LRRC4多抗制备及在构建不同级别脑胶质瘤差异表达中的应用  

Preparation of anti-LRRC4 polyclonal antibody and its application in constructing expression profile of human gliomas with different pathological grades

作　　者：李丹 武明花[1] 陈琼[1] 黄河[1] 黄琛[1] 李伟松[1] 李小玲[1] 李桂源[1] 

机构地区：[1]中南大学肿瘤研究所,长沙410078

出　　处：《中南大学学报（医学版）》2007年第3期373-379,共7页Journal of Central South University ：Medical Science

基　　金：国家重大科学研究计划(2006CB910502;2006CB910504);国家自然科学基金(30600224);湖南省自然科学基金(06JJ20080);中国博士后科学基金(20060400265)~~

摘　　要：目的:制备兔抗脑瘤候选抑瘤基因LRRC4(leucine-rich repeats containing 4)的多克隆抗体,应用该抗体检测LRRC4表达与脑胶质瘤病理分级的相关性。方法:应用DSGene1.1软件分析LRRC4全长蛋白,避开跨膜结构域,选取亲水性及抗原性均好的蛋白序列。构建相应的融合表达载体pGEX-4T-2/276bp,转入E.coliJM109中IPTG诱导表达。融合蛋白经纯化后免疫新西兰兔,收集并纯化抗血清,获得兔抗人LRRC4多抗,应用该抗体通过Western印迹和免疫组织化学检测LRRC4在正常人胚胎各器官组织中的表达及其在正常脑组织与各级脑胶质瘤中的表达差异。结果:制备了效价高、特异性好的兔抗人LRRC4多抗,进一步证实了LRRC4在正常人脑组织中相对特异高表达,在脑胶质瘤中表达下调(22例/37例)或缺失(15例/37例),其表达水平与脑胶质瘤的病理分级相关。结论:兔抗人LRRC4多抗效价高、特异性好,应用该抗体检测到LRRC4的表达水平与脑胶质瘤病理分级密切相关,为LRRC4后续的研究工作奠定了基础。Objective To prepare anti-LRRC4 polyclonal antibody and analyze the correlation between the expression of LRRC4 and pathological grades of gliomas in rabbits. Methods Appropriate protein sequence with good hydrophilicity and antigenicity was chosen by analyzing with DS Gene 1. 1 software. The corresponding nucleic acid sequence amplified by PCR was used to construct a recombinant pGEX-4T-2/276 bp. E. coli JM109 transformed with the recombinant was induced by IPTG to express GST- fusion protein, and the fusion protein expressed as insoluble inclusion bodies. Then the purified inclusion body was used to immunize rabbits. Once the titer of antiserum reached 1：10s by indirect ELISA, the serum was collected and purified. The expression-profile of LRRC4 inembryonic tissues and gliomas with various pathological grades were obtained by western blot and immunohistochemistry with the anti-LRRC4 polyclonal antibody. Results The highly specific anti - LRRC4 polyclonal antibody whose titer reached 1： 10Swas prepared. The relatively specific expression of LRRC4 was detected in the normal brain, but reduced expression or loss of expression in gliomas was also noticed by immunohistochemistry, and there was a correlation between the expression level of LRRC4 and the pathological grade of gliomas. Conclusion The anti-LRRC4 polyclonal antibody with high titer and specificity has been obtained. A correlation between the expression level of LRRC4 and the pathological grade of gliomas is detected, which lays the foundation for advanced research of LRRC4.

关 键 词：LRRC4 多克隆抗体 脑胶质瘤 病理分级 

分 类 号：R739.4[医药卫生—肿瘤]