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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p H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10 L和100 L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p H值及25%乙醇沉淀时所加入无水Na Ac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3~4 h,菌体收量最高;改变细菌灭活时间1 h为6 h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p H值控制在6.8,25%乙醇沉淀时加入无水Na Ac终浓度为0.28 mol/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。
Bacterial culture process and lipopolysaccharide extraction process of glycoprotein B conjugate vaccine were explored and optimized. According to the change of p H value in bacterial culture medium, the intermittent flow plus glucose was added instead of continuous flow plus glucose, the Salmonella paratyphi A strain was cultured by 10 L and 100 L semi-automatic bioreactor, Bacterium inactivation process was improved and optimized. During the extraction of lipopolysaccharide from Parain B, the preparation method of lipopolysaccharide suspension was studied by hot phenol method. The final concentration of phenol and p H value of phenol-water mixed solution and the precipitation of 25% ethanol When added Na Na concentration of anhydrous extraction conditions such as technology, the yield of lipopolysaccharide, nucleic acid content, protein content. Results: Using intermittent flow plus glucose instead of continuous flow, Salmonella paratyphi B could be cultured for 3 ~ 4 h with the highest yield of bacteria. The method of changing bacteria inactivation time for 1 h was 6 h , Can reduce the nucleic acid content of lipopolysaccharide to 14%; the bacterial suspension was used to prepare the bacterial suspension, and the phenol concentration of the bacillus parahaemolyticus LPS was increased from 90% to 95%; the bacterial suspension and phenol mixed solution p H value controlled at 6.8,25% ethanol precipitation by adding anhydrous Na Ac final concentration of 0.28 mol / L, the yield of lipopolysaccharide can reach 6.17%, the nucleic acid content can be controlled at 12%, the protein content can be controlled at 1.2% the following. The establishment of a stable B Paratuberculosis conjugate vaccine bacterial culture and lipopolysaccharide extraction process.