牛源CCR7基因克隆、生物信息学及表达分析

doi:10.16431/j.cnki.1671-7236.2022.04.005

摘要/Abstract

摘要： 【目的】探究CC趋化因子受体7(CC chemokine receptor 7,CCR7)基因编码蛋白的生物学特性及其mRNA在健康和炎性奶牛乳腺组织中的表达量。【方法】使用PCR扩增并克隆中国荷斯坦奶牛CCR7基因CDS区,测序后进行相似性比对及系统进化树构建;使用多种生物信息学软件分析CCR7蛋白的组成、理化性质及结构等;使用实时荧光定量PCR技术检测CCR7基因在健康和炎性奶牛乳腺组织中的表达差异。【结果】中国荷斯坦奶牛CCR7基因CDS区全长1 325 bp,与绵羊的相似性较高(95.2%),亲缘关系较近;与鸡的相似性较低(56.4%),亲缘关系较远。CCR7基因编码379个氨基酸,其中亮氨酸含量最多,缬氨酸含量次之,组氨酸和色氨酸含量最低;CCR7蛋白分子质量为14.56 ku,理论等电点为8.89,平均亲水系数为0.640,是一种跨膜疏水性蛋白。CCR7蛋白信号肽切割位点可能存在于第24和25位氨基酸之间。CCR7蛋白二级结构中α-螺旋占比最大,高达51.72%,三级结构主要为α-螺旋和无规则卷曲。CCR7基因在炎性奶牛乳腺组织中的表达量极显著高于健康乳腺组织(P<0.01),其可能在奶牛乳腺炎的免疫调控中有着重要的作用。【结论】试验成功克隆出牛CCR7基因CDS区序列,并进行了相应的生物信息学分析及组织定量表达,为进一步研究CCR7基因的具体功能提供材料,对于探究CCR7基因在奶牛乳腺炎中的调控功能具有重要的意义。

关键词: 奶牛; CCR7基因; 克隆; 生物信息学分析; 表达

Abstract: 【Objective】 This study was aimed to explore the biological characteristics of the protein encoded by CC chemokine receptor 7(CCR7) gene and the expression of mRNA in breast tissues of healthy and inflammatory dairy cows.【Method】 The CDS region of CCR7 gene in Chinese Holstein dairy cows was amplified by PCR method and cloned,the similarity alignment and phylogenetic tree construction were carried out after sequencing.The composition,physicochemical properties and structure of CCR7 protein were analyzed by bioinformatics softwares.The expression difference of CCR7 gene in breast tissues between healthy and inflammatory dairy cows were detected by Real-time quantitative PCR.【Result】 The full-length of CCR7 gene CDS region in Chinese Holstein dairy cows was 1 325 bp,which had high similarity (95.2%) and close genetic relationship with sheep;And had low similarity (56.4%) and far genetic relationship with chicken.CCR7 gene encoded 379 amino acids,among which the content of leucine was the most,the content of valine was the second,and the content of histidine and tryptophan were the lowest.The molecular weight of CCR7 protein was 14.56 ku,the theoretical isoelectric point was 8.89,and the average hydrophilic coefficient was 0.640,it was a transmembrane hydrophobic protein.The cleavage site of signal peptide might exist between amino acids 24 and 25.The structure of alpha helix was the most in the secondary structure,as high as 51.72%,and the tertiary structure was mainly alpha helix and random coil.The expression of CCR7 gene in breast tissue of inflammatory dairy cows was extremely significantly higher than that in healthy dairy cows (P<0.01),which might play an important role in the immune regulation of dairy cows mastitis.【Conclusion】 The CDS region of bovine CCR7 gene was successfully cloned,and the corresponding bioinformatics analysis and tissue quantitative expression were carried out,which provided materials for further study of the specific functions of CCR7 gene,and was of great significance to explore the regulatory function of CCR7 gene in dairy cow mastitis.

Key words: dairy cows; CCR7 gene; cloning; bioinformatics analysis; expression

中图分类号:

申祥, 陈伶慧, 姚竞杰, 王三虎, 白跃宇, 张晓建. 牛源CCR7基因克隆、生物信息学及表达分析[J]. 中国畜牧兽医, 2022, 49(4): 1244-1252.

SHEN Xiang, CHEN Linghui, YAO Jingjie, WANG Sanhu, BAI Yueyu, ZHANG Xiaojian. Cloning,Bioinformatics and Expression Analysis of Bovine CCR7 Gene[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(4): 1244-1252.

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