乙型副伤寒多糖蛋白结合疫苗培养工艺及脂多糖提取工艺摸索及优化

The Exploration and Optimization of Paratyphoid B Glycoprotein Conjugate Vaccine Cultured Process and the Extraction Process of LPS

对乙型副伤寒多糖蛋白结合疫苗的细菌培养工艺及脂多糖提取工艺进行摸索及优化。根据细菌培养液pH值的变化情况，采用间歇式流加补加葡萄糖代替连续流加补加葡萄糖的方式，利用10L和100L半自动生物反应器对乙型副伤寒沙门氏菌进行培养，同时对细菌灭活工艺改进及优化；在乙型副伤寒脂多糖提取过程中，探讨了热酚法提取脂多糖菌悬液制备方法、苯酚终浓度和苯酚水混合溶液的pH值及25％乙醇沉淀时所加入无水NaAc浓度等工艺提取条件，对脂多糖的收率、核酸含量、蛋白含量的影响。结果：采用间歇式流加葡萄糖代替连续流加的方式，对乙型副伤寒沙门氏菌进行培养，时间可缩短3～4h，菌体收量最高；改变细菌灭活时间1h为6h的方法，可以降低脂多糖的核酸含量到14％；采用研磨菌体制备菌悬液、乙型副伤寒脂多糖热酚法使用的苯酚浓度从90％提高到95％、菌悬液和苯酚混合溶液的pH值控制在6．8，25％乙醇沉淀时加入无水NaAe终浓度为0．28mol／L，脂多糖收率能够达到6．17％，核酸含量可控制在12％以下，蛋白含量可控制在1．2％以下。建立了稳定的乙型副伤寒多糖结合疫苗细菌培养及脂多糖提取工艺。

The bacterial culture and fat polysaccharide extraction process for paratyphoid B glycoprotein conjugate vaccine was explored and optimized. Based on the bacterial culture of changes pH value, and the batch fed with glucose instead of continuous flow added with glucose, Salmonella paratyphi B was cultivated with 10 L and 100 L semi-automatic bioreactor, at the same time conduc- ting bacteria inactivated process improvement and optimization; In paratyphi B fat polysaccharide extraction process, studying hot phenol method and preparing lipopolysaccharide bacteria suspension, pH value of mixed solution of phenol concetration and phenol water, the concentration of anhydrous NaAc when adding 25% ethamol procipitation, the influence on yield of lipopolysaccharide, the nucleic acid content and protein content. Results showed that using intermittent flow plus glucose instead of continuous flow and the way, to cultivate of salmonella paratyphi B, the time can be shortened 3 - 4 h, yield the highest biomass ; Changing the inactivated bacteria for 1 h to 6 h method, can reduce fat polysaccharide nucleic acid content to 14% ;Using milling bacteria suspension system for bacteria and paratyphi B paratyphoid lipopolysaccharide hot phenol method using phenol concentration from 90% to 95% ,the bacterial suspension and phenol mixture of pH control in 6.8,25% ethanol precipitation with anhydrous NaAc concentration is 0.28 mol/L, lipopolysaccharide yield can reach 6. 17%, the nucleic acid content can be controlled under 12% and protein content can be controlled below 1.2%. The stable paratyphi B polysaccharide conjugate vaccine bacterial culture and fat polysaccharide extraction process was established.