柔嫩艾美耳球虫MIF基因克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2023.06.029

摘要/Abstract

摘要： 【目的】克隆柔嫩艾美耳球虫巨噬细胞迁移抑制因子（macrophage migration inhibitory factor，MIF）基因CDS区，探究其生物信息学特征，为后续抗原表位筛选提供理论依据。【方法】采集晋中地区疑似柔嫩艾美耳球虫感染的阳性样本进行PCR鉴定，根据GenBank中公布的柔嫩艾美耳球虫MIF基因序列，利用RT-PCR技术扩增MIF基因CDS区，构建pET28a-EtMIF重组质粒，经PCR和双酶切鉴定后测序并进行序列分析；利用生物信息学软件对其编码蛋白的理化性质、结构和功能等进行预测。【结果】成功克隆了柔嫩艾美耳球虫MIF基因CDS区序列，全长348 bp，共编码115个氨基酸，相对分子质量为1.203×10⁴。系统进化树结果表明，柔嫩艾美耳球虫MIF基因氨基酸序列与毒害艾美耳球虫的亲缘关系最近，相似性为98.3%。MIF蛋白为非跨膜、可溶性、非分泌型蛋白，亚细胞定位主要在细胞质中，共有10个磷酸化位点；含有1个保守结构域，二级结构由无规则卷曲（35.65%）、延伸链（30.43%）、α-螺旋（27.83%）和β-转角（6.09%）组成，三级结构预测结果与二级结构一致。存在3个最佳潜在抗原表位。【结论】本试验成功克隆出柔嫩艾美耳球虫MIF基因，并对其进行了生物信息学分析，为今后MIF基因的功能研究及基因工程疫苗候选分子的筛选提供参考。

关键词: 柔嫩艾美耳球虫; MIF基因; 生物信息学

Abstract: 【Objective】 The purpose of this study was to obtain the CDS region of macrophage migration inhibitory factor (MIF) gene of Eimeria tenella, and explore its bioinformatics characteristics, so as to provide a theoretical basis for subsequent epitope screening.【Method】 The positive samples suspected of Eimeria tenella infection in Jinzhong area were collected for PCR identification.According to the MIF gene sequence of Eimeria tenella published in GenBank, the CDS region of MIF gene was amplified by RT-PCR, and the recombinant plasmid pET28a-EtMIF was constructed.PCR and double enzyme digestion were used for identification, sequencing and sequence analysis.Bioinformatics software was used to predict the physical and chemical properties, structure and function of MIF protein.【Result】 The CDS region of MIF gene in Eimeria tenella was successfully cloned, with a total length of 348 bp, encoding 115 amino acids, and a relative molecular weight was 1.203×10⁴.The phylogenetic tree analysis showed that the amino acid sequences of MIF gene of Eimeria tenella and Eimeria necatrix had the closest relationship, with the similarity of 98.3%.MIF protein was a non-transmembrane, soluble, non-secretory protein with subcellular localization mainly in the cytoplasm and contains 10 phosphorylation sites and a conserved domain.The secondary structure was composed of random coil (35.65%), extended chain (30.43%), alpha helix (27.83%) and beta turn (6.09%).The tertiary structure was predicted to be consistent with the secondary structure.There were three best potential epitopes.【Conclusion】 MIF gene of Eimeria tenella was successfully cloned, and the bioinformatics analysis of this gene was carried out.The results provided a reference for future research on the function of MIF gene and the screening of genetic engineering vaccine candidates.

Key words: Eimeria tenella; MIF gene; bioinformatics

中图分类号:

S852.72

李永彬, 杨惠琳, 宋子豪, 张昱, 杨瑾, 孟佳, 崔小珍, 吕晓玲, 郑明学, 白瑞. 柔嫩艾美耳球虫MIF基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(6): 2450-2459.

LI Yongbin, YANG Huilin, SONG Zihao, ZHANG Yu, YANG Jin, MENG Jia, CUI Xiaozhen, LYU Xiaoling, ZHENG Mingxue, BAI Rui. Cloning and Bioinformatics Analysis of MIF Gene in Eimeria tenella[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(6): 2450-2459.

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