癌基因LMO3的原核表达及多克隆抗体的制备及应用  

Prokaryotic expression,polyclonal antiserum preparation and application of oncogene LMO3

作　　者：惠玲[1] 石洁[1,2,3] 王晓辉[1] 吕同德[1] 杨金升[2] 哈小琴[1] 

机构地区：[1]兰州军区兰州总医院医学实验中心,甘肃兰州730050 [2]兰州军区兰州总医院神经内科,甘肃兰州730050 [3]兰州大学附属第二医院输血科,甘肃兰州730030

出　　处：《中华肿瘤防治杂志》2009年第11期817-820,共4页Chinese Journal of Cancer Prevention and Treatment

基　　金：中国博士后科学基金(20070410230)

摘　　要：目的:制备抗神经特异性表达的癌基因LMO3的多克隆抗体,进行LMO3分子检测和功能研究。方法:采用RT-PCR方法扩增LMO3的基因序列,构建其原核表达载体pET32a-LMO3。在大肠埃希菌中诱导融合蛋白表达,SDS-PAGE分离纯化原核表达的融合蛋白,PBS溶解聚丙烯酰胺凝胶颗粒中的融合蛋白,乳化后免疫新西兰兔制备多克隆抗体。采用免疫印迹、免疫组化等对该抗体的特异性进行鉴定。结果:构建的重组质粒经酶切鉴定和测序证实序列正确。经诱导表达在相对分子质量为37×103处有一条明显的蛋白条带,与预期值一致;免疫动物所得抗血清效价为1∶16 000,该抗体能够识别原核表达的LMO3蛋白及细胞内源性表达的LMO3蛋白;免疫组化显示,LMO3在SHG44胶质瘤细胞株及脑胶质瘤组织中均有表达。结论:制备了能识别天然LMO3分子的抗LMO3的多克隆抗体,为检测LMO3分子的表达和进一步研究其功能提供了有力的工具。OBJECTIVE： To preparate polyclonal antibody of anti-LMO3, an oncogene of the neuronal specific expression in the nervous system, which can be used for further identification and function study of LMO3 molecule. METHODS： LMO3 was ampli- fied by RT-PCR and cloned into the prokaryotic expression vector pET-32a. Recombinant plasmid pET32a-LMO3 was expressed in E. coli after induction with IPTG. A fusion protein was identified and purified by SDS PAGE. The gel slice corresponding to the fu- sion protein was crushed and dissolved by PBS and emulsified with Freund＇s adjuvant. Rabbits were immunized with the emulsified mixture and the collected and purified rabbit antiserum was evalua- ted by Western blot and immuno histochemica] techniques. RE- SULTS：The coding sequence of LMO3 was successfully inserted in- to pET-32a. After induction, a Mr 37 × 10^3 fusion protein was ex pressed and confirmed by SDS-PAGE, which was identical to that expected. The titers of the antiserum were up to 1 ： 16 000. This antibody could specifically recognize the LMO3 protein expressed in the E, coli system and SHG44 cells. Immuno-histochemical staining indicated that LMO3 was expressed in SHG44 cells and glioma. CONCLUSION：Successful preparation of anti-LMO3 polyclonal an tibody provides a useful tool in identification or further functional study of LMO3 molecule.

关 键 词：基因 LMO3 原核表达 抗体 多克隆 

分 类 号：R73-3[医药卫生—肿瘤]