摘要
目的制备针对胚胎小鼠心肌L型钙通道Cav1.3编码的α1D亚基胞内末端的多克隆抗体。方法通过基因重组从小鼠胚胎心脏中获得2种原核表达质粒pGEX-4T-α1DN/α1DC,在大肠杆菌中经异丙基-β-D硫代半乳糖苷(IPTG)诱导表达谷胱甘肽S转移酶(GST)融合蛋白,纯化得到α1D基因N端和C端产物GST-α1DN/α1DC,用此抗原经弗式佐剂、弗式不完全佐剂乳化后免疫新西兰家兔,收集的免疫血清经protein G Agarose亲和纯化后获得IgG型抗α1D蛋白的多克隆抗体。对这2种抗体采用Westernblot法进行特异性检测,ELISA法测定进行效价分析。结果成功构建α1D胞内片段的原核表达载体,表达纯化了GST融合蛋白GST-α1DC/α1DN,制备2种IgG型多克隆抗体,经ELISA检验证实二者效价均较高,滴度在1:256000时仍有信号,其中GST-α1DN的效价更高。用Westernblot法检测发现,2种抗体都均在相对分子质量240000处得到单一的蛋白印迹条带,特异性良好,能识别小鼠心肌细胞中α1D蛋白。结论用重组融合蛋白GST-α1D作为抗原制备出了具有高效价、强特异性的α1D抗体,为探讨α1D蛋白在小鼠胚胎心脏发育过程中分布的时空特征及研究胚胎发育过程中的钙离子相关信号调控活动奠定了基础。
Objective To prepare high titer and specific polyclonal antibody of α1D subunit of L-type calcium channel encoded by Cav1.3 from mouse embryonic heart.Methods Prokaryotic expression plasmids pGEX-4T-α1DN/α1DC was obtained by recombination from mice embroynic heart.The recombinant of pGEX-4T-α1DN/α1DC was transformed into E.coli BL-21,induced and expressed by Isopropyl-β-D-thiogalactopyranoside（IPTG）.Then glutathione S-transferase（GST） fusion proteins that recognize N terminus and C terminus of α1D respectively were highly expressed in E.coli and purified.Rabbits were immunized with purified GST-α1DN/α1DC.IgG type polyclonal antibody of α1D generated from gathered antiserum purified by protein G Agarose.The specificity of 2 kinds of antibody were tested by Western blot and the title of them were identified by enzyme linked immunosorbent assay （ELISA）.Results The prokaryotic expression plasmid pGEX-4T-α1DN/α1DC were recombinated,the GST fusion protein GST-α1DN/α1DC were expressed and purifed and these 2 kinds of polyclonal antibody were prepared successfully.The titer of antibody by ELISA was 1：256 000 and the titer of anti-α1DN was higher than the titer of another.Western blot showed that α1D proteins extracted from mice heart were specifically recognize by either anti-α1DN or anti-α1DC.Conclusions Purified fusion GST-α1D proteins expressed by recombinants of pGEX-4T-α1D were used as antigen and the α1D polyclonal antibody with high titer and strong specificity has been successfully prepared.They will be beneficial to investigate the temporal and spatial expression pattern of α1D protein and to study the regulation of signal transduction concerning calcium ion during mouse embryonic heart development.
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2009年第23期1845-1847,共3页
Journal of Applied Clinical Pediatrics
基金
国家自然科学基金项目资助(30570662)
国家重点基础研究发展计划项目资助(2007104GJB0117)
关键词
胚胎心脏
多克隆抗体
小鼠
embryonic heart
polyclonal antibodies
mouse
