猪瘟活疫苗效力检验替代方法的研究

摘要

目前，我国猪瘟活疫苗效力检验最常用的方法是测定猪瘟疫苗中病毒的兔体感染量（ RID），但是用该方法易受到检验兔品种、个体和饲养环境的影响。为了探索一种不依赖动物的疫苗效力检验新方法，本研究将待检猪瘟活疫苗系列稀释后分别接种易感细胞，使用抗原捕获ELISA、RT－nestPCR和RT－PCR三种方法检测不同稀释度疫苗中活病毒在感染细胞后增殖的子代病毒，计算疫苗病毒的最高细胞感染剂量，建立了猪瘟疫苗病毒的细胞感染量（ CID）的效力检验方法。结果显示：疫苗中活病毒粒子越多，CID就越高；对不同类型猪瘟疫苗的检测结果表明，该方法适用于传代细胞源和细胞源猪瘟活疫苗的效力效检。使用CID和RID两种检测方法同时对12批猪睾丸传代细胞源（传代细胞源）和3批牛睾丸原代细胞源（细胞源）猪瘟活疫苗进行了比对效检，结果表明，用CID测定方法检验，12批猪瘟活疫苗（传代细胞源）每头份均含105 CID，3批猪瘟活疫苗（细胞源）每头份含量均为104 CID；用兔子感染体温测定法，12批猪瘟活疫苗（传代细胞源）每头份含量在2．6×104～3．0×104 RID之间，3批猪瘟活疫苗（细胞源）每头份含7．0×103～8．0×103 RID。试验证明：本实验室建立的CID检测结果与现有质量标准RID的结果存在良好的相关性，能够较好反映疫苗中活病毒的含量，有望成为RID的替代方法。%The potency detection method of C-strain vaccine based on classical swine fever virus rabbit infection dose( RID) is widely used in China, however, this method is greatly influenced by bread,individual difference and housing environment. In order to establish an alternative potency test strategy avoid using experimental animals, the permissive cell was inoculated with serial diluted vaccine, and then the antigen-capture ELISA, RT-nestPCR and traditional RT-PCR were utilized to detect the progeny virus. The highest positive dilution was termed as the indicator of the vaccine’ s potency. The method of cell infection dose( CID) was established. The results showed that the higher live virus particles in vaccine, the higher dose of cell infection virus. Results from the potency tests of different types of C-strain vaccines indicated that the alternative strategy could be applied to potency test of ST cell-line produced C-strain vaccine and bovine testicular cell produced C-strain vaccine. Potency tests of 12 batches of ST cell-line produced C-strain vaccines and 3 batches of bovine testicular cell produced C-strain vaccines were conducted with both the alternative strategy CID and the RID test parallel. The results showed, the positive dilution of all 12 batches of ST cell-line produced C-strain vaccine were 105 CID/dose, and 2.6×104~3.0×104RID/dose. And the positive dilution of 3 batches of bovine testicular cell produced C-strain vaccine were 104 CID/dose and 7.0×103~8.0×103RID/dose. The potency test results of the alternative strategy CID were at certain extent corresponding to that of RID test, and it could reflect the concentration of infectious virus particles in vaccine. The alternative strategy CID is of potentiality to replace the RID test.