Fuqian Zhao1, Suhui He1, Lingge He1, Miaofen Lin1, Sen Wang1, Zhangquan Chen2. 1. Guangdong Key Laboratory for Medical Molecular Diagnostics, Guangdong Medical College, Dongguan 523808, China. 2. Guangdong Key Laboratory for Medical Molecular Diagnostics, Department of Clinical Immunology, Guangdong Medical College, Dongguan 523808, China.
Abstract
OBJECTIVE: To construct a prokaryotic expression system for interleukin-37 (IL-37) and prepare its polyclonal antibody. METHODS: 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. RESULTS: 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. CONCLUSION: The mouse anti-IL-37 antibody with high titer and specificity was successfully prepared.
OBJECTIVE: To construct a prokaryotic expression system for interleukin-37 (IL-37) and prepare its polyclonal antibody. METHODS: 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. RESULTS: 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. CONCLUSION: The mouse anti-IL-37 antibody with high titer and specificity was successfully prepared.