鸡热休克蛋白8基因克隆、生物信息学分析及其在胸腺中表达模式研究

doi:10.16431/j.cnki.1671-7236.2025.01.004

摘要/Abstract

摘要： 【目的】探讨鸡热休克蛋白8(heat shock protein family A member 8,HSPA8)的生物学特性及其在应激状态下胸腺中的表达模式。【方法】试验以固始鸡胸腺为材料,利用PCR技术扩增并克隆HSPA8基因CDS区,通过生物信息学方法分析其生物学特性和结构特点,并检测其在不同组织中的相对表达量。构建断喙应激和热应激模型,采用实时荧光定量PCR技术检测炎症因子和HSPA8基因在不同应激模型鸡胸腺中的表达情况。【结果】鸡HSPA8基因CDS区长度为1 941 bp,共编码氨基酸646个,与红原鸡基因序列相似性为98.92%;HSPA8基因核苷酸序列的系统发育树结果表明,固始鸡与火鸡亲缘关系最近,与哺乳动物次之,与低等鱼类亲缘关系最远;HSPA8蛋白属于酸性蛋白,稳定性和亲水性强,不属于分泌蛋白且无跨膜结构域,有多个磷酸化和糖基化修饰位点,主要在细胞核内发挥作用,具有2个保守性结构域,与DNAJC5、HSPB1、DNAJA1和GAK等蛋白存在相互作用;HSAP8基因在雏鸡各组织中广泛表达,其中在胰腺中表达量最高,在胸肌中表达量最低;应激模型评价结果发现,断喙应激和热应激导致鸡血清皮质酮、白细胞介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)等含量显著增加(P＜0.05),T细胞亚群(CD3⁺)和免疫球蛋白G(IgG)含量有所降低;断喙应激模型鸡胸腺中IL-1β、IL-6、TNF-α和IL-8基因表达量在第1天显著升高(P＜0.05),第5天显著降低(P＜0.05);热应激模型鸡胸腺中IL-1β、IL-6、TNF-α和IL-8基因表达量显著升高(P＜0.05);HSPA8基因在断喙后第1和3天胸腺中表达量显著升高(P＜0.05),第5天显著降低(P＜0.05);在持续热应激组中显著降低(P＜0.05)。【结论】 HSPA8基因参与鸡胸腺应激性免疫应答反应,可作为反映家禽是否处于应激性免疫抑制状态的可靠指标。以上结果为进一步研究HSPA8的免疫调控功能提供参考依据。

关键词: 鸡; HSPA8基因; 生物信息学; 胸腺; 应激调控

Abstract: 【Objective】 The aim of this study was to investigate the biological characteristics of heat shock protein family A member 8 (HSPA8) in chickens and its expression patterns in thymus under stress. 【Method】 In this study,the thymus of Gushi chickens was used as the material,and the CDS region of HSPA8 gene was amplified using PCR and cloned,and its biological and structural characteristics were analyzed by bioinformatics method.The relative expression of HSPA8 gene in different tissues were detected. The models of beak trimming stress and heat stress were constructed,and Real-time quantitative PCR was used to detect the expression of inflammatory factor and HSPA8 gene in thymus of chickens with different stress models. 【Result】 The length of CDS region of chicken HSPA8 gene was 1 941 bp,encoding 646 amino acids,which was 98.92% similar to the gene sequence of Gallus gallus.Phylogenetic tree results of HSPA8 gene sequence showed that Gushi chickens was the closest related to Meleagris gallopavo,followed by mammals,and most distantly related to lower fish.HSPA8 protein was acidic protein with strong stability and hydrophilicity.It didn’t belong to secreted protein and had no transmembrane domain,with multiple phosphorylation and glycosylation modification sites.It mainly functions in the nucleus and had two conserved domains.It interacted with DNAJC5,HSPB1,DNAJA1 and GAK proteins,et al.HSAP8 gene was widely expressed in various tissues and organs of chicks,with the highest expression in pancreas and the lowest expression in pectoral muscle.The results of stress model evaluation showed that beak trimming stress and heat stress resulted in the significant increase of the contents of serum corticosterone,interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) (P＜0.05),and decrease of the contents of T cell subsets (CD3⁺) and immunoglobulin G (IgG).The expressions of IL-1β,IL-6,TNF-α and IL-8 genes in thymus were significantly increased on day 1 (P＜0.05) and significantly decreased on day 5 (P＜0.05),while the expressions of IL-1β,IL-6,TNF-α and IL-8 genes were significantly increased in thymus under heat stress (P＜0.05).The expression of HSPA8 gene in thymus were increased significantly on day 1 and day 3 after beak trimming (P＜0.05),decreased significantly on day 5 (P＜0.05),and decreased significantly in the continuous heat stress group (P＜0.05). 【Conclusion】 HSPA8 gene was involved in the stress immune response of chicken thymus,which could be used as a reliable indicator of whether poultry was in the state of stress immunosuppression.The above results provided a reference for further studying the immune regulatory function of HSPA8 gene.

Key words: chickens; HSPA8 gene; bioinformatics; thymus; regulation of stress

中图分类号:

S831

田慧慧, 王冰欣, 朱召岩, 于燕鸽, 丁梦霞, 田亚东, 蒋瑞瑞, 孙桂荣, 韩瑞丽, 康相涛, 闫峰宾, 郭玉洁. 鸡热休克蛋白8基因克隆、生物信息学分析及其在胸腺中表达模式研究[J]. 中国畜牧兽医, 2025, 52(1): 39-51.

TIAN Huihui, WANG Bingxin, ZHU Zhaoyan, YU Yange, DING Mengxia, TIAN Yadong, JIANG Ruirui, SUN Guirong, HAN Ruili, KANG Xiangtao, YAN Fengbin, GUO Yujie. Cloning,Bioinformatics Analysis of HSPA8 Gene and Its Expression Pattern in Thymus in Chickens[J]. China Animal Husbandry and Veterinary Medicine, 2025, 52(1): 39-51.

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