摘要
目的 构建含人核心结合因子α1（CBFα1）基因的过表达慢病毒载体,并观察在重组慢病毒感染的人牙周膜成纤维细胞（PDLFs）中CBFα1基因的表达情况.方法 采用PCR 技术体外扩增人CBFα1,将扩增产物与慢病毒载体pGC-FU连接,构建重组质粒pGC-FU-hCBFα1,并进行酶切及测序鉴定.利用脂质体转染法将pGC-FU-hCBFα1、pHelper1.0和pHelper2.0的3种质粒共转染293T细胞,包装产生慢病毒.将PDLFs分为未感染组（未感染任何病毒）、阴性对照病毒感染组（加阴性对照病毒感染）、CBFα1重组慢病毒感染组（加CBFα1重组慢病毒感染）,应用免疫细胞化学方法检测目的 基因CBFα1在PDLFs中的表达.结果 重组质粒经测序证实,插入片段与人CBFα1基因序列完全一致.包装慢病毒后检测病毒滴度为2.00×109 TU/ml.免疫细胞化学检测证实了CBFα1在PDLFs中的有效表达.结论 成功构建了携带hCBFα1基因的重组慢病毒,并能有效感染PDLFs,为进一步研究其在牙周组织再生中的生物学功能奠定了基础.
Objective To construct a recombinant lentiviral vector containing human CBFa 1 gene and to investigate its expression in human periodontal ligament fibroblasts. Methods A lentiviral expression vector for human CBFa 1 was constructed by recombinant DNA technique. The reconstructed lentiviral vector containing hCBF a 11 was confirmed by PCR and sequencing, The human periodontal ligament fibroblast （PDLF） 293T cells were cotransfected with lentiviral vector pGC-FU-hCBFa 1, pHelper 1.0 and pHelper2.0 with Lipofectamine 2000. PDLFs were infected with recombinant lentiviral vector containing CBF a 1. Infection efficiency was measured by fluorescent microscope and CBFa 1 expression was detected by immunocytochemical analysis. Results The sequence analysis of recombinant plasmid pGC-FU-hCBFa 1 showed that the human CBFa 1 was inserted into lentiviral vector pGC-FU accurately. The titer of concentrated virus was 2.00 × 109TU/mI. Immunohistochemistry showed that CBFa 1 was expressed in PDLF 293T cells infected with recombinant lentiviral vector for CBF a 1. Conclusion The recombinant lentiviral vector pGC-FU-hCBF a 1 has been constructed and CBF a 1 are expressed in transfectecl PDLFs successfully.
出处
《浙江医学》
CAS
2012年第2期87-90,共4页
Zhejiang Medical Journal
基金
浙江省教育厅资助项目（20070914）
温州市科技局资助项目（Y20070052）
