Beagle狗肾细胞及用于制备乙脑活疫苗的研究

Study on Production Technology of Live Japanese Encephalitis Vaccine Using Beagle Dog Kidney Cells as a Substrate

Ｂｅａｇｌｅ乳狗（５－１０日龄）肾块用热消化法制成细胞批放液氮保存，检定合格后做下列试验：（１）复苏培养长满单层后更换维持液，其细胞能维持４天以上，克服了常规消化培养细胞维持时间短（３６小时）的缺陷。（２）传代１４－２株病毒时，１代病毒的滴度即达６．０４－６．２４ＬｏｇＰＦＵ／ｍｌ，１代后的各代（至１１代）病毒滴度无明显提高。（３）该细胞接种１４－２株病毒时不产生明显的破坏性病变（ＣＰＥ），在收毒前采取冻溶１次比冻溶前的病毒滴度提高０．２７－０．５ＬｏｇＰＦＵ／ｍｌ。以ＢＤＫ细胞传１代的１４－２株病毒作生产毒种，收毒前冻溶１次等工艺法制备五批疫苗，全面检定结果均符合《乙脑活疫苗规程》的质量标准。其中病毒滴度为６．０９－６．２４ＬｏｇＰＦＵ／ｍｌ；免疫效价（ＩＤ５０＝ｍｌ）为１．９－４．０×１０－５，与相同滴度的原毒株１４－２ＰＨＫ苗对照无明显差异（ｔ＝０．９６８Ｐ＞０．０５），表明其免疫原性与原毒株相似。

Kidneys obtained from Beagle dogs of 5-10 days old were prepared by trypsinization at 37℃ into cell suspentions,and the cell lots were stored in liquid nitrogen.The qualified cells were used as follows.1.After the thawed cells were cultivated into confluent monolayer cell cultres,the growth medium was replaced with serum-free maintenance medium,in which the cells could retain growth excellence for at least 4 days.This was advantageous to overcoming the shortcoming of cell maintenance period too short (36h) in cells trypsinized in the usual manner.2.When the strain 14-2 virus was serially undergone in the cells,the virus titers of the strain reached 6.04-6.24 LogPFU/ml in the first passage,and showed no significant increase in the further passages(to passage 11).3.The strain 14-2 showed no apparent CPE in the cells.When the cell cultures were treated one cycle of freezing and thrawing before harvesting of virus,the virus titers increased by 0.27-0.50 LogPFU/ml over the non-treatment. 5 lots of the freeze-dried live JE vaccine were produced using the strain 14-2 virus at BDK passage 1 as a working seed and adopting the technological process of freezing and thawing one times before virus harvest.Complete characterization and control test proved that the quality of vaccine reached the live JE vaccine requirements.Among tests,the virus titers were 6.09-6.24 LogPFU/ml,and immunizing potency(ID50/ml)Were 1.90x10 -5 -4.0x10 -5 ,which showed no significant difference compared with primary strain 14-2 PHK at the identical titer.This suggested the immunogenicity is similar to that of the primary strain.