CN109439661A - 抑制pirB表达的短发卡shRNA、慢病毒及其应用 - Google Patents
抑制pirB表达的短发卡shRNA、慢病毒及其应用 Download PDFInfo
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Abstract
本发明公开了一种抑制pirB表达的短发卡shRNA，其核苷酸序列为SEQ ID No.1所示的碱基序列，本发明还公开了一种pirB基因shRNA敲除慢病毒表达载体，将上述的抑制pirB表达的短发卡shRNA插入慢病毒载体质粒pGLVH3/H1/GFP+Puro所得；以及一种pirB病毒的制备方法制备得到pirB病毒；以及抑制pirB表达的短发卡shRNA或pirB基因shRNA敲除慢病毒表达载体或pirB病毒在制备抑制肺癌增殖或迁移的药物中的应用，为肺癌的治疗应用提供了一种新方法。
Description
技术领域
本发明属于基因工程技术领域，涉及一种抑制pirB表达的短发卡 shRNA，本发明还涉及一种pirB基因shRNA敲除慢病毒表达载体、pirB病毒及其应用。
背景技术
原发性肺癌是我国及至全球发病率最高的肿瘤，也是癌症死因之首。《2015年中国癌症统计》报告估计，中国2015年新增429.2万癌症病例，死亡病例超过281.4万，其中肺癌的发病率和死亡率均占首位。非小细胞性肺癌(NSCLC)是肺癌中最常见的类型，约占肺癌总发病率的75％～80％。目前NSCLC的治疗主要采取综合治疗与个体化治疗相结合的原则，即应用手术、化疗、放疗和分子靶向治疗等手段，达到延长患者生存时间、控制肿瘤进展的目的。
pirB，也称为LILRB3，是一种I型跨膜糖蛋白，由胞外四个免疫球蛋白样(IgG)结构域和胞内四个ITIMs组成。早期研究发现pirB在树突状细胞、 B细胞、粒细胞及肥大细胞等细胞表面高表达，认为其作为抑制性白细胞免疫球蛋白样受体，通过胞外段的IgG样结构域与胞外的配体结合，传导抑制信号介导机体的炎症和免疫反应。但是pirB在肿瘤中的作用鲜有报道。
RNA干扰(RNA interference，RNAi)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，是一种转录后基因沉默的机制。目前RNAi的表达载体有很多中，慢病毒表达系统相比于其他表达系统，具有感染效率高、感染时间长、感染效果稳定，可容纳较大的基因片段，不易诱发宿主免疫反应等优点,已成为当前基因治疗和转基因动物中载体研究的热点。
目前尚无研究表明pirB基因与肺癌发生发展相关，也无文献表明抑制 pirB的表达可以抑制肺癌细胞的增殖、迁移。
发明内容
本发明的目的是提供一种抑制pirB表达的短发卡shRNA，能抑制肺癌细胞的增殖、迁移，为肺癌的治疗应用提供了一种新方法。
本发明的另一目的是提供一种pirB基因shRNA敲除慢病毒表达载体；
本发明的第三个目的是提供一种pirB病毒的制备方法。
本发明的第四个目的是提供抑制pirB表达的短发卡shRNA、pirB基因 shRNA敲除慢病毒表达载体以及pirB病毒的应用。
本发明所采用的第一种技术方案是，抑制pirB表达的短发卡shRNA，其核苷酸序列为SEQ ID No.1所示的碱基序列。
本发明的第二种技术方案是，一种干扰质粒，该质粒含有上述的核苷酸序列。
本发明的第三种技术方案是，一种pirB基因shRNA敲除慢病毒表达载体，将上述的抑制pirB表达的短发卡shRNA插入慢病毒载体质粒 pGLVH3/H1/GFP+Puro所得。
以及重组上述的pirB慢病毒表达载体的构建方法，具体包括：合成用于编码pirB的核苷酸片段；用限制性内切酶用BamHI和EcoRI分别对上述的核苷酸片段和pGLVH3/H1/GFP+Puro载体进行双酶切；将双酶切后的所述核苷酸片段和pGLVH3/H1/GFP+Puro进行连接，得到重组的pirB慢病毒载体。
本发明的第四种技术方案是，一种pirB病毒的制备方法，具体包括：用慢病毒包装质粒pGag/Pol、pRev和pVSV-G对权利要求4所述的重组的pirB 慢病毒载体进行包装，转染293T细胞后，得到pirB病毒，其中，重组的pirB 慢病毒表达载体、pGag/Pol、pRev和pVSV-G的质量比为(1.5-2.5)：(0.5-1.5)： (0.6-1.5)：(0.5-1.4)。
本发明的第五种技术方案是，采用上述一种pirB病毒的制备方法制备得到的pirB病毒。
本发明的第六种技术方案是，抑制pirB表达的短发卡shRNA或pirB基因shRNA敲除慢病毒表达载体或pirB病毒在制备抑制肺癌增殖或迁移的药物中的应用。
本发明的有益效果是：
本发明为制备治疗肺癌的药物提供了一种新的方法，可促进肺癌新药的开发；获得稳定抑制pirB表达的肺癌细胞株，为研究pirB基因功能提供了实验材料，补充了pirB基因在肺癌中的功能研究空白。
本发明的shRNA不仅可以用于制备肺癌的药物，可以用于pirB基因在肺癌发生发展中的作用及机制研究。
附图说明
图1是本发明pGLVH3/H1/GFP+Puro质粒DNA图谱示意图；
图2是本发明shRNAs抑制A549pirB mRNA表达的结果；
图3是本发明shRNAs抑制A549pirB蛋白表达的结果；
图4是本发明抑制pirB的表达后抑制A549增值的结果；
图5是本发明抑制pirB的表达抑制A549细胞迁移的结果；A:显微镜视野；B：A图的数据统计。
具体实施方式
下面结合附图和具体实施方式对本发明进行详细说明。
实施例中未注明具体条件的实验方法及未说明配方的试剂均为按照常规条件，如分子克隆试验指南，第三版。北京:科学出版社2008中所述的条件。
一、肺癌细胞的培养:
肺癌细胞系A549(购买自中国科学院上海生命科学院细胞资源中心)培养于37℃，5％CO2恒温培养箱。对数生长期的细胞用胰酶消化脱壁，之后加入适量培养液并反复吹打以混匀细胞；将得到的细胞悬液移入15ml离心管中，800rPm离心5min；去上清，用5ml培养基将细胞悬起，吹打混匀；加约4ml细胞悬液于装有6ml新鲜培养基的10cm培养皿，放入37℃，5％CO2培养箱中培养，约3天左右传代1次(主要查看细胞有无铺满培养瓶)。
二、pirB基因干扰慢病毒质粒的设计
设计对应于pirB基因的shRNA的寡核苷酸序列(pirB-shRNA:GGAGTACCAACTGGATAAAGAGG)，为67bp，并进行合成。
该shRNAs的寡核苷酸序列为sense是shRNA序列，anti是与其互补的序列)：
pirB-shRNA
Sense:5'-GATCCGGAGTACCAACTGGATAAAGAGGTTCAAGAGACCTCTTTATCCAGTTGGTACTCCTTTTTTG-3'
Anti-sense:5'-AATTCAAAAAAGGAGTACCAACTGGATAAAGAGGTCTCTTGAACCTCTTTATCCAGTTGGTACTCCG-3'；
主要步骤如下：本发明所用的慢病毒载体(pGLVH3/H1/GFP+Puro，见附图1)由吉玛基因公司合成，用BamHI和EcoRI双酶切穿梭质粒(上海吉玛制药技术有限公司，商品号：C06003)，得到载体大片段，将上述模板序列(由pirB shRNA的寡核苷酸sense与anti-sense序列退火形成)与载体大片段连接，得到重组穿梭质粒，测序正确后就构建成功pirB基因shRNA敲除慢病毒表达载体(pGLVH3/H1-pirB-shRNA)。
测序结果(包含插入片段的载体序列)：
pirB-shRNA：
CAACGGTGGCGTGGGTGCACTGGTTTGCTGACGCAACCCCCACTG GTTGGGGCATTGCCACCACTGTCAGCTCCTTTCCGGGACTTTCGCTTTC CCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGT CGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTG GATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCA GCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCG CGTCTCCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCT CCCCGCCTGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGATC TTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACT CCCAACGATGTCAAGAATTGGAACGCTGACGTCATCAACCCGCTCCAA GGAATCGCGGGCCCAGTGTCACTAGGCGGGAACACCCAGCGCGCGTG CGCCCTGGCAGGAAGATGGCTGTGAGGGACAGGGGAGTGGCGCCCTG CAATATTTGCATGTCGCTATGTGTTCTGGGAAATCACCATAAACGTGAA ATGTCTTTGGATTTGGGAATCTTATAAGTTCTGTATGAGACCACTTGGATCCGGAGTACCAACTGG ATAAAGAGGTTCAAGAGACCTCTTTATCCAGTTGGTACTCCTTTTTTGAATTCTTCGATTCTGCTTTTTGCTTCTACTGGGT CTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAG GGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAG TAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAG ACCCTTTCAGTCAGTGTGGAAAATCTCTAGCAGTAGTAGTTCATGTCAT CTTATTATTCAGTATTTATAACTGCAAAGAAA
三、慢病毒pGLVH3/H1-pirB-shRNA的包装
293T细胞在10cm培养皿中培养至80-90％融合时，接种15cm培养皿；倾去培养液，用1ml D-Hank’s solution洗涤细胞两次；加入1ml Trypsin-EDTA solution，混匀后，37℃放置2-3分钟；小心吸去胰酶溶液，加入2ml含10％FBS的DMEM培养液，吹打使细胞形成单细胞悬液；将细胞悬液接种15cm培养皿，加入18ml含10％FBS的DMEM培养液，混匀后37℃、5％CO2培养过夜；在一支无菌的5ml离心管中加入1.5ml无血清 DMEM，按比例加入pirB shRNA的序列的穿梭质粒和包装质粒(pGag/Pol、 pRev、pVSV-G)，混匀质量比为(pGLVH3/H1-pirB-shRNA：pGag/Pol：pRev： pVSV-G＝2：1：1：1)，取另一支无菌的5ml离心管，加入1.5ml无血清DMEM，再加入300ul RNAi-Mate，混匀，室温放置5分钟后将两管混合，室温放置 20～25分钟；除去15cm培养皿中的培养液，加入8ml无血清的DMEM培养液；将转染混合物逐滴加入15cm培养皿中，轻轻地前后摇晃培养皿以混匀复合物，在37℃、5％CO2培养箱中温育4-6小时；吸弃转染液，加入 18ml含10％FBS的DMEM培养液。37℃、5％CO2继续培养72小时；将培养皿中细胞上清液吸到50ml离心管中，4℃，4500rpm，5min；低速离心后，将离心管上清液倒入50ml注射器内，用0.45um过滤器过滤；滤液在离心机中进行超速离心，4℃，20000rpm，3h；将浓缩液收集分装至1.5mlEP 管中，-80℃冰箱保存。
空载系统慢病毒制备同上。
四、获得一种稳定敲除pirB基因的A549细胞株
采用含10％FBS的DMEM培养基培养A549(购自中国科学院上海细胞所)，待细胞密度生长至约60％时进行靶细胞感染。采用MOI值为10的慢病毒感染A549细胞，用含有终浓度为3pg/ml嘌呤霉素的培养基传代筛选得到稳定敲除pirB的A549细胞株。
五、利用定量PCR法和Western blot法检测shRNA对pirB转录水平和蛋白表达的抑制作用
由primer premier设计出检测pirB基因表达量的引物：
pirB-qrtF:GTGGAGCTGGACAGTCAGAG
pirB-qrtR:CAGACAGTGAGGAGGGAGGA
利用trizol法提取稳定敲除pirB的A549细胞株总RNA，消化DNA后，利用反转录试剂盒反转录成cDNA；
定量PCR体系为：10μl的Premix Ex Taq(SYBR qPCR)(2×)，10μmol/L 的Bmi1上下游引物各0.5μl，待检cDNA模板或阴性对照1μl，加灭菌去离子水补足体积至20μl。
PCR反应条件为：95℃预变性30s；然后95℃15s，60℃15s，72℃15s，反应40个循环。
发现敲除pirB的A549细胞株中pirB转录水平的表达量明显下降，如图2所示。
收集细胞蛋白样品进行电泳。电泳时，按每孔50μg蛋白上样量等量上样。以400mA恒压转膜100min。将膜放入1％酪蛋白/TBS中于室温下振荡封闭1h。加入TBST稀释后的pirB抗体(abcam)或对照α-tubulin(Santa Cruz Biotechnology)抗体一抗(pirB抗体按1：2000稀释，抗α-tubulin抗体按1： 2000稀释)，4℃振荡孵育过夜。吸净一抗，TBST漂洗膜10min×3；加入稀释后的二抗(均为1：4000稀释)，水平摇床室温孵育1h。吸净二抗，TBST 漂洗膜，10min×4，混合ECL(A：B＝100：1液)，充分覆盖湿润NC膜表面，静置1min；于暗室中用保鲜膜将NC膜封好，将X光片平放于NC膜上显影并记录数据。结果可见，发现敲除pirB的A549细胞株中pirB蛋白水平的表达量明显下降，如图3所示。
六、肺癌增殖实验
取对数生长期的肺癌细胞接种于96孔板，每孔6000个细胞，每隔24h加入CCK-8在450nm处读数，根据吸光值分析细胞增殖的能力。发现与对照组相比，敲除pirB的肺癌细胞增殖能力降低，如图4所示。
七、肺癌细胞的迁移实验
利用transwell法对肺癌的迁移活性进行研究。抑制pirB的A549细胞株细胞与对照细胞株细胞无血清饥饿24小时，用无血清培养基稀释，种5×105个细胞在transwell小室中(Chemicon公司)，下面加入含10％血清的肺癌细胞培养液，培养8小时后，用棉签擦去上层未侵袭的细胞，用100％甲醇固定10min 后空气中晾干，加入DAPI染核，在荧光显微镜下观察并拍照，随机数10个视野中的细胞数目，敲除pirB的肺癌细胞迁移活性降低，如图5所示。
SEQUENCE LISTING
<110> 西安医学院
<120> 抑制pirB表达的短发卡shRNA、慢病毒及其应用
<130> 2018
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 23
<212> DNA
<213> 人工序列（Artificial sequence）
<400> 1
ggagtaccaa ctggataaag agg 23
Claims (9)
1.抑制pirB表达的短发卡shRNA，其特征在于，其核苷酸序列为SEQ ID No.1所示的碱基序列。
2.一种干扰质粒，其特征在于，该质粒含有如权利要求1所述的核苷酸序列。
3.一种pirB基因shRNA敲除慢病毒表达载体，其特征在于，将如权利要求1所述的抑制pirB表达的短发卡shRNA插入慢病毒载体质粒pGLVH3/H1/GFP+Puro所得。
4.一种重组如权利要求3所述的pirB慢病毒表达载体的构建方法，其特征在于，具体包括：
合成用于编码pirB的核苷酸片段；用限制性内切酶用BamHI和EcoRI分别对权利要求1所述的核苷酸片段和pGLVH3/H1/GFP+Puro载体进行双酶切；将双酶切后的所述核苷酸片段和pGLVH3/H1/GFP+Puro进行连接，得到重组的pirB慢病毒载体。
5.一种pirB病毒的制备方法，其特征在于，具体包括：用慢病毒包装质粒pGag/Pol、pRev和pVSV-G对权利要求4所述的重组的pirB慢病毒载体进行包装，转染293T细胞后，得到pirB病毒。
6.根据权利要求5所述的一种pirB病毒的制备方法，其特征在于，所述重组的pirB慢病毒表达载体、pGag/Pol、pRev和pVSV-G的质量比为(1.5-2.5)：(0.5-1.5)：(0.6-1.5)：(0.5-1.4)。
7.采用权利要求5或6所述一种pirB病毒的制备方法制备得到的pirB病毒。
8.根据权利要求1或3或6或7所述的抑制pirB表达的短发卡shRNA或pirB基因shRNA敲除慢病毒表达载体或pirB病毒在制备抑制肺癌增殖的药物中的应用。
9.根据权利要求1或3或6或7所述的抑制pirB表达的短发卡shRNA或pirB基因shRNA敲除慢病毒表达载体或pirB病毒在制备抑制肺癌迁移的药物中的应用。
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| XUNLEI KANG,ET AL: "Inhibitory leukocyte immunoglobulin-like receptors: Immune checkpoint proteins and tumor sustaining factors.", 《CELL CYCLE》 * |
| ZHAOHUA ZHAO ET AL.: "PirB Overexpression Exacerbates Neuronal Apoptosis by Inhibiting TrkB and mTOR Phosphorylation After Oxygen and Glucose Deprivation Injury.", 《CELL MOL NEUROBIOL》 * |
| 罗琴等: "RNA干扰下调SOX9表达抑制肝癌细胞的增殖", 《肿瘤》 * |
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