抗乙酰胆碱受体单链抗体酵母表达的特性鉴定

Characterization of expressed anti-cetylcholine receptor single chain variable fragment in Pichia pastoris

[目的]探讨毕赤酵母GS115表达的重症肌无力单链抗体A7的一般特性及与大鼠肋间肌乙酰胆碱受体的结合特异性.[方法]采用SDS-聚丙烯酰胺凝胶电泳和蛋白印迹试验,检测已经转化至毕赤酵母GS115中的重症肌无力重组载体pPIC9K-ScFvA7表达的单链抗体A7蛋白的分子质量;应用间接免疫荧光技术确定表达蛋白与大鼠肋间肌乙酰胆碱受体的亲和性.[结果]毕赤酵母GS115培养上清液中有目的蛋白的表达,分子质量约为44ku;间接免疫荧光试验显示,在大鼠肋间肌细胞间有荧光出现.[结论]单链抗体A7可以在毕赤酵母中成功地表达,并且可与大鼠肋间肌乙酰胆碱受体结合.

OBJECTIVE To determine the specificity of expressed single chain variable fragment （ScFv） A 7 of antibody against acetylcholine receptor （AChR） in myasthenia gravis in Pichia molecular weight of expressed protein （ScFvA 7） was analyzed by sodium dodec pa yl storis. METHODS The sulfate-olyacrylamide gel electrophoresis （SDS-AGE） and Western blotting. The affinity of expressed protein to AChR in mouse intercostals muscles was detected using indirect immunofluorescence technology. RESULTS The target protein was secreted in the supernatant of expression culture medium. Its molecular weight was about 44 ku. There was fluorescence between the mouse intercostals muscle cells. CONCLUSION The target protein has been expressed from Pichia pastoris GS 115 successfully and it can bind to the AChR in mouse intercostals muscles.