摘要
【论文特点介绍】既往研究表明角质化细胞生长因子（KGD对急性起病的ALI／ARDS，以及慢性进展的PF均有预防作用。因此，构建KGF、KGFR的重组腺病毒载体对于探索肺部疾病的治疗，探讨KGF，乃至其他生长因子在肺损伤免疫进程中的作用都是一个有力的工具。
Objective To construct the recombinant adenovirus vectors expressing mouse KGF or KGFR.
Methods （1） According to the gene order of mouse KGF and KGFR provided by the gene Bank in National Center for Biotechnology Information, we had designed the sense and the antisense primer, with which we amplified the KGF/KGFR gene by PCR, with cDNA acquired from mouse lung RNA using RT- PCR. （2） pShuttle-CMV with the drug resistance of kanamycin to be the carrying plasmid, we inserted the KGF or KGFR gene into it, to construct the shuttle vector pKGF and pKGFR. （3） We then linearized pKGF and pKGFR, transformed them, respectively, into the E.Coli BJ5183 which contain virus backbones, followed by the step transfection of HEK293 cells after identification of endonuclease Pac I and amplification. Finally, we harvested the virus after amplification, caesium chloride density gradient centrifugation purification and measurement of virus titers.
Results （1） The results of DNA sequencing suggested the success of construction of shuttle vector pKGF and pKGFR. （2） The bacteria could grow in the culture with kanamycin, as well as the agarose gel electrophoresis showed the proper strap, which had shown the success of construction of virus plasmid expressing KGF or KGFR. （3） HFK293 cells showed the pathologic morphologic changes, under microscope, which suggested the synthesis and duplication of recombinant adenovirus. （4） Finally, we had measured the virus titers, which had achieved the requirement of the future experiments in vivo and in vitro.
Conclusion We have successfully constructed the recombinant adenovirus vector of KGF or KGFR, which had supplied a tool to understand more about the role of KGF in pulmonary disease, especially as a cytokine in immune reaction, and even might be a new, a lower cost way to cure PF.
出处
《世界急危重病医学杂志》
2007年第3期1846-1846,共1页
internationl journal of emergency and critical care medicine
