流感病毒超滤工艺的优化
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摘要
目的分析截留相对分子质量不同的超滤膜浓缩对流感病毒纯化效果的影响,以获得杂质含量低的高浓度病毒液。方法将H1N1、H3N2、Bv和By 4个型别流感病毒分别接种9~11日龄鸡胚,经(34±1)℃培养72 h后,收获尿囊液,经连续流离心获得澄清的病毒液后,再进行截留相对分子质量1 000 000、300 000的超滤膜及其两种超滤膜组合的超滤浓缩,测定超滤前后的总蛋白含量、卵清蛋白含量和血凝滴度。结果截留相对分子质量1 000 000的超滤膜超滤后,杂质去除率高,均在80%以上,截留相对分子质量300 000的超滤膜超滤后杂质去除率相对低,但后者病毒回收率较高,二者组合既能降低杂蛋白含量,又可提高病毒回收率。结论流感病毒鸡胚尿囊液采用截留相对分子质量1 000 000+300 000超滤膜组合超滤浓缩,病毒纯化效果较好。
Objective To analyze the effect of concentration by ultrafiltration membrane with various relative molecular mass cutoffs on purification of influenza virus. Methods Four types of influenza viruses(H1 N1,H3 N2,Bv and By)were inoculated into 9-to 11-day-old chick embryo respectively,and cultured at(34 ± 1)℃ for 72 h. The allantoic fluid was harvested,clarified by continuous flow centrifugation,and concentrated by using the ultrafiltration membranes with relative molecular mass cutoffs of 1 000 000 and 300 000 alone or in combination. The total protein and ovalbumin contents and hemagglutination titer before and after ultrafiltration were determined. Results The removal efficiency of foreign matters by ultrafiltration using membrane with relative molecular mass cutoff of 1 000 000 was more than 80%.However,the removal efficiency by the membrane with relative molecular mass cutoff of 300 000 was relatively low,while the virus recovery rate was relatively high. The combination of two methods decreased the foreign protein content and increased the virus recovery rate. Conclusion Influenza virus was effectively purified by concentration of chick embryo allantoic fluid by the combination of ultrafiltration membranes with relative molecular mass cutoffs of 1 000 000 and 300 000.
Objective To analyze the effect of concentration by ultrafiltration membrane with various relative molecular mass cutoffs on purification of influenza virus. Methods Four types of influenza viruses(H1 N1,H3 N2,Bv and By)were inoculated into 9-to 11-day-old chick embryo respectively,and cultured at(34 ± 1)℃ for 72 h. The allantoic fluid was harvested,clarified by continuous flow centrifugation,and concentrated by using the ultrafiltration membranes with relative molecular mass cutoffs of 1 000 000 and 300 000 alone or in combination. The total protein and ovalbumin contents and hemagglutination titer before and after ultrafiltration were determined. Results The removal efficiency of foreign matters by ultrafiltration using membrane with relative molecular mass cutoff of 1 000 000 was more than 80%.However,the removal efficiency by the membrane with relative molecular mass cutoff of 300 000 was relatively low,while the virus recovery rate was relatively high. The combination of two methods decreased the foreign protein content and increased the virus recovery rate. Conclusion Influenza virus was effectively purified by concentration of chick embryo allantoic fluid by the combination of ultrafiltration membranes with relative molecular mass cutoffs of 1 000 000 and 300 000.
引文
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[14]ZHANG Y,WU P,FENG L,et al.Influenza vaccine effectiveness against influenza-associated hospitalization in 2015/16season,Beijing,China[J].Vaccine,2017,35(23):3129-3134.
[15]MARTINEZ-BAZ I,CASADO I,NAVASCUES A,et al.Effect of repeated vaccination with the same vaccine component against 2009 pandemic influenza A(H1N1)virus[J].J Infect Dis,2017,215(6):847-855.
[2]ZHANG Y L,ZHANG H.Vaccinology[M].Beijing:Science Press,2006:1171-1185.(in Chinese)张延龄,张晖.疫苗学[M].北京:科学出版社,2006:1171-1185.
[3]GUO F,TAO R,SHEN Y S,et al.Study of the method of isolated and identified of influenza virus[J].Int J Lab Med,2010,31(4):409-411.(in Chinese)郭芳,陶瑞,沈云松.流感病毒分离鉴定方法研究[J].国际检验医学杂志,2010,31(4):409-411.
[4]ZHU L P,YAN S G.Large scale concentration technology of the microorganism[J].Chin J Microecol,2007,19(2):227-228.(in Chinese)朱丽萍,颜世敢.微生物的规模化浓缩技术[J].中国微生态学杂志,2007,19(2):227-228.
[5]ASANZHANOVA N N,RYSKELDINOVA S Z,CHERVYA-KOVA O V,et al.Comparison of different methods of purification and concentration in production of influenza vaccine[J].Bull Exp Biol Med,2017,164(2):229-232.
[6]TSENG Y F,WENG T C,LAI C C,et al.A fast and efficient purification platform for cell-based influenza viruses by flowthrough chromatography[J].Vaccine,2018,36(22):3146-3152.
[7]NESTOLA P,PEIXOTO C,SILVA R R,et al.Improved virus purification processes for vaccines and gene therapy[J].Biotechnol Bioeng,2015,112(5):843-857.
[8]BASKIN E.Increasing influenza vaccination rates via low cost messaging interventions[J].PLo S One,2018,13(2):e0192594.doi:10.1371/journal.pone.0192594.
[9]WOOLTHUIS R G,WALLINGA J,VAN BOVEN M.Variation in loss of immunity shapes influenza epidemics and the impact of vaccination[J].BMC Infect Dis,2017,17(1):632.
[10]Chinese Pharmacopoeia Commission.Pharmacopoeia of People's Republic of China(VolⅢ)[S].Beijing:China Med Sci Press,2010:162-163.(in Chinese)国家药典委员会.中华人民共和国药典(三部)[S].北京:中国医药科技出版社,2010:162-163.
[11]SCHAFFNER W,CHEN W H,HOPKINS R H,et al.Effective immunization of older adults against seasonal influenza[J].Am J Med,2018.pii:S0002-9343(18)30206-7.doi:10.1016/j.amjmed.2018.02.019.
[12]HARDING A T,HEATON N S.Efforts to improve the seasonal influenza vaccine[J].Vaccines(Basel),2018,6(2).pii:E19.doi:10.3390/vaccines6020019.
[13]NOWAK G J,CACCIATORE M A,LEN-RIOS M E.Understanding and increasing influenza vaccination acceptance:insights from a 2016 national survey of U.S.Adults[J].Int JEnviron Res Public Health,2018,15(4):711.
[14]ZHANG Y,WU P,FENG L,et al.Influenza vaccine effectiveness against influenza-associated hospitalization in 2015/16season,Beijing,China[J].Vaccine,2017,35(23):3129-3134.
[15]MARTINEZ-BAZ I,CASADO I,NAVASCUES A,et al.Effect of repeated vaccination with the same vaccine component against 2009 pandemic influenza A(H1N1)virus[J].J Infect Dis,2017,215(6):847-855.