[Preparation and identification of rabbit anti-mouse coiled-coil domain containing 189(Ccdc189)polyclonal antibody].

Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology Pub Date : 2023-05-01

Huiping Zhou, Fan Yang, Lu Yuan, Changmin Niu, Ying Zheng

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Abstract

Objective To prepare a rabbit anti-mouse coiled-coil domain containing 189 (Ccdc189) polyclonal antibody. Methods The pET-28a-Ccdc189 prokaryotic expression plasmid was constructed and transformed into E.coli BL21. IPTG was used to induce the expression of Ccdc189 prokaryotic protein. Adult male New Zealand rabbits were immunized with purified recombinant protein to obtain rabbit anti-mouse Ccdc189 polyclonal antibody. The specificity of the polyclonal antibody was identified by Western blot analysis, indirect ELISA and immunofluorescence histochemical staining. Results The pET-28a-Ccdc189 recombinant plasmid was successfully constructed and the expression of the Ccdc189 recombinant protein was induced. ELISA revealed that the titer of the polyclonal antibody was 1:1 000 000. Western blot and immunofluorescence staining demonstrated that the Ccdc189 polyclonal antibody could specifically identify the Ccdc189 prokaryotic protein and the Ccdc189 protein in adult wild-type mouse testis. Conclusion A polyclonal antibody with high specificity against mouse Ccdc189 was successfully created.

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[兔抗小鼠线圈结构域189(Ccdc189)多克隆抗体的制备与鉴定]。

目的制备含189 (Ccdc189)的兔抗小鼠线圈结构域多克隆抗体。方法构建pET-28a-Ccdc189原核表达质粒，转染大肠杆菌BL21。利用IPTG诱导Ccdc189原核蛋白的表达。用纯化的重组蛋白免疫成年雄性新西兰兔，获得兔抗小鼠Ccdc189多克隆抗体。经Western blot分析、间接ELISA和免疫荧光组织化学染色鉴定该多克隆抗体的特异性。结果成功构建了pET-28a-Ccdc189重组质粒，并诱导了Ccdc189重组蛋白的表达。ELISA结果显示，该多克隆抗体的效价为1:1 000 000。Western blot和免疫荧光染色表明，Ccdc189多克隆抗体能特异性鉴定Ccdc189原核蛋白和成年野生型小鼠睾丸中的Ccdc189蛋白。结论成功制备了一种具有高特异性的小鼠Ccdc189多克隆抗体。

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Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology

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