绵羊KAP11.1基因克隆、原核表达及皮肤毛囊表达研究

doi:10.16431/j.cnki.1671-7236.2022.03.009

摘要/Abstract

摘要： 【目的】对毛囊角蛋白关联蛋白11.1（keratin associated protein 11.1，KAP11.1）基因进行克隆及原核表达，并对KAP11.1基因在不同品种绵羊皮肤毛囊中表达量进行比较，探究KAP11.1基因在南疆地方绵羊品种间表达差异及其对羊毛品质的影响。【方法】以平原型和田羊、山区型和田羊和卡拉库尔羊体侧皮肤毛囊为研究材料，以GenBank中绵羊KAP11.1基因序列（登录号：HQ595347.1）为参照设计引物，对KAP11.1基因进行PCR扩增，构建pMD19-T-KAP11.1克隆质粒，双酶切鉴定后构建pET-28a （+）-KAP11.1原核重组表达质粒，经PCR和双酶切鉴定后测序并进行序列分析，转化大肠杆菌BL21（DE3）感受态细胞中表达，采用SDS-PAGE和Western blotting检测；利用实时荧光定量PCR技术检测KAP11.1基因在不同绵羊皮肤毛囊中的表达情况。【结果】 3种绵羊KAP11.1基因CDS区序列为480 bp，编码159个氨基酸，为不稳定的疏水蛋白。相似性比对结果发现，与参照基因相比，2种类型和田羊基因序列相似性均为99.79％，均在423 bp处发生突变，由C变为T，卡拉库尔羊基因序列相似性为99.38％，其69 bp处G变为T、93 bp处C变为T、423 bp处C变为T。系统进化树分析发现，3种绵羊和山羊亲缘关系最近，和瘤牛亲缘关系最远。KAP11.1蛋白二级结构主要由无规则卷曲组成。试验成功构建了pET-28a （+）-KAP11.1原核重组表达质粒，并纯化得到19 ku的KAP11.1蛋白。KAP11.1基因在山区型和田羊和卡拉库尔羊皮肤毛囊中的表达量均显著高于平原型和田羊（P<0.05），在山区型和田羊和卡拉库尔羊中差异不显著（P>0.05）。【结论】克隆获得480 bp的绵羊KAP11.1基因CDS区序列，成功构建了pET-28a （+）-KAP11.1原核重组表达质粒，并获得19 ku的KAP11.1蛋白，且KAP11.1基因在3个品种绵羊皮肤毛囊中均有表达。

关键词: 和田羊; 卡拉库尔羊; KAP11.1基因; 克隆; 原核表达; 毛囊

Abstract: 【Objective】 The aim of this study was to clone and prokaryotic express the keratin associated protein 11.1(KAP11.1) gene of hair follicles, compare the expression of KAP11.1 gene in skin follicles of different sheep breeds, and explore the expression difference of KAP11.1 gene in local sheep breeds in Southern Xinjiang and its effect on wool quality.【Method】 The skin follicles on the body side of plain-type Hetian sheep, mountain-type Hetian sheep and Karakul sheep were used as the research materials, and the sequence of KAP11.1 gene in sheep (accession No.:HQ595347.1) in GenBank was used as the reference to design primers.PCR amplification of KAP11.1 gene was carried out to construct pMD19-T-KAP11.1 clone plasmid, and pET-28a(+)-KAP11.1 prokaryotic recombinant expression plasmid was constructed after double-enzyme digestion identification.Sequence analysis was carried out after PCR and double-enzyme digestion identification.The recombinant plasmid was transformed into E.coli BL21(DE3).SDS-PAGE and Western blotting were used for detection.The expression of KAP11.1 gene in skin follicles of different sheep was detected by Real-time quantitative PCR.【Result】 The CDS sequence of KAP11.1 gene in 3 sheep breeds was 480 bp, encoding 159 amino acids, which was an unstable hydrophobic protein.The similarity alignment results showed that compared with the reference gene, the two types of Hetian sheep was 99.79%, and mutations occurred at 423 bp, from C to T.The sequence homology of Karakul sheep was 99.38%, from G to T at 69 bp, from C to T at 93 bp, and from C to T at 423 bp.Phylogenetic tree analysis showed that 3 sheep breeds had the closest genetic relationship with Capra hircus, and the farthest genetic relationship with Bos indicus.The secondary structure of KAP11.1 was mainly composed of random coil.The prokaryotic recombinant expression plasmid pET-28a(+)-KAP11.1 was successfully constructed, and KAP11.1 protein (19 ku) was purified.The expression of KAP11.1 gene in skin follicles of mountain-type Hetian sheep and Karakul sheep were significantly higher than that of plain-type Hetian sheep (P<0.05), and there was no significant difference between mountain-type Hetian sheep and Karakul sheep (P>0.05).【Conclusion】 The 480 bp CDS sequence of KAP11.1 gene in sheep was cloned, the prokaryotic recombinant expression plasmid pET-28a(+)-KAP11.1 was successfully constructed, 19 ku KAP11.1 protein was obtained, and KAP11.1 gene was expressed in skin follicles of 3 sheep breeds.

Key words: Hetian sheep; Karakul sheep; KAP11.1 gene; cloning; prokaryotic expression; skin follicles

中图分类号:

宫淑娟, 史瑞军, 吴庭辉, 李树伟. 绵羊KAP11.1基因克隆、原核表达及皮肤毛囊表达研究[J]. 中国畜牧兽医, 2022, 49(3): 876-886.

GONG Shujuan, SHI Ruijun, WU Tinghui, LI Shuwei. Cloning, Prokaryotic Expression and Skin Follicles Expression of KAP11.1 Gene in Sheep[J]. China Animal Husbandry and Veterinary Medicine, 2022, 49(3): 876-886.

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