CN102876698B - 抑制绵羊成纤维细胞生长因子5表达的试剂及其应用 - Google Patents
抑制绵羊成纤维细胞生长因子5表达的试剂及其应用 Download PDFInfo
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Abstract
本发明公开了抑制绵羊成纤维细胞生长因子5(FGF5)表达的试剂及其应用，所述的试剂包括能够有效抑制绵羊成纤维细胞生长因子5(FGF5)表达的shRNA分子，且具有高度特异性。该试剂能够用于增加绵羊的毛发长度，具有显著的应用价值。
Description
技术领域
本发明属于分子生物学领域，尤其涉及一种抑制绵羊成纤维细胞生长因子5(FGF5)表达的试剂及其应用。
背景技术
羊毛长度是细毛羊的主要经济性状之一。羊毛长度遗传力约为0.56，表明羊毛长度主要决定于遗传因素，即主要受与毛囊生长发育相关基因的调控。成纤维细胞因子5(FGF5)基因表达的抑制或突变失活，能延长毛囊的生长期，显著增加毛发的长度。根据国内外研究报道，目前在狗、猫上都发现了与毛发长度相关的突变基因型。除突变基因型外，可以通过基因敲除或干扰抑制FGF5表达使毛囊生长期延长，增加毛发长度的研究已经成为FGF5研究的热点。RNA干扰技术是最近两年来发展起来的阻断基因表达的新技术，其抑制基因表达的功能得到了充分的证明，并被广泛应用。
针对FGF5的基因结构，设计合成能够有效抑制FGF5基因表达的干扰RNA分子，对深入研究羊毛生长发育的分子调控机制和培育促进羊毛生长的转基因羊生物新品种具有重要的意义。
发明内容
本发明的目的是获得一种能够有效抑制FGF5基因表达试剂，其具有高特异性。为了实现本发明的目的，拟采用如下技术方案：
本发明一方面涉及一种抑制FGF5基因表达试剂，其包括表达载体，所述的载体上包含SEQ ID NO.1#、2#或3#的shRNA正向和反向序列。
在本发明的一个优选实施方式中，所述的shRNA序列通过人工合成获得，退火形成双链后与XhoI和HpaI双酶切线性化的pLL3.7载体连接形成表达载体。
在本发明的一个优选实施方式中，所述的shRNA序列为SEQ ID NO.2，所述的试剂是特异性的。
本发明另一方面还涉及上述试剂的应用，所述的试剂用于抑制FGF5基因的表达，所述的抑制对象包括绵羊或者绵羊细胞。
本发明另一方面还涉及上述试剂在提高羊毛长度中的应用。
在本发明的一个实施方式中，所述的提高羊毛长度是通过抑制绵羊成纤维细胞生长因子5来实现的。
附图说明
图1不同Lv-shRNA-FGF5干扰分子对pLEX-FGF5表达的抑制效果的Western Blotting分析。(图例说明1为Lv-shRNA-FGF5-1#与pLex-FGF5共转染，2为Lv-shRNA-FGF5-2#与pLex-FGF5共转染，3为Lv-shRNA-FGF5-3#与pLex-FGF5共转染，4为Lv-shRNA-Luciferase与pLex-FGF5共转染。其中，A所用一抗为鼠抗HA标签蛋白抗体，二抗为抗鼠的荧光标记抗体；B所用一抗为鼠抗β-actin蛋白抗体，二抗为抗鼠的荧光标记抗体)。
图2Lv-shRNA-FGF5-2#干扰分子抑制FGF5表达的特异性验证(Rescue)。(图例说明：1为pLex-FGF5，2为pLex-FGF5m突变体，3为Lv-shRNA-FGF5-2#与pLex-FGF5共转染，4为Lv-shRNA-FGF5-2#与pLex-FGF5m共转染，5为未转染细胞。其中，A所用一抗为鼠抗HA标签蛋白抗体，二抗为抗鼠的荧光标记抗体；B所用一抗为鼠抗β-actin蛋白抗体，二抗为抗鼠的荧光标记抗体)。
具体实施方式
1抑制FGF5基因转录shRNA干扰分子的设计
以NCBI数据库(登录号：JQ941956)公布的绵羊FGF5基因序列，选择其中相应的靶序列，利用网上在线软件siDESIGN设计针对该基因的干扰序列3个(http://www.dharmcon.com)，以及一个针对Luciferase基因的干扰序列作为对照。序列为：
1#：
正向序列：
5’-T AGATCTACCCGGATGGCAA TTCAAGAGA TTGCCATCCGGGTAGATCT TTTTTT C-3’
反向序列：
5，-TCGAG AAAAAA AGATCTACCCGGATGGCAA TCTCTTGAA TTGCCATCCGGGTAGATCT A-3’
2#：
正向序列：
5’-T GGAAATATTTGCTGTGTCT TTCAAGAGA AGACACAGCAAATATTTCC TTTTTTC-3’
反向序列：
5’-TCGAG AAAAAA GGAAATATTTGCTGTGTCT TCTCTTGAA AGACACAGCAAATATTTCC A-3’
3#：
正向序列：
5’-T CATGCAAGTGCCAAATTTA TTCAAGAGA TAAATTTGGCACTTGCATG TTTTTTC-3’
反向序列：
5’-TCGAG AAAAAA CATGCAAGTGCCAAATTTA TCTCTTGAA TAAATTTGGCACTTGCATG A-3’
Luciferase：
正向序列：
5’-T CGGTATGATTGATGAGAAT TTCAAGAGA ATTCTCATCAATCATACCG TTTTTT C-3’
反向序列：
5’-TCGAG AAAAAA CGGTATGATTGATGAGAAT TCTCTTGAA ATTCTCATCA ATCATACCG A-3’
2干扰分子的合成及表达载体的构建
将以上设计的序列送交上海生物工程公司合成。每个干扰分子合成后的正链和反向互补链分别用双蒸水溶解，各取10ul 10uM的两条shRNA寡核苷酸，加入8μl 10×annealing buffer和12μl灭菌超纯水混合，煮沸10分钟后自然冷却至室温。同时，pLL3.7质粒载体经XhoI和HpaI双酶切后回收线性化载体，与上述shRNA寡核苷酸退火产物4℃过夜连接。连接产物转化DH5a感受态细胞，LB平板氨苄青霉素(100ug/ul)筛选。用载体检测引物及shRNA寡核苷酸引物筛选阳性克隆并送上海生工测序鉴定序列正确后，提取纯化重组质粒用于转染(其中干扰分子shRNA1#，2#和3#序列对应的表达载体分别为Lv-shRNA-FGF5-1#，Lv-shRNA-FGF5-2#和Lv-shRNA-FGF5-3#)。
3细胞水平评价shRNA干扰分子对FGF5基因转录表达的抑制效果
3.1绵羊FGF5表达载体的构建
取细毛羊体侧皮肤组织100mg于液氮中研磨，用Trizol(Invitrogen)法提起总RNA，采用1％的琼脂糖凝胶电泳验证RNA的完整性，-70℃保存备用。根据反转录试剂盒(TaKaRa)说明，以Oligo dT Primer为引物合成cDNA，以该cDNA为模板，以引物FGF5-s为上游，FGF5-as(见表1)为下游进行RT-PCR。扩增产物和pLEX-MCS质粒分别用Not I和Xho I酶切，经胶回收纯化后T4连接酶连接过夜。连接产物转化入100μL大肠杆菌DH5α感受态细胞，提取质粒、酶切、PCR扩增、测序鉴定正确后，纯化重组质粒用于转染。
3.2shRNA表达载体与FGF5表达质粒共转染293T细胞
转染前一天按每孔4×105个/mL的密度将293T细胞接种于六孔板，用2ml含有10％胎牛血清的DMEM培养液培养细胞。24h后细胞达到70％～80％汇合度时，将1μg的shRNA干扰载体(包括无关基因对照质粒)与1μg FGF5表达质粒加入到0.3ml预热的Opti-MEM培养基后，轻轻混匀，同时将6ul的脂质体加入到0.3ml opti-MEM培养基，室温放置5min后，将上述稀释好的DNA与脂质体混合，置于室温孵育20分钟；在此期间，用Opti-MEM培养基清洗待转染细胞一次，将脂质体和DNA复合物加入细胞；将细胞培养板放回37℃培养箱中孵育；2-4小时后，移去脂质体和DNA复合物，在更换为含有10％胎牛血清的DMEM完全培养液。培养48小时后收获细胞，提取蛋白。
3.3利用Western blot观察shRNA的抑制效果
上述转染细胞的提取蛋白，变性后12％SDS-PAGE进行蛋白电泳，转膜、封闭，而后分别加一抗，即鼠抗HA(1∶2000)、鼠抗β-actin(1∶1000)抗体，于4℃孵育过夜。次日，洗涤后加抗鼠的荧光标记抗体二抗(1∶10000)，室温孵育40分钟；洗涤后，通过Odyssey红外荧光扫描成像系统成像，灰度值扫描分析，得出蛋白量的相对值。通过与对照样比较，pLL-FGF5-2#能够有效抑制82％FGF5蛋白的翻译，Lv-shRNA-FGF5-2#能够有效抑制82％FGF5蛋白的表达，见图1。
4shRNA干扰分子抑制FGF5基因转录的特异性评价
4.1绵羊FGF5突变体表达载体pLEX-FGF5m的构建
在FGF5全长CDS序列中找到Lv-shRNA-FGF5-2#干扰分子的靶序列，并按FGF5开放阅读框在靶序列的Seeding region引入4个无义突变位点，保证其编码的氨基酸不变。shRNA-FGF5-2#干扰分子的靶序列：…G GAAATA TTT GCT GTG TCT…突变后序列：…G GAA ATA TTC·GCA GTCTCA…。按突变后的FGF5序列设计一对引物FGF5m-s和FGF5m-as(见表1)，上游引物FGF5m-s包含突变位点，下游引物FGF5m-as设计为上游的反向互补序列，利用重叠延伸PCR法获得无义突变FGF5。反应1以引物FGF5-s为上游，FGF5m-as为下游扩增FGF5m的5′端；反应2以引物FGF5m-s为上游，FGF5-as为下游扩增FGF5m的3′端；反应3以引物FGF5-s为上游，FGF5-as 为下游，含重叠DNA片段的反应1和反应2的产物等比例混合作为模板以扩增全长FGF5m。PCR反应体系(50μL)：10×buffer5μL，引物0.5μL(10pmol/μL)，各1μL，dNTP(10mmol/L)2μL，，pfu酶0.4μL，反转录产物2μL，ddH2O补至50μL。PCR反应程序：94℃3min；94℃30sec；60℃30sec；72℃1min，35个循环；72℃5min；4℃保存。PCR产物用2％的琼脂糖凝胶进行检测。扩增产物和pLex质粒分别用Not I和Xho I酶切，经胶回收纯化后T4连接酶连接过夜。连接产物转化入100μL大肠杆菌DH5α感受态细胞，提取质粒、酶切、PCR扩增、测序鉴定正确后，提取纯化重组质粒用于转染。
4.2shRNA表达载体与FGF5m表达质粒共转染293T细胞
具体方法同3.2.
4.3Lv-shRNA-FGF5-2#干扰分子的特异性验证
上述转染细胞的蛋白提取后，通过western blot对FGF5基因表达进行分析，评价对Lv-shRNA-FGF5-2#干扰分子的特异性。
干扰分子shRNA-FGF5-2#分别和重组pLEX-FGF5、pLEX-FGF5m共转染293T，转染48小时后收集细胞Western鉴定(图2)。从第4泳道能看出Lv-shRNA-FGF5-2#几乎没有干扰突变后的pLEX-FGF5m表达。而第3泳道的结果表明Lv-shRNA-FGF5-2#对重组pLEX-FGF5有极明显的干扰作用，证实干扰分子Lv-shRNA-FGF5-2#的特异性良好。
表1实验引物
当理解的是，本发明的具体实施例仅仅是出于示例性说明的目的，其不以任何方式限定本发明的保护范围，本领域的技术人员可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。
Claims (5)
1.一种抑制FGF5基因表达试剂，其包括表达载体，所述的载体上包含SEQ ID NO.2#的shRNA序列。
2.根据权利要求1所述的试剂，所述的shRNA序列系人工合成获得，退火形成双链后与XhoI和HpaI双酶切线性化的pLL3.7载体连接形成表达载体。
3.权利要求1至2任意一项所述的试剂的应用，所述的试剂用于抑制FGF5基因的表达，所述的抑制对象包括绵羊或者绵羊细胞，所述的应用是非治疗和非诊断目的的。
4.权利要求1至3任意一项所述的试剂在提高羊毛长度中的应用，所述的应用是非治疗目的的。
5.根据权利要求4所述的应用，所述的提高羊毛长度是通过抑制绵羊成纤维细胞生长因子5基因的表达来实现的。
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