Objective To explore the possibility of recombinant porcine trypsin replacing porcine trypsin in the production of freeze-dried human rabies vaccine(hamster kidney cells).Methods SPF-grade hamster kidneys were taken aseptical-ly,and primary hamster kidney(PHK)cells were digested with four different concentrations of recombinant porcine trypsin(0.05,0.10,0.15 and 0.20 mg/mL).The total number of cells,cell viability and cell morphology after 6 days of culture were compared to screen the optimal working concentration of recombinant porcine trypsin.The digestion effects of digestive solution A(0.30％sodium citrate solution containing 0.30 mg/mL recombinant trypsin)and digestive solution B(0.30％sodium citrate solution containing 0.15 mg/mL recombinant trypsin)were compared to select the appropriate digestive solu-tion.Finally,primary PHK cells were digested by different types of trypsin,inoculated with rabies virus aG strain to deter-mine the optimal multiplicity of infection(MOI),and virus titers were compared to evaluated the effects of digestion rea-gents.Results Digestive solution A was more suitable for the digestion of PHK cells,and the optimal concentration was 0.10 mg/mL.A dense monolayer was formed after 6 days of cell culture.The optimal MOI range was 0.10-0.50,under the con-firmed conditions above,the experiments had high repeatability.There was no significant change in cellular pathology detected through microscope.Furthermore,the cells digested by recombi-nant porcine trypsin shed less and the virus harvest liquid was more clear,than those digested by porcine trypsin.In terms of virus titers,there was no significant difference between the two sourses of trypsin digestion(P=0.630,P＞0.05).Conclusion Recombinant porcine trypsin can replace porcine tryp-sin in the preparation of freeze-dried human rabies vaccine(hamster kidney cells),which lays a foundation for the improve-ment of the production process of freeze-dried human rabies vaccine(hamster kidney cells).
Recombinant porcine trypsinPrimary hamster kidney cellRabies virusDigestive solutionPreparation of vaccine
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