Construction Of An Eukaryotic Plasmid Encoding TNFR2Mutation And It’s Effects On Macrophage Inflammatory Responses

AIM:To construct the eukaryotic expression vector of the human TNFR2gene mutation (wide TNFR2196MET and mutation TNFR2196ARG). Transfect the restructured plasmid to macrophages and establish table cell lines.Investigate the effect of tumor necrosis factor receptor superfamily-1B(TNFRSF1B)196gene polymoism(T mutation for G)on TNF/TNFR signal pathway mediated inflammatory response of the macrophage.METHODS:1. Artificially synth TNFR2196MET target fragment, and connected with pMD18-T simple carrier to form a cloning vector. Then through the design of mutagenic primers,PCR site-directed mutagenesis amplified the plasmid containing mutation. Transformed it into competent E.coli and positive clone was selected. Extracted the plasmid(pMD18-T simple-TNFR2196ARG) for sequencing test.2.The plasmids, PMD18-T simple-TNFR2196MET and pMD18-T simple-TNFR2196ARG,were cut by double enzyme HindⅢand EcoR I,including pcDNA6.0. TNFR2196MET and TNFR2196ARG were inserted into pcDNA6.0with T4ligase, and then transformed into competent E.coli and positive clone was selected. The recombinant plasmid,pcDNA6.0-TNFR2196MET和pcDNA6.0-TNFR2196ARG,were confirmed by restriction endonuclease analysed and sequencing test.3.Transfect the plasmid pcDNA6.0-TNFR2196MET, pcDNA6.0-TNFR2196ARG and pcDNA6.0using Lipofectamine2000to RAW264.7cells.After48h use blasticidin resistantly selected4weeks to establish stable cell lines.Observe under inverted microscope the growth status and morphous of RAW264.7cell.Cultivate the cells by enzymatic digestion method. Divided the cells into four groups:contro group, pcDNA6.0empty plasmid group, pcDNA6.0-TNFR2196MET group and pcDNA6.0-TNFR2196ARG group. Using RT-PCR method to detect the changes of TNFR2、 cIAP1and cIAP2mRNA;Western blot method to detect the protein expression of p-JNK cIAP1、cIAP2、TNFR2and the NF-kappa B e; ELISA test the level of sTNFR2、IL-1βand IL-6in clear liquid of cellsRESULTS:1.DNA sequencing showed that restructuring plasmid contains purpose TNFR2, and segments’base sequences was complete, and T successfully mutated for G,without the loss of the bases,2.DNA sequencing and restriction endonuclease digestion analysis indicated that the eukaryotic expression plasmid pcDNA6.0-TNFR2196MET和pcDNA6.0-TNFR2196ARG were constructed successfully.3.Established RAW264.7cell line which stablely expressed plasmid pcDNA6.0-TNFR2196MET and pcDNA6.0-TNFR2196ARG.In pcDNA6.0-TNFR2196ARG group mRNA level of TNFR2、 cIAP1、cIAP2were significantly decreased with pcDNA6.0-TNFR2196MET(P<0.05). Western blot results showed that in pcDNA6.0-TNFR2196ARG group the protein expression of p-JNK、 cIAP1、cIAP2、TNFR2and NF-kappa B were statistically significant decreased with pcDNA6.0-TNFR2196MET group (P<0.05). ELISA showed that in pcDNA6.0-TNFR2196ARG group level of sTNFR2、IL-1β and IL-6were significant decreased with pcDNA6.0-TNFR2196MET (P<0.05). And with the control group and empty plasmid group compared, both pcDNA6.0-TNFR2196MET group and pcDNA6.0-TNFR2196ARG group antiapoptotic and former inflammation factors TNFR2, cIAPl, cIAP2, IL-1β, IL-6, the NF-kappa B and p-JNK expression increased (p<0.05).CONCLUSIONS:1.Construction of pcDNA6.0-TNFR2196METand pcDNA6.0-TNFR2196ARG eukaryotic expression plasmid was successful. Then establish successfully stable cell lines.This study for the next step on angiocardiopathy research laid the experimental foundation.2. The expression of the antiapoptotic and former inflammation factors TNFR2, cIAPl, cIAP2, IL-1β, IL-6, p-JNK, the NF-kappa B TNFR2196ARG in mutant type were decreased with the wild type.TNFR2196ARG through the TNF/TNFR signaling pathways mediated inflammation, may be a chronic inflammation of the molecular mechanism of coronary heart disease.