Production and identification of polyclonal antibody against HSF1 in sheep and immunohistochemical analysis
10.16303/j.cnki.1005-4545.2025.11.13
- VernacularTitle:绵羊HSF1生物信息学分析、多克隆抗体制备及免疫组化分析
- Author:
Wenyu FAN
1
;
Lanying GU
1
;
Qinchuan ZHANG
1
;
Shiyi LI
1
;
Yanbing ZHANG
1
;
Yanming SUN
1
Author Information
1. 石河子大学动物科技学院,新疆石河子 832003
- Publication Type:Journal Article
- Keywords:
sheep;
heat shock factor 1;
polyclonal antibody;
bioinformatics;
immunohistochemistry
- From:
Chinese Journal of Veterinary Science
2025;45(11):2394-2404
- CountryChina
- Language:Chinese
-
Abstract:
Heat shock factor 1(HSF1)is a core transcription factor in cellular stress response and protein homeostasis maintenance,and is widely involved in biological processes such as cell growth,differentiation,and metabolism.This study aims to design antigenic peptides of the sheep HSF1 protein and prepare polyclonal antibodies through bioinformatics analysis and immunologi-cal techniques,and verify their application effects in sheep lung and testis tissues.Firstly,the se-quence,structure,and evolution of the sheep HSF1 gene were analyzed using bioinformatics meth-ods,and key antigenic epitopes were predicted.Then,suitable peptide fragments were selected for synthesis,emulsified with Freund's complete adjuvant or incomplete adjuvant,and used to immu-nize New Zealand rabbits.The specificity and effectiveness of the antibodies were verified by indi-rect ELISA,Western blot,and immunohistochemistry(IHC)experiments.The results showed that the HSF1 protein is 564 amino acids(aa)in length,with a molecular weight of 60.76 kDa,a theo-retical isoelectric point(pI)of 5.32,and is hydrophilic and unstable.Phylogenetic tree analysis revealed that HSF1 is highly conserved among most members of the Bovidae family,as well as in humans,mice,rats,and other species,but with local site differences.Sheep HSF1 is most closely related to goat HSF1,followed by Canadian bighorn sheep and goral.The phosphorylation sites of sheep HSF1 are predominantly concentrated in the middle and C-terminal regions,which is largely consistent with the predicted results for human HSF1,though some local differences exist.The prepared polyclonal antibodies exhibited a titer of over 1:8000 in recognizing the sheep HSF1 pro-tein,and Western blot experiments confirmed clear bands with consistent molecular weight,indica-ting high specificity of the antibodies.Furthermore,the expression and specific distribution of HSF1 were detected in sheep lung and testis tissues.This study successfully prepared polyclonal antibodies based on the sheep HSF1 antigen peptide and,for the first time,detected the immuno-histochemical distribution of HSF1 in the testis and lung tissues of Argali hybrid sheep.This pro-vides an important tool for further exploring the physiological role of HSF1 in Argali hybrid sheep and its potential applications in stress response,disease prevention,and treatment.
