棒狀體相關(guān)蛋白1（rhoptry-associated protein 1，RAP-1）是由棒狀體分泌的與侵襲宿主細(xì)胞相關(guān)的蛋白。本研究以頂復(fù)門原蟲RAP-1基因?yàn)榘袠?biāo)，用最大似然法構(gòu)建了巴貝斯屬、瘧原蟲屬、泰勒蟲屬的RAP-1基因進(jìn)化樹(shù)；應(yīng)用軟件預(yù)測(cè)分析RAP-1蛋白的理化性質(zhì)、親疏水性、跨膜區(qū)以及二、三級(jí)結(jié)構(gòu)，并表達(dá)純化了馬泰勒蟲（Theileria equi）RAP-1重組蛋白。結(jié)果顯示：巴貝斯屬不同蟲株相聚類，測(cè)定序列與馬泰勒蟲聚為一支，又與惡性瘧原蟲相聚類，表明親緣關(guān)系較近。馬泰勒蟲RAP-1蛋白理論等電點(diǎn)為9.85，半衰期為30 h，總平均親水性（GRAVY）為-0.368，無(wú)跨膜區(qū)；RAP-1蛋白二級(jí)結(jié)構(gòu)中，α-螺旋、β-折疊、β-轉(zhuǎn)角和無(wú)規(guī)卷曲占比分別為38.5%、15.5%、27.8%、18.1%；構(gòu)建的三級(jí)結(jié)構(gòu)立體展現(xiàn)了RAP-1蛋白形態(tài)。成功構(gòu)建了原核表達(dá)載體pET-32a-RAP-1；該重組蛋白主要以包涵體形式存在，蛋白大小為65 kDa。RAP-1蛋白可通過(guò)原核表達(dá)系統(tǒng)高效表達(dá)，純化后的產(chǎn)物能被馬泰勒蟲標(biāo)準(zhǔn)陽(yáng)性血清識(shí)別，具有良好的反應(yīng)原性。本研究為深入了解馬泰勒蟲入侵宿主的機(jī)制機(jī)理以及RAP-1蛋白的功能研究奠定了基礎(chǔ)。
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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