Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.
Dev Comp Immunol. 2012 Jul;37(3-4):334-41. doi: 10.1016/j.dci.2011.12.010. Epub 2011 Dec 27.
When using mRNA from gills of normal whiteleg shrimp Penaeus (Litopenaeus) vannamei as the tester and mRNA from yellow head virus (YHV)-infected shrimp as the driver, subtractive suppression hybridization (SSH) revealed that a novel EST clone of 198 bp with a putative C-type lectin-like domain (CTLD) was downregulated in YHV-infected shrimp. The clone nucleotide sequence had 99% identity with one contig MGID1052359 (1,380 bp) reported in an EST database of P. vannamei, and the presence of this target in normal shrimp was confirmed by RT-PCR using primers designed from the MGID1052359 sequence. Analysis of the primary structure of the deduced amino acid (a.a.) sequence of the contig revealed a short portion (40 a.a. residues) at its N-terminus with high similarity to a low density lipoprotein receptor (LDLR) class A domain and another 152 a.a. residues at its C-terminus with high similarity to a C-type lectin domain. Thus, the clone was named LvCTLD and three recombinant proteins (LvCTLD, the LDLR domain and the CTLD domain) were synthesized in a bacterial system based on its sequence. An in vitro encapsulation assay revealed that Sepharose 4B beads coated with rLvCTLD were encapsulated by shrimp hemocytes and that melanization followed by 24 h post-encapsulation. The encapsulation activity of rLvCTLD was inhibited by 100 mM galactose, but not mannose or EDTA. In vivo injection of rLvCTLD or rLvCTLD plus YHV resulted in a significant elevation of PO activity in the hemolymph of the challenged shrimp when compared to shrimp injected with buffer, suggesting that rLvCTLD could activate the proPO system. An ELISA test revealed that rLvCTLD could bind to YHV particles in the presence of shrimp hemolymph. Phylogenetic analysis suggested that the LvCTLD sequence was more closely related to an antiviral gene found in Penaeus monodon (PmAV) than to other reported shrimp lectins. Taken together, we conclude that a novel shrimp LvCTLD is a host recognition molecule involved in the shrimp defense mechanism against YHV via recruitment of hemocytes, probably at the site of viral infection, and via activation of the proPO system.
当使用正常凡纳滨对虾（Litopenaeus）鳃部的 mRNA 作为测试者，并用黄头病毒（YHV）感染的虾的 mRNA 作为驱动者时，消减抑制杂交（SSH）显示，在 YHV 感染的虾中，一个具有假定 C 型凝集素样结构域（CTLD）的新 EST 克隆被下调。该克隆的核苷酸序列与 P. vannamei 的 EST 数据库中报告的一个连续体 MGID1052359（1380bp）具有 99%的同一性，并且使用从 MGID1052359 序列设计的引物通过 RT-PCR 证实了正常虾中存在该靶标。对连续体推导的氨基酸（a.a.）序列的一级结构进行分析，发现其 N 端有一个短片段（40 个 a.a.残基）与低密度脂蛋白受体（LDLR）A 类结构域高度相似，C 端还有 152 个 a.a.残基与 C 型凝集素结构域高度相似。因此，该克隆被命名为 LvCTLD，并根据其序列在细菌系统中合成了三种重组蛋白（LvCTLD、LDLR 结构域和 CTLD 结构域）。体外包封实验表明，用 rLvCTLD 包被的琼脂糖 4B 珠被虾血细胞包封，并在包封后 24 小时内发生黑化。100mM 半乳糖可抑制 rLvCTLD 的包封活性，但甘露糖或 EDTA 则没有抑制作用。与用缓冲液注射的虾相比，体内注射 rLvCTLD 或 rLvCTLD 加 YHV 可导致受挑战虾的血淋巴 PO 活性显著升高，这表明 rLvCTLD 可激活 proPO 系统。ELISA 测试表明，rLvCTLD 可在虾血淋巴存在的情况下结合 YHV 颗粒。系统进化分析表明，LvCTLD 序列与在斑节对虾（PmAV）中发现的抗病毒基因更密切相关，而与其他报道的虾凝集素则不相关。综上所述，我们得出结论，一种新的虾 LvCTLD 是一种宿主识别分子，通过募集血细胞（可能在病毒感染部位）并通过激活 proPO 系统，参与对虾防御 YHV 的机制。
