌细胞、哺乳动物细胞，优选所述受体细胞来源于非人哺乳动物。According to the seventh aspect of the present invention, the present invention also provides a recombinant cell. According to an embodiment of the present invention, the recombinant cell is obtained by transforming the recipient cell with the aforementioned construct. Thus, the recombinant cells of the present invention can express the SCAP gene mutant carried by the construct. According to some embodiments of the present invention, the recombinant cells of the present invention can be used to effectively screen drugs for treating premature myocardial infarction. According to the embodiment of the present invention, the type of recipient cells is not particularly limited, for example, it can be Escherichia coli cells, mammalian cells, preferably the recipient cells are derived from non-human mammals.
下面参考具体实施例，对本发明进行说明，需要说明的是，这些实施例仅仅是说明性的，而不能理解为对本发明的限制。The present invention will be described below with reference to specific embodiments. It should be noted that these embodiments are only illustrative and should not be construed as limiting the present invention.
若未特别指明，实施例中所采用的技术手段为本领域技术人员所熟知的常规手段，可以参照《分子克隆实验指南》第三版或者相关产品进行，所采用的试剂和产品也均为可商业获得的。未详细描述的各种过程和方法是本领域中公知的常规方法，所用试剂的来源、商品名以及有必要列出其组成成分者，均在首次出现时标明，其后所用相同试剂如无特殊说明，均以首次标明的内容相同。Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and can be carried out with reference to the third edition of the "Molecular Cloning Experiment Guide" or related products, and the reagents and products used are also available. commercially acquired. Various processes and methods that are not described in detail are conventional methods well known in the art. The source, trade name and necessary list of components of the reagents used are all indicated when they appear for the first time. Descriptions are the same as those indicated for the first time.
实施例1确定PMI致病突变Embodiment 1 determines PMI pathogenic mutation
1、样本收集1. Sample collection
发明人收集到一个5代的中国早发心肌梗死(在本文中有时也简称为PMI)患者家系，其家系图见图2。如图2所示，如图2所示，●表示患病女性，■表示患病男性；○表示健康女性，□表示健康男性；表示已故女性，表示已故男性；箭头所指为先证者。该家系共有四代人存活，共19个成员，其中早发心肌梗死患者3人(男性2人，女性1人)。The inventor collected a 5-generation pedigree of Chinese patients with premature myocardial infarction (sometimes referred to as PMI in this paper), the pedigree diagram of which is shown in FIG. 2 . As shown in Figure 2, ● means sick women, ■ means sick men; ○ means healthy women, □ means healthy men; means a deceased woman, Indicates a deceased male; the arrow points to the proband. Four generations of the family survived, with a total of 19 members, including 3 patients with premature myocardial infarction (2 males and 1 female).
发明人对所有患者均进行了全面的体格、血液生化及影像学检查。所有的临床诊断或排除诊断均基于心肌梗死诊断的金标准，即：结合患者典型心肌梗死症状及心电图表现的基础上，冠状动脉造影阳性确诊心肌梗死诊断，冠状动脉CT造影阴性作为排除心肌梗死诊断标准。其中，先证者PMI1-1(如图2示)，为一中年女性，于39岁发生急性心肌梗死；心肌梗死发生前3年间，间断出现典型劳力性心绞痛症状，并呈进行性加重，于39岁时发生心肌梗死，冠状动脉造影确诊为心肌梗死，后择期共植入支架4枚；既往有发现高血压、高血脂4年，未规律服药；有高脂血症及早发冠心病家族史。The inventor has all carried out comprehensive physical examination, blood biochemical examination and imaging examination to all patients. All clinical diagnoses or excluded diagnoses are based on the gold standard for the diagnosis of myocardial infarction, that is, based on the typical symptoms of myocardial infarction and ECG findings, a positive coronary angiography can confirm the diagnosis of myocardial infarction, and a negative coronary CT angiography can be used to exclude the diagnosis of myocardial infarction standard. Among them, the proband PMI1-1 (as shown in Figure 2) was a middle-aged female who suffered from acute myocardial infarction at the age of 39. During the 3 years before the occurrence of myocardial infarction, typical symptoms of exertional angina pectoris appeared intermittently, and the symptoms were progressively aggravated. Myocardial infarction occurred at the age of 39. Coronary angiography was diagnosed as myocardial infarction, and a total of 4 stents were implanted at a later date; hypertension and hyperlipidemia were found in the past for 4 years, and he did not take medicine regularly; he had a family with hyperlipidemia and premature coronary heart disease history.
发明人收集获得上述PMI患者家系中尚在世的3个患者以及16个非早发心梗患病者(即家系内正常人)的样本。The inventor collected samples from 3 living patients and 16 non-premature myocardial infarction patients (ie, normal people in the family) in the above-mentioned PMI patient family.
2、全外显子组测序2. Whole exome sequencing
发明人利用AgilentSureSelectHumanAllExonV5试剂盒结合illuminaHiSeq4000平台，采用PE150测序策略，对图2所示的PMI患者家系中的三名患者(PMI1-1、PMI1-3及PMI1-8)和16个家系内正常人进行了全外显子组测序，具体步骤如下：The inventors used the AgilentSureSelectHumanAllExonV5 kit in combination with the illuminaHiSeq4000 platform, and adopted the PE150 sequencing strategy to conduct three patients (PMI1-1, PMI1-3 and PMI1-8) in the PMI patient family shown in Figure 2 and 16 normal persons in the family. Whole-exome sequencing was performed, and the specific steps were as follows:
2.1DNA提取2.1 DNA extraction
采集图2所示PMI患者家系中的三名患者和16个家系内正常人的外周血，利用常规盐析法从外周血样品中提取各家系成员的基因组DNA，并利用分光光度计测量DNA的浓度及纯度，所得各基因组DNA的OD260/OD280均应位于1.7-2.0之间，浓度不少于200ng/微升，总量不少于3微克。The peripheral blood of three patients and normal persons in 16 families of PMI patients shown in Figure 2 was collected, and the genomic DNA of each family member was extracted from the peripheral blood samples by the conventional salting-out method, and the DNA was measured by a spectrophotometer The OD 260 /OD 280 of each genomic DNA obtained should be between 1.7 and 2.0, the concentration should not be less than 200ng/microliter, and the total amount should not be less than 3 micrograms.
2.2外显子捕获与测序2.2 Exon capture and sequencing
利用超声波仪(CovarisS2,Massachusetts,USA)将各基因组DNA样本随机打断成150-200bp左右的片段，随后按照制造商提供的操作说明书，在片段两端分别连接上接头制备DNA文库(可参见：http://www.illumina.com/提供的Illumina/Solexa标准建库说明书，通过参照将其全文并入本文)。Each genomic DNA sample was randomly broken into fragments of about 150-200 bp using a sonicator (CovarisS2, Massachusetts, USA), and then according to the operating instructions provided by the manufacturer, adapters were connected to both ends of the fragments to prepare a DNA library (see: Illumina/Solexa Standard Library Construction Instructions provided at http://www.illumina.com/, which is hereby incorporated by reference in its entirety).
然后，采用AgilentSureSelectHumanAllExonV5试剂盒，将上述制备获得的DNA文库与生物素标记的RNA探针进行液相杂交(95℃5分钟，65℃24小时)，再使用带链霉素的磁珠将DNA-RNA混合物捕获下来，然后用Qiagen提纯试剂盒将DNA洗脱下来，再利用AgilentPCR引物将捕获得到的DNA扩增12个循环，结果，50M区域的DNA序列，20,965个基因的334,378个外显子被捕获下来。Then, using the AgilentSureSelectHumanAllExonV5 kit, the DNA library prepared above was subjected to liquid-phase hybridization with biotin-labeled RNA probes (95°C for 5 minutes, 65°C for 24 hours), and then the DNA- The RNA mixture was captured, and then the DNA was eluted with a Qiagen purification kit, and the captured DNA was amplified for 12 cycles using AgilentPCR primers. As a result, the DNA sequence of the 50M region, 334,378 exons of 20,965 genes were capture it.
进一步，利用安捷伦生物分析仪DNA芯片进行文库质检，合格后即可上机测序，以便获得原始测序数据。其中，测序平台为IlluminaHiseq4000，读取长度为90bp，各样本的平均测序深度最少为150×。Furthermore, the Agilent bioanalyzer DNA chip is used for library quality inspection, and after passing the test, it can be sequenced on the machine to obtain the original sequencing data. Among them, the sequencing platform is IlluminaHiseq4000, the read length is 90bp, and the average sequencing depth of each sample is at least 150×.
3、数据质控、变异检测、注释及变异过滤3. Data quality control, variation detection, annotation and variation filtering
3.1数据质控3.1 Data quality control
Hiseq4000测序得到的原始图像数据rawdata，以fastq文件格式存储(文件名:*.fq)。Rawdata中会包含接头信息，低质量碱基，未测出的碱基(以N表示)，这些信息会对后续的信息分析造成很大的干扰，分析前需要将这些干扰信息去除掉，最终得到的数据即为有效数据。The original image data rawdata obtained by Hiseq4000 sequencing is stored in fastq file format (file name: *.fq). Rawdata will contain linker information, low-quality bases, and undetected bases (indicated by N), which will cause great interference to subsequent information analysis. These interference information need to be removed before analysis, and finally get The data is valid data.
具体地，首先利用IlluminabasecallingSoftware1.7对上述获得的原始测序数据进行处理，即按照以下条件进行过滤去污染：Specifically, first use IlluminabasecallingSoftware1.7 to process the raw sequencing data obtained above, that is, filter and decontaminate according to the following conditions:
A.过滤掉含有接头序列的reads；A. Filter out reads containing adapter sequences;
B.当单端测序read中含有的N的含量超过该条read长度比例的10％时，需要去除此对pairedreads；B. When the N content contained in the single-end sequencing read exceeds 10% of the length ratio of the read, the pair of pairedreads needs to be removed;
C.当单端测序read中含有的低质量(碱基质量值小于5)碱基数超过该条read长度比例的50％时，需要去除此对pairedreads。经过对测序数据的严格过滤，得到高质量的cleandata。C. When the number of low-quality (base quality value less than 5) bases contained in the single-end sequencing read exceeds 50% of the length ratio of the read, the pair of pairedreads needs to be removed. After strict filtering of the sequencing data, high-quality cleandata were obtained.
进而，对产出数据进行统计，包括测序read数量，数据产量，测序错误率，Q20含量，Q30含量，GC含量等。Furthermore, the output data is counted, including the number of sequencing reads, data output, sequencing error rate, Q20 content, Q30 content, GC content, etc.
3.2序列比对3.2 Sequence Alignment
进一步，使用SOAPaligner/SOAP2(可参见：LiR,LiY,KristiansenK,etal,SOAP:shortoligonucleotidealignmentprogram.Bioinformatics2008,24(5):713-714；LiR,YuC,LiY,eaal,SOAP2:animprovedultrafasttoolforshortreadalignment.Bioinformatics2009,25(15):1966-1967，通过参照将其全文并入本文)比对到UCSC人类参考基因组(hg19,build37.1，http://genome.ucsc.edu/)，以便获得比对到基因组上的唯一比对序列。Further, using SOAPaligner/SOAP2 (see: LiR, LiY, KristiansenK, et al, SOAP: shortoligonucleotide alignmentprogram. Bioinformatics2008, 24 (5): 713-714; LiR, YuC, LiY, eaal, SOAP2: animprovedultrafasttoolforshortreadalignment. Bioinformatics2009, 25 (15 ):1966-1967, which is incorporated herein by reference in its entirety) to the UCSC Human Reference Genome (hg19, build37.1, http://genome.ucsc.edu/), in order to obtain the unique Align sequences.
具体地，在制备文库的过程中，由于PCR扩增过程中会存在一些偏差，也就是说有的序列会被过量扩增。这样，在比对的时候，这些过量扩增出来的完全相同的序列就会比对到基因组的相同位置。而这些过量扩增的reads并不是基因组自身固有序列，不能作为变异检测的证据，因此，要尽量去除这些由PCR扩增所形成的duplicates，这一步可以使用picard来完成。对结果应该没有什么影响，GATK后期分析时可以忽略这一部分。并且，在indel附近的比对会出现大量的碱基错配，对碱基质量值进行重新校正和检测变异不利。因此，需要将比对到indel附近的reads进行局部重新比对，将比对的错误率降到最低。GATK分析流程中，RealignerTargetCreator是用于确定要进行重新比对的区域，IndelRealigner是用于对这些区域内进行重新比对。Specifically, in the process of preparing the library, due to some deviations in the PCR amplification process, that is to say, some sequences will be over-amplified. In this way, during alignment, these over-amplified identical sequences will be aligned to the same position in the genome. However, these excessively amplified reads are not the inherent sequence of the genome itself and cannot be used as evidence for mutation detection. Therefore, these duplicates formed by PCR amplification should be removed as much as possible. This step can be done using picard. It should have no effect on the results, and this part can be ignored in the later analysis of GATK. Moreover, there will be a large number of base mismatches in the alignment near the indel, which is unfavorable for recalibrating the base quality value and detecting mutations. Therefore, it is necessary to locally re-align the reads near the indel to minimize the error rate of the alignment. In the GATK analysis process, RealignerTargetCreator is used to determine the areas to be re-aligned, and IndelRealigner is used to re-align within these areas.
3.3变异检测和功能注释3.3 Variant Detection and Functional Annotation
然后利用SOAPsnp(可参见：LiR,LiY,FangX,YangH,etal,SNPdetectionformassivelyparallelwhole-genomeresequencing.GenomeRes2009,19(6):1124-1132，通过参照将其全文并入本文)确定靶区域的基因型。The genotype of the target region was then determined using SOAPsnp (see: LiR, LiY, FangX, YangH, et al, SNP detection formally parallel whole-genomeresequencing. GenomeRes 2009, 19(6): 1124-1132, which is hereby incorporated by reference in its entirety).
具体地，最终的BAM文件用GATK的HaplotypeCaller模块进行SNP/INDEL检测。变异结果利用ANNOVAR进行变异注释，其中，基于dbSNP和1000Genome数据库，进行突变位置，突变类型，保守区域预测。而针对外显子区突变，则基于CDS、RefSeq、数据库和UCSC进行。Specifically, the final BAM file was tested for SNP/INDEL using the HaplotypeCaller module of GATK. The variation results were annotated using ANNOVAR, in which, based on the dbSNP and 1000Genome databases, mutation positions, mutation types, and conserved regions were predicted. For exon mutations, it is based on CDS, RefSeq, databases and UCSC.
3.4变异过滤3.4 Mutation filtering
随后通过dbSNP数据库(http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp135.txt.gz.)、HapMap数据库(ftp://ftp.ncbi.nlm.nih.gov/hapmap)、千人基因组数据库(ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp)、炎黄数据库(http://yh.genomics.org.cn/)、1000Genome(1000GenomesProjectConsortium)等公共数据库的过滤，去掉所有已知的变异以及与临床无关的位点；保留外显子区或剪切位点附近位点，可能导致氨基酸改变；过滤同义突变，保留非同义突变；最后利用PolyPhen和SIFT软件预测氨基酸保守性后，共得到249处SNP变异位点及361处插入/缺失变异位点。Then through dbSNP database (http://hgdownload.cse.ucsc.edu/goldenPath/hg19/database/snp135.txt.gz.), HapMap database (ftp://ftp.ncbi.nlm.nih.gov/hapmap) , Thousand Genomes Database (ftp://ftp.1000genomes.ebi.ac.uk/vol1/ftp), Yanhuang Database (http://yh.genomics.org.cn/), 1000Genome (1000GenomesProjectConsortium) and other public databases Filter to remove all known mutations and clinically irrelevant sites; retain exon regions or sites near splicing sites, which may lead to amino acid changes; filter synonymous mutations and retain non-synonymous mutations; finally use PolyPhen and After predicting the amino acid conservation by SIFT software, a total of 249 SNP variation sites and 361 insertion/deletion variation sites were obtained.
进一步，基于变异位点的特征进行优先选择：(a)选择三个患者共有的同基因、同位点、同突变类型的变异；(b)根据家系图中的疾病的常染色体显性遗传方式(不完全显性)进行筛选；(c)基于文献检索及基因功能信息，进一步筛选经推断与心肌梗死的发生可能相关的位点。Further, priority selection is performed based on the characteristics of the mutation site: (a) select the variation of the same gene, same site, and same mutation type shared by the three patients; (b) according to the autosomal dominant inheritance mode of the disease in the pedigree diagram ( (c) Based on literature search and gene function information, further screen the loci that may be inferred to be related to the occurrence of myocardial infarction.
结果，最终发现位于SCAP基因18号外显子的点突变c.3035C>T(p.A1012V)为PMI的潜在致病基因突变。其中，三个患者均携带上述突变(c.3035C>T)，且都为杂合突变类型，而家系内正常人在此位置均未发生突变。As a result, it was finally found that the point mutation c.3035C>T (p.A1012V) located in exon 18 of the SCAP gene was a potential causative gene mutation for PMI. Among them, three patients all carried the above-mentioned mutation (c.3035C>T), and all of them were heterozygous mutation types, while none of the normal people in the family had mutations at this position.
随后，在图2所示的上述PMI患者家系中对SCAP基因进行突变排查，即针对SCAP基因中的无义突变c.3035C>T进行Sanger测序验证，结果确认，该突变在家系中存在与疾病表型共分离的现象。Subsequently, the mutation screening of the SCAP gene was carried out in the above-mentioned PMI patient family shown in Figure 2, that is, the nonsense mutation c.3035C>T in the SCAP gene was verified by Sanger sequencing. The phenomenon of co-segregation of phenotypes.
由此，初步判定SCAP基因为早发心肌梗死的致病基因，无义突变c.3035C>T(p.A1012V)为该早发心肌梗死家系中患者的致病原因，也即SCAP基因的c.3035C>T(p.A1012V)突变为早发心肌梗死的致病突变。Therefore, it is preliminarily determined that the SCAP gene is the causative gene of premature myocardial infarction, and the nonsense mutation c.3035C>T (p.A1012V) is the cause of the disease in this family of premature myocardial infarction, that is, the c of the SCAP gene. .3035C>T(p.A1012V) mutation is the pathogenic mutation of premature myocardial infarction.
实施例2Sanger法测序验证Example 2 Sanger method sequencing verification
分别对实施例1中所述的早发心肌梗死患者家系中的三名患者(PMI1-1、PMI1-3及PMI1-8)和16个家系内正常人、70个中国汉族人群的早发心肌梗死患者、以及200名家系外正常人的SCAP基因进行检测：针对SCAP基因的外显子设计引物，然后通过PCR扩增、产物纯化和测序的方法获得SCAP有关序列，根据确定序列测定结果验证SCAP基因、SCAP基因的c.3035C>T(p.A1012V)突变与早发心肌梗死之间的相关性。Three patients (PMI1-1, PMI1-3 and PMI1-8) in the premature myocardial infarction patient family described in embodiment 1 and the premature myocardial infarction of normal person in 16 families, 70 Chinese Han population Detect the SCAP gene of infarction patients and 200 normal people outside the family: design primers for the exons of the SCAP gene, then obtain the relevant sequence of SCAP through PCR amplification, product purification and sequencing, and verify SCAP according to the determined sequence determination results The correlation between c.3035C>T(p.A1012V) mutation of SCAP gene and premature myocardial infarction.
具体方法步骤如下：The specific method steps are as follows:
1、DNA提取1. DNA extraction
按照实施例1中所述的提取DNA的方法，分别提取制备受试者外周血中的基因组DNA，备用。According to the method for extracting DNA described in Example 1, the genomic DNA in the peripheral blood of the subject was extracted and prepared respectively for future use.
2、引物设计及PCR反应2. Primer design and PCR reaction
首先，参考人类基因组序列数据库GRCh38.p2，设计得到SEQIDNO：3-48所示的SCAP基因外显子特异性引物，具体序列见下表：First, referring to the human genome sequence database GRCh38.p2, the exon-specific primers of the SCAP gene shown in SEQ ID NO: 3-48 were designed, and the specific sequences are shown in the following table:
接着，分别按照以下配比配制各基因组DNA样本的PCR反应体系以及进行PCR反应：Then, prepare the PCR reaction system of each genomic DNA sample according to the following proportions and perform the PCR reaction:
总反应体系体积为25ul，反应管为0.2ml的Eppendorf离心管，体系中含有ddH2O9.5ul，Mix12.5ul，引物正向、反向各1ul，模板1ul(约50ng)，混匀，迅速短暂离心。在PCR仪上进行扩增。The total reaction system volume is 25ul, and the reaction tube is a 0.2ml Eppendorf centrifuge tube. The system contains ddH2O9.5ul, Mix12.5ul, primer forward and reverse each 1ul, template 1ul (about 50ng), mix well, and centrifuge quickly and briefly . Amplify on a PCR machine.
所有引物均采用TouchdownPCR方法，具体如下：95℃预变性5分钟；95℃变性30秒，68℃退火30秒，72℃延伸30秒，每个循环退火温度降1℃，10个循环降至58℃；然后95℃变性30秒，60℃退火30秒，72℃延伸30秒，30个循环。循环后72℃延伸10分钟。All primers use the Touchdown PCR method, as follows: pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 68°C for 30 seconds, and extension at 72°C for 30 seconds. °C; then denature at 95°C for 30 seconds, anneal at 60°C for 30 seconds, and extend at 72°C for 30 seconds, 30 cycles. The cycle was followed by an extension at 72°C for 10 minutes.
由此，获得各样本的PCR扩增产物。Thus, PCR amplification products of each sample were obtained.
3、测序3. Sequencing
针对步骤2中获得的各待测者的PCR产物，利用MultiScreen-PCRPlates(Millipore,Billerica,MA,USA)真空泵过膜纯化，然后利用BigDyeTerminatorDNA测序试剂盒(version3.1)和3730XL测序仪(AppliedBiosystems,FosterCity,CA,USA)进行直接测序(采用Sanger法进行)。且所有可疑突变均经反向测序确定。其中，纯化、测序过程由北京六合华大基因科技股份有限公司完成。For the PCR product of each test subject obtained in step 2, utilize MultiScreen-PCCRlates (Millipore, Billerica, MA, USA) vacuum pump to pass membrane purification, then utilize BigDyeTerminatorDNA sequencing kit (version3.1) and 3730XL sequencer (AppliedBiosystems, FosterCity, CA, USA) for direct sequencing (using the Sanger method). And all suspicious mutations were confirmed by reverse sequencing. Among them, the purification and sequencing processes were completed by Beijing Liuhe Huada Gene Technology Co., Ltd.
其中，图3显示了上述早发心肌梗死患者家系中患者、家系内正常人以及家系外正常人的SCAP基因c.3035C>T突变位点的代表性Sanger测序验证峰图。由图3可知，该早发心肌梗死患者家系中患者都携带有SCAP基因18号外显子的c.3035C>T突变，而家系内正常人以及家系外正常人都未携带该突变；而在70个早发心梗散发病例中，有一个患者携带位于SCAP基因9号外显子的点突变p.V468A(c.1403T>C)，该突变在早发心肌梗死患者家系中以及家系外正常人中都未出现。Among them, Figure 3 shows the representative Sanger sequencing verification peaks of the c.3035C>T mutation site of the SCAP gene in the family of patients with premature myocardial infarction, normal people in the family, and normal people outside the family. It can be seen from Figure 3 that the patients in the family of patients with premature myocardial infarction all carried the c.3035C>T mutation of exon 18 of the SCAP gene, while neither the normal people in the family nor the normal people outside the family carried the mutation; Among the sporadic cases of premature myocardial infarction, one patient carried a point mutation p.V468A (c.1403T>C) located in exon 9 of the SCAP gene. Neither appeared.
进一步的生物信息学分析显示，SCAP基因的c.3035C>T(p.A1012V)突变，以及p.V468A(c.1403T>C)突变在28,000个东亚人群中为阴性，且在物种间高度保守。Further bioinformatic analysis showed that the c.3035C>T (p.A1012V) mutation and p.V468A (c.1403T>C) mutation of the SCAP gene were negative in 28,000 East Asian populations and were highly conserved among species .
综上，证明SCAP基因为早发心肌梗死的致病基因，SCAP基因的c.3035C>T(p.A1012V)突变为早发心肌梗死的致病突变。In summary, it is proved that the SCAP gene is the causative gene of premature myocardial infarction, and the c.3035C>T (p.A1012V) mutation of the SCAP gene is the causative mutation of premature myocardial infarction.
实施例3检测试剂盒Embodiment 3 detection kit
制备一检测试剂盒，其包含能够检测SCAP基因的c.3035C>T突变(位于18号外显子)的引物，用于筛选易患早发心肌梗死的生物样品，其中这些引物为SCAP基因外显子特异性引物(针对SCAP基因18号外显子)，其序列如实施例1中所述SEQIDNO：37-38所示。Prepare a detection kit, which includes primers capable of detecting the c.3035C>T mutation (located in exon 18) of the SCAP gene, for screening biological samples susceptible to premature myocardial infarction, wherein these primers are SCAP gene overexpression Sub-specific primers (for exon 18 of the SCAP gene), the sequences of which are shown in SEQ ID NO: 37-38 described in Example 1.
利用上述试剂盒筛选易患早发心肌梗死的生物样品的具体步骤为：按照实施例2的步骤1所述的方法提取待测者DNA，以所提取的DNA为模板与上述SCAP基因的外显子特异性引物进行PCR反应，并按照本领域常规方法对PCR产物纯化，将纯化的产物进行测序，然后通过观察测序所得到的序列是否具有c.3035C>T突变，能够有效地检测本发明的SCAP基因突变体在待测者DNA中是否存在，从而能够有效地检测待测者是否易患早发心肌梗死，进一步，能够从待测者中筛选出易患早发心肌梗死的生物样品。The specific steps for using the above-mentioned kit to screen biological samples prone to premature myocardial infarction are: extract the DNA of the test subject according to the method described in step 1 of Example 2, and use the extracted DNA as a template and the expression of the above-mentioned SCAP gene Subspecific primers were used to carry out PCR reaction, and the PCR product was purified according to conventional methods in the art, and the purified product was sequenced, and then by observing whether the sequence obtained by the sequencing had the c.3035C>T mutation, it was possible to effectively detect the Whether the SCAP gene mutant exists in the test subject's DNA can effectively detect whether the test subject is susceptible to premature myocardial infarction, and further, can screen biological samples from the test subject that are prone to premature myocardial infarction.
具体地，利用上述试剂盒对实施例1中所述的早发心肌梗死患者家系中的三名患者(PMI1-1、PMI1-3及PMI1-8)和16个家系内正常人，以及200名家系外正常人的SCAP基因进行c.3035C>T突变检测，结果发现，该早发心肌梗死患者家系中患者都携带有c.3035C>T突变，而家系内正常人以及家系外正常人都未携带该突变。Specifically, three patients (PMI1-1, PMI1-3 and PMI1-8) in the family of patients with premature myocardial infarction described in Example 1, normal persons in 16 families, and 200 family members were tested using the above kit. The c.3035C>T mutation was detected for the SCAP gene of the normal people outside the family. The results showed that all the patients in the family of the patient with premature myocardial infarction carried the c.3035C>T mutation, while the normal people in the family and the normal people outside the family did not. carry this mutation.
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。In the description of this specification, descriptions referring to the terms "one embodiment", "some embodiments", "example", "specific examples", or "some examples" mean that specific features described in connection with the embodiment or example , structure, material or characteristic is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。Although the embodiments of the present invention have been shown and described, those skilled in the art can understand that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the invention is defined by the claims and their equivalents.
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| CN107974502A (en) * | 2016-10-19 | 2018-05-01 | 北京大学人民医院 | A kind of kit for being used to detect NBPF10, TSFM, PRB2 and DIAPH1 gene mutation at the same time |
| CN113588945A (en) * | 2021-06-30 | 2021-11-02 | 杭州迈尔德生物科技有限公司 | Novel coronavirus neutralizing antibody detection reagent and kit based on chemiluminescence |
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| WO2017107545A1 (en) * | 2015-12-23 | 2017-06-29 | 北京大学人民医院 | Scap gene mutant and application thereof |
| CN107974502A (en) * | 2016-10-19 | 2018-05-01 | 北京大学人民医院 | A kind of kit for being used to detect NBPF10, TSFM, PRB2 and DIAPH1 gene mutation at the same time |
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| CN113588945A (en) * | 2021-06-30 | 2021-11-02 | 杭州迈尔德生物科技有限公司 | Novel coronavirus neutralizing antibody detection reagent and kit based on chemiluminescence |
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