重组猪胰酶在冻干人用狂犬病疫苗(地鼠肾细胞)中的应用  

Application of recombinant porcine trypsin in freeze-dried rabies vaccine for human use(hamster kidney cells)

作　　者：肖蕾 刘晓巍 张志刚 马树奇 杨俊杰 徐小明 张艳 李红芳 XIAO Lei;LIU Xiaowei;ZHANG Zhigang;MA Shuqi;YANG Junjie;XU Xiaoming;ZHANG Yan;LI Hongfang(The Second Department of Vaccine,Lanzhou Institute of Biological Products Co.,Ltd.,Center for Gansu Provincial Vaccine Engineering Research,Lanzhou 730046,Gansu Province China)

机构地区：[1]兰州生物制品研究所有限责任公司疫苗二室、甘肃省疫苗工程技术研究中心,甘肃兰州730046

出　　处：《微生物学免疫学进展》2024年第1期17-22,共6页Progress In Microbiology and Immunology

摘　　要：目的 探究重组猪胰酶(recombinant porcine trypsin, RPT)代替猪源胰酶在冻干人用狂犬病疫苗(地鼠肾细胞)生产中的可能性。方法 选用SPF级地鼠,无菌取肾,用4种不同浓度(0.05、0.10、0.15和0.20 mg/mL)的重组猪胰酶消化原代地鼠肾(primary hamster kidney, PHK)细胞,比较细胞总数、细胞活率以及培养6 d的细胞形态,筛选出最佳重组猪胰酶的工作浓度;对消化液A(含0.30%柠檬酸钠的0.30 mg/mL重组猪胰蛋白酶溶液)和消化液B(含0.30%柠檬酸钠的0.15 mg/mL重组猪胰蛋白酶溶液)消化效果进行对比,选择合适的消化液;对P1代PHK细胞接种狂犬病病毒aG株,确定最佳病毒感染复数(multiplicity of infection, MOI);对不同胰酶消化的细胞接种狂犬病病毒aG株,并比较两者病毒滴度。结果 重组猪胰酶对原代PHK细胞消化的最佳浓度为0.10 mg/mL,消化液A更适用于P1代PHK细胞的消化,细胞培养6 d后形成致密单层,胞体饱满;最佳MOI范围为0.10~0.50,且试验具有高度重复性;镜下观察细胞病变无明显变化,但是重组猪胰酶消化后的细胞比猪源胰酶脱落较少,肉眼可见病毒收获液更澄清;2种胰酶消化制备的P1代PHK细胞病毒滴度差异无统计学意义(P=0.630,P>0.05)。结论 重组猪胰酶可以替代猪源胰酶用于制备冻干人用狂犬病疫苗(地鼠肾细胞),为冻干人用狂犬病疫苗(地鼠肾细胞)生产工艺的改进奠定基础。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 aseptically, 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 solution. Finally, primary PHK cells were digested by different types of trypsin, inoculated with rabies virus aG strain to determine the optimal multiplicity of infection(MOI),and virus titers were compared to evaluated the effects of digestion reagents. 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 confirmed conditions above, the experiments had high repeatability. There was no significant change in cellular pathology detected through microscope. Furthermore, the cells digested by recombinant 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 trypsin in the preparation of freeze-dried human rabies vaccine(hamster kidney cells),which lays a foundation for the improvement of the production process of freeze-dried human rabies vaccine(hamster kidney cells).

关 键 词：重组猪胰酶 原代地鼠肾细胞 狂犬病病毒 消化液 疫苗制备 

分 类 号：R373.9[医药卫生—病原生物学]