大鲵虹彩病毒β-丙内酯灭活方法的研究

为探讨β-丙内酯(β-propiolactone,BPL)灭活大鲵虹彩病毒(Giant salamander iridovirus,GSIV)的最适条件,研究了BPL对GSIV的灭活方法。采用终浓度分别为0.025%、0.05%、0.1%、0.2%的BPL灭活细胞培养的GSIV,4℃条件下分别灭活24 h、48 h、72 h、96 h,通过细菌培养、细胞培养、病毒核酸PCR扩增以及鱼体感染试验进行灭活病毒的安全性检验,确定最适灭活条件。试验结果表明,GSIV经终浓度为0.1%的BPL 4℃灭活处理72 h后可完全灭活病毒,灭活病毒无细菌污染,接种对GSIV敏感的鲤上皮瘤细胞系(EPC)细胞无细胞病变效应(CPE)出现,病毒主衣壳蛋白(MCP)基因特异性引物PCR反应未扩增出靶基因产物,灭活病毒对健康大鲵的感染试验未出现疾病症状。灭活效果检测结果表明,与未灭活GSIV相比较,最适灭活条件下的GSIV结构蛋白与抗原性未发生显著变化。结论显示BPL可用来灭活GSIV,本研究确立了BPL灭活GSIV的最适条件,为大鲵虹彩病毒细胞培养灭活疫苗研究奠定了重要基础。

Studies on the method of inactivating giant salamander iridovirus with β-propiolactone

In order to investigate the optimal condition of inactivating the giant salamander iridovirus(GSIV) with β-propiolactone(BPL),the method of inactivating GSIV with BPL was studied.The GSIV cell cultures was inactivated by BPL with different concentrations(0.025%,0.05%,0.1%,0.2%) for different durations(24 h,48 h,72 h and 96 h) at 4 ℃,respectively.Then the efficacy of inactivation of GSIV was tested by means of bacterial culture,cell culture,viral DNA PCR amplification and artificial infection of healthy fish to determine the optimal condition of inactivation.The results showed that the GSIV were inactivated completely with the final concentration of 0.1% of BPL for 72 hours at 4 ℃.There were no bacteria and no cytopathic effect(CPE) observed both in bacteria and cell culture.The PCR amplification with specific primer pair targeted the viral major capsid protein(MCP) generated no product.The artificial infection test in healthy giant salamanders did not induce the clinical signs of the disease.The results demonstrated that the structure protein and the antigenicity of the inactivated GSIV treated under the optimal condition had not changed significantly.This study established the optimal condition of GSIV inactivated by BPL and laid an important foundation for the further preparation of GSIV inactivated vaccine.