用酵母双杂交系统筛选抗人p53单链抗体  

Screening of special sc Fv antibody against human p53 protein by yeast two-hybrid system

作　　者：郑美云[1] 李妙君[1] 沈国婴 林素颖 李恢波 孟春[1] 王航[1] 

机构地区：[1]福州大学生物科学与工程学院,福建福州350108

出　　处：《细胞与分子免疫学杂志》2016年第1期112-117,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：迈新病理基金资助(m1206)

摘　　要：目的采用重叠PCR技术,构建小鼠抗人p53基因的全套单链抗体(sc Fv)文库,利用酵母双杂交系统筛选抗人p53sc Fv。方法构建表达p53诱饵载体p GBKT7-p53,提取P53蛋白免疫的小鼠脾脏组织总RNA,反转录PCR和重叠PCR方法构建VH-linker-VL型sc Fv基因库。将其与载体p GADT7连接,产物转化至含诱饵载体的AH109酵母菌中,于营养缺陷型平板中筛选阳性克隆。采用免疫细胞化学染色方法验证sc Fv与人P53蛋白的亲和力。结果成功构建诱饵质粒p GBKT7-p53并转入酵母AH109细胞,其在酵母细胞中无自激活能力,对宿主菌亦无毒性;成功获取VH-linker-VL型的抗体文库并构建捕获载体,利用酵母双杂交系统成功筛选到抗人P53 sc Fv片段;sc Fv1、sc Fv2和sc Fv3与人P53蛋白间均具有一定亲和力,可用以检测细胞、组织中的P53蛋白。结论利用酵母双杂交系统,获得具有良好的亲和力抗人P53 sc Fv,为筛选sc Fv提供新思路。Objective To construct a mouse single-chain fragment variable（ sc Fv） antibody library specific to human P53 by overlapping PCR method and screen the single chain antibodies against human P53 protein with the yeast two-hybrid（ Y2H） system. Methods The bait vector p GBKT-p53 expressing P53 protein in yeast AH109 cells was constructed by means of genetic engineering method. The total RNA which was extracted from the P53-immunized mouse spleen tissue was used to synthesize the single chain VH-linker-VLfragment by reverse transcription-PCR and overlapping PCR. And then the VH-linker-VLfragment constructed on the vector p GADT7 was transformed into the AH109 cells containing the bait vector. The positive clones were screened on the nutrition auxotrophic media. The characteristics of sc Fv were verified by immunocytochemistry. Results The bait vector p GBKT7-p53 was constructed successfully and transformed into AH109 cells. It had no self-activation in the yeast cells and no toxicity to the host. The library of VH-linker-VLwas successfully obtained. The captured vector harboring VH-linker-VLfragments was constructed. We screened out the human P53 sc Fvs by the Y2 H system. And sc Fv1 /2 /3 was proved to have a good affinity for human P53 protein,so it could be used for the identification of P53 protein in cells and tissues. Conclusion We obtained human P53 sc Fvs with a good affinity for human P53 protein by Y2 H system,which provided a new way for screening sc Fv.

关 键 词：P53 单链抗体 抗体库 酵母双杂交 

分 类 号：Q939[生物学—微生物学] R392.11[医药卫生—免疫学]