Tang Xiaoqian, Zhai Fude, Sheng Xiuzhen, Xing Jing, Zhan Wenbin
Laboratory of Pathology and Immunology of Aquatic Animals, Ocean University of China, 5 Yushan Road, Qingdao 266003, China.
Laboratory for Marine Fisheries Science and Food Production Processes, Qingdao National Laboratory for Marine Science and Technology, No. 1 Wenhai Road, Aoshanwei Town, Jimo, Qingdao 266071, China.
Int J Mol Sci. 2017 Jul 7;18(7):1465. doi: 10.3390/ijms18071465.
Our previous study demonstrated that an integrin β subunit of Chinese shrimp () (FcβInt) plays an important role in white spot syndrome virus (WSSV) infection. In the present work, in order to further elucidate the potential role of FcβInt in WSSV infection, the recombinant extracellular domain of β integringene of (rFcβInt-ER) was expressed in BL21 (DE3), and the eukaryotic expression plasmid PcDNA3.1-FcβInt-ER (PFcβInt-ER) was also constructed. Far-western blotting was performed to determine the binding specificity of rFcβInt-ER to WSSV envelope proteins, and results showed that rFcβInt-ER was able to specifically interact with rVP31, rVP37, rVP110 and rVP187. Moreover, the blocking effects of mouse anti-rFcβint-ER antibodies were both detected in vivo and in vitro. The ELISA and Dot-blotting in vitro assays both showed that mouse anti-rFcβInt-ER antibodies could partially block the binding of WSSV to the hemocyte membrane of . In the in vivo assays, the mortality of shrimp injected with WSSV mixed with anti-rFcβInt-ER antibodies was delayed, and was lower than in the control group. While the shrimp were intramuscularly injected with PFcβInt-ER, transcripts of PFcβInt-ER could be detected in different shrimp tissues within 7 days, and the mortality of shrimp injected with PFcβInt-ER was also delayed and lower compared with the control group post WSSV challenge. Furthermore, gene silencing technology was also used to verify the effect of FcβInt in WSSV infection, and results showed that the expression levels of the WSSV immediate early gene , early gene , and late gene and the mortality of were all significantly decreased in the FcβInt knock-down hemocyctes compared to the control group. Taken together, these results suggest that FcβInt plays important roles in WSSV infection.
我们之前的研究表明，中国对虾（）的一种整合素β亚基（FcβInt）在白斑综合征病毒（WSSV）感染中起重要作用。在本研究中，为了进一步阐明FcβInt在WSSV感染中的潜在作用，在大肠杆菌BL21（DE3）中表达了重组的中国对虾β整合素基因胞外结构域（rFcβInt-ER），并构建了真核表达质粒PcDNA3.1-FcβInt-ER（PFcβInt-ER）。通过Far-western印迹法确定rFcβInt-ER与WSSV包膜蛋白的结合特异性，结果表明rFcβInt-ER能够与rVP31、rVP37、rVP110和rVP187特异性相互作用。此外，在体内和体外均检测了小鼠抗rFcβint-ER抗体的阻断作用。体外ELISA和斑点印迹分析均表明，小鼠抗rFcβInt-ER抗体可部分阻断WSSV与中国对虾血细胞细胞膜的结合。在体内试验中，注射WSSV与抗rFcβInt-ER抗体混合物的对虾死亡率延迟，且低于对照组。当对虾肌肉注射PFcβInt-ER时，在7天内可在不同对虾组织中检测到PFcβInt-ER的转录本，并且在WSSV攻击后，注射PFcβInt-ER的对虾死亡率也延迟且低于对照组。此外，还利用基因沉默技术验证FcβInt在WSSV感染中的作用，结果表明，与对照组相比，FcβInt敲低的血细胞中WSSV立即早期基因、早期基因和晚期基因的表达水平以及对虾的死亡率均显著降低。综上所述，这些结果表明FcβInt在WSSV感染中起重要作用。
