一种鉴别免疫筛选噬菌体克隆真、假阳性的方法

A Method for Confirming the Phages by Immunoscreening

目的探讨及早鉴别用免疫学方法筛选cDNA文库得到的阳性克隆是否真实可靠的方法.方法将阳性噬菌体克隆与宿主菌按一定的比例诱导培养后,取沉淀做SDS-PAGE后行W estern b lot;同时取出一个阳性克隆进行亚克隆,直至3次亚克隆均为全部阳性.结果3次亚克隆均为全部阳性的噬菌体克隆,在W estern b lot鉴定中也呈阳性反应.结论可用W estern b lot方法来鉴别用免疫学方法筛选cDNA文库得到的阳性克隆是否真实可靠.

Objective To find a new method for confirming whether the phages previously in immunescreening cDNA library are true or false. Methods Immunoscreening for the detection of reactive clones in the H128 cDNA expression library, which was constructed in expression vector lambda gt11 with SCLC cell line H128 by using mAb N - 35. Then subcloning was performed after obtaining the positive clones. Additional work was done to identify these clones by PCR and Western blot. After these positive phages were got by immunescreening, they were cultured with their hosts in proportion as 1 ： 50 for some time. Then the precipitate was used for Western blot. At the same time, a positive phages clone was used for sub - cloning till those clones derived from it were all positive through 3 sub - cloning,. Result The true positive clone in 3 sub - cloning was showed as positive reaction by Western blot Conclusion Western blot could be used for confirming the truth of the phages by immunoscreening.