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

Abstract

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

CLC Number:

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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Cloning, Bioinformatics and Expression Analysis of CCR5 Gene in Chinese Holstein Dairy Cows

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