荧光定量PCR法在重组逆转录病毒pLXSN-BDNF滴度测定中的应用  

Determination of recombinant retrovirus pLXSN-BDNF titer with fluorescent quantitative PCR

作　　者：周欣荣[1] 原慧萍[1] 李鸿翼[1] 邵正波[1] 王雅[1] 

机构地区：[1]哈尔滨医科大学附属第二医院眼科,150086

出　　处：《中华微生物学和免疫学杂志》2007年第8期766-770,共5页Chinese Journal of Microbiology and Immunology

基　　金：国家自然科学基金资助项目(30471844);黑龙江省教育厅资助项目(10551187)

摘　　要：目的制备含大鼠脑源性神经营养因子（BDNF）基因的逆转录病毒，建立荧光定量PCR测定滴度的方法。方法扩增BDNF基因，构建重组质粒pLXSN-BDNF，将其转染包装细胞，经G418筛选、病毒浓缩后用荧光定量PCR法和克隆形成法测定滴度。结果经PCR、酶切和测序鉴定，BDNF基因成功插入逆转录病毒载体中，筛选得到稳定的分泌重组病毒细胞系。重组病毒100倍浓缩后经荧光定量PCR法和克隆形成法测定的滴度分别为6．92×10^6拷贝/ml和3．2×10^5CFU/ml，两种方法测得的结果差异有统计学意义（P〈0．05）。结论成功扩增BDNF基因，制备了重组病毒pLX—SN-BDNF，建立了荧光定量PCR检测滴度的方法，为基因治疗青光眼性视神经损伤的实验研究提供重要参考指标。Objective To construct the recombinant retroviral expression vector carrying rat brain-derived neurotrophic factor （BDNF） gene and develop a modified retrovirus, establish the fluorescent quantitative PCR assay for retroviral liter and compare it with clone formation method, laying foundations for development of a stable and effective retroviral expression system for the protection of optic nerve injury respecting glaucoma with gene therapy. Methods The BDNF gene was amplified by RT-PCR from total RNA of the rat hippocamnpal tissue, cloned into pMD-18T simple vector and then subcloned into the retroviral vector pLXSN after sequencing correctly. The plasmid pLXSN-BDNF was transfected by LipofectAM1NE into the packing cell line PA317 and G418- resistant clones were selected. Retrovirus titer was determined by fluorescent quantitative PCR and clone formation method. Results PCR, restriction analysis and DNA sequencing proved that the BDNF gene was inserted into the retroviral vector exactly. The cell line, which can secrete recombinant retrovirus pLXSN-BDNF stably, was established. The liter of recombinant retrovirus was 6.92 ×10^6 copies/ml by fluorescent quantitative PCR, while it was 3.2 ×10^5 CFU/ml by clone formation method after it was concentrated. They were significantly different （ P 〈 0.05）. Conclusion The plasmid pLXSN-BDNF was successfully constructed, and the recombinant retrovirus secreting BDNF had been received. A novel quantitative PCR assay had been developed to replace the standard clone formation method for retroviral liter, which may be useful for gene therapy experiment for curing the optic nerve injury due to glaucoma.

关 键 词：脑源性神经营养因子 逆转录病毒 荧光定量PCR 

分 类 号：R450[医药卫生—治疗学]