Prokaryotic expression and preliminary analysis of immunogenicity of outer mem-brane protein of yak-derived Escherichia coli OmpA
10.16303/j.cnki.1005-4545.2025.03.09
- VernacularTitle:牦牛源大肠杆菌OmpA外膜蛋白的原核表达及免疫效果评价
- Author:
Shinan ZHANG
1
;
Shengyi HAN
;
Tian SHI
;
Shuping LI
;
Guoyuan HU
;
Rui GAO
;
Jiaqi TIAN
;
Wenwen ZHOU
;
Shengqing LI
Author Information
1. 青海大学农牧学院,青海西宁 810016;青海省畜牧兽医科学院,青海 西宁 810016;青海省动物疫病病原诊断与绿色防控技术研究重点实验室,青海西宁 810016
- Publication Type:Journal Article
- Keywords:
yak-derived E.coli;
OmpA protein;
prokaryotic expression;
bioinformatics analysis;
im-mune protection effect
- From:
Chinese Journal of Veterinary Science
2025;45(3):458-465,472
- CountryChina
- Language:Chinese
-
Abstract:
The amino acid sequences of the OmpA protein isolated from Escherichia coli QML2206-1(E.coli QML2206-1)in our laboratory were analyzed for homology with different strains of OmpA proteins using bioinformatics software,and the OmpA protein was analyzed for its physicochemical properties,transmembrane structure and signal peptide prediction,B-cell anti-genic epitope prediction,secondary and tertiary structure prediction.The OmpA gene fragment was ligated with pET-32a vector to construct a prokaryotic expression vector,which was purified by a nickel column affinity purification system after prokaryotic expression and optimization of ex-pression conditions in BL21(DE3).The purified recombinant protein was fully mixed with Freund's adjuvant to immunize mice,and the levels of mouse-specific IgG antibody and the expression levels of cytokines CD4,CD8 and IL-4 in mouse serum were detected by ELISA,and the immuno-protective effect was evaluated by mouse attack protection test.OmpA protein is a hydrophilic protein with no transmembrane structural domains and a secondary structure consisting mainly of irregular coils(47.98％)and α-helices(29.77％),with 12 antigenic epitopes that can bind to anti-bodies produced by B cells.The recombinant protein OmpA with a relative molecular mass of a-bout 55 kDa was successfully obtained by prokaryotic expression,and the highest expression was induced by IPTG concentration of 0.000 4 mmol/L for 6 h at 37 ℃.The serum-specific IgG anti-body potency of recombinant protein immunized mice was up to 1∶32 000;the expression levels of CD4,CD8 and IL-4 in the serum of immunized mice were elevated compared with those of the con-trol group.The survival rate of mice was 80％and 40％after attack with minimum lethal dose(MLD)and 2 times minimum lethal dose(2MLD),respectively.OmpA recombinant protein has good antigenicity and certain immunoprotective effects,and this study provides a technical basis for the next step in the development of a genetically engineered subunit vaccine against yak-appli-cable E.coli based on OmpA protein.
