ETEC F4-F5融合基因表达及间接ELISA方法建立

Development of an indirect ELISA for detecting specific antibody against ETEC F4-F5 fusion protein

表达产肠毒素性大肠杆菌(enterotoxigenic Escherichia coli,ETEC)黏附素F4和F5融合蛋白,建立间接ELISA方法,旨在监测猪场ETEC感染以及疫苗免疫情况。根据GenBank中登录F4和F5基因序列,分析保守区,优化密码子,合成F4-F5串联基因,克隆入原核表达载体,表达重组蛋白His-F4-F5。以纯化的His-F4-F5蛋白作为检测抗原,建立检测ETEC抗体的间接EIISA方法。经酶切和DNA序列分析,成功构建重组菌BL21(pCold I-F4-F5),SDS-PAGE和Western blot分析,融合表达F4-F5相对分子质量为29 000,与ETEC高免血清发生特异性反应,条带单一。经优化筛选的间接ELISA最佳反应条件:抗原包被浓度6ng/孔,待检血清稀释比例1∶200,HRP-羊抗猪二抗的工作浓度1∶15 000,5%脱脂奶封闭l h,底物作用时间15min。本研究建立F4-F5间接ELISA方法具有特异性好、敏感性高、重复性好、稳定性强的特点,是猪场ETEC感染和疫苗免疫效果检测的有效工具,进而指导疫苗使用和新型疫苗研发。

To monitor enterotoxigenic escherichia coli （ETEC） infection and evaluate vaccination efficacy in pig farms, an indirect ELISA was developed based on fusion antigens of F4 F5 from por- cine ETEC. After analysis of F4 and F5 genes from the GenBank,the conserved domains were se- lected to optimize their codons,and the synthesized fusion gene F4-F5 was cloned into prokaryotic expression vector to express recombinant protein of His F4-FS. Restriction endonuclease digestion and DNA sequence analysis showed that the recombinant strain BL21 （pColdI F4-FS） was success- fully constructed, and SDS-PAGE and Western blot assay showed that fusion protein F4-F5 of 29 000 was expressed and could react to rabbit immune serum against ETEC. The working condi- tions of indirect ELISA were as follows：the concentration of coating antigen was 6 ng/well, the di- lution of primary serum and HRP labeled goat anti pig IgG were 1 ： 200 and 1 ： 15 000,respec- tively,5% skimmed milk blocked for 1 h. and the incubation time of coloration was 15 min. This F4-FS indirect ELISA established in our study has good specificity,high sensitivity,good reproducibility,stability,and will be an effective tool to examine ETEC infection and pig guide vaccine immuniza tion in farms.