猪CD8B基因编码区克隆和序列分析

Coding Sequence Cloning and Sequence Analysis of Pig CD8B Gene

本研究旨在克隆猪CD8B基因的编码区及检测其碱基变异,并利用生物信息学对CD8B基因及其编码的蛋白质序列进行分析。提取大白猪脾脏总RNA后,用RT-PCR扩增CD8B基因。结果表明,扩增得到长785 bp猪CD8B基因序列,包含完整的630 bp编码区(共编码209个氨基酸),并检测到1个同义突变(c.204 A/G)。经预测,猪CD8β蛋白的分子式为C1053H1702N296O279S10,分子量为23293.51,理论等电点为10.24,不稳定系数为50.40,平均亲水性系数为-0.041;二级结构以无规卷曲、延伸链为主,含1个Ig V样结构域;存在1个长21个氨基酸残基的信号肽和1个跨膜区;含1个N-糖基化位点和4个O-糖基化位点。同源性分析表明,猪CD8β蛋白与8个哺乳动物(普通牛,水牛,山羊,绵羊,猫,狗,小鼠,大鼠)的相似性在49%以上。分子进化分析表明,猪CD8β蛋白与普通牛、水牛、山羊、绵羊的亲缘关系最近,其次是猫、狗,最远的是小鼠、大鼠。本试验为研究猪CD8B基因的结构和免疫功能提供基础资料。

The purpose of this study was to clone the coding region of pig CD8 B gene and detect its base mutation,and to analyze the CD8 B gene and its encoded protein sequence by bioinformatics.After extracting total RNA from the spleen of Large White pigs,and amplifying the CD8 B gene by reverse transcription-polymerase chain reaction.The results showed that the amplified the long 785 bp sequence of pig CD8 B gene contained a complete630 bp coding region(Encoding a total of 209 amino acids),and a synonymous mutation(c.204 A/G)was detected.It was predicted that the molecular formula of pig CD8βprotein was C1053 H1702 N296 O279 S10,the molecular weight is 23293.51,the theoretical isoelectric point was 10.24,the instability index was 50.40,and the grand average of hydropathicity was-0.041;the mainly secondary structure were random coil and extended strand,and containing one Ig V-like domain;existing one signal peptide with 21 amino acid residues and one transmembrane region;including one N-glycosylation site and four O-glycosylation sites.Homology analysis showed that the similarity between pig CD8βprotein and eight mammals(cattle,buffalo,goat,sheep,cat,dog,mouse and rat)was more than 49%.Molecular evolution analysis showed that pig CD8βprotein was closely related to cattle,buffalo,goat and sheep,followed by cat and dog,and far from mouse and rat.This study provided basic data for the study of the structure and immune function of pig CD8 B gene.