猪支气管败血波氏杆菌菌毛fimD基因的克隆表达及间接ELISA检测方法的建立

Indirect ELISA for Detection of Antibody against Swine Bordetella bronchiseptica Using Recombinant fimD Expressed in E. coli

参照GenBank上支气管败血波氏杆菌的菌毛fimD基因序列(X75811),设计1对引物,从本实验室分离鉴定的猪源支气管败血波氏杆菌中扩增出fimD编码区1 098 bp的片段,将其克隆至原核表达载体pET-28a,转化到大肠杆菌BL21(DE3)中进行融合表达。SDS-PAGE和Western blot检测证实该表达产物以包涵体形式存在,大小约42 ku,与用支气管败血波氏杆菌菌体制备的阳性血清能发生特异性反应。将包涵体变性和复性后包被酶标板建立间接ELISA(fimD-ELISA),特异性良好。用fimD-ELISA检测临床送检的668份血清,阳性率为30.7%。用该法与微量凝集试验平行检测102份血清,fimD-ELISA的敏感性高于微量凝集试验。

According to the Bordetella bronchiseptica fimD sequence（X75811） of GenBank, we designed a pair of primer, and 1 098 bp DNA fragment of fired was amplified from swine Bordetella bronchiseptica identificated in our laboratory. The acquired PCR products were inserted into pET-28a and expressed in BL21 （DE3）. Results of Sodium docecyl sulfate-polyacrylamide gel electrophoresis （SDS-PAGE） and Western blot assay showed that the fimD gene was expressed in the form of inclusion body,and the recombinant protein was about 42 kD, and can react with the swine antisera to Bordetella bronchiseptica. The fusion protein was purified and used as coating antigen to develop the indirect Enzyme-Linked Immunosorbent Assay（ELISA）. When using this ELISA to detect 668 clinical sera, 30.7% positive sera were detected. Parallel examination of the same 102 serum samples was conducted by using the ELISA and micro-agglutination test（MAT）, the result showed that the ELISA is more sensitivity than MAT.