摘要：
本研究对白斑综合征病毒(White Spot Syndrome Virus,WSSV)与传染性皮下及造血组织坏死病毒(Infectious Hypodermal and Haematopoietic Necrosis Virus,IHHNV)能否竞争虾鳃细胞膜上的NM23蛋白受体进行探索.先用蔗糖梯度离心法提纯WSSV的全蛋白,利用病毒覆盖蛋白印迹技术(VOPBA)与对虾鳃细胞膜NM23蛋白作用,将疑似蛋白条带进行LC-MS/MS分析,初步筛选出3种WSSV蛋白,分别为WSSV013、Wsv497和Wsv035,再构建这3种蛋白的原核表达载体,通过VOPBA和免疫共沉淀(co-immunoprecipitation)验证了Wsv497、Wsv035能与NM23蛋白相互作用,WSSV013不能与NM23蛋白相互作用.初步推测WSSV与IHHNV可能竞争对虾鳃细胞膜上的NM23蛋白受体,该结果为今后研究两种病毒竞争细胞膜受体和虾病毒蛋白作用机制提供理论基础.%White spot syndrome and infectious hypodermal and haematopoietic necrosis are primary viral diseases of prawns. Outbreaks of white spot syndrome virus (WSSV) and infectious hypodermal and haematopoietic ne-crosis virus (IHHNV) arise worldwide and have caused serious economic losses in recent years, including in China. Some researchers found that, given the same conditions, WSSV-infected prawn display lower levels of mortality than IHHNV-infected prawn. Our laboratory previously demonstrated that IHHNV capsid protein (CP) has the ability to combine with NM23 protein in prawn gill membrane. NM23, a kind of nucleoside diphosphate kinase (NDPK), has various biological functions and is located mainly in the cytoplasm and nucleus, but is also evident in the cell membrane. We conducted an experiment to investigate whether WSSV and IHHNV compete with NM23 protein receptor sites on the cell membrane of shrimp gill tissue. First, we purified the total WSSV proteins using sucrose gradient centrifugation, and then we used a virus overlay protein binding assay (VOPBA) to detect pro-tein-protein interaction between the total proteins and NM23. Next, three suspected positive proteins were selected by the combined LC-MS/MS technique: WSSV013, Wsv497 and Wsv035. Wsv035, also known as VP110, is one of the capsule membrane proteins of WSSV and is encoded byORF035. It contains a membrane structure domain and a RGD locus (namely, the Arg-Gly-Asp motif). Some scholars, using fluorescence assay and competitive in-hibition tests, found Wsv035 could stick to the host cell: Wsv035 RGD can form RGDT peptides at the cell surface and it plays a role in WSSV infection. There have been few studies of Wsv497 and WSSV013 proteins, however, and thus their distribution, structure and functions are not yet clear. Therefore, we constructed prokaryotic expres-sion vectors of these three proteins, for purifying WSSV013, Wsv497 and Wsv035 in order to interact with NM23 protein, using VOPBA and co-immunoprecipitation, respectively. The results showed that Wsv497 and Wsv035 have the ability to interact with NM23, while WSSV013 showed an invalid effect on NM23. Accordingly, we sur-mise that WSSV and IHHNV have the ability to compete with NM23 protein receptors on the cell membrane of prawn gill tissue. This study adds to theories about the mechanism of competition between WSSV and IHHNV for receptor sites on the cell membrane of prawn gill tissue, and lays a foundation for further studies of protein inter-actions in the context of WSSV and prawn culture.
