科研动态

棒状体相关蛋白1（rhoptry-associated protein 1，RAP-1）是由棒状体分泌的与侵袭宿主细胞相关的蛋白。本研究以顶复门原虫RAP-1基因为靶标，用最大似然法构建了巴贝斯属、疟原虫属、泰勒虫属的RAP-1基因进化树；应用软件预测分析RAP-1蛋白的理化性质、亲疏水性、跨膜区以及二、三级结构，并表达纯化了马泰勒虫（Theileria equi）RAP-1重组蛋白。结果显示：巴贝斯属不同虫株相聚类，测定序列与马泰勒虫聚为一支，又与恶性疟原虫相聚类，表明亲缘关系较近。马泰勒虫RAP-1蛋白理论等电点为9.85，半衰期为30 h，总平均亲水性（GRAVY）为-0.368，无跨膜区；RAP-1蛋白二级结构中，α-螺旋、β-折叠、β-转角和无规卷曲占比分别为38.5%、15.5%、27.8%、18.1%；构建的三级结构立体展现了RAP-1蛋白形态。成功构建了原核表达载体pET-32a-RAP-1；该重组蛋白主要以包涵体形式存在，蛋白大小为65 kDa。RAP-1蛋白可通过原核表达系统高效表达，纯化后的产物能被马泰勒虫标准阳性血清识别，具有良好的反应原性。本研究为深入了解马泰勒虫入侵宿主的机制机理以及RAP-1蛋白的功能研究奠定了基础。

Prokaryotic Expression of RAP-1 Gene of Theileriaequi and

Characteristic Analysis on the Encoded Protein

Rhoptry-associated protein 1（RAP-1）is a protein related to invading the host cells and secreted by the rhoptry. Targeting at the RAP-1 gene of Protozoan protozoa，the gene evolution trees of Babesia，Plasmodium and Theileria were constructed by the maximum likelihood method. The physical and chemical properties，hydrophobicity，transmembrane zone，secondary and tertiary structures of RAP-1 protein were predicted by software，and the recombinant RAP-1 protein of Theileria equi was expressed and purified. The results showed that the genus of Babesia was clustered together，its sequence and that of Theileria equi belonged to the same branch and clustered with the genus of Plasmodium falciparum，indicating that their genus was closely related. The theoretical isoelectric point of RAP-1 protein was 9.85，the half-life was 30 h，the total average hydrophilicity was -0.368，and there was no transmembrane zone. The secondary structure α-helices，β-sheets，β-turns and random coils of RAP-1 protein accounted for 38.5%，15.5%，27.8% and 18.1%，respectively. The form of RAP-1 protein was shown by the three-level structure，and the prokaryotic expression vector pET-32a-RAP-1 was successfully constructed，the recombinant protein mainly existed in the form of inclusion body with the size of 65 kDa. In conclusion，the RAP-1 protein could be highly expressed by prokaryotic expression system，and the purified products could be identified by the standard positive serum of Theileriaequi due to its good reactogenicity. Therefore，a foundation was provided for further studying the mechanism of Theileriaequi invading into hosts and the functions of RAP-1 protein.

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