不同胰蛋白酶在制备腮腺炎减毒活疫苗中的效果比较

Comparison of the effects of different trypsin on the preparation of live attenuated mumps vaccine

目的比较基因重组胰蛋白酶和猪源胰蛋白酶在腮腺炎减毒活疫苗中的使用效果。方法使用浓度为10%、15%、20%基因重组胰蛋白酶制备鸡胚成纤维细胞,通过细胞密度、细胞活性、24 h以及48 h细胞贴壁状态等指标确定基因重组胰蛋白酶使用浓度;与猪源胰蛋白酶进行比较,通过细胞密度、细胞活性、48 h细胞贴壁状态以及病毒滴度等指标比较两种胰蛋白酶的使用效果;使用基因重组胰蛋白酶制备3批腮腺炎减毒活疫苗,检定疫苗的稳定性;规模化放大验证该工艺的稳定性。结果通过研究发现基因重组胰蛋白酶的使用浓度为15%;与猪源胰蛋白酶进行比较,两种胰酶制备的细胞密度水平一致,无显著性差异(P>0.05),48 h后细胞均生长至单层,细胞状态良好,按照同一MOI进行接毒,病毒滴度无显著性差异(P>0.05);使用CF-10进行试验,病毒滴度也无差异(P>0.05);连续3批使用基因重组胰蛋白酶制备的疫苗热稳定性良好,且该工艺可以规模化放大。结论在制备腮腺炎减毒活疫苗中可以使用浓度15%的基因重组胰蛋白酶替代猪源胰蛋白酶,作为鸡胚成纤维细胞消化液使用。

Objective To compare the efficacy of recombinant trypsin and porcine trypsin in live attenuated mumps vaccine.Methods Chicken embryo cells were prepared by using 10%,15%,20%recombinant trypsin,and the concentration of recombinant trypsin was determined by cell density,cell viability,24 h and 48 h cell adhesion.The effects of the two trypsins were compared by cell density,cell activity,48 h cell adherence status and virus titer.Three batches of live attenuated mumps vaccine were prepared with recombinant trypsin,and the stability of the vaccine was tested.The stability of the process was verified by large-scale amplification.Results The concentration of recombinant trypsin was 15%.The results showed that the density levels of the cells prepared by the two trypsins were the same with no significant difference(P>0.05).After 48 h,the cells grew to monolayer,and the cells were in good condition.After being infected according to the same MOI,there was no significant difference in disease progression and virus titer(P>0.05).There was no difference in virus titer when CF-10 was used for process amplification(P>0.05).The vaccine prepared by using recombinant trypsin in three consecutive batches possessed good thermal stability,and the process could be scaled up.