水牛缝隙连接蛋白43基因克隆及表达

本研究克隆了水牛缝隙连接蛋白43(connexin 43,Cx43)基因序列,并运用生物信息学方法对其核苷酸序列的保守性和氨基酸的理化性质、蛋白质结构进行了系统分析,对Cx43基因在水牛不同组织和不同发育阶段卵泡中的表达差异进行了检测。结果表明,应用RT-PCR技术克隆获得了水牛Cx43基因序列,其中编码区全长1152 bp,编码383个氨基酸,蛋白质理论分子质量43.13 ku,等电点为8.88。多重序列比对结果显示,水牛Cx43核苷酸序列与牛、羊、猪、马和人相应序列的同源性分别为99%、98%、94%、93%和92%,系统进化树分析结果推测,Cx43基因在不同物种进化过程中具有高度保守性;对水牛Cx43蛋白的二级和三级结构分析发现,其具有缝隙连接蛋白的特有结构。定量RT-PCR结果显示,Cx43在水牛卵巢组织中相对表达量最高,睾丸、肾脏、心脏和皮肤次之,肝脏和大脑表达量较低。免疫组化结果发现,Cx43蛋白表达随卵泡发育时期的不同而变化,Cx43蛋白随卵泡发育表达逐渐增强。

Cloning and Expression of Buffalo Cx43 Gene

Buffalo Cx43 gene was cloned in the present study, the Cx43 sequence was systemically analysized by bioinformatics techniques. The expression patterns of Cx43 in different tissues and different stages of follicles were also assayed with RT-PCR and immunochemistry methods. The results showed that, a buffalo Cx43 gene fragment was cloned and sequenced, including the whole CDS of 1152 bp (coding 383 amino acids). The molecular weight and isoelectric point of buffalo Cx43 protein were predicted as 43.13 ku and 8.88 respectively. The results of sequence multialigned showed that the sequence of buffalo Cx43 gene shared 99%,98%,94%,93% and 92% homology with Bos taurus,Ovis aries, Sus scrofa,horse and Homo sapiens respectively. Buffalo Cx43 protein was predicted containing special connexin protein domain. In addition, we also analyzed the mRNA expression level of Cx43 gene in buffalo tissues through Real-time fluorescence quantitative-PCR. The results showed the mRNA of Cx43 gene existed in all of the six detected buffalo tissues with the most abundant expression in ovary liver,followed by kidney,heart and skin,the minimal expression was observed in liver. The Cx43 expression was detected in buffalo follicle during various development stages by immunochemistry method. Expression of Cx43 protein increased along with the follicle development. The cloning and analysis of buffalo Cx43 gene laid an important foundation for furthur investigating the function of Cx43 gene.