| 小鼠截短型可溶性腺苷酸环化酶基因的克隆表达及活性鉴定 |
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| 引用本文： | 王文,戚中田.小鼠截短型可溶性腺苷酸环化酶基因的克隆表达及活性鉴定[J].粉末涂料与涂装,2010,23(2). |
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| 作者姓名： | 王文 戚中田 |
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| 作者单位： | 第二军医大学微生物学教研室上海市医学生物防护重点实验室,上海,200433 |
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| 摘 要： | 目的克隆表达小鼠截短型可溶性腺苷酸环化酶(mtsAC)基因,并检测其活性,为后续研究提供技术平台。方法Trizol法提取小鼠睾丸组织总RNA,RT-PCR法扩增mtsAC基因,经测序正确后,克隆至真核表达载体pcDNA4-V5,转染HEK293T细胞,Western blot及免疫荧光染色法检测目的蛋白的表达,并检测HEK293T细胞中cAMP的水平。结果所构建的重组表达质粒pcDNA4-V5-mtsAC经酶切和测序证明构建正确,插入的mtsAC基因长度为1549bp;表达的重组mtsAC蛋白相对分子质量为48000,在胞浆及胞核中呈均一性表达;转染质粒pcDNA4-V5-mtsAC的HEK293T细胞中cAMP的水平较未转染的HEK293T细胞升高约17倍,并可被NaHCO3和CaCl2激活。结论已成功克隆了mtsAC基因,并在HEK293T细胞中表达了具有活性的重组mtsAC蛋白,建立了可用于mtsAC特性研究及男性不育药物筛选的技术平台。
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| 关 键 词： | 可溶性腺苷酸环化酶 克隆 表达 cAMP |
Gene Cloning, Expression and Activity of Mouse Truncated Soluble Adenylyl Cyclase |
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| WANG Wen,QI Zhong-tian.Gene Cloning, Expression and Activity of Mouse Truncated Soluble Adenylyl Cyclase[J].Chinese Journal of Biologicals,2010,23(2). |
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| Authors: | WANG Wen QI Zhong-tian |
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| Affiliation: | WANG Wen,QI Zhong-tian (Department of Microbiology,Second Military Medical University,Shanghai 200433,China) |
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| Abstract: | Objective To clone and express mouse truncated soluble adenylyl cyclase gene, determine the activity of ex-pressed product and provide a technical platform for further study. Methods Total RNA was extracted from mouse testis tissue by Trizol method, based on which mtsAC gene was amplified by RT-PCR, identified by sequencing and cloned into eukaryotic expression vector pcDNA4-V5. The constructed recombinant plasmid was transfected to HEK293T cells, and the expressed protein was identified by Western blot and... |
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| Keywords: | cAMP Soluble adenylyl cyclase Cloning Expression cAMP |
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