Effect of the neukaryotic vector expressing human keratinocyte growth factor (hKGF) gene on rat AEC II proliferation / 重庆医科大学学报

ABSTRACT

Objective:To construct the pEGFP-C2-hKGF vector,a neukaryotic expression plasmid of hKGF gene in order to provide experimental material for the studies of hKGF gene function and hKGF gene therapy afterward.Methods:The hKGF gene was amplified by reverse transcriptage polymerase chain reaction(RT-PCR).Total RNA was extracted from human embryo lung fibroblast(HLF)and then cloned into pMD18-T vector.The recombinant plasmid were screened and extracted.The pEGFP-C2-hKGF vector,an eukaryotic expression plasmid of hKGF gene was constructed after the object hKGF cDNA fragment was inserted into pEGFP-C2 vector and the recombinant were screened,cloned and extracted.The recombinant plasmid vector was transferred into AEC II,and expression of hKGF was assayed by immunohistochemistry.The effect of AEC II proliferation was analyzed by cell count.Results:609bp DNA fragment containing hKGF gene and restriction sites were obtained by RT-PCR.After cloned by pMD18-T vector and certified by restriction analysis and sequencing,pEGFP-C2-hKGF vector was successfully constructed by means of recombinant DNA technology.pEGFP-C2-hKGF encoding the green fluorescent protein(GFP),capable of directing the expression of functional hKGF successfully.Furthermore,hKGF had the effect on AEC II proliferation.Conclusion:The efficient expression vector of hKGF gene is constructed,which can be used to establish the hKGF gene therapy vector.