中国荷斯坦奶牛CCR5基因克隆、生物信息学及表达分析

doi:10.16431/j.cnki.1671-7236.2021.12.005

摘要/Abstract

摘要： 试验旨在克隆中国荷斯坦奶牛CC趋化因子受体5(C-C chemokine receptor type 5,CCR5)基因,对其进行生物信息学分析,并探究CCR5基因在奶牛炎性和健康组织中的表达水平。采用PCR技术扩增并克隆荷斯坦奶牛CCR5基因CDS区全长序列,连接pMD18-T载体并转化大肠杆菌DH5α感受态细胞,通过蓝白斑方法筛选阳性克隆并测序,对序列进行相似性比对及系统进化树构建;应用多种在线生物信息学软件对其编码蛋白进行分析,并利用实时荧光定量PCR方法检测CCR5基因在健康和炎症奶牛乳腺组织中的表达情况。结果显示,中国荷斯坦奶牛CCR5基因CDS区全长1 059 bp,编码352个氨基酸。相似性和遗传进化分析结果显示,奶牛CCR5基因与绵羊的遗传距离最近,高达96.0%,与鸡遗传关系最远,为61.0%,且在不同物种之间CCR5基因高度保守。中国荷斯坦奶牛CCR5蛋白分子质量为40.235 ku,理论等电点(pI)为9.30,为疏水性蛋白但不是分泌蛋白,主要存在于细胞质内;在CCR5蛋白二级结构中α-螺旋和无规则卷曲分别占51.14%和32.95%,三级结构模型预测结果与二级结构一致。实时荧光定量PCR结果显示,CCR5基因在健康奶牛乳腺组织中的表达量极显著低于炎性奶牛乳腺组织(P<0.01),提示其可能参与奶牛乳腺炎的发生过程。本试验结果为进一步研究奶牛CCR5蛋白的功能提供了理论依据,对探究奶牛CCR5基因在奶牛乳腺炎中的调控功能等具有重要意义。

关键词: 中国荷斯坦奶牛; CCR5基因; 克隆; 生物信息学分析; 表达

Abstract: The purpose of this study was to clone C-C chemokine receptor type 5 (CCR5) gene in Chinese Holstein dairy cows, analyze its bioinformatics, and explore the expression in inflammatory and healthy tissues of dairy cows.The full-length CDS sequence of CCR5 gene in Holstein dairy cows was amplified by PCR and cloned, and the pMD18-T vector was connected and transformed into Escherichia coli DH5α competent cells.The positive clones were screened and sequenced by blue-white blot method, and sequence similarity alignment and phylogenetic tree construction were carried out.The encoded proteins were analyzed by a variety of online bioinformatics softwares.Real-time quantitative PCR was used to detect the expression of CCR5 gene in the mammary tissues of healthy and inflammatory dairy cows.The results showed that the total length of CDS region of CCR5 gene in Chinese Holstein dairy cows was 1 059 bp, which encoded 352 amino acids.The results of similarity and genetic evolution analysis showed that the genetic distance of CCR5 gene was 96.0% between Chinese Holstein dairy cows and Ovis aries, and 61.0% between Chinese Holstein dairy cows and Gallus gallus.CCR5 genes were highly conserved among different species.The molecular weight of CCR5 protein was 40.235 ku, and the theoretical isoelectric point (pI) was 9.30.CCR5 protein was hydrophobic protein but not secreted protein, which mainly existed in cytoplasm.In the secondary structure, alpha helix and random coil accounted for 51.14% and 32.95%, respectively.The tertiary structure prediction results were consistent with that of the secondary structure.Real-time quantitative PCR results showed that the expression of CCR5 gene in normal mammary tissue was extremely significantly lower than that in mammary tissue of dairy cows with mastitis (P<0.01), suggesting that CCR5 might be involved in the occurrence of mastitis in dairy cows.The results provided a theoretical basis for further study of the function of CCR5 protein in dairy cows, and had important significance for exploring the regulatory function of CCR5 gene in dairy cows mastitis.

Key words: Chinese Holstein dairy cows; CCR5 gene; cloning; bioinformatics analysis; expression

中图分类号:

陈伶慧, 张文婷, 申祥, 张伟, 郭利亚, 张子敬, 徐萍, 白跃宇, 张晓建. 中国荷斯坦奶牛CCR5基因克隆、生物信息学及表达分析[J]. 中国畜牧兽医, 2021, 48(12): 4372-4381.

CHEN Linghui, ZHANG Wenting, SHEN Xiang, ZHANG Wei, GUO Liya, ZHANG Zijing, XU Ping, BAI Yueyu, ZHANG Xiaojian. Cloning, Bioinformatics and Expression Analysis of CCR5 Gene in Chinese Holstein Dairy Cows[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4372-4381.

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