绵羊SUN2基因生物信息学分析及其在A549细胞中的表达

doi:10.16431/j.cnki.1671-7236.2021.11.003

摘要/Abstract

摘要： 本研究旨在克隆绵羊SUN2（Sad1 and UNC84 domain containing 2）基因并进行生物信息学分析，构建真核表达载体并检测其在A549细胞中的表达。根据绵羊SUN2基因序列（GenBank登录号：XM_015095026.2）设计特异性引物，应用RT-PCR方法扩增并克隆绵羊SUN2基因CDS序列，测序鉴定后对其进行生物信息学分析，并构建pEGFP-C1-SUN2重组质粒。利用脂质体转染方法将pEGFP-C1-SUN2重组质粒转染至A549细胞并检测其表达。结果显示，绵羊SUN2基因CDS区序列全长2 187 bp，编码728个氨基酸，理论分子质量为81.06 ku，分子式为C₃₅₉₃H₅₆₃₉N₁₀₃₁O₁₁₁₀S₁₄，等电点（pI）为6.20，总原子数为11 387，不稳定系数为56.88，属于不稳定蛋白。绵羊SUN2基因序列与山羊、牛、猪、马、家犬、大鼠、小鼠及人的相似性分别为98.9%、97.2%、91.6%、90.6%、87.1%、82.4%、82.1%和86.1%，不同物种间基因相似性较高，进化过程中相对保守。系统进化树分析表明，绵羊SUN2基因与山羊、牛、猪、马和家犬等哺乳动物的遗传距离相对较近，与鸡的遗传距离较远。结构域及跨膜结构域预测显示，绵羊SUN2蛋白含有1个高度保守的SUN结构域，且存在跨膜结构域。信号肽预测显示其不存在信号肽位点，疏水性分析为亲水性蛋白，氨基酸序列二级结构主要为α-螺旋（51.65%），其余为无规则卷曲（31.46%）、延伸链（12.36%）和β-转角（4.53%）。试验成功构建了绵羊SUN2基因真核表达载体pEGFP-C1-SUN2，将其转染至A549细胞并检测到蛋白表达。本试验结果为绵羊SUN2基因功能的研究提供了参考依据，为后续探究SUN2基因在绵羊肺腺瘤病中的作用奠定了基础。

关键词: 绵羊; SUN2基因; 载体构建; 生物信息学分析; 转染

Abstract: This study was aimed to clone Sad1 and UNC84 domain containing 2(SUN2) gene in sheep and conduct bioinformatics analysis, construct eukaryotic expression vector and detect its expression in A549 cells. The specific primers were designed according to sequence of SUN2 gene in sheep (GenBank accession No. :XM_015095026.2), and the CDS sequence of SUN2 gene in sheep was amplified by RT-PCR method and cloned. After sequencing and identification, the sequence were analyzed by bioinformatics, and pEGFP-C1-SUN2 recombinant plasmid was constructed. The pEGFP-C1-SUN2 recombinant plasmid was transfected into A549 cells by liposome transfection method and its expression was detected. The results showed that the total length of SUN2 gene CDS was 2 187 bp, encoding 728 amino acids, the theoretical molecular mass was 81.06 ku, the molecular formula was C₃₅₉₃H₅₆₃₉N₁₀₃₁O₁₁₁₀S₁₄, the isoelectric point was 6.20, the total number of atoms was 11 387, and the instability coefficient was 56.88, which was unstable protein. The similarity of the nucleotide sequence of SUN2 gene between sheep and Capra hircus, Bostaurus, Susscrofa, Equuscaballus, Canis lupus familiaris, Rattus norvegicus, Mus musculus and Homo sapiens were 98.9%, 97.2%, 91.6%, 90.6%, 87.1%, 82.4%, 82.1% and 86.1%, respectively. The gene similarity among different species was relatively high and conservative in the evolution process. The phylogenetic tree results showed that sheep were closely related to Capra hircus, Bos taurus, Sus scrofa, Equus caballus and Canis lupus familiaris, but far related to Gallus gallus. The prediction of structural domain and transmembrane domain showed that SUN2 protein in sheep contained a highly conserved SUN domain, and there was a transmembrane domain. The signal peptide prediction showed that there was no signal peptide site, and the hydrophobicity analysis was a hydrophilic protein. The secondary structure of the amino acid sequence was mainly alpha helix (51.65%), and the rest were random coil (31.46%), extended chain (12.36%) and beta turn (4.53%). The eukaryotic expression vector pEGFP-C1-SUN2 of SUN2 gene in sheep was successfully constructed, which was transfected into A549 cells and the protein expression was detected. The results would provide a reference for the functional study of SUN2 gene in sheep, and laid a foundation for the subsequent exploration of the role of SUN2 gene in ovine pulmonary denocarcinoma.

Key words: sheep; SUN2 gene; vector construction; bioinformatics analysis; transfection

中图分类号:

杜晓悦, 张良, 李慧萍, 刘淑英. 绵羊SUN2基因生物信息学分析及其在A549细胞中的表达[J]. 中国畜牧兽医, 2021, 48(11): 3929-3939.

DU Xiaoyue, ZHANG Liang, LI Huiping, LIU Shuying. Bioinformatics Analysis of SUN2 Gene in Sheep and Its Expression in A549 Cells[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(11): 3929-3939.

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