CAJ | 학술논문

[目的]以原核表达的重组蛋白作为检测抗原，建立间接 ELISA方法用来检测猪血清中APP抗体。[方法]将猪胸膜肺炎放线杆菌 ApxⅡ主要抗原表位区基因片段克隆至原核表达载体 pET-28a（+），构建重组质粒 pET-ApxⅡA1。将重组质粒转化至宿主菌 BL21（DE3）中进行表达。通过 Western-blot分析重组蛋白免疫原性。用纯化的重组蛋白作为包被抗原建立检测猪 APP抗体的间接 ELISA方法，并优化反应条件，确定抗原的最佳包被量和血清的最佳稀释度。[结果]经 PCR双酶切及测序鉴定，重组质粒构建成功，诱导表达纯化后，重组蛋白可被 APP阳性血清特异性的识别，表明表达产物具有良好的免疫原性。用纯化的重组蛋白初步建立了检测 APP抗体的间接ELISA方法，用该方法与商品化 ApxⅣ抗体检测试剂盒分别对94份临床非免疫血清样品进行检测，结果两者的符合率为90.4%。[结论]所建立的间接 ELISA 方法具有良好的特异性和敏感性，为流行病学调查和疫苗免疫效果评估提供了技术手段。
[목적]이원핵표체적중조단백작위검측항원，건립간접 ELISA방법용래검측저혈청중APP항체。[방법]장저흉막폐염방선간균 ApxⅡ주요항원표위구기인편단극륭지원핵표체재체 pET-28a（+），구건중조질립 pET-ApxⅡA1。장중조질립전화지숙주균 BL21（DE3）중진행표체。통과 Western-blot분석중조단백면역원성。용순화적중조단백작위포피항원건립검측저 APP항체적간접 ELISA방법，병우화반응조건，학정항원적최가포피량화혈청적최가희석도。[결과]경 PCR쌍매절급측서감정，중조질립구건성공，유도표체순화후，중조단백가피 APP양성혈청특이성적식별，표명표체산물구유량호적면역원성。용순화적중조단백초보건립료검측 APP항체적간접ELISA방법，용해방법여상품화 ApxⅣ항체검측시제합분별대94빈림상비면역혈청양품진행검측，결과량자적부합솔위90.4%。[결론]소건립적간접 ELISA 방법구유량호적특이성화민감성，위류행병학조사화역묘면역효과평고제공료기술수단。
[Objective] This study aimed to develop an indirect ELISA to detect the antibodies against Actinobacil us pleuropneumoniae (APP) using the recombinant ApxⅡA1 protein expressed in prokaryotic cells as the antigen. [Method] The major epi-tope domain of ApxⅡ was cloned into the prokaryotic expression vector pET-28a (+) to obtain the recombinant plasmid pET-ApxⅡA1, which was then transformed into E. coli BL21 (DE3) for expression. The immunogenicity of the recombinant pro-tein was analyzed by western-blotting. After that, the purified recombinant protein was used as the coating antigen in the indirect ELISA for detecting the antibodies against APP. Final y, the concentration of coated antigen and the dilution of serum were optimized. [Result] Proved by enzyme digestion and sequencing, the recombi-nant plasmid pET-ApxⅡA1 was constructed successful y. The recombinant protein was highly expressed in prokaryotic cells, and Western-blotting analysis showed that it was recognized specifical y by positive serum of APP. The indirect ELISA could detect the antibody against APP with the purified recombinant protein as the coating antigen. The optimal concentration of coated antigen was 1.23 μg/ml and the opti-mal dilution of serum was 1:100. Compared with a commercial ELISA kit detecting antibody against ApxⅣ, the coincidence rate of the indirect ELISA was 90.4%. [Conclusion] Our results indicated that the indirect ELISA is sensitive and specific, and suitable for evaluating the effect of APP vaccine and epidemiological surveys.