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

Gene Cloning, Expression and Activity of Mouse Truncated Soluble Adenylyl Cyclase

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。

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 immunostaining assay then determined for activity by cAMP assay. Results Restriction analysis and sequencing proved that recombinant plasmid pcDNA4-V5-mtsAC was constructed correctly, containing mtsAC gene at a length of 1 549 bp. Re-combinant mtsAC protein, with a relative molecular mass of 48 000, was homogenously expressed in both cytoplasm and karyon of HEK293T cells. The cAMP in HEK293T cells transfected with recombinant plasmid pcDNA4-V5-mtsAC increased by 17 folds as compared with that in untransfected HEK293T cells, which was activated by sodium bicarbonate and calcium chloride. Conclusion The mtsAC gene was successfully cloned, and the recombinant mtsAC protein with activity was expressed in HEK293T cells, which provided a technical platform for study on characters of mtsAC and screening of drugs for male infertility.