CIP29对布鲁氏菌侵袭和巨噬细胞凋亡影响的初步研究  

Effects of CIP29 in macrophage apoptosis and infection of brucella in THP-1 cells

作　　者：吴小艳[1] 赵忠鹏[1] 张良艳[1] 王希良[1] 

机构地区：[1]军事医学科学院微生物与流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《免疫学杂志》2008年第1期106-110,共5页Immunological Journal

基　　金：国家自然科学基金资助项目(30170853)

摘　　要：目的研究CIP29在细胞中的定位及其对巨噬细胞凋亡和布鲁氏菌侵袭的影响。方法通过RT-PCR方法从细胞中获取CIP29基因片段,并将其亚克隆入质粒载体pET28a和eGFP,构建原核表达质粒pET28a-CIP29和真核表达质粒eGFP-CIP29,原核表达质粒用于制备CIP29抗体,用于后续CIP29蛋白的检测;真核表达质粒用于转染巨噬细胞,通过荧光显微镜、Western blot技术检测CIP29在真核细胞中的表达及定位,Annexin-Ⅴ/PI双染检测其对细胞凋亡的影响,布鲁氏菌侵袭试验检测其对布鲁氏菌入侵巨噬细胞的影响。结果获得了CIP29基因,构建的表达质粒pET28a-CIP29和eGFP-CIP29经测序和双酶切鉴定,证实重组表达质粒构建成功。将pET28a-CIP29转化BL21中,CIP29蛋白得到了表达,制备了特异性抗体,经ELISA检测效价达1∶25600。进一步构建真核表达质粒eGFP-CIP29,转染细胞后,荧光显微镜观察结果显示其定位于核内,但也有部分扩散之胞质中,CIP29可诱导细胞的凋亡,但对布鲁氏菌侵袭的影响无统计学意义。结论本研究获得CIP29重组蛋白,并制备了其多克隆抗体,CIP29瞬时高表达诱导巨噬细胞凋亡。Objective To observe CIP29 location in cells and study its function in apoptosis of macrophages for investigating the influence of CIP29 on infection of brucella in eukaryotic cells. Methods CIP29 gene fragment was obtained by RT-PCR from THP-1 cells. The recombinant expression vectors were constructed by inserting the gene into expression vector pET28a and eGFP, and then expressed in E. coli 21 and THP-1, respectively. Rabbits were immunized with the protein and the titers of the antibodies were analyzed. The eukaryotic expression vector was transfected into THP-1 cell to observe the location and expression in cells by fluorescence microscopy and westernblotting. Function of Cfl：＇29 in apoptosis of macrophages and its influence to infection of brucella were evaluated by Annexin-V/PI double staining and related technology. Results A DNA fragment was obtained by RT-PCR and the recombinant plasmids were constructed, which named pE328a-CIP29 and eGFP-CIP29. The titers of the antibodies were 1 ： 25600 detected by ELISA. The eukaryotic expression vector eGFP- CIP29 was expressed in nucleus and diffused to cytoplasm observed by fluorescence microscope. Conclusion CIP29 can induce apoptosis of cells but have no effects to infection of brucella. The expression and specific antibody preparation of human CIP29 gene provide a base for detecting CIP29 molecule.

关 键 词：巨噬细胞 CIP29 布鲁氏菌 凋亡 

分 类 号：R516.7[医药卫生—内科学]