人源程序性死亡配体PD-L2蛋白的原核表达与抗血清制备
Prokaryotic expression and antiserum preparation of human programmed ligand PD-L2 protein

利用原核表达系统获得包涵体表达形式的重组蛋白hPD-L220~123（IgV结构域）和hPD-L220~208（IgV结构域和IgC结构域）；将重组蛋白利用Ni-NTA柱亲和层析纯化与复性后，作为免疫原免疫Balb/c雌性小鼠制备鼠抗人的多抗血清，经ELISA法检测其效价均达到1∶106；Western-blot实验结果表明，制备的多抗血清均能特异性识别hPD-L2蛋白. 以制备的多抗血清进行IHC实验评估与鉴定，结果显示：以hPD-L220~123重组蛋白为免疫原制备的多抗血清阳性较弱且定位模糊，以hPD-L220~208重组蛋白为免疫原制备的多抗血清具有很强的膜定位且背景清晰.
The recombinant proteins hPD-L220-123 (the major antigenic epitope region of human PD-L2) and hPD-L220-208 (the extracellular domain of human PD-L2) were expressed in inclusion bodies. The two recombinant proteins were purified by Ni-NTA agarose resin and refolded. Then，the rat antiserums against the recombinant proteins of hPD-L220-123 and hPD-L220-208 were prepared with titers of 1∶106 measured by indirect ELISA. Western-blot experiments demonstrated that the prepared antiserums can specifically recognize hPD-L2 protein. Finally，lung cancer tissues with high level expression of hPD-L2 were used to assay the location of the prepared antiserums by IHC experiments，the results indicating that the membrane localization of the antiserum prepared by antigen of hPD-L220-123 protein was blur and weak；the membrane localization of the antiserum prepared by antigen of hPD-L220-208 protein had a specific localization，with less background and non-specific staining