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《Journal of Applied Clinical Pediatrics》 2009-23
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Preparation and Identification of Polyclonal Antibody of Mouse Embryonic Cardiac Calcium Channel α1D

ZHAI Su-lan1,2,ZHANG Sha-sha1,ZHAO Zhong-zhong1,QIAN Hui1,ZHANG Zhao1(1.Jiangsu Key Laboratory for Molecular and Medical Biotechnology,College of Life Science,Naijing Normal University,Nanjing 210046,Jiangsu Province,China;2.Department of Pharmacology,Nanjing Medical University,Nanjing 210029,Jiangsu Province,China)  
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.
【Fund】: 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)
【CateGory Index】: R392
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Chinese Journal Full-text Database 1 Hits
1 HUANG Guo-ying(Cardiovascular Heart Center,Children's Hospital Affiliated to Fudan University,Shanghai 200032,China);Role of Cardiac Neural Crest Cells in Pathogenesis of Congenital Heart Disease[J];Journal of Applied Clinical Pediatrics;2009-01
【Secondary Citations】
Chinese Journal Full-text Database 1 Hits
1 ZHAO Xiao-qing~*,(HUANG Guo-ying,) XIE Li-jian,PENG Tao,ZHOU Guo-min.~*Pediatric Heart Center,Children′s Hospital of Fudan University,Shanghai 200032,China;Abnormal development of conotruncal region in Cx43 knockout mice[J];National Medical Journal of China;2005-38
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