稳定表达鸡ST3GalⅠ的BHK-21细胞株的构建及禽流感病毒的增殖  

Construction of BHK-21 cell line stably expressing chicken ST3GalⅠgene and the proliferation of avian influenza virus

作　　者：陈欢[1] 李明义 高轩[1] 

机构地区：[1]河北农业大学动物科技学院,河北保定071000 [2]山东信得科技股份有限公司,山东青岛266101

出　　处：《中国兽医学报》2013年第10期1527-1532,共6页Chinese Journal of Veterinary Science

基　　金：动物疫苗关键生产技术研究与开发资助项目(2009BADBAB04-3)

摘　　要：克隆鸡ST3GalⅠ基因并将其插入真核表达载体pcDNA3.1-EGFP中构建真核表达质粒,转染BHK-21细胞后,通过G418抗性和绿色荧光蛋白标记双重筛选,鉴定1株表达ST3GalⅠ阳性的单克隆细胞株,绿色荧光丰富。经流式细胞仪检测,BHK-21-ST3GalⅠ细胞株中α-2,3连接型受体丰度较亲代BHK-21细胞显著提高。连续传5代10代后仍能检测到其表达,表明ST3GalⅠ基因在细胞中稳定表达。为了检测BHK-21-ST3GalⅠ细胞对H9亚型流感病毒和H5N1Re-5病毒的增殖效果,将H9亚型流感病毒和H5N1Re-5病毒接种BHK-21-ST3GalⅠ细胞和BHK-21细胞,同时设普通胰酶和TPCK-胰酶对照组。接毒72h后收获细胞液,常规方法测定血凝素(HA)滴度。试验结果表明,BHK-21-ST3GalⅠ细胞中禽流感病毒的HA滴度达到1∶256,显著高于BHK-21细胞组,表明其更适于流感病毒的增殖,具备生产流感病毒疫苗的潜力。The chicken ST3Gal I gene was coloned and incorporated into the eukaryotic expression vector pcDNA3.1-EGFP to generate the recombinant plasmid. The recombinant vector pcDNA3.1- EGFP-ST3Gal I was transfected into BHK-21 cells using lipofectamine 2000. We got monoclone cell lines by G418 resistance screening and RT-PCR appraisal. Green fluorescence could be ob- served. BHK-21-ST3Gal I cell expressed higher amounts of 2, 3-1inked receptors than parent BHK-21 cell by flow cytometryt analysis. Continuous passing five and ten generations can still de- tected the expression of ST3Gal I gene. To screen proliferation of avian influenza virus（AIV） H9 Subtype and HSN1 Re-5 in BHK-21- ST3Gal I cell,the different proportion of the H9 subtype and HSN1 Re-5 avian influenza virus was vaccinated to BHK-21-ST3Gal I cells and BHK-21 cells,at the same time we set pancreatic enzyme group and TPCK pancreatic enzyme control group. Har vested cell liquid after 72 hours, conventional method was used for determining hemagglutinin （HA） titer. The experimental results showed that the HA titer of influenza virus of BHK-21- ST3Gal I cells reached 1 -＂ 256, significantly higher than BHK-21 cell group, showing that it is suitable for the flu virus proliferation,displaying a potential of the influenza virus vaccine produc tion.

关 键 词：ST3GalⅠ重组质粒pcDNA3 1-EGFP—ST3GalⅠ 转染 稳定表达 BHK-21-ST3GalⅠ细胞 

分 类 号：S852.65[农业科学—基础兽医学]