鸡白痢沙门菌SpiC蛋白的原核表达及其应用  

Prokaryotic expression and application of Spi C protein of Salmonella pullorum

作　　者：耿士忠 刘欢 焦新安 潘志明 钱珊珊 郭荣显 安树敏 薛颖 

机构地区：[1]扬州大学,江苏省人兽共患病学重点实验室,江苏省动物重要疫病与人兽共患病防控协同创新中心,江苏扬州225009

出　　处：《细胞与分子免疫学杂志》2015年第2期149-152,158,共5页Chinese Journal of Cellular and Molecular Immunology

基　　金：863项目(2011AA10A212);国家自然科学基金(31230070);中国博士后基金(2014M551670)

摘　　要：目的进行鸡白痢沙门菌Spi C蛋白原核表达并建立以Spi C蛋白为检测抗原的间接ELISA。方法以鸡白痢沙门菌S06004基因组DNA为模板,PCR扩增384 bp的spi C基因。将spi C基因克隆至原核表达载体p ET30a中,构建重组原核表达质粒p ET30a-spi C,再转化到表达宿主菌E.coli BL21(DE3)中,异丙基β-D-硫代半乳糖苷(IPTG)诱导表达,SDS-PAGE和Western blot分析鉴定,纯化His-Spi C蛋白,建立Spi C蛋白为检测抗原的间接ELISA并检测临床血清样品。结果通过对诱导后重组菌裂解产物进行SDS-PAGE和Western blot分析,重组菌可以表达相对分子质量(Mr)约19 200的可溶性重组蛋白His-Spi C。重组蛋白GST-Spi C免疫无特定病原体鸡,所获得的高免血清能识别重组蛋白His-Spi C,表明体外表达的重组蛋白His-Spi C有良好的免疫反应原性。所建立的以重组蛋白His-Spi C介导的间接ELISA有较好的特异性,能够区分spi C基因缺失型疫苗候选株免疫与野生菌自然感染。结论重组蛋白His-Spi C呈可溶性表达,具有良好的免疫原性,以重组蛋白His-Spi C建立的间接ELISA,可以作为区分感染和免疫动物(DIVA)鸡白痢疫苗免疫的鉴定方法。Objective To express the SpiC protein of Salmonella pul/orum and establish an indirect ELISA method with SpiC protein as antigen. Methods The 384 bp spiC gene of Salmonella pullorum was amplified by PCR from the genomic DNA and cloned into pET30a vector. The recombinant plasmid pET30a-spiC was transformed into competent E. coil BL21 （DE3） cells and induced by IPTG. The expressed product was analyzed by SDS-PAGE and Western blotting. Indirect ELISA based on purified SpiC protein was applied to detect 144 clinical serum samples. Results SDS-PAGE and Western blotting confirmed that a soluble recombinant His-SpiC protein of 19.2 ku was expressed in BL21 （DE3） cells. SPF chicken antibodies against GST-SpiC could recognize His-SpiC, indicating that His-SpiC had a good immunogenicity. The indirect ELISA that we established using His-SpiC protein as coating antigen for detecting antibodies against SpiC could differentiate infected from vaccinated animals （DIVA）. Conclusion The recombinant His-Spit was successfully expressed and the indirect ELISA with it as coating antigen could be used as DIVA method for the related vaccine of pullorum disease.

关 键 词：鸡白痢沙门菌 来杭鸡 spiC基因 原核表达 间接ELISA 

分 类 号：R446.61[医药卫生—诊断学] Q786[医药卫生—临床医学]