CN101575605A - 视黄酸活性物质转基因斑马鱼生物探测器的生产方法 - Google Patents
视黄酸活性物质转基因斑马鱼生物探测器的生产方法 Download PDFInfo
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Abstract
本发明公开了斑马鱼的一段基因组DNA序列和一种视黄酸活性物质转基因斑马鱼生物探测器，以及这种生物探测器的制作生产方法。本发明克隆了斑马鱼对视黄酸反应的一段DNA序列，并融合在增强黄色荧光蛋白(eYFP)编码序列的5’端，通过显微注射的方法注射斑马鱼单细胞受精卵，经过筛选子代得到了稳定遗传的斑马鱼品系。由于其eYFP的表达强度是受各种视黄类活性物质浓度依赖性的诱导，可以用来筛选探测未知的视黄类物质。这种转基因斑马鱼生物探测器，经济、快速、方便、准确，可以用于药物筛选和环境检测等领域。
Description
技术领域
本发明涉及一种生产新的转基因斑马鱼生物探测器的方法，以及新的斑马鱼基因片段和转基因斑马鱼。
背景技术
视黄酸信号传导途径是脊椎动物体内一种重要的信号传导方式，一般通过视黄类物质起始信号的传递。视黄类物质进入细胞后，通过和结合在特定基因增强子---视黄酸反应元件---上的视黄酸受体(RAR)/视黄酸X受体(RXR)异二聚体的结合，招募辅激活物，激活靶基因的表达，从而行使其生物学功能，如指导胚胎的前后、左右及背腹轴的建立，决定体节和中枢神经系统等的发生。
大量研究表明，正常生理状态下，如果改变视黄酸信号会导致生物体的发育异常，如在胚胎发育过程中如果缺乏RA信号，会导致脑水肿、脊柱裂、无眼及小眼畸形；RA信号过多，导致颅顶骨发育不全(露脑)、小脑畸形、脑突出、小眼畸形和脊柱裂等。在一些病理状态下，视黄类物质可以作为药物来治疗相应的症状，可以通过影响视黄酸信号的强弱来改变其病理状态，现代医学中视黄类物质已被广泛应用于痤疮、银屑病、鱼鳞病、扁平苔癣、鳞状细胞癌、黑色素瘤、白血病、肝癌、胃癌等疾病的治疗中。
建立一种适合的生物学模型来筛选鉴别具有影响视黄酸信号传导的小分子，以及监控环境中视黄酸信号分子的存在，在药物筛选，环境监测，科学研究方面有着现实重要的意义。
斑马鱼是一种热带淡水鱼，来源于印度的热带河流，最初被用于发育生物学和遗传学的研究。由于斑马鱼有着独特的优势特征如：体型较小、很容易饲养繁殖、体外受精、卵生且产卵数目大、胚胎透明、胚胎发育快、胚胎发育不需要亲鱼的照料、性成熟期短、污染物和药物一般可以直接加入斑马鱼胚胎的培养液中，斑马鱼现在已成为研究小分子物质生物活性的一种重要动物模型。
随着荧光蛋白如绿色荧光蛋白(GFP)和黄色荧光蛋白(YFP)等转基因技术成功地在斑马鱼模型上的应用，使用斑马鱼模型，尤其是荧光蛋白转基因斑马鱼胚胎来定性定量的研究小分子物质的生物学活性成为了一种可能。与传统的药物筛选模型相比，转基因斑马鱼胚胎模型有着：1，经济2，快速3，方便4，有效；等优势。
我们选择并克隆受视黄酸直接调控的基因调控序列，接在YFP编码序列的5‘端，通过胚胎显微注射的方法制成转基因斑马鱼品系，胚胎在24hpf(受精后24小时)发出的YFP信号强弱是受外源性视黄类物质浓度依赖性的诱导的。这样我们就成功地研制成了监测鉴定视黄类活性物质的转基因斑马鱼生物监测器。
发明内容
本发明的目的是提供一种制作可以稳定表达增强黄色荧光蛋白(eYFP)的转基因斑马鱼品系的方法，eYFP的表达强度受外源性视黄类物质浓度依赖性的诱导。
本发明的另一个目的涉及基因片段，包括(1)斑马鱼细胞色素基因cyp26a1的2553bp调控序列、(2)eYFP荧光蛋白基因、(3)蛋白入核信号和(4)SV40polyA加尾信号。
附图说明
附图可以更好的说明本发明的意义和效果，而不是对本发明的限定。
图1为克隆的2533bp DNA片段的序列。
图2为胚胎显微注射使用的DNA片段示意图。
图3为培养至18hpf时，视黄酸处理6小时后，视黄酸对胚胎荧光蛋白的浓度依赖性的诱导表达。
具体实施方式
制做转基因斑马鱼生物探测器的方法，包括以下的步骤：
(1)克隆cyp26a1基因ATG 5’上游2533bp的调控序列。
通过mRNA序列与斑马鱼基因组序列的BLASTN程序比对分析，在GENEBANK上找到斑马鱼cyp26a1基因的ATG 5’上游序列，设计对应的正反向引物，两端分别引入了Sac I和Nco I的酶切位点，高保真PCR的方法扩增到片段，此片段连接到pGEMT载体中，经测序鉴定确认此克隆是2533bp的目标片段。
(2)构建表达载体zcyp26a1promoter-eYFP-pA。
用Sac I和Nco I将斑马鱼cyp26a1启动子2533bp片段从载体上切下，装入到pGEM-9zf(-)载体中得到质粒pGEM-zcyp26a1promoter-pA。再用BamH I将克隆到的eYFP片段切下装到pGEM-zcyp26a1promoter-pA载体上，从而得到了pzcyp26a1promoter-eYFP-pA质粒。所得到的重组表达载体pzcyp26a1promoter-eYFP-pA，包含斑马鱼cyp26a1基因的调控序列、eYFP编码序列、蛋白入核信号序列和polyA序列。
(3)线性化表达载体。
将pzcyp26a1promoter-eYFP-pA载体用Sac I和Not I双酶切，琼脂糖凝胶电泳得到两条DNA片段，胶回收其中约4kb长度的片段，制成最终浓度约为50ng/ul的溶液。
(4)显微注射。
收集单细胞时期的斑马鱼受精卵，将约2ul的DNA溶液装入微电极中，显微注射到细胞浆中，每个胚胎约2nl。胚胎培养到性成熟。
(5)转基因斑马鱼的获得。
将F0代与野生型的斑马鱼交配，观察其F1代是否有YFP表达来鉴别出阳性的F1代。再提取胚胎的基因组DNA，用YFP的特异性引物来扩增出对应大小的片段进行测序确认。
<110>胡平
<120>视黄酸活性物质转基因斑马鱼生物探测器的生产方法
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<213>斑马鱼(Danio Rerio.)
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ttacgaggga aaacaagcct cataattgca caaattcaca aataaggcca agtttgaagg 720
ttcgctcagg gtatttggca tgcttgggtc atgccgtaga ataaatatgg caacggttga 780
gtaatgggtt aacatatgaa cgcactgctt actcagaggc atgcctaaaa taccctcaaa 840
catagatgtg tcatgcgtga gtattccccg tcccattgca caggatgaaa ggtgaaatgc 900
agggcaggca cacatcaaag aatgaggaga gggaaaaatt tggagccatc ctgtctcttt 960
aactgactcg cttcccagct tgttttttcc acgttatggc tgtcgtaaaa acagtaattc 1020
agactgcagt gggtgtttgc tcttcatgta ccggtaacct tgctctgctc cgtcactttt 1080
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Claims (4)
1.一种转基因斑马鱼生物探测器的制作方法，其特征在于，包括如下步骤：
1)从斑马鱼基因组中克隆cyp26a1基因ATG 5’上游2533bp的调控序列
2)将此2533bp DNA片段融合在eYFP 编码序列(CDS)的上游构建成表达载体pzcyp26a1promoter-eYFP-pA
3)将线性化的DNA片段进行斑马鱼胚胎显微注射
4)将注射后的胚胎培养至性成熟，并且与野生型斑马鱼进行配对产卵
5)荧光显微镜下筛选阳性的F1代，继续自交传代F2，得到遗传稳定的转基因品系
2.根据权利要求1所述的方法，其特征在于，2533bp DNA序列来源于斑马鱼基因组cyp26a1基因的ATG起始编码子的5’上游。
3.根据权利要求1所述的方法，其特征在于，pzcyp26a1promoter-eYFP-pA 质粒包含2533bp调控序列、eYFP编码序列、蛋白入核信号序列、SV40polyA加尾信号序列。
4.根据权利要求1所述的方法，其特征在于，线性化的DNA片段在斑马鱼受精卵单细胞时期进行显微注射。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191271A (zh) * | 2010-03-18 | 2011-09-21 | 中国科学院动物研究所 | 抗肿瘤重组质粒、基因疫苗及其制备方法和用途 |
| CN112790125A (zh) * | 2021-01-22 | 2021-05-14 | 中国科学院水生生物研究所 | 一种玉兔百褶裙泰狮金鱼的创制方法 |
-
2008
- 2008-08-19 CN CNA2008100219244A patent/CN101575605A/zh active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102191271A (zh) * | 2010-03-18 | 2011-09-21 | 中国科学院动物研究所 | 抗肿瘤重组质粒、基因疫苗及其制备方法和用途 |
| CN112790125A (zh) * | 2021-01-22 | 2021-05-14 | 中国科学院水生生物研究所 | 一种玉兔百褶裙泰狮金鱼的创制方法 |
| CN112790125B (zh) * | 2021-01-22 | 2022-09-06 | 中国科学院水生生物研究所 | 一种玉兔百褶裙泰狮金鱼的创制方法 |
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