【摘要】 目的:用人工合成的大鼠心室肌细胞L-型钙通道4个亚单位的抗原表位短肽免疫家兔,产生可识别该通道不同亚单位的特异性抗体,并进行验证。方法:从大鼠心室肌细胞L-型钙通道4个亚单位的氨基酸序列中筛选出三段短肽,进行人工合成。用化学合成的多肽与破伤风类毒素(TT)以戊二醛法连接,并以此作为抗原免疫家兔制备抗大鼠心室肌细胞L-型钙通道的免疫血清,经分离纯化获得抗体。抗体的验证通过ELISA,Western blot,免疫荧光组织化学技术进行检测。结果:所制备的抗大鼠心室肌细胞L-型钙通道α1C、β2和α2/δ亚单位的抗体滴度分别为1∶51 200~1∶102 400、1∶1 280和1∶1 280,3个抗体结合到大鼠心肌细胞膜蛋白的分子量分别为2358、9和162 kD。免疫荧光组织化学实验显示,抗体均能特异性的结合在培养大鼠心室肌细胞膜上。结论:所制备的抗大鼠心室肌细胞L-型钙通道亚单位抗体能特异性识别大鼠心室肌细胞膜上L-型钙通道的不同亚单位。更多还原

【Abstract】 Objective:To prepare and confirm special antibodies against L-type calcium channel subunits in rat ventricular myocytes through immunizing rabbit with synthetic short peptides that can represent the possible antigen epitope of L-type calcium channel subunits.Methods:Screening three peptides fragments from amino acid sequences of L-type calcium channel subunits in rat ventricular myocytes and synthesizing them,then synthetic peptides were coupled to tetanus toxoid（TT） to form three complexes of peptide-TT.The anti sera were developed by immunizing rabbit with peptide-TT.After extracting and purifying the sera,the specificity of purified anti sera were detected by enzyme-linked immunosorbent assay,Western blot and immunofluorescence technique.Results:The titers of the prepared antibodies of L-type calcium channel subunits α_1C,β₂,α₂δ reached to around 1∶51 200-1∶102 400,1∶1 280 and 1∶1 280,respectively by ELISA.Three antibodies bound in the site of the protein bands of 235,89 and 162 kD respectively by Western blot.In immunofluorescence,the stain was specially collocated with membranes of cultured rat ventricular myocytes.Conclusion:Three prepared antibodies against L-type calcium channel subunits in rat ventricular myocytes can specially recognize respective subunits of L-type calcium channel in rat ventricular myocytes.更多还原