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目的通过凝胶免疫法制备抗狂犬病病毒糖蛋白单链抗体Fv57的多克隆抗体。方法诱导表达Fv57蛋白,SDS-PAGE分离后,分别以KCl和考马斯亮蓝R-250染色,切取含目的蛋白的凝胶,经背部皮下免疫家兔,共免疫4次。第4次免疫后1周采血,分离血清,Western blot分析Fv57蛋白多克隆抗体的特异性,ELISA检测抗体效价。结果表达的Fv57蛋白相对分子质量约26 000;两种染色方法制备的多抗均能与Fv57蛋白特异性结合,与考马斯亮蓝R-250染色法相比,KCl法的非特异性杂带较少,结合效果更好;两种方法制备的多抗效价均在1∶10 000以上。结论 已成功采用凝胶免疫法制备了Fv57多克隆抗体,为检测Fv57与狂犬病病毒糖蛋白的特异性结合奠定了基础。
Objective To prepare polyclonal antibodies against Fv57, an anti-rabies virus glycoprotein single-chain antibody, by gel immunoassay. Methods The Fv57 protein was induced by SDS-PAGE and stained with KCl and Coomassie Brilliant Blue R-250, respectively. The gel containing the target protein was cut out and subcutaneously immunized rabbits for 4 times. One week after the 4th immunization, blood was collected and serum was separated. The specificity of the polyclonal antibody to Fv57 protein was analyzed by Western blot. The antibody titer was detected by ELISA. The results showed that the relative molecular mass of Fv57 protein was about 26 000. The polyclonal antibodies prepared by both methods could specifically bind to Fv57 protein. Compared with the Coomassie Brilliant Blue R-250 staining method, the non-specific heterozygosity of KCl method was less, The binding effect is better; the multi-antibody titer prepared by the two methods is more than 1:10 000. Conclusion Fv57 polyclonal antibody was successfully prepared by gel immunoassay, which laid the foundation for the detection of the specific binding of Fv57 to rabies virus glycoprotein.