Aquatic Animal Health Research Team, Integrative Aquaculture Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi Office, Rama VI Rd., Bangkok, 10400, Thailand; Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand; Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok, 10400, Thailand.
Aquatic Animal Health Research Team, Integrative Aquaculture Biotechnology Research Group, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi Office, Rama VI Rd., Bangkok, 10400, Thailand.
Fish Shellfish Immunol. 2019 Jun;89:108-116. doi: 10.1016/j.fsi.2019.03.052. Epub 2019 Mar 27.
To identify molecules involved in Macrobrachium rosenbergii nodavirus (MrNV) entry into hemocytes of the giant freshwater prawn M. rosenbergii, biotinylated prawn hemocyte membrane proteins were prepared, purified and separated by SDS-PAGE. The proteins were blotted on the nitrocellulose membrane before incubation with the MrNV capsid protein (MrNV-CP) by a VOPBA technique. Subsequent mass spectrometry and analysis of immune-reactive bands represent putative binding partners including transglutaminase (TG), actin, α2-macroglobulin, α1-tubulin, F-ATP synthase β-subunit and a currently uncharacterized protein. The sequence of TG has been characterized and found 5 amino acids differences to a previously reported MrTG (ADX99580), mainly at its N-terminal part and thus, we named it MrTGII (KM008611). Recombinant MrTGII was prepared to produce a polyclonal antibody against it, which was successfully revealed the presence of MrTGII (100 kDa) in prawn hemocyte lysates. Using the pentylamine-biotin incorporation assay, an acyl transfer reaction was observed when hemocyte lysates were added to solutions containing MrNV-CP, suggesting that hemocyte MrTG could use MrNV-CP as the substrate. The expression levels of MrTGII were changed during the course of MrNV infection. By using immunostaining technique, location of MrTGII on the hemocyte surface was confirmed. Specific interaction between MrTGII with MrNV-CP in a dose-dependent manner was confirmed by in vitro ELISA assay. The highest binding activity of MrNV-CP was found with the N-terminal portion of the protein. In vitro neutralization using anti-MrTGII antibody resulted in inhibition of MrNV attachment to the hemocyte surface, accompanied by a dramatic reduction in viral replication. This is the first time that crustacean TG has been shown to be involved in viral entry, in addition to its roles in blood clotting and haematopoiesis.
为了鉴定与罗氏沼虾诺达病毒（MrNV）进入罗氏沼虾血细胞有关的分子，制备、纯化并通过 SDS-PAGE 分离了罗氏沼虾血细胞的生物素化膜蛋白。在与 MrNV 衣壳蛋白（MrNV-CP）孵育之前，将这些蛋白印迹到硝酸纤维素膜上，通过 VOPBA 技术进行孵育。随后的质谱分析和免疫反应带分析代表了潜在的结合伴侣，包括转谷氨酰胺酶（TG）、肌动蛋白、α2-巨球蛋白、α1-微管蛋白、F-ATP 合酶β亚基和一种目前尚未鉴定的蛋白质。TG 的序列已经被表征，并发现与之前报道的 MrTG（ADX99580）有 5 个氨基酸的差异，主要在其 N 端，因此，我们将其命名为 MrTGII（KM008611）。制备了重组 MrTGII 以产生针对它的多克隆抗体，该抗体成功地在虾血细胞裂解物中检测到了 MrTGII（100 kDa）的存在。使用戊胺生物素掺入测定法，当血细胞裂解物被添加到含有 MrNV-CP 的溶液中时，观察到酰基转移反应，表明血细胞 MrTG 可以将 MrNV-CP 用作底物。在 MrNV 感染过程中，MrTGII 的表达水平发生了变化。通过免疫染色技术，确认了 MrTGII 在血细胞表面的位置。通过体外 ELISA 测定证实了 MrTGII 与 MrNV-CP 之间的特异性相互作用呈剂量依赖性。发现 MrNV-CP 与蛋白的 N 端部分具有最高的结合活性。体外中和实验中，抗 MrTGII 抗体的使用导致 MrNV 附着在血细胞表面的抑制，同时病毒复制明显减少。这是首次证明甲壳动物 TG 除了在血液凝固和造血中发挥作用外，还参与病毒进入。
