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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。
Objective: To prepare rabbit anti-human calreticulin antibody and characterize it. Methods: Human calreticulin (CRT) gene was amplified by PCR and cloned into pET32a expression vector. Escherichia coli BL-21 (DE3) was transformed with recombinant plasmid pET32a / hCRT. The recombinant protein was induced with IPTG. The recombinant protein was purified by Ni2 + -NTA agarose gel column affinity chromatography. The purified hCRT was used as immunogen to immunize New Zealand white rabbits to prepare antibody. The antibody titer and specificity were detected by ELISA, Western blot and immunohistochemistry. Results: hCRT recombinant protein was successfully expressed and purified. The purified recombinant hCRT was used to immunize New Zealand rabbits. The titer of the antibody was 1:51 200 by ELISA. Western blot analysis showed that the antibody could specifically bind to hCRT. Immunohistochemical staining results showed that the antibody can recognize natural hCRT. Conclusion: The recombinant hCRT was used as immunogen to prepare high titer and good specificity rabbit anti-hCRT antibody, which laid the foundation for the further detection of calreticulin and its clinical application.