Ag85B真核表达载体的构建及其生物活性的研究  

Construction of eukaryotic expression vectors for Ag85B with biological activity

作　　者：吴学忠[1] 魏海明[2] 刘文涛[2] 张小俊[2] 田志刚[2] 

机构地区：[1]山东省皮肤病性病防治研究所,济南250022 [2]中国科学技术大学生命科学学院免疫研究所,合肥230027

出　　处：《中国麻风皮肤病杂志》2006年第10期800-803,共4页China Journal of Leprosy and Skin Diseases

基　　金：国家自然科学基金杰出青年基金项目(30125038);国家重点基础研究发展计划项目(2001CB510009)

摘　　要：目的:研究Ag85B蛋白促PPD+人外周血单个核细胞的增殖活性。方法:用PCR技术扩增Ag85B基因,构建重组pcDNA3-Ag85B,并将其转染B16细胞株,用RT-PCR鉴定阳性细胞克隆,用Westernblotting鉴定其在阳性细胞克隆内的表达,用3H-TdR掺入法来检测Ag85B的促增殖活性。结果:获得了阳性B16细胞克隆,并鉴定在阳性细胞克隆内有Ag85B成熟蛋白表达;此阳性细胞克隆对PPD+人的PBMC具有较明显地促PBMC增殖活性。结论:转染了pcDNA3-Ag85B的B16阳性细胞克隆对PPD+人的PBMC具有较明显地促PBMC增殖活性,为Ag85B诱发抗肿瘤免疫应答及其机制的研究奠定了基础。Objective： To study the proliferation - activity of Ag85B protein stimulating the PBMCs of PPD^＋ human donors, Methods： The gene encoding Ag85B mature protein was amplified by polymerase chain reaction （PCR）, and recombinant vector （pcDNA3） was reconstructed and transfected into B16 cell lines. The positive cell clones by RT - PCR and its protein expression was measured by Western blotting. The PBMCs＇ proliferation was analyzed by ：^3H - TdR after the stimulation of positive cell clones in the PBMCs of PPD ^＋ and PPD^- human donors. Results： The positive B16 cell clones were obtained and identified by Western blotting the Ag85B mature protein expressed in the clones of positive cells, which could stimulate the PBMCs＇ proliferation - activity in PPD^＋ human donors, Conclusion： These modified B16/pcDNA3 - Ag85B cells significantly enhanced the proliferation of PBMCs of PPD^＋ human donors, which provided foundation to the study of Ag85B antitumor immune activity.

关 键 词：AG85B PCDNA3 B16细胞 增殖活性 

分 类 号：R346[医药卫生—基础医学]