大鼠坐骨神经cDNA文库的抗体筛选法  

SCREENING cDNA LIBRARY OF RAT SCIATIC NERUE WITH ANTIBODIES

作　　者：印其友[1] 谭湘陵[1] 曹铮[1] 张沛云[1] 严志强 顾晓松[1] 

机构地区：[1]南通医学院附属医院外科

出　　处：《南通医学院学报》1998年第1期6-8,共3页ACTA Academiae Medicinae Nantong

基　　金：国家杰出青年科学基金

摘　　要：以成年ＳＤ大鼠坐骨神经损伤后的远段为材料，以λｇｔ１１为载体，构建了ＳＤ大鼠损伤性坐骨神经的ｃＤ－ＮＡ基因文库。将文库以５×１０４／１２０ｍｍ培养皿的密度铺皿培养，８ｈ后将印迹的硝酸纤维素膜与特异性６５ＫＤ单克隆抗体混合物４℃孵育过夜。行免疫组化ＡＢＣ反应，筛选４×１０５克隆后得到３个阳性信号点。挑出相应的克隆，进行第２、３轮筛选，得到表达６５ＫＤ化学诱向因子的单克隆。In an attempt to identify the gene of the novel neurotropic factor of 65KD,provided by injuried nerve, A cDNA library was constructed with the distal stump after sciatic nerve lesion and the vector of λgt11 was used to form an expression library. The infected plate with the density about 4×104 plaques/120mm plate, overlayed with nitrocellous filtres treated by IPTG before, were incubated for about 8 hours at 37℃. Then the filtres absorbed with fusion protein were peeled off the plate followed by washing with PBS buffer, incubating with the nixed monoconal antiboties against the neurotropic factors of 65KD overnight at 4℃。Then the fitres were treated according to the guidances of ABC kit. Three clones were obtained firstly after a screening of 4×105 recombinant. But only one clone was isolated in the followng four cycles of screening. The result showed that the construction of the cDNA library atributes to the study of the mechanism of peripheral nerve regeneration at molecular level.

关 键 词：CDNA文库 抗体筛选 坐骨神经 

分 类 号：R392.11[医药卫生—免疫学]