BTLA蛋白胞外区片段的制备及鉴定  

Preparation and identification of exBTLA protein

作　　者：毛丽伟[1] 倪兵[1] 姜曼[1] 黎万玲[1] 肖宇[1] 吴玉章[1] 

机构地区：[1]第三军医大学全军免疫学研究所,重庆400038

出　　处：《免疫学杂志》2005年第2期100-103,共4页Immunological Journal

摘　　要：目的　克隆人的BTLA蛋白分子的基因胞外区片段(ExtracellularregionofBTLA ,exBTLA) ,并在Flp InCHO系统表达、纯化,用于单克隆抗体的制备或直接进行功能研究。方法　用RT PCR方法制备exBTLA基因,以哺乳细胞高效表达质粒载体pSec WG为载体构建重组融合exBTLA IgFc pSec WG质粒。重组体经双酶切鉴定后,再通过测序鉴定克隆的正确性。采用脂质体法将重组质粒转染Flp InCHO细胞,G蛋白亲和层析法纯化蛋白。用Westernblotting检测BTLA基因胞外区表达的特异性。结果　正确构建了exBTLA pSec WG重组质粒,并在转染细胞所分泌的上清液中检测出了蛋白片段的表达。利用亲和层析法成功纯化出特异的exBTLA蛋白。结论　成功地构建了exBTLA pSec WG重组质粒,纯化出exBTLA蛋白,为下一步mAb制备及其功能研究提供实验依据。Objective To clone human extracellular region of B and T lymphocyte attenuator (exBTLA) gene and purify exBTLA protein which is expressed stably in Flp-In CHO cell lines. Methods The recombinant plasmid exBTLA/pSec/WG was prepared using the exBTLA gene prepared by RT-PCR and the vector pSec/WG,which was highly expressed in mammal cells. After the double-restriction-enzyme digestion, the recombinant was sequenced and the transformed into Flp-In CHO cell lines with Lipofectamine 2000. The culture medium of these recombinant cells was pooled and the target protein was purified with protein G-Sepharose 4B column. The correctness of exBTLA protein expression was checked by Western-blot method. Results The recombinant exBTLA/pSec/WG plasmid was constructed correctly and the expression of exBTLA gene in transformed Flp-In CHO cell lines was determined. The exBTLA protein was purified successfully by protein G-Sepharose 4B column. Conclusion We have constructed the recombinant exBTLA/pSec/WG plasmid and purified the exBTLA protein successfully, which could provide experimental data for the preparation of the mAb and the direct functional analysis of this protein.

关 键 词：BTLA RT-PCR 克隆 

分 类 号：R392.11[医药卫生—免疫学]