摘要
目的 构建表达小鼠KGFR基因的重组腺病毒.方法 提取小鼠肺组织RNA,RT-PCR获得cDNA后,PCR扩增目的 基因片段.将获得的KGFR基因插入载体质粒pShuttle-CMV,构建穿梭质粒pShuttle-CMV-KGFR（pKGFR）.线性化pKGFR后,转化入含Adeasy-1病毒骨架的BJ5183细菌中.筛选正确的同源重组质粒pAdeasy-1-pShuttle-CMV-KGFR（pAd-KGFR）,转染HEK293细胞,产生病毒颗粒Adeasy-1-pShuttle-CMV-KGFR（Ad-KGFR）.进一步,大量扩增病毒,氯化铯梯度离心纯化,测量病毒滴度.结果 ①经限制性内切酶酶切和基因测序鉴定,证实pKGFR构建成功;②限制性内切酶酶切,确定穿梭质粒重组于病毒骨架;显微镜下观察HEK293细胞形态,证实病毒包装复制成功;③病毒滴度2.25×10^10PFU/ml,达到进一步体内、体外实验要求.结论 成功构建的重组腺病毒Ad-KGFR,为了解KGFR在肺部疾病中的作用,及进一步的基因治疗肺纤维化奠定基础.
Objective To construct a recombinant adenovirus vector expressing keratinocyte growth factor receptor （KGFR） gene of mouse. Methods KGFR gene was amplified by polymerase chain reaction （PCR） from mouse cDNA which had been reverse transcripted from RNA harvested from C57BL/ 6 mouse, and was then inserted into the plasmid pShuttle-CMV to construct the shuttle plasmid pShuttle-CMV-KGFR （pKGFR）. After linearized by restriction enzyme, the plasmid was transformed into Escberichia. coli BJ5183 containing adenovirus backbone. The homologous recombinant pAdeasy-1-pShuttle-CMV-KGFR（pAd-KGFR） was identified, linearized, and then transfected into HEK293 cells using lipofectamineTM 2000 to package the adenovirus, Adeasy-l-pShuttle-CMV-KGFR （Ad-KGFR）, followed by further amplification, caesium chloride density gradient eentrifugation purification and measurement of virus titer. Results Firstly, the shuttle plasmid pKGFR was proved to be successfully constructed by gene sequencing and restriction enzyme, as well as the recombinant adenovirus plasmid. Then, the cytopathic effects of HEK293 cells observed under the microscope suggested the successful duplication of the virus. Finally, the plague titration of on HEK293 cells showed virus titers were 2.25 × 10^10 PFU/ml, which was enough concentration for future test in vivo or in vitro. Conclusion The recombinant adenovirus vector of pAd-KGFR is constructed successfully to be used in the future test to investigate the effects of KGFR in pulmonary diseases, and provides attractive therapeutic target in the pulmonary disease.
出处
《中国急救复苏与灾害医学杂志》
2011年第5期412-416,共5页
China Journal of Emergency Resuscitation and Disaster Medicine
关键词
角质化细胞生长因子
腺病毒
小鼠
肺
受体
Keratinocyte growth factor receptor （KGFR）
Lung
Adenovirus
Mouse
