【作者】 卢莉莉；

【导师】 董长垣；

【作者基本信息】 武汉大学 ， 病原生物学， 2004， 硕士

【摘要】 本文发展了一种反向免疫共沉淀分离纯化病毒的新方法，用于从培养物中分离高纯度，高活性的蓝舌病毒。用透射电镜和凝胶过滤层析检测纯化产物病毒的结构完整性和纯度；组织培养技术检测产物的生物学活性并计算纯化得率。结果表明，纯化后的病毒悬液通过凝胶过滤层析仅出现一个紫外吸收峰，其余的未见明显的吸收峰，仅表现为靠近基线的波动；将纯化前后的病毒悬液用磷钨酸负染后直接透射电镜观察，可见到完整的病毒粒子及其干净的背景；在相同条件下，纯化前的BTV-HbC₃对Vero细胞的TCID₅₀是10^-6.6／ml，纯化后的病毒对Vero细胞的TCID₅₀是10^-5.7／ml；选取一定稀释度的复孔计数空斑，依据空斑计算公式求得不同稀释度下纯化前后病毒的滴度，通过比较纯化前后的病毒滴度计算该纯化方法的纯化得率，在10^-3，10^-4，10^-5这三个稀释度下，所计算纯化得率分别为69.6％，70.8％，50.4％。 经蓝舌病毒处理过的细胞（7402细胞，Hep-3B细胞）在普通光学显微镜下即可看到明显的形态改变。细胞在接种病毒24 hr后开始收缩变圆，间隔变大，细胞轮廓增强，胞内有粗大折光颗粒；36hr后细胞逐渐脱落，漂浮，直至破碎死亡。用MTT染色方法检测蓝舌病毒对这两株肝癌细胞株的抑制活性。在10°稀释度的蓝舌病毒作用下测得这两株细胞的抑制率分别为：51.45％和77.69％。 通过PI染色检测经蓝舌病毒处理后不同时间的细胞凋亡情况，其中感染病毒后48hr的7402，Hep-3B细胞的凋亡率分别为20.1％，33.0％。通过Hoechst33258染色后进行共聚焦显微镜观察，结果在这两株细胞中均观察到典型的凋亡细胞，主要表现为凋亡细胞出现核碎裂、边聚，碎裂的细胞核呈致密浓染的颗粒状或块状荧光，与此同时，完整核膜清晰可见。更多还原

【Abstract】 A novel reverse co-immunoprecipitation method was used to get the purified BTV-HbC3 particles and gel filtration, electron microscopy(EM), TCID50 and plaque assay were used to analyze the purification results. Sephadex G-200 gel filtration analysis of the purified sample by absorbance values measured at 280nm , only one steep UV absorbance peak can be seen ; background of electron micrograph of the purified virus is clean and the complete structure of virus particles can be clearly seen; the 50% tissue culture infective dose on Vero cell line is 10-6.6/ml before purification and 10-5.7/ml after purification; purified and unpurified virus liters can be attained from plaque assay of BTV-HbC3 on Hep-3B cell line . Purification recovery rate can be gotten by comparing between virus titers of purified and unpurified. On 10-3, 10-4, 10-5 dilution degrees respectively , the recovery rates are 69.6 %, 70.8 %, 50.4 %. From the results described above we can draw the conclusion that the new method introduced can produce large amounts of high purified viral particles with high infectivity so it can be applied to further research on developing a large scale production system.BTV-HbC3 can selectively replicate in 7402, Hep-3B cell lines and induce cytopathic effects (CPE), and eventually cause complete lysis and death of cells. Between the two human liver cell lines, Hep-3B cells are more susceptible to BTV-HbC3 sinfection, the CPE of which occur quickly and can reach 90% at 36h p.i., while 7402 cells are the less sensitive. The inhibition activity of BTV-HbC3 on the two liver cell lines (7402, Hep-3B ) was tested by MTT. Under the same condition, the inhibition ratio of BTV is 77.69% on Hep-3B; 51.45% on 7402.BTV-HbC3 can induce apoptosis in 7402, Hep-3B cell lines. The result of flow cytometry assay shows the apoptosis ratio of 48hr p.i. is 20.1% on 7402; 33.0% on Hep-3B. The result of confocal microscopy also indicates typical apoptosis induced by BTV-HbC3 in two human liver cell lines.更多还原