武夷黑猪HSPA5基因克隆、生物信息学分析及组织表达研究

doi:10.16431/j.cnki.1671-7236.2025.07.022

摘要/Abstract

摘要： 【目的】对武夷黑猪热休克蛋白A5(heat shock protein family A member 5,HSPA5)进行生物信息学预测与分析，为探究其生物学功能提供参考。【方法】对武夷黑猪HSPA5基因编码区进行克隆并测序,再与不同物种进行相似性比对并构建系统进化树，利用生物信息学方法预测其蛋白的理化特性、结构及功能，最后测定其在武夷黑猪不同组织中的表达量。【结果】武夷黑猪HSPA5基因编码区长1 965 bp，编码654个氨基酸。相似性比对和进化树分析结果表明，武夷黑猪HSPA5氨基酸序列与杜洛克猪转录本变体2和鼠的HSPA5氨基酸序列相似性最高(100%)，且与鼠的亲源关系最近，而与大黄花鱼的亲源关系最远。武夷黑猪HSPA5蛋白为酸性亲水蛋白，含有信号肽序列，在内质网中含量最高。主要由α-螺旋构成，且保守基序位于2个重叠的功能结构域内；含有56个磷酸化位点，76个O-糖基化位点，24个化学因子作用位点。同时具有26个抗原决定簇、11个T细胞抗原表位，13个B细胞抗原表位。与热休克蛋白90(heat shock protein member 90，HSP90)、激活转录因子6(activating transcription factor 6，ATF6)等互作关系密切。实时荧光定量PCR结果表明，HSPA5基因在武夷黑猪肝脏和子宫中的表达量显著高于其他组织(P＜0.05)。【结论】本研究成功克隆了武夷黑猪HSPA5基因，且与鼠的亲源关系最近。武夷黑猪HSPA5蛋白主要定位于细胞质中，包含磷酸化、O-糖基化修饰位点，含有与T或B细胞抗原表位重叠的抗原决定簇序列，与未折叠蛋白反应通路中相关蛋白互作，在肝脏表达量最高。这些特征可为进一步研究HSPA5蛋白生物学功能提供参考。

关键词: 武夷黑猪; HSPA5基因; 生物信息学; 功能; 组织表达

Abstract: 【Objective】 The heat shock protein family A member 5 (HSPA5) of Wuyi Black pig was analyzed through bioinformatics predictions to provide insights on its biological functions. 【Method】 The coding sequence of HSPA5 gene was amplified and sequenced,similarity analysis was performed afterward,then a phylogenetic tree was constructed.The physicochemical properties,structural features,and functional roles of the HSPA5 protein were predicted using bioinformatics software,and its expression in different tissues of Wuyi Black pig were finally measured. 【Result】 The coding sequence of HSPA5 gene in Wuyi Black pig was 1 965 bp,encoding 654 amino acids.The results of similarity and phylogenetic tree analyses showed that the amino acid sequence of HSPA5 in Wuyi Black pig had the highest similarity (100%) with the transcript variant 2 of HSPA5 in Duroc pig and that in mice.It had the closest genetic relationship with Mus musculus and the most distant genetic relationship with Larimichthys crocea.The HSPA5 protein was predicted as an acidic hydrophilic protein containing a signal peptide sequence,with the highest concentration observed in the cytoplasm.It was mainly composed of alpha helix conformation with conserved motifs within two overlapping functional domains.It contained 56 phosphorylation sites,76 O-glycosylation sites and 24 chemical interaction sites,as well as 26 antigenic determinants,11 T-cell epitopes,and 13 B-cell epitopes.It was closely related to interactions with heat shock protein member 90 (HSP90) and activating transcription factor 6 (ATF6).The Real-time quantitative PCR results showed that the expression of HSPA5 gene in liver and uterus of Wuyi Black pig was significantly higher than that in other tissues (P＜0.05). 【Conclusion】 The HSPA5 gene of Wuyi Black pig had been successfully cloned,and had the closest genetic relationship with that of Mus musculus.It was predominantly localized in the cytoplasm,featuring phosphorylation and O-glycosylation modification sites,it also interacted with proteins related to the unfolded protein response pathway.It had the highest expression in liver of Wuyi Black pig.These characteristics could provide valuable references for further studies on the biological functions of HSPA5 protein.

Key words: Wuyi Black pig; HSPA5 gene; bioinformatics; function; tissue expression

中图分类号:

S828.8⁺9

李指全, 傅思静, 徐舒平, 高萌若, 杨涛涛, 张志榜, 李凯, 李鹏成. 武夷黑猪HSPA5基因克隆、生物信息学分析及组织表达研究[J]. 中国畜牧兽医, 2025, 52(7): 3214-3224.

LI Zhiquan, FU Sijing, XU Shuping, GAO Mengruo, YANG Taotao, ZHANG Zhibang, LI Kai, LI Pengcheng. Cloning, Bioinformatics Analysis and Tissue Expression of HSPA5 Gene in Wuyi Black Pig[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(7): 3214-3224.

参考文献

[1] LYER K,CHAND K,MITRA A,et al.Diversity in heat shock protein families:Functional implications in virus infection with a comprehensive insight of their role in the HIV-1 life cycle[J]. Cell Stress & Chaperones:The International Journal of Cell Biology,Stress Physiology and Molecular Chaperones,2021,26(5):743-768.
[2] FEDER M E,HOFMANN G E.Heat-shock proteins,molecular chaperones,and the stress response:Evolutionary and ecological physiology[J].Annual Review of Physiology,1999,61(1):243-282.
[3] TIWARI S,THAKUR R,SHANKAR J.Role of heat-shock proteins in cellular function and in the biology of fungi[J].Biotechnology Research International,2015,2015(2):1-11.
[4] LI T,FU J,CHENG J L,et al.New progresses on cell surface protein HSPA5/BiP/GRP78 in cancers and COVID-19[J].Frontiers in Immunology,2023,14(2023):1166680.
[5] 张志标,余伟,马达,等.热休克蛋白A5对雨蛙肽诱导的胰腺腺泡细胞损伤的作用研究[J].国际消化病杂志,2019,39(5):357-362.ZHANG Z B,YU W,MA D,et al.Effect of HSPA5 on cerulein-induced pancreatic acinar cell injury[J].International Journal of Digestive Diseases,2019,39(5):357-362.(in Chinese)
[6] AMANKWAH Y S,COLLINS P,FLEIFIL Y,et al.Grp94 works upstream of BiP in protein remodeling under heat stress[J].Journal of Molecular Biology,2022,434(19):167762.
[7] SINGH R,KAUR N,CHOUBEY V,et al.Endoplasmic reticulum stress and its role in various neurodegenerative diseases[J].Brain Research,2024,1826(2024):148742.
[8] HUANG L,LIU Q,ZHOU T T,et al.Deficiency of β-arrestin2 alleviates apoptosis through GRP78-ATF6-CHOP signaling pathway in primary Sjögren’s syndrome[J].International Immunopharmacology,2021,101(2021):108281.
[9] XU S,LIU H,WANG C,et al.Dual roles of UPR(er) and UPR(mt) in neurodegenerative diseases[J].Journal of Molecular Medicine,2023,101(12):1499-1512.
[10] LAY A J,DUPUY A,HAGIMOLA L,et al.Endoplasmic reticulum protein 5 attenuates platelet endoplasmic reticulum stress and secretion in a mouse model[J].Blood Advances,2023,7(9):1650-1665.
[11] HAN B,LV Y B,MOSER D,et al.ACE2-independent SARS-CoV-2 virus entry through cell surface GRP78 on monocytes-evidence from a translational clinical and experimental approach[J].EBioMedicine,2023,98(2023):104869.
[12] 国家畜禽遗传资源委员会组.中国畜禽遗传资源志.猪志[M].北京：中国农业出版社,2011.NATIONAL COMMISSION ON ANIMAL GENETIC RESOURCES.Animal Genetic Resources in China.Pigs[M].Beijing：China Agriculture Press,2011.(in Chinese)
[13] CHEN X,CUBILLOS-RUIZ J R.Endoplasmic reticulum stress signals in the tumour and its microenvironment[J].Nature Reviews Cancer,2021,21(2):71-88.
[14] YI S Y,CHEN K,ZHANG L H,et al.Endoplasmic reticulum stress is involved in stress-induced hypothalamic neuronal injury in rats via the PERK-ATF4-CHOP and IRE1-ASK1-JNK pathways[J].Frontiers in Cellular Neuroscience,2019,13(2019):190.
[15] HAAS I G.BiP (GRP78),an essential HSP70 resident protein in the endoplasmic reticulum[J].Cellular and Molecular Life Sciences,1994,50(11-12):1012-1020.
[16] 陈龙冠,覃锦红,黄云娜,等.信号肽优化对重组抗体分泌表达的影响及研究进展[J].中国生物工程杂志,2016,36(3):77-81.CHEN L G,TAN J H,HUANG Y N,et al.Optimized signal peptides sequences for the development of high expressing McAbs[J].Chinese Journal of Bioengineering,2016,36(3):77-81.(in Chinese)
[17] GAUT J R. In vivo threonine phosphorylation of immunoglobulin binding protein(BIP) maps to its protein binding domain[J].Cell Stress and Chaperones,1997,2(4):252-262.
[18] HEINEN R C,DINIZ-MENDES L,SILVA J T,et al.Identification of the divergent calmodulin binding motif in yeast Ssb1/Hsp75 protein and in other HSP70 family members[J].Brazilian Journal of Medical and Biological Research,2006,39(11):1399-1408.
[19] ZHOU C J,LIU Y C,WEI Q,et al.HSPA5 promotes attachment and internalization of Porcine epidemic diarrhea virus through interaction with the spike protein and the Endo-/Lysosomal pathway[J].Journal of Virology,2023,97(6):e54923.
[20] MARCHAN J.In silico identification of epitopes present in human heat shock proteins (HSPs) overexpressed by Tumour cells[J].Journal of Immunological Methods,2019,471(2019):34-45.
[21] WISEMAN R L,MESGARZADEH J S,HENDERSHOT L M.Reshaping endoplasmic reticulum quality control through the unfolded protein response[J].Molecular Cell,2022,82(8):1477-1491.
[22] DONG W G,DU D W,HUANG H.HSPA5 is a prognostic biomarker correlated with immune infiltrates in thyroid carcinoma[J].Endokrynologia Polska,2022,73(4):680-689.
[23] KOTLER J L M,STREET T O.Mechanisms of protein quality control in the endoplasmic reticulum by a coordinated Hsp40-Hsp70-Hsp90 system[J].Annual Review of Biophysics,2023,52(2023):509-524.
[24] SUN M H,JIANG W J,LI X H,et al.ATF6 aggravates apoptosis in early porcine embryonic development by regulating organelle homeostasis under high-temperature conditions[J].Zoological Research,2023,44(5):848-859.
[25] HETZ C,PAPA F R.The Unfolded protein response and cell fate control[J].Molecular Cell:A Journal of Experimental Biology,2018,69(2):169-181.
[26] WEITZMANN A,VOLKMER J,ZIMMERMANN R.The nucleotide exchange factor activity of Grp170 may explain the non-lethal phenotype of loss of Sil1 function in man and mouse[J].Federation of European Biochemical Societies Letters,2006,580(22):5237-5240.
[27] MANN M J,MELENDEZ-SUCHI C,VORNDRAN H E,et al.Loss of Grp170 results in catastrophic disruption of endoplasmic reticulum function[J].Molecular Biology of the Cell,2024,35(4):r59.
[28] 刘婷婷,郭晶晶,陈兆东,等.黑龙江林蛙热休克蛋白HSP70家族基因的鉴定及其在感染下的表达分析[J].生物工程学报,2023,39(4):1710-1730.LIU T T,GUO J J,CHEN Z D,et al.Identification of heat shock protein HSP70 family genes from Rana amurensis and its expression profiles upon infection[J]. Chinese Journal of Biotechnology,2023,39(4):1710-1730.(in Chinese)