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目的确认RTX家族胞外蛋白A(RTX family exoprotein A,RfeA)为2-型猪链球菌(Streptococcus suis serotype 2,SS2)的分泌蛋白。方法通过生物信息学软件分析RfeA的氨基酸序列,预测RfeA在SS2中的分布;以纯化的RfeA重组蛋白为免疫原免疫小鼠,制备RfeA鼠多抗血清,间接ELISA检测抗血清效价;以制备的RfeA抗血清为一抗,利用ELISA方法分析RfeA在05ZYH33株培养上清中的分布,通过实验证实生物信息学预测的结果。结果生物信息学软件分析RfeA氨基酸序列,预测RfeA为SS2分泌性蛋白;ELISA方法分析发现RfeA存在于05ZYH33株培养上清中,是SS2分泌性蛋白。结论实验研究结果与生物信息学预测一致,RfeA为SS2分泌性蛋白的确定为进一步研究RfeA的功能以及在SS2致病过程中扮演的角色奠定了基础。
Objective To confirm that RTX family exoprotein A (RfeA) is a secreted protein of Streptococcus suis serotype 2 (SS2). Methods The amino acid sequence of RfeA was analyzed by bioinformatics software to predict the distribution of RfeA in SS2. The purified recombinant RfeA protein was used as the immunogen to immunize mice, and then the polyclonal antiserum against RfeA was prepared. The antiserum titer was detected by indirect ELISA. RfeA antiserum was used as the primary antibody. The distribution of RfeA in the supernatant of 05ZYH33 strain was analyzed by ELISA. The results of bioinformatics prediction were confirmed by experiments. Results Bioinformatics software analyzed the amino acid sequence of RfeA and predicted that RfeA was SS2 secreted protein. ELISA analysis showed that RfeA was present in the supernatant of 05ZYH33 strain and was a secreted SS2 protein. Conclusion The experimental results are consistent with the prediction of bioinformatics. The determination of SSF secreted by RfeA laid the foundation for the further study on the function of RfeA and its role in the pathogenicity of SS2.