技术领域technical field
本发明涉及基因工程技术领域，具体地说涉及一种特异性抑制MAGEA1基因表达的小干扰RNA及其应用。The invention relates to the technical field of genetic engineering, in particular to a small interfering RNA for specifically inhibiting the expression of MAGEA1 gene and its application.
背景技术Background technique
RNA干扰(RNA interference,RNAi)技术是指是指在进化过程中高度保守的、由双链RNA(double-stranded RNA，dsRNA)诱发的、同源mRNA高效特异性降解的现象，RNA分子通过破坏特定的mRNA，以抑制某种基因表达的生物学过程。该技术具有以下几点优势：1高效性；2特异性；3位置效应；4高稳定性；5可传播性；6浓度时间依赖性。由于使用RNAi技术可以特异性剔除或关闭特定基因的表达，该技术迅速成为基因功能研究和基因治疗研究领域最受关注的研究工具之一，已被广泛用于探索基因功能和传染性疾病及恶性肿瘤的治疗领域。RNA interference (RNA interference, RNAi) technology refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (double-stranded RNA, dsRNA), which is highly conserved in the evolutionary process. Specific mRNA to inhibit the biological process of certain gene expression. The technology has the following advantages: 1 high efficiency; 2 specificity; 3 position effect; 4 high stability; 5 transmissibility; 6 concentration time dependence. Since the use of RNAi technology can specifically knock out or shut down the expression of specific genes, this technology has quickly become one of the most concerned research tools in the field of gene function research and gene therapy research, and has been widely used to explore gene function and infectious diseases and malignant diseases. Therapeutic areas of cancer.
恶性黑色素瘤(malignant melanoma，MM)：是源自于黑色素细胞的一种恶性肿瘤，好发于皮肤，恶性程度高，治疗难度大。虽然黑素瘤仅占全部皮肤肿瘤的4％,但却占皮肤肿瘤死亡人数的80％,是皮肤肿瘤中死亡率最高的一种恶性肿瘤。美国国家癌症研究所(NCI)的数据显示，在2003-2007年间，患病人数为每年20.1人/10,000人。截止到2010年，估计有68130个患者被诊断患有黑色素瘤，其中8700个患者将因此丧生(http://www.cancer.gov/)。恶性黑色素瘤的发生率在全世界范围内正在不断地上升，包括以前发病率低的地区，它已成为一种严重威胁人们健康的恶性疾病。在中国黑色素瘤患者有逐年增高的趋势。北京市八城区统计资料显示，2000年MM发病率为0.2例/10万，到了2004年已达1例/10万。根据中国临床肿瘤协会(CSCO)黑色素瘤专家委员会发表的《中国黑色素瘤诊治指南(2011版)》，我国每年新发病例约2万例，已经成为了严重危及我国人民健康的疾病之一。Malignant melanoma (MM): It is a malignant tumor derived from melanocytes, which tends to occur in the skin, has a high degree of malignancy, and is difficult to treat. Although melanoma only accounts for 4% of all skin tumors, it accounts for 80% of skin tumor deaths, and it is a malignant tumor with the highest mortality rate among skin tumors. According to the National Cancer Institute (NCI), between 2003 and 2007, the number of cases was 20.1 per 10,000 persons per year. As of 2010, an estimated 68,130 patients have been diagnosed with melanoma, of whom 8,700 will die from it (http://www.cancer.gov/). The incidence of malignant melanoma is increasing all over the world, including areas where the incidence rate was low before, and it has become a malignant disease that seriously threatens people's health. The number of melanoma patients in China is increasing year by year. Statistical data from the eight districts of Beijing show that the incidence of MM was 0.2 cases per 100,000 in 2000, and reached 1 case per 100,000 in 2004. According to the "Guidelines for the Diagnosis and Treatment of Melanoma in China (2011 Edition)" issued by the Melanoma Expert Committee of the Chinese Society of Clinical Oncology (CSCO), there are about 20,000 new cases in my country every year, and it has become one of the diseases that seriously endanger the health of our people.
Melanoma antigen family A1(MAGEA1)是MAGE基因家族成员之一，该家族共有12个同源基因，编码蛋白序列具有50％-80％的相似性，而启动子区和第一外显子区差异相对比较大。MAGEA1是1991年由Vander Bruggeon等人从一例患有黑色素肿瘤但是临床表现良好的病人身上所分离得到的。MAGEA1基因所编码的蛋白能够被人体毒性T淋巴细胞克隆(CTL克隆)识别。MAGEA1基因所编码的蛋白可以与SKIP蛋白发生相互作用并且通过招募组蛋白去乙酰酶(HDAC-1),从而调控某些特定基因的转录。MAGEA1染色质定位于人类X染色体q28区域，该基因全长1755bp，主要由3个外显子和2个内含子组成。编码蛋白309个氨基酸长度，一般认为MAGEA1在细胞内主要分布在细胞核和锚定在细胞膜上，正常情况下，MAGEA1仅在雄性生殖系统和胎盘表达，通常在恶性肿瘤细胞中不表达，其原因是该基因的启动予处CpG岛的甲基化。近年来研究发现在多种肿瘤当中，包括黑素瘤、乳腺癌、神经母细胞瘤、膀胱癌以及非小细胞肺癌中也存在MAGE-A1基因的表达，因此MAGEA1被称作癌症生殖(cancer-germline,CG)基因或者肿瘤睾丸抗原(cancer-testis antigen,CTA)基因。MAGEA1编码的蛋白在肿瘤细胞表面可被细胞毒性T淋巴细胞识别，经过抗原递呈以及机体免疫应答，产生体液免疫和细胞免疫的应答效应，因此MAGEA1基因被视为潜在的肿瘤疫苗靶点。Melanoma antigen family A1 (MAGEA1) is one of the members of the MAGE gene family. There are 12 homologous genes in this family, and the coding protein sequences have 50%-80% similarity, while the promoter region and the first exon region are relatively different. bigger. MAGEA1 was isolated in 1991 by Vander Bruggeon et al. from a patient with melanoma but with good clinical performance. The protein encoded by MAGEA1 gene can be recognized by human toxic T lymphocyte clone (CTL clone). The protein encoded by MAGEA1 gene can interact with SKIP protein and regulate the transcription of some specific genes by recruiting histone deacetylase (HDAC-1). The chromatin of MAGEA1 is located in the q28 region of human X chromosome. The gene is 1755bp in length and mainly consists of 3 exons and 2 introns. The encoded protein is 309 amino acids in length. It is generally believed that MAGEA1 is mainly distributed in the nucleus and anchored on the cell membrane in cells. Under normal circumstances, MAGEA1 is only expressed in the male reproductive system and placenta, and is usually not expressed in malignant tumor cells. The reason is that The activation of this gene is subject to the methylation of CpG islands. In recent years, studies have found that MAGE-A1 gene expression also exists in a variety of tumors, including melanoma, breast cancer, neuroblastoma, bladder cancer and non-small cell lung cancer, so MAGEA1 is called cancer reproductive (cancer- germline, CG) gene or tumor-testis antigen (cancer-testis antigen, CTA) gene. The protein encoded by MAGEA1 can be recognized by cytotoxic T lymphocytes on the surface of tumor cells, and through antigen presentation and immune response of the body, it can produce humoral and cellular immune responses. Therefore, the MAGEA1 gene is regarded as a potential tumor vaccine target.
由于当前对MAGEA1的研究进展有限，其在生殖系统和肿瘤中的生物学功能还不是非常清楚，但可以肯定的是，MAGEA1编码的分子参与了多种分子功能调节并发挥一系列作用。下调MAGEA1的表达，可以有效抑制肿瘤细胞的迁移和侵袭能力，目前针对下调MAGEA1的表达研究，特别是特异性抑制MAGEA1表达的小干扰RNA报道较少。Due to the limited research progress on MAGEA1, its biological function in the reproductive system and tumors is not very clear, but it is certain that the molecule encoded by MAGEA1 is involved in the regulation of various molecular functions and plays a series of roles. Down-regulating the expression of MAGEA1 can effectively inhibit the migration and invasion of tumor cells. At present, there are few reports on down-regulating the expression of MAGEA1, especially the small interfering RNA that specifically inhibits the expression of MAGEA1.
发明内容Contents of the invention
本发明的目的在于提供一种能够特异性抑制MAGEA1基因表达的小干扰RNA(siRNA)及其应用。The purpose of the present invention is to provide a small interfering RNA (siRNA) capable of specifically inhibiting the expression of MAGEA1 gene and its application.
本发明根据RNAi发生原理，设计并合成了针对MAGEA1基因的小干扰RNA，其GC含量为42.11％，且正义链和反义链的长度均为19nt。According to the principle of RNAi generation, the present invention designs and synthesizes a small interfering RNA targeting MAGEA1 gene, the GC content of which is 42.11%, and the lengths of both sense strand and antisense strand are 19nt.
进一步地，本发明的小干扰RNA双连体分子的正义链和反义链序列分别为：Further, the sequences of the sense strand and the antisense strand of the small interfering RNA duplex molecule of the present invention are respectively:
正义链：5’GAGUAUGUGAUCAAGGUCAdTdT-3’，(SEQ ID NO.1)Sense strand: 5'GAGUAUGUGAUCAAGGUCAdTdT-3', (SEQ ID NO.1)
反义链：5’-dTdTCUCAUACACUAGUUCCAGU-3’(SEQ ID NO.2)。Antisense strand: 5'-dTdTCUCAUACACUAGUUCCAGU-3' (SEQ ID NO.2).
本发明提供的上述小干扰RNA，能够特异性下调细胞或生物体中MAGEA1基因的表达量。The above-mentioned small interfering RNA provided by the present invention can specifically down-regulate the expression level of the MAGEA1 gene in cells or organisms.
进一步地，本发明的小干扰RNA作用于MAGEA1基因的靶序列为：5’-GAGTATGTGATCAAGGTCA-3’(SEQ ID NO.3)。Further, the target sequence of the small interfering RNA of the present invention acting on the MAGEA1 gene is: 5'-GAGTATGTGATCAAGGTCA-3' (SEQ ID NO.3).
一种编码本发明所述小干扰RNA的DNA序列属于本发明的保护范围。A DNA sequence encoding the small interfering RNA of the present invention belongs to the protection scope of the present invention.
本发明提供一种表达载体，其包含编码上述小干扰RNA的DNA序列。The present invention provides an expression vector, which comprises the DNA sequence encoding the above-mentioned small interfering RNA.
一种宿主细胞，其为可表达本发明所述小干扰RNA的细胞。A host cell, which is a cell capable of expressing the small interfering RNA of the present invention.
一种含有上述小干扰RNA的药物属于本发明的保护范围。A medicine containing the above-mentioned small interfering RNA belongs to the protection scope of the present invention.
进一步地，所述的药物为抗肿瘤的药物。所述的肿瘤为黑色素瘤、乳腺癌、神经母细胞瘤、膀胱癌、非小细胞肺癌、大肠癌、胃癌、肝癌、精原细胞瘤、食道癌、白血病、淋巴瘤、肉瘤。Further, the drug is an anti-tumor drug. The tumors are melanoma, breast cancer, neuroblastoma, bladder cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, liver cancer, seminoma, esophageal cancer, leukemia, lymphoma, and sarcoma.
进一步地，所述的药物为免疫治疗剂。Further, the drug is an immunotherapeutic agent.
一种可表达上述小干扰RNA的细胞系也属于本发明的保护范围。A cell line that can express the above-mentioned small interfering RNA also belongs to the protection scope of the present invention.
本发明提供了上述小干扰RNA在制备调节细胞或生物体中的MAGEA1基因表达药物中的用途。The present invention provides the use of the above-mentioned small interfering RNA in the preparation of medicines for regulating the expression of MAGEA1 gene in cells or organisms.
本发明提供了上述小干扰RNA在制备抑制肿瘤细胞迁移和侵袭的药物中的用途。The present invention provides the use of the above-mentioned small interfering RNA in the preparation of drugs for inhibiting tumor cell migration and invasion.
优选地，所述肿瘤细胞为黑色素瘤细胞、乳腺癌细胞、神经母细胞瘤细胞、膀胱癌细胞、非小细胞肺癌细胞、大肠癌细胞、胃癌细胞、肝癌细胞、精原细胞瘤细胞、食道癌细胞、淋巴瘤细胞、肉瘤细胞。Preferably, the tumor cells are melanoma cells, breast cancer cells, neuroblastoma cells, bladder cancer cells, non-small cell lung cancer cells, colorectal cancer cells, gastric cancer cells, liver cancer cells, seminoma cells, esophageal cancer cells cells, lymphoma cells, sarcoma cells.
本发明提供了上述小干扰RNA在制备治疗疾病的药物中的用途，所述的疾病为黑色素瘤、乳腺癌、神经母细胞瘤、膀胱癌、非小细胞肺癌、大肠癌、胃癌、肝癌、精原细胞瘤、食道癌、白血病、淋巴瘤、肉瘤。The present invention provides the use of the above-mentioned small interfering RNA in the preparation of medicines for treating diseases, and the diseases are melanoma, breast cancer, neuroblastoma, bladder cancer, non-small cell lung cancer, colorectal cancer, gastric cancer, liver cancer, sperm Primary cell tumor, esophageal cancer, leukemia, lymphoma, sarcoma.
有效的靶向治疗是目前国内外不断寻找的辅助治疗方法，本发明提供的靶向针对MAGEA1基因的siRNA转染黑色素瘤细胞后，发现siRNA能够显著下调MAGEA1基因的表达，有效抑制肿瘤细胞的迁移和侵袭能力，对于开发新的抗肿瘤基因药物和肿瘤药物的治疗效果具有重要指导作用，可用于开发制备抗肿瘤药物，将会取得巨大的社会效益和经济价值。Effective targeted therapy is an adjuvant therapy that is constantly being sought at home and abroad. After transfecting melanoma cells with siRNA targeting the MAGEA1 gene provided by the present invention, it is found that the siRNA can significantly down-regulate the expression of the MAGEA1 gene and effectively inhibit the migration of tumor cells It can be used to develop and prepare anti-tumor drugs, and will achieve huge social and economic value.
附图说明Description of drawings
图1为本发明的siRNA对A375细胞MAGEA1基因表达的敲低效果。其中1：未转染siRNA的A375细胞；2：转染阴性对照序列的A375细胞；3：转染siRNA的A375细胞。Figure 1 shows the knockdown effect of siRNA of the present invention on MAGEA1 gene expression in A375 cells. Wherein 1: A375 cells not transfected with siRNA; 2: A375 cells transfected with negative control sequence; 3: A375 cells transfected with siRNA.
图2为A375细胞中MAGEA1基因mRNA敲低水平的电泳检测结果，其中泳道1：分子量标记；泳道2：转染siRNA的A375细胞；泳道3：转染阴性对照序列的A375细胞；泳道4：未转染siRNA的A375细胞。Figure 2 is the electrophoresis detection results of MAGEA1 gene mRNA knockdown level in A375 cells, in which swimming lane 1: molecular weight marker; swimming lane 2: A375 cells transfected with siRNA; swimming lane 3: A375 cells transfected with negative control sequence; A375 cells transfected with siRNA.
图3为A375细胞MAGEA1蛋白敲低水平的Western blot检测，其中泳道1：未转染siRNA的A375细胞；泳道2：转染阴性对照序列的A375细胞；泳道3：转染siRNA的A375细胞。Figure 3 is the Western blot detection of MAGEA1 protein knockdown level in A375 cells, where lane 1: A375 cells not transfected with siRNA; lane 2: A375 cells transfected with negative control sequence; lane 3: A375 cells transfected with siRNA.
具体实施方式Detailed ways
以下实施例用于说明本发明，但不用来限制本发明的范围。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, any modifications or substitutions made to the methods, steps or conditions of the present invention fall within the scope of the present invention.
若未特别指明，实施例中所用的化学试剂均为常规市售试剂，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.
实施例1特异性靶向MAGEA1基因的siRNA的设计合成及转染黑色素瘤细胞Example 1 Design, synthesis and transfection of siRNA specifically targeting the MAGEA1 gene into melanoma cells
1、特异性靶向MAGEA1基因的siRNA的设计1. Design of siRNA specifically targeting MAGEA1 gene
在公共的siRNA设计网站(网址为：http://sirna.wi.mit.edu/home.php；https://www.thermofisher.com/cn/zh/home.html；http://sidirect2.rnai.jp/等)上输入MAGEA1的mRNA序列，按照网站指示预测可以敲低MAGEA1 mRNA表达的siRNA序列。In the public siRNA design website (URL: http://sirna.wi.mit.edu/home.php; https://www.thermofisher.com/cn/zh/home.html; http://sidirect2. rnai.jp/etc.) to input the mRNA sequence of MAGEA1, and follow the website instructions to predict the siRNA sequence that can knock down the expression of MAGEA1 mRNA.
比对多个网站的给出的预测结果，选择在至少同时在两个网站均被预测到的siRNA。Comparing the prediction results given by multiple websites, select siRNAs that are predicted on at least two websites at the same time.
筛选未有文献报道的siRNA序列。本实施例筛选的siRNA序列GC含量为42.11％，且正义链和反义链的长度均为19nt。Screen for siRNA sequences that have not been reported in the literature. The GC content of the siRNA sequence screened in this embodiment is 42.11%, and the lengths of the sense strand and the antisense strand are both 19 nt.
具体来说，其正义链和反义链序列分别为：Specifically, its sense strand and antisense strand sequences are:
正义链：5’GAGUAUGUGAUCAAGGUCAdTdT-3’，(SEQ ID NO.1)Sense strand: 5'GAGUAUGUGAUCAAGGUCAdTdT-3', (SEQ ID NO.1)
反义链：5’-dTdTCUCAUACACUAGUUCCAGU-3’(SEQ ID NO.2)。Antisense strand: 5'-dTdTCUCAUACACUAGUUCCAGU-3' (SEQ ID NO.2).
本发明的小干扰RNA作用于MAGEA1基因的靶序列为：5’-GAGTATGTGATCAAGGTCA-3’(SEQ ID NO.3)。The target sequence of the small interfering RNA of the present invention acting on the MAGEA1 gene is: 5'-GAGTATGTGATCAAGGTCA-3' (SEQ ID NO.3).
SiRNA及阴性对照序列交由广州锐博生物科技有限公司合成。SiRNA and negative control sequences were synthesized by Guangzhou Ruibo Biotechnology Co., Ltd.
2、本发明的siRNA转染黑色素瘤细胞2. Transfection of melanoma cells with siRNA of the present invention
黑色素瘤细胞A375(购自ATCC，American type culture collection)在10cm培养皿中培养至80％后，用胰酶消化，加入6孔板，使每个孔的最终细胞数为5×105个细胞。Melanoma cells A375 (purchased from ATCC, American type culture collection) were cultured to 80% in a 10 cm culture dish, digested with trypsin, and added to a 6-well plate so that the final cell number in each well was 5×105 cells .
转染试剂准备：Transfection reagent preparation:
A.250μl Opti-MEM(赛默飞世尔科技，美国)+5μl siRNA(20uM)，静置5分钟。A. 250 μl Opti-MEM (Thermo Fisher Scientific, USA) + 5 μl siRNA (20uM), let stand for 5 minutes.
B.250μl Opti-MEM(赛默飞世尔科技，美国)+3μl Lipofeactmine 2000(赛默飞世尔科技，美国)，静置5分钟，B.250 μl Opti-MEM (Thermo Fisher Scientific, U.S.)+3 μl Lipofeactmine 2000 (Thermo Fisher Scientific, U.S.), let stand for 5 minutes,
C.将A与B所得试剂混合后，静置20分钟。将混合后的转染试剂加入6孔板中，与A375细胞混匀。37℃,培养48小时后，收集细胞。C. After mixing the reagents obtained from A and B, let it stand for 20 minutes. Add the mixed transfection reagent into the 6-well plate and mix with the A375 cells. After culturing at 37°C for 48 hours, the cells were collected.
实施例2 siRNA转染后鉴定MAGEA1基因的mRNA表达水平Example 2 Identification of the mRNA expression level of the MAGEA1 gene after siRNA transfection
1、实时定量PCR(q-PCR)鉴定实施例1中转染了本发明siRNA的黑色素瘤细胞A375的MAGEA1基因的mRNA表达水平1. Real-time quantitative PCR (q-PCR) identifies the mRNA expression level of the MAGEA1 gene of the melanoma cell A375 transfected with siRNA of the present invention in Example 1
表1 q-PCR反应体系Table 1 q-PCR reaction system
前引物：AAGGTGGCTGATTTGGTTGGTFront primer: AAGGTGGCTGATTTGGTTGGT
后引物：CTGCAAGGACTCAGAGGCTTTBack primer: CTGCAAGGACTCAGAGGCTTT
q-PCR反应程序：95℃5min；95℃15sec，62℃30sec，循环35次。q-PCR reaction program: 95°C for 5min; 95°C for 15sec, 62°C for 30sec, cycle 35 times.
使用实时定量自带软件分析MAGEA1基因表达量的水平，结果见图1。图1显示，A375细胞转染MAGEA1特异性siRNA后，MAGEA1表达量仅相当于转染阴性对照序列(NC)的8.7％。1：未转染siRNA的A375细胞；2：转染阴性对照序列的A375细胞；3：转染siRNA的A375细胞。The real-time quantitative built-in software was used to analyze the expression level of the MAGEA1 gene, and the results are shown in Figure 1. Figure 1 shows that after A375 cells were transfected with MAGEA1-specific siRNA, the expression level of MAGEA1 was only equivalent to 8.7% of the transfected negative control sequence (NC). 1: A375 cells not transfected with siRNA; 2: A375 cells transfected with negative control sequence; 3: A375 cells transfected with siRNA.
将PCR产物进行电泳检测，A375细胞中MAGEA1基因mRNA敲低水平的电泳检测结果见图2，图2显示转染siRNA后，可以有效的敲低MAGEA1基因的表达。The PCR products were detected by electrophoresis, and the results of the electrophoresis detection of MAGEA1 gene mRNA knockdown level in A375 cells are shown in Figure 2. Figure 2 shows that after transfection of siRNA, the expression of MAGEA1 gene can be effectively knocked down.
实施例3 siRNA转染后鉴定MAGEA1蛋白表达水平Example 3 Identification of MAGEA1 protein expression level after siRNA transfection
1、Western blot鉴定MAGEA1基因的蛋白表达水平1. Western blot identification of protein expression level of MAGEA1 gene
样品准备：贴壁的黑色素瘤细胞A375：1×PBS洗两遍，100μl胰酶消化，500μl培养液终止放入1.5ml EP管中。用14000rpm的转速离心5分钟。弃上清，200μl 1×PBS洗一次。加入50μl细胞裂解缓冲液，剧烈震荡，使细胞充分裂解，-20℃保存。用BCA方法检测总蛋白量。5×上样缓冲液稀释到1×，加入到裂解的细胞样品中。95℃热变性5min，随后用11500rpm的转速离心5分钟。Sample preparation: Adherent melanoma cells A375: washed twice with 1×PBS, digested with 100 μl trypsin, terminated with 500 μl culture medium and put into a 1.5ml EP tube. Centrifuge at 14,000 rpm for 5 minutes. Discard the supernatant and wash once with 200μl 1×PBS. Add 50 μl of cell lysis buffer, shake vigorously to fully lyse the cells, and store at -20°C. Total protein was detected by BCA method. 5X Loading Buffer was diluted to 1X and added to the lysed cell sample. Heat denaturation at 95°C for 5 minutes, followed by centrifugation at 11500 rpm for 5 minutes.
2、配置12％SDS-PAGE胶。2. Configure 12% SDS-PAGE glue.
3、蛋白质电泳：点样，每个样品最少20μg，蛋白分子量标记点3μl。恒压100V，30分钟，随后调整电压至120V，1小时。转膜：纤维素膜预先用甲醇浸泡，用预冷的转膜缓冲液进行转膜，恒流300mA，2小时。封闭：将膜放入5％脱脂奶粉溶液(1g脱脂奶粉+20ml 1×TTBS)中，振荡1小时。倒掉脱脂奶粉溶液，加入一抗，振荡1小时。去掉一抗后，用1×TTBS洗三次，每次10分钟。随后加入二抗，振荡1小时。去掉二抗后，用1×TTBS洗三次，每次10分钟。配制显色液并均匀滴在膜上，曝光显影。将三种细胞总蛋白进行Western blot检测，结果见图3，结果显示转染siRNA后，可以有效的敲低MAGEA1蛋白的表达。3. Protein electrophoresis: sample spotting, at least 20 μg for each sample, and 3 μl for protein molecular weight markers. Constant voltage 100V, 30 minutes, then adjust the voltage to 120V, 1 hour. Membrane transfer: The cellulose membrane was pre-soaked in methanol, and then transferred to the membrane with pre-cooled transfer buffer at a constant flow of 300mA for 2 hours. Blocking: Put the membrane into 5% skimmed milk powder solution (1g skimmed milk powder + 20ml 1×TTBS) and shake for 1 hour. Pour off the skimmed milk powder solution, add the primary antibody, and shake for 1 hour. After removing the primary antibody, wash three times with 1×TTBS, 10 minutes each time. The secondary antibody was then added and shaken for 1 hour. After removing the secondary antibody, wash three times with 1×TTBS, 10 minutes each time. Prepare chromogenic solution and evenly drop it on the film, expose and develop. The total proteins of the three cells were detected by Western blot, and the results are shown in Figure 3. The results showed that after transfection with siRNA, the expression of MAGEA1 protein could be effectively knocked down.
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。Although the present invention has been described in detail with general descriptions and specific embodiments above, it is obvious to those skilled in the art that some modifications or improvements can be made on the basis of the present invention. Therefore, the modifications or improvements made on the basis of not departing from the spirit of the present invention all belong to the protection scope of the present invention.
| Application Number | Priority Date | Filing Date | Title |
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| CN201510958796.6ACN105462977B (en) | 2015-12-18 | 2015-12-18 | One species specificity suppresses siRNA and its application of MAGEA1 gene expressions |
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| CN201510958796.6ACN105462977B (en) | 2015-12-18 | 2015-12-18 | One species specificity suppresses siRNA and its application of MAGEA1 gene expressions |
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| WO2006006948A2 (en)* | 2002-11-14 | 2006-01-19 | Dharmacon, Inc. | METHODS AND COMPOSITIONS FOR SELECTING siRNA OF IMPROVED FUNCTIONALITY |
| CN101492729A (en)* | 2008-01-22 | 2009-07-29 | 上海人类基因组研究中心 | Uses of MAGEA1 gene |
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| Publication | Publication Date | Title |
|---|---|---|
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