Preparation Of Polyclonal Antibodies Against JAZF1 And RPS14 Proteins In Broiler Pulmonary Arteries And Analysis Of Their Expression Levels In Broiler Ascites Syndrome

As an important regulator of ribosomal protein S14(RPS14),juxtaposition with another zinc finger protein 1(JAZF1)regulates ribosome biosynthesis and translation of important proteins by maintaining the balance between ribosomal subunit components.The purpose of this study was to investigate the potential role of JAZF1 and RPS14 in broiler ascites syndrome(BAS).Based on the prepared polyclonal antibodies,the expression levels and localization distribution of JAZF1 and RPS14 proteins in important tissues of poultry and mammals were analyzed.In this study,the recombinant expression plasmids PET32a-JAZF1 and PET28a-RPS14 were constructed using TA cloning and subcloning techniques(host strains were Rosetta and BL21).Afterwards,the His-JAZF1 and His-RPS14 fusion proteins(30 k Da and 22 k Da)induced and expressed by isopropyl-β-D-thiogalactoside(IPTG)were purified and used as antigens to prepare high-quality antiserum.Through enzyme-linked immunosorbent assay,western blotting and indirect immunofluorescence and other techniques,the titer and reactogenicity of polyclonal antibodies and the corresponding protein expression changes in key tissues of diseased broilers were analyzed.The results showed that:(1)In this experiment,the expression vectors PET32a-JAZF1 and PET28a-RPS14 were constructed and induced by IPTG for prokaryotic expression.The obtained recombinant proteins were purified and immunized with New Zealand white rabbits,and then the rabbit anti-chicken JAZF1 and RPS14 protein polyclonal antibodies were successfully extracted and isolated.(2)These two antibodies could specifically recognize the JAZF1 and RPS14 proteins in important tissues of broilers,and their titers are as high as 1:102,400 and 1:204,800,respectively.(3)Immunofluorescence showed that JAZF1 and RPS14 proteins were mainly localized in the cytoplasm of pulmonary artery,liver,kidney,heart and lung tissue cells of broilers,ducks,goats,mice and rabbits.(4)The semi-quantitative analysis of the mean fluorescence intensity showed that the expression of these proteins in the tissues of broilers and ducks was more significant than that in mammals(P < 0.01),indicating that this type of antiserum has higher sensitivity and specificity to poultry.(5)Western blot combined with immunofluorescence detection showed that BAS could significantly reduce the expression levels of JAZF1 and RPS14 proteins in tissue cells(P <0.01 or P < 0.05).In short,rabbit anti-chicken JAZF1 and RPS14 polyclonal antibodies were successfully prepared by prokaryotic expression of foreign genes and animal immunization techniques.The expression of JAZF1 and RPS14 proteins during the development of broiler ascites syndrome was investigated using this antibody,and the distribution of JAZF1 and RPS14 proteins in various animal tissues was mapped.This article lays a theoretical foundation for further research on the key role of JAZF1 and RPS14 proteins in broiler ascites syndrome,and also provides a new entry point for the study of the biological functions of JAZF1 and RPS14 proteins.