摘要
目的构建视紫红质启动子(rhodopsin,Rho)驱动的、大鼠视锥视杆细胞同源盒基因(cone-rod homeobox gene,CRX)为目的基因的重组慢病毒载体LV-Rho-EGFP/CRX,研究其在体外的转染效率。方法用PCR法扩增质粒pEGFP-C3/CRX中的EGFP/CRX基因片段,取代慢病毒载体LV-Rho-EGFP中的EGFP片段,构建表达载体LV-Rho-EGFP/CRX,经酶切和双向测序验证。用磷酸钙沉淀法四质粒共转染293T细胞,包装、收集病毒,利用有限稀释法测定病毒滴度。用慢病毒原液感染光感受器细胞来源的人视网膜母细胞瘤细胞株HXO-RB44,观察感染情况。结果成功构建慢病毒表达载体LV-Rho-EGFP/CRX,产生的慢病毒滴度为2×104IU/ml,感染48h后,HXO-RB44细胞内可见强弱不等的绿色荧光。结论重组载体LV-Rho-EGFP/CRX产生的慢病毒能感染光感受器细胞来源的HXO-RB44细胞,但还需提高转染的效率和滴度。
Objective To investigate the transfection efficiency of the recombinant cone-rod hemeobox （CRX） gene-containing lentiviral vector （LV-Rho-EGFP/CRX）drived by the Rhodopsin promoter in vitro. Methods The lentiviral vector（LV-Rho-EGFP/CRX） was reconstructed by replace of the EGFP gene fragment in the lentiviral vectors LV-Rho-EGFP with the EGFP/CRX gene fragment, which was amplified by PCR from pEGFP- C3/CRX vector. It was analyzed by restriction digestion ancl bidirectional sequencing. The recombination lentiviral particles were produced by the packaging 293T cell by using the Ca3 （ PO4 ） 2 method, the culture supernatant was harvested and the titration of them were calculated by the limiting dilution method. The photoreceptor-deriving human retinoblastoma cell line HXO-RB44 cells were transduced by the recombinant lentiviral particles, after 48 h the GFP expression was observed under the fluorescence microscope. Results The recombinant lentiviral vector LV-Rho- EGFP/CRX had been successfully constructed,the titration of lentivirus particles was 2 ×10^4 IU/ml. The HXO-RB44 ceils could be transduced by the recombinant lentiviral particles（2×10^4 IU/ml）. After 48 h coculture, some HXO- RB44 cells could express GFP. Conclusions The recombination lentiviral vector LV-Rho-EGFP/CRX was successfully constructed. The photoreceptor-deriving HXO-RB44 cells could be transduced by lentiviral particles with the titration 2 ×10^4 IU/ml in a certain degree.
出处
《中华临床医师杂志（电子版）》
CAS
2012年第15期103-105,共3页
Chinese Journal of Clinicians(Electronic Edition)
