Construction And Identification Of Expression Plasmid Of IL-17A And Its ShRNA Plasmid Of Mice

PART ONE: CONSTRUCTION AND IDENTIFICATION OFEXPRESSION PLASMID OF IL-17A GENE OF MICEObjective To construct the eukaryotic expression plasmid of IL-17Agene of mice, and to investigate its ability of expression of IL-17A in293Tcells.Methods Design the primer of IL-17A gene according to thesequence of IL-17AmRNA in GenBank, add restriction enzyme sites to theterminations of the primer. The RNA from spleen cells of Balb/c mousewas reversed to cDNA, and the full-length CDS fragment of IL-17A wasamplified, and the target segment was cloned into the T-vector. AfterRestriction analysis and sequencing, successfully recombinational T-vectorwas digested by two restriction enzymes, then the target segment wasretrieved and inserted into the eukaryotic expression vectorpLVX-IRES-ZsGreen1. This gave the recombinational plasmidpLVX-IL-17A-IRES-ZsGreen1. The plasmid was identified by restrictionanalysis and sequencing, and then was transfected into293T cells in mediation of liposome. The transcription levels of IL-17A mRNA weredetected by fluorescent quantitative PCR, while expression levels ofIL-17A protein were detected by ELISA after transfection for24h and48h.Results We can see a segment about500bp when the digestedproducts of T-vector and recombinational plasmid were run with gelelectrophoresis, which was coincided with the target gene. Additionally,the sequence was in accordance with IL-17A gene. For24h and48h afterthe expression vector was transfected into293T cells, IL-17AmRNA levelof cells and IL-17A protein level in serum were significantly increasedwhen compared with that of control group(every P<0.05).Conclusion IL-17A gene was cloned from spleen cells of mice,IL-17A eukaryotic expression plasmid was constructed successfully, andplasmid can be used in the research of the function and molecularmechanism of IL-17A in the glomerular disease. PART TWO: CONSTRUCTION OF EXPRESSION PLASMIDS OFIL-17AshRNA OF MICE AND SCREENING OF EFFECTIVESEQUENCES TO INHIBIT IL-17AEXPRESSIONObjective To construct three expression plasmids of IL-17AshRNAof mice, to investigate their inhibitory effects on IL-17A in293T cells, andscreen the most effective sequence that inhibits IL-17A expression.Methods Three pairs of shRNA chains targeting the IL-17A geneand one pair control shRNA chain were designed using the software onlineprovided by corporate web sites named Invitrogen, and restriction enzymesites were added to the terminations of the chains. The Oligo DNA werefirst synthesized, then annealed to form double strand, and inserted into theexpression vector pLVX-shRNA. Three shRNA plasmids and one controlplasmid were constructed, which were named shRNA1、 shRNA2、shRNA3、 shRNAC, respectively. These plasmids were identified bysequencing, and then were co-transfected into293T cells with IL-17Aexpression vector in part one in mediation of liposome. The transcriptionlevels of IL-17A mRNA were detected by fluorescent quantitative PCR,while expression levels of IL-17A protein were measured by ELISA.Results Restriction analysis and sequencing proved that all therecombinational plasmids were constructed correctly. IL-17AmRNA levelsof total cells were significantly reduced in the group of shRNA1、shRNA2、 shRNA3after co-transfection for24h and48h as compared with theshRNAC group(every P<0.05), and the group of shRNA1had the leastexpression. IL-17A protein levels in the serum of each group were inaccordance with the IL-17AmRNA levels.Conclusion Three eukaryotic recombinational expression plasmidsand one control plasmid of IL-17A shRNA were constructed successfully.All the shRNA expression plasmids can inhibit IL-17A mRNA and proteinexpression in293T cells, and the plasmid shRNA1had the best inhibitoryeffects.