适应H9亚型禽流感病毒增殖的MDCK悬浮细胞株的驯化及其生物学特性

Domestication of MDCK suspension cell lines for the H9 subtype of avian influenza virus proliferation and its biological characteristics evaluation

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。

To provide technology supports of manufacture of vaccine based on this cell lines,this study characterizes the biological features of the suspension-cultured MDCK-sus cell. The MDCK cells were adapted and screened in suspension growth mode, named MDCK-sus. Its growth rate, cell viability and density were tested. The indirect fluorescence assay and flow cytometry were employed to determine the abundance of receptor for influenza virus, SAa2,3Gal and SAa2,6Gal,on the cell membrane of the parent MDCK and MDCK-sus cells. The fluorescence quantitive real -time PCR wa s adopted toquantitive real-time PCR was adopted to analysis the difference in the mRNA levels of ST3Gals and ST6Gals, and the deterruination of the influenza virus HA titer of coagulation was used to compare the parent and suspended MDCK cells. The adherence MDCK cells were successfully converted to serum-free and suspended cultured cells with the maxinmnl cell density of 3.6×10^5 cells in 1 ml, cell viabilityof 99% and growth rate of 0.56 every day. Compared to the parent MDCK cells, the abundance of influenza viruses receptor, SAα2,3 Gal, the expression level of ST3 Gals, and the propagation capability- of avian influenza viruses of the MDCK-sus were higher. HA titer of AH1102 virus strain in MDCK-sus cells was 91g2 in 25 μl. The growth features of the MDCK-sus cell indicate that it is capable of propagation of H9 subtype ilffluenza viruses and can provide technical support for industrial manufacturing of avian influenza vaccine in suspended culture cell.