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
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摘要
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。
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 p H 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 conducting bacteria inactivated process improvement and optimization; In paratyphi B fat polysaccharide extraction process,studying hot phenol method and preparing lipopolysaccharide bacteria suspension,p H value of mixed solution of phenol concetration and phenol water,the concentration of anhydrous Na Ac 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 p H control in 6. 8,25% ethanol precipitation with anhydrous Na Ac 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.
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 p H 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 conducting bacteria inactivated process improvement and optimization; In paratyphi B fat polysaccharide extraction process,studying hot phenol method and preparing lipopolysaccharide bacteria suspension,p H value of mixed solution of phenol concetration and phenol water,the concentration of anhydrous Na Ac 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 p H control in 6. 8,25% ethanol precipitation with anhydrous Na Ac 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.
引文
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[12]邢宇,张丽叶.基于p H的反馈补料方法在谷氨酸发酵中的应用[J].生物工程学报,2011,27(10):1457-1463.
[2]王鲁茜,阚飙.伤寒、副伤寒的全球流行概况及其预防控制[J].疾病监测,2007,22(7):492-494.
[3]高金霞,张静.2004~2007年全国伤寒副伤寒暴发疫情分析[J].现代预防医学,2010,37(23):4401-4404.
[4]阳波,闫梅英,阚飙,等.伤寒副伤寒传播的风险因素及防控措施[J].疾病监测,2012,(6):492-496.
[5]兰惠琴,罗军.甲型副伤寒疫苗研究进展[J].国际生物制品学杂志,2009,32(4):180-183.
[6]Konadu E,Robbins JB,Szu SC,et al.Phase 1 and Phase 2 studies of Salmonella enteric serovar paratyphi A O-specific polysaccharide-tetanus toxoid conjugates in adults,teenagers,and 2-to 4-year-old children in Vietnam.Infection[J].Immunity,2000,68(3):1529-1534.
[7]康洁.3种方法提取大肠杆菌E.coli脂多糖的比较[J].中国农学通报,2010,26(21):12-15.
[8]刘红亮,陈学忠,李克生,等.肠出血性大肠杆菌O157∶H7脂多糖抗原的提取鉴定及间接ELISA法的建立[J].中国人兽共患病学报,2011,27(7):637-6404.
[9]阳幼荣,吴雪琼,张俊仙,等.结核分枝杆菌脂多糖抗原的提取及血清学诊断价值研究[J].武警医学,2011,22(9),774-775.
[10]鲁学萍,杜惠芬,李克生,等.肠炎沙门菌脂多糖抗原的制备及其生物学特性[J].中国生物制品学杂志,2011,24(7):853-856.
[11]杨成兰,罗薇,刘内生,等.丙型副伤寒沙门氏菌脂多糖提取纯化及活性分析简[J].中国畜牧兽医,2014,41(4):75-78.
[12]邢宇,张丽叶.基于p H的反馈补料方法在谷氨酸发酵中的应用[J].生物工程学报,2011,27(10):1457-1463.