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百日咳粘附素(PRN)是无细胞百日咳疫苗的重要保护性抗原之一,优化其浸提工艺对提高PRN产量、纯度乃至百日咳疫苗的质量都具有重要意义.通过中心复合表面设计(CCF)响应面模型和ELISA定量分析相结合的方法研究了影响PRN浸提工艺的关键因素.经两轮CCF响应面优化,确定了PRN浸提的最优工艺参数:温度60℃,菌浓度50 mg/mL,时间90 min,p H 8.5,NaCl 0.15 mol/L;通过Monte Carlo模拟和Plackett Burman模型设计预测和验证了工艺稳健性参数范围:温度58-62℃,菌浓度40-60 mg/mL,时间80-100 min,pH 8.45-8.55,NaCl 0.12-0.18 mol/L.响应面优化后,粗提液PRN产量提高了50%.研究表明,温度对PRN浸提影响最大,其次是pH;菌浓度、时间及NaCl含量的影响相对较小,但与温度、pH有交互作用.图6表3参13
Pertussis adhesin (PRN) is one of the most important protective antigens for acellular pertussis vaccine, and its optimization of its extraction process is of great importance to improve the yield and purity of PRN and even the quality of pertussis vaccine.Central composite surface design (CCF) response Surface model and ELISA quantitative analysis were used to study the key factors influencing the leaching process of PRN.After two rounds of CCF response surface optimization, the optimal technological parameters of PRN leaching were determined: the temperature was 60 ℃, the concentration of bacteria was 50 mg / mL , Time 90 min, p H 8.5, NaCl 0.15 mol / L. The process robustness parameters were predicted and validated by Monte Carlo simulation and Plackett Burman model. The parameters ranged from 58-62 ℃, the concentration of bacteria was 40-60 mg / mL, 80-100 min, pH 8.45-8.55, NaCl 0.12-0.18 mol / L. After optimizing the response surface, the yield of PRN in crude extract increased by 50% .The results showed that temperature had the greatest effect on PRN extraction, followed by pH, , The influence of time and NaCl content is relatively small, but it has interaction with temperature and pH.