Chen Zhang-quan, Huang Zhen, Liang Xiao-dong, He Tian-wen, Lu Tian-tian
Department of Clinical Immunology, Guangdong Medical College, Zhanjiang 524023, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2008 Mar;24(3):250-2.
To prepare the rabbit antibody against the alpha3 domain of the human CD1d (hCD1d-alpha3).
The gene fragment coding for hCD1d-alpha3 was amplified by PCR and cloned into prokaryotic expression vector pET28, then expressed in E.coli BL21(DE3). The recombinant protein hCD1d-alpha3 was purified with Ni(2+)-NTA agarose column and used as immunogen to immunize the rabbit. The titer and specificity of the anti-hCD1d-alpha3 antibody from the rabbit were analyzed by ELISA, Western blot and immunohistochemistry, respectively.
The recombinant hCD1d-alpha3 was successfully expressed and purified, and the polyclonal anit-hCD1d-alpha3 antibody was successfully prepared. The titer of the antiserum was 1:6 400 by ELISA. Western blot analysis showed this antiboday reacted specifically with hCD1d. Immunohistochemistry analysis showed the antibody could recognize the native hCD1d in the human intestinal tissue.
The anti-CD1d-alpha3 antibody from the rabbit with high titer and specificity has been prepared with purified recombinant hCD1d-alpha3 as immunogen, which lays a foundation for further research into detection and functional study of CD1d.
制备抗人CD1d（hCD1d）α3结构域的兔抗体。
通过PCR扩增编码hCD1d-α3的基因片段，并克隆到原核表达载体pET28中，然后在大肠杆菌BL21(DE3)中表达。用Ni(2+)-NTA琼脂糖柱纯化重组蛋白hCD1d-α3，并将其用作免疫原免疫兔子。分别通过ELISA、Western印迹和免疫组织化学分析兔抗hCD1d-α3抗体的效价和特异性。
重组hCD1d-α3成功表达并纯化，成功制备了多克隆抗hCD1d-α3抗体。ELISA检测抗血清效价为1:6400。Western印迹分析表明该抗体与hCD1d特异性反应。免疫组织化学分析表明该抗体可识别人类肠道组织中的天然hCD1d。
以纯化的重组hCD1d-α3为免疫原，成功制备了效价高、特异性强的兔抗CD1d-α3抗体，为进一步研究CD1d的检测和功能奠定了基础。
