| Objective:To study the effect that K15 protein of Kaposi’s sarcoma herpesviruses promotes proliferation and migration in endothelial cell as well as the relationship between those effects and SOCE, and also the molecular mechanisms that K15 protein promotes cell proliferation and migration.Methods:(1) Generation of polyclonal antibody of K15 protein:After select of eighth exon (EX8) sequence of K15P gene as a template, the recombinant plasmid named pQE-80L-K15Pex8 was constructed by ClonExpressTM Ⅱ One Step Cloning Kit. Recombinant protein was induced and expressed with inducer. The location of recombinant protein was identified by 10% SDS-PAGE and the protein concentration was determined. Then three New Zealand white rabbits were immunized with the K1SPex8 to generate polyclonal antibodies against K15P. Indirect Enzyme-linked immunosorbent assay (ELISA) was applied to characterize the titers of the polyclonal antibody. The two recombinant plasmids, pFJ and pFJ-K15P, were transfected HEK 293T cells to produce K15P-Flag fusion protein, which was identified by western blot.(2) Generation of lentivirues:The gene region of K15P and K15P(YF), amplified by ordinary PCR, was cloned into pCDH-CMV-GFP vector with restriction enzyme, Nhe I. The recombinant plasmid was transiently tansfected into HEK 293T cell. The pCDH-CMV-K15P-GFP vector was cotansfected with three assisted plasmids pMDL-PRRE,pRSV-Rev,pMD2.g into HEK 293T cell to make lentivirus and western blot detected the expression of Kl 5P protein.(3) Relationship between the effect of K15 protein and SOCE:Three plasmids, pFJ, pFJ-K15P, pFJ-K15P(YF),were transfected into HEK 293T. The change of intracellular calcium concentration was detected by Leica TCS SP5. At the same time, three kinds of lentiviruses were infected into endothelia cell, EA.hy 926 and the change of intracellular calcium concentration was detected by Leica TCS SP5.The STIM 1 and Orail 1 protein were detected by western blot.(4) Molecular mechanisms of proliferation and migration with K15 protein:The proliferation was detected by CCK-8 kit in HEK 293T and EA.hy 926. Then the change of cell migration was observed by wound scratch assay in EA.hy 926.Results:(1) The concentration of purified recombinant protein was 152.79 p.g/ml. The titer of the polyclonal antibody against K15P was more than 1:6400 with indirect ELISA and could react specifically with the K15P-Flag fusion protein in 293T cells.(2) The lentiviruses vector,K15P and K15P(YF), were successfully constructed and the titer of lentivirus was 4×106 and 2×106 transducing units (TU)/ml obtained for 48 hours after cotansfected. K15 protein was successfully detected by western blot.(3) SOCE was significantly enhanced in the group of K15P compared with the group of pFJ and K15P(YF) in HEK 293T and the same results can get in EA.hy 926. STIM1 protein was significantly reduced in the group of K15P(YF) and there is no change in others. Then the Orail1 protein was significantly increased in the group of K15P compared with other groups.(4) The proliferation and migration were enhanced in the group of K15P compared with other groups in HEK 293T and EA,hy 926.Conclusion:We preliminarily found K15Pex8 protein have immunoreactivity and the rabbit polyclonal antibody against K15P could react with the K15P-Flag fusion protein. Lentiviral vector was successfully constructed and the lentiviral expression system of K15 gene also establishes successfully and efficiently. In HEK 293T and EA.hy 926, K15P protein can enhance the SOCE and the expression of Oraill and there is no change in the expression of STIM1, but K15P(YF) protein reduced the SOCE and the expression of STIM 1 and Oraill. |
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