Construction，expression Of The Inflammatory Factor Daintain/AIF-1(Y54A) Mutant And Its Function In Raw264.7

Daintain,a new bioactive polypeptide was isolated and characterized from porcineintestines by Chen et al in1994. The amino acid sequence is90%homologous withAllograft inflammatory factor-1(AIF-1) which was cloned by Utans et al from activatedmacrophages of rat atherosclerotic allogenic heart grafts. Therefore, we call thepolypeptide as "Daintain/AIF-1". This polypeptide is expressed in macrophages andsecreted from these cells. It has been characterized as a novel multifunctionalinflammatory factor taking part in vasculopathy, allograft rejection, cancer, andautoimmune diseases.Daintain/AIF-1is a17kD cytokine which is consisted with146amino acid. It hasbeen definited as a member of EH domain family with two EF-hand structures. Somestudy reveals one of the EF-hand has the ability of binding Ca2+. MutantDaintain/AIF(D95A) lost the ability of binding Ca2+, which resulted in the loss ofVSMC immigration ability. In addition, Daintain/AIF-1has a motif which can combinewith PDZ domain, tyrosine phosphorylation site(site37,54,103,124), a–KR–KK–GKR–motif.The tyrosine phosphorylation is an important post-translational modification. It playsan important role in cell signal transduction. The frequency of tyrosine phosphorylation incells is very low. This lower level of tyrosine phosphorylation makes the cells sensitivelyand accurately response to stimulation of the internal and external signals.According to the primary structure study of Daintin/AIF-1, we reasonably assue thatDaintin/AIF-1could be phosphorylated or dephosphorylated during carring in functions.Using immunoprecipitation method, we detect Daintin/AIF-1expression inpcDNA-Daintain stable transfected Raw264.7cell line after17-β-estradiol (E2)stimulation. Meanwhile, we detect Daintain/AIF-1tyrosine phosphorylation with the useof antibody PY20after immunoprecipitation. Taking into account the possibility ofDaintain/AIF-1antibody non-specific binding, we also find a positive result in thenegative control. Considering that such factors can not be avoided, we use site-directedmutagenesis method to make further identification of Daintain/AIF-1tyrosine phosphorylation and its phosphorylation sites.By bioinformatics prediction, the Daintain/AIF-154tyrosine residue could play animportant role in Daintain/AIF-1regulation. The main purpose of this study is todetermine the effect of54tyrosine mutated Daintain/AIF-1on macrophages Raw264.7,and to further speculate whether54tyrosine is a phosphorylated site. In this study, thefinished work is mainly the following aspects:1. Enrich protein Daintain/AIF-1specificity in pcDNA-Daintain stable transfectedRaw264.7cell lines with the methods of Immunoprecipitation.2. Detect Daintain/AIF-1’s tyrosine phosphorylation with the use of antibody PY20.3. Use bioinformatics methods to determine the54tyrosine of Daintain/AIF-1playsan important role in Daintain/AIF-1’s functionalregulation.4. Construction of Daintain/AIF-1(Y54A)-PET23a by overlap PCR.5. Purification of Daintain/AIF-1(Y54A).6. Daintain/AIF-1(Y54A) can remain promoting Raw264.7proliferation andreleasing NO.In present study, we find out that there is no difference between Daintain/AIF-1andDaintain/AIF-1(Y54A) when they are acting with Raw264.7. Therefore, the result suggestthat no phosphorylation at54tyrosine in Daintain/AIF-1.