猪 Siglec-10基因的克隆与生物信息学分析

Cloning and Analysis of Porcine Siglec-10 Gene

为了研究猪Siglec-10(pSiglec-10)的分子生物学特征,利用Ensemble网站提供的预测序列(ENSSSCT00000032460)设计一对特异性引物,采用RT-PCR技术,克隆pSiglec-10分子的全长CDS区,经过测序比较鉴定正确后,对该分子的基本特征进行分析;构建截短pSiglec-10的原核表达载体,另将该分子的CDS区全长克隆至真核表达载体pcDNA3.1-3′FLAG,将重组质粒转染至PK-15和Marc-145细胞中分析其表达定位特征。结果克隆的pSiglec-10分子的全长CDS为2 127bp,编码709个氨基酸;pSiglec-10基因序列与猴的同源性最高,同源性达73.6%,与鼠的同源性为66.9%;与人的同源性为62.5%,氨基酸序列的同源性分别为58.9%、69.4%和65.3%;pSiglec-10C端的ITIM区域和磷酸化位点高度保守;pSiglec-10分子具有较好的抗原特性和亲水性;该分子主要表达定位在细胞浆中,在Marc-145细胞中呈不连续的点状分布于细胞浆,细胞核周围荧光强度较强。pSiglec-10分子全长CDS区的克隆和分析将对该分子的功能研究提供一定的理论依据。

In order to explore the functions of pSiglec-10,here we designed a pair of primers according to the predict sequence from Ensemble database to clone whole CDS region of pSiglec-10,then the basic infor-mation of pSiglec-10 was analyzed.A prokaryotic expression vector of truncated pSiglec-10 was recon-structed,the targeting clone was screened correctly and cloned into expression vector pcDNA3.1-3′FLAG, the reconstructing plasmid was transfected into cell PK-15 and Marc-145 to analyze the location of pSiglec-10 in cells.As a result,a full length CDS clone of pSiglec-10 with 2 127 bp was screened correctly,and it has a similarity with human,macaque and mouse in 62.5%,73.6%,66.9%,respectively.The consistency of the amino acid sequence of pSiglec-10 was compared with other three species in 58.9%,69.4%,65.3%, respectively.This molecule expressed in cytoplasm and distributed in discontinuous punctate collection in cell Marc-145,and showed a better antigenicity and hydrophilicity.This research would be a reference to explore the functions of pSiglec-10.