Expression Of Recombinant AnnexinⅡ Protein In E.coli And AnnexinⅡ MRNA Expression In Human Acute Leukemia

Annexin II belongs to a family of widely distributed, phospholipid-binding, calcium-regulated proteins known as the annexins. Annexin II is richly expressed in endothelial cells, mononuclear macrophages, nerve cells and some tumor cells. Through the expression of independent binding sites for both plasminogen and tissue plasminogen activator (t-PA), annexin II assembles these 2 proteins on the cell surface, thereby accelerating the production of plasmin. The current study found that the overexpression of annexin II as the predominant causative factor in the coagulopathy associated with APL. In addition, the study found that annexin II was related with migration and metastasis of tumor and angiogenesis. In our research work, we got recombinant annexin II protein from E.coli, and immunized Balb/c mice with the protein to obtain the specific monoclonal antibodies against annexin II. We used the monoclonal antibodies established a flow cytometry method to detect the expression level of annexin II on the cell surface. Furthermore, in order to explore the functions of annexin II, we measured the annexin II mRNA expression in acute leukemia cell lines as well as bone marrow mononuclear cells from acute leukemia patients with the fluorescence quantitative PCR (Rea1-time PCR) method.The expression of recombinant Annexin II proteinWe transformed the vector that containing full-length cDNA of annexin II into E. coli BL21 (DE3) pLysS. The positive clones were selected and induced by IPTG. The recombinant protein size is 39kDa. Before use it to immune mice, the recombinant protein was purified by Ni-NTA agarose column. After the spleen cells of the mice and hybridoma cells SP2/0 were hybridized, we got two clones that can constantly secreting McAb against annexin II. These two clones were named SZ135 and SZ136, and their titers of McAbs in ascites were 5×10-5and 6×10-5 respectively. The heavy chain of the McAbs both belonged to IgG1 subclass. The concentration of ascites were 0.25mg/ml and 0.5mg/ml after purified by proteinG-Sephrose4B affinity chromatography column. The antibodies could not only identify the recombinant 39kDa annexin II protein but also 36 kDa protein in NB4 and U937 cell lysate.Annexin II mRNA expression in human acute leukemiaReal time PCR was used to examine the expression of annexin II mRNA in different cell lines. It was found that NB4 had the highest quantity of annexin II mRNA, U937 and SHI-1 also had high expression. Among 81 newly diagnosed Als, the annexin II expression level in M3(APL) was highest in all subtypes.Furthermore,M4 and M5 subtypes also had high expression. No statistic difference was found between the M1 and M2 groups with control group. Conclusion: In addition to M3 subtype, M4 and M5 also had high expression of annexin II mRNA and these two types had the highly infiltration capacity. We speculate to adopt Annexin II as a plasminogen, tissue-type plasminogen activator co-receptor, to promote the generation of plasmin, thereby starting proteolytic cascade, selective degradation of extracellular matrix proteins involved in invasion and infiltration process.