干旱区生态环境知识资源中心(Arid): 大鼠Desert Hedgehog基因的克隆和表达

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	大鼠Desert Hedgehog基因的克隆和表达
其他题名	CLONING AND EXPRESSION OF DESERT HEDGEHOG GENE IN THE RAT
	刘靖 1; 赵焕英 2; 赵春礼 2; 段德义 2; 高福禄 3; 徐群渊 2
ISSN	0529-1356
出版年	2006
卷号	37 期号:5 页码:545-548
中文摘要	目的克隆大鼠Desert Hedgehog(DHH)基因，构建其真核表达载体并转染PT67细胞；同时制备DHHcRNA正义及反义探针用于检测其在细胞中的表达。方法提取SD大鼠皋丸总RNA, RT-PCR法扩增DHH-cDNA片段，连接于pGEM-T Easy载体，经测序后构建真核表达载体pLXSN/DHH并转染PT67细胞；重组质粒经限制性内切酶Not I和Nco I酶切、回收后，进行转录标记反应，原位杂交检测DHH在PT67细胞中的表达。结果 RT-PCR扩增得到1 220 bp的片段；成功构建了真核表达载体pLXSN/DHH；制备DHH正义及反义探针浓度分别为150mg/L和80 mg/L; DHH在PT67细胞中有表达。结论克隆的DHH基因与大鼠Sertoli细胞的DHH基因相同，成功标记了特异、敏感的DHHcRNA探针，转染的DHH基因能够在PT67细胞中表达。
英文摘要	Objective In order to investigate the biological roles of Desert Hedgehog (DHH) expressed by Sertoli cells, we planed to clone the gene of DHH from rat, to construct its eukaryiotic expression vector, meanwhile, to label DHH sense and antisense probes and to detect the expression of DFIH gene in PT67 cells. Methods Total cellular RNA of rat’ s testes was abstracted and the cDNA fragment was amplified by RT-PCR. The fragment cDNA was ligated to pGEM-T easy vector by T4 DNA ligationase and sent to test sequence, and its sequence was analyzed. Plasmid pGEM-T easy/DHH and pLXSN plasmids were extracted, digested with EcoR I and BamH I, then ligated by T4 DNA ligationase. The eukaryiotic expression vector-pLXSN-DHH was constructed and then transfected into PT67 cells. The pGEM-T easy-DHH was digested by Not I and Nco I restriction enzymes and DHH cRNA probes were labeled by Dig-cRNA probe labeling kit. The expression of DHH was examined by in suit hybridization. Results The cDNA fragment of 1 220 bp was amplified by RT-PCR. An eukaryiotic expression vector, pLXSN-DHH, was successfully constructed. Anti-sense and sense probes were respectively 150 mg/L and 80 rag/L, and DHH gene was expressed in PT67 cells. Conclusion The DHH gene cloned in present study is same as the DHH gene in Sertoli cells. DHH probes were labeled successfully. The newly constructed vector, pLXSN-DHH, can be transfected into and expressed in PT67 cells.
中文关键词	Desert Hedgehog基因 ; 克隆 ; 表达 ; 逆转录聚合酶链反应 ; 大鼠
英文关键词	Desert Hedgehog gene Clone Expression RT-PCR Rat
语种	中文
国家	中国
收录类别	CSCD
WOS类目	BIOCHEMISTRY MOLECULAR BIOLOGY
WOS研究方向	Biochemistry & Molecular Biology
CSCD记录号	CSCD:2430632
资源类型	期刊论文
条目标识符	http://119.78.100.177/qdio/handle/2XILL650/219902
作者单位	1.河北医科大学组织学与胚胎学教研室, 石家庄, 河北 050017, 中国; 2.首都医科大学北京神经科学研究所, 北京 100069, 中国; 3.承德医学院, 承德, 河北 067000, 中国
推荐引用方式 GB/T 7714	刘靖,赵焕英,赵春礼,等. 大鼠Desert Hedgehog基因的克隆和表达[J],2006,37(5):545-548.
APA	刘靖,赵焕英,赵春礼,段德义,高福禄,&徐群渊.(2006).大鼠Desert Hedgehog基因的克隆和表达.,37(5),545-548.
MLA	刘靖,et al."大鼠Desert Hedgehog基因的克隆和表达".37.5(2006):545-548.