Expression-purification, Anti-inflammatory And Subcellular Location Study Of Annexin B1

Neurocysticercosis (NCC), caused by the larval stage of Taenia solium, is an important parasitic disease of the human nervous system and constitutes a public health problem. Cysticerci locats in a cavity lined by host epitheliod cell originating from small vessels, this epitheliod cell layer, which was named bladder wall (BW), constitutes the host-parasite interface in cysticercosis. Cysticerci may persist in BW for a long period of time, often for years only eliciting slight surrounding host inflammatory reaction. It indicates that living cysticerci may cause an asymptomatic infection through active evasion and suppression of immunity and inflammation.Annexin Bl is a new gene isolated from the cDNA library of cysticercus cellulosae by immunological screening.bioinformatics analysis revealed that it is indeed a nobel member of annexin family. The annexins are a multigene family of proteins characterized by their capacity of reversibly binding to anionic phospholipids in a Ca²⁺-dependent manner. Members of the family have been implicated in a widely distributed in eukaryotes. As the first was found in platyhelminth, studies on annexin Bl can be helpful to make clear what role it plays in the infection,loading and other activies of cysticercus cellulosae, which may provide some new ways to prevent and control this diasease. Furthermore, annexin Bl can also act as a molecule model for studying the evolutionary divergence of annexins. Non-fusion espression plasmid of annexin Bl was constructed, the protein was expressed as soluble form in E.coli. A new purification method for annexin Bl has been developed and established. We make good use of the calcium-dependent phospholipids binding character which is one of the typical features of annexin superfamily. Immunohistochemistry showed that annexin Bl expressed in cystwall and peripheral muscle, especially in the region with high-grade of inflammation.It suggests that the protein possibly secreted into extracellular miliue and decreases infection responsible in order to protect the parasite. So we research the intracellular relocation and the anti-inflammatory function of the protein.The main results of our research are as follows:1. Non-fusion espression vector of annexin Bl was constrcted, the protein was expressed as soluble form in E.coli. A new purification method for annexin Bl has been developed and established. The method makes good use of the calcium-dependent phospholipids binding character which is one of the typicalfeatures of annexin superfamily. The Ca2+-triggered precipitation method which is fast, easy to manipulate is fit for all of the family members.Followed by two steps of chromatography we got the protein which the purity was greater than 95%.2. We examined the effect of recombinant annexin Bl on the ability of cytosolic phospholipase A2 (cPLA2) to hydrolyze phospholipids in vitro. We used phosphatidylserine liposome and phosphatidylcholine liposome as phospholipids substrates and monitored cPLA2 activity with a continuous fluorescence displacement assay. AnxBl can inhibit cPLA2 activity under our experimental conditions and this effect depends on millimolar concentrations of Ca2+ and presence of anionic phospholipids used as substrates, suggesting that anxBl may inhibit cPLA2 activity in vitro by the substrate depletion mechanism.3. In this study, we have constructed the pEGFP- annexin Bl plasmid, and transfected to siHa cells. We study the intracellular relocation of the protein under the confocal microscope after treatment by PMA and ionomycin.To monitor the concentration of intracellular calcium,the cells were loaded the Rhod-2 .Annexin Bl relocation to the plasma membrane stimulate by PMA whereas it relocation to plasma and nuclear membrane. It seems that PMA increase anxnexin Bl binding, which may facilitate the exocytosis.And the reclocation of it to plasma and nuclear membrane stimulated by ionomycin, must be related to the some function of the protein.