摘要
目的 建立产逆转录病毒的PA31 7/HGSTπ细胞株 ,为进一步把耐药基因GSTπ导入造血干细胞以逆转卵巢癌化疗药物对骨髓的毒副作用的研究奠定基础。方法 用限制性内切酶HpaⅠ、BamhⅠ分别双酶切质粒 pLXSH GSTπ和pLXSN ,回收GSTπ小片段和 pLXSN大片段 ,在T4连接酶的作用下 ,构建重组逆转录病毒质粒pLXSN GSTπ ;酶切鉴定后用脂质体转染PA31 7包装细胞 ,G4 1 8筛选获得抗性克隆 ,用Hela细胞测定病毒滴度。结果 (1 )酶切证实pLXSN GSTπ和 pLXSH GSTπ的目的片段完全一致 ,表明重组质粒构建成功 ;(2 )G4 1 8筛选获得多个抗性克隆 ,Hela测定病毒滴度最高为 2 .2× 1 0 3 CFU/ml。结论 本研究成功建立了产病毒包装细胞株PA31 7/HGSTπ ,该细胞株能产生含有耐药基因GSTπ的逆转录病毒 。
Objective To construct the packaging cell line PA317/HGSTπ.Methods Retrivoral vector pLXSH GSTπ and pLXSN were cut respectively with restriction endonucleses HpaⅠand BamhⅠ.The small fragment of GSTπ and the big fragment of pLXSN were ligased to pLXSN GSTπ using T4 ligase.The pLXSN GSTπ was transfected into PA317 and then G418 resistant colonies were isolated.The titer of retroviral was estimated by Hela.Results The small fragment of pLXSN GSTπ and pLXSH GSTπ are fully same.The most high titer of recombinant retroviral is 2.2×10 3 CFU/ml.Conclusion The research showed that the PA317/HGSTπ was constructed,which can producte the recombinant retrovirus for further research of GSTπ in chemotherapy of ovarian cancer?
出处
《重庆医学》
CAS
CSCD
2004年第8期1187-1189,共3页
Chongqing medicine
基金
重庆市科委科研基金资助项目 (2 0 0 0 1 78)
