| Background: TLR4/MD-2 are essential components for LPS receptor to recognize LPS, the recognition of HSP60, EDA, Toxal and some other immune induction of cancer also depend partially on TLR4/MD-2. MD-2 not only combines to TLR4(TLR2), enhancing the signal upon binding their ligands, but also affects TLR4 distribution, meanwhile MD-2 can increase the expression of TLR4 (TLR2) protein, therefore MD-2 is regarded as an important modification factor, which regulates the function of TLR4 (TLR2) directly or indirectly during recognition of ligands, signal transduction and the distribution of receptors. MD-2 and TLR4 might therefore have close relationship with the occurrence of sepsis. Based on preliminary results, we speculated that the expression pattern of TLR4 and MD-2 in perioperative patients without endotoxemia could be affected by operation injury. Research plan: to observe the changes of TLR4/MD-2 in perioperative patients and discuss the clinical significance; to analyze the mechanisms changes in the expression of MD-2 with bioinformatics, and at last the protein expression of MD-2 in E.coli will be prepared for our next research needed.Main methods and material:1. Perioperative TLR4/MD-2 changes: twelve cardiothoracic surgery patients without endotoxemia and other infective complications were chosen. The monocyte was isolated from 11 milliliter of vein blood from patients and the expression of TLR4 and MD-2 were tested by the FCM. The levels of TNF-alpha and IL-10 in serum were examined by radioimmunoassay, and the concentration of the complex of TLR4/MD-2 was determined by ELISA. The plasma LPS was detected with limulus lysate test. Bioinformatics of MD-2: Hydrophilicity, Antigenicity , Accessibility and Flexibility of MD-2 was predicted with GoldKey software. And the average antigen index(AI) was calculated by using Wu's method. The secondary structure prediction and the functional sites analysis was carried out via the online servers of PHD maintained by EMBL and SOPMA(Lyon, France). The isoelectric point (PI) and molecular weight(MW) of MD2,2. GST/MD2 and GST/MD2-C were predicted by the program of Comput PI/MW. By using BLAST, PRS and Superfamily network servers, the domain on TRL4/TLR2 which might bind to MD-2, the proteins that may interact with MD-2 and the molecular structure characteristics of MD-2 were predicted.3. Expression of MD-2 in E.coli and its preliminary separation: the primers were designed according to the pGEX-4T-1 open reading frame. The MD-2 gene was subcoloned into pGEX-4T-1 by PCR through the temple of pEF-BOS/MD-2. The expressive vector of fusion protein of GST and MD-2, pGE/MD-2 ,was cut off 165 bp at the 5'end by the restrict endonucleases BamHI. A new modified expression plasmid pGE/MD-2-C was prepared. At the different temperature various dose of IPTG (isopropyl-β-D-thiogalaactopyranoside) added into LB to induce the expression of fusion protein in the DH-5α. The SDS-PAGE was used to analyze the volume of protein, and the western blot was used to identify the protein. At last, the preliminary fusion protein was separated through the elusion of inclusion body, carbamide denature and grades dialysis.Results: 1. TLR4/MD-2: The levels of LPS and TNF-α varied in normal range, however a marked increased level of IL-10 was found 3 day after the operation(p<0.001). The expression of TLR4 and MD-2 in monocytes elevated profoundly at 1 to 3 day, then restored to normal at day 5 following operation. It is interested that the increased serum level of MD-2 was found at 3 to 5 day after operation, but the TLR4 just has slightly elevated at the third day after operation. Meanwhile the complex receptor of TLR4/MD-2 also increased similar to the changes of the serum MD-2.2. Bio-informatics of MD-2: MD-2 is one kind of compact globin and no proteinase was found. It has two relatively separated domains which bind to specific proteins (C- terminal ) and its ligands (N- terminal) ,respectively. Some important functional sites (such as phospharylation, N-My...
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