Expression Of Recombinant Arresten And Effect Of Polymethoxylated Flavones On Insulin Secretion In Mouse Pancreatic Island β Cell Line INS-1

Malignancies and diabetes are two critical diseases which do serious harm to human healthy, and to each of them there were no efficient therapy and preventing method. So, exploiting new high effective drugs and methods to establish effective prevention measures is a longterm goal for every researcher.Arresten is a 26-kDa anti-angiogenic fragment proteolysis from the a 1 chain of type IV collagen. This molecule inhibits endothelial cell proliferation, migration, tube formation and matrigel neovascularization. It functions as an efficient inhibitor of angiogenesis and tumor growth in mouse models. As a new potential clinical anti-angiogenic factor, Arresten is expected to be a prospective anti-angiogenic drug. By now, many research institutions are looking for a suitable protein expression method in order to satisfy future therapy. In this study, by using primer designing, PCR and restrict digestion, we cloned Arresten gene into prokaryotic and eukaryotic expression plasmid respectively. This lays foundation for constructing suitable Arresten expression system and further study of the relationship between structure and function. We also analyzed the effect of Polymethoxylated Flavones on insulin secretion in mouse pancreatic islandβcell line INS-1.Animal cell culture,MTT,ELISA and radioimmunoassay et al. were adopted in this process. Collectively, these data sheds more light into the research of the antidiabetic mechanism of Polymethoxylated Flavones, also it provides theoretical and technical basis for looking for natural antidiabetic supplements and making full use of resourceful citrus.Result:1. The recombinant Arresten-pET22b (+) prokaryotic expression was successfully constructed. Preservative Arresten was amplified by PCR. PCR product was digested with HindⅢ/BamH I, and ligated into predigested pET22b (+). Recombiant plasmid was identified by restriction PCR, enzyme digestion and sequenceing.2. The recombinant Arresten-pET22b(+)was successfully transformed into E. coli B121 (DE3) using CaCl2 transformation method for expressing, The expression level of soluble Arresten was detected by quantitative SDS-PAGE and Western blotting analysis.This recombinant protein(with His-tag) was isolated using affinity chromatography.3. Recombinant Arresten eukaryotic expression was successfully constructed. Preservative Arresten was was amplified by PCR. After digestion, it was ligated into predigested pEGFP-N1, leaving Arresten on the upstream of GFP. Restriction enzyme digestion and sequenceing were adopted to identify recombinant plasmid.4. The pEGFP-N1-Arresten recombinant vector was used to transfect HEK293 A using liposome. The recombinant protein was successfully expressed in it as Arresten-GFP fusion protein. We successfully obtained a transient expression system of HEK293A.5. Polymethoxylated Flavones was used to stimulate INS-1, and cell viability was analyzed by MTT. Compared with control group, there are no significant difference when the concentration was at the range of 10μg/mL～80μg/mL.6. The result of ELISA, radioimmunoassay and RT-PCR primarily revealed that Polymethoxylated Flavones can not stimulate insulin secretion of INS-1 derectly. Result showed that after 24h affected by Polymethoxylated Flavones, insulin secretion in INS-1 had no obvious change.