一种重组腺病毒载体的扩增、纯化和病毒滴度检测方法

Reproduction,purification and biological activity identification of AxCA-BDNF

目的建立扩增、纯化和生物活性鉴定重组腺病毒AxCA-BDNF的有效方法,为进一步研究应用腺病毒介导的BDNF进行神经系统疾病的基因治疗奠定基础。方法应用人胚肾293细胞作为包装细胞,扩增携带BDNF cDNA表达片段的重组腺病毒载体AxCA-BDNF;利用改良的CsCl超速离心法对其进行纯化;应用病毒空斑试验,对纯化病毒液进行病毒生物活性测定。结果获得了50 μl纯化病毒液;病毒滴度达2.2×108~1.8×1010 pfu/ml。结论通过培养扩增293细胞,可成功扩增重组腺病毒AxCA-BDNF;纯化病毒液量可完全满足体内基因转染实验的需要;病毒空斑实验证明其具有高强度的生物活性。

Objective To find methods for reproduction, purification and biological activity identification of the helper-free replication-defective recombined adenorirus vector containing BDNF cDNA expression cassette (AxCA-BDNF) in order to study gene therapy for nerve system diseases. Methods The cell 293, a human embryonic kidney cell line as a packaging cell was used for culture of recombinant adenovirus vectors containing BDNF gene. AxCA-BDNF was reproduced and purified. After 2 rounds of CsC1 centrification, biological activity identification was carried out using plaques test. Results The purified recombinant adenovirus AxCA-BDNF was obtained with total volume of 50 μl and titer of 2.2×10 2 -1.8×10 2 pfu/ml. Conclusion Culturing and reproduction of cell 239 can reproduce recombined adenovirus AxCA-BDNF successfully. The volume of purified AxCA-BDNF, which has been verified in plaques test to be of high biological activity, can meet the needs of in vivo gene transfection at laboratory study.