本研究通过筛选驯化的MDCK细胞获得无血清悬浮生长MDCK细胞(命名为MDCK-sus),测定该细胞的比生长速率、细胞活力及细胞最大生长密度等生长特性,采用间接免疫荧光法和流式细胞仪检测法比较母本MDCK细胞与MDCK-sus细胞流感病毒受体[唾液酸...本研究通过筛选驯化的MDCK细胞获得无血清悬浮生长MDCK细胞(命名为MDCK-sus),测定该细胞的比生长速率、细胞活力及细胞最大生长密度等生长特性,采用间接免疫荧光法和流式细胞仪检测法比较母本MDCK细胞与MDCK-sus细胞流感病毒受体[唾液酸-α-2,3-半乳糖糖链受体(SAα2,3Gal)和唾液酸-α-2,6-半乳糖糖链受体(SAα2,6Gal)]的丰度,用荧光定量PCR检测比较细胞中α-2,3唾液酸转移酶(ST3Gals)基因与α-2,6唾液酸转移酶(ST6Gals)基因表达水平的差异,通过测定流感病毒血凝HA效价比较母本MDCK细胞与MDCK-sus细胞对流感病毒的增殖能力。结果表明,经驯化获得的适合无血清悬浮培养的MDCK-sus细胞,细胞密度最高可达1ml 3.6×106个细胞,最大比生长速率可达1 d 0.56。MDCK-sus细胞表面流感病毒受体SAα2,3Gal的丰度明显高于母体MDCK细胞,ST3Gal转移酶基因表达水平也明显高于母本MDCK细胞。MDCK细胞驯化后增殖禽流感病毒的能力增强,其中H9亚型禽流感病毒AH1102株在MDCK-sus细胞中HA效价达到9lg2(25μl)。MDCK-sus细胞各种生长特性表明,其适于H9亚型禽流感的繁殖,可以为采用悬浮细胞培养禽流感疫苗的大规模工业化生产提供技术支持。展开更多
To accurately determine the expression and distribution patterns of two infuenza virus receptors （SAa2,3-gal and SAa2,6-gal） in trachea and lung tissues of humans, mice, chickens and ducks, we analyzed lectin immuno...To accurately determine the expression and distribution patterns of two infuenza virus receptors （SAa2,3-gal and SAa2,6-gal） in trachea and lung tissues of humans, mice, chickens and ducks, we analyzed lectin immunofluorescence stainings of various tissue sections qualitatively and quantitatively. Results from the qualitative analysis showed that both influenza virus receptors were expressed in lung tissues of humans, mice, chickens and ducks as well as trachea tissues of mice and ducks. However, SAa2,6-gal receptor was expressed only in the human trachea tissue and SAa2,3-gal receptor was expressed only in the chicken trachea tissue. Results from the quantitative analysis demonstrated that both receptors were expressed in trachea tis- sues of human and mouse, as well as in lung tissues of humans, chickens and ducks. Meanwhile, our results also showed that the expression and distribution of influenza virus receptors in the same tissue were not always uniform, indicating that their distribution and expression in various tissues are not simply the distinction between the presence or absence of receptors, but rather the difference in the amount of expressed receptors.展开更多
The human influenza A （H3N2） virus dominated the 2014-2015 winter season in many countries and caused massive morbid- ity and mortality because of its antigenic variation. So far, very little is known about the anti...The human influenza A （H3N2） virus dominated the 2014-2015 winter season in many countries and caused massive morbid- ity and mortality because of its antigenic variation. So far, very little is known about the antigenic patterns of the recent H3N2 virus. By systematically mapping the antigenic relationships of H3N2 strains isolated since 2010, we discovered that two groups with obvious antigenic divergence, named SW13 （A/Switzerland/9715293/2013-1ike strains） and HK14 （A/Hong Kong/5738/2014-1ike strains）, co-circulated during the 2014-2015 winter season. HK14 group co-circulated with SW13 in Europe and the United States during this season, while there were few strains of HK14 in China's Mainland, where SW13 has dominated since 2012. Furthermore, we found that substitutions near the receptor-binding site on hemagglutinin played an im- portant role in the antigenic variation of both the groups. These findings provide a comprehensive understanding of the recent antigenic evolution of H3N2 virus and will aid in the selection of vaccine strains.展开更多
文摘本研究通过筛选驯化的MDCK细胞获得无血清悬浮生长MDCK细胞(命名为MDCK-sus),测定该细胞的比生长速率、细胞活力及细胞最大生长密度等生长特性,采用间接免疫荧光法和流式细胞仪检测法比较母本MDCK细胞与MDCK-sus细胞流感病毒受体[唾液酸-α-2,3-半乳糖糖链受体(SAα2,3Gal)和唾液酸-α-2,6-半乳糖糖链受体(SAα2,6Gal)]的丰度,用荧光定量PCR检测比较细胞中α-2,3唾液酸转移酶(ST3Gals)基因与α-2,6唾液酸转移酶(ST6Gals)基因表达水平的差异,通过测定流感病毒血凝HA效价比较母本MDCK细胞与MDCK-sus细胞对流感病毒的增殖能力。结果表明,经驯化获得的适合无血清悬浮培养的MDCK-sus细胞,细胞密度最高可达1ml 3.6×106个细胞,最大比生长速率可达1 d 0.56。MDCK-sus细胞表面流感病毒受体SAα2,3Gal的丰度明显高于母体MDCK细胞,ST3Gal转移酶基因表达水平也明显高于母本MDCK细胞。MDCK细胞驯化后增殖禽流感病毒的能力增强,其中H9亚型禽流感病毒AH1102株在MDCK-sus细胞中HA效价达到9lg2(25μl)。MDCK-sus细胞各种生长特性表明,其适于H9亚型禽流感的繁殖,可以为采用悬浮细胞培养禽流感疫苗的大规模工业化生产提供技术支持。
基金supported by the Governor’s Fund of Guizhou Province for Outstanding Individuals in Science,Technology and Education(Grant No. 2010-68)
文摘To accurately determine the expression and distribution patterns of two infuenza virus receptors （SAa2,3-gal and SAa2,6-gal） in trachea and lung tissues of humans, mice, chickens and ducks, we analyzed lectin immunofluorescence stainings of various tissue sections qualitatively and quantitatively. Results from the qualitative analysis showed that both influenza virus receptors were expressed in lung tissues of humans, mice, chickens and ducks as well as trachea tissues of mice and ducks. However, SAa2,6-gal receptor was expressed only in the human trachea tissue and SAa2,3-gal receptor was expressed only in the chicken trachea tissue. Results from the quantitative analysis demonstrated that both receptors were expressed in trachea tis- sues of human and mouse, as well as in lung tissues of humans, chickens and ducks. Meanwhile, our results also showed that the expression and distribution of influenza virus receptors in the same tissue were not always uniform, indicating that their distribution and expression in various tissues are not simply the distinction between the presence or absence of receptors, but rather the difference in the amount of expressed receptors.
基金supported by the National Basic Research Program of China(2015CB910501)the Major National Earmark Project for Infectious Diseases(2014ZX10004002-001)+1 种基金the Key Research Program of the Chinese Academy of Sciences(KJZD-EW-L09-1-2)to Jiang Tai Jiaothe National Natural Science Foundation of China(31470273)to Wu Ai Ping
文摘The human influenza A （H3N2） virus dominated the 2014-2015 winter season in many countries and caused massive morbid- ity and mortality because of its antigenic variation. So far, very little is known about the antigenic patterns of the recent H3N2 virus. By systematically mapping the antigenic relationships of H3N2 strains isolated since 2010, we discovered that two groups with obvious antigenic divergence, named SW13 （A/Switzerland/9715293/2013-1ike strains） and HK14 （A/Hong Kong/5738/2014-1ike strains）, co-circulated during the 2014-2015 winter season. HK14 group co-circulated with SW13 in Europe and the United States during this season, while there were few strains of HK14 in China's Mainland, where SW13 has dominated since 2012. Furthermore, we found that substitutions near the receptor-binding site on hemagglutinin played an im- portant role in the antigenic variation of both the groups. These findings provide a comprehensive understanding of the recent antigenic evolution of H3N2 virus and will aid in the selection of vaccine strains.
