Gene Engeneering Preliminary Studies Of Immunomodulatory Molecule BAFF/BAFF Receptor And Preliminary Analysis Of FADD Mutation Induced Enteritis

Inflammation was part of the complex biological response of vascular tissues to harmful stimuli, such as pathogens, damaged cells, or irritants. Inflammation was a protective attempt by the organism to remove the injurious stimuli and to initiate the healing process. However, as a common basic pathological processes, prolonged inflammation, known as chronic inflammation, leaded to progressive destruction of the tissue and then cause a host of diseases, such as periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer. In this study, target to the inflammatory response, new BAFF inhibitors was developed for the treatment of autoimmune diseases, and for FADD-/-TgD mice, the relationship between FADD phosphorylation and enteritis was explored.1. BAFF, as the new drug target of the treatment of autoimmune diseases, had been studied In-depth. BAFF inhibitors were also associated from belimumab, the earliest specific antibodies against sBAFF drugs, to a variety of other antibodies fusion protein drugs, such as atacicept (TACI-Ig fusion protein) and then blisibimod, which targets to the membrane BAFF and sBAFF. With the deepening of theoretical research, the role of the different forms of BAFF in disease development has gradually been recognized. So from K562 cell line cDNA, pET21a-sBAFF is constructed to express solution BAFF, and then control the pH to mimic the form of BAFF 60-mer; pET21a-mBAFF is constructed, which replace the position 217-224 in the BAFF "Flap zone" with Gly-Gly, prevent the formation of 60-mer and retain their receptor binding activity. Meanwhile signaling through TACI in mature B cells or plasmablasts was only achieved by higher-order BAFF oligomers, all of which were also potent activators of a multimerization-dependent reporter signaling pathway. The gene sequences which encoded TACI CRD2 region and BR3 ligand binding region were obtained from mouse spleen tissues, and then through mimicing the structure of TACI, The pET28a-TACI-CDR2, pET28a-TACI-BR3, pET28a-BR3-TACI were constructed, which expressed TACI CRD2 region and BR3 ligand binding region fuse protein that linked with (GGGGS) 3 linker peptide, with a view to enhance its binding capacity with BAFF multimeric forms.The recombinant plasmids pET21a-sBAFF、pET21a-mBAFF were transformed into BL21(DE3), and induced by IPTG for expression. All the recombinant proteins were expressed. Expression parameters including temperature、IPTG concentration were optimized to improve expression and yield of soluble expression of target proteins. For sBAFF, the optimal induction conditions is 28℃,1 mM IPTG. Low temperature fermentation can increase the production of soluble target proteins. The result show that under the conditions of 28℃,1 mM IPTG, the expression of sBAFF reached about 30.1% of total cellular proteins, and soluble protein content was 25.1%; the expression of mBAFF reached about 17.1% of total cellular proteins, and soluble protein content was 63.4%. The result of TACI-CDR2、TACI-BR3、BR3-TACI expression show that under the conditions of 37℃,1 mM IPTG, the expression of TACI-CDR2、TACI-BR3、BR3-TACI reached 25.4%,23.0%、27.4% of total cellular proteins, and soluble protein content were 68.6%、61.3%、81.9%, respectively. The construction, expression of the BAFF/BAFF receptor derivative lay the solid foundation for further in-depth investigation of biological function of BAFF/BAFF receptor.2. FADD, act as key protein in Fas/FasL signal pathway, and plays an essential role in apoptosis. Fas-associated death domain (FADD) recruits pro-caspase-8 homodimers, which are then autoproteolytically activated. Activated caspase-8 is released into cytoplasm, where it cleaves various proteins including pro-caspase-3, resulting in the caspase cascade and the final apoptosis. Recent studies showed that FADD, a classical adaptor protein involved in apoptosis, has also been reported important in cell proliferation, and this function is highly associated with its phosphorylation in its Ser191 in mice.Through genotyping, FADD-/-TgD mice is obtained. Pathological studies have showed that, intestinal from FADD-/- TgD mice were shorter and thicker compared to control macroscopically, and infiltration of F4/80+and Gr-1+ myeloid cells were detected in the intestinal epithelial cells of FADD’7TgD mice, suggesting that FADD-/- TgD mouse exists enteritis. Quantitative PCR analyzes the mRNA expression level of inflammatory cytokines. As FADD+/TgD a control, the mRNA expression levels of TNFa, INFy, MCP-1, IL17, CXCL12,cPLA2 in FADD-/-TgD small intestine were significantly increased, the mRNA expression levels of TLR4, IL-1β were also increased, the mRNA expression levels of TGFβ levels was down, that provide some idea for the pathogenesis of enteritis.