摘要
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.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2011年第6期641-643,共3页
Chinese Journal of Cellular and Molecular Immunology
基金
广东省湛江市科技招标项目(2007年度)
广东省医学科研基金项目(A2008653)
关键词
钙网蛋白
表达
抗体
制备
兔
calreticulin
expression
antibody
preparation
rabbit
