兔抗重组人钙网蛋白抗体的制备及其特性鉴定

Preparation of rabbit anti-recombinant human calreticulin antibody and its characterization

目的:制备兔抗人钙网蛋白抗体并进行特性鉴定。方法:用PCR法扩增人钙网蛋白(CRT)基因,并克隆至pET32a表达载体中。以重组质粒pET32a/hCRT转化大肠杆菌BL-21(DE3),用IPTG诱导重组蛋白的表达,通过Ni2+-NTA琼脂糖凝胶柱亲和层析纯化目的蛋白。以纯化的hCRT为免疫原免疫新西兰大白兔制备抗体,用ELISA法、Westernblot和免疫组化染色法检测抗体的效价和特异性。结果:成功地表达并纯化hCRT重组蛋白。以纯化的重组hCRT免疫新西兰大白兔制备的抗体,ELISA检测其效价为1∶51 200,Western blot分析显示,该抗体能与hCRT特异性结合。免疫组化染色法检测结果表明,该抗体能识别天然的hCRT。结论:以重组hCRT为免疫原,成功地制备效价高、特异性好的兔抗hCRT抗体,为进一步进行钙网蛋白的检测及其临床应用研究奠定了基础。

AIM： To prepare the rabbit anti-recombinant human calreticulin（hCRT） antibody and its characterization.METHODS： The gene coding for hCRT was amplified by PCR and cloned into prokaryotic expression vector pET32a.Then the recombinant plasmid pET32a/hCRT was transformed into E.coli BL21（DE3） and expressed under IPTG induction.The recombinant hCRT was purified through Ni2＋-NT agarose gel column and the purified hCRT used as immunogen to immunize the rabbit.The titer and specificity of the rabbit anti-hCRT antibody were analyzed by ELISA,Western blot and immunohistochemical staining,respectively.RESULTS： The recombinant hCRT was successfully expressed and purified,and the polyclonal anit-hCRT antibody was successfully prepared.The titer of the antiserum was 1∶51 200 by ELISA.Western blot analysis showed that the antibody reacted specifically to hCRT.Immunohistochemical staining detection manifested the antibody could recognize the native hCRT.CONCLUSION： The rabbit anti-hCRT antibody with high titer and specificity has been successfully prepared,which lays the foundation for further research on detection of hCRT and its clinical application.