Zhao Fuqian, He Suhui, He Lingge, Lin Miaofen, Wang Sen, Chen Zhangquan
Guangdong Key Laboratory for Medical Molecular Diagnostics, Guangdong Medical College, Dongguan 523808, China.
Guangdong Key Laboratory for Medical Molecular Diagnostics, Department of Clinical Immunology, Guangdong Medical College, Dongguan 523808, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2014 May;30(5):513-6.
To construct a prokaryotic expression system for interleukin-37 (IL-37) and prepare its polyclonal antibody.
The gene encoding mature interleukin-37 (IL-37m) was amplified by PCR and subcloned into prokaryotic expression vector pET28a. Then the recombinant plasmid pET28a/IL-37m was transformed into E.coli Rosetta and expressed under IPTG induction. The recombinant IL-37m was purified through Ni²⁺;-NT agarose gel column and the purified recombinant IL-37m was used as immunogen to immunize the BALB/c mouse. The titer and specificity of the mouse anti-IL-37 antibody were analyzed by ELISA, Western blotting and immunohistochemical staining, respectively.
The recombinant IL-37 was successfully expressed and purified, and the mouse anit-IL-37 antibody was successfully prepared. ELISA showed that the titer of the antiserum was 1:128 000. Western blot analysis revealed that the antibody reacted with IL-37 specifically. Immunohistochemical staining detection manifested the antibody could recognize the native IL-37.
The mouse anti-IL-37 antibody with high titer and specificity was successfully prepared.
构建白细胞介素-37（IL-37）的原核表达系统并制备其多克隆抗体。
通过PCR扩增编码成熟白细胞介素-37（IL-37m）的基因，并将其亚克隆到原核表达载体pET28a中。然后将重组质粒pET28a/IL-37m转化到大肠杆菌Rosetta中，在IPTG诱导下表达。重组IL-37m通过Ni²⁺-NT琼脂糖凝胶柱纯化，纯化后的重组IL-37m用作免疫原免疫BALB/c小鼠。分别通过ELISA、Western印迹和免疫组织化学染色分析小鼠抗IL-37抗体的效价和特异性。
重组IL-37成功表达并纯化，成功制备了小鼠抗IL-37抗体。ELISA显示抗血清效价为1:128 000。Western印迹分析表明该抗体与IL-37特异性反应。免疫组织化学染色检测表明该抗体能够识别天然IL-37。
成功制备了效价高且特异性强的小鼠抗IL-37抗体。
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