Hc-hrg-1参与捻转血矛线虫血红素运输的功能研究

Heme transport is essential for development of Haemonchus contortus. Genes involved in this pathway could provide effective drug targets for development of new drugs. However, researches about heme transport pathway in H. contortus is rare. We already cloned a novel gene Hc-hrg-1 in the process of studying on the development of H. contortus. The results of primary bioinformatic analysis of Hc-hrg-1 showed that it was a homologous gene of Ce-hrg-1, which could encode a transmembrane protein with an important role in heme transport in Caenorhabditis elegans. The putative Hc-hrg-1 encoded protein also had a transmembrane binding site, thus indicating that Hc-hrg-1 may also play an important role in heme transport, therefore affect the development of H. contortus. Our research aims to analyze the heme binding ability of reconstructive Hc-HRG-1 expressed via E. coli BL21system and heme binding assay in vitro, and verify the function of the gene through rescue assay of heme synthesize defectient mutant strain of yeast. Over-expression of Hc-hrg-1 in C. elegans and rescue assay of Ce-hrg-1 mutant strain will be applied to further characterize gene function. Together with RNAi and other methods, including expression pattern analysis, ZnMP uptake assay and pathogenicity test, we would like to finally clarify the exact function of Hc-hrg-1 and explain the role this gene played in heme transport pathway of H. contortus. The prospective results would lay a foundation for the explanation of heme transport pathway in H. contortus or even in parasitic nematodes, and provide a possible new drug target for anthelmintic development.

捻转血矛线虫血红素运输途径是其生长发育必需的代谢通路，能为新型环保高效抗虫药的开发提供有效的药物靶位点，但目前相关研究极少。前期研究获得的新基因Hc-hrg-1与秀丽线虫血红素运输跨膜蛋白基因Ce-hrg-1高度同源，且其编码蛋白存在保守的跨膜结合位点，推测Hc-HRG-1与Ce-HRG-1具有相似的生物学功能。本项目拟采用体外表达系统和血红素结合试验分析Hc-HRG-1与血红素的结合能力；并通过酵母血红素合成缺陷株拯救试验初步验证Hc-hrg-1的功能；进而利用秀丽线虫过表达和突变株拯救试验探究Hc-hrg-1可能的生物学特性；最终回归捻转血矛线虫本体，利用RNAi技术，结合时空特异性分析、ZnMP摄取和致病力试验，阐明Hc-hrg-1参与捻转血矛线虫血红素运输的生物学功能。研究结果将为捻转血矛线虫乃至寄生性线虫的血红素运输分子机制研究奠定基础，并为新型抗虫药物的研发提供新靶标。

捻转血矛线虫是反刍动物寄生性线虫，吸食宿主血液为生，因此需面对从宿主红细胞分解代谢过程中释放出来的高浓度血红素。游离的血红素易产生强氧化自由基，氧化细胞膜的蛋白和脂质，甚至是DNA分子，而溶酶体可通过关键基因调控血红素稳态。因此，合理调控血红素转运及代谢对寄生虫的生存和寄生阶段的建立至关重要，了解这种调控机制可为药物和疫苗开发以及疾病的治疗提供潜在靶标。本研究在捻转血矛线虫血红素转运机制探索过程中扩增得到一个新的血红素应答基因Hc-hrg-1，cDNA全长393 bp，编码130个氨基酸，DNA全长2998 bp，含4个外显子和3个内含子。该基因与秀丽线虫同源基因Ce-hrg-1位于同一分支，且在寄生线虫中高度保守；具有三个跨膜结构域，其中的组氨酸残基和FSRRY残基可能与血红素相关。对Hc-hrg-1进行表达特性分析，结果显示其在感染性三期幼虫中转录水平最高，且在血红素的缺乏环境下该基因转录水平会显著升高；利用IHF研究Hc-HRG-1在成虫中的定位发现其主要表达在肠道以及性腺；亚细胞定位结果则显示在HEK29T细胞中，该基因定位于溶酶体。这说明Hc-hrg-1作为血红素应答基因，主要在血红素聚积的肠道发挥作用，具体细胞器可能为溶酶体。利用模式生物秀丽线虫RNAi验证Hc-hrg-1参与血红素运输的功能，发现异源干扰不会使线虫产生明显的性状改变，但会导致血红素类似物ZnMP蓄积在肠道的溶酶体中。构建酵母血红素合成缺陷株hem1Δ并进行Hc-hrg-1拯救试验，结果显示Hc-HRG-1的表达能促进酵母从环境中摄取血红素，并拯救缺失株的生长。通过共定位及Co-IP试验，发现Hc-HRG-1与溶酶体V型ATP酶V0功能域的C型亚单位（Hc-ATP6V0C）存在互作，且第二个和第三个跨膜结构域是互作的关键功能域。本研究初步明确了Hc-hrg-1在溶酶体中参与血红素转运的分子机制，为后续进一步探明线虫血红素转运机制以及疫苗药物的开发奠定了良好基础。