摘要
目的应用慢病毒介导单纯疱疹病毒胸苷激酶(HSV-TK)基因转导大鼠9L胶质瘤细胞。方法构建慢病毒表达质粒pLenti6/V5-TK并以PCR、酶切及测序等方法鉴定。利用ViraPowerTM慢病毒表达系统,将重组质粒与包装混合物(Packaging Mix)共同转染293T细胞生产假病毒颗粒,转染48 h后收集培养上清,用其感染大鼠9L胶质瘤细胞,以抗稻瘟菌素(BSD)筛选出9L-TK细胞。RT-PCR、Western blot方法检测TK基因在9L-TK细胞中的表达。用MTT方法测试更昔洛韦(GCV)对体外培养的9L-TK细胞杀伤效果。结果构建的重组质粒pLenti6/V5-TK经PCR、酶切及测序鉴定正确;该质粒与包装质粒共转染293T细胞获取的慢病毒滴度达2.7×105转导单位(TU)/ml。经BDS筛选获得的9L-TK细胞RT-PCR及Western blot结果显示TK基因表达阳性,且GCV对该细胞作用敏感。结论成功利用慢病毒实现了TK基因转导大鼠9L胶质瘤细胞。
Objective To transfer herpes simplex viral thymidine kinase gene into rat glioma 9L cells using lentivirus. Methods The lentivirus-expressing plasmid pLenti6N5-TK was constructed and identified by PCR restriction enzymes and DNA sequence analysis. The identified pLenti6N5-TK plasmid and the ViraPowerTM Packaging Mix were cotransfected into 293T cells to produce a replicationincompetent lentivirus. The cultured supernatant was collected 48 h later, and the lentivirus was added into rat glioma 9L cells and selected with Blasticidin （BSD） for the stably transduce 9L-TK cells. The expression ofTK gene in 9L-TK cells was detected by RT-PCR and Western blotting. The killing activities of ganciclovir （GCV） for 9L-TK cells were assayed by MTT. Results The TK gene sequence of pLenti6N5-TK plasmid was consistent with Gen Bank V00470 by PCR, enzyme digestion and sequencing. The electrophoresis results of constructed plasmid by means of PCR and restriction enzymes were coincident with expectation. The titer of cotransfected lentivirus was 2.7xl 0s transducing units （TU）/ml. RT-PCR and Western blot results showed that the TK gene and TK protein were positively expressed in 9L-TK cells. The 9L-TK cells were sensitive to GCV. Conclusion The lentivirus-mediated gene transfer system can successfully transfer TK gene into rat glioma 9L cells.
出处
《中国微侵袭神经外科杂志》
CAS
2007年第8期358-362,共5页
Chinese Journal of Minimally Invasive Neurosurgery
基金
广东省自然科学基金(001122)
全军医学科研"十五"计划项目(01MA038)
