Prokaryotic expression of eIF3i gene encoding protein from lamb testicular cells and the preparation and application of its polyclonal antibody
10.16303/j.cnki.1005-4545.2025.07.16
- VernacularTitle:羔羊睾丸细胞eIF3i基因编码蛋白的原核表达及多克隆抗体的制备与应用
- Author:
Qin WU
1
;
Shuaibin WU
;
Simei XIAN
;
Youci LONG
;
Weihao ZHENG
;
Mengyi YU
;
Jing LI
Author Information
1. 贵州大学动物科学学院,贵州贵阳 550025
- Publication Type:Journal Article
- Keywords:
lamb testis cells;
eIF3i gene;
prokaryotic expression;
polyclonal antibody
- From:
Chinese Journal of Veterinary Science
2025;45(7):1478-1484
- CountryChina
- Language:Chinese
-
Abstract:
The eIF3i gene was amplified from lamb testicular(LT)cells by PCR and cloned into pCold vector to construct the pCold-eIF3i plasmid.Plasmid PCR,double enzyme digestion and se-quencing were used to verify the results.The recombinant eIF3i protein was induced under the op-timized expression conditions.The expression and reactogenicity of the target protein were detected by SDS-PAGE and Western blot.New Zealand white rabbits were immunized with purified recom-binant eIF3i protein combined with Freund's complete and incomplete adjuvants for three times.Se-rum samples were collected after immunization.Indirect ELISA was used to detect antiserum titer,and Western blot was used to analyze antibody specificity.Indirect immunofluorescence assay(IFA)was used to detect the application effect of antibodies.The results showed that the size of LT eIF3i gene was 978 bp.The optimal expression conditions for the eIF3i recombinant protein were as follows:IPTG concentration of 0.2 mmol/L,temperature of 37 ℃,and induction time of 8 h.The recombinant eIF3i protein was expressed as an inclusion body with a size of about 36 kDa.The titer of polyclonal antibody against eIF3i protein was 1∶51 200.Western blot and IFA showed that the prepared polyclonal antibody against eIF3i protein had good reactivity and specificity.In conclusion,we successfully prepared rabbit anti-eif3i polyclonal antibody and confirmed that it could specifically recognize endogenous eIF3i protein,which laid a foundation for further study on the biological function of eIF3i protein.
