LKB1(Thr336)磷酸化多克隆抗体的制备及其应用

目的制备LKB1(Thr336)磷酸化多克隆抗体,并以其检测细胞中LKB1的表达。方法用生物信息学方法选取LKB1Thr336位点附近10个氨基酸,且使Thr336位点磷酸化,并铰链到BSA上。以此为抗原免疫家兔,制备抗血清,纯化后通过ELISA法检测抗体效价,Westernblot法检测抗体特异性,并用LKB1抗体和p-LKB1(Thr336)抗体分别检测正常细胞及肿瘤细胞中LKB1的表达。结果纯化后的p-LKB1(Thr336)抗体效价为1∶1000;LKB1抗体不仅识别His-LKB1蛋白,而且识别p-LKB1(Thr336)抗原,而p-LKB1(Thr336)抗体仅识别p-LKB1(Thr336)抗原;在正常细胞HL7702和HEK293中,LKB1表达较多,而在HeLa细胞中,LKB1基本没有表达;在这3种细胞中,p-LKB1(Thr336)基本没有表达;在肿瘤细胞S180、COS-7和Caco2中,LKB1和p-LKB1(Thr336)均有表达,p-LKB1表达相对较多。结论已制备了特异性的p-LKB1(Thr336)多克隆抗体,LKB1-Thr336位点的磷酸化可能是某些肿瘤发生的特征,有望成为肿瘤诊断的指标之一。

Preparation and Application of Phosphorylated LKBI(Thr336) Polyclonal Antibody

Objective To prepare phosphorylated LKB1(Thr336)polyclonal antibody for determination of expressed LKB1 in various cells.Methods The Thr at site 336(Thr336)of LKB1 was phosporylated and the neighboring 10 amino acids were cho-sen to hinge on BSA by bioinformatic methods.The constructed peptide was used as an antigen to immunize rabbits,and the antisera was purified then determined for titer by ELISA and for specificity by Western blot.Determine the expressions of LKB1 in normal and tumor cells with prepared p-LKB1(Thr336)antibody,using LKB1 antibody as control.Results The purified p-LKB1(Thr336)an-tibody reached a titer of 1 ∶ 1 000.LKB1 antibody recognized not only His-LKB1 protein,but also p-LKB1(Thr336) antigen.However,p-LKB1(Thr336) antibody recognized only p-LKB1(Thr336) antigen.LKB1 showed high expressions in HL7702 and HEK293 cells while few expression in HeLa cells.However,few expression of p-LKB1(Thr336)was observed in the 3 kinds of cells.Both LKB1 and p-LKB1(Thr336) were expressed in S180,COS-7 and Caco2 cells,of which the expression level of p-LKB1(Thr336)was relatively high.Conclusion Specific p-LKB1(Thr336)polyclonal antibody was prepared.The phosphorylation of LKB1(Thr336)might be used as an index for diagnosis of tumors.