[Construction and identification of the eukaryotic expression vector pIRES2-EGFP-IL-1ra-Fcepsilon].
The cDNA of IgE constant domain of rat was cloned from the spleen of allergy asthma rat by RT-PCR. The IL-1ra segment was obtained from intermediate vector pBV220-IL-1ra. By overlap extension PCR, the fusion gene IL-1ra-Fcepsilon was cloned, then inserted into the eukaryotic expression plasmid pIRES2-EGFP to obtain a recombinant expression plasmid pIRES2-EGFP-IL-1ra-Fcepsilon. The recombinant expression plasmid was transfected into 293T cells using lipofectamin and instillated into the rat lung through trachea. The expression of IL-1ra-Fcepsilon was identified by Western blot, RT-PCR, and this protein could inhibit the activity of IL-1 in vitro. Green fluorescent protein could be detected in the transfected 293T cells and the rat lungs at different times. The research paved the way for the gene therapy of allergy asthma.
Liu
Zhong Cheng
ZC
Institute of Basic Medical Sciences, Academy of Military Medical Sciences, Beijing.
Wang
Yuan Yuan
YY
Zou
Min Ji
MJ
Wang
Jia Xi
JX
Xu
Dong Gang
DG
D000818:Animals; D002460:Cell Line; D005260:Female; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics*; D006801:Humans; D007073:Immunoglobulin E / Q000737:chemistry; D007140:Immunoglobulin Fab Fragments / Q000235:genetics*; D053590:Interleukin 1 Receptor Antagonist Protein / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D007140:Immunoglobulin Fab Fragments; D053590:Interleukin 1 Receptor Antagonist Protein; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D007073:Immunoglobulin E