重组牛分枝杆菌CFP-10蛋白间接ELISA检测方法的建立

Development of an indirect ELISA using the recombinant culture filter protein 10 from Mycobacterium bovis

【目的】建立检测牛分枝杆菌的间接ELISA方法,快速区分致病性与非致病性牛分枝杆菌,为净化牛结核病提供技术支撑。【方法】以体外扩增表达并纯化的重组牛分枝杆菌CFP-10蛋白为包被抗原,建立检测牛分枝杆菌的间接ELISA,并应用方阵法优化间接ELISA的检测条件。【结果】重组CFP-10蛋白间接ELISA的最佳检测条件为:以8μg/mL重组CFP-10蛋白包为被抗原,37℃下包被1 h,再以5%脱脂奶封闭1 h,血清(一抗)经1∶200倍稀释后作用1 h,然后以1∶500倍稀释的辣根过氧化物酶标记羊抗兔IgG(二抗)作用1 h。该间接ELISA的阴阳临界值为0.281,仅与牛分枝杆菌呈阳性反应,与牛布鲁氏杆菌、牛口蹄疫病毒无交叉反应;其敏感性能检测1∶320倍稀释的血清,批内与批间的变异系数均小于5.0%。【结论】建立的重组牛分枝杆菌CFP-10蛋白间接ELISA具有特异、敏感等优点,临床上可用于致病性牛分枝杆菌的检测。

[Objective] An indirect enzyme-linked immunosorbent assay （ELISA） with the recombinant culture filter protein 10 （CFP-10） from Mycobacterium bovis（M.bovis） was developed to detect M.bovis as a rapid and sensitive method.[Method]The CFP-10 gene from M.bovis was amplified and transferred to competent cell.The expressed CFP-10 gene protein from recombinant was purified and used as the coating antigen in this ELISA.The best conditions for this ELISA were tested with CFP-10 recombinant protein as an antigen.[Result]The best coating concentration of the CFP-10 protein was 8 μg/mL at 37 ℃ for 1 h and it was enclosed with 5％ skimmed milk for 1 h.The best antibody （serum） was diluted to 200 and the goat-rabbit antibody IgG labeled with enzyme HRP was diluted to 500 before incubated at 37℃ for 1 h.The critical value was 0.281.It did not cross with the serum from brucellosis,foot and mouth disease,and it was only positive to M.bovis.The sensitive test of 1∶320 dilution of serum showed the variable coefficient within and among the batch was less than 5.0％.[Conclusion] Due to its specificity and sensitivity,this indirect ELISA assay established here will be a good method for detecting M.bovis in future.