摘要
罗氏沼虾肌肉白浊病(whitish muscle disease of Macrobrachium rosenbergii)是一种发生在罗氏沼虾苗种阶段的流行病,发病虾苗出现肌肉白浊、白斑或白尾症状,死亡率高达60%以上。作者所在实验室在确定其病原是罗氏沼虾诺达病毒(Macrobrachium rosenbergii Nodavirus,MrNV)的基础上,用MrNV免疫BALB/c小鼠,取小鼠脾细胞与SP2/0鼠骨髓瘤细胞融合,用间接ELISA筛选阳性孔,经有限稀释法克隆,得到12株能特异分泌抗MrNV的单克隆抗体(Mab)的杂交瘤细胞。注射小鼠,制备腹水单克隆抗体,ELISA效价为1∶105～106。亚型鉴定结果表明,有6株单抗为IgG1型,4株单抗为IgG2a型,2株单抗为IgG2b型。12株单抗与对虾白斑综合征病毒(WSSV)和桃拉综合征病毒(TSV)均无交叉反应。挑选效价高的腹水单抗2B5,建立了检测罗氏沼虾诺达病毒的三抗体夹心酶联免疫吸附测定(TAS-ELISA)法,该方法检测灵敏度达0.98ng左右。Wesrternblot分析表明,2B5能与MrNV43kD的外膜蛋白特异性结合。
Whitish muscle diseases of freshwater giant prawn, Macrobrachium rosenbergii, is a new epidemic disease in the larvae and post-larvae of M. Rosenbergii in recent years. The clinical sign of the diseam is a whitish or opacity appearance of the muscles in abdomen. Mortalities may reach more than 60%, sometimes even reach 100%. The pathogen of the disease is confirmed as Macrobrachium rosenbergii Nodavirus（MrNV）, an icosahedral and non-enveloped virus sized from 25 - 26 nm with two linear ssRNA sized 3.0 kb and 1.3 kb respectively. MrNV was the first nodavirus founded in crustacean. To develop a rapid method for MrNV detection, monoclonal antibody against MrNV was prepared and used for virus detection. Briefly, mouse myeloma .cells（SP2/0） were fused with spleen cells from BALB/c immunized with purified MrNV from diseased postlarvae（PL）. Positive colonies were screened by indirect ELISA with the plates coated with purified MrNV and cloned 2 - 3 times with limited dilution method. Twelve positive hybridoma cell lines secreting monoclonal antibodies （Mabs） against MrNV were at last obtained and used for ascites preparation. The titres of positive ascites Mabs were ranged from 1 ： 105 to 1 ： 106 by indirect ELISA. These twelve Mabs were isotyped by Southern Biotechnology clonotyping system, and the results showed that six Mabs were IgG1, four IgG2a and two IgG2b, respectively. No cross reactions, were found when the twelve Mabs were used for indirect ELISA with white spot syndrome virus （WSSV） and Taura syndrome virus（TSV）. A triple antibody sandwich enzyme-linked immunosorbent assay（TAS-ELISA） was developed with the plates pre-coated with rabbit polyclonal antibodies against MrNV for virus capturing, followed by MrNV and samples incubaticn, Mabs and goat-anti-mouse-IgG antibodies labeled with HRP. Mab 2B5, characterized as Mab against 43 kD coat protein of MrNV by Western blot, was selected for TAS-ELISA. The results showed that TAS-ELISA can be used successfully for minimum virus detection with the sensitivity of 0.98 ng MrNV, or with the viral titer of 1：40960 when typical diseased PL homogenization supematants were used for TAS-ELISA. TAS-ELISA could be considered as a useful technique for MrNV detection as well as for epidemiological studies for its rapid and inexpensive cost with high specificity and sensitivity.
出处
《水产学报》
CAS
CSCD
北大核心
2005年第4期529-533,共5页
Journal of Fisheries of China
基金
中国水产科学研究院科研基金(2003-4-4)
浙江省自然科学基金(M303493)
