逆转录聚合酶链反应检测小鼠MN/CA9基因的表达

Detection of expression of mouse MN/CA9 gene with reverse transcriptase-polymerase chain reaction

目的:克隆、分析ICR小鼠MN/CA9基因序列,并检测MN/CA9基因在ICR小鼠各组织中的表达。方法:分别取ICR小鼠小肠、肝、肾、脾、胃、胸腺、心脏、卵巢、肺、胰腺、肌肉、皮肤、子宫和膀胱新鲜组织,采用胍尼啶异硫氢酸呱酚氯仿提取法(GIT)提取总RNA。合成cDNA的第1链和第2链,连接EcoRⅠ载体,EcoRⅠ末端磷酸化,XhoⅠ消化,将cDNA构建到ZAPexpressvector进行包装、种植培养,筛选、切取噬菌体,使用引物扩增后进行DNA序列分析。用逆转录聚合酶链反应检测小鼠MN/CA9基因在上述组织中的表达。结果:使用人类MN/CA9基因片段做探针,用放射性同位素32P标记探针,筛选1·47×103大肠埃希菌落,杂交后发现一阳性cDNA信号,序列测定确定其含1671bp核苷酸序列(已在GenBank登录,登录号AB086322),这个序列与人类的MN/CA9(基因序列号Z54349)有69·1%的同源性。在引物P521-P1193区间,MN/CA9基因在小鼠小肠、子宫、肌肉、胰腺、心脏、肺、胸腺、脾、肾、卵巢、胃和膀胱组织表达均较强,在皮肤和肝脏不表达。结论:MN/CA9基因在ICR小鼠组织中的表达与人类基本相同,可以用ICR小鼠进行MN/CA9基因的研究。

Objective To clone and analyze the MN/CA9 gene sequence in ICR mice and detect the expressions of MN/CA9 gene in various tissues of ICR mice. Methods The fresh tissues of small intesine, uterrus, skin, tousle, liver, pancreas, heart, lung, thymus, spleen, kidney, ovary, stomach , urinary bladder from ICR mice were obtained, the total RNA was extracted by GIT method, the 1st strand and 2nd strand of cDNA were synthesized, the EcoR Ⅰ adapters were lingated, the EcoR Ⅰ ends were phosphorylated, digested with Xho Ⅰ , cDNA was ligated into the ZAP expression vector, packaged, planted, screened. The expressions of MN/CA 9 gene in various tissues in mice were detected by RT-PCR. Results A fragment of human MN/CA9 gene was used as probe, and 1.47×10^3 clones were screened with radioactive isotopic ^32P labeled probe, after hybridizations, one positive signal of cDNA was detected and the complete nucleotide sequence contained 1 671 bp was determined （GenBank： AB086322）, The nucleotide similarity between mouse and human cDNA （GenBank： Z54349） was 69. 1%. The MN/CA9 gene detected by RT-PCR assay （primer： P521-P1193） strongly expressed in small intesine, uterus, tousle, pancreas, heart, lung, thymus, spleen, kidney, ovary, stomach, and urinary bladder, meanwhile did not express in skin and liver. Conclusion The expressions of MN/CA9 gene in some tissues of ICR mice are similar to that of human, it can be used to further functional analysis of MN/CA9 gene.