3株嗜吞噬细胞无形体AnkA蛋白原核表达及免疫原性差异性研究

Prokaryotic expression and immunogenicity of recombinant AnkA protein from 3 Anaplasma phagocytophilumisolates

目的研究分析3株嗜吞噬细胞无形体AnkA蛋白的免疫原性。方法利用原核表达PCR扩增了嗜吞噬细胞无形体Webster株,斯洛文尼亚1 567株,美国96HE58株ankA基因部分序列,构建pET-30a-AnkA表达载体,转化至大肠杆菌BL21中,以IPTG诱导高效表达融合蛋白,经飞行质谱鉴定氨基酸序列,免疫BALB/c小鼠制备多克隆抗体。Western blot分析3株菌AnkA重组蛋白与免疫小鼠血清抗体相互间的免疫反应以及交叉反应性。同时检测3株菌AnkA重组蛋白与我国无形体病人血清抗体反应性。结果表达的重组蛋白主要以可溶性形式存在,小鼠免疫获得高效价抗AnkA IgG抗体,3株菌重组蛋白免疫抗体间存在免疫交叉。3株菌重组蛋白与我国无形体病人血清抗体存在免疫反应。结论成功原核表达并纯化了3株嗜吞噬细胞无形体AnkA蛋白,重组蛋白与我国无形体病人阳性血清存在免疫反应。3株嗜吞噬细胞无形体AnkA重组蛋白与免疫小鼠多克隆抗体间存在交叉反应。

In order to investigate the immunogenicity of recombinant AnkA proteins from 3 A. phagocytophilum iso- lates, the partial ankA fragments of its strain Webster, strain Slovenia 1 567, and strain 96HE58 were amplified by PCR re- spectively and then inserted into the vector pET-30a and transformed into Escherichia coli BL21 to express with IPTG induc- tion. The amino acid sequences of the expression products were identified by mass spectrum analysis. Polyclonal antibodies a- gainst the 3 recombinant AnkA proteins were produced by immuned mouse with the purified fusion proteins. The immunoreac- tivity of the 3 AnkA recombinant proteins was analyzed by Western blot using the serum from immunized mouse and HGA pos- itive serum from human. The recombinant AnkA proteins were efficiently expressed. High titer antibodies against the 3 AnkA recombinant proteins were successfully produced and cross reacted among the 3 AnkA recombinant proteins and their corre- sponding antibodies. Apparent reaction was also observed among the 3 AnkA recombinant proteins and Chinese human HGA positive serum. Here we concluded that there was no immunogenicity difference among the 3 recombinant AnkA proteins of A. phagocytophilum strain Webster, strain Slovenia 1 567, and strain 96HE58.