马身猪CD63基因CDS区的克隆、表达及生物信息学分析

Molecular Cloning,Expression Pattern and Bioinformatics Analysis of CD63 Gene in Mashen Pig

为了克隆马身猪CD63基因的CDS区,检测其在不同组织中的表达规律,预测编码蛋白的结构与功能,该研究对山西省地方品种马身猪CD63基因CDS区进行扩增和克隆,采用qRT-PCR技术检测CD63基因在心脏、肝脏、脾脏、肾脏、腰大肌、背部皮下脂肪等组织中的表达规律,采用生物信息学方法分析CD63蛋白的生物学特性。结果表明,该研究成功克隆出猪CD63基因的CDS区,全长714 bp,已上传NCBI数据库,登录号为MN082631。该基因在所检测的组织中均有表达,且在不同组织中的表达量有显著差异(P<0.05),脂肪组织中表达量最高,其次是肝脏、肌肉、脾脏,肾脏中的表达量最低。经生物信息学预测,该基因编码的蛋白质含237个氨基酸,属于中性稳定四跨膜蛋白,有3个可能的N-糖基化位点,其中2个位点位于第3个与第4个跨膜结构域之间的胞外区内,另一个在第1个与第2个跨膜结构域之间的胞外区内;可能存在Ser磷酸化位点6个,Thr磷酸化位点4个,Tyr磷酸化位点1个;CD63蛋白为疏水性蛋白,α-螺旋、延伸、β-折叠和无规则卷曲分别占58.23%、11.39%、5.49%和24.89%。猪CD63蛋白氨基酸序列与野骆驼的相似性最高,其次是羊驼、长江江豚和抹香鲸,同时与骆驼科的野骆驼和羊驼的遗传距离最近,符合生物进化规律。该试验成功克隆了马身猪CD63基因CDS区,并进行了生物信息学分析,为深入探讨CD63基因生物学功能提供参考。

The aim of this study was to clone the coding sequence(CDS)of CD63 gene in Mashen pig,to detect the expression patterns of CD63 in different tissues,and to predict the structures and functions of their coding proteins.The full-length CDS of CD63 gene of Mashen pig,a local pig breed of Shanxi Province,was cloned and the mRNA expression patterns of CD63 gene in heart,liver,spleen,kidney,psoas magnus and back subcutaneous fat were detected by quantitative real-time PCR(qRT-PCR),and the biological characteristics of CD63 protein was analyzed by bioinformatics methods.The CDS of CD63 gene was successfully cloned in this study.The length of CDS region of CD63 gene is 714 bp,and which has been uploaded into NCBI database,and the GenBank accession number is MN082631.CD63 was expressed in all tissues detected in this study,and the differences of the expression amount among different tissues were significant(P<0.05).The highest abundance of CD63 was in adipose tissue,followed by liver,muscle and spleen,and the lowest expression amount was in kidney.Bioinformatics analysis predicted that CD63 was a neutral stable four-transmembrane protein,which consisted of 237 amino acids.CD63 had three possible N-linked glycosylation sites,two of which were located in the extracellular region between the third and fourth transmembrane domains,and the other was in the first and second transmembrane domains.In addition,there were 6 Ser phosphorylation sites,4 Thr phosphorylation sites and 1 Tyr phosphorylation site in CD63 protein.Hydrophobic analysis showed that the CD63 protein is a hydrophobic protein,and theα-helix,extension,β-sheet and random coil in the porcine CD63 protein accounted for 58.23%,11.39%,5.49%and 24.89%,respectively.The highest similarity of porcine CD63 amino acid sequence is to that of Camelus feru,followed by Vicugna pacos,Neophocaena asiaeorientalis asiaeorientalis and Physeter catodon.And the genetic distance based on amino acid sequence of CD63 protein between pig and Camelus feru or Vicugna pacos which belong to camelid family is closest,which is consistent with species evolution.In this study,the CD63 gene CDS of Mashen pig was successfully cloned and analyzed by bioinformatics,which provided reference for further exploration of the biological function of CD63 gene.