鼠疫耶尔森菌carf1基因的克隆表达及其产物免疫学评价

Clone expression of the cafl gene of Yersinia pestis and immunological evaluation on recombinant F1 antigen

目的表达具有免疫学活性重组F1抗原（rF1），并以其构建检测鼠疫抗体胶体金试纸条。方法将去掉信号肽编码序列的caf1基因片段与载体质粒pET32a（＋）通过BamHl和NotⅠ双酶切位点进行连接，将重组质粒[caf1-pET32a（＋）]转化入BL21（DE3）中进行诱导表达，表达产物经亲和层析纯化，以纯化rF1及天然F1抗原制备双检测鼠疫抗体胶体金标试纸条，并对浙江省528份人血清标本进行检测。结果含caf1-pET32a（＋）的BL21（DE3），经诱导产生相对分子质量（Mτ）约为35．5×10^3的rF1融合蛋白；rF1融合蛋白检测鼠疫抗体的敏感性等同甚至超越天然F1抗原；在528份人血清标本的检测中，rF1与天然F1的符合率为97．9％（κ=0．466），有较好一致性。结论制备的rF1，具有良好免疫学活性，该rF1可替代天然F1抗原，用于鼠疫免疫学检测。

Objective To get recombinant F1 antigen （rF1） and to construct the detection dipstick of plague antibody. Methods The call gene removing the signal peptide coding sequence was cloned into plasmid pET32a （ ＋ ） by double-digested sites of BamHI and Not I. Recombinant plasmid caf1-pET32a （＋） was transformed into BL21 （DE3） and the rF 1 was expressed. Expression products were purified by affinity chromatography. Dual detection dipstick of plague antibody was constructed with purified rF1 and natural F1, and evaluated with 528 human serum samples of Zhejiang province. Results The fusion protein rF1 of 35.5 KD was expressed by BL21 strains containing cafl-pET32a （＋）. The sensitivity of rF 1 showed equivalent to or higher than the natural F1 antigen in detecting plague antibody. It seemed that there was a better consistency of 97.9% （κ = 0.466） when 528 human sera was detected by rF1 and natural F1. Conclusion We successfully extracted the rF 1 with good immunological activity that might be used to detecting Yersinia pestis.