家兔支气管败血波氏杆菌重组百日咳杆菌粘附素（PRN）蛋白的原核表达及其间接ELISA方法的建立

目的表达支气管败血波氏杆菌（Bordetella bronchiseptica,Bb）PRN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prn基因序列（AY376325）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pGEX-4T-1中,以E.coli BL21（DE3）为表达菌株进行诱导表达,以纯化重组蛋白PRN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等,建立检测支气管败血波氏杆菌抗体的间接ELISA方法。结果成功克隆了prn全基因序列,并在E.coliBL21（DE3）中获得高效表达,经SDS-PAGE、Western blot分析显示重组蛋白PRN具有良好的抗原性。应用重组蛋白PRN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PRN抗原的包被浓度为500ng/mL,最适血清稀释度为1∶40。结论建立的ELISA检测方法,不仅为Bb抗体检测提供了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。

Prokaryotic Expression of Rabbits Bordetella Bronchiseptica Pertactin Recombinant Protein and Development of an Indirect ELISA for Detection of Its Antibodies

Objective To get Bordetella bronchiseptic（Bb） expressing recombinant protein pertactin（PRN）and establish an indirect ELISA for detection of rabbit Bordetella bronchiseptic antibodies.Methods According to a pair of designed specific primers,PRN gene of rabbit Bordetella bronchiseptica was amplified by PCR and inserted into prokaryotic expression vector pGEX-4T-1.The recombinant expression plasmids were transfected into BL21（DE3） strain.The optimal concentration of coated antigen recombinant protein PRN and serum dilution was determined to develop the ELISA technique.Results PRN 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 PRN determined in this experiment was 500 ng /mL,and the optimal testing serum dilution was 1：40.Conclusions The PRN indirect ELISA developed in this study 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.