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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。
OBJECTIVE: To immunize rabbits with artificial epitopes of 4 subunits of L-type calcium channel in rat ventricular myocytes to generate specific antibodies that can recognize different subunits of this channel and to verify them. Methods: Three short peptides were selected from the amino acid sequences of 4 subunits of L-type calcium channel in rat ventricular myocytes and synthesized. Antibodies were isolated and purified by immunization of rabbits with chemically synthesized peptides linked to tetanus toxoid (TT) by glutaraldehyde method and immunization of rabbits with anti-rat serum of L-type calcium channel. Antibody validation was detected by ELISA, Western blot, and immunofluorescence histochemistry. Results: The antibody titers of α1C, β2 and α2 / δ subunits of L-type calcium channel in rat ventricular myocytes were 1:51 200 ~ 1:102 400, 1:1 280 and 1: 280 respectively The molecular weights of the three antibodies that bind to the membrane proteins of rat myocardial cells were 2358, 9 and 162 kD, respectively. Immunofluorescence histochemistry experiments showed that the antibodies were able to bind specifically to rat ventricular myocytes membrane. CONCLUSION: The prepared L-type calcium channel subunit antibody against rat ventricular myocytes can specifically recognize different subunits of L-type calcium channel in rat ventricular myocyte membrane.