马泰勒虫AMA1基因的真核表达与鉴定

Eukaryotic expression and identification of AMA1 gene in Theileria equi

以GenBank中马泰勒虫(Theileria equi,T.equi)美国WA株基因组序列(XM_004833042.1)为目标,选取目前公开的所有顶复门原虫泰勒虫属顶膜抗原1(apical membrane antigen 1,AMA1)基因进行本地BLAST比对。针对目的基因片段设计特异性引物后,以T.equi新疆分离株为模板对AMA1目的基因片段进行扩增克隆,并对T.equi AMA1蛋白进行真核表达以及多克隆抗体制备,最后通过间接免疫荧光和Western blot鉴定真核重组蛋白。结果显示,成功扩增出T.equi新疆株AMA1基因,与T.equi美国WA株AMA1基因相比,两者进化关系最为接近,核苷酸和氨基酸同源性分别为76.34%和78.64%。成功在HEK293T细胞中表达AMA1真核重组蛋白,约为55 kDa,将AMA1原核重组蛋白免疫小鼠后,所产生的多克隆抗体效价为1∶819200。Western blot结果显示,免疫小鼠产生的多克隆抗体可以识别AMA1真核重组蛋白,表明重组蛋白具有较好的抗原性。本试验完成了T.equi AMA1真核重组蛋白的鉴定和多克隆抗体的制备,为后续开展T.equi保护性抗原研究提供了候选靶点,为进一步探究T.equi入侵宿主细胞机制奠定理论基础。

The genome sequence of Theileria equi(T.equi)WA strain in GenBank(XM004833042.1)was used as the target,and all the currently available AMA1 genes of Theileria were selected for local BLAST comparison.The specific primers were designed for the target gene fragments,and then the eukaryotic expression of T.equi AMA1 protein was carried out and polyclonal antibody was prepared.Finally,the eukaryotic recombinant protein was identified by indirect immunofluorescence and Western blot.The results showed that the T.equi AMA1 gene of Xinjiang strain was amplified successfully.Compared with the AMA1 gene of T.equi WA strain from USA,the evolutionary relationship between the two strains was the closest,and the homology of nucleotide as well as amino acid was 76.34% and 78.64%,respectively.The AMA1 eukaryotic recombinant protein was successfully expressed in HEK293 T cells,which size was about 55 kDa.After immunizing mice with the AMA1 prokaryotic recombinant protein,the produced polyclonal antibody titers was1:819200.The results of Western blot showed that the polyclonal antibody produced by immunized mice could recognize the AMA1 eukaryotic recombinant protein,indicating that the recombinant protein showed a specific antigenicity.This experiment completed the identification of T.equi AMA1 eukaryotic recombinant protein and the preparation of polyclonal antibody,which provided a candidate target for the subsequent research of T.equi protective antigen and laid a theoretical foundation for further investigation of the invasion mechanism of T.equi into host cells.