1.Gene Cloning、Expression And Purification Of Human TNFR1-PLAD And Its Mutants As Well As Ientification Of Their Biological Activity2.Gene Cloning And Expression Of Murine TNF-α And Its Mutants

The extracellular domain of TNFR1contains four cysteine-rich domains (CRDs),The CRD1-containing region was necessary and sufficient for homotypic assembly oftrimeric TNFR complexes that bind TNF-α and mediate signaling. Because itpromoted receptor trimer assembly in the absence of ligand, this domain was termedthe pre-ligand binding assembly domain (PLAD). In our previous study, TNF-αbinding peptide (TBP) and TNFR blocking peptide (TRBP) were screened from aphage random peptide library, using soluble hrTNF-α and soluble TNFRI as a specificbait, respectively. The blocking effects of these peptides on the biological actions ofTNF-α were shown in vivo and vitro. However, the peptide has short half-life andfiltering out easily through renal glomerulus, it’s hard to make full use of theirpharmaceutical effect. Therefore, it is necessory to obtain the key peptide which werenecessary and sufficient for homotypic assembly of trimeric PLAD complexes bymolecule cloning technique, and then to connect the key peptode with TBP or TRBPin order to prolong the half-life of TBP or TRBP. This study will provides somebasis to develop TNF-αrelated anti-inflammation drugs in future. With the improvement of peoples lives, obesity has become more and morecommon.There are now more overweight than underweight people worldwide. Withthe prevalence of obesity, the incidence of disorders related obesity, such as metabolicsyndrome, non-alcoholic fatty liver disease as well as type2diabetes mellitus, is alsoincreasing. Insulin resistance plays a crucial part in the pathogenesis of all thesedisorders. However, the cellular mechanisms involved in insulin resistance are stillpoorly understood. TNF-α as a adipokine is secreted by adipose tissue and is involvedin a variety of physilogical and pathological process, such as regulating appetite,insulin sensitivity and inflammation. TNF-α can be divided into two types: secretoryTNF-α (sTNF-α) and transmembrane TNF-α (tmTNF-α), both of them can bind TNFreceptor, but resultes in different effects. In our previous study, tmTNF-α can killcancer cells which resistant to sTNF-α, and sTNF-α mainly induce necrosis,tmTNF-α induce apoptosis. We supposed that tmTNF-α and sTNF-α may playdifferent roles in insulin resistant. In this study, to compare the effects and molecularmechanism of two types of TNF-α in insulin resisitants induced by high level ofglucose. we cloned and constructed the plasmid pIRES2-GFP-wtTNF, pIRES2-GFP-tmTNF-α (only produce tm TNF-α) as well as pIRES2-GFP-sTNF-α (only secretsTNF-α), and stably and respectively transfected the plasmids into3T3-L1cells, andthen identified the protein expression.