多房棘球蚴亮氨酸氨基肽酶优势抗原表位的鉴定

Identification of the Dominant Antigen Epitopes of Leucine Aminopeptidase in Echinococcus multilocularis

目的根据预测多房棘球蚴亮氨酸氨基肽酶(Leucine aminopeptidase,LAP)抗原表位的结果,进一步鉴定其优势的T细胞和B细胞抗原表位。方法构建重组质粒pCzn1-LAP后进行蛋白原核表达与纯化,将纯化的LAP蛋白进行动物免疫后,利用脾脏淋巴细胞增殖、流式细胞术、ELISA pot、ELISA等方法鉴定优势抗原表位。最后,利用SWISS-MODEL软件对LAP蛋白3D结构建模,PyMOL软件对鉴定的LAP优势抗原表位分布进行准确定位。结果pCzn1-LAP菌株诱导表达的蛋白约为57 kDa,蛋白经过复性后成功纯化。流式细胞术鉴定的Th1型优势抗原表位为LAP 79-63、LAP 396-410和LAP 106-120;Th2型优势抗原表位为LAP 13-28、LAP 495-510和LAP 396-410。ELISA pot鉴定的Th1型优势抗原表位为LAP 396-410、LAP 504-518和LAP 106-120;Th2型优势抗原表位为LAP 504-518、LAP 313-328和LAP 396-410。BALB/c小鼠血清的抗原表位特异性ELISA筛选出的优势B表位结果为LAP 464-479、LAP 495-510和LAP 313-328。LAP蛋白3D结构建模成功,LAP优势抗原表位分布于表面居多。结论多房棘球蚴LAP蛋白优势T细胞和B细胞抗原表位鉴定成功,使得研制抗多房棘球蚴感染的多价表位疫苗成为可能。

The present study aimed to identify dominant T cell and B cell epitopes in leucine aminopeptidase of Echinococcus multilocularis. Prokaryotic expression of LAP protein was performed, and the purified LAP protein was used to immunize mice. Then an lymphocyte-proliferation assay, flow cytometry, ELISA pot, and ELISA were used to identify the dominant antigen epitopes. Finally, the 3 D structure was modeled with SWISS-MODEL software, and PyMOL software was used to accurately locate LAP’s dominant epitopes. The expressed LAP protein was approximately 57 kDa, and the protein was successfully purified after renaturation. The dominant antigen epitopes of Th1 were identified by flow cytometry as LAP79-63, LAP396-410, and LAP106-120. The epitopes of Th2 were LAP13-28, LAP495-510, and LAP396-410. The dominant antigen epitopes identified by ELISA pot were LAP396-410, LAP504-518, and LAP106-120. The epitopes of Th2 were LAP504-518, LAP313-328, and LAP396-410. The predominant B cell epitopes identified by the ELISA were LAP464-479, LAP495-510, and LAP313-328. Bioinformatics analysis showed that the antigenic peptides were mostly located on the surface of the protein. The dominant T cell and B cell epitopes of LAP were successfully identified and may contribute to the development of a multivalent epitope vaccine against Echinococcus multilocularis infection.