【作者】 沈锦玉； 尹文林； 潘晓艺； 郝贵杰； 徐洋； 何天雷； 王友慧； 叶金云；

【Author】 SHENG Jinyu1, YIN Wenlin1, PAN Xiaoyi1, HAO Guijie1, XU Yang1, HE Tianlei2, WANG Youhui1, YE Jinyun1 (Zhejiang Institute of Freshwater Fisheries,Huzhou 313001, China)

【机构】 浙江省淡水水产研究所； 浙江金大地龟鳖养殖有限公司；

【摘要】 以纯化的中华鳖血清中免疫球蛋白IgM为免疫原,利用淋巴细胞杂交瘤技术,共制备了3株针对中华鳖免疫球蛋白的特异性单克隆抗体,并建立了由单抗3H3介导的三抗体夹心ELISA(TAS-ELISA)方法进行疫苗免疫效果的评价。将分离自中华鳖主要细菌性疾病如肠道出血性败血症、鳖穿孔病(疖疮病)、红脖子红底板病、鳖腐皮病的四株病原菌,在3.5%福尔马林溶液中24h全部灭活;经灭活的四株菌加蜂胶作为佐剂,制成总含菌量为6×109个/mL的四价菌苗。小批量免疫50只体重约100～150g/只的健康中华鳖,后肢肌肉注射,免疫剂量为0.2mL/只;并设置对照组,后肢肌肉注射等量的生理盐水,共接种20只健康中华鳖。分别于免疫前及免疫后10d、20d、30d、40d、50d、60d、80d和100d时,随机采集3只受免中华鳖和1只对照组中华鳖的血液并制备血清,用所建立的单抗介导的TAS-ELISA方法测定血清中抗体ELISA效价。结果表明:当中华鳖免疫后40d,血清中抗体ELISA效价达到最高,为1:1.28×104。免疫后50d将受免中华鳖分成四组,分别用四种菌株以5LD50(9×106个/mL)攻击,0.5mL/只,进行攻毒实验,免疫保护率均达到100%,而对照组死亡率均为100%。这些结果表明,用蜂胶作为免疫佐剂进行鳖用四价疫苗的免疫,取得了较好的免疫效果。同时用上述制备的鳖用四价疫苗开展鳖生产性免疫预防试验,试验表明鳖存活率有较大提高,能有效地防治养殖鳖主要病害的发生。更多还原

【Abstract】 The IgM in serum of turtles was purified to prepare antigen for the immunizing of 8-week-old female BALB/c mice. Spleen cells collected from immunized mice after the forth immunization were fused with SP2/0 myeloma cells. Indirect Enzyme-Linked Immunosorbent Assay ( ELISA) was used to screen hybridoma cells and limited dilution method was performed to subclone the positive clones. After three cycles of subcloning, three McAbs against the IgM were selected, and were designated as 4F2,3H3 and 3B7 respectively. The three McAbs in culture liquid were proved to have high ELISA titers between 1:3200-1:6400. The 3H3 was chosed to prepare ascites and the ELISA titer was 1:512000. A triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) was developed with the plates pre-coated with the bacteria vaccine for combining the serum of immunized turtles, followed by McAb(3H3) and goat-anti-mouse-IgG antibodies labeled with HRP. Four strains of Aeromonas sobria, isolated mainly from bacterial diseases of cultivated soft-shelled turtle, were inactivated with 3.5% formalin for 24 hours. Propolis adjuvant was chosen as immune adjuvant of four inactivated bacteria vaccines above with concentration 6×109cfu/ml.The fifty healthy turtles were immunized with the vaccines above by intramuscular injection in hind leg. The immunizing dose was 0.2mL per turtle. The blank group turtles were injected with the same dose with saline water. The immune sera of three turtles were collected on 10-100 days after immunization. The ELISA titers of the multi-clone antibody against the vaccines were evaluated TAS-ELISA above. The results showed that the ELISA titer was 1:1.28×104 reaching a peak on the 40th day after immunization. The Relative Survival Percent (RSP, %) of turtles were 100% on the 50th day after immunization. The vaccination tests and field prevention trials against bacterial diseases of soft-shelled turtle(Trionyx sinensis)were carried out in several turtle farms from 2005-2006. The results showed the vaccine had a good protection and a practical prospect.更多还原