小鼠角质细胞生长因子重组腺病毒载体的构建

Construction of recombinant adenovirus vector of mouse keratinocyte growth factor

目的探讨构建表达小鼠角质细胞生长因子（KGF）基因重组腺病毒载体的方法。方法提取小鼠肺组织RNA，经逆转录-聚合酶链反应（RT—PCR）获得cDNA后，PCR扩增目的基因片段。将获得的KGF基因插入载体质粒pShuttle—CMV，构建穿梭质粒pShuttle—CMV—KGF（pKGF）。线性化pKGF后，转化人含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—KGF（pAd—KGF），转染HEK293细胞，产生病毒颗粒AdEasy-1-pShuttle—CMV—KGF（Ad—KGF）。进一步大量扩增病毒，氯化铯梯度离心纯化，测定病毒滴度。制备病毒Ad—GFP为对照，显微镜下观察转染率。结果①经限制性内切酶酶切和基因测序鉴定，证实pKGF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；⑧病毒滴度3．0×10^10pfu／ml，达到进一步体内、外实验要求。结论成功构建了重组腺病毒Ad—KGF，为了解KGF在肺部疾病中的作用以及进一步的基因治疗肺纤维化方法奠定了基础。

Objective To construct a recombinant adenovirus vector expressing keratinocyte growth factor （KGF） gene in mouse. Methods KGF gene amplified from mouse cDNA by polymerase chain reaction （PCR）. It was reverse transcripted from RNA that had been harvested from C57BL/6 mouse, and then inserted into the plasmid pShuttle- CMV to construct the shuttle plasmid pShuttle- CMV- KGF （pKGF）. After linearized by restriction enzyme, the plasmid was transformed into E. Coli BJ5183 containing adenovirus backbone. The homologous recombinant pAdeasy - 1 - pShuttle - CMV - KGF （pAd - KGF） was identified, linearized, and then transfected into HEK293 cells using the lipofectamineTM 2000 to package the adenovirus, Adeasy - 1 - pShuttle - CMV - KGF （Ad - KGF）, followed by further amplification, caesium chloride density gradient centrifugation purification and measurement of virus titer. Ad -GFP was used as control, and its transfection efficacy was observed. Results ①The shuttle plasmid pKGF was proved to be successfully constructed by gene sequencing and restriction enzyme, as well as the recombinant adenovirus plasmid. ② The cytopathic effects of HEK293 cells observed under the microscope suggested that the duplication of the virus was successful. ③The plague titration of HEK293 cells showed virus titers were 3.0× 10^10 pfu/ml, the concentration of which was adequate for future test in vivo or in vitro. Conclusion The harvest of recombinant adenovirus vector of Ad -KGF, is the first step for the future test to investigate the effects of KGF in pulmonary diseases, and the possible gene therapy to treat pulmonary fibrosis.