家兔支气管败血波氏杆菌重组蛋白DNT1的原核表达及其间接ELISA方法的建立

摘要

Objective To get expression of Bordetella bronchiseptic recombinant protein DNT1 and establish an indirect ELISA for detection of recombinant protein DNT1. Method According to a pair of specific primers designed to amplify DNT gene of Bordetella bronchiseptica in swines promulgated by Genbank, the fragment of the target gene was amplified by PCR and then constructed with prokaryotic expression vector PET-28a ( + ). The recombinant expression plasmids were transfected into BL21 ( DE3 ) strain. The optimal concentration of coated antigen recombinant protein DNT1 and serum dilution was determined to develop the ELISA technique. Results DNT1 gene of Bordetella bronchiseptica was successfully cloned and expressed. The SDS-PAGE and Western blot analysis unfolded the excellent immunogenicity of the recombinant proteins which were used as coating antigens to develop the ELISA method for Bb-specific antibody diagnosis.The peridium consistency of the recombinant protein DNT1 determined in this experiment was 6. 25 μg/mL, and the optimal testing serum dilution was 1: 100. Concision The development of DNT1 indirect ELISA offers a simple and practical way for monitoring antibody of Bb, and provides much information for laying a basis for development of a Bb diagnosis kit.%目的 表达支气管败血波氏杆菌(Bordetella bronchiseptica,Bb)DNT蛋白,并以此建立检测Bb抗体的间接ELISA方法.方法 参照GenBank公布的猪源支气管败血波氏杆菌dnt基因序列(AB020025)针对其N-端设计了一对特异性引物,PCR扩增出相应的核苷酸片段.将PCR扩增产物连接至原核表达载体pET-28a(+)载体中,以E.coli BL21(DE3)为表达菌株进行诱导表达,以纯化重组蛋白DNT1作为诊断抗原,通过探索最佳抗原包被量和抗体血清稀释倍数,建立检测支气管败血波氏杆菌重组蛋白DNTI抗体的ELISA方法.结果 成功克隆了dntN-端的基因序列,并在E.coli BL21(DE3)中获得高效表达,经SDS-PAGE、Western blot分析显示重组蛋白DNT1具有良好的抗原性.应用重组蛋白DNTI为抗原建立了检测Bb血清抗体的间接ELISA诊断方法.试验确定重组蛋白DNT1抗原的包被浓度为6.25μg/mL,最适血清稀释度为1:100.结论 建立的ELISA检测方法,不仅为Bb抗体检测提供了一种比较实用的血清学检测手段,也为进一步开发Bb检测试剂盒奠定了基础.