Taengchaiyaphum Suparat, Nakayama Hideki, Srisala Jiraporn, Khiev Ratny, Aldama-Cano Diva January, Thitamadee Siripong, Sritunyalucksana Kallaya
Shrimp-pathogen Interaction (SPI) laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Yothi Office, Rama VI Rd., Bangkok, 10400, Thailand.
Department of Environmental Science, Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki, Japan.
Dev Comp Immunol. 2017 Nov;76:56-64. doi: 10.1016/j.dci.2017.05.016. Epub 2017 May 22.
To improve the efficacy of WSSV protection, multimeric (tetrameric) recombinant VP28 (4XrVP28) was produced and tested in comparison with those of monomeric VP28 (1XrVP28). In vitro binding of either 1XrVP28 or 4XrVP28 to shrimp hemocyte surface was evident as early as 10 min after protein inoculation. Similar results were obtained in vivo when shrimp were injected with recombinant proteins that the proteins bound to the hemocyte surface could be detected since 5 min after injection. Comparison of the WSSV protection efficiencies of 1XrVP28 or 4XrVP28 were performed by injection the purified 1XrVP28 or 4XrVP28 (22.5 μg/shrimp) and WSSV inoculum (1000 copies/shrimp) into shrimp. At 10 dpi, while shrimp injected with WSSV inoculum reached 100% mortality, shrimp injected with 1XrVP28 + WSSV or 4XrVP28 + WSSV showed relative percent survival (RPS) of 67% and 81%, respectively. PCR quantification revealed high number of WSSV in the moribund shrimp of WSSV- and 1XrVP28+WSSV-injected group. In contrast, lower number of WSSV copies were found in the survivors both from 1XrVP28+WSSV- or 4XrVP28+WSSV- injected groups. Histopathological analysis demonstrated the WSSV infected lesions found in the moribund from WSSV-infected group and 1XrVP28+WSSV-injected group, but less or none in the survivors. ELISA demonstrated that 4XrVP28 exhibited higher affinity binding to rPmRab7, a WSSV binding protein essential for WSSV entry to the cell than 1XrVP28. Taken together, the protection against WSSV in shrimp could be improved by application of multimeric rVP28.
为提高对白斑综合征病毒（WSSV）的保护效果，制备了多聚体（四聚体）重组VP28（4XrVP28），并与单体VP28（1XrVP28）进行比较测试。早在蛋白质接种后10分钟，1XrVP28或4XrVP28与虾血细胞表面的体外结合就很明显。当给虾注射重组蛋白时，在体内也得到了类似结果，即自注射后5分钟起就能检测到结合在血细胞表面的蛋白。通过向虾注射纯化的1XrVP28或4XrVP28（22.5微克/只虾）和WSSV接种物（1000个拷贝/只虾），对1XrVP28或4XrVP28的WSSV保护效率进行比较。在感染后第10天，注射WSSV接种物的虾死亡率达到100%，而注射1XrVP28 + WSSV或4XrVP28 + WSSV的虾相对存活率（RPS）分别为67%和81%。PCR定量分析显示，在注射WSSV和1XrVP28 + WSSV组的濒死虾中WSSV数量很多。相比之下，在注射1XrVP28 + WSSV或4XrVP28 + WSSV组的存活虾中发现的WSSV拷贝数较少。组织病理学分析表明，在WSSV感染组和注射1XrVP28 + WSSV组的濒死虾中发现了WSSV感染病变，但在存活虾中较少或没有。酶联免疫吸附测定（ELISA）表明，4XrVP28与rPmRab7（WSSV进入细胞所必需的一种WSSV结合蛋白）的亲和力结合高于1XrVP28。综上所述，应用多聚体rVP28可提高虾对WSSV的保护作用。
