| 草鱼(Ctenopharyngodon idellus)出血病二价细胞灭活疫苗体液免疫效果及免疫保护率评价研究 |
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| 引用本文： | 郝贵杰,林锋,黄爱霞,叶霆,姚嘉赟,潘晓艺,袁雪梅,徐磊,胡金春,沈锦玉.草鱼(Ctenopharyngodon idellus)出血病二价细胞灭活疫苗体液免疫效果及免疫保护率评价研究[J].海洋与湖沼,2022,53(6):1467-1475. |
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| 作者姓名： | 郝贵杰 林锋 黄爱霞 叶霆 姚嘉赟 潘晓艺 袁雪梅 徐磊 胡金春 沈锦玉 |
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| 作者单位： | 农业农村部淡水渔业健康养殖重点实验室 浙江省鱼类健康与营养重点实验室 浙江省淡水水产研究所 浙江湖州 313001;衢州市水产技术推广中心 浙江衢州 324000 |
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| 基金项目： | 蓝色粮仓科技创新项目,2019YFD0900104号;浙江省公益技术研究农业项目,2016C32076号。 |
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| 摘 要： | 草鱼出血病是由草鱼呼肠孤病毒(GCRV)引起的草鱼养殖业一种严重的病毒性出血病,给草鱼养殖产业造成重大损失。该病毒是一种双链分节段RNA病毒,根据GCRV多个病毒分离株基因序列分析及临床发病特点,共有三种基因型GCRV,其中基因Ⅰ型和Ⅱ型较为流行。通过细胞分离培养基因Ⅰ型(GCRV-ZV8909)和Ⅱ型(GCRV-HZ13) GCRV,研制出草鱼出血病二价细胞灭活疫苗,通过绝对定量的方法,测定基因Ⅰ型和Ⅱ型的病毒含量分别为2.0×109及1.5×106 copies/mL。用生理盐水将其分别进行50倍稀释和100倍稀释,包括原液及生理盐水对照共4组,分别腹腔注射免疫(20±5)g健康草鱼,剂量均为0.2mL/尾,每组40尾,各组于免疫后3、5、7、14、21、28、42、56、70、84 d分别取3尾采集血液制备血清。分别利用经原核表达并纯化制备两种基因型GCRV的VP7蛋白以及实验室保存的草鱼IgM单抗,建立了三抗体夹心酶联免疫方法(TAS-ELISA)分析评价疫苗体液免疫效果。结果表明,草鱼经腹腔注射不同剂量的细胞灭活疫苗后,与对照组相比,各免疫组血清抗体均有不同程度的提高,且原液组与50倍稀释组基本持平,但都高于100倍稀释组,各免疫组在免疫后5d即可检测到两种基因型GCRV抗体,且抗体水平持续到84d,但两种基因型抗体水平达到高峰的时间不一致,抗基因Ⅰ型GCRV抗体达到高峰是在28d,抗基因Ⅱ型GCRV抗体是在14d达到高峰。两种基因型抗体水平达到高峰时的血清效价分别为1︰800、1︰600。免疫保护率实验结果表明,原液组保护率最高,为67%,50倍稀释组为60%,100倍稀释组只有33%。
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| 关 键 词： | 草鱼呼肠孤病毒(GCRV) 三抗体夹心酶联免疫方法(TAS-ELISA) 体液免疫 免疫保护率 |
| 收稿时间： | 2022/3/31 0:00:00 |
| 修稿时间： | 2022/6/10 0:00:00 |
EVALUATION OF HUMORAL IMMUNE EFFICACY AND PROTECTION RATE OF BIVALENT CELL INACTIVATED VACCINE AGAINST GRASS CARP HEMORRHAGIC DISEASE |
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| HAO Gui-Jie,LIN Feng,HUANG Ai-Xi,YE Ting,YAO Jia-Yun,PAN Xiao-Yi,YUAN Xue-Mei,XU Lei,HU Jin-Chun,SHEN Jin-Yu.EVALUATION OF HUMORAL IMMUNE EFFICACY AND PROTECTION RATE OF BIVALENT CELL INACTIVATED VACCINE AGAINST GRASS CARP HEMORRHAGIC DISEASE[J].Oceanologia Et Limnologia Sinica,2022,53(6):1467-1475. |
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| Authors: | HAO Gui-Jie LIN Feng HUANG Ai-Xi YE Ting YAO Jia-Yun PAN Xiao-Yi YUAN Xue-Mei XU Lei HU Jin-Chun SHEN Jin-Yu |
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| Institution: | Key Laboratory of Healthy Freshwater Fisheries Aquaculture, Ministry of Agriculture and Rural Affairs, Key Laboratory of Fish Health and Nutrition of Zhejiang Province, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China;Quzhou Aquatic Technology Extension Center, Quzhou 324000, China |
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| Abstract: | Grass Carp haemorrhagic disease is a serious viral haemorrhagic disease causing great threat to Grass Carp culture. The pathogen of gohemorrhage of Grass Carp is Grass Carp reovirus (GCRV), which is a double-stranded partited RNA virus. According to the characteristics of gene sequence of GCRV isolates, there are three GCRV genotypes in clinical practice, among which genotype I and II are more popular. In this study, bivalent cell inactivated vaccine of grass carp hemorrhagic disease was developed by cell isolation and culture of type I (GCRV-ZV8909) and type II (GCRV-HZ13) GCRV. The contents of genotype I and II were 2.0×109 and 1.5×106Scopies/mL, respectively. They were 50 times diluted and 100 times diluted with normal saline, including the original solution and normal saline control, the experiment was divided into four groups. There are 40 healthy grass carps with 20g±5g/tail in each group, they were intraperitoneally injected and the dose was 0.2 mL/tail. Three samples of blood were collected from each group at 3d, 5d, 7d, 14d, 21d, 28d, 42d, 56d, 70d and 84d respectively to prepare serum. The VP7 protein of GCRV were expressed in prokaryotic expression and purified respectively. Three antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) was established to evaluate the humoral immune effect of the vaccine combined grass carp IgM monoclonal antibody. Results show that the serum antibody of grass carp after injecting of different doses of cells inactivated vaccine has varying degrees of increase compared with the control group. The original solution group was the same as the 50 times diluted group, but is higher than 100 times dilution group. The two genotypes of GCRV antibodies could be detected 5 days after immunization in each immune group, and the antibody level lasted until 84d, but the peak time of the two genotypes of GCRV antibodies was not consistent, the peak time of the antibody against genotype I GCRV was 28d, and the peak time of the antibody against genetype II GCRV was 14d. The serum titers of the antibody levels peaked at 1:800 and 1:600 for the two genotypes, respectively. The results of immune protection rate experiment showed that the original solution group had the highest protection rate of 67%, 50 times dilution group was 60%, 100 times dilution group only 33%. |
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| Keywords: | grass carp reovirus (GCRV) triple antibody sandwich enzyme-linked immunoassay (TAS-ELISA) humoral immunity immune protection rate |
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